Applicability of molecular assays for detection and typing of herpes simplex viruses in encephalitis cases

Abstract

Herpes simplex viruses (HSVs) cause a latent infection in humans which is mainly associated with characteristic cold sores or fever blisters and genital blisters. Large segments of the world population are suffering from the HSV infection and early diagnosis as well as treatments are needed to avoid further complications. HSV surveillance is very sparse, especially from developing countries including India. The aim of the present study is to develop and evaluate molecular assays for rapid detection and typing of HSV. In the present study, viral DNA was extracted from cerebro-spinal fluid from HSV suspected encephalitis patients. The conventional multiplex PCR for HSV-1 and HSV-2 was optimized and their comparative analysis was done with Real-Time qPCR for detection and typing of HSV. Out of 137 clinical samples, eleven samples (8.03%) were diagnosed as HSV positive by Real-Time qPCR while ten (7.3%) by conventional multiplex PCR which were further typed as subtyping HSV-1 (nine) and HSV-2 (two). Real-Time qPCR is highly sensitive and able to detect 9.4 × 10¹ to 3.1 × 10⁶ copies/ml of HSV DNA. Conventional PCR was found to be having 99.21% specificity with 100% sensitivity. The positive predictive value was 90.91% whereas negative predictive value was 100%. Logistic regression indicates blisters with pain and skin rash as the most significant symptoms associated with HSV infection. The present study could be applied for rapid, specific, sensitive and cost-effective diagnosis of HSV-1 and HSV-2 thereby helpful in better patient management through early detection and treatment of HSV.

Keywords: Encephalitis; HSV-1; HSV-2; Herpes viruses; Multiplex PCR; Real-time qPCR.

Fig. 1

Multiplex subtyping of conventional PCR on clinical samples. Examination of PCR product in 2% (w/v) agarose gel electrophoresis. Lane 1 1 kb ladder, Lane 2 negative control, Lane 3 positive control, Lane 4–7 CLINICAL samples; Lane 4 show HSV-2 positive while Lane 6,7 show HSV-1 positive samples

Fig. 2

Amplification curve for RT-qPCR detecting HSV. Normalized fluorescence is the ∆R_n showing variation in fluorescence signal of reporter dye (FAM) normalized to the fluorescence signal of internal dye (JOE). The curve was plotted between ∆R_n and cycle of amplification. C_t is the cycle at which the fluorescence meets the threshold in the amplification curve

Fig. 3

The graph showing standard curve of C_t-value vs concentration. DNA copy number is denoted as concentration. Red dots represent the samples and blue dots denote the standards all forming a straight line