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.

Plasma Metabolomics Reveals Distinct Biological and Diagnostic Signatures for Melioidosis.

Rationale: The global burden of sepsis is greatest in low-resource settings. Melioidosis, infection with the gram-negative bacterium Burkholderia pseudomallei, is a frequent cause of fatal sepsis in endemic tropical regions such as Southeast Asia. Objectives: To investigate whether plasma metabolomics would identify biological pathways specific to melioidosis and yield clinically meaningful biomarkers. Methods: Using a comprehensive approach, differential enrichment of plasma metabolites and pathways was systematically evaluated in individuals selected from a prospective cohort of patients hospitalized in rural Thailand with infection. Statistical and bioinformatics methods were used to distinguish metabolomic features and processes specific to patients with melioidosis and between fatal and nonfatal cases. Measurements and Main Results: Metabolomic profiling and pathway enrichment analysis of plasma samples from patients with melioidosis (n = 175) and nonmelioidosis infections (n = 75) revealed a distinct immuno-metabolic state among patients with melioidosis, as suggested by excessive tryptophan catabolism in the kynurenine pathway and significantly increased levels of sphingomyelins and ceramide species. We derived a 12-metabolite classifier to distinguish melioidosis from other infections, yielding an area under the receiver operating characteristic curve of 0.87 in a second validation set of patients. Melioidosis nonsurvivors (n = 94) had a significantly disturbed metabolome compared with survivors (n = 81), with increased leucine, isoleucine, and valine metabolism, and elevated circulating free fatty acids and acylcarnitines. A limited eight-metabolite panel showed promise as an early prognosticator of mortality in melioidosis. Conclusions: Melioidosis induces a distinct metabolomic state that can be examined to distinguish underlying pathophysiological mechanisms associated with death. A 12-metabolite signature accurately differentiates melioidosis from other infections and may have diagnostic applications.

Sensitivity and specificity of DPP® Fever Panel II Asia in the diagnosis of malaria, dengue and melioidosis.

Introduction. Rapid diagnostic tests (RDTs) that can facilitate the diagnosis of a panel of tropical infectious diseases are critically needed. DPP® Fever Panel II Asia is a multiplex lateral flow immunoassay comprising antigen and IgM panels for the diagnosis of pathogens that commonly cause febrile illness in Southeast Asia.Hypothesis/Gap Statement. Accuracy of DPP® Fever Panel II Asia has not been evaluated in clinical studies.Aim. To evaluate the sensitivity and specificity of DPP® Fever Panel II Asia for malaria, dengue and melioidosis.Methodology. We conducted a cohort-based case-control study. Both cases and controls were derived from a prospective observational study of patients presenting with community-acquired infections and sepsis in northeast Thailand (Ubon sepsis). We included 143 and 98 patients diagnosed with malaria or dengue based on a positive PCR assay and 177 patients with melioidosis based on a culture positive for Burkholderia pseudomallei. Controls included 200 patients who were blood culture-positive for Staphylococcus aureus, Escherichia coli or Klebsiella pneumoniae, and cases of the other diseases. Serum samples collected from all patients within 24 h of admission were stored and tested using the DPP® Fever Panel II Asia antigen and IgM multiplex assays. We selected cutoff values for each individual assay corresponding to a specificity of ≥95 %. When assessing diagnostic tests in combination, results were considered positive if either individual test was positive.Results. Within the DPP® Fever Panel II Asia antigen assay, a combination of pLDH and HRPII for malaria had a sensitivity of 91 % and a specificity of 97 %. The combination of dengue NS1 antigen and dengue antibody tests had a sensitivity of 61 % and a specificity of 91 %. The B. pseudomallei CPS antigen test had a sensitivity of 27 % and a specificity of 97 %. An odds ratio of 2.34 (95 % CI 1.16-4.72, P=0.02) was observed for the association between CPS positivity and mortality among melioidosis patients.Conclusion. The performance of the DPP® Fever Panel II Asia for diagnosis of malaria was high and that for dengue and melioidosis was relatively limited. For all three diseases, performance was comparable to that of other established RDTs. The potential operational advantages of a multiplex and quantitative point-of-care assay are substantial and warrant further investigation.

Evaluation of antigen-detecting and antibody-detecting diagnostic test combinations for diagnosing melioidosis.

BACKGROUND: Melioidosis, an infectious disease caused by Burkholderia pseudomallei, is endemic in many tropical developing countries and has a high mortality. Here we evaluated combinations of a lateral flow immunoassay (LFI) detecting B. pseudomallei capsular polysaccharide (CPS) and enzyme-linked immunosorbent assays (ELISA) detecting antibodies against hemolysin co-regulated protein (Hcp1) or O-polysaccharide (OPS) for diagnosing melioidosis. METHODOLOGY/PRINCIPAL FINDINGS: We conducted a cohort-based case-control study. Both cases and controls were derived from a prospective observational study of patients presenting with community-acquired infections and sepsis in northeast Thailand (Ubon-sepsis). Cases included 192 patients with a clinical specimen culture positive for B. pseudomallei. Controls included 502 patients who were blood culture positive for Staphylococcus aureus, Escherichia coli or Klebsiella pneumoniae or were polymerase chain reaction assay positive for malaria or dengue. Serum samples collected within 24 hours of admission were stored and tested using a CPS-LFI, Hcp1-ELISA and OPS-ELISA. When assessing diagnostic tests in combination, results were considered positive if either test was positive. We selected ELISA cut-offs corresponding to a specificity of 95%. Using a positive cut-off OD of 2.912 for Hcp1-ELISA, the combination of the CPS-LFI and Hcp1-ELISA had a sensitivity of 67.7% (130/192 case patients) and a specificity of 95.0% (477/502 control patients). The sensitivity of the combination (67.7%) was higher than that of the CPS-LFI alone (31.3%, p<0.001) and that of Hcp1-ELISA alone (53.6%, p<0.001). A similar phenomenon was also observed for the combination of CPS-LFI and OPS-ELISA. In case patients, positivity of the CPS-LFI was associated with a short duration of symptoms, high modified Sequential (sepsis-related) Organ Failure Assessment (SOFA) score, bacteraemia and mortality outcome, while positivity of Hcp1-ELISA was associated with a longer duration of symptoms, low modified SOFA score, non-bacteraemia and survival outcome. CONCLUSIONS/SIGNIFICANCE: A combination of antigen-antibody diagnostic tests increased the sensitivity of melioidosis diagnosis over individual tests while preserving high specificity. Point-of-care tests for melioidosis based on the use of combination assays should be further developed and evaluated.

Comparative clinical characteristics and outcomes of patients with community acquired bacteremia caused by Escherichia coli, Burkholderia pseudomallei and Staphylococcus aureus: A prospective observational study (Ubon-sepsis).

BACKGROUND: Community acquired bacteremia (CAB) is a common cause of sepsis in low and middle-income countries (LMICs). However, knowledge about factors associated with outcomes of CAB in LMICs is limited. METHODOLOGY/PRINCIPAL FINDINGS: A prospective observational study (Ubon-sepsis) of adults admitted to a referral hospital with community-acquired infection in Northeastern Thailand was conducted between March 1, 2013 and February 1, 2017. In the present analysis, patients with a blood culture collected within 24 hours of admission that was positive for one of the three most common pathogens were studied. Clinical features, management, and outcomes of patients with each cause of CAB were compared. Of 3,806 patients presenting with community-acquired sepsis, 155, 131 and 37 patients had a blood culture positive for Escherichia coli, Burkholderia pseudomallei and Staphylococcus aureus, respectively. Of these 323 CAB patients, 284 (89%) were transferred from other hospitals. 28-day mortality was highest in patients with B. pseudomallei bactaeremia (66%), followed by those with S. aureus bacteraemia (43%) and E. coli (19%) bacteraemia. In the multivariable Cox proportional hazards model adjusted for age, sex, transfer from another hospital, empirical antibiotics prior to or during the transfer, and presence of organ dysfunction on admission, B. pseudomallei (aHR 3.78; 95%CI 2.31-6.21) and S. aureus (aHR 2.72; 95%CI 1.40-5.28) bacteraemias were associated with higher mortality compared to E. coli bacteraemia. Receiving empirical antibiotics recommended for CAB caused by the etiologic organism prior to or during transfer was associated with survival (aHR 0.58; 95%CI 0.38-0.88). CONCLUSIONS/SIGNIFICANCE: Mortality of patients with CAB caused by B. pseudomallei was higher than those caused by S. aureus and E. coli, even after adjusting for presence of organ dysfunction on admission and effectiveness of empirical antibiotics received. Improving algorithms or rapid diagnostic tests to guide early empirical antibiotic may be key to improving CAB outcomes in LMICs.

Effectiveness of a multifaceted prevention programme for melioidosis in diabetics (PREMEL): A stepped-wedge cluster-randomised controlled trial.

BACKGROUND: Melioidosis, an often-fatal infectious disease caused by the environmental Gram-negative bacillus Burkholderia pseudomallei, is endemic in tropical countries. Diabetes mellitus and environmental exposure are important risk factors for melioidosis acquisition. We aim to evaluate the effectiveness of a multifaceted prevention programme for melioidosis in diabetics in northeast Thailand. METHODOLOGY/PRINCIPAL FINDINGS: From April 2014 to December 2018, we conducted a stepped-wedge cluster-randomized controlled behaviour change trial in 116 primary care units (PCUs) in Ubon Ratchathani province, northeast Thailand. The intervention was a behavioural support group session to help diabetic patients adopt recommended behaviours, including wearing rubber boots and drinking boiled water. We randomly allocated the PCUs to receive the intervention starting in March 2016, 2017 and 2018. All diabetic patients were contacted by phone yearly, and the final follow-up was December 2018. Two primary outcomes were hospital admissions involving infectious diseases and culture-confirmed melioidosis. Of 9,056 diabetics enrolled, 6,544 (72%) received a behavioural support group session. During 38,457 person-years of follow-up, we observed 2,195 (24%) patients having 3,335 hospital admissions involved infectious diseases, 80 (0.8%) melioidosis, and 485 (5%) deaths. In the intention-to-treat analysis, implementation of the intervention was not associated with primary outcomes. In the per-protocol analysis, patients who received a behavioural support group session had lower incidence rates of hospital admissions involving infectious diseases (incidence rate ratio [IRR] 0.89; 95%CI 0.80-0.99, p = 0.03) and of all-cause mortality (IRR 0.54; 95%CI 0.43-0.68, p<0.001). However, the incidence rate of culture-confirmed melioidosis was not significantly lower (IRR 0.96, 95%CI 0.46-1.99, p = 0.66). CONCLUSIONS/SIGNIFICANCE: Clear benefits of this multifaceted prevention programme for melioidosis were not observed. More compelling invitations for the intervention, modification of or addition to the behaviour change techniques used, and more frequent intervention may be needed. TRIAL REGISTRATION: This trial is registered with ClinicalTrials.gov, number NCT02089152.

Utility of qSOFA and modified SOFA in severe malaria presenting as sepsis.

Sepsis can be caused by malaria infection, but little is known about the utility of the quick Sequential (Sepsis-Related) Organ Failure Assessment (qSOFA) and SOFA score in malaria. We conducted a prospective observational study from March 2013 to February 2017 to examine adults admitted with community-acquired infection in a tertiary-care hospital in Ubon Ratchathani, Northeast Thailand (Ubon-sepsis). Subjects were classified as having sepsis if they had a modified SOFA score ≥2 within 24 hours of admission. Serum was stored and later tested for malaria parasites using a nested PCR assay. Presence of severe malaria was defined using modified World Health Organization criteria. Of 4,989 patients enrolled, 153 patients (3%) were PCR positive for either Plasmodium falciparum (74 [48%]), P. vivax (69 [45%]), or both organisms (10 [7%]). Of 153 malaria patients, 80 were severe malaria patients presenting with sepsis, 70 were non-severe malaria patients presenting with sepsis, and three were non-severe malaria patients presenting without sepsis. The modified SOFA score (median 5; IQR 4-6; range 1-18) was strongly correlated with malaria severity determined by the number of World Health Organization severity criteria satisfied by the patient (Spearman's rho = 0.61, p<0.001). Of 80 severe malaria patients, 2 (2.5%), 11 (14%), 62 (77.5%) and 5 (6%), presented with qSOFA scores of 0, 1, 2 and 3, respectively. Twenty eight-day mortality was 1.3% (2/153). In conclusion, qSOFA and SOFA can serve as markers of disease severity in adults with malarial sepsis. Patients presenting with a qSOFA score of 1 may also require careful evaluation for sepsis; including diagnosis of cause of infection, initiation of medical intervention, and consideration for referral as appropriate.

Sensitivity and specificity of a lateral flow immunoassay (LFI) in serum samples for diagnosis of melioidosis.

Background: Culture is the gold standard for the diagnosis of melioidosis, an infection caused by Burkholderia pseudomallei. Here we evaluate a lateral flow immunoassay (LFI) to detect B. pseudomallei capsular polysaccharide (CPS) in serum samples. Methods: Patients with culture from any clinical specimen positive for B. pseudomallei were selected as cases. Patients who were blood culture positive for Staphylococcus aureus, Escherichia coli or Klebsiella pneumoniae as well as those who were malaria or dengue polymerase chain reaction assay positive were selected as controls. Results: The sensitivity of the LFI was 31.3% (60/192 case patients [95% confidence interval {CI} 24.8 to 38.3]) and the specificity was 98.8% (559/566 control patients [95% CI 97.4 to 99.5]) in serum samples. Conclusions: Although LFI may have limited sensitivity in serum, it can rapidly diagnose melioidosis in resource-limited settings.

Antibiotic use in poultry: a survey of eight farms in Thailand.

OBJECTIVE: To investigate antibiotic use in poultry farms in Thailand and estimate the total amount of antibiotics used annually in Thai production of chicken meat. METHODS: In a single province, we surveyed eight farms in which chickens were raised for meat and interviewed the farms' owners in 2016. The antibiotic use for each chicken was defined as the amount of antibiotic given to the chicken over its entire lifetime divided by the target weight of the chicken at the time of its slaughter. Assuming that the results were nationally representative, we estimated annual antibiotic use on all Thai chickens raised for meat. FINDINGS: No use of antibiotics for growth promotion was reported. Five farms raised 1-kg chickens for company A and reportedly used no antibiotics unless the chickens were sick. The other three farms raised 3-kg chickens for company B and reported routine use of antibiotics for prophylaxis. Per kg final weight, each chicken raised for company B was reportedly routinely given a mean of 101 mg of antibiotics - that is, 33 mg of amoxicillin, 29 mg colistin, 19 mg oxytetracycline, 18 mg doxycycline and 2 mg tilmicosin. The total amount of antibiotic used on all Thai chickens raised for meat in 2016 was estimated to be 161 tonnes. CONCLUSION: Each year in Thailand, many tonnes of antibiotics are probably routinely used in raising chickens for meat. Labels on retail packs of meat should include data on antibiotic use in the production of the meat.

Presence of Burkholderia pseudomallei in Soil and Paddy Rice Water in a Rice Field in Northeast Thailand, but Not in Air and Rainwater.

Environmental Burkholderia pseudomallei has been postulated to be aerosolized during ploughing and heavy rain, and could result in inhalational melioidosis. Here, we determined the presence of B. pseudomallei in soil, paddy field water (PFW), air, and rainwater samples in a single rice paddy field in Ubon Ratchathani, northeast Thailand. In 2012, we collected 100 soil samples during the dry season, 10 PFW samples during the monsoon season, 77 air samples during ploughing (N = 31) and heavy rains (N = 46), and 60 rainwater samples during 12 rain events. We found that 32 soil samples (32%), six PFW samples (60%), and none of the air and rainwater samples were culture positive for B. pseudomallei. Other soil bacteria were isolated from air and rainwater samples. Mean quantitative count of B. pseudomallei estimated from two culture-positive PFW samples was 200 colony forming units/mL. Our findings suggest that the risk of melioidosis acquisition by inhalation in Thailand might be low.

Evolution of the Staphylococcus argenteus ST2250 Clone in Northeastern Thailand Is Linked with the Acquisition of Livestock-Associated Staphylococcal Genes.

Staphylococcus argenteus is a newly named species previously described as a divergent lineage of Staphylococcus aureus that has recently been shown to have a global distribution. Despite growing evidence of the clinical importance of this species, knowledge about its population epidemiology and genomic architecture is limited. We used whole-genome sequencing to evaluate and compare S. aureus (n = 251) and S. argenteus (n = 68) isolates from adults with staphylococcal sepsis at several hospitals in northeastern Thailand between 2006 and 2013. The majority (82%) of the S. argenteus isolates were of multilocus sequence type 2250 (ST2250). S. aureus was more diverse, although 43% of the isolates belonged to ST121. Bayesian analysis suggested an S. argenteus ST2250 substitution rate of 4.66 (95% confidence interval [CI], 3.12 to 6.38) mutations per genome per year, which was comparable to the S. aureus ST121 substitution rate of 4.07 (95% CI, 2.61 to 5.55). S. argenteus ST2250 emerged in Thailand an estimated 15 years ago, which contrasts with the S. aureus ST1, ST88, and ST121 clades that emerged around 100 to 150 years ago. Comparison of S. argenteus ST2250 genomes from Thailand and a global collection indicated a single introduction into Thailand, followed by transmission to local and more distant countries in Southeast Asia and further afield. S. argenteus and S. aureus shared around half of their core gene repertoire, indicating a high level of divergence and providing strong support for their classification as separate species. Several gene clusters were present in ST2250 isolates but absent from the other S. argenteus and S. aureus study isolates. These included multiple exotoxins and antibiotic resistance genes that have been linked previously with livestock-associated S. aureus, consistent with a livestock reservoir for S. argenteus These genes appeared to be associated with plasmids and mobile genetic elements and may have contributed to the biological success of ST2250.IMPORTANCE In this study, we used whole-genome sequencing to understand the genome evolution and population structure of a systematic collection of ST2250 S. argenteus isolates. A newly identified ancestral species of S. aureus, S. argenteus has become increasingly known as a clinically important species that has been reported recently across various countries. Our results indicate that S. argenteus has spread at a relatively rapid pace over the past 2 decades across northeastern Thailand and acquired multiple exotoxin and antibiotic resistance genes that have been linked previously with livestock-associated S. aureus Our findings highlight the clinical importance and potential pathogenicity of S. argenteus as a recently emerging pathogen.

Colony morphology variation of Burkholderia pseudomallei is associated with antigenic variation and O-polysaccharide modification.

Burkholderia pseudomallei is a CDC tier 1 select agent that causes melioidosis, a severe disease in humans and animals. Persistent infections are common, and there is currently no vaccine available. Lipopolysaccharide (LPS) is a potential vaccine candidate. B. pseudomallei expresses three serologically distinct LPS types. The predominant O-polysaccharide (OPS) is an unbranched heteropolymer with repeating d-glucose and 6-deoxy-l-talose residues in which the 6-deoxy-l-talose residues are variably replaced with O-acetyl and O-methyl modifications. We observed that primary clinical B. pseudomallei isolates with mucoid and nonmucoid colony morphologies from the same sample expressed different antigenic types distinguishable using an LPS-specific monoclonal antibody (MAb). MAb-reactive (nonmucoid) and nonreactive (mucoid) strains from the same patient exhibited identical LPS banding patterns by silver staining and indistinguishable genotypes. We hypothesized that LPS antigenic variation reflected modification of the OPS moieties. Mutagenesis of three genes involved in LPS synthesis was performed in B. pseudomallei K96243. Loss of MAb reactivity was observed in both wbiA (encoding a 2-O-acetyltransferase) and wbiD (putative methyl transferase) mutants. The structural characteristics of the OPS moieties from isogenic nonmucoid strain 4095a and mucoid strain 4095c were further investigated. Utilizing nuclear magnetic resonance (NMR) spectroscopy, we found that B. pseudomallei 4095a and 4095c OPS antigens exhibited substitution patterns that differed from the prototypic OPS structure. Specifically, 4095a lacked 4-O-acetylation, while 4095c lacked both 4-O-acetylation and 2-O-methylation. Our studies indicate that B. pseudomallei OPS undergoes antigenic variation and suggest that the 9D5 MAb recognizes a conformational epitope that is influenced by both O-acetyl and O-methyl substitution patterns.

Clinical and molecular epidemiology of Staphylococcus argenteus infections in Thailand.

Molecular typing of 246 Staphylococcus aureus isolates from unselected patients in Thailand showed that 10 (4.1%) were actually Staphylococcus argenteus. Contrary to the suggestion that S. argenteus is less virulent than S. aureus, we demonstrated comparable rates of morbidity, death, and health care-associated infection in patients infected with either of these two species.

Development of a prototype lateral flow immunoassay (LFI) for the rapid diagnosis of melioidosis.

Burkholderia pseudomallei is a soil-dwelling bacterium and the causative agent of melioidosis. Isolation of B. pseudomallei from clinical samples is the "gold standard" for the diagnosis of melioidosis; results can take 3-7 days to produce. Alternatively, antibody-based tests have low specificity due to a high percentage of seropositive individuals in endemic areas. There is a clear need to develop a rapid point-of-care antigen detection assay for the diagnosis of melioidosis. Previously, we employed In vivo Microbial Antigen Discovery (InMAD) to identify potential B. pseudomallei diagnostic biomarkers. The B. pseudomallei capsular polysaccharide (CPS) and numerous protein antigens were identified as potential candidates. Here, we describe the development of a diagnostic immunoassay based on the detection of CPS. Following production of a CPS-specific monoclonal antibody (mAb), an antigen-capture immunoassay was developed to determine the concentration of CPS within a panel of melioidosis patient serum and urine samples. The same mAb was used to produce a prototype Active Melioidosis Detect Lateral Flow Immunoassay (AMD LFI); the limit of detection of the LFI for CPS is comparable to the antigen-capture immunoassay (∼0.2 ng/ml). The analytical reactivity (inclusivity) of the AMD LFI was 98.7% (76/77) when tested against a large panel of B. pseudomallei isolates. Analytical specificity (cross-reactivity) testing determined that 97.2% of B. pseudomallei near neighbor species (35/36) were not reactive. The non-reactive B. pseudomallei strain and the reactive near neighbor strain can be explained through genetic sequence analysis. Importantly, we show the AMD LFI is capable of detecting CPS in a variety of patient samples. The LFI is currently being evaluated in Thailand and Australia; the focus is to optimize and validate testing procedures on melioidosis patient samples prior to initiation of a large, multisite pre-clinical evaluation.

Melioidosis caused by Burkholderia pseudomallei in drinking water, Thailand, 2012.

We identified 10 patients in Thailand with culture-confirmed melioidosis who had Burkholderia pseudomallei isolated from their drinking water. The multilocus sequence type of B. pseudomallei from clinical specimens and water samples were identical for 2 patients. This finding suggests that drinking water is a preventable source of B. pseudomallei infection.

Prevalence of melioidosis in patients with suspected pulmonary tuberculosis and sputum smear negative for acid-fast bacilli in northeast Thailand.

The clinical and radiological features of pulmonary melioidosis can mimic tuberculosis. We prospectively evaluated 118 patients with suspected pulmonary tuberculosis who were acid-fast bacilli (AFB) smear negative at Udon Thani Hospital, northeast Thailand. Culture of residual sputum from AFB testing was positive for Burkholderia pseudomallei in three patients (2.5%; 95% confidence interval [CI] 0.5-7.3%). We propose that in melioidosis-endemic areas, residual sputum from AFB testing should be routinely cultured for B. pseudomallei.

Rapid detection of Burkholderia pseudomallei in blood cultures using a monoclonal antibody-based immunofluorescent assay.

Melioidosis is a severe bacterial infection caused by Burkholderia pseudomallei. Rapid antimicrobial therapy is necessary to improve patient outcome, which is aided by direct detection of B. pseudomallei in clinical samples. A drawback for all antigen assays is that the number of B. pseudomallei in blood usually falls below the achievable level of detection. We performed a prospective cohort study of 461 patients with 541 blood cultures to evaluate the utility of a pre-incubation step prior to detection of B. pseudomallei using a monoclonal antibody-based immunofluorescent assay (Mab-IFA). The Mab-IFA was positive in 74 of 76 patients with melioidosis (sensitivity = 97.4%), and negative in 385 patients who did not have blood cultures containing B. pseudomallei (specificity = 100%). The Mab-IFA could be a valuable supplementary tool for rapid detection. We recommend the use of the Mab-IFA to test blood cultures that flag positive in regions where melioidosis is endemic.