Dataset Information

FISHing Mycobacterium tuberculosis Complex by Use of a rpoB DNA Probe Bait.

ABSTRACT: Routine staining of sputum specimens does not identify acid-fast bacilli as Mycobacterium tuberculosis with utmost precision, limiting its usability as a confirmatory test for pulmonary tuberculosis. We have combined Ziehl-Neelsen staining and fluorescence in situ hybridization (FISH) to detect M. tuberculosis in sputum specimens. We have developed a new fluorescent oligonucleotide rpoBMTC probe (5'-Alexa-555-AGCGGGGTGATGTCAACCCAG-3') targeting the M. tuberculosis complex rpoB gene. In silico alignment yielded 100% match for M. tuberculosis complex mycobacteria, 66.6% to 47.6% for other bacteria, and no significant hits for viruses and eukaryotes. Negative binding of rpoBMTC probe to the top six respiratory tract bacterial pathogens and to Mycobacterium abscessus and Mycobacterium avium experimentally confirmed its specificity. As for sensitivity, rpoBMTC-FISH detected 103 CFU/ml M. tuberculosis as confirmed by successful detection of M. tuberculosis in artificially seeded sputum samples. The application of rpoBMTC-FISH to 116 routine sputum specimens yielded a detection of M. tuberculosis in all of the 31 Ziehl-Neelsen-positive and culture-positive specimens, and no detection of M. tuberculosis in the 85 M. tuberculosis-negative specimens. These data established the proof of concept that rpoBMTC-FISH alone or combined with Ziehl-Neelsen staining can specifically "FISH out" M. tuberculosis complex mycobacteria in sputum samples collected from patients suspected of pulmonary mycobacteriosis. We are implementing this probe for the routine and specific detection of M. tuberculosis complex bacteria in sputum exhibiting acid-fast mycobacteria.

SUBMITTER: Loukil A

PROVIDER: S-EPMC6156301 | biostudies-literature | 2018 Oct

REPOSITORIES: biostudies-literature

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FISHing Mycobacterium tuberculosis Complex by Use of a rpoB DNA Probe Bait.

Loukil Ahmed A Kirtania Prithwiraj P Bedotto Marielle M Drancourt Michel M

Journal of clinical microbiology 20180925 10

Routine staining of sputum specimens does not identify acid-fast bacilli as Mycobacterium tuberculosis with utmost precision, limiting its usability as a confirmatory test for pulmonary tuberculosis. We have combined Ziehl-Neelsen staining and fluorescence in situ hybridization (FISH) to detect M. tuberculosis in sputum specimens. We have developed a new fluorescent oligonucleotide rpoBMTC probe (5'-Alexa-555-AGCGGGGTGATGTCAACCCAG-3') targeting the M. tuberculosis com ...[more]

PMID: 30068538

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Project description:Discordant results between genotypic drug susceptibility testing (gDST) and phenotypic DST (pDST) for Mycobacterium tuberculosis isolates with disputed (discordance between gDST and pDST results) mutations affect rifampin (RIF)-resistant (RR) and multidrug-resistant (MDR) tuberculosis (TB) treatments due to a lack of practical clinical guidelines. To investigate the role of disputed rpoB mutations in M. tuberculosis and TB treatment outcomes, initial isolates of 837 clinical RR- or MDR-TB cases confirmed during 2014 to 2018 were retested using agar-based RIF pDST and rpoB gene sequencing. MICs were determined for isolates with disputed rpoB mutations. Disputed rpoB mutations were identified in 77 (9.2%) M. tuberculosis isolates, including 50 (64.9%) and 14 (18.2%) phenotypically RIF- and rifabutin (RFB)-resistant isolates, respectively. The predominant single mutations were those encoding L533P (a change of L to P at position 533) (44.2%) and L511P (20.8%). Most of the isolates harboring mutations encoding L511P (87.5%), H526N (100%), D516Y (70.0%), and L533P (63.6%) had MICs of ≤1 mg/liter, whereas isolates harboring the mutation encoding H526L (75%) had a MIC of >1 mg/liter. Of the 63 cases with treatment outcomes available, 11 (17.5%) cases died, 1 (1.6%) case transferred out, and 51 (81%) cases had favorable outcomes, including 8 and 20 cases treated with standard-dose RIF- and RFB-containing regimens, respectively. Excluding cases that transferred out or received no or 1-day treatment, we observed statistically significant differences between the outcomes using active and inactive fluoroquinolones (FQs) (P = 0.008, odds ratio = 0.05 [95% confidence interval, 0.01 to 0.38]) in 57 cases (where active means a case susceptible to the drug and inactive means a case resistant to the drug or drug not used). We concluded that disputed rpoB mutations are not rare. Depending on the resources available, sequencing and/or MIC testing is recommended for better management of RR- and MDR-TB cases.

Dataset Information

FISHing Mycobacterium tuberculosis Complex by Use of a rpoB DNA Probe Bait.

Publications

FISHing Mycobacterium tuberculosis Complex by Use of a <i>rpoB</i> DNA Probe Bait.

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