Improvement of Mycobacterium tuberculosis detection in sputum using DNA extracted by sonication

Abstract Tuberculosis (TB) is one of the infectious diseases with high mortality in the world. DNA amplification techniques have been used to overcome barriers to the diagnosis of this disease. However, the success of these methodologies is highly dependent on the DNA obtained from the sample. This study was carried out to verify whether the DNA extracted by sonication (in house method) could yield suitable DNA for amplification by real-time PCR (qPCR). Sixty sputum samples were submitted to DNA extraction using sonication compared to a commercial method (Detect-TB kit, Labtest/MG-Brazil). All DNA samples were amplified by qPCR for IS6110 region (IS6110-qPCR/SYBR Green assay). Out of 60 samples, 40 were positive for TB; of these, all had positive results when extracted by sonication (100%) and 80% when extracted by the commercial method. The limit of detection (LOD) of Mycobacterium tuberculosis (H37Rv strain) by qPCR was 14CFU/mL when the DNA was extracted by sonication, compared to countless colonies when extracted by commercial kit. In conclusion, the sonication protocol (without purification step) proved to be a simple, fast, and suitable method for obtaining DNA for use in qPCR from sputum samples.

Detection of Drug Resistant Mycobacterium Tuberculosis Strains Using Kit SIRE Nitratase®: a Multicenter Study

Abstract (1) Background: The Commercial Kit SIRE Nitratase® PlastLabor, is a drug susceptibility test kit used to detect Mycobacterium tuberculosis resistance to first-line TB treatment drugs. The present study aimed at evaluating its performance in a multicenter study. (2) Methods: To determine its accuracy, the proportion methods in Lowenstein Jensen medium or the BACTECTMMGITTM960 system was used as a gold standard. (3) Results: The study revealed that the respective accuracies of the kit with 190 M. tuberculosis clinical isolates, using the proportion methods in Lowenstein Jensen medium or BACTECTMMGITTM960 system as a gold standard, were 93.9% and 94.6%, 96.9% and 94.6%, 98.0% and 97.8%, and 98.0% and 98.9%, for streptomycin, isoniazid, rifampicin, and ethambutol, respectively. (4) Conclusion: Thus, the kit can rapidly screen resistance to streptomycin, isoniazid, rifampicin, and ethambutol. Additionally, it does not require sophisticated equipment; hence, it can be easily used in the laboratories of low and middle income countries.

Prevalence of hepatitis C virus and human immunodeficiency virus in a group of patients newly diagnosed with active tuberculosis in Porto Alegre, Southern Brazil

BACKGROUND Porto Alegre is the Brazilian state capital with second highest incidence of tuberculosis (TB) and the highest proportion of people infected with human immunodeficiency virus (HIV) among patients with TB. Hepatitis C virus (HCV) infection increases the risk of anti-TB drug-induced hepatotoxicity, which may result in discontinuation of the therapy. OBJECTIVES The aim of this study was (i) to estimate prevalence of HCV and HIV in a group of patients newly diagnosed with active TB in a public reference hospital in Porto Alegre and (ii) to compare demographic, behavioural, and clinical characteristics of patients in relation to their HCV infection status. METHODS One hundred and thirty-eight patients with TB were tested for anti-HCV antibody, HCV RNA, and anti-HIV1/2 antibody markers. HCV RNA from real-time polymerase chain reaction (PCR)-positive samples was submitted to reverse transcription and PCR amplification. The 5′ non-coding region of the HCV genome was sequenced, and genotypes of HCV isolates were determined. FINDINGS Anti-HCV antibody, HCV RNA, and anti-HIV antibodies were detected in 27 [20%; 95% confidence interval (CI), 13-26%], 17 (12%; 95% CI, 7-18%), and 34 (25%; 95% CI, 17-32%) patients, respectively. HCV isolates belonged to genotypes 1 (n = 12) and 3 (n = 4). Some characteristics were significantly more frequent in patients infected with HCV. Among them, non-white individuals, alcoholics, users of illicit drugs, imprisoned individuals, and those with history of previous TB episode were more commonly infected with HCV (p < 0.05). MAIN CONCLUSIONS HCV screening, including detection of anti-HCV antibody and HCV RNA, will be important to improving the management of co-infected patients, given their increased risk of developing TB treatment-related hepatotoxicity.