Nontuberculous Mycobacteria DNA Detection

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General Information

Lab Name
Nontuberculous Mycobacteria DNA Detection
Lab Code
NTMDNA
Epic Name
Nontuberculous Mycobacteria DNA Detection
Description

Detection and identification of Nontuberculous Mycobacteria DNA

While Mycobacterium tuberculosis complex members are the most commonly known human pathogens, Non-tuberculosis Mycobacteria (also known as environmental Mycobacteria, atypical Mycobacteria and Mycobacteria other than tuberculosis (MOTT)) are also implicated in tuberculosis-like disease, localized lymphadenitis, gastrointestinal disease, and disseminated infections. Rapid and accurate identification of pathogenic Mycobacteria important for correct therapeutic intervention against an infectious disease. Acid fast bacilli can be very difficult to grow due to their nutritional requirements. Some specimens may never reveal the presence of a pathogen because of low abundance and/or lack of viability. The use of PCR to detect Mycobacterial DNA extracted directly from clinical specimens facilitates the identification of these pathogens.

For an accurate detection of the presence of Mycobacteria in clinical specimens, the UWMC Molecular Diagnosis Section utilizes a combination of several techniques. The detection of Mycobacterium avium complex, for example, relies on fluorescence resonance energy transfer (FRET) probes in a nested PCR protocol that targets the heat shock protein 65 gene (hsp65). Utilization of MAI complex specific fluorescent probes on a real-time PCR platform facilitates rapid and highly sensitive detection of MAI complex DNA in clinical specimens.

For other Non-tuberculous Mycobacteria (including rapidly growing mycobacteria (RGM)) and Mycobacterium leprae, detection and speciation involves a multi-locus PCR strategy. Identifications are performed by DNA sequencing, which provides direct, unambiguous data and can distinguish medically relevant subspecific phylogenetic lineages.

Fluorescence Resonance Energy Transfer (FRET) methodology, using hybridization probes targeted to species-specific hsp65 sequences, can be used to distinguish between the most frequently isolated Mycobacteria species (M. abscessus, M. avium, M. chelonae, M. fortuitum, M. gordonae, M. haemophilum, M. intracellulare, M. kansasii, M. marinum and M. tuberculosis). Each of these species have their own characteristic melting points. They are distinguished from other Mycobacteria isolates and closely related species that are commonly isolated in the clinical laboratory. The “MAC” probe sets were designed specifically to detect and identify Mycobacterium avium complex.

References

Selvarangan R, et al. Characterization of a novel group of mycobacteria and proposal of Mycobacterium sherrisii sp. nov. J Clin Microbiol 2004, 42:52-9. 14715731

Forms & Requisitions

Molecular Microbiology Order Form

Synonyms
AFB PCR, Atypical Mycobacteria PCR, broad range AFB PCR, broad range Mycobacteria PCR, hsp65, M. avium complex, MAC, MAI PCR, Molecular AFB, Molecular Mycobacteria, Molecular Mycobacterium, MOTT, Mycobacterial identification, Mycobacterial sequencing, Mycobacterium identification, Mycobacterium leprae, Mycobacterium sequencing, Nontuberculous Mycobacterium PCR, rpoB, universal AFB PCR, universal Mycobacterium PCR
Components
Code Name
NTMSUM Nontuberculous PCR: Summary
NT16RS Nontuberculous PCR: Detection, 16S rDNA
NT16ID Nontuberculous PCR: 16S Identification
NTHSRS Nontuberculous PCR: Detection, hsp65
NTHSID Nontuberculous PCR: hsp65 Identification
NTRPRS Nontuberculous PCR: Detection, rpoB
NTRPID Nontuberculous PCR: rpoB Identification
NTMSI Nontuberculous PCR: Specimen Description
NTMSPI Nontuberculous PCR: External Identifier
NTMSR Nontuberculous PCR: Special Requests
NTMSC Nontuberculous PCR: Specimen Comments
NTMNAE Nontuberculous PCR: Specimen DNA Extraction
NTMREV Nontuberculous PCR: Pathologist Review
NTMME Nontuberculous PCR: Method Note

Interpretation

Guidelines
Method

DNA extraction, nucleic acid purification, polymerase chain reaction (PCR), sequencing

Reference Range
See individual components
Interferences and Limitations

Indeterminate Results Interpretation

This Nontuberculous Mycobacteria DNA Detection test can detect and identify Nontuberculous Mycobacteria with 3 gene targets: 16S, hsp65, and rpoB.

The presence of non-Mycobacterial DNA in these targets can interfere with our ability to rule out Nontuberculous Mycobacteria.

In this scenario, the corresponding target will be reported as "Indeterminate. Unable to rule out due to interfering templates."

  • 16S: indeterminate results may be resolved by additional testing if it is noted in the results.
    Resolution may require Bacterial DNA Detection by PCR (BCTDNA) and may also require reflexive 16S rRNA Next Generation Sequencing (NGS16S) if there is evidence of multiple bacterial templates.
  • rpoB: is a more specific target for Nontuberculous Mycobacteria. There currently is not any additional testing available to resolve and indeterminate result for rpoB.
  • hsp65: is the most specific target for Nontuberculous Mycobacteria. There currently is not any additional testing available to resolve and indeterminate result for hsp65.

If there is not any interference seen in a target it will be reported as "Not Detected. Not applicable."

References

Selvarangan R, et al. Characterization of a novel group of mycobacteria and proposal of Mycobacterium sherrisii sp. nov. J Clin Microbiol 2004, 42:52-9. 14715731

Ordering & Collection

Specimen Type
Tissue (Fresh frozen or paraffin-embedded), Fluid (see Acceptable Specimens for details)
Collection

Acceptable specimens are listed below. Please see Molecular Microbiology Specimen Submission for complete specimen collection and handling instructions.

Shipping/Handling

  • Fresh frozen tissue/fluid specimens should be collected into a DNA free container labeled with at least two identifiers and be submitted and maintained on dry ice.
  • Formalin Fixed Paraffin-embedded tissues (FFPE, PET) can be sent ambient or with ice packs during warmer summer months to prevent melting.

Acceptable Specimens

*Mycobacterium avium complex DNA Detection [MAVDNA] can be ordered on sputum

**Fungal PCR reflex NGS [FUNDNA] and Fungal DNA Detection by PCR (without reflex to NGS) [NRFDNA] may have interference due to some lots of eSwabs which have been found to contain Saccharomyces cerevisiae DNA, resulting in false positive detection. Clinical correlation and/or retesting with a different collection method is advised. The detection of S. cerevisiae from eSwab specimens can interfere with our ability to rule out other fungal DNA.

    Unacceptable Specimens

    • Blood, serum, plasma, stool/rectal swabs
    • No citrated or heparinized solutions
    • Tissues floating in formalin
    • Swab/fluid collected in tube containing agar

    Optimal Quantity:

    • Fresh Tissue: 0.3-1.0 cm^3
    • Fluid: 0.2-1 mL
    • Formalin Fixed Paraffin-embedded Tissue (FFPE/PET): blocks are preferred over scrolls/unstained slides and will be sent back to client upon completion of testing
    • Scrolls/unstained slides: cross-sectional area >1cm^2 send 10 sections of 10µm thickness, if <1cm^2 send 20 sections if available

    Please note: We do not need a separate specimen aliquot for each test ordered. Only a single specimen aliquot or block of optimal quantity is necessary for performing multiple tests. If multiple aliquots or blocks of optimal quantity are sent, up to 2 will be pooled.

    Fresh tissue is the optimal specimen of choice, as it reduces the chance of introducing exogenous DNA templates or microorganisms during embedding/fixation. Formalin fixation dramatically reduces the sensitivity of the assays due to reduced template yield and quality.

    Forms & Requisitions

    Molecular Microbiology Order Form

    Handling Instructions

    Please see Molecular Microbiology Specimen Submission for complete specimen collection and handling instructions.

    Quantity
    Requested: See "Collection" for Optimal Quantity
    Minimum: Specimens below optimal quantity are acceptable for testing, however, diagnostic yield is generally proportional to specimen size.

    Processing

    Receiving Instructions

    UWMC/HMC: Store and send fresh tissue/fluid specimens refrigerated, if specimen storage and transport will exceed 8 hours, freeze at -20°C. Freeze all fresh tissue/fluid specimens at -20°C upon arrival in UW Molecular Microbiology.

    _____________________________________________________________________

    Specimen Processing Technicians: If NTMDNA is ordered on sputum please cancel/credit as YSTYP. Then order MAVDNA if the following verbiage is noted on the requisition (see below). If the following verbiage is not noted on requisition/order form, client needs to be contacted to add on MAVDNA.

    Requisition Verbiage If one of the following boxes is checked off, then cancel/credit as YSTYP and order MAVDNA:

    [ ] AFB (if sputum sample, only TBCDNA and MAVDNA will be performed)
    OR
    [ ] Nontuberculous Mycobacteria PCR NTMDNA, Not Acceptable NTMDNA: Sputum (see MAVDNA)

    Misc Sendout

    Performance

    Lab Department
    Micro Molecular Diag(MMD)
    Frequency
    Fresh frozen tissues/fluids result in 2-3 business days after receipt of specimen. Formalin Fixed Paraffin-embedded tissues result in 3-4 business days after receipt of specimen.
    Available STAT?
    No
    Performing Location(s)
    UW-MT Microbiology, Molecular Diagnostics
    206-520-4600

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    Shipping Address

    Attn: Molecular Microbiology
    UW CLSPS
    1601 Lind Ave SW Room 117
    Renton, WA 98057
    Phone: 206-520-4600
    Alternate phone: 206-598-6147

    Performing Lab Address

    Clinical Microbiology Lab, NW177
    University of Washington Medical Center
    1959 NE Pacific Street
    Seattle, WA 98195
    Phone: 206-598-5735
    Alternate phone: 206-598-6147

    Contact Information

    Please e-mail us with any questions or comments you may have. Your inquiry will be answered as soon as possible.

    email: molmicdx@uw.edu

    The Molecular Microbiology lab is open from Monday-Friday, 7am-4pm PDT.

    Billing inquiries and requests for faxed reports can be made to our Client Services Department at (206) 520-4600 or (800) 713-5198.

    For results or other inquiries, we can be reached by phone at the following numbers:

    • Phone: (206) 598-5735
    • Alternate phone: (206) 598-6147
    • FAX: (206) 520-4903

    For assistance during weekends, holidays and after hours, please contact Lab Medicine Resident at (206) 598-6190

    Billing & Coding

    CPT Codes
    87551
    LOINC
    14974-0
    Interfaced Order Code
    UOW4316