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microbial diseases laboratory

Test Name 

Mycobacterium tuberculosis complex: Molecular Detection of Drug Resistance by Pyrosequencing Assay

Test Methodology

Pyrosequencing (PSQ) is the method used to identify mutations associated with drug resistance.

The following molecular targets associated with drug resistance are detected:

First-line drug resistance (default):

Isoniazid:  katG, inhA promoter, ahpC promoter, and fabG1

Rifampin:  rpoB

Second-line drug resistance (reflex or special request):

Quinolones:  gyrA

Injectable drugs (kanamycin, amikacin, capreomycin):  rrs (upon request)

Please contact the laboratory for more information on pyrosequencing method.

Reflex Testing Criteria

If mutations associated with resistance are detected in the first-line targets, the second-line PSQ panel testing will be performed automatically. Alternatively, second-line PSQ testing can be performed in parallel or alone upon submitter request.

Pre-Approval Required

Pre-approval is required for the following: 

  • Specimen is AFB-negative 
  • Submission of sterile body fluids/specimens
  • Duplicate submission, i.e., samples from the same patient collected within 2 months from prior collection date.

Supplemental Information and Required Form(s)

Form LAB 444 (PDF) ā€œMycobacteriology-Drug Susceptibility Testing of MTBC (CA only)ā€

Instructions of completing this form.

Required Specimens/Sample Type(s)

Sediments or cultures are acceptable.

Processed sediment. Concentrated specimens from pulmonary and extra-pulmonary sources, including sputum, bronchial alveolar lavage, pleural fluid, CSF, abscess, gastric aspirate, urine, 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-positive (at least 1+); smear-negative specimens need approval prior to submission.

Culture on solid or in liquid media.

Minimum Volume Required

Sediments: 0.5mL; 1.0 mL is preferred

Culture in broth: 0.5 mL; 1.0 mL is preferred

Culture on solid media: visible growth is desired

Specimen Stability

Sediments:
Room Temp: not acceptable
Refrigerated (2 ā€“ 8Ā°C): up to 30 days
Frozen (ā‰¤ -70Ā°C): up to 120 days

Cultures:
Room Temp: up to 30 days
Refrigerated: 2 ā€“ 8Ā°C: up to 6 months; isolate may be subcultured
Frozen: Subculture prior to submission

Rejection Criteria

  • Specimens with insufficient or conflicting labelling
  • Leaking specimens
  • Specimens with insufficient quantities
  • Specimens that are too old
  • Specimens 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 2 months from prior collection date, if no prior approval was received. 

Storage/Transport Conditions

Sediment: Refrigerated or frozen.  

Cultures: Grow isolate under appropriate conditions. Isolates may be stored under refrigeration or ambient temperature 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 diagnostic specimen with cold packs (do not freeze). Ship cultures at ambient temperature (do not freeze). 

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 

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.

Specimen Collection Instructions, if applicable

Not applicable

Turnaround Time

1 to 3 days

Interferences & Limitations

Inhibitory substances in the specimen may prevent successful DNA amplification and pyrosequencing.

Reference Range

Not applicable

Additional Information

The assay is indicated for the patients suspected of having drug-resistant tuberculosis and patients who are especially susceptible to treatment failure. The test can be performed on cultures of M. tuberculosis complex mixed with non-TB mycobacteria or other bacteria.

The samples will be forwarded to CDC if further characterization is needed. 

MDL Point of Contact

For pre-approval or questions regarding submissions, please email CDPHTBDST@cdph.ca.gov or call the MMPDS Section at (510) 412-3929.

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