Fungal DNA Detection by PCR (without reflex to NGS)

In many specimens, fungal elements can be seen by microscopy of tissue sections but are very difficult to grow due to their fastidious nature, or are not viable as a result of antifungal therapy.

Some specimens may never reveal the presence of a fungal pathogen because of low abundance and/or lack of viability. Often, fungal elements are seen on paraffin embedded tissue when a fresh tissue specimen is no longer available.

The use of PCR technology to detect fungal DNA within the total DNA extracted directly from clinical specimens facilitates the identification of these pathogens. Using both fungal 28S and Internal Transcribed Spacer sequence (ITS) DNA as targets we have set up PCR assays to detect fungal pathogens. Sequences of amplified DNA are analyzed to identify the fungal pathogen present in the specimen.

Reflexive Testing

When suspected pathogenic microorganisms are detected, identification procedures are performed, as appropriate for the organism and specimen. Without reflex to Next Generation Sequencing, the individual species comprising a polymicrobial specimen cannot be resolved or reported. See, Fungal PCR reflex NGS [FUNDNA] for the assay WITH reflex to NGS.

• Coccidioides DNA detection by PCR [COCDNA]
• Cryptococcus DNA detection by PCR [CRYDNA]
• Mucorales DNA detection by PCR [MUCDNA]
• Histoplasma DNA detection by PCR [HISDNA]
• Pneumocystis DNA detection by PCR [PNEDNA]

Incidental Finding Reflexes

• Acanthamoeba and Balamuthia species PCR [AMBPCR]
• Leishmania species DNA detection by PCR [LSHDNA]
• Toxoplasma DNA detection by PCR [TOXDNA]

Multiple Template Reflexes, if requested by ordering physician

• Fungal DNA Detection by ITS Next Gen Seq [NGSITS]

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.