In-Depth Molecular Characterization of Mycobacterium tuberculosis from New Delhi – Predominance of Drug Resistant Isolates of the ‘Modern’ (TbD1−) Type

BACKGROUND: India has the highest estimated burden of tuberculosis in the world, accounting for 21% of all tuberculosis cases world-wide. However, due to lack of systematic analysis using multiple markers the available information on the genomic diversity of Mycobacterium tuberculosis in India is limited. METHODOLOGY/PRINCIPAL FINDINGS: Thus, 65 M. tuberculosis isolates from New Delhi, India were analyzed by spoligotyping, MIRU-VNTR, large deletion PCR typing and single nucleotide polymorphism analysis (SNP). The Central Asian (CAS) 1 _DELHI sub-lineage was the most prevalent sub-lineage comprising 46.2% (n = 30) of all isolates, with shared-type (ST) 26 being the most dominant genotype comprising 24.6% (n = 16) of all isolates. Other sub-lineages observed were: East-African Indian (EAI)-5 (9.2%, n = 6), EAI6_BGD1 (6.2%, n = 4), EAI3_IND, CAS and T1 with 6.2% each (n = 4 each), Beijing (4.6%, n = 3), CAS2 (3.1%, n = 2), and X1 and X2 with 1 isolate each. Genotyping results from five isolates (7.7%) did not match any existing spoligopatterns, and one isolate, ST124, belonged to an undefined lineage. Twenty-six percent of the isolates belonged to the TbD1+ PGG1 genogroup. SNP analysis of the pncA gene revealed a CAS-lineage specific silent mutation, S65S, which was observed for all CAS-lineage isolates (except two ST26 isolates) and in 1 orphan. Mutations in the pncA gene, conferring resistance to pyrazinamide, were observed in 15.4% of all isolates. Collectively, mutations in the rpoB gene, the katG gene and in both rpoB and katG genes, conferring resistance to rifampicin and isoniazid, respectively, were more frequent in CAS1_DELHI isolates compared to non-CAS_DELHI isolates (OR: 3.1, CI95% [1.11, 8.70], P = 0.045). The increased frequency of drug-resistance could not be linked to the patients' history of previous anti-tuberculosis treatment (OR: 1.156, CI95% [0.40, 3.36], P = 0.79). Fifty-six percent of all new tuberculosis patients had mutations in either the katG gene or the rpoB gene, or in both katG and rpoB genes. CONCLUSION: CAS1_DELHI isolates circulating in New Delhi, India have a high frequency of mutations in the rpoB and katG genes. A silent mutation (S65S) in the pncA gene can be used as a putative genetic marker for CAS-lineage isolates

Adverse Effects and Choice between the Injectable Agents Amikacin and Capreomycin in Multidrug-Resistant Tuberculosis

Background: The prolonged use of injectable agents in an MDR-TB regimen is recommended by the WHO despite association with ototoxicity and nephrotoxicity.Objective: We undertook this study to look at the relative adverse effects of capreomycin and amikacin.Methods: We reviewed the case notes of 100 consecutive patients treated at 4 MDR-TB treatment centres in the UK.Results: The median total duration of treatment with an injectable agent was 178 (IQR 109-192, n=73) days for those with MDR-TB, 179 (104-192, n=12) days for those with MDR-TB plus fluoroquinolone resistance and 558 (324-735, n=8) days for those with XDR-TB. Injectable use was longer for those started with capreomycin at 183 (IQR 123-197) days compared to 119 (IQR 83-177) days with amikacin (p=0.002). Excluding XDR-TB, 51 (51/85, 60%) patients were treated with an injectable for over 6 months and 12 (12/85, 14%) for over 8 months. 40 % of all patients discontinued the injectable due to hearing loss. 55% of patients experienced ototoxicity: 5 times (hazard ratio (HR) 5.2, CI 1.2-22.6, p=0.03) more likely in those started on amikacin compared to treatment with capreomycin only. Amikacin was associated with less hypokalemia than capreomycin (Odds ratios: 0.28 (0.11-0.72)), with 5 (5/37, 14%) patients stopping capreomycin due to recurrent electrolyte loss. There was no difference in the number experiencing a creatinine rise of > 1.5 times baseline.Conclusion: Hearing loss is frequent in this cohort, though significantly lower in those starting capreomycin which should be given greater consideration as a first line agent

Validation of the ALS Assay in Adult Patients with Culture Confirmed Pulmonary Tuberculosis

BACKGROUND: We have earlier shown that Bacille Calmette-Guérin (BCG) vaccine-specific IgG Antibodies in Lymphocyte Supernatant (ALS) can be used for diagnosis of active tuberculosis (TB) in adults and children. METHODOLOGY/PRINCIPAL FINDINGS: The ALS method was validated in a larger cohort (n = 212) of patients with suspicion of pulmonary TB using multiple antigens (BCG, LAM, TB15.3, TB51A, CFP10-ESAT6-A, CFP, CW) from Mycobacterium tuberculosis. The sensitivity and specificity of the ALS assay was calculated using non-TB patients as controls. The sensitivity and the specificity were highest with BCG vaccine (90% and 88% respectively) followed by LAM (89% and 87% respectively). Simultaneous assessment of multiple antigen-specific antibodies increased sensitivity (91%) and specificity (88%). Using higher lymphocyte count in smaller volume of culture media increased detection and reduced the assay duration to ∼30 hrs. Twenty one patients with clinical findings strongly suggestive of TB finally diagnosed as non-TB patients were positive by the ALS assay, of which 9 (43%) were positive for 7 antigens and 19 (90%) for at least 3 antigens. CONCLUSIONS/SIGNIFICANCE: Our findings show that simultaneous detection of antigens improves the diagnostic potential of the ALS assay; the modified method increases sensitivity and can provide results in <48 hours, and enable detection of some cases of pulmonary TB that are not detectable by standard methods

Activation of an NLRP3 Inflammasome Restricts Mycobacterium kansasii Infection

Mycobacterium kansasii has emerged as an important nontuberculous mycobacterium pathogen, whose incidence and prevalence have been increasing in the last decade. M. kansasii can cause pulmonary tuberculosis clinically and radiographically indistinguishable from that caused by Mycobacterium tuberculosis infection. Unlike the widely-studied M. tuberculosis, little is known about the innate immune response against M. kansasii infection. Although inflammasome activation plays an important role in host defense against bacterial infection, its role against atypical mycobacteria remains poorly understood. In this report, the role of inflammasome activity in THP-1 macrophages against M. kansasii infection was studied. Results indicated that viable, but not heat-killed, M. kansasii induced caspase-1-dependent IL-1β secretion in macrophages. The underlying mechanism was found to be through activation of an inflammasome containing the NLR (Nod-like receptor) family member NLRP3 and the adaptor protein ASC (apoptosis-associated speck-like protein containing a CARD). Further, potassium efflux, lysosomal acidification, ROS production and cathepsin B release played a role in M. kansasii-induced inflammasome activation. Finally, the secreted IL-1β derived from caspase-1 activation was shown to restrict intracellular M. kansasii. These findings demonstrate a biological role for the NLRP3 inflammasome in host defense against M. kansasii

Nontuberculous mycobacteria, Zambia

Clinical relevance of nontuberculous mycobacteria (NTM) isolated from 180 chronically ill patients and 385 healthy controls in Zambia was evaluated to examine the contribution of these isolates to tuberculosis (TB)-like disease. The proportion of NTM-positive sputum samples was significantly higher in the patient group than in controls; 11% and 6%, respectively (p<0.05). NTM-associated lung disease was diagnosed for 1 patient, and a probable diagnosis was made for 3 patients. NTM-positive patients and controls were more likely to report vomiting and diarrhea and were more frequently underweight than the NTM-negative patients and controls. Chest radiographs of NTM-positive patients showed deviations consistent with TB more frequently than those of controls. The most frequently isolated NTM was Mycobacterium avium complex. Multiple, not previously identified mycobacteria (55 of 171 NTM) were isolated from both groups. NTM probably play an important role in the etiology of TB-like diseases in Zambia