microbial diseases laboratory branch

Test Name

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

Eligible 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	The 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. This is a Laboratory Developed Test (LDT). The following molecular targets associated with drug resistance are detected by the WGS-DST assay: First-line drugs: Isoniazid: katG, inhA/fabG1* Rifampin: rpoB Ethambutol: embA, embB Pyrazinamide: pncA Second-line drugs: Ethionamide: ethA, inhA, fabG1* Fluoroquinolones (moxifloxacin, levofloxacin): gyrA, gyrB 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 * Variants in fabG1 are reported under inhA gene numbering per WHO v.2 (2023) guidance. 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. Please contact the laboratory for more information on the method and analysis.
Reflex Testing Criteria	Mixed/contaminated cultures will be reflexed to targeted Next Generation Sequencing (tNGS)-based DST.
Pre-Approval Required	Pre-approval is not required except when requesting testing for duplicate submissions, i.e., samples from the same patient collected within 3 months of the 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. Please indicate if WGS-DST is requested for PZA resistance prediction only (e.g. in case phenotypic testing is unavailable or results need confirmation).
Required Specimen Type(s)	Culture on LJ (solid) or in liquid (BACTEC MGIT) media. Isolates submitted on other types of media are not acceptable. Pure cultures with visible biomass are required. Mixed or non-viable cultures, as well as primary specimens, are 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).
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/biomass Samples that are too old (past 12 weeks from subculture date) Samples improperly stored or transported Specimen/sample types not meeting acceptance criteria Cultures on not acceptable media Samples submitted in flasks 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	Testing subject to CLIA regulations. Minimum label requirement on the specimen includes patient name and one other identifier (e.g. preferably submitter sample ID, or patient medical record number, or patient date of birth) on the specimen container and the test requisition.
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	WGS-DST results may be delayed or unattainable if a mixed culture is submitted. 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	Mycobacterial, Mycotic, and Parasitic Diseases Section (510) 412-3929 Monday–Friday 8 AM to 5 PM PT Email: CDPHTBDST@cdph.ca.gov