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HIV Medicine
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Number of copies Log10 10 1.0 50 1.7 100 2.0 500 2.7 1,000 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 a decrease in CD4 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, i.e. 5.0 logs (sometimes even above 50,000 copies/ml), is generally considered to be high; a value below 10,000 copies/ml (sometimes less than 5,000 copies/ml), low. However, these thresholds are not absolute and can only provide points of reference. The level of the plasma viremia can have very different effects on the immune status of individuals. There are some patients whose CD4 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. Viral load is probably lower overall in women than in men. In a meta-analysis, the difference was 41 % or 0.23 logs (95 % confidence interval 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 discussion. Methods Three methods or assays are currently used to measure viral load: Reverse Transcription Polymerase Chain Reaction (RT-PCR); branched-chain DNA (b-DNA); and, occasionally, Nucleic Acid Sequence-Based Amplification (NASBA). These methods differ both in levels of detection and in the linear range within which measurement is reliable or reproducible (see Table 11.1 below). In all methods, the minute amount of viral RNA must first be amplified to enable measurement. In the case of PCR and NASBA, the viral RNA is transformed in several enzymatic steps and then amplified to measurable amounts. B-DNA does not require this enzymatic step; signal amplification occurs via binding of branched DNA fragments to viral RNA. The actual procedure of PCR is based on real-time detection (TagMan-PCR, Roche) and possess a linear scale from 40-10,000,000 RNA copies/ml. Thus, replacing the ultrasensitive method, which was necessary in the earlier PCR versions (for example Cobas Amplicor). Although intra-assay variability is good for all three methods and one can expect reproducible values, variations in measurements 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 who, after hearing mere numbers, frequently worry unnecessarily or become falsely optimistic should be made aware of this. Considerable differences exist between the results of the three methods (Coste 1996). It is therefore not favorable to change from one method to another. The results obtained by b-DNA are often 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, for example, 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 superior in sensitively measuring even unusual HIV subtypes. All assays have a linear dynamic range, outside of which precise numbers are not possible. The following rule applies: 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). Table 11.1: Methods of measurement, linear range and level of detection should be clearly indicated for the clinician on every test result Company Roche/Abbott Bayer Siemens Organon Method RT-PCR b-DNAn NucliSens HIV-1 QT Linear range of assay 40 - 10,000,000 75 - 500,000 40 - 10,000,000 Comparability Values possibly higher than b-DNA Values possibly lower than PCR values Values approx. same as PCR Advantages Higher specificity, possibly less false positive results than b-DNA (subtypes A-F) Equally good for all subtypes (A-G), technically relatively simple Equally good for all subtypes (A-G), large linear range Influencing factors Apart from methodological variability, a host of other factors may influence levels of viral load, including for example vaccinations and concurrent infections. During active opportunistic infections, viral load is often particularly high. One study showed a 5- to 160-fold elevation during active tuberculosis (Goletti 1996). Viral load can also increase significantly during syphilis (Buchacz 2004). In these situations, determining the viral load does not make much sense. Following immunization 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 for up to four weeks following immunization. It should be noted that not every increase is indicative of virological treatment failure and resistance. Slight transient increases, called blips, are usually of no consequence (see "Goals and Principles of Therapy"). The possibility of mixing up samples always has to be considered. Unexpected results should be double-checked with the laboratory in the first instance, and if no cause is found there, then they should be repeated - people make mistakes. Viral kinetics on HAART 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 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 already reduced by 99 % after two weeks (Perelson 1997). In one large cohort, the viral load in 84 % of patients was already below 1,000 copies/ml after four weeks. The decrease follows biphasic kinetics. In the first phase, within the first three to six weeks, an extremely rapid drop occurs, followed by a longer phase during which the viral load decreases slowly (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 1,000 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. Figure 1: Typical biphasic decrease in viral load on HAART. 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. HAART was not changed. Numerous studies have focused on whether durable treatment success can be predicted early in treatment (Demeter 2001, Kitchen 2001, Lepri 2001, Thiabaut 2000). In a study on 124 patients, a decrease of less than 0.72 logs after one week was predictive of virological treatment failure in more than 99 % of patients (Polis 2001). 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. In the first few months, we measure viral load every four weeks until it has dropped below the level of detection - the most important goal! After this, viral load can be measured every three months. In case of rebound, closer monitoring becomes necessary. Following initiation of therapy, viral load should be below 5,000 copies/ml after one month. Higher values are predictive of failure to reach levels below detection (Maggiolo 2000). Viral load can also be measured 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 routine. Practical tips for dealing with viral load (see "Goals and Principles") § 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 4 weeks with new HAART, until the viral load is below the level of detection. § Then measure viral loads on successful HAART 3 monthly . § Without HAART, measurement every three months is also sufficient. § Don't measure shortly after vaccinations or with concurrent infections. § Unexpected results should be rechecked after 2-4 weeks. § Remember differences between HIV subtypes (in some cases it may be useful to use another method). CD4 cells CD4 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-cell level is the most important parameter or surrogate marker in HIV medicine. It allows for a reliable estimation of the individual risk of developing AIDS. Every HIV patient should have had a CD4-cell measurement within the last six months! Two reference values are generally accepted: above 400-500 CD4 cells/µl, severe AIDS-related diseases are very rare; below 200 CD4 cells/µl, the risk of AIDS-related morbidity increases significantly with increased duration of immunosuppression. However, most AIDS-related illnesses only occur below 100 CD4 cells/µl. Several points should be considered when measuring CD4 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 only one (experienced) laboratory. The same applies to viral load as to CD4 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 cells/µl, the 95 % confidence interval was between 118 and 337 cells/µl (Hoover 1993). Figure 2. Example of variations in absolute CD4 cells/µl over a period of four years. The viral load was continuously below 50 copies/ml; HAART remained unchanged. Measurement of CD4 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 greater decreases in CD4 cells. In such cases, the relative values (CD4 percentages) and the CD4/CD8 ratio (ratio of CD4 to CD8 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 cells/µl, more than 29 % is to be expected, with less than 200 CD4 cells/µl 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 cells, any decisions involving treatment should be carefully considered - if in doubt, it is better to check the values one more time! The remaining differential blood count should also be scrutinized carefully: is leucopenia or leukocytosis present? Clinicians sometimes forget that the result of the CD4-cell count is of great importance to the patient.and oftens associated with a great deal of stress for many patients. Insensitively informing the patient of a supposedly bad result can lead to reactive depression. From the start, patients must be informed about the possible physiological and method-related variability of laboratory tests. A drop from 1,200 to 900 cells/µl is often of no importance, but for the patient, this information can be a "disaster!" 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. In our opinion,. once CD4-cell counts are within the normal range with adequate viral suppression, half-yearly measurements are adequate. Indeed, in such cases, the probability of CD4 cells dropping to values below 350/µl is extremely low (Phillips 2003). Influencing factors Several other factors influence CD4 counts apart from laboratory-related variables. These include concurrent infections, leucopenia of varying etiology, spleenectomy and steroids or other immunosuppressive therapies. Extreme exertion, surgical procedures or pregnancy can also lead to lower values. Even diurnal variation occurs; CD4 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 cells on HAART Similarly to viral load, a biphasic increase in CD4 cells occurs following the initiation of HAART (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 1,000 patients, the CD4 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 cells/µl per month (Le Moing 2002). The initial rapid increase in CD4 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 load suppression, or whether a plateau is reached after three to four years, beyond which there is no further improvement (Smith 2004, Viard 2004). Several factors can influence the extent of immune reconstitution during HAART. The degree of viral suppression is crucial - the lower the viral load, the more pronounced the effect (Le Moing 2002). The absolute increase is higher if CD4-cell counts were high at the start of HAART (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 cells is likely to be (Kolte 2002)., CD4 cells in older patients do not increase as much as those in younger ones, due to age-related degeneration of the thymus (Viard 2001). However, we have seen both 20-year-old patients with very poor CD4-cell count recovery and 60-year-old patients with very good, above average increases in CD4 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 the ddI+tenofovir combination are associated with less immune reconstitution than others. Immunosuppressive concurrent medications should also be considered (see "Goals and Principles of Therapy"). Beyond the measurement of the CD4-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 (Gorochov 1998, Lederman 2001, Lange 2002, review in 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 (few) patients, who are at risk of developing opportunistic infections despite good CD4 counts. Practical tips for dealing with CD4-cell counts § 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 cells are sufficient. § In the presence of good viral suppression, CD4 cells (not viral load!) may also be checked less frequently. § CD4-cell count and viral load should be discussed with the physician. Do not leave patients alone with their results. Other routine checks - what else should be monitored? Besides the CD4-cell count and viral load, several other parameters should be monitored in the HIV 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 and, depending on the problem, further investigations are required. Additional tests may also be necessary. A complete physical examination should be performed regularly, and this often leads to the discovery of important findings such as Kaposi lesions, condyloma or mycoses (thrush!). The lower the CD4 cell count, the more frequently patients should be examined. 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 cells 2-4 x 2-4 x Lipids 1-2 x 1 x Physical examination 2-4 x 1-2 x Gynecological examination 1 x 1 x Fundoscopy if CD4 cells < 200/µl 2-4 x 4 x In patients with less than 200 CD4 cells/µl, we recommend fundoscopy every three to six months to exclude CMV retinitis. Close cooperation with an HIV-experienced ophthalmologist is important. The better the CD4 cells, the less often fundoscopy is requiredy - 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 (see also the European guidelines: http://hiv.net/link.php?id=185). Many experts now also recommend rectal examination (including proctoscopy) for the early detection of precancerous lesions and anal cancer. However, such guidelines or recommendations are interpreted very differently. In our experience, 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 also "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. Therapeutic drug monitoring (TDM) Individual plasma levels of many antiretroviral drugs may vary considerably for differing reasons (e.g. compliance, metabolism, absorption). But, sufficient plasma levels are essential for success of virological 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). 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. The measurement of drug concentrations in serum or plasma (therapeutic drug monitoring, TDM) has therefore become an important tool for monitoring therapy. The best reviews are to be found in Back 2002, Burger 2002, and Clevenbergh 2004. Due to the increasing complexities of antiretroviral combinations, TDM of protease inhibitors and NNRTIs will probably become more important in the future. Several problems associated with TDM are limiting its broader use. The measurement of nucleoside analogs, for example, is senseless since they are converted to the active metabolites only intracellularly. Intracellular measurements are difficult and will not be available in routine clinical practice. 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 are available, proving the clinical value of TDM, both the measurement and interpretation of the results should be left to specialized centers. TDM is currently recommended in the following situations: § Complex drug combinations § Concomitant medications that could lead to interactions or reduced efficacy § Suspected absorption problems § Pregnancy References 1. 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. http://amedeo.com/lit.php?id=10860900 2. Back D, Gatti G, Fletcher C, et al. Therapeutic drug monitoring in HIV infection: current status and future directions. AIDS 2002, Suppl 1:S5-37. Review. http://amedeo.com/lit.php?id=12035820 3. 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. 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