Evaluating PCR, culture & histopathology in the diagnosis of female genital tuberculosis.

Background & objectives: Genital tuberculosis (GTB) is one of the major causes for severe tubal disease leading to infertility. Unlike pulmonary tuberculosis, the clinical diagnosis of GTB is difficult because in majority of cases the disease is either asymptomatic or has varied clinical presentation. Routine laboratory values are of little value in the diagnosis. An absolute diagnosis cannot be made from characteristic features in hysterosalpingogram (HSG) or laparoscopy. Due to the paucibacillary nature of GTB, diagnosis by mycobacterial culture and histopathological examination (HPE) have limitations and low detection rate. The objective of this study was to evaluate the efficacy of PCR technique, culture and histopathological examination in the diagnosis of GTB in female infertility. Methods: This study included 72 infertile women who met the inclusion and exclusion criteria. After a detailed history and clinical examination all patients were subjected to investigations including pelvic sonogram, HSG and laparoscopy. Endometrial samples from were allocated for AFB smear, culture and HPE examination. Only 49 samples were available for PCR using IS 6110 and TRC4 primers. In seven patients peritoneal fluid was also taken for culture and PCR. Based on the clinical profile and laparoscopic findings, a diagnostic criteria was derived to suspect GTB. Specific diagnostic tests were evaluated against this diagnostic criterion. Results: Laparoscopy was suggestive of tuberculosis in 59.7 per cent of cases, AFB smear was positive in 8.3 per cent, culture was positive in 5.6 per cent, HPE positive in 6.9 per cent and PCR was positive in 36.7 per cent of cases. Based on the diagnostic criteria, GTB was suspected in 28 of the 49 cases. On evaluating against the diagnostic criteria, the sensitivity of PCR, HPE and culture were 57.1, 10.7, 7.14 per cent respectively. The concordance of results between the clinical criteria and specific diagnostic tests were analysed by Kappa measure of agreement. The culture and HPE showed mild agreement with the clinical criteria, whereas PCR showed a moderate agreement. PCR was positive in Two of the 21 cases in whom GTB was not suspected. False positive PCR in these two cases were ruled out by multiple areas of sampling and re-sampling in one case. The PCR results were negative in 12 of the 28 cases. PCR using TRC4 primers had a higher sensitivity (46.4%) than IS 6110 primers (25%) in detecting clinically suspected GTB. Interpretation & conclusions: Our results showed that conventional methods of diagnosis namely, HPE, AFB smear and culture have low sensitivity. PCR was found to be useful in diagnosing early disease as well as confirming diagnosis in clinically suspected cases. False negative PCR was an important limitation in this study.

Simple direct drug susceptibility tests on sputum samples for early detection of resistance in tubercle bacilli.

BACKGROUND: Direct sensitivity test either by sputum concentrate (DS) or swab method (DSM) set up along with the primary culture would avoid the delay of four or more weeks required for the indirect test. A comparison of these two methods against the standard indirect sensitivity method under routine laboratory conditions is necessary to prove their merit. METHOD: Smear positive sputum samples were aliquoted and sensitivity tests were set up by both the direct methods as also an indirect test set up from the primary culture of the same sample. RESULTS: The agreement with the indirect test results for isoniazid (INH) ranged from 97-98% for the DS method and 93-97% for the DSM method. The corresponding figures were 96-98% by the DS and 94-99% by the DSM method for rifampicin (R). The agreement was less satisfactory for ethambutol (Emb). CONCLUSION: This study showed that direct sensitivity tests such as DS and DSM methods can detect most of the cultures resistant to INH and R (MDR) from the time growth appears on the primary culture, even as early as the second week of setting up the tests.

Evaluation of bacteriological diagnosis of smear positive pulmonary tuberculosis under programme conditions in three districts in the context of dots implementation in India.

Objective: To study the smear and culture positivity rates in pulmonary tuberculosis patients declared as smear positive in the districts of North Arcot (Tamil Nadu), Raichur (Karnataka) and Wardha (Maharashtra) in India in order to evaluate the diagnosis of pulmonary tuberculosis at the field level under programme conditions. Methods: Two specimens of sputum from each of 320 patients in North Arcot, 314 patients in Raichur and 302 patients from Wardha district, all of whom had been reported as smear-positive at the field level, were examined by smear and culture. Findings: The proportion of specimens found to be smear-negative was 4.7% in North Arcot and 5.7% in Raichur as against 38.7% in Wardha. The proportions of culture negative specimens were 5.7% and 6.3% respectively in North Arcot and Raichur, while it was 35.6% at Wardha. The difference in the smear and culture negativity between Wardha and the other two districts was highly significant. Conclusions: The study revealed an unacceptably high level of false positives in sputum smear microscopy in the Wardha district. This could be attributed to the absence of systematic and intensive training in smear examination consequent to the non-implementation of the DOTS strategy in this district and a high standard of training offered in the RNTCP implemented districts.

Computerized tomography detects pulmonary lesions in children with normal radiographs diagnosed to have tuberculosis.

This report is based on observations during the conduct of a larger study to develop diagnostic criteria for childhood tuberculosis (TB). Of 201 children confirmed to have pulmonary or lymph node TB, 84 had normal chest radiographs. Computerized tomography (CT) of the chest was performed in nine of them, seven of whom had normal chest radiographs while two had visible calcification. Eight of the nine children had definitive lesions detected by computerized tomography of the chest. While five children had primarily hilar lymph node enlargement, three had pulmonary parenchymal lesions. The use of more sensitive diagnostic tests like computed tomography helps to detect tuberculosis lesions not otherwise visualized on chest radiographs. This report highlights the difficulty in excluding active tuberculosis in children. More studies are required on the role of CT scans in the diagnosis of tuberculosis in children.

Drug resistance in tuberculosis in India.

The current global concern in the treatment of tuberculosis (TB) is the emergence of resistance to the two most potent drugs viz., isoniazid and rifampicin. The level of initial drug resistance is an epidemiological indicator to assess the success of the TB control programme. Though drug resistance in TB has frequently been reported from India, most of the available information is localized, sketchy or incomplete. A review of the few authentic reports indicates that there is no clear evidence of an increase in the prevalence of initial resistance over the years. However, a much higher prevalence of acquired resistance has been reported from several regions, though based on smaller numbers of patients. A strong TB control programme and continuous surveillance studies employing standardized methodology and rigorous quality control measures will serve as useful parameters in the evaluation of current treatment policies as well as the management of multidrug resistant (MDR) TB cases.

Seroprevalence of human immunodeficiency virus (HIV) infection among tuberculosis patients in Tamil Nadu.

BACKGROUND & OBJECTIVES: The dual epidemic of HIV and tuberculosis is a cause for concern in those countries where these two infections are prevalent in epidemic proportions. We undertook a survey at two sites in North Arcot district of Tamil Nadu in 1992-1993, to know the seroprevalence of HIV infection among tuberculosis patients. The objective of this study was to re-examine the prevalence of HIV infection among tuberculosis patients in a repeat survey. METHODS: The study was undertaken in four centres: District Tuberculosis Centre (DTC), Vellore, Tuberculosis Sanatorium, Pennathur (Vellore), District TB Centre (DTC), Kancheepuram and the Government Thiruvotteswarar Tuberculosis Hospital (GTTH), Chennai in the northern part of Tamil Nadu during 1997-1998. A total of 2361 newly diagnosed TB patients were registered in this study. HIV serology after pre-test counseling was done along with sputum examination for acid-fast bacillus by smear and culture for mycobacteria for all patients. RESULTS: The overall HIV seroprevalence among TB patients was 4.7 per cent. The highest HIV seropositivity rate was found among patients aged 30-39 yr (10.6%). HIV seroprevalence showed a wide variation among the different centres ranging from 0.6. per cent in DTC, Kancheepuram to 9.4 per cent in Pennathur Sanatorium, Vellore. Sputum smear positivity was 88 per cent among the HIV-negative and 83 per cent among HIV-positive tuberculosis patients. INTERPRETATION & CONCLUSION: HIV infection is on the rise among TB patients in Tamil Nadu. Acid-fast smear microscopy is adequate for the diagnosis of pulmonary tuberculosis, and drug resistance among HIV positive patients is not a major problem at this point of time; hence antituberculosis regimens recommended by the Revised National Tuberculosis Control Program (RNTCP) can be used to treat HIV positive patients with tuberculosis.

Nocardia bacteraemia in an HIV positive patient - A case report.

Nocardiosis has been recognized in recent times as an unusual opportunistic infection associated with HIV. Bacteraemia due to this pathogen is even rarer and only few cases have been reported in the literature. We report here a case of pulmonary nocardiosis with bacteraemia, which was initially diagnosed as pulmonary tuberculosis. A high index of suspicion is required to diagnose this infection as the clinical presentation and radiographic features mimic pulmonary tuberculosis.

Status of drug resistance in tuberculosis after the introduction of rifampicin in India.

The current threat in tuberculosis treatment lies on the fact of emergence of strains resistant to two most antituberculous drugs, isoniazid and rifampicin. Drug resistance to TB may be classified as primary and acquired. Causes of drug resistance are inefficient administration of effective treatment, poor case handling, use of sub-standard drugs, ignorance of healthcare workers, etc. Multidrug resistant TB (MDR-TB) prevalence (median) in new case is highest (14.1%) in Estonia. Studies undertaken in different regions in India by Tuberculosis Research Centre (TRC) during 1997-2000 revealed acquired MDR-TB resistance levels of 25-100%. The key to successful prevention of the emergence of drug resistance remains adequate case finding, prompt and correct diagnosis and effective treatment of infective patients.

Comparison of different methods of assessing in vitro resistance of Mycobacterium tuberculosis to rifampicin.

BACKGROUND & OBJECTIVES: Definitions of in vitro resistance to rifampicin in strains of Mycobacterium tuberculosis by different methods have not been consistent, leading to variations in the interpretation and validity of results. This study compared three methods of defining in vitro resistance to rifampicin. METHODS: (i) A total of 598 clinical isolates of M. tuberculosis were concurrently compared by the minimal inhibitory concentration (MIC) and the proportion method on Lowenstein-Jensen medium; (ii) 54 strains tested by the MIC method were retested by the proportion method and the BACTEC radiometric method; and (iii) 72 strains which yielded an MIC of 64 mg/l by the MIC method were retested by the same method. RESULTS: Out of 598 cultures tested by the MIC and the proportion methods, identical classification as susceptible or resistant was observed in 99.7 per cent. A 100 per cent agreement was observed when 54 strains were tested by the MIC, proportion and BACTEC radiometric methods. When 72 strains with an MIC of 64 mg/l were retested by the same method, 61 (85%) yielded a lower MIC, 9 (12%) gave the same MIC while 2 (3%) yielded a higher MIC of 128 mg/l, reflecting perhaps the inherent limitations of the variations in the inoculum size. INTERPRETATION & CONCLUSION: All 3 definitions of resistance, viz., an MIC of 128 mg/l, a proportion of 1 per cent or more on 40 mg/l by the proportion method, both on L-J medium and a growth of 1 per cent or more on 2 mg/l by the radiometric method were found to be equally satisfactory.

Use of vancomycin in the culture of Mycobacterium tuberculosis from gastric lavage.

BACKGROUND & OBJECTIVES: Earlier studies from the Tuberculosis Research Centre, Chennai, on culture of Mycobacterium tuberculosis from gastric lavage (GL) specimens in selective Kirchner's medium (SK) resulted in a loss of 60 per cent culture results due to contamination with aerobic spore bearers (ASB). Addition of vancomycin to SK (SKV) effectively reduced the contamination rate to 20 per cent. The objective of the present study was to further reduce the contamination by collecting the specimens in bottles containing vancomycin, thus providing continuous exposure of the sample to the drug, which is bactericidal to ASB. METHODS: One thousand GL specimens collected from children in vancomycin containing bottles were decontaminated and cultured in SK medium, with and without vancomycin, subcultured on Lowenstein Jensen (LJ) medium and the culture results compared. RESULTS: The contamination of cultures in SK and SKV was 15 and 4 per cent respectively when the specimens were collected in bottles containing vancomycin compared to 60 and 20 per cent contamination reported in the earlier studies. INTERPRETATION & CONCLUSION: The reduced contamination in SK and SKV is most likely due to the collection of samples in vancomycin containing bottles. Although a concurrent comparison of samples processed in vancomycin free conditions would have been ideal, it could not be done due to practical difficulties. The study thus confirms the value of vancomycin as a major deterrent for contamination due to aerobic spores and better results can be obtained if vancomycin is used in sample collection bottles, transport media and liquid culture media used in mycobacteriology laboratories particularly in humid and tropical environment.

Evaluation of various methods of susceptibility to ofloxacin in strains of Mycobacterium tuberculosis.

A comparison of three methods of susceptibility testing was undertaken on 30 susceptible and 25 resistant strains of Mycobacterium tuberculosis to determine an acceptable in vitro definition of resistance of ofloxacin. The strains were tested by the proportion method on Lowenstein Jensen (L-J) and 7H11 media and also by the BACTEC radiometric method. Using a criterion of 1 per cent or more growth at a concentration of 2 mg/1, there was a 100 per cent agreement with the conventional MIC method by the proportion tests on L-J as well as on 7H11 media. The BACTEC radiometric method, at the same concentration, yielded 98 per cent agreement. Thus, any of these methods could be used depending upon the infrastructure available.

A histological spectrum of host responses in tuberculous lymphadenitis.

A total of 446 lymph node biopsy specimens showing histological evidence of tuberculosis were classified into four groups based on the organization of the granuloma, the type and numbers of participating cells and the nature of necrosis. These were, hyperplastic (22.4%)--a well-formed epithelioid cell granuloma with very little necrosis, reactive (54.3%)--a well-formed granuloma consisting of epithelioid cells, macrophages, lymphocytes and plasma cells with fine, eosinophilic caseation necrosis, hyporeactive (17.7%)--a poorly organized granuloma with macrophages, immature epithelioid cells, lymphocytes and plasma cells and coarse, predominantly basophilic caseation necrosis and nonreactive (3.6%)--unorganized granuloma with macrophages, lymphocytes, plasma cells and polymorphs with non caseating necrosis. Though the number of bacilli in the sections differed in each group, there were no differences in culture positivity, Mantoux reaction or the clinical features. It is likely that the spectrum of histological responses seen in tuberculous lymphadenitis is the end result of different pathogenic mechanisms underlying the disease.

Early results from indirect drug susceptibility test for tubercle bacilli.

The indirect susceptibility test results on L-J medium for tubercle bacilli against streptomycin, isoniazid and rifampicin were read at the end of 2 wk and compared with the results at 4 wk. It was found that drug resistance could be correctly predicted in over 70 per cent of cultures including multi-drug resistant tuberculosis (MDR TB) strains at the end of 2 wk. The susceptibility to para-nitrobenzoic acid (PNB) read at 2 wk was able to distinguish non-tuberculous mycobacteria from Mycobacterium tuberculosis cultures. The early detection of resistance by this procedure requires only minimum inputs, and can benefit the majority of patients harbouring drug resistant tubercle bacilli.

Evaluation of the BACTEC radiometric method in the early diagnosis of tuberculosis.

A comparison of the BACTEC radiometric method with the conventional culture and drug susceptibility testing methods on isolates from clinical specimens in pulmonary and extrapulmonary tuberculosis, childhood TB and TB in HIV-infected individuals was undertaken. In the case of pulmonary TB, the rate of isolation of positive cultures was significantly faster with the BACTEC method, with 87 per cent of the positives being obtained by 7 days, and 96 per cent by 14 days. However, while there was no difference in the total number of positive cultures by the two methods in smear positive pulmonary tuberculosis, in smear negative pulmonary TB, the BACTEC method yielded more number of positive cultures. In extrapulmonary TB, HIV-TB and childhood TB, although the BACTEC method did not yield additional positives, the detection of positives was considerably faster than by the conventional methods, in which the degree of growth was also scanty. The agreement in drug susceptibility tests was 94 per cent for streptomycin and isoniazid, 99 per cent for rifampicin and 91 per cent for ethambutol. Further, most of the drug susceptibility test results became available within 8 days by the BACTEC method. By facilitating early diagnosis, the BACTEC method may prove to be cost effective in a population with a high prevalence of tuberculosis, particularly in the extrapulmonary and paucibacillary forms of the disease.

Action of metronidazole in combination with isoniazid & rifampicin on persisting organisms in experimental murine tuberculosis.

To study the activity of metronidazole on persisting tubercle bacilli, BALB/c mice were infected with Mycobacterium tuberculosis and, after 14 days, treated with isoniazid (H) or rifampicin (R) or isoniazid + rifampicin (HR) for 2 months. An untreated group and a group treated with metronidazole (M) alone served as controls. At the end of 2 months, M was added to the H, R, and HR regimen in half the mice, and the treatment was continued for 1 more month in all mice. At the end of treatment, no viable organisms were detected in the lung or spleen of mice treated with HR or HRM regimens. In contrast, compared to the mice treated with R alone, the log10 colony forming units (cfu) of mice treated with RM were lower by 1.84 and 0.52 in the lung and spleen, respectively. Similarly, compared to the H group, the log10 cfu were lower by 0.67 in the spleen of mice treated with HM, and no additional effect due to M was seen in the lung. Three months after stopping treatment, viable organisms were isolated from both the organs of all the groups. However, the log10 cfu in the lung and spleen for the groups with metronidazole were below the log10 cfu for the respective single or 2 drug groups, except the log10 cfu in the lung for the RM group. These findings suggest that metronidazole, given with bactericidal drugs such as rifampicin and isoniazid may be of value in eliminating persisting tubercle bacilli, but further studies are warranted.

Bioluminescence assay of adenosine triphosphate in drug susceptibility testing of Mycobacterium tuberculosis.

Twenty three clinical isolates M. tuberculosis and the reference strain, M. tuberculosis H37Rv were tested for their susceptibility to trifluoperazine (TFP) by the standard broth dilution method and the bioluminescence assay. The results showed that in 15 of the 23 isolates, the minimal inhibitory concentration (MIC) was identical in both the methods and in the remaining 8 isolates the difference in the MIC values between the methods, was less than two fold and was not significant. The findings suggest that the measurement of adenosine triphosphate (ATP) by bioluminescence assay can be employed as an alternative method for the rapid screening of clinical isolates for their susceptibility to anti-mycobacterial agents.

Restriction fragment length polymorphism of Mycobacterium tuberculosis strains from various regions of India, using direct repeat probe.

Intraspecies differentiation was studied on 68 M. tuberculosis strains obtained from 6 states of India by restriction fragment length polymorphism (RFLP) using a direct repeat probe (DR probe) hybridised with Alu I digest of DNA. Most strains showed polymorphism based patterns that comprised between 2 to 7 bands and were grouped into 26 RFLP types. Of the 11 strains tested from Amritsar, 8 were RFLP type 5; the remaining 3 were of type 11 and were exclusively confined to this region. The strains from other regions were more heterogeneous. We confirm that DR-associated RFLP can be an excellent tool for the differentiation of M. tuberculosis strains. Depending on their geographical origin, these strains can be differentiated to a large extent by DR fingerprinting.

In vitro susceptibility of clinical isolates of Mycobacterium tuberculosis to cefadroxil--a cephalosporin antibiotic.

The bactericidal activity (BA) of cefadroxil, a semisynthetic cephalosporin antibiotic, against M. tuberculosis H37Rv was studied in Middlebrook 7H9 medium. Cefadroxil showed good BA (average fall of viable counts = log10 0.32 colony forming units/ml/day) against the log phase culture of M. tuberculosis H37Rv. Its minimal inhibitory concentration (MIC) and minimal bactericidal concentration (MBC) were found to be 15 micrograms/ml or less. The MIC of cefadroxil for 29 clinical isolates of M. tuberculosis and a laboratory strain, M. tuberculosis H37Rv was also determined by agar dilution method using Middlebrook 7H11 agar as a screening procedure. The MIC of cefadroxil was found to be 10 micrograms/ml or less for M. tuberculosis H37Rv and 16 (55.1%) of 29 clinical isolates tested. The MIC for 3 of 10 drug sensitive and 9 of 19 drug resistant isolates was 40 or more, a concentration much higher than the peak plasma concentration (28 micrograms/ml) attained in human beings. The higher MIC observed in 12 of 29 clinical isolates irrespective of their susceptibility pattern requires further studies to assess the usefulness of cefadroxil in the treatment of tuberculosis.