|
.gif)
|
|
Detection and differentiation of herpes simplex virus types 1 and 2 by a duplex LightCycler PCR that incorporates an internal control PCR reaction.
Whiley DM, Mackay IM, Syrmis MW, Witt MJ, Sloots TP
J Clin Virol 2004;30:32-38
and
Duplex real-time polymerase chain reaction assay for detection and quantification of herpes simplex virus type 1 and herpes simplex virus type 2 in genital and cutaneous lesions
Filen F, Strand A, Allard A, Blomberg J, Herrmann B.
Sex Transm Dis 2004;31:331-36
|
| by |
Sepehr N. Tabrizi Ph.D.
Senior Scientist
Department of Microbiology and Infectious Diseases
The Royal Women’s Hospital
Carlton, Victoria.
Australia |
|
Herpes simplex virus (HSV) is one of the most common viral agents infecting humans. HSV infection can occur in all ages and produce a spectrum of clinical manifestations ranging from mucocutaneous infection, to infection of the central nervous system, possibly resulting in encephalitis with high rates of morbidity and mortality.
Laboratory methods used in diagnosis of HSV infections have improved greatly over time. Generally, serological detection assays cannot discriminate between previous and current infection, therefore, presence of HSV infection has been more reliably performed using conventional assays such as cell culture and immunofluorescent staining using monoclonal antibodies. However, these assays are labor intensive and time consuming. More recent diagnostic tools include amplification technology assays, such as polymerase chain reaction (PCR). The introduction of these assays has allowed a more accurate and timely management of HSV infections with enhanced sensitivity. Recently, with the advent of real-time PCR, simultaneous detection, typing and quantitation has been possible in a short time frame, often 1-2 hours. Viral typing and quantification can provide further knowledge about the course of infection and post antiviral monitoring.
There is currently a need for standardized commercially available kits for HSV molecular detection, typing, and qunatitation, and in the absence of such assays, laboratories are forced to develop in-house assays. Two papers by Filen el al. and Whiley et al. describe real-time PCR assays and are the topic for this commentary.
Filen et al. describes a duplex real-time PCR assay using two sets of primers and probes based on 5’ nuclease chemistry for type detection and quantification utilizing a 216 bp target sequence within the junction between glycoprotein G and J. They report an analytical sensitivity of 1-5 copies per reaction, as well as higher sensitivity (by 27%) when compared to cell culture. Two samples, generating a culture positive and negative PCR, were also reported. This could be due to differential sampling or inhibition. An inhibition control was not included in the PCR reactions; therefore, it may be possible that samples generating a negative PCR could be due to inhibition.
Whiley et al. describe a real-time PCR with degenerate base primers for detection of a 260 bp region within the conserved region of glycoprotein D gene, and separate primer and probes for detection of an internal control (ERV-3). Detection and typing was done using melt curve analysis of the FRET probes. Comparison of this method to an alternative PCR, targeting the HSV DNA polymerase, achieved similar sensitivity. As the authors indicate, incorporation of an internal control can reduce the overall sensitivity of the assay. Although primer concentration was in excess for the HSV amplification, samples containing low copy number HSV targets could give false negative results. In addition, incorporation of an internal control target and probes would be best done if amplification targets of HSV and internal control were of similar product size. As this study reports, amplification of an internal control target that is half the amplicon size of the target gene could result in false negative results in samples containing low-level inhibition. One possible way to address this is to spike a separate reaction with the same target, i.e., HSV 1 or 2 and assess inhibition in this way. Although the authors do not report on quantification of HSV, similar to the study by Filen et al., their assay could also quantitate the viral particles in the clinical samples.
Even if samples are not inhibitory, one aspect, which may need to be explored, is whether samples have been collected adequately, in particular the mucocutaneous samples. Examination of such sample adequacy can be performed by detection of host cell DNA, for example beta-globin gene, which is present at one copy per cell, by PCR [1].
While these two studies have utilized different targets for detection and typing of HSV, both assays offer specific real-time HSV detection, typing, and quantitation in clinical samples. In addition, both assays provide enough detail for implementation into a diagnostic laboratory. However, a concern with real-time PCR was highlighted in a recent report, which showed that a single nucleotide polymorphism within the amplicon’s probe binding region, which are used to distinguish HSV-1 from HSV-2, can decrease assay sensitivity [2].
References:
1. Bauer BA, Ting Y, Greer CE, Chambers et al. Manos MM. Genital human papillomavirus infection in female university students as determined by a PCR-based method. JAMA 1991; 265:472-477.
2. Stevenson J, Hymas W, Hillyard D. Effect of sequence polymorphisms on performance of two real-time PCR assays for detection of herpes simplex virus.J Clin Microbiol. 2005; 43:2391-8.
|
