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Eligible Submitters |
Test is available to all pre-approved submitters within the state of California. Submissions from clinical laboratories are routed through the corresponding county public health laboratories (PHL) unless a specific exception has been made. 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.
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Test Methodology
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The targeted Next Generation Sequencing-based drug susceptibility testing (tNGS-DST) assay is used for the rapid molecular detection of drug resistance in Mycobacterium tuberculosis complex (MTBC) directly from processed human specimens or cultures for diagnostic purposes. The tNGS-DST assay is performed using custom primer set for targeted amplification of MTBC genomic loci associated with resistance, followed by short-read Illumina sequencing and analysis using a bioinformatics pipeline developed in-house. Downstream bioinformatics analysis detects mutations and generates predicted drug interpretations. The tNGS data can also be used for MTBC ID confirmation. This is a Laboratory Developed Test (LDT).
The following molecular targets associated with drug resistance are detected by the tNGS-DST assay:
First-line drugs:
Isoniazid: katG, inhA/fabG1*
Rifampin: rpoB
Ethambutol:
embB
Pyrazinamide:
pncA
Second-line drugs:
Ethionamide: inhA/fabG1*
Fluoroquinolones (moxifloxacin, levofloxacin): gyrA, gyrB Kanamycin & amikacin: rrs, eis Capreomycin: rrs
Novel and repurposed drugs:
Bedaquiline:
Rv0678, atpE
Clofazimine:
Rv0678
Linezolid:
rrl, rplC
*Variants in fabG1 are reported under inhA gene numbering per WHO v.2 (2023) guidance. The exact coordinates of targets covered by tNGS-DST assay are provided on the test FAQ webpage.
The tNGS-DST assay additionally provides MTBC DNA presence confirmation and differentiation of
Mycobacterium bovis species from non-M. bovis MTBC.
Please contact the laboratory for more information on the method and analysis.
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Reflex Testing
Criteria |
All sediments and mixed/non-viable cultures submitted for tNGS-DST undergo MTBC RT-PCR prescreening (BD MAX – BioGX MTBC NAAT, performed as an in-house Laboratory Developed Test).
- If the sample is PCR-negative, or PCR-positive with a Ct value above MDL’s validated threshold (indicating low MTBC load), tNGS will not be performed, and a final RT-PCR-only report is issued.
- If the sample has an acceptable Ct value for reflex to tNGS-DST, a preliminary RT-PCR report is issued, followed by a final report containing both RT-PCR and tNGS results upon test completion.
The MDL prescreening MTBC RT-PCR does not detect rifampin resistance and is not a substitute for the GeneXpert MTB/RIF assay.
Successful amplification by GeneXpert MTB/RIF does not guarantee that a sample will meet acceptance criteria for tNGS-DST at MDL.
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Pre-Approval Required |
Pre-approval is not required except when requesting testing for the following:
- AFB smear-negative pulmonary processed specimens (smear-negative pulmonary specimens without NAAT will not be accepted; specimens that are both smear-negative and MTBC NAAT-negative will not be accepted)
- AFB smear-negative extrapulmonary processed specimens (NAAT result not required, but pre-approval required)
- Sterile body fluids (regardless of AFB status)
- Processed specimens with volume below 0.5 mL
- Duplicate submission, i.e., samples from the same patient collected within 3 months from prior collection date.
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Supplemental Information and Required Form(s) |
Use MDL Lab Web Portal for ordering the test, select Test Requisition form “Mycobacteriology-Drug Susceptibility Testing of MTBC (CA only)"– Form 444". For submitters who do not have access to the MDL LWP; please contact MDL.Submissions@CDPH.ca.gov for additional information.
Please include submitter’s test results, including NAAT status, on the submittal form.
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Required Specimen Type(s)
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Sediments or mixed/nonviablecultures are acceptable; pure cultures will automatically be routed to Whole Genome Sequencing (WGS)-DST.
Processed specimens (sediments). Concentrated specimens from pulmonary and extra-pulmonary sources, including sterile body fluids, urine, and processed tissues are acceptable. Submission of primary unprocessed specimens is NOT acceptable for tNGS-DST.
Submission of specimens from sterile body sites requires pre-approval.
Sediment should be confirmed to be AFB smear-positive (graded at least 1+); preapproval is required for smear-negative specimens.
Culture on solid (LJ slants) or in liquid (BACTEC MGIT) media. tNGS is reserved for mixed or non-viable cultures. Sequencing-based DST for pure cultures will be performed by the WGS-DST method.
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Minimum Volume Required |
Sediments: 0.5 mL; 1.0 mL is preferred
Culture in broth: 0.5 mL; 1.0 mL is preferred
Culture on solid media: visible growth
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Specimen Stability
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Sediments:
Room Temp: not acceptable
Refrigerated (2 – 8°C): up to 120 days
Frozen(≤ -70°C): up to 1 year
Cultures:
Refrigerated to Room Temperature (2 – 25°C): up to 12 weeks from subculture date.
Frozen (0°C and below): subculture prior to submission.
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Rejection Criteria
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- Samples with insufficient or conflicting labelling
- Leaking samples
- Samples with insufficient quantities
- Samples that are too old
- Samples improperly stored or transported
- Stool specimens
- Formalin fixed tissue and other specimen/sample types not meeting acceptance criteria
- 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
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Storage/Transport Conditions
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Sediment: Refrigerated or frozen (see specimen stability)
Cultures: Grow isolate under appropriate conditions. Isolates may be stored under refrigeration or at ambient temperature (2° - 25°C) prior to shipping. Frozen cultures should be subcultured prior to shipping.
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Transport Medium,
if applicable
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Not applicable
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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.
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Shipping Instructions and Specimen Handling Requirements
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Ship primary specimens (sediments) with cold packs, or if stored frozen, primary specimens can be shipped on dry ice.
Cultures may be shipped with cold packs or at ambient temperature. Do not freeze. Ensure that culture isolates are sent in tubes containing a screw-top cap that is additionally sealed with parafilm or tape for additional protection against leaking. Do not submit cultures in flasks.
Please note that inappropriate storage temperatures in transit may affect specimen integrity which can impact the ability to obtain sequencing results. Specimens known to be improperly stored and/or transported may be rejected.
Ship Monday through Thursday only; do not ship on Fridays. MDL is closed Saturdays and Sundays. Use standard overnight shipping. Do not place more than one specimen in a specimen bag.
Follow the appropriate DOT/IATA approved shipping procedures. All the suspected MTBC human specimens should be shipped as a Biological Substance, Category B (UN3373). Suspected or confirmed MTBC cultures should be shipped as a Category A Infectious Substance, Affecting Humans (UN2814).
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
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Specimen Collection Instructions, if applicable
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Not applicable |
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Turnaround Time |
7-14 calendar days
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Interferences & Limitations |
Inhibitory substances in the specimen may prevent successful DNA amplification and sequencing. The MTBC tNGS-DST assay is not a presence/absence test. The inability to generate successful sequences in targeted drug-resistance associated loci or a negative MTBC ID result does not preclude the presence of MTBC.
Absence of reportable mutations does not rule out the presence of resistance-conferring mutations elsewhere in the genome. Heteroresistance below the level of detection of this assay may contribute to predicted false-susceptible results.
MTBC tNGS-DST test results should not be used as the sole criterion for the diagnosis of drug susceptible or resistant tuberculosis but can be used in conjunction with other clinical data.
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Reference Range |
Not applicable
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Additional Information |
This assay is indicated for patients suspected of having drug-resistant tuberculosis and patients who are especially susceptible to treatment failure or drug intolerance.
Samples that are tested at MDL may be forwarded to the CDC if further characterization is needed or requested.
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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
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