Characterisation & drug susceptibility patterns of extrapulmonary mycobacterial isolates

Characterisation & drug susceptibility patterns of extrapulmonary mycobacterial isolates

Sachdeva, Ranjana

Background & objectives: There is a paucity of information on extrapulmonary tuberculosis as much of the attention is focussed on pulmonary tuberculosis. This prospective study aimed at identification and characterisation of mycobacterial isolates from extra pulmonary sites and the evaluation of the drug susceptibility patterns of Mycobacterium tuberculosis isolates from extrapulmonary sites using the conventional method and the E-test.

Methods: A total of 350 specimens from patients of extrapulmonary tuberculosis with varied presentation, were studied. Speciation and characterisation of isolates were done on the basis of growth and biochemical characteristics. Drug susceptibility testing for M. tuberculosis isolates was done by proportion method for isoniazid, rifampicin, ethambutol and pyrazinamide, whereas resistance ratio method was used for streptomycin. E-test (AB Biodisk, Sweden) was carried out to compare susceptibility patterns of the M. tuberculosis isolates for isoniazid and rifampicin with the conventional method.

Results: Thirty two of 350 (9.14%) patients clinically suspected to have extrapulmonary tuberculosis were culture positive for mycobacteria. On characterisation, 20 of the 32 isolates were identified as M. tuberculosis and 12 as non-tubercular mycobacteria (NTM) with 5 of the 12 being Mycobacterium avium complex. Among M. tuberculosis isolates both initial and acquired resistance was highest for streptomycin followed by isoniazid, rifampicin and ethambutol. No strain showed resistance to pyrazinamide. Two strains were found to be multidrug resistant. Drug susceptibility patterns by conventional method corroborate with the E-test results.

Interpretation & conclusion: This study shows that the characterisation and species identification of mycobacterial isolates along with drug susceptibility testing help in better understanding of extrapulmonary tuberculosis. E-test had the advantage of being rapid and simple without need for additional equipment.

Key words Characterisation – drug susceptibility – E-test – extrapulmonary tuberculosis

Tuberculosis can present in pulmonary or extrapulmonary forms. Extrapulmonary tuberculosis has varied presentations including lymphadenitis, cold abscess, infertility and meningitis, but most cases are diagnosed and empirically treated on the basis of clinical presentation’. Comprehensive studies that include all forms of extrapulmonary tuberculosis are lacking. The occurrence of extrapulmonary tuberculosis in immunocompromised individuals, and the increased identification of non-tubercular mycobacteria (NTM) as pathogenic agents underlines the importance of undertaking studies on extrapulmonary tuberculosis. The problem of drug resistance has been reported both in developing and developed countries2. Since the early 1990s, several outbreaks of multidrug resistant (MDR) tuberculosis have been reported in different regions of the world as a consequence of the inappropriate use of antitubercular drugs3.

This prospective study aimed to identify the causative mycobacterial species from patients clinically suspected to have extrapulmonary tuberculosis and to characterise these isolates. Considering the present scenario of emerging trends of drug resistance, the study also aimed to test the drug susceptibility patterns of the first line antitubercular drugs against Mycobacterium tuberculosis isolates. Since routinely used conventional methods are labour intensive and time consuming, evaluation of E-test as a newer method for drug susceptibility testing for extrapulmonary isolates of M. tuberculosis was carried out.

Material & Methods

Included in the study were 350 specimens from patients clinically suspected to have extrapulmonary tuberculosis who presented at the University College of Medical Sciences and Guru Tegh Bahadur Hospital, Delhi, between April 1999 and March 2000. The specimens were from 96 patients clinically suspected to have tubercular lymphadenitis, 84 pelvic inflammatory disease with infertility, 46 cold abscess, 45 tubercular osteomyelitis, 28 urinary tract tuberculosis and 51 patients of tubercular peritonitis, pleuritis or meningitis. Specimens were collected and transported in clean, leakproof and labelled containers to the laboratory. These were processed on the same day for both direct microscopy and culture.

Direct microscopy: Smears were stained by the Zeihl Neelson (ZN) method and reported according to CDC guidelines, 1985(4).

Microbiological culture: Specimens from sites not contaminated by commensals were handled aseptically and inoculated directly to culture media [Lowenstein Jensen (LJ) slopes (HiMedia, Mumbai, India)] after concentration. Specimens from sites contaminated with commensals were decontaminated and concentrated by previously described standardised procedures before inoculating onto culture medial.

Culture on LJ slopes: Deposit was inoculated with a sterile Pasteur pipette over the surface of slopes of LJ media in duplicate. The slopes were examined weekly for appearance of growth. This was checked for morphology by a ZN stained smear. Incubation was continued with negative cultures for 8 wk before discarding.

M. tuberculosis and non-tubercular mycobacteria were identified on the basis of colony morphology, pigment production and biochemical tests including niacin test, aryl sulphatase test, catalase production, nitrate reduction, thiophen-2-carboxylic acid hydrazide susceptibility, tellurite reduction, Tween hydrolysis, pyrazinamidase production and growth on MacConkey’s mediums.

Drug susceptibility testing-conventional method: All M. tuberculosis isolates were tested for their susceptibility to isoniazid (H), rifampicin (R), ethambutol (E) and pyrazinamide (Z) by the proportion method and for streptomycin (S) by the resistance ratio method as described by Canetti et al6.

Briefly, the proportion method used for H, R, E, Z consists of calculating the proportion of resistant bacilli present in a strain.

LJ slopes containing 7 ml of media were used. The drug was dissolved in distilled water and incorporated in the medium before coagulation. The control medium without drug was prepared at the same time as the drug containing media. Subsequently the media distributed in slopes was coagulated at 85 deg C for 50 min.

For pyrazinamide, the medium was acidified by adding IN HCl to the medium before inspissation until the pH was 4.850.05.

The concentrations of the drugs ((mu)g/ml) used were isoniazid: 0.2, rifampicin: 40, ethambutol: 2, and pyrazinamide: 100. The bacterial suspension of the test strain was made in a flat-bottomed bottle containing 30 glass beads 3 mm in diameter and shaken for 20-30 sec with 5 ml distilled water. The opacity of the bacterial suspension was adjusted by the addition of distilled water and compared to that of a standard suspension containing I mg/ml of tubercle bacilli.

A wire loop (0.7 mm wire diameter) with an internal diameter of 3 mm that delivered 0.01ml was used to prepare dilutions of 10^sup -2^ and 10^sup -4^ mg/ml so that the inocula were 10^sup -4^ mg and 10^sup -6^ mg of bacilli respectively for each slope. Two slopes of media without drug and two slopes with drug were inoculated with a loopful of each dilution. These slopes were then incubated at 37 deg C. The results were read for the first time on day 28. The ratio between the average number of colonies on the drug containing slopes to the number of colonies on the control slopes is the proportion of resistant bacilli existing in the strain. If, according to the criteria indicated below, the result of the reading made on day 28 was resistant, no further readings of the test for the drug were taken and the strain was classified as resistant. If the result on day 28 was sensitive, a second reading was made on day 42 to provide a definitive result. The criteria for resistance were any strain showing a proportion of resistant bacilli equal to or superior to that indicated below was classified as resistant to the corresponding drug. Critical proportion for resistance: 1 per cent for isoniazid and rifampicin; and 10 per cent for ethambutol and pyrazinamide.

The susceptibility to streptomycin was studied by the resistance ratio method. With a 22 standard wire gauge (SWG) nichrome loop, a representative sweep from the growth was taken (approximately 2 mg moist weight of bacilli) and discharged in 0.4 ml of sterile distilled water with 6 glass beads in a 7 ml bottle. The suspension was prepared by shaking it for I min on a mechanical shaker, and then, with a 3 min external diameter 27 SWG nichrome loop, a loopful of the suspension was spread on the surface of each slope for the sensitivity test. As a control, a drug free slope was set up for each strain tested. The standard sensitive strain, H37Rv, was tested in each set of tests. The slopes were incubated at 370C. A reading was made at 2 wk to give a preliminary indication of the presence of resistant strains, but the definitive reading was made at 4 wk, and a report that a strain was sensitive was not given earlier. For all tests, growth was defined as the presence of 20 or more colonies. The resistance ratio (RR) is the minimal concentration inhibiting growth of the test strain divided by the minimal concentration inhibiting growth of the standard sensitive strain, H37Rv, in the same set of tests.

Concentrations of streptomycin (before inspissation) in the test medium for the test strain were 8, 16, 32, 64, and 128 (mu)g/ml, while for H37Rv concentrations were 2, 4, 8, and 16 (mu)g/ml.

Definition of resistance: Sensitive : a RR of 2 or less, Resistant; a RR of 8 or more or a RR of 4 followed by a RR of 4 or more in a further test done from the control slope7.

Patients who were positive of M. tuberculosis were further interviewed for information on previous antitubercular treatment and depending upon the history, were divided into two groups. Group I comprised subjects who had no treatment history, and Group II those who had history of complete/ incomplete treatment with antitubercular drugs. These groups were used to calculate the initial and acquired drug resistance patterns respectively. Although exhaustive and thorough history of past treatment was taken, it is a common occurrence that the patient might conceal previous treatment history or sometimes may be unaware of it. This is the reason for calculating initial and not primary resistance on the basis of Group I.

Drug susceptibility testing E-test: All the strains positive for M. tuberculosis were tested for their susceptibility to isoniazid and rifampicin by the E-test method (AB Biodisk, Sweden) as per the manufacturer’s instructions. This test was performed concurrently with the conventional drug susceptibility test.

Results A total of 350 specimens from extrapulmonary sotes were included in the study. Among these, 229 (65.4%) specimens were from individuals in the 13-39 yr age groups followed by 55 (15.7%) in the paediatric age group (6-12 yr), 44 (12.6%) from individuals in the 40-59 yr age group and the remaining 22 (96.3%) from the elderly (>60 yr) age group. Number of cultures positive were 32 of 350 (9.14%). On characterisation, 20 of the 32 isolates were identified as M. tuberculosis and 12 as NTM (Table I). Among culture positive specimens, prior direct smear examination afeter ZN staining has been positive in 2 specimens – one pus and one endometrial biopsy specimen. Both these isolates were M. tuberculosis.

Drug susceptibility testing was done for the M. tuberculosis strains only. Group I which comprised 70 per cent of the subjects who had no treatment history was used to calculate the initial drug resistance while Group II, comprising 30 per cent of the subjects who had history of complete/ incomplete treatment with antitubercular drugs was used for calculating acquired drug resistance (Table II). One patient in Group II with history of complete treatment two years earlier, may possibly represent a case of relapse. The M. tuberculosis strain isolated from this patient was susceptible to all first line antitubercular drugs. Overall 10 of the 20 M. tuberculosis isolates showed resistance to one or more drugs. Six of 10 resistant isolates were from Group I and 4 belonged to Group II. Resistance to streptomycin was highest followed by isoniazid, rifampicin and ethambutol. No strain showed resistance to pyrazinamide. Also, there was no isolate with triple drug resistance. Streptomycin resistance was found in every resistant strain.

There were two multidrug resistant strains both showing four-drug resistance (H, R, S and E). One was isolated in a patient of Group I, while the other was isolated from a patient in Group II who had history of monotherapy, possibly rifampicin as there was history of passing yellowish-orange urine after drug intake. The results of the E-test corroborated with those of the conventional methods.


Tuberculosis is a major cause of morbidity in India. Recently various studies have reported the resurgence of tuberculosis in various parts of the world compounded with the problem of extrapulmonary tuberculosis, non-tubercular mycobacterial species, drug resistance and spread of the HIV pandemic1. The exact magnitude of the problem of extrapulmonary tuberculosis is not known. The present study found a culture positivity rate of 9.14 per cent in specimens from a wide range of sites in patients clinically suspected to have extrapulmonary tuberculosis. This is unlike the findings of previous studies that included either highly selected site-specific specimen groups, or were based only on clinical features7,8.

The findings that 65.4 per cent of the patients were in the 13-39 yr age group and the most common presentation of lymph node enlargement correlates with the earlier findings1,9. Another common presentation was infertility. Of the two patients presenting with pleural effusion, one also showed pulmonary tuberculosis on chest X-ray and sputum culture positivity for acid fast bacilli. Such patients, according to WHO, with both pulmonary or extrapulmonary tuberculosis are considered as patients of extrapulmonary tuberculosis and were hence included in the study3.

Twenty of the 32 isolates were identified as All. tuberculosis (62.5%) and 12 as NTM, of which 5 were M. avium complex (MAC). An interesting observation was that isolates representing all the groups of Runyon classification9 were found. In an earlier study in Delhi on the incidence of tubercular lymphadenopathy, 78.9 per cent isolates were identified as M. tuberculosis, 10 per cent as M. fortuitum, 7.9 per cent as M. scrofulaceum and 2.8 per cent as M. kansasil8. Other studies from different parts of the world7,10,11 have reported MAC as the commonest (63-80%) isolate from patients with lymphadenitis followed by M. scrofulaceum. The higher incidence of MAC isolates in previous studies7,11,11 is explained by the fact that M. tuberculosis complex is a more frequent cause of tuberculosis in regions like India where tuberculosis is rampant.

Overall 50 per cent strains showed resistance to one or more drugs in our study. Resistance to streptomycin was highest followed by isoniazid, rifampicin and ethambutol. No strain showed resistance to pyrazinamide as has been documented by various workers12-14. Our results concur with those of another study carried out in east Delhi12 while studies from other regions13,14 showed resistance to isoniazid being the highest.

The figures for acquired resistance when compared with 1999 figures15 (4-54% for isoniazid, 0-15% for rifampicin, 0-19% for streptomycin and 0-14% for ethambutol), are higher for streptomycin and rifampicin resistance. Since these figures relate to pulmonary tuberculosis, comparisons are not strictly valid. Otherwise, these higher values could be because of regional/local variation or could represent a changing or increasing trend in this direction. There were two multidrug resistant strains, both showing four drug resistance (H, R, S and E). Prevalence of MDR tuberculosis has been shown to vary widely over different regions, with the highest rates being found in Nepal (48%), Gujarat (India) (34%), New York City (USA) (30%), Bolivia (15 %) and South Korea (15%)15. A study in East Delhi in 1996 found presence of MDR tuberculosis in 5.7 per cent patients12. As these figures are based on isolates from patients of pulmonary tuberculosis, direct comparison is not possible. Drug susceptibility patterns to isoniazid and rifampicin by the conventional method showed complete association with E-test results. Moreover, the time required was shortened by E-test, taking 1-2 wk (mean 10 days) as compared to the conventional method which took 2-3 wk. This observation needs to be confirmed by larger studies.

E-test has many advantages over the laborious, conventional method in being rapid and simple without the need for additional equipment16-18. However, the higher cost of the E-test could be.a limitation for routine use.

Thus, the findings of the present study show that characterisation and species identification of mycobacterial isolates along with drug susceptibility testing help in better understanding of extrapulmonary tuberculosis.


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Ranjana Sachdeva, D.V. Gadre & Vibha Talwar

Department of Microbiology, University of Medical Sciences & Guru Tegh Bahadur Hospital, Dehli, India

Received September 4, 2001

Reprint requests Dr Ranjan Seva, Det of Microbiology, University College of Medical Sciences & GTB Hospital, Dilshad Garden, Delhi 110092, India

Copyright Indian Council of Medical Research Mar 2002

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