PREVALENCE, CHARACTERIZATION AND CLINICAL EVALUATION OF INDIGENOUS MYCOBACTERIUM TUBERCULOSIS

Abstract

A total six thousands five hundred and seventy three (6573) indigenous pulmonary and extra- pulmonary specimens were collected from tuberculosis suspicious patients of 17-67 years age group during November, 2004 to December, 200. The sputum, pus and bronchial washings were collected from five different sources, labeled and processed for initial screening. One hundred and seventy two (172) 2.616% of total (6573) tuberculosis diagnosed (AFB positive) patients were selected from six different sources. The patients were selected, regardless of their age, gender and previous therapeutic profile. The specimen comprised of 85% sputum, 10.5% puss and 4.5% bronchial washing. We considered 29.% female and 71% males with 84.% pulmonary (sputum, bronchial washing & puss) and 16% extra-pulmonary (puss & bronchial washing) specimens. Sixty six (66) resistant Mycobacterium tuberculosis strains were further studied to determine the highest level of resistance (in % age) . The clinical isolates were collected from cultured growth on Lowenstein Jensen media supplemented with antitubercular drugs at minimum inhibitory concentration (MIC) level. The parameters of study were the pattern of sensitivity/ resistance of mycobacterial TB against rifampicin, isoniazid, ethambutol and pyrazinamide, overall pattern of resistance, resistance percentages with respect of number of colonies, overall trend of resistance during Jan. - Dec. 2005, resistance pattern in percentage against five different levels (μg/ml) above their respective critical concentrations, therapeutical interpretation of drugs to evaluate the pharmacological credibility and molecular study of Pnc A gene of Mycobacterium tuberculosis responsible of resistance against pyrazinamide. The data obtained from this study showed 37 (21.5%) strains resistant and 135 (78.5%) strains sensitive to rifampicin, 25 (14.5%) strains resistant and 147 (85.5%) strains were sensitive to isoniazid, 10 (5.8%) resistant and 162 (94.2%) strains founded sensitive to Ethambutol, 47 (27.3%) resistant and 125 (72.7%) strains were founded sensitive to Pyrazinamide of total 172 clinical isolates of Mycobacterium tuberculosis. The resistance of Mycobacterium tuberculosis noted on basis of growth pattern (number of colonies) over the mycobacterial specific Lowenstein Jensen medium. Overall mono-resistance pattern was observed as 25.71% resistant to rifampicin, 8.57% resistant to isoniazid, 2.85% resistant to ethambutol and 62.85% resistant to pyrazinamide out of 20.34% mono-resistant isolates of total 172 Mycobacterium tuberculosis strains. Poly resistance profile obtained was as 19.35% Mycobacterium TB strains resistant to rifampicin & isoniazid, 22.58% resistant to isoniazid & pyrazinamide, 3.22% resistant to ethambutol & pyrazinamide, 6.45% resistant to isoniazid & pyrazinamide, 22.58% resistant to rifampicin, isoniazid and pyrazinamide, 3.22% resistant to rifampicin, ethambutol and pyrazinamide and 22.58% resistant to all of the four 1st line drugs. The resistant Mycobacterium TB having an ultimate highest level of resistance against the first line antitubercular drugs. Which were interpreted therapeutically to study the pharmacological suitability of dosage and regimen. It was observed that no any rifampicin strain inhibited at 1st and 2nd drug levels. 40.54% resistant Mycobacterium -TB strains inhibited at 3rd rifampicin level of 120ug/ml. Practically it is not feasible to maintain a plasma concentration higher than therapeutic range of 6.5±3.5ug/ml (Joel et al., 2001). It was observed that no any isoniazid strain inhibited at 1st, 2nd and 3rd drug levels. There 28% resistant Mycobacterium-TB strains inhibited at 4th isoniazid level 9ug/ml. Maximally plasma concentration that can be maintained in body is - 4ug/ml (Richard et al., 2006), therefore it can not be used in actual practice. It was observed that no any ethambutol strain inhibited at 1stand 2nd drug levels 2ug/ml and 4ug/ml. 50% resistant Mycobacterium TB strains inhibited at 3rd level of 6ug/ml. The maximum plasma concentration (Cmax) that can be maintained in tuberculosis patient during treatment protocol are described by other researchers as 3-5ug/ml (Bertram G. Katzung, 2004), 2-5ug/ml (Leon et al., 2004) and 4- 6ug/ml (Richard et al., 2006). It was observed that no any pyrazinamide resistant strain inhibited at 1st and 2nd drug levels100ug/ml and 200ug/ml. 27.66% pyrazinamide resistant Mycobacterium TB strains were inhibited at 3rd pyrazinamide level of 300ug/ml. The maximum plasma concentration than can be maintained in human body reported by different researchers are 9- 12ug/ml (Joel et al, 2001), 19ug/ml (Leon et al., 2004), 30-50ug/ml (Bertram, 2004), 37-40ug/ml (Richard et al., 2006). The genomic DNA of pyrazinamide resistant Mycobacterium TB extracted by mechanical method and examined on gel. PCR for Mycobacterium TB is specific for Mycobacterium TB complex DNA. By using the SSCP (Single Strand Conformational Polymorphism), we were able to show most divers pattern. The resistant 17.44% showed different pattern than sensitive samples. Which indicate the mutation in this domain, while 9.88% did not show any difference in mobility in comparison to sensitive samples.