MOLECULAR DIAGNOSIS AND EPIDEMIOLOGY OF FUNGAL-INFECTIONS

A variety of methods are utilized for DNA strain subtyping of Candida spp. because no 'gold standard' exists. Random amplified polymorphic D NA (RAPD) or restriction enzyme analysis (REA) are useful to determine the source of an outbreak, but more reproducible and discriminatory m ethods such as Southern hybridization and pulsed field gel electrophor esis (PFGE) may be required. When applied to some nocosomial Candida i nfections, multiple strains and species have been identified. Microevo lution of yeast species occurs and epidemiologically related isolates may show minor pattern differences, creating uncertainty as to whether they are distinct strains. Approximately 1000 isolates of Aspergillus fumigatus from environmental and clinical sources were typed by REA p robed with an A. fumigatus-specific retrotransposon-like sequence. Pat ients with no symptom of aspergillosis may carry several strains, wher eas patients with pulmonary aspergillosis may carry one or two strains ; nocosomial transmission of aspergillosis was proven in 39% of the pa tients studied; any given environmental strain can be infectious; the environmental population of A. fumigatus is extremely diverse and no s pecific niche was found in the hospital. A PCR assay was designed to t arget conserved 18S-ribosomal DNA (rDNA) sequences shared by most fung i and a 687 bp product was amplified from 25 medically important fun g al species. Studies with blood, cerebrospinal fluid and sputum specime ns from patients with mycoses indicated that the PCR assay is more sen sitive in diagnosing invasive fungal infections than blood culture met hods. More specific identification is obtainable with genus/species-sp ecific probes designed from within the PCR-amplified sequences for C, albicans, C. krusei, C. lusitaniae, Pneumocystis carinii, Cryptococcus neoformans, Aspergillus/Penicillium spp. and C. glabrata/Saccharomyce s cerevisiae. A. fumigatus and A. niger were differentiated by denatur ing gradient gel electrophoresis. In situ hybridization (ISH) detected a 648 bp fragment of the 18S rDNA of C. neoformans and a 568 bp fragm ent of the alkaline proteinase gene of A. fumigatus in tissues from ex perimentally infected animals. In ISH, the entire process can be autom ated, making this procedure rapid and easy The difficulty in establish ing a diagnosis of invasive candidiasis has prompted the quest for a c linically useful PCR test for candidaemia. The universal fungal oligon ucleotide primer pair, ITS3 and ITS4, amplifies portions of the 5.8S a d 28S rDNA subunits, and the ITS2 region. Although rRNA genes are high ly conserved, the ITS regions are distinctive. DNA probes were designe d from ITS2 that were specific for 16 different Candida species. Simpl e, rapid sample preparation was suitable for PCR analysis of BacT/Aler t blood culture bottles. Sample preparation, PCR, and EIA detection of the amplicon from five different Candida species was accomplished in 7 h, 2.5 days sooner than by conventional culture methods. As well as saving time, minor yeast species among a major species, or among bacte ria, were simultaneously detected. PCR-EIA using a microtitration plat e format had sensitivity 10-times greater than that obtained with ethi dium bromide-stained agarose gels. Taqman combines in one step PCR, pr obe hybridization, and fluorescent signal generation. Taqman PCR had s ensitivity equivalent to PCR-EIA and required only 5 h, including samp le preparation.