1661. Advancing Heteroresistance Detection in Tuberculosis using Single Molecule-Overlapping Read (SMOR) Analysis
Session: Poster Abstract Session: Mycobacterial Infections
Saturday, October 5, 2013
Room: The Moscone Center: Poster Hall C
Background: The incidence of drug-resistant (DR) tuberculosis (TB) continues to increase worldwide. Undetected heteroresistance in the Mycobacterium tuberculosis (Mtb) populations at treatment initiation may play a role in the development of DR-TB. Current minor DR component detection levels are limited to ~1%, using phenotypic drug susceptibility testing. By that point during an infection, it is likely too late to prevent DR-TB and treatment failure.

Methods: We have developed a method of detecting resistant Mtb sub-populations consisting of 0.1% or less of the total Mtb population. Detection of minor components in complex biological mixtures has radically advanced with the emergence of next-generation sequencing. Low-level detection from sequence data, however, is not trivial, primarily due to the error rates in sequencing. The error associated with the respective sequencing platform, as well as the GC content of the organism, sets the limit of discerning actual minor component from error. However, the use of “single molecule-overlapping reads” (SMOR) analysis for determination of actual mutation ratios in target loci (e.g., antibiotic resistance genes) leads to an increase in heteroresistance detection sensitivity and lower error bias.  Using the Illumina Miseq platform, we sequenced amplicons from several different in vitro mixtures of DR and susceptible Mtb strains to explore the use of SMOR for identifying heteroresistance.

Results: We calculated the average combined amplification and sequencing error rate for Mtb (a high GC organism) to be 0.51% per position across the amplicons tested. When employing SMOR, the theoretical limit of detection of a minor component would be 2.6x10-6, readily allowing for detection of minor components below 0.51%. In this study, we were able to detect a 0.3% artificial mixture of SNP alleles in the inhA promoter at a frequency of 3.07x10-3, which was at least two orders of magnitude more frequent than identifiable sequence errors.

Conclusion: The use of SMOR will allow us to follow the evolution of heteroresistance, determine its clinical relevance and ultimately could help clinicians develop appropriate treatment strategies to suppress minor component resistant sub-populations before they become clinically significant.

Rebecca E. Colman, PhD1, James A. Schupp, BS MBA1, David Smith, BS1, Paul S. Keim, PhD1,2, Faramarz Valafar, PhD3, Timothy C. Rodwell, MD PhD MPH4, Antonio Catanzaro, MD5 and David M. Engelthaler, MS1, (1)Translational Genomics Research Institute, Flagstaff, AZ, (2)Northern Arizona University, Flagstaff, AZ, (3)San Diego State Univ, San Diego, CA, (4)University of California San Diego, San Diego, CA, (5)University of California - San Diego, La Jolla, CA


R. E. Colman, None

J. A. Schupp, None

D. Smith, None

P. S. Keim, None

F. Valafar, None

T. C. Rodwell, None

A. Catanzaro, None

D. M. Engelthaler, None

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