Detection and subtyping of Herpes simplex virus in clinical samples by LightCycler PCR, enzyme immunoassay and cell culture.

Institute of Medical & Veterinary Science, Adelaide, South Australia.
BMC Microbiology (Impact Factor: 2.98). 07/2002; 2:12. DOI: 10.1186/1471-2180-2-12
Source: PubMed

ABSTRACT Prompt laboratory diagnosis of Herpes simplex virus (HSV) infection facilitates patient management and possible initiation of antiviral therapy. In our laboratory, which receives various specimen types for detection of HSV, we use enzyme immunoassay (EIA) for rapid detection and culture of this virus. The culture of HSV has traditionally been accepted as the diagnostic 'gold standard'. In this study, we compared the use of real time PCR (LightCycler) for amplification, detection and subtyping of specific DNA with our in-house developed rapid and culture tests for HSV.
The LightCycler PCR (LC-PCR) detected and subtyped HSV in 99% (66/67) of HSV positive specimens, compared to 81% (54/67) by rapid antigen EIA or 57% (36/63) by culture. A specimen was considered positive when two or more tests yielded HSV identifications or was culture positive. Discordant results were confirmed with an in-house developed PCR-ELISA or DNA sequence analysis. The typing results obtained with the LC-PCR and by culture amplified test were completely concordant.
This study showed that the LC-PCR provided a highly sensitive test for simultaneous detection and subtyping of HSV in a single reaction tube. In addition to increased sensitivity, the LightCycler PCR provided reduced turn-around-times (2 hours) when compared to enzyme immunoassay (4 hours) or culture (4 days).

  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: We characterize a novel probe binding-site polymorphism detectable solely by melt curve analysis using the Roche LightCycler real-time PCR HSV 1/2 ASR assay. The frequency of this novel (47(o)C) and previously described intermediate (60-62(o)C) melt curves was 0.016% and 4.9%, respectively.
    Journal of clinical microbiology 12/2013; · 4.16 Impact Factor
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: Antiretroviral therapy (ART) reduces transmission of HIV-1. However, genital HIV-1 can be detected in patients on ART. We analyzed factors associated with genital HIV-1 shedding among high-risk women on ART in Burkina Faso. Plasma (PVL) and enriched cervico-vaginal lavage HIV-1 RNA were measured every 3-6 months for up to 8 years. Random-effects logistic and linear regression models were used to analyze associations of frequency and quantity of genital HIV-1 RNA with behavioral and biological factors, adjusting for within-woman correlation. The lower limit of detection of HIV-1 RNA in plasma and eCVL samples was 300 copies/ml. 188 participants initiated ART from 2004 to 2011. PVL was detectable in 16% (171/1050) of visits, in 52% (90/174) of women. Cervico-vaginal HIV-1 RNA was detectable in 16% (128/798) of visits with undetectable plasma HIV-1 RNA, in 45% (77/170) of women. After adjusting for PVL, detectable cervico-vaginal HIV-1 RNA was independently associated with abnormal vaginal discharge, and use of nevirapine or zidovudine vs. efavirenz and stavudine respectively; longer time on ART and hormonal contraception were not associated with increased shedding. The presence of bacterial vaginosis, Herpes Simplex Virus-2 (HSV-2) DNA, and the use of nevirapine vs efavirenz were independently associated with an increased quantity of cervico-vaginal HIV-1 RNA. Certain ART regimens, abnormal vaginal discharge, bacterial vaginosis and genital HSV-2 are associated with HIV-1 cervico-vaginal shedding or quantity in women on ART after adjusting for PVL. This may reduce the effectiveness of ART as prevention in high-risk populations.
    JAIDS Journal of Acquired Immune Deficiency Syndromes 11/2013; · 4.65 Impact Factor
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: Since the type of herpes simplex virus (HSV) infection affects prognosis and subsequent counseling, type-specific testing to distinguish HSV-1 from HSV-2 is always recommended. Although PCR has been the diagnostic standard method for HSV infections of the central nervous system, until now viral culture has been the test of choice for HSV genital infection. However, HSV PCR, with its consistently and substantially higher rate of HSV detection, could replace viral culture as the gold standard for the diagnosis of genital herpes in people with active mucocutaneous lesions, regardless of anatomic location or viral type. Alternatively, antigen detection--an immunofluorescence test or enzyme immunoassay from samples from symptomatic patients--could be employed, but HSV type determination is of importance. Type-specific serology based on glycoprotein G should be used for detecting asymptomatic individuals but widespread screening for HSV antibodies is not recommended. In conclusion, rapid and accurate laboratory diagnosis of HSV is now become a necessity, given the difficulty in making the clinical diagnosis of HSV, the growing worldwide prevalence of genital herpes and the availability of effective antiviral therapy.
    Virology journal. 05/2014; 11(1):83.

Full-text (2 Sources)

Available from
May 20, 2014