Detection, quantification and genotyping of Herpes Simplex Virus in cervicovaginal secretions by real-time PCR: a cross sectional surveyReportar como inadecuado




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Virology Journal

, 2:61

First Online: 11 August 2005Received: 17 June 2005Accepted: 11 August 2005

Abstract

BackgroundHerpes Simplex Virus HSV Genital Ulcer Disease GUD is an important public health problem, whose interaction with HIV results in mutually enhancing epidemics. Conventional methods for detecting HSV tend to be slow and insensitive. We designed a rapid PCR-based assay to quantify and type HSV in cervicovaginal lavage CVL fluid of subjects attending a Genito-Urinary Medicine GUM clinic. Vaginal swabs, CVL fluid and venous blood were collected. Quantitative detection of HSV was conducted using real time PCR with HSV specific primers and SYBR Green I. Fluorogenic TaqMan Minor Groove Binder MGB probes designed around a single base mismatch in the HSV DNA polymerase I gene were used to type HSV in a separate reaction. The Kalon test was used to detect anti-HSV-2 IgG antibodies in serum. Testing for HIV, other Sexually Transmitted Infections STI and related infections was based on standard clinical and laboratory methods.

ResultsSeventy consecutive GUM clinic attendees were studied. Twenty-seven subjects 39% had detectable HSV DNA in CVL fluid; HSV-2 alone was detected in 19 70% subjects, HSV-1 alone was detected in 4 15% subjects and both HSV types were detected in 4 15% subjects. Eleven out of 27 subjects 41% with anti-HSV-2 IgG had detectable HSV-2 DNA in CVL fluid. Seven subjects 10% were HIV-positive. Three of seven 43% HIV-infected subjects and two of five subjects with GUD 40% were secreting HSV-2. None of the subjects in whom HSV-1 was detected had GUD.

ConclusionQuantitative real-time PCR and Taqman MGB probes specific for HSV-1 or -2 were used to develop an assay for quantification and typing of HSV. The majority of subjects in which HSV was detected had low levels of CVL fluid HSV, with no detectable HSV-2 antibodies and were asymptomatic.

List of AbbreviationsCDPI3tripeptide 1,2-dihydro-3H-pyrrolo 3,2-eindole-7-carboxylate

CtCycle threshold

CVLCervicovaginal lavage

DNADeoxyribonucleic acid

EIAEnzyme immunoassay

ELISAEnzyme-Linked Immunosorbent Assay.

gGglycoprotein G

GUDGenital Ulcer Disease

GUMGenito-Urinary Medicine

HBsAgHepatitis B surface Antigen

HIVHuman Immunodeficiency Virus

HSVHerpes Simplex Virus

IgImmunoglobulin

MAbMonoclonal antibodies

MGBMinor Groove Binder

MRCMedical Research Council

PBSPhosphate Buffered Saline

PCRPolymerase Chain Reaction

qPCRquantitative Polymerase Chain Reaction

SNPSingle Nucleotide Polymorphism

STDSexually Transmitted Disease

STISexually Transmitted Infection

STITm Melting temperature

Electronic supplementary materialThe online version of this article doi:10.1186-1743-422X-2-61 contains supplementary material, which is available to authorized users.

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Autor: Esther AN Aryee - Robin L Bailey - Angels Natividad-Sancho - Steve Kaye - Martin J Holland

Fuente: https://link.springer.com/







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