Objective: To compare the performance of nested polymerase chain reaction (NPCR) with that of cultures in the detection of the Mycobacterium tuberculosis complex in pulmonary and extrapulmonary specimens. Methods: We analyzed 20 and 78 pulmonary and extrapulmonary specimens, respectively, of 67 hospitalized patients suspected of having tuberculosis. An automated microbial system was used for the identification of Mycobacterium spp. cultures, and M. tuberculosis IS6110 was used as the target sequence in the NPCR. The kappa statistic was used in order to assess the level of agreement among the results. Results: Among the 67 patients, 6 and 5, respectively, were diagnosed with pulmonary and extrapulmonary tuberculosis, and the NPCR was positive in all of the cases. Among the 98 clinical specimens, smear microscopy, culture, and NPCR were positive in 6.00%, 8.16%, and 13.26%, respectively. Comparing the results of NPCR with those of cultures (the gold standard), we found that NPCR had a sensitivity and specificity of 100% and 83%, respectively, in pulmonary specimens, compared with 83% and 96%, respectively, in extrapulmonary specimens, with good concordance between the tests (kappa, 0.50 and 0.6867, respectively). Conclusions: Although NPCR proved to be a very useful tool for the detection of M. tuberculosis complex, clinical, epidemiological, and other laboratory data should also be considered in the diagnosis and treatment of pulmonary and extrapulmonary tuberculosis.

Keywords: Tuberculosis/diagnosis; Tuberculosis/microbiology; Mycobacterium tuberculosis; Polymerase chain reaction.

Objective: To develop a system for the molecular diagnosis of tuberculosis by polymerase chain reaction (PCR), constructing primers based on the difference in gene organization of the intergenic region of phospholipase C (plcB-plcC region), which differentiates Mycobacterium tuberculosis from other mycobacteria. Methods: A PCR product of the expected size (432 bp) was obtained from M. tuberculosis and M. africanum only. A total of 33 mycobacterial isolates and 273 clinical samples from patients suspected of having tuberculosis were examined. These were used in the comparative study of the PCR technique versus culture. Results: For PCR versus culture, the data showed 93.8% accuracy (p < 0.0001), 93.1% sensitivity (CI: 88.7-96.0), and 96.4% specificity (CI: 96.1-99.4). The Kappa value (0.82) shows that there was a near-perfect concordance between the two tests. Conclusion: The use of the plcB-plcC region in PCR amplification was found to be an important and reliable tool for the specific diagnosis of tuberculosis in the samples analyzed.

Keywords: Polymerase chain reaction; Diagnosis; Tuberculosis; Mycobacterium tuberculosis

Objective: To evaluate the accuracy of bacteriological methods and of polymerase chain reaction (with primers specific for IS6110 of the Mycobacterium tuberculosis complex) in testing sputum samples from indigenous (Amerindian) and non-indigenous patients. Methods: A total of 214 sputum samples (154 from indigenous patients and 60 from non-indigenous patients) were analyzed in order to determine the accuracy of smear microscopy (direct and concentrated versions) for acid-fast bacilli, culture, and polymerase chain reaction. Results: Both microscopy methods presented low sensitivity in comparison with culture and polymerase chain reaction. Specificity ranged from 91% to 100%, the concentrated acid-fast smear technique being the least specific. Nontuberculous mycobacteria were isolated three times more frequently in samples from indigenous patients than in those from non-indigenous patients. False-positive and false-negative polymerase chain reaction results were more common in the indigenous population. Conclusion: Positivity and isolation of nontuberculous mycobacteria in the acid-fast smear in conjunction with polymerase chain reaction positivity raise the following hypotheses: nontuberculous mycobacteria species with DNA regions homologous to, or even still possessing, the M. tuberculosis IS6110 exist in the Amazon; colonization of the oropharynx or of a tuberculous lesion accelerates the growth of the nontuberculous mycobacteria present in the sputum samples, making it impossible to isolate M. tuberculosis; A history of tuberculosis results in positivity for M. tuberculosis DNA. The absence of bacteriological positivity in the presence of polymerase chain reaction positivity raises questions regarding the inherent technical characteristics of the bacteriological methods or regarding patient history of tuberculosis.

Keywords: Tuberculosis, pulmonary/diagnosis; Mycobacterium tuberculosis, Polymerase chain reaction/methods; Indians, south american

Background: Streptococcus pneumoniae is the most common etiologic agent of community-acquired respiratory infections. In recent years, S. pneumoniae resistance to antimicrobial agents has increased. Minimum inhibitory concentration (MIC) is routinely used to determine resistance. Polymerase chain reaction (PCR) detects the genes responsible for Streptococcus pneumoniae resistance to penicillin within approximately 8 hours. Objective: To compare the PCR and MIC methods in determining Streptococcus pneumoniae resistance to penicillin. Method: A total of 153 Streptococcus pneumoniae samples, isolated from various anatomical sites, were evaluated in order to detect mutations in the genes encoding pbp1a, pbp2a and pbp2x, which are responsible for Streptococcus pneumoniae penicillin resistance. A correlation was found between mutations and penicillin MIP, as determined by the agar diffusion method. Results: Overal Streptococcus pneumoniae resistance to penicillin was 22.8% (16.3% intermediate resistance and 6.5% high resistance). In a statistically significant finding, we observed no mutations in the penicillin-sensitive samples and only one mutation, typically in the gene encoding pbp2x, among the samples with intermediate resistance, whereas mutations in all three genes studied were observed in the high-resistance samples. Conclusion: For determining Streptococcus pneumoniae resistance to penicillin, PCR is a rapid method of detection that could well be used in clinical practice.

Keywords: Streptococcus pneumoniae. Penicillin resistance. Polymerase chain reaction/methods.

This study aimed to assess drug resistance in Mycobacterium tuberculosis strains isolated from sputum samples. To that end, sputum samples were collected from 263 patients suspected of having tuberculosis. All subjects lived in the Complexo de Manguinhos, which is located in the city of Rio de Janeiro, Brazil. Cultures testing positive between October of 2000 and December of 2002 were tested to determine strain susceptibility to isoniazid, rifampicin, streptomycin, ethionamide, and ethambutol. Of the 75 patients diagnosed with tuberculosis, resistance to at least one of the drugs was found in 16 (21.4%). Of those 16 patients, 8 (50%) were new cases, and 8 (50%) had previously been treated. Multidrug-resistant tuberculosis was identified in 8 (10.6%) of the 75 patients, being associated with previous treatment in 6 (8%). The incidence of multidrug-resistant tuberculosis might have been underestimated, since M. tuberculosis was not isolated from all of the samples testing positive for acid-fast bacilli. However, at least, our findings shed some light on the problem.

Keywords: Tuberculosis; Drug resistance; Drug resistance, multiple.

Objective: To evaluate the performance of nested PCR (nPCR) in detecting the Mycobacterium tuberculosis complex in blood samples of patients suspected of having TB, in order to determine its potential for use as an auxiliary tool in the laboratory diagnosis of TB in children. Methods: Detection of the M. tuberculosis complex in blood samples using as a target the insertion sequence IS6110 of the genomic DNA of the bacillus. Blood samples of 120 patients were evaluated. All of the patients were under 15 years of age at the time of their treatment at public hospitals in the city of Recife, Brazil (between January of 2003 and August of 2005). Attending physicians at the hospitals diagnosed TB based on the criteria recommended by the American Thoracic Society. The nPCR amplified a 123-bp fragment with outer oligonucleotides (IS1/IS2) and, in the subsequent reaction, using inner oligonucleotides (IS3/IS4), generating an 81-bp amplicon. Results: Active or latent TB was found in 65 patients, TB was ruled out in 28 suspected cases, and 27 patients were TB-free (controls). The sensitivity of nPCR was 26.15% and was significantly higher for the extrapulmonary form of the disease (55.56%) than for the pulmonary form (18.18%). The specificity was 92.73%. Conclusions: Despite the difficulties in diagnosing TB in children and the low number of cases evaluated in the present study, nPCR in blood samples proved to be a rapid and specific technique, albeit one with low sensitivity. In order to establish its true usefulness in the diagnosis of paucibacillary forms, especially extrapulmonary TB, further studies need to be carried out with a larger sample of children and analyzing biological specimens other than blood.

Keywords: Tuberculosis; Diagnosis; Blood; Polymerase chain reaction.

To describe some aspects of tuberculosis in a low-income community (the Complexo de Manguinhos, in Rio de Janeiro, Brazil), a retrospective study was carried out. Of the 290 cases reported in the 2000-2002 period, 75.8% were new cases. The annual incidence rates were 157/100,000 (2000), 205/100,000 (2001), and 145/100,000 (2002). Although there was a tendency toward a decrease in the number of cases over the period studied, the difference was not significant, suggesting that tuberculosis continues to be endemic in the area. Therefore, despite the existence of local public health care services, more efficient strategies should be implemented in order to increase the effectiveness of tuberculosis control programs in the area.

Keywords: Tuberculosis/epidemiology; Poverty areas; Mycobacterium tuberculosis.

We report the case of a 74-year-old male patient with a one-year history of chest pain in the suprasternal notch associated with erythema, edema and drainage of purulent material from a fistulous lesion. The patient was HIV-negative with no history of TB. A CT scan of the chest showed an osteolytic lesion in the sternum, and a biopsy revealed caseous granuloma, which, in the microbiological evaluation, was negative for fungi and acid-fast bacilli. The diagnosis of sternal osteomyelitis caused by Mycobacterium tuberculosis was confirmed using PCR.

Keywords: Thorax; Mycobacterium infections; Tuberculosis, osteoarticular; Sternum; Diagnosis.

Objective: To determine the drug resistance profile of Mycobacterium tuberculosis in Mozambique. Methods: We analyzed secondary data from the National Tuberculosis Referral Laboratory, in the city of Maputo, Mozambique, and from the Beira Regional Tuberculosis Referral Laboratory, in the city of Beira, Mozambique. The data were based on culture-positive samples submitted to first-line drug susceptibility testing (DST) between January and December of 2011. We attempted to determine whether the frequency of DST positivity was associated with patient type or provenance. Results: During the study period, 641 strains were isolated in culture and submitted to DST. We found that 374 (58.3%) were resistant to at least one antituberculosis drug and 280 (43.7%) were resistant to multiple antituberculosis drugs. Of the 280 multidrug-resistant tuberculosis cases, 184 (65.7%) were in previously treated patients, most of whom were from southern Mozambique. Two (0.71%) of the cases of multidrug-resistant tuberculosis were confirmed to be cases of extensively drug-resistant tuberculosis. Multidrug-resistant tuberculosis was most common in males, particularly those in the 21-40 year age bracket. Conclusions: M. tuberculosis resistance to antituberculosis drugs is high in Mozambique, especially in previously treated patients. The frequency of M. tuberculosis strains that were resistant to isoniazid, rifampin, and streptomycin in combination was found to be high, particularly in samples from previously treated patients.

Keywords: Extensively drug-resistant tuberculosis; Tuberculosis; Tuberculosis, multidrug-resistant.

Objective: To estimate the prevalence of latent Mycobacterium tuberculosis infection (LTBI) in renal transplant recipients and to assess sociodemographic, behavioral, and clinical associations with positive tuberculin skin test (TST) results. Methods: This was a cross-sectional study of patients aged ≥ 18 years who underwent renal transplantation at the Renal Transplant Center of the Federal University of Minas Gerais Hospital das Clínicas, located in the city of Belo Horizonte, Brazil. We included renal transplant recipients who underwent the TST between January 2011 and July 2013. If the result of the first TST was negative, a second TST was administered. Bivariate and multivariate analyses using logistic regression were used to determine factors associated with positive TST results. Results: The sample included 216 patients. The prevalence of LTBI was 18.5%. In the multivariate analysis, history of contact with a tuberculosis case and preserved graft function (estimated glomerular filtration rate ≥ 60 mL/min/1.73 m2) were associated with positive TST results. TST induration increased by 5.8% from the first to the second test, which was considered significant (p = 0.012). Conclusions: The prevalence of LTBI was low in this sample of renal transplant recipients. The TST should be administered if renal graft function is preserved. A second TST should be administered if the first TST is negative.

Keywords: Tuberculosis; Tuberculin test; Immunocompromised host.

Objective: To determine the prevalence of and the factors associated with latent Mycobacterium tuberculosis infection (LTBI) in prisoners in the state of Minas Gerais, Brazil. Methods: This was a cross-sectional cohort study conducted in two prisons in Minas Gerais. Tuberculin skin tests were performed in the individuals who agreed to participate in the study. Results: A total of 1,120 individuals were selected for inclusion in this study. The prevalence of LTBI was 25.2%. In the multivariate analysis, LTBI was associated with self-reported contact with active tuberculosis patients within prisons (adjusted OR = 1.51; 95% CI: 1.05-2.18) and use of inhaled drugs (adjusted OR = 1.48; 95% CI: 1.03-2.13). Respiratory symptoms were identified in 131 (11.7%) of the participants. Serological testing for HIV was performed in 940 (83.9%) of the participants, and the result was positive in 5 (0.5%). Two cases of active tuberculosis were identified during the study period. Conclusions: Within the prisons under study, the prevalence of LTBI was high. In addition, LTBI was associated with self-reported contact with active tuberculosis patients and with the use of inhaled drugs. Our findings demonstrate that it is necessary to improve the conditions in prisons, as well as to introduce strategies, such as chest X-ray screening, in order to detect tuberculosis cases and, consequently, reduce M. tuberculosis infection within the prison system.

Keywords: Prisons; Tuberculin test; Latent tuberculosis, HIV.

Objective: Virulent strains of the Mycobacterium tuberculosis complex, under certain appropriate conditions, grow as characteristic ropes, bundles or serpentine cords known as cord factor or growth in cords. The objective of the present study was to evaluate cord factor detection as a method of achieving presumptive identification of the M. tuberculosis complex, comparing it to conventional typing tests. Methods: A total of 743 strains were analyzed from January of 2002 to December of 2005 in the Mycobacteria Sector of the Adolfo Lutz Institute, located in the city of Santos, Brazil. Samples were obtained from clinical specimens collected from patients with respiratory symptoms treated at basic health clinics in the greater metropolitan area of Santos. Ziehl-Neelsen-stained smears were prepared, 301 (40.5%) in MB/BacT broth and 442 (59.5%) on solid media, either Lowenstein-Jensen or Ogawa-Kudoh. Results: The sensitivity, specificity, positive predictive value and negative predictive value obtained during the performance comparison of the two methods (cord factor detection and conventional typing) using both isolation media were, respectively, 98.5, 88, 97 and 93%. The method was more sensitive on solid medium (100%), and the difference in sensitivity between the two media types was only 2.7%. Conclusions: Taking into consideration the results obtained, we conclude that, in laboratories with a high incidence of M. tuberculosis complex isolation and limited economic resources, cord factor detection is a fast and valid criterion for identifying these mycobacteria using liquid or solid medium. It also enables subsequent conclusive identification tests, as well as additional sensitivity tests when necessary.

Keywords: Laboratory techniques and procedures; Mycobacterium tuberculosis; Cord factors

Objective: To analyze and compare the mutations in two different regions of the katG gene, which is responsible for isoniazid (INH) resistance. Methods: We analyzed 97 multidrug-resistant Mycobacterium tuberculosis strains isolated in cultures of sputum samples obtained from the Professor Hélio Fraga Referral Center, in Brasília, Brazil. Another 6 INH-sensitive strains did not present mutations and were included as controls. We used PCR to amplify two regions of the katG gene (GenBank accession no. U06258)-region 1, (from codon 1 to codon 119) and region 2 (from codon 267 to codon 504)-which were then sequenced in order to identify mutations. Results: Seven strains were resistant to INH and did not contain mutations in either region. Thirty strains carried mutations in region 1, which was characterized by a high number of deletions, especially at codon 4 (24 strains). Region 2 carried 83 point mutations, especially at codon 315, and there was a serine-to-threonine (AGC-to-ACC) substitution in 73 of those cases. The analysis of region 2 allowed INH resistance to be diagnosed in 81.4% of the strains. Nine strains had mutations exclusively in region 1, which allowed the proportion of INH-resistant strains identified to be increased to 90.6%. Conclusions: The number of mutations at codon 315 was high, which is consistent with cases described in Brazil and in other countries, and the analysis of region 1 resulted in a 9.2% increase in the rate at which mutations were identified.

Keywords: Isoniazid; Mutation; Tuberculosis; Drug resistance, multiple.

Objective: To determine the drug resistance profile of Mycobacterium tuberculosis in the state of Mato Grosso do Sul, Brazil, between 2000 and 2006. Methods: Descriptive study of reported tuberculosis cases in the Brazilian Case Registry Database. We included only those cases in which M. tuberculosis culture was positive and sensitivity to drugs (rifampicin, isoniazid, streptomycin and ethambutol) was tested. Löwenstein-Jensen and Ogawa-Kudoh solid media were used for cultures, as was an automated liquid medium system. Sensitivity tests were based on the proportion method. Results: Among the 783 cases evaluated, males predominated (69.7%), as did patients in the 20-49 year age bracket (70%), a diagnosis of pulmonary tuberculosis (94.4%) and positive HIV serology (8.6%); 645 (82.4%) were new cases, and 138 (17.6%) had previously been treated. Resistance to at least one drug was found in 143 cases (18.3%). The primary resistance (PR) rate was, respectively, 8.1%, 1.6%, 2.8% and 12.4%, for monoresistance, multidrug resistance (MDR), other patterns of resistance and resistance to at least one drug, whereas the acquired resistance (AR) rate was 14.5%, 20.3%, 10.9% and 45.7%, respectively, and the combined resistance (CR) rate was 9.2%, 4.9%, 4.2% and 18.3%, respectively. In PR, streptomycin was the most common drug, whereas isoniazid was the most common in AR and CR (7.2% and 3.7%, respectively). Conclusions: These high levels of resistance undermine the efforts for tuberculosis control in Mato Grosso do Sul. Acquired MDR was 12.7 times more common than was primary MDR, demonstrating that the previous use of drug therapy is an indicator of resistance. These levels reflect the poor quality of the health care provided to these patients, showing the importance of using the directly observed treatment, short course strategy, as well as the need to perform cultures and sensitivity tests for the early diagnosis of drug resistance.

Keywords: Tuberculosis; Drug resistance, multiple; Mycobacterium tuberculosis.

Objective: Mycobacteriosis is frequently diagnosed among HIV-infected patients. In Mozambique, where few patients are under antiretroviral therapy and the prevalence of tuberculosis is high, there is need for better characterization of mycobacteria at the species level, as well as for the identification of patterns of resistance to antituberculous drugs. Methods: We studied a sample of 503 HIV-infected individuals suspected of having pulmonary tuberculosis. Of those 503, 320 tested positive for mycobacteria through sputum smear microscopy or culture of bronchoalveolar lavage fluid. Results: Acid-fast bacilli were observed in the sputum of 73% of the individuals presenting positive cultures. Of 277 isolates tested, only 3 were nontuberculous mycobacteria: 2 were identified as Mycobacterium avium and one was identified as M. simiae. Strains initially characterized as M. tuberculosis complex through polymerase chain reaction restriction analysis (PRA) of the hsp65 gene were later confirmed as such through PRA of the gyrB gene. Among the M. tuberculosis isolates, resistance patterns were as follows: to isoniazid, 14%; to rifampin, 6%; and multidrug resistance, 5%. Previously treated cases showed significantly higher rates of resistance to first-line antituberculous drugs. The most common radiological pattern was interstitial infiltrate (in 67%), followed by mediastinal lymph node enlargement (in 30%), bronchiectasis (in 28%), miliary nodules (in 18%) and cavitation (in 12%). Patients infected with nontuberculous mycobacteria presented clinical profiles indistinguishable from those of other patients. The median CD4 lymphocyte count in this group was 134 cells/mm3. Conclusions: There is a strong association between tuberculosis and AIDS in Mozambique, as expected in a country with a high prevalence of tuberculosis. Although drug resistance rates are high, the isoniazid-rifampin regimen continues to be the appropriate choice for initial therapy.

Keywords: Tuberculosis; Mycobacteria, atypical; HIV; Acquired immunodeficiency syndrome; Drug Resistance, Multiple; Mozambique.

This retrospective molecular study involving restriction fragment length polymorphism, using insertion sequence 6110 as a marker, was conducted in order to provide an initial insight into the genetic diversity of Mycobacterium tuberculosis strains isolated in the slums of the Complexo de Manguinhos, located in the city of Rio de Janeiro, Brazil. Of the 67 strains evaluated, 23 (34.3%) were found to belong to clusters (total clusters, 10). Household and social chains of transmission were associated with clustering, in 20% and 60%, respectively. Living in the Conjunto Habitacional Programado 2 slum was associated with clustering. Although not significant, it is relevant that 26% of the clustered strains presented primary resistance. These findings, although possibly underestimating the prevalence due to the failure to analyze all strains, could help improve the local tuberculosis control program.

Keywords: Tuberculosis; Epidemiology, molecular; Mycobacterium tuberculosis/transmission; Polymorphism, Restriction Fragment Length.

Objective: To evaluate the incidence of Mycobacterium tuberculosis infection, using tuberculin skin test, among community health agents (CHAs) monitoring TB patients in the city of Cachoeiro de Itapemirim, Brazil. Methods: We included 30 CHAs acting in the Family Health Program and 30 of their family members residing in the same household. The tuberculin skin test results of each CHA were compared with those of the corresponding family member. Results: Of the 30 CHAs, 27 (90.0%) were female, compared with 23 (76.7%) of the 30 family members (p = 0.299). The mean age of the CHA group and of the family member group was, respectively, 36.8 and 39.7 years. No statistically significant difference was found between the groups regarding the level of education. Regarding M. tuberculosis exposure, the same number of participants in the two groups reported having known or had contact with a TB patient (17 individuals; 56.7%). There was a statistically significant difference regarding positive tuberculin skin test results (26.7% in the CHA group and 3.3% in the family member group; p = 0.011). Conclusions: M. tuberculosis infection was significantly higher among CHAs than among their family members, fueling the debate on the occupational risk involved in the activities of these professionals.

Keywords: Community health aides; Tuberculosis; Tuberculin test.

Introduction: The teaching hospital, HUCFF, Southeastern Brazil, annually receives three hundred tuberculosis cases. Medical students have their practice at that hospital. Objective: To determine the prevalence of Mycobacterium tuberculosis (Mtb) infection among the students of the UFRJ School of Medicine. Design: Cross-sectional study of medical students at different levels of their training programs. Information about social and demographic characteristics, BCG vaccination history, and potential exposures to tuberculosis were obtained with a standardized questionnaire. Tuberculin skin testing was used to determine the prevalence of tuberculosis infection. Results: The prevalence of tuberculin skin testing positivity among medical students increased as they advanced to higher training levels (4.6%, 7.8%, 16.2%, respectively; P = 0.002). The risks were greatest during the years of clinical training, when medical students had more contact with patients. Conclusions: Medical students in the HUCFF/UFRJ may be at an increased risk of M. tuberculosis infection. A program of routine tuberculin skin testing, combined with interventions to confirm TST conversion and to reduce the risk of nosocomial transmission in the workplace is needed.

Keywords: Nosocomial infection. Medical students. Risk factors. Cross-sectional studies.