Determination of ethambutol MICs for Mycobacterium tuberculosis and Mycobacterium avium isolates by resazurin microtitre assay

Objectives: To test susceptibilities of Mycobacterium tuberculosis (MTB) isolates to ethambutol by the Lowenstein-Jensen (LJ) proportion method and resazurin microtitre assay (REMA) and to evaluate REMA for the determination of ethambutol MICs for MTB and Mycobacterium avium isolates. Methods: A total of 50 MTB and 20 M. avium isolates were tested to determine the MICs of ethambutol by REMA and agar dilution method. MTB isolates were also tested by the LJ proportion method. Results: REMA provided ethambutol susceptibility results for all the isolates within 8-9 days. For MTB isolates, REMA showed 96.7% sensitivity, 100.0% specificity and 98.0% accuracy when LJ proportion results were taken as 'gold standard'. For both MTB and M. avium isolates, the MICs determined by REMA were lower than those determined in agar medium, indicating that MIC values determined by REMA are closer to the actual MICs for the isolates. Conclusions: REMA can be used as a rapid and inexpensive method for mycobacterial drug susceptibility testing against ethambutol. In comparison with the agar method, the MICs determined by REMA can more accurately be correlated with achievable plasma concentrations of antimycobacterial agents

Antimycobacterial activity of econazole against multidrug-resistant strains of Mycobacterium tuberculosis

This study evaluated the antimycobacterial activity of econazole against multidrug-resistant (MDR) strains of Mycobacterium tuberculosis. The minimum inhibitory concentration (MIC90) and minimum bactericidal concentration (MBC>99.99) against MDR strains were found to be 0.120-0.125μg/mL and 0.125-0.150μg/mL, respectively, demonstrating the antimycobacterial potential of econazole

Drug susceptibility profiles of Mycobacterium tuberculosis isolates from Gulbarga, South India

Aim of the work: Drug resistance surveillance is a useful tool to assess the effective functioning of tuberculosis (TB) control program. This study was undertaken to know the first line anti tuberculosis drug susceptibility profile of Mycobacterium tuberculosis clinical isolates from the Gulbarga district of South India. Methods: Drug susceptibility test was performed for 102 clinical isolates of M. tuberculosis belonging to new (n = 62), treated (n = 22) and unknown treatment category (n = 18) of TB. All the isolates were tested for susceptibility to first line anti-tuberculosis drugs by minimum inhibitory concentration (MIC) and resistance ratio method (for streptomycin). Results: The susceptibility profile of M. tuberculosis to all five first line anti-tubercular drugs was found to be 60.78% (62/102). Overall, multi drug resistance (resistance to at least isoniazid and rifampicin) was observed in 8.82% (9/102) isolates and was found to be higher for treated cases (18.18%). Conclusions: High level of drug resistance observed in new cases for isoniazid, rifampicin and ethambutol suggests need for the implementation of drug resistance surveillance studies in order to document the success of the tuberculosis control program in reducing the level of drug resistance

Molecular typing of Mycobacterium tuberculosis isolates from a rural area of Kanpur by spoligotyping and mycobacterial interspersed repetitive units (MIRUs) typing

Molecular typing of Mycobacterium tuberculosis isolates has greatly facilitated the understanding of epidemiology of tuberculosis (TB). This study was done to characterize prevalent genotypes of M. tuberculosis on a collection of 97 isolates based on spoligotyping and mycobacterial interspersed repetitive units-variable number of tandem repeats (MIRU-VNTR) typing in rural area of Kanpur, North India. In this area different types of interventions are being undertaken and follow-up studies are progressing. Predominant spoligotypes prevalent in this region belonged to Central Asian-Delhi family (CAS1_Del) (37%), East African-Indian family (11%), T1 family (8%) and Beijing (4%) family. Highly distinct MIRU-VNTR genotypes were obtained. Significant spoligotypes such as Beijing and CAS1_Del type were further divided into subtypes with MIRU-VNTR. This preliminary study reveals that CAS is the most predominant family in this rural area of Kanpur. If confirmed in other areas, this combined approach of molecular typing can be preferably be used as first line tool for studying linkage and transmission dynamics of TB in India

Identification of Mycobacterium tuberculosis genes preferentially expressed during human infection

The identification of Mycobacterium tuberculosis genes, specifically expressed during infection is a key step in understanding molecular mechanism of mycobacterial pathogenesis. Such genes likely encode proteins required for mycobacterium’s survival and progressive infection within the host. In this study,we applied in-vivo-induced antigen technology (IVIAT) to M. tuberculosis and identified 11 putative in-vivo induced genes encoding for immunogenic proteins of diverse functions; these included transcriptional regulators (Rv1460 and Rv2565), biosynthesis and macromolecule metabolism (leuD, guaB1, plcC, hupB and glyS), polyketide synthases (pks6 and pks9), cell processes (ctpA) and one with unknown function (Rv3701c). Quantitative real time-PCR analysis of these genes in the specimens obtained from TB patients demonstrated induced expression of eight genes as compared with bacteria grown in-vitro. In addition, distribution of these genes in different strains of M. tuberculosis was analyzed using PCR and their nucleotide sequence alignments and they were found to be widely distributed among M. tuberculosis isolates including multiple-drug resistant (MDR) and extensively-drug resistant (XDR). This study identified several antigenic determinants of M.tuberculosis expressed during infection, which might help pathogens adapt to or counter hostile environments and suggesting their role during disease process