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.

Figures

FIG. 1.

Scatter plot of observed versus expected HIV-1 RNA levels, expressed in log10 copies, estimated by HIV-1 RNA Light Cycler assay in HIV-1-negative CVS spiked with a known amount of HIV-1 subtype B, C, and D from an HIV-1 RNA PeliCheck-02 reference panel.

FIG. 2.

Differences between the mean loads measured by the branched DNA assay and real-time RT-PCR by using the Bland-Altman plot analysis. The mean difference (bias) is indicated by a solid line, and the 95% confidence interval (±2 standard deviations) is represented by dotted lines. The mean difference ± standard deviation was 0.07 ± 0.41 log10 copies/ml.

FIG. 3.

Genital HIV-1 RNA shedding in HSV-2 DNA-shedding and -nonshedding women coinfected with HSV-2 and HIV-1. Genital HIV-1 RNA loads from HSV-2 DNA-shedding and HSV-2-nonshedding women, expressed in log10 copies/ml, are represented by triangles. Means of log10 copies/ml HIV-1 RNA load are indicated in each group by a plain bar. HIV-1 non-RNA-shedding women were given a log-transformed value of zero.