Molecular Genetic Analysis of Multi-drug Resistance in Indian Isolates of Mycobacterium tuberculosis

A total of 116 isolates from patients attending the out-patient department at the All India Institute of Medical Sciences, New Delhi and the New Delhi Tuberculosis Centre, New Delhi, India were collected. They were analyzed for resistance to drugs prescribed in the treatment for tuberculosis. The drug resistance was initially determined by microbiological techniques. The Bactec 460TB system was employed to determine the type and level of resistance in each isolate. The isolates were further characterized at molecular level. The multi-drug loci corresponding to rpo b, gyr A, kat G were studied for mutation(s) by the polymerase chain reaction-single strand conformational polymorphism (PCR-SSCP) technique. The SSCP positive samples were sequenced to characterize the mutations in rpo b, and gyr A loci. While previously reported mutations in the gyr A and rpo b loci were found to be present, several novel mutations were also scored in the rpo b locus. Interestingly, analysis of the gyr A locus showed the presence of point mutation(s) that could not be detected by PCR-SSCP. Furthermore, rifampicin resistance was found to be an important marker for checking multi-drug resistance (MDR) in clinical isolates of Mycobacterium tuberculosis. This is the first report on molecular genetic analysis of MDR tuberculosis one from India, highlights the increasing incidence of MDR in the Indian isolates of M. tuberculosis.

Predictive value of the acid fast smear for detection of Mycobacterium tuberculosis in respiratory specimens in a Reference Center of HIV/Aids in Rio de Janeiro, Brazil

In order to evaluate the predictive value of acid fast bacilii (AFB) smear for the diagnosis of Mycobacterium tuberculosis in respiratory specimens in a setting with a high prevalence of Aids and an unknown prevalence of nontuberculous mycobacteria (NTM), we retrospectively examined specimens cultured for mycobacteria between 1 September 1993 and 30 September 1994 and medical records of patients with positive culture in a General Hospital, Aids reference in Rio de Janeiro, Brazil. Seventy three per cent (1517/2077) of samples were respiratory specimens and mycobacteria were recovered from 20.6% (313/1517) of these. M. tuberculosis was identified in 94.2% (295/313) and NTM in 5.8% (18/313). The yield of positive AFB smear and of positive culture was 6.1% (93/1517) and 20.6% (313/1517), respectively. The positive predictive value (PPV) of AFB for M. tuberculosis was 98.4% in expectorated sputum and 96.4% in bronchoalveolar lavage. Forty four percent (130/295) of specimens with positive culture for M. tuberculosis and 66.7% (12/18) for NTM were from patients HIV positive. The conclusion was that in our study population, the PPV of AFB for M. tuberculosis in respiratory specimens was high and the prevalence of NTM was low despite the high prevalence of HIV positive.

Standartization of broth microdilution method for Mycobacterium tuberculosis

Indirect drug susceptibility tests of Mycobacterium tuberculosis was done to investigate the accuracy and feasibility of a broth microdilution method (BMM) for determining minimal inhibitory concentrations of conventional drugs against M. tuberculosis. Test drugs included isoniazid (H), rifampicin (R), ethambutol (E), streptomycin (S) and pyrazinamide (Z). Fifty isolates of M. tuberculosis from patients who had never received drug therapy, and H37Rv strain for control, were evaluated in the system. When comparing this method with the gold standard proportional method in Lowenstein-Jensen medium, sensitivity of 100% for all drugs and specifities of 91, 100, 96, 98 and 85% were observed respectively for H, R, E, S and Z. The BMM was read faster (14-20 days) than the proportional method (20-28 days). The microdilution method evaluated allows the testing of multiple drugs in multiple concentrations. It is easy to perform and does not require special equipment or expensive supplies. In contrast to radiometric method it does not use radioactive material.

Risk Factors for tuberculosis among human immunodeficiency virus-infected persons. A case-control study in Belo Horizonte, Minas Gerais, Brazil (1985-1996)

The objective of this study was to identify tuberculosis risk factors and possible surrogate markers among human immunodeficiency virus (HIV)-infected persons. A retrospective case-control study was carried out at the HIV outpatient clinic of the Universidade Federal de Minas Gerais in Belo Horizonte. We reviewed the demographic, social-economical and medical data of 477 HIV-infected individuals evaluated from 1985 to 1996. The variables were submitted to an univariate and stratified analysis. Aids related complex (ARC), past history of pneumonia, past history of hospitalization, CD4 count and no antiretroviral use were identified as possible effect modifiers and confounding variables, and were submitted to logistic regression analysis by the stepwise method. ARC had an odds ratio (OR) of 3.5 (CI 95% - 1.2-10.8) for tuberculosis development. Past history of pneumonia (OR 1.7 - CI 95% 0.6-5.2) and the CD4 count (OR 0.4 - CI 0.2-1.2) had no statistical significance. These results show that ARC is an important clinical surrogate for tuberculosis in HIV-infected patients. Despite the need of confirmation in future studies, these results suggest that the ideal moment for tuberculosis chemoprophylaxis could be previous to the introduction of antiretroviral treatment or even just after the diagnosis of HIV infection.

Flow cytometry as a tool to identify Mycobacterium tuberculosis interaction with the immune system and drug susceptibility

Flow cytometric analysis is a useful and widely employed tool to identify immunological alterations caused by different microorganisms, including Mycobacterium tuberculosis. However, this tool can be used for several others analysis. We will discuss some applications for flow cytometry to the study of M. tuberculosis, mainly on cell surface antigens, mycobacterial secreted proteins, their interaction with the immune system using inflammatory cells recovered from peripheral blood, alveolar and pleura spaces and the influence of M. tuberculosis on apoptosis, and finally the rapid determination of drug susceptibility. All of these examples highlight the usefulness of flow cytometry in the study of M. tuber-culosis infection.

Mutations in the rpoB gene of rifampicin-resistant Mycobacterium tuberculosis strains isolated in Brazil and France

We evaluated the mutations in a 193bp of the rpoB gene by automated sequencing of rifampicin (RMP)-resistant and susceptible Mycobacterium tuberculosis strains isolated from Brazil (25 strains) and France (37 strains). In RMP-resistant strains, mutations were identified in 100% (16/16) from France and 89% (16/18) from Brazil. No mutation was detected in the 28 RMP-susceptible strains. Among RMP-resistant or RMP-susceptible strains deletion was observed. A double point mutation which had not been reported before was detected in one strain from France. Among French resistant strains mutations were found in codons 531 (31.2%), 526, 513 and 533 (18.7% each). In Brazilian strains the most common mutations were in codons 531 (72.2%), 526 (11.1%) and 513 (5.5%). The heterogeneity found in French strains may be related to the fact that most of those strains were from African or Asian patients.

Transmission of tuberculosis in Havana, Cuba: a molecular epidemiological study by IS6110 restriction fragment length polymorphism typing

The combination of molecular and conventional epidemiological methods has improved the knowledge about the transmission of tuberculosis in urban populations. To examine transmission of tuberculosis in Havana, Cuba, with DNA fingerprinting, we studied 51 out of 92 Mycobacterium tuberculosis strains isolated from tuberculosis patients who resided in Havana and whose infection was culture-confirmed in the period from September 1997 to March 1998. Isolates from 28 patients (55%) had unique IS6110 restriction fragment length polymorphism (RFLP) patterns, while isolates from 23 others (45%) had identical patterns and belonged to 7 clusters. Three clusters consisting of six, five and two cases were each related to small outbreaks that occurred in a closed setting. Three other clustered cases were linked to a large outbreak that occurred in another institution. Younger patients were more correlated to clustering than older ones. The finding that 45% of the isolates had clustered RFLP patterns suggests that recent transmission is a key factor in the tuberculosis cases in Havana. The IS6110 RFLP typing made it possible to define the occurrence of outbreaks in two closed institutions.

Inhibition of the polymerase chain reaction by sputum samples from tuberculosis patients after processing using a silica-guanidiniumthiocyanate DNA isolation procedure

With the objective to evaluate PCR-mediated detection of Mycobacterium tuberculosis DNA as a diagnostic procedure for diagnosis of tuberculosis in individuals attending ambulatory services in Primary Health Units of the City Tuberculosis Program in Rio de Janeiro, Brazil, their sputum samples were collected and treated with a DNA extraction procedure using silica-guanidiniumthiocyanate. This procedure has been described to be highly efficient for extraction of different kind of nucleic acids from bacteria and clinical samples. Upon comparing PCR results with the number of acid-fast bacilli, no direct relation was observed between the number of bacilli present in the sample and PCR positivity. Part of the processed samples was therefore spiked with pure DNA of M. tuberculosis and inhibition of the PCR reaction was verified in 22 out of 36 (61%) of the samples, demonstrating that the extraction procedure as originally described should not be used for PCR analysis of sputum samples.

Direct Sensitivity Test of the MB/BacT System

In order to evaluate the direct-method test of sensitivity to drugs used in the principal tuberculosis treatment regimes, in the Organon Teknika MB/BacT system, we tested 50 sputum samples positive to microscopy taken from patients with pulmonary tuberculosis and with clinical indications for an antibiogram, admitted sequentially for examination during the routine of the reference laboratory. The material was treated v/v with 23% trisodium phosphate solution, incubated for 24 h at 35°C, and neutralized v/v with 20% monosodium phosphate solution. The material was then centrifuged and the sediment inoculated into flasks containing Rifampin - 2 µg/ml, Isoniazid - 0.2 µg/ml, Pyrazinamide - 100 µg/ml, Ethambutol - 2.5 µg/ml, Ethionamide - 1.25 µg/ml, and Streptomycin - 2 µg/ml. The tests were evaluated using the indirect method in the BACTEC 460 TB (Becton Dickinson) system as the gold standard. The results showed that the Rifampin test performed best, i.e., 100% sensitivity at 95% Confidence Interval (82.2-100) and 100% specificity at 95% Confidence Interval (84.5-100), followed by Isoniazid and Pyrazinamide. In this experiment, 92% of the materials showed a final reading in 30 days; this period represents the time for primary isolation as well as the results of the sensitivity profile, and is within Centers for Disease Control and Prevention recommendations regarding time for performance of the antibiogram. The inoculated flasks showed no contamination during the experiment. The MB/BacT is shown to be a reliable, rapid, fully automated nonradiometric system for the tuberculosis antibiogram.

Comparison of the Proportion Method With Mycobacteria Growth Indicator Tube and E-test for Susceptibility Testing of Mycobacterium tuberculosis

The aim of this study was to investigate the correlation between proportion method with mycobacteria growth indicator tube (MGIT) and E-test for Mycobacterium tuberculosis. Forty clinical isolates were tested. MGIT and E-test with the first line antituberculous drugs correlated with the proportion method. Our results suggested that MGIT and E-test methods can be routinely used instead of the proportion method.

Hantavirus pulmonary syndrome in Uberaba, Minas Gerais, Brazil

This report describes the epidemiological and clinical-evolutive characteristics of eight patients with hantavirus pulmonary syndrome (HPS) in Uberaba, Minas Gerais, Brazil. A positive history of contact with rodents was present in 100% of the cases. The time between the onset of symptoms and hospital care was, on average, 3.6 days. All patients showed clinical and laboratory findings suggestive of HPS. Elevated urea and creatinine levels were observed in 6 (75%) cases, PO2 was < 60 mmHg in 100% of the cases, and a chest X-ray demonstrated a bilateral interstitial-alveolar infiltrate. The diagnosis was confirmed by the detection of IgM antibodies against Sin Nombre virus by ELISA. Three patients died as a direct consequence of HPS.

IS6110 fingerprinting of sensitive and resistant strains (1991-1992) of Mycobacterium tuberculosis in Colombia

The standardized method to study the polymorphism of IS 6110 was used to characterize 53 isolates of Mycobacterium tuberculosis obtained during 1991-1992 from 14 regions in Colombia. In Valle region cluster rate was 25% (4/16). The mean number of IS6110 band was 10 ± 3. Similarity between strains was of 60% in 81% of strains and this tended to be correlated with geographic origin. For the first time M. tuberculosis without IS6110 bands in restriction fragment length polymorphism analysis was found in Colombia. Additional studies are necessaries in order to best characterize the situation in relation to human immunodeficiency virus epidemic and recent changes in tuberculosis control program.

Kinetics of T cell-activation molecules in response to Mycobacterium tuberculosis antigens

The phenotypic features acquired subsequent to antigen-specific stimulation in vitro were evaluated by means of the kinetic expressions of CD69 and CD25 activation molecules on T lymphocytes and assayed by flow cytometry in response to PPD, Ag85B, and ferritin in PPD-positive healthy control individuals. In response to PHA, CD69 staining on both CD4+ and CD8+ T cells became initially marked after 4 h, peaked at 24 h, and quickly decreased after 120 h. For CD25, a latter expression was detected around 8 h, having increased after 96 h. As expected, the response rate to the mycobacterial antigens was much lower than that to the mitogen. Positive staining was high after 96 h for CD25 and after 24 h for CD69. CD69 expression was significantly enhanced (p < 0.05) on CD8+ as compared to CD4+ T cells. High levels were also found between 96-120 h. Regarding Ag85B, CD25+ cells were mostly CD4+ instead of CD8+ T cells. Moreover, in response to ferritin, a lower CD25 expression was noted. The present data will allow further characterization of the immune response to new mycobacterial-specific antigens and their evaluation for possible inclusion in developing new diagnostic techniques for tuberculosis as well in a new vaccine to prevent the disease.