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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.

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.

Background Lower respiratory infections constitute a major disease burden worldwide. Treatment is usually empiric and targeted towards typical bacterial pathogens. Understanding the prevalence of pathogens not covered by empirical treatment is important to improve diagnostic and treatment algorithms. Methods A prospective observational study in peri-urban communities of Yangon, Myanmar was conducted between July 2018 and April 2019. Sputum specimens of 299 adults presenting with fever and productive cough were tested for Mycobacterium tuberculosis (microscopy and GeneXpert MTB/RIF [Mycobacterium tuberculosis/resistance to rifampicin]) and Burkholderia pseudomallei (Active Melioidosis Detect Lateral Flow Assay and culture). Nasopharyngeal swabs underwent respiratory virus (influenza A, B, respiratory syncytial virus) polymerase chain reaction testing. Results Among 299 patients, 32% (95% confidence interval [CI] 26 to 37) were diagnosed with tuberculosis (TB), including 9 rifampicin-resistant cases. TB patients presented with a longer duration of fever (median 14 d) and productive cough (median 30 d) than non-TB patients (median fever duration 6 d, cough 7 d). One case of melioidosis pneumonia was detected by rapid test and confirmed by culture. Respiratory viruses were detected in 16% (95% CI 12 to 21) of patients. Conclusions TB was very common in this population, suggesting that microscopy and GeneXpert MTB/RIF on all sputum samples should be routinely included in diagnostic algorithms for fever and cough. Melioidosis was uncommon in this population.

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.

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.

A 33-year-old man from Ghana who had diabetes had chronic osteomyelitis of the femoral shaft develop. Tissue samples from surgical debridement grew Burkholderia pseudomallei. He received meropenem, followed by oral trimethoprim/sulfamethoxazole and doxycycline, and fully recovered without complications. Our case report extends the range of countries in Africa as sources of culture-confirmed melioidosis.

Background We aimed to measure 13-valent pneumococcal conjugate vaccine (PCV13) impact on various pneumococcal endpoints with minimal baseline data. Methods PCV13 was introduced in October 2013 in Lao People’s Democratic Republic. We implemented studies to evaluate PCV13 impact: retrospective record review of pneumonia cases for three years pre-PCV13; prospective study of acute respiratory infection (ARI) for 4.5 years post-PCV to demonstrate effectiveness against hypoxic pneumonia; invasive pneumococcal disease (IPD) surveillance (2004-2019); carriage in children hospitalised with ARI (2013-2019); and community carriage surveys pre- and two years post-PCV13. Results Annual incidence of hospitalized pneumonia pre-PCV13 in children 2-59 months was 1,530 (95% confidence interval [CI] 1,477-1,584) per 100,000. Vaccine effectiveness against hypoxic pneumonia was 37% (95% CI 6-57%). In children Conclusions Despite limited baseline data in Laos, we found evidence of PCV13 impact using a variety of methods. Our approach could be used in similar settings to augment existing WHO guidelines. Key messages A combination of methods may be useful to determine the impact of vaccine introduction in countries with limited surveillance.

OBJECTIVES: Pneumococcal conjugate vaccines (PCVs) are effective in reducing pneumococcal disease. We measured 13-valent PCV (PCV13) effect on different pneumococcal outcomes using diverse studies in Lao People's Democratic Republic. METHODS: Studies included: pre-PCV13 population-based record review of hospitalized childhood pneumonia cases; acute respiratory infection (ARI) study post-PCV13 to demonstrate effectiveness (VE) against hypoxic pneumonia; invasive pneumococcal disease (IPD) surveillance in all ages (2004-2018); carriage studies in children hospitalized with ARI (2013-2019); community carriage surveys pre- and post-PCV13. RESULTS: Annual pneumonia incidence rate in children pre-PCV13 was 1,530 (95% confidence interval [CI] 1,477-1,584) per 100,000. Adjusted VE against hypoxic pneumonia was 37% (95% CI 6-57%). For IPD, 85% (11/13) of cases were due to vaccine-types pre-PCV13, and 43% (3/7) post-PCV13 in children aged

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.

To investigate the current epidemiology of melioidosis in Yangon, Myanmar, between June 2017 and May 2019 we conducted enhanced surveillance for melioidosis in four tertiary hospitals in Yangon, where the disease was first discovered in 1911. Oxidase-positive Gram-negative rods were obtained from the microbiology laboratories and further analysed at the Department of Medical Research. Analysis included culture on Ashdown agar, the three disc sensitivity test (gentamicin, colistin and co-amoxiclav), latex agglutination, API 20 NE, antibiotic susceptibility testing, and a subset underwent molecular confirmation with a Burkholderia pseudomallei specific assay. Twenty one of 364 isolates (5.7%) were confirmed as B. pseudomallei and were mostly susceptible to the antibiotics used in standard therapy for melioidosis. Ten patients were from Yangon Region, nine were from Ayeyarwaddy region, and one each was from Kayin and Rakhine States. A history of soil contact was given by seven patients, five had diabetes mellitus and one had renal insufficiency. The patients presented with septicaemia (12 cases), pneumonia (three cases), urinary tract infection (two cases) and wound infection (four cases). Eighteen patients survived to hospital discharge. This study highlights the likelihood that melioidosis may be far more common, but underdiagnosed, in more rural parts of Myanmar as in other countries in SE Asia.

IntroductionEmpiric data on indirect (herd) effects of pneumococcal conjugate vaccines (PCVs) in settings with low or heterogeneous PCV coverage are limited. The indirect effects of PCV, which benefits both vaccinated and non-vaccinated individuals, are mediated by reductions in vaccine-type (VT) carriage (a prerequisite for disease). The aim of this study among hospitalised children in Lao People’s Democratic Republic (Lao PDR) is to determine the effectiveness of a 13-valent PCV (PCV13) against VT pneumococcal nasopharyngeal carriage (direct effects) and the association between village-level PCV13 coverage and VT carriage (indirect effects).MethodsPneumococcal nasopharyngeal carriage surveillance commenced in December 2013, shortly after PCV13 introduction (October 2013). We recruited and swabbed children aged 2–59 months admitted to hospital with acute respiratory infection. Pneumococci were detected using lytA quantitative real-time PCR and serotyped using microarray. PCV13 status and village-level PCV13 coverage were determined using written immunisation records. Associations between both PCV13 status and village-level PCV13 coverage and VT carriage were calculated using generalised estimating equations, controlling for potential confounders.ResultsWe enrolled 1423 participants and determined PCV13 coverage for 368 villages (269 863 children aged under 5 years). By 2017, median village-level vaccine coverage reached 37.5%, however, the IQR indicated wide variation among villages (24.1–56.4). Both receipt of PCV13 and the level of PCV13 coverage were independently associated with a reduced odds of VT carriage: adjusted PCV13 effectiveness was 38.1% (95% CI 4.1% to 60.0%; p=0.032); and for each per cent increase in PCV13 coverage, the estimated odds of VT carriage decreased by 1.1% (95% CI 0.0% to 2.2%; p=0.056). After adjustment, VT carriage decreased from 20.0% to 12.8% as PCV13 coverage increased from zero to 60% among under 5.ConclusionsDespite marked heterogeneity in PCV13 coverage, we found evidence of indirect effects in Lao PDR. Individual vaccination with PCV13 was effective against VT carriage.

Background Burkholderia pseudomallei is a Gram-negative bacterium found in soil and water in many tropical countries. It causes melioidosis, a potentially fatal infection first described in 1911 in Myanmar. Melioidosis is a common cause of sepsis and death in South and South-east Asia, but it is rarely diagnosed in Myanmar. We conducted a nationwide soil study to identify areas where B. pseudomallei is present. Methodology/Principal findings We collected soil samples from 387 locations in all 15 states and regions of Myanmar between September 2017 and June 2019. At each site, three samples were taken at each of three different depths (30, 60 and 90 cm) and were cultured for B. pseudomallei separately, along with a pooled sample from each site (i.e. 10 cultures per site). We used a negative binomial regression model to assess associations between isolation of B. pseudomallei and environmental factors (season, soil depth, soil type, land use and climate zones). B. pseudomallei was isolated in 7 of 15 states and regions. Of the 387 sites, 31 (8%) had one or more positive samples and of the 3,870 samples cultured, 103 (2.7%) tested positive for B. pseudomallei. B. pseudomallei was isolated more frequently during the monsoon season [RR-2.28 (95% CI: 0.70–7.38)] and less in the hot dry season [RR-0.70 (95% CI: 0.19–2.56)] compared to the cool dry season, and in the tropical monsoon climate zone [RR-2.26; 95% CI (0.21–6.21)] compared to the tropical dry winter climate zone. However, these associations were not statistically significant. B. pseudomallei was detected at all three depths and from various soil types (clay, silt and sand). Isolation was higher in agricultural land (2.2%), pasture land (8.5%) and disused land (5.8%) than in residential land (0.4%), but these differences were also not significant. Conclusion/Significance This study confirms a widespread distribution of B. pseudomallei in Myanmar. Clinical studies should follow to obtain a better picture of the burden of melioidosis in Myanmar., Author summary Melioidosis is caused by a soil dwelling bacterium called Burkholderia pseudomallei. It is a potentially fatal disease even if appropriate antibiotics are given. The disease was first described in Rangoon (now Yangon) in 1911 but it has rarely been reported in Myanmar over the past century even though it is now known to be a common cause of community-acquired infection in neighbouring countries in South and Southeast Asia. The true melioidosis burden in Myanmar is obscured by a lack of awareness among clinicians and limited diagnostic facilities to identify the causative organism. To explore this issue, we conducted a nationwide study to assess the presence of B. pseudomallei in soil samples collected from all fifteen states and regions of Myanmar. Our study revealed a widespread distribution of the organism in the Myanmar environment. This finding contributes to the understanding of the global distribution of B. pseudomallei and should alert clinicians throughout Myanmar to consider melioidosis as a potential cause of fever, sepsis, pneumonia and abscesses.

Background: Blood cultures are one of the most important specimens processed by microbiology laboratories. Currently, there is insufficient guidance on how to transport blood cultures in low- and middle- income countries with tropical climates where delays maybe unavoidable. To fill this knowledge gap, this study investigated the effects of storage for different times and temperatures on the isolation of important bacterial pathogens. Methods and materials: In three laboratories located in Cambodia, Laos and Thailand, paediatric blood culture bottles were spiked with approximately 3 CFU/ml of Escherichia coli, Staphylococcus aureus, Streptococcus pneumoniae, Streptococcus agalactiae, or Haemophilus influenzae. The spiked bottles were stored at 25 °C, in a cool box at ambient temperature or at 40 °C; for 0 h, 6 h, 12 h or 24 h before testing by one of four blood culture systems (two manual and two automated). All bottles were incubated until positive or for at least five days, with all growth identified. Results: No significant differences in positive bottle yields were detected when bottles were stored for 6 h (240/270, 88.9%), 12 h (233/269, 86.6%), at 25 °C (243/270, 90.0%); or in a cool box (232/269, 86.2%), when compared to no storage (80/90, 88.9%; p values > 0.05). However, significantly lower yields compared to no storage were detected when stored for 24 h (212/270, 78.5%; p = 0.030) or at 40 °C (210/270, 77.8%; p = 0.021); with the lowest yield obtained for bottles stored for 24 h at 40 °C (59/90, 65.6%; p < 0.001). The median time to positivity from inoculation increased with increased storage time; rising from 13.0 h (IQR 12.3–16.4) with no storage to 28.1 h (IQR 26.8-30.9) following 24 h of storage (p for trend < 0.001). Conclusion: Blood culture bottles should be incubated with minimal delay to maximise pathogen recovery and timely result reporting, however, this study provides some reassurance that unavoidable delays in incubating blood cultures in tropical climates can be managed to minimise negative impacts. If delays to incubation are unavoidable and are ≥12 h, transportation at a temperature not exceeding 25 °C and blind sub-cultures prior to incubation should be considered.

BackgroundBlood cultures are one of the most important tests performed by microbiology laboratories. Many hospitals, particularly in low and middle-income countries, lack either microbiology services or staff to provide 24 h services resulting in delays to blood culture incubation. There is insufficient guidance on how to transport/store blood cultures if delays before incubation are unavoidable, particularly if ambient temperatures are high. This study set out to address this knowledge gap.MethodsIn three South East Asian countries, four different blood culture systems (two manual and two automated) were used to test blood cultures spiked with five common bacterial pathogens. Prior to incubation the spiked blood culture bottles were stored at different temperatures (25 °C, in a cool-box at ambient temperature, or at 40 °C) for different lengths of time (0 h, 6 h, 12 h or 24 h). The impacts of these different storage conditions on positive blood culture yield and on time to positivity were examined.ResultsThere was no significant loss in yield when blood cultures were stored ConclusionBlood cultures should be incubated with minimal delay to maximize pathogen recovery and timely result reporting, however, this study provides some reassurance that unavoidable delays can be managed to minimize negative impacts. If delays to incubation ≥ 12 h are unavoidable, transportation at a temperature not exceeding 25 °C, and blind sub-cultures prior to incubation should be considered.

Background No studies have explored the association between pneumococcal nasopharyngeal density and severe pneumonia using the World Health Organization (WHO) 2013 definition. In Lao People’s Democratic Republic (Lao PDR), we determine the association between nasopharyngeal pneumococcal density and severe pneumonia in children. Methods A prospective observational study was undertaken at Mahosot Hospital, Vientiane, from 2014 to mid-2018. Children Results Of 1268 participants with ARI, 32.3% (n = 410) had severe pneumonia and 36.9% (n = 468) had pneumococcal carriage. For pneumococcal carriers, pneumococcal density was positively associated with severe pneumonia (adjusted odds ratio, 1.4 [95% confidence interval, 1.1–1.8]; P = .020). Conclusions Among children with ARIs and pneumococcal carriage, pneumococcal carriage density was positively associated with severe pneumonia in Lao PDR. Further studies may determine if pneumococcal density is a useful marker for pneumococcal conjugate vaccine impact on childhood pneumonia.

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, where most diagnosed cases have originated., 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 strains within Vientiane Capital, 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 that it is widespread in the environment and that exposure rates in urban Vientiane are likely higher than previously thought. Whole-genome comparative analysis demonstrated that Lao B. pseudomallei isolates are highly genetically diverse, suggesting that 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, 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 that 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 demonstrate the epidemiological and phylogeographical insights that can be gained from WGS.

Background: Pneumococcal pneumonia is a leading cause of childhood mortality. Pneumococcal conjugate vaccines (PCVs) have been shown to reduce hypoxic pneumonia in children. However, there are no studies from Asia examining the effectiveness of PCVs on hypoxic pneumonia. We describe a novel approach to determine the effectiveness of the 13-valent PCV (PCV13) against hypoxia in children admitted with pneumonia in the Lao People's Democratic Republic. Methods: A prospective hospital-based, test-negative observational study of children aged up to 59 months admitted with pneumonia to a single tertiary hospital in Vientiane was undertaken over 54 months. Pneumonia was defined using the 2013 WHO definition. Hypoxia was defined as oxygen saturation

Melioidosis was first identified in Myanmar in 1911 but for the last century it has remained largely unreported there. Burkholderia pseudomallei was first isolated from the environment of Myanmar in 2016, confirming continuing endemicity. Recent genomic studies showed that B. pseudomallei originated in Australia and spread to Asia, with phylogenetic evidence of repeated reintroduction of B. pseudomallei across countries bordered by the Mekong River and the Malay Peninsula. We present the first whole-genome sequences of B. pseudomallei isolates from Myanmar: nine clinical and seven environmental isolates. We used large-scale comparative genomics to assess the genetic diversity, phylogeography and potential origins of B. pseudomallei in Myanmar. Global phylogenetics demonstrated that Myanmar isolates group in two distantly related clades that reside in a more ancestral Asian clade with high amounts of genetic diversity. The diversity of B. pseudomallei from Myanmar and divergence within our global phylogeny suggest that the original introduction of B. pseudomallei to Myanmar was not a recent event. Our study provides new insights into global patterns of B. pseudomallei dissemination, most notably the dynamic nature of movement of B. pseudomallei within densely populated Southeast Asia. The role of anthropogenic influences in both ancient and more recent dissemination of B. pseudomallei to Myanmar and elsewhere in Southeast Asia and globally requires further study.

Melioidosis is endemic in many rural areas in Southeast Asia where facilities for culture and identification of Burkholderia pseudomallei are often limited. We performed a prospective observational study in patients presenting with fever to Mahosot Hospital, the primary referral hospital in Laos, to establish whether the detection of abscesses on ultrasound could support a presumptive diagnosis of melioidosis. All patients underwent ultrasound examination to detect abscesses in the liver, spleen, prostate, or, if indicated, subcutaneous tissue. We enrolled 153 patients, including 18 patients with melioidosis. Of these, 11 (61%) had an abscess at one or more sites, including five (28%) with splenic and/or liver abscesses. Absence of abscesses cannot rule out melioidosis, but the positive predictive value of abscesses for melioidosis was high at 93% (88–96%). Therefore, in endemic areas, the presence of abscesses in febrile patients should prompt empiric antibiotic therapy for melioidosis even in the absence of culture confirmation.

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.

Background Klebsiella pneumoniae is a leading cause of bloodstream infection (BSI). Strains producing extended-spectrum beta-lactamases (ESBLs) or carbapenemases are considered global priority pathogens for which new treatment and prevention strategies are urgently required, due to severely limited therapeutic options. South and Southeast Asia are major hubs for antimicrobial-resistant (AMR) K. pneumoniae and also for the characteristically antimicrobial-sensitive, community-acquired “hypervirulent” strains. The emergence of hypervirulent AMR strains and lack of data on exopolysaccharide diversity pose a challenge for K. pneumoniae BSI control strategies worldwide. Methods We conducted a retrospective genomic epidemiology study of 365 BSI K. pneumoniae from seven major healthcare facilities across South and Southeast Asia, extracting clinically relevant information (AMR, virulence, K and O antigen loci) using Kleborate, a K. pneumoniae-specific genomic typing tool. Results K. pneumoniae BSI isolates were highly diverse, comprising 120 multi-locus sequence types (STs) and 63 K-loci. ESBL and carbapenemase gene frequencies were 47% and 17%, respectively. The aerobactin synthesis locus (iuc), associated with hypervirulence, was detected in 28% of isolates. Importantly, 7% of isolates harboured iuc plus ESBL and/or carbapenemase genes. The latter represent genotypic AMR-virulence convergence, which is generally considered a rare phenomenon but was particularly common among South Asian BSI (17%). Of greatest concern, we identified seven novel plasmids carrying both iuc and AMR genes, raising the prospect of co-transfer of these phenotypes among K. pneumoniae. Conclusions K. pneumoniae BSI in South and Southeast Asia are caused by different STs from those predominating in other regions, and with higher frequency of acquired virulence determinants. K. pneumoniae carrying both iuc and AMR genes were also detected at higher rates than have been reported elsewhere. The study demonstrates how genomics-based surveillance—reporting full molecular profiles including STs, AMR, virulence and serotype locus information—can help standardise comparisons between sites and identify regional differences in pathogen populations.

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.

Highlights • Chloramphenicol resistant strains of Neisseria meningitidis are present in three countries across Southeast Asia. • These strains are all closely related to each other and to resistant strains previously observed in Vietnam and France. • This lineage has acquired other antimicrobial resistance genes during its spread., Objectives Invasive disease caused by Neisseria meningitidis is a significant health concern globally, but our knowledge of the prevailing serogroups, antimicrobial susceptibility patterns, and genetics of N. meningitidis in Southeast Asia is limited. Chloramphenicol resistance in N. meningitidis has rarely been reported, but was first described in isolates from Vietnam in 1998. We aimed to characterise eight chloramphenicol resistant meningococcal isolates collected between 2007 and 2018 from diagnostic microbiology laboratories in Cambodia, Thailand and the Lao People's Democratic Republic (Laos). Methods Whole-genome sequencing was used to generate genome sequences from 18 meningococcal isolates including the eight chloramphenicol resistant isolates. We identified antimicrobial resistance genes present in these strains, and examined the phylogenetic relationships between strains. Results The eight resistant strains all contain the same chloramphenicol resistance gene first described in 1998, and are closely related to each other. Strains resistant to penicillin, tetracycline, and ciprofloxacin were also observed, including a chloramphenicol-resistant strain which has acquired penicillin and ciprofloxacin resistance. Conclusions This study suggests that chloramphenicol-resistant N. meningitidis is more widespread than previously thought, and that the previously-identified resistant lineage is now found in multiple countries in Southeast Asia.

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.

Low-resource settings are disproportionately burdened by infectious diseases and antimicrobial resistance. Good quality clinical bacteriology through a well functioning reference laboratory network is necessary for effective resistance control, but low-resource settings face infrastructural, technical, and behavioural challenges in the implementation of clinical bacteriology. In this Personal View, we explore what constitutes successful implementation of clinical bacteriology in low-resource settings and describe a framework for implementation that is suitable for general referral hospitals in low-income and middle-income countries with a moderate infrastructure. Most microbiological techniques and equipment are not developed for the specific needs of such settings. Pending the arrival of a new generation diagnostics for these settings, we suggest focus on improving, adapting, and implementing conventional, culture-based techniques. Priorities in low-resource settings include harmonised, quality assured, and tropicalised equipment, consumables, and techniques, and rationalised bacterial identification and testing for antimicrobial resistance. Diagnostics should be integrated into clinical care and patient management; clinically relevant specimens must be appropriately selected and prioritised. Open-access training materials and information management tools should be developed. Also important is the need for onsite validation and field adoption of diagnostics in low-resource settings, with considerable shortening of the time between development and implementation of diagnostics. We argue that the implementation of clinical bacteriology in low-resource settings improves patient management, provides valuable surveillance for local antibiotic treatment guidelines and national policies, and supports containment of antimicrobial resistance and the prevention and control of hospital-acquired infections.

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.