Fungal PCR reflex NGS

General Information

Lab Name

Lab Code

FUNDNA

Epic Name

Fungal DNA PCR w/ reflex to NGS

Description

Fungal PCR reflex NGS

Rapid and accurate species-level identification of pathogenic fungi can inform appropriate antifungal therapy. Traditional culture-based methods require organism growth, which may required extended incubation. In some cases, even when fungal elements are seen by direct staining, fastidious fungi may be non-viable or difficult to recover, particularly when fresh tissue is no longer available and only paraffin-embedded tissue can be tested.

PCR amplification of microbial DNA followed by 28S rRNA and ITS gene sequencing bypasses the requirement for organism growth, enabling faster turnaround time and more accurate identification of molds and yeasts compared to conventional biochemical and phenotypic methods.

Selection of this test will reflex to Fungal DNA Detection by ITS Next Gen Seq [NGSITS] if multiple fungal templates are detected by the ITS gene.

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. Reflex tests include:

Trichophyton mentagrophytes genotype identification
The Trichophyton mentagrophytes (TM) complex includes over 20 genotypes that are indistinguishable by traditional phenotypic methods. The UW laboratory reflexively analyzes ITS sequences to identify two of the most clinically significant genotypes: genotype VII (TMVII), which is associated with sexually transmitted dermatophytosis, and genotype VIII (TMVIII; Trichophyton indotineae), which is associated with drug-resistant tinea corporis and tinea cruris. When T. mentagrophytes complex is detected by fungal PCR, additional genotype information will be reported if TMVII or TMVIII are identified.

Incidental Finding Reflexes

Forms & Requisitions

Molecular Microbiology Order Form

Synonyms

28s PCR, 28S sequencing, broad range fungal PCR, broad range fungi PCR, dermatophyte, fungal 28S, fungal identification, fungal ITS, Fungal PCR, fungal sequencing, fungi identification, Fungi PCR, fungi sequencing, genotype 7, genotype 8, genotype VII, genotype VIII, indotineae, ITS PCR, ITS sequencing, molecular fungal, molecular fungi, tissue, TM 7, TM 8, TM7, TM8, TMVII, TMVIII, Trichophyton, 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 (sputum is acceptable for pathogen specific testing only).
- 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: Conditionally acceptable
- Not acceptable for broad range PCR testing: 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
- Acceptable for pathogen specific testing including Mycobacterium avium complex DNA Detection [MAVDNA]
eSwabs*, UTM (universal transport media), body fluid/bone marrow in EDTA (not peripheral blood)
*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 containers 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/NW: 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.

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?

Performing Location(s)

CL	Infectious Disease Division (IDD) - Molecular Microbiology 206-520-4600 Shipping Address Attn: Molecular Microbiology UW CLSPS 1601 Lind Ave SW Room 171A Renton, WA 98057 Phone: 206-598-5735 Performing Lab Address UW Medicine Central Laboratory IDD-Molecular Microbiology 1601 Lind Ave SW Room 485 Renton, WA 98057 Phone: 206-485-9108	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 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