Nontuberculous Mycobacteria DNA Detection

Interpretation

Guidelines

• Molecular Microbiology Acceptable Specimens
• Molecular Microbiology Specimen Submission
• Specialty testing for Molecular Microbiology

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

• Fresh frozen tissue
• Fresh frozen fluid: any body fluid is acceptable if it is not listed under Unacceptable Specimens.
  • Common examples include: cerebrospinal fluid, pleural fluid, pericardial fluid, urine, bronchial lavage, joint fluid, bone marrow, vitreous fluid, etc.
• Formalin Fixed Paraffin-embedded tissues (FFPE, PET): blocks, scrolls, and unstained slides. Note that fresh tissue is considered the optimal specimen of choice, as it reduces the chance of introducing exogenous DNA templates or microorganisms during embedding/fixation, and because formalin fixation dramatically reduces the sensitivity of molecular assays due to reduced template yield and quality.
• Sputum: acceptable - except for Bacterial PCR reflex NGS [BCTDNA]/Bacterial DNA Detection by PCR (without reflex to NGS) [NRBDNA], Fungal PCR reflex NGS [FUNDNA]/Fungal DNA Detection by PCR (without reflex to NGS) [NRFDNA], and Nontuberculous Mycobacteria DNA Detection [NTMDNA]* assays
• eSwabs**, UTM (universal transport media), body fluid/bone marrow in EDTA (not including blood)
• Sodium polyanethol sulfonate (SPS, Wampole Isolator Tubes) acceptable with disclaimer

*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 ---------------------------------------- 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