Enzyme Cascade:




When (1,3)-ß-D-glucan is present in a sample, it activates Factor G, a serine protease zymogen. The activated Factor G converts the inactive proclotting enzyme to the active clotting enzyme, which in turn cleaves pNA from the chromogenic peptide substrate, Boc-Leu-Gly-Arg-pNA, creating a chromophore that absorbs at 405 nm. The reagent is used in the Fungitell kinetic assay, to detect the rate of optical density increase in a sample. This rate is interpreted against a standard curve to produce estimates of (1,3)-ß-D-glucan concentration in the sample.

Similar to enzyme immunoassays, the Fungitell assay is performed in microplates and read in an incubating reader. It is a homogeneous assay, and does not require any washing steps. It can be completed in approximately one hour.

In May, 2004, the FDA cleared the Fungitell kit for marketing, to assist in the diagnosis of fungal infections.

Clinical Significance
There is an increasing incidence of fungal infections by both primary and opportunistic pathogens, especially in immunocompromised patients. Invasive fungal diseases, as opportunistic infections, are common among hematological malignancy and AIDS patients and account for a growing number of nosocomial infections, particularly among organ transplant recipients and other patients receiving immunosuppressive treatments.

Common primary human fungal pathogens are Candida spp. and Aspergillus spp., opportunistic fungal pathogens, including Fusarium spp., Trichosporon spp., Saccharomyces cerevisiae, Acremonium, Coccidioides immitis, Histoplasma capsulatum, Sporothrix schenckii, Blastomyces dermatitidis and Pneumocystis carinii. The (1,3)-ß-D-glucan produced by these organisms can be detected by the Fungitell assay.

Normal human serum contains low levels of (1,3)-ß-D-glucan, typically 10-40 pg/mL, presumably from commensal yeasts present in the alimentary canal and gastrointestinal tract. Most pathogenic fungi have (1,3)-ß-D-glucan in their cell walls and minute quantities are sloughed into the circulation during the life cycle. Thus, (1,3)-ß-D-glucan appears in the serum in cases of invasive fungal infection (IFI). Monitoring serum glucanemia for evidence of elevated and rising levels provides a convenient surrogate marker for IFI. Levels above 80 pg/mL, in at-risk patients, are considered positive.

The Fungitell assay is indicated for presumptive diagnosis of fungal infection. It should be used in conjunction with other diagnostic procedures. The Fungitell assay does not detect certain fungal species such as the genus Cryptococcus, which produces very low levels of (1,3)-ß-D-glucan. This assay also does not detect the Zygomycetes, such as Absidia, Mucor, and Rhizopus, which are not known to produce (1,3)-ß-D-glucan.

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Patient Samples 
Serum samples should be collected in sterile vacuum tubes, allowed to clot, and then centrifuged to separate the clot from the serum, i.e., for approximately 1800 rpm for 10 minutes in a clinical centrifuge. Remove the serum sample under asceptic conditions and aliquot to storage vials. Each patient test well requires 5 µL of serum.

Serum is the only sample type that is currently approved for use with the Fungitell assay. Serum that is hemolyzed, lipemic, or visually icteric or turbid is not suitable for use with the Fungitell assay.

Because a fungal infection is a dynamic process repeat testing, typically 2-3 times per week, improves sensitivity. Time courses of serum (1,3)-ß-D-glucan levels through the course of an infection have been shown to exhibit a rising level which falls in response to efficacious therapy.

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reference VALUES

Negative	Less than 60 pg/mL
Indeterminate	60-79 pg/mL
Positive	>/= 80 pg/mL

The Fungitell assay is indicated for presumptive diagnosis of fungal infection. It should be used in conjunction with other diagnostic procedures. The Fungitell assay does not detect certain fungal species such as the genus Cryptococcus, which produces very low levels of (1,3)-ß-D-glucan. This assay also does not detect the Zygomycetes, such as Absidia, Mucor, and Rhizopus, which are not known to produce (1,3)-ß-D-glucan.

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