Updates to the CDC MDDR Service
April 18, 2017
Beverly Metchock, DrPH, D(ABMM)Team Lead, Reference Laboratory
Division of TB Elimination
National Center for HIV/AIDS, Viral Hepatitis, STD and TB Prevention
Division of TB Elimination
MDDR Service at CDC Clinical/Programmatic Use
Make rapid confirmation of MDR TB available Make laboratory testing data available to clinicians about SLD
resistance in cases of Rifampin‐ R or MDR TB Testing Criteria
Isolate or NAAT (+) sediment (not raw specimen) High‐risk patients (Rifampin‐R, MDR TB)
From population with high rates of drug resistance Exposed to drug resistant case Failing therapy
Cases of public health importance Known Rifampin resistance
Conventional or molecular test performed by submitter Mixed or non‐viable culture Other reasons
Molecular Analysis(PSQ;
PSQ then Sanger; Sanger)*
Conventional DST
Molecular Results (Interim Report[s])
Isolate or NAAT(+) Sediment Received for MDDR
Molecular + Conventional DST Results (Final Report)
2‐3 day turn‐around time
~35 day turn‐around time
*based on information supplied on request form
CDC’s Molecular Detection of Drug Resistance Service
http://www.cdc.gov/tb/topic/laboratory/MDDRUsersGuide.pdf
0100200300400500600700800900
1000
2009
2010
2011
2012
2013
2014
2015
2016
DST onlyMDDR only (DNA)DST and MDDRPZA onlyTotal
State Public Health Laboratory Submissions to RLT for Drug Susceptibility Studies
Number of submissions
Future of MDDR Improving current assay performance Evaluate our data / results to improve assay performance
Case – “discordant” rpoB results
Isolate sent to CA MDL DST Reference Lab for PSQ (INH and RIF) and DST katGmutation (Ser315Thr) ‐ INH resistant No mutation in rpoB – Probably RIF Susceptible DST pending
Patient from Myanmar HIV (+) TB of the spine (paraspinal abscess/ osteo) Treated twice in past for pulmonary and spinal TB
RTMCC requested isolate to be sent to CDC for full panel MDDR (Sanger sequencing)
Ile572Phe mutation
Outside the 81‐bp region known as the rifampin resistance determining region or RRDR (codons 507 to 533 [E.coli]) Same as ILe491Phe mutation (codons 426 to 452 [TB])
Missed by Xpert MTB/RIF assay Missed by CA and CDC PSQ assays* Primers used in our Sanger assay allow detection 3 previous isolates received for MDDR
“Rare” but recent report that 30% MDR TB strains in Swaziland have this mutation (NEJM. 2015. 372(12):1181)
* CDC PSQ panel updated and this mutation can now be detected
Future of MDDR Improving current assay performance Incorporate additional loci (validation complete) ahpC, fabG1, rpoB 176 (146) for PSQ and SS rpoB 572 for PSQ gyrB for SS
MDDR Version 3(Isolates, NAAT+ Sediments, NAAT+ extractions by IDPB from fixed tissue)
• rpoB (81bp region)+ Val176 +Ile172• inhA (‐15, ‐8)• katG (Ser315)• fabG1 (mabA) Leu203• ahpC (promoter)• embB (Met306, Gly406)• pncA• gyrA +gyrB QRDR• rrs (nt1401/1402,1484)
• eis (promoter region)• tlyA (coding region)
• Rifampin• Isoniazid• Isoniazid• Isoniazid• Isoniazid• Ethambutol• Pyrazinamide• Fluoroquinolones• Amikacin, Kanamycin,
Capreomycin• Kanamycin• Capreomycin
PSQ
Sanger
Future of MDDRImproving current assay performanceAdjusting PSQ inhA primers to improve assay performance: in progress
Comparison of Pyrosequencing and Sanger Sequencing Success Rates for Rifampin and Isoniazid
rpoB1* rpoB2* katG inhA
PSQ sediments 66.3% 66.7% 64.5% 38.0%
PSQ isolates 95.4% 97.4% 97.4% 97.4%
SS sediments 68.9% N/A 74.3% 68.9%
SS isolates 99.0% N/A 99.0% 99.5%
March 2015 through April 2016; 774 samples; analysis by Mallory Little
* PSQ rpoB assay requires 2 sequencing reactions to cover RRDR
Future of MDDRTransition to eLIMS reporting Encrypted e‐mail Web portal
Movement toward standardized reporting as global efforts align Transition to MTBC numbering system for
rpoB (from E. coli numbering system)
Renumbering the rpoB RRDR codons to reflect the true codon number
*Codon numbering is based on E. coli (e.g. Ser531 is actually Ser450 in MTBC)
Leu533 (E.coli)=Leu452 (MTB actual)
Gly507 (E.coli)=Gly426 (MTB actual)
Future of MDDREvaluation of next generation sequencing (NGS) platform for current assay Also look at other genetic
loci associated with resistance
Future of MDDRDefine most efficient workflow / testing algorithm for isolates and NAAT+ sediments What is best algorithm for isolates? Sediments? Potential for incorporation of whole genome
sequencing (WGS) Operationalize the use of minimum inhibitory concentration (MIC) testing
RLT Staff(past and current)
RLT Staff(past and current)
The findings and conclusions in this report are those of the authors and do not necessarily represent the official position of the Centers for Disease Control and Prevention.
AcknowledgementsDTBE Laboratory Branch
National Center for HIV/AIDS, Viral Hepatitis, STD & TB Prevention
Division of Tuberculosis Elimination
[email protected](404) 639‐1285