the example of HIV, HBV, HCV and CMV

Journal of Antimicrobial Chemotherapy (2002) 49, 713–721 DOI: 10.1093/jac/dkf050

Review Viral genome quantification as a tool for improving patient management: the example of HIV, HBV, HCV and CMV Annemarie Berger and Wolfgang Preiser* Institute for Medical Virology, Klinikum der J. W. Goethe-Universität, Paul Ehrlich-Strasse 40, D-60596 Frankfurt am Main, Germany In recent years, new therapeutic options have led to enormous improvements in the management of certain chronic viral infections. Nevertheless, it has also become clear that such treatments require careful consideration and follow up. At the same time, a number of new technologies have been developed to measure quantitatively the concentration of viral genome in the patient’s body fluids. Initially, these tests yielded important insights into the pathogenesis of viral infections and, in the case of human immunodeficiency virus (HIV), in fact revolutionized our understanding of its natural history. In addition, however, such ‘viral load’ tests have become vital tools in patient management; formerly pure research tools, they are now widely used in routine virological diagnosis, and a number of commercial assays have become available. In clinical virology, viral load testing serves four purposes: for diagnosis; to assess the patient’s prognosis; as therapeutic markers to monitor the effect of antiviral treatment; and to estimate the patient’s infectivity, i.e. the risk of transmission. In this review paper, we summarize the current role of viral genome quantification in the clinical management of patients infected with HIV, hepatitis B virus, hepatitis C virus and those at risk of developing human cytomegalovirus-related diseases.

Introduction Viral infections, including those taking a chronic course, are becoming increasingly amenable to specific antiviral treatment. Antiviral agents have considerably improved the prognosis for many human immunodeficiency virus (HIV)infected patients, at least in societies that can afford it, as is evident from the annual numbers of AIDS cases and deaths due to HIV disease reported from industrialized countries (Figure 1).1 There have also been significant advances in the management of patients with chronic hepatitis B virus (HBV) or hepatitis C virus (HCV) infections and of immunocompromised patients at risk of developing human cytomegalovirus (CMV)-related diseases. However, current antiviral therapy is not without problems. As well as drug toxicity, undesired side effects (often leading to non-compliance) and cost, the development of antiviral resistance is a major obstacle to achieving a sustained

treatment response. Therefore, not only must the decision of whom to treat, when to start and what drug or combination of drugs to use be based on as much relevant information as can possibly be obtained, but each individual patient must be monitored for his or her treatment response in order to be able to react accordingly. While the aim of antiviral treatment is to maintain or to improve the patient’s state of health and is therefore based on clinical observations, the assessment of therapeutic success often cannot be made on clinical grounds alone. This is particularly so when the therapeutic goal is to prevent the development of severe illness in the future; here, markers are needed on a continuous basis to guide therapeutic decisions. Such surrogate markers may be CD4 lymphocyte counts in HIV infection or liver enzyme levels in chronic hepatitis, although increasingly virus quantification is employed as a more direct measure of virus replication. There are a number of conventional, non-molecular methods for quantifying

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*Corresponding author. Tel: +49-69-6301-4303; Fax: +49-69-6301-6477; E-mail: [email protected] . ... ... ... .... ... ... ... ... ... ... ... ... ... .... ... ... ... ... ... ... ... ... ... ... .... ... ... ... ... ... ... ... ... ... .... ... ... ... ... ... ... ... ... ... .... ... ... ... ... ... ... ... ... ... .... ... ... ... ... ... ... ... ... ... .... ... ... ... ... ... ... ... ... ... .... ... ... ... ... ... ... ... ... .

713 © 2002 The British Society for Antimicrobial Chemotherapy

A. Berger and W. Preiser

Figure 1. AIDS cases and deaths, 1991–2000, in Western Europe. Data reported by 31 December 2000: AIDS cases by year of diagnosis adjusted for reporting delays; AIDS deaths by year of report. Bars, AIDS deaths; line, new AIDS cases. Source: European Centre for the Epidemiological Monitoring of AIDS.1

viruses from clinical materials, such as quantitative virus culture or quantitative viral antigen detection; while these may be extremely useful in some contexts, they may also be either impossible (HBV, HCV) or cumbersome (HIV) to perform, too slow (CMV), or of insufficient sensitivity (HBV, CMV) to be clinically useful. Methods for the detection of viral genome (nucleic acid testing, NAT) have considerably extended the diagnostic repertoire of virological laboratories in recent years, proving superior to conventional techniques in many circumstances. Table 1 gives an overview of currently accepted applications of viral NAT. In addition to qualitative analysis and genotyping, the quantitative detection of viral genome, often re-

ferred to as ‘viral load’ (VL) testing, has now firmly established itself in routine diagnostic virology. While initially used mainly for research purposes, the ability to accurately determine VL has led to great advances in our understanding of the natural history and pathogenesis of a number of virus infections. Assays for the quantification of viral genome (VL tests) have become an integral part of patient management, particularly in the context of antiviral therapy. They meet the increasing need for virological assays that combine maximum sensitivity with a high prognostic predictive value regarding disease development, to address the questions of when to start antiviral therapy and how to assess its success. Viral genome quantification is employed for four routine diagnostic indications: as a diagnostic marker, as a prognostic marker for assessing disease progression, as a therapeutic marker for monitoring the efficacy of anti(retro)viral chemotherapy and for predicting treatment failure, and to assess an individual’s infectivity, i.e. the risk of transmission.2 In addition, the ability to accurately determine VL has led to great advances in our understanding of the natural history and pathogenesis of a number of virus infections. Extensive up-to-date reviews cover the available methodologies (Table 2).3,4 Almost always, some form of amplification must or should be utilized, as the concentrations of viral genome present are too small to be detectable without it. Furthermore, there is an ongoing push for ever greater sensitivity of quantitative NAT, i.e. for reducing the lower limit of sensitivity even further, to as little as 50 copies of viral genome per millilitre of patient sample (plasma) in the case of currently available ‘ultra’sensitive HIV VL tests.

Table 1. Currently established clinical uses of viral NAT Detection of viral genome (qualitative: ‘yes or no’ result): presence of viral genome as marker of infection virus not or hardly cultivable in vitro or detectable by other methods; e.g. HBV, HCV, papilloma viruses, HIV small sample volume or low infectious dose (resulting in insufficient sensitivity of virus isolation or antigen testing), e.g. ocular, amniotic or cerebrospinal fluid for herpes simplex, varicella zoster, JC, CMV and (prenatally) rubella virus when antibody testing fails; e.g. acute infection (‘diagnostic window’), passively acquired antibodies (e.g. newborn babies of HIV- or HCV-infected mothers), immunosuppressed patients to exclude infectivity, e.g. of blood and blood products Quantification of viral genome (VL testing: ‘how much?’) diagnostic marker, e.g. CMV VL in suspected CMV disease prognostic marker, e.g. HIV VL testing therapeutic marker for the monitoring of anti(retro)viral chemotherapy, e.g. patients with chronic HIV, HBV or HCV infection undergoing therapy to assess infectivity; e.g. risk of vertical (mother-to-child) transmission of HIV Characterization of viral genome (genotyping to detect virus mutants or strains) prognostic marker (different virulence of different variants or strains), e.g. HCV, HBV epidemiology, e.g. to elucidate chains of infection, e.g. HBV, HCV, HIV, rotavirus therapeutic marker for the monitoring of anti(retro)viral chemotherapy: development of resistance-associated mutations?, e.g. HIV, HBV

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Viral genome quantification and patient management Table 2. Currently available methods for the detection and quantification of viral nucleic acid sequences (i) No amplification Southern (DNA) or northern (RNA) blot hybridization dot blot hybridization in situ hybridization (ii) With amplification (a) signal amplification branched DNA signal amplification (bDNA) ‘hybrid capture’—signal amplification (b) target sequence amplification PCR and reverse transcription (RT)–PCR ligase chain reaction (LCR) nucleic acid sequence-based amplification (NASBA, TMA), self-sustained sequence replication (3SR)

Table 3. Commercially available test systems for the quantification of HIV, HCV, HBV and CMV, and their basic characteristics: sample volume, sensitivity (lower limit of detectability), linear range and ability to quantify different viral types and subtypes Target amplification methods Virus HIV sample volume sensitivity (copies/mL) linear range (sub)type detection HCV sample volume sensitivity (IU/mL) linear range subtype detection HBV sample volume sensitivity (copies/mL) linear range CMV sample volume sensitivity (copies/mL) linear range

Signal amplification methods

q-PCR

LCx

NASBA

bDNA

hybrid capture

regular sensitive 200 µL 500 µL 500 50 7.5 × 105 1 × 10 5 HIV-1 excl. O

1000 µL 50 1 × 106 HIV-1

100–2000 µL 40 10 × 109 HIV-1 excl. O

1000 µL 50 5 × 105 HIV-1

n.a. n.a. n.a. n.a.

n.a. n.a. n.a. n.a.

200 µL 600 8.5 × 105 all known subtypes

n.a. n.a. n.a. n.a.

n.a. n.a. n.a. n.a.

50 µL 500 8 × 106 all known subtypes

200 µL 200 2 × 105

n.a. n.a. n.a.

n.a. n.a. n.a.

10 µL 0.7 × 106 5 × 107

n.a. n.a. n.a. n.a. regular 30 µL 0.5 pg/mL 6000 pg/mL

n.a. n.a. n.a. n.a. sensitive 1000 µL 4.700 5.6 × 107

200 µL plasma 400 2 × 105

n.a. n.a. n.a.

n.a. n.a. n.a.

n.a. n.a. n.a.

3.5 mL whole blood 700 10 7

n.a., not available; excl., excluding.

Assays employing target [PCR and nucleic acid sequencebased amplification (NASBA, TMA)] or signal [branched chain signal amplification (bDNA) and the hybrid capture system (HCS)] amplification techniques are now commercially available for the quantification of viral burden in patients infected with HIV, HBV, HCV and CMV. Table 3 lists the assays on the market (although these are not necessarily available in all countries) and their basic characteristics. In this review, we will attempt to define briefly the current role of genome quantification in clinical virology for each of these four viruses.

HIV type 1 (HIV-1) Although a valuable marker of disease stage and immunological function, the CD4+ lymphocyte count on its own has been shown to be inadequate as a prognostic and therapeutic marker, and should be supplemented by information on the level of viral burden. A number of assays are available for quantifying HIV-1 RNA levels in plasma, and guidelines have been issued for the reporting of test results.5 Owing to increasing concern about falsely low HIV-1 VL results obtained when patients with non-clade-B subtypes are

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A. Berger and W. Preiser Table 4. Influence of viral load on the risk of mother-to-child transmission of HIV Reference

No. of mother–child pairs

Fang et al.11

30

Dickover et al.12

97

Sperling et al.13

419

HIV viral load (copies/mL)

Prophylaxis

Transmission rate (%)

100 000 80 000 5000

none none none none none none none zidovudine placebo zidovudine placebo zidovudine

3 80 10 20 50 66 100 3.7 12.5 9.1 19.6 7.5

tested (HIV-1-B accounting for the vast majority of infections in industrialized countries), great efforts have gone into improving the detection of various subtypes. Nevertheless, the commercial test systems are still unsuitable for HIV-1-O and HIV-2 strains (Table 3).

Management of HIV-infected patients HIV-1 VL testing has now been widely introduced into the management of HIV-1-infected patients in affluent countries.6 Several prospective analyses of HIV-1-infected adults have established the importance of HIV-1 VL and CD4+ lymphocyte count as independent predictors of HIV-1 disease progression. Whilst the CD4+ lymphocyte count is more a marker of short-term risk than a long-term predictor of progression, the HIV-1 VL is important both as a short-term marker of current disease progression risk and as a long-term predictor.7 Quantitative NAT has made previously used methods obsolete.8 HIV-1 VL is one of the markers used to guide initiation of highly active antiretroviral therapy (HAART). The aim of HAART should be to suppress viral replication as fully as possible, in order to avoid the emergence of drug-resistant virus mutants and to attain durable virological and clinical responses, monitored by serial HIV-1 VL testing. To take account of the extreme reduction in HIV-1 VL aimed for, highly (ultra) sensitive HIV quantification assays have been developed, permitting a lower detection limit of as low as 50 HIV-1 RNA copies/mL. However, even a sustained ‘optimal’ response (i.e. HIV-1 VL persistently below 50 copies/mL) does not indicate eradication of the virus. For clinical studies, HIV-1 VL is now recognized and widely used as a surrogate marker, reflecting traditional endpoints such as survival time, time to onset of disease, etc.9,10

Estimation of infectivity Although the risk of vertical HIV transmission from mother to child is apparently correlated with the exposure dose and thus maternal VL, no reliable threshold value of HIV-1 RNA to differentiate between transmitters and non-transmitters has so far been determined (Table 4). In children with vertically acquired HIV infection, best diagnosed by (qualitative) NAT, the measurement of VL is less reproducible than in adults, and the magnitude of spontaneous biological variation is higher.14 The efficacy of horizontal transmission of HIV depends upon a variety of factors, including the size of the inoculum, in part determined by VL. Therefore, VL testing in a needlestick ‘donor’ may be useful to assess the risk to the recipient more accurately.

HBV While active HBV infection is defined by the presence of HBV surface antigen (HBsAg) in plasma, the secretory version of HBV core protein, HBeAg, serves as a marker for viral replication and can easily be detected by enzyme immunoassay. However, the absence of HBeAg in serum or plasma does not exclude viral replication.15 We detected HBV DNA in 37% (n = 73) of 196 HBeAg-negative, HBsAg-positive (and anti-HBe-positive) patients using a hybridization assay (>4700 copies/mL = 0.018 pg/mL).2 Reasons may be the relatively low sensitivity of current HBeAg tests or mutations in the pre-core region of the HBV core gene inducing a stop codon that inhibits the production of HBeAg but does not influence viral replication. Nevertheless, there is a strong correlation between the detection of HBeAg and the HBV DNA level: significantly higher HBV DNA concentrations (P < 10–6, Wilcoxon–Mann–Whitney test) are observed in HBeAg-positive than in HBeAg-negative patients.

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Management of HBV-infected patients The correlation between HBV DNA serum levels and severity of liver disease is rather low. However, serial quantifications of HBV DNA levels during antiviral therapy, although more cumbersome than HBeAg testing, permit the early identification of non-responders, thus avoiding ineffective and expensive therapy.16 Quantitative HBV NAT also plays a role in assessing patients with isolated antibody reactivity to HBV core antigen (anti-HBc), some of whom may be highly viraemic.17 In patients undergoing antiviral therapy against chronic HBV infection, HBV DNA quantification is useful to assess treatment response, and serves as a surrogate marker for the emergence of resistant viral strains in patients treated with the nucleoside analogue lamivudine (3TC) or famciclovir. This is particularly relevant in the context of liver transplantation for HBV-related hepatic damage, where re-infection of the transplanted organ poses a serious problem.

Estimation of infectivity An important indication for HBV VL testing is the assessment of the infectivity of HBV carriers. Without intervention, >90% of HBeAg-positive female chronic HBV carriers transmit the virus to their infants;18 of these, 85–90% develop chronic HBV infection, in most cases asymptomatic, themselves, thus perpetuating the infection in high-endemicity settings. Immediate post-partum immunization of the infant can efficiently prevent transmission. For the reasons outlined above, HBeAg-negative individuals carrying HBV mutants may also reach high titres. However, HBV DNA quantification does not yet form an integral part of the management of pregnancies of HBV carrier women. HBeAg-positive physicians will not be allowed to exercise so-called ‘exposure-prone’ procedures (EPP), i.e. operations, etc., carrying a higher risk of blood contact. However, HBeAg-negative health care workers have on occasion transmitted HBV infection. A more reliable estimate of the infectivity can be obtained by testing serum concentrations of HBV DNA.19 In Germany, health care workers with no more than 105–106 HBV genome copies/mL used to be allowed to continue to practice without restrictions;20 however, more recent recommendations stipulate that only those immune to HBV should perform EPP.21 In the UK, a cut-off level of 103 genomes/mL of plasma is now in place for those undertaking EPP.22 When applying such guidelines and regulations, it has to be borne in mind that different assays may give different results that are not always readily comparable.23

HCV Chronic infection develops in the majority of those infected with HCV and is characterized by persistent viraemia. This is

diagnosed by the detection of HCV RNA, which has become the standard diagnostic marker for active infection, besides antibody serology, as a marker of exposure. While recent reports have suggested a role for HCV core antigen testing for the detection of recent HCV infection before antibody seroconversion, when there is a high virus titre, its sensitivity is insufficient to monitor chronically HCVinfected patients.49 In contrast, a number of highly sensitive assays for HCV RNA have been developed,24 some of which are marketed and widely used. Whereas, perhaps surprisingly for an RNA virus, storage conditions and speed of transport do not seem to be major confounding factors,25 results obtained by different methods and by different laboratories are not easily comparable. The recent introduction of a WHO standard for HCV RNA26 will hopefully lead to improvements in this respect.

Management of HCV-infected patients There does not seem to be a reliable association between serum HCV RNA levels and severity of liver disease in infected individuals. High fluctuations of HCV RNA serum levels were reported by several studies. Likewise, combined population data indicate that the likelihood of progression appears to be independent of genotype or VL, but increases with alcohol intake, male sex, age >40 years at infection and co-infection with HIV or HBV.27 Although of limited clinical relevance for prognosis, HCV VL is a valuable predictive marker for the outcome of antiviral therapy. Patients with a high VL (i.e. >2 million copies/ mL) at baseline frequently have a poorer response to interferon therapy. Under effective antiviral treatment, a significant reduction of HCV load is observed after 1–2 weeks; in the majority of cases the HCV RNA level falls to below the detection limit of PCR (100–1000 copies/mL). Therefore, a follow up of HCV load at regular intervals is now part of routine patient management to assess therapeutic success. However, HCV NAT has to extend beyond the end of the treatment phase (6–12 months) to assess sustained viral clearance.

Estimation of infectivity The average rate of HCV acquisition among infants born to HCV-positive, HIV-negative women is 5–6% (range 0–25%).28,29 Maternal VL is an important risk factor for vertical transmission: mother-to-infant transmission is more likely if the maternal serum HCV RNA level is >106–107 copies/mL.30 Co-infection with HIV leads to higher transmission rates of 14–17% (range 5–36%), probably because of the increase in maternal HCV RNA levels seen in co-infected subjects.31

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A. Berger and W. Preiser Transmission from HCV-infected surgeons to patients, although rare on the whole, has been documented repeatedly. Therefore, VL testing is now part of several national guidelines regarding the issue of HCV-infected health care workers.32

Human CMV CMV establishes a lifelong state of persistent (‘latent’) infection in between 60% and >80% of humans, from which it is able to reactivate, potentially causing severe disease, particularly in immunocompromised hosts. Bone marrow transplant (BMT) recipients, for instance, have a 15–35% chance of developing CMV disease and, before the advent of HAART, about one in three AIDS patients would suffer CMV retinitis.33 CMV VL has been shown to be correlated with the risk of developing clinical CMV disease for a number of risk groups, such as HIV-infected individuals,34 recipients of solid organ transplants35 and infants with intrauterine infection.36 Despite a growing number of available drugs with potent anti-CMV activity, the antiviral treatment of established CMV disease is still problematic. On the other hand, the prophylactic administration of anti-CMV agents to patient groups known to be at higher risk of CMV-associated disease is able to prevent the development of CMV disease in most cases. However, this means considerable ‘over-treatment’ of patients who would never develop the disease. Anti-CMV prophylaxis poses problems of practicability, cost and, most importantly, drug toxicity. It has been shown that in BMT recipients, the prophylactic use of ganciclovir, while reducing the incidence of CMV disease, does not confer an overall benefit: owing to its bone marrow toxicity, the neutropenic phase is prolonged and the incidence of bacterial and fungal infections is thus increased.37,38

Management of patients at risk for human CMV disease For the reasons outlined above, alternative management strategies (Table 5) are increasingly used nowadays, particularly in BMT units. So-called suppressive or pre-emptive (also termed ‘early’) therapy is based upon the regular monitoring for evidence of active CMV replication, through the sensitive detection of (actively replicating) CMV; if and when active infection is found, this is treated with anti-CMV agents, before it leads to overt disease. This approach avoids the toxicity and cost of anti-CMV prophylaxis given to all patients, while aiming to intervene before overt disease develops. While non-molecular techniques (such as rapid viral culture or antigen detection in blood leucocytes) may suffer from low sensitivity in some patient groups such as BMT recipients,48 highly sensitive qualitative CMV NAT has a comparatively low positive predictive value regarding the development of disease, due to the ubiquitous nature of the virus and frequent asymptomatic reactivation or even the detection of latent virus. Thus, many currently employed nonquantitative surveillance methods still lead to substantial over-treatment.39 The quantification of CMV in clinical samples as a marker of the degree of viral replication allows better identification of patients at high risk of CMV-related disease.40,41 In BMT recipients, CMV VL testing as part of routine surveillance can further refine pre-emptive therapy.42 In recipients of solid organ transplants, CMV infection is common, but often without clinical correlation; genome quantification allows more accurate assessment of the situation.50 One particular area of interest is immune reconstitution in AIDS patients with CMV retinitis started on HAART; in most cases, CMV-specific immunity is regained sufficiently to allow cessation of secondary anti-CMV prophylaxis, but this is not invariably so.43 Here, the determination of CMV VL together with HIV-1 VL may be helpful in deciding whether or not to discontinue anti-CMV drugs.

Table 5. Options for the management of CMV infection and disease in high-risk patient groups (i) Antiviral treatment of manifest CMV-associated disease avoids ‘unnecessary’ exposure to antiviral drugs often without success (ii) Prophylactic therapy: all patients at risk avoids CMV disease exposes patients to antiviral drugs who would not develop disease anyway (iii) Suppressive/pre-emptive therapy based upon detection of active CMV infection in peripheral body sites (suppressive therapy, e.g. from urine, swabs) or systemically (pre-emptive therapy, e.g. from blood) designed to combine the advantages of (i) (limiting treatment to those at higher risk, i.e. avoiding unnecessary exposure to antiviral drugs) with those of (ii) (avoiding CMV-associated disease by preventing the development of overt disease by treating infection rather than disease) requires reliable, fast and sensitive methods for regular surveillance of patients at risk to detect active infection

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Viral genome quantification and patient management Ideally, defining a ‘threshold value’ of CMV load might allow a distinction between commonly occurring, clinically irrelevant CMV infection (due to true latency, persistence or asymptomatic reactivation) and levels of active CMV replication likely to lead to clinical disease. However, such a ‘threshold level’ not only needs to be defined separately for each risk group, but also has to take into account patient factors, such as pre-conditioning and immunosuppressive medication in transplant recipients, as well as assay-related factors,44 e.g. the use of whole blood or the leucocyte fraction (potentially containing latent virus) or plasma (detecting only extracellular virus as a marker of active viral replication). Furthermore, the design of studies to define an assay’s predictive value as regards CMV disease has become difficult, if not impossible, as it would be unethical to withhold timely treatment, on the basis of positive CMV surveillance results, for a potentially life-threatening disease. Besides its roles in diagnosis and as a prognostic marker, CMV VL testing is useful for monitoring the response to therapeutic interventions. The development of antiviral resistance following the initiation of anti-CMV therapy is not infrequent and poses a potentially important problem;45 therefore, CMV VL testing also serves a useful function as a surrogate marker for the emergence of antiviral drug resistance.

Practical problems still waiting to be resolved are virus strain-related differences in quantitative results and a relatively low degree of intra- and inter-assay, as well as interlaboratory, reproducibility.44 Further progress in assay design, increasingly available international quantification standards46 and newly developed proficiency testing programmes 47 will hopefully lead to improvements here. In addition, VL testing will be used increasingly for other situations and viruses. One example is assessing the risk of transplant recipients for developing post-transplant lymphoproliferative disease by monitoring their Epstein–Barr virus VL.

Conclusions

5. Centers for Disease Control and Prevention. (2001). Guidelines for laboratory test result reporting of human immunodeficiency virus type 1 ribonucleic acid determination: recommendations from a CDC working group. MMR Morbidity and Mortality Weekly Report 50, 1–14.

Viral genome quantification has within a few years become an integral part of the clinical management of patients suffering from infection with HIV, HBV, HCV or human CMV. To appreciate the speed with which this development has taken place, it is worth bearing in mind that PCR was only developed little more than 10 years ago. Since then, the modern molecular biological techniques have evolved from being elusive and research tools into widely used and also rather reliable virological routine diagnostic tools. Besides providing prognostic information on individual cases, particularly for HIV and human CMV, genome quantification plays a most important role in monitoring of the patient’s response to antiviral treatment. VL testing assesses the success of antiviral therapy, including, but unable to distinguish between, different factors involved. These include treatment failure due to viral (development of antiviral resistance) and host factors (one of which is non-compliance). Several studies have proven the value of VL determination as a surrogate marker for clinical markers of therapeutic success. The increasing availability and clinical use of potent anti(retro)viral chemotherapy has sparked the development of a variety of commercial assays for viral genome quantification. Furthermore, hitherto less well established uses of VL testing include the assessment of an individual’s infectivity, be it in the health care setting (transmission risk of blood-borne viruses) or in infected pregnant women.

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