We developed a microtitration plate enzyme immunoassay to detect PCR-amplified DNA from Candida species. Nucleotide sequences derived from the internal transcribed spacer (ITS) region of fungal rDNA were used to develop species-specific oligonucleotide probes for Candida albicans, C. tropicalis, C. parapsilosis, and C. krusei. No cross-hybridization was detected with any other fungal, bacterial, or human DNAs tested. In contrast, a C. (Torulopsis) glabrata probe cross-reacted with Saccharomyces cerevisiae DNA but with no other DNAs tested. Genomic DNA purified from C. albicans blastoconidia suspended in blood was amplified by PCR with fungus-specific universal primers ITS3 and ITS4. With the C. albicans-specific probe labeled with digoxigenin, a biotinylated capture probe, and streptavidin-coated microtitration plates, amplified DNA from as few as two C. albicans cells per 0.2 ml of blood could be detected by enzyme immunoassay.

Random amplified polymorphic DNA (RAPD) was used to better characterize the genotypic relatedness among medically important Candida species. By using short oligomer primers (10-mers) with arbitrarily chosen sequences in the polymerase chain reaction, distinctive and reproducible sets of polymerase chain reaction products were observed for isolates of C. albicans, C. lusitaniae, C. tropicalis, and Torulopsis (Candida) glabrata. The RAPD analysis differentiated a physiologically homogeneous panel of C. parapsilosis into three distinct groups and showed genetic diversity within C. haemulonii. Intraspecies DNA-length polymorphisms were seen for RAPD profiles derived from different isolates of each species. Analysis of RAPDs from a panel of C. albicans, which included 16 laboratory derivatives of two reference strains, showed that the profiles of unrelated strains differed and that the derivatives of each reference strain were identifiable. Minor differences in the RAPD profiles, suggestive of mutations that had occurred during the long-term maintenance of the strains, were detected. Because of its ease and reliability, RAPD analysis should be useful in providing genotypic characters for taxonomic descriptions, for confirming the identities of stock isolates, for typing Candida species in epidemiologic investigations, and for use in the rapid identification of pathogenic fungi.

Fifty-five epidemiologically linked Aspergillus fumigatus isolates obtained from six nosocomial outbreaks of invasive aspergillosis were subtyped by sequencing the polymorphic region of the gene encoding a putative cell surface protein, Afu3g08990 (denoted as CSP). Comparative sequence analysis showed that genetic diversity was generated in the coding region of this gene by both tandem repeats and point mutations. Each unique sequence in an outbreak cluster was assigned an arbitrary number or CSP sequence type. The CSP typing method was able to identify "clonal" and genotypically distinct A. fumigatus isolates, and the results of this method were concordant with those of another discriminatory genotyping technique, the Afut1 restriction fragment length polymorphism typing method. The novel single-locus sequence typing (CSP typing) strategy appears to be a simple, rapid, discriminatory tool that can be readily shared across laboratories. In addition, we found that A. fumigatus isolates substructured into multiple clades; interestingly, one clade consisted of isolates predominantly representing invasive clinical isolates recovered from cardiac transplant patients from two different outbreak situations. We also found that the A. fumigatus isolate Af293, whose genome has been sequenced, possesses a CSP gene structure that is substantially different from those of the other A. fumigatus strains studied here, highlighting the need for further taxonomic study.Conidia of Aspergillus fumigatus become airborne easily, and subsequent inhalation of these infectious particles is believed to be the route of transmission for invasive aspergillosis. Accordingly, several nosocomial outbreaks of invasive aspergillosis have been reported with strong evidence suggesting that such infections are acquired from the environment of the hospital (10,31,35,43). The Centers for Disease Control and Prevention (CDC) recommends that in the setting of a nosocomial aspergillosis outbreak and in the presence of continuing evidence of Aspergillus infection in the hospital population, an environmental assessment should be undertaken to determine and eliminate the source of infection (51). In such nosocomial outbreak investigations, Aspergillus strain typing methods can indicate the source and/or route of infection by determining whether epidemiologically related isolates are also genetically related. Once the source is identified, corrective measures can then be undertaken to eliminate the implicated source to contain the infection. Several molecular methods have been evaluated for A. fumigatus strain typing, and these methods include randomly amplified polymorphic DNA typing (1), sequence-specific DNA primer analysis (32), polymorphic microsatellite markers (6,7,12), and analysis of hybridization profiles with a dispersed, repetitive DNA probe Afut1 (Afut1 restriction fragment length polymorphism [Afut1 RFLP]) (11,36). Although a combination of typing methods appears to have more utility in strain typing, polymorphic microsatellite markers, and Af...

In the present investigation, 49 Aspergillus fumigatus isolates obtained from four nosocomial outbreaks were typed by Afut1 restriction fragment length polymorphism (RFLP) analysis and three PCR-based molecular typing methods: random amplified polymorphic DNA (RAPD) analysis, sequence-specific DNA primer (SSDP) analysis, and polymorphic microsatellite markers (PMM) analysis. The typing methods were evaluated with respect to discriminatory power (D), reproducibility, typeability, ease of use, and ease of interpretation to determine their performance and utility for outbreak and surveillance investigations. Afut1 RFLP analysis detected 40 types. Thirty types were observed by RAPD analysis. PMM analysis detected 39 allelic types, but SSDP analysis detected only 14 types. All four methods demonstrated 100% typeability. PMM and RFLP analyses had comparable high degrees of discriminatory power (D ‫؍‬ 0.989 and 0.988, respectively). The discriminatory power of RAPD analysis was slightly lower (D ‫؍‬ 0.971), whereas SSDP analysis had the lowest discriminatory power (D ‫؍‬ 0.889). Overall, SSDP analysis was the easiest method to interpret and perform. The profiles obtained by PMM analysis were easier to interpret than those obtained by RFLP or RAPD analysis. Bands that differed in staining intensity or that were of low intensity were observed by RAPD analysis, making interpretation more difficult. The reproducibilities with repeated runs of the same DNA preparation or with different DNA preparations of the same strain were high for all the methods. A high degree of genetic variation was observed in the test population, but isolates were not always similarly divided by each method. Interpretation of band profiles requires understanding of the molecular mechanisms responsible for genetic alternations. PMM analysis and Afut1 RFLP analysis, or their combination, appear to provide the best overall discriminatory power, reproducibility, ease of interpretation, and ease of use. This investigation will aid in planning epidemiologic and surveillance studies of A. fumigatus.