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 were systematically evaluated in patients from a prospective cohort of individuals hospitalized in rural Thailand with infection. Statistical and bioinformatics methods were used to distinguish metabolomic features and processes specific to melioidosis patients, and between fatal and non-fatal cases. Measurements and Main Results: Metabolomic profiling and pathway enrichment analysis of plasma samples of melioidosis (n=175) and non-melioidosis 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 lipid metabolism such as sphingomyelins and ceramide species. We derived a 12-metabolite classifier to distinguish melioidosis from other infections, with an area under the receiver operating characteristic curve of 0.87 in a second validation set of patients. Melioidosis non-survivors (n=94) had a significantly disturbed metabolome compared to survivors (n=81) with increased leucine, isoleucine and valine metabolism, and elevated circulating free fatty acids and acylcarnitines. A limited 8-metabolite panel shows promise as an early prognosticator of mortality in melioidosis. Conclusions: Melioidosis induces a distinct metabolomic state that can be leveraged to distinguish underlying pathophysiological mechanisms leading to increased risk of death. A twelve-metabolite signature accurately differentiates melioidosis from other infections and may have diagnostic applications. This article is open access and distributed under the terms of the Creative Commons Attribution 4.0 International License (https://creativecommons.org/licenses/by/4.0/

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

Longitudinal profiling of plasma cytokines in melioidosis and their association with mortality: a prospective cohort study

Objectives: To characterize plasma cytokine responses in melioidosis and analyse their association with mortality.Methods: A prospective longitudinal study was conducted in two hospitals in Northeast Thailand to enrol 161 individuals with melioidosis, plus 13 uninfected healthy individuals and 11 uninfected individuals with diabetes to act as controls. Blood was obtained from all individuals at enrolment (day 0), and at days 5, 12 and 28 from surviving melioidosis patients. Interferon-γ (IFN-γ), interleukin-1β (IL-1β), IL-2, IL-4, IL-6, IL-8, IL-10, IL-12p70, IL-13, IL-17A, IL-23, and tumour necrosis factor-α (TNF-α) were assayed in plasma. The association of each cytokine and its dynamics with 28-day mortality was determined.Results: Of the individuals with melioidosis, 131/161 (81%) were bacteraemic, and 68/161 (42%) died. On enrolment, median levels of IFN-γ, IL-6, IL-8, IL-10, IL-23 and TNF-α were higher in individuals with melioidosis compared with uninfected healthy individuals and all but IFN-γ were positively associated with 28-day mortality. Interleukin-8 provided the best discrimination of mortality (area under the receiver operating characteristic curve 0.78, 95% CI 0.71–0.85). Over time, non-survivors had increasing IL-6, IL-8 and IL-17A levels, in contrast to survivors. In joint modelling, temporal trajectories of IFN-γ, IL-6, IL-8, IL-10 and TNF-α predicted survival.Conclusions: In a severely ill cohort of individuals with melioidosis, specific pro- and anti-inflammatory and T helper type 17 cytokines were associated with survival from melioidosis, at enrolment and over time. Persistent inflammation preceded death. These findings support further evaluation of these mediators as prognostic biomarkers and to guide targeted immunotherapeutic development for severe melioidosis

Longitudinal profiling of plasma cytokines in melioidosis and their association with mortality: a prospective cohort study

Objectives: To characterize plasma cytokine responses in melioidosis and analyse their association with mortality.Methods: A prospective longitudinal study was conducted in two hospitals in Northeast Thailand to enrol 161 individuals with melioidosis, plus 13 uninfected healthy individuals and 11 uninfected individuals with diabetes to act as controls. Blood was obtained from all individuals at enrolment (day 0), and at days 5, 12 and 28 from surviving melioidosis patients. Interferon-γ (IFN-γ), interleukin-1β (IL-1β), IL-2, IL-4, IL-6, IL-8, IL-10, IL-12p70, IL-13, IL-17A, IL-23, and tumour necrosis factor-α (TNF-α) were assayed in plasma. The association of each cytokine and its dynamics with 28-day mortality was determined.Results: Of the individuals with melioidosis, 131/161 (81%) were bacteraemic, and 68/161 (42%) died. On enrolment, median levels of IFN-γ, IL-6, IL-8, IL-10, IL-23 and TNF-α were higher in individuals with melioidosis compared with uninfected healthy individuals and all but IFN-γ were positively associated with 28-day mortality. Interleukin-8 provided the best discrimination of mortality (area under the receiver operating characteristic curve 0.78, 95% CI 0.71–0.85). Over time, non-survivors had increasing IL-6, IL-8 and IL-17A levels, in contrast to survivors. In joint modelling, temporal trajectories of IFN-γ, IL-6, IL-8, IL-10 and TNF-α predicted survival.Conclusions: In a severely ill cohort of individuals with melioidosis, specific pro- and anti-inflammatory and T helper type 17 cytokines were associated with survival from melioidosis, at enrolment and over time. Persistent inflammation preceded death. These findings support further evaluation of these mediators as prognostic biomarkers and to guide targeted immunotherapeutic development for severe melioidosis

Graham Bickley in uniform

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 ELISA that a hemolysin-coregulated protein (Hcp1) is a promising target for serodiagnosis of melioidosis. In this study, we have 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: (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 for other infections was 91.8%. The results of the Hcp1-ICT demonstrated 92.4% agreement with the Hcp1-ELISA with kappa value of 0.829 and is much improved when compared with the current serological method, 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 resource-limited areas