Detection of Japanese Encephalitis Virus RNA in Human Throat Samples in Laos \u2013 A Pilot study

Tehmina Bharucha,Xavier De Lamballerie,Audrey Dubot-Pérès,Géraldine Piorkowski,Malee Seephonelee,Sayaphet Rattanavong,Marc Lecuit,Onanong Sengvilaipaseuth,Christopher Gorman,Jean-David Pommier,Manivanh Vongsouvath,Malavanh Vongsouvath,Paul N Newton

doi:10.1038/s41598-018-26333-4

Abstract

Japanese encephalitis virus (JEV) is the most commonly identified cause of acute encephalitis syndrome (AES) in Asia. The WHO recommended test is anti-JEV IgM-antibody-capture-enzyme-linked-immunosorbent-assay (JEV MAC-ELISA). However, data suggest this has low positive predictive value, with false positives related to other Flavivirus infections and vaccination. JEV RT-PCR in cerebrospinal fluid (CSF) and/or serum is highly specific, but is rarely positive; 0–25% of patients that fulfil the WHO definition of JE (clinical Acute Encephalitis Syndrome (AES) and JEV MAC-ELISA positive). Testing other body fluids by JEV RT-qPCR may improve the diagnosis. As a pilot study thirty patients admitted to Mahosot Hospital 2014–2017, recruited to the South-East-Asia-Encephalitis study, were tested by JEV MAC-ELISA and two JEV real-time RT-PCR (RT-qPCR) assays (NS2A and NS3). Eleven (36.7%) were JEV MAC-ELISA positive. Available CSF and serum samples of these patients were JEV RT-qPCR negative but 2 (7%) had JEV RNA detected in their throat swabs. JEV RNA was confirmed by re-testing, and sequencing of RT-qPCR products. As the first apparent report of JEV RNA detection in human throat samples, the provides new perspectives on human JEV infection, potentially informing improving JEV detection. We suggest that testing patients’ throat swabs for JEV RNA is performed, in combination with molecular and serological CSF and serum investigations, on a larger scale to investigate the epidemiology of the presence of JEV in human throats. Throat swabs are an easy and non-invasive tool that could be rolled out to a wider population to improve knowledge of JEV molecular epidemiology.

Highlights

Evidence continues to implicate Japanese encephalitis virus (JEV) as a major cause of encephalitis in Asia, with recent evidence of possible autochthonous transmission in Africa[1,2,3]
All cerebrospinal fluid (CSF) and serum samples were negative by JEV RT-qPCR, but 2 (7%) patients had JEV RNA detected in their throat swabs, patient 5 and patient 10 (Table 1)
We provide strong evidence of JEV RNA detection in throat swab from two patients with clinical presentation of AES

Summary

Introduction

Evidence continues to implicate Japanese encephalitis virus (JEV) as a major cause of encephalitis in Asia, with recent evidence of possible autochthonous transmission in Africa[1,2,3]. The conventional mainstay of JEV encephalitis diagnosis is serology, with serum and cerebrospinal fluid (CSF) anti-JEV IgM antibody capture enzyme-linked immunosorbent assays (JEV MAC-ELISA) recommended by the World Health Organization (WHO)[4]. There are concerns about the accuracy of JEV MAC-ELISA for diagnosing JEV. Low positive predictive value of JEV MAC-ELISA has been reported[5], and there are recognised difficulties with false positives related to vaccination and in areas where other Flavivirus infections are endemic[6,7]. JEV RT-PCR testing of CSF and serum samples has low sensitivity, rarely positive in patients at presentation °Anti-JEV IgM detection using JE Detect IgM Antibody Capture ELISA Kit (InBios, Washington, USA). Syndrome (AES) and JEV MAC-ELISA positivity)[5,9]. Other Flaviviruses, such as West Nile virus, Zika virus and Dengue virus, have been detected in urine, and JEV RNA has recently been detected in the urine of two patients[10,11,12,13]

Methods

Results

Conclusion