R. Prabhakar

IS6110 restriction fragment length polymorphism typing of clinical isolates of Mycobacterium tuberculosis from patients with pulmonary tuberculosis in Madras, South India

SETTING Madras, India. OBJECTIVE To explore the utility of a standardized IS6110/PvuII deoxyribonucleic acid (DNA) fingerprinting restriction fragment length polymorphism (RFLP) typing method for distinguishing between isolates of Mycobacterium tuberculosis, and to assess the potential for distinguishing between relapse versus reinfection rates. DESIGN To assess RFLP heterogeneity in the population, initial isolates, obtained from the sputum of tuberculous 98 patients in diagnosis and follow-up during short-course chemotherapy, were stored and compared. To assess the frequency of disparity between the RFLP type of the initial isolate and one obtained after successful completion of chemotherapy, either during relapse or as an isolated positive culture, 124 isolates comprising 62 such pairs were coded and compared both blind and after decoding. RESULTS Although a wide variety of DNA band patterns (fingerprints) was present, the isolates from 39 (40%) of the patients showed a single copy of IS6110. Only 15 pairs of coded initial and follow-up isolates could be identified as having the same band pattern when isolates with zero or single bands were excluded. Nevertheless, after decoding, in a retrospective analysis that included all isolates, those isolates that bacteriologically defined a patients relapse more often showed RFLP type identity with the initial isolate (19 of 30 comparisons) than did isolates that were obtained as isolated positive cultures (3 of 32 comparisons) (X2 P < 0.001). Tests of sensitivity to chemotherapeutic drugs, catalase activity and resistance to thiophene-2-carboxylic acid hydrazide were of minimal value in discriminating between isolates. CONCLUSIONS Despite the high frequency of single- and zero-band isolates in this population, the discriminatory power of RFLP typing with IS6110 is sufficiently high to be useful for clinical and epidemiological studies.

Association of pulmonary tuberculosis and HLA in south India.

In 204 patients with smear-positive pulmonary tuberculosis HLA-A10, B8 and DR2 were more frequently found than in 404 control subjects (p = 0.01); the greatest attributable risk (0.29) was associated with HLA-DR2. The radiographic extent of disease was also associated with HLA-DR2 (p = 0.0001). In 152 patients with smear-negative pulmonary tuberculosis, the frequencies of HLA-A10 and B8, but not DR2, were greater in the control subjects (p = 0.001 and 0.01 respectively). HLA-DR2 may be involved in the pathogenesis of advanced pulmonary tuberculosis. Study of endogamous, genetically disparate populations (caste) revealed other HLA associations (A3, B12 and DR4) unique to them, suggesting that genes linked with the HLA complex might also be significant in the pathogenesis of tuberculosis.

A Controlled Trial of Ivermectin and Diethylcarbamazine in Lymphatic Filariasis

Ivermectin is a new antifilarial drug that can be given in a single oral dose. To compare the efficacy and side effects of ivermectin with those of diethylcarbamazine, the standard antifilarial treatment, we conducted a randomized, double-blind trial in 40 South Indian men with lymphatic filariasis caused by Wuchereria bancrofti. Patients were randomly assigned to one of three treatments: a single low dose of ivermectin (mean [+/- SE], 21.3 +/- 0.7 micrograms per kilogram of body weight; n = 13) followed by placebo for 12 days; a single high dose of ivermectin (mean, 126.2 +/- 3.7 micrograms per kilogram; n = 13) followed by placebo for 12 days; or diethylcarbamazine for 13 days (6 mg per kilogram per day for 12 days preceded by 3 mg per kilogram for 1 day; n = 14). Eleven patients were initially assigned to receive placebo and after five days were reassigned to one of the three treatment groups. At day 12 there was complete clearance of microfilariae from the blood in all 26 men who took ivermectin and in 11 of the 14 men who took diethylcarbamazine. At six months the numbers of detectable microfilariae (as a percentage of the pretreatment values) were 18.3 percent after low-dose ivermectin and 19.5 percent after high-dose ivermectin, as compared with 6.0 percent after diethylcarbamazine (P less than 0.05). The side effects were confined to the first five days and were similar in the three treatment groups. We conclude that in lymphatic filariasis, the clinical response to a single dose of ivermectin compares favorably with that after the standard 12-day course of diethylcarbamazine. Given the practical advantages of single-dose administration, ivermectin should become a useful medication for the control of bancroftian filariasis.

Value of bronchoalveolar lavage and gastric lavage in the diagnosis of pulmonary tuberculosis in children

OBJECTIVE The aim of our study was to find out if bronchoalveolar lavage (BAL) would be better than gastric lavage for the isolation of Mycobacterium tuberculosis from pediatric patients with suspected pulmonary tuberculosis. DESIGN 50 children with suspected pulmonary tuberculosis at a mean age of 5.1 years (range 7 months to 12 years) were studied. Early morning gastric lavage was collected. Flexible bronchoscopy and bronchoalveolar lavage was performed under local anaesthesia after obtaining informed consent from the parents. The BAL fluid and gastric lavage specimens were subjected to smear examination for acid-fast bacilli (AFB) and culture for mycobacteria using established methods. RESULTS Of the 50 cases, M. tuberculosis was grown in 6 BAL samples (12%) and 16 gastric lavage samples (32%) making a total of 17 culture proven cases (34%). Out of the 6 BAL positive cases, gastric lavage was also positive in 5 cases. CONCLUSION We conclude that gastric lavage is better than BAL for bacteriologic confirmation of pulmonary tuberculosis in children. The overall bacteriologic yield combining both procedures was 34% while gastric lavage alone was positive in 32% of the cases.

Critical assessment of smear-positive pulmonary tuberculosis patients after chemotherapy under the district tuberculosis programme

This is a status report of a retrospectively assembled cohort of 3357 smear-positive patients initiated on anti-tuberculosis chemotherapy in the North Arcot district between April 1986 and March 1988. The patients were contacted once at their homes between November 1988 and June 1989 (6 and 36 months after start of treatment), and information on their status, including death, could be obtained from 76% of them. Regimens were selected by the patients. 2306 (69%) had accepted short course regimens (SCC) and 1051 (31%) had been started on standard chemotherapy (non-SCC), 43% and 35% in SCC and non-SCC respectively had completed 80% or more of their treatment. Overall mortality was 28%. Of those remaining, 31% had active disease and were excreting bacilli, among which 65% of the cultures were resistant to isoniazid and 12% to rifampicin. Combined resistance to isoniazid and rifampicin was seen in 4% and to isoniazid and streptomycin was seen in 19%. A significant finding was that even among those who had taken less than 50% of their treatment, 56% were bacteriologically negative. However, inadequate or irregular chemotherapy resulted in over four times the mortality and about twice the rate of smear positivity as compared with those taking adequate chemotherapy. No comparisons are made between patients on short-course and standard regimens as the patients selected their treatment and the groups are not comparable.

Short course chemotherapy for tuberculous lymphadenitis in children.

OBJECTIVE--To assess the efficacy of a short course chemotherapy regimen for treating tuberculosis of the lymph nodes in children. DESIGN--Open, collaborative, outpatient clinical trial. SETTING--Outpatient department of the Tuberculosis Research Centre, paediatric surgery departments of the Institute of Child Health and Hospital for Children and the Government Stanley Hospital, Madras, South India. PATIENTS--Children aged 1-12 years with extensive, multiple site, superficial tuberculous lymphadenitis confirmed by biopsy (histopathology or culture). INTERVENTIONS--Patients were treated with a fully supervised intermittent chemotherapy regimen consisting of streptomycin, rifampicin, isoniazid, and pyrazinamide three times a week for two months followed by streptomycin and isoniazid twice a week for four months on an outpatient basis. Surgery was limited to biopsy of nodes for diagnosis and assessment. MAIN OUTCOME MEASURES--Response to chemotherapy was assessed by regression of lymph nodes and healing of sinuses and abscesses during treatment and follow up. Compliance with treatment and frequency of adverse reactions were also estimated. RESULTS--197 Patients were admitted to the study and 168 into the analysis. The regimen was well tolerated and compliance was good with 101 (60%) patients receiving the prescribed chemotherapy within 15 days of the stipulated period of six months. Those whose chemotherapy extended beyond that period received the same total number of doses. Clinical response was favourable in most patients at the end of treatment. Sinuses and abscesses healed rapidly. Residual lymphadenopathy (exceeding 10 mm diameter) was present in 50 (30%) patients at the end of treatment; these nodes were biopsied. Fresh nodes, increase in size of nodes, and sinuses and abscesses occurred both during treatment and follow up. After 36 months of follow up after treatment only 5 (3%) patients required retreatment for tuberculosis. CONCLUSION--Tuberculous lymphadenitis in children can be successfully treated with a short course chemotherapy regimen of six months.

Three chemotherapy studies of tuberculous meningitis in children.

Chemotherapy studies were undertaken in 180 patients with tuberculous meningitis. They were treated for 12 months with 1 of 3 regimens: the first consisted of streptomycin, isoniazid and rifampicin daily for the first 2 months, followed by ethambutol plus isoniazid for 10 months; in the second, pyrazinamide was added for the first 2 months, and in the third, rifampicin was reduced to twice weekly in the first 2 months. Steroids were prescribed for all the patients in the initial weeks of treatment. Approximately 50% of the patients were aged less than 3 years. On admission, 13% of the patients were classified as stage I, 77% as stage II and 9% as stage III. Cerebrospinal fluid (CSF) culture results were available for all the 180 patients and M. tuberculosis was isolated in 59 (33%). CSF smear results for acid fast bacilli were available only for the 103 patients admitted to the second and the third studies, and of these in 60 (58%) the CSF was positive either by smear or culture. The response to therapy was similar in the 3 studies. Despite administration of rifampicin for 2 months, the mortality was high. In all, 27% of the patients died of tuberculous meningitis, 39% had neurological sequelae and 34% recovered completely. There was a strong association between the stage on admission and the mortality rate, the deaths being highest in stage III. In the first study, when isoniazid was prescribed daily in a dosage of 20 mg/kg, 39% of the patients developed jaundice; however, when the dosage was reduced to 12 mg/kg, the incidence fell to 16%. In the third study, where rifampicin was administered twice a week, the incidence of jaundice was much lower (5%).

Pulmonary function in healthy young adult Indians in Madras.

Forced vital capacity, forced expiratory volume in one second, functional residual capacity, residual volume, total lung capacity, and single breath diffusing capacity measurements (effective alveolar volume, carbon monoxide transfer factor, and transfer coefficient) were measured in 247 young healthy adults (130 male, 117 female) aged 15-40 years living in Madras. Subjects were of Dravidian stock, living at sea level with rice as their staple diet. Regression equations were derived for men and women for predicting normal pulmonary function for young adults in South India. The values were similar to those reported for subjects from Western India and lower than those reported for North Indians and caucasians.

Effect of storage of sputum specimens at room temperature on smear and culture results

A study of 41 specimens of sputum stored for up to 28 days at room temperature in a tropical country showed that smear results were not affected by storage, the positivity being 83% before storage and 80-83% after. The culture positivity was 88% before storage, and 83%, 68%, 22%, 13% and 0% after 3, 7, 14, 21 and 28 days of storage respectively. The reduction in positivity on storage attains significance at 7 days (P = 0.05). Another study involved 163 specimens of sputum and storage for 3, 5 or 7 days. Each specimen was examined before storage and after 2 periods of storage, at random. The smear results were again not affected. There was, however, significant loss of viability, the proportion culture positive being reduced from 92% before storage to 83% at 3 days (P = 0.05), 71% at 5 days (P less than 0.01) and 63% at 7 days (P less than 0.001). The contamination rate was 5%, 7%, 12% and 18% respectively. It is concluded that sputum should not be stored at room temperature for longer than 3 days for culture but it can be stored for 4 weeks without any loss of smear-positivity.

Species level identification of non-tuberculous mycobacteria from South Indian BCG trial area during 1981

In 1981, non-tuberculous mycobacteria (NTM) were obtained from 8.6% of 16 907 sputum specimens in a trial in the Chingleput district of Madras state to test the efficacy of BCG vaccination in the prevention of tuberculosis, but from only 0.6% of 672 autoclaved specimens cultured as part of a quality control procedure. This finding suggested that the NTM were truly derived from the sputum of the BCG trial subjects. The mycobacterial species could be identified in 966 (96.6%) of the first 1000 isolates of NTM: 54.6% were potential pathogens and 73.0% were slow growing. The species isolated most frequently were M. avium/intracellulare (22.6% of all NTM), M. terrae (12.5%) and M. scrofulaceum (10.5%). Those species accounting for 8-5% of all NTM were M. fortuitum, M. chelonei, M. flavescens, M. gordonae and M. vaccae.