Fungal diagnostics

Abstract

Early diagnosis of fungal infection is critical to effective treatment. There are many impediments to diagnosis such as a diminishing number of clinical mycologists, cost, time to result, and requirements for sensitivity and specificity. In addition, fungal diagnostics must meet the contrasting needs presented by the increasing diversity of fungi found in association with the use of immunosuppressive agents in countries with high levels of medical care and the need for diagnostics in resource-limited countries where large numbers of opportunistic infections occur in patients with AIDS. Traditional approaches to diagnosis include direct microscopic examination of clinical samples, histopathology, culture, and serology. Emerging technologies include molecular diagnostics and antigen detection in clinical samples. Innovative new technologies that use molecular and immunoassay platforms have the potential to meet the needs of both resource-rich and resource-limited clinical environments.

Figure 1.

Ribosomal subunit organization in eukaryotes with variable regions. Eukaryotic ribosomal subunit genes are typically organized in repeats of the 18s rDNA (small subunit), ITS1 (internal transcribed spacer region 1), 5.8s rDNA, ITS2 (internal transcribed spacer region 2), and the 28s rDNA (large subunit). Conserved priming sites exist at the end of the 28s subunit and the beginning of the 28s subunit, and at the end of the D1/D2 region within the 28s subunit. Additional sites exist within the 5.8s subunit and throughout the large and small ribosomal subunits. The variable regions, which can provide information that can discriminate to the species level, depending on genus, include ITS1, ITS2, and D1/D2. The ITS region can be covered in a single PCR reaction and by double-stranded sequencing, and is roughly 450–750 bp in length, depending on species. The D1/D2 region is in a similar size range.