PLoS ONE (Jan 2020)

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

  • 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

DOI
https://doi.org/10.1371/journal.pone.0232343
Journal volume & issue
Vol. 15, no. 5
p. e0232343

Abstract

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BackgroundDrug 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.MethodsWe 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.ResultsWe 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.ConclusionThis rapid AFB smear DNA-based method for determining drug resistance may be useful for the diagnosis and surveillance of drug-resistant tuberculosis.