Molecular prediction of antimicrobial resistance in MTBC
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Molecular prediction of antimicrobial resistance of M. tuberculosis complex (MTBC) cultures through the analysis of whole genome sequencing data. This test is meant to supplement and not replace, phenotypic antimicrobial susceptibility testing. Resistance or susceptibility can be predicted to the first line antimicrobials isoniazid, rifampin, ethambutol and pyraziminade, as well as the second line antimicrobials moxifloxacin, ofloxacin, streptomycin, amikacin, capreomycin, and kanamycin.
- Tuberculosis (TB)
This test will be performed on cultures previously identified as MTBC and it is not appropriate for clinical samples. This method can be performed on solid or liquid media growth, though solid media is optimal. For cultures on solid media, visible growth that is less than 6 weeks old is required, and isolated colonies on plated media are preferred. Liquid cultures must be flagged as positive on the Bactec MGIT 960 and have a minimum volume of 4 ml. Cultures with inadequate growth or volume, and cultures contaminated with other bacterial or mycobacterial species will be rejected.
N/A
Ship cultures at room temperature (DO NOT freeze) for overnight delivery, and prior to Wednesday each week to ensure receipt by Friday.
Shipping of specimens shall be done by a TDG certified individual in accordance with TDG regulations. For additional information regarding classification of specimens for the purposes of shipping, consult either Part 2 Appendix 3 of the TDG Regulations or section 3.6.2 of the IATA Dangerous Goods Regulations as applicable.
This test will be automatically performed on cultures submitted to the NRCM for phenotypic MTBC first or second line antimicrobial susceptibility testing. This test can also be performed on cultures that are not submitted for phenotypic antimicrobial susceptibility testing if phenotypic antimicrobial susceptibility testing results are submitted with the culture.
Notification of sample submission must be emailed or faxed prior to shipping isolates. Samples should be shipped attention: Melissa Rabb, Head, Reference and Diagnostic Services, NRCM/NML at 204-789-6081 / 204-789-6038. A requisition for the NRCM must be completed and signed off by the supervisor/designate of the submitting laboratory and include the source of specimen, patient gender, date of birth, clinical history, submitting laboratory identifier and submitter information. Please also include isolate characteristics: microscopy, pigmentation, culture characteristics, growth rate/temperature, identification, and susceptibility results, if available. Requested testing for urgent submissions should be accompanied with justification.
Cultures will be rejected if appropriate documentation and justification is incomplete or missing.
This test predicts antimicrobial resistance by screening whole genome sequences against a list of high confidence mutations (1) using the bioinformatics tool Mykrobe Predictor (2). Antimicrobial resistance in M. tuberculosis is largely due to single nucleotide polymorphisms that are clonally inherited (3,4,5). For each antimicrobial, prediction of drug resistance and mutations (or absence of mutations) detected in genetic regions associated with resistance to that antimicrobial will be provided. Mutations detected in promotor regions will be reported as: wild type nt [position relative to promoter] mutated nt (example C-15T). Mutations detected in coding regions will be reported with the one letter amino acid code as: wild type AA [M. tuberculosis codon number] mutated AA (example S315T). Mutations in rpoB will be reported with M. tuberculosis codon numbers and not the E. coli codon numbering previously provided (6).
Prediction of first line antimicrobials will be provided for isolates predicted to be sensitive, or phenotypically sensitive to the first line antimicrobials. For isolates that are predicted to be resistant or phenotypically resistant to isoniazid, prediction of resistance to moxifloxacin and ofloxacin will be provided. Prediction results for all first and second line antimicrobials will be provided upon request, or for isolates that are predicted to be resistant or phenotypically resistant to two or more first line antimicrobials. The absence of a mutation does not exclude the possibility of antimicrobial resistance. Phenotypic testing remains the gold standard for the determination of antimicrobial resistance, please correlate these results with phenotypic testing. Mutations detected by this test will be used by the NRCM to confirm phenotypic antimicrobial resistance.
Routine testing from culture: 20 calendar days from the date of specimen receipt.
- Walker TM, Kohl TA, Omar SV, Hedge J, Del Ojo Elias C, Bradley P, et al. Whole-genome sequencing for prediction of Mycobacterium tuberculosis drug susceptibility and resistance: a retrospective cohort study. Lancet Infect Dis. 2015;15: 1193–1202. doi:10.1016/S1473-3099(15)00062-6.
- Bradley P, Gordon NC, Walker TM, Dunn L, Heys S, Huang B, et al. Rapid antibiotic-resistance predictions from genome sequence data for Staphylococcus aureus and Mycobacterium tuberculosis. Nat Commun. 2015;6: 10063. doi:10.1038/ncomms10063.
- Zhang Y, Yew W-W. Mechanisms of drug resistance in Mycobacterium tuberculosis: update 2015. Int J Tuberc Lung Dis. 2015;19: 1276–1289. doi:10.5588/ijtld.15.0389.
- Bishai WR, Cohen KA, Pym AS. Molecular Basis of Drug Resistance in Mycobacterium tuberculosis. Microbiology Spectrum. 2014;2. doi:10.1128/microbiolspec.MGM2-0036-2013.
- Hameed HMA, Islam MM, Chhotaray C, Wang C, Liu Y, Tan Y, et al. Molecular Targets Related Drug Resistance Mechanisms in MDR-, XDR-, and TDR-Mycobacterium tuberculosis Strains. Front Cell Infect Microbiol. 2018;8: 114. doi:10.3389/fcimb.2018.00114.
- Andre E. Consensus numbering system for the rifampicin resistance-associated rpoB gene mutations in pathogenic mycobacteria. Clin Microbiol Infect. 2016; doi:10.1016/j.cmi.2016.09.006.