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MDL Test Name Mycobacterium tuberculosis complex: Drug Resistance Detection & Species Identification by Whole Genome Sequencing

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Test Name

Mycobacterium tuberculosis complex: Drug Resistance Detection & Species Identification by Whole Genome Sequencing

Elegible Submitters	Test is available to all pre-approved submitters within the state of California. It is preferred that submissions from clinical laboratories are routed through the corresponding county public health laboratories (PHL). For submissions from out-of-state, please refer to the APHL TB DST Reference Center website. For any questions on service eligibility, please contact CDPHTBDST@cdph.ca.gov.
Test Methodology	Whole Genome Sequencing-based drug susceptibility testing (WGS-DST) assay is used for the rapid molecular detection of drug-resistance in Mycobacterium tuberculosis complex (MTBC) cultures isolated from patients for diagnostic purposes. The WGS-DST assay leverages Illumina next generation sequencing technology for sequencing the entire genome of the microorganism. Downstream bioinformatics analysis detects variants in specific loci known to be associated with drug resistance in MTBC. The WGS data can also be used for MTBC species confirmation and genotyping. The following molecular targets* associated with drug resistance are detected by the WGS-DST assay: First-line drugs: Isoniazid: katG, fabG1, inhA Rifampin: rpoB Ethambutol: embA, embB Pyrazinamide: pncA Second-line drugs: Ethionamide: ethA, fabG1, inhA Fluoroquinolones (moxifloxacin, levofloxacin): gyrA, gyrB Injectable drugs: Kanamycin & amikacin: rrs, eis Capreomycin: rrs, tlyA Novel and repurposed drugs: Bedaquiline: Rv0678, atpE, pepQ, mmpL5, mmpS5 Clofazimine: Rv0678, pepQ, mmpL5, mmpS5 Linezolid: rrl, rplC * All targets tested by WGS include full-length gene and promoter regions. WGS-DST assay additionally provides identification for Mycobacterium tuberculosis and M. bovis species and further differentiates BCG strain of M. bovis. For detailed information on assay design, please see the Summary of WGS/tNGS-DST reporting language and bioinformatics pipeline (PDF). Please contact the laboratory for more information on the method and analysis.
Reflex Testing Criteria	If mutations of uncertain significance or a mutation associated with resistance are detected by WGS-DST, phenotypic DST will be performed for the corresponding drug if available at MDL, unless already performed.
Pre-Approval Required	Pre-approval is not required except when requesting testing for the following: Duplicate submission, i.e., samples from the same patient collected within 3 months from prior collection date.
Supplemental Information and Required Form(s)	Use MDL Lab Web Portal for ordering the test, select Test Requisition “Mycobacteriology-Drug Susceptibility Testing of MTBC (CA only)-444”. Barcode submittal form (PDF) is currently not available for ordering WGS-DST assay. Please contact the laboratory if experiencing difficulties submitting the order. Please include submitter’s test results on the submittal form.
Required Specimen Type(s)	Culture on LJ (solid) or in BACTEC MGIT (liquid) media. Isolates submitted on other types of media will be subcultured at MDL which will extend turnaround time. Pure cultures with visible biomass are required. Submissions of mixed or non-viable cultures, as well as primary specimens, is NOT acceptable for WGS-DST.
Minimum Volume Required	Culture in broth: 0.5 mL; 1.0 mL is preferred. Culture should have visible growth (recommended amount: equivalent to approximately 10ul of spun down biomass in a microcentrifuge tube). Culture on solid media: visible growth (preferably equivalent to heaping 1 µl loopful of culture). Cultures with insufficient amount of biomass would need to be incubated at MDL further, which will extend turnaround time.
Specimen Stability	Cultures: Room Temp (20° - 25°C): up to 12 weeks from subculture date. Refrigerated (2 – 8°C): up to 12 weeks from subculture date. Frozen (0°C and below): subculture prior to submission. Subculture prior to submission if culture was not kept at appropriate temperature or if age exceeds 12 weeks and submit fresh culture once sufficient growth is obtained.
Rejection Criteria	Samples with insufficient or conflicting labelling Leaking samples Samples with insufficient quantities Samples that are too old Samples improperly stored or transported Specimen/sample types not meeting acceptance criteria Duplicate submission, i.e., samples from the same patient collected within 3 months from prior collection date, if no prior approval was received
Storage/Transport Conditions	Cultures: Grow isolate under appropriate conditions. Isolates may be stored under refrigeration or at ambient temperature (2° - 25°C) prior to shipping.
Transport Medium, if applicable	Not applicable
Specimen Labeling	Two identifiers, including patient full name, and at least one other identifier (specimen accession number, date of birth, medical record number)
Shipping Instructions and Specimen Handling Requirements	Ship at temperature 2° - 25°C (do not freeze). Follow the appropriate DOT/IATA approved shipping procedures. Suspected or confirmed MTBC cultures should be shipped as a Category A Infectious Substance, Affecting Humans (UN2814). Ensure that culture isolates are sent in media flask or tubes containing a screw-top cap that is additionally sealed with parafilm or tape for additional protection against leaking. Ship to: California Department of Public Health Microbial Diseases Laboratory ATTN: TB Drug Susceptibility Testing Unit 850 Marina Bay Parkway, Specimen Receiving Room B106 Richmond, CA 94804
Specimen Collection Instructions, if applicable	Not applicable
Turnaround Time	7-14 days
Interferences & Limitations	Results may be delayed or unattainable if mixed culture is submitted. Submission of culture on the media types other than listed under sample requirements or submission of cultures with scant growth will delay testing and availability of the results. A negative result (i.e. no mutations detected) does not rule out the presence of resistance-conferring mutations elsewhere in the genome. Heteroresistance below the level of detection of this assay may also contribute to false-negative results.
Reference Range	Not applicable
Additional Information	The assay is indicated for patients suspected of having drug-resistant tuberculosis and patients who are especially susceptible to treatment failure Isolates that are tested at MDL will be forwarded to the CDC if further characterization is needed or requested. If TB WGS genotyping is needed, please indicate it as a requested test on the form 444; additional information is available under the corresponding test on the service menu.
MDL Point of Contact	For questions regarding submissions, please email CDPHTBDST@cdph.ca.gov or call the Mycobacterial, Mycotic, and Parasitic Diseases Section (MMPDS) at (510) 412-3929

Frequently Asked Questions (FAQs) for MTBC WGS- & tNGS-based drug susceptibility and WGS genotyping

MDL Procedure Change Notification: Pyrazinamide (PZA) Drug Susceptibility Testing for TB

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