Cryptococcus DNA detection by PCR

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.

Cryptococcus PCR detects Cryptococcus neoformans and Cryptococcus gattii with higher specificity and sensitivity than the broad-range fungal PCR. The analytical sensitivity is 1 genome per PCR reaction.

Heitman et al. (2005) Deciphering the model pathogenic fungus Cryptococcus neoformans Nature Reviews Microbiology, 3, pp 753

//en.wikipedia.org/wiki/Cryptococcus

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.