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Molecular Detection of Drug Resistance (MDDR) service

What technology will be used? 

Conventional PCR and DNA sequencing will be performed.  Utilization of DNA sequencing technology was chosen as the platform for multiple reasons.  First, the platform is semi-automated.  In addition, the assay provides rapid results with extensive information regarding the specific mutations as well as some evidence of mixed populations of M. tuberculosis.  Another benefit is the ease of expansion; as new mutations associated with resistance are defined, additional loci can be added to the sequencing panel relatively quickly.

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What genetic loci will be sequenced as part of the MDDR service?

The sequencing panel was selected to be able to detect resistance associated mutations for the first- and second-line anti-tuberculosis drugs that define MDR- and XDR-TB. (MDR-TB is defined as resistance to at least  RMP and INH; XDR-TB is defined as MDR-TB plus resistance to a FQ and at least one of the second-line anti-TB injectable drugs: KAN, CAP or AMK).  Specific regions (loci) associated with genes previously reported to confer resistance will be sequenced including “hot spots†in rpoB (81 bp region associated with  RMP resistance), inhA (promoter region) and katG (associated with INH resistance), gyrA (associated with resistance to FQs), rrs (associated with resistance to KAN, AMK, and CAP), tlyA (associated with CAP resistance), and eis (promoter region associated with KAN resistance). In addition, loci associated with resistance to EMB (embB) and PZA (pncA) will be sequenced. MDDR results will be issued in an interim report as soon as they are available.  All isolates will also undergo conventional DST using agar proportion to determine phenotypic resistance to first- and second-line drugs (RIF, INH, ethambutol, streptomycin, ofloxacin, ciprofloxacin, KAN, CAP, AMK, ethionamide, and Para-aminosalicylic).   PZA testing will be performed by the MGIT 960 method.   Molecular and conventional results will be analyzed and released in a final report.

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How was the MDDR service validated in the LB at CDC?

Retrospective phase:

254 isolates from both US- and foreign-born patients referred to CDC for DST between the years 2000-2008 were selected for sequencing based on conventional DST results.   The collection included 163 (52%) MDR-TB, 10 (3%) XDR-TB, and 58 (18%) pan-susceptible isolates.  If sequencing results differed from conventional DST, the conventional DST and sequencing was repeated, as needed, for confirmation. 

Prospective phase:

85 consecutive isolates submitted June-August 2009 were simultaneously tested using MDDR and conventional methods to detect drug resistance.  During this phase of validation, unidirectional workflow was established, , appropriate testing algorithms were developed, and QC and QA measures implemented.  The specificity of MDDR for MTB complex was verified by completing the molecular assay on representative examples of nontuberculous mycobacteria species and members of the M. tuberculosis complex. 

Performance:

Combined sensitivity and specificity for both the retrospective and prospective validation phases for MTB isolates were calculated using conventional drug susceptibility results (agar proportion method) as the gold standard. 

Drug Mutation Sensitivity (%) Specificity (%)
RIF rpoB 96.1 97
INH inhA + katG 88.6 98.7
FQ gyrA 82.2 97
KAN rrs + eis 86.8 96.9
AMK rrs 87.9 99
CAP rrs + tlyA 44.6 85.9
PZA pncA 84.6 85.1
EMB embB 78.6 93.1

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What are the expected limitations to MDDR?

The limitations of the molecular service can be attributed to gaps in knowledge. One limitation is that the clinical relevance of some mutations remains unknown.  Many times sequencing will identify point mutations known to be associated with resistance to a particular drug; however, sometimes the association of detected mutations with resistance will be unknown due to insufficient genetic data.  An additional limitation is that not all mechanisms of resistance are known.  Therefore, if no mutation is detected by the molecular assay, resistance can not be ruled out.  Conventional DST results are essential to confirm susceptibility to individual drugs.

 
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