Early detection of drug-resistant mycobacterium tuberculosis
Inventors
Engelthaler, David • Allender, Christopher • Schupp, James • Metcalfe, John
Assignees
Translational Genomics Research Institute TGen • University of California San Francisco UCSF • University of California San Diego UCSD
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Publication Number
US-12522877-B2
Publication Date
2026-01-13
Expiration Date
Abstract
The present invention relates to oligonucleotides, methods, and kits for detecting an antibiotic-resistant subpopulation within a heteroresistant population of Mycobacterium tuberculosis in a sample. An amplicon of a target locus is obtained from the sample. The target locus comprises a region of interest which comprises one or more minor variants associated with the antibiotic resistance. The target locus is selected from the group consisting of: Rv0678, pepQ, atpE, ddn, fbiA, fbiB, fbiC, fgd, fgd1, and fgd2. The region of interest is interrogated to detect the one or more minor variants and thus, the antibiotic-resistant subpopulation of M. tuberculosis.
Core Innovation
The disclosed invention relates to oligonucleotides, primers, methods, and kits for detecting and/or quantifying a drug-resistant subpopulation of Mycobacterium tuberculosis in a sample obtained from a subject. The method generates one or more amplicons from the sample using primer pairs that produce a region of interest in Rv0678, and the drug-resistant subpopulation comprises at least one minor variant of a polymorphism associated with drug-resistance in the region of interest in Rv0678.
Minor variants of drug-resistance-associated polymorphisms are detected and/or quantified from sequencing of the one or more amplicons. The approach emphasizes rapid detection of very low minor components within heteroresistant Mycobacterium tuberculosis populations by interrogating polymorphisms in loci including Rv0678 and additional loci such as pepQ, atpE, ddn, fbiA, fbiB, fbiC, fgd, fgd1, and fgd2, where the polymorphisms include SNPs and indels.
The disclosure further includes sequence-based interrogation and quantification rules for calling minor variants, including sequencing and detecting/quantifying minor variants in the polymorphisms and calling a minor variant when independent sequencing reads are identical. The disclosed method can optionally predict phenotypic resistance to bedaquiline and/or nitroimidazole based on a micro-heteroresistance threshold, and relates the detected minor variants and resulting drug-resistance determination to therapeutic agent customization using listed therapeutic agents and combinations.
Claims Coverage
Independent claim clm-00001 defines a method for detecting and/or quantifying a drug-resistant Mycobacterium tuberculosis subpopulation by generating Rv0678-targeted amplicons with specified primer pairs, sequencing the amplicons, and detecting/quantifying minor polymorphism variants whose presence indicates the subpopulation. The independent claim is refined by multiple dependent claims that specify variant types and positions, tighten criteria for minor-variant calling, and further add phenotype prediction and therapy customization.
Rv0678-targeted minor-variant detection to indicate a drug-resistant subpopulation
A method of detecting and/or quantifying a drug-resistant subpopulation of Mycobacterium tuberculosis in a sample obtained from a subject, comprising generating one or more amplicons from the sample using specified primer pairs, wherein each amplicon comprises a region of interest in Rv0678, and wherein the drug-resistant subpopulation comprises at least one minor variant of a polymorphism associated with drug-resistance in at least one region of interest in Rv0678; sequencing the one or more amplicons; and detecting and/or quantifying the at least one minor variant, wherein the presence of the at least one minor variant indicates the presence of the drug-resistant subpopulation in the sample.
Therapeutic customization based on detected drug-resistant subpopulation
The method further includes administering to a subject a customized therapeutic agent based on the drug resistance of an M. tuberculosis subpopulation in a sample from the subject, where the agent is selected from an antibiotic, PA-824, OPC-67683, SQ109, TMC207, NAS-21, NAS-91, or combinations thereof.
Quantifying low-abundance minor variants
The method includes a minor Mycobacterium tuberculosis variant that is present at 0.1% or less of the total Mycobacterium tuberculosis population.
Specific Rv0678 insertion/deletion minor-variant positions
The method defines the at least one minor variant as including an insertion or deletion in Rv0678 at specified positions 132, 136, 137, 138, 139, 192, or 193, or a combination of these.
Calling a minor variant when independent sequencing reads are identical
The method sequences two complementary strands of each amplicon, detects and/or quantifies minor variants in the polymorphisms, and calls a minor variant only when the independent sequencing reads are identical.
Phenotypic resistance prediction using a micro-heteroresistance threshold
The method further predicts phenotypic Mycobacterium tuberculosis resistance to bedaquiline, nitroimidazole, or both using a micro-heteroresistance threshold.
Overall, the claim set centers on Rv0678-targeted amplicon generation with specified primer pairs, sequencing, and detection/quantification of minor drug-resistance polymorphism variants whose presence indicates a drug-resistant Mycobacterium tuberculosis subpopulation. Dependent claims further specify quantitative low-abundance thresholds, define variant types and positions in Rv0678, require stringent minor-variant calling based on identical independent sequencing reads, and optionally link micro-heteroresistance to phenotypic resistance prediction and customized therapeutic agent selection.
Stated Advantages
Enables detection and/or quantification of a drug-resistant Mycobacterium tuberculosis subpopulation by detecting and/or quantifying minor polymorphism variants.
Supports detection of very low minor components within heteroresistant Mycobacterium tuberculosis populations, including minor variants present at 0.1% or less.
Tightens minor-variant calling by requiring that independent sequencing reads be identical.
Allows prediction of phenotypic resistance to bedaquiline and/or nitroimidazole based on a micro-heteroresistance threshold.
Enables customized therapeutic agent selection based on the detected drug resistance of an M. tuberculosis subpopulation.
Documented Applications
Tracking detection and/or quantification of bedaquiline-related drug resistance emergence and micro-heteroresistance dynamics using serial patient isolates described in the disclosure.
Programmatic cohort analysis of bedaquiline resistance acquisition associated with prior clofazimine exposure as described in the disclosure.
Predicting phenotypic resistance to bedaquiline and/or nitroimidazole and using that information for therapy customization using the listed therapeutic agents.
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