Prospective use of RFLP analysis on amplified Cryptococcus neoformans URA5 gene sequences for rapid identification of varieties and serotypes in clinical samples.

Clinical isolates of Cryptococcus neoformans, whole blood, cerebrospinal fluid, bronchoalveolar lavage fluid from patients with positive cryptococcal antigen latex-agglutination test, and spiked clinical material from healthy individuals, were tested by polymerase chain reaction (PCR) with primers amplifying C. neoformans URA5 gene sequences. To test compatibility of different DNA extraction protocols with the PCR-restriction fragment length polymorphism (RFLP) assay, a commercial DNA extraction kit (XTRAX; Gull Laboratories, UT, USA) was used alongside with the hexadecyltrimethylammonium bromide (CTAB) method on spiked biological fluids. Both methods extracted DNA from spiked clinical samples containing C. neoformans (8 +/- 2 cells ml(-1)) and generated amplification products suitable for restriction enzyme analysis. Alu I digestion differentiated the two varieties of C. neoformans. Three distinct RFLP patterns were obtained upon restriction with MspI corresponding to serotypes A, AD and B, C and D. URA5 PCR followed by RFLP analysis, coupled with a sensitive in-house or commercially available DNA extraction method from clinical samples, could be successfully incorporated into rapid routine diagnostic strategies. It could also provide an expeditious tool for epidemiology-based population genetics studies.

Identification of medically significant fungal genera by polymerase chain reaction followed by restriction enzyme analysis.

Rapid non-culture-dependent assays for identification of fungi quicken diagnosis and prompt treatment of invasive fungal disease. Fungal DNA extracts from pure cultures of the most frequently isolated fungal pathogens belonging to the Genera Aspergillus, Candida and Cryptococcus along with less common pathogenic Genera were amplified with the general fungal primer pair internal transcribed spacer-1/4. Subsequently, the amplicon was digested with the restriction endonucleases MspI, HaeIII, HinfI and EcoRI in order to generate genus- or species-specific patterns for identification of the fungus. HinfI produced indistinguishable fingerprints for all Aspergillus species tested. MspI produced species-specific patterns for: Cryptococcus neoformans, Cryptococcus non-neoformans, Candida albicans and Candida tropicalis. EcoRI succeeded in differentiating penicillia from aspergilli and cryptococci from Candida spp. It is concluded that this procedure can differentiate genera and occasionally species of medically important fungi and that following the necessary validation experiments, it can be used directly on clinical samples to assist prompt diagnosis of systemic fungal infections.