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Master of Public Health

Resistance-conferring mutations on whole-genome sequencing of fluoroquinolone-resistant and -susceptible M. tuberculosis isolates: a proposed threshold for identifying resistance

AUTHORS

Maruri F , Guo Y , Blackman A , van der Heijden YF , Rebeiro PF , Sterling TR , . Clinical infectious diseases : an official publication of the Infectious Diseases Society of America. 2020 4 29; ().

ABSTRACT

BACKGROUND: Fluoroquinolone resistance in Mycobacterium tuberculosis is conferred by DNA gyrase mutations, but not all fluoroquinolone-resistant M. tuberculosis isolates have mutations detected. The optimal allele frequency threshold to identify resistance-conferring mutations by whole-genome sequencing is unknown.

METHODS: Phenotypically ofloxacin-resistant and lineage-matched ofloxacin-susceptible M. tuberculosis isolates underwent whole-genome sequencing at an average coverage depth of 868 reads. Polymorphisms within the quinolone resistance determining region (QRDR) of gyrA and gyrB were identified. The allele frequency threshold using the Genome Analysis Toolkit (GATK) pipeline was ~8%; allele-level data identified the predominant variant allele frequency and mutational burden (i.e., sum of all variant allele frequencies in the QRDR) in gyrA, gyrB, and gyrA + gyrB for each isolate. Receiver operating characteristic (ROC) curves assessed the optimal measure of allele frequency and potential thresholds for identifying phenotypically- resistant isolates.

RESULTS: Of 42 ofloxacin-resistant M. tuberculosis isolates, area under the ROC curve was highest for predominant variant allele frequency, so that measure was used to evaluate optimal mutation detection thresholds. Areas under the ROC curve for 8%, 2.5%, and 0.8% thresholds were 0.8452, 0.9286, and 0.9069, respectively. Sensitivity and specificity were (69%,100%) for 8%; (86%,100%) for 2.5%;(91%,91%) for 0.8%. The sensitivity of the 2.5% and 0.8% thresholds were significantly higher than the 8% threshold(P=0.016 and P=0.004, respectively) but not significantly different between one another(P=0.5).

CONCLUSIONS: A predominant mutation allele frequency threshold of 2.5% had the highest area under the ROC curve for detecting DNA gyrase mutations that confer ofloxacin resistance, and was therefore the optimal threshold.

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