Institute for Medical Virology, Klinikum der J. W. Goethe-Universität, Paul Ehrlich-Strasse 40, D-60596 Frankfurt am Main, Germany
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Abstract |
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Introduction |
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While the aim of antiviral treatment is to maintain or to improve the patients 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 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 referred to as viral load (VL) testing, has now firmly established itself in routine diagnostic virology.
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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 individuals 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 ultrasensitive HIV VL tests.
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HIV type 1 (HIV-1) |
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Owing to increasing concern about falsely low HIV-1 VL results obtained when patients with non-clade-B subtypes are 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
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HBV |
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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, 8590% 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 105106 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
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HCV |
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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 HCV-infected 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 12 weeks; in the majority of cases the HCV RNA level falls to below the detection limit of PCR (1001000 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 (612 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 56% (range 025%).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 >106107 copies/mL.30 Co-infection with HIV leads to higher transmission rates of 1417% (range 536%), probably because of the increase in maternal HCV RNA levels seen in co-infected subjects.31
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
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Human CMV |
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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 non-quantitative surveillance methods still lead to substantial over-treatment.39
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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 assays 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.
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Conclusions |
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Besides providing prognostic information on individual cases, particularly for HIV and human CMV, genome quantification plays a most important role in monitoring of the patients 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 individuals infectivity, be it in the health care setting (transmission risk of blood-borne viruses) or in infected pregnant women.
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 inter-laboratory, reproducibility.44 Further progress in assay design, increasingly available international quantification standards46 and newly developed proficiency testing programmes47 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 EpsteinBarr virus VL.
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Footnotes |
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References |
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