BackgroundMelioidosis is an increasingly recognised cause of sepsis and death across South East Asia and Northern Australia, caused by the bacterium Burkholderia pseudomallei. Risk factors include diabetes, alcoholism and renal disease, and a vaccine targeting at-risk populations is urgently required. A better understanding of the protective immune response in naturally infected patients is essential for vaccine design.MethodsWe conducted a longitudinal clinical and immunological study of 200 patients with melioidosis on admission, 12 weeks (n = 113) and 52 weeks (n = 65) later. Responses to whole killed B. pseudomallei were measured in peripheral blood mononuclear cells (PBMC) by interferon-gamma (IFN-γ) ELIspot assay and flow cytometry and compared to those of control subjects in the region with diabetes (n = 45) and without diabetes (n = 43).ResultsWe demonstrated strong CD4+ and CD8+ responses to B. pseudomallei during acute disease, 12 weeks and 52 weeks later. 28-day mortality was 26% for melioidosis patients, and B. pseudomallei-specific cellular responses in fatal cases (mean 98 IFN-γ cells per million PBMC) were significantly lower than those in the survivors (mean 142 IFN-γ cells per million PBMC) in a multivariable logistic regression model (P = 0.01). A J-shaped curve association between circulating neutrophil count and mortality was seen with an optimal count of 4000 to 8000 neutrophils/μl.Melioidosis patients with known diabetes had poor diabetic control (median glycated haemoglobin HbA1c 10.2%, interquartile range 9.2–13.1) and showed a stunted B. pseudomallei-specific cellular response during acute illness compared to those without diabetes.ConclusionsThe results demonstrate the role of both CD4+ and CD8+ T-cells in protection against melioidosis, and an interaction between diabetes and cellular responses. This supports development of vaccine strategies that induce strong T-cell responses for the control of intracellular pathogens such as B. pseudomallei.

A recent modelling study estimated that there are 2800 deaths due to melioidosis in Thailand yearly. The Thailand Melioidosis Network (formed in 2012) has been working closely with the Ministry of Public Health (MoPH) to investigate and reduce the burden of this disease. Based on updated data, the incidence of melioidosis is still high in Northeast Thailand. More than 2000 culture-confirmed cases of melioidosis are diagnosed in general hospitals with microbiology laboratories in this region each year. The mortality rate is around 35%. Melioidosis is endemic throughout Thailand, but it is still not uncommon that microbiological facilities misidentify Burkholderia pseudomallei as a contaminant or another organism. Disease awareness is low, and people in rural areas neither wear boots nor boil water before drinking to protect themselves from acquiring B. pseudomallei. Previously, about 10 melioidosis deaths were formally reported to the National Notifiable Disease Surveillance System (Report 506) each year, thus limiting priority setting by the MoPH. In 2015, the formally reported number of melioidosis deaths rose to 112, solely because Sunpasithiprasong Hospital, Ubon Ratchathani province, reported its own data (n = 107). Melioidosis is truly an important cause of death in Thailand, and currently reported cases (Report 506) and cases diagnosed at research centers reflect the tip of the iceberg. Laboratory training and communication between clinicians and laboratory personnel are required to improve diagnosis and treatment of melioidosis countrywide. Implementation of rapid diagnostic tests, such as a lateral flow antigen detection assay, with high accuracy even in melioidosis-endemic countries such as Thailand, is critically needed. Reporting of all culture-confirmed melioidosis cases from every hospital with a microbiology laboratory, together with final outcome data, is mandated under the Communicable Diseases Act B.E.2558. By enforcing this legislation, the MoPH could raise the priority of this disease, and should consider implementing a campaign to raise awareness and melioidosis prevention countrywide.

Melioidosis, caused by Burkholderia pseudomallei, is a potentially lethal infection with no licensed vaccine. There is little understanding of why some exposed individuals have no symptoms, while others rapidly progress to sepsis and death, or why diabetes confers increased susceptibility. We prospectively recruited a cohort of 183 acute melioidosis patients and 21 control subjects from Northeast Thailand and studied immune parameters in the context of survival status and the presence or absence of diabetes. HLA-B*46 (one of the commonest HLA class I alleles in SE Asia) and HLA-C*01 were associated with an increased risk of death (odds ratio 2.8 and 3.1 respectively). Transcriptomic analysis during acute infection in diabetics indicated the importance of interplay between immune pathways including those involved in antigen presentation, chemotaxis, innate and adaptive immunity and their regulation. Survival was associated with enhanced T cell immunity to nine of fifteen immunodominant antigens analysed including AhpC (BPSL2096), BopE (BPSS1525), PilO (BPSS1599), ATP binding protein (BPSS1385) and an uncharacterised protein (BPSL2520). T cell immunity to GroEL (BPSL2697) was specifically impaired in diabetic individuals. This characterization of immunity associated with survival during acute infection offers insights into correlates of protection and a foundation for design of an effective multivalent vaccine.

BackgroundThe environmental bacterium Burkholderia pseudomallei causes the infectious disease melioidosis with a high case-fatality rate in tropical and subtropical regions. Direct pathogen detection can be difficult, and therefore an indirect serological test which might aid early diagnosis is desirable. However, current tests for antibodies against B. pseudomallei, including the reference indirect haemagglutination assay (IHA), lack sensitivity, specificity and standardization. Consequently, serological tests currently do not play a role in the diagnosis of melioidosis in endemic areas. Recently, a number of promising diagnostic antigens have been identified, but a standardized, easy-to-perform clinical laboratory test for sensitive multiplex detection of antibodies against B. pseudomallei is still lacking.Methods and Principal FindingsIn this study, we developed and validated a protein microarray which can be used in a standard 96-well format. Our array contains 20 recombinant and purified B. pseudomallei proteins, previously identified as serodiagnostic candidates in melioidosis. In total, we analyzed 196 sera and plasmas from melioidosis patients from northeast Thailand and 210 negative controls from melioidosis-endemic and non-endemic regions. Our protein array clearly discriminated between sera from melioidosis patients and controls with a specificity of 97%. Importantly, the array showed a higher sensitivity than did the IHA in melioidosis patients upon admission (cut-off IHA titer ≥1:160: IHA 57.3%, protein array: 86.7%; p = 0.0001). Testing of sera from single patients at 0, 12 and 52 weeks post-admission revealed that protein antigens induce either a short- or long-term antibody response.ConclusionsOur protein array provides a standardized, rapid, easy-to-perform test for the detection of B. pseudomallei-specific antibody patterns. Thus, this system has the potential to improve the serodiagnosis of melioidosis in clinical settings. Moreover, our high-throughput assay might be useful for the detection of anti-B. pseudomallei antibodies in epidemiological studies. Further studies are needed to elucidate the clinical and diagnostic significance of the different antibody kinetics observed during melioidosis.

We undertook a prospective study to quantitate Burkholderia pseudomallei in blood, urine, respiratory secretions, and pus [corrected] obtained from 414 patients with melioidosis. The median was count 1.1, 1.5 x 10(4), 1.1 x 10(5), and 1.1 x 10(7) CFU/mL in these sample types, respectively. This provides important insights into the likely feasibility of future studies such as expression microarray analysis using clinical material.