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Abstract Background Previous studies of T cell activation in dengue infection have focused on restriction of specific T cell receptors (TCRs) and classical MHC molecules. However, bystander T cell activation, which is TCR independent, occurs via cytokines in other viral infections, both in vitro and in vivo, and enables T cells to bypass certain control checkpoints. Moreover, clinical and pathological evidence has pointed to cytokines as the mediators of dengue disease severity. Therefore, we investigated bystander T cell induction by dengue viral antigen. Results Whole blood samples from 55 Thai schoolchildren aged 13-14 years were assayed for in vitro interferon-gamma (IFN-γ) induction in response to inactivated dengue serotype 2 antigen (Den2). The contribution of TCR-dependent and independent pathways was tested by treatment with cyclosporin A (CsA), which inhibits TCR-dependent activation of T cells. ELISA results revealed that approximately 72% of IFN-γ production occurred via the TCR-dependent pathway. The major IFN-γ sources were natural killer (NK) (mean ± SE = 55.2 ± 3.3), CD4+T (24.5 ± 3.3) and CD8+T cells (17.9 ± 1.5), respectively, as demonstrated by four-color flow cytometry. Interestingly, in addition to these cells, we found CsA-resistant IFN-γ producing T cells (CD4+T = 26.9 ± 3.6% and CD8+T = 20.3 ± 2.1%) implying the existence of activated bystander T cells in response to dengue antigen in vitro. These bystander CD4+ and CD8+T cells had similar kinetics to NK cells, appeared after 12 h and were inhibited by anti-IL-12 neutralization indicating cytokine involvement. Conclusions This study described immune cell profiles and highlighted bystander T cell activation in response to dengue viral antigens of healthy people in an endemic area. Further studies on bystander T cell activation in dengue viral infection may reveal the immune mechanisms that protect or enhance pathogenesis of secondary dengue infection.

The incidence of rabies in Thailand reached its peak in 2018 with 18 human deaths. Preexposure prophylaxis (PrEP) vaccination is thus recommended for high-risk populations. WHO has recently recommended that patients who are exposed to a suspected rabid animal and have already been immunized against rabies should receive a 1-site intradermal (ID) injection of 0.1 mL on days 0 and 3 as postexposure prophylaxis (PEP). In Thailand, village health and livestock volunteers tasked with annual dog vaccination typically receive only a single lifetime PrEP dose and subsequent boosters solely upon confirmed animal bites. However, the adequacy of a single PrEP dose for priming and maintaining immunity in this high-risk group has not been evaluated. Therefore, our study was designed to address two key questions: (1) sufficiency of single-dose PrEP-to determine whether a single ID PrEP dose provides adequate long-term immune protection for high-risk individuals exposed to numerous dogs during their vaccination duties. (2) Booster efficacy for immune maturation-to investigate whether one or two additional ID booster doses effectively stimulate a mature and sustained antibody response in this population. The level and persistence of the rabies antibody were determined by comparing the immunogenicity and booster efficacy among the vaccination groups. Our study demonstrated that rabies antibodies persisted for more than 180 days after cost-effective ID PrEP or the 1st or the 2nd single ID booster dose, and adequate antibody levels were detected in more than 95% of participants by CEE-cELISA and 100% by indirect ELISA. Moreover, the avidity maturation of rabies-specific antibodies occurred after the 1st single ID booster dose. This smaller ID booster regimen was sufficient for producing a sufficient immune response and enhancing the maturation of anti-rabies antibodies. This safe and effective PrEP regimen and a single visit involving a one-dose ID booster are recommended, and at least one one-dose ID booster regimen could be equitably implemented in at-risk people in Thailand and other developing countries. However, an adequate antibody level should be monitored before the booster is administered., (© 2024. The Author(s).)

Melioidosis, infection caused by Burkholderia pseudomallei, is characterized by robust innate immune responses. We have previously reported associations of TLR1 single nucleotide missense variant rs76600635 with mortality and of TLR5 nonsense variant rs5744168 with both bacteremia and mortality in single-center studies of patients with melioidosis in northeastern Thailand. The objective of this study was to externally validate the associations of rs76600635 and rs5744168 with bacteremia and mortality in a large multicenter cohort of melioidosis patients. We genotyped rs76600635 and rs5744168 in 1,338 melioidosis patients enrolled in a prospective parent cohort study conducted at nine hospitals in northeastern Thailand. The genotype frequencies of rs76600635 did not differ by bacteremia status (P = 0.27) or 28-day mortality (P = 0.84). The genotype frequencies of rs5744168 did not differ by either bacteremia status (P = 0.46) or 28-day mortality (P = 0.10). Assuming a dominant genetic model, there was no association of the rs76600635 variant with bacteremia (adjusted odds ratio [OR], 0.75; 95% CI, 0.54-1.04, P = 0.08) or 28-day mortality (adjusted OR, 0.96; 95% CI, 0.71-1.28, P = 0.77). There was no association of the rs5744168 variant with bacteremia (adjusted OR, 1.24; 95% CI, 0.76-2.03, P = 0.39) or 28-day mortality (adjusted OR, 1.22; 95% CI, 0.83-1.79, P = 0.21). There was also no association of either variant with 1-year mortality. We conclude that in a large multicenter cohort of patients hospitalized with melioidosis in northeastern Thailand, neither TLR1 missense variant rs76600635 nor TLR5 nonsense variant rs5744168 is associated with bacteremia or mortality.

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.

Diagnosis of non-tuberculous mycobacterial (NTM) infection is difficult due to low sensitivity and time-consuming laboratory tests. Current serological assays fail in tropical countries due to high antibody background. This study aimed to investigate an appropriate method for detecting anti-glycopeptidolipid (GPL)-core antibodies to diagnose NTM infection in Thailand. Heparinized plasma samples were collected from 20 patients with NTM-pulmonary disease (NTM-PD) and 22 patients with disseminated NTM (dNTM) for antibody detection by ELISA. The results were compared with those from patients with tuberculosis, other bacterial pulmonary infections and healthy controls. Among the different antibody isotypes, anti-GPL-core IgA exhibited the highest suitability. Therefore, anti-GPL-core IgA and its subclass IgA2 were further investigated. A significant increase in antibody levels was observed during the active infection stage, whereas NTM-PD with culture conversion at the 6-month follow-up showed reduced IgA levels. The diagnostic cut-off for IgA and IgA2 was newly defined as 1.4 and 1.0 U/ml, respectively. Using our IgA cut-off, the sensitivity and specificity for diagnosing NTM-PD were 77.3% and 81.4%, respectively. The new IgA cut-off demonstrated significantly improved specificity compared to the manufacturer's cut-off. Thus, serological detection of anti-GPL-core IgA, with a cut-off of 1.4 U/ml, can be a valuable tool for supporting NTM diagnosis in Thailand., (© 2023. The Author(s).)

Recent evidence has demonstrated that both acute and chronic exposure to particulate air pollution are risk factors for respiratory tract infections and increased mortality from sepsis. There is therefore an urgent need to establish the impact of ambient particulate matter (PM) on innate immune cells and to establish potential strategies to mitigate against adverse effects. PM has previously been reported to have potential adverse effects on neutrophil function. In the present study, we investigated the impact of standard urban PM (SRM1648a, NIST) and PM 2.5 collected from Chiang Mai, Thailand, on human peripheral blood neutrophil functions, including LPS-induced migration, IL-8 production, and bacterial killing. Both NIST and the PM 2.5 , being collected in Chiang Mai, Thailand, increased IL-8 production, but reduced CXCR2 expression and migration of human primary neutrophils stimulated with Escherichia coli LPS. Moreover, PM-pretreated neutrophils from vitamin D-insufficient participants showed reduced E. coli-killing activity. Furthermore, in vitro vitamin D3 supplementation attenuated IL-8 production and improved bacterial killing by cells from vitamin D-insufficient participants. Our findings suggest that provision of vitamin D to individuals with insufficiency may attenuate adverse acute neutrophilic responses to ambient PM., (© 2023. Springer Nature Limited.)

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., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Kaewarpai, Wright, Yimthin, Phunpang, Dulsuk, Lovelace-Macon, Rerolle, Dow, Hantrakun, Day, Lertmemongkolchai, Limmathurotsakul, West and Chantratita.)

Melioidosis is an often fatal infection in tropical regions caused by an environmental bacterium, Burkholderia pseudomallei Current recommended melioidosis treatment requires intravenous β-lactam antibiotics such as ceftazidime (CAZ), meropenem (MEM) or amoxicillin-clavulanic acid (AMC) and oral trimethoprim-sulfamethoxazole. Emerging antibiotic resistance could lead to therapy failure and high mortality. We performed a prospective multicentre study in northeast Thailand during 2015-2018 to evaluate antibiotic susceptibility and characterize β-lactam resistance in clinical B. pseudomallei isolates. Collection of 1,317 B. pseudomallei isolates from patients with primary and relapse infections were evaluated for susceptibility to CAZ, imipenem (IPM), MEM and AMC. β-lactam resistant isolates were confirmed by broth microdilution method and characterized by whole genome sequence analysis, penA expression and β-lactamase activity. The resistant phenotype was verified via penA mutagenesis. All primary isolates were IPM-susceptible but we observed two CAZ-resistant and one CAZ-intermediate resistant isolates, two MEM-less susceptible isolates, one AMC-resistant and two AMC-intermediate resistant isolates. One of 13 relapse isolates was resistant to both CAZ and AMC. Two isolates were MEM-less susceptible. Strains DR10212A (primary) and DR50054E (relapse) were multi-drug resistant. Genomic and mutagenesis analyses supplemented with gene expression and β-lactamase analyses demonstrated that CAZ-resistant phenotype was caused by PenA variants: P167S (N=2) and penA amplification (N=1). Despite the high mortality rate in melioidosis, our study revealed that B. pseudomallei isolates had a low frequency of β-lactam resistance caused by penA alterations. Clinical data suggest that resistant variants may emerge in patients during antibiotic therapy and be associated with poor response to treatment., (Copyright © 2021 Fen et al.)

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., Competing Interests: Conflicts of interests We declare that we have no competing interests.

Infections by Acinetobacter species are recognized as a serious global threat due to causing severe disease and their high levels of antibiotic resistance. Acinetobacter baumannii is the most prevalent pathogen in the genus, but infection by Acinetobacter nosocomialis has been reported widely. Diagnosis of patients with A. baumannii infection is often misdiagnosed with other Acinetobacter species, especially A. nosocomialis. This study investigated whether there were significant differences in clinical outcomes between patients infected with A. baumannii versus A. nosocomialis in Northeast Thailand, and to characterize serological responses to infection with these pathogens. The results show that A. baumannii had higher levels of multidrug resistance. Despite this, clinical outcomes for infection with A. baumannii or A. nosocomialis were similar with mortalities of 33% and 36%, respectively. Both pathogens caused community-acquired infections (A. baumannii 35% and A. nosocomialis 29% of cases). Plasma from uninfected healthy controls contained IgG antibody that recognized both organisms, and infected patients did not show a significantly enhanced antibody response from the first week versus 2 weeks later. Finally, the patterns of antigen recognition for plasma IgG were similar for patients infected with A. baumannii or A. nosocomialis infection, and distinct to the pattern for patients infected with non-Acinetobacter. In conclusion, our data revealed that infection with A. nosocomialis was associated with a similarly high level of mortality as infection with A. baumannii, the high rate of community-acquired infection and antibodies in uninfected individuals suggesting that there is significant community exposure to both pathogens. IMPORTANCE Bacterial infections by Acinetobacter species are global threats due to their severity and high levels of antibiotic resistance. A. baumannii is the most common pathogen in the genus; however, infection by A. nosocomialis has also been widely reported but is thought to be less severe. In this study, we have prospectively investigated 48 reported cases of A. baumannii infection in Northeast Thailand, and characterized the serological responses to infection. We found that 14 (29%) of these infections were actually caused by A. nosocomialis. Furthermore, the incidence of antibiotic resistance among A. nosocomialis strains, APACHE II scores, and mortality for patients infected with A. nosocomialis were much higher than published data. Both A. baumannii and A. nosocomialis had unexpectedly mortality rates of over 30%, and both pathogens caused a high rate of community-acquired infections. Importantly, background antibodies in uninfected individuals suggest significant community exposure to both pathogens in the environment.

Zinc deficiency impairs the antibody-mediated immune response and is common in children from lower-income countries. This study aimed to investigate the impact of different zinc supplementation regimens (7, 10 or 20 mg/day elemental zinc)-therapeutic dispersible zinc tablets (TZ), daily multiple micronutrient powder (MNP), daily preventive zinc tablets (PZ) and placebo powder (control)-and compare between baseline and endline antibody production against pathogenic Escherichia coli in Laotian children (aged 6-23 months). Fifty representative plasma samples of each treatment group were randomly selected from 512 children to determine anti-E. coli IgG antibody levels and avidity. Of the 200 children, 78.5% had zinc deficiency (plasma zinc concentration < 65 µg/dL) and 40% had anaemia before receiving zinc supplementation. aAfter receiving the TZ, MNP or PZ regimen, the plasma anti-E. coli IgG levels were significantly increased compared with baseline; the effect on the antibody level was more pronounced in children with zinc deficiency. Interestingly, there was increased anti-E. coli IgG avidity in the control and PZ groups. This study suggests that PZ might be the optimal zinc supplementation regimen to increase both the quantity and quality of antibody responses in children with zinc deficiency. Clinical trial registration: https://clinicaltrials.gov/ct2/show/NCT02428647 (NCT02428647, 29/04/2015)., (© 2022. The Author(s).)

The complement system is required for innate immunity against Acinetobacter baumannii , an important cause of antibiotic resistant systemic infections. A. baumannii strains differ in their susceptibility to the membrane attack complex (MAC) formed from terminal complement pathway proteins, but the reasons for this variation remain poorly understood. We have characterized in detail the complement sensitivity phenotypes of nine A. baumannii clinical strains and some of the factors that might influence differences between strains. Using A. baumannii laboratory strains and flow cytometry assays, we first reconfirmed that both opsonization with the complement proteins C3b/iC3b and MAC formation were inhibited by the capsule. There were marked differences in C3b/iC3b and MAC binding between the nine clinical A. baumannii strains, but this variation was partially independent of capsule composition or size. Opsonization with C3b/iC3b improved neutrophil phagocytosis of most strains. Importantly, although C3b/iC3b binding and MAC formation on the bacterial surface correlated closely, MAC formation did not correlate with variations between A. baumannii strains in their levels of serum resistance. Genomic analysis identified only limited differences between strains in the distribution of genes required for serum resistance, but RNAseq data identified three complement-resistance genes that were differentially regulated between a MAC resistant and two MAC intermediate resistant strains when cultured in serum. These data demonstrate that clinical A. baumannii strains vary in their sensitivity to different aspects of the complement system, and that the serum resistance phenotype was influenced by factors in addition to the amount of MAC forming on the bacterial surface., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Kamuyu, Ercoli, Ramos-Sevillano, Willcocks, Kewcharoenwong, Kiratisin, Taylor, Wren, Lertmemongkolchai, Stabler and Brown.)

Melioidosis is an often lethal tropical disease caused by the Gram-negative bacillus, Burkholderia pseudomallei . The study objective was to characterize transcriptomes in melioidosis patients and identify genes associated with outcome. Whole blood RNA-seq was performed in a discovery set of 29 melioidosis patients and 3 healthy controls. Transcriptomic profiles of patients who did not survive to 28 days were compared with patients who survived and healthy controls, showing 65 genes were significantly up-regulated and 218 were down-regulated in non-survivors compared to survivors. Up-regulated genes were involved in myeloid leukocyte activation, Toll-like receptor cascades and reactive oxygen species metabolic processes. Down-regulated genes were hematopoietic cell lineage, adaptive immune system and lymphocyte activation pathways. RT-qPCR was performed for 28 genes in a validation set of 60 melioidosis patients and 20 healthy controls, confirming differential expression. IL1R2, GAS7, S100A9, IRAK3, and NFKBIA were significantly higher in non-survivors compared with survivors ( P < 0.005) and healthy controls ( P < 0.0001). The AUROCC of these genes for mortality discrimination ranged from 0.80-0.88. In survivors, expression of IL1R2 , S100A9 and IRAK3 genes decreased significantly over 28 days ( P < 0.05). These findings augment our understanding of this severe infection, showing expression levels of specific genes are potential biomarkers to predict melioidosis outcomes.

Antibody therapy may be an alternative treatment option for infections caused by the multi-drug resistant (MDR) bacterium Acinetobacter baumannii. As A. baumannii has multiple capsular serotypes, a universal antibody therapy would need to target conserved protein antigens rather than the capsular polysaccharides. We have immunized mice with single or multiple A. baumannii strains to induce antibody responses to protein antigens, and then assessed whether these responses provide cross-protection against a collection of genetically diverse clinical A. baumannii isolates. Immunized mice developed antibody responses to multiple protein antigens. Flow cytometry IgG binding assays and immunoblots demonstrated improved recognition of both homologous and heterologous clinical strains in sera from mice immunized with multiple strains compared to a single strain. The capsule partially inhibited bacterial recognition by IgG and the promotion of phagocytosis by human neutrophils. However, after immunization with multiple strains, serum antibodies to protein antigens promoted neutrophil phagocytosis of heterologous A. baumannii strains. In an infection model, mice immunized with multiple strains had lower bacterial counts in the spleen and liver following challenge with a heterologous strain. These data demonstrate that antibodies targeting protein antigens can improve immune recognition and protection against diverse A. baumannii strains, providing support for their use as an antibody therapy., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Kamuyu, Suen Cheng, Willcocks, Kewcharoenwong, Kiratisin, Taylor, Wren, Lertmemongkolchai, Stabler and Brown.)

As the capacity for generating large-scale molecular profiling data continues to grow, the ability to extract meaningful biological knowledge from it remains a limitation. Here, we describe the development of a new fixed repertoire of transcriptional modules, BloodGen3, that is designed to serve as a stable reusable framework for the analysis and interpretation of blood transcriptome data. The construction of this repertoire is based on co-clustering patterns observed across sixteen immunological and physiological states encompassing 985 blood transcriptome profiles. Interpretation is supported by customized resources, including module-level analysis workflows, fingerprint grid plot visualizations, interactive web applications and an extensive annotation framework comprising functional profiling reports and reference transcriptional profiles. Taken together, this well-characterized and well-supported transcriptional module repertoire can be employed for the interpretation and benchmarking of blood transcriptome profiles within and across patient cohorts. Blood transcriptome fingerprints for the 16 reference cohorts can be accessed interactively via:  https://drinchai.shinyapps.io/BloodGen3Module/ ., (© 2021. The Author(s).)

Detection of IgA antibody against Mycobacterium avium complex (MAC) glycopeptidolipid (GPL) has recently been shown to improve the diagnosis of MAC pulmonary disease but has yet to be tested in disseminated Non-tuberculous mycobacteria (NTM) infection. In this study, we address the diagnostic efficacies of an anti-GPL-core ELISA kit in disseminated lymphadenopathy patients positive for NTM culture and anti-IFN-γ autoantibodies. The study was conducted in a tertiary referral center in northeastern Thailand and patients with NTM, tuberculosis, melioidosis, and control subjects were enrolled. Plasma immunoglobulin A (IgA) and G (IgG) antibodies against GPL-core were detected in the subjects and the specificity and sensitivity of the assay was assessed. Anti-GPL-core IgA and IgG levels were significantly higher in NTM patients than other groups (p < 0.0001). Diagnostic efficacy for NTM patients using anti-GPL-core IgA cut-off value of 0.352 U/ml showed good sensitivity (91.18%) and intermediate specificity (70.15%). Using a cut-off value of 4.140 AU/ml for anti-GPL-core IgG showed the same sensitivity (91.18%) with increased specificity (89.55%) and an 81.58% positive predictive value. Most patients with moderate levels (4.140-7.955 AU/ml) of anti-GPL-core IgG had rapidly growing mycobacteria (RGM) infection. Taken together, the detection of anti-GPL-core antibodies could provide a novel option for the diagnosis and management of disseminated NTM infected patients., Competing Interests: The authors have declared that no competing interests exist.

Background: Burkholderia pseudomallei is an environmental bacterium that causes melioidosis. A facultative intracellular pathogen, B. pseudomallei can induce multinucleated giant cells (MNGCs) leading to plaque formation in vitro. B. pseudomallei can switch colony morphotypes under stress conditions. In addition, different isolates have been reported to have varying virulence in vivo, but genomic evolution and the relationship with plaque formation is poorly understood., Methodology/principle Findings: To gain insights into genetic underpinnings of virulence of B. pseudomallei, we screened plaque formation of 52 clinical isolates and 11 environmental isolates as well as 4 isogenic morphotype isolates of B. pseudomallei strains K96243 (types II and III) and 153 (types II and III) from Thailand in A549 and HeLa cells. All isolates except one environmental strain (A4) and K96243 morphotype II were able to induce plaque formation in both cell lines. Intracellular growth assay and confocal microscopy analyses demonstrated that the two plaque-forming-defective isolates were also impaired in intracellular replication, actin polymerization and MNGC formation in infected cells. Whole genome sequencing analysis and PCR revealed that both isolates had a large genomic loss on the same region in chromosome 2, which included Bim cluster, T3SS-3 and T6SS-5 genes., Conclusions/significance: Our plaque screening and genomic studies revealed evidence of impairment in plaque formation in environmental isolates of B. pseudomallei that is associated with large genomic loss of genes important for intracellular multiplication and MNGC formation. These findings suggest that the genomic and phenotypic differences of environmental isolates may be associated with clinical infection., Competing Interests: The authors have declared that no competing interests exist.

Background: Zinc deficiency impairs immune function and is common among children in South-East Asia., Objectives: The effect of zinc supplementation on immune function in young Laotian children was investigated., Methods: Children (n = 512) aged 6-23 mo received daily preventive zinc tablets (PZ; 7 mg Zn/d), daily multiple micronutrient powder (MNP; 10 mg Zn/d, 6 mg Fe/d, plus 13 other micronutrients), therapeutic dispersible zinc tablets only in association with diarrhea episodes (TZ; 20 mg Zn/d for 10 d after an episode), or daily placebo powder (control). These interventions continued for 9 mo. Cytokine production from whole blood cultures, the concentrations of T-cell populations, and a complete blood count with differential leukocyte count were measured at baseline and endline. Endline means were compared via ANCOVA, controlling for the baseline value of the outcome, child age and sex, district, month of enrollment, and baseline zinc status (below, or above or equal to, the median plasma zinc concentration)., Results: T-cell cytokines (IL-2, IFN-γ, IL-13, IL-17), LPS-stimulated cytokines (IL-1β, IL-6, TNF-α, and IL-10), and T-cell concentrations at endline did not differ between intervention groups, nor was there an interaction with baseline zinc status. However, mean ± SE endline lymphocyte concentrations were significantly lower in the PZ than in the control group (5018 ± 158 compared with 5640 ± 160 cells/μL, P = 0.032). Interactions with baseline zinc status were seen for eosinophils (Pixn = 0.0036), basophils (Pixn = 0.023), and monocytes (P = 0.086) but a significant subgroup difference was seen only for eosinophils, where concentrations were significantly lower in the PZ than in the control group among children with baseline plasma zinc concentrations below the overall median (524 ± 44 compared with 600 ± 41 cells/μL, P = 0.012)., Conclusions: Zinc supplementation of rural Laotian children had no effect on cytokines or T-cell concentrations, although zinc supplementation affected lymphocyte and eosinophil concentrations. These cell subsets may be useful as indicators of response to zinc supplementation.This trial was registered at clinicaltrials.gov as NCT02428647., (Published by Oxford University Press on behalf of the American Society for Nutrition 2020.)

Type 2 diabetes mellitus (T2DM) is an important risk factor for development of tuberculosis (TB). Our previous study showed glibenclamide, an anti-diabetic drug used to control blood glucose concentration, reduced interleukin (IL)-8 secretion from primary human monocytes challenged with M. tuberculosis (Mtb). In mice infected with Mtb, IL-1β is essential for host resistance through the enhancement of cyclooxygenase that limits excessive Type I interferon (IFN) production and fosters Mtb containment. We hypothesize that glibenclamide may also interfere with monocyte mediated immune responses against Mtb and alter the balance between IL-1β and IFNα-mediated immunity. Purified monocytes from non-diabetic and diabetic individuals were infected with Mtb or M. bovis BCG. We demonstrate that monocytes from diabetes patients who were being treated with glibenclamide showed reduced IL-1β and IL-8 secretion when exposed to Mtb. Additionally, these responses also occurred when monocytes from non-diabetic individuals were pre-treated with glibenclamide in vitro. Moreover, this pre-treatment enhanced IFNa1 expression but was not involved with prostaglandin E2 (PGE2) expression in response to Mtb infection. Taken together, our data show that glibenclamide might exacerbate susceptibility of diabetes patients to Mtb infection by reducing IL-1β and IL-8 production by monocytes., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Melioidosis is caused by Burkholderia pseudomallei and is predominantly seen in tropical regions. The clinical signs and symptoms of the disease are nonspecific and often result in misdiagnosis, failure of treatment, and poor clinical outcome. Septicemia with septic shock is the most common cause of death, with mortality rates above 40%. Bacterial culture is the gold standard for diagnosis, but it has low sensitivity and takes days to produce definitive results. Early laboratory diagnosis can help guide physicians to provide treatment specific to B. pseudomallei In our study, we adapted host gene expression signatures obtained from microarray data of B. pseudomallei -infected cases to develop a real-time PCR diagnostic test using two differentially expressed genes, AIM2 ( a bsent i n m elanoma 2) and FAM26F ( fam ily with sequence similarity 26 , member F ). We tested blood from 33 patients with B. pseudomallei infections and 29 patients with other bacterial infections to validate the test and determine cutoff values for use in a cascading diagnostic algorithm. Differentiation of septicemic melioidosis from other sepsis cases had a sensitivity of 82%, specificity of 93%, and negative and positive predictive values (NPV and PPV) of 82% and 93%, respectively. Separation of cases likely to be melioidosis from those unlikely to be melioidosis in nonbacteremic situations showed a sensitivity of 40%, specificity of 54%, and NPV and PPV of 44% and 50%, respectively. We suggest that our AIM2 and FAM26F expression combination algorithm could be beneficial for early melioidosis diagnosis, offering a result within 24 h of admission., (Copyright © 2020 American Society for Microbiology.)

Patients with beta-thalassaemia increase the risk of bacterial infections, particularly Burkholderia pseudomallei (Bp), the causative agent of melioidosis in Thailand. Impaired immune cell functions may be the cause of this susceptibility, but detailed mechanisms have not been defined. In this study, we observed impaired production of IFN-gamma and IL-10 by whole blood from beta-thalassaemia patients upon stimulation with a range of bacteria-derived stimuli. In contrast, IFN-gamma response via TCR and plasma IgG specific for Bp were still intact. Importantly, mRNA expression of heme oxygenase 1 (HO-1), a potential modulator of immune function, was increased in whole blood from beta-thalassaemia patients, either with or without stimulation with Bp in vitro. Induction of HO-1 by hemin or CoPP in vitro reduced production of IFN-gamma and IL-10 from healthy human PBMCs and decreased bacterial clearance activity of whole blood from healthy controls and beta-thalassaemia, while inhibition of HO-1 by SnPP enhanced both functions in healthy controls. These results were confirmed to some extent in purified human monocytes of healthy controls. Our results suggest a mechanism that excess hemin of beta-thalassaemia patients is a significant cause of immune suppression via HO-1 induction and may underlie the susceptibility of these individuals to severe bacterial infection.

The anti-interferon-gamma (IFN-gamma) autoantibody is a known cause of opportunistic non-tuberculous mycobacterial (NTM) infection in adults. Diagnosis of those patients is difficult due to the low sensitivity of bacterial culture, and because detection of the neutralizing autoantibody needs special laboratory devices. We conducted a retrospective review of indirect and inhibitory ELISA, both used for detection of anti-IFN-gamma auto-antibody in 102 patients with lymphadenopathies. We assessed hospital records of NTM isolation and/or diagnosis of NTM infection. The review revealed the compatible sensitivity and superior specificity and predictive values for inhibitory ELISA over against indirect ELISA-the latter achieving 100% specificity and positive predictive value for diagnosis of NTM infection in patients with lymphadenopathies. The results confirm functional assays that show plasma samples from NTM-infected patients with positive results by either indirect and/or inhibitory ELISA are IFN-gamma neutralizing autoantibodies. The inhibitory titer of anti-IFN-gamma auto-antibody can be used to distinguish patients with active from inactive NTM infection. Inhibitory ELISA is thus a practical, rapid, high performance tool for routine detection of anti-IFN-gamma autoantibody and NTM infection diagnosis before confirmation, enabling a timely therapeutic strategy for active infection treatment.

Diabetes mellitus (DM) patients are at an increased risk of complications following influenza-virus infection, seasonal vaccination (SV) is recommended. However, SV with trivalent influenza vaccine (TIV) can induce antibody and type-I interferon (IFN) responses, and the effect of anti-DM treatment on these responses is incompletely understood. We evaluated the antibody response and IFN-α expression in individuals with and without type 2 DM (T2DM) following SV, and examined the effects on anti-DM treatment. TIV elicited sero-protection in all groups, but antibody persistency was <8 months, except for the antibody response to B-antigens in non-DM. T2DM impaired the IgG avidity index, and T2DM showed a significantly decreased response against H1N1 and H3N2, in addition to delaying and reducing haemagglutination-inhibition persistency against influenza B-antigens in DM groups treated with metformin (Met-DM) or glibenclamide (GB-DM). Following TIV, the Met-DM and GB-DM groups exhibited reduced IFN-α expression upon stimulation with whole- and split-virion influenza vaccines. Suppression of IFN-α expression in the Met-DM group was associated with a reduction in the mechanistic target of rapamycin complex-1 pathway and impaired IgG avidity index. Thus, single-dose TIV each year might not be suitable for T2DM. Our data could aid the development of an efficacious influenza vaccine for T2DM.

Environmental enteric dysfunction (EED) may be ameliorated by zinc supplementation. The objective of this study was to investigate the impact of different forms of zinc supplementation on biomarkers of EED (i.e., plasma citrulline, kynurenine, and tryptophan concentrations and the kynurenine:tryptophan [KT] ratio) among young Laotian children. In a double-blind randomized controlled trial, 3,407 children aged 6-23 months were randomized into one of four groups: daily preventive zinc dispersible tablets (PZ; 7 mg zinc), daily multiple micronutrient powders (MNP; 10 mg zinc, 6 mg iron, and 13 other micronutrients), therapeutic zinc supplements for diarrhea treatment (TZ; 20 mg/day for 10 days), or daily placebo powder, and followed up for ∼36 weeks. Plasma samples at baseline and endline for 359 children were analyzed for citrulline, kynurenine, and tryptophan concentrations. At baseline, the prevalence of stunting and zinc deficiency was 37% and 76.5%, respectively. The mean plasma citrulline, kynurenine, and tryptophan concentrations were 24.6 ± 5.4 µmol/L, 3.27 ± 0.83 µmol/L, and 72.3 ± 12.9 µmol/L, respectively; the mean KT ratio (×1,000) was 45.9 ± 12.0. At endline, neither plasma citrulline, kynurenine, or tryptophan concentrations, nor the KT ratio differed among intervention groups ( P > 0.05). In this population, PZ, MNP, and TZ had no overall effect on plasma concentrations of citrulline, kynurenine, and tryptophan, or the KT ratio. The need remains to better understand the etiology of EED, and the development of biomarkers to diagnose EED and evaluate the impact of interventions.

The Burkholderia genus encompasses many Gram-negative bacteria living in the rhizosphere. Some Burkholderia species can cause life-threatening human infections, highlighting the need for clinical interventions targeting specific lipopolysaccharide proteins. Burkholderia cenocepacia O -linked protein glycosylation has been reported, but the chemical structure of the O -glycan and the machinery required for its biosynthesis are unknown and could reveal potential therapeutic targets. Here, using bioinformatics approaches, gene-knockout mutants, purified recombinant proteins, LC-MS-based analyses of O -glycans, and NMR-based structural analyses, we identified a B. cenocepacia O -glycosylation ( ogc ) gene cluster necessary for synthesis, assembly, and membrane translocation of a lipid-linked O -glycan, as well as its structure, which consists of a β-Gal-(1,3)-α-GalNAc-(1,3)-β-GalNAc trisaccharide. We demonstrate that the ogc cluster is conserved in the Burkholderia genus, and we confirm the production of glycoproteins with similar glycans in the Burkholderia species: B. thailandensis , B. gladioli , and B. pseudomallei Furthermore, we show that absence of protein O- glycosylation severely affects bacterial fitness and accelerates bacterial clearance in a Galleria mellonella larva infection model. Finally, our experiments revealed that patients infected with B. cenocepacia , Burkholderia multivorans , B. pseudomallei , or Burkholderia mallei develop O- glycan-specific antibodies. Together, these results highlight the importance of general protein O- glycosylation in the biology of the Burkholderia genus and its potential as a target for inhibition or immunotherapy approaches to control Burkholderia infections.

Germinal center (GC) B cells at viral replication sites acquire specificity to poorly immunogenic but conserved influenza hemagglutinin (HA) epitopes. Here, high-throughput epitope mapping of local GC B cells is used to identify conserved HA epitope selecting cross-reactive antibodies that mediate heterosubtypic protection. A distinct feature of this epitope is an occlusion in the naive trimeric HA structure that is exposed in the post-fusion HA structure to occur under low pH conditions during viral replication. Importantly, systemic immunization by the post-fusion HA antigen results in GC B cells targeting the occluded epitope, and induces a class of protective antibodies that have cross-group specificity and afford protection independent of virus neutralization activity. Furthermore, this class of broadly protective antibodies develops at late time points and persists. Our results identify a class of cross-protective antibodies that are selected at the viral replication site, and provide insights into vaccine strategies using the occluded epitope.

Introduction: Anti-interferon-gamma (IFN- γ ) autoantibodies are increasingly recognized as a cause of adult-onset immunodeficiency (AOID) worldwide. These patients are susceptible to various intracellular pathogens especially nontuberculous mycobacteria. Most of the patients have a refractory clinical course. Herein, we report the use of immunotherapy with pulse intravenous cyclophosphamide (IVCY) in patients who had progressive, refractory Mycobacterium abscessus infection., Method: We included patients, seen at Srinagarind Hospital, Thailand, infected with M. abscessus , who had received ≥3 courses of parenteral antibiotics within the last 12 months and who received pulse IVCY with a tapering dose of prednisolone., Results: There were 8 AOID patients who met the criteria and received pulse IVCY between January 2011 and December 2015. One patient was lost to follow-up after 5 courses of IVCY: he had died at home 3 months later. Five patients had favorable outcomes: 2 were able to discontinue NTM therapy, and 3 had stable disease and were on NTM treatment without hospitalization for parenteral antibiotics. Two patients relapsed and needed hospitalization. The IFN- γ Ab titers among the 7 patients were significantly decreased during treatment, and the median initial antibody titer started at 200,000 and then decreased to 5,000 after 2 years of treatment ( P < 0.0001). The antibody titer reduction among responsive vs. nonresponsive patient was significantly different after 6 months of treatment: the median antibody titer was 5,000 and 100,000, respectively ( P = 0.0467)., Conclusion: IVCY therapy might be an alternative treatment for AOID patients infected with M. abscessus and refractory to antimycobacterial therapy.

Background: Zinc is an essential nutrient that is required for children's normal growth and resistance to infections, including diarrhea and pneumonia, two major causes of child mortality. Daily or weekly preventive zinc supplementation has been shown to improve growth and reduce the risk of infection, while therapeutic zinc supplementation for 10-14 days is recommended for the treatment of diarrhea. The overall objective of the present study is to compare several regimens for delivering zinc to young children, both for the prevention of zinc deficiency and the treatment of diarrhea., Methods: The present study is a community-based, randomized controlled trial in the Lao People's Democratic Republic (PDR). Three thousand, four hundred children 6-23 months of age will be randomized to one of four intervention groups (daily preventive zinc dispersible tablet, daily preventive multiple micronutrient powder, therapeutic zinc dispersible tablet for diarrhea, or placebo control); interventions will be delivered for 9 months and outcomes measured at pre-determined intervals. Primary outcomes include physical growth (length and weight), diarrhea incidence, hemoglobin and micronutrient status, and innate and adaptive immune function. Secondary outcomes include mid-upper-arm circumference, neuro-behavioral development, hair cortisol concentrations, markers of intestinal inflammation and parasite burden. Incidence of adverse events and the modifying effects of inherited hemoglobin disorders and iron status on the response to the intervention will also be examined. We will estimate unadjusted effects and effects adjusted for selected baseline covariates using ANCOVA., Discussion: Many countries are now rolling out large-scale programs to include therapeutic zinc supplementation in the treatment of childhood diarrhea, but few have established programs demonstrated to be effective in the prevention of zinc deficiency. This study will address how best to deliver supplemental zinc to prevent zinc deficiency and reduce the severity of diarrhea-related health complications., Trial Registration: Trial registration identifier (NCT02428647) ; Date of registration: April 29, 2015., Competing Interests: Competing interestsKBH is an employee of Nutrition International, formerly the Micronutrient Initiative and GAR was an employee of the Mathile Institute for the Advancement of Human Nutrition at the time of the initial proposal writing. KHB works for the Bill & Melinda Gates Foundation, which provided part of the financial support. The spouse of SYH works for the Bill & Melinda Gates Foundation. KRW, SK, MAB, GMH, SS, KR, CBS, CPL, CDA, GL, SF and RMB declare that they have no competing interests., (© The Author(s). 2018.)

Tuberculosis (TB) is a global public health problem, which is caused by Mycobacterium tuberculosis (Mtb). Type 2 diabetes mellitus (T2DM) is one of the leading predisposing factors for development of TB after HIV/AIDS. Glibenclamide is a widely used anti-diabetic drug in low and middle-income countries where the incidence of TB is very high. In a human macrophage cell line, glibenclamide, a K + ATP-channel blocker, promoted alternative activation of macrophages by enhancing expression of the M2 marker CD206 during M2 polarization. M2 macrophages are considered poorly microbicidal and associated with TB susceptibility. Here, we investigated the effect of glibenclamide on M1 and M2 phenotypes of primary human monocytes and further determined whether specific drug treatment for T2DM individuals influences the antibacterial function of monocytes in response to mycobacterial infection. We found that glibenclamide significantly reduced M1 (HLA-DR + and CD86 + ) surface markers and TNF-α production on primary human monocytes against mycobacterial infection. In contrast, M2 (CD163 + and CD206 + ) surface markers and IL-10 production were enhanced by pretreatment with glibenclamide. Additionally, reduction of bactericidal activity also occurred when primary human monocytes from T2DM individuals who were being treated with glibenclamide were infected with Mtb in vitro , consistent with the cytokine responses. We conclude that glibenclamide reduces M1 and promotes M2 polarization leading to impaired bactericidal ability of primary human monocytes of T2DM individuals in response to Mtb and may lead to increased susceptibility of T2DM individuals to TB and other bacterial infectious diseases.

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., (Copyright © 2018 Phokrai et al.)