Enzyme-linked immunosorbent assay (ELISA) using a glycolipid antigen for the serodiagnosis of melioidosis

Abstract The serodiagnosis of melioidosis is commonly performed with tests using protein or polysaccharide as antigen. However, due to the low sensitivity, specificity and difficulty in the preparation of the antigens, more simple, precise and reproducible diagnostic tests were required. A purified glycolipid antigen (GL) which is a specific lipid component of Burkholderia pseudomallei has been used in an ELISA. With this antigen, specific immunoglobulin G (IgG) was detected in 49 out of 50 melioidosis sera. IgG was also detected in 2 out of 185 (Japanese) and 16 out of 181 (Vietnamese) control sera. Thus, the sensitivity was 98.0%, and specificity was 98.9% and 91.1% in the Japanese and Vietnamese sera, respectively. When the ELISA and indirect haemagglutination (IHA) tests were combined, a sensitivity of 100% and specificity of 97.8% were achieved. The advantages of the glycolipid antigen are ease of preparation, stability, high sensitivity and specificity.     

T-Cell Responses Are Associated with Survival in Acute Melioidosis Patients

Background

Melioidosis 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.

Methods

We 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).

Results

We 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 HbA_1c 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.

Conclusions

The 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.     

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

BackgroundMelioidosis, 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 findingsFrom 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/SignificanceClear 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 registrationThis trial is registered with ClinicalTrials.gov, number {"type":"clinical-trial","attrs":{"text":"NCT02089152","term_id":"NCT02089152"}}NCT02089152.     

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)

BackgroundCommunity 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 findingsA 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/SignificanceMortality 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.     

Feasibility of Modified Surviving Sepsis Campaign Guidelines in a Resource-Restricted Setting Based on a Cohort Study of Severe S. Aureus Sepsis

Background

The Surviving Sepsis Campaign (SSC) guidelines describe best practice for the management of severe sepsis and septic shock in developed countries, but most deaths from sepsis occur where healthcare is not sufficiently resourced to implement them. Our objective was to define the feasibility and basis for modified guidelines in a resource-restricted setting.

Methods and Findings

We undertook a detailed assessment of sepsis management in a prospective cohort of patients with severe sepsis caused by a single pathogen in a 1,100-bed hospital in lower-middle income Thailand. We compared their management with the SSC guidelines to identify care bundles based on existing capabilities or additional activities that could be undertaken at zero or low cost. We identified 72 patients with severe sepsis or septic shock associated with S. aureus bacteraemia, 38 (53%) of who died within 28 days. One third of patients were treated in intensive care units (ICUs). Numerous interventions described by the SSC guidelines fell within existing capabilities, but their implementation was highly variable. Care available to patients on general wards covered the fundamental principles of sepsis management, including non-invasive patient monitoring, antimicrobial administration and intravenous fluid resuscitation. We described two additive care bundles, one for general wards and the second for ICUs, that if consistently performed would be predicted to improve outcome from severe sepsis.

Conclusion

It is feasible to implement modified sepsis guidelines that are scaled to resource availability, and that could save lives prior to the publication of international guidelines for developing countries.     

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

BackgroundMelioidosis, 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 findingsWe 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/SignificanceA 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.