Adequacy of examining one sputum specimen in tuberculosis drug resistance surveys.

OBJECTIVE/BACKGROUND: Collection of one spot and one morning sputum specimen is recommended for tuberculosis (TB) drug resistance surveys. This was a retrospective analysis of Mycobacterium tuberculosis cultures isolated from two spot sputum specimens collected from smear positive TB patients in a TB drug resistance survey. It was conducted to understand the value of a second specimen. METHODS: A TB drug resistance survey was conducted in the state of Tamil Nadu, India, to estimate the prevalence of drug resistance among new sputum smear-positive (NSP) and previously treated (PT) patients diagnosed in Revised National Tuberculosis Control Program microscopy centers. A total of 2425 patients (1524 NSP and 901 PT cases) were enrolled in the study. From these patients, two spot sputum specimens (C and D) were collected within a period of 2h. No preservative was added to sputum. The samples were transported at ambient conditions without cold storage to the central laboratory for culture of M. tuberculosis. Culture yield from each sample was computed and analyzed. RESULTS: The proportion of cultures retrieved from C and D specimens among NSP cases (89.3% and 89.7%) and PT cases (90.8% and 90.3%) were similar. The culture grades of C and D samples were comparable (chi-square test, 3560.135; p<.001) and the agreement was moderate (kappa test, 0.454). CONCLUSION: The findings of the study reveal the adequacy of single spot sputum specimen from smear positive pulmonary TB patients for bacteriological examination in a quality-assured TB laboratory to determine precisely the level of drug resistance in a province of India.

Overview on mechanisms of isoniazid action and resistance in Mycobacterium tuberculosis.

Isoniazid (INH) is one of the most active compounds used to treat tuberculosis (TB) worldwide. In addition, INH has been used as a prophylactic drug for individuals with latent Mycobacterium tuberculosis (MTB) infection to prevent reactivation of disease. Importantly, the definition of multidrug resistance (MDR) in TB is based on the resistance of MTB strains to INH and rifampicin (RIF). Despite its simple chemical structure, the mechanism of action of INH is very complex and involves several different concepts. Many pathways pertaining to macromolecular synthesis are affected, notably mycolic acid synthesis. The pro-drug INH is activated by catalase-peroxidase (KatG), and the active INH products are targeted by enzymes namely, enoyl acyl carrier protein (ACP) reductase (InhA) and beta-ketoacyl ACP synthase (KasA). In contrast, INH is inactivated by arylamine N-acetyltransferases (NATs). Consequently, the molecular mechanisms of INH resistance involve several genes in multiple biosynthetic networks and pathways. Mutation in the katG gene is the major cause for INH resistance, followed by inhA, ahpC, kasA, ndh, iniABC,fadE, furA, Rv1592c and Rv1772. The recent association of efflux genes with INH resistance has also gained considerable attention. Interestingly, substitutions have also been observed in nat, fabD, and accD recently in resistant isolates. Understanding the mechanisms operating behind INH action and resistance would enable better detection of INH resistance. This information would aid novel drug design strategies. Herein we review all mechanisms known to potentially contribute to the complexity of INH action and mechanisms of resistance in MTB, with insights into methods for detection of INH resistance as well as their limitations.

Randomized clinical trial of thrice-weekly 4-month moxifloxacin or gatifloxacin containing regimens in the treatment of new sputum positive pulmonary tuberculosis patients.

BACKGROUND: Shortening tuberculosis (TB) treatment duration is a research priority. This paper presents data from a prematurely terminated randomized clinical trial, of 4-month moxifloxacin or gatifloxacin regimens, in South India. METHODS: Newly diagnosed, sputum-positive HIV-negative pulmonary TB patients were randomly allocated to receive gatifloxacin or moxifloxacin, along with isoniazid and rifampicin for 4 months with pyrazinamide for first 2 months (G or M) or isoniazid and rifampicin for 6 months with ethambutol and pyrazinamide for first 2 months (C). All regimens were administered thrice-weekly. Clinical and bacteriological assessments were done monthly during treatment and for 24 months post-treatment. The Data and Safety Monitoring Board recommended termination of the trial due to high TB recurrence rates in the G and M regimens. RESULTS: Of 416 patients in intent-to-treat analysis, 6 (5%) of 124, 2 (2%) of 110 and 2 (2%) of 137 patients with drug-susceptible TB in the G, M and C arms respectively had unfavorable response at the end of treatment; during the next 24 months, 17 (15%) of 115, 11 (11%) of 104 and 8 (6%) of 132 patients respectively, had TB recurrence. Of 38 drug-resistant patients 1 of 8 and 3 of 26 in the G and C arms respectively had unfavourable response at the end of treatment; and TB recurrence occurred in 2 of 7 and 2 of 23 patients, respectively. The differences in TB recurrence rates between the G and C arms was statistically significant (p = 0.02). Gastro-intestinal symptoms occurred in 23%, 22% and 9% of patients in the G, M and C arms respectively, but most reactions were mild and manageable with symptomatic measures; 1% required regimen modification. CONCLUSIONS: 4-month thrice-weekly regimens of gatifloxacin or moxifloxacin with isoniazid, rifampicin and pyrazinamide, were inferior to standard 6-month treatment, in patients with newly diagnosed sputum positive pulmonary TB. TRIAL REGISTRATION: Clinical Trials Registry of India CTRI/2012/10/003060.

Evaluation of a diagnostic algorithm for sputum smear-negative pulmonary tuberculosis in HIV-infected adults.

BACKGROUND: The Revised National TB Control Program bases diagnosis of tuberculosis (TB) on sputum smear examination and response to a course of antibiotics, whereas World Health Organization recommends early chest radiography [chest x-ray (CXR)] for HIV-infected symptomatic patients. We evaluated the utility of initial CXR in the diagnostic algorithm for symptomatic HIV-infected patients with negative sputum smears. METHODS: HIV-infected ambulatory patients with cough or fever of ≥2 weeks and 3 sputum smears negative for acid-fast bacilli were enrolled in Chennai and Pune, India, between 2007 and 2009. After a CXR and 2 sputum cultures, a course of broad-spectrum antibiotics was given and patients were reviewed after 14 days. Sensitivity, specificity, positive and negative predictive values of symptoms, CXR, and various combinations for diagnosing pulmonary tuberculosis (PTB) were determined, using sputum culture as gold standard. RESULTS: Five hundred four patients (330 males; mean age: 35 years; median CD4: 175 cells per cubic millimeter) were enrolled. CXR had a sensitivity and specificity of 72% and 57%, respectively, with positive predictive value (PPV) of 21% and negative predictive value (NPV) of 93% to diagnose PTB. TB culture was positive in 49 of 235 patients (21%) with an abnormal initial CXR and 19 of 269 patients (7%) with a normal CXR (P < 0.001). Sensitivity and specificity of cough ≥2 weeks for predicting PTB was 97% and 6%, with PPV and NPV of 14% and 94%, respectively. CONCLUSIONS: Although moderately sensitive, basing a diagnosis of TB on initial CXR leads to overdiagnosis. An absence of weight loss had a high NPV, whereas none of the combinations had a good PPV. A rapid and accurate diagnostic test is required for HIV-infected chest symptomatic.

Characterization and phylogenetic analysis of antituberculous compound producing actinomycete strain D25 isolated from Thar Desert soil, Rajasthan.

During the course of the anti-infective drug discovery programme, actinomycete strain D25 was recovered from the Thar Desert soil, Rajasthan, India. Actinomycin type of compound isolated from the strain D25 showed promising activity against multi drug resistant and extensively drug resistant M. tuberculosis isolates. The present study reports the characteristics and phylogenetic status of the actinomycete strain D25. Phenotypic and cell wall characteristics revealed that the strain belongs to the genus Streptomyces. Further 16s rRNA analysis confined the genus Streptomyces with 97% similarity to the closely related species Streptomyces althioticus KCTC 9752. The 16s rRNA sequence was submitted to GenBank with the accession number JN604533.1. According to Bossard et al. (2003) strain D25 was found to be a novel species of the genus Streptomyces from Thar Desert soil, Rajasthan.

Binding of activated isoniazid with acetyl-CoA carboxylase from Mycobacterium tuberculosis.

AccD6 (acetyl coenzyme A (CoA) carboxylase), plays an important role in mycolic acid synthesis of Mycobacterium tuberculosis (Mtb). Induced gene expression by isoniazid (isonicotinylhydrazine - INH), anti-tuberculosis drug) shows the expression of accD6. It is our interest to study the binding of activated INH with the AccD6 model using molecular docking procedures. The study predicts a primary binding site for activated INH (isonicotinyl acyl radical) in AccD6 as a potential target.

Pulmonary tuberculosis: a public health problem amongst the Saharia, a primitive tribe of Madhya Pradesh, Central India.

OBJECTIVES: To estimate the prevalence of pulmonary tuberculosis (TB) disease amongst the Saharia, a 'primitive' tribe of Madhya Pradesh, Central India. METHODS: A community-based cross-sectional TB prevalence survey was undertaken in the Saharia, a 'primitive' tribal community of Madhya Pradesh. A representative random sample of villages predominated by tribal populations was chosen from the selected block of Sheopur District. All eligible individuals were screened for chest symptoms related to TB. Sputum samples were collected from all eligible individuals, transported to the laboratory, and examined by Ziehl-Neelsen smear microscopy and solid media culture methods. RESULTS: Of the 11,468 individuals eligible for screening, 11,116 (96.9%) were screened for symptoms. The overall prevalence of pulmonary TB disease was 1518 per 100,000 population. Prevalence increased with age and the trend was statistically significant (p<0.001). The prevalence of pulmonary TB was also significantly higher in males (2156/100,000) than females (933/100,000) (p<0.001). CONCLUSION: The findings suggest that TB disease remains a major public health problem in the Saharia 'primitive' tribal community of Madhya Pradesh, Central India.

Insight to pyrazinamide resistance in Mycobacterium tuberculosis by molecular docking.

Pyrazinamide (PZA) - an important drug in the anti-tuberculosis therapy, activated by an enzyme Pyrazinamidase (PZase). The basis of PZA resistance in Mycobacterium tuberculosis was owing to mutation in pncA gene coding for PZase. Homology modeling of PZase was performed using software Discovery Studio (DS) 2.0 based on the crystal structure of the PZase from Pyrococcus horikoshii (PDB code 1im5), in this study. The model comprises of one sheet with six parallel strands and seven helices with the amino acids Asp8, Asp49, Trp68, Lys96, Ala134, Thr135 and Cys138 at the active site. Five mutants were generated with Gly at position 8, Thr at position 96, Arg at position 104, Tyr and Ser at position 138. The Wild-type (WT) and five mutant models were docked with PZA. The results indicate that the mutants Lys96Thr, Ser104Arg Asp8Gly and Cys138Tyr may contribute to higher level drug resistance than Cys138Ser. These models provide the first in-silico evidence for the binding interaction of PZA with PZase and form the basis for rationalization of PZA resistance in naturally occurring pncA mutant strains of M. tuberculosis.

Low frequency of moaA3 gene among the clinical isolates of Mycobacterium tuberculosis from Tamil Nadu and Pondicherry--south eastern coastal states of India.

BACKGROUND: Comparative genomic analysis of M. tuberculosis H37Rv and M. bovis BCG have shown that 16 RDs (Regions of Differences) are deleted in BCG and have shown six deletion regions in M. tuberculosis H37Rv. RD1, is present in M. tuberculosis but is absent in all M. bovis BCG sub-strains. A study from Kerala, a south-western coastal state of India aimed to find out differences in RD1 region showed for the first time the presence of moaA3 gene in majority of their clinical isolates, that was absent in type strain H37Rv. We attempted to find out such polymorphism between type strains and the clinical isolates within RD1, targeting moaA3 gene among the clinical isolates of Tamil Nadu & Pondicherry, south-eastern coastal states of India METHODS: One hundred and sixteen clinical isolates of M. tuberculosis were included in the study. PCR using RD1DLa and RD1DRa primers was carried out to amplify a 652 bp fragment, encoding for cfp10 and esat 6 proteins of RD1. A second PCR using primers designed from the surrounding regions of moaA3 gene was done to confirm the presence of the full Open Reading Frame (ORF) in clinical isolates. RESULTS: In M. tuberculosis H37Rv the expected 652 bp band was present. In BCG it was absent as expected, but a 386 bp fragment was amplified. Around 12/116 (10.3%) of our clinical isolates showed both 652 and 386 bp fragments. The additional 386 bp amplicon is a part of the moaA3 gene which codes for molybdopterin cofactor protein A in M. bovis. The second PCR amplified the flanking sequence of moaA3 and yielded the expected amplicon of 1254 bp in all those 10.3% of clinical isolates which had the 386 bp fragment. However the earlier study carried out in Kerala, reported the presence of moaA3 gene in majority (97%) of their clinical isolates. CONCLUSION: This finding showed that there was regional variation presenting polymorphism in moA3 gene, among the strains of M. tuberculosis and further strengthens the speculation of genetic differences among the strains of Kerala and Tamil Nadu & Pondicherry, the South Indian states.

Prevalence of pulmonary tuberculosis amongst the tribal population of Madhya Pradesh, central India.

BACKGROUND: This was a prevalence survey of pulmonary tuberculosis (PTB) disease in the tribal population of Madhya Pradesh state, central India. METHODS: A community-based cross-sectional tuberculosis (TB) disease prevalence survey was undertaken among adults aged > or = 15 years in the tribal population of Madhya Pradesh. A multistage stratified cluster sampling was adopted. A representative random sample of villages predominated by tribal populations was selected from 11 districts. All eligible individuals were questioned for chest symptoms relating to TB. Sputum samples were collected from all eligible individuals, transported to the laboratory, and examined by Ziehl-Neelsen (ZN) smear microscopy and solid media culture methods. RESULTS: Of the 23,411 individuals eligible for screening, 22,270 (95.1%) were screened for symptoms. The overall proportion of symptomatic individuals was 7.9%. Overall prevalence (culture and/or smear positive) of PTB was 387 [95% confidence interval (CI): 273-502] per 100,000 population. The prevalence increased with age and was also significantly higher among males (554/100,000; 95% CI: 415-693) as compared with females (233/100,000; 95% CI: 101-364) (P < 0.001). CONCLUSION: The findings suggest that the TB situation amongst the tribal population is not that different from the situation among the non-tribal population in the country. However, TB remains a major public health problem amongst the tribal population and there is a need to maintain and further strengthen TB control measures on a sustained and long-term basis.