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 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

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.

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: 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: The rapid differentiation between Mycobacterium tuberculosis and nontuberculous mycobacteria is fundamental for patients co-infected with tuberculosis and HIV. To that end, we use two methods in our laboratory: detection of cord factor and PCR-restriction enzyme analysis (PRA). The objective of this study was to evaluate the accuracy of a screening test on solid medium as a rapid method for the presumptive identification of M. tuberculosis complex, considering costs and turnover time. Methods: A total of 152 strains were submitted to a combined screening test, consisting of the detection of cord factor under microscopy (Ziehl-Neelsen staining) and evaluation of the macroscopic aspect of colonies, as well as to PRA, which was used as the gold standard. Costs were estimated by calculating the price of all of the materials needed for each test. Results: The overall accuracy of cord factor detection alone was 95.4% (95% CI: 90.7-98.1%), and that of the combined screening test was 99.3% (95% CI: 96.4-100%). Cord factor detection costs US$ 0.25, whereas the PRA costs US$ 7.00. Results from cord factor detection are ready in 2 days, whereas PRA requires 4 days to yield results. Conclusions: The presumptive identification of M. tuberculosis using the macroscopic evaluation of colonies combined with cord factor detection under microscopy is a simple, rapid and inexpensive test. We recommend the combined screening test to rapidly identify M. tuberculosis in resource-poor settings and in less well-equipped laboratories while awaiting a definite identification by molecular or biochemical methods.

Keywords: Tuberculosis; Mycobacterium/classification; Polymerase chain reaction; Diagnostic tests, routine.

Objective: To describe the clinical manifestations of patients with pulmonary infection caused by mycobacteria of the Mycobacterium abscessus complex (MABSC), and to compare these manifestations with those of patients infected with other nontuberculous mycobacteria (NTM). Methods: This was a retrospective cohort study involving 43 patients divided into two groups: the MABSC group, consisting of patients with pulmonary infection caused by MABSC (n = 17); and the NTM group, consisting of patients with pulmonary infection caused by NTM other than MABSC (n = 26). Patients were previously treated with a regimen of rifampin, isoniazid, pyrazinamide, and ethambutol before the diagnosis of NTM was confirmed by two culture-positive sputum samples. The nucleotide sequences of the hsp65, 16S rRNA, and/or rpoB genes were analyzed to identify the mycobacteria. Data were collected on demographic, clinical, and radiological characteristics, as well as on treatment responses and outcomes. Results: Loss of appetite was the only clinical manifestation that was significantly more common in the MABSC group than in the NTM group (p = 0.0306). The chance of having to use a second treatment regimen was almost 12 times higher in the MABSC group than in the NTM group. Treatment success was significantly higher in the NTM group than in the MABSC group (83.2% vs. 17.6%; p < 0.0001). The chance of recurrence was approximately 37 times higher in the MABSC group than in the NTM group. Conclusions: In the study sample, treatment response of pulmonary disease caused by MABSC was less favorable than that of pulmonary disease caused by other NTM.

Keywords: Nontuberculous mycobacteria/classification; Nontuberculous mycobacteria/drug effects; Lung diseases.

Objective: Although Mycobacterium avium complex (MAC) lung disease has been shown to be associated with lung cancer and hematologic malignancies, there have been few studies of its relationships with other types of cancer. The aim of this study was to assess the effect that coexisting advanced extrapulmonary solid tumors have on the progression of MAC lung disease. Methods: This was a retrospective study of patients diagnosed with MAC lung disease, on the basis of the American Thoracic Society (ATS) criteria, between October of 2005 and March of 2019. The patients were divided into three groups: those with advanced-stage cancer (A-SC group); those with early-stage cancer (E-SC group); and those without cancer (control group). Progression of MAC lung disease was defined as exacerbation seen on imaging. Patient characteristics and the time to progression were compared among the three groups. Results: A total of 286 patients met the ATS diagnostic criteria for MAC lung disease, and 128 of those were excluded. Of the remaining 158 patients, 20 (7.0%) were in the A-SC group, 36 (12.6%) were in the E-SC group, and 102 (35.7%) were in the control group. The median time to progression in the A-SC, E-SC, and control groups was 432, 3,595, and 2,829 days, respectively (p < 0.01). A proportional hazards model showed that the significant predictors of MAC lung disease progression were advanced-stage cancer (hazard ratio [HR] = 6.096; 95% CI: 2.688-13.826; p < 0.01), cavitary lesions (HR = 2.750; 95% CI: 1.306-5.791; p < 0.01), and a high Nodule-Infiltration-Cavity-Ectasis score (HR = 1.046; 95% CI: 1.004-1.091; p = 0.033). Conclusions: A coexisting advanced extrapulmonary solid tumor could hasten the progression of MAC lung disease.

Keywords: Nontuberculous mycobacteria; Mycobacterium avium complex; Neoplasms; Radiography.

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.

Objective: To determine the current use and potential acceptance (by tuberculosis experts worldwide) of novel rapid tests for the diagnosis of tuberculosis that are in line with World Health Organization target product profiles. Methods: A multilingual survey was disseminated online between July and November of 2016. Results: A total of 723 individuals from 114 countries responded to the survey. Smear microscopy was the most commonly used rapid tuberculosis test (available to 90.9% of the respondents), followed by molecular assays (available to 70.7%). Only a small proportion of the respondents in middle- and low-income countries had access to interferon-gamma-release assays. Serological and lateral flow immunoassays were used by more than a quarter (25.4%) of the respondents. Among the respondents who had access to molecular tests, 46.7% were using the Xpert assay overall, that proportion being higher in lower middle-income countries (55.6%) and low-income countries (76.6%). The data also suggest that there was some alignment of pricing for molecular assays. Respondents stated they would accept novel rapid tuberculosis tests if available, including molecular assays (acceptable to 86.0%) or biomarker-based serological assays (acceptable to 81.7%). Simple biomarker-based assays were more commonly deemed acceptable in middle- and low-income countries. Conclusions: Second-generation molecular assays have become more widely available in high- and low-resource settings. However, the development of novel rapid tuberculosis tests continues to be considered important by tuberculosis experts. Our data also underscore the need for additional training and education of end users.

Keywords: Tuberculosis/diagnosis; Surveys and questionnaires; Income; Mycobacterium tuberculosis/isolation & purification; Molecular diagnostic techniques/methods; Serologic tests/methods.

Objectives: To evaluate the performance of a computer program designed to facilitate the detection of pulmonary nodules using multidetector computed tomography (MDCT) scans of the chest. Methods: We evaluated 24 consecutive MDCT scans of the chest at the Fleury Diagnostic Imaging Center during the period from October 7 to October 19 of 2006, using a 64-channel CT scanner. The study comprised 12 females and 12 males, ranging from 35 to 77 years of age (mean, 57.9 years). Double reading and a computer-aided diagnosis (CAD) system were used in order to perform two independent analyses of the data. The nodules found using both methods were recorded, and the data were compared. Results: The total sensitivity of CAD for the detection of nodules was 16.5%, increasing to 55% when nodules <4 mm in diameter were excluded. Sensitivity by diameter was 6.5% for nodules <4 mm, 45% for nodules of 4-6 mm, 100% for nodules of 6 mm-1 cm, and 0% for nodules >1 cm. More than 99% of true nodules detected by CAD were registered in the image double-reading process. Conclusions: In this preliminary 24-case study, the sensitivity of computer program tested was not significantly greater than that of the double-reading process that is routinely performed in this facility.

Keywords: Image interpretation, computer-assisted; Coin lesion, pulmonary; Tomography, X-ray computed.

Coccidioidomycosis, a fungal illness acquired by the inhalation of arthroconidia of Coccidioides sp., was first described in 1894. Coccidioidomycosis is mainly restricted to areas with arid climate, alkaline soil and low rainfall. Consequently, most of the reported cases in Brazil have occurred in the northeastern region. We report three cases of pulmonary coccidioidomycosis occurring between 2005 and 2006 in an endemic area in the state of Ceará, Brazil. The three patients were immunocompetent adult males, hunters of armadillos (Dasypus novemcinctus), with complaints of cough, fever, dyspnea and pleuritic pain. All three patients presented pulmonary involvement, and one also presented cutaneous lesions. Chest X-rays and CT scans of the patients revealed characteristic coccidioidomycosis lesions. The diagnosis was confirmed by serological testing. All of the patients evolved to cure after antifungal treatment.

Keywords: Coccidioidomycosis; Lung diseases, fungal; Coccidioides.

Objective: To investigate the significance of cellular immune markers, as well as that of collagen and elastic components of the extracellular matrix, within granulomatous structures in biopsies of patients with pulmonary or extrapulmonary sarcoidosis. Methods: We carried out qualitative and quantitative evaluations of inflammatory cells, collagen fibers, and elastic fibers in granulomatous structures in surgical biopsies of 40 patients with pulmonary and extrapulmonary sarcoidosis using histomorphometry, immunohistochemistry, picrosirius red staining, and Weigert's resorcin-fuchsin staining. Results: The extrapulmonary tissue biopsies presented significantly higher densities of lymphocytes, macrophages, and neutrophils than did the lung tissue biopsies. Pulmonary granulomas showed a significantly higher number of collagen fibers and a lower density of elastic fibers than did extrapulmonary granulomas. The amount of macrophages in the lung samples correlated with FVC (p < 0.05), whereas the amount of CD3+, CD4+, and CD8+ lymphocytes correlated with the FEV1/FVC ratio and VC. There were inverse correlations between TLC and the CD1a+ cell count (p < 0.05), as well as between DLCO and collagen/elastic fiber density (r = −0.90; p = 0.04). Conclusions: Immunophenotyping and remodeling both showed differences between pulmonary and extrapulmonary sarcoidosis in terms of the characteristics of the biopsy samples. These differences correlated with the clinical and spirometric data obtained for the patients, suggesting that two different pathways are involved in the mechanism of antigen clearance, which was more effective in the lungs and lymph nodes.

Keywords: Sarcoidosis; Granulomatous disease, chronic; Extracellular matrix; Immunophenotyping; Respiratory function tests.

In view of the fact that the World Health Organization has recommended the use of the mycobacteria growth indicator tube (MGIT) 960 system for the diagnosis of tuberculosis and that there is as yet no evidence regarding the clinical impact of its use in health care systems, we conducted a pragmatic clinical trial to evaluate the clinical performance and cost-effectiveness of the use of MGIT 960 at two health care facilities in the city of Rio de Janeiro, Brazil, where the incidence of tuberculosis is high. Here, we summarize the methodology and preliminary results of the trial. (ISRCTN.org Identifier: ISRCTN79888843 [http://isrctn.org/])

Keywords: Controlled clinical trial; Tuberculosis; Diagnostic tests, routine.

Objective: To evaluate the impact of the use of the molecular test for Mycobacterium tuberculosis and its resistance to rifampin (Xpert MTB/RIF), under routine conditions, at a referral hospital in the Brazilian state of Bahia. Methods: This was a descriptive study using the database of the Mycobacteriology Laboratory of the Octávio Mangabeira Specialized Hospital, in the city of Salvador, and georeferencing software. We evaluated 3,877 sputum samples collected from symptomatic respiratory patients, under routine conditions, between June of 2014 and March of 2015. All of the samples were submitted to sputum smear microscopy and the Xpert MTB/RIF test. Patients were stratified by gender, age, and geolocation. Results: Among the 3,877 sputum samples evaluated, the Xpert MTB/RIF test detected M. tuberculosis in 678 (17.5%), of which 60 (8.8%) showed resistance to rifampin. The Xpert MTB/RIF test detected M. tuberculosis in 254 patients who tested negative for sputum smear microscopy, thus increasing the diagnostic power by 59.9%. Conclusions: The use of the Xpert MTB/RIF test, under routine conditions, significantly increased the detection of cases of tuberculosis among sputum smear-negative patients.

Keywords: Tuberculosis/diagnosis; Molecular diagnostic techniques; Sputum.

Background: The appearance of tuberculosis/human immunodeficiency virus co-infection and the growing number of diseases caused by nontuberculous mycobacteria, as well as the confusion that these can cause in relation to emerging multidrug-resistant strains, require more accurate and rapid laboratory results, not only in the isolation of strains but also in their identification. Objective: A comparative study evaluating a new tool of molecular identification, which uses a genetic probe based on the 16S rDNA sequence of the Mycobacterium tuberculosis gene (Gen-Probe Accuprobe® Gen Probe, Inc.), and the classic methodology. Method: Fifty-five Mycobacterium strains, isolated from the sputum of patients treated at a tuberculosis reference clinic, were selected for study. Subcultures were performed in three tubes: one submitted to genetic identification, one analyzed through classical tests (production and accumulation of niacin; growth in the Lowenstein-Jensen medium with the inhibitor agents p-nitrobenzoic acid and thiophene-2-carboxylic acid hydrazide added), and one held in reserve. Results: The probe identified 51 cases as belonging to the M. tuberculosis complex (one associated with M. kansasii) and the other 4 as nontuberculous mycobacteria, later identified as M. kansasii (3) and M. avium (1). Using traditional methods, 47 samples were identified as belonging to the M. tuberculosis complex, 4 were classified as fitting the profile of nontuberculous mycobacteria (in agreement with the genetic probe results), and 4 were unidentified, 1 of which presented the exact characteristics that 2 mycobacterium species have in common. Conclusion: The benefits of the molecular biology technique justify its implementation and routine use, in combination with classical methods, in a high-traffic clinic where complex cases of tuberculosis are treated.

Keywords: Mycobacterium tuberculosis. Molecular probe techniques.

We compared the nitrate reductase assay with the proportion method, which is considered the gold standard, in 57 Mycobacterium tuberculosis strains isolated from patients treated at the Federal University of Minas Gerais Hospital das Clínicas, located in the city of Belo Horizonte, Brazil. For rifampin and isoniazid, the sensitivity, specificity, and accuracy of the nitrate reductase assay were all 100%, whereas they were 100%, 88.9%, and 66.7%, respectively, for streptomycin and 98.0%, 100%, and 98.2%, respectively, for ethambutol. The mean time to results was ten days. In the study sample, the nitrate reductase assay proved highly accurate and showed excellent concordance with the gold standard.

Keywords: Mycobacterium tuberculosis; Microbial sensitivity tests; Tuberculosis, multidrug-resistant; Nitrate reductase.