https://imcjms.com/public Ibrahim Medical College Journal of Medical Science https://imcjms.com/public/registration/journal_full_text/69 Tue, 02 Aug 2016 12:06:05 +0000 The present study was undertaken to determine the drug resistance pattern of M. tuberculosis isolated from 225 pulmonary and 45 extrapulmonary tuberculosis cases. The samples were cultured on Lowenstein Jensen (L-J) media for isolation of M. tuberculosis. Drug resistance to first line anti tubercular drugs- namely isoniazid (INH), rifampicin (RIF), Ethambutol (ETH) and streptomycin (SM) were determined by indirect proportion method. The overall drug resistance of M. tuberculosis was 53.6% to any of the first line anti tubercular drugs. Rate of multi drug resistant tuberculosis (MDR-TB) among the untreated cases was 4.2%, while it was 36.0% in previously treated cases. It was found that 83.3% rifampicin resistant M. tuberculosis was cross resistant to one or more of other first line anti-tubercular drugs, while cross resistance of INH, ETH and SM resistant isolates was much low. The present study revealed that high level of drug resistance exists to individual anti tubercular drugs and MDR-TB is an emerging problem, particularly in treated cases. Rifampicin resistance could be used as a surrogate marker for drug resistance to other first line anti tubercular drugs. Address for Correspondence:Prof. J. Ashraful Haq, Professor, Department of Microbiology, Ibrahim Medical College, 122 Kazi Nazrul Islam Avenue, Shahbag, Dhaka-1000. e-mail: jahaq54@yahoo.com Introduction In Bangladesh, TB remains a major public health problem. Over 300,000 new cases of TB and 70,000 deaths are estimated to occur per year in Bangladesh and the country ranks 6th out of the 22 highest TB burden countries of the world.2 The estimated incidence and prevalence rate of all forms of TB were 223 and 387 per 100,000 population respectively. The estimated death rate was 45 per 100,000 population. Globally the median prevalence of drug resistance to any drug in untreated cases was the highest (19.8%) in South East Asia (SEA) followed by Western Pacific (11.4%) and Europe (8.4%). The median prevalence of drug resistance to any drug in treated cases was the highest (63.3%) in the Eastern Mediterranean followed by SEA (39.9%) and (in Europe (15.9%). The rate of MDR- TB ranged from 4.7%-48.3% in above regions.5 The present study was undertaken to determine the rate of drug resistance of MTB to first line anti-tubercular agents in patients attending tertiary care hospitals of Dhaka city. The study also investigated the concomitant resistance of MTB among rifampicin resistant MTB. Materials and Methods   The early morning sputum samples were collected in clean, sterile wide mouthed container closed with lid. The quantity of sputum collected from each patient was 2 – 5 ml. The LN aspirates were collected aseptically in 50 ml of sterile Falcon tubes containing 3 ml sterile distilled water in each container. The containers were labeled with patient’s name, identification number and date. The samples were brought to the department of Microbiology, BIRDEM, Dhaka as soon as possible, where necessary laboratory tests were done after processing the samples in Class 2 bio-safety cabinet. The processed products of the samples were kept in 3 different eppendorf tubes for: a) Ziehl-Neelsen (ZN) stain, b) culture of mycobacteria on Lowenstein Jensen (L-J) media and c) rapid detection of mycobacteria by PCR method. Smear was stained by ZN method for the detection of acid fast bacilli (AFB). Culture was done by inoculating it on L-J media and incubating it at 370C for isolation of mycobacterium. The culture bottles were examined weekly for 8 weeks for the evidence of growth. On appearance of visible colonies, the colony morphology, rate of growth and pigment production were noted. The growth of M. tuberculosis was identified by staining of colonies with ZN stain and confirmed by necessary biochemical tests.6 Drug susceptibility test   The number of colonies on control and drug containing media were counted and the percentage of the resistant organisms was calculated as follows: If the percentage of resistant organism was 1% or more, then the isolate was considered resistant to the specific drug. A set of tubes with and without drugs were incubated with reference strain M. tuberculosis H37Rv as a quality control. Results Table-1 shows the results of culture of the study samples. Out of the total 300 samples, sputum sample was 255 of which 180 (70.59%) were culture positive, 40 (15.69%) were culture negative and 35 (13.72%) became contaminated. Among the 45 lymph node aspirates 20 (44.45%) were culture positive, 15 (33.33%) were culture negative and 10 (22.22%) became contaminated. Table-2 shows the species distribution of culture positive mycobacteria in sputum and LN aspirates. Out of 180 culture positive isolates from sputum 176 (97.8%) were M. tuberculosis and 4 (2.2%) were mycobacterium other than tuberculosis (MOTT). Out of 20 isolates from lymph node aspirates 16 (80.0%) were M. tuberculosis and 4 (20.0%) were MOTT. Table-1: Results of culture of study samples   Table-2: Species distribution of culture positive Mycobacteria in sputum and LN aspirates  Overall susceptibility pattern of M. tuberculosis and MOTT to first line anti-TB drugs are depicted in Table-3. Out of 192 M. tuberculosis isolates 89 (46.35%) were sensitive to all of the four first line anti-TB drugs and 103 (53.65%) were resistant to any of the four first line anti-TB drugs. In case of the MOTT, all 8 (100%) were resistant to any of the first line anti-TB drugs. Table-3: Overall susceptibility pattern of M. tuberculosis and MOTT to first line anti-TB drugs       Table-5 shows resistance pattern of 167 M. tuberculosis isolates to 4 first line anti-TB drugs in untreated cases. Out of the total 167 isolates, 53 (31.74%) were resistant to one drug, 19 (11.38%) were resistant to two drugs, 3 (1.80%) were resistant to three drugs and 3 (1.80%) were resistant to four drugs. Table-6 shows resistance pattern of M. tuberculosis to four first line anti-TB drugs in previously treated cases. Out of the total 25 isolates, 4 (16.0%) were resistant to one drug, 13 (52.0%) were resistant to two drugs, 1 (4.0%) was resistant to three drugs and 7 (28.0%) were resistant to four drugs. Table-5: Resistant pattern of M. tuberculosis to 4 first line anti-tubercular drugs isolated from untreated tuberculosis cases (n=167)     Table 7 shows the rate of MDR-TB in untreated and treated pulmonary TB cases. Among the untreated cases, MDR-TB was 4.2% while it was 36.0% among the treated cases. The rate was significantly higher in previously treated group. The rate of concomitant resistance pattern of RIF resistant M. tuberculosis to INH, ETH and SM are described in Table-8. It was observed that 83.3% RIF resistant M. tuberculosis isolates were resistant to other three drugs. The association of RIF resistance with resistance to other three drugs were significantly associated (p<.05). The concomitant resistance of INH, ETH and SM resistant M. tuberculosis to any other three drugs were 55.5-74.3% and the co-resistance was not significantly associated (P>0.05). Table-7: Rate of isolation of MDR-TB from untreated and treated pulmonary tuberculosis cases      Table-9: Rate of concomitant resistance of RIF / INH / ETH /SM sensitive M. tuberculosis to corresponding drugs Table-9 shows the concomitant resistance rate of M. tuberculosis to any three first line anti-TB drugs which were sensitive to RIF, INH, ETH or SM. Rate of resistance to three other drugs ranged from 34.78% to 43.21% among RIF, INH, ETH or SM sensitive isolates. Discussion Monitoring of drug resistance pattern, early diagnosis and initiating prompt treatment has been the mainstay to interrupt the transmission of tuberculosis. In this context, the present study was designed to determine the drug resistance pattern of mycobacterium. In the present study, about 70.0% sputum samples yielded positive culture results on L-J media. Various authors have reported similar culture positivity rate in L-J media which ranged from 59.72 to 87.2%.8-11 However, the culture positivity rate was only 44.0% in lymph node aspirate samples. The failure to isolate mycobacteria in about 30-56% sputum and lymph node aspirates was due to contamination of media or damage to organisms during decontamination process. Previous studies reported the contamination rate from 1.2% to 27.2%.9-13 Therefore, the isolation rate of mycobacteria can be increased if contamination is prevented and sample processing procedure is further improved. Out of the 200 isolates of mycobacteria, 96.0% were M. tuberculosis and 4.0% were MOTT. Earlier, a study in Dhaka by Miah et al. reported 95.3% isolates as M. tuberculosis and 4.7% as MOTT.3 The resistance pattern of first line anti-tubercular drugs observed in the present study among untreated cases was almost similar to the resistance pattern reported previously in 2000 and 2007.3,4 Almost similar rate of resistance was observed in other neighboring countries.14,15 In the present study, out of 30 RIF resistant M. tuberculosis, 83.3% were also concomitantly or cross resistant to other three first line anti-tubercular drugs (p<0.05; Table-8). On the other hand, of the 50 INH resistant M. tuberculosis, 64.0% were concomitantly or cross resistant to other three first line anti tubercular drugs (p>0.05) while for ETH and SM the rate was 74.3% and 55.5% respectively. Resistance to RIF in M. tuberculosis occurs in a high frequency and mono resistance to RIF is rare, whereas mono resistance to INH is common.17 It has been proposed that resistance to RIF can be used as a surrogate marker for MDR-TB as nearly 90% of the RIF resistant strains are also INH resistant.17,18 It is to be noted that only 43.21% M. tuberculosis isolates which were sensitive to RIF, was concomitantly resistant to other 3 drugs (Table-9). This indicates that a sensitive M. tuberculosis isolates (sensitive to RIF, INH, ETH and SM) could be resistant to any of the three other first line anti-TB drugs and it could not therefore, predict that if an isolate sensitive to any single first line drug would simultaneously be sensitive to other three drugs.   1.    World Health Organization Report 2010: Global tuberculosis control. World Health Organization, 1211 Geneva 27, Switzerland. 2010; 5-7. 3.    Miah MR, Ali MS, Saleh AA, Sattar H. Primary drug resistance pattern of mycobacterium tuberculosis in Dhaka, Bangladesh. Bangladesh Med Res Council Bull 2000; 26: 33-40. 5.    World Health Organization: Global tuberculosis control: surveillance, planning and financing. World Health Organization, 1211 Geneva 27, Switzerland 2006a; 362. 7.    Velayati AA, Masjedi MR, Farnia P, Tabarsi P, Ghanavi J, Ziazarifi AH, et al. Emergence of new forms extensively drug-resistant tuberculosis bacilli: super resistant strains in Iran. Chest 2009; 136: 420-25. 9.    Hanna BA, Ebrahimzadeh A, Elliot LB, Morgan MA, Novak SM, Rusch-Gerdes S, et al. Multicentre evaluation of BACTEC MGIT 960 system for recovery of mycobacteria. J Clin Microbiol 1999; 37: 748-52. 11.  Uddin MN, UddinMJ, Mondol MEA, Islam SMJ, Wadud ABM. Comparison of conventional and automated culture system for isolation of Mycobacterium tuberculosis. JAFMC Bangladesh 2009; 5: 14-17. 13.  Chien HP, Yu MC, WU MH, Lin TP, Luh KT. Comparison of the BACTEC MGIT 960 with Lowenstein Jensen media for recovery of mycobacteria from clinical specimens. Int J Tuberc Lung DIS 2000; 4: 866-70. 15.  Iqbal R, Shabbir I, Khan SU, Saleem S, Munir K. Multidrug resistance tuberculosis in Lahore. Pak J Med Res 2008: 47: 22-25. 17.  Somoskovi A, Parsons LM and Salfinger M. The molecular basis of resistance to isoniazid, rifampicin and pyrazinanide in Mycobacterium tuberculosis. Respir Res 2001; 2: 164-68. 2026 Ibrahim Medical College. All rights reserved.