Nontuberculous Mycobacteria DNA Detection

Detection and identification of Nontuberculous Mycobacteria DNA.

While Mycobacterium tuberculosis complex members are the most commonly known human pathogens, Non-tubercolosis 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. 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 tuberculosis and Mycobacterium avium complexes, 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 MTB and MAI complex specific fluorescent probes on a real-time PCR platform facilitates rapid and highly sensitive detection of MTB and 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.

In many specimens acid fast bacilli can be seen microscopy of tissue sections but are very difficult to grow due to their fastidious nature, or are not viable as a result of antimicrobial therapy. 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 this DNA extracted directly from clinical specimens facilitates the identification of these pathogens.

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."

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
• 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 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.

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)