This study was a subcomponent of a prospective microscopy study undertaken in Bhubaneswar, the state capital of Odisha from January 2014 to September 2014 in collaboration with state and district TB department, Government of Odisha. During this period, suspected TB patients attending Designated Microscopy Center (DMC) of Capital hospital, Bhubaneswar were interviewed with the standard questionnaire to determine their history of anti-TB drug therapy and were classified as new or old cases based on treatment history. Patients unwilling to take part in the study or denying signing the consent form were not included in the study. Sputum specimens of 850 suspected TB patients were collected from the DMC after the DMC laboratory technician finished his/her smearing for Ziehl-Neelsen (ZN) microscopy. The sputum specimens were transported same day from DMC to the NRL of RMRC, Bhubaneswar for AFB microscopy by Ziehl-Neelsen (ZN) and Auramin staining. All the sputum specimens irrespective of their AFB status were processed for solid culture by modified Petroff’s method up to May 2014 after which they were processed by N-acetyl-L-cysteine-Sodium hydroxide (Nalc-NaOH) method due to introduction of Line Probe Assay (LPA) in the laboratory. After processing, a loopfull of deposit was inoculated on two LJ slopes and one LJ slope with p-Nitrobenzoic Acid (PNB). The culture slants were incubated at 37°C. All the slopes were observed for occurrence of growth daily for first week and then at weekly intervals for eight weeks. The M. tuberculosis isolates were confirmed by the rate of growth, optimum temperature of growth, colony morphology, pigmentation, growth in PNB, catalase and niacin test. Drug susceptibility was carried out as per RNTCP guidelines following proportion method [9]. Briefly colonies from surface of LJ medium were transferred into sterile test tubes containing 6-8 glass beads. The suspension was adjusted to 1 McFarland standard. The final concentration of drugs were; 0.2μg/ml for Isoniazid (H), 40μg/ ml for Rifampicin (R), 2μg/ml for Ethambutol (E) and 4μg/ml for Streptomycin (S). H37Rv was used as internal control for drug susceptibility testing. During this period the laboratory passed external quality testing by Supra National Reference Laboratory, National Institute for Research in Tuberculosis (NIRT), Chennai. All the activities like reagent and media preparation were carried out as per standard operating procedures. The ethical committee of RMRC Bhubaneswar approved the study protocol.

Out of the 850 cases, 161 patients showed positive growth on LJ medium. Out of these culture positives, 53 were smear negative and 10 were scanty and rest were having higher grades of AFB load in the specimen [Table/Fig-1]. The age of the 161 patients ranged between 10 and 82 years (Mean ± SD, 39.84 ± 15.05, Median 40). Among them 72.0% were males and 72.7% were newly diagnosed cases [Table/Fig-2]. The age of newly diagnosed cases (n=117) ranged between 10 and 82 years (Mean ± SD, was 40.45 ± 16.09, Median 40), and previously treated cases (n=44) ranged between 19 and 63 years (Mean ± SD, 38.20 ± 11.85, Median 40). On drug susceptibility testing of these 161 cases, three cases showed resistance to rifampicin and isoniazid. The single MDR-TB case detected among the 117 newly diagnosed cases was an 80 year male, who showed resistance to Streptomycin (S), Isoniazid (H), Rifampicin (R), and Ethambutol (E). Thus, the prevalence of MDR-TB among new sputum positive pulmonary TB patients was 0.85%. The prevalence of MDR-TB observed among 44 previously treated cases were 4.54%. Prevalence of mono resistance to streptomycin, isoniazid, rifampicin and ethambutol in new cases were 3.41%, 2.56%, 0 and 0.85% respectively [Table/Fig-3]. While mono resistance to streptomycin, isoniazid, rifampicin and ethambutol in previously treated cases were 2.27%, 13.6%, 2.27% and 0 respectively and one showed resistance to both streptomycin and isoniazid in previously treated as well as in new case.

The emergence of MDR-TB is a challenge to TB control programmes globally. India is one of the highest MDR-TB burden countries in the world with an estimated 99,000 incidence of MDR-TB cases. In order to plan effective control measures for MDR-TB, data from various areas across the country are needed. The prevalence of MDR-TB varies between 0.07 to 5.7% across India [2,3,5–8,10] and our findings of 0.85% also falls in this range. However few areas like urban Mumbai reported very high prevalence of 24% of MDR-TB in new cases [4]. In the present study it was observed that out of 161 symptomatic pulmonary TB patients found positive by LJ culture, 18.9% were smear negatives and 6.2% were having very low levels of mycobacteria (scanty smears) proving the superiority of culture over microscopy. The study reported mono resistance of 3.41%, 2.56%, 0 and 0.85% in new and 2.27%, 13.6%, 2.27% and 0 in previously treated TB cases to streptomycin, isoniazid, rifampicin and ethambutol respectively. The observed 0.85% prevalence of MDR TB in new cases was slightly higher than the previous studies [7,8] that reported 0.7% and 0 prevalence in Odisha. Prevalence of 4.1% MDR-TB in the present study of previously treated individuals was also lower than the earlier study in Odisha that reported 8.1% MDR-TB [8]. It was advocated that, in areas reporting high MDR- TB prevalence may be due to high transmission from primary DR-TB cases rather than failure of Directly Observed Treatment Short-course (DOTS) [11]. A study carried out in Sahariya tribe of Madhya Pradesh showed that in spite of high TB prevalence (3000/1,00,000 population), MDR-TB prevalence was 2.2% in newly diagnosed cases [12]. In order to maintain the low levels of MDR-TB, it will be necessary to diagnose DR-TB cases early and render DOTS Plus treatment promptly. In the present scenario with the availability of Cartridge Based Nucleic Acid Amplification Tests (CB- NAAT) to district level hospitals, the diagnosis of MDR-TB will be accelerated resulting in early DOTS plus treatment initiation and blocking of DR-TB transmission.

The major limitation of the present study is the small sample size and therefore, it is not representative of the population at large. In fact, this limitation was observed in most previous studies on MDR-TB.

Our findings suggest, the persistence of low level of MDR-TB prevalence in the state and good adherence to the basic DOTS. This low level can be maintained by early diagnosis of MDR-TB cases by CB NAAT in district level and prompt initiation of DOTS Plus regimen in the state of Odisha.