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Molecular Detection of Drug Resistance (MDDR) by DNA Sequencing

Introduction

What is the need for MDDR?

The ability to rapidly and accurately detect drug resistance in Mycobacterium tuberculosis Complex (MTBC) clinical isolates is critical for the appropriate treatment of patients suffering from tuberculosis (TB) and the effectiveness of TB control programs.  Efforts to treat patients and control the spread of tuberculosis can be hindered by the emergence of MTBC clinical isolates resistant to both first and second line anti-tuberculosis drugs.  Additionally, the slow growth rate of MTBC and inherent difficulties associated with conventional drug susceptibility testing methods often serve as impediments to obtaining timely results. To address these issues, the Laboratory Branch (LB) of the Division of Tuberculosis Elimination (DTBE) at the CDC is implementing a molecular testing service using DNA sequencing for the identification of drug resistance associated mutations in isolates of  MTBC.  This service (MDDR) will allow rapid confirmation of MDR TB through the identification of genetic mutations associated with rifampin  (RMP) and isoniazid (INH) resistance.  In addition, genetic loci associated with resistance to the most effective second-line drugs, fluroquinolones (FQ) and the injectables amikacin (AMK), kanamycin (KAN), and capreomycin (CAP), as well as pyrazinamide (PZN) and ethambutol (EMB) will be examined.   

All testing and reporting will be CLIA compliant. http://www.cdc.gov/ncidod/srp/2008-2010_CLIA_Certificate_ocr.pdf

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What is known about the genetic basis of resistance in M. tuberculosis?

The phenotypic drug resistance of clinical isolates of MTBC as determined by conventional methods (e.g, broth and agar proportion.) is explained by the presence of mutations in specific genes.  These mutations often consist of only a single nucleotide change in the DNA sequence (i.e., point mutation).  For example, >95% of clinical isolates that are resistant to  RMP have a single point mutation in an 81-bp region of the rpoB gene know as the  RMP resistance determining region (RRDR) (1).  Mutations in this region affect binding of  RMP to the target; thus, conferring resistance.  Similarly, 70-90% of INH resistant isolates can be detected by sequencing the inhA promoter region, the inhA gene, and the katG gene (1)INH resistance can be attributed to mutations in the inhA promoter region which lead to overproduction of the drug target and mutations within katG which inhibit activation of the INH prodrug.  Rapid detection of the presence of these mutations in rpoB, inhA, and katG can indicate that the isolate is resistant to  RMP and/or INH weeks before conventional DST results would typically be available.

Though the genetic basis of resistance for some of the first and second line anti-tuberculosis drugs has been identified, some resistant isolates have unexplained mechanisms of resistance.  As a result, the interpretation of molecular assays examining mutations associated with resistance must be done carefully and with a thorough understanding of the limitations of the test results.  Although the presence of a mutation indicates that a clinical isolate is most likely resistant to the drug of interest; the absence of a mutation is not confirmation of drug susceptibility. 

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