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

Abstract

Background: Herpes 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.

Results: Seventy 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.

Conclusion: Quantitative 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.

Figure 1

R = A/G; M = A/C; N = A/T/G/C. Control HSV-1 and -2 DNA was obtained from 2003 Quality Control Molecular Diagnostics 2003 Proficiency panel (Block 6, Kelvin Campus, West of Scotland Science Park, Glasgow UK). The reference sequences were based on Blast results from NCBI. The T_m of the PCR product (146 b.p.) which was amplified during quantitation with SYBR Green I is shown against its complementary sequence with the SNP position marked in bold. The probe and sequence ascertainment of types were in agreement. * T_m was unable to distinguish dual infection in this assay. -n.c. = not confirmed by sequencing.

Figure 2

Dissociation curves of amplicons used to identify HSV-1, HSV-2 and dual positive samples. No curves were observed in HSV negative subjects. T_m was recorded and compared with probe binding and sequence results. Collectively nine different peaks with T_m in the range 86.6 – 88.0°C could be observed. Three T_m representative of HSV-1, HSV-2 and dual positive samples confirmed by sequencing are shown with T_m indicated.