Evaluation of the Cepheid herpes simplex virus typing real-time PCR assay using dermal and genital specimens

Abstract

The Cepheid herpes simplex virus (HSV) (Cepheid, Sunnyvale, CA) typing multiplex real-time polymerase chain reaction (PCR) assay was evaluated for its ability to detect HSV in dermal and genital specimens stored in M5 media. Swab specimens (n = 114) for HSV testing were placed in M5 media and split between our laboratory and a highly experienced reference laboratory. Aliquots for testing with the Cepheid assay were processed using a simple boil-and-go procedure and then run in a SmartCycler® II (Cepheid). Aliquots tested at the reference laboratory were processed using a MagNA Pure LC DNA extractor (Roche Molecular Systems, Alameda, CA) and tested by the Roche HSV real-time PCR assay. Both laboratories detected 35 positives. Of the positive specimens, the Cepheid assay typed 16 as HSV 1 and 19 as HSV 2; the reference laboratory typed 15 as HSV 1, 19 as HSV 2, and 1 as HSV indeterminate. Our results demonstrate that the Cepheid real-time PCR assay, using specimens subjected to minimal specimen processing, performed as well as the Roche real-time PCR assay, using DNA extracts, for the detection of HSV DNA in genital and dermal specimens.

Introduction

Herpes simplex virus (HSV) is divided into 2 antigenic types: HSV 1 and HSV 2 (Corey, 2005). Both HSV 1 and HSV 2 are capable of producing a spectrum of infections associated with skin, mucous membranes, eye, central nervous system, and, occasionally, the internal organs. Like all members of the Herpesviridae family, HSV 1 and HSV 2 induce lifelong latent infection in their human hosts and can induce recurrent infections. The course of an HSV infection and the symptoms produced depend upon the site involved, the age and immune status of the host, and the antigenic type of the virus. With the advent of the ability to successfully treat HSV 1 and HSV 2 infections with antiviral drugs, demand has increased for laboratory tests that can quickly and reliably diagnose these infections. Polymerase chain reaction (PCR) has been used for many years for the detection of HSV 1 and HSV 2 in clinical samples (Aslanzadeh et al., 1992, Cone et al., 1994, Guffond et al., 1994, Lakeman and Whitley, 1995). In the early days of PCR, it (and the associated specimen processing) was an extremely expensive and laborious procedure. However, with advances in specimen processing procedures, advances in nucleic acid amplification technology (such as real-time PCR), and competition between the manufacturers of these products, the cost and complexity of nucleic acid amplification testing associated with HSV have dropped significantly. Real-time PCR is one of the most significant advances in nucleic acid amplification testing. Real-time PCR allows simultaneous amplification and detection in a single reaction tube, without opening the tube. Because of the decreasing costs and complexity associated with real-time PCR, it is becoming increasingly popular in clinical laboratories as a replacement for convention culture of dermal and genital specimens for HSV. Several reports have been published documenting the use of real-time PCR for the detection of HSV in various types of clinical specimens (Espy et al., 2000, Koenig et al., 2001, Schmutzhard et al., 2004, van Doornum et al., 2003). These real-time PCR assays have been shown to have sensitivities at least equal to, and many times superior to, cell culture for the detection of HSV in clinical specimens.

Although there are many reports pertaining to real-time PCR for the detection of HSV in various clinical samples, there is very little available on the recently released Cepheid HSV typing analyte-specific reagent (ASR) (Cepheid, Sunnyvale, CA). The Cepheid HSV typing ASR is a TaqMan®-based multiplex real-time PCR assay with the specific amplification and detection reagents provided in a lyophilized bead. The bead contains primers and probes specific for HSV 1, HSV 2, and internal control DNA (control DNA also included in the bead). The aim of our study is to determine how well the Cepheid HSV typing primer and probe set ASR performed on swab-collected genital or dermal specimens stored in M5 viral collection and transport media. Dermal and genital specimens for HSV testing are commonly received in the laboratory in M5 media. To evaluate this real-time PCR assay, we used 114 genital or dermal swab specimens placed in M5 media for the study as well as purified HSV control DNA. The specimens in M5 media were split, with 1 aliquot being tested with the Cepheid HSV typing ASR, and the remainder was sent to a highly experienced reference laboratory for HSV testing using a different real-time PCR assay. To speed up the assay time, we chose to use only boil-and-go specimen processing to generate extracts to be used in the Cepheid HSV typing ASR. Using the control HSV DNA, we undertook experiments to estimate the PCR efficiency, and sensitivity, of the Cepheid HSV typing ASR using M5-based samples.