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

• Published in: PLoS ONE
• Main Authors: Mazhgan Rowneki, Naomi Aronson, Peicheng Du, Paige Sachs, Robert Blakemore, Soumitesh Chakravorty, Shawn Levy, Angela L Jones, Geetika Trivedi, Sheilla Chebore, Dennis Addo, Denis K Byarugaba, Panganani Dalisani Njobvu, Frederick Wabwire-Mangen, Bernard Erima, Eric S Ramos, Carlton A Evans, Braden Hale, James D Mancuso, David Alland
• Format: Article
• Language: English
• Published: Public Library of Science (PLoS) 2020-01-01
• Online Access: https://doi.org/10.1371/journal.pone.0232343

<h4>Background</h4>Drug susceptibility testing for Mycobacterium tuberculosis (MTB) is difficult to perform in resource-limited settings where Acid Fast Bacilli (AFB) smears are commonly used for disease diagnosis and monitoring. We developed a simple method for extraction of MTB DNA from AFB smears for sequencing-based detection of mutations associated with resistance to all first and several second-line anti-tuberculosis drugs.<h4>Methods</h4>We isolated MTB DNA by boiling smear content in a Chelex solution, followed by column purification. We sequenced PCR-amplified segments of the rpoB, katG, embB, gyrA, gyrB, rpsL, and rrs genes, the inhA, eis, and pncA promoters and the entire pncA gene.<h4>Results</h4>We tested our assay on 1,208 clinically obtained AFB smears from Ghana (n = 379), Kenya (n = 517), Uganda (n = 262), and Zambia (n = 50). Coverage depth varied by target and slide smear grade, ranging from 300X to 12000X on average. Coverage of ≥20X was obtained for all targets in 870 (72%) slides overall. Mono-resistance (5.9%), multi-drug resistance (1.8%), and poly-resistance (2.4%) mutation profiles were detected in 10% of slides overall, and in over 32% of retreatment and follow-up cases.<h4>Conclusion</h4>This rapid AFB smear DNA-based method for determining drug resistance may be useful for the diagnosis and surveillance of drug-resistant tuberculosis.