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To characterize the full spectrum of the causal agents involved and their potential sources, partial DNA sequences from three loci (RPB2, EF-1␣, and nuclear ribosomal rRNA) totaling 3.48 kb were obtained from 91 corneal and 100 isolates from the patient's environment (e.g., contact lens and lens cases). We also sequenced a 1.8-kb region encoding the RNA polymerase II second largest subunit (RPB2) from 126 additional pathogenic isolates to better understand how the keratitis outbreak isolates fit within the full phylogenetic spectrum of clinically important fusaria. These analyses resulted in the most robust phylogenetic framework for Fusarium to date. In addition, RPB2 nucleotide variation within a 72-isolate panel was used to design 34 allele-specific probes to identify representatives of all medically important species complexes and 10 of the most important human pathogenic Fusarium in a single-well diagnostic assay, using flow cytometry and fluorescent microsphere technology. The multilocus data revealed that one haplotype from each of the three most common species comprised 55% of CDC's corneal and environmental isolates and that the corneal isolates comprised 29 haplotypes distributed among 16 species. The high degree of phylogenetic diversity represented among the corneal isolates is consistent with multiple sources of contamination.

Members of the Fusarium solani species complex (FSSC) are increasingly implicated as the causative agents of human mycoses, particularly in the expanding immunocompromised and immunosuppressed patient populations. Best known as ubiquitous plant pathogens and saprotrophs, the FSSC comprises over 45 phylogenetically distinct species distributed among three major clades. To identify which species are associated with human infections, we generated multilocus haplotypes based on four partial gene sequences from 471 isolates. Of these, 278 were from human patients, 21 were from hospital environments, and 172 were from other sources. Phylogenetic trees inferred from an ergosterol biosynthesis gene (erg-3) were highly discordant with those inferred from the three other partial gene sequences; therefore, this partition was analyzed separately. Multilocus analysis showed that isolates from humans were restricted to but spread throughout clade 3 of the FSSC phylogeny, comprising at least 18 phylogenetically distinct species. The majority (74.5%) of the clinical isolates, however, were associated with four major lineages, designated groups 1 to 4. Groups 1 and 2 were strongly supported as phylogenetic species, whereas groups 3 and 4 were not. Although isolates from ocular infections were found in all four groups, they had a significant tendency to belong to group 3 (P < 0.001). Human clinical isolates shared identical multilocus haplotypes with isolates from plants, other animals, and from hospital environments, suggesting potential nosocomiality. The major finding of this study is that FSSC-associated mycoses of humans and other animals have origins in a broad phylogenetic spectrum, indicating widespread ability to cause infection in this diverse species complex.Fusarium solani is one of the most frequently isolated fungi from soil and plant debris and is also associated with serious invasive mycoses in immunocompromised and immunosuppressed patients (3,18). This species, as defined based on morphology, is actually a diverse complex of over 45 phylogenetic and/or biological species (13 and this study), termed the Fusarium solani species complex (FSSC). These morphologically similar species are generally identified broadly under the name F. solani (12). They are ubiquitous in soil and decaying plant material, where they act as decomposers, but they are also host-specific pathogens of a number of agriculturally important plants, including pea, cucurbits, and sweet potato. Moreover, they are increasingly associated with opportunistic infections of humans and other animals, causing systemic infections with a high mortality rate (8), as well as localized infections in the skin and other body parts (5, 6). In immunocompetent patients, FSSC isolates are mainly known from mycotic keratitis subsequent to traumatic introduction of inoculum. Neutropenic patients, a category of particularly strongly immunocompromised patients, are susceptible to dissemination of infection from superficial or subcutaneous initiation; such infections ar...

Fusarium oxysporum is a phylogenetically diverse monophyletic complex of filamentous ascomycetous fungi that are responsible for localized and disseminated life-threatening opportunistic infections in immunocompetent and severely neutropenic patients, respectively. Although members of this complex were isolated from patients during a pseudoepidemic in San Antonio, Tex., and from patients and the water system in a Houston, Tex., hospital during the 1990s, little is known about their genetic relatedness and population structure. This study was conducted to investigate the global genetic diversity and population biology of a comprehensive set of clinically important members of the F. oxysporum complex, focusing on the 33 isolates from patients at the San Antonio hospital and on strains isolated in the United States from the water systems of geographically distant hospitals in Texas, Maryland, and Washington, which were suspected as reservoirs of nosocomial fusariosis. In all, 18 environmental isolates and 88 isolates from patients spanning four continents were genotyped. The major finding of this study, based on concordant results from phylogenetic analyses of multilocus DNA sequence data and amplified fragment length polymorphisms, is that a recently dispersed, geographically widespread clonal lineage is responsible for over 70% of all clinical isolates investigated, including all of those associated with the pseudoepidemic in San Antonio. Moreover, strains of the clonal lineage recovered from patients were conclusively shown to genetically match those isolated from the hospital water systems of three U.S. hospitals, providing support for the hypothesis that hospitals may serve as a reservoir for nosocomial fusarial infections. Members of the phylogenetically diverse monophyleticFusarium oxysporum complex (FOC) are best known as cosmopolitan soilborne plant pathogens that are responsible for economically devastating vascular wilts of an enormous range of agronomically important plant hosts (6). Members of the FOC are also frequently isolated from nonplant sources, particularly from the soil but also from air and animals. Over the past 2 decades, however, fusaria have emerged as opportunistic pathogens causing life-threatening disseminated infections in immunocompromised patients (3). In patients who are persistently neutropenic, deeply invasive fusarial infections cause 100% mortality (18). Most localized and disseminated cases of fusariosis are caused by members of the Fusarium solani species complex, followed by members of the FOC (1). Fortunately, the recent development of one strain of F. oxysporum as a model system will greatly facilitate the molecular genetic dissection of fungal virulence determinants during plant and animal pathogenesis (24).Although molecular epidemiological studies have been completed for nosocomial fusariosis (1, 25), most of the analyses were conducted on members of the F. solani species complex.

To date, three species of Phaeoacremonium have been associated with phaeohyphomycosis. These are P. parasiticum (formerly Phialophora parasitica), P. inflatipes, and P. rubrigenum. Numerous unknown isolates resembling Phaeoacremonium spp. have in recent years been isolated from human patients as well as from woody plants that appear to be the main environmental source of these fungi. Nine new Phaeoacremonium species, of which six were obtained as etiologic agents of human opportunistic infection, are reported. They can be identified based on their cultural and morphological characters, and the identifications are strongly supported in phylogenetic analyses of partial sequences of the actin, ␤-tubulin, and calmodulin genes. A multiple-entry electronic key based on morphological, cultural, and ␤-tubulin sequence data was developed to facilitate routine species identification. Reexamination of all isolates of P. inflatipes associated with human disease showed them to be misidentified and to belong to the new taxa described here.

Candida dubliniensis is a newly described species that is closely related phylogenetically to Candida albicans and that is commonly associated with oral candidiasis in human immunodeficiency virus-positive patients. Several recent studies have attempted to elucidate phenotypic and genotypic characteristics of use in separating the two species. However, results obtained with simple phenotypic tests were too variable and tests that provided more definitive data were too complex for routine use in the clinical laboratory setting. The objective of this study was to determine if reproducible identification of C. dubliniensis could be obtained with commercial identification kits. The substrate reactivity profiles of 80 C. dubliniensis isolates were obtained by using the API 20C AUX, ID 32 C, RapID Yeast Plus, VITEK YBC, and VITEK 2 ID-YST systems. The percentages of C. dubliniensisisolates capable of assimilating or hydrolyzing each substrate were compared with the percentages from the C. albicans profiles in each kit's database, and the results were expressed as percentC. dubliniensis and percent C. albicans. Any substrate that showed >50% difference in reactivity was considered useful in differentiating the species. In addition, assimilation of methyl-α-d-glucoside (MDG), d-trehalose (TRE), and d-xylose (XYL) by the same isolates was investigated by the traditional procedure of Wickerham and Burton (L. J. Wickerham and K. A. Burton, J. Bacteriol. 56:363–371, 1948). At 48 h (the time recommended by the manufacturer for its new database), we found that the assimilation of four carbohydrates in the API 20C AUX system could be used to distinguish the species, i.e., glycerol (GLY; 88 and 14%), XYL (0 and 88%), MDG (0 and 85%), and TRE (15 and 97%). Similarly, results with the ID 32 C system at 48 h showed that XYL (0 and 98%), MDG (0 and 98%), lactate (LAT; 0 and 96%), and TRE (30 and 96%) could be used to separate the two species. Phosphatase (PHS; 9 and 76%) and α-d-glucosidase (23 and 94%) proved to be the most useful for separation of the species in the RapID Yeast Plus system. While at 24 h the profiles obtained with the VITEK YBC system showed that MDG (10 and 95%), XYL (0 and 95%), and GLY (26 and 80%) could be used to separate the two species, at 48 h only XYL (6 and 95%) could be used to separate the two species. The most useful substrates in the VITEK 2 ID-YST system were TRE (1 and 89%), MDG (1 and 99%), LAT (4 and 98%), and PHS (83 and 1%). While the latter kit was not yet commercially available at the time of the study, it would appear to be the most valuable for the identification of C. dubliniensis. Although assimilation of MDG, TRE, and XYL proved to be the most useful for species differentiation by the majority of commercial systems, the results with these carbohydrates by the Wickerham and Burton procedure were essentially the same for both species, albeit following protracted incubation. Thus, it is the rapidity of the assimilation achieved with the commercial systems that allows the differentiation of C. dubliniensis from C. albicans.

To evaluate procedures used for epidemiologic analysis of outbreaks of aspergillosis, we analyzed a collection of 35 Aspergillus fumigatus isolates using three typing methods: isoenzyme analysis (IEA), random amplified polymorphic DNA (RAPD) analysis, and restriction endonuclease analysis (REA). Twenty-one isolates were from a single hospital, with four isolates coming from different patients. Three clinical isolates came from a different hospital, and 11 clinical or environmental isolates were derived from a culture collection. With IEA, the patterns of alkaline phosphatase, esterase, and catalase discriminated nine types. In contrast, 22 types were obtained with five different RAPD primers, and 21 types could be detected with three of these (R108, R151, and UBC90). Restriction endonuclease analysis of genomic DNA, digested with either XbaI, XhoI, or SalI, detected 3, 17, and 13 different REA types, respectively, and 22 types were identified by combining the data from the XhoI and SalI REAs. Twenty-eight types were obtainable with a combination of REA, IEA, and RAPD patterns. Overall, the results pointed to substantial genetic variation among the isolates. Though two isolates had markedly distinct genotypes, their morphologic features and exoantigens were consistent with their being A. fumigatus. The analysis will help in planning epidemiologic studies of aspergillosis.

During October and November 1993, four patients contracted Acremonium kiliense endophthalmitis at one ambulatory surgical center. We hypothesized that the source was environmental and conducted a matched case-control study, environmental evaluation, and observational studies. Case and control patients were similar in clinical characteristics. However, case patients all had surgery on the first operative day of the week or had surgery significantly sooner after the operating room opened than did controls (a median of 46 vs. 150 minutes afterward; P = .03). An environmental review revealed the ventilation system was switched on 5-30 minutes before procedures began on the first operative day of the week, and air was filtered before but not after humidification. Cultures of the humidifier water in the ventilation system yielded A. kiliense phenotypically identical to isolates from case patients. Our data suggest that switching on the ventilation system each week aerosolized a reservoir of A. kiliense and caused infection of patients. We believe this is the first reported outbreak of fungal endophthalmitis traced to an environmental source, and it underscores the importance of utilizing established hospital infection control practices in the outpatient setting.