Mycobacterium Tuberculosis In Sputum Samples Research Articles

Rapid detection of Mycobacterium tuberculosis in sputa by the amplification of IS6110.

A polymerase chain reaction (PCR) assay for the rapid detection of Mycobacterium tuberculosis in sputum samples was studied. The target DNA was a 123-base pair (bp) fragment of IS6110, which was repeated in the M.tuberculosis genome and was specific for the M.tuberculosis complex. Glass beads (2mm diameter) and lysozyme were used to lyse the mycobacteria and DNA was extracted by the phenol-extraction method. The amplified PCR product was detected by examination of ethidium-bromide-stained agarose gel and by hybridization with an oligonucleotide alkali-phosphatase-labeled probe. A total of 70 samples were tested. PCR was positive in all 13 smear and culture-positive samples, in 5 of 8 smear-negative and culture-positive samples, and in 1 of 49 smear and culture negative samples. The overall sensitivity and specificity were 85.7% and 98%, respectively. Thus, IS6110 as a PCR target was found to be very useful for the rapid diagnosis of M.tuberculosis infection and start of anti-tuberculous chemotherapy.

Detection of Mycobacterium tuberculosis in sputum samples by polymerase chain reaction using a simplified procedure

A repetitive sequence of Mycobacterium tuberculosis DNA was amplified by polymerase chain reaction (PCR), from sputum samples, for the diagnosis of pulmonary tuberculosis. The method of heating the sample in a boiling water bath to break down the bacterial cell wall and to release the DNA was compared with that of enzymatic lysis of bacteria and then phenol-chloroform extraction of DNA. Heating the sample was the better method with a sensitivity of approximately 10 microorganisms. A total of 78 sputum specimens prepared by heating were examined by PCR, and the results were compared with the results of acid-fast stained smears, cultures, and clinical data. M. tuberculosis was detected by PCR in all smear- and culture-positive and smear-negative, culture-positive cases. Additionally, PCR was capable of detecting four of nine cases which were smear and culture negative but clinically suspected of tuberculosis. DNA amplification by PCR is a sensitive and specific method for the diagnosis of tuberculosis, and with this simplified DNA isolation procedure it can be used in routine clinical practice.

Detection ofMycobacterium tuberculosisin Sputum Samples Using a Polymerase Chain Reaction

A polymerase chain reaction (PCR) assay for the rapid detection of Mycobacterium tuberculosis in sputum samples is described. The target DNA is a 123-base pair (bp) segment of IS6110, which is repeated in the M. tuberculosis chromosome and is specific for the M. tuberculosis complex. Methodology used to lyse the mycobacteria, extract the DNA, and amplify the 123-bp target DNA is presented. The amplified PCR product is detected by examination of ethidium-bromide-stained acrylamide gels. An internal control using the same primers as the target DNA has been constructed to assess the efficacy of each individual reaction. Of 162 sputum samples tested, 82 were smear-positive for acid-fast bacilli. Of the 94 specimens from patients in whom pulmonary tuberculosis was diagnosed, 51 were culture-positive, smear-positive, or both. Fifty of these were PCR positive. Of the 42 specimens from patients with nontuberculous mycobacterial pulmonary disease, 41 were PCR negative. All 26 specimens from patients without mycobacterial infection were PCR negative. This assay provides a sensitive and specific means for the laboratory diagnosis of tuberculosis within 48 h that is relatively simple to perform.

Clinical profiling and use of loop-mediated isothermal amplification assay for rapid detection of <i>Mycobacterium tuberculosis</i> from sputum

Tuberculosis is a global health problem and the situation is worsening with newer incidences of drug resistance and HIV association. Diagnosis of tuberculosis can be done by many methods and test, culture of sputum being the ideal one. Nucleic acid amplification (NAA) assay are more time efficient one, that amplify and detect specific nucleic acid sequences allows rapid, sensitive and specific detection of M. tuberculosis in sputum samples. The present study intends to compile the clinical presentations of the pulmonary tuberculosis (PTB) patients and to evaluate the efficacy of in-house loop-mediated isothermal amplification (LAMP) in detecting Mycobacterium tuberculosis in sputum samples by comparing with microscopy and culture. Two hundred two sputum samples were collected from 202 patients at National Tuberculosis Center, Bhaktapur, Nepal. Complete clinical profiling, epidemiological data and record on BCG vaccination were noted and the samples were subjected for microscopy, culture and in-house LAMP with six primers specific for 16S RNA gene of Mycobacterium tuberculosis. Of the 176 cases of clinical profiling, productive cough was most common symptom in 147 (83.52%), followed by chest pain 136 (77.27%), fever 133 (75.56%) and haemoptysis 61 (34.66%). There was a statistically significant association between BCG vaccination and development of TB (chi(2)=5.33, P=0.02). Of 202 cases, 115 (56.93%) were chest X-ray positive, 101(50%) were direct smear-positive and 100 (49.51%) were culture positive. LAMP had a sensitivity of 97% and specificity of 94.12% while comparing with culture. In addition, its sensitivity and specificity were 91.09% and 89.11% respectively with reference to microscopy. As in our previous study, overall, the result of present study further confirms that the in-house LAMP is a simple, rapid, sensitive and specific DNA amplification technique for PTB diagnosis. Because of rapidity of microscopy and specificity of culture, in-house LAMP assay can be used as a very powerful and useful supplementary tool with complete clinical profiling of the patients for rapid diagnosis of TB in both AFB-positive and negative cases who are suspected as PTB in disease endemic country like Nepal.