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Journal of Clinical Microbiology, February 2006, p. 423-432, Vol. 44, No. 2
0095-1137/06/$08.00+0     doi:10.1128/JCM.44.2.423-432.2006
Copyright © 2006, American Society for Microbiology. All Rights Reserved.

Real-Time PCR Quantification of Genital Shedding of Herpes Simplex Virus (HSV) and Human Immunodeficiency Virus (HIV) in Women Coinfected with HSV and HIV

Université Paris V, Equipe "Immunité et Biothérapie Muqueuses," Unité INSERM Internationale U743 ("Immunologie Humaine"), Centre de Recherches Biomédicales des Cordeliers & Laboratoire de Virologie, Hôpital Européen Georges Pompidou, Paris, France,¹ Centre National de Référence des Maladies Sexuellement Transmissibles et du SIDA de Bangui & Unité de Recherches et d'Intervention sur les Maladies Sexuellement Transmissibles et du SIDA, Faculté des Sciences de la Santé, Bangui, Central African Republic,² Clinical Research Unit, Department of Infectious and Tropical Diseases, London School of Hygiene and Tropical Medicine, London, United Kingdom,³ West African Project To Combat AIDS and STD, Accra, Ghana,⁴ Centre for International Health, University of Sherbrooke, Quebec, Canada,⁵ Centre Médical, Institut Pasteur, Paris, France⁶

Received 5 August 2005/ Returned for modification 15 September 2005/ Accepted 16 November 2005

The accuracy and usefulness of laboratory-developed real-time PCR procedures using a Light Cycler instrument (Roche Diagnostics) for detecting and quantifying human immunodeficiency virus type 1 (HIV-1) RNA and DNA as well as herpes simplex virus type 1 (HSV-1)/HSV-2 DNA in cervicovaginal secretions from women coinfected with HIV and HSV were evaluated. For HIV-1, the use of the NEC152 and NEC131 primer set and the NEC-LTR probe in the long terminal repeat gene allowed us to detect accurately the majority of HIV-1 subtypes of group M circulating in sub-Saharan Africa, including subtypes A, B, C, D, and G as well as circulating recombinant forms 02 and 11. The detection threshold of real-time PCR for HIV in cervicovaginal lavage samples was 5 copies per assay for both RNA and DNA; the intra- and interassay coefficients of variation of C_T values were 1.30% and 0.69% (HIV-1 RNA) and 1.84% and 0.67% (HIV-1 DNA), respectively. Real-time PCR for HSV using primers and probe targeting the HSV DNA polymerase gene allowed both detection and quantification of HSV DNA and also differentiation between HSV-1 and HSV-2 genotypes. The detection threshold of real-time PCR for HSV was 5 copies per assay; the intra- and interassay coefficients of variation of C_T values were 0.96% and 1.49%, respectively. Both manual and automated silica-based procedures were appropriate for combined extraction of HIV and HSV genomes from female genital secretions. Taken together, these findings indicate that real-time PCR may be used as a unique nucleic acid amplification procedure to detect and quantify HIV and HSV genomes in cervicovaginal secretions and thus to assess at reduced costs the genital shedding of both viruses in women included in intervention studies.

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