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Increased colonization by antimicrobial-resistant organisms is closely associated with international travel. This study investigated the diversity of mobile genetic elements involved with antimicrobial resistance (AMR) gene carriage in extended-spectrum beta-lactamase (ESBL)-producing Escherichia coli that colonized travellers to Laos. Long-read sequencing was used to reconstruct complete plasmid sequences from 48 isolates obtained from the daily stool samples of 23 travellers over a 3 week period. This method revealed a collection of 105 distinct plasmids, 38.1 % (n=40) of which carried AMR genes. The plasmids in this population were diverse, mostly unreported and included 38 replicon types, with F-type plasmids (n=23) the most prevalent amongst those carrying AMR genes. Fine-scale analysis of all plasmids identified numerous AMR gene contexts and emphasized the importance of IS elements, specifically members of the IS6/IS26 family, in the evolution of complex multidrug resistance regions. We found a concerning convergence of ESBL and colistin resistance determinants, with three plasmids from two different F-type lineages carrying bla CTX-M and mcr genes. The extensive diversity seen here highlights the worrying probability that stable new vehicles for AMR will evolve in E. coli populations that can disseminate internationally through travel networks.

Background: Early and accurate diagnosis of acute infection has important consequences for antibiotic stewardship, resource allocation, and clinical outcomes. However, limited sensitivity and specificity of current diagnostics lead to the erroneous prescription of antibiotics. Host-response-based diagnostics have the potential to address these challenges, but accuracy varies widely across heterogeneous global patient populations. Methods: We performed a multi-cohort analysis of 4,200 unique samples across 69 retrospective blood transcriptome datasets from 20 countries. These samples were collected from patients with acute bacterial or viral infections representing a broad spectrum of biological (age, sex, race, ethnicity, pathogen, host genetics), clinical (severity, day of presentation), and technical (Affymetrix, Agilent, Illumina) heterogeneity. We also enrolled patients with infectious diseases in two cohorts from Laos and Nepal. Findings: Current host-response-based gene signatures distinguished intracellular bacterial infection from viral infections with substantially lower accuracy. Using 69 datasets, divided into training and validation datasets, we identified an 8-gene signature that distinguished intracellular or extracellular bacterial infections from viral infections with an area under the receiver operating characteristic curve (AUROC) >0.91 (85.9% specificity, 90.2% sensitivity). In two prospective cohorts from Nepal and Laos, profiled using Fluidigm RT-PCR, the 8-gene classifier distinguished bacterial infections from viral infections with an AUROC of 0.94 (87.9% specificity, 91% sensitivity). Interpretation: The 8-gene signature met the target product profile (90% sensitivity, 80% specificity) proposed by the WHO and others for distinguishing bacterial and viral infections. The 8-gene signature should be considered for further validation and implementation. Funding Information: PK is funded in part by the Bill and Melinda Gates Foundation (OPP1113682); the National Institute of Allergy and Infectious Diseases (NIAID) grants 1U19AI109662, U19AI057229, and 5R01AI125197; Department of Defense contracts W81XWH-18-1-0253 and W81XWH1910235; and the Ralph & Marian Falk Medical Research Trust. DAR is supported by NIH/NIAID U19 AI109761 and the Thomas C. and Joan M. Merigan Endowment at Stanford University. LOMWRU is funded by the Wellcome Trust of Great Britain. AMR is funded by the National Science Foundation Graduate Research Fellowship and the Stanford Graduate Fellowship. This research was funded in whole, or in part, by the Wellcome Trust [Grant number 220211]. Declaration of Interests: AMR, SJP, TES, DAR, and PK are named as inventors on a patent application describing the 8-gene set, which has been licensed to Inflammatix. TES and SAT are employees of Inflammatix, and TES and PK are shareholders in Inflammatix. Inflammatix had no role in the design, funding, or reporting of this work. SD is currently employed by FIND. The other authors declare no conflicts of interest. Ethics Approval Statement: Ethical clearance was granted by the former Faculty of Medical Sciences Ethical Review Committee (now University of Health Sciences Ethics Committee), National University of Laos, the Oxford University Tropical Ethics Committee, and the Stanford University Institutional Review Board.

Bartonella species are fastidious Gram negative vector-borne bacteria with a wide range of mammalian reservoirs. While it is understood that some species Bartonella are human pathogens, the extent of human exposure to Bartonella species (both pathogenic and non-pathogenic) has yet to be fully understood. To this end, residual sera from participants enrolled in undifferentiated fever studies in Cambodia, Ghana, Laos, and Peru were screened for the presence of IgG antibodies against B. quintana and B. henselae, using the FOCUS diagnostics Dual Spot- Bartonella IgG Immunofluorescence assay. Forty-eight patients with suspected or confirmed B. bacilliformis exposure or infection in Peru, were screened to assess cross-reactivity of the FOCUS assay for IgG against other Bartonella species. Ten of 13 patients with confirmed B. bacilliformis infection were Bartonella-specific IgG positive and overall, 36/48 of the samples were positive. Additionally, 79/206, 44/200, 101/180, and 57/100 of samples from Peru, Laos, Cambodia, and Ghana, respectively were Bartonella-specific IgG positive. Further, ectoparasites pools from Cambodia, Laos, and Peru were tested using quantitative real-time PCR (qPCR) for the presence of Bartonella DNA. Of the sand-fly pools collected in Peru, 0/196 were qPCR positive; 15/140 flea pools collected in Cambodia were qPCR positive; while 0/105 ticks, 0/22 fleas, and 0/3 louse pools collected in Laos tested positive for Bartonella DNA. Evidence of Bartonella in fleas from Cambodia supports the possibility that humans are exposed to Bartonella through this traditional vector. However, Bartonella species were not found in fleas, ticks, or lice from Laos, or sandflies from Peru. This could account for the lower positive serology among the population in Laos and the strictly localized nature of B. bacillformis infections in Peru. Human exposure to Bartonella species and Bartonella as a human pathogen warrants further investigation.

Increased colonisation by antimicrobial resistant organisms is closely associated with international travel. This study investigated the diversity of mobile genetic elements involved with antimicrobial resistance (AMR) gene carriage in extended-spectrum beta-lactamase (ESBL) -producing Escherichia coli that colonised travellers to Laos. Long-read sequencing was used to reconstruct complete plasmid sequences from 49 isolates obtained from the daily stool samples of 23 travellers over a three-week period. This method revealed a collection of 105 distinct plasmids, 38.1% of which carried AMR genes. The plasmids in this population were diverse, mostly unreported and included 38 replicon types, with F-type plasmids (n=22) the most prevalent amongst those carrying AMR genes.Fine-scale analysis of all plasmids identified numerous AMR gene contexts and emphasised the importance of IS elements, specifically members of the IS6/IS26 family, in the creation of complex multi-drug resistance regions. We found a concerning convergence of ESBL and colistin resistance determinants, with three plasmids from two different F-type lineages carrying blaCTX-M and mcr genes. The extensive diversity seen here highlights the worrying probability that stable new vehicles for AMR will evolve in E. coli populations that can disseminate internationally through travel networks.Impact StatementThe global spread of AMR is closely associated with international travel. AMR is a severe global concern and has compromised treatment options for many bacterial pathogens, among them pathogens carrying ESBL and colistin resistance genes. Colonising MDR organisms have the potential to cause serious consequences. Infections caused by MDR bacteria are associated with longer hospitalisation, poorer patient outcomes, greater mortality, and higher costs compared to infections with susceptible bacteria.This study elucidates the numerous different types of plasmids carrying AMR genes in colonising ESBL-producing E. coli isolates found in faecal samples from in travellers to Vientiane, Laos. Here we add to known databases of AMR plasmids by adding these MDR plasmids found in Southeast Asia, an area of high AMR prevalence. We characterised novel AMR plasmids including complex ESBL (blaCTX-M) and colistin (mcr) resistance co-carriage plasmids, emphasising the potential exposure of travellers to Laos to a wide variety of mobile genetic elements that may facilitate global AMR spread. This in-depth study has revealed further detail of the numerous factors that may influence AMR transfer, therefore potential routes of AMR spread internationally, and is a step towards finding methods to combat AMR spread.Data SummaryLong-read sequencing data is available through National Center for Biotechnology Information under the BioProject PRJNA853172. Complete plasmid sequences have been uploaded to GenBank with accession numbers in supplementary S1. The authors confirm all supporting data, code and protocols have been provided within the article or through supplementary data files.

Conventional disease surveillance for shigellosis in developing country settings relies on serotyping and low-resolution molecular typing, which fails to contextualise the evolutionary history of the genus. Here, we interrogated a collection of 1,804 Shigella whole genome sequences from organisms isolated in four continental Southeast Asian countries (Thailand, Vietnam, Laos, and Cambodia) over three decades to characterise the evolution of both S. flexneri and S. sonnei. We show that S. sonnei and each major S. flexneri serotype are comprised of genetically diverse populations, the majority of which were likely introduced into Southeast Asia in the 1970s–1990s. Intranational and regional dissemination allowed widespread propagation of both species across the region. Our data indicate that the epidemiology of S. sonnei and the major S. flexneri serotypes were characterised by frequent clonal replacement events, coinciding with changing susceptibility patterns against contemporaneous antimicrobials. We conclude that adaptation to antimicrobial pressure was pivotal to the recent evolutionary trajectory of Shigella in Southeast Asia., Hao Chung The et al. analyze 1,804 Shigella genome sequences from organisms isolated in four Southeast Asian countries over three decades to study the evolution of both S. flexneri and S. sonnei. This study suggests that adaptation to antimicrobial pressure may have played a pivotal role in the recent evolutionary trajectory of Shigella in Southeast Asia.

Background: Streptococcus agalactiae is a normal commensal of the human gastro-intestinal and female genital tracts. It causes serious disease in neonates and pregnant women, as well as non-pregnant adults. Food-borne outbreaks have also been described. A link between invasive Group B streptococcus (GBS) infection in humans caused by S. agalactiae serotype III-4, sequence type 283 (ST283) and the consumption of raw fresh-water fish was first described in Singapore in 2015. Case presentation: We report the simultaneous occurrence of acute fever and myalgia in two sisters who were visiting Laos. Both were found to have invasive GBS ST283 infection, confirmed by blood culture. Infection was temporally linked to fish consumption. They responded well to intravenous antibiotics within 48 hours. Conclusions: Food-borne transmission of Streptococcus agalactiae is an important and under-recognised source of serious human disease throughout Southeast Asia and possibly beyond.

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. Translations For the Khmer, Lao, Vietnamese and Burmese translation of the abstract see Supplementary Materials section.

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.

ObjectivesTo review the scientific evidence base on antimicrobial use (AMU) and antimicrobial resistance (AMR) in human and animal sectors in the Lao PDR (Laos).MethodsWe reviewed all publications from July 1994 (the first article describing AMR in Laos) to December 2020. Electronic searches were conducted using Google Scholar and PubMed with specific terms relating to AMR and AMU in Lao, French and English languages.FindingsWe screened 1,357 peer-reviewed and grey reports by title and abstract and then full articles/reports. Of 80 included, 66 (83%) related to human health, nine (11%) to animal health, four (5%) to both animal and human health and one (1%) to the environment. Sixty-two (78%) were on AMR and 18 (22%) on AMU. Extended spectrum beta lactamase-producing Escherichia coli was the greatest concern identified; the proportion of isolates increased fivefold from 2004 to 2016 (2/28 (7%) to 27/78 (35%)) from blood cultures submitted to the Microbiology Laboratory, Mahosot Hospital, Vientiane. Carbapenem resistant Escherichia coli was first identified in 2015. Methicillin-resistant Staphylococcus aureus (MRSA) was uncommon, with 15 cases of MRSA from blood cultures between its first identification in 2017 and December 2020. AMR patterns of global antimicrobial resistance surveillance system (GLASS) target pathogens from livestock were less well documented. There were few data on AMU in human health and none on AMU in livestock. The first hospital AMU survey in Laos showed that 70% (1,386/1,981) of in-patients in five hospitals from 2017 to 2018 received antimicrobial(s). Antibiotic self-medication was common.ConclusionAMR in Laos is occurring at relatively low proportions for some GLASS pathogens, giving the country a window of opportunity to act quickly to implement strategies to protect the population from a worsening situation. Urgent interventions to roll out new guidelines with enhanced one-health antibiotic stewardship, reduce antibiotic use without prescriptions, enhance surveillance and improve understanding of AMU and AMR are needed.

Background Antimicrobial resistance is highly prevalent in low-income and middle-income countries. International travel contributes substantially to the global spread of intestinal multidrug-resistant Gram-negative bacteria. Hundreds of millions of annual visitors to low-income and middle-income countries are all exposed to intestinal multidrug-resistant Gram-negative bacteria resulting in 30–70% of them being colonised at their return. The colonisation process in high-exposure environments is poorly documented because data have only been derived from before travel and after travel sampling. We characterised colonisation dynamics by exploring daily stool samples while visiting a low-income and middle-income countries. Methods In this prospective, daily, real-time sampling study 20 European visitors to Laos volunteered to provide daily stool samples and completed daily questionnaires for 22 days. Samples were initially assessed at Mahosot Hospital, Vientiane, Laos, for acquisition of extended-spectrum β-lactamase-producing (ESBL) Gram-negative bacteria followed by whole-genome sequencing of isolates at MicrobesNG, University of Birmingham, Birmingham, UK. The primary outcome of the study was to obtain data on the dynamics of intestinal multidrug-resistant bacteria acquisition. Findings Between Sept 18 and Sept 20, 2015, 23 volunteers were recruited, of whom 20 (87%) European volunteers were included in the final study population. Although colonisation rates were 70% at the end of the study, daily sampling revealed that all participants had acquired ESBL-producing Gram-negative bacteria at some point during the study period; the colonisation status varied day by day. Whole-genome sequencing analysis ascribed the transient pattern of colonisation to sequential acquisition of new strains, resulting in a loss of detectable colonisation by the initial multidrug-resistant Gram-negative strains. 19 (95%) participants acquired two to seven strains. Of the 83 unique strains identified (53 Escherichia coli, 10 Klebsiella spp, and 20 other ESBL-producing Gram-negative bacteria), some were shared by as many as four (20%) participants. Interpretation To our knowledge, this is the first study to characterise in real-time the dynamics of acquiring multidrug-resistant Gram-negative bacterial colonisation during travel. Our data show multiple transient colonisation events indicative of constant microbial competition and suggest that travellers are exposed to a greater burden of multidrug-resistant bacteria than previously thought. The data emphasise the need for preventing travellers' diarrhoea and limiting antibiotic use, addressing the two major factors predisposing colonisation. Funding The Finnish Governmental Subsidy for Health Science Research, The Scandinavian Society for Antimicrobial Chemotherapy, the Sigrid Jusélius Foundation, Biotechnology and Biological Sciences Research Council; Wellcome Trust, Medical Research Council; The Royal Society; Joint Programming Initiative on Antimicrobial Resistance, and European Research Council.

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.

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.

Leptospirosis is a globally important cause of acute febrile illness, and a common cause of non-malarial fever in Asia, Africa, and Latin America. Simple rapid diagnostic tests (RDTs) are needed to enable health-care workers, particularly in low resource settings, to diagnose leptospirosis early and give timely targeted treatment. This study compared four commercially available RDTs to detect human IgM against Leptospira spp. in a head-to-head prospective evaluation in Mahosot Hospital, Lao PDR. Patients with an acute febrile illness consistent with leptospirosis (N = 695) were included in the study during the 2014 rainy season. Samples were tested with four RDTs: (“Test-it” [Life Assay, Cape Town, South Africa; N = 418]; “Leptorapide” [Linnodee, Ballyclare, Northern Ireland; N = 492]; “Dual Path Platform” [DPP] [Chembio, Medford, NY; N = 530]; and “SD-IgM” [Standard Diagnostics, Yongin, South Korea; N = 481]). Diagnostic performance characteristics were calculated and compared with a composite reference standard combining polymerase chain reaction (PCR) (rrs), microscopic agglutination tests (MATs), and culture. Of all patients investigated, 39/695 (5.6%) were positive by culture, PCR, or MAT. The sensitivity and specificity of the RDTs ranged greatly from 17.9% to 63.6% and 62.1% to 96.8%, respectively. None of the investigated RDTs reached a sensitivity or specificity of > 90% for detecting Leptospira infections on admission. In conclusion, our investigation highlights the challenges associated with Leptospira diagnostics, particularly in populations with multiple exposures. These findings emphasize the need for extensive prospective evaluations in multiple endemic settings to establish the value of rapid tools for diagnosing fevers to allow targeting of antibiotics.

Objectives The aim of this study was to determine the antimicrobial susceptibility of Neisseria gonorrhoeae isolates in the Lao People’s Democratic Republic (Laos). Methods A total of 165 gonococcal isolates (1.3%) were obtained from 12 281 genital samples routinely submitted to a diagnostic laboratory in Vientiane, Laos, between 2011 and 2015. Susceptibility to five antibiotics was determined by the standard disk diffusion method for 158 of the isolates. Results Rates of resistance to penicillin (by β-lactamase production), tetracycline and ciprofloxacin were 89.9%, 99.4% and 84.8%, respectively. All isolates were susceptible to ceftriaxone and spectinomycin. Conclusions The situation in Laos is similar to that in neighbouring countries; this fortunately means that the latest Lao national guidelines for treating gonorrhoea should still be effective.

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. (C) 2019 The Authors. Published by Elsevier Inc.

International audience; 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.

Respiratory diseases are a major contributor to morbidity and mortality in many tropical countries, including Lao PDR. However, little has been published regarding viral or bacterial pathogens that can contribute to influenza-like illness (ILI) in a community setting. We report on the results of a community-based surveillance that prospectively monitored the incidence of ILI and its causative pathogens in Vientiane capital in Lao PDR. A cohort of 995 households, including 4885 study participants, were followed-up between May 2015 and May 2016. Nasopharyngeal swabs, throat swabs, and sputum specimens were collected from ILI cases identified through active case-finding. Real-Time PCR was used to test nasopharyngeal swabs for 21 respiratory pathogens, while throat and sputum samples were subjected to bacterial culture. Generalized linear mixed models were used to assess potential risk factors for associations with ILI. In total, 548 episodes of ILI were reported among 476 (9.7%) of the study participants and 330 (33.2%) of the study households. The adjusted estimated incidence of ILI within the study area was 10.7 (95%CI: 9.4-11.9) episodes per 100 person-years. ILI was significantly associated with age group (p

To help address the limitations of operating conventional microbiological culture incubators in low resource environments, a new incubator design was developed and tested to meet the requirements of operation in laboratories without reliable power (power outages up to 12 contiguous hours) or climate control (ambient indoor temperatures from 5 °C to 45 °C). The device is designed to enable adherence to incubation temperatures recommended for growth detection, identification, and drug susceptibility testing (DST) of human pathogenic bacteria. During power outages, stable temperatures are maintained in the device's internal sample compartment by employing phase change material (PCM) as a bi-directional thermal battery to maintain incubation temperature. Five prototypes were tested in a laboratory setting using environmental test chambers and programmable power supplies, and three were field tested in the Lao PDR in situations of intended use. The prototypes successfully held their temperature to within ±1 °C in both laboratory environmental chamber testing as well as during the field test. The results indicate that the device will maintain stable culture temperatures across periods of intermittent power supply, while enabling normal workflow of this could greatly increase the availability of microbiological culture for diagnosis and antimicrobial resistance (AMR) monitoring.

Background Murine typhus, or infection with Rickettsia typhi, is a global but neglected disease without randomized clinical trials to guide antibiotic therapy. Methods A prospective, open, randomized trial was conducted in nonpregnant, consenting inpatient adults with rapid diagnostic test evidence of uncomplicated murine typhus at 2 hospitals in Vientiane, Laos. Patients were randomized to 7 days (D7) or 3 days (D3) of oral doxycycline or 3 days of oral azithromycin (A3). Primary outcome measures were fever clearance time and frequencies of treatment failure and relapse. Results Between 2004 and 2009, the study enrolled 216 patients (72 per arm); 158 (73.2%) had serology/polymerase chain reaction (PCR)–confirmed murine typhus, and 52 (24.1%) were R. typhi PCR positive. The risk of treatment failure was greater for regimen A3 (22.5%; 16 of 71 patients) than for D3 (4.2%; 3 of 71) or D7 (1.4%; 1 of 71) (P < .001). Among R. typhi PCR–positive patients, the area under the time-temperature curve and the fever clearance time were significantly higher for A3 than for D3 (1.8- and 1.9-fold higher, respectively; P = .005) and D7 (1.5- and 1.6-fold higher; P = .02). No patients returned with PCR-confirmed R. typhi relapse. Conclusion In Lao adults, azithromycin is inferior to doxycycline as oral therapy for uncomplicated murine typhus. For doxycycline, 3- and 7-day regimens have similar efficacy. Azithromycin use in murine typhus should be reconsidered. Investigation of genomic and phenotypic markers of R. typhi azithromycin resistance is needed. Clinical Trial Registration ISRCTN47812566., A clinical trial of murine typhus, or infection with Rickettsia typhi, in Lao adults suggests that azithromycin is inferior to doxycycline for the oral therapy of uncomplicated murine typhus. Three and 7 days of doxycycline have similar efficacy.

Burkholderia pseudomallei causes significant global morbidity and mortality, with the highest disease burden in parts of Asia where culture-based diagnosis is often not available. We prospectively evaluated the Active Melioidosis Detect (AMD; InBios International, USA) lateral flow immunoassay (LFI) for rapid detection of B. pseudomallei in turbid blood cultures, pus, sputum, sterile fluid, urine, and sera. The performance of this test was compared to that of B. pseudomallei detection using monoclonal antibody latex agglutination (LA) and immunofluorescence assays (IFA), with culture as the gold standard. AMD was 99% (99/100; 95% confidence interval, 94.6 to 100%) sensitive and 100% (308/308; 98.8 to 100%) specific on turbid blood culture bottles, with no difference from LA or IFA. AMD specificity was 100% on pus (122/122; 97.0 to 100%), sputum (20/20; 83.2 to 100%), and sterile fluid (44/44; 92 to 100%). Sensitivity on these samples was as follows: pus, 47.1% (8/17; 23.0 to 72.2%); sputum, 33.3% (1/3; 0.84 to 90.6%); and sterile fluid, 0% (0/2; 0 to 84.2%). For urine samples, AMD had a positive predictive value of 94% (32/34; 79.7 to 98.5%) for diagnosing melioidosis in our cohort. AMD sensitivity on stored sera, collected prospectively from melioidosis cases during this study, was 13.9% (5/36; 4.7% to 29.5%) compared to blood culture samples taken on the same day. In conclusion, AMD is an excellent tool for rapid diagnosis of melioidosis from turbid blood cultures and maintains specificity across all sample types. It is a promising tool for urinary antigen detection, which could revolutionize diagnosis of melioidosis in resource-limited settings. Further work is required to improve sensitivity on nonblood culture samples.

Burkholderia pseudomallei, causative agent of the often fatal disease melioidosis, dwells in tropical soils and has been found in freshwater bodies. To investigate whether rivers are potential habitats or carriers for B. pseudomallei and to assess its geographical distribution in Laos, we studied 23 rivers including the Mekong, applying culture-based detection methods and PCR to water filters and streambed sediments. B. pseudomallei was present in 9% of the rivers in the dry season and in 57% in the rainy season. We found the pathogen exclusively in Southern and Central Laos, and mainly in turbid river water, while sediments were positive in 35% of the B. pseudomallei-positive sites. Our results provide evidence for a heterogeneous temporal and spatial distribution of B. pseudomallei in rivers in Laos with a clear north-south contrast. The seasonal dynamics and predominant occurrence of B. pseudomallei in particle-rich water suggest that this pathogen is washed out with eroded soil during periods of heavy rainfall and transported by rivers, while river sediments do not seem to be permanent habitats for B. pseudomallei. Rivers may thus be useful to assess the distribution and aquatic dispersal of B. pseudomallei and other environmental pathogens in their catchment area and beyond.

Burkholderia pseudomallei is the cause of melioidosis, a severe and potentially fatal disease of humans and animals. It is endemic in northern Australia and Southeast Asia and is found in soil and surface water. The environmental distribution of B. pseudomallei worldwide and within countries where it is endemic, such as the Lao People's Democratic Republic (Laos), remains unclear. However, this knowledge is important to our understanding of the ecology and epidemiology of B. pseudomallei and to facilitate public health interventions. Sensitive and specific methods to detect B. pseudomallei in environmental samples are therefore needed. The aim of this study was to compare molecular and culture-based methods for the detection of B. pseudomallei in soil and surface water in order to identify the optimal approach for future environmental studies in Laos. Molecular detection by quantitative real-time PCR (qPCR) was attempted after DNA extraction directly from soil or water samples or after an overnight enrichment step. The positivity rates obtained by qPCR were compared to those obtained by different culture techniques. The rate of detection from soil samples by qPCR following culture enrichment was significantly higher (84/100) than that by individual culture methods and all culture methods combined (44/100; P < 0.001). Similarly, qPCR following enrichment was the most sensitive method for filtered river water compared with the sensitivity of the individual methods and all individual methods combined. In conclusion, molecular detection following an enrichment step has proven to be a sensitive and reliable approach for B. pseudomallei detection in Lao environmental samples and is recommended as the preferred method for future surveys.

Objectives To compare two molecular assays (rrs quantitative PCR (qPCR) versus a combined 16SrRNA and LipL32 qPCR) on different sample types for diagnosing leptospirosis in febrile patients presenting to Mahosot Hospital, Vientiane, Laos. Methods Serum, buffy coat and urine samples were collected on admission, and follow-up serum ∼10 days later. Leptospira spp. culture and microscopic agglutination tests (MAT) were performed as reference standards. Bayesian latent class modelling was performed to estimate sensitivity and specificity of each diagnostic test. Results In all, 787 patients were included in the analysis: 4/787 (0.5%) were Leptospira culture positive, 30/787 (3.8%) were MAT positive, 76/787 (9.7%) were rrs qPCR positive and 20/787 (2.5%) were 16SrRNA/LipL32 qPCR positive for pathogenic Leptospira spp. in at least one sample. Estimated sensitivity and specificity (with 95% CI) of 16SrRNA/LipL32 qPCR on serum (53.9% (33.3%–81.8%); 99.6% (99.2%–100%)), buffy coat (58.8% (34.4%–90.9%); 99.9% (99.6%–100%)) and urine samples (45.0% (27.0%–66.7%); 99.6% (99.3%–100%)) were comparable with those of rrs qPCR, except specificity of 16SrRNA/LipL32 qPCR on urine samples was significantly higher (99.6% (99.3%–100%) vs. 92.5% (92.3%–92.8%), p Conclusions Serum and urine are better samples for qPCR than buffy coat, and 16SrRNA/LipL32 qPCR performs better than rrs qPCR on urine. Quantitative PCR on admission is a reliable rapid diagnostic tool, performing better than MAT or culture, with significant implications for clinical and epidemiological investigations of this global neglected disease.

Cryptococcosis is amongst the most important invasive fungal infections globally, with cryptococcal meningitis causing an estimated 180,000 deaths each year in HIV infected patients alone. Patients with other forms of immunosuppression are also at risk, and disease is increasingly recognized in apparently immunocompetent individuals. Cryptococcus neoformans var. grubii (serotype A, molecular type VNI) has a global distribution and is responsible for the majority of cases. Here, we used the consensus ISHAM Multilocus Sequence Typing (MLST) for C. neoformans to define the population structure of clinical isolates of Cryptococcus neoformans var. grubii from Vietnam (n=136) and Laos (n=81). We placed these isolates into the global context using published MLST data from 8 other countries (total N = 669). We observed a phylo-geographical relationship in which Laos was similar to its Southeast Asian neighbor Thailand in being dominated (83%) by Sequence Type (ST) 4 and its Single Locus Variant ST6. On the other hand, Vietnam was uniquely intermediate between Southeast Asia and East Asia having both ST4/ST6 (35%) and ST5 (48%) which causes the majority of cases in East Asia. Analysis of genetic distance (Fst) between different populations of Cryptococcus neoformans var. grubii supported the intermediate nature of the population from Vietnam. A strong association between ST5 and infection in apparently immunocompetent, HIV-uninfected patients was observed in Vietnam (OR: 7.97, [95%CI: 3.18-19.97], p < 0.0001). Our study emphasizes that Vietnam, with its intermediate Cryptococcus neoformans var. grubii population structure, provides the strongest epidemiological evidence of the relationship between ST5 and infection of HIV-uninfected patients. Human population genetic distances within the region suggest these differences in CNVG population across Southeast Asia are driven by ecological factors rather than host factors.Author summaryCryptococcus neoformans is a yeast that causes meningitis in people, usually with damaged immune systems. There are >180,000 deaths in HIV-infected patients each year, most occurring where there are the highest HIV/AIDS disease burdens. Vietnam and Laos have contributed significantly to clinical trials aiming to improve the treatment of cryptococcal meningitis, but the relationship of isolates from these countries to the global population is not yet described. Here, we address this knowledge gap by using Multilocus Sequence Typing to study the population of Cryptococcus neoformans var. grubii (CNVG) in Laos and Vietnam, with the specific aim of incorporating these populations into the wider global context. We found that, in most countries, a single lineage (family) of strains was responsible for most disease. The Vietnamese CNVG population was unusual in that 2 main lineages circulated at the same time. The Vietnamese CNVG population occupies a middle ground between Thailand/Laos in the west and China in the east. The differences in population structure moving from West to East are probably due to ecological differences. Disease in HIV uninfected patients was almost always due to members of a single family of strains (ST5).

This is the first report of the molecular epidemiology of Staphylococcus aureus from skin and soft tissue infections (SSTI) in Laos. We selected a random sample of 96 S. aureus SSTI isolates received by the Microbiology Laboratory, Mahosot Hospital, Vientiane, between July 2012 and June 2014, including representation from seven referral hospitals. Isolates underwent susceptibility testing by Clinical and Laboratory Standards Institute methods, spa typing and DNA microarray analysis, with whole genome sequencing for rare lineages. Median patient age was 19.5 years (interquartile range 2–48.5 years); 52% (50) were female. Forty-three spa types, representing 17 lineages, were identified. Fifty-eight percent (56) of all isolates encoded Panton-Valentine leukocidin (PVL), representing six lineages: half of these patients had abscesses and three had positive blood cultures. The dominant lineage was CC121 (39; 41%); all but one isolate encoded PVL and 49% (19) were from children under five. Staphyococcus argenteus was identified in six (6%) patients; mostly adults > 50 years and with diabetes. Six isolates (6%) belonged to rare lineage ST2885; two possibly indicate cross-infection in a neonatal unit. One isolate from a previously undescribed lineage, ST1541, was identified. Antibiotic resistance was uncommon except for penicillin (93; 97%) and tetracycline (48; 50%). Seven (7%) isolates were methicillin-resistant S. aureus (MRSA), belonging to ST239-MRSA-III, CC59-MRSA-V(T) Taiwan Clone, ST2250-MRSA-IV, ST2885-MRSA-V and CC398-MRSA-V. Globally widespread CC5 and CC30 were absent. There are parallels in S. aureus molecular epidemiology between Laos and neighboring countries and these data highlight the prominence of PVL and suggest infiltration of MRSA clones of epidemic potential from surrounding countries.

Orientia tsutsugamushi, which requires specialized facilities for culture, is a substantial cause of disease in Asia. We demonstrate that O. tsutsugamushi numbers increased for up to 5 days in conventional hemocultures. Performing such a culture step before molecular testing could increase the sensitivity of O. tsutsugamushi molecular diagnosis.

Leptospirosis is an important zoonosis worldwide, with infections occurring after exposure to contaminated water. Despite being a global problem, laboratory diagnosis remains difficult with culture results taking up to 3 months, serology being retrospective by nature, and polymerase chain reaction showing limited sensitivity. Leptospira have been shown to survive and multiply in blood culture media, and we hypothesized that extracting DNA from incubated blood culture fluid (BCF), followed by quantitative real-time polymerase chain reaction (qPCR) could improve the accuracy and speed of leptospira diagnosis. We assessed this retrospectively, using preincubated BCF of Leptospira spp. positive (N= 109) and negative (N= 63) febrile patients in Vientiane, Lao PDR. The final method showed promising sensitivities of 66% (95% confidence interval [CI]: 55-76) and 59% (95% CI: 49-68) compared with direct or direct and indirect testing combined, as the respective reference standards (specificities > 95%). Despite these promising diagnostic parameters, a subsequent prospective evaluation in a Lao hospital population (N= 352) showed that the sensitivity was very low (∼30%) compared with qPCR on venous blood samples. The disappointingly low sensitivity does suggest that venous blood samples are preferable for the clinical microbiology laboratory, although BCF might be an alternative if leptospirosis is only suspected postadmission after antibiotics have been used.

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.

International audience; The emergence of multidrug-resistant (MDR) typhoid is a major global health threat affecting many countries where the disease is endemic. Here whole-genome sequence analysis of 1,832 Salmonella enterica serovar Typhi (S. Typhi) identifies a single dominant MDR lineage, H58, that has emerged and spread throughout Asia and Africa over the last 30 years. Our analysis identifies numerous transmissions of H58, including multiple transfers from Asia to Africa and an ongoing, unrecognized MDR epidemic within Africa itself. Notably, our analysis indicates that H58 lineages are displacing antibiotic-sensitive isolates, transforming the global population structure of this pathogen. H58 isolates can harbor a complex MDR element residing either on transmissible IncHI1 plasmids or within multiple chromosomal integration sites. We also identify new mutations that define the H58 lineage. This phylogeographical analysis provides a framework to facilitate global management of MDR typhoid and is applicable to similar MDR lineages emerging in other bacterial species.

Background Mycetoma is a neglected, chronic, localized, progressively destructive, granulomatous infection caused either by fungi (eumycetoma) or by aerobic actinomycetes (actinomycetoma). It is characterized by a triad of painless subcutaneous mass, multiple sinuses and discharge containing grains. Mycetoma commonly affects young men aged between 20 and 40 years with low socioeconomic status, particularly farmers and herdsmen. Methodology / Principal Findings A 30 year-old male farmer from an ethnic minority in Phin District, Savannakhet Province, Lao PDR (Laos) developed a painless swelling with multiple draining sinuses of his right foot over a period of approximately 3 years. X-ray of the right foot showed osteolysis of tarsals and metatarsals. Aerobic culture of sinus discharge yielded large numbers of Staphylococcus aureus and a slow growing Gram-positive rod. The organism was subsequently identified as Nocardia aobensis by 16S ribosomal RNA gene sequencing. The patient received antimicrobial treatment with amikacin and trimethoprim-sulfamethoxazole according to consensus treatment guidelines. Although slight improvement was noted the patient left the hospital after 14 days and did not take any more antibiotics. Over the following 22 weeks the swelling of his foot subsequently diminished together with healing of discharging sinuses. Conclusion This is the first published case of Actinomycetoma caused by Nocardia aobensis and the second case of Actinomycetoma from Laos. A treatment course of only 14 days with amikacin and trimethoprim-sulfamethoxazole was apparently sufficient to cure the infection, although long-term treatment up to one year is currently recommended. Treatment trials or prospective descriptions of outcome for actinomycetoma should investigate treatment efficacy for the different members of Actinomycetales, particularly Nocardia spp., with short-term and long-term treatment courses., Author Summary Mycetoma is a neglected tropical disease caused either by fungi or aerobic actinomycetes. It predominantly affects poor people in remote communities, where access to health care is very limited and people usually rely on traditional treatment. The incidence of mycetoma in the Lao PDR is not known, although the first case of mycetoma from the northern part of the country was published only recently. The present case report is the second case from the Lao PDR and the first published case of actinomycetoma caused by Nocardia aobensis. Data from the literature and the present case suggest that far shorter courses of antibiotics than those usually recommended might be sufficient to treat actinomycetoma caused by Nocardia spp., although these observations need to be confirmed by further studies. Shorter treatment courses would reduce drug side effects and the financial burden on patients. The latter is particularly important for low-income countries like the Lao PDR with very limited resources for health care.

Infection by Shigella spp. is a common cause of dysentery in Southeast Asia. Antimicrobials are thought to be beneficial for treatment; however, antimicrobial resistance in Shigella spp. is becoming widespread. We aimed to assess the frequency and mechanisms associated with decreased susceptibility to azithromycin in Southeast Asian Shigella isolates and use these data to assess appropriate susceptibility breakpoints. Shigella isolates recovered in Vietnam and Laos were screened for susceptibility to azithromycin (15 μg) by disc diffusion and MIC. Phenotypic resistance was confirmed by PCR amplification of macrolide resistance loci. We compared the genetic relationships and plasmid contents of azithromycin-resistant Shigella sonnei isolates using whole-genome sequences. From 475 available Shigella spp. isolated in Vietnam and Laos between 1994 and 2012, 6/181 S. flexneri isolates (3.3%, MIC ≥ 16 g/liter) and 16/294 S. sonnei isolates (5.4%, MIC ≥ 32 g/liter) were phenotypically resistant to azithromycin. PCR amplification confirmed a resistance mechanism in 22/475 (4.6%) isolates ( mphA in 19 isolates and ermB in 3 isolates). The susceptibility data demonstrated the acceptability of the S. flexneri (MIC ≥ 16 g/liter, zone diameter ≤ 15 mm) and S. sonnei (MIC ≥ 32 g/liter, zone diameter ≤ 11 mm) breakpoints with a S. sonnei isolates on at least seven occasions between 2000 and 2009 but failed to become established. While the proposed susceptibility breakpoints may allow better recognition of resistant isolates, additional studies are required to assess the impact on the clinical outcome. The potential emergence of azithromycin resistance highlights the need for alternative options for management of Shigella infections in countries where Shigella is endemic.