Fungal PCR reflex NGS
General Information
Lab Name
Fungal PCR reflex NGS
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:
- 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]
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
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
Method
DNA extraction, nucleic acid purification, polymerase chain reaction (PCR), sequencing
Reference Range
See individual componentsGuidelines
Ordering & Collection
Specimen Type
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
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.
Performance
LIS Dept Code
Micro Molecular Diag (MMD)
Performing Location(s)
| CL |
Infectious Disease Division (IDD) - Molecular Microbiology
206-520-4600 Shipping Address Attn: Molecular Microbiology Performing Lab Address UW Medicine Central Laboratory |
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:
For assistance during weekends, holidays and after hours, please contact Lab Medicine Resident at (206) 598-6190 |
|---|
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