Zoonotic Pathogens in Wildlife Traded in Markets for Human Consumption, Laos

We tested animals from wildlife trade sites in Laos for the presence of zoonotic pathogens. Leptospira spp. were the most frequently detected infectious agents, found in 20.1% of animals. Rickettsia typhi and R. felis were also detected. These findings suggest a substantial risk for exposure through handling and consumption of wild animal meat

Viral RNA Degradation Makes Urine a Challenging Specimen for Detection of Japanese Encephalitis Virus in Patients With Suspected CNS Infection: JEV RNA Not Detected in Urine of Suspected Central Nervous System Infections.

International audienceBackground: Japanese encephalitis virus (JEV) is a leading cause of central nervous system (CNS) infections in Asia and results in significant morbidity and mortality. JEV RNA is rarely detected in serum or cerebrospinal fluid (CSF), and diagnosis of JEV infection is usually based on serological tests that are frequently difficult to interpret. Unlike serum or CSF, urine is relatively easy to obtain, but, to date, there has been minimal work on the feasibility of testing urine for JEV RNA.Methods: We investigated the use of lysis buffer and a Microsep device to optimize urine storage for detection of JEV RNA by reverse transcription real-time polymerase chain reaction (RT-qPCR). The best of the studied methods was then evaluated in consecutive patients admitted to the hospital with suspected CNS infections in Laos.Results: We demonstrated degradation of JEV RNA in urine after even short storage periods at 4°C or -80°C. Although there was no advantage in using a Microsep concentration device alone, immediate addition of lysis buffer to fresh urine improved the detection of JEV RNA at the limit of detection.Conclusions: In 2 studies of 41 patients with acute encephalitis syndrome, 11 (27%) were positive for JEV IgM in CSF and/or serum, and 2 (4.9%) were JEV RT-qPCR positive from throat swabs. JEV RNA was not detected in any of these patients' urine samples. However, lysis buffer was only used during a prospective study, that is, for only 17/41 (41%) patient urine samples. Our findings suggest a need for larger studies testing urine for JEV RNA, with urine collected at different times from symptom onset, and using lysis buffer, which stabilizes RNA, for storage

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

International audienceJapanese 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 SouthEast -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. 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 1–3. Among the key factors in its persistent role as a public health problem are the limitations of existing diagnostic tests, our understanding of its epidemiology and inadequate implementation of vaccination programmes. 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. However, 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. Diagnosis of JEV by Reverse-Transcription (RT)-PCR is highly specific, and enables improved understanding of the molecular epidemiology of JEV 8. However, JEV RT-PCR testing of CSF and serum samples has low sensitivity , rarely positive in patients at presentation (0–25% that fulfil the WHO definition of Acute Encephaliti

Large-Scale Survey for Tickborne Bacteria, Khammouan Province, Laos

We screened 768 tick pools containing 6,962 ticks from Khammouan Province, Laos, by using quantitative real-time PCR and identified Rickettsia spp., Ehrlichia spp., and Borrelia spp. Sequencing of Rickettsia spp.–positive and Borrelia spp.–positive pools provided evidence for distinct genotypes. Our results identified bacteria with human disease potential in ticks in Laos

Development of an improved RT-qPCR Assay for detection of <i>Japanese encephalitis virus</i> (JEV) RNA including a systematic review and comprehensive comparison with published methods - Fig 1

<p>Standard curves of the 1) Pyke, 2) NS2A assays and 3) NS3 assays with G3-RP9-190 on (A) Day 1 and repeated on (B) Day 2. Result of the RT-qPCR run, ‘Cq’, is plotted against the ‘log starting copies number’, at the RNA dilutions detected: Pyke assay 1:10 serial dilutions of G1-769 in triplicate at 10⁻³ to 10⁻⁶; NS2A assay 1:10 serial dilutions of G1-769 in triplicate at 10⁻³ to 10⁻⁷; and NS3 with G3-RP-190 10⁻⁴ to 10⁻⁷. Efficiency = 10^−1/slope-1. R² = Correlation Coefficient. RT-qPCR performed with Fastvirus kit (TaqMan® Fast Virus 1-Step) with a reaction volume of 50μL, sample volume of 30μl, and primer and probe concentrations of 600nM and 300nM respectively. Thermocycling conditions were 50°C for 5 minutes, 95°C for 20 seconds and 45 x (95°C for 15 seconds + x°C for 60 seconds). The optimal annealing temperature ‘x°C’ was different for each assay: 62°C, 60°C and 56°C for the Pyke, NS2A and NS3 assays respectively.</p

Primers and probes evaluated during the study.

<p>Primers and probes evaluated during the study.</p

Evaluation of the three optimised RT-qPCR assays using patient samples with Central Nervous System (CNS) infections.

<p>Patients with RT-qPCR ‘positive’ test results.</p

Optimisation experiments performed for the three best performing RT-qPCR systems^*.

<p>Optimisation experiments performed for the three best performing RT-qPCR systems^{<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0194412#t003fn001" target="_blank">*</a>}.</p

Cq results for validation of the optimised JEV RT-qPCR assays.

<p>Cq results for validation of the optimised JEV RT-qPCR assays.</p

Standard reaction mixes and cycling conditions.

<p>Standard reaction mixes and cycling conditions.</p