Category Archives: Part 2 – Antiretroviral Therapy

ART 2011
Managing Side Effects
Lipodystrophy Syndrome
Mitochondrial Toxicity of NRTIs
HIV Resistance and Viral Tropism Testing

6.11. Monitoring

– Christian Hoffmann, Christian Noah –

Which parameters should be included in routine laboratory monitoring of HIV-positive patients? What results can be expected? This section deals with viral load, CD4 T cells, routine checks, and plasma levels. Resistance and tropism tests are the subject of a separate chapter (see HIV Resistance Testing). For the tests to be performed on initial presentation see The New Patient.

Viral Load

Viral load is the amount of HIV RNA in the blood. Alongside the CD4 T cell count, viral load has become the most important surrogate marker for HIV infection (Hughes 1997, Mellors 1997, Lyles 2000, Ghani 2001, Phillips 2004). It provides information on how high the risk is for disease progression and whether antiretroviral therapy is indicated; it is the critical value in determining the success of therapy. Viral load assays measure the amount of HIV RNA (viral genetic material), which correlates directly with the number of virions. The units are viral copies/ml (or genome equivalents). This is reported either as a direct whole number or as a logarithmic number. A change of one or more logs refers to the change in viral load by one or more decimal powers. Many labs provide both values, the number and the log. There is no standardized international unit/ml as in used in hepatitis B or C.

Number of copies Log10
10 1.0
50 1.7
100 2.0
500 2.7
1000 3.0
10,000 4.0
50,000 4.7
100,000 5.0
1,000,000 6.0

Assessment

The higher the viral load, the higher the risk of decrease in CD4 T cells, with subsequent disease progression or occurrence of AIDS-related illnesses (Mellors 1997, Lyles 2000, Phillips 2004). A viral load above 100,000 copies/ml (sometimes even above 50,000 copies/ml) is considered to be high; a value below 10,000 copies/ml (sometimes below 5000 copies/ml), low. However, these thresholds are not absolute and only provide points of reference.

The effects of plasma viremia on immune status can vary greatly between individuals. There are some patients whose CD4 T cells remain stable for relatively long periods despite having a high viral load, while others experience a rapid drop, although the viral load is relatively low. Even in the so-called elite controllers in which the viral load is undetectable without ART a slow but constant drop in the CD4 cells can be observed (Stellbrink 2008).

Viral load is probably lower in women than in men. In a meta-analysis, the difference was 41% or 0.23 logs (95% CI 0.16-0.31 logs) (Napravnik 2002). The reason for this phenomenon remains unclear and whether it should have an impact on the indication for treatment is still the subject of debate.

Methods

Three methods or assays are currently used to measure viral load: Reverse Transcription-Polymerase Chain Reaction (RT-PCR); branched-chain DNA (bDNA); and, occasionally, Nucleic Acid Sequence-Based Amplification (NASBA). These three methods differ both in levels of detection and in the linear range within which measurement is reliable or reproducible (see Table 11.1). In the case of PCR and NASBA, the viral RNA is transformed in several enzymatic steps and then amplified to measurable amounts. Detection occurs after binding of marked DNA fragments. bDNA does not require an enzymatic step; signal amplification occurs via binding of branched DNA fragments to viral RNA.

The market for assay systems is very dynamic. New assay systems will become available, existing ones further developed. Siemens, for example, offers an RT-PCR in addition to bDNA technology. Roche concentrates on RT-PCR and is working on additional functions such as “dual-target detection” for more successful results. This means that not one section of the viral RNA, like before, but two sections can be duplicated at the same time. If duplication fails in one section on account of the high variability of the HIV genome (the result in this case would be incorrect negative), it will be duplicated in the second section. Besides already established manufacturers, newer companies such as Qiagen are trying to gain market share. Experience will show whether their testing systems are reliable or not.

Recent further developments also concern a reduction below detection level which is at 20 copies/ml in the most sensitive tests. Clinical relevance of a viral load below 50 copies/ml is questionable due to lack of data. It should be noted that a higher sensitivity can lead to insecurity in patients and clinicians and to more frequent control tests.

Although intra-assay variability is fairly good for all three methods, methodological variations should be carefully considered. Differences of less than 0.5 logs are not considered significant. A decrease from 4.3 to 3.9 logs, for example (corresponding to a decrease from approximately 20,000 to 8,000 viral copies/ml) does not necessarily signify a drop in viral load. The same holds for increases in viral load. Changes of up to threefold can therefore be irrelevant. Patients should be made aware of this.

Considerable differences exist between the methods (Coste 1996) and to change from one method to another is therefore generally not advisable. The results obtained by bDNA are usually lower than the PCR by a factor of 2. Different subtypes are also detected with varying success according to the method employed (Parekh 1999). One should be particularly cautious in patients from Africa and Asia with non-B subtypes in whom the viral load at first presentation can be unexpectedly low. In such cases, use of a different assay may actually be indicated. However, newer versions with improved primers are probably superior in measuring even unusual HIV subtypes with adequate sensitivity.

All assays have a linear dynamic range, outside of which precise numbers are not so reliable. The following rule applies: use one method, one laboratory. The laboratory should be experienced and routinely perform a sufficiently large number of tests. Measurement should take place as soon as possible after blood withdrawal, and correct collection and shipping of centrifuged plasma is also important (contact the laboratory ahead of time on these issues).

Tabelle 11.1. Methods of measurement.
Company

Test

Technology

Detection limit (co-pies/ml)

Linear Range (copies/ml)

Roche Diagnostics

COBAS TaqMan HIV-1 Test; v2.0

RT-PCR

20

20–10,000,000

Siemens Healthcare Diagnostics

Versant HIV-1 RNA 1.0 Assay (kPCR)

RT-PCR

37

37–11,000,000

Abbott Molecular

Abbott RealTime HIV-1

RT-PCR

40

40–10,000,000

Siemens Healthcare Diagnostics

Versant HIV-1 RNA 3.0 Assay (bDNA)

bDNA

65

50–500,000

Biomérieux

NucliSENS EasyQ HIV v. 2.0

NASBA

250

25–7,900,000

Influencing factors

Apart from methodological variability a host of other factors may influence levels of viral load including vaccinations and concurrent infections. During active OIs viral load is often high. One study showed a 5- to 160-fold elevated viral load during active tuberculosis (Goletti 1996). Viral load can also increase significantly during syphilis and declines after successful treatment (Buchacz 2004, Kofoed 2006, Palacios 2007). In a large retrospective study, 26% of transient viremia in patients on ART were caused by intercurrent infections (Easterbrook 2002). In these situations, determining the viral load does not make much sense.

Following immunizations, for instance for influenza (O’Brien 1995) or pneumococcus (Farber 1996), the viral load may be transiently elevated (Kolber 2002). As the peak occurs one to three weeks after immunization, routine measurements of viral load should be avoided within four weeks of immunization. It should be noted that not every increase is indicative of virologic treatment failure and resistance. Slight transient increases in viral load, or blips, are usually of no consequence, as numerous studies in the last few years have shown (see chapter on Goals and Principles of Therapy). The possibility of mixing up samples always has to be considered. Unusually implausible results should be double-checked with the laboratory, and if no cause is found there, they need to be monitored – people make mistakes. Should there be any doubt on an individual result; the lab should be asked to repeat the measurement from the same blood sample.

Viral kinetics on ART

The introduction of viral load measurement in 1996-1997 fundamentally changed HIV therapy. The breakthrough studies by David Ho and his group showed that HIV infection has significant in vivo dynamics (Ho 1995, Perelson 1996). The changes in viral load on antiretroviral therapy clearly reflect the dynamics of the process of viral production and elimination. The concentration of HIV-1 in plasma is usually reduced by 99% as early as two weeks after the initiation of ART (Perelson 1997). In one large cohort, the viral load in 84% of patients was already below 1000 copies/ml after four weeks. The decrease in viral load follows biphasic kinetics. In the first phase, i.e., within the first three to six weeks, an extremely rapid drop occurs, followed by a longer phase during which the viral load gradually decreases further (Wu 1999).

The higher the viral load at initiation of therapy, the longer it takes to drop below the level of detection. In one study, the range was between 15 days with a baseline viral load of 1000 and 113 days with a baseline of 1 million viral copies/ml (Rizzardi 2000). The following figure shows a typical biphasic decrease in viral load after initial high levels.

Numerous studies have focused on whether durable treatment success can be predicted early (Thiabaut 2000, Demeter 2001, Kitchen 2001, Lepri 2001). In a study on 124 patients, a decrease of less than 0.72 logs after one week was predictive of virologic treatment failure in more than 99% of patients (Polis 2001). According to another prospective study, it is possible to predict virologic response at 48 weeks even after 7 days (Haubrich 2007). However, this has little clinical relevance, and in our opinion it is pointless to start measurement of viral load only one or two weeks after initiation of therapy.

 

Figure 1: Typical biphasic decrease in viral load on ART. Viral load was initially very high, and reached a level below 50 copies/ml only at week 32. Note the temporary increase at week 24, which is possibly due to methodological variability. ART was not changed.

We recommend to measure viral load every four weeks until it has dropped to below detection of 20-50 copies/ml. Once that is achieved, measurement every three to four months is enough. Eventually, longer intervals are possible (Chaiwarith 2010). In case of rebound, closer monitoring becomes necessary. Within the first 4 weeks of therapy initiation the viral load should be reduced by a factor of 100, after 3-4 months (6 months if viral load was high) it should be below the level of detection.

Viral load can also be measured fairly reliably in body fluids other than blood or plasma (for example cerebrospinal, vaginal or seminal fluid). However, such tests are usually performed for scientific purposes and are not officially licensed for other reasons.

Practical tips for dealing with viral load (see chapter Goals and Principles of Therapy)

  • Use only one assay, if possible.
  • Use only one experienced laboratory, if possible, no home-brewed assays.
  • Watch for assay variability (up to half a log) and explain this to the patient.
  • Monitor viral load every four weeks with new ART until the viral load is below the level of detection (50 copies/ml).
  • Then measure viral load sparingly – on successful ART every three months may be sufficient.
  • Not on ART, measurement every three months is usually sufficient.
  • Do not measure shortly after vaccinations or with concurrent infections.
  • Implausible results should be rechecked after 2-4 weeks.
  • Consider differences between subtypes (in some cases it may be useful to use another method).

CD4 T cells

CD4 T cells are T lymphocytes that express the CD4 receptor on their surface. This lymphocyte subpopulation is also referred to as T helper cells. Alongside viral load, measurement of the CD4 T cell level is the most important parameter or surrogate marker in HIV medicine. It allows for a reliable estimate of the individual risk of developing AIDS. All HIV-positive patients should have a CD4 T cell measurement every six months. Two reference values are generally accepted: above 400-500 CD4 T cells/µl, severe AIDS-related diseases are very rare; below 200 CD4 T cells/µl, the risk of AIDS-related morbidity increases significantly with increased duration of immunosuppression. Most AIDS-related illnesses occur below 100 CD4 T cells/µl.

Several points should be considered when measuring CD4 T cells (usually by flow cytometry). Blood samples should be processed within 18 hours. The lower normal values are between 400 and 500 cells/µl, depending on the laboratory. Samples should always be sent to the same (experienced) laboratory. The same applies for viral load as for CD4 T cells: the higher the level, the greater the variability. Differences of 50-100 cells/µl are not unusual. In one study, the 95% confidence intervals with a real value of 500 cells/µl were between 297 and 841 cells/µl. At 200 CD4 T cells/µl, the 95% confidence interval was between 118 and 337 cells/µl (Hoover 1993).

Measurement of CD4 T cells should only be repeated in the case of highly implausible values. As long as the viral load remains below the level of detection, there is no need to be concerned even with decreases in CD4 T cells. In such cases, the relative values (CD4 percentages) and the CD4/CD8 ratio (ratio of CD4 to CD8 T cells) should be referred to; these are usually more robust and less prone to fluctuation. As a general point of reference, with values above 500 CD4 T cells/µl, fluctuations of more than 29% are to be expected, with less than 200 CD4 T cells/µl fluctuations of less than 14%. Individual laboratories may define the normal ranges for the relative values and the ratio differently. If there are considerable discrepancies between absolute and relative CD4 T cells, any decisions involving treatment should be carefully considered – if in doubt, it is better to check the values again. The remaining differential blood count should also be scrutinized carefully – is leucopenia or leukocytosis present?

Clinicians sometimes forget that the result of the CD4 T cell count is of existential importance for the patient. To go to the doctor and discuss the test results can involve a great deal of stress for many patients. Insensitively informing the patient of a supposedly bad result can lead to further negative results. From the start, patients must be informed about the possible physiological and method-related variability of laboratory tests. In the case of unexpectedly good results, every effort should be made to contain euphoria. In the long run, this saves time and discussions, and the patient is spared unnecessary ups and downs. We do not consider it advisable for non-physician personnel (without extensive HIV experience) to inform patients of results.

 

Figure 2: Slow decline of the absolute and relative (dashed line) CD4 T cells/µl over almost ten years in a treatment-naïve patient. Note the variations in the absolute numbers.

Once CD4 T cell counts within the normal range are reached in addition to adequate viral suppression, measurements every six months should suffice, in our opinion. The probability of CD4 T cells dropping to values below 350/µl is extremely low in such cases (Phillips 2003). Patients who might sometimes insist on more frequent monitoring of immune status can be assured that there are usually no detrimental changes in the CD4 T cell count as long as HIV remains suppressed.

Influencing factors

Several other factors can influence CD4 T cell counts apart from laboratory-related variables. These include concurrent infections, leucopenia of varying etiology and steroids or other immunosuppressive therapies. Extreme exertion, surgical procedures or pregnancy can also lead to lower values. Even diurnal variation occurs; CD4 T cells are lower at noon, and highest in the evening around 8 p.m. (Malone 1990). Psychological stress seems to play a negligible role, even though patients often assume the contrary.

Kinetics of CD4 T cells on ART

Similarly to viral load, a biphasic increase in CD4 T cells occurs following the initiation of ART (Renaud 1999, Le Moing 2002), with a rapid increase within the first three to four months and a much slower rise thereafter. In a study of almost 1000 patients, the CD4 T cell count increased by 21/µl per month during the first three months. In the following 21 months, this rate was only 5.5 CD4 T cells/µl per month (Le Moing 2002). The initial rapid increase in CD4 T cells is probably due to redistribution, which is followed by the new production of naïve T cells (Pakker 1998). Diminished apoptosis may also play a role (Roger 2002).

It is still being debated whether the immune system steadily continues its recovery even after a long period of viral suppression, or whether a plateau is reached after three to four years beyond which there is less improvement (Smith 2004, Viard 2004).

Several factors can influence the extent of immune reconstitution during ART. The degree of viral suppression is crucial – the lower the viral load, the more pronounced the effect (Le Moin  2002). The absolute increase is higher if CD4 T cell counts were high at the start of ART (Kaufmann 2000). Naïve T cells still present at initiation of therapy are a particularly important factor for long-term immune reconstitution (Notermans 1999).

Age is also important (Grabar 2004). The larger the thymus and the more active the process of thymopoiesis, the more significant the rise in CD4 T cells is likely to be (Kolte 2002); due to age-related degeneration of the thymus, CD4 T cells in older patients do not increase as much as those in younger ones (Viard 2001). However, we have seen both 20 year-old patients with very poor CD4 T cell count recovery and 60 year-old patients with very good, above average increases in CD4 T cells. The regenerative capacity of the human immune system seems to vary considerably, and no method to date has been capable of reliably predicting this capacity.

It is possible that some antiretroviral therapies such as ddI+tenofovir are associated with less immune reconstitution than others. In addition, current studies evaluate if immune reconstitution is better during treatment with CCR5 antagonists. Immunosuppressive concurrent medications should also be considered (see chapter on Goals and Principles of Therapy).

Practical tips for dealing with CD4 T cells

  • As with viral load: use only one (experienced) laboratory.
  • The higher the values, the greater the variability (consider numerous factors) – compare the relative (percentage) values and CD4/CD8 ratio with previous results.
  • Do not disconcert the patient when there are apparent decreases – if viral suppression is sufficient, the drop is usually not HIV-related. Only highly implausible results should be repeated.
  • If the viral load is below the level of detection, three-monthly measurements of CD4 T cells are sufficient.
  • In the presence of good viral suppression and normal CD4 T cells, CD4 T cells (not viral load) may also be checked less frequently.
  • CD4 count and viral load should be discussed with the physician. Do not leave patients alone with their results.

Beyond the measurement of the CD4 T cell count and lymphocyte subpopulations, a number of other assays allow detailed testing of the qualitative or functional capacity of the immune system, for example in response to specific antigens (Telenti 2002). These often cumbersome methods are not currently necessary for routine diagnostics and their use remains questionable. However, they could one day help to better describe individual immune status and, for example, identify those patients who are at risk of developing opportunistic infections despite good CD4 cell counts.

Routine checks – What else should be monitored?

Besides the CD4 T cell count and viral load several other parameters should be monitored in the HIV-positive patient. The following recommendations apply to clinically asymptomatic patients with normal results on routine laboratory evaluation, who have been on stable treatment for several months or who are not taking antiretroviral therapy. Of course, if treatment is started or changed or if the patient develops complaints more frequent monitoring is required. Depending on the problem additional tests may be necessary.

A complete physical examination should be performed regularly, and this often leads to the discovery of important findings such as Kaposi lesions or mycoses (thrush). The lower the CD4 T cells, the more frequently patients should be examined.

In patients with less than 200 CD4 T cells/µl, we usually perform fundoscopies every three to six months to exclude CMV retinitis. Close cooperation with an HIV-experienced ophthalmologist is essential. The better the CD4 T cells, the less often fundoscopies are necessary – in our opinion when CD4 counts have normalized these can be stopped completely. In contrast, regular gynecological examinations with PAP smears are recommended regardless of CD4 count. Many experts now also recommend rectal examination (including proctoscopy) for the early detection of precancerous lesions and anal cancer.

However, such guidelines or recommendations can be interpreted very differently. In our opinion in cases of good immune status unless there is a specific suspicion routine X-rays, ultrasound examinations (exception: patients with chronic hepatitis, as hepatocellular carcinoma is not rare in such cases), multiple serologies or lactate measurements are not necessary. An annual ECG is only indicated in our view in patients with a specific risk profile (see chapter on HIV and Cardiac Disease). The tuberculin test (the Mendel-Mantoux skin test with 5 IE once a year) should only be repeated if it is negative initially.

Table 11.2. Minimal evaluations per year in stable asymptomatic patients.
 

Patient on ART

per year

Untreated

per year

Blood count, LDH, ALT, AST, creatinine, bilirubin, AP, lipase, GGT, glucose

4-6 x

2-4 x

Viral load

4 x

2-4 x

CD4 T cells

2-4 x

2-4 x

Lipids

1-2 x

1 x

Physical examination, urine status

2-4 x

1-2 x

Gynecological examination

1 x

1 x

Funduscopy if CD4 T cells <200/µl

1-2 x

4 x

With regard to the growing age of the HIV population, it is essential not to forget cancer screening. In many countries, for example, colonoscopy is recommended for early detection of colorectal cancer for every individual older than 50-55 years (colonoscopy should be performed every 10 years). For further information see WHO website, http://www.who.int/cancer/detection/en/

Therapeutic Drug Monitoring (TDM)

Plasma levels of many antiretroviral drugs may vary considerably for diverse reasons (e.g., compliance, metabolism, absorption). Measurement of drug concentrations in serum or plasma is also referred to as therapeutic drug monitoring (TDM).

Sufficient plasma levels are essential for success of virologic treatment (Acosta 2000). In the VIRADAPT Study adequate PI concentrations were even more crucial than knowledge of resistance mutations (Durant 2000). The importance of sufficient plasma levels has also been shown for NNRTIs (Marzolini 2001, Veldkamp 2001). This information however dates to the early years of ART.

Whether TDM still improves virologic response today, is not clearly validated (Kredo 2009). Only a few large randomized studies exist that have provided data regarding this question (Review: Liu 2010). One of the few randomized studies could only show a trend to virologic (Best 2007). TDM remained without any effect in another study with patients receiving boosted PIs (Demeter 2009).

On the other hand, very high plasma levels correlate with a higher rate of side effects. Reported renal problems with indinavir (Dielemann 1999), gastrointestinal disturbances with ritonavir (Gatti 1999), hepatotoxicity with nevirapine (Gonzalez 2002) or CNS problems with efavirenz (Marzolini 2001) were all associated with high plasma levels. For this reason, TDM will remain a tool for therapy observation: not every interaction between antiretroviral drugs or with concomitant drugs has been investigated.

Measurement of plasma levels may currently be reasonable in the following situations (German-Austrian ART guidelines):

  • Complex drug combinations including boosted PIs
  • Patients with very high or low body weight
  • Side effects
  • Treatment failure (resistance?)
  • Suspected absorption or adherence problems
  • Severe liver or renal diseases
  • Art with children, pregnancy
  • Once daily regimen
  • Use of new drugs (unknown interactions)

Several problems associated with TDM limit its broader use. The measurement of NRTIs, for example, is not possible since they are converted to the active metabolites only intracellularly. Intracellular measurements are difficult and are not available in routine clinical practice. There is no valid data available for new antiretroviral agents such as T-20, raltegravir or maraviroc.

Measuring NNRTIs or PIs may therefore currently determine levels of only one component of a failing combination. Further problems include not only viral strains with different levels of resistance, different inhibitory concentrations, variable protein binding, and time-dependent variability of plasma levels, but also methodological problems with the assays, as well as the lack of clearly defined limits. Many uncertainties thus remain in the assessment of therapeutic drug plasma levels. Until data from randomized studies is available, proving the clinical value of TDM, both the measurement and interpretation of results should be left to specialized centers.

References

Acosta EP, Kakuda TN, Brundage RC, Anderson PL, Fletcher CV. Pharmacodynamics of HIV type 1 protease inhibitors. Clin Infect Dis 2000, Suppl 2:S151-9.

Best BM, Goicoechea M, Witt MD, et al. A randomized controlled trial of therapeutic drug monitoring in treatment-naive and -experienced HIV-1-infected patients. J AIDS 2007;46:433-42.

Buchacz K, Patel P, Taylor M, et al. Syphilis increases HIV viral load and decreases CD4 cell counts in  HIV-infected patients with new syphilis infections.  AIDS 2004, 18:2075-2079.

Chaiwarith R, Praparattanapan J, Salee P, et al. Frequency of HIV-RNA Monitoring: impact on outcome of antiretroviral therapy. Abstract 500, 17th CROI 2010, San Francisco.

Clevenbergh P, Mouly S, Sellier P, et al. Improving HIV infection management using antiretroviral plasma drug levels monitoring: a clinician’s point of view. Curr HIV Res 2004, 2:309-21.

Coste J, Montes B, Reynes J, et al. Comparative evaluation of three assays for the quantitation of HIV type 1 RNA in plasma. J Med Virol 1996, 50:293-302.

Demeter LM, Hughes MD, Coombs RW, et al. Predictors of virologic and clinical outcomes in HIV-1-infected patients receiving concurrent treatment with indinavir, zidovudine, and lamivudine. ACTG Protocol 320. Ann Intern Med 2001, 135: 954-64.

Demeter LM, Jiang H, Mukherjee AL, et al. A randomized trial of therapeutic drug monitoring of protease inhibitors in antiretroviral-experienced, HIV-1-infected patients. AIDS 2009, 23:357-68.

Dieleman JP, Gyssens IC, van der Ende ME, de Marie S, Burger DM. Urological complaints in relation to indinavir plasma concentrations in HIV-infected patients. AIDS 1999, 13:473-8.

Durant J, Clevenbergh P, Garraffo R, et al. Importance of protease inhibitor plasma levels in HIV-infected patients treated with genotypic-guided therapy: pharmacological data from the Viradapt Study. AIDS 2000, 14:1333-9.

Easterbrook PJ, Ives N, Waters A, et al. The natural history and clinical significance of intermittent viraemia in patients with initial viral sup-pression to < 400 copies/ml. AIDS 2002; 16:1521-7.

Farber CM, Barath AA, Dieye T. The effects of immunization in HIV type 1 infection. N Engl J Med 1996, 335:817; discussion 818-9.

Gatti G, Di Biagio A, Casazza R, et al. The relationship between ritonavir plasma levels and side-effects: implications for therapeutic drug monitoring. AIDS 1999, 13:2083-9.

Ghani AC, de Wolf F, Ferguson NM, et al. Surrogate markers for disease progression in treated HIV infection. J Acquir Immune Defic Syndr 2001; 28: 226-31.

Goletti D, Weissman D, Jackson RW, et al. Effect of Mycobacterium tuberculosis on HIV replication. Role of immune activation. J Immunol 1996, 157:1271-8.

Gonzalez de Requena D, Nunez M, Jimenez-Nacher I, Soriano V. Liver toxicity caused by nevirapine. AIDS 2002, 16:290-1.

Grabar S, Kousignian I, Sobel A, et al. Immunologic and clinical responses to highly active antiretroviral therapy over 50 years of age. Results from the French Hospital Database on HIV. AIDS 2004, 18:2029-2038.

Haubrich R, Riddler S, Ribaudo H, et al. Initial viral decay to assess the relative antiretroviral potency of PI-, NNRTI-, and NRTI-sparing regimens for first line therapy of HIV-1 infection: ACTG 5160s. Abstract 137, 14th CROI 2007, Los Angeles. Abstract: http://www.retroconference.org/2007/Abstracts/28232.htm

Ho DD, Neumann AU, Perelson AS, et al. Rapid turnover of plasma virions and CD4 lymphocytes in HIV-1 infection. Nature 1995, 373:123-6.

Hoover DR. Would confirmatory retesting of CD4+ cells to verify AIDS status be too expensive? J Acquir Immune Defic Syndr 1993, 6:537-9.

Hughes MD, Johnson VA, Hirsch MS, et al. Monitoring plasma HIV-1 RNA levels in addition to CD4+ lymphocyte count improves assessment of antiretroviral therapeutic response. ACTG 241 Protocol Virology Substudy Team. Ann Intern Med 1997; 126: 929-38.

Kaufmann GR, Bloch M, Zaunders JJ, Smith D, Cooper DA. Long-term immunological response in HIV-1-infected subjects receiving potent antiretroviral therapy. AIDS 2000, 14: 959-69.

Kitchen CM, Kitchen SG, Dubin JA, Gottlieb MS. Initial virological and immunologic response to HAART predicts long-term clinical outcome. Clin Infect Dis 2001; 33: 466-72.

Kofoed K, Gerstoft J, Mathiesen LR, Benfield T. Syphilis and human immunodeficiency virus (HIV)-1 coinfection: influence on CD4 T-cell count, HIV-1 viral load, and treatment response. Sex Transm Dis 2006;33:143-8.

Kolber MA, Gabr AH, De La Rosa A, et al. Genotypic analysis of plasma HIV-1 RNA after influenza vaccination of patients with previously undetectable viral loads. AIDS 2002, 16: 537-42.

Kolte L, Dreves AM, Ersboll AK, et al. Association between larger thymic size and higher thymic output in HIV-infected patients receiving HAART. J Infect Dis 2002, 185:1578-85.

Kredo T, Van der Walt JS, Siegfried N, Cohen K. Therapeutic drug monitoring of antiretrovirals for people with HIV. Cochrane Database Syst Rev 2009, CD007268. Review.

Le Moing V, Thiebaut R, Chene G, et al. Predictors of long-term increase in CD4(+) cell counts in HIV-infected patients receiving a protease inhibitor-containing antiretroviral regimen. J Infect Dis 2002, 185: 471-80.

Lepri AC, Miller V, Phillips AN, et al. The virological response to HAART over the first 24 weeks of therapy according to the pre-therapy viral load and the weeks 4-8 viral load. AIDS 2001, 15: 47-54.

Liu X, Ma Q, Zhang F. Therapeutic drug monitoring in highly active antiretroviral therapy. Expert Opin Drug Saf 2010, 9:743-58.

Lyles RH, Munoz A, Yamashita TE, et al. Natural history of HIV type 1 viremia after seroconversion and proximal to AIDS in a large cohort of homosexual men. J Infect Dis 2000, 181:872-880.

Maggiolo F, Migliorino M, Pirali A. Duration of viral suppression in patients on stable therapy for HIV-1 infection is predicted by plasma HIV RNA level after 1 month of treatment. J Acquir Immune Defic Syndr 2000, 25:36-43.

Malone JL, Simms TE, Gray GC, et al. Sources of variability in repeated T-helper lymphocyte counts from HIV type 1-infected patients: total lymphocyte count fluctuations and diurnal cycle are important. J Acquir Immune Defic Syndr 1990, 3:144-51.

Marzolini C, Telenti A, Decosterd LA, et al. Efavirenz plasma levels can predict treatment failure and central nervous system side effects in HIV-1-infected patients. AIDS 2001, 15: 71-5.

Mellors JW, Munoz AM, Giorgi JV, et al. Plasma viral load and CD4+ lymphocytes as prognostic markers of HIV-1 infection. Ann Intern Med 1997, 126:946-954.

Napravnik S, Poole C, Thomas JC, Eron JJ Jr. Gender difference in HIV RNA levels: a meta-analysis of published studies. J Acquir Immune Defic Syndr 2002, 31:11-9.

Notermans DW, Pakker NG, Hamann D, et al. Immune reconstitution after 2 years of successful potent ART in previously untreated HIV type 1-infected adults. J Infect Dis 1999, 180: 1050-6.

O’Brien WA, Grovit-Ferbas K, Namazi A, et al. HIV-type 1 replication can be increased in peripheral blood of seropositive patients after influenza vaccination. Blood 1995, 86:1082-9.

Pakker NG, Notermans DW, de Boer RJ, et al. Biphasic kinetics of peripheral blood T cells after triple combination therapy in HIV-1 infection: a composite of redistribution and proliferation. Nat Med 1998, 4: 208-14.

Palacios R, Jimenez-Onate F, Aguilar M, et al. Impact of syphilis infection on HIV viral load and CD4 cell counts in HIV-infected patients. J Acquir Immune Defic Syndr 2007;44:356-9.

Parekh B, Phillips S, Granade TC, et al. Impact of HIV type 1 subtype variation on viral RNA quantitation. AIDS Res Hum Retroviruses 1999, 15:133-42.

Perelson AS, Essunger P, Cao Y, et al. Decay characteristics of HIV-1-infected compartments during combination therapy. Nature 1997, 387:188-91.

Perelson AS, Neumann AU, Markowitz M, Leonard JM, Ho DD. HIV-1 dynamics in vivo: virion clearance rate, infected cell life-span, and viral generation time. Science 1996, 271:1582-6.

Phillips A, CASCADE Collaboration. Short-term risk of AIDS according to current CD4 cell count and viral load in antiretroviral drug-naive individuals and those treated in the monotherapy era. AIDS 2004, 18:51-8.

Phillips AN, Youle M, Lampe F, et al. CD4 cell count changes in individuals with counts above 500 cells/mm and viral loads below 50 copies/ml on antiretroviral therapy. AIDS 2002; 16: 1073-5.

Polis MA, Sidorov IA, Yoder C, et al. Correlation between reduction in plasma HIV-1 RNA concentration 1 week after start of antiretroviral treatment and longer-term efficacy. Lancet 2001,  358: 1760-5

Renaud M, Katlama C, Mallet A, et al. Determinants of paradoxical CD4 cell reconstitution after protease inhibitor-containing antiretroviral regimen. AIDS 1999, 13:669-76.

Rizzardi GP, DeBoer RJ, Hoover S, et al. Predicting the duration of antiretroviral treatment needed to suppress plasma HIV-1 RNA. J Clin Invest 2000, 105:777-782.

Roger PM, Breittmayer JP, Durant J, et al. Early CD4(+) T cell recovery in HIV-infected patients receiving effective therapy is related to a down-regulation of apoptosis and not to proliferation. J Infect Dis 2002, 185: 463-70.

Smith CJ, Sabin CA, Youle MS, et al. Factors influencing increases in CD4 cell counts of HIV-positive persons receiving long-term highly active antiretroviral therapy. J Infect Dis 2004, 190:1860-8.

Smith CJ, Staszewski S, Sabin CA, et al. Use of viral load measured after 4 weeks of highly active antiretroviral therapy to predict virologic outcome at 24 weeks for HIV-1-positive individuals. J AIDS 2004, 37:1155-1159.

Stellbrink HJ, Schewe CK, Hoffmann C, Wolf E. Is there a harmless level of plasma viremia in untreated HIV infection? CD4+ T cells in the long-term follow-up of elite controllers and controls. Abstract 351, 14th CROI 2008, Boston

Telenti A. New developments in laboratory monitoring of HIV-1 infection. Clin Microbiol Infect 2002, 8:137-43.

Thiebaut R, Morlat P, Jacqmin-Gadda H, et al. Clinical progression of HIV-1 infection according to the viral response during the first year of antiretroviral treatment. AIDS 2000, 14: 971-8.

Veldkamp AI, Weverling GJ, Lange JM, et al. High exposure to nevirapine in plasma is associated with an improved virological response in HIV-1-infected individuals. AIDS 2001; 15: 1089-95.

Viard JP, Burgard M, Hubert JB, et al. Impact of 5 years of maximally successful highly active antiretroviral therapy on CD4 cell count and HIV-1 DNA level. AIDS 2004, 18:45-9.

Viard JP, Mocroft A, Chiesi A, et al. Influence of age on CD4 cell recovery in HIV-infected patients receiving HAART: evidence from the EuroSIDA study. J Infect Dis 2001, 183: 1290-4.

Walter EA, Gilliam B, Delmar JA, et al. Clinical implications of identifying non-B subtypes of HIV type 1 infection. Clin Infect Dis 2000, 31:798-802.

Wu H, Kuritzkes DR, McClernon DR, et al. Characterization of viral dynamics in HIV type 1-infected patients treated with combination antiretroviral therapy: relationships to host factors, cellular restoration, and virologic end points. J Infect Dis 1999, 179: 799-807.

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Filed under 6.11. Monitoring, 6.9. Salvage Therapy, Part 2 - Antiretroviral Therapy

6.12. Prevention, compliance, costs

– Christian Hoffmann –

In the following, some aspects of antiretroviral therapy will be discussed in more detail that have only been mentioned briefly in previous chapters.

Prevention

About 30 years after AIDS was first described a prophylactic vaccination remains a distant prospect. In 2007 two highly promising vaccine studies were prematurely interrupted. It seems doubtful now that a vaccine to effectively prevent HIV infection will be discovered anytime soon – the moderate but surprisingly successful RV144 vaccine study will not change that (Rerks-Ngarm 2009, see chapter on Preventive Vaccination). Several experts believe that a promising vaccine candidate does not exist presently (Desrosiers 2008, Nathanson 2008) while others say it is time to get used to the fact that a vaccine may never come. Neither blind hope nor time schedules have proved very helpful. Some vaccine studies up until now can in fact be regarded as counter-productive, fatiguing both sponsors and the community.

Considering all this, prevention will continue to be the central means of controlling the HIV epidemic. However, common prevention strategies focused on the ABC guidelines (abstinence, be faithful, condom use) have reached their limits. In 2009 UNAIDS declared a worldwide increase of 2.2 million new infections. In every major city in the US or in Europe syphilis endemics in HIV-infected patients have been reported. In Germany, for example, the number of new infections among men who have sex with men (MSM) has been almost continuously rising since 2001.

It seems clear that advertisements and brochures alone are not the solution, especially when these simple publicity mechanisms are not maintained. High-risk groups are not being reached effectively. Prevention remains an arduous business and success is not immediately visible nor is it profitable. Sexual behavior is not easily modified by a few advertisements or brochures.

For some time, preventive medicine in HIV has been taking completely new and sometimes unusual paths to reach focus groups. Terms such as serosorting, seropositioning, dipping or strategic positioning show that the medical world is learning to face reality. People have sex and not everyone cares about, follows, or can follow guidelines. Recent studies with serosorting, choosing sexual partners according to perceived HIV serostatus, show that new prevention strategies can be developed (Morin 2008).

The following focuses mainly on medical prevention strategies. In 2010 there have been groundbreaking new findings in this area regarding PrEP and microbicides.

ART & Prevention

Antiretroviral therapy is an important contribution to prevention, possibly the most important (Hosseinipour 2002). As these studies show:

  • In a group of 415 HIV-discordant couples in Uganda 90 new infections were diagnosed over a period of 30 days. Not a single infection was caused by an infected partner with a viral load below 1500 copies/ml. With every additional log of HIV RNA, infection risk increased by a factor of 2.45 (Quinn 2000).
  • In a study in Thailand with 493 HIV-discordant couples, the factor was 1.81. No infection from a partner with less than 1094 copies/ml was recorded (Tovanabutra 2002).
  • In a study in Spain with 393 heterosexual HIV-discordant couples, a transmission rate of 8.6% was observed between 1991 and 2003. No infection was recorded with infected partners who were receiving combination ART.
  • In a group of 534 MSM in San Francisco, infectiousness based on the probability of transmission per couple decreased by 60% between 1994 and 1998 (Porco 2004). The HIV incidence decreased in spite of the reported higher number of partners and risk contacts, even though not all of the HIV-infected partners were on antiretroviral therapy.
  • In a Spanish study with 62 HIV-discordant couples (22 HIV-infected women, 40 HIV-infected men, all of them on ART), 76 “natural” pregnancies were diagnosed. Not a single HIV infection of a non-infected partner was recorded (Barreiro 2006).

The above-mentioned clinical studies show clearly that the lower the viral load in the plasma, the less infectious the patient. In an ongoing meta-analysis in 11 cohorts with 5021 heterosexual couples (and 461 HIV transmissions) the transmission rate of patients on ART was 0.46 per 100 person years (5 cases). Not one transmission was detected from anyone who was below 400 copies/ml (Attia 2009).

Test and treat?

At the end of 2008 a statistical paper caused great discussion. A research group led by the Director of WHO Kevin De Cock calculated how to, at least theoretically, curtail and even eliminate the worldwide HIV epidemic (Granich 2008). For this ambitious goal they concentrated totally on the preventive effect of antiretroviral therapies. They compared the common treatment strategy used today, beginning ART only on symptomatic patients or on those who have less than a certain number of CD4 T cells, to a theoretical strategy that seems simple enough. Every person is tested for HIV once a year and if found positive, starts ART immediately, irrespective of CD4 T cells or viral load. The study was based on population data in South Africa, where 17% of the adult population is HIV-infected and on data from a successful intervention in Malawi. Other preconditions of the calculation model are that infectiousness of treated versus not-treated patients was estimated at 1%. The case-reproduction number, the so called R0 number of new infections caused by one infection, was crucial for this calculation. The corresponding simple assumption that R0 of <1 is required in order to reduce the incidence and to eventually eliminate HIV means that an incidence rate of less than one new case per 1000 person years was determined in order to eliminate HIV.

Reality shows different results. At present, every untreated HIV-infected individual causes another 7 HIV infections (R0=7) in the course of their lifetime. R0 could be reduced to 4 if every person received regular treatment with therapy starting at 200 CD4 T cells/µl, or even to 3 if therapy starts at 350 CD4 T cells/µl. However, an R0 reduction to less than 1 is impossible by this method and curtailing the epidemic with ART alone remains unrealistic. This could change however, with regular testing and immediate treatment of positively-diagnosed individuals – elimination of the epidemic could be possible by 2020, even in a country as severely affected as South Africa. Compared with common practice today where ART is begun only at a certain level of CD4 T cells, immediate treatment could reduce AIDS mortality to half today’s number by 2050. Calculations showed that this initially more expensive strategy could start to be cost-saving by around 2032.

The comments to the WHO publication ranged from “provocative” (Cohen 2008) to “extremely radical” (Garnett 2008). Critics raised concerns over the risks and the absence of ethics (would all actors agree? Could a restricted individual autonomy be achieved? Can changes in sexual habits be maintained?), medical (compliance problems, the dangers of possible resistance, the side effects and “overtreatment” – starting too early) as well as financial (South Africa would have to triple their financial commitments) considerations.

Such calculations are not new. Other groups have arrived at similar results in the past (Velasco-Hernandez 20002, Montaner 2006). What is new is that antiretroviral therapies today are potentially more user-friendly and such programs are probably easier to put into practice than just a few years ago.

In addition, people are realizing that the current preventive measures can only improve slowly and that neither vaccines nor microbicides can be expected in the near future. At present, approximately 80% of the population in sub-Saharan Africa is not aware of their infection. More than 90% do not know if their sexual partners are infected – an invitation for further spread of the epidemic.

Juggling figures like this may seem unhelpful at first. Despite all objections regarding methodological, ethical, financial or logistic considerations, etc., facing 2.2 million new infections per year, a number that is not likely to decline much (if at all) in the near future, and the failure of several vaccine and prevention studies, one thing has become clear. Antiretroviral therapy has turned into one of the most important components of prevention.

Such initiatives like that of the WHO must be continued, and new and unusual strategies must be continually developed. It cannot do harm to bring more therapy to the 6.7 million people, worldwide, the number who by the end of 2007 desperately needed ART and were not receiving it (see chapter on Global Access).

ART & viral load in other body fluids

Do viral load in plasma and viral load in other body fluids correlate?  Here are some data:

  • In a study from Italy the viral load on PI-containing ART regimens decreased by several logs in plasma as well as in semen (Liuzzi 1999).
  • In a Swiss study with 114 male patients with plasma viremia under 400 copies/ml on ART, only 2 (2%) isolated viral loads were detected in semen, compared to 67% in untreated control groups.
  • In 205 HIV-infected women with plasma viremia under 400, 400-9999 and over 10,000 copies/ml, the rate of detectable HIV-1 RNA in the genital tract was 3, 17 and 48%, respectively (Cu-Uvin 2000). In 7 ART-naïve women, the viral load decreased by 0.7–2.1 logs within the first 14 days of ART. Similar results were achieved with 11 Brazilian female patients (Vettore 2006).
  • In a group of 290 women with plasma viremia under 500 copies/ml, 44 (15%) had detectable HIV-1 RNA in cervical smears (Neely 2007). In comparison to PI-containing ART the risk with NNRTIs was double.
  • In a study with 34 females with plasma viremia below 80 copies/ml, all treated with ART for at least 6 months, only one woman showed a viral load over 80 copies/ml in cervical vaginal fluid (CVF) compared to 7 rebounds in plasma (Kwara 2008).
  • Out of 122 samples of cervical vaginal lavage the viral load in the lavage correlated highly with plasma viral load (Fiore 2003). However, in 25% of cases, virus was found in the lavage even when plasma viremia findings proved negative.
  • In a study with 233 MSM (1996–1997), far less virus was found in anorectal smears of those treated with ART. Among those patients with less than 50 copies/ml in plasma, 1/54 (2%) HIV-1 RNA was detected in the anorectal smear. However, in 14/50 (28%) HIV-1 DNA was detected.
  • Among 255 MSM receiving ART with a plasma viral load below 40 copies/ml, 7 patients (3%) showed an isolated viral load in semen (Marcelin 2009). These 7 patients had been on ART for some time and treated with agents detected in semen.
  • In a prospective study on 25 Canadian patients on ART, a viral load in semen was found in 19/116 (14%) samples (Sheth 2009). There was no reference to drug concentration  in seminal fluid.

In conclusion, in most cases, viral load in plasma parallels viral load in other bodily fluids. If the viral load in plasma decreases, so does the RNA in semen or the vaginal fluid within a short time. “Below the limit of detection in plasma” also means “below the limit of detection in other bodily fluids”. In most cases. There are exceptions: in the studies above the variation was between 1 and 14%. Although there are implications that the detected virus in semen is not completely infectious (Nunnari 2002), one cannot rule out the patient being potentially infectious even on successful ART.

Putting together these facts with clinical data, transmission with a low viral load seems unlikely. To date, only a few cases have been recorded in which transmission has taken place despite effective ART (Stürmer 2008). These cases show that there is in fact a residual risk. The question is how to manage that risk.

The EKAF paper

In January 2008 a paper was released by the “Eidgenössische Kommission für Aids-Fragen” (EKAF), the Swiss AIDS commission. Just the title of this paper caused a great stir: “HIV-infected individuals without other STDs on effective antiretroviral therapy are not sexually infectious.” The original manuscript can be found at http://www.saez.ch/pdf_d/2008/2008-05/2008-05-089.PDF.

EKAF concluded that HIV-infected individuals do not transmit the disease under three conditions:

1. ART is adhered to and monitored by a clinician

2. The viral load has been below detection for at least six months

3. There is no other STD

This first official statement from public authorities on this subject had a major impact. Despite its caveats, critics feared that this publication could be misunderstood as an all-clear signal resulting in people being less careful and unnecessarily exposing themselves to risks of HIV infection.

Critics say that the data is not sufficient, especially for the risk of anal sexual contacts. The probability of infection is certainly under 1:100,000, but nevertheless not zero (Wilson 2009). The preventive effect of ART may be endangered by higher risk taking. According to mathematical models, a 10% rise in risk behavior could counter the effects of ART (Blower 2001, Law 2001). However, a meta-analysis came to the conclusion that ART does not increase risk behavior of the patient, even if the viral load is below detection (Crepaz 2004).

HIV clinicians must be prepared for this discussion. Patients are asking more questions: do I have to use a condom for the rest of my life? Here, it is better to give individualized advice. It depends greatly on the non-infected partner as well, as he or she should not be pressured. On the other hand, information of this type can be a relief for many patients and their partners. The EKAF paper may also motivate high-risk patients to finally start antiretroviral treatment (preventing more infections rather than causing new ones initially feared by the release of the paper).

However, it must be repeated that the EKAF statement refers only to stable relationships. Safer sex is still recommended, especially with occasional sexual contacts to avoid other sexually transmittable diseases.

Medical prevention strategies besides ART

Circumcision

Circumcision of the male foreskin reduces the risk of infection for several diseases in unprotected sexual intercourse (Weiss 2006). At least three randomized trials with heterosexual males in Uganda, Kenya and South Africa demonstrated this in recent years for HIV as well. Remarkably similar results were achieved (Table 12.1).

Table 12.1. Large randomized studies on circumcision.
Place

Reference

n

Main Results

Reduction of Transmission risk

Kenya
(Bailey 2007)

2784

Two–Year HIV Incidence 2.1%  (95% CI 1.2-3.0) vs 4.2% (95% CI 3.0-5.4)

53-60%

Uganda
(Gray 2007)

4996

Over 24 months HIV incidence
0.66 vs 1.33/100 person years

51-60%

South Africa
(Auvert 2005)

3274

Over 18 months HIV incidence
0.85 vs 2.10/100 person years

60-61%

TR = Transmission Risk, partly different definition/calculation

A meta-analysis of these studies shows a relative risk of 0.44 for circumcision (Mills 2008). The NNT (number needed to treat) required to prevent an event reached a relatively low number of 72.

The effect of circumcision is explained by the presence of CD4-positive Langerhans cells and primary HIV target cells in the male foreskin. Circumcision reduces the frequency of genital HSV-2 infection (Tobian 2008), which however does not explain the protective effect (Gray 2009). An estimated 2 million HIV infections in Africa alone could be prevented in the next few years (Williams 2006). The WHO recommends circumcision as a preventive means for heterosexual men. A favourable side effect is that fewer HPV-infections are transferred (Serwadda 2010)

Circumcision, however, is not without risk. Complications (infections, postoperative bleeding) occur in 3-4% of cases (Gray 2007). Sexual behavior after circumcision, ethics and logistical problems are only a few aspects (Lie 2006). It must be noted that circumcision reduces the risk for male but not for female partners. The randomized study in Uganda showed a slight increase in infections of the female partners of circumcised males (Waver 2008). This can be mainly explained by couples probably having sexual intercourse earlier than recommended. Several weeks of no-sex are stipulated after the operation.

Is there a protective effect for MSM after circumcision? If there is, the data is less clear compared to the results for heterosexual men. A meta-analysis of 15 greatly varying studies with 53,567 MSM (52% with circumcision) showed no significant difference between circumcised and uncircumcised males (Millet 2008). Another newer study confirms these results (Sanchez 2011).

Preventive treatment of HSV and other diseases

Genital infections clearly increase the risk of acquiring HIV. This applies especially to the human herpes virus 2 (HSV-2). According to a meta-analysis, the risk of HIV increases with HSV-2-seropositivity: when HSV-2 antibodies are detected in the blood, the risk increases in male patients by 2.7 fold and in female patients by 3.1 fold (Freeman 2006). A considerable amount of new HIV infections are due to HSV coinfection, with an estimated 38–69% in female patients and 8–49% in male patients.

Is a reduction of the HIV transmission rate in HIV-negative persons possible by suppression of HSV-2? HPTN 039, a double-blind, randomized, Phase III trial investigated (Celum 2008). In total, 1871 MSM from the USA and Peru and 1380 women from Zimbabwe, Zambia and South Africa received 400 mg acyclovir or placebo twice daily. Enrolled subjects were all HIV-negative and HSV-2-positive at the beginning of the trial. Although less HSV ulcers were observed in the active group, the trial failed to show a decline in HIV incidence in the acyclovir-group, with 3.9/100 person-years compared to 3.3/100 in the placebo group. These disappointing results were confirmed by the Mwanza trial with 821 women in Tanzania, in which again no decline was observed (Watson-Jones 2008). It is still not clear why, however, resistances against acyclovir is unlikely (Watson-Jones 2010).

According to current data, preventing HIV infection with HSV therapy using acyclovir has proven unsuccessful. The prophylactic use of azithromycin, to prevent bacterial STDs also showed no protective effect against HIV (Kaul 2004).

Can the transmission rate be reduced if the HIV-infected partner is treated with acyclovir? A huge study enrolling 3408 discordant African couples showed no effect of the transmission rate, albeit the clearly reduced rate of genital HSV ulcers (Celcum 2010). However, this study did show an interesting side effect, that there is a slight but measurable effect with acyclovir and its derivatives regarding HIV viral load. Compared to placebo, a decline of 0.25 logs was observed. This effect slightly decreased the risk of HIV progression in therapy naïve patients (Lingappa 2010). The transmission rate was obviously not influenced by the reduction in viral load. Resistances were not induced by acyclovir (Baeten 2011).

Antiviral effects were also observed in several other randomized studies. The viral load in blood and cervicovaginal fluids was reduced by 0.26 to 0.53 logs by using acyclovir or valacyclovir (Delany 2009, Nagot 2007, Zuckermann 2007, Baeten 2008, Dunne 2008, Paz-Bailey 2009). These studies may possibly lead to the development of new acyclovir derivatives with improved antiviral potency, provided they respond well to HIV (Vanpouille 2010).

Microbicides, lubricants, diaphragms

Microbicides are chemical agents, mostly of topical application, in the form of gels that kill or immobilize HIV and other diseases. Presently heterogenic mechanisms are being examined. Among them are inactivated agents that inhibit docking to the target cell or antiviral agents. It is required that microbicides are not only inexpensive, easy to apply and non-toxic, but also effective against other STDs, as these increase the risk of HIV-transmission. The CAPRIAL trial (see below) has led to a noticeable revival in this field of research.

Inactive microbicides: Up to now, there is no product that has delivered convincing protective effects in clinical studies. HIV transmission risk in fact increased with nonoxynol-9 (Van Damme 2002) or cellulose sulfate (van Damme 2008).  PRO 2000, which initially seemed promising, had no effect (McCormack 2010). Application of diaphragma and/or lubricants in addition to condomes had no protective effect, as one randomized study showed (Padian 2007).

Antiretroviral Microbicides: A breakthrough in research of microbicides was achieved in the CAPRISA trial in September 2010. CAPRISA was a doubleblind study, in which 889 HIV-negative women in South Africa used 1% tenofovir gel (Abdol Karim 2010). Compared to placebo, HIV incidence was reduced from 9.1 to 5.6/100 years. Transmission risk for women applying the gel regularly was reduced by 54%. This first success (“proof of concept”) has led to a focus on antiretroviral substances in the research of microbicides, such as tenofovir and even the more experimental NNRTIs dapivirine and MIV-150, as well as maraviroc and raltegravir (Review: Mertenskötter 2011).

PrEP (Pre-exposure Prophylaxis)

In the HIV setting, PrEP is an oral prophylactic antiretroviral treatment. Like malaria prophylaxis, it is taken before exposure. PrEP trials are currently being conducted in high-risk groups (i.e., commercial sex workers). Most trials use tenofovir, either alone or in combination with FTC. Such studies, however, have been regarded with criticism. Pressured by activists and others, a study with Cambodian sex workers was interrupted in 2004 and others in Cameroon and Nigeria in 2005 (Cohen 2004, Sing 2005). The researchers involved were accused of not providing sufficient information to the participants and of discontinuing treatment once the study was over.

A similar breakthrough, like the one with microbicides by the CAPRISA trial, was seen with PrEP in the end of 2010. In the iPrEx study, 2499 MSM from six countries received either TDF+FTC or placebo. After a median of 1.2 years, 36 versus 64 infections were observed and the risk for infection was reduced by 44%. Apart from slightly more cases of nausea and weight loss in the verum group, there were no differences. Only in 3/34 patients infected in the verum group, tenofovir or FTC were detected in plasma. One may argue that complete protection is not secured: However, with an alarmingly high number of 2.2 million new infections worldwide, PrEP remains an approach that must be pursued.

Table 12.2: Large randomized study on PrEP, March 2011
Country

n

Risk group, kind of  PrEP

Status, first results

Thailand

2.400

IDU: tenofovir

fully recruited, 2012

Africa, Partners PrEP Study

4.758

discordant couple: tenofovir, Tenofovir+FTC

fully recruited, 2012

Africa, FEM PrEP

3.900

women: tenofovir+FTC

49 % recruited, 2013

Africa,  VOICE/ MTN 003

5.000

women: tenofovir, tenofovir+FTC, vaginal tenofovir gel

65 % recruited, 2013

Physicians must be prepared for inquiries on PrEP, although some questions still remain that have not been answered by the above study.

How should PrEP be administered? Who will receive the treatment? And who will cover the expense? What about development of resistance in unidentified HIV infection? Will this decrease the use of condoms? Will PrEP be sold on the black market in the near future (and so, with limited adherence programs)? These are only a few aspects to be considered. In Switzerland as well as at the EMA, a commission dealing with these questions has already been set up, although the benefits of PrEP have not yet been scientifically proven.

In conclusion, with dramatically high numbers of continuing infections worldwide, prevention must strike new paths. Strictly Propagating safer sex alone is not enough. Among medical approaches, the use of antiretroviral therapy is the most imaginative strategy right now. The EKAF paper will continue to be discussed. Like it or not, microbicides and PrEP will have a lasting effect on HIV prevention. Patients will be asking for it.

Compliance

Compliance is the Achilles’ heel of every antiretroviral therapy and non-compliance the main, if not the major factor for developing resistance and treatment failure (Turner 2000). Partial viral suppression with insufficient drug levels is ideal conditions under which resistance grows. There is no doubt – ART must be taken regularly, correctly or not at all. Taking either more than 90% or less than 69% of the treatment are both associated with a lower risk of resistance (Sethi 2003). Compliance is defined as a patient’s consent and acceptance of therapy. In the mid-90s a new term “adherence”, from the English language, was adopted. Since then, the more politically correct term – “adherence” is frequently used. This term refers to both physician and patient working together to set up a treatment concept acceptable to both parties and emphasizes that responsibility for a failure of the therapy is not only the patient’s fault.

Adherence includes all factors that influence staying on a regimen, in terms of acceptability. Whichever term is used, three facts remain:

  1. The success of a treatment is endangered if medication is taken irregularly
  2. Clinicians tend to overestimate a patient’s compliance
  3. Compliance diminishes with the complexity of the treatment

Not only drug consumers, those dependent on alcohol or patients with side effects are considered “risky patients” when it comes to adherence. In several studies, depressed patients, patients living alone and younger patients have been identified as problem groups (Murri 2001, Frank 2002, Glass 2006). Positive factors are the physician’s experience, the patient’s confidence in the positive effects of ART, and social support. Race, sex or stage of disease does not seem to be relevant. The individual’s general view of illness and health, accepting modern medicine and fear of side effects are further considerations. However, all these factors vary greatly, and in the end, adherence is difficult to predict in individual cases (Lerner 1998). The physician must rely on experience and intuition.

The importance of taking drugs regularly has been demonstrated in numerous studies. In one study with 99 patients, in which compliance was evaluated via an electronic monitoring system, the rate of viral treatment failure was only 22% in patients with a compliance level of at least 95% (95% of doses taken). Failure rates of 61% and as much as 80% were measured with a patient’s adherence between 80-94% and < 80% (Paterson 2000). However, it must be taken into consideration that this much-cited study is outdated. Newer drugs, such as darunavir, with longer half-lives, higher resistance barriers and better overall pharmacokinetics may forgive a clearly higher non-compliance (Nelson 2010). In the previously mentioned study, clinicians misjudged their patient’s compliance in 41% of the cases. Nurses did better – judging incorrectly in only 30% of the cases (Paterson 2000). Adherence tended to be overestimated in other studies as well (Miller 2002). The importance of adherence was demonstrated in patients with directly observed therapy (DOT) or directly administered ART (DAART), applied in some penal institutions in the USA. In institutions in Florida, 100% of the patients in a DOT study achieved a viral load below 400 copies/ml after 48 weeks, compared to 81% in an unmonitored control group (Fischl 2001). According to one randomized study, response improved in drug-addicted patients receiving DAART (Altice 2007). More recent data indicate that effects of PI based regimen (given as DAART) are marginal and disappear rapidly as soon as the patient is on his own (Gross 2009).

Poor compliance not only leads to virologic failure. It also bears immunological consequences. In an analysis of two prospective studies, patients with a compliance of 100%, 80-99% and < 79% experienced a reduction in viral load by 2.77, 2.33 and 0.67 logs after a year. At the same time, the CD4 T cell count increased by 179, 159 and 53 cells/µl, respectively (Mannheimer 2002).

Moreover, poor adherence has clinical effects beyond surrogate markers. In a Spanish study, patients who did not take more than 10% of their drugs showed a four-fold increase of mortality risk (Garcia 2002). This data has been confirmed in other studies (Maher 1999, Hogg 2000, Wood 2004). Hospital stays are also less frequent in patients with high adherence to ART (Paterson 2000). In addition, it should be considered that non-adherent patients increase the risk of transmission of primary resistant viruses. The basic mechanisms for development of resistance should be explained to patients. One must emphasize that, in contrast to other chronic illnesses, resistance mutations do not disappear once they have developed. Diabetes and hypertension make effective examples. These diseases may “tolerate” forgetting some tablets occasionally, but HIV is different. Blood glucose and blood pressure levels can easily be lowered again the next day, but with HIV this strategy may not work. Even short-term lapses can have irreversible consequences. And every new occurrence of resistance complicates therapy. Patients have to be made aware of these dangers. Such conversations should be repeated from time to time and become a standard component of routine care. Cooperation with special treatment discussion groups offered by patient-centered support organizations can be useful. The 12-step table below provides additional suggestions. In addition, a number of strategies on improving adherence have been investigated. They range from employment of additional nurses to telephoning patients regularly. This last one, telephone reminders, appears to not have an influence on compliance (Collier 2005).

If adherence remains poor

Despite all efforts, some patients will not succeed in improving their adherence. Physicians and other healthcare providers should not take this personally or feel offended should a patient not want to participate in the advances of medicine. Although it may be difficult to accept the patient’s views on life, disease and treatment, healthcare providers must keep tolerance and acceptance as key components in their interactions with patients. Some providers, especially those who treat selective patient populations in university settings, tend to forget the reality of routine medical practice. Rigidly upholding the principles of modern medicine usually does not help here and putting patients under pressure achieves even less. It is important to clearly outline and explain, advise, help, question and listen.

The question of whether noncompliant patients should continue to be treated with antiretroviral therapy is not always easy to address. On the one hand, there are patients who benefit even from suboptimal therapy; on the other hand, drugs are expensive and should not be prescribed too readily. When resources are limited, available drugs should be distributed with care. Restraint should be applied until the reason for poor compliance is understood.

Twelve steps to improve compliance

  1. Every patient should receive a written (understandable by the patient) treatment plan, which should be reviewed at the end of the visit. It should include a telephone number to call in case of problems or questions, accessible evenings and weekends would be even better.
  2. Patient and clinician should agree on the treatment plan. The patient’s concerns, questions and criticisms should be discussed.
  3. The patient should have the impression that the treatment regimen is not randomly chosen, but tailored to his/her individual needs.
  4. The explanation of a new or modified treatment plan takes time, and should not be rushed – all questions should be answered.
  5. The reasons why adherence is so important should be explained. It makes sense to repeat such conversations – they should not only take place when initiating or modifying treatment, but should be part of routine care.
  6. Possible side effects should be explained, as well as what can be done to alleviate them.
  7. Support groups and other types of assistance should be named and offered.
  8. It is important to tell the patient to come back if any problems are encountered with ART – it is better to try to solve them together than have the patient try to deal with them alone at home.
  9. The patient should know that the treatment regimen must be taken in its entirety (avoid, “Last month I left out the big tablets”).
  10. Prescriptions should be documented, in order to get a rough idea of adherence. Irregularities should be addressed openly. Pills counted, bottles checked?
  11. During all stages of therapy, the patient should be informed of treatment success as seen by reduction of viral load and rise in CD4 count.
  12. Ensure clinical vigilance to detect the early signs of depression and treat appropriately.

Duesbergians – a sect of HIV medicine

Patients who principally refuse antiretroviral treatment form a special case. These patients are frequently under treatment by (shockingly misdirected) doctors, who call themselves “Duesbergians” (after the US virologist and AIDS dissident Peter Duesberg, who denies any association between AIDS and illness). In such cases, it can be very difficult to leave patients to their fate. Informative consultations should be as detailed as possible and preferably documented in writing. Below, an example:

An approximately 40-year-old patient with a long history of untreated HIV, 30 CD4 T cells/µl and cerebral toxoplasmosis (TE), which improved significantly after 4 weeks of acute treatment (the last MRI still showed scattered lesions) introduced his case to the HIV outpatient department. Clinically, he was relatively well and fully oriented and due for discharge that day. In a conversation, the patient categorically refused to start the urgently recommended antiretroviral therapy. His Duesbergian physician had advised him against HIV therapy (“You can die from AZT, and the other drugs are not much better, etc”). He refused antibiotics on principal as well. This was why the patient would not continue the TE maintenance therapy, which had made him suffer from diarrhea (NB, probably cryptosporidiosis), skin problems (seborrhoic dermatitis, thrush), and extreme loss of weight (MAC?) since his first day in hospital. It was very important for him to have a break from all medication.

In such cases, we make sure the patients sign the information sheets. Every patient is allowed to and should decide for himself (if fully cognizant and capable) – they must know and be fully informed about what they are doing. It is important to give the patient control: if they changes their mind, they may return!

In our experience, arguing with medical Duesbergians leads to nothing at all. This sect has a very restricted view of the world and stick to their repetitive –mantra-like arguments. Discussing with them is time consuming and a waste of energy.

Fortunately, these cases have become rarer. The initial widespread scepticism towards ART has decreased significantly, due to its overwhelming success in the last few years. Concerning Peter Duesberg, it has become quiet, at least as far as his HIV activities goes. The sect is in decline.

Costs

Antiretroviral treatment is expensive. A health provider needs to be informed about costs for drugs.

In Germany, for example, the price for individual drugs range between €300 (Epivir®) and over €2,400 (Fuzeon®) per month, common threefold therapies range between €14,000 and €24,000 per year. Even within drug classes, there are astonishing differences. Crixivan® (hardly used today) is relatively cheap, while Aptivus® is more than three times the price. Even within primary therapies recommended in guidelines there are great price variations: PIs are almost double the price of NNRTIs in many countries. In Germany, annual costs for Kivexa®+Sustiva® are €6,400 cheaper than for Truvada®+Prezista®/r. If the integraseinhibitor Isentress®, the most expensive drug licensed for primary therapy is used instead of Prezista®,the difference is almost € 8.800 per year. A salvage therapy for a patient with multiresistant virus can amount to as much as €50,000 and more per year. Even though prices alone should not influence the choice of therapy, it is still important for the physician to be aware of the costs.

It is difficult to comprehend the price policy intended by pharmaceutical companies. The reason why prices for directly competing agents (3TC and FTC) are almost exactly the same, whilst prices for other agents of the same drug class differ by 200-300%, cannot be explained by development costs alone. There is no doubt. People are making money with ART and the market is full of competitors – monopolies and patents are being protected.

Despite all the criticism and price discussions, two facts can not be forgotten:

First, the high development costs for new medicines can rise to a billion dollars or more. Most agents never make it to the market. Even a licensed drug such as T-20 may never recoup its development costs. According to Roche, development and research alone chewed up 600 million dollars. To cover such production costs, thousands of patients worldwide would have to be treated with T-20 for several years – a very unrealistic scenario.

Second, there is hardly a more effective therapy than antiretroviral therapy. US estimations assume an expenditure of between $13,000 and $23,000 per additional QALY (quality-adjusted life year) (Freedberg 2001). Compared to many other therapies this is relatively cheap. ART reduces the cost of expensive treatment of opportunistic infections, inpatient and outpatient care. In one German study, between 1997 and 2001 total annual spending per patient decreased from €35,865 to €24,482 (Stoll 2002). Many patients return to work, resulting in an overall economic gain for society (Sendi 1999).

Nevertheless, ART is expensive. Therefore, it should be expected from patients to use up remaining packets of drugs, etc. if the reasons for a change in therapy are not urgent. Concerns of pill reduction or doubts about long-term toxicity should be part of an ongoing discussion with patients. All patients need to be made aware of the costs of medication –to make them aware of the value of the therapy.

Initially, ART should be prescribed for a month. This way, mountains of unused pills will not be wasted, if signs of intolerability or complicated adverse events set in. If response to ART is positive and its effects constant, prescriptions can then be done for a period of three months.

Many companies now offer three-month supply packages. This practice has not been without criticism. In any case, prescriptions of longer than a three months supply should be avoided.

In the future, we all need to be more aware of the costs for ART. The patents for AZT, ddI, 3TC, d4T and abacavir, but also saquinavir will disappear or have already gone. Efavirenz and nevirapine will soon follow. It will be interesting to watch price developments when generics come to the market, as they have in resource-limited settings

References

Abdool Karim Q, Abdool Karim SS, Frohlich JA, et al. Effectiveness and safety of tenofovir gel, an antiretroviral microbicide, for the prevention of HIV infection in women. Science 2010, 329:1168-74.

Altice FL, Maru DS, Bruce RD, et al. Superiority of directly administered antiretroviral therapy over self-administered therapy among HIV-infected drug users: a prospective, randomized, controlled trial. CID 2007;45:770-8.

Attia S, Egger M, Müller M, Zwahlen M, Low N. Sexual transmission of HIV according to viral load and antiretroviral therapy: systematic review and meta-analysis. AIDS. 2009 Apr 17.

Auvert B, Taljaard D, Lagarde E, et al. Randomized, controlled intervention trial of male circumcision for reduction of HIV infection risk: the ANRS 1265 Trial. PLoS Med 2005; 2: 298.

Baeten JM, Lingappa J, Beck I, et al. Herpes simplex virus type 2 suppressive therapy with acyclovir or valacyclovir does not select for specific HIV-1 resistance in HIV-1/HSV-2 dually infected persons. J Infect Dis 2011, 203:117-21.

Baeten JM, Strick LB, Lucchetti A, et al. Herpes simplex virus (HSV)-suppressive therapy decreases plasma and genital HIV-1 levels in HSV-2/HIV-1 coinfected women: a randomized, placebo-controlled, cross-over trial. J Infect Dis 2008, 198:1804-8.

Bailey RC, Moses S, Parker CB, et al. Male circumcision for HIV prevention in young men in Kisumu, Kenya: a randomized controlled trial. Lancet 2007;369:643-56.

Barreiro P, del Romero J, Leal M, et al. Natural pregnancies in HIV-serodiscordant couples receiving successful antiretroviral therapy. J AIDS 2006;43:324-6.

Blower SM, Aschenbach AN, Gershengorn HB, Kahn JO. Predicting the unpredictable: transmission of drug-resistant HIV. Nat Med 2001, 7:1016-20.

Castilla J, Del Romero J, Hernando V, Marincovich B, Garcia S, Rodriguez C. Effectiveness of highly active antiretroviral therapy in reducing heterosexual transmission of HIV. J Acquir Immune Defic Syndr 2005; 40: 96-101.

Celum C, Wald A, Hughes J, et al. Effect of aciclovir on HIV-1 acquisition in herpes simplex virus 2 seropositive women and men who have sex with men: a randomized, double-blind, placebo-controlled trial. Lancet 2008, 371:2109-19.

Celum C, Wald A, Lingappa JR, et al. Acyclovir and transmission of HIV-1 from persons infected with HIV-1 and HSV-2. NEJM 2010, 362:427-39.

Cohen J. Cambodian leader throws novel prevention trial into limbo. Science 2004, 305:1092.

Cohen J. Treat Everyone Now? A ‘Radical’ Model to Stop HIV’s Spread. Science. 2008;322:1453.

Collier AC, Ribaudo H, Mukherjee AL, et al. A randomized study of serial telephone call support to increase adherence and thereby improve virologic outcome in persons initiating antiretroviral therapy. J Infect Dis 2005, 192:1398-406.

Crepaz N, Hart TA, Marks G. Highly active antiretroviral therapy and sexual risk behavior: a meta-analytic review. JAMA 2004, 292:224-36.

Cu-Uvin S, Caliendo AM, Reinert S, et al. Effect of HAART on cervicovaginal HIV-1 RNA. AIDS 2000, 14: 415-21.

De Cock KM , Gilks CF, Lo YR, et al. Can antiretroviral therapy eliminate HIV transmission? Lancet. 2008. Nov 25.

Delany S, Mlaba N, Clayton T, et al.  Impact of aciclovir on genital and plasma HIV-1 RNA in HSV-2/HIV-1 co-infected women: a randomized placebo-controlled trial in South Africa. AIDS 2009, 23:461-9.

Desquilbet L, Deveau C, Goujard C, et al. Increase in at-risk sexual behaviour among HIV-1-infected patients followed in the French PRIMO cohort. AIDS 2002, 16:2329-33.

Desrosiers R. Scientific obstacles to an effective HIV vaccine. Abstract 91, 15th CROI 2008, Boston.

Dunne EF, Whitehead S, Sternberg M, et al. Suppressive acyclovir therapy reduces HIV cervicovaginal shedding in HIVand HSV-2-infected women, Chiang Rai, Thailand. J AIDS 2008, 49:77–83.

Fiore JR, Suligoi B, Saracino A, et al. Correlates of HIV-1 shedding in cervicovaginal secretions and effects of antiretroviral therapies. AIDS 2003;17:2169-76.

Fischl M, Castro J, Monroig R, et al. Impact of directly observed therapy on long-term outcomes in HIV clinical trials. Abstract 528, 8th CROI 2001, Chicago, USA.

Frank I. Once-daily HAART: toward a new treatment paradigm. J AAIDS 2002, 31 Suppl 1:S10-5, discussion S24-5. Review.

Freedberg KA, Losina E, Weinstein MC, et al. The cost effectiveness of combination antiretroviral therapy for HIV disease. N Engl J Med 2001;344:824-31.

Freeman EE, Weiss HA, Glynn JR, Cross PL, Whitworth JA, Hayes RJ. Herpes simplex virus 2 infection increases HIV acquisition in men and women: systematic review and meta-analysis of longitudinal studies. AIDS 2006;20:73-83.

Friedland GH, Williams A. Attaining higher goals in HIV treatment: the central importance of adherence. AIDS 1999, 13 Suppl 1: S61-72.

Garcia de Olalla P, Knobel H, Carmona A, et al. Impact of adherence and HAART on survival in HIV-infected patients. J AIDS 2002, 30:105-10.

Garcia-Lerma JG, Otten RA, Qari SH, et al. Prevention of rectal SHIV transmission in macaques by daily or intermittent prophylaxis with emtricitabine and tenofovir. PLoS Med 2008;5:

Garnett GP , Baggaley RF. Treating our way out of the HIV pandemic: could we, would we, should we? Lancet. 2008. Nov 25.

Glass TR, De Geest S, Weber R, et al. Correlates of Self-Reported Nonadherence to Antiretroviral Therapy in HIV-Infected Patients: The Swiss HIV Cohort Study. J AIDS 2006, 41:385-392.

Granich RM , Gilks CF, Dye C, et al. Universal voluntary HIV testing with immediate antiretroviral therapy as a strategy for elimination of HIV transmission: a mathematical model. Lancet. 2008. Nov 25.

Grant RM, Lama JR, Anderson PL, et al. Preexposure chemoprophylaxis for HIV prevention in men who have sex with men. N Engl J Med 2010, 363:2587-99.

Gray R, Kigozi G, Serwadda D, et al. Randomized trial of male circumcision for HIV prevention in Rakai, Uganda, Abstract 155LB, 14th CROI 2007, Los Angeles.

Gray RH, Serwadda D, Tobian AA, et al. Effects of genital ulcer disease and herpes simplex virus type 2 on the efficacy of male circumcision for HIV prevention: Analyses from the Rakai trials. PLoS Med 2009, 6:e1000187.

Gross R, Tierney C, Andrade A, et. Modified directly observed antiretroviral therapy compared with self-administered therapy in treatment-naive HIV-1-infected patients: a randomized trial. Arch Intern Med 2009, 169:1224-32.

Hogg R, Yip B, Chan K. Non-adherence to triple combination therapy is predictive of AIDS progression and death in HIV-positive men and women. Abstract TuOrB419, 13th International AIDS Conference 2000, Durban, South Africa.

Hosseinipour M, Cohen MS, Vernazza PL, Kashuba AD. Can antiretroviral therapy be used to prevent sexual transmission of HIV type 1? Clin Infect Dis 2002, 34:1391-5.

Kaul R, Kimani J, Nagelkerke NJ, et al. Monthly antibiotic chemoprophylaxis and incidence of sexually transmitted infections and HIV-1 infection in Kenyan sex workers: a randomized controlled trial. JAMA 2004;291:2555-62.

Kwara A, Delong A, Rezk N, et al. Antiretroviral drug concentrations and HIV RNA in the genital tract of HIV-infected women receiving long-term highly active antiretroviral therapy. Clin Infect Dis 2008;46:719-25.

Lampinen TM, Critchlow CW, Kuypers JM, et al. Association of antiretroviral therapy with detection of HIV-1 RNA and DNA in the anorectal mucosa of homosexual men. AIDS 2000;14:

Law MG, Prestage G, Grulich A, Van de Ven P, Kippax S. Modelling the effect of combination antiretroviral treatment on HIV incidence. AIDS 2001, 15:1287-94.

LeGoff J, Weiss HA, Gresenguet G, et al. Cervicovaginal HIV-1 and herpes simplex virus type 2 shedding during genital ulcer disease episodes. AIDS 2007;21:1569-78.

Lerner BH, Gulick RM, Dubler NN. Rethinking nonadherence: historical perspectives on triple-drug therapy for HIV disease. Ann Intern Med 1998, 129:573-8.

Lie RK, Emanuel EJ, Grady C. Circumcision and HIV prevention research: an ethical analysis. Lancet 2006; 368: 522-5.

Lingappa JR, Baeten JM, Wald A, et al. Daily aciclovir for HIV-1 disease progression in people dually infected with HIV-1 and herpes simplex virus type 2: a randomised placebo-controlled trial. Lancet 2010, 375:824-33.

Liu A, Vittinghoff E, Irby R, et al. BMD Loss in HIV– Men Participating in a TDF PrEP Clinical Trial in San Francisco. Abstract 93, 18th CROI 2011, Boston.

Liuzzi G, Chirianni A, Bagnarelli P, Clementi M, Piazza M. A combination of nucleoside analogues and a protease inhibitor reduces HIV-1 RNA levels in semen: implications for sexual transmission of HIV infection. Antivir Ther.1999, 4:95-9.

Maher K, Klimas N, Fletcher MA. Disease progression, adherence, and response to protease inhibitor therapy for HIV infection in an Urban Veterans Affairs Medical Center. J Acquir Immune Defic Syndr 1999,  22:358-63.

Mannheimer S, Friedland G, Matts J, et al. The consistency of adherence to antiretroviral therapy predicts biologic outcomes for HIV-infected persons in clinical trials. Clin Infect Dis 2002, 34: 1115-21.

Marcelin AG, Tubiana R, Lambert-Niclot S, et al, and the Pitie-Salpetriere AMP a Risque Viral Study Group. Detection of HIV-1 RNA in seminal plasma samples from treated patients with undetectable HIV-1 RNA in blood plasma. Abstract 51, 16th CROI 2009 Montréal.

McCormack S, Ramjee G, Kamali A, et al. PRO2000 vaginal gel for prevention of HIV-1 infection (Microbicides Development Programme 301): a phase 3, randomised, double-blind, parallel-group trial. Lancet 2010, 376:1329-37.

Mertenskötter T, Kaptur PE. Update on microbicide research and development – seeking new HIV prevention tools for woman. Eur J Med Res 2011, 16:1-6.

Miller LG, Liu H, Hays RD, et al. How well do clinicians estimate patients´ adherence to combination antiretroviral therapy? J Gen Intern Med 2002; 17: 1-11.

Millett GA, Flores SA, Marks G, Reed JB, Herbst JH. Circumcision status and risk of HIV and sexually transmitted infections among men who have sex with men: a meta-analysis. JAMA 2008, 300:1674-84.

Mills E, Cooper C, Anema A, Guyatt G. Male circumcision for the prevention of heterosexually acquired HIV infection: a meta-analysis of randomized trials involving 11,050 men. HIV Med 2008, 9:332-5. Review.

Montaner JS, Hogg R, Wood E, Kerr T, Tyndall M, Levy AR, Harrigan PR. The case for expanding access to highly active antiretroviral therapy to curb the growth of the HIV epidemic. Lancet 2006, 368:531-536.

Morin SF, Shade SB, Steward WT, et al. A behavioral intervention reduces HIV transmission risk by promoting sustained serosorting practices among HIV-infected men who have sex with men. J AIDS 2008, 49:544-51.

Mulligan K, Glidden D, Gonzales P, et a l. Effects of FTC/TDF on bone mineral density in seronegative men from 4 continents: DEXA results of the Global iPrEx Study. Abstract 94LB, 18th CROI 2011, Boston.

Murri R, Ammassari A, De Luca A, et al. Self-reported nonadherence with antiretroviral drugs predicts persistent condition. HIV Clin Trials 2001, 2:323-9.

Nagot N, Ouedraogo A, Foulongne V, et al. Reduction of HIV-1 RNA levels with therapy to suppress herpes simplex virus. NEJM 2007;356:790-9.

Nathanson N. AIDS vaccine at the crossroads. Abstract 92, 15th CROI 2008, Boston.

Neely MN, Benning L, Xu J, et al. Cervical shedding of HIV-1 RNA among women with low levels of viremia while receiving highly active antiretroviral therapy. J AIDS 2007;44:38-42.

Nelson M, Girard PM, Demasi R, et al. Suboptimal adherence to darunavir/ritonavir has minimal effect on efficacy compared with lopinavir/ritonavir in treatment-naive, HIV-infected patients: 96 week ARTEMIS data. J Antimicrob Chemother 2010, 65:1505-9.

Nunnari G, Otero M, Dornadula G, et al. Residual HIV-1 disease in seminal cells of HIV-1-infected men on suppressive HAART: latency without on-going cellular infections. AIDS 2002;16:39-45.

Padian NS, van der Straten A, Ramjee G, et al. Diaphragm and lubricant gel for prevention of HIV acquisition in southern African women: a randomized controlled trial. Lancet 2007;370:251-61.

Paterson DL, Swindells S, Mohr J. Adherence to protease inhibitor therapy and outcomes in patients with HIV infection. Ann Intern Med 2000, 133:21-30.

Paz-Bailey G, Sternberg M, Puren AJ, et al. Improvement in healing and reduction in HIV shedding with episodic acyclovir therapy as part of syndromic management among men: a randomized, controlled trial. J Infect Dis 2009, 200:1039-49.

Porco TC, Martin JN, Page-Shafer KA, et al. Decline in HIV infectivity following the introduction of HAART. AIDS 2004, 18:81-8.

Quinn TC, Wawer MJ, Sewankambo N, et al. Viral load and heterosexual transmission of HIV type 1. N Engl J Med 2000, 342:9219.

Rerks-Ngarm S, Pitisuttithum P, Nitayaphan S, et al. Vaccination with ALVAC and AIDSVAX to prevent HIV-1 infection in Thailand. N Engl J Med 2009, 361:2209-20.

Sánchez J, Sal Y Rosas VG, Hughes JP, et al. Male circumcision and risk of HIV acquisition among MSM. AIDS 2011, 25:519-23.

Schwarze S. Getretener Quark wird breit, nicht stark: Was man von den “AIDS-Skeptikern” wirklich lernen kann.

Schwebke JR. Abnormal vaginal flora as a biological risk factor for acquisition of HIV infection and sexually transmitted diseases. J Infect Dis 2005, 192:1315-7.

Sendi PP, Bucher HC, Harr T, et al. Cost effectiveness of HAART in HIV-infected patients. Swiss HIV Cohort Study. AIDS 1999, 13:1115-22.

Serwadda D, Wawer MJ, Makumbi F, et al. Circumcision of HIV-infected men: effects on high-risk human papillomavirus infections in a randomized trial in Rakai, Uganda. J Infect Dis 2010, 201:1463-9.

Sethi AK, Celentano DD, Gange SJ, Moore RD, Gallant JE. Association between adherence to antiretroviral therapy and HIV drug resistance. Clin Infect Dis 2003;37:1112-8.

Sheth P, Kovacs C, Kemal K, et al, and the Toronto Mucosal HIV Res Group. Persistent HIV RNA shedding in semen despite effective ART. Abstract 50, 16th CROI 2009 Montréal.

Siegfried N, Muller M, Deeks J, et al. HIV and male circumcision–a systematic review with assessment of the quality of studies. Lancet Infect Dis 2005; 5: 165-73.

Singh JA, Mills EJ. The abandoned trials of pre-exposure prophylaxis for HIV: what went wrong? PLoS Med 2005, 2:e234.

Stoll M, Claes C, Schulte E, et al. Direct costs for the treatment of HIV-infection in a German cohort after the introduction of HAART. Eur J Med Res 2002, 7:463-471

Stone A, Jiang S. Microbicides: stopping HIV at the gate. Lancet 2006; 368: 431-3.

Stürmer M, Doerr HW, Berger A, Gute P. Is transmission of HIV-1 in non-viraemic serodiscordant couples possible? Antivir Ther 2008, 13:729-32.

Tobian A, Serwadda D, Quinn T, et al. Trial of male circumcision: prevention of HSV-2 in men and vaginal infections in female partners, Rakai, Uganda. Abstract 28LB, 15th CROI 2008, Boston.

Tovanabutra S, Robison V, Wongtrakul J, et al. Male viral load and heterosexual transmission of HIV-1 subtype E in northern Thailand. J Acquir Immune Defic Syndr. 2002. 29:275-83.

Turner BJ. Adherence to antiretroviral therapy by HIV-infected patients. J Infect Dis 2002; 185 Suppl 2: S143-51.

Van Damme L, Govinden R, Mirembe FM, et al. Lack of effectiveness of cellulose sulfate gel for the prevention of vaginal HIV transmission. N Engl J Med 2008, 359:463-72.

Van Damme L, Ramjee G, Alary M, et al. Effectiveness of COL-1492, a nonoxynol-9 vaginal gel, on HIV-1 transmission in female sex workers: a randomized controlled trial. Lancet 2002; 360: 971-7.

Van de Perre P, Segondy M, Foulongne V, et al. Herpes simplex virus and HIV-1: deciphering viral synergy. Lancet Infect Dis 2008, 8:490-7.

Vanpouille C, Lisco A, Derudas M, et al. A new class of dual-targeted antivirals: monophosphorylated acyclovir prodrug derivatives suppress both human immunodeficiency virus type 1 and herpes simplex virus type 2. J Infect Dis 2010, 201:635-43.

Velasco-Hernandez JX, Gershengorn HB, Blower SM. Could widespread use of combination antiretroviral therapy eradicate HIV epidemics? Lancet Infect Dis 2002, 2:487-93.

Vernazza PL, Troiani L, Flepp MJ, et al. Potent antiretroviral treatment of HIV-infection results in suppression of the seminal shedding of HIV. The Swiss HIV Cohort Study. AIDS 2000;14:117-21.

Vettore MV, Schechter M, Melo MF, Boechat LJ, Barroso PF. Genital HIV-1 viral load is correlated with blood plasma HIV-1 viral load in Brazilian women and is reduced by antiretroviral therapy. J Infect 2006;52:290-3.

Watson-Jones D, Wald A, Celum C, et al. Use of acyclovir for suppression of human immunodeficiency virus infection is not associated with genotypic evidence of herpes simplex virus type 2 resistance to acyclovir: analysis of specimens from three phase III trials. J Clin Microbiol 2010, 48:3496-503.

Watson-Jones D, Weiss HA, Rusizoka M, et al. Effect of herpes simplex suppression on incidence of HIV among women in tanzania. N Engl J Med 2008, 358:1560-71.

Wawer M, Kigozi G, Serwadda D, et al. Trial of male circumcision in hiv+ men, rakai, uganda: effects in HIV+ men and in women partners. Abstract 33LB, 15th CROI 2008, Boston.

Weiss HA, Thomas SL, Munabi SK, Hayes RJ. Male circumcision and risk of syphilis, chancroid, and genital herpes: a systematic review and meta-analysis. Sex Transm Infect 2006; 82: 101-9

Williams BG, Lloyd-Smith JO, Gouws E, et al. The potential impact of male circumcision on HIV in Sub-Saharan Africa. PLoS Med 2006; 3:

Wilson DP, Law MG, Grulich AE, et al. Relation between HIV viral load and infectiousness: a model-based analysis. Lancet 2008, 372:314-20.

Wood E, Hogg RS, Yip B, et al. The impact of adherence on CD4 cell count responses among HIV-infected patients. J Acquir Immune Defic Syndr 2004, 35:261-8.

Yang O, Daar E, Jamieson B, et al. HIV-1 Clade B superinfection: evidence for differential immune containment of distinct clade b strains. J Virol 2005; 79:860-8

Zuckerman RA, Lucchetti A, Whittington WL, et al. Herpes simplex virus (HSV) suppression with valacyclovir reduces rectal and blood plasma HIV-1 levels in HIV-1/HSV-2-seropositive men: a randomized, double-blind, placebo-controlled crossover trial. J Infect Dis 2007, 196: 1500–8.

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Filed under 6.12. Prevention, compliance, costs, 6.9. Salvage Therapy, Part 2 - Antiretroviral Therapy

6.13. Global Access to HIV Treatment

– Rob Camp –

We all know this data, and we gloss over it every time we go to an international meeting:

•  Some 7,000 people become infected with HIV every day.

•  Approximately 5.25 million people are currently receiving ART, while at least 14.6 million people are still in need.

•  For every 2 people put on treatment, 5 more become infected.

•  With universal access, approximately 6.7 million people would receive life-saving ART, 2.6 million new infections could be averted and 1.3 million lives saved. Based on global goals and targets for 2015, it is estimated that an investment of US$25.1 billion/yr will be required for the global AIDS response in low- and middle-income countries (www.who.org, accessed 19.04.10).

In the developing world, the price of ART has fallen drastically in recent years. Even so their cost remains an obstacle to access for many millions. Moreover, the health infrastructure required to deliver ART and maintain adherence is lacking in many places. Access to drugs depends not only on financial and human resources. It depends also on people being aware of their HIV status, knowledgeable about treatment, and empowered to seek it. Thus public information and education are important elements in widening access, alongside efforts to build or strengthen health services. Stigma has been and remains a major stumbling block in wanting, seeking and taking the treatment regimen correctly. The campaign for universal access to life-saving drugs for HIV and AIDS, started originally by grassroots AIDS activists, is today a major focus of attention of UN agencies and other influential organizations at national and global levels.

The Declaration of Commitment on HIV/AIDS, unanimously endorsed by the UN General Assembly in 2001, embraced equitable access to care and treatment as a fundamental component of a comprehensive and effective global HIV response. Since then many countries, through the support of intergovernmental organizations and donors, have definitively demonstrated the ability to deliver HIV treatment in very resource-limited settings. Access to treatment has helped mobilize communities in response to HIV, preserved the health and viability of households vulnerable to HIV, and strengthened HIV prevention efforts in many parts of the world.

In the goal to reach universal access to HIV prevention, treatment, care and support, leadership at national level is required to establish policies that support treatment scale-up by:

•  increasing the number of people who choose to know their HIV status;

•  reducing HIV stigma;

• building human capacity to sustain treatment through training and better use of current human resources;

• improving supply management and integrating HIV care with other health services.

In 2011, the international community recommitted to the goal of universal access. This time, countries committed to achieving universal access by 2015. The goal of universal access is also part of Millennium Development Goal (MDG) 6 which includes the goal of halting and beginning to reverse the spread of HIV/AIDS by 2015.

The updated 2011-2015 global health strategy was released in June 2011. This strategy outlines four key targets that countries need to achieve if universal access and MDG 6 are to be realised: reduce new infections by 50 percent among young people (15-24 years), reduce TB-related mortality by 50 percent, eliminate new infections in children, and reduce HIV-related mortality.

Major Players

PEPFAR Update

The President’s Emergency Plan for AIDS Relief (PEPFAR) was launched in 2003 to combat global HIV/AIDS, and is the largest commitment by any nation to combat a single disease in history. During PEPFAR’s initial phase covering 2004-2008, the United States invested nearly $19 billion in PEPFAR (defined to include bilateral HIV/AIDS and tuberculosis programs, as well as contributions to the Global Fund to Fight AIDS, Tuberculosis and Malaria). For FY 2011, $5.56 billion was enacted for bilateral HIV/AIDS programs, $1.05 billion for the Global Fund; the line item for bilateral TB programs has not been agreed upon yet.

PEPFAR and the fight against HIV/AIDS is now the cornerstone of the US Global Health Initiative, which commits $63 billion over six years to support countries in improving and expanding access to health services. As part of the Global Health Initiative, PEPFAR is moving from its initial emergency focus to a heightened emphasis on sustainability, and serves as a platform for expanded responses to a broad range of global health needs. Through its partnerships with 31 countries, as of September 2010, PEPFAR directly supported ART for over 3.2 million men, women and children. PEPFAR partnerships have directly supported care for nearly 11 million people affected by HIV/AIDS.

In 2010, PEPFAR directly supported prevention of mother-to-child transmission programs that allowed nearly 100,000 infants of HIV-infected mothers to be born without HIV, adding to the nearly 340,000 infants born without HIV due to PEPFAR support during 2004-2009. This of course, is not the limit as it is estimated that less than 25% of pregnant HIV-infected women get the care they need (beyond point-of-care transmission prevention). In FY 2010, PEPFAR also directly supported HIV counseling and testing for nearly 33 million people, providing what may be an important entry point to prevention, treatment, and care.

2004 – 2011 PEPFAR Funding ($ in USD millions)
Program

2004

2005

2006

2007

2008

2009

2010

2011

Total

Bilateral HIV/AIDS Programs1

1,643

2,263

2,654

3,699

5,028

5,503

5,542

5,549

26,332

GFATM

547

347

545

724

840

1,000

1,050

1,050

5,053

Bilateral TB Prog’s

87

94

91

95

163

177

243

ND

950

TOTAL PEPFAR (w/o malaria)

2,277

2,705

3,290

4,518

6,031

6,680

6,835

6,599*

32,335

1 Bilateral HIV/AIDS Programs includes funding for bilateral country/regional programs, UNAIDS, IAVI, Microbicides and NIH HIV/AIDS research.
*Possibly not the final total. For final US spending on PEPFAR 2012, please see Fiscal Year 2012 Budget Tracker at http://www.kff.org/globalhealth/8045.cfm.
Note: All funding amounts have been rounded to the nearest million $ so the numbers shown in the table may not sum to the totals.

GFATM

The Global Fund to Fight AIDS, Tuberculosis and Malaria is an international financing institution that invests the world’s money to save lives. To date, it has committed US$ 22.4 billion in 150 countries to support large-scale prevention, treatment and care programs against the three diseases. Round 11 of the grants cycle is now open until 15 Dec 2011. Right now, however, the Fund is unable to provide any accurate forecast or give assurances regarding the level of resources that will actually be available, since very few pledges have been confirmed.

The actual amount available will be influenced by three key considerations: 1) the level and timing of donor pledges, 2) new pledges to be announced at the time of the mid-term replenishment review in March 2012 and 3) the extent of any savings that the Global Fund will achieve through the stringent application of “value for money” and new performance-based funding principles, which can be seen at http://www.theglobalfund.org/en/application/process/eligibility/.

“More donor assistance is urgently needed to close the financing gap in health in the poorest countries of the world… Assistance from developed nations should increase from the current levels of about US$ 6 billion per year globally to US$ 27 billion by 2007 and US$ 38 billion by 2015”, according to the Commission on Macroeconomics and Health, 2003. As can be seen from the chart above, if PEPFAR represents 50% of the Global total, we are far from the target, about 1/3 of where we need to be.

The Global Fund is meant to constitute a major source of this funding. To operate effectively, the Global Fund requires strong and consistent financial commitments from all of its stakeholders. It is thus essential that substantial new pledges are received to finance additional grants and to continue successful programs.

Pledges and contributions from donors are received on an ad-hoc voluntary basis (www.globalfund.org, accessed 24.05.10). The Global Fund is relatively transparent and has an architecture of application, funding structure, management of performance and future planning that is inclusive and as broad-based as a top-down organism from Geneva that disburses money locally can be (www.globalfund.org/en/grantarchitecture). That much being said, the Fund often has to deal with scandals that stop funding from any one or group of countries temporarily. In 2011, we saw this happen with some European countries in what was a fairly small local abuse of funds report (Germany, Sweden and the Netherlands have all re-committed after temporary halts). For other reasons, like the economic downturn that started in 2008 and that shows little sign of abating in many places, Spain and Italy have not contributed the Global Fund for 2011 as of 8 September, which leaves a funding gap of close to half a billion euros. Other European countries with less commitments (Portugal, Ireland, Switzerland) have also not contributed this year.

The World Bank

The World Bank supplies more than just money for drugs – large chunks of its investment is intended for infrastructure of health systems (i.e., child health, health system performance, etc). This money, at least that specifically under AIDS, started to fall in 2004. Although the single year with most money was 2007, it did little to change the overall 3-year rolling average of being half now what it was 6 years ago. The 2010 moneys are at about the 1996 investment level. Which does not mean that infrastructure money may not be coming in under other themes – like malaria, nutrition or tuberculosis (http://search.worldbank.org/projects?qterm=HIV, accessed on 20.05.10). They also lend, offer technical support and analytic work. For example, in light of the current economic crisis, the Bank is supporting countries through technical assistance and collaborative efforts for a joint response by assessing the fiscal implications of scaling up national AIDS programs in Botswana, South Africa, Uganda and Swaziland. They lent some $55 million to African HIV programs in 2010, and expect to lend some $90 million in 2011.

UNAIDS

UNAIDS provides technical support to countries to assist them with expertise and planning for their national AIDS programs, to help ‘make the money work’ for the people on the ground, those that need it most. UNAIDS tracks, evaluates and projects the financial resource requirements at global, regional and country levels to generate reliable and timely information on the epidemic and the response. Based on these evaluations, UNAIDS produces guidelines and progress reports. Much of the international data we juggle is set and approved by UNAIDS. At the 2011 IAS meeting in Rome, they set out a plan of how to move forward in middle- to low-income countries in the face of the current economic downturn. The total investment needs should be met by a combination of sources, each of which has the potential to increase: The first is domestic public investment within low and middle-income countries, which can increase as a result of economic growth in those countries, as well as an increase in the historically relatively low level of priorisation to the AIDS response in their domestic budget. This, while a great idea, is not something that can be “turned on” overnight and while the lobbying for this goes on, people need treatment. A second source is to promote the potential for private financing and philanthropic foundations in low and middle-income countries. A third source would be an increase in the level of donor financing, compatible with a movement in the direction of meeting the target of devoting 0.7% of GDP in OECD countries to development assistance. Needed: Low- to middle-income countries’ domestic health budgets have to average at least 15% of government revenue (as in the Abuja Declaration in Africa). Another promising approach would be to expand innovative mechanisms like indirect taxation (airline tickets, mobile phone usage, exchange rate transactions) to support global health initiatives, and ensure that HIV benefits from these in relation to disease burden. The larger community must continue to support and strengthen existing financial mechanisms, including the Global Fund and relevant UN organizations.

The Bill and Melinda Gates Foundation

The largest private philanthropic organization to date is located in Seattle, US, “focusing on improving people’s health and giving them the chance” to emerge from “hunger and extreme poverty.” They have approximately 957 employees with an endowment of USD 36.3 billion. They have committed USD 25.364 billion since inception and in 2010 committed grants to the tune of USD 2.6 billion in over 100 countries. Much of these moneys are for non-AIDS-specific works, including development (reducing poverty and hunger). In health, they fight and prevent enteric and diarrheal diseases, HIV/AIDS, malaria, pneumonia, TB, neglected and infectious diseases, working on integrated heath solutions, improving delivery of existing tools and supporting research and development in new interventions like vaccines, drugs and diagnostics (http://www.gatesfoundation.org). They have supported the Global Fund with some $650 million as of mid-2010.

Drugs available from whom and where

FDA’s qualification of generics

Generic drugs are important options that allow greater access to health care. Generic drugs approved by FDA have the same high quality, strength, purity and stability as brand-name drugs. And, the generic manufacturing, packaging, and testing sites must pass the same quality standards as those of brand name drugs.

For PEPFAR use, all drugs need FDA approval. As of 2 September 2011, FDA had approved some 132 generic drugs for use in the PEPFAR program that are approved in as short a time as two to six weeks. While quality, strength, purity and stability are guaranteed, administration, delivery and correct use is another issue. For example, a drug approved in May 2010 was a fixed-dose combination of d4T and 3TC. And there the rub. Generics companies (in the case of FDA for PEPFAR, to date there are eleven Indian generics companies, 1 South African company, one company from China and one from the US) copy what is easiest and cheap, not necessarily the most innovative or optimal treatments only. We must continue to try to remind the generics companies that what is best for the patient will continue selling for years, while (hopefully) a less-than-optimal combination like 3TC+d4T has a limited life-time, and can do a lot of harm via side effects along the way.

Lopinavir/r is the first PI approved for generic licensing although there are eight approved PIs on the market in the Global North. Aurobindo got approval for a 25 mg version of ritonavir in early 2009, what could be a very interesting option in boosting in the future. Matrix got an FDA approval of a ritonavir 50 mg version (with lopinavir) on the same day. Matrix and Emcure both have approval for atazanavir (2010).

There are a handful of generics companies with an abacavir approval. As HLA testing for abacavir HSR is not easily available in the Global South, it is very important to train both the medical profession as well as users on diagnosis of HSR and what to do if it occurs, and the importance of never re-starting it once HSR is suspected, things that from an international regulatory agency would be hard to monitor. And although REMS programs from FDA or EMA would accept information on side effects from the Global South (which has up to 5 times the amount of people on drug), they probably contribute little to the overall numbers and thus better definition of safety of these drugs.

In 2011 (until 18 August), FDA has issued one warning letter to one generics company that manufactures HIV products. The letter was unclear about if it was related to an HIV product or not.

In total, in 2011, 400 FDA inspectors will perform more than 2,200 drug-related inspections. FDA takes many different enforcement actions.

WHO-approved generics

Prequalification and quality assurance of antiretroviral products – a fundamental human right

WHO’s Prequalification Programme conducts evaluation and inspection activities and builds national capacity for manufacturing and monitoring high-quality medicines. WHO began reviewing HIV antiretroviral drugs for prequalification in 2001.

In 2005–2006, WHO conducted a quality assurance survey of antiretroviral medicines in Cameroon, the Democratic Republic of the Congo, Kenya, Nigeria, Uganda, United Republic of Tanzania and Zambia. Of the 395 samples tested, none had quality deficiencies that would pose a risk to the people taking them. The results of this and future surveys on drug quality are key to ensuring that the pace of scaling up treatment does not compromise the quality of the medicines available.

Invitations to manufacturers to submit an expression of interest (EOI) for product evaluation are issued not only for HIV/AIDS-related care and treatment products, but also for antimalarial medicines, antituberculosis medicines, influenza-specific antiviral medicines and reproductive health products.

On the WHO List of Prequalified Medicinal Products is an extended list of 290 products (http://apps.who.int/prequal/, accessed 9.09.2011) for HIV/AIDS, made by both originator companies and generics companies. Prequalification may be better described as pre-, on-going, and post-qualification, as they do inspections at all these time points. On this list is atazanavir, which has WHO approval from a BMS manufacturing facility in the US as well as an Indian generics company. Neither Brazil nor Thailand have pre-approved drugs on either list (FDA or WHO) because although they both have and produce generic HIV drugs, they do so only for domestic use.

On the list are many drugs for OIs (acyclovir, ceftriaxone, ciprofloxacin, amongst others). WHO also approves medicines quality control laboratories (QCLs): 21 QCLs are currently prequalified all around the world.

Antiretroviral therapy in low- and middle-income countries by region, December 2009

Region

Estimated number of people receiving ART

Estimated number of people needing ART

ART coverage

Sub-Saharan Africa

3 911 000

10 600 000

37%

Eastern and southern Africa

3 203 000

7 700 000

41%

West and central Africa

709 000

2 900 000

25%

Latin America and the Caribbean

478 000

950 000

50%

Latin America

425 000

840 000

51%

The Caribbean

52 400

110 000

48%

East, South and South-East Asia

739 000

2 400 000

31%

Europe and Central Asia

114 000

610 000

19%

North Africa and the Middle East

12 000

100 000

11%

Total

5 254 000

14 600 000

36%

Source: Towards universal access: scaling up priority HIV/AIDS interventions in the health sector. Progress report 2010 (WHO, UNICEF, UNAIDS), p.53.

With the new threshold of starting treatment at 350 CD4s, even with more people starting treatment, the percentage of those treated who should be on treatment has actually fallen.

A chasm

Improved treatment in line with scientific evidence and recognized international standards of care

Médecins Sans Frontières (MSF, Doctors without Borders) serves approximately 210,000 of the ~5 million people today on treatment, and because they are on the front lines in clinics and health centers in more than 70 countries, their advocacy is not of the ivory tower type. Due to the implementation of ART, they have seen first hand the reduction in mortality for both adults and children, the lowering of incidence of TB as well as the importance of supporting HIV prevention by lowering incidence. They believe that not continuing to invest today in improved treatment and protocols will cost lives down the road, increase a double standard in HIV care and lead to increased costs later. They believe that there is a clear risk that donors may not continue to support or try to delay the implementation of proven and recommended medical strategies for the sake of short-term savings. They recommend:

• Supporting initiation of ART at a CD4 T cell threshold of 350/μl to reduce the incidence of TB and other OIs and improve survival rates, reducing the need for costly and complex acute care.

• Implementing a tenofovir-based first-line regimen will allow patients to stay on their first regimen as long as possible with fewer side effects and delay the need for more costly second-line regimens.

• Providing access to viral load testing to support adherence and detect treatment failure earlier, thereby preventing resistance and needless switching to expensive sub-optimal second-line treatment.

• Supporting innovation that can lead to further improvement and simplification of HIV treatment in resource-poor settings.

According to MSF, most people with HIV/AIDS in need of treatment in the world will die within three years if they do not gain access to treatment now.

How to ensure that prices of drugs and diagnostics remain reasonable?

The international community needs to support policies that will enable funds to stretch as far as possible to meet needs and contain costs in the short- and long-term by ensuring a competitive supply for drugs.

In accordance with the Doha Declaration on TRIPS and Public Health, governments can authorize governmental use or compulsory licenses to ensure generic production of patented products (as in Brazil and Thailand).

Companies and governments can support the Medicines Patent Pool for antiretroviral medicines that originated at UNITAID (www.unitaid.eu), now a freestanding organization at www.medicinespatentpool.org. This mechanism brings together patents held by different owners and makes them available to others for generic production and further development. MSF has started a ‘Make It Happen Campaign”. Gilead was the first company to sign on, in July 2011. They are currently negotiating with F. Hoffmann-La Roche, Sequoia Pharmaceuticals, the US National Institutes of Health and ViiV Healthcare. BI and BMS have just started to negotiate. This Pool could save lower income countries more than $1 billion a year in drug costs.

Prices of first-line regimens in low-income countries

The median price paid for tenofovir+3TC+efavirenz (prequalified by WHO) in low-income countries in June 2011 ranged from US$ 143 per person per year for the two pill dose to US$ 173 for the fixed-dose combination. The weighted average median price of the four combinations most widely used in first-line treatment (representing 86% of the prescribed first-line treatments in low-income countries) was US$ 170 per person per year in 2007. The decline in drug prices between 2004 and 2007 can be attributed to the scaling up of treatment programs, increased competition between a growing number of products prequalified by WHO, new pricing policies by pharmaceutical companies and successful negotiations between the William J. Clinton Foundation (CHAI) and major generic manufacturers. While combinations with d4T are cheaper, and studies continue to be carried out with even-lower doses, etc, the long-term side effects do not outweigh the harm, and programs should move to tenofovir-containing regimens as soon as they can.

Second-line regimens

Second-line regimens are still significantly more expensive than first-line regimens in low- and middle-income countries. In 2011, the median cost of a regimen of AZT/3TC+atazanavir/r, a newly indicated second-line regimen, was US$ 442 in low-income countries and up to six times that in middle-income countries. The actual prices paid for second-line regimens vary significantly between countries. For example, South Africa pays an average price of US$ 1,600 per person per year for ddI+abacavir+lopinavir/r, whereas El Salvador paid US$ 3,448 per person per year for the same regimen in 2007.

In the UK, a recent study showed that first-line treatments can last 8 years or longer, but by then, of those who start, more than 25% of people will have failed (UK Chic 2010). If ARV access started in earnest in 2002, we are at the 8-year mark. What to do with the approximately 1 million people who must need to move to a new regimen? And of the regimens that fail, NNRTIs fail at a rate almost three times higher than the rate of PIs. Most people in resource-limited settings are on an NNRTI containing regimen. MSF estimates that regimen failure is “largely under-diagnosed” due to limited lab facilities for viral load testing, which can only lead to resistance and harder-to-construct post-first-line regimens.

How to expand treatment to more people plus switch those currently failing to an effective regimen, all within a framework of cutting back on donor spending?

As the absolute numbers of people who need access to second-line regimens continue to grow, addressing the high cost of second-line regimens will become increasingly important to ensure the most cost-effective use of available resources. A third-line treatment is currently US$ 2766 in low-income, US$ 5870 in middle-income countries.

Future Funding

As funding stalls, major funders – US, UK, Netherlands, France, Germany, Norway and Sweden – may be becoming fatigued. In 2001 at the UNGCP meeting, recipient countries were asked to dedicate 15% of their national budgets to health, agreed to n theory by the Abuja Declaration. Only 8 countries have done so. More than half of African countries spend less than the UN minimum of 34 US$ per capita.

New strategies have to be developed – small taxes on currency transactions, etc. Small airline ticket tax from many countries (see above). Product (Red) is a fund-raiser of the Global Fund that coordinates profits from sales from partner businesses and has recently reached the USD 170 million mark.

As mainstays of program centers are at best maintaining previous amounts (flat-funding, like PEPFAR) or unclear about their budgets for this year (GFATM), it is important that we all contribute, both economically and using advocacy, about how to continue to arrive to the amount needed (USD 25.1 billion/yr).

Europe gets involved

The European Union can impact access to medicines for developing countries through its policies, legislation and bilateral and regional trade agreements. The EU can adopt appropriate measures to improve access to existing medical tools (medicines, diagnostics, vaccines) as well as stimulate the research and development of better tools for people in resource-starved countries. The Working Group on Innovation, Access to Medicines and Poverty-Related Diseases will create a meaningful dialogue between Members of the European Parliament, the European Commission, and civil society.

More problems than solutions

It is quite possibly easier to flag the difficulties than offer or implement solutions, but one survey was recently carried out, this time by Oxfam. In a recent analysis of meeting the Millenium Development Goals, they criticized Europe overall as not valuing poor people “enough to … guarantee … by making aid commitments legally binding. … This year alone, the EU is 19 billion euros short of its targets … enough to have saved 3 million lives in poor countries.” They have started a Robin Hood tax (a tiny tax on bankers that could generate billions of US$). In their change.org blog, they say that donors are not transparent with how their money is spent and where. For example, they highlight Kenya, where PEPFAR purportedly spent over one 500 million dollars on AIDS programs in 2008. A representative of the Kenyan Ministry of Health says that they cannot get “a list of partners, where they are working, how much they are spending, on what” from the US (http://globalpoverty.change.org/blog/view/how_transparent_is_us_foreign_aid). Which makes an integrated strategy on AIDS hard to accomplish. Without being too blithe, imagine that happening in the North: we knew how much we are spending, but not on what!

The unconscionable health gap: a global plan for justice

In the Lancet, Lawrence Gostin outlined a plan for health access for all (Gostin 2010). Despite robust international norms, health disparities render a person’s likelihood of survival drastically different depending on where she or he is born. WHO urges “closing the health gap in a generation” through action on the social determinants of health.

International health assistance has quadrupled over two decades rising to US$21.8 billion in 2007 (Ravishankar 2009). This level of funding might seem impressive but sits modestly beside the annual $1.5 trillion spent globally on military expenditures (2.43% of global gross domestic product), and $300 billion in agricultural subsidies.

Foreign aid simply is not predictable and scalable to needs and often reflects donors’ geostrategic interests rather than the key determinants of health. Developed countries recognize the health gap, but are resistant to taking bold remedial action.

If the health gap is unfair and unacceptable, how can the international community be galvanized to make a genuine difference? A global plan for justice would be a voluntary compact between states and their partners. It would simply encourage WHO to exercise its constitutional powers and leadership.

A global plan for justice would set achievable funding targets for a global health fund to be distributed according to need (Ooms 2008). Although WHO would negotiate the funding levels, developed countries could donate, for example, 0.25% of gross national income (GNI) per annum, in addition to current foreign assistance.

A global plan for justice would guarantee a universal package of essential services, comprising three core components: essential vaccines and medicines, basic survival needs, and adaption to climate change.

The international community must do more than lament ongoing, unconscionable health inequalities. It must act boldly and with a shared voice, such as through a global plan for justice. If the world does not act, the avoidable suffering and early death among the world’s least healthy people will continue unabated—a breach of social justice that is no longer ethically acceptable (Gostin 2010).

The amount of people who need access to ART in the next few years – in the short term – will grow substantially. Because of this, we need to keep up the pressure on all actors – donor organisations as well as individual nations, manufacturers, health care workers and affected communities of all sizes – to do their part in order to provide the most current and useful treatment strategies possible, to whole populations. In order to achieve this, we can not sit idly by and hope for the best – we must continue to push that boulder up the hill for as long as it takes so everyone who needs it has access to treatment and care as early and for as long as necessary.

 

Because global access is such a moving target, all references are web-based and in the text.

References

Collaborative Group on HIV Drug Resistance and UK CHIC Study Group. Long-term probability of detecting drug-resistant HIV in treatment-naïve patients initiating combination antiretroviral therapy. Clin Infect Dis 2010, 50: 1275-85.

Gostin L. The unconscionable health gap: a global plan for justice. Lancet 2010, 375:1504-5.

Harrigan RP. HIV drug resistance over the long haul. Clin Infect Dis 2010, 50: 1286-87.

Ooms G, Hammonds R. Correcting globalization in health: transnational entitlements versus the ethical imperative of reducing aid-dependency. Public Health Ethics 2008; 1: 154-170.

Ravishankar N, Gubbins P, Cooley RJ, et al. Financing of global health: tracking development assistance for health from 1990 to 2007. Lancet 2009; 373: 2113-2124.

Greener R. Financing the response to HIV in low- and middle-income countries: how it is affected by the economic crisis? UNAIDS presentation at IAS Rome, 20 July 2011.

Utw.msfaccess.org, Untangling the web of antiretroviral price reductions, 14th edition, Medecins Sans Frontieres, July 2011.

Links

Europe is Missing in Action, www.oxfam.org/en/pressroom/pressrelease/2010-06-17/europe-missing-action-when-should-be-leading-poverty-charge, 17 June 2010

FDA enforcement actions in the year 2008, http://www.fda.gov/downloads/ICECI/EnforcementActions/EnforcementStory/UCM129812.pdf.

http://www.globalfund.org/en/applicantsimplementers/resources/?lang=en

PEPFAR, http://www.pepfar.gov/press/80064.htm

 “Punishing Success? Early Signs of a Retreat from Commitment to HIV/AIDS Care and Treatment”, November 2009, http://www.msf.org/msfinternational/ accessed online 29.04.10.

www.msfaccess.org/main/access-patents/european-parliament-working-group/about-working-group/

World Health Assembly. Reducing health inequities through action on the social determinants of health, http://apps.who.int/gb/ebwha/pdf_files/EB124/B124_R6-en.pdf, accessed Jan 4, 2010

UNAIDS, http://www.unaids.org/en/resources/presscentre/pressreleaseandstatementarchive/2011/june/20110610psdeclaration/

http://www.un.org/en/ga/aidsmeeting2011/

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Filed under 6.13. Global Access to HIV Treatment, 6.9. Salvage Therapy, Part 2 - Antiretroviral Therapy

Lipodystrophy Syndrome

– Georg Behrens, Reinhold E.Schmidt –

Background

The HIV lipodystrophy syndrome including metabolic complications and altered fat distribution is a possible side effect of HIV therapy. Fortunately, modern therapy regimens are much less likely to lead to fat tissue abnormalities. The metabolic abnormalities may harbor a significant risk of developing cardiovascular disease. In addition, several studies report a reduced quality of life in patients with body habitus changes leading to a reduced treatment adherence. Despite the impact of lipodystrophy syndrome on HIV management, little is known about the pathogenesis, its prevention, diagnosis and treatment. Current data indicate a rather multifactorial pathogenesis where HIV infection, ART, and patient-related factors are all major contributors. The lack of a clear and easy definition reflects the clinical heterogeneity, limits a clear diagnosis and impairs the comparison of results among clinical studies. Therapeutic and prevention strategies have so far been of only limited clinical success, where avoiding the use of thymidine analogues appears to be most effective in avoiding peripheral fat loss. General recommendations include dietary changes and lifestyle modifications, altering antiretroviral therapy (replacing protease inhibitors with NNRTIs or replacing d4T and AZT with abacavir or tenofovir), and finally, the use of metabolically active drugs. Here we summarize the pathogenesis, diagnosis and treatment options of the HIV lipodystrophy syndrome.

Clinical manifestation

Lipodystrophy was originally described as a condition characterized by regional or generalized loss of subcutaneous fat. The non-HIV-associated forms, such as congenital or familial partial lipodystrophy, have a very low prevalence. Generally, these forms are associated with complex metabolic abnormalities and are difficult to treat. The term “lipodystrophy syndrome” was introduced to describe a complex medical condition including an apparent abnormal fat redistribution and metabolic disturbances in HIV patients receiving protease inhibitor therapy (Carr 1998). Now, years after its first description, there is still no consensus on a case definition for lipodystrophy syndrome in HIV. Thus, the diagnosis of lipodystrophy in clinical practice often relies on a more individual interpretation than on an evaluated classification. Finally, changes in fat distribution have to be considered as being part of a rather dynamic process. In most cases, peripheral lipoatrophy is clinically diagnosed when significant fat loss of about 30% has already occurred.

HIV-associated lipodystrophy includes both clinical and metabolic alterations. The most prominent clinical sign is a loss of subcutaneous fat (lipoatrophy) in the face (periorbital, temporal), limbs, and buttocks. Prospective studies in patients on thymidine alanogues have demonstrated an initial increase in limb fat during the first months of therapy, followed by a progressive decline over the ensuing years (Mallon 2003), which is mostly persistent (Grunfeld 2010). Peripheral fat loss can be accompanied by an accumulation of visceral fat, which can cause mild gastrointestinal symptoms. Initially truncal fat increases on therapy and then remains stable (Mallon 2003). Visceral obesity, as a singular feature of abnormal fat redistribution, appears to occur in only a minority of patients. Fat accumulation may also be found as dorsocervical fat pads (buffalo hump) or within the muscle and the liver. Female HIV-positive patients sometimes complain about painful breast enlargement, attributed to the lipodystrophy syndrome. Whether gynecomastia in male patients is a component of the syndrome remains unclear. There is now accumulating evidence that the major clinical components – lipoatrophy, central adiposity and the combination of both – result from different pathogenetic developmental processes.

The prevalence of a clinically evident lipodystrophy syndrome was estimated to be between 30 and 50% based on cross-sectional studies before 2005. A prospective study over an 18-month period after initiation of therapy revealed a prevalence of 17% but current HIV therapy combinations can be expected to lead to a lower incidence. More recent studies estimated the annual incidence rates of detectable but clinically inapparent peripheral fat loss (-20%) with about 5-10% in patients receiving a nuke-backbone with tenofovir, abacavir, 3TC or FTC. Lipodystrophy, and in particular lipoatrophy, has been observed most frequently in patients receiving a combination regimen of nucleoside analogues (particularly thymidine analogues) and protease inhibitors, although almost all antiretroviral drug combinations can be associated with fat redistribution. The risk of the syndrome increases with the duration of treatment, the age of the patient and the level of immunodeficiency. Children can be affected, like adults, with clinical fat redistribution shortly after initiation or change of ART. The evolution of the individual clinical components of the lipodystrophy syndrome is variable. The nucleoside analogue linked most strongly to lipoatrophy is d4T, particularly when used in combination with ddI. Tenofovir combined with 3TC and efavirenz is associated with less loss of limb fat than d4T in a similar combination in therapy-naïve HIV patients (Gallant 2004).

Frequently, complex metabolic alterations are associated with these body shape alterations. These include peripheral and hepatic insulin resistance, impaired glucose tolerance, type 2 diabetes, hypertriglyceridemia, hypercholesterolemia, increased free fatty acids (FFA), and decreased high-density lipoprotein (HDL). Often these metabolic abnormalities appear or deteriorate before the manifestation of fat redistribution. The prevalence of insulin resistance and glucose intolerance has been reported in the literature at 20 to 50% depending on the study design and measurement methods and it increases with age (Hasse 2011). Frank diabetes is less frequent with a prevalence of between 1 and 6%. The incidence rate were highest at the time when PIs were introduced around 2000 but remain elevated as compared to seronegative control groups (Capeau 2011). Lipodystrophic patients present with the highest rates of metabolic disturbances.

Hyperlipidemias are a frequently observed side effect of antiretroviral therapy, especially in combinations that include PIs. Newer drugs such as maraviroc or raltegravir and also second-generation NNRTIs such as rilpivirine seem to cause only minor disturbances in lipid metabolism (DeJesus 2010). Given that many HIV-infected patients present with already decreased HDL levels, these are not further reduced by antiretroviral drugs, but usually improve to some degree, particularly when NNRTIs such as nevirapine are used. Hypertriglyceridemia, especially in patients with evidence of body fat abnormalities, is the leading lipid abnormality either alone or in combination with hypercholesterolemia. Several weeks after initiation or change of HIV therapy, lipid levels usually reach a plateau and remain stable. Part of this increase can be considered as reconstitution of health, as some patients return to the lipid levels they had before seroconversion. All protease inhibitors can potentially lead to hyperlipidemia, although to different extents. For example, atazanavir and darunavir appear to be less frequently associated with dyslipidemia and insulin resistance. In contrast, ritonavir often leads to hypertriglyceridemia correlating to the drug levels. Lopinavir leads to an approximate 18% mean increase in total cholesterol and 40% mean increase in triglycerides in patients on first line therapy.

The therapy-induced dyslipidemias are characterized by increased low-density lipoproteins (LDL) and triglyceride-rich very low density lipoproteins (VLDL). Detailed characterization revealed an increase of apoplipoprotein B, CIII and E. Raised levels of lipoprotein(a) have been described in protease inhibitor recipients. Mild hypercholesterolemia can occur during therapy with efavirenz but is not typical with nevirapine. D4T-based ART is associated with early and statistically significant increases in total triglycerides and cholesterol or NRTIs. Several studies suggest that tenofovir exerts a moderate lipid-lowering effect on both total and LDL cholesterol as well as HDL cholesterol (Randell 2010). It is important to note that HIV infection itself is associated with disturbed lipid metabolism. During disease progression, total cholesterol, LDL, and HDL levels decline and the total triglyceride level rises. The latter is presumably caused by increased cytokine concentrations (TNFα, IFNg) and an enhanced lipogenesis in addition to impaired postprandial triglyceride clearance.

ART, lipodystrophy syndrome and cardiovascular risk

The fat redistribution and disturbances in glucose and fat metabolism resemble a clinical situation that is known as the “metabolic syndrome” in HIV-negative patients. This condition includes symptoms such as central adipositas, insulin resistance and hyperinsulinemia, hyperlipidemia (high LDL, Lp(a) hypertriglyceridemia and low HDL) and hypercoagulopathy. Given the well-established cardiovascular risk resulting from this metabolic syndrome, there is growing concern about a potential therapy-related increased risk of myocardial infarction in HIV-positive patients. These fears are further sustained by reports of arterial hypertension on ART, a high rate of smoking among HIV patients and increased levels of tissue plasminogen activator (tPA) and plasminogen activator inhibitor-1 (PAI-1) in patients with lipodystrophy. Although many of the mainly retrospective studies dealing with this issue are inconclusive, data from a large international study (the D:A:D study) provide evidence for an increased relative risk of myocardial infarction during the first 7 years of ART (Friis-Møller 2003, El-Sadr 2006). The incidence of myocardial infarction increased from 1.39/1,000 patient years in those not exposed to ART, to 2.53/1,000 patient years in those exposed for < 1 year, to 6.07/1,000 patient years in those exposed for ≥ 6 years (RR compared to no exposure: 4.38, p = 0.0001). After adjustment for other potential risk factors, there was a 1.17-fold increased risk of myocardial infarction per additional year of combined ART exposure. It is, however, of note that older age, male gender, smoking, diabetes mellitus, and pre-existing coronary artery disease were still associated with a higher risk of sustaining cardiovascular events than ART in this study. Analysis of this cohort later provided evidence that PIs (particularly indinavir and lopinavir), abacavir and triglycerides contribute to this increased risk (Friis-Moller 2007, D:A:D Study Group 2008, Worm 2010, Worm 2011). Several other cohort studies, although not all (Lang 2011), confirmed the association of abacavir use and myocardial infarction (Behrens 2010). According to a meta-analysis (Cruciani 2011) considering prospective, controlled studies about the use of abacavir, the rate of myocardial infarction in these mostly young patients with a rather low cardiovascular risk profil was not significantly different to control regimens. Currently, it seems reasonable to consider alternatives for abacavir only in patients with a high cardiovascular risk (Framingham risk score >20%).

Although the CHD risk profile in D:A:D patients worsened over time, the risk of myocardial infarction decreased over time after controlling for these changes. Several other studies used ultrasonography to measure the thickness of the carotid intima media or endothelial function to predict the cardiovascular risk. Some of these investigations found abnormal test results (e.g., reduced flow-mediated dilation) that correlated either with the use of PIs or the presence of dyslipidemia (Currier 2005). Interestingly, HIV infection may lead to endothelial dysfunction and an unfavorable pro-atherogenic profile (Grunfeld 2009). While there is some indication of an increased rate of coronary artery disease with ART, particularly in combinations containing abacavir (D:A:D 2008), the benefit of suppressed viral replication and improved immune function resulting in reduced morbidity and mortality, clearly argues for the use of antiretroviral drugs according to current international guidelines. It seems obvious however, that pre-existing cardiovascular risk factors in individual patients need to be considered more carefully before starting or switching ART.

Recommendations such as the National Cholesterol Education Program (NECP) have been proposed for non-HIV-infected patients with similar risk profiles. These guidelines are being considered for HIV patients as well (Schambelan 2002, Grinspoon 2005). According to these recommendations, the overall cardiovascular risk in HIV-infected patients can be determined from specific risk factors by using the Framingham equation. Prediction of coronary heart disease using this equation, however, may have some limitations. A 10-year CHD risk estimation at any time point is determined by the individual’s past and expected future lipid levels (best assessed as area under the curve). Hyperlipidemia in many treated HIV-infected patients, however, does not follow the 10-year time course seen in the uninfected population due to therapy changes that may lower total cholesterol, increase HDL, and improve atherogenic risk (Behrens 2005). Thus, the validity of this calculation for long-term cardiovascular risk assessment in young patients with changing lipid levels and medication regimens requires further studies, but it seems helpful to identify patients with increased myocardial risk.

Clearly, more clinical studies are necessary to assess whether these recommendations are also applicable in the presence of HIV and to determine the clinical value of lipid lowering drug therapy in these patients. Most importantly, the information about drug interactions of lipid lowering and antiretroviral drugs is still incomplete. The accumulation of pre-existing and drug-related risk factors will get more clinical attention, because, by improving the HIV-associated morbidity and mortality, ART consequently increases an additional relevant cardiovascular risk factor: the age of patients who are effectively treated with antiretroviral drugs.

Pathogenesis

For a better understanding of the pathogenesis of complex metabolic abnormalities, it is useful to separate individual aspects of the lipodystrophy syndrome: adipocytes/fat redistribution, lipid metabolism, and carbohydrate metabolism. This is because it is very likely that the lipodystrophy syndrome is not a stereotypic syndrome but rather an amalgam of miscellaneous clinical features, with perhaps multifactorial causes. Studies published in recent years provide evidence for two fundamental assumptions: firstly, lipoatrophy and lipoaccumulation result from divergent or only partially overlapping pathogenetic reasons. Secondly, NRTIs, NNRTIs, PIs, and even drugs within each class contribute to the lipodystrophy syndrome and its individual features by different possibly overlapping mechanisms.

NRTIs and lipodystrophy

The patterns of fat redistribution in patients who are only taking NRTIs are unlike those observed in patients on PI therapy. Peripheral fat loss is the major symptom observed in NRTI therapy (particularly d4T and AZT), although a few clinical studies have described a minimal intra-abdominal fat increase in these patients, which is clearly less than on PIs. In ACTG 5142, in which patients receiving efavirenz (+2 NRTIs) or lopinavir (+2 NRTIs) where compared, a 20% percent reduction in peripheral fat tissue was significantly more frequently found in patients receiving d4T or AZT (Haubrich 2009). Given that, commonly, only a mild increase in triglycerides has been observed, exclusive NRTI therapy seems to be of minor impact on lipid metabolism, but is also not a preferred choice of therapy. Postprandially elevated FFA in patients with lipodystrophy, together with in vitro experiments, have led to the hypothesis that NRTIs could impair fatty acid binding proteins (FABP) that are responsible for cellular fat uptake and intracellular fat transport.

It is well established that long-term NRTI therapy can cause mitochondrial toxicity. The clinical manifestation of this presents in symptoms such as hepatic steatosis, severe hyperlactatemia, and polyneuropathy. As an explanation for these symptoms, the “pol-γ hypothesis” has been proposed, which was later extended to reveal the lipoatrophy observed under NRTIs (Brinkman 1999). To maintain an adequate bioenergetic level for accurate cell function, all metabolically active cells depend on a persistent polymerase γ-mediated mitochondrial (mt) DNA synthesis. Mitochondria require a constant supply of nucleosides for this process. The mitochondrial DNA polymerase γ retains both DNA- as well as RNA-dependent DNA polymerase activity. The latter is perhaps responsible for the HIV reverse transcriptase activity and therefore its susceptibility for interactions with NRTIs. Experimental data reveals that, for NRTI uptake into mitochondria, the subsequent phosphorylation and then incorporation into the DNA, certain pharmacodynamic requirements need to be fulfilled. These requirements, including thymidine kinase activity and deoxynucleotide transport specificity of the mitochondrial membrane, are apparently different for AZT and d4T, which partially explains the prevailing association between lipoatrophy and d4T therapy. The postulated mechanisms of NRTI-induced mitochondrial dysfunction consist of competitive inhibition, incorporation into the mtDNA resulting in mtDNA depletion, impairment of mitochondrial enzymes, uncoupling of oxidative phosphorylation and induction of apoptosis. Depletion of mtDNA and structural changes in the mitochondria, resulting in increased rates of apoptosis in subcutaneous adipocytes, have been confirmed in other studies. Despite the experimental link between mitochondrial toxicity and fat tissue as one potential target organ, the degree to which mitochondrial damage contributes to fat distribution abnormalities and its specificity remains unknown. Most likely, additional factor may be relevant given that mtDNA loss and mitochondrial dysfunction has been found in fat tissue of therapy-naïve patients (Garrabou 2011). Also, mt DNA measurement in PBMCs from the blood may be of little relevance for toxicity in fat tissue. In contrast, mitochondrial damage is widely believed to be responsible for other NRTI-related side effects, such as myopathy, hyperlactatemia, microvesicular steatosis, and steatohepatitis with lactic acidosis.

Protease inhibitors and lipodystrophy

PIs account for the majority of metabolic abnormalities associated with the lipodystrophy syndrome. Numerous studies report increases in the levels of total triglycerides and triglyceride-rich lipoproteins (VLDL) accompanied by raised LDL levels after initiation of PI therapy (Walli 1998, Behrens 1999). Conversely, these parameters improve substantially in most studies after discontinuation of the PI or on switching to abacavir or nevirapine. The hyperlipidemic changes are frequently associated with hyperinsulinemia and/or insulin resistance.

It has been proposed, based on in vitro experiments, that PIs such as saquinavir, indinavir, and ritonavir are able to inhibit proteasomal degradation of apolipoprotein B leading to intracellular stockpiling of this lipoprotein and excessive release in response to FFA (Liang 2001). Using stable isotopes in vivo, other authors demonstrate a dramatic increase in FFA turnover together with increased lipolysis and decreased clearance of triglyceride-rich VLDL and chylomicrons (Shekar 2002). These conditions point towards an impaired postprandial insulin-mediated lipid metabolism, since insulin, on the one hand, normally inhibits lipolysis and, on the other hand, increases uptake of FFA, triglyceride synthesis, and fat oxidation in favor of glucose oxidation.

It remains unclear whether impaired insulin action eventually leads to dyslipidemia, or whether hyperlipidemia is responsible for reduced insulin function and insulin resistance in the periphery. Presumably, both mechanisms are important given that some PIs (e.g., indinavir) have been shown to induce insulin resistance without changes occurring in lipid metabolism after short-term administration (Noor 2001, Noor 2002), whereas other PIs (e.g., ritonavir) have been demonstrated to cause mainly hypertriglyceridemia due to increased hepatic synthesis without major changes occurring in glucose metabolism (Purnell 2000).

It is reasonable to speculate that lipid abnormalities and, in particular increased FFA levels, contribute substantially to the peripheral and central insulin resistance of skeletal muscles and the liver, presumably due to the increased storage of lipids in these organs (Gan 2002). Given this hypothesis, the visceral adiposity could reflect the adaptation of the body in response to raised FFA concentrations and an attempt to minimize the lipotoxic damage to other organs.

Several in vitro experiments have indicated that almost all PIs can potentially lead to insulin resistance in adipocytes. Short-term administration of indinavir caused an acute and reversible state of peripheral insulin resistance in healthy volunteers, which was determined in an euglycemic-hyperinsulinemic clamp. These effects are most likely caused by the inhibition of glucose transport mediated by GLUT-4, the predominant transporter involved in insulin-stimulated cellular glucose uptake in humans (Murata 2002). A common structural component found in most PIs has been proposed to cause GLUT-4 inhibition. In some patients with lipodystrophy, additional impairment of glucose phosphorylation may contribute to insulin resistance (Behrens 2002). This is presumably due to an impaired insulin-mediated suppression of lipolysis and subsequently increased FFA levels (Behrens 2002, van der Valk 2001) and accumulation of intramyocellular lipids. Peripheral insulin resistance may also account for an increase in the resting energy expenditure in HIV lipodystrophy and a blunted insulin-mediated thermogenesis.

Indinavir may also induce insulin resistance by inhibiting the translocation, processing or phosphorylation of the sterol regulatory element-binding protein 1c (SREBP-1c). Either directly or via the peroxisome proliferator activated receptor g (PPARg), SREBP-1 regulates FFA uptake and synthesis, adipocyte differentiation and maturation, and glucose uptake by adipocytes. Similarly, the function of these factors has been proposed to be disturbed in inherited forms of lipodystrophy. Finally, hypoadiponectinemia, as found in patients with abnormal fat distribution, may contribute to insulin resistance (Addy 2003). Genetically, host factors interfering with drug metabolism (Domingo 2011) and additional predisposing factors in mechanistically plausible and other genes (Montes 2010, Pinti 2010 Wangsomboonsiri 2010) appear to contribute.

Diagnosis

Both the lack of a formal definition and uncertainty about the pathogenesis and possible long-term consequences leads to a continuing discussion about appropriate guidelines for the assessment and management of HIV lipodystrophy syndrome and its metabolic abnormalities. Outside clinical studies, the diagnosis relies principally on the occurrence of apparent clinical signs and the patient reporting them. A standardized data collection form may assist in diagnosis (Grinspoon 2005). This appears sufficient for the routine clinical assessment, especially when the body habitus changes develop rather rapidly and severely. For clinical investigations however, especially in epidemiological and interventional studies, more reliable measurements are required. A recent multicenter study to develop an objective and broadly applicable case definition proposes a model including age, sex, duration of HIV infection, HIV disease stage, waist-to-hip ratio, anion gap, serum HDL cholesterol, trunk-to-peripheral-fat ratio, percentage leg fat, and intra-abdominal to extra-abdominal fat ratio. Using these parameters, the diagnosis of lipodystrophy had a 79% sensitivity and 80% specificity (Carr 2003). Although this model is largely for research and contains detailed body composition data, alternative models and scoring systems, incorporating only clinical and metabolic data, also gave reasonable results (for more information, see http://www.med.unsw.edu.au/nchecr).

Despite individual limitations, several techniques are suitable for measuring regional fat distribution. These include dual energy x-ray absorptiometry (DEXA), computer tomography (CT), magnetic resonance imaging (MRI) and sonography. Anthropometric measurements are safe, portable, cheap and much easier to perform than imaging techniques. Waist circumference or sagittal diameter are more sensitive and specific measures than waist-to-hip ratio. Repeated measurements of skin fold thickness can be useful for individual long-term monitoring but need to be performed by an experienced person.

The main imaging techniques (MRI, CT, DEXA) differentiate tissues on the basis of density. Single-slice measurements of the abdomen and extremities (subcutaneous adipose tissue = SAT, visceral adipose tissue = VAT) and more complex three-dimensional reconstructions have been used to calculate regional or total body fat. Limitations of these methods include most notably their expense, availability and radiation exposure (CT). Consequently, CT and MRI should only be considered in routine clinical practice for selected patients (e.g., extended dorso-cervical fat pads, differential diagnosis of non-benign processes and infections).

DEXA is appropriate for examining appendicular fat, comprised almost entirely of SAT, and has been successfully employed in epidemiological studies. However, SAT and VAT cannot be distinguished by DEXA, which therefore limits the evaluation of changes in truncal fat. Application of sonography to measure specific adipose compartments, including those in the face, requires experienced investigators and has been minimally applied in HIV infection so far. Bioelectrical impedance analysis estimates the whole body composition and cannot be recommended for measurement of abnormal fat distribution.

Patients should routinely be questioned and examined for cardiovascular risk factors, such as smoking, hypertension, adiposity, type 2 diabetes, and family history. For an accurate assessment of blood lipid levels, it is recommended to obtain blood after a fasting of at least 8 hours. Total cholesterol and triglycerides together with LDL and HDL cholesterol should be obtained prior to the initiation of, or switch to, any new antiretroviral therapy and repeated 3 to 6 months later. Fasting glucose should be assessed with at least a similar frequency. The oral glucose tolerance test (OGTT) is a reliable and accurate instrument for evaluating insulin resistance and glucose intolerance. An OGTT may be indicated in patients with suspected insulin resistance such as those with adipositas (BMI > 27 kg/m2), a history of gestational diabetes and a fasting glucose level of 110 to 126 mg/dl (impaired fasting glucose). The diagnosis of diabetes is based on fasting glucose levels > 126 mg/dl, glucose levels of > 200 mg/dl independent of fasting status, or a 2-hour OGTT glucose level above 200 mg/dl. Screening of HbA1c appears to be less reliable as in sero-negative patients (Kim 2009, Eckhardt 2011). Additional factors that could lead to or assist in the development of hyperlipidemia and/or insulin resistance always need to be considered (e.g., alcohol consumption, thyroid dysfunction, liver and kidney disease, hypogonadism, concurrent medication such as steroids, b-receptor blockers, thiazides, etc.).

Therapy

So far, most attempts to improve or even reverse the abnormal fat distribution by modification of the antiretroviral treatment have shown only modest clinical success. In particular, peripheral fat loss appears to be resistant to most therapeutic interventions. The metabolic components of the syndrome may be easier to improve (Table 1). Thus, preventing lipoatrophy by avoiding thymidine analogues (AZT, d4T) is the main goal (Behrens 2008). For more detailed recommendations for improving fat redistribution and treating dyslipidemia, please see the guidelines of the European AIDS Clinical Society (www.eacs.eu). These guidelines emphasize that all traditional cardiovascular risk factors, such as arterial hypertension, hyperlipidemia and type 2 diabetes should be assessed and considered for intervention.

Lifestyle changes

Dietary interventions are commonly accepted as the first therapeutic option for hyperlipidemia, especially hypertriglyceridemia. Use of NCEP guidelines may reduce total cholesterol and triglycerides by 11 and 21%, respectively. Whenever possible, dietary restriction of total fat to 25-35% of the total caloric intake should be a part of any treatment in conjunction with lipid-lowering drugs. Consultation with professional and experienced dieticians should be considered for HIV-infected patients and their partners. Patients with excessive hypertriglyceridemia (>1,000 mg/dl) may benefit from a very low fat diet and alcohol abstinence to reduce the risk of pancreatitis, especially if there is a positive family history or concurrent medications that may harbor a risk of developing pancreatitis. Regular exercise may have beneficial effects, not only on triglycerides and insulin resistance, but probably also on fat redistribution (reduction in truncal fat and intramyocellular fat) and should be considered in all HIV-infected patients (Driscoll 2004a). All patients should be advised and supported to give up smoking in order to reduce cardiovascular risk. Cessation of smoking is more likely to reduce cardiovascular risk than any choice or change of ART or use of any lipid-lowering drug (Petoumenos 2010).

Table 1: Therapeutic options for HIV-associated lipodystrophy and related metabolic complications
Lifestyle changes  (reduce saturated fat and cholesterol intake, increase physical activity, stop smoking)

Change antiretroviral therapy: replacement of PI, d4T (Zerit®) or AZT (Retrovir™)

Statins: Atorvastatin (Sortis®), Pravastatin (Pravasin®), Fluvastatin (Lescol®)

Fibrates: Gemfibrozil (Gevilon®) or Bezafibrat (Cedur®)

Metformin (Glucophage®)

Thiazolidinediones: pioglitazone (Actos®)

Recombinant human growth hormones (e.g., Serostim®) or analogues (e.g. Tesamorelin®)

Surgical intervention

Specific interventions

Given the extensive indications that PIs are the culprits that substantially contribute to metabolic side effects, numerous attempts have been made to substitute the PI component of a regimen with nevirapine, efavirenz, or abacavir. Similarly, given the close association of d4T-based therapy with lipoatrophy, replacement of this thymidine nucleoside analogue by, for example, abacavir or tenofovir has been evaluated in several studies. Indeed, these “switch studies” have demonstrated substantial improvement, although not normalization, of serum lipids (total and LDL cholesterol, triglycerides) and/or insulin resistance in many patients. In patients with hyperlipidemia, substitution of PIs with alternative PIs that have less metabolic side effects (e.g., atazanavir) has also been proven to be a successful strategy (Mallolas 2009, Moebius 2005). Protease inhibitor cessation has not been shown to improve lipoatrophy. However, stopping administration of the thymidine nucleoside analogue d4T or AZT usually leads to a slow recovery (over months and years) measured by DEXA and moderate clinical increase in limb fat (Moyle 2006, Tebas 2009). Under restricted inclusion criteria and study conditions, most patients maintain complete viral suppression after changes to the ART regimen, but not all of these studies included control groups with unchanged antiretroviral therapy. Recently, a pilot study evaluating the effect of uridine (NucleomaxX®) on lipoatrophy in HIV-infected patients continuing their ART regimen described a significant increase in subcutaneous fat after only three months, but this effect was not confirmed in a larger randomized trial (MyComsey 2010). .

The most advantageous changes of metabolic parameters have been observed by replacement of the PI with nevirapine or abacavir. This option is, however, not always suitable, and the clinical benefit of effective viral suppression and improved immune function needs to be considered in view of drug history, current viral load, and resistance mutations. When options are limited, antiretroviral drugs that may lead to elevation of lipid levels should not be withheld for fear of further exacerbating lipid disorders.

Lipid-lowering agents should be considered for the treatment of severe hypertriglyceridemia, elevated LDL or a combination of both. The clinical benefit, however, of lipid lowering or insulin-sensitizing therapy in HIV patients with lipodystrophy remains to be demonstrated. In light of the potentially increased cardiovascular risk to recipients of antiretroviral therapy, the AIDS Clinical Trials Group (ACTG) published recommendations based on the National Cholesterol Education Program (NCEP) for primary and secondary prevention of coronary artery disease in seronegative patients. In addition, more detailed recommendations by the European AIDS Clinical Society have been published to provide guidelines for physicians actively involved in HIV care that will be regularly updated (www.eacs.eu). However, these recommendations should be considered as being rather preliminary, given the so far limited numbers, size and duration of the clinical studies they are based on. It appears reasonable to measure fasting lipid levels annually before and 3-6 months after ART is initiated or changed. Whenever possible, the ART least likely to worsen lipid levels should be selected for patients with dyslipidemia.

The decision on lipid-lowering therapy can be based on estimating the 10-year risk for myocardial infarction according to the Framingham equation (http://hin.nhlbi.nih.gov/atpiii/calculator.asp). In case of a more than 20% risk, dietary interventions and change of antiviral therapy should be considered. In patients with frank diabetes or coronary heart disease (CHD), lipid lowering drugs are recommended. The EACS guidelines (2011) recommend to target total cholesterol levels of < 190 mg/dl (<5 mmol/l) and for patients with type 2 diabetes and CHD levels < 155 mg/dl (<4 mmol/l). For LDL-cholesterol levels one should aim for levels < 115 mg/dl (<3 mmol/l), and if possible, in patients with type 2 diabetes or CHD, levels < 80 mg/dl (< 2mmol/l).

HMG-CoA reductase inhibitors have been successfully used in combination with dietary changes in HIV-positive patients with increased total and LDL cholesterol. These drugs may decrease total and LDL cholesterol by about 25% (Grinspoon 2005). Many of the statins (as well as itraconazole, erythromycin, diltiazem, etc.) share common metabolization pathways with PIs via the cytochrome P450 3A4 system, thereby potentially leading to additional side effects due to increased plasma levels of statins which can then cause liver and muscle toxicity. They can be combined with ezetimibe in order to improve their lipid lowering effect (Negredo 2006). Based on limited pharmacokinetic and clinical studies, atorvastatin (Sortis®), fluvastatin (Lescol®), and pravastatin (Pravasin®), carefully administered at increasing doses, are the preferred agents for a carefully monitored therapy in HIV-infected patients on ART. Lovastatin (Mevinacor®) and simvastatin (Zocor®) should be avoided due to their potential interaction with PIs.

Fibric acid analogues such as gemfibrozil or fenofibrate are particularly effective in reducing the triglyceride levels by up to 50% (Rao 2004, Miller 2002) and should be considered in patients with severe hypertriglyceridemia (>1000 mg/dl). Fibric acid analogues retain a supportive effect on lipoprotein lipase activity and can thereby lower LDL levels. Despite their potentially synergistic effect, co-administration of fibric acid analogues and statins in patients on ART should only be used carefully in selected individuals, since both can cause rhabdomyolysis. Niacinic acid has been shown to only minimally improve the hyperlipidemia induced by ART. It does, however, increase peripheral insulin resistance (Gerber 2004). Extended-release niacin (Niaspan®) has been shown to have beneficial effects mainly on triglycerides and was well tolerated at a dose of 2,000 mg daily in a study with 33 individuals (Dube 2005). Similarly, polyunsaturated fatty acids could be beneficial in patients with hypertriglyceridemia (De Truchis 2007). Finally, it should be stressed that the long-term effects of lipid-lowering agents and their impact on cardiovascular outcomes, especially in HIV-positive patients with moderate or severe hypertriglyceridemia, are unknown.

Metformin has been evaluated for the treatment of lipodystrophy syndrome. Some studies have revealed a positive effect on the parameters of insulin resistance and the potential reduction of intra-abdominal (and subcutaneous) fat, although not clinically obvious. Together with exercise training, metformin has been described to reverse the muscular adiposity in HIV-infected patients (Driscoll 2004b). Metformin, like all biguanides, can theoretically precipitate lactic acidosis and should thus be used with caution. Use of metformin should be avoided in patients with creatinine levels above 1.5 mg/dl, increased aminotransferase levels, or hyperlactatemia. Thiazolidinediones, such as rosiglitazone (Avandia®) or pioglitazone (Actos®), exhibit the potency to improve insulin sensitivity via stimulation of the PPARγ and other mechanisms. However, both drugs have been associated with significant side effects and can currently not be recommended for HIV-infected patients. Rosiglitazone has been successfully used to treat abnormal fat distribution in genetic lipodystrophies. Three published studies on HIV patients, however, revealed no or only minimal improvement in abnormal fat distribution. But, insulin sensitivity was increased at the expense of increased total cholesterol and triglycerides (Carr 2004, Hadigan 2004, Sutinen 2003, Cavalcanti 2005, Sheth 2010). Thus, rosiglitazone cannot be recommended for general treatment of lipoatrophy in HIV (Grinspoon 2005). It also reduces the bioavailability of nevirapine, although not of efavirenz and lopinavir (Oette 2005). A randomized double-blind placebo-controlled trial (ANRS 113) revealed a significant increase in subcutaneous fat 48 weeks after treatment with pioglitazone 30 mg once daily without demonstrating negative effects on lipid parameters (Slama 2008). The peripheral fat increase was most pronounced in patients, which stopped thymidine analogue therapy (Tungsiripat 2010), but because of side effects pioglitazone cannot be recommended to treat HIV-associated lipoatrophy.

Recombinant growth hormone (Serostim®) at doses of 4-6 mg/d sc over 8-12 weeks has been demonstrated in small studies to be a successful intervention for reducing visceral fat accumulation, but it also reduces subcutaneous fat (Kotler 2004). Unfortunately, these improvements have been shown to consistently reverse after the discontinuation of growth hormone therapy. Studies with lower maintenance doses have not been performed yet. The possible side effects associated with growth hormone therapy include arthralgia, peripheral edema, insulin resistance and hyperglycemia. Alternatively, a stabilized analogue of the growth hormone-releasing factor (Tesamorelin®), administered subcutaneously, can lead to reduction in visceral fat accumulation with less side effects (Falutz 2007) and was recently approved by the FDA.

Surgical intervention (liposuction) for the treatment of local fat hypertrophy has been successfully performed, but appears to be associated with an increased risk of secondary infection (Guaraldi 2011), and recurrence of fat accumulation is possible. For the treatment of facial lipoatrophy, repeated subcutaneous injection of agents such as poly-L-lactic acid (Sculptra®, New-Fill®), a resorbable molecule that promotes collagen formation, has been effectively used in HIV patients (Valantin 2003, Lafaurie 2003¸ Guaraldi 2005, Mest 2004, Casavantes 2004, Behrens 2008). In 2004, Sculptra®was approved by the FDA as an injectable filler to correct facial fat loss in people with HIV. We recommend consultation with experienced specialists for surgical treatments and injection therapy. Further evaluation in long-term follow-up studies is necessary to fully assess the value of these methods.

References

Addy CL, Gavrila A, Tsiodras S, et al. Hypoadiponectinemia is associated with insulin resistance, hypertriglyceridemia, and fat redistribution in HIV–infected patients treated with HAART. J Clin Endocrinol Metab 2003; 88:627-36.

Behrens GMN. Treatment option for lipodystrophy in HIV-positive patients. Expert Opin Pharmacother 2008; 9:39-52.

Behrens G, Dejam A, Schmidt H, et al. Impaired glucose tolerance, beta cell function and lipid metabolism in HIV patients under treatment with protease inhibitors. AIDS 1999; 13:F63-70.

Behrens GMN. Cardiovascular risk and body-fat abnormalities in HIV-infected adults. N Engl J Med 2005; 352:1721-1722.

Behrens GMN, Boerner AR, Weber K, et al. Impaired glucose phosphorylation and transport in skeletal muscle causes insulin resistance in HIV-1 infected patients with lipodystrophy. J Clin Invest 2002, 110:1319-1327.

Behrens GM, Reiss P. Abacavir and cardiovascular risk. Curr Opin Infect Dis 2010;23:9-14.

Brinkman K, Smeitink JA, Romijn JA, Reiss, P. Mitochondrial toxicity induced by nucleoside-analogue reverse-transcriptase inhibitors is a key factor in the pathogenesis of antiretroviral-therapy-related lipodystrophy. Lancet 1999; 354:1112-1115.

Calza L, Manfredi R, Chiodo F. Statins and fibrates for the treatment of hyperlipidaemia in HIV-infected patients receiving HAART. AIDS 2003; 17: 851-859.

Capeau J, Bouteloup V, Raffi F et al. Diabetes Mellitus in Treated HIV-infected Patients: Incidence over 10 Years in 1046 patients from the ANRS CO8 APROCO-COPILOTE Cohort. Abstract 850, 18th CROI 2011, Boston.

Carr A, Emery S, Law M, Puls R, Lundgren JD, Powderly WG. An objective case definition of lipodystrophy in HIV-infected adults: a case-control study. Lancet 2003; 361:726-35.

Carr A, Samaras K, Burton S, et al. A syndrome of peripheral lipodystrophy, hyperlipidaemia and insulin resistance in patients receiving HIV protease inhibitors. AIDS 1998; 12:F51-8.

Carr A, Workman C, Carey D, et al. No effect of rosiglitazone for treatment of HIV-1 lipoatrophy: randomised, double-blind, placebo-controlled trial. Lancet 2004; 363:429-38.

Casavantes LC, Gottlieb M. Bio-Alcamid, a high-volume injectable posthesis for facial reconstruction in HIV-related lipoatrophy: a report on 100 patients. Antivir Ther 2004; 9:L37.

Cavalcanti R, Kain K, Shen S, Raboud J, Walmsley S. A randomized placebo controlled trial of rosoglitazone for the treatment of HIV lipodystrophy. Abstract 854, 12th CROI 2005, Boston.

Cruciani M, Zanichelli V, Serpelloni G, et al. ABACAVIR use and cardiovascular disease events: a meta-analysis of published and unpublished data. AIDS. 2011 Jun 29. PMID:21716077

Currier JS, Kendall MA, Zackin R, et al. Carotid artery intima-media thickness and HIV infection: traditional risk factors overshadow impact of protease inhibitor exposure. AIDS 2005; 19: 927-33.

D:A:D Study Group. Use of nucleoside reverse transcriptase inhibitor and risk of myocardial infarction in HIV-infected patients enrolled in the D:A:D study: a mulit-cohort collaboration. Lancet 2008; 371: 1417-26.

De Truchis P, Kirstetter M, Perier A, et al. Reduction in triglyceride level with N-3 polyunsaturated fatty acids in HIV-infected patients taking potent antiretroviral therapy: a randomized prospective study. J Acquir Immune Defic Syndr 2007, 44: 278-85.

DeJesus E, Cohen C, Lennox J, et al. Metabolic profiles and body composition changes in treatment-naive HIV-infected patients treated with raltegravir 400 mg twice-daily vs efavirenz 600 mg each bedtime combination therapy: 96-week follow-up. Abstract 720, 17th CROI 2010b, San Francisco

Domingo P, Cabeza MC, Pruvost A et al. Association of thymidylate synthase genep olymorphisms with stavudine triphosphate intracellular levels and lipodystrophy. Antimicrob Agents Chemother 2011; 55:1428-35.

Driscoll SD, Meininger GE, Lareau MT, et al. Effects of exercise training and metformin on body composition and cardiovascular indices in HIV-infected patients. AIDS 2004, 18: 465-473.

Eckhardt B, Holzman R, Kwan C, Baghdadi J, Aberg J. Glycated Hemoglobin A1C as Screening for Diabetes Mellitus in HIV-infected Individuals. Abstract 849, 18th CROI 2011, Boston.

El Sadr WM, Lundgren JD, Neaton JD, et al. CD4+ count-guided interruption of antiretroviral treatment. N Engl J Med; 2006; 355:2283-2296.

Falutz J, Allas S, Blot K, et al. Metabolic effects of a growth hormone-releasing factor in patients with HIV. N Engl J Med 2007; 357:2359-70.

Friis-Moller N, Sabin CA et al. for the Data Collection on Adverse Events of Anti-HIV Drugs (DAD) Study Group.Combination antiretroviral therapy and the risk of myocardial infarction. N Engl J Med 2003; 349: 1993-2003

Friis-Moller N, Reiss P, El-Sadr W et al. Class of antiretroviral drugs and the risk of myocardial infarction. N Engl J Med. 2007; 356:1723-35.

Gan SK, Samaras K, Thompson CH, et al. Altered myocellular and abdominal fat partitioning predict disturbance in insulin action in HIV protease inhibitor-related lipodystrophy. Diabetes 2002; 51:3163-9.

Garrabou G, López S, Morén C et al. Mitochondrial damage in adipose tissue of untreated HIV-infected patients. AIDS 2011, 25:165-70.

Gerber MT, Mondy KE, Yarasheski KE, et al. Niacin in HIV-infected individuals with hyperlipidemia receiving potent antiretroviral therapy. Clin Infect Dis 2004; 39: 419-25.

Grinspoon S, Carr A. Cardiovascular risk and body-fat abnormalities in HIV-infected adults. N Engl J Med 2005; 352:48-62.

Grunfeld C et al. HIV infection is an independent risk factor for atherosclerosis similar in magnitude to traditional cardiovascular disease risk factors. Abstract 146, 16th CROI 2009, Montreal.

Grunfeld C, Saag M, Cofrancesco J Jr et al. Study of Fat Redistribution and Metabolic Change in HIV Infection (FRAM). Regional adipose tissue measured by MRI over 5 years in HIV-infected and control  participants indicates persistence of HIV-associated lipoatrophy. AIDS 2010; 24:1717-26.

Guaraldi G, Orlando G, De Fazio D, et al. Comparison of three different interventions for the correction of HIV-associated facial lipoatrophy: a prospective study. Antivir Ther 2005; 10: 753-9.

Guaraldi G, Fontdevila J, Christensen LH et al. Surgical correction of HIV-associated facial lipoatrophy. AIDS 2011; 25:1-12.

Hadigan C, Yawetz S, Thomas A, et al. Metabolic effects of rosiglitazone in HIV lipodystrophy: a randomized, controlled trial. Ann Intern Med 2004; 140:786-94.

Hasse B, Ledergerber B, Egger M et al. Aging and Non-HIV-associated Co-morbidity in HIV+ Persons: The SHCS. Abstract 792, 18th CROI 2011, Boston.

Haubrich RH, Riddler S, Dirienzo G, et al. Metabolic outcomes in a randomized trial of nucleoside, nonnucleoside and protease inhibitor-sparing regimens for initial HIV treatment. AIDS 2009, May 4. [Epub ahead of print]

Kim PS, Woods C, Georgoff P, Crum D, Rosenberg A, Smith M, Hadigan C. A1C underestimates glycemia in HIV infection. Diabetes Care. 2009; 32:1591-3.

Kotler DP, Muurahainen N, Grunfeld C, et al. Effects of growth hormone on abnormal visceral adipose tissue accumulation and dyslipidemia in HIV-infected patients. J Acquir Immune Defic Syndr 2004; 35: 239-52.

Lafaurie M, Dolivo M, Boulu D, et al. Treatment of facial lipoatrophy with injections of polylactic acid in HIV-infected patients. Abstract 720, 10th CROI 2003; Boston.

Liang JS, Distler O, Cooper DA, et al. HIV protease inhibitors protect apolipoprotein B from degradation by the proteasome: A potential mechanism for protease inhibitor-induced hyperlpidemia. Nat Med 2001; 7:1327-1331.

Mallolas J, Podzamczer D, Milinkovic A, et al. Efficacy and safety of switching from boosted lopinavir to boosted atazanavir in patients with virological suppression receiving a LPV/r-containing HAART: the ATAZIP study. J Acquir Immune Defic Syndr 2009; 51: 29-36.

Mallon PWG, Miller J, Cooper DA, Carr A. Prospective evaluation of the effects of antiretroviral therapy on body composition in HIV-1-infected men starting therapy. AIDS 2003; 17:971-79.

McComsey G, Walker U, Budhathoki C, et al. Uridine supplementation in the management of HIV lipoatrophy: Results of ACTG 5229. Abstract 131, 17th CROI 2010, San Francisco

Mest DR, Humble G. Safety and efficacy of intradermal poly-L-lactic acid (SculptraTM) injections in patients with HIV-associated facial lipoatrophy. Antivir 2004; 9:L36.

Miller J, Brown D, Amin J, et al. A randomized, double-blind study of gemfibrozil for the treatment of protease inhibitor-associated hypertriglyceridaemia. AIDS 2002; 16: 2195-2200.

Möbius U, Lubach-Ruitman M, Castro-Frenzel B, et al. Switching to atazanavir improves metabolic disorders in patients with severe hyperlipidemia. J Acquir Immune Defic Syndr 2005; 39:174-180.

Montes AH, Valle-Garay E, Suarez-Zarracina T et al. The MMP1 (-16071G/2G) single nucleotide polymorphism associates with the HAART-related lipodystrophic syndrome. AIDS 2010; 24:2499-506.

Moyle GJ, Sabin CA, Cartledge J, et al. A randomized comparative trial of tenofovir DF or abacavir as replacement for a thymidine analogue in persons with lipoatrophy. AIDS 2006; 20: 2043-50.

Murata H, Hruz PW, Mueckler M. Indinavir inhibits the glucose transporter isoform Glut4 at physiologic concentrations. AIDS 2002; 16:859-863.

Negredo E, Molto J, Puig J, et al. Ezetimibe, a promising lipid-lowering agent for the treatment of dyslipidaemia in HIV-infected patients with poor response to statins. AIDS 2006; 20: 2159-64.

Noor MA, Lo JC, Mulligan K, et al. Metabolic effects of indinavir in healthy HIV-seronegative men. AIDS 2001; 15:F11-F18.

Noor MA, Seneviratne T, Aweeka FT, et al. Indinavir acutely inhibits insulin-stimulated glucose disposal in humans: a randomized, placebo-controlled study. AIDS 2002; 16:F1-F8.

Oette M, Kurowski M, Feldt T, et al. Impact of rosiglitazone treatment on the bioavailability of antiretroviral compounds in HIV-positive patients. J Antimicrob Chemother 2005; 56:416-419.

Petoumenos K, Worm S, Reiss P, et al. Rates of cardiovascular disease following smoking cessation in patients with HIV infection: results from the D:A:D study. Abstract 124, 17th CROI 2010, San Francisco

Pinti M, Gibellini L, Guaraldi G et al.  Upregulation of nuclear-encoded mitochondrial LON protease in HAART-treated HIV-positive patients with lipodystrophy: implications for the pathogenesis of the disease. AIDS 2010, 24:841-50.

Rao A, D’Amico S, Balasubramanyam A, Maldonado M. Fenofibrate is effective in treating hypertriglyceridemia associated with HIV lipodystrophy. Am J Med Sci 2004; 327: 315-318.

Randell PA, Jackson AG, Zhong L, Yale K, Moyle GJ. The effect of tenofovir disoproxil fumarate on whole-body insulin sensitivity, lipids and adipokines in healthy volunteers. Antiviral Therapy 2010; 15:227-233

Schambelan M, Benson CA, Carr A, et al. Management of metabolic complications associated with antiretroviral therapy for HIV-1 infection: recommendations of an International AIDS-Society-USA panel. J AIDS 2002; 31:257-275.

Sekhar RV, Jahoor F, White AC, et al. Metabolic basis of HIV-lipodystrophy syndrome. Am J Physiol Endocrinol Metab. 2002;283:E332-7.

Sheth SH, Larson RJ. The efficacy and safety of insulin-sensitizing drugs in HIV-associated lipodystrophy syndrome: a meta-analysis of randomized trials. BMC Infect Dis. 2010;10:183.

Slama L, Lanoy E, Valentin MA et al. Slama L, Lanoy E, Valentin MA, et al. Effect of pioglitazone on HIV-1-related lipodystrophy: a randomized double-blind placebo-controlled trial (ANRS 113). Antivir Ther 2008, 13:67-76.

Sutinen J, Hakkinen AM, Westerbacka J, et al. Rosiglitazone in the treatment of HAART-associated lipodystrophy – a randomized double-blinded placebo controlled study. Antivir Ther 2003; 8:199-207.

Tebas P, Zhang J, Hafner R, et al. Peripheral and visceral fat changes following a treatment switch to a non-thymidine analogue or a nucleoside-sparing regimen in HIV-infected subjects with peripheral lipoatrophy: results of ACTG A5110. J Antimicrob Chemother 2009; 63: 998-1005.

Tungsiripat M, Bejjani DE, Rizk N et al. Rosiglitazone improves lipoatrophy in patients receiving thymidine-sparing regimens. AIDS 2010;24:1291-8.

Valantin MA, Aubron-Olivier C, Ghosn J, et al. Polylactic acid implants (New-Fill) to correct facial lipoatrophy in HIV-infected patients: results of the open-label study VEGA. AIDS 2003; 17: 2471-7.

van der Valk M, Bisschop PH, Romijn JA, et al. Lipodystrophy in HIV-1-positive patients is associated with insulin resistance in multiple metabolic pathways. AIDS 2001; 15:2093-2100.

Walli R, Herfort O, Michl GM, et al. Treatment with protease inhibitors associated with peripheral insulin resistance and impaired oral glucose tolerance in HIV-1-infected patients. AIDS 1998; 12:F167-73.

Wangsomboonsiri W, Mahasirimongkol S, Chantarangsu S et al. Association between HLA-B*4001 and lipodystrophy among HIV-infected patients from Thailand who received a stavudine-containing antiretroviral regimen. Clin Infect Dis 2010; 50:597-604.

Worm SW, Kamara DA, Reiss P et al. Elevated triglycerides and risk of myocardial infarction in HIV-positive persons. AIDS. 2011;25:1497-504

Worm SW, Sabin C, Weber R, et al. Risk of myocardial infarction in patients with HIV infection exposed to specific individual antiretroviral drugs from the 3 major drug classes: the data collection on adverse events of anti-HIV drugs (D:A:D) study. J Infect Dis 2010;201:318-30.

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Filed under 8. Lipodystrophy Syndrome, Part 2 - Antiretroviral Therapy

Managing Side Effects

– Christiane Schieferstein-Knauer, Thomas Buhk –

Patients on ART commonly suffer from side effects. As a result, treatment of HIV infection has become a complicated balancing act between the benefits of durable HIV suppression and the risks of drug toxicity. Adherence problems, regimen changes or even withdrawal from therapy are often the result of drug toxicity. In former times at least 25% of patients stopped therapy within the first year on ART because of side effects (d’Arminio Monforte 2000, Yuan 2006). Between 2003 and 2007, the rate was still about 20% (Cicconi 2010). Only over the last three years tolerability of ART has been improved, thanks to new drugs becoming available. Treatment cessation due to side effects has become less frequent (Carr 2009).

Factors for poor or non-adherence include poverty, intravenous drug abuse, young age, Afro American origin, hepatitis co-infection and regular alcohol consumption (Robison 2008, Hendershot 2009). The patient should be counseled in detail on the potential side effects, so that he or she is in a position to recognize them and to contact their physician in time. This can save lives, for example in the case of abacavir hypersensitivity reaction, or prevent the irreversible damage of some side effects, such as polyneuropathy. Being prepared for the occurrence of possible problems and providing potential solutions improves both the acceptance of treatment and adherence. This information needs to be presented by the provider to the patient in a user-friendly and accessible manner – the extensive package inserts tend to frighten patients. It must be stressed that the majority of patients are able to tolerate ART well, even for years. Nevertheless, the regular monitoring of treatment by an HIV clinician, even in asymptomatic patients, is recommended through at least quarterly visits, and even more frequently at the beginning of any new regimenwhen visit schedules may be weekly or fortnightly. Standard evaluations include a thorough history (including allergies and other side effects), a physical examination and measurement of vital signs and body weight. Routine investigations include a full blood count, liver, pancreas and renal function tests, electrolytes (plus phosphate in patients on tenofovir) as well as fasting cholesterol, triglycerides and glucose levels. A urine dipstick can detect proteinuria in patients on tenofovir.

It is often difficult to distinguish between symptoms related to HIV infection and those caused by ART. An accurate history considering the intensity, variation and reproducibility of complaints is mandatory.

Gastrointestinal side effects

Gastrointestinal (GI) problems are the most common side effects even if they have fortunately become less frequent, as older NRTIs like AZT or ddI are no longer part of the current recommendations for ART (Robinson 2008, Chubineh 2008). GI side effects appear more frequently during the early stages of therapy. Typical symptoms include abdominal discomfort, loss of appetite, diarrhea, nausea and vomiting, heartburn, abdominal pain, gas in the abdomen or intestines and constipation. Diarrhea occurs frequently with all PIs, also but more rarely with 3TC.

In addition to the often considerable impact on everyday life, gastrointestinal side effects can lead to dehydration, malnutrition with accompanying weight loss, and low plasma drug levels with the risk of development of resistant viral strains.

In most cases, symptoms occur early on in therapy. Patients should be informed that these side effects usually resolve after some weeks (4–6) of treatment. If gastrointestinal side effects appear for the first time after longer periods on ART, other causes such as gastritis and infectious diarrhea need to be considered.

Nausea and vomiting

If administration on an empty stomach leads to nausea and vomiting, most drugs can be taken together with meals. Only the NNRTI efavirenz has to be administered on an empty stomach; small quantities of low-fat salty crackers may lessen nausea. Ginger, peppermint or chamomile teas or sweets may also be helpful, as well as more frequent but smaller meals. Care should be taken with fatty foods and dairy products. Coffee, smoking, alcohol, aspirin and very spicy foods should be avoided.

If treatment is necessary, metoclopramide has been proven to be useful. Dimenhydrinate, cimetidine, ranitidine or ondansetrone can also be taken. Antiemetic drugs can not only be administered if the patient is feeling ill, but taken regularly prophylactically, ideally 30 to 45 minutes before taking ART. If taken on a regular basis, attention should be paid to side effects such as dyskinesia. After a few weeks, doses can generally be slowly reduced. If nausea persists for more than two months, a change of treatment should be considered – otherwise adherence problems will certainly occur.

Diarrhea

In patients with acute or severe diarrhea, the priority is to treat dehydration and loss of electrolytes. Other causes such as GI infection or lactose intolerance should be excluded. Difficult-to-digest foodstuffs (particularly those high in fat or glucose) should be avoided and those that are easy to digest (e.g., potatoes, rice, noodles) eaten instead. It makes sense to remember approved “homespunremedies (see table 1). If significant dehydration and loss of electrolytes occur, coke and salty crackers, sports drinks, herbal teas or electrolyte solutions may be taken. Oral rehydration solution can be easily made from the juice of 5 oranges, 800 ml of boiled water or tea (cooled to room temperature), a teaspoon of iodized salt and two tablespoons of sugar.

Oat bran tablets have been proven to be useful and cheap for PI-associated diarrhea. They are taken together with antiretroviral therapy (daily dose 1500 mg). Pancrelipase, a synthetic pancreatic enzyme, has also been shown to be effective for PI-associated diarrhea.

PI-associated diarrhea can also be alleviated by calcium (Turner 2004), taken as calcium carbonate, at a dose of 500 mg BID. However, as calcium binds with many other agents, it should be taken 2 hours apart from HIV medication.

Oral supplements of glutamine (10–30 g/day) or alanyl–glutamine (up to 44 g/day) alleviate diarrhea and can also boost levels of antiretroviral drugs in the blood (Bushen 2004, Heiser 2004). Glutamine can be purchased in drugstores or ordered online. The probiotics Saccharomyces boulardii and Lactobacillus acidophilus are used in infectious diarrhea and for the prevention of antibiotic-associated diarrhea. They can sometimes ameliorate medication-associated diarrhea. Alternatively, psyllium may be effective. It should not be taken together with loperamide or opium tincture, or at the same time as HIV medication. Charcoal tablets might be helpful.

The cornerstone of symptomatic treatment is loperamide which inhibits bowel movement (initially 2–4 mg, followed by 2 mg, up to a maximum of 16 mg daily). If loperamide is not effective, opium tincture is an alternative (initially 5 drops, maximum 15 to 20 drops), and attention should be paid to the risk of intestinal obstruction, especially if overdosed. In some cases, a combination of different antidiarrheal drugs may be appropriate.

Table 1: “Approved” homespun remedies
Pectin in apples (raw with paring), bananas (purée), carrots (purée, cooked, soup), St. John’s bread  (oatmeal gruel or rice gruel with St. John’s flour). Pectin is a dietary fiber, which is not digested, it binds water and toxic agents and lessens the diarrhea.

Gruel

Soups made of oatmeal or rice gruel

Tanning agents

Black or green tea, dried blueberries (tea, powder), dark chocolate

Hepatotoxicity

ART led to a substantial reduction in the number of deaths related to AIDS. However this has been accompanied by an increase in liver-related morbidity and mortality which now is the most common cause of deaths among HIV – infected patiens not related to AIDS (Joshi 2011, Price 2010).

Elevated liver enzymes are common with ART, and severe hepatotoxicity occurs in up to 10% of patients (Price 2010). Liver failure is rare (Nunez 2005). Hepatotoxicity occurs more often in patients with pre-existing liver dysfunction (Soriano 2008). Severe, sometimes fatal liver damage has been associated with nevirapine, ritonavir and tipranavir, with several fatalities linked to nevirapine and tipranavir (Bjornsson 2006, Rachlis 2007, Chan-Tack 2008). Case reports also exist about liver failure occurring on darunavir, indinavir, lopinavir, ritonavir, tipranavir, atazanavir, efavirenz, nelfinavir and different NRTIs (Carr 2001, Clark 2002, Nunez 2010). Liver enzyme elevation up to severe hepatotoxicity has also been reported for maraviroc and raltegravir.

Risk factors for severe hepatotoxicity are elevated liver enzymes before initiating treatment, chronic hepatitis B or C, concomitant hepatotoxic medication, PI therapy, older age, higher BMI, female gender, thrombocytopenia, high alcohol intake, high viral load or renal dysfunction (Sulkowski 2002, Servoss 2006, Nunez 2010). Patients with pre-existing liver disease should use these drugs only along with strict monitoring. Four possible mechanism of hepatotoxicity are: Hypersensitivity reaction, mitochondrial toxicity/steatohepatitis, direct drug toxicity/drug metabolism or immune reconstitution syndrome. These hepatotoxic reactions occur at different time points for different drug classes.

Hypersensitivity reactions are typical for NNRTIs, not dose related and symptoms resolve usually after stopping the drug (Joshi 2011). They occur within the first 4 to 12 weeks. There are black box warnings for hypersensitivity for nevirapine, abacavir as well as the CCR5-inhibitor maraviroc. NNRTIs can also cause direct drug toxicity which appears within a couple of months (Price 2010).

Nucleoside analogs lead to hepatic steatosis, which is probably caused by mitochondrial toxicity and usually occurs after more than 6 months on treatment. PIs and boosted atazanavir, indinavir and tipranavir can lead to hepatotoxicity at any stage during the course of treatment – once again, patients with chronic viral hepatitis are particular at risk (Sulkowski 2004). One possible cause is immune reconstitution syndrome while on ART, with increased cytolytic activity against hepatitis virus infected liver cells usually within the first two months on ART accompanied by a decline in HIV-RNA and a rise in CD4 T cell count (Price 2010).

NNRTIs

Liver toxicity occurs more commonly on nevirapine than on other antiretroviral drugs. Clinically asymptomatic and symptomatic liver toxicity, including rapidly occurring fatal liver failure have been observed (Bjornsson 2006). Serious and fatal liver toxicity has been reported even during post-exposure prophylaxis (PEP), but not after single-dose nevirapine (McKoy 2009). Therefore the use of nevirapine in  occupational or non-occupational PEP is contraindicated. Symptomatic hepatotoxicity seems to depend on different risk factors, such as female gender, a body mass index lower than 18.5 (Sanne 2005) or chronic hepatitis C (Torti 2007).

A higher risk of serious liver toxicity was also observed in patients with higher CD4 T cell counts prior to initiation of therapy. A retrospective analysis of the Boehringer Ingelheim database showed a higher risk for females with CD4 T cell counts > 250 cells/µl (males: > 400/µl). Although these findings could not be confirmed by other studies (Manfredi 2006, Peters 2010), the Indications and Usage section of the Viramune® label advises against starting nevirapine treatment above these CD4 T cell counts unless the benefits clearly outweigh the risks. The increased risk seems to be particular to ART-naïve patients. Virologically suppressed patients switching to nevirapine seem not to have a significantly higher risk (Mallolas 2006, De Lazarri 2008). These findings were confirmed by an evaluation of seven observational clinical cohorts. Initiating nevirapine in antiretroviral-experienced patients with high CD4 cell counts was well tolerated provided there is no detectable viremia (Kesselring 2009). For pregnant women, data are inconsistent. One study showed a significant association between CD4 T cell count and hepatotoxicity with nevirapine (Jamisse 2007) whereas another study did not (Ouyang 2010). However, pregnancy itself is significantly associated with increased hepatotoxocity (Ouyang 2009).

NNRTIs should be used with caution in patients with HCV coinfection. They should be avoided in patients with liver cirrhosis Child–Pugh class B or C (Nunez 2010). Liver toxicity occurs usually early during ART (within 18 weeks of starting) and may progress to liver failure despite laboratory monitoring, which is not characteristic of other antiretrovirals. If liver enzymes increase to > 3.5 times upper limit of normal (ULN) during treatment, nevirapine should be stopped immediately. If liver enzymes return to baseline and if the patient has had no clinical signs or symptoms of hepatitis, rash, flu-like symptoms, fever or other findings suggestive of organ dysfunction, it may, on a case-by-case basis, be possible to reintroduce NVP. However, frequent monitoring is mandatory in such cases. If liver function abnormalities recur, nevirapine should be permanently discontinued. If clinical hepatitis (anorexia, nausea, jaundice, etc.) occurs, nevirapine must be stopped immediately and never readministered.

In patients treated with efavirenz minor enzyme elevations are generally safe and usually resolve so that a treatment change usually is not necessary (Gutierrez 2008, Kontorinis 2003). This also applies to tenofovir (Lattuada 2008).

Protease inhibitors

Atazanavir and indinavir inhibit the hepatic enzyme UDP-glucuronosyltransferase, increasing the level of bilirubin in up to 50% of patients (Torti 2009). Hyperbilirubinemia is not usually associated with signs or symptoms of hepatocellular injury. It clinically resembles Gilbert’s syndrome. The levels of bilirubin return to normal following discontinuation of the drugs. If bilirubin is only mildly elevated (3 – 5 times ULN) and the serum liver enzyme levels are normal, treatment change is not mandatory. If the bilirubin is constantly markedly elevated, medication should be discontinued: no one knows about the long-term consequences of hyperbilirubinemia (Sulkowski 2004). Pre-existing liver fibrosis or cirrhosis seem to not increase substantially the risk of severe transaminase elevations with atazanavir/r in co-infected patients (HIV plus hepatitis) (Pineda 2008). In patients with end-stage liver disease, unboosted atazanavir did not worsen liver disease; in fact, atazanavir allowed patients to maintain or gain immunovirological eligibility for orthopic liver transplantation (Guaraldi 2009).

Tipranavir/r is associated with a risk of transaminase elevations. In the RESIST trials, grade 3 or 4 transaminase elevations were significantly more common in tipranavir/r than in all other boosted PIs (Hicks 2006). From June 2005 to March 2007, twelve cases of liver-associated deaths were identified (Chan-Tack 2008). Tipranavir/r should not be administered to patients with hepatic impairment Child-Pugh Class B or C. Extreme caution should be exercised when administering it to patients with mild hepatic impairment or patients with chronic hepatitis, as treatment-experienced patients with chronic hepatitis B or C co-infection or elevated transaminases are at approximately 2-fold elevated risk for developing grade 3 or 4 transaminase elevations or hepatic decompensation. Frequent monitoring is mandatory in such cases.

Besides serological tests for viral hepatitis, an abdominal ultrasound should be performed in order to recognize structural liver dysfunction early, e.g., non-alcoholic steatohepatitis or liver cirrhosis, before initiating ART. Liver function should be monitored biweekly at the start of treatment with nevirapine and PIs and even more frequently in patients with pre-existing liver disease. Monthly tests are generally sufficient for all other drugs. If liver enzymes (ALT, AST) are moderately elevated (< 3.5 times ULN) in the absence of clinical symptoms, treatment can be continued under close monitoring. If liver enzymes are elevated to more than 3.5 times ULN, additional diagnostic tests should be performed, including an abdominal ultrasound. In cases of co-infection with hepatitis B or C, treatment of these conditions should be considered. With other pre-existing liver conditions, it may be useful to determine drug plasma levels. Discontinuation of treatment may not be necessary except in the case of nevirapine (see above).

If liver enzymes are elevated in a later phase of therapy (after more than 6 months after initiation), a thorough investigation including serology for viral hepatitis, CMV, and EBV, as well as an abdominal ultrasound, should be performed. Lactic acidosis, hypersensitivity reactions to abacavir and other hepatotoxic drugs should also be considered. A liver biopsy can reveal macro- and microvesicular steatosis and mitochondrial alterations in NRTI-induced steatosis and is therefore helpful to identify a nucleoside-induced hepatopathy and to distinguish it from other causes of liver injury.

In patients with HCV co-infection, hepatitis C should, if possible, be treated before the initiation of ART (see chapter on Hepatitis C). In HBV co-infection, the ART regimen should include FTC or 3TC with tenofovir. Patients with pre-existing liver dysfunction should undergo drug plasma level monitoring, especially during treatment with PIs. Doses can be adjusted according to the plasma levels to help keep the patient on therapy.

Finally, drug interactions and hepatotoxicity related to other drugs or herbal medication taken concomitantly, should not be overlooked (Van den Bout-van den Beukel 2008).

Renal problems

Renal problems occur in particular with tenofovir as well as with atazanavir and the nowadays rarely used PI Indinavir. Indinavir and Atazanavir cause neprohlithiasis through excretion of unchanged drug in the urine. (see Chapter HIV and Kidney)

Tenofovir

Tenofovir is a potentially nephrotoxic drug. Although experience over several years shows that severe renal toxicity occurs rarely, Tenofovir has an effect on renal function. One study showed that elevations in serum creatinine occurred in 2.2% of patients (Nelson 2007). In ART-naïve patients initiating ART with tenofovir was associated with a greater decline in renal function and a higher risk of proximal tubular dysfunction: 4.8% of patients on tenofovir had a more than 50% decline of GFR compared to 2.9% without tenofovir (Horberg 2010). A meta-analysis of 17 studies confirmed an association with a statistically significant loss of renal function with tenofovir, however the clinical magnitude of this effect was modest (Cooper 2010).

Severe cases have been reported with acute renal failure, proximal tubulopathy with Fanconi’s syndrome and nephrogenic diabetes insipidus and rarely hypophosphatemic osteomalacia (Rollot 2003, Saumoy 2004). Renal toxicity occurs after some months, rarely at the beginning of therapy. Risk factors include a relatively high TDF exposure due to pre-existing renal impairment, low body weight or  co-administration of nephrotoxic drugs (Nelson 2007).

PIs can interact with the renal transport of organic anions, leading to proximal tubular intracellular accumulation of tenofovir (Izzedine 2004 and 2007, Rollot 2003).  Whether  combination with boosted PI has a higher risk for nephrotoxicity is unclear. Some studies did not observe increased tenofovir-associated nephrotoxicity in patients with concomitant PI therapy (Antoniou 2005, Crane 2007) others showed that treatment with TDF and a boosted PI was associated with greater declines in renal function compared with tenofovir and NNRTI-based regimens (Goicoechea 2008, Gallant 2009).

Furthermore, extensive pre-treatment with nucleoside reverse transcriptase inhibitors seems to be another risk factor (Saumoy 2004). However, even in patients without any predisposing factors, nephrotoxicity may occur (Barrios 2004).

In case of renal dysfunction, especially in patients with low body weight, tenofovir should be avoided, or the dosing interval should be adjusted. The manufacturer recommends administering TDF every 48h in patients with a creatinine clearance of between 30 and 49 ml/min. In case of severe renal dysfunction (creatinine clearance < 30 ml/min) it should not be administered. Normal creatinine levels may be misleading especially in subjects with low body weight, which is why creatinine clearance should be measured before initiating tenofovir treatment. Renal function tests including creatinine, urea, creatinine clearance, proteinuria, glycosuria, blood and urine phosphate should be monitored every other week.

The majority of the incident renal dysfunction in tenofovir patients is related to preexisting renal disorders (Brennan 2011). Therefore it is not recommended for use in patients with pre-existing renal insufficiency. It should also be avoided with concomitant or recent use of nephrotoxic agents such as aminoglycosides, amphotericin B, foscarnet, ganciclovir, pentamidine, vancomycin, cidofovir or interleukin-2. Usually, abnormalities resolve upon discontinuation of the drug (Izzedine 2004, Roling 2006).

Neurological side effects

Most important neurological side effects are peripheral polyneuropathy caused by NRTI and CNS side effects caused by Efavirenz; see also chapter neuromuscular disorders.

Peripheral polyneuropathy

Peripheral polyneuropathy (PNP) is mainly caused by NRTIs that are no longer prescribed as first- or second-line drugs in most Western Countries, although still frequently used in Africa or Asia, such as ddI, d4T or AZT. Because of their continued use in poor-resource areas, we will review the symptoms and possibilities for palliation. PNP usually presents with a distal symmetrical distribution and sensorimotor paralysis. Patients complain of paresthesia and pain (“tingling”) in hands and feet and perioral dysesthesia. The symptoms often begin gradually after several months of therapy. HIV infection itself can lead to PNP, but the drug-induced form becomes apparent much earlier and may develop within a shorter period of time. Patients must be informed that they should consult their treating physician as soon as possible if these complaints develop. Additional risk factors for polyneuropathy, such as vitamin B12 deficiency, alcohol abuse, diabetes mellitus, malnutrition, or treatment with other neurotoxic drugs, e.g., INH, should be addressed as well. In any case, the nucleoside analogs ddI and d4T have been dropped from first-line therapy recommendations (ddC is no longer manufactured). If possible they should be avoided for salvage therapy as well. Symptoms frequently improve within the first two months following discontinuation of the drugs responsible, but may initially increase in intensity and are not always fully reversible. Because treatment is difficult, and there is no specific therapy, it is extremely important that peripheral polyneuropathy is recognized early by the doctor, resulting in a rapid change of treatment. The causative agent needs to be stopped.

An easy test, in practice, is to test vibration with a tuning fork. A 64-Hz tuning fork (Rydel-Seiffer) is applied to the appropriate bony surface (e.g., distal hallux, medial malleolus or lateral malleolus) bilaterally. The patient is asked to report the perception of both the start of the vibration sensation and the cessation of vibration on dampening. As the intensity of the vibration starts to diminish the two triangles move closer together again. The intensity at which the patient no longer detects the vibration is read as the number adjacent to the intersection. It can thus be quantified and compared to the results of other tests. Through this simple method first signs of polyneuropathy can be recognized easily and early.

Apart from symptomatic treatment with metamizole, acetaminophen (paracetamol), carbamazepine, amitriptyline, gabapentine and opioids, methods such as acupuncture or transcutaneous nerve stimulation have been tried with varying success. Vitamin B supplementation can help to improve peripheral polyneuropathy faster. Tight shoes or long periods of standing or walking should be avoided; cold showers may relieve pain before going to bed.

 

CNS side effects

In up to 40% of patients, treatment with efavirenz may lead to CNS side effects such as dizziness, insomnia, nightmares, mood fluctuations, depression, depersonalization, paranoid delusions, confusion and suicidal ideation. These side effects are observed mainly during the first days and weeks of treatment. Discontinuation of therapy becomes necessary in approximately 3% of patients. There is an association between high plasma levels of efavirenz and the occurrence of CNS symptoms (Marzolini 2001). Genetic predisposition also seems to play a role. Different variations described in the enzyme system CYP2B6 may be responsible for the elimination of efavirenz (Haas 2004). Certain genetic variations more frequent in Afro-Americans than in Europeans raise the levels of efavirenz (Wyen 2007). High plasma levels can also be caused by medication interactions, so a thorough drug history should be taken; perceptions of drug tolerance by the patients can play an important role. It has been shown that efavirenz changes the sleeping pattern (Moyle 2006).

Patients should be informed thoroughly about the nature of these side effects and that they are usually expected to resolve after a relatively short period of time. Driving cars or operating machinery can be impaired in the first weeks. Treatment with efavirenz should not be started before exams or other important events.

If the CNS side effects persist for more than two to four weeks, it is reasonable to prescribe 200 mg pills, so that the dose can be divided into a 400 mg night dose and a 200 mg morning dose. With this schedule, we observed a reduction in unpleasant CNS side effects in 50% of patients in our center. The daily dose should not be reduced from 600 mg to 400 mg because of the higher risk of therapy failure and development of drug resistance.

Lorazepam can diminish the CNS side effects, and haloperidol can be given for panic attacks and nightmares, but both drugs should be restricted to severe cases, because of their side effects and addictive potency (lorazepam). If they persist with efavirenz even after splitting the dosage as described above for more than six weeks, efavirenz should be replaced.

CNS side effects are possible with etravirine, too (Madruga 2007), although they are less intensive and less frequent. Depression, insomnia and even psychosis rarely occur or get worse on 3TC or abacavir therapy. If the patient complains of CNS-related side effects, 3TC or abacavir should be considered as a possible cause (Foster 2004).

HIV infection itself may cause neurocognitive impairment, for which the earliest possible start of ART is a good preventative measure (Fessel 2009).

Allergic Reactions

Allergic reactions are frequent during HIV therapy. They occur with all NNRTIs, as well as with the nucleoside analog abacavir and the PIs fosamprenavir, tipranavir, atazanavir and darunavir. Because fosamprenavir, tipranavir and darunavir are sulfonamides, they should be given with caution to patients with sulfonamide allergies. When there are limited alternative treatment options, desensitization may permit continued use of fosamprenavir or darunavir in patients with a history of allergy (Marcos Bravo 2009). Atazanavir-associated macular or maculopapular rash is reported in about 6% of patients and is usually mild, so that treatment withdrawal is not necessary (Ouagari 2006).

NNRTIs

Nevirapine may cause a rash in 15 to 30% of patients, leading to discontinuation in about 5%. The rash is seen less frequently during efavirenz and etravirine therapy, where only rarely patients discontinue the drug (Carr 2001). With etravirine, fatal cases of toxic epidermal necrolysis have been reported as well as hypersensitivity reactions which were sometimes accompanied by hepatic failure (Borrás-Blasco 2008). It should be immediately discontinued when signs and symptoms of severe skin or hypersensitivity reactions develop.

The NNRTI allergy is a reversible, systemic reaction and typically presents as an erythematous, maculopapular, pruritic and confluent rash, distributed mainly over the trunk and arms. Fever may precede the rash. Further symptoms include myalgia (sometimes severe), fatigue and mucosal ulceration. The allergy usually begins in the second or third week of treatment. Women are more often and more severely affected (Bersoff-Matcha 2001). If symptoms occur later than 8 weeks after initiation of therapy, other drugs should be suspected. Severe reactions such as Stevens Johnson Syndrome, toxic epidermal necrolysis (Lyell’s syndrome) or anicteric hepatitis are rare.

Treatment should be discontinued immediately in cases with mucous membrane involvement, blisters, exfoliation, hepatic dysfunction (transaminases > 5 times the upper limit of normal) or fever > 39°C.

Approximately 50% of NNRTI allergies resolve with continuation of therapy. Antihistamines may be helpful. Prophylactic treatment with glucocorticosteroids or antihistamines has no protective effect; rashes were even more common in some studies (Montaner 2003, The Grupo Estudio 2004). Following a severe allergic reaction, the drug responsible for the reaction should never be given again.

Abacavir hypersensitivity

Abacavir causes a hypersensitivity reaction (HSR), which may be life-threatening if not recognized in time. It occurs in approximately 4-8% of Caucasian patients (Hughes 2008). A higher rate is noted in patients on a once-daily regime, in art-naïve patients, in patients with a nevirapine allergy, and in acute HIV infection. In over 90% of cases, the HSR occurs after a median of 8 days, and within the first 6 weeks. Hypersensitivity reaction to abacavir is strongly associated with the presence of the HLA-B*5701 allele, which has a prevalence of approximately 6% in Caucasians, and a very low prevalence in black population (Orkin 2010). The prospective PREDICT study involving 1,956 patients from 19 countries showed that HLA-B*5701 screening reduced the risk of hypersensitivity reaction to abacavir (Mallal 2008). HLA-B*5701 screening should be incorporated into routine care for patients who may require abacavir (Phillips 2009). It can prevent significant HSR-related costs and is likely to lead to overall net savings (Wolf 2010). Nevertheless, HLA-B*5701-negative patients should be informed about HSR, as it can rarely present also in these patients.

The rash associated with the abacavir HSR is often discrete, in contrast to the skin reactions caused by nevirapine or efavirenz; in 30% of patients it may not occur at all. 80% of patients have fever. In addition to general malaise (which grows worse day to day), other frequent symptoms include gastrointestinal side effects such as nausea, vomiting, diarrhea and abdominal pain. Respiratory symptoms, such as dyspnea, cough and sore throat, are rare. Changes in the blood count, elevation of liver transaminases, alkaline phosphatase, creatinine and LDH may accompany the HSR. There is usually no eosinophilia. One case of Stevens Johnson Syndrome has been described (Bossi 2002).

The simultaneous start of abacavir with NNRTIs is unfavorable because of the difficulties of differentiating between allergic reactions to NNRTIs and HSR. If abacavir is part of the initial therapy and flu-like symptoms occur, it is difficult to distinguish between immune reconstitution syndrome (IRIS) and HSR; HSR is diagnosed clinically. The differential diagnosis from an intercurrent infection is often difficult. Criteria in favor of HSR include the development of symptoms within the first 6 weeks of treatment, deterioration with each dose taken and the presence of gastrointestinal side effects. If abacavir is discontinued in time, the HSR is completely reversible within a few days. HSR may be fatal if not diagnosed. Following discontinuation of abacavir, further supportive treatment includes intravenous hydration and possibly steroids.

If the suspicion of HSR is only vague, and abacavir is not stopped, the patient should be seen or spoken to daily, to be able to react immediately in case of clinical worsening. Once the diagnosis of HSR has been established and abacavir stopped, rechallenge with abacavir can be fatal and is strictly contraindicated. If there is only a vague suspicion of HSR and abacavir stopped, rechallenge under in-patient conditions is possible. Whenever treatment is interrupted, it needs to be noted that the HSR can occur for the first time after restarting treatment, even without a prior HSR.

Treatment with abacavir requires detailed counseling on the possible occurrence and symptoms of the HSR. Patients should know whom to contact in case of possible HSR. It is important, however, to emphasise to patients that unnecessary discontinuation must also be avoided. Due to the implementation of routine HLA-B*5701 screening the diagnosis of HSR is becoming increasingly rare.

Avascular necrosis

The incidence ofasymptomatic avascular necrosis is approximately 4.4% in HIV-positive patients, significantly more frequent than in the general population (Lawson-Ayayin 2005, Cazanave 2008). The postulated association with PIs has not been confirmed (Loiseau-Peres 2002). Risk factors for avascular necrosis are alcohol abuse, hyperlipidemia, steroid treatment, hypercoagulability, hemoglobinopathy, trauma, nicotine abuse and chronic pancreatitis. Virological (viral load) or immunological parameters are not associated with a risk of developing avascular necrosis (Mondy 2003).

The most common site of the necrosis is the femoral head and, less frequently, the head of the humerus. Initially, patients complain of pain when bearing weight on the affected joint, with symptoms worsening over the following days and weeks. The initial stages may be asymptomatic, but are followed by severe bone pain and reduced mobility. Necrosis of the femoral head produces pain in the hip or groin, which may radiate to the knee.

All patients on ART, especially those with additional risk factors like steroids should be monitored closely when hip pain occurs for the first time. Even in subjects with moderate bone or joint pain, an MRI should be performed early on, as this is more sensitive than conventional radiography. Early diagnosis and treatment can spare patients pain, loss of mobility and surgical intervention.

If the diagnosis is confirmed, patients should be referred to an orthopedic surgeon as soon as possible. Different treatment strategies are available for reducing bone and joint damage as well as pain, depending on the stage of disease, localization and grade of severity. In the early stages, reduced weight bearing with crutches is often sufficient. Surgical core decompression is an option: several holes are drilled in the femoral neck or head, causing new blood vessels to develop and thereby reducing the pressure within the bone. In the more advanced stages, the chances of success decrease with the size of the necrosis. The alternative, osteotomy, has the disadvantage of reducing the mobility of patients over long periods of time. In severe cases, a total endoprothesis (TEP) is usually necessary.

Further risk factors need to be identified and eliminated. If possible, steroids should be discontinued. Sufficient data are missing as to whether treatment modification on non–PI therapy is successful (Mondy 2003). Physiotherapy is recommended. Non-steroidal anti-inflammatory drugs (e.g., ibuprofen) are the treatment of choice for analgesia.

Osteopenia and Osteomalacia

HIV-infected individuals have a lower bone density than uninfected individuals (Loiseau-Peres 2002). Bone density is determined by the measurement of X-ray absorption (e.g., DEXA scan). Results are given as the number of standard deviations (the T-score) from the mean value in young, healthy individuals. Values between -1 and -2.5 standard deviations (SD) are referred to as osteopenia, values above -2.5 SD as osteoporosis. Osteomalacia is the softening of the bones. Osteopenia and osteomalacia may occur in combination. In addition to HIV infection, other factors such as malnutrition, diminished fat tissues, steroid treatment, hypogonadism, immobilization and treatment with PIs and (N)NRTIs, seem to play a role in the pathogenesis of this disorder (Herzman 2009). One study showed a loss of bone mineral density after antiretroviral therapy initiation, independent of which antiretroviral regimen was given (Brown 2009). For the association between tenofovir and bone metabolism see chapter Rheumatic disorders and bone disorders ).

Osteopenia and osteoporosis are often asymptomatic. Osteoporosis occurs mainly in the vertebrae, lower arms and hips. A bone fracture in a HIV patient should always make one suspect osteopenia or osteoporosis.

The following tests should be performed in all patients with AIDS: a lumbar spine X-ray in the standard anteroposterior and lateral views, bone density measurement (DEXA scan) of the lumbar spine and hip; and laboratory blood tests, including calcium, phosphate and alkaline phosphatase. Osteopenia should be treated with 1000 I.E. vitamin D daily and a calcium-rich diet or calcium tablets at a dose of 1200 mg/day. Patients should be advised to exercise and offered methods on how to give up alcohol and nicotine. In cases of osteoporosis, bisphosphonates (e.g., alendronate at 70 mg QW) should be added (McComsey 2007, Huang 2009). The tablets should be taken on an empty stomach 30 minutes before breakfast, and an upright position should be maintained for at least 30 minutes. No calcium should be taken on this day. Antiretroviral therapy should not be taken together with calcium. Because testosterone suppresses osteoclasts, hypogonadism should be treated. Alcohol and smoking should be avoided; regular exercise is an essential part of the therapy.

Enfuvirtide (T-20)

The most common side effect of T-20 is an injection site reaction (ISR) with erythema, induration, nodules, pruritus, ecchymosis, pain and discomfort. Almost every patient is affected, most of them, however, only mildly. ISR rarely limits treatment, and only 3-7% of patients discontinue therapy (Lazzarin 2003). The practitioner and the patient have to get used to the injection technique and the management of ISRs. Good injection technique (see Table 2), may be most effective in minimizing the incidence and severity, as well as the incidence of associated events, including infections. The appropriate management of ISR can lessen the reaction (Clotet 2004). Desensitization therapy is available for the skin rash that occurs rarely with T-20 (Shahar 2005). Patients traveling to foreign countries should be prepared for questions about their injection material. Taking along a medical certificate stating that the patient is on injection therapy can help to avoid unpleasant situations.

Table 2: Suggestions for prevention and management of injection site reactions (ISR) and other injection-related adverse events (Clotet 2004)
Good injection technique

  • Ensure solution is at room temperature
  • Avoid muscle by bevelling needle at 45–90 degrees, depending on body habitus
  • Inject slowly
  • Maintain sterile technique (wash hands, use gloves, clean injection area and vial caps with alcohol swabs, never touch needle)
  • Feel for hard, subcutaneous bumps, avoid injecting into sites of previous ISR
  • Avoid indurated or erythematous areas
  • Avoid injections on the belt line
  • Rotate sites (abdomen, thighs, arms) and never inject two consecutive doses into the same place
  • Gentle manual massage after every injection

Interventions for ISR

1. Injection pain

  • Topical anesthetic (e.g. lidocaine gel)
  • Oral analgesics pre-injection (e.g. ibuprofen or metamizole)
  • Numb area with ice or a cool pack before injecting

2. Management of pruritus

  • Oral antihistamines
  • Emollient creams or lotions (non-alcohol based and fragrance-free)

Changes in blood count

HIV infection itself may cause pancytopenia. A very low CD4 T cell count may therefore be rarely due to a severe leukopenia. In this case, the percentage of the CD4 T cells and the CD4/CD8 ratio is normal.

Some antiretroviral drugs (especially AZT) are myelosuppressive, especially with respect to red cells, and lead to anemia (de Jesus 2004). Most commonly affected are patients with advanced HIV infection and pre-existing myelosuppression, on chemotherapy or co-medication with other myelotoxic drugs such as cotrimoxazole, pyrimethamine, amphotericin B, ribavirin, and interferon, or with other antiretroviral drugs.

5 to 10% of patients taking AZT develop anemia – usually during the first 3 months of therapy, but sometimes even after years on treatment (Carr 2001). AZT should be discontinued in severe cases, and a blood transfusion may be necessary. MCV is always elevated, even in patients on AZT without anemia, and is therefore a measure of adherence. It sometimes makes sense to change from Combivir® to the single drugs Retrovir® and Epivir® in anemic patients, because of the lower AZT dose in Retrovir® (250 mg) compared to Combivir® (300 mg). Because there are many alternatives to this third-line myelotoxic drug we see no reason to give high cost medications like erythropoietin.

Due to drug-induced neutropenia, it is possible that despite viral suppression the CD4 T cells remain low after an initial rise. In these cases treatment should be changed to less myelotoxic antiretroviral drugs and AZT should be avoided. Leukopenia may also occur on abacavir, tenofovir or indinavir. A low CD4 T cell count is also seen on combination of tenofovir and ddI.

For thrombocytopenia see also chapter HIV asscociated thrombocytopenia.

Increased bleeding episodes

HIV-infected patients with hemophilia A or B, after some weeks of treatment with PIs, may have increased episodes of spontaneous bleeding into joints and soft tissues. Rarely, intracranial and gastrointestinal bleeding has occurred. The etiology is unclear (Review: Wilde 2000).

During clinical trials with tipranavir/r, the manufacturer received 14 reports of intracranial hemorrhage, among them 8 fatal cases, in 13 out of 6,840 HIV-1 infected individuals. Most of them occurred more than one year after initiating therapy. So far, there have been no more spontaneous reports of intracranial hemorrhage on marketed tipranavir. Many of the patients affected had other risk factors for intracranial hemorrhage such as CNS lesions, head trauma, recent neurosurgery, coagulaopathy, hypertension or alcohol abuse, or were receiving anticoagulant or antiplatelet agents. Tipranavir was observed in vitro to inhibit human platelet aggregation (Graff 2007). No pattern of abnormal hematologic or coagulation parameters was observed. Therefore, routine measurement of coagulation parameters is not indicated. Tipranavir/r should be avoided if possible in patients with the above mentioned risk factors. This applies also for patients on antiplatelet agents or anticoagulants. Patients should be informed about the possible risk of intracranial hemorrhage.

Lactic acidosis

Lactic acidosis is a rare but life-threatening complication due to mitochondrial toxicity. It occurs most frequently on treatment with d4T and ddI, and less so in patients on AZT, abacavir and 3TC (Garrabou 2009). Risk factors are obesity, female sex, pregnancy and therapy with ribavirin or hydroxyurea, a diminished creatinine clearance and a low CD4 T cell nadir (Bonnet 2003, Butt 2003, Wohl 2006).

Cases of severe lactic acidosis can occur without prior symptomatic hyperlactatemia. Lactate levels do not need to be monitored routinely, as increases are not predictive and may lead to unnecessary changes in treatment (Brinkman 2001, Vrouenraets 2002). In contrast, lactate levels should be tested immediately in symptomatic patients complaining of fatigue, sudden weight loss, abdominal disturbances, nausea, vomiting or sudden dyspnea, in pregnant women on NRTI treatment and in patients on NRTIs post-lactic acidosis (Carr 2003).

For clinical symptoms, pathogenesis, and treatment please see chapter on Mitochondrial Toxicity.

References

Antoniou T, Raboud J, Chirhin S, et al. Incidence of and risk factors for tenofovir-induced nephrotoxicity: a retrospective cohort study. HIV Med 2005; 6:284-90.

Barrios A, Garcia-Benayas T, Gonzalez-Lahoz J, et al. Tenofovir-related nephrotoxicity in HIV-infected patients. AIDS 2004; 18:960-3.

Bersoff-Matcha SJ, Miller WC, Aberg JA, et al. Sex differences in nevirapine rash. CID 2001; 32:124-9.

Bjornsson E, Olsson R. Suspected drug-induced liver fatalities reported to the WHO database. Dig Liver Dis 2006, 38:33-8.

Bonnet F, Bonarek M, Morlat P, et al. Risk factors for lactic acidosis in HIV-infected patients treated with nucleoside reverse-transcriptase inhibitors: a case-control study. Clin Infect Dis 2003; 36:1324-8.

Borrás-Blasco J, Navarro-Ruiz A, Borrás C, et al. Adverse cutaneous reactions associated with the newest antiretroviral drugs in patients with human immunodeficiency virus infection. J Antimicrob Chemother 2008; 62:879-88.

Bossi P, Roujeau JC, Bricaire F, et al. Stevens-johnson syndrome associated with abacavir therapy. Clin Infect Dis 2002; 35:902.

Brannan A, Evans D, Maskew M et al. Relationship betwee renal dysfunction, nephrotoxicity and death among HIV adults on tenofovir. AIDS 2011; 25:1603-1609.

Brinkman K. Management of hyperlactatemia: no need for routine lactate measurements. AIDS 2001; 15:795-7.

Brown TT, McComsey GA, da Silva BA, et al. Loss of Bone mineral density after antiretroviral therapy initiation, independent of antiretroviral regimen. JAIDS 2009, 51:554-561.

Bushen OY, Davenport JA, Bezerra Lima A, et al. Diarrhea and reduced levels of antiretroviral drugs: improvement with glutamine or alanyl-glutamine in a randomized controlled trial in Northeast Brazil. Clin Infect Dis 2004, 38:1764–70

Butt AA. Fatal lactic acidosis and pancreatitis associated with ribavirin and didanosine therapy. AIDS Read 2003; 13:344-8.

Carr A, Cooper DA. Adverse effects of antiretroviral therapy. Lancet 2001; 356:1423-30.

Carr A, Morey A, Mallon P, et al. Fatal portal hypertension, liver failure, and mitochondrial dysfunction after HIV-1 nucleoside analogue-induced hepatitis and lactic acidaemia. Lancet 2001; 357:1412-4.

Carr A. Lactic acidemia in infection with HIV. Clin Infect Dis 2003; 36:S96-S100.

Carr A and Amin J. Efficacy and tolerability of initial antiretroviral therapy: a systematic review. AIDS 2009, 23:343-353.

Cazanave C, Dupon M,  Lavignolle-Aurillac, et al. Reduced bone mineral density in HIV-infected patients: prevalence and associated factors. AIDS 2008, 22:395-402.

Chan-Tack KM, Struble KA, Birnkrant DB. Intracranial hemorrhage and liver-associated deaths associated with tipranavir/ritonavir: review of cases from the FDA’s Adverse Event Reporting System. AIDS Patient Care STDS 2008; 11:843-50.

Chubineh S, McGowan J. Nausea and vomiting in HIV: a symptom review. Int J STD AIDS 2008; 11;723-8.

Cicconi P, Cozzi-Lepri A, Castagna A, et al. Insights into reasons for discontinuation according to year of starting first regimen of highly antiretroviral therapy in a cohort of antiretroviral-naive patients. HIV Medicine 2010, 11:104-113.

Clark SJ, Creighton S, Portmann B, et al. Acute liver failure associated with antiretroviral treatment for HIV: a report of six cases. J Hepatol 2002, 36:295-301

Clotet B, Raffi F, Cooper D, et al. Clinical management of treatment-experienced, HIV infected patients with the fusion inhibitor enfuvirtide: consensus recommendations. AIDS 2004; 18:1137–1146.

Cooper RD, Wiebe N, Smith N, et al. Systematic review and meta-analysis: renal safety of tenofovir disoproxil fumarate in HIV-infected patients. Clin Infect Dis 2010; 51:496-505.

Crane HM, Kestenbaum B, Harrington RD, et al. Amprenavir and didanosine are associated with declining kidney function among patients receiving tenofovir. AIDS 2007, 21:1431-1439.

D’Arminio Monforte A, Lepri AC, Rezza G, et al. Insights into the reasons for discontinuation of the first highly active antiretroviral therapy regimen in a cohort of antiretroviral naïve patients. AIDS 2000; 14:499-507.

De Jesus E, Herrera G, Teofilo E, et al. Abacavir versus zidovudine combined with lamivudine and efavirenz, for the treatment of antiretroviral-naive HIV-infected adults. Clin Infect Dis 2004; 39:1038-46.

De Lazzari E, León A, Arnaiz JA, et al. Hepatotoxicity of nevirapine in virologically suppressed patients according to gender and CD4 cell counts. HIV Med 2008, 9:221-6.

Fessel WJ. Impaired neurocognition in HIV-infected patients: antecedents and treatment. AIDS 2009, 23:1731-1733.

Foster R, Taylor C, Everall IP. More on abacavir-induced neuropsychiatric reactions. AIDS 2004; 18:2449.

Gallant JE, Moore RD. Renal function with use of a tenofovir-containing initial antiretroviral regimen. AIDS 2009; 23: 1971-1975.

Garrabou G, Morén C, Miró O, et al. Genetic and functional mitochondrial assessment of HIV-infected patients developing HAART-related hyperlactatemia. JAIDS 2009, 52:443-451.

Goicoechea M, Liu S, Best B, et al. Greater tenofovir-associated renal function decline with protease inhibitor-based versus NNRTI-based therapy. J Infect Dis 2008; 1:102-8.

Graff J, von Hentig N, Kuczka K, et al. Significant effects of tipranavir on platelet aggregation and thromboxane B2 formation in vitro and in vivo. J Antimicrob Chemother 2008; 61:394-9.

Guaraldi G, Cocchi S, Motta A, et al. Efficacy and safety of atazanavir in patients with end-stage liver disease. Infection 2009;37:250-5.

Gutierrez S, Guillemi S, Jahnke N, et al. Tenofovir-based rescue therapy for advanced liver disease in 6 patients coinfected with HIV and hepatitis B virus and receiving lamivudine. Clin Infect Dis 2008; 46:e28-e30.

Haas DW, Heather JR, Richard BK, et al. Pharmacogenetics of efavirenz and central nervous system side effects: an Adult AIDS Clinical Trials Group study. AIDS 2004; 18:2391–2400.

Heiser CR, Ernst JA, Barrett JT, et al. Probiotics, soluble fiber, and L-Glutamine (GLN) reduce nelfinavir (NFV)- or lopinavir/ritonavir (LPV/r)-related diarrhea. J Int Assoc Physicians AIDS Care 2004; 3:121-9.

Hendershot CS, Stoner SA, Simoni JM, et al. Alcohol use and antiretroviral adherence: review and meta-analysis. JAIDS 2009, 52:180-202.

Herzmann C and Arastéh K. Efavirenz induced osteomalacia. AIDS 2009, 23:274-275.

Hicks CB, Cahn P, Cooper DA, et al. Durable efficacy of tipranavir-ritonavir in combination with an optimised background regimen of antiretroviral drugs for treatment-experienced HIV-1-infected patients at 48 weeks in the Randomized Evaluation of Strategic Intervention in multi-drug resistant patients with Tipranavir (RESIST) studies: an analysis of combined data from two randomised open-label trials. Lancet 2006; 9534:466-75

Horberg M, Tang B, Towner W, et al. Impact of tenofovir on renal function in HIV-infected, antiretroviral-naive patients. J Acquir Immune Defic Syndr. 2010;53:62-9.

Huang J, Meixner L, Fernandez S, et al. A double-blinded, randomized controlled trial of zoledronate therapy for HIV-associated osteopenia and osteoporosis. AIDS 2009; 23:51-7.

Hughes CA, Foisy MM, Dewhurst N, et al. Abacavir hypersensitivity reaction: an update. Ann Pharmacother 2008; 3:387-96.

Izzedine H, Isnard-Bagnis C, Hulot JS, et al. Renal safety of tenofovir in HIV treatment-experienced patients. AIDS 2004; 18:1074-6.

Izzedine H and Deray G. The nephrologist in the HAART era. AIDS 2007; 21:409-421.

Jamisse L, Balkus J, Hitti J, et al. Antiretroviral-associated toxicity among HIV-1-seropositive pregnant women in Mozambique receiving nevirapine-based regimens. J Acquir Immune Defic Syndr. 2007;1;44:371-6.Joshi D, O’Grady J, Dieterich D, et al. Increasing burden of liver disease in patients with HIV infection. Lancet 2011;377:1198-209.

Kesselring AM, Wit FW, Sabin CA; et al. Risk factors for treatment-limiting toxicities in patients starting nevirapine-containing antiretroviral therapy. AIDS 2009; 23:1689-1699.

Kontorinis N, Dieterich DT. Toxicity of nonnucleoside analogue reverse transcriptase inhibitors. Semin Liver Dis 2003; 23:173-182.

Lattuada E, Lanzafame M, Carolo G, et al. Does tenofovir increase efavirenz hepatotoxicity? AIDS 2008; 22:995.

Lawson-Ayayin S, Bonnet F, Bernardin E, et al. Avascular necrosis in HIV-Infected patients: A case-control study from the Aquitaine Cohort, 1997–2002, France. Clin Infect Dis 2005; 40:1188–93.

Lazzarin A, Clotet B, Cooper D, et al. Efficacy of enfuvirtide in patients infected with drug-resistant HIV-1 in Europe and Australia. N Engl J Med 2003; 348:2186-95.

Loiseau-Peres S, Delaunay C, Poupon S, et al. Osteopenia in patients infected by the HIV. A case control study. Joint Bone Spine 2002; 69:482-5.

Madruga JV, Cahn P, Grinsztejn B, et al. Efficacy and safety of TMC125 (etravirine) in treatment-experienced HIV-1-infected patients in DUET-1: 24-week results from a randomised, double-blind, placebo-controlled trial. Lancet 2007; 9581:29-38.

Mallolas J. Nevirapine-associated hepatotoxicity in virologically suppressed patients—role of gender and CD4+ cell counts. AIDS Rev 2006; 8:238-9.

Mallal S, Phillips E, Carosi G, et al. HLA B5701 Screening for hypersensitivity to abacavir. N Engl J Med 2008, 358:568-79.

Manfredi R, Calza L. Nevirapine versus efavirenz in 742 patients: no link of liver toxicity with female sex, and a baseline CD4 cell count greater than 250 cells/microl. AIDS 2006; 20:2233-6.

Marcos Bravo MC, Ocampo Hermida A, Martinez Vilela J, et al. Hypersensitivity reaction to darunavir and desensitization protocol. J Investig Allergol Clin Immunol 2009; 19:250-1.

Marzolini C, Telenti A, Decosterd LA, et al. Efavirenz plasma levels can predict treatment failure and central nervous system side effects in HIV-1-infected patients. AIDS 2001; 15:71-75.

McComsey GA, Kendall MA, Tebas P, et al. Alendronate with calcium and vitamin D supplementation is safe and effective for the treatment of decreased bone mineral density in HIV. AIDS 2007, 21:2473-2482.

McKoy JM, Bennett CL, Scheetz MH, et al. Hepatotoxicity associated with long- versus short-course HIV-prophylactic nevirapine use: a systematic review and meta-analysis from the Research on Adverse Drug events And Reports (RADAR) project. Drug Saf 2009; 32:147-58.

Montaner JS, Cahn P, Zala C, et al. Randomized, controlled study of the effects of a short course of prednisone on the incidence of rash associated with nevirapine in patients infected with HIV-1. J AIDS 2003; 33:41-6.

Mondy K, Tebas P. Emerging bone problems in patients infected with HIV. Clin Infect Dis 2003; 36:S101-5.

Moyle G , Fletcher C, Brown H, et al. Changes in sleep quality and brain wave patterns following initiation of an efavirenz-containing triple antiretroviral regimen. HIV Med 2006, 7:243-7.

Nelson MR, Katlama C, Montaner JS, et al. The safety of tenofovir disoproxil fumarate for the treatment of HIV infection in adults: the first 4 years. AIDS 2007; 21:1273-81.

Nunez M, Soriano V. Hepatotoxicity of antiretrovirals: incidence, mechanisms and management. Drug Saf 2005, 28:53-66.

Orkin C, Wang J, Bergin C, et al. An epidemiologic study to determine the prevalence of the HLA-B*5701 allele among HIV-positive patients in Europe. Pharmacogenet Genomics 2010; 20:307-14.

Ouagari Z, Tubiana R, Mohand HA, et al. Skin rash associated with atazanavir: report of three cases. AIDS 2006, 20:1207-8.

Ouyang DW, Shapiro DE, Lu M, et al.  Increased risk of hepatotoxicity in HIV-infected pregnent women receiving antiretroviral therapy independent of nevirapine exposure. AIDS 2009, 23:2425-2430.

Ouyang DW, Brogly SB, Lu M, et al. Lack of increased hepatotoxicity in HIV-infected pregnant women receiving nevirapine compared with other antiretrovirals. AIDS 2010; 24:109-14.

Peters PJ, Stringer J, McConnell MS, et al. Nevirapine-associated hepatotoxicity was not predicted by CD4 count ≥250 cells/μL among women in Zambia, Thailand and Kenya. HIV Med 2010; 11:650-60. Phillips E, Mallal S. Successful translation of pharmacogenetics into the clinic: the abacavir example. Mol Diagn Ther 2009;13:1-9.

Pineda JA, Santos J, Rivero A, et al. Liver toxicity of antiretroviral combinations including atazanavir/ritonavir in patients co-infected with HIV and hepatitis viruses: impact of pre-existing liver fibrosis. J Antimicrob Chemother 2008; 4:925-32.

Price JC, Thio CL. Liver Disease in the HIV–Infected Individual Clinical Gastroenterology and Hepatology 2010; 1002-1012.

Rachlis A, Clotet B, Baxter J, et al. Saftey, tolerability and efficacy of darunavir (TMC114) with low-dose ritonavir in treatment-experienced, hepatitis B od C co-infected patients in POWER1 and 3. HIV Clin Trials 2007; 4:213-220.

Robison LS, Westfall AO, Saag MS, et al. Short-term discontinuation of HAART regimens more common in vulnerable patient populations. AIDS Res. 2008; 24: 1347-1355.

Roling J, Schmid H, Fischereder M, et al. HIV-associated renal diseases and highly active antiretroviral therapy-induced nephropathy. Clin Infect Dis 2006; 42:1488-95.

Rollot F, Nazal EM, Chauvelot-Moachon L, et al. Tenofovir-related Fanconi syndrome with nephrogenic diabetes insipidus in a patient with AIDS: the role of lopinavir-ritonavir-didanosine. Clin Infect Dis 2003; 37:e174-6.

Sanne I, Mommeja-Marin H, Hinkle J, et al. Severe hepatotoxicity associated with nevirapine use in HIV-infected Subjects. J Infect Dis 2005; 191 :825-829.

Saumoy M, Vidal F, Peraire J, et al. Proximal tubular kidney damage and tenofovir: a role for mitochondrial toxicity? AIDS 2004; 18:1741-2.

Servoss JC, Kitch DW, Andersen JW, et al. Predictors of antiretroviral-related hepatotoxicity in the adult AIDS Clinical Trial Group (1989-1999). J Acquir Immune Defic Syndr 2006; 1;43:320-3.

Shahar E, Moar C, Pollack S. Successful desensitization of enfuvirtide-induced skin hypersensitivity reaction. AIDS 2005; 19:451.

Soriano V, Puoti M, Garcia-Gasco P, et al. Antiretroviral drugs and liver injury. AIDS 2008, 22:1-13.

Sulkowski MS, Thomas DL, Mehta SH, et al. Hepatotoxicity associated Nevirapine or Efavirenz-containing antiretroviral therapy: role of Hepatitis C and B Infection. Hepatology 2002; 35:182-9.

Sulkowski MS. Drug-induced liver injury associated with antiretroviral therapy that includes HIV-1 protease inhibitors. Clin Infect Dis 2004; 38:S90-7.

Sulkowski MS, Mehta SH, Chaisson RE, et al. Hepatotoxicity associated with protease inhibiotor-based antiretroviral regimens with or without concurrent ritonavir. AIDS 2004; 18:2277-84.

The Grupo Estudio Syndrome Immunodeficiencies Adquirida 26/02 Study Group. Failure of cetirizine to prevent nevirapine-associated rash: A double-blind placebo-controlled trial for the GESIDA 26/01 Study. J AIDS 2004; 37:1276-1281.

Torti C, Costarelli S, De Silvestri A, et al. Analysis of severe hepatic events associated with nevirapine-containing regimens: CD4+ T-cell count and gender in hepatitis C seropositive and seronegative patients. Drug Saf 2007, 30:1161-9.

Torti C, Lapadula G, Antinori A, et al. Hyperbilirubinemia during atazanavir treatment in 2,404 patients in the Italian atazanavir expanded access program and MASTER Cohorts. Infection 2009; 37:244-9.

Turner MJ, Angel JB, Woodend K. The efficacy of calcium carbonate in the treatment of protease inhibitor-induced persistent diarrhea in HIV-infected patients. HIV Clin Trials 2004; 5:19-2.

Van den Bout – van den Beukel CJ, Bosch ME, Burger D, et al.  Toxic lopinavir concentrations in an HIV-1 infected patient taking herbal medications. AIDS 2008; 22:1243-4.

Vrouenraets SM, Treskes M, Regez RM, et al. Hyperlactataemia in HIV-infected patients: the role of NRTI-treatment. Antivir Ther 2002; 7:239-44.

Wilde JT. Protease inhibitor therapy and bleeding. Haemophilia 2000; 6:487-90.

Wohl DA, McComsey G, Tebas P, et al. Current concepts in the diagnosis and management of metabolic complications of HIV infection and its therapy. Clin Infect Dis 2006, 43:645-53.

Wolf E, Blankenburg M, Bogner JR  et al. Cost impact of prospective HLA-B*5701-screening prior to abacavir/lamivudine fixed dose combination use in Germany. Eur J Med Res. 2010;15:145-51.

Wyen C, Hendra H, Vogel M, et al. Impact of CYP2B6 983T>C polymorphism on non-nucleoside reverse transcriptase inhibitor plasma concentrations in HIV-infected patients. J Antimicrob Chemother 2008, 61:914-8.

Yuan Y, L’italien G, Mukherjee J, et al. Determinants of discontinuation of initials highly active antiretroviral therapy regimens in a US HIV-infected patient cohort. HIV Med 2006;7:156-62.


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Filed under 7. Managing Side Effects, Part 2 - Antiretroviral Therapy

HIV Resistance and Viral Tropism Testing

– Patrick Braun and Eva Wolf –

The goal of antiretroviral therapy is to achieve maximum suppression of viral replication. Viral blips while on suppressive ART are relatively common and are mostly owed to random biological and statistical fluctuations. However, patients with repeated episodes of detectable viremia – suggesting ongoing viral replication rather than virus release from latent reservoirs due to immune activation – are at increased risk for the development of drug resistance. The level of viral load while on therapy is the best predictor of subsequent virological failure which is increased at viral load levels between 100 and 300 copies/ml (Nettles 2005, Delaguerre 2009, Garcia-Gasco 2008).

The rapid development of resistant variants is due to the high turnover of HIV – in an untreated HIV-infected patient approximately 10 million new viral particles are produced every day (Perelson 1996) – and the exceptionally high error rate of HIV reverse transcriptase. This leads to a high mutation rate and constant production of new viral strains, even in the absence of treatment. In the presence of antiretroviral drugs, the development of HIV-1 resistance depends on the selection of resistance-associated mutations. If a virus has acquired one or more resistance-associated mutations leading to reduced drug sensitivity, the mutant virus attains a replication advantage in comparison to wild-type virus when exposed to drugs. The development of resistant viral strains is one of the main reasons for virological failure of antiretroviral therapy. However, with the strategic use of the newer drug classes, effective regimens are available even in salvage situations.

The discussion about genotypic resistance in this chapter focuses on the methods of resistance testing, on mutation patterns emerging on ART, and their interpretations and clinical relevance. Most data are derived from patients with subtype B viruses, representing the main subtype in North America, Australia and Europe, but only about 12% of the global HIV-1 epidemic. During recent years, non-B subtype viruses have been investigated, some with different resistance pathways and patterns (Snoeck 2006).

Assays for resistance testing

There are two established assays for measuring resistance or sensitivity of HIV to specific antiretroviral drugs – the genotypic and the phenotypic resistance tests (Wilson 2003). Assays accredited by the FDA are:

  • HIV-1 TruGene™ (Siemens Healthcare Diagnostics)
  • ViroSeq™/ABI Prism®
  • 3100 Genetic Analyzer (Abbott Molecular/Applera Corporation of Applied Biosystems and Celera).

Standard (population-based) genotypic tests can only detect viral mutants when these comprise at least 20% of the total virus population. Ultrasensitive methods (allele-specific real-time PCR, single genome sequencing) with detection limits of <0.1–5% are available only in few laboratories. The clinical relevance of minority populations remains a controversial issue. However, there is evidence especially for minor variants with NNRTI mutations (Li 2011).

Commercial phenotypic resistance tests include:

  • Antivirogram™ (Virco)
  • PhenoSense™ (Monogram Biosciences)
  • PhenoTecT™ (InPheno)
  • Phenoscript™ (Viralliance)

The cost of genotyping ranges from 260 to 400 Euros, depending on the assay and laboratory used. It is approximately twice that much for phenotyping. The drawback of both methods is that a minimum amount of virus is necessary in order to perform the test. Depending on the method and on the laboratory 100-1,000 copies/ml are required for detection of resistance. Tables 1 and 2 show the advantages and disadvantages of phenotypic and genotypic resistance analyses.

Phenotyping

Phenotypic resistance tests involve direct quantification of drug sensitivity. Viral replication is measured in cell culture under the selective pressure of increasing concentrations of antiretroviral drugs and is compared to viral replication of wild-type virus.

Drug concentrations are expressed as IC50 values (50% inhibitory concentration), the concentration of drug required to inhibit viral replication in cell cultures by 50%. The sensitivity of the virus is expressed as the IC50 divided by the IC50 of a wild-type reference virus (fold-change value also reported as resistance factor) and compared to the so-called cut-off value. The cut-off value indicates by how much the IC50 of an HIV isolate can be increased in comparison to that of the wild-type and still be classified as sensitive. The determination of the cut-off is crucial for the interpretation of the results.

Table 1. Advantages and disadvantages of phenotypic resistance analysis.
Phenotypic resistance analysis
Advantages Disadvantages
  • Direct measure of drug susceptibility
  • Measure of drug susceptibility feasible irrespective of the presence of unknown resistance mutations
  • Considers the complexity of resistance patterns and the presence of re-sensitizing mutations
  • Only detection of viral mutants comprising ≥20-30% of the total virus population
  • Clinical cut-offs not available for all drugs
  • Expensive (reimbursement by health insurance often not guaranteed)
  • Time-consuming (several weeks)
  • HIV-1 subtyping not possible
  • Interactions between antiviral drugs are not reflected in the test results
  • Test results are not affected by amino acid exchanges, which are only an intermediate step to resistance

Cut-offs: technical, biological and clinical

Three different cut-offs are currently being used.

The technical cut-off is a measure of the methodological variability of the assay.

The biological cut-off involves the inter-individual variability of wild-type virus isolates from ART-naïve HIV-positive patients. If the IC50 is below the biological cut-off, virological success is very likely. However, an IC50 above the biological cut-off does not allow prediction of the virologic response to a drug.

In contrast, the clinical cut-off indicates up to what levels of IC50 virologic effectiveness can still be expected. Complete resistance to a drug (i.e., to protease inhibitors) generally evolves gradually with the acquisition of several amino acid changes.

In general, lower and upper clinical cut-offs are defined. The lower clinical cut-off is the fold-change in IC50 which indicates slightly reduced virological response. A fold-change above the upper clinical cut-off indicates resistance, and a fold-change between the two cut-offs indicates partial resistance. Due to limited clinical experience cut-off data is often lacking for recently approved drugs. In these cases, interpretations are based on biological cut-offs.

In the phenotypic analysis, mutations that do not confer resistance by themselves but provide evidence for transmitted, emerging or reverting resistance have no influence on the measure of resistance.

Genotyping

Background & nomenclature

The HIV genome consists of 2 RNA (ribonucleic acid) strands containing the genetic information of the virus. Within the nucleotide sequence of the HIV genome, a group of three nucleotides, called a codon, code for a particular amino acid in the protein sequence. Resistance mutations are described using a number for each gene, showing the position of the relevant codon, and two letters, the letter preceding the number corresponding to the amino acid specified by the codon at this position in the wild-type virus, while the letter after the number describes the amino acid that is produced from the mutated codon.

A change in the nucleotide sequence of a codon is called a mutation. ‘Silent’ mutations code for the same amino acid. ‘Lethal’ mutations cause a defective protein structure leading to a stop of the viral replication cycle. Only those mutations that code for a different amino acid that leads to a change in the protein structure are clinically relevant. This affects protein function and can contribute to the development of resistance to antiretroviral agents. M184V indicates a mutation in codon 184 of the reverse transcriptase gene leading to a valine for methionine substitution in the reverse transcriptase enzyme and rendering the virus resistant to 3TC and FTC.

Genotypic assays are based on the analysis of mutations associated with resistance. These are determined by the direct sequencing of the amplified HIV genome or by specific hybridization techniques with wild-type or mutant oligonucleotides. For therapeutic decision making, sequencing of the pol region, which encodes for the viral enzymes protease, reverse transcriptase and integrase, and sequencing of the env region, which encodes for the glycoproteins of the viral envelope, gp41 and gp120, are of relevance. Other gene regions, in particular RNase H and gag, are reported to be associated with phenotypic drug resistance. However, sequencing of these regions has only been performed in the context of research and is not part of routine diagnostics.

The interpretation of genotypic resistance patterns is based on the correlation between genotype, phenotype and clinical response. There is data available from in vitro studies, clinical studies, clinical observations and duplicate testing, in which genotypically localized mutations have been investigated for phenotypic resistance.

Table 2. Advantages and disadvantages of genotypic resistance analysis.
Genotypic resistance analysis
Advantages Disadvantages
  • Quickly performed (results within days)
  • Widely used (no specific safety requirements for laboratory)
  • Listing of all changes in the nucleotide sequence
  • Detection of any mutation – with either evidence of resistance, emerging resistance or reverting resistance
  • HIV-1 subtyping possible
  • In general reimbursement by health insurance (i.e., sequencing of the protease and the RT genes)
  • Indirect measurement of resistance
  • Only detection of viral mutants comprising ≥20-30% of the total virus population
  • Complex resistance patterns are often difficult to interpret
  • Unknown mutations are not considered for interpretation
  • Interpretation systems must be updated regularl

Rules-based interpretation systems

For the phenotypic interpretation of genotypic mutation patterns rules-based interpretation systems are commonly available. Expert panels such as HIV-GRADE have developed algorithms based on the literature and clinical outcomes. Table 3 shows an overview of widely used interpretation systems.

Several commercial providers of resistance assays have integrated interpretation guidelines into their systems (e.g., virco®Type HIV-1 from Virco or GuideLines© (TruGene™) from Siemens Healthcare Diagnostics).

Data-based interpretation systems and virtual phenotype

Contrary to the knowledge-based interpretation algorithms developed by experts, data-based interpretation systems like geno2pheno or vircoType™ are mathematical approaches to predicting (“virtual”) phenotype from genotypic information. The virtual phenotype is characterized by the fact that phenotypic information is derived from genotype without performing a phenotypic resistance test in the laboratory. Phenotypic estimates derive from large databases of paired genotypic and phenotypic information.

The interpretation system geno2pheno, available free of charge, uses machine learning techniques such as decisiontrees and support vector machines (Beerenwinkel 2003).

The vircoType™ interpretation is based on a multiple linear regression model, which is applied to a database consisting of more than 61,000 (as of August 2010) matched genotype/phenotype pairs: For every drug the fold-change in IC50 is a function of all mutations of the patient’s virus that contribute to specific drug resistance. To account for synergistic and antagonistic effects between mutations, specific pairs of mutations are included in the model. According to their relevance, drug-specific weight factors are attributed to individual mutations or pairs of mutations. Weight factors are positive for mutations or pairs that contribute to resistance, negative for mutations or pairs with a resensitizing effect.

Table 3. Genotypic resistance interpretation systems: an overview.
Interpretation system Interpretation Available free of charge Internet address:http://www.
HIV-GRADE (07/2010), Germany Rules-based Yes hiv-grade.de
Rega V8.0.2 (HIV-1&2) (06/2009), Belgium Rules-based Yes regaweb.med.kuleuven.be/software/rega_algorithm
HIVdb Version 6.0.9 (08/2010), USA Rules-based Yes hivdb.stanford.edu/ (no www)
ANRS (HIV1&2) V19 (07/2010), France Rules-based Yes hivfrenchresistance.org/index.html
EuResistEuResist Network GEIE Data-based Yes euresist.org
MGRM GeneSure®  MG(Monogram Bioscience) Rules- and data-based No monogramvirology.com/hiv-tests/resistance-testing/genotype/genosuremg/
geno2phenoGermany Data-based(virtual phenotype) Yes geno2pheno.org/
virco®Type HIV-1 (Virco) Data-based(virtual phenotype) No vircolab.com

Methods of tropism testing

To enter the target cell, HIV binds to the CD4-receptor and so called chemokine co-receptors, of which the most important are CCR5 and CXCR4. Dependent on the use of co-receptors (“tropism”) the virus is classified as CCR5- (“R5”-) tropic or CXCR4- (“X4”-) tropic. Viral strains using both co-receptors are called dual-tropic. Since tropism tests cannot distinguish between dual-tropic viral isolates and a mixture of R5- and X4-tropic viral isolates, the term dual/mixed (D/M) tropic is used.

Analogous to resistance testing, tropism testing can be performed genotypically or phenotypically. Due to its use in clinical trials, Trofile™ is the best-known phenotypic tropism test. The original standard test had a sensitivity limit of 5 to 10%. With the enhanced sensitivity TrofileTM assay (ESTA) minor virus populations can be detected that comprise less than 1% of the total virus population. Another phenotypic test is Phenoscript® ENV (EuroFins/VIRalliance). An 85% agreement between both assays was reported (Skrabal 2007).

For genotypic tropism analysis, the V3 domain of the gp120 gene – which is crucial for co-receptor binding and encodes for the viral tropism – is sequenced. Web-based bioinformatic tools are used to predict viral tropism from the respective nucleotide sequence. These tools have implemented methods like the charge rule, support vector machines or decision trees (Skrabal 2007, Garrido 2008, Obermeier 2008). Tropism prediction tools for genotypic sequences can be found at the following web addresses:

The interpretation with the coreceptor tool of geno2pheno is widely used and shows good concordance with ESTA (Prosperi 2010). In contrast to phenotypic analysis, genotypic analysis cannot distinguish between X4-tropic and dual-tropic or mixed populations. The result of the geno2pheno co-receptor tool is the so-called false positive rate (FPR), which is the probability of classifying an R5-virus falsely as X4. A false positive rate of 0.1% means that X4-tropism is very likely, whereas a FPR of 90% means that X4-tropism is very unlikely because an X4-prediction would be false with a 90% probability.

The current FPR cut-offs in international guidelines are ≤10-12% for X4-prediction and ≥20% for R5-prediction. Phenotypic testing is recommended for indeterminate results. For tropism testing from proviral DNA, which is used in case of undetectable viral load or low level viremia, the same FPR can be used. There are discussions about further reducing the cut-offs (Walter 2009, Vandekerckhove 2011).

As for genotypic resistance testing, a distinction is made between standard population sequencing (detecting X4-tropic virus variants if they comprise at least 20% of the total virus population) and ultrasensitive methods (such as ultra-deep sequencing (UDS) with detection limits of a few percent or less).

In a study using Maraviroc+Atazanavir/r in ART-naive patients, ESTA was used for tropism testing. All samples were analyzed using population sequencing and UDS, each with a FPR of 5.75%. Using ESTA, R5-tropic virus was found in 123 samples (69%); D/M-tropic virus was detected in 39 samples (22%). In 16 samples, tropism testing failed. Using population sequencing, R5-tropic virus was found in 82% of samples, X4-tropic virus was found in 15%. In 3% of samples genotyping was not successful. The concordance for R5-tropic virus between population sequencing and UDS was 95%. Of samples classified as R5-tropic by population sequencing, only 3% (3 of 114) harbored X4-tropic virus of more than 2%. For all failing ESTA measurements viral tropism was determined with population sequencing (Portsmouth 2010a).

The advantages of genotypic tropism testing are its wide availability and the rapid results. Analyses that have correlated genotypic and phenotypic tropism results with virologic response showed that the two methods can be considered as equivalent (Braun 2009, Harrigan 2009). As a consequence, genotypic tropism testing has been included in national and international guidelines on the management of HIV-1 tropism testing (Vanderkerckhove 2011).

A key advantage of genotypic tropism testing is its feasibility in samples with undetectable plasma viral load. This may be important in patients suffering from side effects from a virological..xxx. By sequencing proviral DNA, good concordance has been shown between TrofileTM results and the genotypic tropism predictions (Obermeier 2008). Genotyping of proviral DNA is of clinical importance in successfully treated patients requiring treatment change due to side effects. Recently, TrofileTM has also become available for the testing of proviral DNA.

In the European guidelines concerning the use of tropism testing both the enhanced sensitivity Trofile assay and V3 loop population sequencing were recommended. However, the choice of the test should be based on the local capacity, logistics, cost and turnaround time. The preferred method of analysis for patients with a viral load between 50 – 1000 copies/ml is the V3 loop population sequencing.

Table 4. Advantages (+) and disadvantages (-) of genotypic and phenotypic tropism testing, (Examples using geno2pheno and Trofile™).
Phenotypic tropism testESTA ™

  • Phenotypic analysis using the complete gp160
  • Result derives from cell culture
Genotypic tropism testgeno2pheno

  • Genotypic analysis based on V3 sequence
  • Prediction of tropism using bioinformatics tools
+ Validated by clinical data+ Differentiation of R5-, X4- and D/M(dual/mixed)-tropic HIV-  Commercial test / expensive

–  Result within about 3-4 weeks

–  Required viral load of ≥500 – 1,000

copies/ml when using RNA

+ Feasible in case of low/undetectable

plasma viral load when using proviral

DNA

+   Validated by clinical data+   Result based on the exclusion ofX4-tropic virus+   Feasible in molecular biology

laboratories

+   Widely available / less expensive

+   Result within about 5 days

–    Required viral load of ≥500 – 1,000

copies/ml when using RNA

+  Genotyping of proviral DNA in case of

low or undetectable viral load

Mechanisms of resistance

NRTIs are prodrugs that only become effective after being intracellularly converted to triphosphates. Nucleotide analogs require only two instead of three phosphorylation steps. Phosphorylated NRTIs compete with naturally occurring dNTPs (deoxynucleotide triphosphates). The incorporation of a phosphorylated NRTI into the proviral DNA blocks elongation of the DNA resulting in interruption of the chain. There are two main biochemical mechanisms that lead to NRTI resistance (De Mendoza 2002):

Sterical inhibition is caused by mutations enabling reverse transcriptase to recognize structural differences between NRTIs and dNTPs. Incorporation of NRTIs is then prevented in favor of dNTPs, e.g. in the presence of mutations M184V, Q151M, L74V, or K65R (Naeger 2001, Clavel 2004).

Phosphorolysis via ATP (adenosine triphosphate) or pyrophosphate leads to the excision of the NRTIs already incorporated into the growing DNA chain. This is the case with M41L, D67N, K70R, L210W, T215Y and K219Q (Meyer 2000). Phosphorolysis leads to cross-resistance between NRTIs, the degree of which may differ between agents (AZT, d4T > abacavir > ddI > 3TC). Contrary to the excision mutations, K65R leads to a decreased excision of all NRTIs when compared to wild-type, resulting in a greater stability once incorporated. For K65R, the combined effect of its opposing mechanisms (decreased incorporation and decreased excision) results in a decreased susceptibility to NRTIs but an increased susceptibility to AZT (White 2005).

NNRTIs also inhibit the viral enzyme reverse transcriptase (RT). NNRTIs are small molecules that bind to the hydrophobic pocket close to the catalytic domain of the RT. Mutations at the NNRTI binding site reduce the affinity of the NNRTIs to the RT and thus lead to a loss of antiviral activity due to NNRTI treatment failure. Whereas a single mutation can confer resistance to first generation NNRTIs, resistance patterns are more complex for second generation NNRTIs (Vingerhoets 2008, Molina 2008).

PIs hinder the cleavage of viral precursor gag-pol-polyprotein by the HIV protease, thereby producing immature, non-infectious viral particles. PI resistance usually develops slowly, as several mutations must first accumulate. This is also referred to as the genetic barrier. For PIs, a distinction is made between major (or primary) and minor (or secondary) mutations.

Table 5. PI-specific resistance mutations.
Major mutations           
D30N, V32I, M46I/L, I47V/A, G48V/M/A/L/S/T/Q, I50V/L, F53L, I54V/A/M/L/T/S, L76V, V82A/C/F/L/M/S/T, I84V/A/C, N88S, L90M
Minor mutations (a selection)
L10F/I/R/V/Y, V11I, L23I, L24I, D30 other than N, V32 other than I,  L33F/I, E35G, K43T, M46V, I47 other than V/A, I50 other than V/L, F53L/Y, Q58E, A71V/T/I/L, G73C/A/T/S, T74P, L76 other than V, N83D, I84other than V/A/C, N88D/G/T, L89V
(HIV Drug Resistance Database, Sequence Analyses Program, version 6.0.9, 2010-08-24; http://hivdb.stanford.edu/pages/documentPage/PI_mutationClassification.html)

Major mutations are responsible for phenotypic resistance. They are selected early in the process of resistance to a drug and/or are located within the active site of the target enzyme, the HIV protease. They reduce the ability of the protease inhibitor to bind to the enzyme. Major or primary mutations may also lead to reduced activity of the protease.

Minor mutations (often referred to as secondary mutations) are located outside the active site and usually occur after major mutations. Minor mutations are commonly found at polymorphic sites of non-B subtypes. Minor mutations compensate for the reduction in viral fitness caused by major mutations (Nijhuis 1999, Johnson 2007b). Mutations at positions 20, 36, 63, and 77 are polymorphisms which are observed without specific selective drug pressure particularly in non-B subtypes. Their contribution to resistance is minor and depends on the presence of other mutations.

Entry inhibitors prevent HIV from entering target cells. The first step in cell entry occurs when the HIV envelope glycoprotein gp120 binds to the CD4-receptor leading to conformational changes in gp120 and enabling the binding of the V3 loop of gp120 to the chemokine co-receptors, CCR5 or CXCR4, of the target cell. Interactions between the two heptad repeat regions HR1 and HR2 within the transmembrane glycoprotein subunit gp41 lead to a conformational change in gp41 and enable fusion of the viral and cellular membranes.

CCR5 antagonists bind to the CCR5 co-receptor and thereby impede interaction with the viral surface protein gp120 necessary for entry into the target cell.

The fusion inhibitor T-20, a synthetic peptide consisting of 36 amino acids, mimics the C-terminal HR2 domain of gp41 and competitively binds to HR1. Thus, interactions between HR1 and HR2 are blocked and the conformational change of gp41 that is necessary for fusion of virions to host cells is inhibited. A single amino acid substitution in HR1 can reduce the efficacy of T-20.

 

Integrase inhibitors prevent insertion of HIV DNA into the human DNA genome. The primary role of viral integrase is to catalyze the insertion of the viral cDNA into the genome of infected cells. Integrase inhibitors like raltegravir or elvitegravir block the strand transfer step. They bind to the catalytic pocket of the integrase and are transported as a component of the DNA/integrase pre-integration complex into the cell nucleus where strand transfer activity of integrase is inhibited. The selection of key mutations in the integrase gene confers resistance to integrase inhibitors. Strand transfer as well as the preceding step of 3’ processing (cleavage of the terminal dinucleotides from both 3’ ends of viral cDNA to which integrase binds) can be affected by these mutations. Different resistance pathways have been observed. The accumulation of additional mutations leads to a further decrease in susceptibility (Fransen 2008, Miller 2008).

Transmission of resistant HIV strains

The prevalence of mutations already present in treatment-naïve patients differs among demographic regions. Prevalence – of more than 20% – has been observed in large US cities with significant populations of homosexual men and a long history of access to antiretroviral treatment. Data on the incidence and prevalence of primary drug resistance which were published before 2007 should be interpreted with caution, since a consensus definition of transmitted genotypic drug resistance had not been established at that time. In 2007, an international research group agreed upon criteria defining mutations indicative for transmitted drug resistance. The corresponding list of mutations was again updated in 2009 (Bennett 2009). This standardization allows for comparisons of epidemiological data across geographic regions and periods of time.

In the German seroconverter study of the Robert Koch Institute the prevalence of resistance mutations was 12.4% between 1996 and 2007. Although the proportion of isolates with primary resistance remained stable during the observation period, the proportion of NRTI resistant virus populations decreased (to 7.5%), while there was a trend to more NNRTI resistance (3.5%) (Bartmeyer 2010). In chronically-infected patients of the RESINA study, the proportion with primary resistance was 14% between 2001 and 2007 (Oette 2008).

The European CATCH study, a substudy of SPREAD (Strategy to Control Spread of HIV Drug Resistance), determined a proportion of 10.4% with primary resistance out of 2,208 new HIV diagnoses between 1996 and 2002 (Wensing 2005). Whereas NRTI resistance decreased over time, NNRTI resistance increased. PI resistance remained relatively constant. Primary resistance was observed especially in subtype B infections (70% of all new diagnoses). However, primary resistance increased also in non-B subtypes.

European-wide data from the years 2006-2007 derive from SPREAD (Strategy to Control Spread of HIV Drug Resistance), a program established to monitor primary resistance in newly infected patients and ART-naïve patients. 9.7% of 1630 newly diagnosed HIV patients were infected with virus harboring at least one resistance mutation. The proportion of isolates with NRTI, NNRTI and PI resistance was 5.7%, 3.9% and 1.7%, respectively. Two-class resistance was present in less than one percent (Frentz 2011).

Ultrasensitive methods such as allele-specific real-time PCR (AS-PCR) or ultra-deep sequencing detect resistance mutations more often than conventional sequencing methods. In a Swiss study, M184V and/or K103N quasi-species were detected as minor variants in 18% (13/74) of patients with primary HIV infection and documented wild-type virus (Metzner 2007a). In a study from Atlanta focusing on L90M, M41L, K70R, K103N, Y181C, M184V, T215F and T215Y, resistance mutations were detected in 33/205 acutely or chronically infected patients (16%) (Johnson 2007a). In a British study investigating 165 anonymized samples from the years 2003-2006, drug resistance was detected in 13% of samples when using the standard assay compared to 19% when using an assay more sensitive for K103N, Y181C or M184V. In particular, the proportion of M184V isolates increased from 0.6% to 8%. The prevalence of drug resistance was almost the same for treatment-naive patients with either primary or chronic HIV infection (19% and 20%) confirming data showing that  primary resistance can persist for a long time (Buckton 2010, Pao 2004). In a Spanish study a (partial) reversion of transmitted drug resistance was observed in only 3 of 10 seroconverters after a median time of 41 months (De Mendoza 2005b). Contrary to K103N or M184V, K65R is a rare primary mutation. K65R was observed in only 4/194 patients (2%) as a minority variant at initiation of treatment (Metzner 2007b).

Transmitted resistance mutations can limit further treatment options and reduce treatment response rates (Little 2002, Wittkop 2010). This was also confirmed by a meta-analysis for minor NNRTI resistant virus variants (10 studies, 985 patients) (Li 2011). However, with special regard to existing resistance, treatment success is often possible (Oette 2006, Reuter 2008).

In early 2005, one patient from New York caused a sensation: he was infected with a multidrug resistant virus with a replication capacity comparable to that of wild-type virus. As a consequence, his remaining treatment options were very limited. Even though the transmission of multidrug resistant virus and rapid clinical progression are rare events, this case report demonstrates the possible clinical consequences of primary drug resistance (Markowitz 2005). In 2010 the transmission of a virus resistant to integrase inhibitors was reported for the first time. The virus also harboured NRTI, NNRTI and PI resistance mutations. Therefore the author recommended sequencing the integrase gene in case of transmitted multidrug resistance (Young 2010).

Table 6. Prevalence of resistance prior to initiation of therapy (a selection).
Author Region Period Patient population N Primary resistance
Bartmeyer 2010 Germany 1996-2007 Seroconverters 1298 12.4%
De Mendoza 2005 Spain 1997-2004 Seroconverters 198 12.1%
Recordon 2007 France 1996-2005 Seroconverters 194 15.7%*
Little 2002 USA 1995-2000 Seroconverters 377 22.7%
Chaix 2007 France 2005-2006 Seroconverters + chronically infected 289 10.4%
Frentz 2011 Europe 2006-2007 Newly diagnosed 1630 9.7%
Truong 2006 San Francisco 2004 Newly diagnosed 129 13.2%
Jayaraman 2006 Canada 1999-2003 Newly diagnosed 768 10.2%
Nkengafac 2007 Cameroon 2005-2006 Newly diagnosed 180 7.8%
Oette 2008 Germany 2001-2004 Chronically infected 1373 14%
Cane 2005 Great Britain 1996-2005 Chronically infected 2357 14.2%
 *(18.2% subtype B, 8.3% non-B)

Clinical studies

The clinical importance of resistance testing before making changes to therapy has been demonstrated in several prospective, controlled studies using genotypic tests such as VIRADAPT, CPCRA 046 or Havana (Durant 1999, Baxter 2000, Tural 2002) as well as in studies using phenotypic tests like VIRA 3001 (Cohen 2002). Patients whose physicians had access to information about existing mutations before the therapy was changed usually had more significant decreases in their viral loads than patients in whom ART was changed without knowledge of the resistance profile.

With regard to the increased number of NRTIs, NNRTIs or PIs with different resistance profiles, the clinical relevance of resistance testing might be even greater today. For ethical reasons no longer justifiable are studies that prospectively examine the benefits of resistance analysis, i.e. for regimens including new drug classes such as integrase inhibitors or CCR5 antagonists.

A resistance test before ART initiation is part of routine diagnosis in regions where the transmission of resistant HIV viruses is observed. The impact of transmitted HIV resistance on the initial success of ART was investigated in a retrospective analysis of the Eurocoord chain project. Of 10,458 patients initiated on ART in 1998, blood samples from the period before therapy were retrospectively examined for resistance. The initial regimens’ activities were essential for durable therapeutic success. Patients who were treated with only partially active regimens had a 2.6-fold higher risk of treatment failure (Wittkop 2010).

Resistance testing at time of treatment initiation and at time of virological failure is an integral part of national and international guidelines for the management and treatment of HIV infection.

Interpretation of genotypic resistance profiles

The algorithms cited in the following chapter are only indicative. Treatment decisions should not be made based on these data alone. We recommend the use of a resistance interpretation system listed in Table 3.

NRTIs

For several NRTIs such as 3TC and for NNRTIs a high degree of resistance develops following just a single mutation. For this reason, such drugs should only be used as part of highly effective regimens. However, the 3TC-specific mutation, M184V, also reduces viral replication capacity (often referred to as reduced viral fitness) by 40-60% (Miller 2003, Deval 2004). After 52 weeks with 3TC monotherapy, the viral load remained 0.5 log below the initial levels despite early development of the M184V mutation (Eron 1995). When compared to treatment interruptions, continuous monotherapy with 3TC delays virological and immunological deterioration (Castagna 2006). FTC has nearly the same genotypic and phenotypic resistance pattern as 3TC (Borroto-Esoda 2007).

M184I is often detected before M184V, but is then quickly replaced by M184V (Schuurmann 1995). Depending on the co-medication, M184V is more common on 3TC than on FTC, especially in combination with tenofovir (Svicher 2010). However, in the HEAT study which compared Tenofovir+FTC+lopinavir/r with Abacavir/3TC/lopinavir/r, M184V was more common on FTC (Smith 2008).

T69I is a rare mutation, which is observed in 0.5% of treated patients and 0.2% of ART-naive patients. This mutation causes high-level resistance to 3TC, FTC, and possibly also against tenofovir (Svicher 2010).

Thymidine analog mutations, known as TAMs, include the mutations M41L, D67N, K70R, L210W, T215Y and K219Q, which were first observed on treatment with AZT (Larder 1989), but were also selected on d4T (Loveday 1999).

There are two mutation pathways: the so-called TAM-1 path with 41L, 210W and 215Y, and the TAM-2 path with D67N, K70R, T215F, and K219Q/E (Flandre 2004). Depending on the individual TAMs and their combination, AZT resistance factors and the degree of resistance vary largely. The variation of the corresponding d4T resistance factor is much smaller. However, for full resistance the resistance factor is much lower for d4T than for AZT. This demonstrates that resistance factors of different drugs can not be compared. On AZT- and d4T-based regimens the TAM-1 pathway is more commonly observed (Cozzi-Lepri 2009).

The term NAMs (nucleoside analog mutations) is also used, as these mutations are associated with cross-resistance to all other nucleoside analogs, with the exception of 3TC and FTC (Harrigan 2000). In particular, the combination of certain TAMs can largely affect the effectiveness of abacavir, ddI and tenofovir (Table 7). The presence of L210W reduces the virological response to tenofovir (Antinou 2003). As with abacavir and ddI, TAMs do not arise on tenofovir, but can be re-selected.

The V75T mutation, which is associated with an approximately 5-fold increase in resistance to d4T and ddI, is only rarely observed (Lacey 1994).

Under failing therapy with abacavir or ddI the mutation L74V/I usually occurs. More rarely, the mutation K65R can occur. Y115F is a specific abacavir-associated resistance mutation.

Tenofovir primarily selects for the K65R mutation and leads to an (intermediate) resistance to tenofovir, abacavir, ddI, 3TC, FTC, and possibly d4T (Shafer 2002, Garcia-Lerma 2003). Although K65R may emerge on abacavir, K65R was rarely seen before the introduction of tenofovir. The reason is that with combination therapies containing AZT, the incidence of the K65R mutation is lower. Prior to tenofovir, abacavir was mainly used as part of the combination AZT+3TC+abacavir (Trizivir®).

K65R seldom emerges in the presence of TAMs. Since K65R and TAMs represent two antagonistic resistance pathways (see Mechanisms of resistance), K65R is only rarely observed on the same genome together with TAMs, and almost never together with L74V (Wirden 2005). Corresponding to observations made in large clinical trials using tenofovir within divergent (PI- or NNRTI-containing) treatment regimens, the incidence of K65R stabilized at ≤5%. However, virological failure of other triple-nuke combinations such as tenofovir+3TC+abacavir or tenofovir+3TC+ddI was often associated with the development of the K65R (Gallant 2003, Landman 2003, Jemsek 2004). The main reason for the high failure rate seems to be the low genetic barrier of these regimens: the emergence of the K65R induces a loss of sensitivity to all three drugs.

K65R increases sensitivity to AZT and induces a resensitization to AZT in the presence of (few) TAMs (White 2005, Underwood 2005). Vice versa, TAMs reduce the K65R-associated resistance to tenofovir, abacavir, and ddI (Parikh 2007).

As with M184V, the mutation K65R leads to a reduction in the viral replication capacity (RC), which is not the case with TAMs or the L74V/I. The median RCs for viruses with M184V/I (n=792), K65R (n=72) or L74V/I (n=15) alone were 68% (p<0.0001), 72% (p<0.0001) and 88% (p=0.16), respectively (McColl 2005). If both mutations K65R and M184V were present, an RC of only 29% was observed (Miller 2003, Deval 2004).

Less frequently than K65R, the mutation K70E was observed on failing therapy with tenofovir, particularly in NRTI-based regimens with abacavir and 3TC (Delaugerre 2008). M70E and K65R may be observed simultaneously, but it is unlikely that these mutations emerge on the same genome (Lloyd 2005). There is one case report of the development of K70E and M184V during therapy with tenofovir and FTC, which were then replaced by K70G and M184V. Both mutations were located on the same genome and conferred phenotypic resistance to 3TC, FTC, abacavir, ddI, and tenofovir, but not to AZT or d4T (Bradshaw 2007).

The 3TC-associated mutation, M184V, as well as the L74V mutation and the NNRTI-specific mutations, L100I and Y181C, may have an antagonistic effect on the further development of resistance (Vandamme 1999).

M184V induces resensitization to AZT, resulting in a 50-60% reduction of IC50. Resensitization to d4T results in a 30% reduction of IC50. However, resensitization is of clinical relevance only if there are no more than three other AZT- or d4T-associated mutations present (Shafer 1995, Underwood 2005). In one genotypic and phenotypic resistance study consisting of 9,000 samples, a combination of M41L, L210W and T215Y decreased the susceptibility to AZT by more than 10-fold in 79% of cases. If the M184V mutation was also present, only 52% had a more than 10-fold decreased susceptibility to AZT (Larder 1999). The M184V mutation also increases sensitivity to tenofovir (Miller 2001, Miller 2004a). In contrast, the presence of M184V plus multiple NAMs or mutations at positions 65, 74 or 115 increases resistance to abacavir (Harrigan 2000, Shafer 2003).

So-called multidrug resistance (MDR) to all nucleoside analogs – except 3TC and probably FTC – is established if one of the following combinations occurs: T69SSX, i.e., the T69S mutation plus an insertion of 2 amino acids (SS, SG or SA) between positions 69 and 70, plus an AZT-associated mutation or Q151M, plus another MDR mutation (V75I, F77L or F116Y) (Masquelier 2001).

The MDR mutation Q151M is relatively uncommon, with a prevalence of less than 5%. Q151M alone leads to intermediate resistance to AZT, d4T, ddI and abacavir and involves only a minor loss of tenofovir activity. Q151M combined with mutations at positions 75, 77, and 116 confers high-grade resistance to AZT, ddI, d4T and abacavir and intermediate resistance to tenofovir (Shafer 2003). Instead, the T69SSX insertion induces an approximately 20-fold increase in the resistance to tenofovir (Miller 2001, Miller 2004a).

The insertion T69SSX together with the mutation M184V, as well as the mutation Q151M together with M184V, leads to a 70% reduction in viral replication capacity (Miller 2003, Deval 2004).

The L74V mutation emerges after exposure to ddI or abacavir and leads to a 2-5 fold increase in the resistance to ddI (Winters 1997). L74V/I with or without M184V leads to a reduction in IC50 of about 70%; phenotypic susceptibility increases by a factor of 3 (Underwood 2005).

In large patient cohorts, quantitative measurements of sensitivity have shown that up to 29% of NRTI-experienced patients have a hypersusceptibility to NNRTIs (i.e., a reduction in the inhibitory concentration by a factor of 0.3-0.6). A reduction in AZT or 3TC sensitivity correlates inversely with an increased NNRTI susceptibility (Shulman 2000). The reverse transcriptase mutations T215Y, H208Y and V118I seem predictive for EFV hypersusceptibility. A database analysis of pair-wise genotypes and phenotypes showed NNRTI hypersusceptibility for TAMs and for non-thymidine analog-associated NAMs. Hypersusceptibility for efavirenz was detected for 1-2 TAMs, multiple TAMs plus M184V and for non-thymidine analog-associated NAMs like K65R, T69X, M184V and in particular for K65R+M184V (Whitcomb 2000, Shulman 2004, Coakley 2005a). However, these results have not influenced treatment strategies so far.

NNRTIs

First generation NNRTIs (efavirenz, nevirapine)

NNRTI resistance mutations may occur individually or in combination. A single mutation can confer high-level resistance to one or more NNRTIs.

The relatively frequent K103N mutation leads to a 20- to 50-fold increase in resistance to efavirenz and nevirapine (Petropolus 2000). V106M is more frequent in subtype C viruses and leads to a 30-fold increase in nevirapine resistance. V106M is associated with high-level resistance not only to nevirapine but also to efavirenz (Grossman 2004). Y181C/I causes a 30-fold increase in nevirapine resistance, and response to efavirenz is only temporary. G190A is associated with a high degree of nevirapine resistance and an intermediate resistance to efavirenz. G190S and Y188C/L/H are mutations that result in a high degree of resistance against both drugs (Shafer 2003, De Mendoza 2002).

A98G/S alone (more common in subtype C) or V108I alone are usually not clinically relevant. Mutations like K101E or L101P can confer intermediate resistance to first generation NNRTIs. V106A confers a more than 30-fold resistance to nevirapine. In contrast to subtype B virus, V106M more frequently emerges in subtype C virus; V106M confers resistance against both drugs (Grossman 2004).

Continued use of first generation NNRTIs is not recommended in the presence of respective mutations, because further mutations may be selected, which may influence the effectiveness of second generation NNRTIs.

Second generation NNRTIs

Etravirine is effective against variants with single NNRTI mutations like K103N, Y188L and/or G190A (Andries 2004). Compared to earlier NNRTIs, etravirine has a higher genetic barrier, probably due to flexible binding to the reverse transcriptase site. High-level resistance is usually seen with more than two mutations (Mills 2007, Katlama 2007, Vingerhoets 2007). In a selection experiment, the dominant viral population harbored, after several in vitro passages, the mutations V179F (a new variant at this position) and Y181C. Other mutations that have been selected in vitro are L100I, E138K, Y188H, G190E, M230L, and V179I (Brilliant 2004, Vingerhoets 2005). The frequently occurring mutation K103N does not affect the effectiveness of etravirine (Vingerhoets 2006).

In the DUET studies, the following resistance mutations were identified: V90I, A98G, L100I, K101E/H/P, V106I, E138A, V179D/F/T, Y181C/I/V, G190A/S and M230L. Based on these mutations, an etravirine resistance score weighting the individual NNRTI mutations was developed. The main criteria were the impact of the baseline mutations on virological response at 24 weeks and the correlation between respective mutations and the fold-change in IC50. A weighting factor of 3 was attributed to Y181I/V (with fold-changes of 13 and 17 in site-directed mutants), followed by a weighting factor of 2.5 for L100I, K101P, Y181C, and M230L. The mutations E138A, V106I, G190S, and V179F received a weighting factor of 1.5 and the other mutations were weighted with 1. Total scores of 0-2, 2.5-3.5 and ≥4 corresponded to 74%, 52% and 38% virological response rates in the DUET studies (Vingerhoets 2008).

In a panel of 4248 NNRTI-resistant clinical HIV-1 isolates, the mutations with the highest weight, Y181I and Y181V, had a low prevalence of 1.5% and 0.9%, respectively. The mutation Y181C, which is selected more frequently in patients taking nevirapine than with efavirenz, had a prevalence of 32% (Vingerhoets 2008).

Monogram has developed a weighted score including 37 mutations. Mutations with the highest level of resistance, i.e. L100I, K101P and Y181C/I/V, received a score of 4. E138A/G, V179E, G190Q, M230L and K238N received a score of 3; 101E, V106A / I, E138K, V179L, Y188L and G190S received a score of 2. V90I, A98G, K101H, K103R, V106M, E138Q, V179D/F/I/M/T, Y181F, V189I, G190A/E/T, H221Y, P225H, and K238T contributed with a score of 1. A loss of efficacy is likely with a total score of 4 or higher (Haddad 2010).

The effectiveness of rilpivirine does not seem to be impaired by single NNRTI resistance mutations such as K103N, V106A, G190S/A; In vitro no resistant variants were selected in the presence of 40 nM rilpivirine over a period of 30 days. When using 10 nM, up to eight mutations were selected within 8 days, including L100L/I, V106V/I, Y181Y/C and M230M/I; the respective increased IC50 was 4.

In a clinical study involving treatment-naïve patients without any (known) NNRTI mutations at baseline, eight emerging mutations were observed during treatment with rilpivirine: L100I, K101E, K103N, E108I, E138K/R, Y181C and M230L (Molina 2008).

In two phase III studies in which rilpivirine was tested against efavirenz, virological failure was more frequent on rilpivirine (10.5% versus 5.7%). Furthermore, the development of resistance mutations was more common in patients failing on rilpivirine (63% versus 54%). The most common mutations were E138K (45%), K101E (13%), H221Y (10%), V189I (8%), Y181C (8%) and V90I (8%). In 46%, 31% and 23% of resistant isolates respectively, 1, 2 or 3 NNRTI mutations were detected. Cross-resistance to etravirine was commonly observed among patients failing rilpivirine (>90%), cross-resistance to efavirenz was rather unlikely.

Besides NNRTI mutations, NRTI mutations were also more frequent among treatment failures on rilpivirine (68% versus 32%) – with primarily M184I on rilpivirine and M184V on efavirenz (Eron 2010).

Protease Inhibitors

The spectrum of PI mutations is very large. Although there is a moderate to high degree of cross-resistance between PIs, the primary mutations are relatively specific for the individual drugs. If treatment is changed early on to another PI combination, i.e., before the accumulation of multiple mutations, the subsequent regimen may still be successful. Most data on primary mutations that are selected for early on in the presence of a PI are derived from studies using unboosted PIs. In first-line therapy with boosted lopinavir, fosamprenavir, saquinavir, atazanavir or darunavir, the emergence of major PI mutations is rare (Eron 2006, Walmsley 2007, Clumeck 2007, Gathe 2008, Lataillade 2008, Molina 2008). The development of primary PI resistance in patients failing boosted PI therapy has been observed in few cases (Lanier 2003, Conradie 2004, Friend 2004, Coakley 2005b, Lataillade 2008).

First-generation PIs

Nelfinavir has a specific resistance profile, with the D30N primary mutation and further secondary mutations, resulting in a relatively low degree of cross-resistance to other PIs (Larder 1999). Virological failure with nelfinavir can also be associated with the emergence of L90M (Craig 1999). In subtype B viruses, treatment with nelfinavir generally leads to the emergence of D30N or M46I plus N88S. In subtype C, G and AE viruses, however, the mutations L90M and I84V occur more frequently. A reason for these different resistance pathways is the prevalence of natural polymorphisms: whereas M36I is present in only 30% of subtype B viruses, it is present in 70–100% of non-B subtypes (Gonzales 2004, Grossman 2004b, Sugiura 2002, Snoeck 2006).

Unboosted saquinavir primarily selects for G48V which leads to a 10-fold decrease in the susceptibility to saquinavir. G48V in combination with L90M reduces susceptibility to an even higher degree (Jakobson 1995). In general, several mutations including I84V/A are required to affect efficacy of ritonavir-boosted saquinavir (Valer 2002). One study re-evaluated the genotypic interpretation of saquinavir resistance in a retrospective analysis of 138 PI-experienced patients. Here, the presence of 3 to 4 mutations out of L10F/I/M/R/V, I15A/V, K20I/M/R/T, L24I, I62V, G73ST, 82A/F/S/T, I84V, and L90M was identified as being most strongly associated with reduced virological response (Marcelin 2007a). In contrast, L76V can lead to a clinically relevant re-sensitization for saquinavir (Braun 2007).

Fosamprenavir/r: In patients with virological failure while on amprenavir, the following mutations have been selected: I54L/M, I50V or V32I plus I47V, often together with the mutation M46I (Maguire 2002). The Zephir study evaluated virological response to treatment with fosamprenavir/r in 121 patients. With less than three mutations of L10I/F/R/V, L33F, M36I, M46I/L, I54L/M/T/V, I62V, L63P, A71I/L/V/T, G73A/C/F/T, V82A/F/S/T, I84V and L90M, viral load was reduced by 2.4 logs 12 weeks after treatment initiation compared to only -0.1 log with 4 or more mutations. At least 80% of patients with a maximum of 3 mutations reached a viral load below 400 copies/ml, compared to 35-45% of patients with 4-7 mutations and only 10% of patients with at least 8 mutations (Pellegrin 2005). In a retrospective study in 73 patients receiving fosamprenavir/r, the mutations L10F/I/V, L33F, M36I, I54L/M/V/A/T/S, I62V, V82A/F/C/G, I84V and L90M were associated with reduced virological response. In a univariate analysis the most striking mutations were I54L/M/V/A/T/S, V82A/F/C/G, and L90M: in the case of two mutations, virological response was reduced, while three mutations conferred resistance. N88S/D was associated with an increased response (Masquelier 2006). L76V may arise on fosamprenavir or on lopinavir (Muller 2004).

As with other boosted protease inhibitors major PI mutations occur very rarely on lopinavir-based first-line therapy. Few case reports of primary lopinavir resistance have been published. In one patient, virological failure was associated with the occurrence of the V82A followed by the mutations V32I, M46M/I and I47A (Friend 2004).

Lopinavir/r: The response in patients who had been exposed to first generation PIs correlated with the number of any of the following mutations: L10F/I/R/V, K20M/R, L24I, M46I/L, F53L, I54L/T/V, L63P, A71I/L/T/V, V82A/F/T, I84V, and L90M. Five mutations or less resulted in an increase in the IC50 by a median factor of 2.7; with 6-7 mutations this factor was 13.5, and with at least 8 mutations it was 44 (Kempf 2001).

A different algorithm to predict lopinavir resistance also includes mutations at novel amino acid positions. Viruses with any 7 mutations out of L10F/I, K20I/M, M46I, L, I50V, I54A/M/S/T/V, L63T, V82A/F/S as well as G16E, V32I, L33F, E34Q, K43T, I47V, G48M/V, Q58E, G73T, T74S, and L89I/M display approximately a 10-fold increase in the IC50. Mutations at positions 50, 54 and 82 particularly affect the phenotypic resistance (Parkin 2003, Jimenez 2005).

In vivo selection of lopinavir resistance was described in 54 PI-experienced patients failing treatment with lopinavir. Mutations at positions 82, 54 and 46 frequently emerged. Mutations such as L33F, I50V or V32I together with I47V/I were selected less frequently. New mutations at positions 84, 90 and 71 were not observed (Mo 2005).

The mutation I47A, which has rarely been observed since the availability of lopinavir, was identified to be associated with lopinavir resistance. I47A reduces the binding affinity to lopinavir and results in an 86- to >110-fold loss of sensitivity. In contrast, I47A leads to saquinavir hypersusceptibility due to an enhanced binding affinity to saquinavir (Kagan 2005).

On failing lopinavir monotherapy, the occurrence of the mutation L76V was reported in subtype CRF02 (Delaugerre 2007). The mutation L76V, selected for by lopinavir and rarely by amprenavir, is associated with resistance to lopinavir, amprenavir und darunavir, but can lead to resensitization to atazanavir, saquinavir and tipranavir – even in the presence of 5-10 PI mutations which normally confer broad PI cross-resistance (Müller 2004, De Meyer 2006b, Braun 2007).

Atazanavir is an aza-peptidomimetic PI. The resistance profile differs in part from that of other PIs. In patients in whom first-line treatment with atazanavir failed, the mutation I50L – often combined with A71V, K45R, and/or G73S – was primarily observed. On the one hand, I50L leads to a loss of sensitivity to atazanavir; on the other hand, I50L leads to an increased susceptibility to other PIs. Mutants harboring I50L plus A71V showed a 2- to 9-fold increased binding affinity to the HIV protease. Even in the presence of other major and minor PI-mutations I50L can increase susceptibility to other PIs (Colonno 2002, Weinheimer 2005, Yanchunas 2005). In PI-experienced patients, the I50L mutation was selected for in only one third of patients failing atazanavir (Colonno 2004).

The accumulation of PI mutations such as L10I/V/F, K20R/M/I, L24I, L33I/F/V, M36I/L/V, M46I/L, M48V, I54V/L, L63P, A71V/T/I, G73C/S/T/A, V82A/F/S/T, L90M, and in particular, I84V, leads to a loss of sensitivity to atazanavir. In the expanded access program using unboosted atazanavir the number of the respective PI mutations correlated with the change in viral load. For unboosted atazanavir, the threshold for resistance is generally met if 3-4 PI mutations are present; for ritonavir-boosted atazanavir, the genetic barrier is higher (Colonno 2004, Gianotti 2005).

The Reyaphar Score, developed by Pellegrin and colleagues for predicting response to ritonavir-boosted atazanavir, includes mutations at 12 positions (L10I/F/R/V, K20I/M/R, L241, M461/L, 154L/M/T/V, Q58E, L63P, A71I/L/V/T, G73A/C/F/T, V771, V82A/F/S/T, 184V and L90M). With less than 5 Reyaphar mutations, the average viral load reduction at 12 weeks was 1.4 logs, compared to only 0.5 log with more than 5 mutations (Pellegrin 2006).

Second-generation PIs

Tipranavir, the first non-peptidic protease inhibitor, shows good efficacy against viruses with multiple PI mutations. Even with reduced susceptibility to darunavir about half of the 586 isolates proved susceptible to tipranavir (De Meyer 2006a). In vitro, L33F and I84V are the first mutations selected by tipranavir, but the loss in sensitivity is only two-fold. Selection experiments ended up with viral isolates harbouring 10 mutations (L10F, I13V, V32I, L33F, M36I, K45I, I54V, A71V, V82L, I84V) resulting in an 87-fold reduced sensitivity (Doyon 2005).

Due to these and other experiments some mutations were regarded as key mutations, the so-called PRAMs (protease inhibitor-associated resistance mutations) which include the following mutations: L33I/V/F, V82A/F/L/T, I84V and L90M. Resistance analyses showed that reduced sensitivity should be expected with at least three PRAMs. However, a sufficient short term viral load reduction of 1.2 logs was seen after two weeks on treatment with boosted tipranavir plus an optimized backbone in patients with at least three PRAMs, compared to only 0.2–0.4 log with amprenavir, saquinavir or lopinavir plus optimized backbone (Cooper 2003, Johnson 2008, Mayers 2004).

In the re-analysis of the Phase II and III trials, some PRAMs have been confirmed, but new resistance mutations were also identified (Kohlbrenner 2004). Resistance mutations in clinical isolates of tipranavir-experienced patients included L10F, I13V, K20M/R/V, L33F, E35G, M36I, K43T, M46L, I47V, I54A/M/V, Q58E, H69K, T74P, V82L/T, N83D, and I84V (Croom 2005).

Hence the “unweighted” tipranavir mutation score was developed, involving 21 protease mutations at 16 positions (I10V, I13V, K20M/R/V, L33F, E35G, M36I, N43T, M46L, I47V, I54A/M/V, Q58E, H69K , T74P, V82L / T, N83D and I84V) (Baxter 2006).

This score was followed by a “weighted” tipranavir score based on clinical data of the RESIST trials (Scherer 2007). The respective model includes mutations of the unweighted score plus five mutations which were related to an increased tipranavir susceptibility. Weight factors were assigned to the mutations according to their contribution to resistance. The weights of the mutations add up to the weighted tipranavir score. The major mutations I47V, I54A/M/V, Q58E, T74P, V82L/T, and N83D contribute significantly to resistance and have a greater weight than minor mutations like I10V, M36I, N43T, and M46L. L24I, I50L/V, I54L and L76V are mutations conferring an increase in sensitivity and carry a negative weight. The mutations L33F as well as I13V and H69K, the most commonly observed in non-B subtypes were removed from this score. Other national resistance algorithms differ particularly in the weighting of mutations (Table 9).

Darunavir has also shown good activity against a wide spectrum of PI-resistant viruses. In vitro, resistance to darunavir develops more slowly than seen with nelfinavir, amprenavir or lopinavir.

After several passages in vitro, further mutations were selected in addition to R41T and K70E, leading to a reduced replication fitness. A mutant virus with a more than 10-fold loss in darunavir susceptibility showed a corresponding loss in saquinavir susceptibility, but not for the other PIs (atazanavir was not tested). This means that primary darunavir failure is not necessarily associated with complete cross-resistance to first generation PIs (De Meyer 2003+2005).

Eleven mutations at 10 positions were associated with a diminished response to boosted darunavir: V11I, V32I, L33F, I47V, I50V/L, I54L/M, T74P, L76V, I84V and L89V. With three or more of these mutations, the response rate was reduced.

Individual mutations appear to influence susceptibility to darunavir in varying degrees. I50V has the highest impact, followed by I54M, L76V and I84V; V32I, and L33F, and I47V have less influence. The weakest impact was associated with V11I, I54L, G73S and L89V. This weighting needs to be validated.

New mutations that have occurred on treatment failure with darunavir are V32I, L33F, I47V, I54L, and L89V. The median increase in darunavir IC50 was 8.14. Approximately 50% of these isolates were sensitive to tipranavir. The median change in the tipranavir IC50 was 0.82. Conversely, over 50% of isolates with reduced tipranavir susceptibility were still sensitive to darunavir (De Meyer 2006a, De Meyer 2006b, Prezista US Product Information 2006, Johnson 2008). Based on an analysis of the POWER and DUET data, the mutation V82A is positively associated with response to DRV (De Meyer 2009).

A database analysis of 50,000 paired geno- and phenotypes showed that for darunavir-resistant samples (n=2141) between 2006 and 2009 the median for darunavir resistance factor increased from 38 to 50, whereas the tipranavir resistance factor decreased from 7.6 to 4.3. During this period, an increase of darunavir-associated mutations was observed, probably due to the increased use of the substance: I50V rose from 11 to 15%, I54L from 17 to 33% and L76V from 5 to 9%, respectively. The three mutations E35N, I47A and V82L were associated with resistance to both drugs. L10F, V82F and G48M were associated with darunavir resistance; I54S, I84V and I84C were associated with tipranavir resistance. Due to these at least partially different mutation patterns the sequencing of both drugs may be feasible in specific situations (Stawiski 2010).

Fusion inhibitors

The gp41 genome consisting of 351 codons has positions of high variability and well-conserved regions. Polymorphic sites are observed in all regions of gp41. The heptad repeat 2 (HR2) region has the highest variability. Primary resistance to T-20, the only fusion inhibitor thus far approved, is a rare phenomenon (Wiese 2005).

A loss of efficacy is generally accompanied by the appearance of mutations at the T-20 binding site which is the heptad repeat 1 (HR1) region of gp41. Especially affected are the HR1-positions 36 to 45, such as G36D/E/S, 38A/M/E, Q40H/K/P/R/T, N42T/D/S, N43D/K, or L45M/L.

The fold change in IC50, which ranges from £10 to several hundred, depends on the position of the mutation and the substitution of the amino acid. The decrease in susceptibility is greater for double mutations than for a single mutation. For double mutations like G36S+L44M, N42T+N43K, N42T+N43S or Q40H+L45M, a fold-change of >250 has been observed. Additional mutations in HR2 also contribute to T-20 resistance (Sista 2004, Mink 2005). In clinical isolates harbouring G36D as a single mutation, a 4- to 450-fold decrease in susceptibility was found. In the isolate showing a 450-fold decrease in susceptibility a heterozygote change at position 126 in HR2 was observed (N/K). Other mutations in the gp41 gene were found at positions 72, 90 and 113 (Sista 2004, Monachetti 2004, Loutfy 2004).

In one small study, 6 of 17 patients with virological failure additionally developed the mutation S138A in the HR2 region of gp41 – mostly combined with a mutation at position 43 in the HR1 region and a range of HR2 sequence changes at polymorphic sites (Xu 2004).

The replication capacity (RC) in the presence of HR1 mutations is markedly reduced when compared to wild-type virus with a relative order of RC wild-type > N42T > V38A > N42T, N43K » N42T, N43S > V38A, N42D » V38A, N42T. Viral fitness and T-20 susceptibility are inversely correlated (r=0.99, p<0.001) (Lu 2004).

CCR5 Antagonists

CCR5 antagonists are to be used in patients with exclusively R5-tropic virus. In the presence of X4-or dual-tropic virus, their use is not recommended. In about 80% of treatment-naïve patients and 50-60% of treatment-experienced patients R5-tropic virus is detected. The detection of solely X4-tropic virus is unlikely but possible (Demarest 2004, Brumme 2005, Moyle 2005, Wilkinson 2006, Hunt 2006, Coakley 2006, Melby 2006). The probability of X4-tropic virus populations is higher with reduced absolute and relative CD4 cell counts, both in treatment-naive and treatment-experienced patients (Brumme 2005, Hunt 2006). For treatment-naïve patients with a CD4 cell count of less than 200/μl, in only 62% of cases an R5-tropic virus population was detected (Simon 2010).

There are two ways to build up resistance to CCR5 antagonists: a receptor switch from R5- to X4-tropic or dual-tropic viruses or the emergence of mutations that enable the virus to use the CCR5 molecules for entry into in the cell in the presence of CCR5 antagonists.

In approximately one third of patients on a failing regimen with maraviroc, a shift from R5- to X4-tropic virus was reported. In individual cases, a receptor-shift was observed in the control arm as well. Retrospective studies using more sensitive methods have shown that some patients harbored minor X4 variants already at baseline (Heera 2008, Greaves 2006, Mori 2007, Lewis 2007).

On failing treatment with maraviroc or vicriviroc (no longer being investigated) different mutations in the V3 loop of the HIV-1 envelope protein gp120 are detected. Respective resistance patterns were not uniform and included mutations outside the V3 loop. The frequency and clinical relevance of these env mutations is still part of clinical research and resistance analysis is not yet routine. Some of the detected mutations were not associated with an increase in IC50. Instead, phenotypic resistance was characterized by dose-response curves that display a reduction in the maximal inhibition (Mori 2008, McNicholas 2009). Reduced maximal inhibition in phenotypic susceptibility assays indicates that viral strains resistant to the CCR5 antagonist maraviroc utilize inhibitor-bound receptors for entry (Landovitz 2006, Westby 2007, Johnson 2008). Cross-resistance between maraviroc and vicriviroc has been described after several in vitro passages, but cross-resistance to other CCR5 antagonists or complete class resistance, such as TAK-652, remains to be determined. R5-tropic virus with resistance to maraviroc may be suppressed by using monoclonal antibodies, such as PRO 140. In contrast to maraviroc or vicriviroc, PRO 140 binds extracellularly to the CCR5 co-receptor. Therefore, cross-resistance between PRO 140 and maraviroc or vicriviroc is unlikely (Jacobson 2009).

Integrase inhibitors

Sequence analysis of viruses from treatment-naïve patients showed that the integrase gene is very polymorphic, but most of the relevant positions for resistance, such as 148 and 155, are conserved (Hackett 2008). Resistance analysis is currently indicated only in the case of virologically failing therapy with an integrase inhibitor therapy.

The key mutation N155H was observed in two patients on primary therapy with raltegravir, tenofovir and 3TC, in one of the cases along with other integrase resistance mutations. In other patients only 3TC mutations were detected. No further raltegravir mutations were observed after week 48 (Markowitz 2007, Rockstroh 2011). In pre-treated patients three raltegravir resistance pathways have been observed. Key mutations are N155H, Q148K/R/H and less frequently Y143R/C. Mutations observed along with N155H were L74M, E92Q, T97A, V151I, G163R, G163K, or S230R. In the presence of Q148K/R/H the following mutations may occur: L74M, T97A, E138A, E138K, G140A, G140S and G163R, whereas mutations at positions 140 prevail.

The mutations N155H and Q148K/R/H do not occur on the same viral genome; this also applies for E92Q and mutations at position 148. Virus variants harboring N155H and secondary mutations are often replaced by variants with higher replicative fitness harboring Q148H + G140S (Fransen 2008, Miller 2008). In order to preserve the efficacy of second generation integrase inhibitors, raltegravir should be discontinued after the first key mutation has occurred.

The key mutation Y143H/R/C involves the mutations E92Q, T97A, V151I, G163R or S230R (Cooper 2007, Fransen 2008, Steigbigel 2008, Hazuda 2007).

The accumulation of additional mutations after the emergence of key mutations causes an increase in resistance and, depending on the pattern of mutations, also an increase in viral fitness. This is particularly true for the mutation Q148H (Goethals 2008, Hatano 2008). It is important to ensure that raltegravir is not used as functional monotherapy in patients with existing resistance mutations. The genetic barrier of raltegravir is therefore not as high as that of a boosted PIs, which, unlike raltegravir, can also be used as monotherapy in special cases (Gatell 2009).

Mutations occurring on failing therapy with elvitegravir were E92Q, E138K, Q148R/H/K and N155H. E92Q is often associated with the compensatory mutation L68V (Goodman 2008). A high level of cross-resistance between raltegravir and elvitegravir is caused by Q148H/R+G140S (McColl, 2007, DeJesus 2007). Response to raltegravir after failing elvitegravir is unlikely (Goodman 2008, Waters 2009). This has been confirmed by case reports (DeJesus 2007).

The integrase inhibitor dolutegravir (DTG, formerly S/GSK1349572), which is currently being investigated in phase II/III studies, shows promising results. Compared to raltegravir and elvitegravir this integrase inhibitor probably has a higher genetic barrier. Depending on the resistance pattern, little or no cross-resistance has been observed in vitro (Lalezari, 2009, Sato 2009).

In the Viking study, 27 patients with raltegravir-specific resistance mutations and a viral load >1,000 copies/ml were treated with dolutegravir 50 mg QD. At day 11, 21/27 patients had a viral load of <400 copies/ml or a viral load reduction of at least 0.7 log. Resistance mutations at positions 143 and 155 had no impact on the effectiveness, in contrast to mutations at position 148 in combination with two other secondary mutations. With a higher dose of dolutegravir (50 mg twice daily), the resistance can be overcome at least temporarily (Eron 2010 +2011).

Summary

Resistance and tropism tests belong to the standard diagnostic tools in the management of HIV infection. Primary resistant viral variants can be observed in about 10% of treatment-naïve patients in regions that have access to antiretroviral drugs. Resistance testing prior to initiating ART results in significantly better response rates. The emergence of viral mutants is one of the main causes of virological treatment failure. With the aid of HIV resistance tests, antiretroviral treatment strategies can be improved. Pharmacoeconomic studies have shown that genotypic resistance tests are cost-effective both in treatment-experienced and in ART-naïve patients (Weinstein 2001, Corzillius 2004, Sax 2005). HIV treatment guidelines recommend the use of resistance testing. New classes such as integrase inhibitors and CCR5 antagonists should be included in the evaluation of resistance. However, genotyping including sequence analyses of the integrase or envelope genomes is only partially covered by public health insurance in several countries.

Both, genotypic and phenotypic resistance tests as well as genotypic and phenotypic tropism tests show good intra- and inter-assay reliability. The interpretation of genotypic resistance profiles has become very complex and requires constant updating of respective guidelines. The determination of the thresholds associated with clinically relevant phenotypic drug resistance is crucial for the effective use of (virtual) phenotypic testing.

As for of resistance testing, genotyping has become the preferred method of tropism testing in clinical practice. With the co-receptor tool of geno2pheno viral tropism can be predicted.

Even while treatment failure requires the consideration of all causal factors, such as patient adherence to the regimen, metabolism of drugs and drug levels, resistance testing and measurement of viral tropism are of great importance in antiretroviral therapy. Finally, it needs to be emphasized that even with the benefit of well-interpreted resistance tests only experienced HIV practitioners should start, stop or change antiretroviral therapy keeping in mind the clinical and the psychosocial situation of the patient.

Resistance Tables

All tables are based on different rules-based interpretation systems, such as HIV-GRADE (http://www.hiv-grade.de), the ANRS-AC 11 Resistance Group (http://www.hivfrenchresistance.org/) and the Drug Resistance Mutations Group of the International AIDS Society-USA (Johnson 2010) as well as the references mentioned in the text.

These tables are not exhaustive and should not replace the communication between the practitioner and the laboratory experts in resistance interpretation.

Table 7. Mutations on the reverse transcriptase gene leading to NRTI resistance.
RTI Resistance mutations
Zidovudine (AZT) T215 Y/F (esp. with other TAMs)≥3 of the following: M41L, D67N, K70R, L210W, K219Q/EQ151M (esp. with A62V/F77L/F116Y) or T69SSX (insertion)*(Potential resensitizing effect associated with K65R, L74V, Y181C and M184V)
Stavudine (d4T) V75M/S/A/TT215Y/F (usually in combination with other TAMs)≥3 TAMs*Q151M (esp. with A62V/F77L/F116Y) or K65R or T69SSX (insertion)*

(Potential resensitizing effect associated with L74V, Y181C and M184V)

Abacavir M184V + 3 of the following: M41L, D67N, L74I, L210W, T215Y/F, 219Q/E≥5 of the following: M41L, D67N, L74I, L210W, T215Y/F, 219Q/EK65R or Y115F or L74VQ151M (esp. with A62V, F77L, F116Y) or T69SSX (insertion)*
Lamivudine (3TC) M184V/I or T69SSX (insertion)* or K65R (resistance possible)
Emtricitabine (FTC) M184V/I or T69SSX (insertion)* or K65R (resistance possible)
Didanosine (ddI) L74V, esp. with T69D/N or TAMsQ151M (esp. with A62V/F77L/F116Y) or T69SSX (insertion)*K65RT215Y/F and ≥2 of the following: M41L, D67N, K70R, L210W, K219Q/E
Tenofovir DF T69SSX (insertion)*≥3 TAMs with M41L or L210W (only partial resistance)≥3 – 5 of the following: M41L, E44D, D67N, T69D/N/S,  L210W, T215Y/F, K219Q/EK65R or K70E/G

(Potential resensitizing effect associated with L74V and M184V)

TAMs = thymidine analog mutations
* T69SSX in combination with T215Y/F and other TAMs leads to a high degree of resistance to all NRTIs and tenofovir
Table 8. Mutations on the reverse transcriptase-gene leading to NNRTI resistance (mutations associated with a high degree of resistance in bold).

NNRTIs

Resistance mutations

Efavirenz

L100l or K101E or K103N/H/S/T or V106M

V108I (with other NNRTI mutations)

Y181C(I) or Y188L/C/(H) or G190S/A (C/E/Q/T/V)

P225H (with other NNRTI mutations)

Nevirapine

A98G (esp. for HIV-1 subtype C) or L100l

K101E/P/Q or K103N/H/S/T or V106A/M or V108I

Y181C/I/V or Y188C/L/H or G190A/S (C/E/Q/T/V)

Etravirine

≥2*-3 mutations of (V90I, A98G, L100I, K101E/H/P, V106I, E138A/G/K/Q, V179D/F/T, Y181C/I/V, G190A/S, F227C, M230L)

*in combination with a bold mutation


Table 9. Mutations on the protease gene leading to PI resistance.

PIs

Relevant resistance mutations and patterns

Further mutations associated with resistance

Saquinavir

I84V/A, 48V/M

≥3 of the following: L10F/I/M/R/V, K20I/M/ R/T, L24I, I62V, G73CST, 82A/F/S/T and L90M or

≥4 of the following: L10I/R/V, I54V/L, A71V/T, V77I, V82A/ F/S/T and L90M

Possible L76V-associated resensitizing effect

≥2 PRAMs*

Nelfinavir

D30N

l84A/V

N88S/D

L90M

V82A/F/S/T and at least 2 of the following: L10I, M36I, M46l/L, I54V/L/M/T, A71V/T, V77I

≥2 PRAMs*

Fosamprenavir/r

I50V

L76V together with other PI mutations

V32I plus I47V

≥6 of the following: L10F/I/V, K20M/R, E35D, R41K, I54V/L/M, L63P, V82A/F/T/S, I84V or

≥3 of the following: L10I/F/R/ V, L33F, M36I, M46I/L, I54L/ M/T/V, I62V, L63P, A71I/L/V/T, G73A/C/F/T, V82A/F /S/T, I84V and L90M) or

≥3 of the following: L10F/I/V, L33F, M46I/L, I47V, I54L/M/V/A/T/S, A71V, G73S/A/C/T, V82A/F/C/G and L90M

≥2 PRAMs*

Lopinavir/r

I47A+V32I

≥3 of the following: M46I, I47A/V, L50V, I54A/M/V, L76V, V82FATS, I84V

5-7 of the following: L10F/I/R/V, K20M/R, L24l, V32I, L33F, M46l/L, I47V/A, I50V, F53L, l54L/T/V, L63P, A71l/L/V/T, G73S, V82A/F/T, l84V, L90M

≥2 PRAMs*

Atazanavir

and

Atazanavir/r (300/100 mg QD)

I50L – frequently in combination with A71V –

≥4 of the following: L10I/F, K20R/M/I, L24I, V32I, L33I/F/V, M46I, M48V, I54V/M/A, A71V, G73C/S/T/A, V82A/F/S/T, I84V, N88S and L90M

(Possible L76V-associated resensitizing effect)

N88S

≥2 PRAMs*

Tipranavir

≥7 mutations/points of the following: K20M/R/V, L33F, E35G, N43T, M46L, I47V, I54A/M/V, Q58E, H69K, T74P, V82L/T, N83D and I84V; V82L/T and I84V with twofold points score

Score >10 of the following: I10V (+1), L24I (-2), M36I (+2), N43T (+2), M46L (+1), I47V (+6), I50L/V (-4) I54A/M/V (+3), I54L (-7) Q58E (+5), T74P (+6), L76V (-2), V82L/T (+5), N83D (+4), I84V (+ 2)

Possible L76V-associated resensitizing effect

Further resistance-ass. mutations: I54S, I84C

6 mutations/points of the following: K20M/R/V, L33F, E35G, N43T, M46L, I47V, I54A/M/V, Q58E, H69K, T74P, V82L/T, N83D and I84V; V82L/T and I84V with twofold points score

Score 3-10 from I10V (+1), L24I (-2), M36I (+2), N43T (+2), M46L (+1), I47V (+6), I50L/V (-4) I54A/M/V (+3), I54L (-7) Q58E (+5), T74P (+6), L76V (-2),.V82L/T (+5), N83D (+4), I84V (+ 2)

Darunavir/r

≥4 of the following: V11I, V32I, L33F, I47V, I50V, I54L/M, T74P, L76V, I84V, L89V

(with V32I, I50V, I54M, L76V and I84V having a higher impact)

Further resistance-ass. mutations:

L10F, E35N, I47A, V82L, G48M, V82F

≥3 of the following: V11I, V32I, L33F, I47V, I50V, I54L/M, T74P, L76V, I84V, L89V (with I50V, I54M, L76V and I84V having a higher impact)

*PRAMs (protease inhibitor –resistance-associated mutations) include the following mutations: L33I/F/V, V82A/F/S/T, I84V and L90M. They lead to high PI cross-resistance.

Table 10. Mutations leading to entry inhibitor resistance.

Fusion inhibitor

Resistance mutations

T-20

G36A/D/E/S/V or I37V oder 38A/M/E/K/V or Q39R

Q40H/K/P/R/T or N42T/D/S or N42T+(N43S/N43K)

N43D/KH/S or L44M or L44M+ G36S or L45M/L/Q

CCR5 antagonists

 

Maraviroc

Individual mutations described as resistance-associated; no consistent pattern

The reduction in susceptibility is generally higher for double than for single mutations.

Table 11. Mutations on the integrase gene leading to raltegravir resistance (and probably cross resistance to elvitegravir)

Integrase inhibitors

Resistance mutations (Resistance pathways and key mutations)

Other mutation- and resistance-profiles conferring resistance

Raltegravir

Q148H/G/K/R/E

N155H

Y143H/R/C

The appearance of additional mutations produces an increase in the level of resistance.

E157Q

T66I and E92Q

 

References

Andries K, Azijn H, Thielemans T, et al. TMC125, a novel next-generation NNRTI active against nonnucleoside reverse transcriptase inhibitor-resistant HIV type 1. Antimicr Ag Chemoth 2004; 48: 4680-6.

Antoniou T, Park-Wyllie L, Tseng AL. Tenofovir: A nucleotide analog for the management of HIV infection. Pharmacotherapy 2003; 23:29-43.

Bacheler L, Winters B, Harrigan R, et al. Estimation of phenotypic clinical cut-offs for virco®Type HIV-1 through meta analyses of clinical trial and cohort data. Antiviral Therapy 2004; 9:S154. http://www.aegis.com/conferences/hivdrw/2004/Session_6.pdf

Bartmeyer B, Kuecherer C, Houareau C, et al. Prevalence of Transmitted Drug Resistance and Impact of Transmitted Resistance on Treatment Success in the German HIV-1 Seroconverter Cohort. PLoS ONE 5: e12718.

Baxter JD, Mayers DL, Wentworth DN, et al. A randomized study of antiretroviral management based on plasma genotypic antiretroviral resistance testing in patients failing therapy.  AIDS 2000; 14:F83-93.

Baxter JD, Schapiro JM, Boucher CA, et al. Genotypic changes in human immunodeficiency virus type 1 protease associated with reduced susceptibility and virologic response to the protease inhibitor tipranavir. J Virol 2006; 80: 10794-801.

Beerenwinkel N, Däumer M, Oette M, et al. Geno2pheno: estimating phenotypic drug resistance from HIV-1 genotypes. Nucleic Acids Research 2003; 13: 3850-3855.

Bennet DE, Camacho RJ, Ortelea D, Kuritzkes DR, Fleury H., et al. (2009) Drug resistance mutations for surveillance of transmitted HIV – 1 drug resistance : 2009 update. PLoS One: e4724.

Borroto-Esoda K, Parkin N, Miller MD. A comparison of the phenotypic susceptibility profiles of emtricitabine and lamivudine. Antivir Chem Chemother 2007;18:297-300.

Bradshaw D, Malik S, Booth C,,et al. Novel drug resistance pattern associated with the mutations K70G and M184V in human immunodeficiency virus type 1 reverse transcriptase. Antimicrob Agents Chemother 2007, 51:4489-4491.

Braun P, Hoffmann D, Wiesmann, et al. Clinically relevant resensitisation  of protease inhibitors saquinavir and atazanavir by L76V mutation in multidrug-resistant HIV-1 infected patients. Abstract 129, XVI IHDRW 2007, Barbados.

Braun P, Wolf E, Hower M, et al. Genotypic and phenotypic HIV Tropism testing predicts the outcome of Maraviroc regimens. Abstract 47, XVIII IHDRW 2009, Fort Myers/Antiviral Therapy Vol. 14, Suppl. 1, 2009 (p. 51).

Brillant J, Klumpp K, Swallow S, Cammack N, Heilek-Snyder G. In vitro resistance development for a second-generation NNRTI: TMC125. Antivir Ther 2004; 9:S20.

Brumme ZL, Gondrich J, Mayer HB, et al. Molecular and clinical epidemiology of CXCR4-using HIV-1 in a large population of antiretroviral-naive individuals. J Infect Dis 2005; 192:466-474.

Buckton AJ, et al. Increased detection of the HIV-1 reverse transcriptase M184V mutation using mutation-specific minority assays in a UK surveillance study suggests evidence of unrecognised transmitted drug resistance. HIV Medicine 2010, pub ahead of print

Cane P, Chrystie I, Dunn D, et al. Time trends in primary resistance to HIV drugs in the UK: multicentre observational study. BMJ 2005; 331: 1368.

Castagna A, Danise A, Menzo S, et al. Lamivudine monotherapy in HIV-1-infected patients harbouring a lamivudine-resistant virus: a randomized pilot study (E-184V study). AIDS 2006; 20: 795-803.

Chaix ML, Fichou J, Deveau C, et al. Stable frequency of HIV-1 transmitted drug resistance over a decade (1996–2006) in France is likely explained by the increase of chronically treated patients in virological success? Antiviral Therapy 2007; 12:S49 (Abstract 42).

Chapman TM, Plosker GL, Perry CM. Fosamprenavir: a review of its use in the management of antiretroviral therapy-naive patients with HIV infection. Drugs 2004; 64: 2101-24.

Cheng Y, Prusoff WH. Relationship between the inhibition constant (K1) and the concentration of inhibitor which causes 50 per cent inhibition (I50) of an enzymatic reaction. Biochem Pharmacol 1973, 22: 3099–108.

Clavel F, Hance AJ. HIV drug resistance. N Engl J Med 2004; 350: 1023-35.

Clumeck N, Van Lunzen J, Chiliade P, et al. ARTEMIS – Efficacy and safety of lopinavir (BID vs QD) and darunavir (QD) in antiretroviral-naive patients 11th EACS, Madrid, 2007, Abstract LBPS7/5.

Coakley E, Benhamida J, Chappey C, et al. An evaluation of tropism profiles and other characteristics among 3988 individuals screened from A4001026, A4001027 (MOTIVATE 1) and A4001028 (MOTIVATE 2) studies for maraviroc. Abstract 8, 2nd Int Worksh Targ HIV Entry 2006, Boston, MA.

Coakley E, Parkin N. Contribution of non-thymidine analog nucleoside RT inhibitor associated mutations to phenotypic hypersusceptibility to efavirenz. Abstract 704, 12th CROI 2005a, Boston, USA. Abstract: http://www.retroconference.org/2005/cd/Abstracts/25329.htm

Coakley E, Mass M, Parkin N. Atazanavir resistance in a protease inhibitor-naïve patient treated with atazanavir/ritonavir associated with development of high-level atazanavir resistance and the N88S mutation in protease. Abstract 716, 12th CROI 2005b, Boston, USA.

Cohen CJ, Hunt S, Sension M, et al. A randomized trial assessing the impact of phenotypic resistance testing on antiretroviral therapy. AIDS 2002; 16: 579-88.

Colonno R, Rose R, McLaren C, et al. Identification of I50L as the signature atazanavir-resistance mutation in treatment-naive HIV-1-infected patients receiving ATV-containing regimens. J Infect Dis 2004a; 189:1802-10.

Colonno RJ, McLaren C, Kelleher T. Pathways to Atazanavir resistance in treatment-experienced patients on Atazanavir containing regimens. Abstract/Poster 3.1, 2nd European HIV Drug Resistance Workshop 2004b, Rome, Italy.

Colonno RJ, Friborg J, Rose RE, et al. Identification of amino acid substitutions correlated with reduced atazanavir susceptibility in patients treated with atazanavir-containing regimens. Antiviral Ther 2002; 7:S4. Abstract 4.

Conradie F, Sanne I, Venter W, et al. Failure of lopinavir-ritonavir containing regimen in an antiretroviral-naive patient. AIDS 2004; 18:1041-1085.

Cooper D, Hall D, Jayaweera D, et al. Baseline phenotypic susceptibility to tipranavir/ritonavir is retained in isolates from patients with multiple protease inhibitor experience (BI 1182.52). Abstract 596, 10th CROI 2003, Boston, USA.

Cooper D et al. Results from BENCHMRK-1, a phase III study evaluating the efficacy and safety of MK-0518, a novel HIV-1 integrase inhibitor, in patients with triple-class resistant virus. Abstract 105aLB, 14th CROI 2007, Los Angeles, USA.

Corzillius M, Mühlberger N, Sroczynski G, et al. Cost effectiveness analysis of routine use of genotypic antiretroviral resistance testing after failure of antiretroviral treatment for HIV. Antivir Ther 2004; 9:27-36.

Cozzi-Lepri A et al. Rate of accumulation of thymidine analogue mutations in patients continuing tor receive viroloically failing regimens containing zidovudine or stavudine: implications for antiretrovirla therapy programs in resource limited settings. J Infectious Dis 2009; 200: 687-97

Craig C, Goddard C, Whittaker L, et al. HIV-1 genotype and phenotype during dual therapy (NV15436 sub-study). Abstract 103, 7th ECCATH 1999, Lisbon, Portugal.

Craig C, Lewis M, Simpson, et al.  Week 48 results from the phase III study A4001026 (MERIT) – ‘time to loss of virological response’ virology analysis of failures in the enhanced Trofile – censored populations. Abstract 46, XVIII IHDRW 2009, Fort Myers/Antiviral Therapy Vol. 14, Suppl. 1, 2009 (p. 50)

Croom KF, Keam SJ. Tipranavir: a ritonavir-boosted protease inhibitor. Drugs 2005; 65: 1669-77.

DAIG, Deutsche AIDS-Gesellschaft. Deutsch-Österreichische Leitlinien zur antiretroviralen Therapie der HIV-1-Infektion, Stand März 2010. http://www.daignet.de/site-content/hiv-therapie/leitlinien-1

da Silva D, Van Wesenbeeck L, Breilh D, et al. HIV-1 resistance patterns to integrase inhibitors in antiretroviral-experienced patients with virological failure on raltegravir-containing regimens. J Antimicrob Chemother. 2010; 65(6):1262-9.

DeJesus E, Cohen C, Elion R, et al. First report of raltegravir (RAL, MK-0518) use after virologic rebound on elvitegravir (EVT, GS 9137). Abstract TUPEB032, 4th IAS Conference 2007, Sydney, Australia.

Delaugerre C, Flandre P, Chaix ML, et al. Protease gene mutations in a trial comparing first-line lopinavir/ritonavir monotherapy to lopinavir/ritonavir + zidovudine/lamivudine (MONARK-TRIAL). Antiviral Therapy 2007; 12: S84 (Abstract 75).

Delaugerre C, Flandre P, Chaix ML, et al. Protease inhibitor resistance analysis in the MONARK trial comparing first-line lopinavir-ritonavir monotherapy to lopinavir-ritonavir plus zidovudine and lamivudine triple therapy. Antimicrob Agents Chemother. 2009; 53(7):2934-9.

Delaugerre C, Flandre P, Marcelin AG, et al. National survey of the prevalence and conditions of selection of HIV-1 reverse transcriptase K70E mutation. J Med Virol 2008, 80:762-765.

De Mendoza C, Gallego O, Soriano V. Mechanisms of resistance to antiviral drugs – clinical implications. AIDS Rev 2002; 4: 64-82.

De Mendoza C, Rodriguez C, Corral A, et al. Evidence for a different transmission efficiency of viruses with distinct drug-resistant genotypes. Abstract 130, XII Int HIV Drug Resistance Workshop 2003, Los Cabos, Mexico. http://www.mediscover.net/Journals_PDF/Session5.pdf

De Mendoza C, Rodriguez C, Colomina J, et al. Resistance to nonnucleoside reverse-transcriptase inhibitors and prevalence of HIV type 1 non-B subtypes are increasing among persons with recent infection in Spain. Clin Infect Dis 2005a; 41: 1350-4.

De Mendoza C, Rodriguez C, Corral A, et al. Long-term persistence of drug resistance mutations after HIV seroconversion. Abstract PE3.5/3. 10th European AIDS Conference (EACS) 2005b, Dublin, Ireland.

De Meyer S, Van Marck H, Veldemann J, et al. Antiviral activity of TMC114, a potent next-generation protease inhibitor, against >4000 recent recombinant clinical isolates exhibiting a wide range of (protease inhibitor) resistance profiles. Antiviral Therapy 2003; 8:S20

De Meyer S, Azijn H, Surleraux D, et al. TMC114, a novel HIV type 1 protease inhibitor active against protease inhibitor-resistant viruses, including a broad range of clinical isolates. Antimicrob Agents Chemother 2005; 49:2314-21.

De Meyer S, Cao-Van K, Lathouwers E, Vangeneugden T, de Bethune M. Phenotypic and genotypic profiling of TMC114, lopinavir and tipranavir against PI-resistant HIV-1 clinical isolates. Abstract 43, 4th European HIV Drug Resistance Workshop 2006a, Monte Carlo, Monaco.

De Meyer S, Dierynck I, Lathouwers E, et al. Identification of mutations predictive of a diminished response to darunavir/ritonavir: Analysis of data from treatment-experienced patients in POWER 1, 2, 3 and DUET-1 and 2. Abstract 54, 6th Eur HIV Drug Resistance Workshop 2008, Budapest, Hungary.

De Meyer S, Hill A, De Baere I, et al. Effect of baseline susceptibility and on-treatment mutations on TMC114 and control PI efficacy: preliminary analysis of data from PI-experienced patients from POWER 1 and POWER 2. Abstract 157, 13th CROI 2006b, Denver, Colorado, USA.

De Meyer S, Descamps D, Van Baelen B, et al. Confirmation of the negative impact of protease mutations I47V, I54M, T74P and I84V and the positve impact of protease mutation  V82A on virological response to darunavir/ritonavir. Abstract 126, XVIII IHDRW, 2009, Fort Myers.

Deval J, White KL, Miller MD, et al. Mechanistic basis for reduced viral and enzymatic fitness of HIV-1 reverse transcriptase containing both K65R and M184V mutations. J Biol Chem 2004;279:509-16.

Doyon L, Tremblay S, Bourgon L, Wardrop E, Cordingley MG. Selection and characterization of HIV-1 showing reduced susceptibility to the non-peptidic protease inhibitor tipranavir. Antiviral Res 2005; 68: 27-35.

Drake JW. Rates of spontaneous mutation among RNA viruses. PNAS 1993; 90:4171-4175.

Durant J, Clevenbergh P, Halfon P, et al. Drug-resistance genotyping in HIV-1 therapy: the VIRADAPT randomised controlled trial. Lancet 1999; 353:2195-99.

Eberle J, Goebel FD, Postel N, et al. Amino acid changes in the HIV-1/gp41 HR1 region associated with ongoing viral replication selected by T-20 (enfuvirtide) therapy. Abstract/Poster 43, 3rd European Conference on Viral Diseases 2004, Regensburg, Germany.

Eron J, Durant J, Poizot, et al. Activity of a next generation integrase inhibitor (INI), S/GSK 1349572, in subjects with HIV exhibiting raltegravir resistence : initial results of VIKING study (ING 112961). Abstract MOAB0105, 18th IAC 2010, Vienna,  Austria.

Eron JJ, Benoit SL, Jemsek J, et al. Treatment with lamivudine, zidovudine, or both in HIV-positive patients with 200 to 500 CD4+ cells per cubic millimeter. N Engl J Med 1995; 333:1662-1669.

Eron J Jr, Yeni P, Gathe J Jr, et al. The KLEAN study of fosamprenavir-ritonavir versus lopinavir-ritonavir, each in combination with abacavir-lamivudine, for initial treatment of HIV infection over 48 weeks: a randomised non-inferiority trial. Lancet 2006;368:476-482.

Eron  J, Kumar P, Lazzarin A, et al. DTG in subjects with HIV exhibiting RAL resistance: Functional monotherapy results of VIKING study cohort II. Abstract 151LB, 18th CROI, 2011, Boston, MA.

Flandre P, Parkin NT, Petropoulos C, et al. Competing occurrence and mutation pathways of nucleoside reverse transcriptase inhibitor associated mutations. Abstract 645, 11th CROI 2004, San Francisco, CA.

Fransen S, Gupta S, Danovich R, et al. Loss of raltegravir susceptibility in treated patients is conferred by multiple non-overlapping genetic pathways. Abstract 7, XVII International HIV Drug Resistance Workshop 2008; Sitges, Spain.

Frentz D. Recent dynmics of transmitted drug resistance in Europe: SPREAD Programme 2006-2007. Abstract O-13. 9th European Workshop on HIV & Hepatits Treatment Strategies  & Antiviral Drug Resistance, 2011. Paphos, Cyprus

Friend J, Parkin N, Liegler T, et al. Isolated lopinavir resistance after virological rebound of a ritonavir/lopinavir-based regimen. AIDS 2004; 18:1965-70.

Gallant JE, Rodriguez A, Weinberg W, et al. Early non-response to tenofovir DF + abacavir and lamivudine in a randomized trial compared to efavirenz (EFV) + ABC and 3TC: ESS30009 unplanned interim analysis. LB H-1722a, 43rd ICAAC 2003, Chicago, USA.

Garcia-Lerma JG, MacInnes H, Bennett D, et al. A novel genetic pathway of HIV type 1 resistance to stavudine mediated by the K65R mutation. J Virol. 2003; 77:5685-5693. http://intapp.medscape.com/px/medlineapp/getdoc?pmi=12719561&cid=med

Garcia-Gasco P, Maida I, Blanco F, et al. Episodes of low-level viral rebound in HIV-infected patients on antiretroviral therapy: frequency, predictors and outcome. J Antimicrob Chemother 2008; 61:699-704.

Garrido C, Roulet V, Chueca N, et al. Evaluation Evaluation of eight different bioinformatics tools to predict viral tropism in different human immunodeficiency virus type 1 subtypes. J Clin Microbiol. 2008, 46:887-91.

Gathe J, da Silva BA, Loutfy M et al. Primary efficacy results at week 48: phase 3, randomized, open-label study of lopinavir/ritonavir tablets once daily versus twice daily co-administered with tenofovir DF + emtricitabine in antiretroviral-naive HIV-infected subjects. Abstract 775, 15th CROI 2008, Boston.

Gatell JM. The use of integrase inhibitors in treatment-experienced patients. Eur J Med Res. 2009;14 Suppl 3:30-5.

Gianotti N, Seminari E, Guffanti M, et al. Evaluation of atazanavir Ctrough, atazanavir genotypic inhibitory quotient, and baseline HIV genotype as predictors of a 24-week virological response in highly drug-experienced, HIV-infected patients treated with unboosted atazanavir. New Microbiol 2005; 28: 119-25.

Goethals O, Clayton R, Wagemans E, et al. Resistance mutations in HIV-1 integrase selected with raltegravir or elvitegravir confer reduced susceptibility to a diverse panel of integrase inhibitors. Abstract 9, XVII International HIV Drug Resistance Workshop 2008, Sitges.

Gonzalez LM, Brindeiro RM, Aguiar RS, et al. Impact of nelfinavir resistance mutations on in vitro phenotype, fitness, and replication capacity of HIV type 1 with Subtype B and C Proteases. Antimicr Agents Chemother 2004; 48: 3552-55.

Goodman D, Hluhanich R, Waters J, et al. Integrase inhibitor resistance involves complex interactions among primary and secondary resistance mutations: a novel mutation L68V/I associates with E92Q and increases resistance. Abstract 13, XVII Int HIV Drug Resistance Workshop 2008; Sitges.

Grant GM, Liegler T, Spotts G, et al. Declining nucleoside reverse transcriptase inhibitor primary resistance in San Francisco, 2000-2002. Abstract 120, XII International HIV Drug Resistance Workshop, 2003, Los Cabos, Mexico.

Grossman Z, Istomin V, Averbuch D, et al.  Genetic variation at NNRTI resistance-associated positions in patients infected with HIV-1 subtype C. AIDS 2004a; 18: 909-15.

Hackett Jr J, Harris B,  Holzmayer V, et al. Naturally occurring polymorphisms in HIV-1 Group M, N, and O integrase: implications for integrase inhibitors, Abstract 872, 15th CROI 2008, Boston, :A, USA.

Haddad M, Stawiski E, Benhamida J, Coakley, et al. Improved genotypic algorithm for predicting Etravirine susceptibility:Comprehensive list of mutations identified through correlation with matched phenotype. Abstract 574, 17th CROI 2010, San Francisco, CA, USA.

Harrigan PR, Stone C, Griffin P, et al. Resistance profile of the HIV type 1 reverse transcriptase inhibitor abacavir (1592U89) after monotherapy and combination therapy. J Infect Dis 2000; 181:912-920.

Harrigan P, McGovern R, Dong W, et al. Screening for HIV tropism using population based V3 genotypic analysis: a retrospective virological outcome analysis using stored plasma screening samples from MOTIVATE-1. Abstract 15, XVIII IHDRW 2009, Fort Myers/Antiviral Therapy 14, Suppl. 1 (p. 17).

Hatano H, Lampiris H, Huang W, et al. Virological and immunological outcomes in a cohort of patients failing integrase inhibitors. Abstract 10, XVII International HIV Drug Resistance Workshop 2008, Sitges.

Hazuda DJ, Miller MD, Nguyen BY, Zhao J for the P005 Study Team. Resistance to the HIV-integrase inhibitor raltegravir: analysis of protocol 005, a Phase II study in patients with triple-class resistant HIV-1 infection. Abstract 8, XVI IHDRW 2007, Barbados,West Indies.

Heera J, Saag M, Ive P, et al. Virological Correlates associated with treatment failure at week 48 in the phase 3 study of maraviroc in treatment-naive patients. Abstract 40LB, 15th CROI, 2008; Boston, Massachusetts.

Hunt PW, Harrigan PR, Huang W, et al. Prevalence of CXCR4 tropism among antiretroviral-treated HIV-1-infected patients with detectable viremia. J Infect Dis 2006;194:926-930.

Jacobsen H, Yasargil K, Winslow DL, et al. Characterization of HIV type 1 mutants with decreased sensitivity to proteinase inhibitor Ro 31-8959. Virology 1995; 206:527-534.

Jacobson J, Thompson M, Fischl M, et al. Phase 2a study of PRO 140 in HIV-infected adults. Abstract H-1229, 49th ICAAC 2009, San Francisco, CA.

Jayaraman G, Goedhuis N, Brooks J, et al. Trends in transmission of HIV-1 drug resistance among newly diagnosed, antiretroviral treatment naive HIV-infected individuals in Canada (1999-2003). Reviews in Antiviral Therapy 2006; 4: 50 (Abstract 50).

Jimenez JL, Resino S, Martinez-Colom A, et al. Mutations at codons 54 and 82 of HIV protease predict virological response of HIV-infected children on salvage lopinavir/ritonavir therapy. J Antimicrob Chemother 2005.

Johnson JA, Li JF, Wei X, Lipscomb J, Smith A, Heneine H. Sensitive testing demonstrates a high prevalence of transmitted drug resistance among conventionally genotyped wildtype HIV-1 infections. Antiviral Therapy 2007a; 12 (5):S46 (Abstract 39).

Johnson VA, Brun-Vézinet F, Bonaventura C, et al. Update of the Drug Resistance Mutations in HIV-1: 2010. Top HIV Med 2009, 18 (5):156-163. Article: http://www.iasusa.org/resistance_mutations/mutations_figures.pdf

Kagan RM, Shenderovich MD, Heseltine PN, Ramnarayan K. Structural analysis of an HIV-1 protease I47A mutant resistant to the protease inhibitor lopinavir. Protein Sci 2005; 14: 1870-8.

Katlama C, Campbell T, Clotet B, et al. DUET-2: 24 week results of a phase III randomised double-blind trial to evaluate the efficacy and safety of TMC125 versus placebo in 591 treatment-experienced HIV-1 infected patients. Abstract WESS204:2, 4th IAS 2007, Sydney.

Kempf DJ, Isaacson JD, King MS, et al. Identification of genotypic changes in HIV protease that correlate with reduced susceptibility to the protease inhibitor lopinavir among viral isolates from protease inhibitor-experienced patients. J Virol 2001, 75:7462-9.

Kohlbrenner V, Hall D, Schapiro J, et al. Development of a tipranavir mutation score: analysis of protease mutations associated with phenotypic drug susceptibility and antiviral response in Phase II clinical trials. Antivir Ther 2004;9:S143 (Abstract 19).

Kuecherer C, Poggensee C, Korn K, et al. High level of resistant HIV-1 in newly diagnosed patients both with documented seroconversion and with unknown date of infection. Abstract 10, 4th European HIV Drug resistance workshop 2006, Monte Carlo, France.

Lafeuillade A, Tardy JC. Stavudine in the face of cross-resistance between HIV-1 nucleoside reverse transcriptase inhibitors: a review. AIDS Rev 2003, 5:80-6.

Lalezari J, Sloan L, Dejesus E, et al. Potent antiviral activity of S/GSK1349572, a next generation integrase inhibitor (INI), in INI-naïve HIV-1-infected patients. Abstract TUAB105, 5th IAS  2009, Cape Town, South Africa.

Landman R, Descamps D, Peytavin G, et al. Early virologic failure and rescue therapy of tenofovir, abacavir, and lamivudine for initial treatment of HIV-1 infection: TONUS study. HIV Clin Trials 2005; 6: 291-301.

Landovitz R, Faetkenhauer G, Hoffmann C, et al. Characterization of susceptibility profiles for the CCR5 antagonist vicriviroc in treatment-naive HIV-infected subjects. Abstract 18, XV International HIV Drug Resistance Workshop 2006, Sitges.

Lanier ER, Irlbeck D, Liao Q et al. Emergence of resistance-associated mutations over 96 weeks of therapy in subjects initiating ABC/3TC + d4T, EFV or APV/r. Abstract H-910, 43rd ICAAC 2003, Chicago, USA.

Larder B, de Vroey V, Dehertogh P, et al. Predicting HIV-1 phenotypic resistance from genotype using a large phenotype-genotype relational database. Abstract 106, 7th ECCATH 1999, Lisbon, Portugal.

Larder BA, Kemp SD. Multiple mutations in HIV-1 reverse transcriptase confer high-level resistance to zidovudine (AZT). Science 1989, 246:1155-1158.

Larder BA, Bloor S. Analysis of clinical isolates and site-directed mutants reveals the genetic determinants of didanosine resistance. Antivir Ther 2001; 6:38.

Lataillade M , Molina J M, Thiry I et al. The CASTLE study 48 week results: the impact of HIV subtypes and baseline resistance on treatment outcomes and the emergence of resistance. Antiviral Therapy 2008; 13 Suppl 3: A135 (Abstract 123)

Lewis M, Simpson P, Fransen S, et al. CXCR4-using virus detected in patients receiving maraviroc in the Phase III studies MOTIVATE 1 and 2 originates from a pre-existing minority of CXCR4-using virus. Antiviral Therapy 2007; 12:S65 (Abstract 56).

Li J, Paredes R, Ribaudo H et al, Minority HIV-1 drug resistance mutations and the risk of initial ART failure: A systematic review and pooled analysis. Abstract 614, 18th CROI, 2011, Boston.

Little SJ, Holle S, Routy JP, et al. Antiretroviral-drug resistance among patients recently infected with HIV. N Engl J Med 2002; 347:385-394. http://content.nejm.org/cgi/content/short/347/6/385

Loveday C, Devereux H, Huckett L, Johnson M. High prevalence of multiple drug resistance mutations in a UK HIV/AIDS patient population. AIDS 1999; 13: 627-628.

Loutfy MR, Montaner JSG, Raboud JM, et al. Genotypic resistance assay for entire gp-41 sequence with identification of gp-41 polymorphisms in enfuvirtide-naive patients and new gp-41 mutations in patients failing enfuvirtide. Abstract WeOrB1292, 15th IAC 2004, Bangkok, Thailand.

Lu J, Sista P, Giguel F, Greenberg M, Kuritzkes DR. Relative replicative fitness of HIV type 1 mutants resistant to enfuvirtide. J Virol 2004; 78: 4628-37.

Maguire M, Shortino D, Klein A, et al. Emergence of resistance to protease inhibitor amprenavir in HIV type 1-infected patients: selection of four alternative viral protease genotypes and influence of viral susceptibility to coadministered reverse transcriptase nucleoside inhibitors. Antimicrob Agents Chemother 2002; 46:731–8.

Malet I, Delelis O, Valantin MA, et al. Mutations associated with failure of raltegravir treatment affect integrase sensitivity to the inhibitor in vitro. Antimicrob Agents Chemother 2008;52:1351-8.

Marcelin AG, Lamotte C, Delaugerre C, et al. Genotypic inhibitory quotient as predictor of virological response to ritonavir-amprenavir in HIV type 1 protease inhibitor-experienced patients. Antimicrob Agents Chemother 2003; 47: 594-600.

Marcelin AG, Flandre P, de Mendoza C, et al. Clinical validation of saquinavir/ritonavir genotypic resistance score in protease-inhibitor-experienced patients. Antivir Ther. 2007a; 12:247-52.

Marcelin AG et al. Mutations associated with response to boosted tipranavir in HIV-1-infected PI-experienced patients. Abstract 612, 14th CROI 2007b, Los Angeles, California.

Markowitz M, Mohri H, Mehandru S, et al. Infection with multidrug resistant, dual-tropic HIV-1 and rapid progression to AIDS: a case report. Lancet 2005; 365: 1031-8.

Markowitz M, Nguyen BY, Gotuzzo E, et al.  Rapid onset and durable antiretroviral effect of raltegravir (MK-0518), a novel HIV-1 integrase inhibitor, as part of combination ART in treatment-naive HIV-1 infected patients: 48-week results. Abstract TUAB104, 4th IAS 2007, Sydney, Australia.

Masquelier B, Race E, Tamalet C, et al. Genotypic and phenotypic resistance patterns of HIV type 1 variants with insertions or deletions in the reverse transcriptase (RT): multicenter study of patients treated with RT inhibitors. Antimicrob Agents Chemother 2001, 45:1836-42.

Masquelier B., Assoumou KL, Descamps D, et al. Clinically validated mutation scores for HIV-1 resistance to fosamprenavir/ritonavir. J Antimicrob Chemother 2008, 61:1362-1368.

Mayers D, Leith J, Valdez H, et al. Impact of three or four protease mutations at codons 33, 82, 84 and 90 on 2 week virological responses to tipranavir, lopinavir, amprenavir and saquinavir all boosted by ritonavir in Phase 2B trial BI 1182.51. Antivir Ther 2004;9:S163.

McColl D, Parkin NT, Miller M, Mertenskötter T. Charakterisierung von klinischen Virus-Isolaten mit L74V oder K65R in einer großen Resistenzdatenbank. Abstract P126, 10. Deutscher und 16. Österreichischer AIDS Kongress 2005, Vienna, Austria.

McColl DJ, Fransen S, Gupta S, et al. Resistance and cross-resistance to first generation integrase inhibitors: insights from a phase II study of elvitegravir (GS-9137). Antiviral Therapy 2007; 12:S11. Abstract 9.

McNicholas P. Clonal analysis of the gp120 V3 loop from clinical isolates displaying phenotypic resistance to Vicriviroc. Abstract H-906, 49th  ICAAC 2009, San Francisco, CA.

Melby T, Despirito M, Demasi R, et al. HIV-1 Co-receptor tropism in triple-class-experienced patients: baseline correlates and relationship to enfuvirtide response. Abstract 223, 13th CROI 2006, Denver, Colorado, USA.

Metzner KJ, Rauch P, von Wyl V, et al. Prevalence of minority quasispecies of drug resistant HIV-1 in patients with primary HIV-1 infection in Zurich in the years 2002–2006. Antiviral Therapy 2007a; 12:S47 (Abstract 40).

Metzner KJ1, Walter H, P Rauch P, et al. The K65R mutation is rarely detected as a minority quasispecies in therapy-naive, chronically HIV-1-infected persons. Antiviral Therapy 2007b; 12:S52 (Abstract 45).

Meyer PR, Matsuura SE, Schinazi RF, So AG, Scott WA. Differential removal of thymidine nucleotide analogues from blocked DNA chains by HIV reverse transcriptase in the presence of physiological concentrations of 2’-deoxynucleoside triphosphates. Antimic Agents Chemother 2000; 44:3465-72.

Miller MD, Danovich RM, Ke Y, et al. Longitudinal analysis of resistance to HIV-1 integrase inhibitor raltegravir: results from P005 a phase II study in treatment-experienced patients. Abstract 6, 17th IHDRW 2008, Sitges, Spain.

Miller MD, Margot N, Lu B, et al. Genotypic and phenotypic predictors of the magnitude of response to tenofovir disoproxil fumarate treatment in antiretroviral-experienced patients. J Infect Dis 2004; 189:837-46.

Miller MD, Margot NA, Hertogs K, Larder B, Miller V. Antiviral activity of tenofovir (PMPA) against nucleoside-resistant clinical HIV samples. Nucleosides Nucleotides Nucleic Acids 2001; 20:1025-8.

Miller MD, White KL, Petropoulos CJ, et al. Decreased replication capacity of HIV-1 clinical isolates containing K65R or M184V RT mutations. Abstract 616, 10th CROI 2003, Boston, USA.

Mills A, P. Cahn P, Grinsztejn B, et al. DUET-1: 24 week results of a phase III randomised double-blind trial to evaluate the efficacy and safety of TMC125 versus placebo in 612 treatment-experienced HIV-1 infected patients. Abstract WESS204:1, 4th IAS 2007, Sydney.

Mink M, Mosier SM, Janumpalli S, et al. Impact of HIV type 1 gp41 amino acid substitutions selected during enfuvirtide treatment on gp41 binding and antiviral potency of enfuvirtide in vitro. J Virol 2005, 79: 12447-12454. Abstract: http://jvi.asm.org/cgi/content/abstract/79/19/12447

Mo H, King MS, King K, et al. Selection of resistance in protease inhibitor-experienced, HIV type 1-infected subjects failing lopinavir- and ritonavir-based therapy: Mutation patterns and baseline correlates. J Virol 2005, 79: 3329-38. http://jvi.asm.org/cgi/content/abstract/79/6/3329

Molina J-M, Andrade-Villanueva J, Echevarria J, et al. Once-daily atazanavir/ritonavir versus twice-daily lopinavir/ritonavir, each in combination with tenofovir and emtricitabine, for management of antiretroviral-naive HIV-1-infected patients: 48 week efficacy and safety results of the CASTLE study. Lancet 2008, 372: 646-655

Molina JM, Cordes C, Ive P, et. al. Efficacy and safety of TMC278 in treatment-naïve, HIV-infected patients: week 96 data from TMC278-C204.  Journal of the International AIDS Society 2008, 11(Suppl 1):P2

Mori J, Lewis M, Simpson P, et al. Characterization of maraviroc resistance in patients failing treatment with CCR5-tropic virus in MOTIVATE 1 and MOTIVATE. Abstract 51, VI. EHDRW 2008.

Mori J, Mosley M, Lewis M, et al. Characterization of maraviroc resistance in patients failing treatment with CCR5-tropic virus in MOTIVATE 1 and MOTIVATE 2. Abstract 10, 16th IHDRW 2007; Barbados, West Indies.

Moyle GJ, Wildfire A, Mandalia S, et al. Epidemiology and predictive factors for chemokine receptor use in HIV-1 infection. J Inf Dis 2005;191:866-872.

Mueller SM,  Daeumer M, Kaiser R, et al. Susceptibility to saquinavir and atazanavir in highly protease inhibitor (PI) resistant HIV-1 is caused by lopinavir-induced drug resistance mutation L76V. Antiviral Therapy 2004; 9:S44 (Abstract 38).

Naeger LK, Margot NA, Miller MD. Increased drug susceptibility of HIV-1 reverse transcriptase mutants containing M184V and zidovudine-associated mutations: analysis of enzyme processivity, chain-terminator removal and viral replication. Antivir Ther 2001; 6:115-26.

Nettles RE, Kieffer TL, Kwon P, et al. Intermittent HIV-1 viremia (Blips) and drug resistance in patients receiving HAART. JAMA 2005, 293:817-829.

Nettles RE, Kieffer TL, Simmons RP, et al. Genotypic resistance in HIV-1-infected patients with persistently detectable low-level viremia while receiving highly active antiretroviral therapy. Clin Infect Dis 2004, 39:1030-1037.

Nijhuis M, Schuurman R, de Jong D, et al. Increased fitness of drug resistant HIV-1 protease as a result of acquisition of compensatory mutations during suboptimal therapy. AIDS 1999; 13:2349-59.

Nkengafac AD, Tina S, Sua F, et al. Molecular epidemiology and prevalence of drug resistance-associated mutations in newly diagnosed HIV-1 patients in Cameroon. Antiviral Therapy 2007; 12:S50 (Abstract 43).

Obermeier MJ, Berg T, Sichtig N, et al. Determination of HIV-1 co-receptor usage in German patients – comparison of genotypic methods with the TROFILE phenotypic assay. Abstract P201, 9th International Congress on Drug Therapy in HIV Infection.

Oette M. HAART in patients with primary HIV drug resistance: The RESINA Study (oral presentation). 2. HIV Resistenzworkshop 2008, Berlin, Germany.

Oette M, Kaiser R, Daumer M, et al. Primary HIV drug resistance and efficacy of first-line antiretroviral therapy guided by resistance testing. J AIDS 2006; 41: 573-81.

S Palleja, C Cohen, J Gathe, et al. Efficacy of TBR 652, a CCR5 antagonist, in HIV-1-infected, ART-experienced, CCR5 antagonist-naive patients. Abstract 53, 17th CROI 2010, San Francisco, CA, USA. February 16-19, 2010.

Pao D, Andrady U, Clarke J, et al. Long-term persistence of primary genotypic resistance after HIV-1 seroconversion. J AIDS 2004; 37: 1570-3.

Parikh UM, Zelina S, Sluis-Cremer N, Mellors JW. Molecular mechanisms of bidirectional antagonism between K65R and thymidine analog mutations in HIV-1 reverse transcriptase. AIDS 2007;21:1405-14.

Parkin NT, Chappey C, Petropoulos CJ. Improving lopinavir genotype algorithm through phenotype correlations: novel mutation patterns and amprenavir cross-resistance. AIDS 2003; 17: 955-962.

Pellegrin I, Breilh D, Coureau G, et al. Interpretation of genotype and pharmacokinetics for resistance to fosamprenavir-ritonavir-based regimens in antiretroviral-experienced patients. Antimicrob Agents Chemother 2007;51:1473-80.

Pellegrin I, Breilh D, Ragnaud JM, et al. Virological responses to atazanavir-ritonavir-based regimens: resistance-substitutions score and pharmacokinetic parameters (Reyaphar study). Antivir Ther 2006; 11:421-9.

Perelson AS, Neumann AU, Markowitz M, et al. HIV-1 dynamics in vivo: virion clearance rate, infected cell life-span, and viral generation time. Science 1996; 271:1582-1586.

Petropoulos CJ, Parkin NT, Limoli KL, et al. A novel phenotypic drug susceptibility assay for HIV type 1. Antimicrob Agents Chemother 2000; 44:920-8.

Portsmouth S, Valluri S, Daeumer M, et al. Population and ultra-deep sequencing for tropism determination are correlated with Trofile ES: genotypic re-analysis of the A4001078 maraviroc study. Journal of the International AIDS Society 2010a, 3(Suppl 4):P128.

Portsmouth S, Chapman D, Lewis M, et al. Virologic outcome by V3 loop genotypic population sequencing and 454  “deep” sequencing  in clade B and non-B virus in Merit at 48 and 96 weeks, Abstract TUPE 0134, 18th IAC 2010b, Vienna, Austria.

Prosperi MC, Bracciale L, Fabbiani M, et al. Comparative determination of HIV-1 co-receptor tropism by Enhanced Sensitivity Trofile, gp120 V3-loop RNA and DNA genotyping. Retrovirology 2010; 7:56.

Recordon-Pinson P, O Peuchant O, S Capdepont S, et al. HIV-1 transmission dynamics in recent seroconverters: relationship with transmission of drug resistance and viral diversity. Antiviral Therapy 2007; 12:S43 (Abstract 36).

Reeves JD, Su Z, Krambrink A et al. Response to vicriviroc in HIV-infected, treatment-experienced individuals using an enhanced version of the Trofile HIV co-receptor tropism assay [Trofile (ES)]: reanalysis of ACTG 5211 results. 17th IHDRW, Sitges, 2008, Abstract 88

Reuter S, Oette M., Kaiser R, et al. First-line HAART guided by genotypic resistance testing – long-term follow-up data from the RESINA-study. Abstract MOPDA201, XVII  IAS 2008; Mexico City, Mexico.

Rimsky L, Eron J, Clotet B, et al. Characterization of the resistance profile of TMC278: 48-week analysis of the phase 3 studies ECHO and THRIVE. Abstract H-1810, 50th ICAAC 2010, Boston.

Rockstroh J, Lennox J, DeJesus E, et al. Raltegravir demonstrates durable virologic suppression and superior immunologic response with a favorable metabolic profile through 3 years of treatment: 156-week results from STARTMRK. Abstract 542, 18th CROI, 2011, Boston, MA.

Ross L, Parkin L, Chappey C, et al. HIV clinical isolates containing mutations representative of those selected after first line failure with unboosted GW433908 remain sensitive to other protease inhibitors. Abstract 19, XII Int HIV Drug Resist Workshop 2003, Los Cabos, Mexico.

Sato A, Seki T, Kobayashi M, et al. In vitro passage of drug resistant HIV-1 against a next generation integrase inhibitor (INI), S/GSK1349572. Abstract H-932/415, 49th ICAAC, San Francisco, CA.

Sax PE, Islam R, Walensky RP, et al. Should resistance testing be performed for treatment-naive HIV-infected patients? A cost-effectiveness analysis. Clin Infect Dis 2005; 41: 1316-23.

Schnell T, Schmidt B, Moschik G, et al. Distinct cross-resistance profiles of the new protease inhibitors amprenavir, lopinavir, and atazanavir in a panel of clinical samples. AIDS 2003; 17:1258-61.

Schuurmann R, Nijhuis M, van Leeuwen R, et al. Rapid changes in human immodeficiency virus typ 1 RNA load and appearance of drug-restistant virus populations in persons treated with lamivudine (3TC). J Inf Dis1995, 171: 1411–1419.

Shafer RW, Iversen AK, Winters MA, et al. Drug resistance and heterogeneous long-term virologic responses of HIV type 1-infected subjects to zidovudine and didanosine combination therapy. J Infect Dis 1995; 172:70-78.

Shafer R. Genotypic Testing for HIV-1 Drug Resistance (2003). http://hivdb.stanford.edu/modules/lookUpFiles/pdf/GenotypicResistance.pdf

Shulman NS, Bosch RJ, Mellors JW, Albrecht MA, Katzenstein DA. Genetic correlates of efavirenz hypersusceptibility. AIDS 2004; 18: 1781-5.

Smith K,  Fine D, Patel P, et al.Efficacy and Safety of Abacavir/Lamivudine Compared to Tenofovir/Emtricitabine in Combination with Once-daily Lopinavir/Ritonavir through 48 Weeks in the HEAT Study. Abstract 774, 15th CROI 2008; Boston, MA;

Simon B, Grabmeier-Pfistershammer K, Rieger A, et al. HIV coreceptor tropism in antiretroviral treatment-naive patients newly diagnosed at a late stage of HIV-Infection. AIDS 2010, 24:2051-2058.

Snoeck J, Kantor R, Shafer RW, et al. Discordances between interpretation algorithms for genotypic resistance to protease and reverse transcriptase inhibitors of HIV are subtype dependent. Antimic Agents Chemoth 2006; 50:694-701.

Sista PR, Melby T, Davison D, et al. Characterization of determinants of genotypic and phenotypic resistance to enfuvirtide in baseline and on-treatment HIV-1 isolates. AIDS 2004; 18: 1787-94.

Skrabal K, Low AJ, Dong W, et al. Determining human immunodeficiency virus coreceptor use in a clinical setting: degree of correlation between two phenotypic assays and a bioinformatic model. J Clin Microbiol 2007, 45:279-84.

Stawiski E, Paquet A, Napolitano C, et al. Identification of novel mutations strongly associated with darunavir and tipranavir resistance and their trends in a commercial database. Abstract H-912, 50th ICAAC 2010, Boston, MA, USA.

Steigbigel R et al. Results from BENCHMRK-2, a phase III study evaluating the efficacy and safety of MK-0518, a novel HIV-1 integrase inhibitor, in patients with triple-class resistant virus. Abstract 789, 15th CROI 2008, Boston, MA, USA.

Svicher V, Alteri V, Artese A, et al. Different Evolution of Genotypic Resistance Profiles to Emtricitabine Versus Lamivudine in Tenofir – Containing Regimens, J AIDS 2010, 55; 336-344.

Swenson L, Dong W, Mo T, et al. Quantification of HIV tropism by “deep” sequencing shows a broad distribution of prevalence of X4 variants in clinical samples that is associated with virological outcome. Abstract 680, 16th CROI 2009,  Montreal, Canada.

Tural C, Ruiz L, Holtzer C, et al. Clinical utility of HIV-1 genotyping and expert advice: the Havana trial. AIDS 2002; 16:209-18. Abstract:

Truong HH, Grant RM, McFarland W, et al. Routine surveillance for the detection of acute and recent HIV infections and transmission of antiretroviral resistance. AIDS 2006; 20:2193-7.

Underwood M, St Clair M, Ross L, et al. Cross-resistance of clinical Samples with K65R, L74V, and M184V Mutations. Abstract 714, 12th CROI, Boston, MA, USA. http://www.retroconference.org/2005/cd/Abstracts/25534.htm

Valer L, De Mendoza C, De Requena DG, et al. Impact of HIV genotyping and drug levels on the response to salvage therapy with saquinavir/ritonavir. AIDS 2002; 16:1964-6.

Vandamme AM, Van Laethem, de Clercq E. Managing resistance to anti-HIV drugs. Drugs 1999; 57:337-361.

Vandekerckhove LPR, A M J Wensing AMJ, Kaiser R, et al. European guidelines on the clinical management of HIV-1 tropism testing. Lancet Infect Dis. 2011; 11(5):394-407.

Vingerhoets J, Azijn H, Fransen E, et al. TMC125 displays a high genetic barrier to the development of resistance: evidence from in vitro selection experiments. J Virol 2005; 79:12773-82.

Vingerhoets J, Janssen K, Welkenhuysen-Gybels J, et al. Impact of baseline K103N or Y181C on the virological response to the NNRTI TMC125:  analysis of study TMC125-C223. Abstract 17, XV International HIV Drug Resistance Workshop 2006, Sitges, Spain

Vingerhoets J, Buelens A, Peeters M, et al. Impact of baseline NNRTI mutations on the virological response to TMC125 in the phase III clinical trials DUET-1 and DUET-2. Antiviral Therapy 2007;12:S34 (Abstract 32).

Vingerhoets J, Peeters M, Azijn H, et al. An update of the list of NNRTI mutations associated with decreased virologic response to etravirine (ETR): multivariate analyses on the pooled DUET-1 and DUET-2 clinical trial data. Abstract 24, XVIIth Int Drug Res Workshop 2008.

Walmsley S, Ruxrungtham K, Slim J, et al. Saquinavir/r (SQV/r) BiD versus lopinavir/r (LPV/r) BiD, plus emtricitabine/tenofovir (FTC/TENOFOVIR) QD as initial therapy in HIV-1 infected patients: the GEMINI study. Abstract PS1/4, 11th European AIDS Conference 2007, Madrid.

Walter H, Eberle J, Mueller H, et al.  2009. German Guidelines for tropism testing. Available at: http://www.daignet.de/site-content/hiv-therapie/leitlinien-1/Leitlinien%20zur%20Topismus_Testung%20Stand%20Juni%202009.pdf; last access 5 Aug 2011.

Waters J, Margot N, Hluhanich R, et al. Evolution of resistance to the HIV integrase inhibitor elvitegravir can involve genotypic switching among primary INI resistance patterns. Abstract 116, XVIII IHDRW, 2009, Fort Myers.

Weber J, Chakraborty B, Weberova J, Miller MD, Quinones-Mateu ME. Diminished replicative fitness of primary human immunodeficiency virus type 1 isolates harboring the K65R mutation. J Clin Microbiol 2005; 43: 1395-400.

Weinheimer S, Discotto L, Friborg J, Yang H, Colonno R. Atazanavir signature I50L resistance substitution accounts for unique phenotype of increased susceptibility to other protease inhibitors in a variety of HIV type 1 genetic backbones. Antimicrob Agents Chemother 2005; 49:3816-24.

Weinstein MC, Goldie SJ, Losina E, et al. Use of genotypic resistance testing to guide HIV therapy: clinical impact and cost-effectiveness. Ann Intern Med 2001; 134:440-50.

Wensing AM, Boucher CA. Worldwide transmission of drug-resistant HIV. AIDS Rev 2003; 5:140-55.

Westby M, Smith-Burchnell C, Mori J, et al. Reduced maximal inhibition in phenotypic susceptibility assays indicates that viral strains resistant to the CCR5 antagonist maraviroc utilize inhibitor-bound receptor for entry. J Virol 2007;81:2359-71.

Whitcomb JM, Huang W, Limoli K, et al. Hypersusceptibility to non-nucleoside reverse transcriptase inhibitors in HIV-1: clinical, phenotypic and genotypic correlates. AIDS 2002; 16:F41-7.

White KL, Margot NA, Ly JK, et al. A combination of decreased NRTI incorporation and decreased excision determines the resistance profile of HIV-1 K65R RT. AIDS 2005; 19:1751-60.

Wiese N, Müller H, Hingst K, et al. Primary resistance mutations and polymorphisms in gp41-sequences of HIV-1 B-and non-B subtypes from Fuzeon-naïve patients. Abstract P174, 10. Deutscher und 16. Österreichischer AIDS Kongress 2005, Wien.

Wilkin T, Su Z, Kuritzkes D, et al. Co-receptor tropism in patients screening for ACTG 5211, a phase 2 study of vicriviroc, a CCR5 inhibitor. Abstract 655, 13th CROI 2006, Denver, Colorado, USA.

Wilson JW. Update on antiretroviral drug resistance testing: Combining laboratory technology with patient care. AIDS Read 2003; 13:25-38. http://www.medscape.com/viewarticle/448717

Wirden M, Malet I, Derache A, et al. Clonal analyses of HIV quasispecies in patients harbouring plasma genotype with K65R mutation associated with thymidine analogue mutations or L74V substitution. AIDS 2005; 19:630-2.

Wittkop L, (on behalf of the EuroCoord-CHAIN Joint Project Team) Impact of transmitted drug resistence (TDR)on virological and immunological response to initial combination antiretroviral therapy (cART) – EuroCoord – CHAIN joint project (2010). Abstract THLBB108, 18th IAC 2010, Vienna.

Xu L, Pozniak A, Wildfire A, et al. Emergence and evolution of enfuvirtide resistance following long-term therapy involves heptad repeat 2 mutations within gp41. Antimicrob Agents Chemother 2005; 49:1113-9.

Young B, Fransen S, Greenberg K et al.Transmission of integrase strand-transfer inhibitor multi-drug resistant HIV: case report and natural history of response to raltegravir-containing antiretroviral therapy. Abstract TUPE0163, 18th IAC 2010, Vienna.


Table 9. Mutations on the protease gene leading to PI resistance.

PIs

Relevant resistance mutations and patterns

Further mutations associated with resistance

Saquinavir

I84V/A, 48V/M

≥3 of the following: L10F/I/M/R/V, K20I/M/ R/T, L24I, I62V, G73CST, 82A/F/S/T and L90M or

≥4 of the following: L10I/R/V, I54V/L, A71V/T, V77I, V82A/ F/S/T and L90M

Possible L76V-associated resensitizing effect

≥2 PRAMs*

Nelfinavir

D30N

l84A/V

N88S/D

L90M

V82A/F/S/T and at least 2 of the following: L10I, M36I, M46l/L, I54V/L/M/T, A71V/T, V77I

≥2 PRAMs*

Fosamprenavir/r

I50V

L76V together with other PI mutations

V32I plus I47V

≥6 of the following: L10F/I/V, K20M/R, E35D, R41K, I54V/L/M, L63P, V82A/F/T/S, I84V or

≥3 of the following: L10I/F/R/ V, L33F, M36I, M46I/L, I54L/ M/T/V, I62V, L63P, A71I/L/V/T, G73A/C/F/T, V82A/F /S/T, I84V and L90M) or

≥3 of the following: L10F/I/V, L33F, M46I/L, I47V, I54L/M/V/A/T/S, A71V, G73S/A/C/T, V82A/F/C/G and L90M

≥2 PRAMs*

Lopinavir/r

I47A+V32I

≥3 of the following: M46I, I47A/V, L50V, I54A/M/V, L76V, V82FATS, I84V

5-7 of the following: L10F/I/R/V, K20M/R, L24l, V32I, L33F, M46l/L, I47V/A, I50V, F53L, l54L/T/V, L63P, A71l/L/V/T, G73S, V82A/F/T, l84V, L90M

≥2 PRAMs*

Atazanavir

and

Atazanavir/r (300/100 mg QD)

I50L – frequently in combination with A71V –

≥4 of the following: L10I/F, K20R/M/I, L24I, V32I, L33I/F/V, M46I, M48V, I54V/M/A, A71V, G73C/S/T/A, V82A/F/S/T, I84V, N88S and L90M

(Possible L76V-associated resensitizing effect)

N88S

≥2 PRAMs*

Tipranavir

≥7 mutations/points of the following: K20M/R/V, L33F, E35G, N43T, M46L, I47V, I54A/M/V, Q58E, H69K, T74P, V82L/T, N83D and I84V; V82L/T and I84V with twofold points score

Score >10 of the following: I10V (+1), L24I (-2), M36I (+2), N43T (+2), M46L (+1), I47V (+6), I50L/V (-4) I54A/M/V (+3), I54L (-7) Q58E (+5), T74P (+6), L76V (-2), V82L/T (+5), N83D (+4), I84V (+ 2)

Possible L76V-associated resensitizing effect

Further resistance-ass. mutations: I54S, I84C

6 mutations/points of the following: K20M/R/V, L33F, E35G, N43T, M46L, I47V, I54A/M/V, Q58E, H69K, T74P, V82L/T, N83D and I84V; V82L/T and I84V with twofold points score

Score 3-10 from I10V (+1), L24I (-2), M36I (+2), N43T (+2), M46L (+1), I47V (+6), I50L/V (-4) I54A/M/V (+3), I54L (-7) Q58E (+5), T74P (+6), L76V (-2),.V82L/T (+5), N83D (+4), I84V (+ 2)

Darunavir/r

≥4 of the following: V11I, V32I, L33F, I47V, I50V, I54L/M, T74P, L76V, I84V, L89V

(with V32I, I50V, I54M, L76V and I84V having a higher impact)

Further resistance-ass. mutations:

L10F, E35N, I47A, V82L, G48M, V82F

≥3 of the following: V11I, V32I, L33F, I47V, I50V, I54L/M, T74P, L76V, I84V, L89V (with I50V, I54M, L76V and I84V having a higher impact)

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Filed under 10. HIV Resistance and Viral Tropism Testing, Part 2 - Antiretroviral Therapy

Mitochondrial Toxicity of NRTIs

– Nils Venhoff and Ulrich A. Walker –

Introduction

Two years after the introduction of PIs into the armamentarium of ART, reports of HIV-infected individuals experiencing clinically relevant changes in body metabolism began to surface. These metabolic symptoms were initially summarized under the term “lipodystrophy” (Carr 1998). Nowadays this lipodystrophy syndrome is understood as the result of overlapping yet distinct effects of the different drug components in ART. The main pathogenetic mechanism through which nucleoside analogs are thought to contribute to metabolic changes and organ toxicities is mitochondrial toxicity (Brinkman 1999).

Pathogenesis of mitochondrial toxicity

NRTIs are prodrugs (Kakuda 2000) – they require activation in the cell through phosphorylation before they are able to inhibit their target, HIV reverse transcriptase. In addition to impairing the HIV replication machinery, the NRTI-triphosphates also inhibit a human polymerase called gamma-polymerase, which is responsible for the replication of mitochondrial DNA (mtDNA), a small circular molecule normally present in multiple copies in each mitochondrion and in hundreds of copies in most human cells. Thus, the inhibition of gamma-polymerase by NRTIs leads to a decline (depletion) in mtDNA content (Lewis 2003). The biological task of mtDNA is to encode for enzyme subunits of the respiratory chain, which is located in the inner mitochondrial membrane. Therefore, by causing mtDNA depletion, NRTIs also lead to a defect in the respiratory chain function.

An intact respiratory chain is the prerequisite for numerous metabolic pathways. The main task of the respiratory chain is to oxidatively synthesize ATP, our chemical currency of energy. In addition, the respiratory chain consumes NADH and FADH as end products of fatty acid oxidation. This explains the micro- or macrovesicular accumulation of intracellular triglycerides, which often accompanies mitochondrial toxicity. Last but not least, a normal respiratory function is also essential for the synthesis of DNA, because the de novo synthesis of pyrimidine nucleosides depends on an enzyme located in the inner mitochondrial membrane. This enzyme is called dihydroorotate dehydrogenase (DHODH) (Löffler 1997). The clinical implications of this are detailed below.

The onset of mitochondrial toxicity follows several principles (Walker 2002a):

1. Mitochondrial toxicity is concentration-dependent. High NRTI concentrations cause a more pronounced mtDNA depletion compared to low concentrations. The clinical dosing of some nucleoside analogs is close to the limit of tolerance with respect to mitochondrial toxicity.

2. Mitochondrial toxicity is time dependent and develops with prolonged NRTI exposure. Changes in mitochondrial metabolism are observed only if the amount of mtDNA depletion exceeds a certain threshold. As a consequence, long-term NRTI exposure may lead to mitochondrial effects despite relatively low NRTI concentrations and the onset of toxicity is typically not observed in the first few months of ART.

3. There are significant differences in the relative potencies of nucleoside and nucleotide analogs in their ability to interact with gamma-polymerase. The hierarchy of gamma-polymerase inhibition for the active NRTI metabolites has been determined as follows: zalcitabine (ddC, HIVID®) > didanosine (ddI, Videx®) > stavudine (d4T, Zerit®) > lamivudine (3TC, Epivir®) ≥ abacavir (ABC, Ziagen®) ≥ tenofovir (TDF, Viread®) ≥ emtricitabine (FTC, Emtriva®).

4. Zidovudine (AZT, Retrovir®) may be peculiar because its active triphosphate is only a weak inhibitor of gamma-polymerase. However, another mechanism can explain how zidovudine might cause mtDNA depletion independent from gamma-polymerase inhibition. AZT is an inhibitor of mitochondrial thymidine kinase type 2 (TK2), and, as such, interferes with the synthesis of natural pyrimidine nucleotides, thus potentially impairing the formation of mtDNA (McKee 2004). Indeed, inborn defects of TK2 are known to cause mtDNA depletion in human muscle tissue (Saada 2001). It has also been demonstrated that AZT can be non-enzymatically converted into d4T within the body, at least within some cells (Becher 2003, Bonora 2004).

5. Mitochondrial toxicity is tissue-specific. Tissue specificity is explained by the fact that the uptake of the NRTI prodrugs into cells and their mitochondria as well as activation by phosphorylation may be different in different cell types.

6. There may be additive or synergistic mitochondrial toxicities if two or more NRTIs are used in combination.

7. Data suggest that mitochondrial transcription may also be impaired without mtDNA alterations (Galluzzi 2005, Mallon 2005). However, the mechanism and clinical significance of this are not yet understood.

Clinical manifestations

MtDNA depletion may manifest clinically in one or several target tissues (Figure 1). In the liver mitochondrial toxicity is associated with increased lipid deposits, resulting in micro or macrovesicular steatosis. Steatosis may be accompanied by elevated liver transaminases. Such steatohepatitis may progress to liver failure and lactic acidosis, a potentially fatal, but fortunately rare complication.

Although steatohepatitis and lactic acidosis were already described in the early 90s in patients receiving didanosine monotherapy (Lambert 1990), mitochondrial liver toxicity is now observed with treatment with all NRTIs that have a relatively strong potential to inhibit gamma-polymerase, especially with the so called “d-drugs” ddI, d4T (and formerly, ddC). Liver complications have also been described with AZT. It has been demonstrated in the hepatic tissue of HIV patients that each of the d-drugs leads to a time dependent mtDNA depletion. On electron microscopy, morphologically abnormal mitochondria were observed.

Figure 1: Organ manifestations of mitochondrial toxicity.

A typical complication of mitochondrial toxicity is an elevation in serum lactate. Such hyperlactatemia was more frequently described with prolonged d4T treatment (Carr 2000, Saint-Marc 1999), especially when combined with ddI. The toxicity of ddI is also increased through interactions with ribavirin and hydroxyurea. The significance of asymptomatic hyperlactatemia is unclear. When elevated lactate levels are associated with symptoms, these are often non-specific such as nausea, right upper quadrant abdominal tenderness or myalgias. In the majority of cases, levels of bicarbonate and the anion gap (Na+ –  (HCO3 + Cl)) are normal, although liver transaminases are mildly increased in the majority of cases (Lonergan 2000). Therefore, the diagnosis relies on the logistically more cumbersome direct determination of serum lactate. In order to avoid artifacts, venous blood must be drawn without the use of a tourniquet from resting patients. The blood needs to be collected in fluoride tubes and transported to the laboratory on ice for immediate analysis. Non-mitochondrial causes must also be considered in the differential diagnosis of lactic acidosis (Table 1) and underlying organ toxicities should be looked for. The incidence of lactic acidosis is low; it was estimated as 1 per 1000 patient years in NRTI exposed patients (Imhof 2005). In South Africa, much higher incidence rates (10.6 cases per 1,000 patient years) have been reported (Bolhaar 2007).

Mitochondrial myopathy in antiretrovirally treated HIV+ patients was first described with high dose AZT (Arnaudo 1991). Skeletal muscle weakness may manifest under dynamic or static exercise. The serum CK is often normal or only minimally elevated. Muscle histology helps to distinguish this form of NRTI toxicity from HIV myopathy, which may also occur simultaneously. On histochemical examination, the muscle fibers of the former are frequently negative for cytochrome c-oxidase and carry ultrastructurally abnormal mitochondria, whereas those of the latter are typically infiltrated by CD8-positive T lymphocytes. Exercise testing may detect a low lactate threshold and a reduced lactate clearance, but in clinical practice these changes are difficult to distinguish from lack of aerobic exercise (detraining).

In a murine model of cardiomyopathy, nine weeks of oral AZT and ddC induced a mitochondrial lesions with mtDNA depletion, diminished respiratory chain function and ultrastructural abnormalities of mitochondria (Balcarek 2010).

Table 1: Causes of hyperlactatemia/ lactic acidosis.
Type A lactic acidosis Type B lactic acidosis
(Tissue hypoxia)ShockCarbon monoxide poisoningHeart failure (Other mechanisms)Thiamine deficiencyAlkalosis (pH >7.6)Epilepsy

Adrenalin (iatrogenic, endogenous)

Liver failure

Neoplasm (lymphoma, solid tumors)

Intoxication (nitroprusside, methanol, methylene glycol, salicylates)

Fructose

Rare enzyme deficiencies

mtDNA mutations

mtDNA depletion

Prolonged treatment with d-drugs can also lead to a predominantly symmetrical, sensory and distal polyneuropathy of the lower extremities (Moyle 1998, Simpson 1995). An elevated serum lactate level can help distinguish this axonal neuropathy from its HIV-associated phenocopy, although in most cases the lactate level is normal. The differential diagnosis may also take into account the fact that the mitochondrial polyneuropathy mostly occurs weeks or months after initiation of the d-drugs. In contrast, the HIV-associated polyneuropathy generally does not worsen and may indeed improve with prolonged antiretroviral treatment. In mice AZT and ddC induce a mitochondrial neurotoxicity with ddC predominantly affecting the peripheral and AZT mainly the central nervous system (Venhoff 2010). This is consistent with findings in patients (Tardieu 2005, Moyle 1998). Although the toxic effects of AZT on the central nervous system have not been studied well in humans, they are plausible because AZT penetrates well into the CNS.

In its more narrow sense, the term lipodystrophy denotes a change in the distribution of body fat. Some individuals affected with lipodystrophy may experience abnormal fat accumulation in some body areas (most commonly in the abdomen or in the dorsocervical region), whereas others may develop fat wasting (Bichat’s fat pad in the cheeks, temporal fat, or subcutaneous fat of the extremities). Both fat accumulation and fat loss may occur simultaneously in the same individuals. Fat wasting (also called lipoatrophy) is partially reversible and generally observed not earlier than one year after the initiation of antiretroviral therapy. In the affected subcutaneous tissue, ultrastructural abnormalities of mitochondria and reduced mtDNA levels have been identified, in particular in subjects treated with d4T (Walker 2002b). In vitro and in vivo analyses of fat cells have also demonstrated diminished intracellular lipids, reduced expression of adipogenic transcription factors (PPAR-gamma and SREBP-1), and increased apoptotic indices. NRTI treatment may also impair some endocrine functions of adipocytes. For example, NRTIs may impair the secretion of adiponectin and through this mechanism may promote insulin resistance. D4T has been identified as a particular risk factor, but other NRTIs such as zidovudine may also contribute. When d4T is replaced by another NRTI, mtDNA levels and apoptotic indices improve (McComsey 2005), along with an objectively measurable, albeit small increase of subcutaneous adipose tissue (McComsey 2004). In contrast, switching away from protease inhibitors did not ameliorate lipoatrophy or adipocyte apoptosis. Taken together, the available data indicate a predominant effect of mitochondrial toxicity in the pathogenesis of lipoatrophy.

Some studies have suggested an effect of NRTIs on the mtDNA levels in blood (Coté 2003, Miro 2003). The functional consequence of such mitochondrial toxicity on lymphocytes is still unknown. In this context, it is important to note that a delayed loss of CD4 and CD8 lymphocytes was observed when ddI plasma levels were increased by co-medication with TDF or when ddI was given to subjects with low body weight (Negredo 2004). Recent in vitro investigations with exposure of mitotically stimulated T lymphocytes to only slightly supratherapeutic concentrations of ddI also detected a substantial mtDNA depletion with a subsequent late onset decline of lymphocyte proliferation and increased apoptosis (Setzer 2005a, Setzer 2005b). Thus, mitochondrial toxicity is the most likely explanation for the late onset decline of lymphocytes observed with ddI. The data suggest that the mitochondrial toxicity of NRTIs on lymphocytes has immunosuppressive properties.

Asymptomatic elevations in serum lipase are not uncommon under ART, but of no value in predicting the onset of pancreatitis (Maxson 1992). The overall frequency of pancreatitis has been calculated as 0.8 cases/100 years of NRTI-containing ART. Clinical pancreatitis is associated with the use of ddI in particular. DdI re-exposure may trigger a relapse and should be avoided. A mitochondrial mechanism has been cited to explain the onset of pancreatitis, but this assumption remains unproven.

Furthermore an elevation of serum urate was observed on therapy with dideoxynucleosides (ddI and d4T). Impaired ATP production as a result of mitochondrial toxicity may increase urate production in the purine nucleotide cycle (Walker 2006b).

After long and controversial discussions, several studies now provide evidence that TDF does cause mitochondrial damage to the kidney. TDF is a nucleotide analog reverse transcriptase inhibitor (Viread®) which has been associated with cases of renal dysfunction, Fanconi’s syndrome and cases of osteomalacia in animals (Tenofovir review team 2001) and patients (Gupta 2008, Wanner 2009). Patients treated with TDF often present with elevated serum alkaline phosphatase and hypophosphatemia due to a diminished renal phosphate resorption (Kinai 2005). Also osteomalacia was observed in patients treated with TDF, especially when combined with lopinavir/r (Parsonage 2005, Wanner 2009). TDF is taken up into the proximal renal tubules by human renal organic anion transporters (hOATs) 1 and 3. Despite the fact that TDF only has a low potency to impair the polymerase gamma, the hOATs may generate high intratubular tenofovir concentrations that then interfere with the replication of mtDNA (Cote 2006). Decreased mtDNA levels have been found in renal biopsies from patients exposed to TDF+ddI, an NRTI combination that is no longer recommended (Cote 2006). In rats, TDF induced an organ-specific nephrotoxicity with mtDNA depletion and tubular dysfunction of mtDNA-encoded respiratory chain subunits (Lebrecht 2009). It is therefore not recommended to use TDF in patients with established renal dysfunction.

The use of AZT to avoid vertical HIV transmission diminishes mtDNA levels in the placenta, as well as in the peripheral cord blood of perinatally exposed newborns (Divi 2004, Shiramizu 2003, Gingelmaier 2009). AZT was found to be incorporated into mtDNA in pregnant monkeys treated with AZT plus 3TC prior to delivery and mtDNA depletion was found in skeletal muscle, heart and brain (Gerschenson 2004) in which perinatally acquired lesions were shown to persist for months after cessation of NRTI exposure in some models.

Mitochondrial symptoms and abnormal cerebral imaging were found at increased frequency in infants perinatally exposed to NRTIs (Blanche 1999, Tardieu 2005). Hyperlactatemia is not infrequently observed and may persist for several months after delivery (Noguera 2003). Other clinical trials in contrast did not detect an increased perinatal risk in association with perinatal AZT prophylaxis although key parameters of mitochondrial dysfunction were not assessed. Long-term follow-up data are urgently needed (Venhoff 2006). The available information however does not justify deviating from the currently recommended strategy to use AZT to prevent vertical HIV transmission as part of a combination therapy for the mother.

Monitoring

There is currently no method to reliably predict the mitochondrial risk of an individual patient. Routine screening of asymptomatic NRTI-treated subjects with lactate levels is not warranted, since elevated lactate levels in asymptomatic subjects are not predictive of clinical mitochondrial toxicity (McComsey 2004). Quantification of mtDNA levels in PBMCs is not reliable. The [13C]methionine breath test can be used to analyze the oxidative capacity of liver mitochondria, but results may be confounded by several factors other than HIV or ART (Sternfeld 2009). Quantifying mtDNA within affected tissues is likely to be more sensitive; however this form of monitoring is invasive and not evaluated with regard to clinical endpoints.

Once symptoms are established, histological examination of a biopsy may contribute to the correct diagnosis. The following findings in tissue biopsies point towards a mitochondrial etiology: ultrastructural abnormalities of mitochondria, diminished histochemical activities of cytochrome c oxidase, the detection of intracellular and more specifically microvesicular steatosis, and the so-called ragged-red fibers.

Treatment & prophylaxis of mitochondrial toxicity

Drug interactions

Drug interactions may precipitate mitochondrial symptoms and must be taken into account. The mitochondrial toxicity of ddI for example is augmented through drug interactions with ribavirin, hydroxyurea and allopurinol (Ray 2004). When ddI is combined with TDF, the dose of ddI must be reduced to 250 mg once daily. The thymidine analog brivudine is a herpes virostatic that may sensitize for NRTI-related mitochondrial toxicity because one of its metabolites is an inhibitor of DHODH (see below). Brivudine should therefore not be combined with antiretroviral pyrimidine analogues.

Mitochondrial toxins

An impairment of mitochondrial metabolism may also result from ibuprofen, valproic acid and acetyl salicylic acid as these agents impair the mitochondrial utilization of fatty acids. Numerous cases have been described in which a life-threatening lactic acidosis was triggered by valproic acid both in HIV-infected patients and in patients with inherited mutations of mtDNA. Acetyl salicylic acid may damage mitochondria and such damage to liver organelles may result in Reye’s syndrome. Amiodarone and tamoxifen also inhibit the mitochondrial synthesis of ATP. Acetaminophen and other drugs impair the antioxidative defense (glutathione) of mitochondria, allowing for their free radical-mediated damage. Aminoglycoside antibiotics and chloramphenicol not only inhibit the protein synthesis of bacteria, but under certain circumstances may also impair the peptide transcription of mitochondria as our bacteria-like endosymbionts. Adefovir and cidofovir are also inhibitors of gamma-polymerase. Alcohol is a mitochondrial toxin and should be avoided. Lastly, TDF nephrotoxicity may be precipitated by coadministation with lopinavir/r (Wanner 2009). Lopinavir/r increases tenofovir serum levels and also may inhibit MDR4, with both mechanisms then contributing to intratubular tenofovir accumulation.

The most important clinical intervention is probably the discontinuation of the NRTI responsible for mitochondrial toxicity. Several studies have demonstrated that switching from stavudine (Zerit®) to a less toxic alternative led to an objective and progressive improvement in lipoatrophy (Martin 2004, McComsey 2004, Moyle 2004). In contrast, a switch from protease inhibitors to NRTIs was not associated with an improvement of lipoatrophy. These findings stress the importance of mitochondrial toxicity in the pathogenesis of fat abnormalities.

Uridine

When toxic NRTIs can not be avoided, the supplementation of uridine is currently a very promising strategy to treat mitochondrial toxicity. As outlined above, any respiratory chain impairment also results in the inhibition of DHODH, an essential enzyme for the synthesis of uridine and its derived pyrimidines (Figure 2). This decrease in intracellular pyrimidine pools leads to a relative excess of the exogenous pyrimidine nucleoside analogs, with which they compete at gamma-polymerase. A vicious circle is closed and contributes to mtDNA depletion. By supplementing uridine either prophylactically or therapeutically, this depletion may be interrupted, resulting in increased mtDNA levels (Setzer 2008). Indeed, uridine abolishes all the effects of mtDNA depletion in hepatocytes and normalizes lactate production, cell proliferation, the rate of cell death and intracellular steatosis (Walker 2003). In d4T-exposed adipocytes uridine was able to normalize mitochondrial function and lipid metabolism (Walker 2006a). New data indicate that uridine is able to also prevent ddC-induced hepatotoxicity (Lebrecht 2007), and AZT-induced myopathy (Lebrecht 2008), cardiomyopathy and neuropathy (Venhoff 2010) in mice.

The oral substitution of uridine as a pyrimidine precursor is well tolerated by humans, even at high doses (Kelsen 1997, van Groeningen 1986). Mitocnol, a food supplement, was shown to have a more than 8-fold uridine bioavailability over conventional uridine (Venhoff 2005).

Figure 2: Suggested mechanism of mitocnol (NucleomaxXTM) in the prevention and treatment of mitochondrial toxicity.

A randomized placebo-controlled double-blind trial found that mitocnol improved subcutaneous fat in lipoatrophic subjects under continued therapy with d4T or AZT (Sutinen 2007). In a second study mitocnol was found to be efficacious with regard to patient- and physician-assessed lipoatrophy scores (McComsey 2008). ACTG 5229, a prospective, 1:1 randomized, placebo-controlled multicenter trial evaluated the effectiveness of uridine in improving limb fat in HIV-infected individuals with lipoatrophy on a tNRTI-containing regimen (stratified by AZT or d4T). Primary endpoint was change in limb fat from baseline to week 48. Despite a small improvement in limb fat after 24 weeks compared to placebo, the effect was not sustained through 48 weeks of uridine (McComsey 2010). These results dashed the hopes in uridine as a treatment option of tNRTI-associated lipoatrophy.

Mitochondrial steatohepatitis is antagonized by mitocnol in animal models, as well as in HIV-positive patients (Walker 2004, Banasch 2006, Lebrecht 2007). In AZT-induced myopathy in mice mitocnol attenuated mtDNA depletion and muscle atrophy (Lebrecht 2008). Furthermore, mitochondrial cardiomyopathy and neuropathy was successfully prevented by mitocnol in an animal model (Balcarek 2010, Venhoff 2010).

Mitocnol is well tolerated and adverse events have not been observed so far. In one study, a clinically insignificant HDL decline was noted, while in another HDL cholesterol was unchanged (McComsey 2008). There are no known negative interactions of uridine with antiretroviral treatment (Koch 2003, McComsey 2005, Sommadossi 1988, Sutinen 2007). In Europe and North America, mitocnol is available as a dietary supplement called NucleomaxX® and can be acquired in pharmacies and the internet (www.nucleomaxX.com).

Hyperlactatemia

With symptomatic hyperlactatemia and with lactic acidosis, NRTIs should be immediately discontinued (Brinkman 2000).

With respect to mtDNA depletion vitamin supplements were not found to be effective either in vitro or in clinical studies (Venhoff 2002, Walker 1995). In animals and humans mitocnol improves hyperlactatemia (Lebrecht 2007, Sutinen 2007). NRTI re-exposure may be possible after normalization of lactate (Lonergan 2003). The supportive treatment of hyperlactatemia and lactic acidosis is summarized in Table 2.

Table 2: Supportive treatment of lactate elevation in HIV-infected patients (non-pregnant adults).
Lactate 2-5 mmol/L + symptoms Lactate > 5 mmol/L or lactic acidosis
Discontinue mitochondrial toxinsConsider vitamins and

NucleomaxX® (36g TID on 3 consecutive days/month)

Discontinue NRTIs and all mitochondrial toxinsIntensive care

Maintain hemoglobin >100 g/L

Avoid vasoconstrictive agents

Oxygen

Correct hypoglycemia

Bicarbonate controversial – 50-100 mmol if pH <7.1

Coenzyme Q10  (100 mg TID)

Vitamin C (1 g TID)

Thiamine (Vit. B1, 100 mg TID)

Riboflavin (Vit. B2, 100 mg QD)

Pyridoxine (Vit. B6, 60 mg QD)

L-acetyl carnitine (1 g TID)

NucleomaxX (36 g TID until lactate <5 mmol/L)

References

Arnaudo E, Dalakas M, Shanske S, Moraes CT, DiMauro S, Schon EA. Depletion of muscle mitochondrial DNA in AIDS patients with zidovudine-induced myopathy. Lancet 1991; 337: 508-510.

Balcarek K, Venhoff N, Deveaud C, et al. Role of Pyrimidine Depletion in the Mitochondrial Cardiotoxicity of Nucleoside Analogue Reverse Transcriptase Inhibitors. J Acquir Immune Defic Syndr 2010.

Banasch M, Goetze O, Knyhala K, et al. Uridine supplementation enhances hepatic mitochondrial function in thymidine-analogue treated HIV-infected patients. AIDS 2006; 20: 1554-1556.

Becher F, Pruvost AG, Schlemmer DD, et al. Significant levels of intracellular stavudine triphosphate are found in HIV-infected zidovudine-treated patients. AIDS 2003; 17: 555-561.

Blanche S, Tardieu M, Rustin P, et al. Persistent mitochondrial dysfunction and perinatal exposure to antiretroviral nucleoside analogues. Lancet 1999; 354: 1084-1089.

Bolhaar MG, Karstaedt AS. A high incidence of lactic acidosis and symptomatic hyperlactatemia in women receiving highly active antiretroviral therapy in Soweto, South Africa. Clin Infect Dis 2007;45:254-260.

Bonora S, Boffito M, D’Avolio A, et al. Detection of stavudine concentrations in plasma of HIV-infected patients taking zidovudine. AIDS 2004; 18: 577-578.

Brinkman K, Smeitink JA, Romijn JA, Reiss P. Mitochondrial toxicity induced by nucleoside-analogue reverse-transcriptase inhibitors is a key factor in the pathogenesis of antiretroviral-therapy-related lipodystrophy. Lancet 1999; 354: 1112-1115.

Brinkman K, Vrouenraets S, Kauffman R, Weigel H, Frissen J. Treatment of nucleoside reverse transcriptase inhibitor-induced lactic acidosis. AIDS 2000; 14: 2801-2802.

Carr A, Miller J, Law M, Cooper DA. A syndrome of lipoatrophy, lactic acidaemia and liver dysfunction associated with HIV nucleoside analogue therapy: contribution to protease inhibitor-related lipodystrophy syndrome. AIDS 2000; 14: F25-F32.

Carr A, Samaras K, Burton S, et al. A syndrome of peripheral lipodystrophy, hyperlipidaemia and insulin resistance in patients receiving HIV protease inhibitors. AIDS 1998; 12: F51-F58.

Cote HC, Magil AB, Harris M, et al. Exploring mitochondrial nephrotoxicity as a potential mechanism of kidney dysfunction among HIV-infected patients on highly active antiretroviral therapy. Antivir Ther 2006; 11: 79-86.

Coté HC, Yip B, Asselin JJ, et al. Mitochondrial:nuclear DNA ratios in peripheral blood cells from human immunodeficiency virus (HIV)-infected patients who received selected HIV antiretroviral drug regimens. J Infect Dis 2003; 187: 1972-1976.

Divi RL, Walker VE, Wade NA, et al. Mitochondrial damage and DNA depletion in cord blood and umbilical cord from infants exposed in utero to Combivir. AIDS 2004; 18: 1013-1021.

Galluzzi L, Pinti M, Troiano L, et al. Changes in mitochondrial RNA production in cells treated with nucleoside analogues. Antivir Ther 2005; 10: 191-195.

Gerschenson M, Nguyen V, Ewings EL, et al. Mitochondrial toxicity in fetal Erythrocebus patas monkeys exposed transplacentally to zidovudine plus lamivudine. AIDS Res Hum Retroviruses 2004; 20: 91-100.

Gingelmaier A, Grubert TA, Kost BP, et al. Mitochondrial toxicity in HIV type-1-exposed pregnancies in the era of highly active antiretroviral therapy. Antivir Ther 2009; 14: 331-338.

Gupta SK. Tenofovir-associated Fanconi syndrome: review of the FDA adverse event reporting system. AIDS Patient Care STDS 2008; 22: 99-103.

Imhof A, Ledergerber B, Günthard HF et al. Risk factors for and outcome of hyperlactatemia in HIV-infected persons: is there a need for routine lactate monitoring? Clin Infect Dis 2005;41:721-728.

Kakuda TN. Pharmacology of nucleoside and nucleotide reverse transcriptase inhibitor-induced mitochondrial toxicity. Clin Ther 2000; 22: 685-708.

Kelsen DP, Martin D, O’Neil J, et al. Phase I trial of PN401, an oral prodrug of uridine, to prevent toxicity from fluorouracil in patients with advanced cancer. J Clin Oncol 1997; 15: 1511-1517.

Kinai E, Hanabusa H. Renal tubular toxicity associated with tenofovir assessed using urine-beta 2 microglobulin, percentage of tubular reabsorption of phosphate and alkaline phosphatase levels. AIDS 2005; 19: 2031-2033.

Koch EC, Schneider J, Weiss R, Penning B, Walker UA. Uridine excess does not interfere with the antiretroviral efficacy of nucleoside analogue reverse transcriptase inhibitors. Antivir Ther 2003; 8: 485-487.

Lambert JS, Seidlin M, Reichman RC et al. 2′,3′-dideoxyinosine (ddI) in patients with the acquired immunodeficiency syndrome or AIDS-related complex. A phase I trial. N Engl J Med 1990; 322: 1333-1340.

Lebrecht D, Deveaud C, Beauvoit B, Bonnet J, Kirschner J, Walker UA. Uridine supplementation antagonizes zidovudine-induced mitochondrial myopathy and hyperlactatemia in mice. Arthritis Rheum 2008; 58: 318-326.

Lebrecht D, Vargas Infante YA, Setzer B, Kirschner J, Walker UA. Uridine supplementation antagonizes zalcitabine-induced microvesicular steatohepatitis in mice. Hepatology 2007; 15: 72-79.

Lebrecht D, Venhoff AC, Kirschner J, Wiech T, Venhoff N, Walker UA. Mitochondrial tubulopathy in tenofovir-DF treated rats. J Acquir Immune Defic Syndr 2009; 1: 258-63.

Lewis W, Day BJ, Copeland WC. Mitochondrial toxicity of NRTI antiviral drugs: an integrated cellular perspective. Nature Reviews Drug Discovery 2003; 2: 812-822.

Löffler M, Jöckel J, Schuster G, Becker C. Dihydroorotat-ubiquinone oxidoreductase links mitochondria in the biosynthesis of pyrimidine nucleotides. Mol Cell Biochem 1997; 174: 125-129.

Lonergan JT, Barber RE, Mathews WC. Safety and efficacy of switching to alternative nucleoside analogues following symptomatic hyperlactatemia and lactic acidosis. AIDS 2003; 17: 2495-2499.

Lonergan JT, Behling C, Pfander H, Hassanein TI, Mathews WC. Hyperlactatemia and hepatic abnormalities in 10 human immunodeficiency virus-infected patients receiving nucleoside analogue combination regimens. Clin Infect Dis 2000; 31: 162-166.

Mallon PW, Unemori P, Sedwell R, et al. In vivo, nucleoside reverse-transcriptase inhibitors alter expression of both mitochondrial and lipid metabolism genes in the absence of depletion of mitochondrial DNA. J Infect Dis 2005; 191: 1686-1696.

Martin A, Smith DE, Carr A, et al. Reversibility of lipoatrophy in HIV-infected patients 2 years after switching from a thymidine analogue to abacavir: the MITOX Extension Study. AIDS 2004; 18: 1029-1036.

Maxson CJ, Greenfield SM, Turner JL. Acute pancreatitis as a common complication of 2′,3′-dideoxyinosine therapy in the acquired immunodeficiency syndrome. Am J Gastroenterol 1992; 87: 708-713.

McComsey GA, O’Riordan M, Setzer B, Lebrecht D, Baron E, Walker UA. Uridine supplementation in HIV lipoatrophy: pilot trial on safety and effect on mitochondrial indices. Eur J Clin Nutr 2008; 62: 1031-1037.

McComsey GA, Paulsen DM, Lonergan JT, et al. Improvements in lipoatrophy, mitochondrial DNA levels and fat apoptosis after replacing stavudine with abacavir or zidovudine. AIDS 2005; 19: 15-23.

McComsey GA, Ward DJ, Hessenthaler SM, et al. Improvement in lipoatrophy associated with highly active antiretroviral therapy in human immunodeficiency virus-infected patients switched from stavudine to abacavir or zidovudine: the results of the TARHEEL study. Clin Infect Dis 2004; 38: 263-270.

McComsey GA, Yau L. Asymptomatic hyperlactataemia: predictive value, natural history and correlates. Antivir Ther 2004; 9: 205-212.

McComsey GA, Walker UA, Budhathoki CB et al. Uridine supplementation in the treatment of HIV lipoatrophy: results of ACTG 5229. AIDS 2010;24:2507-15McKee EE, Bentley AT, Hatch M, Gingerich J, Susan-Resiga D. Phosphorylation of thymidine and AZT in heart mitochondria: elucidation of a novel mechanism of AZT cardiotoxicity. Cardiovasc Toxicol 2004; 4: 155-167.

Miro O, Lopez S, Pedrol E, et al. Mitochondrial DNA depletion and respiratory chain enzyme deficiencies are present in peripheral blood mononuclear cells of HIV-infected patients with HAART-related lipodystrophy. Antivir Ther 2003; 8: 333-338.

Moyle G, Lysakova L, Brown S, et al. A randomized open-label study of immediate versus delayed polylactic acid injections for the cosmetic management of facial lipoatrophy in persons with HIV infection. HIV Med 2004; 5: 82-87.

Moyle GJ, Sadler M. Peripheral neuropathy with nucleoside antiretrovirals: risk factors, incidence and management. Drug Safety 1998; 19: 481-494.

Negredo E, Moltó J, Burger D, et al. Unexpected CD4 cell count decline in patients receiving didanosine and tenofovir-based regimens despite undetectable viral load. AIDS 2004; : 459-463.

Noguera A, Fortuny C, Sanchez E, et al. Hyperlactatemia in human immunodeficiency virus-infected children receiving antiretroviral treatment. Pediatr Infect Dis J 2003; 22: 778-782.

Parsonage MJ, Wilkins EG, Snowden N, Issa BG, Savage MW. The development of hypophosphataemic osteomalacia with myopathy in two patients with HIV infection receiving tenofovir therapy. HIV Med 2005; 6: 341-346.

Ray AS, Olson L, Fridland A. Role of purine nucleoside phosphorylase in interactions between 2′,3′-dideoxyinosine and allopurinol, ganciclovir, or tenofovir. Antimicrob Agents Chemother 2004; 48: 1089-1095.

Saada A, Shaag A, Mandel H, Nevo Y, Eriksson S, Elpeleg O. Mutant mitochondrial thymidine kinase in mitochondrial DNA depletion myopathy. Nat Genet 2001; 29: 342-344.

Saint-Marc T, Touraine JL. The effects of discontinuing stavudine therapy on clinical and metabolic abnormalities in patients suffering from lipodystrophy. AIDS 1999; 13: 2188-2189.

Setzer B, Lebrecht D, Walker UA. Pyrimidine nucleoside depletion sensitizes to the mitochondrial hepatotoxicity of the reverse transcriptase inhibitor stavudine. Am J Pathol 2008; 172: 681-690.

Setzer B, Schlesier M, Thomas AK, Walker UA. Mitochondrial toxicity of nucleoside analogues in primary human lymphocytes. Antivir Ther 2005a; 10: 327-334.

Setzer B, Schlesier M, Walker UA. Effects of of didanosine-related depletion of mtDNA in human T lymphocytes. J Infect Dis 2005b; 191: 848-855.

Shiramizu B, Shikuma KM, Kamemoto L, et al. Placenta and cord blood mitochondrial DNA toxicity in HIV-infected women receiving nucleoside reverse transcriptase inhibitors during pregnancy. J Acquir Immune Defic Syndr 2003; 32: 370-374.

Simpson DM, Tagliati M. Nucleoside analogue-associated peripheral neuropathy in human immunodeficiency virus infection. J Acquir Immune Defic Syndr 1995; 9: 153-161.

Sommadossi JP, Carlisle R, Schinazi RF, Zhou Z. Uridine reverses the toxicity of 3′-azido-3′-deoxythymidine in normal human granulocyte-macrophage progenitor cells in vitro without impairment of antiretroviral activity. Antimicrob Agents Chemother 1988; 32: 997-1001.

Sternfeld T, Lorenz A, Schmid M, et al. [(13)C]Methionine breath test as a marker for hepatic mitochondrial function in HIV-infected patients. AIDS Res Hum Retroviruses 2009; 25:1243-8.

Sutinen J, Walker UA, Sevastianova K, et al. Uridine supplementation for the treatment of antiretroviral therapy-associated lipoatrophy: a randomized, double-blind, placebo-controlled trial. Antivir Ther 2007; 12: 97-105.

Tardieu M, Brunelle F, Raybaud C, et al. Cerebral MR imaging in uninfected children born to HIV-seropositive mothers and perinatally exposed to zidovudine. AJNR Am J Neuroradiol 2005; 26: 695-701.

Tenofovir review team. Memorandum. www fda gov 2001.

van Groeningen CJ, Leyva A, Kraal I, Peters GJ, Pinedo HM. Clinical and pharmacokinetic studies of prolonged administration of high-dose uridine intended for rescue from 5-FU toxicity. Cancer Treatment Reports 1986; 70: 745-750.

Venhoff N, Setzer B, Lebrecht D, Walker UA. Dietary supplements in the treatment of NRTI-related mitochondrial toxicity. AIDS 2002; 16: 800-802.

Venhoff N, Walker UA. Mitochondrial disease in the offspring as a result of antiretroviral therapy. Expert Opin Drug Saf 2006; 5: 373-381.

Venhoff N, Zilly M, Lebrecht D, et al. Uridine pharmacokinetics of Mitocnol, a sugar cane extract. AIDS 2005; 19: 739-740.

Venhoff N, Lebrecht D, Deveaud C, et al. Oral uridine supplementation antagonizes the peripheral neuropathy and encephalopathy induced by antiretroviral nucleoside analogues. AIDS 2010; 24:345-52.

Walker UA, Auclair M, Lebrecht D, Kornprobst M, Capeau J, Caron M. Uridine abrogates the adverse effects of antiretroviral pyrimidine analogues on adipose cell functions. Antivir Ther 2006a; 11: 25-34.

Walker UA, Bickel M, Lütke Volksbeck SI, et al. Evidence of nucleoside analogue reverse transcriptase inhibitor-associated genetic and structural defects of mitochondria in adipose tissue of HIV-infected patients. J Acquir Immune Defic Syndr 2002a; 29: 117-121.

Walker UA, Byrne E. The therapy of respiratory chain encephalomyopathy: a critical review of the past and current perspective. Acta Neurol Scand 1995; 92: 273-280.

Walker UA, Hoffmann C, Enters M, Thoden J, Behrens G, Mitzel SL. High serum urate in HIV-infected persons: the choice of the antiretroviral drug matters. AIDS 2006b; 20: 1556-1558.

Walker UA, Langmann P, Miehle N, Zilly M, Klinker H, Petschner F. Beneficial effects of oral uridine in mitochondrial toxicity. AIDS 2004; 18: 1085-1086.

Walker UA, Setzer B, Venhoff N. Increased long-term mitochondrial toxicity in combinations of nucleoside analogue reverse-transcriptase inhibitors. AIDS 2002b; 16: 2165-2173.

Walker UA, Venhoff N, Koch E, Olschweski M, Schneider J, Setzer B. Uridine abrogates mitochondrial toxicity related to nucleoside analogue reverse transcriptase inhibitors in HepG2 cells. Antivir Ther 2003; 8: 463-470.

Wanner DP, Tyndall A, Walker UA. Tenofovir induced osteomalacia. Clin Exp Rheumatol 2009; 27: 1001-1003.

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