Direct detection of Mycobacterium tuberculosis rifampin resistance in bio-safe stained sputum smears

Abstract

Direct smear microscopy of sputum forms the mainstay of TB diagnosis in resource-limited settings. Stained sputum smear slides can serve as a ready-made resource to transport sputum for molecular drug susceptibility testing. However, bio-safety is a major concern during transport of sputum/stained slides and for laboratory workers engaged in processing Mycobacterium tuberculosis infected sputum specimens. In this study, a bio-safe USP (Universal Sample Processing) concentration-based sputum processing method (Bio-safe method) was assessed on 87 M. tuberculosis culture positive sputum samples. Samples were processed for Ziehl-Neelsen (ZN) smear, liquid culture and DNA isolation. DNA isolated directly from sputum was subjected to an IS6110 PCR assay. Both sputum DNA and DNA extracted from bio-safe ZN concentrated smear slides were subjected to rpoB PCR and simultaneously assessed by DNA sequencing for determining rifampin (RIF) resistance. All sputum samples were rendered sterile by Bio-safe method. Bio-safe smears exhibited a 5% increment in positivity over direct smear with a 14% increment in smear grade status. All samples were positive for IS6110 and rpoB PCR. Thirty four percent samples were RIF resistant by rpoB PCR product sequencing. A 100% concordance (κ value = 1) was obtained between sequencing results derived from bio-safe smear slides and bio-safe sputum. This study demonstrates that Bio-safe method can address safety issues associated with sputum processing, provide an efficient alternative to sample transport in the form of bio-safe stained concentrated smear slides and can also provide information on drug (RIF) resistance by direct DNA sequencing.

Aptamer-Based TB Antigen Tests for the Rapid Diagnosis of Pulmonary Tuberculosis: Potential Utility in Screening for Tuberculosis

“…Bacterial growth was detected on the basis of turbidity of the liquid medium, and the presence of Mtb was confirmed by MPT64 strip test (SD Bioline), ZN staining, and IS 6110 PCR ( M. tuberculosis complex specific PCR) as described . For DNA isolation, 500 μL of the liquid culture was pelleted at ∼8800 × g for 10 min in a 1.5 mL microcentrifuge tube at room temperature and processed for DNA isolation and PCR. − The other part (0.5 mL) of sputum was liquefied as described . Briefly, 0.5 mL of sputum was liquefied using 2 volumes of 0.1% Dithiothreitol (DTT), followed by centrifugation at ∼8800 × g for 10 min.…”

Impact of Demographic Characteristics and Therapy on Tuberculosis Incident Cases

“…Newer assays are available for detecting the multiple and extensively drug-resistant Mycobacterium tuberculosis. Desikan [94] and Lavania, et al [96] argued that the DNA amplification and oligonucleotide probe hybridization, Line Probe Assay (LPA), and multiple drugresistant TB (MTBDR) are viable diagnostic tools for detecting multiple drug-resistant in some TB patients. After diagnosing patients with TB, the health care providers have prescribed appropriate drug regimens to treat their TB patients.…”

Detection of drug resistant Mycobacterium tuberculosis by high-throughput sequencing of DNA isolated from acid fast bacilli smears

“…AFB smears and then used in TB DST analysis using molecular and other DNA sequencing based methods [5][6][7][8][9][10][11][12]. However, most of these studies involved small sample sizes (<100 slides) and only demonstrated the performance of their assays with one or two TB specific gene targets.…”