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.

γδ T Cells Mediate Protection Against Neutrophil-associated Lung Inflammation in Pulmonary Melioidosis.

Pulmonary melioidosis is a severe tropical infection caused by Burkholderia pseudomallei and is associated with high mortality despite early antibiotic treatment. γδ T cells have been increasingly implicated as drivers of the host neutrophil response during bacterial pneumonia, but their role in pulmonary melioidosis is unknown. Here, we report that in patients with melioidosis, a lower peripheral blood γδ T cell concentration is associated with higher mortality even when adjusting for severity of illness. γδ T cells were also enriched in the lung and protected against mortality in a mouse model of pulmonary melioidosis. γδ T cell deficiency in infected mice induced an early recruitment of neutrophils to the lung, independent of bacterial burden. Subsequently, γδ T cell deficiency resulted in increased neutrophil-associated inflammation in the lung as well as impaired bacterial clearance. Additionally, γδ T cells influenced neutrophil function and subset diversity in the lung after infection. Our results indicate that γδ T cells serve a novel protective role in the lung during a severe bacterial pneumonia by regulating excessive neutrophil-associated inflammation.

Associations between clinical data, vaccination status, antibody responses, and post-COVID-19 symptoms in Thais infected with SARS-CoV-2 Delta and Omicron variants: a 1-year follow-up study.

BACKGROUND: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), which causes coronavirus disease 2019 (COVID-19), led to a global pandemic from 2020. In Thailand, five waves of outbreaks were recorded, with the fourth and fifth waves driven by the Delta and Omicron variants, resulting in over 20,000 new confirmed cases daily at their peaks. METHODS: This cross-sectional study investigated the associations between clinical symptoms, vaccination status, antibody responses, and post-COVID-19 sequelae in COVID-19 patients. Plasma samples and clinical data were collected from participants admitted to hospitals in Thailand between July 2021 and August 2022, with follow-ups conducted for one year. The study included 110 participants infected with either the Delta (n = 46) or Omicron (n = 64) variants. Virus genotypes were confirmed by RT-PCR of nasal swab RNA and partial nucleotide sequencing of the S gene. IgG and IgA antibody levels against the receptor-binding domain (RBD) of SARS-CoV-2 Delta and Omicron variants were measured in plasma samples using ELISA. RESULTS: Pneumonia was found to be associated with Delta variant infections, while sore throat, congestion or runny nose, and headache were linked to Omicron infections. Vaccination with fewer than two doses and diabetes mellitus were significantly associated with higher disease severity. Specific IgG and IgA antibodies against the RBD of the Delta variant generally rose by day 14 and were maintained for up to two months, whereas the pattern of antibody response to the Omicron variant was less clear. Antibody risings were found to be positively associated with pneumonia, certain underlying conditions (obesity, hypertension, dyslipidemia, and diabetes mellitus), and age ≥ 60 years. Delta variant infections were associated with forgetfulness, hair loss, and headache during the 1-year post-infection period. Females were more likely to experience hair loss, forgetfulness, and joint pain, while older age was associated with joint pain. CONCLUSIONS: This study enhances our understanding of SARS-CoV-2 infections in Thais, particularly concerning the Delta and Omicron variants. The findings can inform public health planning and response strategies for future outbreaks of SARS-CoV-2 or other emerging viral diseases.

Prospective Analysis of Antibody Diagnostic Tests and TTS1 Real-Time PCR for Diagnosis of Melioidosis in Areas Where It Is Endemic.

Melioidosis is a tropical infectious disease caused by Burkholderia pseudomallei. Melioidosis is associated with diverse clinical manifestations and high mortality. Early diagnosis is needed for appropriate treatment, but it takes several days to obtain bacterial culture results. We previously developed a rapid immunochromatography test (ICT) based on hemolysin coregulated protein 1 (Hcp1) and two enzyme-linked immunosorbent assays (ELISAs) based on Hcp1 (Hcp1-ELISA) and O-polysaccharide (OPS-ELISA) for serodiagnosis of melioidosis. This study prospectively validated the diagnostic accuracy of the Hcp1-ICT in suspected melioidosis cases and determined its potential use for identifying occult melioidosis cases. Patients were enrolled and grouped by culture results, including 55 melioidosis cases, 49 other infection patients, and 69 patients with no pathogen detected. The results of the Hcp1-ICT were compared with culture, a real-time PCR test based on type 3 secretion system 1 genes (TTS1-PCR), and ELISAs. Patients in the no-pathogen-detected group were followed for subsequent culture results. Using bacterial culture as a gold standard, the sensitivity and specificity of Hcp1-ICT were 74.5% and 89.8%, respectively. The sensitivity and specificity of TTS1-PCR were 78.2% and 100%, respectively. The diagnostic accuracy was markedly improved if the Hcp1-ICT results were combined with TTS1-PCR results (sensitivity and specificity were 98.2% and 89.8%, respectively). Among patients with initially negative cultures, Hcp1-ICT was positive in 16/73 (21.9%). Five of the 16 patients (31.3%) were subsequently confirmed to have melioidosis by repeat culture. The combined Hcp1-ICT and TTS1-PCR test results are useful for diagnosis, and Hcp1-ICT may help identify occult cases of melioidosis.

A 2-Biomarker Model Augments Clinical Prediction of Mortality in Melioidosis.

BACKGROUND: Melioidosis, infection caused by Burkholderia pseudomallei, is a common cause of sepsis with high associated mortality in Southeast Asia. Identification of patients at high likelihood of clinical deterioration is important for guiding decisions about resource allocation and management. We sought to develop a biomarker-based model for 28-day mortality prediction in melioidosis. METHODS: In a derivation set (N = 113) of prospectively enrolled, hospitalized Thai patients with melioidosis, we measured concentrations of interferon-γ, interleukin-1ß, interleukin-6, interleukin-8, interleukin-10, tumor necrosis factor-ɑ, granulocyte-colony stimulating factor, and interleukin-17A. We used least absolute shrinkage and selection operator (LASSO) regression to identify a subset of predictive biomarkers and performed logistic regression and receiver operating characteristic curve analysis to evaluate biomarker-based prediction of 28-day mortality compared with clinical variables. We repeated select analyses in an internal validation set (N = 78) and in a prospectively enrolled external validation set (N = 161) of hospitalized adults with melioidosis. RESULTS: All 8 cytokines were positively associated with 28-day mortality. Of these, interleukin-6 and interleukin-8 were selected by LASSO regression. A model consisting of interleukin-6, interleukin-8, and clinical variables significantly improved 28-day mortality prediction over a model of only clinical variables [AUC (95% confidence interval [CI]): 0.86 (.79-.92) vs 0.78 (.69-.87); P = .01]. In both the internal validation set (0.91 [0.84-0.97]) and the external validation set (0.81 [0.74-0.88]), the combined model including biomarkers significantly improved 28-day mortality prediction over a model limited to clinical variables. CONCLUSIONS: A 2-biomarker model augments clinical prediction of 28-day mortality in melioidosis.

A Rapid Immunochromatography Test Based on Hcp1 Is a Potential Point-of-Care Test for Serological Diagnosis of Melioidosis.

Melioidosis is a fatal infectious disease caused by the environmental bacterium Burkholderia pseudomallei It is highly endemic in Asia and northern Australia but neglected in many other tropical countries. Melioidosis patients have a wide range of clinical manifestations, and definitive diagnosis requires bacterial culture, which can be time-consuming. A reliable rapid serological tool is greatly needed for disease surveillance and diagnosis. We previously demonstrated by enzyme-linked immunosorbent assay (ELISA) that a hemolysin-coregulated protein (Hcp1) is a promising target for serodiagnosis of melioidosis. In this study, we developed a rapid immunochromatography test (ICT) using Hcp1 as the target antigen (Hcp1-ICT). We evaluated this test for specific antibody detection using serum samples obtained from 4 groups of human subjects, including the following: (i) 487 culture-confirmed melioidosis patients from four hospitals in northeast Thailand; (ii) 202 healthy donors from northeast Thailand; (iii) 90 U.S. healthy donors; and (iv) 207 patients infected with other organisms. Compared to culture results as a gold standard, the sensitivity of ICT for all hospitals was 88.3%. The specificities for Thai donors and U.S. donors were 86.1% and 100%, respectively, and the specificity for other infections was 91.8%. The results of the Hcp1-ICT demonstrated 92.4% agreement with the Hcp1-ELISA results with a kappa value of 0.829, indicating that the method is much improved compared with the current serological method, the indirect hemagglutination assay (IHA) (69.5% sensitivity and 67.6% specificity for Thais). The Hcp1-ICT represents a potential point-of-care (POC) test and may be used to replace the IHA for screening of melioidosis in hospitals as well as in resource-limited areas.

Dysfunctional host cellular immune responses are associated with mortality in melioidosis.

Melioidosis is a tropical infection caused by the intracellular pathogen Burkholderia pseudomallei, an underreported and emerging global threat. As melioidosis-associated mortality is frequently high despite antibiotics, novel management strategies are critically needed. Therefore, we sought to determine whether functional changes in the host innate and adaptive immune responses are induced during acute melioidosis and are associated with outcome. Using a unique whole blood stimulation assay developed for use in resource-limited settings, we examined induced cellular functional and phenotypic changes in a cohort of patients with bacteremic melioidosis prospectively enrolled within 24 h of positive blood culture and followed for 28 days. Compared to healthy controls, melioidosis survivors generated an IL-17 response mediated by Th17 cells and terminally-differentiated effector memory CD8+ T cells (P < .05, both), persisting to 28 days after enrolment. Furthermore, melioidosis survivors developed polyfunctional cytokine production in CD8+ T cells (P < .01). Conversely, a reduction in CCR6+ CD4+ T cells was associated with higher mortality, even after adjustments for severity of illness (P = 0.004). Acute melioidosis was also associated with a profound acute impairment in monocyte function as stimulated cytokine responses were reduced in classical, intermediate and non-classical monocytes. Impaired monocyte cytokine function improved by 28-days after enrolment. These data suggest that IL-17 mediated cellular responses may be contributors to host defense during acute melioidosis, and that innate immune function may be impaired. These insights could provide novel targets for the development of therapies and vaccine targets in this frequently lethal disease.

Statin Use and Reduced Risk of Pneumonia in Patients with Melioidosis: A Lung-Specific Statin Association.

Rationale: 3-Hydroxy-3-methylglutaryl coenzyme A reductase inhibitor (statin) use is associated with a lower risk of incident pneumonia and, less robustly, with nonpulmonary infections. Whether statin use is associated with a lower risk of pneumonia than other clinical presentations of infection with the same pathogen is unknown. Objectives: To assess whether preadmission statin use is associated with a lower risk of pneumonia than nonpneumonia presentations among patients hospitalized with Burkholderia pseudomallei infection (melioidosis). Methods: We performed a secondary analysis of a prospective multicenter cohort study of patients hospitalized with culture-confirmed B. pseudomallei infection (melioidosis). We used Poisson regression with robust standard errors to test for an association between statin use and pneumonia. We then performed several sensitivity analyses that addressed healthy user effect and indication bias. Results: Of 1,372 patients with melioidosis enrolled in the parent cohort, 1,121 were analyzed. Nine hundred eighty (87%) of 1,121 were statin nonusers, and 141 (13%) of 1,121 were statin users. Forty-six (33%) of 141 statin users presented with pneumonia compared with 432 (44%) of 980 statin nonusers. Statin use was associated with a lower risk of pneumonia in unadjusted analysis (relative risk, 0.74; 95% confidence interval, 0.58-0.95; P = 0.02) and, after adjustment for demographic variables, comorbidities, environmental exposures, and symptom duration (relative risk, 0.73; 95% confidence interval, 0.57-0.94; P = 0.02). The results of sensitivity analyses, including active comparator analysis and inverse probability of treatment weighting, were consistent with the primary analysis. Conclusions: In hospitalized patients with melioidosis, preadmission statin use was associated with a lower risk of pneumonia than other clinical presentations of melioidosis, suggesting a lung-specific protective effect of statins.

Dysregulated immunologic landscape of the early host response in melioidosis.

Melioidosis, a neglected tropical infection caused by Burkholderia pseudomallei, commonly presents as pneumonia or sepsis with mortality rates up to 50% despite appropriate treatment. A better understanding of the early host immune response to melioidosis may lead to new therapeutic interventions and prognostication strategies to reduce disease burden. Whole blood transcriptomic signatures in 164 patients with melioidosis and in 70 patients with other infections hospitalized in northeastern Thailand enrolled within 24 hours following hospital admission were studied. Key findings were validated in an independent melioidosis cohort. Melioidosis was characterized by upregulation of interferon (IFN) signaling responses compared with other infections. Mortality in melioidosis was associated with excessive inflammation, enrichment of type 2 immune responses, and a dramatic decrease in T cell-mediated immunity compared with survivors. We identified and independently confirmed a 5-gene predictive set classifying fatal melioidosis (validation cohort area under the receiver operating characteristic curve 0.83; 95% CI, 0.67-0.99). This study highlights the intricate balance between innate and adaptive immunity during fatal melioidosis and can inform future precision medicine strategies for targeted therapies and prognostication in this severe infection.

Dynamics of Different Classes and Subclasses of Antibody Responses to Severe Acute Respiratory Syndrome Coronavirus 2 Variants after Coronavirus Disease 2019 and CoronaVac Vaccination in Thailand.

The humoral immune response plays a key role in protecting the population from SARS-CoV-2 transmission. Patients who recovered from COVID-19 as well as fully vaccinated individuals have elevated levels of antibodies. The dynamic levels of the classes and subclasses of antibody responses to new variants that occur in different populations remain unclear. We prospectively recruited 60 participants, including COVID-19 patients and CoronaVac-vaccinated individuals, in Thailand from May to August 2021. Plasma samples were collected on day 0, day 14, and day 28 to determine the dynamic levels of the classes and subclasses of plasma antibodies against the receptor-binding domain (RBD) in the spike protein (S) of four SARS-CoV-2 strains (Wuhan, Alpha, Delta, and Omicron) via enzyme-linked immunosorbent assay. Our results indicated that the patients with SARS-CoV-2 infections had broader class and subclass profiles as well as higher levels of anti-S RBD antibodies to the Wuhan, Alpha, and Delta strains than did the CoronaVac-vaccinated individuals. The median antibody levels increased and subsequently declined in a month in the COVID-19 patients and in the vaccinated group. Correlations of the classes and subclasses of antibodies were observed in the COVID-19 patients but not in the vaccinated individuals. The levels of all of the anti-S RBD antibodies against the Omicron variant were low in the patients and in the vaccinated individuals. Our study revealed distinct antibody profiles between the two cohorts, suggesting different pathways of immune activation. This could have an impact on protection from infections by new variants of concern (VOC). IMPORTANCE The antibody responses to new SARS-CoV-2 variants that occur in different populations remain unclear. In this study, we recruited 60 participants, including COVID-19 patients and CoronaVac-vaccinated individuals, in Thailand and determined the dynamic levels of the IgG, IgA, IgM, and IgG subclasses of antibodies against the spike protein (S) of four SARS-CoV-2 strains. Our results showed that the patients with SARS-CoV-2 infections had broader profiles and higher levels of antibodies to the Wuhan, Alpha, and Delta strains than did the CoronaVac-vaccinated individuals. The antibody levels of both groups increased and subsequently decreased within 1 month. Higher and functional correlations of these antibodies were observed in the COVID-19 patients. The levels of all anti-S RBD antibodies against the Omicron variant were low in patients and vaccinated individuals. Our study revealed distinct antibody responses between the two groups, suggesting different pathways of immune response, which may have an impact on protection from infections by new SARS-CoV-2 variants.

Phenotypic and genetic alterations of Burkholderia pseudomallei in patients during relapse and persistent infections.

The bacterium Burkholderia pseudomallei is the causative agent of melioidosis, a severe tropical disease associated with high mortality and relapse and persistent infections. Treatment of melioidosis requires prolonged antibiotic therapy; however, little is known about relapse and persistent infections, particularly the phenotypic and genetic alterations of B. pseudomallei in patients. In this study, we performed pulsed-field gel electrophoresis (PFGE) to compare the bacterial genotype between the initial isolate and the subsequent isolate from each of 23 suspected recurrent and persistent melioidosis patients in Northeast Thailand. We used whole-genome sequencing (WGS) to investigate multilocus sequence types and genetic alterations of within-host strain pairs. We also investigated the bacterial phenotypes associated with relapse and persistent infections, including multinucleated giant cell (MNGC) formation efficiency and intracellular multiplication. We first identified 13 (1.2%) relapse, 7 (0.7%) persistent, and 3 (0.3%) reinfection patients from 1,046 survivors. Each of the 20 within-host strain pairs from patients with relapse and persistent infections shared the same genotype, suggesting that the subsequent isolates arise from the infecting isolate. Logistic regression analysis of clinical data revealed regimen and duration of oral antibiotic therapies as risk factors associated with relapse and persistent infections. WGS analysis demonstrated 17 within-host genetic alteration events in 6 of 20 paired isolates, including a relatively large deletion and 16 single-nucleotide polymorphism (stocktickerSNP) mutations distributed across 12 genes. In 1 of 20 paired isolates, we observed significantly increased cell-to-cell fusion and intracellular replication in the second isolate compared with the initial isolate from a patient with persistent infection. WGS analysis suggested that a non-synonymous mutation in the tssB-5 gene, which encoded an essential component of the type VI secretion system, may be associated with the increased intracellular replication and MNGC formation efficiency of the second isolate of the patient. This information provides insights into genetic and phenotypic alterations in B. pseudomallei in human melioidosis, which may represent a bacterial strategy for persistent and relapse infections.

IL-1R2-based biomarker models predict melioidosis mortality independent of clinical data.

Introduction: Melioidosis is an often-fatal tropical infectious disease caused by the Gram-negative bacillus Burkholderia pseudomallei, but few studies have identified promising biomarker candidates to predict outcome. Methods: In 78 prospectively enrolled patients hospitalized with melioidosis, six candidate protein biomarkers, identified from the literature, were measured in plasma at enrollment. A multi-biomarker model was developed using least absolute shrinkage and selection operator (LASSO) regression, and mortality discrimination was compared to a clinical variable model by receiver operating characteristic curve analysis. Mortality prediction was confirmed in an external validation set of 191 prospectively enrolled patients hospitalized with melioidosis. Results: LASSO regression selected IL-1R2 and soluble triggering receptor on myeloid cells 1 (sTREM-1) for inclusion in the candidate biomarker model. The areas under the receiver operating characteristic curve (AUC) for mortality discrimination for the IL-1R2 + sTREM-1 model (AUC 0.81, 95% CI 0.72-0.91) as well as for an IL-1R2-only model (AUC 0.78, 95% CI 0.68-0.88) were higher than for a model based on a modified Sequential Organ Failure Assessment (SOFA) score (AUC 0.69, 95% CI 0.56-0.81, p < 0.01, p = 0.03, respectively). In the external validation set, the IL-1R2 + sTREM-1 model (AUC 0.86, 95% CI 0.81-0.92) had superior 28-day mortality discrimination compared to a modified SOFA model (AUC 0.80, 95% CI 0.74-0.86, p < 0.01) and was similar to a model containing IL-1R2 alone (AUC 0.82, 95% CI 0.76-0.88, p = 0.33). Conclusion: Biomarker models containing IL-1R2 had improved 28-day mortality prediction compared to clinical variable models in melioidosis and may be targets for future, rapid test development.

Characteristics and One Year Outcomes of Melioidosis Patients in Northeastern Thailand: A Prospective, Multicenter Cohort Study.

Background: Melioidosis is a neglected tropical infection caused by the environmental saprophyte Burkholderia pseudomallei. Methods: We conducted a prospective, observational study at nine hospitals in northeastern Thailand, a hyperendemic melioidosis zone, to define current characteristics of melioidosis patients and quantify outcomes over one year. Findings: 2574 individuals hospitalised with culture-confirmed melioidosis were screened and 1352 patients were analysed. The median age was 55 years, 975 (72%) were male, and 951 (70%) had diabetes. 565 (42%) patients presented with lung infection, 1042 (77%) were bacteremic, 442 (33%) received vasopressors/inotropes and 547 (40%) received mechanical ventilation. 1307 (97%) received an intravenous antibiotic against B. pseudomallei. 335/1345 (25%) patients died within one month and 448/1322 (34%) of patients died within one year. Most patients had risk factors for melioidosis, but patients without identified risk factors did not have a reduced risk of death. Of patients discharged alive, most received oral trimethoprim-sulfamethoxazole, which was associated with decreased risk of post-discharge death; 235/970 (24%) were readmitted, and 874/1015 (86%) survived to one year. Recurrent infection was detected in 17/994 patients (2%). Patients with risk factors other than diabetes had increased risk of death and increased risk of hospital readmission. Interpretation: In northeastern Thailand patients with melioidosis experience high rates of bacteremia, organ failure and death. Most patients discharged alive survive one year although all-cause readmission is common. Recurrent disease is rare. Strategies that emphasize prevention, rapid diagnosis and intensification of early clinical management are likely to have greatest impact in this and other resource-restricted regions. Funding: US NIH/NIAID U01AI115520.

Distinct classes and subclasses of antibodies to hemolysin co-regulated protein 1 and O-polysaccharide and correlation with clinical characteristics of melioidosis patients.

Melioidosis is a tropical infectious disease caused by Burkholderia pseudomallei that results in high mortality. Hemolysin co-regulated protein 1 (Hcp1) and O-polysaccharide (OPS) are vaccine candidates and potential diagnostic antigens. The correlation of classes/subclasses of antibodies against these antigens with clinical characteristics of melioidosis patients is unknown. Antibodies in plasma samples from melioidosis patients and healthy donors were quantified by ELISA and compared with clinical features. In melioidosis patients, Hcp1 induced high IgG levels. OPS induced high IgG and IgA levels. The area under receiver operating characteristic curve (AUROCC) to discriminate melioidosis cases from healthy donors was highest for anti-Hcp1 IgG (0.92) compared to anti-Hcp1 IgA or IgM. In contrast, AUROCC for anti-OPS for IgG (0.91) and IgA (0.92) were comparable. Anti-Hcp1 IgG1 and anti-OPS IgG2 had the greatest AUROCCs (0.87 and 0.95, respectively) compared to other IgG subclasses for each antigen. Survivors had significantly higher anti-Hcp1 IgG3 levels than non-survivors. Male melioidosis patients with diabetes had higher anti-OPS IgA levels than males without diabetes. Thus, diverse and specific antibody responses are associated with distinct clinical characteristics in melioidosis, confirming the diagnostic utility of these responses and providing new insights into immune mechanisms.

Validation of a monoclonal antibody-based immunofluorescent assay to detect Burkholderia pseudomallei in blood cultures.

BACKGROUND: Identification of Burkholderia pseudomallei, the cause of melioidosis, using routine methods takes several days. Use of a monoclonal antibody-based immunofluorescent assay (IFA) on positive blood cultures may speed diagnosis. METHODS: We tested the diagnostic accuracy of the IFA on 545 blood cultures positive for Gram-negative organisms at Udon Thani Hospital, Thailand, between June 2015 and August 2016. RESULTS: Sensitivity of the IFA was 100% and specificity was 99.6%. The median decrease in time to pathogen identification between the IFA result and routine methods was 28 h (IQR 25-51), p<0.0001. CONCLUSIONS: The IFA accurately expedites the diagnosis of melioidosis.