Evaluating the anti Mycobacterium tuberculosis activity of Alpinia galanga (L.) Willd. axenically under reducing oxygen conditions and in intracellular assays.

BACKGROUND: In tuberculosis (TB), the steadily increasing bacterial resistance to existing drugs and latent TB continue to be major concerns. A combination of conventional drugs and plant derived therapeutics can serve to expand the antimicrobial spectrum, prevent the emergence of drug resistant mutants and minimize toxicity. Alpinia galanga, used in various traditional medicines, possesses broad spectrum antibacterial properties. The study was undertaken to assess the antimycobacterial potential of A. galanga in axenic (under aerobic and anaerobic conditions) and intracellular assays. METHODS: Phytochemical analysis was done using HPTLC. The acetone, aqueous and ethanolic extracts (1, 10, 25, 50 and 100 µg/ml) of A. galanga were tested axenically using Microplate Alamar Blue Assay (MABA) against Mycobacterium tuberculosis (M.tb) H37Rv and three drug sensitive and three multi drug resistant clinical isolates. The activity of the extracts was also evaluated intracellularly in A549 cell line against these strains. The extracts active under intracellular conditions were further tested in an axenic setup under reducing oxygen concentrations using only H37Rv. RESULTS: 1´ acetoxychavicol acetate, the reference standard used, was present in all the three extracts. The acetone and ethanolic extracts were active in axenic (aerobic and anaerobic) and intracellular assays. The aqueous extract did not demonstrate activity under the defined assay parameters. CONCLUSION: A. galanga exhibits anti M.tb activity with multiple modes of action. Since the activity of the extracts was observed under reducing oxygen concentrations, it may be effective in treating the dormant and non-replicating bacteria of latent TB. Though the hypothesis needs further testing, A. galanga being a regular dietary component may be utilized in combination with the conventional TB therapy for enhanced efficacy.

Estimating fitness by competition assays between drug susceptible and resistant Mycobacterium tuberculosis of predominant lineages in Mumbai, India.

BACKGROUND: Multi Drug Resistant Tuberculosis (MDR TB) is a threat to global tuberculosis control. A significant fitness cost has been associated with DR strains from specific lineages. Evaluation of the influence of the competing drug susceptible strains on fitness of drug resistant strains may have an important bearing on understanding the spread of MDR TB. The aim of this study was to evaluate the fitness of MDR TB strains, from a TB endemic region of western India: Mumbai, belonging to 3 predominant lineages namely CAS, Beijing and MANU in the presence of drug susceptible strains from the same lineages. METHODOLOGY: Drug susceptible strains from a single lineage were mixed with drug resistant strain, bearing particular non synonymous mutation (rpoB D516V; inhA, A16G; katG, S315T1/T2) from the same or different lineages. Fitness of M.tuberculosis (M.tb) strains was evaluated using the difference in growth rates obtained by using the CFU assay system. CONCLUSION/SIGNIFICANCE: While MANU were most fit amongst the drug susceptible strains of the 3 lineages, only Beijing MDR strains were found to grow in the presence of any of the competing drug susceptible strains. A disproportionate increase in Beijing MDR could be an alarm for an impending epidemic in this locale. In addition to particular non synonymous substitutions, the competing strains in an environment may impact the fitness of circulating drug resistant strains.

Drug resistance mutations and heteroresistance detected using the GenoType MTBDRplus assay and their implication for treatment outcomes in patients from Mumbai, India.

BACKGROUND: Only 5% of the estimated global multidrug resistant TB (MDRTB) load is currently detected. Endemic Mumbai with increasing MDR would benefit from the introduction of molecular methods to detect resistance. METHODS: The GenoType MTBDRplus assay was used to determine mutations associated with isoniazid and rifampicin resistance and their correlation with treatment outcomes. It was performed on a convenience sample comprising 88 onset and 67 fifth month isolates for which phenotypic drug susceptibility testing (DST) was determined by the Buddemeyer technique for an earlier study. Simultaneous presence of wild type and mutant bands was referred to as "mixed patterns" (heteroresistance). RESULTS: Phenotypically 41 isolates were sensitive; 11 isoniazid, 2 rifampicin, 2 pyrazinamide and 5 ethambutol monoresistant; 16 polyresistant and 78 MDR. The agreement between both methods was excellent (kappa = 0.72-0.92). Of 22 rifampicin resistant onset isolates, the predominant rpoB mutations were the singular lack of WT8 (n = 8) and mixed D516V patterns (n = 9). Of the 64 rifampicin resistant fifth month isolates, the most frequent mutations were in WT8 (n = 31) with a further 9 showing the S531L mutation. Mixed patterns were seen in 22 (34%) isolates, most frequently for the D516V mutation (n = 21). Of the 22 onset and 35 fifth month katG mutants, 13 and 12 respectively showed the S315T1 mutation with loss of the WT. Mixed patterns involving both S315T1 and S315T2 were seen in 9 and 23 isolates respectively. Seventeen of 23 and 23/35 inhA mutant onset and fifth month isolates showed mixed A16G profiles. Additionally, 10 fifth month isolates lacked WT2. Five onset and 6 fifth month isolates had both katG and inhA mutations. An association was noted between only katG but not only inhA resistance and poor outcome (p = 0.037); and additional resistance to ethambutol (p = 0.0033). More fifth month than onset isolates had mixed profiles for at least 1 gene (p = 0.000001). CONCLUSIONS: The use of the assay to rapidly diagnose MDR could guide simultaneous first- and second-line DST, and reduce the delay in administering appropriate regimens. Furthermore, detection of heteroresistance could prevent inaccurate "cured" treatment outcomes documented through smear microscopy and permit more sensitive detection of neonascent resistance.

Chest X-rays and associated clinical parameters in pulmonary tuberculosis cases from the National Tuberculosis Programme, Mumbai.

The study was carried out in pulmonary tuberculosis (PTB) patients from the local Tuberculosis control programme, Mumbai, India. It examined features of chest X-rays and their correlation with clinical parameters for possible application in suspected multidrug resistant TB (MDRTB) and to predict outcome in new and treatment failure PTB cases. X-ray features (infiltrate, cavitation, miliary shadows, pleural effusion, mediastinal lymphadenopathy and extent of lesions) were analyzed to identify associations with biological/clinical parameters through univariate and multivariate logistic regression. Failures demonstrated associations between extensive lesions and high glycosylated hemoglobin (GHb) levels (P=0.028) and male gender (P=0.03). An association was also detected between cavitation and MDR (P=0.048). In new cases, bilateral cavities were associated with MDR (P=0.018) and male gender (P=0.01), low body mass index with infiltrates (P=0.008), and smoking with cavitation (P=0.0238). Strains belonging to the Manu1 spoligotype were associated with mild lesions (P=0.002). Poor outcome showed borderline significance with extensive lesions at onset (P=0.053). Furthermore, amongst new cases, smoking, the Central Asian Strain (CAS) spoligotype and high GHb were associated with cavitation, whereas only CAS spoligotypes and high GHb were associated with extensive lesions. The study highlighted associations between certain clinical parameters and X-ray evidence which support the potential of X-rays to predict TB, MDRTB and poor outcome. The use of X-rays as an additional tool to shorten diagnostic delay and shortlist MDR suspects amongst nonresponders to TB treatment should be explored in a setting with limited resources coping with a high MDR case load such as Mumbai.

Drug-resistant tuberculosis in Mumbai, India: An agenda for operations research.

Operations research (OR) is well established in India and is also a prominent feature of the global and local agendas for tuberculosis (TB) control. India accounts for a quarter of the global burden of TB and of new cases. Multidrug-resistant TB is a significant problem in Mumbai, India's most populous city, and there have been recent reports of totally resistant TB. Much thought has been given to the role of OR in addressing programmatic challenges, by both international partnerships and India's Revised National TB Control Programme. We attempt to summarize the major challenges to TB control in Mumbai, with an emphasis on drug resistance. Specific challenges include diagnosis of TB and defining cure, detecting drug resistant TB, multiple sources of health care in the private, public and informal sectors, co-infection with human immunodeficiency virus (HIV) and a concurrent epidemic of non-communicable diseases, suboptimal prescribing practices, and infection control. We propose a local agenda for OR: modeling the effects of newer technologies, active case detection, and changes in timing of activities, and mapping hotspots and contact networks; modeling the effects of drug control, changing the balance of ambulatory and inpatient care, and adverse drug reactions; modeling the effects of integration of TB and HIV diagnosis and management, and preventive drug therapy; and modeling the effects of initiatives to improve infection control.