|Year : 2018 | Volume
| Issue : 1 | Page : 32-37
Prevalence and first-line drug sensitivity trends of Mycobacterium tuberculosis at a tertiary center in North-East India
Amit Banik, Niladrisekhar Das, Valarie Wihiwot Lyngdoh, Anil Chandra Phukan, Vikramjeet Dutta
Department of Microbiology, North Eastern Indira Gandhi Regional Institute of Health and Medical Sciences (NEIGRIHMS), Shillong, India
|Date of Submission||02-Oct-2017|
|Date of Acceptance||21-Dec-2017|
|Date of Web Publication||21-Mar-2018|
Room# 29, GB Pant Hospital, Andaman and Nicobar Islands Institute of Medical Sciences, Port Blair, Andaman and Nicobar
Source of Support: None, Conflict of Interest: None
Setting Tuberculosis (TB) still remains one of the major health problems facing humans. India accounts for almost a quarter of TB cases worldwide. The scenario is worsening owing to multidrug resistant-tuberculosis (MDR-TB). Assessment of local prevalence rates and detection of MDR-TB are important to rationalize therapy and prevent spread of resistant strains in community.
Objective The study was undertaken to understand drug sensitivity patterns of tubercle bacillus and assess resistance trends in Meghalaya.
Designs Specimens were screened for acid-fast bacilli, decontaminated by N-acetyl cysteine–sodium hydroxide method, and subsequently inoculated onto Lowenstein–Jensen media. Characteristic growth was biochemically identified as TB bacillus. Drug sensitivity assessment to first-line anti-TB drugs was performed by proportion method and sensitivity patterns noted.
Results Among 103 specimens received, 23 showed acid-fast bacilli. Male to female ratio was 3 : 2. Fourteen (13.6%) pure isolates of Mycobacterium spp. were obtained. Biochemically 10 isolates were confirmed as M. tuberculosis. Drug sensitivity profile revealed highest mono resistance to isoniazid and streptomycin. Two (20%) isolates were MDR.
Conclusion The study gives a brief overview of the menace of TB in Meghalaya. The study results provide valuable information about presence of primary MDR-TB and provide basis for future larger field surveys.
Keywords: Lowenstein–Jensen medium, multidrug-resistant tuberculosis, Meghalaya, tuberculosis, Ziehl–Neelsen staining
|How to cite this article:|
Banik A, Das N, Lyngdoh VW, Phukan AC, Dutta V. Prevalence and first-line drug sensitivity trends of Mycobacterium tuberculosis at a tertiary center in North-East India. Egypt J Chest Dis Tuberc 2018;67:32-7
|How to cite this URL:|
Banik A, Das N, Lyngdoh VW, Phukan AC, Dutta V. Prevalence and first-line drug sensitivity trends of Mycobacterium tuberculosis at a tertiary center in North-East India. Egypt J Chest Dis Tuberc [serial online] 2018 [cited 2020 Apr 3];67:32-7. Available from: http://www.ejcdt.eg.net/text.asp?2018/67/1/32/228132
| Introduction|| |
Tuberculosis (TB) is a disease of great antiquity and has almost certainly caused more suffering and death than any other infection . It is a major public health problem owing to its high risk of person-to-person transmission, morbidity, and mortality. TB still remains one of the major health miseries facing humans, particularly in developing countries. It is the second most common cause of death from infectious disease at global level, the first being HIV/AIDS Most deaths of TB occur in the developing countries and it affects the young ones in their productive years of life . India, Indonesia, China, Nigeria, Pakistan, and South Africa were the top six countries with the largest number of incident cases in 2015. India records the highest burden of both TB and multidrug resistant-tuberculosis (MDR-TB) . Estimates reveal that ∼40% of Indian population is infected with TB bacillus. The estimates of TB for India has been revised upward based on the newer evidences gained. Moreover, these revisions are interim in nature, with further changes likely when India conducts its first national TB prevalence survey in 2017–2018 . The situation has worsened owing to the emergence of MDR-TB and HIV-TB coinfection. High prevalence of MDR-TB in certain groups and regions has been a subject of major concern for public health administrators and clinicians .
Besides disease burden, TB also causes an enormous socioeconomic burden. It primarily affects people in most productive years of life, commonly among the poorest and marginalized sections of community. Almost 70% of patients with TB fall within the 15–54 years age group. Direct and indirect costs of TB are huge. On average, each TB case incurs an economic burden of around US$ 12 235 and a health burden of around US$ 4.1 disease-adjusted life years. Similarly, a death from TB in India incurs an average burden of around US$ 67 305 and around US$ 21.3 disease-adjusted life years .
Assessment of local prevalence rates and detection of MDR-TB is important to devise optimum drug therapy and prevent spread of resistant strains in community. Data on drug resistance profile from North-East India are insufficient and inadequate. This region is unique with respect to its ethnicity, geographical location, topography, and climatic condition unlike the rest of India. The study was therefore undertaken with the intention of determining the prevalence of primary TB, understanding drug susceptibility pattern of Mycobacterium tuberculosis isolates to first-line antitubercular drugs and assessing drug-resistance trends of strains prevalent in Meghalaya.
| Materials and methods|| |
The present study was conducted in the Department of Microbiology, NEIGRIHMS Hospital, Shillong, from May 2013 to October 2014. The hospital is the only tertiary-level hospital in the state of Meghalaya. A total of 103 clinical specimens received from patients with newly suspected TB attending the hospital were included in the study. Patients with any prior history of antituberculous medications were excluded. Pulmonary specimens included sputum, bronchoalveolar lavage, and gastric lavage fluids. Extrapulmonary specimens included sterile body fluids and aspirated pus.
Specimens were collected in sterile, leak-proof, disposable, appropriately labeled plastic containers without any fixatives. The specimens were appropriately processed in a class II biosafety cabinet. Smears were made on new clean grease-free glass slides and subjected to Ziehl–Neelsen staining. Samples were decontaminated by the N-acetyl cysteine–sodium hydroxide method. Adequate amount (2–3 ml) of sputum sample was mixed with double the volume of freshly prepared 4% NaOH solution in a graduated centrifuge tube. Tubes were allowed to stand for 15 min at room temperature for decontamination. Contents were then centrifuged in aerosol-free centrifuge cups at 3000g for 15 min. It was ensured that the contact period with the digestant was limited to 30–35 min. Roughly, 6–8 ml of sterile distilled water was added to the tubes, mixed thoroughly and centrifuged again at 3000g for 15 min. The supernatant was decanted, and the sediment obtained was inoculated onto two Lowenstein–Jensen (LJ) media slants (HiMedia Laboratories, Mumbai, India). Samples from sterile sites or obtained by sterile procedures were directly inoculated on to the slants without decontamination. Culture slants were examined on alternate days in first week, and weekly thereafter for a period of 8 weeks . All LJ slants growing characteristic rough, tough, and buff colored colonies were selected for acid-fast smear and identification for M. tuberculosis employing standard biochemical tests, for example, heat-stable catalase test, nitrate reduction test, and niacin accumulation test.
Drug susceptibility testing (DST) was done by employing LJ slants with TB drugs first-line kit SL023-KT (HiMedia Laboratories). DST of M. tuberculosis isolates was performed by the simplified economic variant of proportion method. Five first-line drugs with critical concentrations were tested along with two control slants, namely, isoniazid (H: 0.2 μg/ml), rifampicin (R: 40 μg/ml), ethambutol (E: 2 μg/ml), pyrazinamide (Z: 200 μg/ml), and streptomycin (S: 4 μg/ml) ([Figure 1]). A loopful of bacterial suspension matching McFarlands standard no. 1 opacity with a concentration of 1 mg/ml of tubercle bacilli and two appropriate dilutions (10−2 and 10−4 of undiluted suspension) of the bacilli were inoculated on drug-containing and drug-free media with a 3 mm diameter 24 SWG loop. The inoculated slants were incubated at 37°C. DST slants were read at 28 days and again at 42 days. Growth was recorded as follows: confluent growth=3+, more than 100 colonies=2+, and 1–100 colonies=recording of actual number of colonies. The ratio of number of colonies observed on the drug-containing media to drug-free medium indicates proportion of resistant bacilli present in the strain. Below a certain proportion (critical proportion=1%), the strain is classified as sensitive, and above as resistant to each individual first-line antituberculous drug . To validate the studies, reference strain of M. tuberculosis H37Rv was used as control. The study was approved by the Institutional Ethics Committee, and proper informed consent was taken from each patient.
|Figure 1 Lowenstein–Jensen control and drug slants with first-line antitubercular drugs showing characteristic Mycobacterium tuberculosis colonies. C, control; ETH, ethambutol; INH, isoniazid; PZA, pyrazinamide; RIF, rifampicin; STR, streptomycin.|
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| Results and observations|| |
A total of 103 clinical specimens were received from newly suspected TB patients attending NEIGRIHMS hospital during May 2013 to October 2014. Overall, 100 were pulmonary specimens, and the remaining three were extrapulmonary specimens (two synovial fluid specimens and one pus specimen). The distribution of different clinical wards and outpatient departments from where specimens were received is depicted in [Table 1]. This included 63 (61.1%) male and 40 (38.9%) female patients. The ratio of male to female was ∼3 : 2. The mean age of patients was 37.28±16.64 years (range: 8–83 years). Age wise and sex wise distribution of the study population is depicted in [Table 2]. Acid-fast bacilli (AFB) were detected in 23 specimens with various grades of bacillary loads as shown in [Table 3]. A total of 22 cases were from pulmonary specimens and one from a synovial fluid specimen. Fourteen (13.6%) pure isolates of Mycobacterium spp. were obtained from 103 patient specimens received. These included five (35%) from female and nine (65%) from male patients. The average time to detection for culture positive isolates was 22.9 days, with the fastest isolation recorded in just 12 days and longest at 36 days.
|Table 1 Distribution of specimens from various clinical departments (n=103)|
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|Table 2 Age, sex wise distribution of patients from whom specimens were obtained (n=103)|
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The correlation between smear results and culture positivity is depicted below in [Table 4]. Among 14 pure isolates of Mycobacterium spp. on whom biochemical tests were conducted, 10 isolates were confirmed to be M. tuberculosis isolates.
Drug sensitivity testing was done using simplified economic variant of proportion method on LJ medium slants for five first-line antitubercular drugs (HiMedia Laboratories). Mono resistance was detected for each of the five drugs tested. Combined drug resistance was also observed.
Resistance to an individual drug was highest for isoniazid (60%), followed by streptomycin (30%). Least resistance was seen for rifampicin, ethambutol, and pyrazinamide (20% each). Combined resistance to isoniazid and rifampicin was seen in two isolates of 10 (20%) designated as MDR-TB strains. Resistance to combination of five, four, and two drugs was seen in one (10%) isolate each respectively and resistance to a solitary drug was noted in four (40%) isolates. Mono resistance was noted mostly with isoniazid (three isolates) and with a single isolate for pyrazinamide. Similarly, three (30%) isolates were pan sensitive. The distribution of the drug resistant pattern is shown in [Table 5].
|Table 5 Drug susceptibility test results for Mycobacterium tuberculosis isolates (n=10)|
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| Discussion|| |
This study claims to be the first one to have established and performed DST by conventional solid media culture in North-East India, and results obtained are one of the first from a hospital-based study about culture and drug sensitivity testing of M. tuberculosis in the state of Meghalaya.
In our study, we observed a male preponderance among the cases in a ratio of 3 : 2 compared with females. Although this may not be significant, it is consistent with another study that showed ratio of 3.7 : 1 . The reason for this may be the more number of hours of outdoor exposure and more challenging and hazardous working environments for men. In our study, of all suspected TB cases, 67.5% females and 66.67% males were from the 21 to 50 years age group as against 56% and 70%, respectively, in the study from South India . This can be explained by the fact that people of this age bracket are economically most productive, employed in all kinds of jobs, and come in contact with possible infectious environments. Furthermore, the authors noted the rates increased consistently till 45–49 year age group for females, thereafter remaining a plateau, a fact that is corroborated by our study too.
Smear microscopy is an important adjunct of TB diagnosis, particularly in low-resource settings. In the present study, randomly selected new patients having clinical suspicion of TB were evaluated by microscopy for the presence of AFB by Ziehl–Neelsen staining method. Amongst 103 samples received, 23 (22.3%) samples yielded AFB in smears. Twelve among these were subsequently culture positive and another 11 could not grow on culture slants. Seven among these smear positive but culture negative specimens were lost owing to contamination with other bacteria and fungi and liquefaction of media within the first week of incubation. The remaining four cases that were smear positive but turned out to be culture negative may be because the smear demonstrated dead bacilli or the patients may have been already started on antitubercular drugs. However, in low-resource settings, where nonavailability of culture facilities is a deterrent, empirical or even antitubercular therapy can be started based on the positive smear microscopy reports.
Conventional culture on LJ medium using N-acetyl cysteine–sodium hydroxide decontamination method yielded pure growth in 14 (13.6%) specimens. Twelve among these were smear positive and two were smear negative; the negative smear result being explained by very low bacillary counts in the samples. This fact validates that culture is indeed more sensitive than smear microscopy and rightly considered as the gold standard . The yield of positive viable cultures in our study was 13.6%. This figure is close to the data revealed by Sethi et al.  (12.9%) from a tertiary care center in Chandigarh.
Contamination rate observed in our study was 6.79%. Widespread variation exists in the contamination rates for conventional culture media. Studies by Uddin et al.  (5.07%), Roberts et al.  (5.2%), Tortoli et al.  (8.90%), Hanna et al.  (21.1%) have shown moderate to high contamination rates whereas studies by Scarparo et al.  (4.3%) and Somoskovi et al.  (1.2%) have documented low or minimal contamination rates.
In our study, drug sensitivity testing for five first-line antitubercular drugs revealed individual drug resistance to isoniazid in six (60%) isolates, streptomycin in three (30%), and two (20%) each for rifampicin, ethambutol, and pyrazinamide. Combined isoniazid and rifampicin resistance strains, that is, MDR-TB strains, were found in two (20%) isolates, which is very high for new suspected TB cases considering Indian scenario, as WHO reports put a figure of 2.5% for the same. Even the primary rifampicin resistance at national level is 6% compared with 20% recorded in our study . However, as the sample size was small, this may not truely reflect actual disease prevalence and drug resistant trends in this part of the country.
Sethi et al.  observed primary drug resistance to isoniazid at 26.4%, streptomycin at 28.1%, ethambutol at 14.9%, and rifampicin at 9.9%. Proportion of MDR-TB isolates was 9.9% in new cases (primary drug resistance) and 27.6% in old cases (acquired drug resistance). Menon et al.  pegged acquired MDR at 47.5% from a study in Mumbai. Similarly, prevalence of acquired drug resistance ranged from 25 to 100% in Indian studies . The results of our study when compared with these are very high. Such variation may be because of varied geographical distribution, circulating strain patterns, and demographic, ethnic, and epidemiological differences.
Jain et al.  reported 42.8% resistance to isoniazid and 29.7% resistance to rifampicin. Overall, 27% showed multidrug resistance. Resistance to any drug was observed at 54.4%, which is 70% in our study. Interestingly, according to this study, figures for mono resistance to isoniazid and rifampicin and MDR have all shown a steady declining trend from 2009 to 2012.
A review  of the Indian situation by some researchers from Tuberculosis Research Centre, Chennai, concluded that the magnitude of drug-resistance problem is principally owing to acquired resistance. They had shown that in Gujarat, resistance to rifampicin has increased from 2.8 to 37.3%, to isoniazid from 34.5 to 55.8%, and magnitude of MDR-TB was of the order of 30% over a period of 6–7 years. Similarly, Institute of Thoracic Medicine, Chennai, showed multidrug resistance of 20.3% in various TB centers of Tamil Nadu. Primary combined resistance of isoniazid and rifampicin in different parts of India varies widely as shown by Abraham et al.  (4.8%), Paramasivan et al.  (23.4%, North Arcot) and (18.4%, Pondicherry), Kalo et al.  (30.8%), Sophia et al.  (13.7%), Moitra et al.  (13.4%), and Prasad et al.  (15.6%), the figures of which are close to our results.
Mathuria et al.  in a multicentric study at three different centers reported primary resistance to isoniazid ranging from 14.3 to 37.5%. Similarly primary resistance to rifampicin varied from 7.1 to 25 and 7.1 to 13.3% for MDR. Agatha et al.  observed individual primary resistance of 11.4, 14.3, 12.9, and 5.7% to isoniazid, rifampicin, streptomycin, and ethambutol, respectively. All isoniazid resistant isolates were from MDR-TB. In our study, the high rate of resistance obtained may be because some patients might have been unaware of previous treatment leading to acquired resistance or may have defaulted very early in treatment regime and the same information was concealed on subsequent visits.
| Conclusion|| |
The study uncovers the presence of highly resistant pattern of antitubercular drug sensitivity in Meghalaya. Besides unravelling the presence of a high degree of primary MDR, the study also highlights the need for introduction and development of more robust culture and drug sensitivity facilities for TB. The information obtained may not reflect the actual prevalence of the disease burden and resistance patterns in this region, but it would surely help plan larger in-depth studies with a large sample size to define the same. This would help bring out newer epidemiological and clinically relevant knowledge to devise novel ways to decimate the huge socioeconomic effect of this dreaded disease.
Financial support and sponsorship
Aid in part from RNTCP, India.
Conflicts of interest
There are no conflicts of interest.
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[Table 1], [Table 2], [Table 3], [Table 4], [Table 5]