Detection of herpes simplex virus type 1, herpes simplex virus type 2 and varicella-zoster virus in skin lesions. Comparison of real-time PCR, nested PCR and virus isolation

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Abstract

Background: Herpes simplex virus type 1 (HSV-1), herpes simplex virus type 2 (HSV-2) and varicella-zoster virus (VZV) cause a wide range of signs and symptoms, varying from trivial mucocutaneous lesions to life-threatening infections, especially in immuno-suppressed patients. Since antiviral drugs are available, rapid and sensitive laboratory diagnosis of these virus infections is important. Objective: To set up and evaluate HSV-1, HSV-2 and VZV qualitative real-time PCR on the Lightcycler system and to compare the results with those of the ‘in-house’ nested PCR and virus isolation. Study design: 110 consecutive samples from dermal or genital lesions from patients suspected of having HSV infections and another 110 samples from patients with suspected VZV infections were tested with real-time PCR, nested PCR and virus isolation. Results: 24 samples (22%) were positive for HSV-1 by virus isolation and nested PCR, whereas 26 (24%) were positive by real-time PCR. HSV-2 was detected in 28 samples (25%) by virus isolation, in 41 (37%) by nested PCR and in 40 (36%) by real-time PCR. VZV was isolated in 15 samples (14%) and VZV DNA was detected in 51 samples (46%) by nested PCR as well as by real-time PCR. Nucleic acid amplification increased the detection rate of HSV-2 and VZV DNA in particular compared to virus isolation. No significant difference in sensitivity was found between real-time PCR and nested PCR. Conclusion: Real-time PCR has the advantage of rapid amplification, a reduced risk for contamination and it is a suitable method for diagnosis of VZV and HSV in specimens from skin lesions.

Introduction

Herpes simplex virus type 1 (HSV-1), type 2 (HSV-2) and varicella-zoster virus (VZV) are ubiquitous and important pathogens among humans. Infections with these viruses can cause a wide range of signs and symptoms, varying from trivial mucocutanous lesions to life-threatening infections of the brain (Dwyer and Cunningham, 2002). Since effective antiviral drugs including acyclovir, valacyclovir and famciclovir are available (Hammer and Inouye, 1997), rapid and sensitive laboratory diagnosis of these virus infections is important.

The most common diagnostic methods for the detection of mucocutanous HSV or VZV infections comprise virus isolation and antigen detection by immunofluorescence (IF) using monoclonal antibodies (Lakeman, 1997). Virus isolation requires special facilities and skilled personnel, as well as fresh specimens with viable viruses (Storch, 2001) and long time for detection, especially in the case of VZV. Shell vial culture is a more rapid alternative to culture although not always as sensitive. PCR has become an important tool for the detection of HSV (Aurelius et al., 1991) and VZV (Cinque et al., 1996) in cerebrospinal fluid. In many laboratories, nested PCR is used for the detection of herpesviruses. Recently, real-time PCR assays have been developed for the detection of HSV and VZV (Espy et al., 2000b, Kessler et al., 2000, Nicoll et al., 2001). Real-time PCR is a fluorescent-based technology, which is performed in a closed system.

The aim of this study was to develop a qualitative real-time PCR on the LightCycler system for the rapid and simple detection of HSV-1, HSV-2 and VZV DNA in skin lesions. The real-time PCR assays were evaluated using stored clinical samples from patients with suspected mucocutanous HSV-1, HSV-2 and VZV infections. The results were compared to the results of the accredited routine diagnostic methods for HSV and VZV used in the laboratory, ‘in-house’ nested PCR and virus isolation.

Section snippets

Specimens

One hundred and ten consecutive clinical samples from dermal or genital lesions of patients with suspected mucocutanous HSV infections and another 110 consecutive clinical samples from skin lesions of patients with suspected mucocutanous VZV infections were used. Virus isolation was performed in the diagnostic laboratory and the specimens had been stored at −70 °C until tested in the present study by PCR.

Swabs were collected in Virocult® viral transport medium (AB BIODISK, Stockholm, Sweden). In

Virus isolation, nested PCR and real-time PCR

The results of this study are summarized in Fig. 1 and Table 3. HSV-1 was detected in 26 (24%) of 110 clinical samples by real-time PCR and in 24 (22%) by nested PCR and virus isolation. No specimens were virus isolation or nested PCR positive and real-time PCR negative. HSV-2 was detected in 40 (36%) of 110 clinical samples by real-time PCR, in 41 (37%) by nested PCR and in 30 (27%) by virus isolation. No sample was isolation positive and PCR negative. One sample was nested PCR positive and

Discussion

In the present study, real-time PCR assays for the detection of HSV-1, HSV-2 and VZV, respectively, in skin lesions have been developed on the LightCycler instrument. After evaluation of real-time and ‘in-house’ nested PCR using 110 consecutive clinical specimens from skin lesions for each virus, the results were compared to available results of virus isolation, the routine method used in the diagnostic laboratory.

The comparison of the three methods showed that the detection rate of VZV in skin

Acknowledgements

This research was supported by a scholarship from the Swedish Institute, Stockholm, Sweden and by a grant from the Karolinska Institute, Stockholm, Sweden.

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