title: Real-time PCR quantification of genital shedding of herpes simplex virus (HSV) and human immunodeficiency virus (HIV) in women coinfected with HSV and HIV.
abstract: 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.
authors.affiliation: Unité INSERM Internationale U743 ("Immunologie Humaine"), Equipe Immunité et Biothérapie Muqueuse, Centre de Recherches Biomédicales des Cordeliers, Paris, France.
authors.forename: Jérôme
authors.initials: J
authors.lastname: Legoff
authors.forename: Hicham
authors.initials: H
authors.lastname: Bouhlal
authors.forename: Gérard
authors.initials: G
authors.lastname: Grésenguet
authors.forename: Helen
authors.initials: H
authors.lastname: Weiss
authors.forename: Nzambi
authors.initials: N
authors.lastname: Khonde
authors.forename: Hakim
authors.initials: H
authors.lastname: Hocini
authors.forename: Nathalie
authors.initials: N
authors.lastname: Désiré
authors.forename: Ali
authors.initials: A
authors.lastname: Si-Mohamed
authors.forename: Jean
authors.initials: J
authors.lastname: de Dieu Longo
authors.forename: Cécile
authors.initials: C
authors.lastname: Chemin
authors.forename: Eric
authors.initials: E
authors.lastname: Frost
authors.forename: Jacques
authors.initials: J
authors.lastname: Pépin
authors.forename: Jean-Elie
authors.initials: JE
authors.lastname: Malkin
authors.forename: Philippe
authors.initials: P
authors.lastname: Mayaud
authors.forename: Laurent
authors.initials: L
authors.lastname: Bélec
mesh_terms: D021121:Branched DNA Signal Amplification Assay; D002584:Cervix Uteri / Q000378:metabolism / Q000821:virology; D004279:DNA, Viral / Q000032:analysis; D005260:Female; D015658:HIV Infections / Q000821:virology; D015497:HIV-1 / Q000235:genetics / Q000302:isolation & purification* / Q000502:physiology; D006558:Herpes Genitalis / Q000821:virology; D018258:Herpesvirus 2, Human / Q000235:genetics / Q000302:isolation & purification* / Q000502:physiology; D006801:Humans; D016133:Polymerase Chain Reaction / Q000379:methods*; D011533:Proviruses / Q000235:genetics / Q000302:isolation & purification; D012367:RNA, Viral / Q000097:blood / Q000235:genetics / Q000302:isolation & purification; D015203:Reproducibility of Results; D014621:Vagina / Q000378:metabolism / Q000821:virology; D019562:Viral Load; D017201:Virus Shedding
chemical_list: D004279:DNA, Viral; D012367:RNA, Viral