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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.

SUBMITTER: Dhull D

PROVIDER: S-EPMC6917685 | biostudies-literature | 2019 Dec

REPOSITORIES: biostudies-literature

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

Dhull Divya D Sharma Vikrant V Sharma Yashika Y Kaushik Samander S

Virusdisease 20191205 4

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 detect ...[more]

PMID: 31897415

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Project description:Specific and sensitive reverse transcription-PCR (RT-PCR) assays were developed for the detection of eastern, western, and Venezuelan equine encephalitis viruses (EEE, WEE, and VEE, respectively). Tests for specificity included all known alphavirus species. The EEE-specific RT-PCR amplified a 464-bp region of the E2 gene exclusively from 10 different EEE strains from South and North America with a sensitivity of about 3,000 RNA molecules. In a subsequent nested PCR, the specificity was confirmed by the amplification of a 262-bp fragment, increasing the sensitivity of this assay to approximately 30 RNA molecules. The RT-PCR for WEE amplified a fragment of 354 bp from as few as 2,000 RNA molecules. Babanki virus, as well as Mucambo and Pixuna viruses (VEE subtypes IIIA and IV), were also amplified. However, the latter viruses showed slightly smaller fragments of about 290 and 310 bp, respectively. A subsequent seminested PCR amplified a 195-bp fragment only from the 10 tested strains of WEE from North and South America, rendering this assay virus specific and increasing its sensitivity to approximately 20 RNA molecules. Because the 12 VEE subtypes showed too much divergence in their 26S RNA nucleotide sequences to detect all of them by the use of nondegenerate primers, this assay was confined to the medically important and closely related VEE subtypes IAB, IC, ID, IE, and II. The RT-PCR-seminested PCR combination specifically amplified 342- and 194-bp fragments of the region covering the 6K gene in VEE. The sensitivity was 20 RNA molecules for subtype IAB virus and 70 RNA molecules for subtype IE virus. In addition to the subtypes mentioned above, three of the enzootic VEE (subtypes IIIB, IIIC, and IV) showed the specific amplicon in the seminested PCR. The practicability of the latter assay was tested with human sera gathered as part of the febrile illness surveillance in the Amazon River Basin of Peru near the city of Iquitos. All of the nine tested VEE-positive sera showed the expected 194-bp amplicon of the VEE-specific RT-PCR-seminested PCR.

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

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

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