Quantitative Molecular Virology in Patient Management

76 J Clin Pathol 2000;53:76–83 Quantitative molecular virology in patient J Clin Pathol: first published as 10.1136/jcp.53.1.76 on 1 January 2000. Downloaded from management Wolfgang Preiser, Bianca Elzinger, Nicola S Brink The detection of viral genome (that is, viral and speculate on the possible future role of nucleic acid, either DNA or RNA) has gained quantitative genomic testing for viral infec- enormous importance over the last decade in tions. the diagnosis and management of viral infections and diseases (table 1). Nucleic acid testing (NAT) has proven its superiority over Detection and quantification of viral more conventional laboratory techniques in genome: general considerations several areas, for example: In principle, molecular biological assays that + for viruses that cannot be cultured in test for nucleic acid consist of three compo- conventional cell culture systems or those nents: that are hazardous to isolate; + “front end”: sample preparation, often + for clinical sample material of small encompassing extraction and purification of volume or containing only small amounts of nucleic acids from the clinical material; virus; + “middle part”: hybridisation to the target nucleic acid sequence; with genome amplifi- + where antibody seroconversion is delayed after an acute infection; cation techniques this is where multiplica- tion of the target sequence occurs; + in the immunocompromised patient who “back end”: detection and possible quantifi- may have a suboptimal antibody response; + cation of target nucleic acid sequence; signal for the diagnosis of congenital or perinatally + enhancement techniques use this step for acquired viral infections. signal amplification. As well as qualitative analysis, the characteri- One of the most important objectives of the sation of viruses by analysis of the amplified front end is the removal of inhibitors of the region of the genome allows the detection and subsequent steps; this is particularly important identification of viral types or subtypes and with amplification techniques employing enzy- mutants. This is now used to monitor patients matic reactions which may be inhibited by on highly active antiretroviral therapy substances such as haemoglobin, heparin, and (HAART), based on the multitude of resist- bilirubin. Another important feature is the http://jcp.bmj.com/ ance associated mutations that have been char- eYciency of nucleic acid recovery. Some acterised in HIV infected patients receiving protocols “enrich” nucleic acid before 1 antiretroviral treatment. This allows a more extraction—for example, by centrifugation of rational therapeutic approach. Sequence analy- the specimen to concentrate its virus content. sis has also proved to be a valuable epidemio- The front end must be chosen according to the logical tool—for example, to investigate possi- type of clinical specimen (whole blood, ble links between cases of infection, as in plasma, urine, and so on), its quantity and 2 outbreaks of hepatitis B and hepatitis C quality, and must also take into account the on September 28, 2021 by guest. Protected copyright. infection.3 type of viral nucleic acid to be detected (DNA In addition to qualitative analysis and or RNA) and its location (intracellular or genome sequencing, methods have been devel- extracellular). In the context of introducing oped for the quantification of viral genomes. molecular techniques into a routine diagnostic While initially largely applied in the research service, ease of handling, speed, number of field, quantitative NAT has recently been samples to be extracted per run, reproduc- introduced into routine diagnostic virology. In ibility, and cost are obviously important this review, we will briefly introduce the diVer- considerations. The traditional method of ent methods available for viral genome quanti- phenol-chloroform extraction has in some fication, define their place in clinical virology, instances been replaced by faster and more Department of easily handled methods, for example those Virology, Royal Free based on nucleic acid binding to silica. and University College Table 1 Detection of viral genome (qualitative): clinical Following sample preparation and extraction applications Medical School and of the nucleic acid, diVerent techniques are University College + Detection of viruses that cannot easily be cultured: for available for detection and quantification of the London Hospitals NHS example, hepatitis B, human papillomavirus, HIV,hepatitis C target DNA or RNA. Quantification tech- Trust, Windeyer + Where maximum sensitivity for viral detection is required: niques include: Building, 46 Cleveland for example, ocular, amniotic, cerebrospinal fluid for presence of Street, London herpes simplex, varicella-zoster, JC virus, cytomegalovirus, and + target amplification techniques, for example W1P 6DB, UK rubella virus polymerase chain reaction based assays and W Preiser + Where antibody testing is inappropriate: for example, in a nucleic acid sequence based amplification primary infection (before antibody seroconversion; where B Elzinger (NASBA); and NSBrink passively acquired antibody may complicate the diagnosis in babies born to HCV or HIV infected mothers or in + signal amplification techniques, for example immunosuppressed patients branched DNA assays and hybrid capture Correspondence to: + To exclude infectivity: for example of blood and blood products Dr Brink assays. Quantitative molecular virology 77 AP J Clin Pathol: first published as 10.1136/jcp.53.1.76 on 1 January 2000. Downloaded from PCR products Na+ OH– AP probe "capture" denaturing hybridisation B B SA SA AP black 96 well microtitre plate B LIGHT SA photons per second addition of Dioxetane substrate Figure 1 Principle of the enzyme linked oligonucleotide assay (ELONA) for quantification of PCR products. AP,alkaline phosphatase; B, biotin; PCR, polymerase chain reaction; SA, streptavidin. Quantification of viral genome: specific the same test run with the samples to be methods analysed and may be “external” or “internal” TARGET AMPLIFICATION: POLYMERASE CHAIN standards, depending on whether they undergo REACTION amplification in separate reaction tubes or Methods that amplify the target sequence rely whether they are added to each sample in the on enzymatic actions to increase specifically same test tubes. A combination of internal and the number of target sequence copies in the external calibrators may also be used. Com- sample. This requires a careful laboratory set petitive PCR is based on the comparison of the up and stringent practices to prevent contami- amounts of unknown target sequence pro- nation by carryover of the end product duced in relation to a known quantity of a (containing possibly several million times the competitive template.4 Competitive PCR based http://jcp.bmj.com/ original amount of target sequence) into other quantification assays have been developed for a samples or reagents, causing false positive variety of viruses including cytomegalovirus, results. Kaposi’s sarcoma associated herpesvirus The most widely used technique is the (HHV-8),5 and hepatitis C virus (HCV).6 polymerase chain reaction (PCR). Briefly, a Methods of signal detection play an integral DNA target sequence—in the case of RNA role in the accuracy of PCR based quantifica- viruses (or viral messenger RNA) the RNA is tion assays. The optimal time for signal detec- first reverse transcribed into complementary tion is to measure the amount of amplicon on September 28, 2021 by guest. Protected copyright. DNA (reverse transcription or RT-PCR)—is generated before the “plateau” phase of ampli- amplified exponentially by a series of duplicat- fication is reached—that is, while the reaction is ing reactions. Methods of genome quantifica- still in its exponential phase and the amplicon tion using PCR based assays include: concentration proportional to the amount of + limiting end point dilutions of the PCR target sequence at the start. This determines products; the range over which accurate quantification + amplification with external standards; can be achieved (“dynamic range”, mostly + amplification with internal standards; expressed in log10 steps). Detection is often + amplification using a combination of inter- achieved in a microtitre plate format by means nal and external standards; of specific binding of enzyme labelled reagents + competitive amplification methods.4 to the amplified sequence; these can be Quantification of viral genome by “limit complementary nucleic acid sequences dilution” measures the amount of target (probes), anti-nucleic acid antibodies or a sequence present in a sample by subjecting it at biotin–streptavidin system. Finally, an enzy- diVerent dilutions to PCR and then detecting matic reaction leads to a quantifiable colori- the presence of amplicons by electrophoresis metric or chemiluminescent signal (analogous on an ethidium bromide stained agarose gel. to the enzyme linked immunosorbent assay This method of viral genome quantification is (ELISA) for the detection of antigen-antibody labour intensive and slow. Quantification may reactions).7 This is illustrated in fig 1. also be achieved by means of standards Recent additions to quantitative PCR meth- (calibrators) of known concentration, allowing ods are the LightCycler™ technology (Idaho the construction of a calibration curve against Technologies Inc, Roche Molecular Biochemi- which the signal obtained from the samples is cals) and TaqMan® chemistry (Perkin Elmer plotted. The calibrators are normally run on Biosystems), combining

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