|Year : 2010 | Volume
| Issue : 3 | Page : 99-103
Drug susceptibility profiles of Mycobacterium tuberculosis isolates at Mangalore
Sevitha Bhat1, M Radhakrishna1, Mohan Kotian2, Sunil Rao2
1 Department of Microbiology, Kasturba Medical College, Mangalore, Karnataka, India
2 Department of Microbiology, Yenepoya Medical College, Mangalore, Karnataka, India
|Date of Web Publication||8-May-2012|
Department of Microbiology, Kasturba Medical College, Light House Hill Road, Mangalore - 575 001, Karnataka
Background and Objectives: Tuberculosis (TB) remains one of the major causes of death from a single infectious agent worldwide. Its resurgence in 1990s is primarily due to co-infection with HIV and the emergence of multi-drug-resistant (MDR) strains. Our objectives in this study were demonstration and grading of acid-fast bacilli in smears from sputum specimens of clinically newly diagnosed pulmonary TB patients, isolation of the organism, speciation and drug susceptibility testing of Mycobacterium tuberculosis isolates to isoniazid (H), rifampicin (R), streptomycin (S), and ethambutol (E). Materials and Methods: Sputum specimens were collected from 150 patients. Smear examination was done after Ziehl-Neelsen staining. The specimens were cultured onto Lowenstein Jensen media after Petroff's method of concentration. The growth was identified as M. tuberculosis with standard tests. Sensitivity of 50 isolates of tubercle bacilli to anti-TB drugs H, R, S, E were determined by Resistance-Ratio method. Results: Out of 150 sputum specimens examined, 62(41.3%) were smear positive. Out of these 62,56 grew on culture. 50 isolates of M. tuberculosis were picked up for drug susceptibility testing. Total of 31 (62%) were resistant to S, 14(28%) to H, 9(18%) to R, 6(12%) were resistant to E and 2 strains (4%) were resistant to H and R. Conclusion: From the small cohort, incidence of primary MDR-TB was found to be 4% in this region, which is within the expected range.
Keywords: Drug resistance, isoniazid, multi-drug-resistant tuberculosis, rifampicin
|How to cite this article:|
Bhat S, Radhakrishna M, Kotian M, Rao S. Drug susceptibility profiles of Mycobacterium tuberculosis isolates at Mangalore. Indian J Med Sci 2010;64:99-103
|How to cite this URL:|
Bhat S, Radhakrishna M, Kotian M, Rao S. Drug susceptibility profiles of Mycobacterium tuberculosis isolates at Mangalore. Indian J Med Sci [serial online] 2010 [cited 2013 Jun 19];64:99-103. Available from: http://www.indianjmedsci.org/text.asp?2010/64/3/99/95928
| ¤ Introduction|| |
Tuberculosis (TB) is an infectious bacterial disease caused by Mycobacterium tuberculosis. It commonly affects the lungs. It is transmitted from person to person via droplets from the throat and lungs of people with active respiratory disease. TB remains one of the major causes of death from a single infectious agent worldwide.
The disease is generally endemic in developing countries. Its resurgence in 1990s is primarily due to co-infection with HIV and the emergence of multi-drug-resistant (MDR) strains. 
Drug-resistant TB has been defined as a case of TB excreting bacilli resistant to one or more anti TB drugs. Multi-drug-resistant tuberculosis (MDR-TB) is defined as disease due to M. tuberculosis that is resistant to isoniazid and rifampicin with or without resistance to other drugs.  Extensively drug-resistant tuberculosis (XDR-TB) bacilli is defined as a disease due to M. tuberculosis that is resistant to isoniazid and rifampicin along with resistance to any Quinolones and one of the three injectable second-line drugs. 
Drug resistance may be of two types: primary and acquired. Primary drug resistance may be defined as drug resistance in a patient who has not received any antitubercular treatment in the past and resistance that develops in a patient who has received prior chemotherapy is acquired resistance.
MDR-TB is suspected if the sputum is persistently positive for AFB with clinical or radiological deterioration after multiple courses of treatment including 4 months of WHO retreatment regimens under DOTS.  The gold standard for diagnosis of drug resistance in TB is in-vitro study of resistance in well-standardized laboratories. The prevalence of MDR-TB mirrors the functional state and efficacy of TB control programs and realistic attitude of community toward implementation of programs.  Current estimates report the prevalence of primary and acquired MDR-TB in India as 3.4% and 25%, respectively. 
The present study was undertaken to demonstrate acid-fast bacilli (AFB) in smears of sputum samples received from clinically newly diagnosed pulmonary TB patients, to speciate the isolates as M. tuberculosis and to determine their drug susceptibility pattern by the resistance ratio method.
| ¤ Materials and Methods|| |
Patients attending the hospital, with symptoms and signs suggestive of pulmonary TB were screened.
Early morning coughed out sputum specimens were collected from these patients in sterile wide mouth screw capped bottles. Specimens were collected from 150 patients. All bacteriological investigations were carried out as per WHO/IUATLD guidelines.
Sensitivity or resistance was determined by the ratio of MIC of test organism to that of MIC of H 37 RV. , MIC is lowest drug concentration showing less than 20 colonies.
- Smear examination-Direct thin smears were prepared from the purulent portion of sputum on clean glass-slide and stained by Ziehl-Neelsen's method.  The typical morphology was noted and smears were graded as per WHO recommendations. 
- Culture-Concentration method for culture was followed as per Petroff's method.  The deposit was inoculated onto the entire surface of Lowenstein-Jensen (L.J.) media slopes using Pasteur pipette. Cultures were observed once daily for a week and then twice a week for a period of 8 weeks. At the end of 8 th week, the appearance of growth was noted.
The isolates were characterized as M. tuberculosis complex by the following tests: 
Colony morphology, pigment production, acid fastness, rate of growth, niacin test, nitrate reduction test.
- Drug susceptibility testing of M. tuberculosis Susceptibility of 50 isolates of tubercle bacilli to antituberculous drugs - isoniazid, rifampicin, streptomycin, and ethambutol were estimated by resistance ratio method. This method is based on comparison under standard conditions of test strain with that of standard strain of M. tuberculosis H 37 RV. Doubling concentrations of drugs were incorporated in L.J. medium. A total of six concentrations of drugs were incorporated. Streptomycin: 2, 4, 8,1 6, 32, 64 μg/ml, isoniazid: 0.03, 0.06, 0.12, 0.25, 0.5, 1 μg/ml, rifampicin: 2, 4, 8, 16, 32, 64, 128 μg/ml, and ethambutol: 0.25, 0.5, 1, 2, 4, 8, 16 μg/ml.
For all RR 8:1 = Resistant, 4:1 = Repeat test if again 4:1 = Resistant, 2:1 = Sensitive
| ¤ Results|| |
Out of the 150 sputum specimens of clinically diagnosed pulmonary TB patients, 62 (41.3%) were positive for AFB and 88 (58. 7%) were smear negative.
Out of the 150 specimens cultured on L.J. media, 80 showed growth and 8 slopes were contaminated; thus, the contamination rate was 5.3%. Out of the 62 AFB smear-positive specimens, 56 (90.3%) specimens were grown in culture, 2 showed no growth, and 4 were contaminated. Of the 88 smear-negative specimens, 60 (68.18%) were culture negative, 24 (27.27%) showed growth, and 4 (4.54%) were contaminated.
Identification of 50 positive cultures: All the 50 isolates gave positive niacin test and nitrate reduction test.
Susceptibilities of 50 isolates of tubercle bacilli to streptomycin, isoniazid, rifampicin, and ethambutol were estimated by resistance - ratio method [Table 1].
|Table 1: Sensitivity and drug resistance pattern of 50 strains of Mycobacterium tuberculosis|
Click here to view
| ¤ Discussion|| |
TB is caused by M. tuberculosis and the control of the disease is hampered by widespread emergence of drug resistance in the pathogen. Early information on drug susceptibility would greatly facilitate an effective treatment of TB.
The present study included 150 clinically newly diagnosed pulmonary TB patients. In our study, we noted a higher incidence of TB in the age group 21-40 years (56%). In our study, 65.3% of the patients were males. Previous studies showed a male preponderance, 90% and 71.9%, respectively. ,
The sensitivity of most common procedure Z-N staining ranges from 22-78%. In our study, out of 150 sputum specimens, 62 were smear positive for AFB (41.3%),which is consistent with other studies. 
Drug resistance is a global phenomenon. In the 1960s and 1970s, single and MDR TB was low. According to WHO global surveys (1985-1994), the level of primary drug resistance to INH was 0-16.9, to Streptomycin was 0-23.5%, to Rifampicin was 0-3%, and to Ethambutol was 0-4.2%% (reported from Kenya, India, Haiti). The rate of MDR-TB was 0-10.8%, which was low. 
Information on the prevalence of TB and extent of drug resistance in M. tuberculosis on a national basis is not available in India.
Surveys conducted at North Arcot district, Tamil Nadu, and Pondicherry revealed that the former had an primary resistance of 25% to one or more drugs and latter 13%. 
A study conducted in Mumbai showed that amongst 52% sputum positive cases, 15% were resistant to H, 66.8% to R, 8.4% to E and 72.2% to Z. In a study conducted in L.T.M. Medical College, Mumbai, of the 100 patients tested for drug resistant bacilli, 6 had MDR bacilli. 
In our study, out of the 50 isolates of M. tuberculosis isolated, a total of 9 strains (18%) of M. tuberculosis were sensitive to all the four frontline anti TB drugs. 16 strains (32%) were resistant to S, 5(10%) to H, 3(6%) to R and 3 (6%) strains were resistant to E alone and 2 strains (4%) were resistant to H and R.
A study conducted by TRC, Chennai and NTI, Bangalore, using WHO/IUATLD guidelines between 1999 and 2002, in six districts showed that the incidence of primary MDR ranged from 0.7% to 2.8% and acquired MDR-TB 6% to 100%. 
From the small cohort, incidence of primary MDR-TB was found to be 4% in this region.
Detection of drug susceptibility and resistance to M. tuberculosis is of paramount importance for the successful treatment of TB and for health care planning for the future.
| ¤ References|| |
|1.||Cohn DL, Bustreo F, Raviglione MC. Drug resistant Tuberculosis. Review of world-wide situation and WHO/IUATLD Global Surveillance Project. Clin Infect Dis 1997;24(Suppl):121-30. |
|2.||Prasad R. MDR-TB-Current status. Indian J Tub 2005;52:121-36. |
|3.||Van Rie A, Enarson D. XDR-TB, an indicator of public health negligence. Lancet 2006;368:1554-6. |
|4.||Pablos-Méndez A, Raviglione MC, Laszlo A, Binkin N, Rieder HL, Bustreo F, et al. Global Surveillance for antituberculosis drug resistance; 1994-1997. N Engl J Med 1998;338:1641-9. |
|5.||Smithwick RW. Laboratory manual for acid fast microscopy. 2 nd ed. Atlanta: Centre for Disease Control; 1975. |
|6.||Harries A, Maher D, Graham S. TB/HIV A Clinical Manual. 2 nd ed. Geneva: WHO/HTM/TB/2004. p. 51-3. |
|7.||Kent TK, Kubica GP. Public health mycobacteriology. A guide for level III Laboratory. Atlanta: Centre for Disease Control; 1985. |
|8.||Witebsky FG, Kruczak-Filipor P. Identification of Mycobacteria by conventional methods. Clin Lab Med 1996;16:569-601. |
|9.||Allen BW,Baker FJ. In Mycobacteria - Isolation, Identification and sensitivity testing. Allen and Baker, editors. London: Butterworth and Co.; 1968. p.35. |
|10.||Gupta RR, Singhal B, Sharma TN, Gupta RB, Prevalence of initial drug resistance in tuberculosis patients attending a chest hospital. Ind. J Med. Res 1993; 97: 102-103. |
|11.||Neville K, Bromberg A, Bromberg R, Bonk S, Hanna BA, Rom WN. The third epidemic-multidrug-resistant tuberculosis. Chest 1994;105:45-8. |
|12.||Goble M, Iseman MD, Madsen LA, Waite D, Ackerson L, Horsburgh CR Jr. Treatment of 171 patients with Pulmonary Tuberculosis resistant to H and R. N Engl J Med 1993;328:527-32. |
|13.||Chowgule RV, Deodar L. Pattern of secondary acquired drug resistance to antituberculosis drug in Mumbai, India. Indian J Chest Dis Allied Sci 1998;40:23-31. |
|14.||Espinal MA, Laszlo A, Simonsen L, Boulahbal F, Kim SJ, Reniero A, et al. Global trends in resistance to antituberculosis drugs. N Engl J Med 2001;344:1294-303. |
|15.||Paramasivan CN, Bhaskaran K, Venkatraman, Chandrashekharan V. Surveillance of drug resistance in tuberculosis in Tamilnadu. Indian J Tuberc 2000;47:27-33. |
|16.||Sphia V, Balasangameshwara VH, Kumar P. Initial drug resistance among TB patients under DOTS programme in Bangalore city. Indian J Tuberc 2004;52:17-21. |