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Detection of Low-Level Mixed-Population Drug Resistance in Mycobacterium tuberculosis Using High Fidelity Amplicon Sequencing.

Colman RE, Schupp JM, Hicks ND, Smith DE, Buchhagen JL, Valafar F, Crudu V, Romancenco E, Noroc E, Jackson L, Catanzaro DG, Rodwell TC, Catanzaro A, Keim P, Engelthaler DM - PLoS ONE (2015)

Bottom Line: We describe the method using standardized mixtures of DNA from resistant and susceptible Mtb isolates and the assay's performance for detecting ultra-rare DR subpopulations in DNA extracted directly from clinical sputum samples.SMOR analysis enables rapid near real-time detection and tracking of previously undetectable DR sub-populations in clinical samples allowing for the evaluation of the clinical relevance of low-level DR subpopulations.This will provide insights into interventions aimed at suppressing minor DR subpopulations before they become clinically significant.

View Article: PubMed Central - PubMed

Affiliation: Translational Genomics Research Institute, Flagstaff, AZ, United States of America.

ABSTRACT
Undetected and untreated, low-levels of drug resistant (DR) subpopulations in clinical Mycobacterium tuberculosis (Mtb) infections may lead to development of DR-tuberculosis, potentially resulting in treatment failure. Current phenotypic DR susceptibility testing has a theoretical potential for 1% sensitivity, is not quantitative, and requires several weeks to complete. The use of "single molecule-overlapping reads" (SMOR) analysis with next generation DNA sequencing for determination of ultra-rare target alleles in complex mixtures provides increased sensitivity over standard DNA sequencing. Ligation free amplicon sequencing with SMOR analysis enables the detection of resistant allele subpopulations at ≥0.1% of the total Mtb population in near real-time analysis. We describe the method using standardized mixtures of DNA from resistant and susceptible Mtb isolates and the assay's performance for detecting ultra-rare DR subpopulations in DNA extracted directly from clinical sputum samples. SMOR analysis enables rapid near real-time detection and tracking of previously undetectable DR sub-populations in clinical samples allowing for the evaluation of the clinical relevance of low-level DR subpopulations. This will provide insights into interventions aimed at suppressing minor DR subpopulations before they become clinically significant.

No MeSH data available.


Related in: MedlinePlus

Minor subpopulation detection in two sputum samples from Moldova.Resistant and erroneous allele frequencies from three resistance SNP loci in the inhA promoter are shown. Patient 21–0067 with 0.05% resistant allele and patient 22–0129 with 11.39% resistant allele at inhA -15, compared to erroneous and resistant alleles below 0.01% at the other two SNP positions.
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pone.0126626.g005: Minor subpopulation detection in two sputum samples from Moldova.Resistant and erroneous allele frequencies from three resistance SNP loci in the inhA promoter are shown. Patient 21–0067 with 0.05% resistant allele and patient 22–0129 with 11.39% resistant allele at inhA -15, compared to erroneous and resistant alleles below 0.01% at the other two SNP positions.

Mentions: In order to demonstrate the practical utility of SMOR analysis with clinical samples, DNA from a convenient sample of 12 sputum samples collected from M. tuberculosis patients in Moldova were subjected to SMOR analysis. Due to the limited amount of DNA available from these samples, an increased number of PCR amplification cycles were necessary to yield sufficient amplicon material for sequencing (see Methods section). One sample failed to amplify, presumably due to insufficient input target DNA. SMOR analysis was used to characterize all 36 resistance loci in the 11 remaining samples. The genotypic resistance profile closely matched the phenotypic resistance profile (S7 Table). Initial Kanamycin discordance for sample 21–0100 was resolved by expanding the SMOR analysis to include position -37 in the eis promoter, the G-T mutation, a previously published resistance conferring SNP[45]. Where limited discordance was identified between SMOR and phenotypic DST (sample 21–0017), the SMOR call was determined by the major population being resistant or susceptible (S8 Table). This major population call was confirmed by separate pyrosequencing of target loci (data not shown). Within the analyzed clinical samples, limited minor subpopulations of resistance SNPs were identified. For example, resistance allele subpopulations at 11% and 0.05% of the same inhA promoter resistance locus (inhA -15) were found in two of the clinical samples, whereas resistance alleles at the other two known SNP loci (inhA -8, and -17) within that gene were consistent with erroneous allele calls (~0.01%) (Fig 5). Furthermore, patient 22–0129 had a resistance allele in katG315 at the same allelic frequency (11.7%).


Detection of Low-Level Mixed-Population Drug Resistance in Mycobacterium tuberculosis Using High Fidelity Amplicon Sequencing.

Colman RE, Schupp JM, Hicks ND, Smith DE, Buchhagen JL, Valafar F, Crudu V, Romancenco E, Noroc E, Jackson L, Catanzaro DG, Rodwell TC, Catanzaro A, Keim P, Engelthaler DM - PLoS ONE (2015)

Minor subpopulation detection in two sputum samples from Moldova.Resistant and erroneous allele frequencies from three resistance SNP loci in the inhA promoter are shown. Patient 21–0067 with 0.05% resistant allele and patient 22–0129 with 11.39% resistant allele at inhA -15, compared to erroneous and resistant alleles below 0.01% at the other two SNP positions.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4430321&req=5

pone.0126626.g005: Minor subpopulation detection in two sputum samples from Moldova.Resistant and erroneous allele frequencies from three resistance SNP loci in the inhA promoter are shown. Patient 21–0067 with 0.05% resistant allele and patient 22–0129 with 11.39% resistant allele at inhA -15, compared to erroneous and resistant alleles below 0.01% at the other two SNP positions.
Mentions: In order to demonstrate the practical utility of SMOR analysis with clinical samples, DNA from a convenient sample of 12 sputum samples collected from M. tuberculosis patients in Moldova were subjected to SMOR analysis. Due to the limited amount of DNA available from these samples, an increased number of PCR amplification cycles were necessary to yield sufficient amplicon material for sequencing (see Methods section). One sample failed to amplify, presumably due to insufficient input target DNA. SMOR analysis was used to characterize all 36 resistance loci in the 11 remaining samples. The genotypic resistance profile closely matched the phenotypic resistance profile (S7 Table). Initial Kanamycin discordance for sample 21–0100 was resolved by expanding the SMOR analysis to include position -37 in the eis promoter, the G-T mutation, a previously published resistance conferring SNP[45]. Where limited discordance was identified between SMOR and phenotypic DST (sample 21–0017), the SMOR call was determined by the major population being resistant or susceptible (S8 Table). This major population call was confirmed by separate pyrosequencing of target loci (data not shown). Within the analyzed clinical samples, limited minor subpopulations of resistance SNPs were identified. For example, resistance allele subpopulations at 11% and 0.05% of the same inhA promoter resistance locus (inhA -15) were found in two of the clinical samples, whereas resistance alleles at the other two known SNP loci (inhA -8, and -17) within that gene were consistent with erroneous allele calls (~0.01%) (Fig 5). Furthermore, patient 22–0129 had a resistance allele in katG315 at the same allelic frequency (11.7%).

Bottom Line: We describe the method using standardized mixtures of DNA from resistant and susceptible Mtb isolates and the assay's performance for detecting ultra-rare DR subpopulations in DNA extracted directly from clinical sputum samples.SMOR analysis enables rapid near real-time detection and tracking of previously undetectable DR sub-populations in clinical samples allowing for the evaluation of the clinical relevance of low-level DR subpopulations.This will provide insights into interventions aimed at suppressing minor DR subpopulations before they become clinically significant.

View Article: PubMed Central - PubMed

Affiliation: Translational Genomics Research Institute, Flagstaff, AZ, United States of America.

ABSTRACT
Undetected and untreated, low-levels of drug resistant (DR) subpopulations in clinical Mycobacterium tuberculosis (Mtb) infections may lead to development of DR-tuberculosis, potentially resulting in treatment failure. Current phenotypic DR susceptibility testing has a theoretical potential for 1% sensitivity, is not quantitative, and requires several weeks to complete. The use of "single molecule-overlapping reads" (SMOR) analysis with next generation DNA sequencing for determination of ultra-rare target alleles in complex mixtures provides increased sensitivity over standard DNA sequencing. Ligation free amplicon sequencing with SMOR analysis enables the detection of resistant allele subpopulations at ≥0.1% of the total Mtb population in near real-time analysis. We describe the method using standardized mixtures of DNA from resistant and susceptible Mtb isolates and the assay's performance for detecting ultra-rare DR subpopulations in DNA extracted directly from clinical sputum samples. SMOR analysis enables rapid near real-time detection and tracking of previously undetectable DR sub-populations in clinical samples allowing for the evaluation of the clinical relevance of low-level DR subpopulations. This will provide insights into interventions aimed at suppressing minor DR subpopulations before they become clinically significant.

No MeSH data available.


Related in: MedlinePlus