TY - JOUR
T1 - Detection of drug resistant Mycobacterium tuberculosis by high-throughput sequencing of DNA isolated from acid fast bacilli smears
AU - Rowneki, Mazhgan
AU - Aronson, Naomi
AU - Du, Peicheng
AU - Sachs, Paige
AU - Blakemore, Robert
AU - Chakravorty, Soumitesh
AU - Levy, Shawn
AU - Jones, Angela L.
AU - Trivedi, Geetika
AU - Chebore, Sheilla
AU - Addo, Dennis
AU - Byarugaba, Denis K.
AU - Njobvu, Panganani Dalisani
AU - Wabwire-Mangen, Frederick
AU - Erima, Bernard
AU - Ramos, Eric S.
AU - Evans, Carlton A.
AU - Hale, Braden
AU - Mancuso, James D.
AU - Alland, David
N1 - Publisher Copyright:
This is an open access article, free of all copyright, and may be freely reproduced, distributed, transmitted, modified, built upon, or otherwise used by anyone for any lawful purpose. The work is made available under the Creative Commons CC0 public domain dedication.
PY - 2020/5
Y1 - 2020/5
N2 - Background 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. Methods 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. Results 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. Conclusion This rapid AFB smear DNA-based method for determining drug resistance may be useful for the diagnosis and surveillance of drug-resistant tuberculosis.
AB - Background 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. Methods 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. Results 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. Conclusion This rapid AFB smear DNA-based method for determining drug resistance may be useful for the diagnosis and surveillance of drug-resistant tuberculosis.
UR - http://www.scopus.com/inward/record.url?scp=85084406892&partnerID=8YFLogxK
U2 - 10.1371/journal.pone.0232343
DO - 10.1371/journal.pone.0232343
M3 - Article
C2 - 32384098
AN - SCOPUS:85084406892
SN - 1932-6203
VL - 15
JO - PLoS ONE
JF - PLoS ONE
IS - 5
M1 - e0232343
ER -