Simplified detection of Mycobacterium tuberculosis in sputum using smear microscopy and PCR with molecular beacons

Abstract

The prompt diagnosis of smear-negative cases is a prerequisite to controlling tuberculosis (TB). Several new laboratory approaches, including nucleic acid amplification (NAA), are being evaluated in various disease settings to meet this challenge. However, NAA needs simplification before it is widely accepted. Furthermore, a supporting smear result improves confidence in and reliability of PCR. In this context, an asymmetric devR PCR assay using two molecular beacon probes for visual or fluorimetric end-point detection of Mycobacterium tuberculosis was developed. The assays reproducibly detected 25 fg M. tuberculosis DNA versus 100 fg by conventional gel electrophoresis (henceforth referred to as gel assay). The devR and IS6110 PCR assays were blindly evaluated on sputum specimens obtained from a directly observed-treatment short-course centre. Universal sample processing (USP) smear microscopy and culture were used as a supportive test and the 'gold' standard, respectively. Among the 148 specimens analysed, 120 were M. tuberculosis culture-positive. Amongst the 122 direct smear-negative samples, 96 were culture-positive, of which 61 were detected by USP smear microscopy. All 35 USP smear-negative samples were positive by three or more PCR methods. devR PCR had a sensitivity of 92.5 % in the fluorimetric assay versus 86.7 % by visual inspection and 90.8 % by the gel method. IS6110 PCR performed at almost equivalent levels. devR visual and fluorimetric assays considered together yielded an increased sensitivity of 95 % without compromising on a specificity of 92.9 %. The results suggest that the USP smear test is useful for diagnosing direct smear-negative TB and judiciously restricting PCR testing to only smear-negative samples. When used together, these tests can provide rapid diagnosis of smear-negative TB in a costeffective manner.

Direct Detection of Rifampin and Isoniazid Resistance in Sputum Samples from Tuberculosis Patients by High-Resolution Melt Curve Analysis

“…The assay was validated on archived DNA samples isolated directly from 99 M. tuberculosis culture-positive sputum specimens, of which 84 were SSM negative. Please note that these 99 archived DNA samples were a subset of 148 specimens analyzed in a previous study (49). The details of sample selection and their characteristics are explained in Fig.…”

“…S1 in the supplemental material. These sputum samples were collected between 2004 and 2006 after obtaining informed consent from subjects who were attending the DOTS Centre at the National Institute for Tuberculosis and Respiratory Diseases (NITRD) and documented to be treatment naive (49). For this study, the results of M. tuberculosis culture and direct-smear microscopy were used for sample selection and assessment (Fig.…”

Diagnosis of Mycobacterium tuberculosis

“…Even though nucleic acid amplification test techniques can amplify a small amount of genetic material, the sample must still contain a certain number of TB bacteria to be effective, and this collection is not always possible, particularly with nonpulmonary TB (Haldar et al, 2007). Therefore, it has been suggested that nucleic acid amplification tests should be combined with other diagnostic tests (for example, tests detecting INF-γ) in order to increase the sensitivity of the test (Dinnes et al, 2007).…”

Comparison of Different DNA Extraction Protocols for Molecular Diagnosis of Mycobacterium Tuberculosis

“…The former method has the disadvantage of low sensitivity and the latter method has to be incubated for more than one month before a final diagnosis ( Soini et al, 2001). So, the use of Polymerase Chain Reaction (PCR) enabled a fast and sensitive diagnostic method for identification of M.Tuberculosis especially of samples of low bacilli load (Haldar et al, 2007). A major problem facing the early diagnosis of TB caused by the conflicting data of DNA extraction protocols.…”