Detection and subtyping of Herpes simplex virus in clinical samples by LightCycler PCR, enzyme immunoassay and cell culture

Andreas Nitsche,Tuckweng Kok,Julie Burrows,Belinda Bayly,Geoff Higgins,Elise Walker

doi:10.1186/1471-2180-2-12

Andreas Nitsche, Tuckweng Kok + Show 4 more

Open Access

https://doi.org/10.1186/1471-2180-2-12

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Abstract

BackgroundPrompt laboratory diagnosis of Herpes simplex virus (HSV) infection facilitates patient management and possible initiation of antiviral therapy. In our laboratory, which receives various specimen types for detection of HSV, we use enzyme immunoassay (EIA) for rapid detection and culture of this virus. The culture of HSV has traditionally been accepted as the diagnostic 'gold standard'. In this study, we compared the use of real time PCR (LightCycler) for amplification, detection and subtyping of specific DNA with our in-house developed rapid and culture tests for HSV.ResultsThe LightCycler PCR (LC-PCR) detected and subtyped HSV in 99% (66/67) of HSV positive specimens, compared to 81% (54/67) by rapid antigen EIA or 57% (36/63) by culture. A specimen was considered positive when two or more tests yielded HSV identifications or was culture positive. Discordant results were confirmed with an in-house developed PCR-ELISA or DNA sequence analysis. The typing results obtained with the LC-PCR and by culture amplified test were completely concordant.ConclusionsThis study showed that the LC-PCR provided a highly sensitive test for simultaneous detection and subtyping of HSV in a single reaction tube. In addition to increased sensitivity, the LightCycler PCR provided reduced turn-around-times (2 hours) when compared to enzyme immunoassay (4 hours) or culture (4 days).

Highlights

Prompt laboratory diagnosis of Herpes simplex virus (HSV) infection facilitates patient management and possible initiation of antiviral therapy
Laboratory diagnosis of HSV infection has relied on virus isolation in cell cultures and rapid tests viz. enzyme immunoassays (EIA) [7], immunofluorescence [4] or nucleic acid amplification by PCR [10]
Sensitivity Limiting dilutions of HSV-1 and HSV-2 stock cultures were prepared in 105 A549 epithelial cells followed by DNA extraction and subsequently tested in the LightCycler

Summary

Introduction

Prompt laboratory diagnosis of Herpes simplex virus (HSV) infection facilitates patient management and possible initiation of antiviral therapy. Herpes simplex virus (HSV) infection in adults is usually benign (e.g. oral cold sores) [18] When it occurs in critical anatomical sites, for example ocular or central nervous system, or acquired by the neonate during parturition, the sequelae may lead to serious complications [16,17]. In our laboratory, which provides a diagnostic service for hospital inpatients and outpatients as well as private physicians, genital, ocular and cutaneous specimens are regularly submitted for routine HSV detection. These are tested by rapid EIA and inoculated into cell culture with subsequent detection and subtyping of viral isolates by specific monoclonal antibodies

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