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Background: Burkholderia thailandensis is an environmental bacteria closely related to Burkholderia pseudomallei that rarely causes infection in humans. Some environmental isolates have shown to express a capsular polysaccharide known as B. thailandensis capsular variant (BTCV), but human infection has not previously been reported. Although B. thailandenisis has been identified in environmental samples in Laos before, there have not been any human cases reported., Case: A 44-year-old man presented to a district hospital in Laos with a short history of fever and pain in his left foot. Physical examination identified a deep soft-tissue abscess in his left foot and an elevated white blood count. A deep pus sample was taken and melioidosis was suspected from preliminary laboratory tests. The patient was initially started on cloxacillin, ceftriaxone and metronidazole, and was then changed to ceftazidime treatment following local melioidosis treatment guidelines., Laboratory Methods: A deep pus sample was sent to Mahosot Hospital microbiology laboratory where a mixed infection was identified including Burkholderia sp. Conventional identification tests and API 20NE were inconclusive, and the B. pseudomallei -specific latex agglutination was positive. The isolate then underwent a Burkholderia species specific PCR which identified the isolate as B. thailandensis. The isolate was sent for sequencing on the Illumina NovaSeq 6000 system and multi-locus sequence typing analysis identified the isolate had the same sequence type (ST696) as B. thailandensis E555, a strain which expresses a B. pseudomallei -like capsular polysaccharide., Conclusion: This is the first report of human infection with B. thailandensis in Laos, and the first report of any human infection with the B. thailandensis capsular variant. Due to the potential for laboratory tests to incorrectly identify this bacteria, staff in endemic areas for B. thailandensis and B. pseudomallei should be aware and ensure that appropriate confirmatory methods are used to differentiate between the species., Competing Interests: No competing interests were disclosed., (Copyright: © 2024 Vannachone S et al.)

Background: Parasitological investigation of bone marrow, splenic or lymph node aspirations is the gold standard for the diagnosis of visceral leishmaniasis (VL). However, this invasive test requires skilled clinical and laboratory staff and adequate facilities, and sensitivity varies depending on the tissue used. The direct agglutination test (DAT) is a serological test that does not need specialised staff, with just minimal training required. While previous meta-analysis has shown DAT to have high sensitivity and specificity when using parasitology as the reference test for diagnosis, meta-analysis of DAT compared to other diagnostic techniques, such as PCR and ELISA, that are increasingly used in clinical and research settings, has not been done., Methods: We conducted a systematic review to determine the diagnostic performance of DAT compared to all available tests for the laboratory diagnosis of human VL. We searched electronic databases including Medline, Embase, Global Health, Scopus, WoS Science Citation Index, Wiley Cochrane Central Register of Controlled Trials, Africa-Wide Information, LILACS and WHO Global Index. Three independent reviewers screened reports and extracted data from eligible studies. A meta-analysis estimated the diagnostic sensitivity and specificity of DAT., Results: Of 987 titles screened, 358 were selected for full data extraction and 78 were included in the analysis, reporting on 32,822 participants from 19 countries. Studies included were conducted between 1987-2020. Meta-analysis of studies using serum and DAT compared to any other test showed pooled sensitivity of 95% (95%CrI 90-98%) and pooled specificity of 95% (95%CrI 88-98%). Results were similar for freeze-dried DAT and liquid DAT when analysed separately. Sensitivity was lower for HIV-positive patients (90%, CrI 59-98%) and specificity was lower for symptomatic patients (70%, CrI 43-89%). When comparing different geographical regions, the lowest median sensitivity (89%, CrI 67-97%) was in Western Asia (five studies)., Conclusions: This systematic review and meta-analysis demonstrates high estimated pooled sensitivity and specificity of DAT for diagnosis of VL, although sensitivity and specificity were lower for different patient groups and geographical locations. This review highlights the lack of standardisation of DAT methods and preparations, and the lack of data from some important geographical locations. Future well-reported studies could provide better evidence to inform test implementation for different patient populations and use cases., Prospero Registration: CRD42021240830., (© 2023. The Author(s).)

Wild animal trade for human consumption is a global issue, involving complex interactions between economics, culture, food security and conservation. Whilst being a biodiversity issue, it is also a major public health concern, with recent epidemics and pandemics of zoonotic pathogens linked to interactions with wildlife. At three time points, between March 2017 and June 2018, a longitudinal sero-survey of 150 market vendors from three wet markets in Laos (selling vegetables, domestic animal meat and/or wildlife meat) was conducted to determine if vendors had been differentially exposed to three endemic bacterial pathogens - Orientia tsutsugamushi, Rickettsia typhi , and Leptospira spp. A total of 367 serum samples were tested by IgG enzyme-linked immunosorbent assay (ELISA) and immunofluorescence assay (IFA, for scrub typhus group (STG) and typhus group (TG) only). Among vendors, 32.7% were IgG-positive for at least one pathogen, 13.3% sero-converted during the study. Multi-season occupancy modelling for STG indicated a significantly higher prevalence of STG IgG in vegetable vendors (27.3%) and wildlife vendors (28.4%) than in domestic animal meat vendors (6.9 %, p=0.05), and higher in Phonsavanh market (OR=9.6, p=0.03) compared to Lak Sao and Salavan markets. Estimated mean incidence was 57 cases per 10,000 per 7.5-month period. For TG, vendor age had a significant effect on prevalence (OR=1.04, p=0.006), estimated mean incidence was 64 cases per 10,000 per season (7.5-month period). Despite individuals selling domestic meat having a higher prevalence of Leptospira infections than those that did not (11.6% versus 4.5%), the difference was not significant. Whilst this study has a number of limitations, including vendors changing what food types they sold and no investigation of exposure outside of markets, the finding that the risk of exposure of vendors to zoonotic pathogens may be associated with types of food sold for human consumption warrants further investigation., Competing Interests: Declarations of interest: none

Japanese encephalitis virus is a leading cause of neurological infection in the Asia-Pacific region with no means of detection in more remote areas. We aimed to test the hypothesis of a Japanese encephalitis (JE) protein signature in human cerebrospinal fluid (CSF) that could be harnessed in a rapid diagnostic test (RDT), contribute to understanding the host response and predict outcome during infection. Liquid chromatography and tandem mass spectrometry (LC-MS/MS), using extensive offline fractionation and tandem mass tag labeling (TMT), enabled comparison of the deep CSF proteome in JE vs other confirmed neurological infections (non-JE). Verification was performed using data-independent acquisition (DIA) LC-MS/MS. 5,070 proteins were identified, including 4,805 human proteins and 265 pathogen proteins. Feature selection and predictive modeling using TMT analysis of 147 patient samples enabled the development of a nine-protein JE diagnostic signature. This was tested using DIA analysis of an independent group of 16 patient samples, demonstrating 82% accuracy. Ultimately, validation in a larger group of patients and different locations could help refine the list to 2-3 proteins for an RDT. The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium via the PRIDE partner repository with the dataset identifier PXD034789 and 10.6019/PXD034789.

Background: The mainstay of diagnostic confirmation of acute Japanese encephalitis (JE) involves detection of anti-JE virus (JEV) immunoglobulin M (IgM) by enzyme-linked immunosorbent assay (ELISA). Limitations in the specificity of this test are increasingly apparent with the introduction of JEV vaccinations and the endemicity of other cross-reactive flaviviruses. Virus neutralization testing (VNT) is considered the gold standard, but it is challenging to implement and interpret. We performed a pilot study to assess IgG depletion prior to VNT for detection of anti-JEV IgM neutralizing antibodies (IgM-VNT) as compared with standard VNT., Methods: We evaluated IgM-VNT in paired sera from anti-JEV IgM ELISA-positive patients (JE n=35) and negative controls of healthy flavivirus-naïve (n=10) as well as confirmed dengue (n=12) and Zika virus (n=4) patient sera. IgM-VNT was subsequently performed on single sera from additional JE patients (n=76)., Results: Anti-JEV IgG was detectable in admission serum of 58% of JE patients. The positive, negative and overall percentage agreement of IgM-VNT as compared with standard VNT was 100%. A total of 12/14 (86%) patient samples were unclassified by VNT and, with sufficient sample available for IgG depletion and IgG ELISA confirming depletion, were classified by IgM-VNT. IgM-VNT enabled JE case classification in 72/76 (95%) patients for whom only a single sample was available., Conclusions: The novel approach has been readily adapted for high-throughput testing of single patient samples and it holds promise for incorporation into algorithms for use in reference centres., (© The Author(s) 2022. Published by Oxford University Press on behalf of Royal Society of Tropical Medicine and Hygiene.)

Background: Encephalitis is a worldwide public health issue, with a substantially high burden among children in southeast Asia. We aimed to determine the causes of encephalitis in children admitted to hospitals across the Greater Mekong region by implementing a comprehensive state-of-the-art diagnostic procedure harmonised across all centres, and identifying clinical characteristics related to patients' conditions., Methods: In this multicentre, observational, prospective study of childhood encephalitis, four referral hospitals in Cambodia, Vietnam, Laos, and Myanmar recruited children (aged 28 days to 16 years) who presented with altered mental status lasting more than 24 h and two of the following minor criteria: fever (within the 72 h before or after presentation), one or more generalised or partial seizures (excluding febrile seizures), a new-onset focal neurological deficit, cerebrospinal fluid (CSF) white blood cell count of 5 per mL or higher, or brain imaging (CT or MRI) suggestive of lesions of encephalitis. Comprehensive diagnostic procedures were harmonised across all centres, with first-line testing was done on samples taken at inclusion and results delivered within 24 h of inclusion for main treatable causes of disease and second-line testing was done thereafter for mostly non-treatable causes. An independent expert medical panel reviewed the charts and attribution of causes of all the included children. Using multivariate analyses, we assessed risk factors associated with unfavourable outcomes (ie, severe neurological sequelae and death) at discharge using data from baseline and day 2 after inclusion. This study is registered with ClinicalTrials.gov, NCT04089436, and is now complete., Findings: Between July 28, 2014, and Dec 31, 2017, 664 children with encephalitis were enrolled. Median age was 4·3 years (1·8-8·8), 295 (44%) children were female, and 369 (56%) were male. A confirmed or probable cause of encephalitis was identified in 425 (64%) patients: 216 (33%) of 664 cases were due to Japanese encephalitis virus, 27 (4%) were due to dengue virus, 26 (4%) were due to influenza virus, 24 (4%) were due to herpes simplex virus 1, 18 (3%) were due to Mycobacterium tuberculosis, 17 (3%) were due to Streptococcus pneumoniae, 17 (3%) were due to enterovirus A71, 74 (9%) were due to other pathogens, and six (1%) were due to autoimmune encephalitis. Diagnosis was made within 24 h of admission to hospital for 83 (13%) of 664 children. 119 (18%) children had treatable conditions and 276 (42%) had conditions that could have been preventable by vaccination. At time of discharge, 153 (23%) of 664 children had severe neurological sequelae and 83 (13%) had died. In multivariate analyses, risk factors for unfavourable outcome were diagnosis of M tuberculosis infection upon admission (odds ratio 3·23 [95% CI 1·04-10·03]), coma on day 2 (2·90 [1·78-4·72]), supplementary oxygen requirement (1·89 [1·25-2·86]), and more than 1 week duration between symptom onset and admission to hospital (3·03 [1·68-5·48]). At 1 year after inclusion, of 432 children who were discharged alive from hospital with follow-up data, 24 (5%) had died, 129 (30%) had neurological sequelae, and 279 (65%) had completely recovered., Interpretation: In southeast Asia, most causes of childhood encephalitis are either preventable or treatable, with Japanese encephalitis virus being the most common cause. We provide crucial information that could guide public health policy to improve diagnostic, vaccination, and early therapeutic guidelines on childhood encephalitis in the Greater Mekong region., Funding: Institut Pasteur, Institut Pasteur International Network, Fondation Merieux, Aviesan Sud, INSERM, Wellcome Trust, Institut de Recherche pour le Développement (IRD), and Fondation Total., Competing Interests: Declaration of interests PB is currently an employee of GSK, this employment has no relation with the study presented here that was initiated at the time he was head of the virology unit at Pasteur Institute in Cambodia. All other authors declare no competing interests., (Copyright © 2022 The Author(s). Published by Elsevier Ltd. This is an Open Access article under the CC BY-NC-ND 4.0 license. Published by Elsevier Ltd.. All rights reserved.)

The environmental distribution of Burkholderia pseudomallei, the causative agent of melioidosis, remains poorly understood. B. pseudomallei is known to have the ability to occupy a variety of environmental niches, particularly in soil. This paper provides novel information about a putative association of soil biogeochemical heterogeneity and the vertical distribution of B. pseudomallei. We investigated (1) the distribution of B. pseudomallei along a 300-cm deep soil profile together with the variation of a range of soil physico-chemical properties; (2) whether correlations between the distribution of B. pseudomallei and soil physico-chemical properties exist and (3) when they exist, what such correlations indicate with regards to the environmental conditions conducive to the occurrence of B. pseudomallei in soils. Unexpectedly, the highest concentrations of B. pseudomallei were observed between 100 and 200 cm below the soil surface. Our results indicate that unravelling the environmental conditions favorable to B. pseudomallei entails considering many aspects of the actual complexity of soil. Important recommendations regarding environmental sampling for B. pseudomallei can be drawn from this work, in particular that collecting samples down to the water table is of foremost importance, as groundwater persistence appears to be a controlling factor of the occurrence of B. pseudomallei in soil., (© 2022. The Author(s).)

Background: Scrub typhus (ST) and murine typhus (MT) are common but poorly understood causes of fever in Laos. We examined the spatial and temporal distribution of ST and MT, with the intent of informing interventions to prevent and control both diseases., Methodology and Principle Findings: This study included samples submitted from 2003 to 2017 to Mahosot Hospital, Vientiane, for ST and MT investigation. Serum samples were tested using IgM rapid diagnostic tests. Patient demographic data along with meteorological and environmental data from Laos were analysed. Approximately 17% of patients were positive for either ST (1,337/8,150 patients tested) or MT (1,283/7,552 patients tested). While both diseases occurred in inhabitants from Vientiane Capital, from the univariable analysis MT was positively and ST negatively associated with residence in Vientiane Capital. ST was highly seasonal, with cases two times more likely to occur during the wet season months of July-September compared to the dry season whilst MT peaked in the dry season. Multivariable regression analysis linked ST incidence to fluctuations in relative humidity whereas MT was linked to variation in temperature. Patients with ST infection were more likely to come from villages with higher levels of surface flooding and vegetation in the 16 days leading up to diagnosis., Conclusions: The data suggest that as cities expand, high risk areas for MT will also expand. With global heating and risks of attendant higher precipitation, these data suggest that the incidence and spatial distribution of both MT and ST will increase., Competing Interests: The authors have declared that no competing interests exist.

Antimicrobial resistant (AMR) Enterobacterales are widely distributed among the healthy population of the Indochinese peninsula, including Laos. However, the local reservoir of these pathogens are currently not known and possible sources such as agricultural settings and food have rarely been analyzed. In this work, we investigated the extended-spectrum cephalosporin- (ESC-) and colistin-resistant Escherichia coli strains (CST-R- Ec ) isolated from the gut of local people, feces of poultry, and from chicken meat (60 samples each group) in Laos. Whole-genome sequencing (WGS) analysis based on both short- and long-read sequencing approaches were implemented. The following prevalence of ESC-R- Ec and CST-R- Ec were recorded, respectively: local people (70 and 15%), poultry (20 and 23.3%), and chicken meat (21.7 and 13.3%). Core-genome analysis, coupled with sequence type (ST)/core-genome ST (cgST) definitions, indicated that no common AMR- Ec clones were spreading among the different settings. ESC-R- Ec mostly possessed bla CTX-M-15 and bla CTX-M-55 associated to IS Ecp1 or IS 26 . The majority of CST-R- Ec carried mcr-1 on IncX4, IncI2, IncP1, and IncHI1 plasmids similar or identical to those described worldwide; strains with chromosomal mcr-1 or possessing plasmid-mediated mcr-3 were also found. These results indicate a high prevalence of AMR- Ec in the local population, poultry, and chicken meat. While we did not observe the same clones among the three settings, most of the bla CTX-Ms and mcr-1/-3 were associated with mobile-genetic elements, indicating that horizontal gene transfer may play an important role in the dissemination of AMR- Ec in Laos. More studies should be planned to better understand the extent and dynamics of this phenomenon., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Moser, Kuenzli, Campos-Madueno, Büdel, Rattanavong, Vongsouvath, Hatz and Endimiani.)

Bloodstream infections cause substantial morbidity and mortality. However, despite clinical suspicion of such infections, blood cultures are often negative. We investigated blood cultures that were negative after 5 days of incubation for the presence of bacterial pathogens using specific (Rickettsia spp. and Leptospira spp.) and a broad-range 16S rRNA PCR. From 190 samples, 53 (27.9%) were positive for bacterial DNA. There was also a high background incidence of dengue (90/112 patient serum positive, 80.4%). Twelve samples (6.3%) were positive for Rickettsia spp., including two Rickettsia typhi. The 16S rRNA PCR gave 41 positives; Escherichia coli and Klebsiella pneumoniae were identified in 11 and eight samples, respectively, and one Leptospira species was detected. Molecular investigation of negative blood cultures can identify potential pathogens that will otherwise be missed by routine culture. Patient management would have been influenced in all 53 patients for whom a bacterial organism was identified, and 2.3-6.1% of patients would likely have had an altered final outcome. These findings warrant further study, particularly to determine the cost-benefit for routine use, ways of implementation, and timing of PCR for organisms such as Rickettsia and Leptospira, which are important pathogens in rural Asia.

Melioidosis is a disease of significant public health importance that is being increasingly recognized globally. The majority of cases arise through direct percutaneous exposure to its etiological agent, Burkholderia pseudomallei In the Lao People's Democratic Republic (Laos), the presence and environmental distribution of B. pseudomallei are not well characterized, though recent epidemiological surveys of the bacterium have indicated that B. pseudomallei is widespread throughout the environment in the center and south of the country and that rivers can act as carriers and potential sentinels for the bacterium. The spatial and genetic distribution of B. pseudomallei within Vientiane Capital, from where the majority of cases diagnosed to date have originated, remains an important knowledge gap. We sampled surface runoff from drain catchment areas throughout urban Vientiane to determine the presence and local population structure of the bacterium. B. pseudomallei was detected in drainage areas throughout the capital, indicating it is widespread in the environment and that exposure rates in urban Vientiane are likely more frequent than previously thought. Whole-genome comparative analysis demonstrated that Lao B. pseudomallei isolates are highly genetically diverse, suggesting the bacterium is well-established and not a recent introduction. Despite the wide genome diversity, one environmental survey isolate was highly genetically related to a Lao melioidosis patient isolate collected 13 years prior to the study. Knowledge gained from this study will augment understanding of B. pseudomallei phylogeography in Asia and enhance public health awareness and future implementation of infection control measures within Laos. IMPORTANCE The environmental bacterium B. pseudomallei is the etiological agent of melioidosis, a tropical disease with one model estimating a global annual incidence of 165,000 cases and 89,000 deaths. In the Lao People's Democratic Republic (Laos), the environmental distribution and population structure of B. pseudomallei remain relatively undefined, particularly in Vientiane Capital from where most diagnosed cases have originated. We used surface runoff as a proxy for B. pseudomallei dispersal in the environment and performed whole-genome sequencing (WGS) to examine the local population structure. Our data confirmed that B. pseudomallei is widespread throughout Vientiane and that surface runoff might be useful for future environmental monitoring of the bacterium. B. pseudomallei isolates were also highly genetically diverse, suggesting the bacterium is well-established and endemic in Laos. These findings can be used to improve awareness of B. pseudomallei in the Lao environment and demonstrates the epidemiological and phylogeographical insights that can be gained from WGS., (Copyright © 2020 Rachlin et al.)

In the basin of Mekong, over 70 million people rely on unimproved surface water for their domestic requirements. Surface water is often contaminated with fecal matter and yet little information exists on the underlying mechanisms of fecal contamination in tropical conditions at large watershed scales. Our objectives were to (1) investigate the seasonality of fecal contamination using Escherichia coli as fecal indicator bacteria (FIB), and (2) establish links between the fecal contamination in stream water and its controlling factors (hydrology and land use). We present the results of (1) a sampling campaign at the outlet of 19 catchments across Lao PDR, in both the dry and the rainy seasons of 2016, and (2) a 10-day interval monitoring conducted in 2017 and 2018 at three point locations of three rivers (Nam Ou, Nam Suang, and Mekong) in northern Lao PDR. Our results show the presence of fecal contamination at most of the sampled sites, with a seasonality characterized by higher and extreme E. coli concentrations occurring during the rainy season. The highest E. coli concentrations, strongly correlated with total suspended sediment concentrations, were measured in catchments dominated by unstocked forest areas, especially in mountainous northern Lao PDR and in Vientiane province.

We report 16 Burkholderia pseudomallei genomes, including 5 new multilocus sequence types, isolated from rivers in Laos. The environmental bacterium B. pseudomallei causes melioidosis, a serious infectious disease in tropical and subtropical regions. The isolates are geographically clustered in one clade from around Vientiane, Laos, and one clade from further south., (Copyright © 2021 Liechti et al.)

Introduction: In rural and difficult-to-access settings, early and accurate recognition of febrile children at risk of progressing to serious illness could contribute to improved patient outcomes and better resource allocation. This study aims to develop a prognostic clinical prediction tool to assist community healthcare providers identify febrile children who might benefit from referral or admission for facility-based medical care., Methods and Analysis: This prospective observational study will recruit at least 4900 paediatric inpatients and outpatients under the age of 5 years presenting with an acute febrile illness to seven hospitals in six countries across Asia. A venous blood sample and nasopharyngeal swab is collected from each participant and detailed clinical data recorded at presentation, and each day for the first 48 hours of admission for inpatients. Multianalyte assays are performed at reference laboratories to measure a panel of host biomarkers, as well as targeted aetiological investigations for common bacterial and viral pathogens. Clinical outcome is ascertained on day 2 and day 28.Presenting syndromes, clinical outcomes and aetiology of acute febrile illness will be described and compared across sites. Following the latest guidance in prediction model building, a prognostic clinical prediction model, combining simple clinical features and measurements of host biomarkers, will be derived and geographically externally validated. The performance of the model will be evaluated in specific presenting clinical syndromes and fever aetiologies., Ethics and Dissemination: The study has received approval from all relevant international, national and institutional ethics committees. Written informed consent is provided by the caretaker of all participants. Results will be shared with local and national stakeholders, and disseminated via peer-reviewed open-access journals and scientific meetings., Trial Registration Number: NCT04285021., Competing Interests: Competing interests: None declared., (© Author(s) (or their employer(s)) 2021. Re-use permitted under CC BY. Published by BMJ.)

Although Japanese encephalitis virus (JEV) infection is an important cause of acute febrile illness in Lao PDR (Laos), patient outcome has not been evaluated. We prospectively followed up 123 JEV-infected patients (70 children < 15 years and 53 adults ≥ 15 years) admitted at Mahosot Hospital, Vientiane, from 2003 to 2013. Japanese encephalitis virus infection was diagnosed by the detection of anti-JEV IgM in cerebrospinal fluid and/or IgM seroconversion. Neurological sequelae were assessed using the Liverpool Outcome Score (LOS), total (maximum score = 75), and final (maximum score = 5). The median (interquartile range [IQR]) age of the patients was 12.0 (7.5-18.8) years, and 57% were male. The median (IQR) duration of patients' follow-up was 4.5 (3.2-7.3) years. Of all patients, 10/123 (8.1%) died during hospitalization, and 13/123 (10.6%) died at home after discharge, giving a mortality of 18.7% (23/123) (33 [26.8%] patients were lost to follow-up). The frequency of neurological sequelae at the last follow-up was 61.2% (48.4% in adults and 69.4% in children, P = 0.135). The proportion of patients with severe and moderate functional impairment at the last follow-up was significantly higher in children (25%) than in adults (6.5%), P = 0.042. Half of the patients who were still alive at the last follow-up (67) and for whom LOS data were available (22) had improvements in their total and final LOS between discharge and the last follow-up. The total and final LOS at discharge were not significantly different between children and adults, but total LOS at the last follow-up was significantly higher in adults than in children (median [IQR]: 74.5 [73-75] versus 73.0 [73-75], P = 0.019).

Wet markets are a critical part of South-East Asian culture and economy. However, their role in circulation and transmission of both endemic and emerging disease is a source of concern in a region considered a hotspot of disease emergence. In the Lao People's Democratic Republic (Lao PDR, Laos), live and dead wild animals are frequently found in wet markets, despite legislation against the bushmeat trade. This is generally considered to increase the risk of disease transmission and emergence, although whether or not wildlife vendors themselves have indeed increased incidence of zoonotic disease has rarely been assessed. In preparation for a future longitudinal study of market vendors investigating vendors' exposure to zoonotic pathogens, we conducted a pilot survey of Lao market vendors of wildlife meat, livestock meat and vegetables, to identify demographic characteristics and potential control groups within markets. We also investigated baseline risk perception for infectious diseases among market vendors and assessed the association between risk perception and risk mitigation behaviours. The surveys conducted with 177 vendors revealed similar age, sex, ethnic background and geographical origin between vendor types, but differences in professional background and work history for livestock meat vendors. The perception of disease risk was very low across all vendors, as was the reported use of personal protective equipment, and the two appeared unrelated. Personal risk discounting and assumptions about transmission routes may explain this lack of association. This information will help inform the development of future research, risk communication and risk mitigation policy, especially in the light of the COVID-19 pandemic., (© 2020 The Authors. Zoonoses and Public Health published by Wiley-VCH GmbH.)

Background: Central nervous system (CNS) infections are important contributors to morbidity and mortality and the causative agents for ~50% patients are never identified. The causative agents of some CNS infections have distinct spatial and temporal patterns., Methodology/principal Findings: Here we present the results of a spatial epidemiological and ecological analysis of CNS infections in Lao PDR (2003-2011). The data came from hospitalizations for suspected CNS infection at Mahosot Hospital in Vientiane. Out of 1,065 patients, 450 were assigned a confirmed diagnosis. While many communities in Lao PDR are in rural and remote locations, most patients in these data came from villages along major roads. Japanese encephalitis virus ((JEV); n = 94) and Cryptococcus spp. (n = 70) were the most common infections. JEV infections peaked in the rainy season and JEV patients came from villages with higher surface flooding during the same month as admission. JEV infections were spatially dispersed throughout rural areas and were most common in children. Cryptococcus spp. infections clustered near Vientiane (an urban area) and among adults., Conclusions/significance: The spatial and temporal patterns identified in this analysis are related to complex environmental, social, and geographic factors. For example, JEV infected patients came from locations with environmental conditions (surface water) that are suitable to support larger mosquito vector populations. Most patients in these data came from villages that are near major roads; likely the result of geographic and financial access to healthcare and also indicating that CNS diseases are underestimated in the region (especially from more remote areas). As Lao PDR is undergoing major developmental and environmental changes, the space-time distributions of the causative agents of CNS infection will also likely change. There is a major need for increased diagnostic abilities; increased access to healthcare, especially for rural populations; and for increased surveillance throughout the nation., Competing Interests: The authors have declared that no competing interests exist.

Although there has been an increasing incidence of bacteremia caused by extended-spectrum beta-lactamase (ESBL)-producing Enterobacteriaceae (ESBL-E) across South East Asia, there are sparse data from the Lao PDR, where laboratory capacity for antimicrobial resistance surveillance is limited. We, therefore, retrospectively reviewed bacteremia caused by ESBL-producing Escherichia coli and Klebsiella pneumoniae between 2010 and 2014 at Mahosot Hospital, Vientiane, Lao PDR. Clinical and laboratory data relating to all episodes of ESBL-E bacteremia were reviewed over the 5-year period and compared with non-ESBL-E bacteremia. Blood cultures positive for E. coli or K. pneumoniae were identified retrospectively from laboratory records. Clinical and laboratory data were extracted from research databases and case notes and analyzed using STATA. Between 2010 and 2014, we identified 360 patients with E. coli ( n = 249) or K. pneumoniae ( n = 111) bacteremia, representing 34.8% of all patients with clinically significant bacteremia . Seventy-two (20%) isolates produced ESBL; E. coli accounted for 15.3% (55/360) and K. pneumoniae for 4.7% (17/360), respectively. The incidence of ESBL-producing E. coli bacteremia rose during the study period. By multiple logistic analysis, reported antibiotic use in the previous week was significantly associated with ESBL positivity ( P < 0.001, odds ratio 3.89). Although multiresistant, most ESBL-producing E. coli and K. pneumoniae remained susceptible to meropenem (65/65; 100%) and amikacin (64/65; 98.5%). We demonstrated an alarming increase in the incidence of ESBL-E as a cause of bacteremia in Vientiane during the study period. This has implications for empiric therapy of sepsis in Laos, and ongoing surveillance is essential.

Typhoid conjugate vaccines represent a new tool for typhoid control. However, incidence data are needed to inform decisions about introduction. We sought to estimate typhoid and paratyphoid fever incidence in Vientiane, the capital and largest city of the Lao People's Democratic Republic (Lao PDR). We did a representative cluster survey of health-seeking behavior for fever in Vientiane from January 15, 2019 through January 26, 2019. Multipliers derived from the survey were applied to data from Salmonella Typhi and Salmonella Paratyphi A bloodstream infection surveillance from Mahosot Hospital, Vientiane, for the period of January 1, 2015 through December 31, 2017, to estimate enteric fever incidence. A total of 336 households representing 1,740 persons were enrolled in the healthcare utilization survey, and multipliers were derived based on responses to questions about healthcare seeking in the event of febrile illness. Of 7,997 Vientiane residents receiving blood cultures over the 2-year surveillance period at Mahosot Hospital, we identified 16 (0.2%) with Salmonella Typhi and six (< 0.1%) with Salmonella Paratyphi A bloodstream infection. After applying multipliers, we estimated that the annual incidence of typhoid was 4.7 per 100,000 persons and paratyphoid was 0.5 per 100,000 persons. During the study period, the incidence of typhoid and paratyphoid fever was low in Vientiane. Ongoing surveillance is warranted to identify increases in future years. Similar studies elsewhere in the Lao PDR would be useful to understand the wider enteric fever situation in the country.

Although typhoid is endemic to Southeast Asia, very little is known about the disease in Laos. Typhoid vaccination is not included in the national immunization program. Although sanitation has improved, one million people still do not have access to basic clean water sources. We describe the epidemiology and antimicrobial susceptibility patterns of Salmonella enterica serovar Typhi ( S . Typhi) infection in Laos based on isolates accrued over 18 years at Mahosot Hospital, Vientiane. All blood cultures collected from patients presenting with fever submitted to the Microbiology Laboratory at Mahosot Hospital (February 2000-December 2018) were included. This included patients from Vientiane and four provincial hospitals and one typhoid outbreak investigation. A total of 913 (1.5%) of 60,384 blood cultures were positive for S. Typhi. The majority of isolates with data available (712/898, 79.3%) were susceptible to all antibiotics tested, with 59 (6.5%) multidrug-resistant (MDR) isolates, mostly from one outbreak. Of 854 isolates, 12 (1.4%) were fluoroquinolone resistant. Patient admissions peaked between March and June at the end of the dry season. Although there are key limitations, these data give the first detailed epidemiological evidence of typhoid in Laos. However, estimates will be greatly influenced by access to blood culture services and health-seeking behavior. Although typhoid multidrug resistance and fluoroquinolone resistance are not currently major issues in Laos, continued surveillance and improved antibiotic stewardship are necessary to forestall worsening of the situation. Cost-effectiveness analysis is needed to inform decisions regarding typhoid vaccine introduction.

Determining the etiological basis of central nervous system (CNS) infections is inherently challenging, primarily due to the multi-etiological nature. Using RNA sequencing, we aimed to identify microbes present in cerebrospinal fluid (CSF) of two patients suffering CNS infection, previously diagnosed with Cryptococcus sp. and Streptococcus pneumoniae infection, respectively. After meta-transcriptomic analysis, and confirmation with real-time PCR, hepatitis B virus (HBV) was detected in the CSF of two patients diagnosed with CNS syndrome. Phylogenetic analysis of the partial HBV genomes from these patients showed that they belonged to genotypes B and C and clustered with other viruses of Asian origin. In countries with high levels of HBV endemicity, the virus is likely to be found in patients diagnosed with CNS infections, although whether it contributes to symptoms and pathology, or is simply a coincidental infection, is unknown and merits further investigation., (Copyright © 2019 The Authors. Published by Elsevier Inc. All rights reserved.)

Background: Melioidosis is difficult to diagnose clinically and culture of Burkholderia pseudomallei is the current, imperfect gold standard. However, a reliable point-of-care test (POCT) could enable earlier treatment and improve outcomes., Methods: We evaluated the sensitivity and specificity of the Active Melioidosis Detect™ (AMD) rapid test as a POCT and determined how much it reduced the time to diagnosis compared with culture., Results: We tested 106 whole blood, plasma and buffy coat samples, 96 urine, 28 sputum and 20 pus samples from 112 patients, of whom 26 (23.2%) were culture-positive for B. pseudomallei. AMD sensitivity and specificity were 65.4 and 87.2%, respectively, the latter related to 10 weak positive reactions on urine samples, considered likely false positives. The positive predictive value was 60.7%, negative predictive value was 89.3% and concordance rate between operators reading the test was 95.7%; time to diagnosis decreased by a median of 23 h., Conclusions: Our findings confirm that a strongly positive AMD result can reduce the time to diagnosis of melioidosis. However, the AMD currently has a disappointing overall sensitivity, especially with blood fractions, and specificity problems when testing urine samples., (© The Author(s) 2019. Published by Oxford University Press on behalf of Royal Society of Tropical Medicine and Hygiene. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Trade of bushmeat and other wildlife for human consumption presents a unique set of challenges to policy-makers who are confronted with multiple trade-offs between conservation, food security, food safety, culture and tradition. In the face of these complex issues, risk assessments supported by quantitative information would facilitate evidence-based decision making. We propose a conceptual model for disease transmission risk analysis, inclusive of these multiple other facets. To quantify several processes included in this conceptual model we conducted questionnaire surveys with wildlife consumers and vendors in semi-urban centers in Lao People's Democratic Republic (Lao PDR, Laos) and direct observations of consumer behaviors. Direct observation of market stalls indicated an estimated average of 10 kg bushmeat biomass per stall per hour. The socio-demographic data suggested that consumption of bushmeat in urban areas was not for subsistence but rather driven by dietary preference and tradition. Consumer behavioral observations indicated that each animal receives an average of 7 contacts per hour. We provide other key parameters to estimate the risk of disease transmission from bushmeat consumption and illustrate their use in assessing the total public health and socio-economic impact of bushmeat consumption. Pursuing integrative approaches to the study of bushmeat consumption is essential to develop effective and balanced policies that support conservation, public health, and rural development goals., (Copyright © 2019 The Authors. Published by Elsevier B.V. All rights reserved.)

Cryptococcosis causes approximately 180 000 deaths each year in patients with human immunodeficiency virus (HIV). Patients with other forms of immunosuppression are also at risk, and disease is increasingly recognized in apparently immunocompetent individuals. Cryptococcus neoformans var. grubii, responsible for the majority of cases, is distributed globally. We used the consensus ISHAM Multilocus sequence typing (MLST) scheme to define the population structure of clinical C. neoformans var. grubii isolates from Laos (n = 81), which we placed into the global context using published MLST data from other countries (total N = 1047), including a reanalysis of 136 Vietnamese isolates previously reported. We observed a phylogeographical relationship in which the Laotian population was similar to its neighbor Thailand, being dominated (83%) by Sequence Types (ST) 4 and 6. This phylogeographical structure changed moving eastwards, with Vietnam's population consisting of an admixture of isolates dominated by the ST4/ST6 (35%) and ST5 (48%) lineages. The ST5 lineage is the predominant ST reported from China and East Asia, where it accounts for >90% of isolates. Analysis of genetic distance (Fst) between different populations of C. neoformans var. grubii supports this intermediate structure of the Vietnamese population. The pathogen and host diversity reported from Vietnam provide the strongest epidemiological evidence of the association between ST5 and HIV-uninfected patients. Regional anthropological genetic distances suggest diversity in the C. neoformans var. grubii population across Southeast Asia is driven by ecological rather than human host factors. Where the ST5 lineage is present, disease in HIV-uninfected patients is to be expected., (© The Author(s) 2018. Published by Oxford University Press on behalf of The International Society for Human and Animal Mycology.)

During 2003-2011, we recruited 1,065 patients of all ages admitted to Mahosot Hospital (Vientiane, Laos) with suspected central nervous system (CNS) infection. Etiologies were laboratory confirmed for 42.3% of patients, who mostly had infections with emerging pathogens: viruses in 16.2% (mainly Japanese encephalitis virus [8.8%]); bacteria in 16.4% (including Orientia tsutsugamushi [2.9%], Leptospira spp. [2.3%], and Rickettsia spp. [2.3%]); and Cryptococcus spp. fungi in 6.6%. We observed no significant differences in distribution of clinical encephalitis and meningitis by bacterial or viral etiology. However, patients with bacterial CNS infection were more likely to have a history of diabetes than others. Death (26.3%) was associated with low Glasgow Coma Scale score, and the mortality rate was higher for patients with bacterial than viral infections. No clinical or laboratory variables could guide antibiotic selection. We conclude that high-dependency units and first-line treatment with ceftriaxone and doxycycline for suspected CNS infections could improve patient survival in Laos.