Literature review > Issue_4 > Review van Doornum et al. 

This paper describes a comparison of four real-time PCR amplification assays with culture for the detection of HSV-1, HSV-2, CMV, and VZV in a variety of clinical specimens from different patient groups. Standard laboratory technique for the detection of these herpes viruses is cell culture, the recognition of characteristic cytopathic effects, and identification using specific antibodies. For more rapid detection, the authors used a modification of the cell culture technique in which the inocula were centrifuged onto the cell monolayers and fluorescent antibodies were added after 48 hours. However, negative specimens were held for up to 14 days before being reported as negative. DNA amplification methods have been shown not only to provide more rapid results for both positive and negative specimens, but also to increase the sensitivity of viral detection compared to culture [1-7], and in some situations, have largely replaced conventional culture. The PCR amplification assays described in this paper were real-time TaqMan assays. An internal control molecule, used to monitor efficiency of specimen processing and the presence of inhibitors, was incorporated into each specimen at the time of DNA extraction, which was performed on an automated instrument. Each sample was extracted and tested in duplicate for the target herpes virus and the internal control DNA and considered positive only if both aliquots were positive.

This was a well designed and well written paper. The real-time PCR assays described were sensitive, specific, and the internal control was useful. Samples for which the internal control did not amplify were repeated. Even though, after repeat analysis, only 0.52% of specimens were classified as "no amplification possible", the authors did not report how many samples were originally unsatisfactory for internal control amplification and which type of samples these were. There was no explanation as to why the specimens, which were obtained from a variety of sources, were not all tested by every assay. In fact, of 711 specimens included, only 86 were analyzed for CMV even though 275 throat swabs were collected from transplant patients to screen for CMV.

The data clearly showed that all four of the PCR assays were more sensitive than the culture method. Samples for which results between culture and PCR were discrepant were retested by both methods. The authors did not report how many samples were originally discrepant and therefore, how many were retested, and whether the results of retesting changed any results. After retesting, the remaining discrepant samples were not analyzed using a third method. Instead, the performance of one method was calculated based on the results of the other method. Among the discrepant samples, it would have been interesting to learn which types of specimens, if any, were more likely to be positive by PCR only. Quantification of the viruses was achieved only through reporting of threshold cycles and not absolute numbers. The commercially available electron microscopy-counted HSV and CMV standards used to generate the curves for quantification did not appear reliable. However, sensitivity of the HSV assays was established through an EU QC program.

Real-time PCR assays are usually more sensitive and can provide results more quickly than culture, attributes that are useful for clinical diagnosis. However, the cost of reagents and supplies required for PCR are approximately twice the cost of those needed for culture and the equipment costs (ABI Prism 7700 Sequence Detection System and Roche MagnaPure Isolation station) are very expensive. While DNA amplification may be the only suitable virologic test for HSV in some clinical settings, such as the diagnosis of neonatal herpes or suspected central nervous system HSV infection [1], culture will most likely remain the standard technique until another sensitive, rapid, and inexpensive method is developed.

References:

1. Lakeman FD, Whitley RJ. Diagnosis of herpes simplex encephalitis: application of polymerase chain reaction to cerebrospinal fluid from brain-biopsied patients and correlation with disease. J Infect Dis 1995;171:857-63.

2. Safrin S, Shaw H, Bolan G, Cuan J, Chiang CS. Comparison of virus culture and the
polymerase chain reaction for diagnosis of mucocutaneous herpes simplex virus infection. Sex Transm Dis 1997;24:176-80.

3. Slomka MJ, Emery L, Munday PE, Moulsdale M, Brown DW. A comparison of PCR with virus isolation and direct antigen detection for diagnosis and typing of genital herpes. J Med Virol 1998;55:177-83.

4. Waldhuber MG, Denham I, Wadey C, Leong-Shaw W, Cross GF. Detection of herpes
simplex virus in genital specimens by type-specific polymerase chain reaction. Int J STD
AIDS 1999;10:89-92.

5. Scoular A, Gillespie G, Carman WF. Polymerase chain reaction for diagnosis of genital herpes in a genitourinary medicine clinic. Sex Transm Inf. 2002;78:21-25.

6. Aldea C, Alvarez CP, Folgueira L, Delgado R, 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;40:1060-1062.

7. Wald A, Huang M-L, Carrell D, Selke S, 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.

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