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Cryptococcus DNA detection by PCR

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General Information

Lab Name
Cryptococcus DNA detection by PCR
Lab Code
CRYDNA
Epic Name
Cryptococcus DNA detection by PCR
Description

Detection and differentiation of Cryptococcus neoformans and Cryptococcus gattii DNA

Cryptococcus is a genus of fungus of which Cryptococcus neoformans is the most medically important species that primarily affects immunocompromised individuals. More recently genetic evidence for two distinct species, Cryptococcus neoformans and Cryptococcus gattii has been reported among strains of C. neoformans. C. gattii can infect even non-immunocompromised individuals and has been recognized as an emerging pathogen in US Northwest and adjoining Canadian state of British Columbia. It is therefore critical to correctly identify the exact type of C. neoformans to completely understand a given case of cryptococcal infection.

Histology (mucicarmine stain), serology and culture are routinely used for a diagnosis. However, culture is not always successful or not possible in many cases where histology suggests the presence of Cryptococcus. PCR can be particularly useful in such cases to confirm or rule out a Cryptococcus neoformans infection and also determine the exact species i.e. C. neoformans or C. gattii.

A PCR assay was developed that exploits the inter-species sequence diversity within the ribosomal intergenic regions (IGS) or the non-transcribed spacer (NTS) region.

Traditional targets such as 28S D1-D2 region and the ITS sequences do not exhibit sufficient sequence heterogeneity to distinguish the different C. neoformans species variants. Primers were designed to allow a heminested PCR assay that will amplify DNA from all C. neoformans species variants. Identification of the particular species type will depend on the DNA sequence of the fragment.

References
Forms & Requisitions

Molecular Microbiology Order Form

Synonyms
Cryptococcus detection, Cryptococcus gattii identification, Cryptococcus gattii PCR, Cryptococcus gattii sequencing, Cryptococcus identification, Cryptococcus neoformans identification, Cryptococcus neoformans PCR, Cryptococcus neoformans sequencing, Cryptococcus sequencing, Filobasidiella bacillispora, Fungal sequencing, molecular Cryptococcus, Synonyms: Cryptococcus PCR, universal PCR
Components
Code Name
CRYRS Cryptococcus PCR: Detection, nested IGS
CRYID Cryptococcus PCR: Identification, nested IGS
CRYSI Cryptococcus PCR: Specimen Description
CRYSPI Cryptococcus PCR: External Identifier
CRYSR Cryptococcus PCR: Special Requests
CRYSC Cryptococcus PCR: Specimen Comments
CRYNAE Cryptococcus PCR: Specimen DNA Extraction
CRYREV Cryptococcus PCR: Pathologist Review
CRYME Cryptococcus PCR: Method Note

Interpretation

Guidelines
Method

DNA extraction, nucleic acid purification, polymerase chain reaction (PCR), sequencing

Reference Range
See individual components
References

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

*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 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: 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?
    No
    Performing Location(s)
    UW-MT Microbiology, Molecular Diagnostics
    206-520-4600

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    Shipping Address

    Attn: Molecular Microbiology
    UW CLSPS
    1601 Lind Ave SW Room 117
    Renton, WA 98057
    Phone: 206-520-4600
    Alternate phone: 206-598-6147

    Performing Lab Address

    Clinical Microbiology Lab, NW177
    University of Washington Medical Center
    1959 NE Pacific Street
    Seattle, WA 98195
    Phone: 206-598-5735
    Alternate phone: 206-598-6147

    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
    • 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
    81649-6
    Interfaced Order Code
    UOW4434