Fungal PCR reflex NGS
General Information
- Lab Name
- Fungal PCR reflex NGS
- Lab Code
- FUNDNA
- Epic Name
- Fungal DNA detection by PCR
- Description
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, including evaluation of polymicrobial specimens using Next Generation Sequencing.
- 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]
- Fungal DNA Detection by ITS Next Gen Seq [NGSITS]
Incidental Finding Reflexes
- Forms & Requisitions
- Synonyms
- 28s PCR, 28S sequencing, broad range fungal PCR, broad range fungi PCR, fungal 28S, fungal identification, fungal ITS, Fungal PCR, fungal sequencing, fungi identification, Fungi PCR, fungi sequencing, ITS PCR, ITS sequencing, molecular fungal, molecular fungi, universal fungal PCR, universal fungi PCR
- Components
-
Code Name FUNSUM Fungal PCR: Summary FU28RS Fungal PCR: Detection, 28S rDNA FU28ID Fungal PCR: 28S Identification FUITRS Fungal PCR: Detection, ITS rDNA FUITID Fungal PCR: ITS Identification FUSI Fungal PCR: Specimen Description FUSPI Fungal PCR: External Identifier FUSR Fungal PCR: Special Requests FUSC Fungal PCR: Specimen Comments FUNAE Fungal PCR: Specimen DNA Extraction FUREV Fungal PCR: Pathologist Review FUME Fungal PCR: Method Note
Interpretation
- Guidelines
- Method
DNA extraction, nucleic acid purification, polymerase chain reaction (PCR), sequencing
- Reference Range
- See individual components
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. 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.
- 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.
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
- 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.
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: 206-598-6147Performing Lab Address
Clinical Microbiology Lab, NW177
University of Washington Medical Center
1959 NE Pacific Street
Seattle, WA 98195
Phone: 206-598-5735
Alternate phone: 206-598-6147Contact 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
- 87801
- LOINC
- 42805-2
- Interfaced Order Code
- UOW4304