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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.
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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 DeeplexĀ® Myc-TB (GenoScreen, Lille, France) and custom primers for targeted amplification of MTBC genome 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.
The following molecular targets* associated with drug resistance are detected by the tNGS-DST assay:
First-line drugs:
Isoniazid: katG, fabG1, inhA
Rifampin: rpoB
Ethambutol:
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
Clofazimine:
Rv0678
Linezolid:
rrl, rplC
* 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.
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.
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Reflex Testing
Criteria |
None
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Pre-Approval Required |
Pre-approval is not required except when requesting testing for the following:
- AFB smear-neagtive specimen
- Cepheid XpertĀ® MTB/RIF PCR cycle threshold (Ct) value averaged across probes above 28
- Sterile body fluids or processed tissues
- Processed specimen volume below 1.0 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) |
Instructions for using the MDL Lab Web Portal (ETOR) can be found on MDL services
website.
Please include submitterās test results on the submittal form.
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Required Specimen Type(s)
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Sediments or cultures are acceptable; however, cultures will automatically be routed to Whole Genome Sequencing (WGS)-DST unless the culture meets criteria described below (or other extenuating circumstance communicated to the submitter).
Processed specimens (sediments). Concentrated specimens from pulmonary and extra-pulmonary sources, including sputum, bronchial alveolar lavage, abdominal fluid, CSF, abscess, and tissues are acceptable.
Unprocessed clinical specimens should not be submitted. 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 prior to submission.
Culture on solid (LJ) 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.
Submission of primary unprocessed specimens is NOT acceptable for tNGS-DST.
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Minimum Volume Required |
Sediments: 1.0 mL
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 ā 8Ā°C): up to 120 days
Frozen(ā¤ -70Ā°C): up to 1 year
Cultures:
Refrigerated to Room Temperature (approximately 2 ā 25Ā°C): up to 12 weeks from subculture date.
Frozen (0Ā°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
- Formalin fixed tissue and other 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
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Storage/Transport Conditions
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Sediment: Refrigerated or frozen
Cultures: Grow isolate under appropriate conditions. Isolates may be stored under refrigeration or at ambient temperature (2Ā° - 25Ā°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 |
Two identifiers, including patient full name, and at least one other identifier (specimen accession number (preferred), date of birth, medical record number)
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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 media flask or tubes containing a screw-top cap that is additionally sealed with parafilm or tape for additional protection against leaking.
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.
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 |
7-10 days
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Interferences & Limitations |
Inhibitory substances in the specimen may prevent successful DNA amplification and sequencing. MTBC tNGS-DST assay is not a presence/absence test. Inability to generate successful sequences in targeted drug-resistance associated loci or negative MTBC ID result do 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 |
For questions regarding submissions, please email
CDPHTBDST@cdph.ca.gov or call the Mycobacterial, Mycotic, and Parasitic Diseases Section (MMPDS) at (510) 412-3929
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