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.

Molecular characterization and mapping of glucose-6-phosphate dehydrogenase (G6PD) mutations in the Greater Mekong Subregion.

BACKGROUND: Plasmodium vivax malaria elimination can only be achieved by the deployment of 8-aminoquinolines (primaquine and tafenoquine) in combination with ACT to kill both blood and liver-stage parasites. However, primaquine and the other 8-aminoquinolines cause dose-dependent haemolysis in subjects with G6PD deficiency, an X-linked disorder of red blood cells that is very common in populations living in tropical and subtropical areas. In order to inform safer use of 8-aminoquinolines in the Greater Mekong Subregion, a multi-centre study was carried out to assess the prevalence of G6PD deficiency and to identify the main G6PD variants in samples collected in Cambodia, Lao PDR, Myanmar, Thailand and Vietnam. METHODS: Blood samples were collected in the five countries during National Malaria Surveys or during Population Surveys. During Population Surveys samples were characterized for G6PD phenotype using the Fluorescent Spot Test. Samples were then genotyped for a panel of G6PD mutations. RESULTS: G6PD deficiency was found to be common in the region with an overall mean prevalence of deficient or mutated hemizygous males of 14.0%, ranging from a mean 7.3% in Thailand, 8.1% in Lao PDR, 8.9% in Vietnam, 15.8% in Myanmar and 18.8% in Cambodia. Mahidol and Viangchan mutations were the most common and widespread variants found among the nine investigated. CONCLUSIONS: Owing to the high prevalence of G6PD deficiency in the Greater Mekong Subregion, strategies for vivax malaria elimination should include point-of-care G6PD testing (both qualitative and quantitative) to allow safe and wide treatment with 8-aminoquinolines.

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.

A case-control study of the causes of acute respiratory infection among hospitalized patients in Northeastern Laos.

With the advent of highly sensitive real-time PCR, multiple pathogens have been identified from nasopharyngeal swabs of patients with acute respiratory infections (ARIs). However, the detection of microorganisms in the upper respiratory tract does not necessarily indicate disease causation. We conducted a matched case-control study, nested within a broader fever aetiology project, to facilitate determination of the aetiology of ARIs in hospitalised patients in Northeastern Laos. Consenting febrile patients of any age admitted to Xiengkhuang Provincial Hospital were included if they met the inclusion criteria for ARI presentation (at least one of the following: cough, rhinorrhoea, nasal congestion, sore throat, difficulty breathing, and/or abnormal chest auscultation). One healthy control for each patient, matched by sex, age, and village of residence, was recruited for the study. Nasopharyngeal swabs were collected from participants and tested for 33 pathogens by probe-based multiplex real-time RT-PCR (FastTrack Diagnostics Respiratory pathogen 33 kit). Attributable fraction of illness for a given microorganism was calculated by comparing results between patients and controls (= 100 * [OR - 1]/OR) (OR = odds ratio). Between 24th June 2019 and 24th June 2020, 205 consenting ARI patients and 205 matching controls were recruited. After excluding eight pairs due to age mismatch, 197 pairs were included in the analysis. Males were predominant with sex ratio 1.2:1 and children < 5 years old accounted for 59% of participants. At least one potential pathogen was detected in 173 (88%) patients and 175 (89%) controls. ARI in admitted patients were attributed to influenza B virus, influenza A virus, human metapneumovirus (HMPV), and respiratory syncytial virus (RSV) in 17.8%, 17.2%, 7.5%, and 6.5% of participants, respectively. SARS-CoV-2 was not detected in any cases or controls. Determining ARI aetiology in individual patients remains challenging. Among hospitalised patients with ARI symptoms presenting to a provincial hospital in Northeastern Laos, half were determined to be caused by one of several respiratory viruses, in particular influenza A virus, influenza B virus, HMPV, and RSV.

Viral RNA Degradation Makes Urine a Challenging Specimen for Detection of Japanese Encephalitis Virus in Patients With Suspected CNS Infection.

BACKGROUND: 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.

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.

Development of an improved RT-qPCR Assay for detection of Japanese encephalitis virus (JEV) RNA including a systematic review and comprehensive comparison with published methods.

BACKGROUND: Japanese encephalitis virus (JEV) is a major cause of encephalitis in Asia, and the commonest cause of mosquito-borne encephalitis worldwide. Detection of JEV RNA remains challenging due to the characteristic brief and low viraemia, with 0-25% of patients positive, and the mainstay of diagnosis remains detection of anti-JEV IgM antibody. METHODS: We performed a systematic review of published RT-PCR protocols, and evaluated them in silico and in vitro alongside new primers and probes designed using a multiple genome alignment of all JEV strains >9,000nt from GenBank, downloaded from the NCBI website (November 2016). The new assays included pan-genotype and genotype specific assays targeting genotypes 1 and 3. RESULTS: Ten RT-qPCR assays were compared, a pre-existing in-house assay, three published assays and six newly designed assays, using serial RNA dilutions. We selected three assays, one published and two novel assays, with the lowest limit of detection (LOD) for further optimisation and validation. One of the novel assays, detecting NS2A, showed the best results, with LOD approximately 4 copies/ reaction, and no cross-reaction on testing closely related viruses in the JEV serocomplex, West Nile Virus and St. Louis Virus. The optimised assays were validated in consecutive patients with central nervous system infections admitted to hospitals in Laos, testing paired CSF and serum samples. CONCLUSIONS: We succeeded in developing a JEV specific RT-qPCR assay with at least 1 log10 improved sensitivity as compared to existing assays. Further evaluation is required, field-testing the assay in a larger group of patients.