Burkholderia pseudomallei Bacteria in Ornamental Fish Tanks, Vientiane, Laos, 2023.

In 2019, a melioidosis case in Maryland, USA, was shown to have been acquired from an ornamental fish tank contaminated with Burkholderia pseudomallei bacteria, likely derived from Southeast Asia. We investigated the presence of B. pseudomallei in ornamental fish tanks in the endemic area of Vientiane, Laos.

Melioidosis in Patients with COVID-19 Exposed to Contaminated Tap Water, Thailand, 2021.

In September 2021, a total of 25 patients diagnosed with COVID-19 developed acute melioidosis after (median 7 days) admission to a COVID-19 field hospital in Thailand. Eight nonpotable tap water samples and 6 soil samples were culture-positive for Burkholderia pseudomallei. Genomic analysis suggested contaminated tap water as the likely cause of illness.

Nasopharyngeal Pneumococcal Colonization Density Is Associated With Severe Pneumonia in Young Children in the Lao People's Democratic Republic.

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 <5 years admitted with acute respiratory infections (ARIs) were included. Clinical and demographic data were collected alongside nasopharyngeal swabs for pneumococcal quantification by lytA real-time quantitative polymerase chain reaction. Severe pneumonia was defined using the 2013 WHO definition. For pneumococcal carriers, a logistic regression model examined the association between pneumococcal density and severe pneumonia, after adjusting for potential confounders including demographic and household factors, 13-valent pneumococcal conjugate vaccine status, respiratory syncytial virus co-detection, and preadmission antibiotics. 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.

Melioidosis Manifesting as Chronic Femoral Osteomyelitis in Patient from Ghana.

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.

Using Land Runoff to Survey the Distribution and Genetic Diversity of Burkholderia pseudomallei in Vientiane, Laos.

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

Impact of delays to incubation and storage temperature on blood culture results: a multi-centre study.

BACKGROUND: Blood 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. METHODS: In 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. RESULTS: There was no significant loss in yield when blood cultures were stored < 24 h at 25 °C, however, storage for 24 h at 40 °C decreased yields and longer storage times increased times to detection. CONCLUSION: Blood 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.

Population-Based Estimate of Melioidosis, Kenya.

Melioidosis is thought to be endemic, although underdiagnosed, in Africa. We identified 5 autochthonous cases of Burkholderia pseudomallei infection in a case series in Kenya. Incidence of B. pseudomallei bacteremia in Kenya's Kilifi County is low, at 1.5 cases per million person-years, but this result might be an underestimate.

Management of Central Nervous System Infections, Vientiane, Laos, 2003-2011.

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.

The global impact and cost-effectiveness of a melioidosis vaccine.

BACKGROUND: Every year, 90,000 people may die from melioidosis. Vaccine candidates have not proceeded past animal studies, partly due to uncertainty around the potential market size. This study aims to estimate the potential impact, cost-effectiveness and market size for melioidosis vaccines. METHODS: Age-structured decision tree models with country-specific inputs were used to estimate net costs and health benefits of vaccination, with health measured in quality-adjusted life years (QALYs). Four target groups of people living in endemic regions were considered: (i) people aged over 45 years with chronic renal disease, (ii) people aged over 45 years with diabetes, (iii) people aged over 45 years with diabetes and/or chronic renal disease, (iv) everyone aged over 45 years. Melioidosis risk was estimated using Bayesian evidence synthesis of 12 observational studies. In the base case, vaccines were assumed to have 80% efficacy, to have 5-year mean protective duration and to cost USD10.20-338.20 per vaccine. RESULTS: Vaccination could be cost-effective (with incremental cost-effectiveness ratio below GDP per capita) in 61/83 countries/territories with local melioidosis transmission. In these 61 countries/territories, vaccination could avert 68,000 lost QALYs, 8300 cases and 4400 deaths per vaccinated age cohort, at an incremental cost of USD59.6 million. Strategy (ii) was optimal in most regions. The vaccine market may be worth USD268 million per year at its threshold cost-effective price in each country/territory. CONCLUSIONS: There is a viable melioidosis vaccine market, with cost-effective vaccine strategies in most countries/territories with local transmission.

Microbiology Investigation Criteria for Reporting Objectively (MICRO): a framework for the reporting and interpretation of clinical microbiology data.

BACKGROUND: There is a pressing need to understand better the extent and distribution of antimicrobial resistance on a global scale, to inform development of effective interventions. Collation of datasets for meta-analysis, mathematical modelling and temporo-spatial analysis is hampered by the considerable variability in clinical sampling, variable quality in laboratory practice and inconsistencies in antimicrobial susceptibility testing and reporting. METHODS: The Microbiology Investigation Criteria for Reporting Objectively (MICRO) checklist was developed by an international working group of clinical and laboratory microbiologists, infectious disease physicians, epidemiologists and mathematical modellers. RESULTS: In keeping with the STROBE checklist, but applicable to all study designs, MICRO defines items to be included in reports of studies involving human clinical microbiology data. It provides a concise and comprehensive reference for clinicians, researchers, reviewers and journals working on, critically appraising, and publishing clinical microbiology datasets. CONCLUSIONS: Implementation of the MICRO checklist will enhance the quality and scientific reporting of clinical microbiology data, increasing data utility and comparability to improve surveillance, grade data quality, facilitate meta-analyses and inform policy and interventions from local to global levels.

Azithromycin Resistance in Shigella spp. in Southeast Asia.

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 <3% discrepancy. Phylogenetic analysis demonstrated that decreased susceptibility has arisen sporadically in Vietnamese 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.

Evaluation of a Rapid Diagnostic Test for Detection of Burkholderia pseudomallei in the Lao People's Democratic Republic.

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.

Clostridium difficile infection in the Lao People's Democratic Republic: first isolation and review of the literature.

BACKGROUND: Current knowledge of the epidemiology of Clostridium difficile infection in Asia, and in particular the Greater Mekong Subregion, is very limited. Only a few studies from Thailand and Vietnam have been reported from the region with variable testing methods and results, and no studies from Lao People's Democratic Republic (PDR). Therefore we investigated the presence of C. difficile in a single centre in the Lao PDR and determined the ribotypes present. METHOD: Seventy unformed stool samples from hospital inpatients at Mahosot Hospital, Vientiane, were tested for the presence of C. difficile using selective differential agar and confirmed by latex agglutination. C. difficile isolates were further characterised by ribotyping and toxin gene detection. RESULTS: C. difficile was isolated from five of the 70 patients, and five different ribotypes were identified (014, 017, 020, QX 107 and QX 574). CONCLUSION: This is the first isolation of C. difficile from human stool samples in the Lao PDR. These results will add to the limited amount of data on C. difficile in the region. In addition, we hope this information will alert clinicians to the presence of C. difficile in the country and will help inform future investigations into the epidemiology and diagnosis of C. difficile in Lao PDR.

Survival and Growth of Orientia tsutsugamushi in Conventional Hemocultures.

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.

A retrospective analysis of melioidosis in Cambodian children, 2009-2013.

BACKGROUND: Melioidiosis, infection by Burkholderia pseudomallei, is an important but frequently under-recognised cause of morbidity and mortality in Southeast Asia and elsewhere in the tropics. Data on the epidemiology of paediatric melioidosis in Cambodia are extremely limited. METHODS: Culture-positive melioidosis cases presenting to Angkor Hospital for Children, a non-governmental paediatric hospital located in Siem Reap, Northern Cambodia, between 1st January 2009 and 31st December 2013 were identified by searches of hospital and laboratory databases and logbooks. RESULTS: One hundred seventy-three evaluable cases were identified, presenting from eight provinces. For Siem Reap province, the median commune level incidence was estimated to be 28-35 cases per 100,000 children <15 years per year. Most cases presented during the wet season, May to October. The median age at presentation was 5.7 years (range 8 days-15.9 years). Apart from undernutrition, co-morbidities were rare. Three quarters (131/173) of the children had localised infection, most commonly skin/soft tissue infection (60 cases) or suppurative parotitis (51 cases). There were 39 children with B. pseudomallei bacteraemia: 29 (74.4%) of these had clinical and/or radiological evidence of pneumonia. Overall mortality was 16.8% (29/173) with mortality in bacteraemic cases of 71.8% (28/39). At least seven children did not receive an antimicrobial with activity against B. pseudomallei prior to death. CONCLUSIONS: This retrospective study demonstrated a considerable burden of melioidosis in Cambodian children. Given the high mortality associated with bacteraemic infection, there is an urgent need for greater awareness amongst healthcare professionals in Cambodia and other countries where melioidosis is known or suspected to be endemic. Empiric treatment guidelines should ensure suspected cases are treated early with appropriate antimicrobials.

Melioidosis diagnostic workshop, 2013.

Melioidosis is a severe disease that can be difficult to diagnose because of its diverse clinical manifestations and a lack of adequate diagnostic capabilities for suspected cases. There is broad interest in improving detection and diagnosis of this disease not only in melioidosis-endemic regions but also outside these regions because melioidosis may be underreported and poses a potential bioterrorism challenge for public health authorities. Therefore, a workshop of academic, government, and private sector personnel from around the world was convened to discuss the current state of melioidosis diagnostics, diagnostic needs, and future directions.

A novel technique for detecting antibiotic-resistant typhoid from rapid diagnostic tests.

Fluoroquinolone-resistant typhoid is increasing. An antigen-detecting rapid diagnostic test (RDT) can rapidly diagnose typhoid from blood cultures. A simple, inexpensive molecular technique performed with DNA from positive RDTs accurately identified gyrA mutations consistent with phenotypic susceptibility testing results. Field diagnosis combined with centralized molecular resistance testing could improve typhoid management and surveillance in low-resource settings.

Colonization with Enterobacteriaceae producing ESBLs in children attending pre-school childcare facilities in the Lao People's Democratic Republic.

OBJECTIVES: Intestinal carriage constitutes an important reservoir of antimicrobial-resistant bacteria, with some of the highest rates reported from Asia. Antibiotic resistance has been little studied in Laos, where some antibiotics are available without restriction, but others such as carbapenems are not available. PATIENTS AND METHODS: We collected stools from 397 healthy children in 12 randomly selected pre-school childcare facilities in and around Vientiane. Colonization with ESBL-producing Enterobacteriaceae (ESBLE) and carbapenemase-producing Enterobacteriaceae (CPE) was detected using a disc diffusion screening test and ESBLE were characterized using WGS. Risk factor data were collected by questionnaire. RESULTS: Ninety-two children (23%) were colonized with ESBLE, mainly Escherichia coli carrying blaCTX-M and Klebsiella pneumoniae carrying blaSHV or blaCTX-M, which were frequently resistant to multiple antibiotic classes. Although residence in Vientiane Capital, foreign travel, higher maternal level of education, antibiotic use in the preceding 3 months and attending a childcare facility with a 'good' level of hygiene were all associated with ESBLE colonization on univariable analysis, a significant association remained only for antibiotic use when a stepwise approach was used with a multivariate random-effects model. WGS analysis suggested transmission in both childcare facilities and community settings. CONCLUSIONS: The high prevalence of paediatric colonization with ESBLE in Laos, one of the highest reported in Asia, is probably the result of inappropriate antibiotic use. Paediatric colonization with CPE was not identified in this study, but it is important to continue to monitor the spread of antibiotic-resistant Enterobacteriaceae in Laos.

Evaluation of Molecular Methods To Improve the Detection of Burkholderia pseudomallei in Soil and Water Samples from Laos.

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.