SUMMARY Burkholderia pseudomallei and Burkholderia mallei present significant biosafety challenges due to their high pathogenicity, environmental resilience, intrinsic antimicrobial resistance, and potential use as bioterrorism agents. This review examines key aspects of laboratory management and infection control for these organisms, focusing on inconsistencies in biosafety protocols and risk classifications across regions. We synthesize current evidence on biocontainment requirements, disinfection strategies, and personal protective equipment (PPE), with particular emphasis on sustainable practices for laboratories in low-resource settings. Although laboratory-acquired infections are rare, their potential severity underscores the importance of stringent safety measures. Critical gaps remain in our understanding of infectious dose, the effectiveness of post-exposure prophylaxis (PEP), and the development of risk assessment frameworks. We advocate for harmonized global biosafety standards and targeted research on transmission dynamics and inactivation protocols. These priorities are essential to enhance laboratory safety, especially in endemic areas, and to inform coherent international policy on B. pseudomallei and B. mallei containment and management.

Abstract Acute respiratory infections (ARI) remain a major cause of child mortality in low- and middle-income countries. However, the risk factors for ARI are poorly understood in low-income settings, and ARI aetiology is changing, driven by vaccination. There are very limited data from Lao PDR (Laos) on ARI aetiology and risk factors to support health policy decisions. This study aimed to investigate the aetiology of hospitalised ARI, and describe risk factors associated with hospitalised ARI, in children under 5 years of age in Laos. We conducted a case–control study at Mahosot Hospital, Laos, enrolling children under five years of age hospitalised with ARI, and community controls matched on age, sex and time of recruitment. Demographics and clinical characteristics were collected, and throat swabs taken. Swabs were analysed using probe-based real-time polymerase chain reaction (PCR) to detect bacterial and viral microorganisms. Risk factors for ARI were determined through regression analysis, and microorganism-specific attributable fractions (AF) were calculated to estimate each microorganism’s contribution to hospitalised ARI. We enrolled 307 cases and 564 controls over 12 months in 2016/17. Microorganisms were detected in 93.8% of cases and 58.9% of controls. Respiratory syncytial virus (RSV) was the leading viral cause of hospitalised ARI, attributed to 29.6% of cases, followed by influenza viruses (11.6%). H. influenzae was attributed to 40.8% of cases. RSV exhibited clear seasonality, peaking during the wet season. Exclusive breastfeeding for 3 months (OR: 0.62; 95% CI 0.45–0.86), and being up to date with pneumococcal conjugate vaccination (odds ratio: 0.6; 95% CI 0.41–0.80), were associated with a lower risk of hospitalised ARI; while low birth weight (OR: 2.91; 95% CI 1.63–5.28), and household smoking (OR: 3.07; 95% CI 2.25–4.18), were associated with increased risk. RSV and H. influenzae remain major causes of ARI in Laos. The findings highlight the potential benefit of tailoring interventions to the local context, including vaccination and risk mitigation strategies, to reduce the burden of ARI in Laos and other low and middle-income countries.

Background Melioidosis, caused by Burkholderia pseudomallei , is a severe tropical infection with diverse clinical manifestations and high mortality if untreated. Although endemic in Southeast Asia and northern Australia, its burden is increasingly recognized in South Asia, where the disease remains underdiagnosed and is frequently misclassified as tuberculosis or other infections. This review synthesizes the contemporary scientific evidence and expert consensus emerging from the 4th South Asian Melioidosis Congress held in Guwahati, Assam, India, in November 2025. Methods We conducted an integrated narrative synthesis of the scientific content presented at the Congress, including plenary lectures, oral and poster presentations, panel discussions, and case-based deliberations. Topics encompassed epidemiology, clinical manifestations, laboratory diagnostics, genomics, treatment strategies, outbreak investigations, antimicrobial resistance, and One Health perspectives across South and Southeast Asia. Results The Congress highlighted expanding geographic recognition of melioidosis across India and neighbouring countries, with reported cases likely representing only a small fraction of the true disease burden. Clinical discussions emphasized marked heterogeneity in presentation and strong associations with diabetes mellitus, environmental exposure, and the monsoon season. Advances in diagnostics included broader implementation of MALDI-TOF mass spectrometry, molecular assays, and rapid diagnostic tests, although significant gaps remain in laboratory capacity, surveillance, and clinical recognition. Genomic investigations demonstrated substantial strain diversity while identifying conserved virulence determinants that may support surveillance and outbreak investigations. Participants also underscored the importance of multidisciplinary collaboration, standardized diagnostic practices, and regional data sharing to improve case detection and patient outcomes. Conclusions Melioidosis remains an underrecognized but increasingly important public health challenge in South Asia. Strengthening surveillance systems, expanding diagnostic capacity, improving clinician awareness, fostering regional laboratory networks, and adopting coordinated One Health strategies are essential to reduce morbidity and mortality. The Congress also reinforced the need for sustained advocacy toward recognition of melioidosis as a neglected tropical disease.

Melioidosis, caused by the Gram-negative bacterium Burkholderia pseudomallei, is an infectious disease with high rates of morbidity and mortality, which primarily affects low- and middle-income countries in South and Southeast Asia and Australia. The clinical manifestations of this disease are nonspecific and, therefore, rapid laboratory diagnosis is especially critical as appropriate management requires specific antimicrobials. This article aims to provide an overview of the current diagnostic methodologies, emerging technologies, susceptibility testing, and future perspectives for laboratory diagnosis of melioidosis. By examining conventional culture methods, mass spectrometry, antimicrobial susceptibility testing, antigen detection, molecular diagnostics, and serological assays, this article highlights the current challenges in accurately and cost-effectively diagnosing melioidosis in diverse clinical and resource-limited settings. A detailed analysis of current and future diagnostic methodologies will offer valuable insights for clinicians, researchers, and public health professionals. This review aims to influence clinical and laboratory guidelines for diagnosing melioidosis and future research directions.

Mycoplasma pneumoniae has been described worldwide as an important cause of community-acquired pneumonia. From December 2013 to December 2014, 461 children admitted to Mahosot Hospital, Vientiane, Laos, with acute respiratory infection were investigated for upper respiratory microorganisms using probe-based real-time polymerase chain reaction (PCR) (FTD33). M. pneumoniae was detected by FTD33 in the upper respiratory tract of three patients, two girls and one boy, 5.7 and 3.9 years old and 13.6 years old, respectively. They presented with clinical features compatible with M. pneumoniae infection. They improved without M. pneumoniae directed therapy. The two girls were also positive for other potential pathogens. The boy had abnormal pulmonary auscultation, and one of the girls had significant anaemia. These results suggest that enhancement of diagnostic systems for M. pneumoniae detection is needed to improve understanding of the epidemiology of M. pneumoniae infection in Laos.

Objectives:ESBLs are an important cause of third generation cephalosporin resistance in Enterobacterales. However, there is a paucity of data on ESBLs in blood stream infections (BSI) in Laos. The aim of this study was to investigate the presence of ESBL-producing Escherichia coli (ESBLEC) and ESBL-producing Klebsiella pneumoniae (ESBLKP) in blood cultures submitted to Mahosot Hospital, Laos and how these have changed over 18 years. Methods and materials:This retrospective observational study included blood cultures from patients presenting with fever to Mahosot Hospital between 2000 and 2018. Full identification and antibiotic susceptibility testing was carried out on positive bottles. ESBL production was determined using the double-disc method. Patient clinical and residence data were included in univariable and multivariable analyses to identify risk factors for having an ESBL. Results:From 52 249 blood culture sets collected over the 18-year period, 222 (0.42%) were positive for an ESBLEC or ESBLKP. The proportion of E. coli and K. pneumoniae isolates that were ESBL positive increased from 3.2% in 2000 to 35.4% in 2018. While there was a steady increase seen in ESBLEC over the 18-year period, ESBLKP have remained at ∼7% of K. pneumoniae isolates. Most ESBLs were community acquired. From univariable analysis, factors positively associated with an ESBL isolate included chronic renal failure, renal stones and having taken an antibiotic in the week before the blood sample. Conclusion:With a rising trend of ESBLEC in Laos, controlling unregulated antibiotic usage in the community will be pivotal to stopping further increases.

Background/Objectives: Pneumococcal serotypes 1 and 5 are associated with invasive pneumococcal disease (IPD). However, data on the circulation of these serotypes in Asia following the introduction of the pneumococcal conjugate vaccine (PCV) is limited. The Lao People’s Democratic Republic (Lao PDR) introduced PCV13 into its national immunisation programme in 2013. We undertook a serosurvey to assess the IgG responses to serotypes 1 and 5 from a convenience sample of children aged under 5 years in Vientiane, Lao PDR. Methods: This cross-sectional analysis used a convenience sample of the close contacts of children under five years old who had been hospitalised with acute respiratory infections between 2013 and 2016 in Vientiane, Lao PDR. Serotype-specific IgG concentrations to serotypes 1 and 5 were measured using a modified WHO ELISA method. Results: A total of 214 participants were included, 130 of whom were unvaccinated and 84 were vaccinated with PCV13. Compared to unvaccinated participants, a higher number of PCV-vaccinated participants met the IgG threshold for IPD (≥0.35 μg/mL) [41.5% (54/130) vs. 71.4% (60/84)] for serotype 1. In contrast, for serotype 5, a similar number of participants in the PCV-vaccinated and unvaccinated group met the IgG threshold for IPD (85.7% (72/84) vs. 82.3% (107/130). Among unvaccinated children, serotype 1 IgG levels peaked at 12 and 23 months at 0.49 µg/mL (95% CIs: 0.25–0.96), while serotype 5 IgG levels were similar across age groups, ranging from 0.55 to 0.79 µg/mL. Conclusions: Our findings indicate the considerable circulation of serotypes 1 and 5 within the community in Lao PDR. Ongoing surveillance is important for informing PCV vaccination strategies.

There is growing evidence on the importance of bacterial/viral interaction in the course of pneumonia. In Laos, no study has investigated respiratory pathogen co-detection. We conducted a study at Mahosot Hospital in Vientiane to determine whether bacterial/viral co-detection and pneumococcal density are associated with severe pneumonia. Between December 2013 and December 2016, 934 under 5 years old hospitalized children with ARI were enrolled. Swabs from the upper respiratory tract were collected and analyzed by real-time PCR. The most common co-detected microorganisms were Streptococcus pneumoniae/Haemophilus influenzae (24%), Respiratory Syncytial Virus (RSV)/S. pneumoniae (12%) and RSV/H. influenzae (16%). Pneumococcal density was 4.52 times higher in influenza virus positive participants. RSV/S. pneumoniae and RSV/H. influenzae co-detections were positively associated with severe pneumonia in univariate analysis (OR 1.86, 95%CI:1.22-2.81, p = 0.003 and OR 2.09, 95%CI:1.46-3.00), but not confirmed in adjusted analysis (aOR 0.72, 95%CI:0.38-1.6, p = 0.309 and aOR 1.37, 95%CI:0.73-2.58). In RSV positive patients, there was no association between pneumococcal density and severe pneumonia. Our findings confirmed an association between pneumococcal density and influenza but not RSV severe pneumonia in young children. Results highlight the complexity of the interaction of viral/bacterial pathogens, which might not have a simple synergistic action in the evolution of pneumonia.

The global priorities in the field of infectious diseases are constantly changing. While emerging viral infections have regularly dominated public health attention, which has only intensified after the COVID-19 pandemic, numerous bacterial diseases have previously caused, and continue to cause, significant morbidity and mortality-deserving equal attention. Three potentially life-threatening endemic bacterial diseases (leptospirosis, melioidosis, and rickettsioses) are a huge public health concern especially in low- and middle-income countries. Despite their continued threat, these diseases do not receive proportionate attention from global health organizations and are not even included on the WHO list of neglected tropical diseases (NTDs). This, in turn, has led to a vicious circle of neglect with continued, yet conceivably preventable, hospitalizations and deaths each year especially in the vulnerable population. This is a call from a group of multi-institutional experts on the urgent need to directly address the circle of neglect and raise support in terms of funding, research, surveillance, diagnostics, and therapeutics to alleviate the burden of these 3 diseases.

Burkholderia pseudomallei and Burkholderia cepacia are Gram-negative, soil-dwelling bacteria that are found in a wide variety of environmental niches. While B. pseudomallei is the causative agent of melioidosis in humans and animals, members of the B. cepacia complex typically only cause disease in immunocompromised hosts. In this study, we report the identification of B. cepacia strains isolated from either patients or soil in Laos and Thailand that express a B. pseudomallei-like 6-deoxyheptan capsular polysaccharide (CPS). These B. cepacia strains were initially identified based on their positive reactivity in a latex agglutination assay that uses the CPS-specific monoclonal antibody (mAb) 4B11. Mass spectrometry and recA sequencing confirmed the identity of these isolates as B. cepacia (formerly genomovar I). Total carbohydrates extracted from B. cepacia cell pellets reacted with B. pseudomallei CPS-specific mAbs MCA147, 3C5, and 4C4, but did not react with the B. pseudomallei lipopolysaccharide-specific mAb Pp-PS-W. Whole genome sequencing of the B. cepacia isolates revealed the presence of genes demonstrating significant homology to those comprising the B. pseudomallei CPS biosynthetic gene cluster. Collectively, our results provide compelling evidence that B. cepacia strains expressing the same CPS as B. pseudomallei co-exist in the environment alongside B. pseudomallei. Since CPS is a target that is often used for presumptive identification of B. pseudomallei, it is possible that the occurrence of these unique B. cepacia strains may complicate the diagnosis of melioidosis. IMPORTANCE Burkholderia pseudomallei, the etiologic agent of melioidosis, is an important cause of morbidity and mortality in tropical and subtropical regions worldwide. The 6-deoxyheptan capsular polysaccharide (CPS) expressed by this bacterial pathogen is a promising target antigen that is useful for rapidly diagnosing melioidosis. Using assays incorporating CPS-specific monoclonal antibodies, we identified both clinical and environmental isolates of Burkholderia cepacia that express the same CPS antigen as B. pseudomallei. Because of this, it is important that staff working in melioidosis-endemic areas are aware that these strains co-exist in the same niches as B. pseudomallei and do not solely rely on CPS-based assays such as latex-agglutination, AMD Plus Rapid Tests, or immunofluorescence tests for the definitive identification of B. pseudomallei isolates.

Background Burkholderia thailandensis is an environmental bacteria closely related to Burkholderia pseudomallei that rarely causes infection in humans. Some environmental isolates have shown to express a capsular polysaccharide known as B. thailandensis capsular variant (BTCV), but human infection has not previously been reported. Although B. thailandednisis has been identified in environmental samples in Laos before, there have not been any human cases reported. Case A 44-year-old man presented to a district hospital in Laos with a short history of fever and pain in his left foot. Physical examination identified a deep soft-tissue abscess in his left foot and an elevated white blood count. A deep pus sample was taken and melioidosis was suspected from preliminary laboratory tests. The patient was initially started on cloxacillin, ceftriaxone and metronidazole, and was then changed to ceftazidime treatment following local melioidosis treatment guidelines. Laboratory methods A deep pus sample was sent to Mahosot Hospital microbiology laboratory where a mixed infection was identified including Burkholderia sp. Conventional identification tests and API 20NE were inconclusive, and the B. pseudomallei-specific latex agglutination was positive. The isolate then underwent a Burkholderia species specific PCR which identified the isolate as B. thailandensis. The isolate was sent for sequencing on the Illumina NovaSeq 6000 system and multi-locus sequence typing analysis identified the isolate had the same sequence type (ST696) as B. thailandensis E555, a strain which expresses a B. pseudomallei-like capsular polysaccharide. Conclusion This is the first report of human infection with B. thailandensis in Laos, and the first report of any human infection with the B. thailandensis capsular variant. Due to the potential for laboratory tests to incorrectly identify this bacteria, staff in endemic areas for B. thailandensis and B. pseudomallei should be aware and ensure that appropriate confirmatory methods are used to differentiate between the species.

We review the discovery of the tropical infectious disease melioidosis by Alfred Whitmore, a pathologist from England, and his assistant from India, C.S. Krishnaswami. We discuss how the subsequent disappearance of melioidosis from the medical literature of Burma holds parallels with the current neglect and under recognition of the disease. We urge global and national public health authorities to add melioidosis to existing neglected tropical diseases surveillance systems.

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.

Background:Burkholderia thailandensis is an environmental bacteria closely related to Burkholderia pseudomallei that rarely causes infection in humans. Some environmental isolates have shown to express a capsular polysaccharide known as B. thailandensis capsular variant (BTCV), but human infection has not previously been reported. Although B. thailandenisis has been identified in environmental samples in Laos before, there have not been any human cases reported. Case:A 44-year-old man presented to a district hospital in Laos with a short history of fever and pain in his left foot. Physical examination identified a deep soft-tissue abscess in his left foot and an elevated white blood count. A deep pus sample was taken and melioidosis was suspected from preliminary laboratory tests. The patient was initially started on cloxacillin, ceftriaxone and metronidazole, and was then changed to ceftazidime treatment following local melioidosis treatment guidelines. Laboratory methods:A deep pus sample was sent to Mahosot Hospital microbiology laboratory where a mixed infection was identified including Burkholderia sp. Conventional identification tests and API 20NE were inconclusive, and the B. pseudomallei-specific latex agglutination was positive. The isolate then underwent a Burkholderia species specific PCR which identified the isolate as B. thailandensis. The isolate was sent for sequencing on the Illumina NovaSeq 6000 system and multi-locus sequence typing analysis identified the isolate had the same sequence type (ST696) as B. thailandensis E555, a strain which expresses a B. pseudomallei-like capsular polysaccharide. Conclusion:This is the first report of human infection with B. thailandensis in Laos, and the first report of any human infection with the B. thailandensis capsular variant. Due to the potential for laboratory tests to incorrectly identify this bacteria, staff in endemic areas for B. thailandensis and B. pseudomallei should be aware and ensure that appropriate confirmatory methods are used to differentiate between the species.

Melioidosis is an emerging tropical infectious disease with a rising global burden caused by the environmental bacterium Burkholderia pseudomallei . It is endemic in Southeast and South Asia, including Bangladesh. A rare aminoglycoside-susceptible B. pseudomallei isolate (Y2019) has recently been reported from a melioidosis patient in Dhaka, Bangladesh. To understand the geographical origins of Y2019, we subjected it and 10 other isolates from Bangladesh to whole-genome sequencing. In a phylogenetic tree with a global set of B. pseudomallei genomes, most Bangladeshi genomes clustered tightly within the Asian clade. In contrast, Y2019 was closely related to ST881 isolates from Sarawak, Malaysian Borneo, a gentamicin-sensitive sequence type, suggesting infection in Borneo. Y2019 also contained the same gentamicin sensitivity conferring nonsynonymous mutation in the drug efflux pump encoding the amrB gene. In the absence of a full travel history, whole-genome sequencing and bioinformatics tools have revealed the likely origin of this rare isolate.

In a clinical isolate of Burkholderia pseudomallei from Hainan, the association between the emergence of ceftazidime resistance and a novel PenA P174L allele was identified for the first time, providing an understanding of one mechanism by which ceftazidime resistance arises in B. pseudomallei.

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 IS 6/ IS 26 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.

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

No studies have explored the association between pneumococcal nasopharyngeal density and severe pneumonia using the World Health Organization 2013 definition. Among children with acute respiratory infections and pneumococcal carriage, pneumococcal carriage density was positively associated with severe pneumonia in Laos. 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.