Variations in Antimicrobial Activities of Human Monocyte-Derived Macrophage and Their Associations With Tuberculosis Clinical Manifestations.

Macrophages play a significant role in preventing infection through antimicrobial activities, particularly acidification, and proteolysis. Mycobacterium tuberculosis (Mtb) infection can lead to diverse outcomes, from latent asymptomatic infection to active disease involving multiple organs. Monocyte-derived macrophage is one of the main cell types accumulating in lungs following Mtb infection. The variation of intracellular activities of monocyte-derived macrophages in humans and the influence of these activities on the tuberculosis (TB) spectrum are not well understood. By exploiting ligand-specific bead-based assays, we investigated macrophage antimicrobial activities real-time in healthy volunteers (n = 53) with 35 cases of latent TB (LTB), and those with active TB (ATB), and either pulmonary TB (PTB, n = 70) or TB meningitis (TBM, n = 77). We found wide person-to-person variations in acidification and proteolytic activities in response to both non-immunogenic IgG and pathogenic ligands comprising trehalose 6,6'-dimycolate (TDM) from Mtb or ß-glucan from Saccharamyces cerevisiase. The variation in the macrophage activities remained similar regardless of stimuli; however, IgG induced stronger acidification activity than immunogenic ligands TDM (P = 10-5, 3 × 10-5 and 0.01 at 30, 60, and 90 min) and ß-glucan (P = 10-4, 3 × 10-4 and 0.04 at 30, 60, and 90 min). Variation in proteolysis activity was slightly higher in LTB than in ATB (CV = 40% in LTB vs. 29% in ATB, P = 0.03). There was no difference in measured antimicrobial activities in response to TDM and bacterial killing in macrophages from LTB and ATB, or from PTB and TBM. Our results indicate that antimicrobial activities of monocyte-derived macrophages vary among individuals and show immunological dependence, but suggest these activities cannot be solely responsible for the control of bacterial replication or dissemination in TB.

The APOA5-rs662799 Polymorphism Is a Determinant of Dyslipidemia in Vietnamese Primary School Children.

Apolipoprotein A-V encoded by apolipoprotein 5 (APOA5) gene plays an important role in lipid metabolism, especially in the regulation of plasma triglycerol levels. The study aimed to evaluate the association of the APOA5-rs662799 polymorphism with dyslipidemia in Vietnamese children and the potential modification of obesity-related traits (body mass index, waist circumference, hip circumference, and waist-to-hip ratio) on this association. A case-control study was conducted with a total of 154 dyslipidemia cases and 389 controls at the age of 6 to 10 recruited at 31 primary schools in Hanoi city of Vietnam. Genotype for APOA5-rs662799 polymorphism was determined by the restriction fragment length polymorphism analysis. The association of APOA5-rs662799 polymorphism with dyslipidemia adjusting for age, sex, residence, and obesity-related traits was analyzed by binary logistic regression analysis. The results showed that in comparison with T/T and T/C carriers, the C/C carriers had a higher concentration of serum TAG in cases (p =0.049). Carriers of the C allele (C/C + T/C) had higher risk for developing dyslipidemia and hypertriglyceridemia than subjects with T/T genotype (odds ratio, OR = 1.7, p =0.0062 and OR = 1.6, p = 0.026, respectively). The association remained significant after adjusting for age, gender, residence, and obesity status (OR = 1.75, p = 0.006 and OR = 1.53, p = 0.049, respectively) or other obesity-related traits. The study suggested that the APOA5-rs662799 polymorphism may be a determinant of dyslipidemia and hypertriglyceridemia in Vietnamese children, independent of obesity-related traits.

Comparison of the Mycobacterium tuberculosis molecular bacterial load assay, microscopy and GeneXpert versus liquid culture for viable bacterial load quantification before and after starting pulmonary tuberculosis treatment.

Molecular bacterial load assay (MBLA) rapidly quantifies viable Mycobacterium tuberculosis (Mtb) and may be useful for monitoring treatment response and treatment efficacy. We conducted a prospective study in 56 adults with pulmonary tuberculosis from whom 244 sputum samples were collected before and during the first month of treatment. We evaluated MBLA for early monitoring of bacterial burden and investigated bactericidal activities of first-line therapy in patients infected with drug susceptible and resistant isolates. Mtb loads measured by MBLA and culture were correlated after one-week (r = 0.56) and one-month (r = 0.73) of treatment. Correlations between culture and GeneXpert or microscopy were weaker during treatment. Mtb load by MBLA declined more rapidly than GeneXpert after one-week (2.73 Ct, P < 0.001; 0.95 Ct, P = 0.297, respectively) and one-month (8.94 Ct, P < 0.001; 6.78 Ct, P < 0.001). Mtb loads in multidrug resistant (MDR) infections were significantly greater than in both sensitive and poly/mono-resistance after one-week (P < 0.02) and one-month treatment (P = 0.001). MBLA performed better than GeneXpert and microscopy in comparison to culture for quantifying viable Mtb during treatment. It can be used for monitoring bacterial load during TB treatment, facilitating early detection of treatment failure thus improving outcomes.

Development of ligand-coated beads to measure macrophage antimicrobial activities.

BACKGROUND INFORMATION: After macrophage recognises and phagocytoses the microorganism, their phagosome undergoes a maturation process, which creates a hostile environment for the bacterium. The lumen is acidified, and proteolysis occurs to kill and degrade pathogen for further antigen presentation. It is important to understand the association between the macrophage intracellular activities and the outcome of infection. Different methods have been developed to measure the phagosome dynamics of macrophages, but there are still limitations. RESULTS: We used Mycobacterium tuberculosis (Mtb) antigens, the causative agent of tuberculosis (TB), as a model of infectious disease. Adopting a fluorescent bead-based assay, we developed beads coated with trehalose 6,6'dimycolate (TDM) from Mtb cell wall and ß-glucan from yeast cell wall to measure the macrophage phagosomal activities using a microplate reader. We examined the consistency of the assay using J774 cells and validated it using human monocyte-derived macrophages (hMDM) from healthy volunteers and TB patients. There was a decreased pH and increased proteolysis in the lumen of J774 cells after phagocytosing the ligand-coated beads. J774 macrophage showed no difference in the acidification and proteolysis in response to control IgG beads, TDM and ß-glucan beads. hMDM from healthy volunteers or TB patients showed heterogeneity in the intracellular activities when treated with ligand-coated beads. CONCLUSIONS AND SIGNIFICANCE: The beads coated with specific ligands from Mtb worked well in both macrophage cell line and human primary macrophages, which can be exploited to further study the phagosomal function of macrophage in TB. Our bead model can be applied to different ligands from other pathogens, which could extend the understanding of the associations between macrophage antimicrobial functions and outcomes of infectious diseases and the possible cellular mechanisms involved.

Virulence of Mycobacterium tuberculosis Clinical Isolates Is Associated With Sputum Pre-treatment Bacterial Load, Lineage, Survival in Macrophages, and Cytokine Response.

It is uncertain whether differences in Mycobacterium tuberculosis (Mtb) virulence defined in vitro influence clinical tuberculosis pathogenesis, transmission, and mortality. We primarily used a macrophage lysis model to characterize the virulence of Mtb isolates collected from 153 Vietnamese adults with pulmonary tuberculosis. The virulence phenotypes were then investigated for their relationship with sputum bacterial load, bacterial lineages, bacterial growth, and cytokine responses in macrophages. Over 6 days of infection, 34 isolates (22.2%) showed low virulence (< 5% macrophages lysed), 46 isolates (30.1%) showed high virulence (≥90% lysis of macrophages), and 73 isolates (47.7%) were of intermediate virulence (5-90% macrophages lysed). Highly virulent isolates were associated with an increased bacterial load in patients' sputum before anti-tuberculosis therapy (P = 0.02). Isolate-dependent virulence phenotype was consistent in both THP-1 and human monocyte-derived macrophages. High virulence isolates survived better and replicated in macrophages one hundred fold faster than those with low virulence. Macrophages infected with high virulence isolates produced lower concentrations of TNF-α and IL-6 (P = 0.002 and 0.0005, respectively), but higher concentration of IL-1ß (P = 5.1 × 10-5) compared to those infected with low virulence isolates. High virulence was strongly associated with East Asian/Beijing lineage [P = 0.002, Odd ratio (OR) = 4.32, 95% confident intervals (CI) 1.68-11.13]. The association between virulence phenotypes, bacterial growth, and proinflammatory cytokines in macrophages suggest the suppression of certain proinflammatory cytokines (TNF-α and IL-6) but not IL-1ß allows better intracellular survival of highly virulent Mtb. This could result in rapid macrophage lysis and higher bacterial load in sputum of patients infected with high virulence isolates, which may contribute to the pathogenesis and success of the Beijing lineage.

Influence of Stress and Antibiotic Resistance on Cell-Length Distribution in Mycobacterium tuberculosis Clinical Isolates.

Mycobacterial cellular variations in growth and division increase heterogeneity in cell length, possibly contributing to cell-to-cell variation in host and antibiotic stress tolerance. This may be one of the factors influencing Mycobacterium tuberculosis persistence to antibiotics. Tuberculosis (TB) is a major public health problem in developing countries, antibiotic persistence, and emergence of antibiotic resistance further complicates this problem. We wanted to investigate the factors influencing cell-length distribution in clinical M. tuberculosis strains. In parallel we examined M. tuberculosis cell-length distribution in a large set of clinical strains (n = 158) from ex vivo sputum samples, in vitro macrophage models, and in vitro cultures. Our aim was to understand the influence of clinically relevant factors such as host stresses, M. tuberculosis lineages, antibiotic resistance, antibiotic concentrations, and disease severity on the cell size distribution in clinical M. tuberculosis strains. Increased cell size and cell-to-cell variation in cell length were associated with bacteria in sputum and infected macrophages rather than liquid culture. Multidrug-resistant (MDR) strains displayed increased cell length heterogeneity compared to sensitive strains in infected macrophages and also during growth under rifampicin (RIF) treatment. Importantly, increased cell length was also associated with pulmonary TB disease severity. Supporting these findings, individual host stresses, such as oxidative stress and iron deficiency, increased cell-length heterogeneity of M. tuberculosis strains. In addition we also observed synergism between host stress and RIF treatment in increasing cell length in MDR-TB strains. This study has identified some clinical factors contributing to cell-length heterogeneity in clinical M. tuberculosis strains. The role of these cellular adaptations to host and antibiotic tolerance needs further investigation.

Number and timing of antenatal HIV testing: evidence from a community-based study in Northern Vietnam.

BACKGROUND: HIV testing for pregnant women is an important component for the success of prevention of mother-to-child transmission of HIV (PMTCT). A lack of antenatal HIV testing results in loss of benefits for HIV-infected mothers and their children. However, the provision of unnecessary repeat tests at a very late stage of pregnancy will reduce the beneficial effects of PMTCT and impose unnecessary costs for the individual woman as well as the health system. This study aims to assess the number and timing of antenatal HIV testing in a low-income setting where PMTCT programmes have been scaled up to reach first level health facilities. METHODS: A cross-sectional community-based study was conducted among 1108 recently delivered mothers through face-to-face interviews following a structured questionnaire that focused on socio-economic characteristics, experiences of antenatal care and HIV testing. RESULTS: The prevalence of women who lacked HIV testing among the study group was 10% while more than half of the women tested had had more than two tests during pregnancy. The following factors were associated with the lack of antenatal HIV test: having two children (aOR 2.1, 95% CI 1.3-3.4), living in a remote rural area (aOR 7.8, 95% CI 3.4-17.8), late antenatal care attendance (aOR 3.6, 95% CI 1.3-10.1) and not being informed about PMTCT at their first antenatal care visits (aOR 7.4, 95% CI 2.6-21.1). Among women who had multiple tests, 80% had the second test after 36 weeks of gestation. Women who had first ANC and first HIV testing at health facilities at primary level were more likely to be tested multiple times (OR 2.9 95% CI 1.9-4.3 and OR = 4.7 95% CI 3.5-6.4), respectively. CONCLUSIONS: Not having an HIV test during pregnancy was associated with poor socio-economic characteristics among the women and with not receiving information about PMTCT at the first ANC visit. Multiple testing during pregnancy prevailed; the second tests were often provided at a late stage of gestation.