Background: Various primers are being tested for the detection of Mycobacterium tuberculosis DNA. The accuracy of the polymerase chain reaction (PCR) depends on the target sequence used and whether the test will be performed in culture or in clinical specimens. Objectives: To identify DNA sequences, specifically those commonly reported as targets for diagnosis of tuberculosis (TB), in clinical samples of M. tuberculosis strains. Method: Eighty-one clinical samples from suspected TB patients were initially processed and submitted to bacilloscopy (smear) and culture, and PCR was performed with specific primers for the following targets: IS 6110, 65 kDa, 38 kDa and MPB64. Results: Smear and culture results were negative in 24 samples, as was the PCR. The 19 samples testing smear positive, as well as the isolated strains, were 100% positive on PCR, with the exception of the 89.4% result from PCR with MPB64 primers. In the 38 smear negative and culture positive samples, PCR results were inconsistent. The primers specific for amplifying the 123 bp IS 6110 fragment gave the highest positivity (92.1%), diagnostic agreement (0.9143), co-positivity (94.7%) and co-negativity (100%). Conclusion: The IS 6110, 38 kDa, MPB64 and 65 kDa sequences were found in the genome of all M. tuberculosis strains isolated in patients from the state of Amazonas. The protocol for processing the clinical samples prior to PCR analysis and the specific primers used to amplify the 123bp IS 6110 fragment showed a greater efficiency in diagnosing pulmonary (paucibacillary) tuberculosis in comparison to published data.

Keywords: Primers/PCR. 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

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 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 describe baseline characteristics of outpatients with a positive RT-PCR for SARS-CoV-2 and to define whether "red flags" (new-onset fever, dyspnea, and chest pain) can predict clinical worsening during the isolation period. Methods: This was an epidemiological, observational, descriptive study. Between March and September of 2020, all outpatients who tested positive for SARS-CoV-2 at a tertiary medical center located in Santiago de Chile were included. Demographic variables, comorbidities, red flags, and other symptoms were compiled using follow-up surveys at specific time points. The risk of clinical worsening (hospitalization) and adjusted hazard ratios (HRs) were calculated. Results: A total of 7,108 patients were included. The median age was 38 years (range, 0-101), and 52% were men. At baseline, 77% of the patients reported having characteristic symptoms of SARS-CoV-2 infection. The most prevalent onset symptoms were headache (53%), myalgia (47%), and fever (33%). According to the follow-up surveys, the incidence of symptoms decreased during the isolation period; however, 28% of the patients still presented with symptoms on day 14. The risk of hospitalization for patients with new-onset fever and dyspnea during the follow-up period was HR = 7.43 (95% CI, 3.85-14.3, p<0.01) and HR = 5.27 (95% CI, 1.52-18.30; p < 0.01 for both), respectively. New-onset chest pain showed no association with clinical worsening. Conclusions: In this sample of outpatients with a recent diagnosis of SARS-CoV-2 infection, a survey-based monitoring of symptoms was useful to identify those at risk of clinical worsening. New-onset fever and dyspnea during the isolation period were considered as red flags associated with clinical worsening and warrants prompt medical evaluation.

Keywords: Ambulatory care; COVID-19; Follow-up studies; Hospitalization; Outpatients.