Engagement of a community advisory group to shape and build up participation in TB research.

It is essential that communities at risk from TB are involved in TB research. Community advisory groups (CAGs) are one mechanism for involving communities in research and creating platforms for discussions between researchers and community members. We organised a CAG meeting with community members and people with lived experience in Ho Chi Minh City, Vietnam, to explore the community's knowledge about TB and their perspectives on different diagnostic tests in Vietnam, a low-middle-income country with a high TB burden. Researchers shared basic information and addressed questions about TB. CAG members commented on preference of TB screening tests, and suggested that chest X-rays and blood tests were more acceptable than sputum tests because of the difficulty in sputum expectoration. In addition, clinical studies that required fewer visits to the hospitals would be preferred, even if this meant a greater reliance on blood sampling.

Il est essentiel que les communautés exposées au risque de TB soient impliquées dans la recherche sur la TB. Les groupes consultatifs communautaires (CAG, pour l'anglais « community advisory groups ¼) constituent un mécanisme permettant d'impliquer les communautés dans la recherche et de créer des plateformes de discussion entre les chercheurs et les membres de la communauté. Nous avons organisé une réunion du CAG avec des membres de la communauté et des personnes ayant une expérience vécue à Ho Chi Minh Ville, au Viêt Nam, afin d'explorer les connaissances de la communauté sur la TB et leurs perspectives sur les différents tests de diagnostic au Viêt Nam, un pays à revenu faible et moyen où la charge de la TB est élevée. Les chercheurs ont partagé des informations de base et répondu à des questions sur la TB. Les membres du CAG ont fait part de leur préférence pour les tests de dépistage de la TB et ont suggéré que les radiographies pulmonaires et les analyses de sang étaient plus acceptables que les tests d'expectoration en raison de la difficulté d'expectoration des crachats. En outre, les études cliniques qui nécessitent moins de visites dans les hôpitaux seraient préférées, même si cela implique une plus grande dépendance à l'égard des prélèvements sanguins.

Value of lipocalin 2 as a potential biomarker for bacterial meningitis.

OBJECTIVES: Central nervous system (CNS) infections are common causes of morbidity and mortality worldwide. We aimed to discover protein biomarkers that could rapidly and accurately identify the likely cause of the infections, essential for clinical management and improving outcome. METHODS: We applied liquid chromatography tandem mass spectrometry on 45 cerebrospinal fluid (CSF) samples from a cohort of adults with and without CNS infections to discover potential diagnostic biomarkers. We then validated the diagnostic performance of a selected biomarker candidate in an independent cohort of 364 consecutively treated adults with CNS infections admitted to a referral hospital in Vietnam. RESULTS: In the discovery cohort, we identified lipocalin 2 (LCN2) as a potential biomarker of bacterial meningitis (BM) other than tuberculous meningitis. The analysis of the validation cohort showed that LCN2 could discriminate BM from other CNS infections (including tuberculous meningitis, cryptococcal meningitis and virus/antibody-mediated encephalitis), with sensitivity of 0.88 (95% confident interval (CI), 0.77-0.94), specificity of 0.91 (95% CI, 0.88-0.94) and diagnostic odds ratio of 73.8 (95% CI, 31.8-171.4). LCN2 outperformed other CSF markers (leukocytes, glucose, protein and lactate) commonly used in routine care worldwide. The combination of LCN2, CSF leukocytes, glucose, protein and lactate resulted in the highest diagnostic performance for BM (area under the receiver operating characteristics curve, 0.96; 95% CI, 0.93-0.99). Data are available via ProteomeXchange with identifier PXD020510. CONCLUSIONS: LCN2 is a sensitive and specific biomarker for discriminating BM from a broad spectrum of other CNS infections. A prospective study is needed to assess the diagnostic utility of LCN2 in the diagnosis and management of CNS infections.

A polymorphism in human MR1 is associated with mRNA expression and susceptibility to tuberculosis.

The MR1 antigen-presenting system is conserved among mammals and enables T cells to recognize small molecules produced by bacterial pathogens, including Mycobacterium tuberculosis (M.tb). However, it is not known whether MR1-mediated antigen presentation is important for protective immunity against mycobacterial disease. We hypothesized that genetic control of MR1 expression correlates with clinical outcomes of tuberculosis infection. We performed an MR1 candidate gene association study and identified an intronic single-nucleotide polymorphism (rs1052632) that was significantly associated with susceptibility to tuberculosis in a discovery and validation cohort of Vietnamese adults with tuberculosis. Stratification by site of disease revealed that rs1052632 genotype GG was strongly associated with the development of meningeal tuberculosis (odds ratio=2.99; 95% confidence interval (CI) 1.64-5.43; P=0.00006). Among patients with meningeal disease, absence of the G allele was associated with an increased risk of death (hazard ratio=3.86; 95% CI 1.49-9.98; P=0.005). Variant annotation tools using public databases indicate that rs1052632 is strongly associated with MR1 gene expression in lymphoblastoid cells (P=0.004) and is located within a transcriptional enhancer in epithelial keratinocytes. These data support a role for MR1 in the pathogenesis of human tuberculosis by revealing that rs1052632 is associated with MR1 gene expression and susceptibility to tuberculosis in Vietnam.

MARCO variants are associated with phagocytosis, pulmonary tuberculosis susceptibility and Beijing lineage.

Macrophage receptor with collagenous structure (MARCO) has an important role in the phagocytosis of Mycobacterium tuberculosis (M. tuberculosis). We hypothesized that MARCO polymorphisms are associated with phagocytosis, tuberculosis (TB) disease susceptibility and presentation, and infecting lineage. We used a human cellular model to examine how MARCO genotype mediates the immune response; a case-control study to investigate tuberculosis host genetic susceptibility; and a host-pathogen genetic analysis to study host-pathogen interactions. Two MARCO heterozygous (AG) genotypes (single-nucleotide polymorphisms rs2278589 and rs6751745) were associated with impaired phagocytosis of M. tuberculosis trehalose 6,6'-dimycolate-cord factor and ß-glucan-coated beads in macrophages. The heterozygous genotypes of rs2278589 and rs6751745 were also associated with increased risk of pulmonary TB (PTB; rs2278589, P=0.001, odds ratio (OR)=1.6; rs6751745, P=0.009, OR=1.4), and with severe chest X-ray abnormalities (P=0.007, OR=1.6). These two genotypes were also associated with the Beijing lineage (rs2278589, P=0.001, OR=1.7; rs6751745, P=0.01, OR=1.5). Together, these results suggest that MARCO polymorphisms may regulate phagocytosis of M. tuberculosis and susceptibility and severity of PTB. They also suggest MARCO genotype and Beijing strains may interact to increase the risk of PTB.

TLR9 gene region polymorphisms and susceptibility to tuberculosis in Vietnam.

Humans exposed to Mycobacterium tuberculosis (Mtb) show variation in susceptibility to infection and differences in tuberculosis (TB) disease outcome. Toll-like receptor 9 (TLR9) is a pattern recognition receptor that mediates recognition of Mtb and modulates Mtb-specific T-cell responses. Using a case-population design, we evaluated whether single nucleotide polymorphisms (SNPs) in the TLR9 gene region are associated with susceptibility to pulmonary or meningeal TB as well as neurologic presentation and mortality in the meningeal TB group. In a discovery cohort (n = 352 cases, 382 controls), three SNPs were associated with TB (all forms, p < 0.05) while three additional SNPs neared significance (0.05 < p < 0.1). When these six SNPs were evaluated in a validation cohort (n = 339 cases, 367 controls), one was significant (rs352142) while another neared significance (rs352143). When the cohorts were combined, rs352142 was most strongly associated with meningeal tuberculosis (dominant model; p = 0.0002, OR 2.36, CI 1.43-3.87) while rs352143 was associated with pulmonary tuberculosis (recessive model; p = 0.006, OR 5.3, CI 1.26-31.13). None of the SNPs were associated with mortality. This is the first demonstration of an association between a TLR9 gene region SNP and tuberculous meningitis. In addition, this extends previous findings that support associations of TLR9 SNPs with pulmonary tuberculosis.

A polymorphism in human CD1A is associated with susceptibility to tuberculosis.

CD1 proteins are antigen-presenting molecules that evolved to present lipids rather than peptides to T cells. However, unlike major histocompatibility complex genes, CD1 genes show low rates of polymorphism and have not been clearly associated with human disease. We report that an intronic polymorphism in CD1A (rs411089) is associated with susceptibility to tuberculosis in two cohorts of Vietnamese adults (combined cohort odds ratio 1.78; 95% confidence interval: 1.24-2.57; P=0.001). These data strengthen the hypothesis that CD1A-mediated lipid antigen presentation is important for controlling tuberculosis in humans.

Epiregulin (EREG) variation is associated with susceptibility to tuberculosis.

Although host genetics influences susceptibility to Mycobacterium tuberculosis, the human genes regulating pathogenesis remain largely unknown. We used M. tuberculosis-stimulated macrophage gene expression profiling in conjunction with a case-control genetic association study to discover epiregulin (EREG), as a novel candidate tuberculosis (TB) susceptibility gene. Using a genome-wide association study dataset, we found that among the 21 genes with greater than 50-fold induction, EREG had the most polymorphisms associated with TB. We genotyped haplotype-tagging polymorphisms in discovery (N = 337 cases, N = 380 controls) and validation (N = 332 cases) datasets and an EREG polymorphism (rs7675690) was associated with susceptibility to TB (genotypic comparison; corrected P = 0.00007). rs7675690 was also associated more strongly with infections caused by the Beijing lineage of M. tuberculosis when compared with non-Beijing strains (controls vs Beijing, OR 7.81, P = 8.7 × 10(-5); non-Beijing, OR 3.13, P = 0.074). Furthermore, EREG expression was induced in monocytes and peripheral blood mononuclear cells stimulated with M. tuberculosis as well as TLR4 and TLR2/1/6 ligands. In murine macrophages, EREG expression induced by M. tuberculosis was MYD88- and TLR2-dependent. Together, these data provide the first evidence for an important role for EREG as a susceptibility gene for human TB.

A polymorphism in human TLR2 is associated with increased susceptibility to tuberculous meningitis.

Tuberculous meningitis (TBM) results from the haematogenous dissemination of Mycobacterium tuberculosis from the lung to the brain. Dissemination is believed to occur early during infection, before the development of adaptive immunity. Toll-like receptor 2 (TLR2) mediates recognition of M. tuberculosis and initiates the innate immune response to infection. We hypothesized that polymorphisms in the TLR2 gene influence bacterial dissemination and the development of TBM. A case-control study was designed to test the hypothesis. Cases of bacteriologically confirmed pulmonary tuberculosis (TB) (n=183) and TBM (n=175), and cord blood controls (n=389) were enrolled in Vietnam. TLR2 genotype 597CC was associated with susceptibility to TB (odds ratio (OR)=2.22, 95% confidence interval (CI): 1.23-3.99). The association was found with meningeal rather than pulmonary TB (TBM vs control, OR=3.26, 95% CI: 1.72-6.18), and was strongest when miliary TB was found on chest radiography (controls vs TBM with miliary TB, OR=5.28, 95% CI: 2.20-12.65). Furthermore, the association increased with the severity of neurologic symptoms (grade I TBM, OR=1.93, 95% CI: 0.54-6.92; grade II, OR=3.32, 95% CI: 0.84-13.2; and grade III, OR=5.70, 95% CI: 1.81-18.0). These results demonstrate a strong association of TLR2 SNP T597C with the development of TBM and miliary TB and indicate that TLR2 influences the dissemination of M. tuberculosis.

Polymorphisms of the gene coding for copper/zinc superoxide dismutase (SOD1) in patients with Japanese encephalitis.

Japanese encephalitis is the commonest form of encephalitis globally. Most cases develop characteristic encephalitis but some also present with flaccid paralysis. The paralysis is secondary to damage at the alpha motor neurone, the site that is also damaged in amyotrophic lateral sclerosis (ALS). The gene coding for superoxide dismutase 1 (SOD1) is thought to be involved in ALS and may also be linked to susceptibility to Japanese encephalitis. To investigate this possibility, polymorphisms in the SOD1 gene were investigated, in 61 cases of Japanese encephalitis, 61 matched controls and 171 population controls, in Vietnam. Novel polymorphisms, found only in three of the cases and one of the population controls, may be involved with susceptibility to Japanese encephalitis and potentially to other flavivirus infections that lead to damage to the cells of the anterior horn. Further research on this possible association is required.