Literature review > Issue_1 > Review Aldea et al. 

One goal of a modern diagnostic virology laboratory is to provide rapid viral diagnoses to effect the clinical management of viral illnesses and minimize empirical antibiotic exposure for viral infections that are clinically misdiagnosed as bacterial infections [1]. Towards this end, the increased use of real-time polymerase chain reaction (PCR) amplification of viral nucleic acid has lead to a revolution in diagnostic virology, with the promise of providing sensitive and specific viral diagnoses within hours rather than days. "Real-time PCR" refers to the continuous monitoring of nucleic acid amplification and detection in a closed system with quantification of the continuously monitored fluorescent output signal.

The paper by Aldea and coworkers, evaluated a real-time PCR assay for the rapid detection of human herpes simplex virus (HSV) in genital lesions [2]. A rapid and sensitive assay for the detection of HSV infection is of critical importance for diagnosing central nervous system and neonatal infections; however, the diagnosis of HSV genital ulcer disease does not generally have a similar urgency. So, why is this paper of interest to the clinician and laboratory professional?

There are three reasons: First, the authors applied a simple and relatively inexpensive assay format for real-time PCR using LightCycler™ technology, which adds to the growing literature on the potential utility of this and similar technologies for the diagnostic virology laboratory. Second, they compared the real-time PCR detection of HSV DNA to the more traditional culture isolation of this virus - the gold standard for HSV diagnosis - and underscored the increased sensitivity of real-time PCR over viral culture. Unfortunately, a comparison was not done between real-time DNA PCR and the rapid immunofluorescent antigen (IFA) detection of HSV from ulcer scrapings, which also provides an answer within the same time frame as real-time PCR - within a few hours after the specimen reaches the laboratory - but with a lower sensitivity for HSV than either culture or real-time DNA PCR. Finally, the detection of HSV by real-time PCR is applicable to viral diagnostics in the developing world because of superior sensitivity of real-time PCR compared to viral culture [3].

Several different assay chemistries with a similar detection method are in use for real-time PCR detection. For example: (i) DNA binding fluorophores (LightCylcer™); (ii) 5'-endonuclease with a fluorogenic reporter and quencher oligoprobe (TaqMan®); (iii) adjacent linear and hairpin oligoprobes (molecular beacons); and (iv) self-fluorescing amplicons (sunrise primers, Amplifluor™ hairpin primers, and scorpion primers) [4]. All of these PCR detection methods rely on the measurement of fluorescence during the PCR reaction with the amount of fluorescence being proportional to the amount of PCR product produced. Aldea and coworkers used the simplest and cheapest assay format (method (i)), which is based on an intercalating dye (SYBR green I) specific for double stranded DNA or amplified product. The advantage of this format is that the reaction is conducted in a closed capillary tube, there is no need for any additional fluorescence-labeled oligonucleotides, and the specificity of the product can be verified by melting-curve analysis. In this regard, the authors demonstrated adequate sensitivity, specificity and quantification of the HSV DNA PCR amplification, although the assay was not able to differentiate HSV-1 from HSV-2. This is not a major limitation but would require a further type-specific HSV PCR or IFA staining of cultured virus.

Other real-time PCR technologies are based on the use of additional fluorescence-labeled oligonucleotides. Fluorescence is released (eliminating fluorescence resonance energy transfer (FRET) quenching) after cleavage of the probe (hydrolysis based on the 5'-3' nuclease repair function of the recombinant Taq DNA dependent DNA polymerase from the thermophilic archaea bacterium Thermus aquaticus, method (ii)). Other fluorescent probes provide similar detection but rely on the probes hybridization to the exponentially produced amplicons, where the hybridized probe has a greater fluorescence than the free probe in solution (Eclipse Probes), or hybridization of one or two oligonucleotide probes to the amplicon (method (iii)). The additional sequence-specific probes increase the specificity of the PCR product. The remaining technology uses primers with attached fluorescence-labeled tails that hybridize to amplified targets and are the basis of self-probing amplicons that may eventually provide for more rapid assays, perhaps within minutes (method (iv)).

There are several advantages and limitations to real-time PCR [5]. Advantages includes: wide dynamic range over 7 to 8 log10 nucleic acid copies/mL; high sensitivity to <5-10 copies/reaction; high precision with < 2% CV; no post-PCR analysis; and minimized risk for amplicon contamination because the amplification and detection occur in a sealed reaction vessel. High throughput and simultaneous detection of different targets through multiplexing is also possible. The limitations include: external standards used for calibration; increased risk of false-negative values without use of an internal standard; carry-over contamination of amplicons; and because specific knowledge of target oligonucleotide sequence is required, false-negative results may arise with an unexpected viral pathogen or high variability in the expected target sequence. As such, there will be a continued role for traditional viral culture in reference viral diagnostic laboratories; e.g., the initial isolation by tissue cell culture and subsequent identification of the new human coronavirus that causes the severe acute respiratory syndrome (SARS).

Importantly, real-time PCR does not provide direct information about the infectivity of the viral pathogen in question nor on transmissibility. Although Aldea and co-workers addressed the issue of HSV culture-negative/HSV DNA PCR-positive specimens, Wald and coworkers extended this comparison and performed the largest published evaluation of HSV detection by DNA PCR and culture [3]. In the Wald et al. study of >36,000 specimens from 296 subjects, HSV was detected by real-time PCR, (method (ii)) in 12.1% of specimens and isolated by culture in only 3.0% of specimens - a four-fold greater sensitivity by real-time PCR detection. For HSV DNA in amounts ?104 copies/mL of swab specimens, 45% of samples resulted in virus isolation compared to only 6% of samples with fewer copies of HSV DNA. Another important observation was a seasonal variation in culture positives with as much as a 50% reduction in virus isolation by culture - but not detection by real-time PCR - attributable to a seasonal effect on specimen viability; that is, collection and transportation of specimens in warm ambient temperatures decreased the recovery of infectious virus. This observation strongly supports the use of real-time PCR-based detection methods, rather than virus isolation, for most routine diagnostic situations and for maximizing HSV detection from genital ulcers in tropical climates.

Acknowledgement: Alex Ryncarz, PhD, for helpful comments and discussion.

References:

1. Boissinot M, Bergeron MG. Toward rapid real-time molecular diagnostic to guide smart use of antimicrobials. Curr Opin Microbiol 2002;5:478-82

2. Aldea C, Alvarez CP, Folgueira L, Delgado R and Otero JR. Rapid detection of herpes simplex virus DNA in genital ulcers by real-time PCR using SYBR green I dye as the detection signal. J Clin Microbiol 2002;40:1060-2

3. Wald A, Huang ML, Carrell D, Selke S and Corey L. Polymerase chain reaction for detection of herpes simplex virus (HSV) DNA on mucosal surfaces: comparison with HSV isolation in cell culture. J Infect Dis 2003;188:1345-51

4. Mackay IM, Arden KE and Nitsche A. Real-time PCR in virology. Nucleic Acids Res 2002;30:1292-305

5. Klein D. Quantification using real-time PCR technology: applications and limitations. Trends Mol Med 2002;8:257-60

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