Molecular typing of clinical and environmental isolates of Cryptococcus gattii species complex from southern California, United States.

BACKGROUND: Two species complexes (SC) cause the majority of human Cryptococcus infections: Cryptococcus neoformans SC and Cryptococcus gattii SC. Infection is typically thought to be acquired following environmental exposure. In an urban setting, parks and other public spaces are a likely source of contact with C. gattii SC. OBJECTIVES: The goals of this study were to describe the genetic diversity of C. gattii SC in the California environment, to determine the extent of environmental exposure in publicly accessed areas and to correlate the genotypes of environmental C. gattii SC isolates with those from patients in southern California. METHODS: Specimens from trees and soil from 13 parks and public areas of seven California counties were examined for C. gattii SC isolates. Isolates were sequence typed and compared to sequence types from human clinical isolates from the same area. RESULTS: Multilocus sequence typing identified C. gattii sensu stricto (VGI molecular type) as well as Cryptococcus bacillisporus (VGIII molecular type). Several C. bacillisporus but none of the C. gattii sensu stricto isolates shared sequence types with human clinical isolates from southern California. CONCLUSIONS: C. gattii SC colonies exist in some California public parks. The presence of identical STs in environmental and human isolates of C. bacillisporus is suggestive of an arboreal origin of human infections. Two new tree species were documented as hosts for C. gattii SC in California, adding to the four species previously identified.

Isolation of azole-resistant Aspergillus fumigatus from the environment in the south-eastern USA.

Background: Azole resistance in isolates of the fungus Aspergillus fumigatus has been associated with agricultural use of azole fungicides. Environmental isolation of resistant isolates has been reported in Asia, Africa, Europe and South America. Objectives: To determine whether A. fumigatus isolates containing TR34/L98H or TR46/Y121F/T289A can be found in fields in the USA treated with agricultural azoles. Methods: Crop debris was collected and screened for A. fumigatus. All A. fumigatus isolates were screened for azole resistance. The CYP51A gene of azole-resistant isolates was sequenced. The population structure of a subset of isolates was determined using microsatellite typing. Results: This article identifies azole-resistant A. fumigatus isolates containing the TR34/L98H mutation in an experimental peanut field that had been treated with azole fungicides. Conclusions: These findings suggest the development of resistance to azole antifungals in A. fumigatus may be present where agricultural azoles are used in the USA.

Detection of mucormycetes and other pathogenic fungi in formalin fixed paraffin embedded and fresh tissues using the extended region of 28S rDNA.

Molecular methods of detection based on DNA-sequencing of the internal transcribed spacer 1 and 2 (ITS1 and ITS2) or 5΄ end region of 28S (D1-D2 region) of ribosomal RNA gene (rDNA) have been used extensively for molecular identification and detection of fungal infections. However, these regions are not always informative for identification of mucormycetes and other rare fungal pathogens as they often contain large introns, heterogenic regions, and/or cannot be PCR-amplified using broad range fungal PCR primers. In addition, because of the difficulties of recovering intact fungal DNA from human specimens, smaller regions of DNA are more useful for the direct detection of fungal DNA in tissues and fluids. In this study, we investigated the utility of 12F/13R PCR primers targeting a 200-230 bp region of the extended 28S region of rDNA for molecular identification of fungal DNA in formalin fixed paraffin embedded tissues and other clinical specimens. We demonstrated that this region can be successfully used for identification of all genera and some species of clinically relevant mucormycetes, as well as other medically important fungi, such as Aspergillus, Fusarium, Coccidioides, and Cryptococcus. We also demonstrated that PCR amplification and direct sequencing of the extended 28S region of rDNA was more sensitive compared to targeting the ITS2 region, as we were able to detect and identify mucormycetes and other fungal pathogens in tissues from patients with histopathological and/or culture evidence of fungal infections that were negative with PCR using ITS-specific primers.

Valley fever: finding new places for an old disease: Coccidioides immitis found in Washington State soil associated with recent human infection.

We used real-time polymerase chain reaction and culture to demonstrate persistent colonization of soils by Coccidioides immitis, an agent of valley fever, in Washington State linked to recent human infections and located outside the endemic range. Whole-genome sequencing confirmed genetic identity between isolates from soil and one of the case-patients.

Whole-genome analysis of Exserohilum rostratum from an outbreak of fungal meningitis and other infections.

Exserohilum rostratum was the cause of most cases of fungal meningitis and other infections associated with the injection of contaminated methylprednisolone acetate produced by the New England Compounding Center (NECC). Until this outbreak, very few human cases of Exserohilum infection had been reported, and very little was known about this dematiaceous fungus, which usually infects plants. Here, we report using whole-genome sequencing (WGS) for the detection of single nucleotide polymorphisms (SNPs) and phylogenetic analysis to investigate the molecular origin of the outbreak using 22 isolates of E. rostratum retrieved from 19 case patients with meningitis or epidural/spinal abscesses, 6 isolates from contaminated NECC vials, and 7 isolates unrelated to the outbreak. Our analysis indicates that all 28 isolates associated with the outbreak had nearly identical genomes of 33.8 Mb. A total of 8 SNPs were detected among the outbreak genomes, with no more than 2 SNPs separating any 2 of the 28 genomes. The outbreak genomes were separated from the next most closely related control strain by ∼136,000 SNPs. We also observed significant genomic variability among strains unrelated to the outbreak, which may suggest the possibility of cryptic speciation in E. rostratum.

Coccidioides immitis identified in soil outside of its known range - Washington, 2013.

Coccidioidomycosis ("valley fever") is caused by inhaling spores of the soil-dwelling fungi Coccidioides immitis or Coccidioides posadasii. Most infections are subclinical. When clinical manifestations do occur (typically 1-4 weeks after exposure), they are similar to those associated with influenza or community-acquired pneumonia. Disseminated disease is rare. Residual pulmonary nodules can lead to chronic lung disease. Fluconazole or other triazoles often are used for treatment, but mild cases often resolve without specific therapy. A total of 17,802 cases were reported in the United States in 2012.

Multilocus sequence typing of Histoplasma capsulatum in formalin-fixed paraffin-embedded tissues from cats living in non-endemic regions reveals a new phylogenetic clade.

Infections caused by Histoplasma capsulatum are found most often in endemic regions of North, Central, and South America. H. capsulatum has been divided into eight geographic clades by multi-locus sequence typing (MLST). Recently, one isolate and five formalin-fixed paraffin-embedded (FFPE) tissue samples were received from six of 15 suspected cases of histoplasmosis in cats residing in areas not known to be endemic for H. capsulatum. Polymerase chain reaction (PCR) amplification and sequence analysis of the rDNA ITS-2 region confirmed the diagnosis of H. capsulatum. Since these cases were not, as noted, from the accepted endemic areas, it was of interest to understand the molecular epidemiology of these isolates. Results of molecular analysis indicated that the H. capsulatum recovered from the cats were most closely related to the North American-1 clade, but clustered separately outside this clade, suggesting that the H. capsulatum infecting the animals may represent a separate clade or phylogenetic species. This study also demonstrated the utility of obtaining valuable molecular subtype data directly from archived FFPE tissue blocks, particularly when a fungus culture was not performed or is otherwise unavailable.

Molecular analyses of Fusarium isolates recovered from a cluster of invasive mold infections in a Brazilian hospital.

BACKGROUND: Invasive fusariosis (IF) is a rare but often fatal fungal infection in immunosuppressed patients. In 2007, cases of IF above the expected epidemiologic baseline were detected in the hematology ward of a hospital in Rio de Janeiro, Brazil. Possible sources of infection were investigated by performing environmental sampling and patient isolate collection, followed by molecular typing. Isolates from dermatology patients with superficial fusariosis were included in the study for comparison to molecular types found in the community. METHODS: Environmental sampling focused on water-related sources in and around the hematology ward. Initially, we characterized 166 clinical and environmental isolates using the Fusarium translation elongation factor 1α (EF-1α) genetic locus. Isolates included 68 collected from water-related sources in the hospital environment, 55 from 18 hematology patients, and 43 from the skin/nails of 40 outpatients seen at the hospital dermatology clinic. Multi-locus sequence typing was performed on Fusarium solani species complex (FSSC) species 1 and 2 isolates to investigate their relatedness further. RESULTS: Most of the hematology samples were FSSC species 2, with species type FSSC 2-d the most commonly isolated from these patients. Most of the outpatient dermatology samples were also FSSC 2, with type 2-d again predominating. In contrast, environmental isolates from water sources were mostly Fusarium oxysporum species complex (FOSC) and those from air samples mostly Fusarium incarnatum-equiseti species complex (FIESC). A third of the environmental samples were FSSC, with species types FSSC 1-a and FSSC 1-b predominating. CONCLUSIONS: Fusarium isolate species types from hematology patient infections were highly similar to those recovered from dermatology patients in the community. Four species types (FSSC 1-a, 1-b, 2-d and 2-f) were shared between hematology patients and the environment. Limitations in environmental sampling do not allow for nosocomial sources of infection to be ruled out. Future studies will focus on environmental factors that may have influenced the prevalence of FSSC fusariosis in this hematology ward.

Low cost fabrication of a superhydrophobic V-grooved polymer surface.

Engineering of polymer surfaces to control their wetting properties has shown a wide range of potential applications. In this paper we show low cost fabrication of a superhydrophobic polymer surface via a hierarchical combination of hot embossing, O2 reactive ion etching (RIE) and deposition of a hydrophobic silane. The hot embossing and O2 RIE were used to produce respective micro and nanoscale surface roughness which is a requirement for obtaining superhydrophobic surfaces, while the deposition of a hydrophobic silane modified surface chemistry. In order to increase the water/air interface in the Cassie-Baxter composite wetting model, a brass mold with microscale V-grooves was used for hot embossing. Images of droplets in both static water contact angles and dynamic impact tests with the surface clearly show that the wetting state follows the Cassie-Baxter wetting model. The results of this study indicate that our design of the dual level surface roughness and the fabrication process allow for low cost and easy production of a highly superhydrophobic surface.

Use of terbinafine in the treatment protocol of intestinal Cryptococcus neoformans in a dog.

A 2.5 yr old sexually intact male vizsla was admitted to the Iowa State University Veterinary Teaching Hospital for persistent diarrhea, weight loss, and panhypoproteinemia. Examination revealed an emaciated condition and melena. Two masses were palpated in the cranial abdomen. Hematology and serum biochemistry exhibited a regenerative anemia and confirmed the presence of panhypoproteinemia, suggestive of a protein-losing eneteropathy. Distinct areas of thickened intestinal wall and enlarged mesenteric lymph nodes were found on abdominal ultrasound. Cytology from those nodes showed the presence of suspected Cryptococcus spp., and infection was confirmed utilizing a cryptococcal antigen titer. Medical therapy with lipid-complexed amphotericin B and fluconazole was unsuccessful. Two surgical procedures were performed to remove the affected areas of intestine and lymph nodes, but the disease persisted as evidenced by a persistently elevated cryptococcal antigen titer. Terbinafine was prescribed, which resulted in complete resolution of clinical signs and a steadily decreasing cryptococcal antigen titer. Very few cases of intestinal cryptococcosis have been reported. In this case, infection resulted in a protein-losing enteropathy. In addition, this article describes the use of terbinafine in the treatment of intestinal cryptococcal infection in the dog, which has not been previously reported.

Global population structure of Aspergillus terreus inferred by ISSR typing reveals geographical subclustering.

BACKGROUND: Aspergillus terreus causes invasive aspergillosis (IA) in immunocompromised individuals and can be the leading cause of IA in certain medical centers. We examined a large isolate collection (n = 117) for the presence of cryptic A. terreus species and employed a genome scanning method, Inter-Simple Sequence Repeat (ISSR) PCR to determine A. terreus population structure. RESULTS: Comparative sequence analyses of the calmodulin locus revealed the presence of the recently recognized species A. alabamensis (n = 4) in this collection. Maximum parsimony, Neighbor joining, and Bayesian clustering of the ISSR data from the 113 sequence-confirmed A. terreus isolates demonstrated that one clade was composed exclusively of isolates from Europe and another clade was enriched for isolates from the US. CONCLUSIONS: This study provides evidence of a population structure linked to geographical origin in A. terreus.

Factors Influencing Distribution of Coccidioides immitis in Soil, Washington State, 2016.

Coccidioides immitis and Coccidioides posadasii are causative agents of Valley fever, a serious fungal disease endemic to regions with hot, arid climate in the United States, Mexico, and Central and South America. The environmental niche of Coccidioides spp. is not well defined, and it remains unknown whether these fungi are primarily associated with rodents or grow as saprotrophs in soil. To better understand the environmental reservoir of these pathogens, we used a systematic soil sampling approach, quantitative PCR (qPCR), culture, whole-genome sequencing, and soil chemical analysis to identify factors associated with the presence of C. immitis at a known colonization site in Washington State linked to a human case in 2010. We found that the same strain colonized an area of over 46,000 m2 and persisted in soil for over 6 years. No association with rodent burrows was observed, as C. immitis DNA was as likely to be detected inside rodent holes as it was in the surrounding soil. In addition, the presence of C. immitis DNA in soil was correlated with elevated levels of boron, calcium, magnesium, sodium, and silicon in soil leachates. We also observed differences in the microbial communities between C. immitis-positive and -negative soils. Our artificial soil inoculation experiments demonstrated that C. immitis can use soil as a sole source of nutrients. Taken together, these results suggest that soil parameters need to be considered when modeling the distribution of this fungus in the environment. IMPORTANCE Coccidioidomycosis is considered a highly endemic disease for which geographic range is likely to expand from climate change. A better understanding of the ecological niche of Coccidioides spp. is essential for generating accurate distribution maps and predicting future changes in response to the changing environment. Our study used a systematic sampling strategy, advanced molecular detection methods, and soil chemical analysis to identify environmental factors associated with the presence of C. immitis in soil. Our results demonstrate the fungus can colonize the same areas for years and is associated with chemical and microbiological soil characteristics. Our results suggest that in addition to climate parameters, soil characteristics need to be considered when building habitat distribution models for this pathogen.

Evaluation of the Specificity of Two Enzyme Immunoassays for Coccidioidomycosis by Using Sera from a Region of Endemicity and a Region of Nonendemicity.

Coccidioidomycosis (CM), a serious life-threatening fungal infection endemic to arid regions of the western United States and Mexico, can be challenging to diagnose in a timely manner. Commercially developed enzyme immunoassays (EIAs) (from Meridian Biosciences and Immuno-Mycologics [IMMY]) have provided faster, simpler means for serodiagnosis; however, independent evaluations have questioned EIA specificity, particularly IgM-positive/IgG-negative results. This study was conducted to evaluate EIA specificity among persons residing in Puerto Rico (n = 534), where CM is not endemic (who were not likely to have been exposed to Coccidioides spp.), compared to blood bank donors residing in Arizona (n = 1,218), where CM is endemic. Upon comparing serum reactivity between Puerto Rico and Arizona, the Meridian EIA showed a significant difference in IgG reactivity (0.37% versus 3.6%; P < 0.001) but not IgM reactivity (3.4% versus 2.4%; P = 0.31). No IgM-/IgG-reactive sera were detected among sera from Puerto Rico, compared to 7 (0.57%) sera from Arizona. Similar results were observed using the IMMY EIA, although significantly (P = 0.03) fewer IgM-reactive sera from Arizona were observed, compared to the Meridian EIA. EIA-reactive sera were also evaluated by immunodiffusion before and after 3- to 4-fold concentration of the sera. These results demonstrate that elevated IgG EIA reactivity is present in sera from healthy individuals in regions of endemicity and that IgM EIA reactivity observed in sera from individuals residing outside regions of endemicity is most likely nonspecific. Other criteria, including clinical and microbiological evaluations, should be taken into account when interpreting results from surveillance studies and other reporting measures.

Gastrointestinal basidiobolomycosis treated with posaconazole.

A 67 year-old Caucasian male from Arizona presented with indolent symptoms of intestinal obstruction and hydronephrosis, found at surgery to be caused by a mass involving the terminal ileum and cecum, extending into the posterior abdominal wall and obstructing the right ureter. Histopathology was diagnostic of basidiobolomycosis. PCR of tissue and sequencing identified the fungus as, Basidiobolus ranarum. During one year of posaconazole treatment, the residual mass shrank, hydronephrosis was relieved and peripheral eosinophilia resolved.

Ruptured mycotic aortic aneurysm in a sooty mangabey (Cercocebus atys).

Mycotic aortic aneurysm is a local, irreversible dilatation of the aorta associated with destruction of the vessel wall by infection and is a grave clinical condition associated with high morbidity and mortality in humans. Rupture of aortic aneurysms can be spontaneous, idiopathic, or due to severe trauma, and the condition has been associated with bacterial and, rarely, fungal infections in humans and animals. Here, we describe a case of ruptured spontaneous aortic aneurysm associated with zygomycetic infection in a 21-y-old female sooty mangabey. The animal did not present with any significant clinical signs before being found dead. At necropsy, she was in good body condition, and the thoracic cavity had a large amount of clotted blood filling the left pleural space and surrounding the lung lobes. Near the aortic arch, the descending thoracic aorta was focally perforated (diameter, approximately 0.15 cm), and clotted blood adhered to the tunica adventitia. The aortic intima had multiple, firm, pale-yellow nodules (diameter, 0.25 to 0.5 cm). Histopathologically, these nodules consisted of severe multifocal pyogranulomatous inflammation intermixed with necrosis, fibrin, and broad, infrequently septate, thin-walled fungal hyphae. Immunohistochemistry revealed fungal hyphae characteristic of Mucormycetes (formerly Zygomycetes), and PCR analysis identified the organism as Basidiobolus spp. Dissemination of the fungus beyond the aorta was not noted. Spontaneous aortic aneurysms have been described in nonhuman primates, but this is the first reported case of a ruptured spontaneous aortic aneurysm associated with entomophthoromycetic infection in a sooty mangabey.

Production and evaluation of reagents for detection of Histoplasma capsulatum antigenuria by enzyme immunoassay.

The detection of urinary Histoplasma capsulatum polysaccharide antigen (HPA) by enzyme immunoassay (EIA) has proven useful for the presumptive diagnosis of histoplasmosis in AIDS patients. Assay limitations include (i) detection of a largely uncharacterized antigen and (ii) difficulty in reproducibly generating antibodies for use in the EIA. To improve antibody production for use in this test and to better understand the antigen being detected, we compared rabbit antibodies elicited using various immunization schedules, routes, and H. capsulatum-derived antigens. Antibodies were evaluated by EIA for their ability to detect purified H. capsulatum C antigen (C-Ag) and antigenuria. Reported as enzyme immunoassay (EI) units (the A(450) with antigen divided by the A(450) without antigen), results demonstrated that intravenous immunization of rabbits with whole, killed yeast-phase cells (yeast-i.v. regimen) produced antibodies giving the highest EI values in the C-Ag EIA (mean EI units +/- standard deviation, 14.9 +/- 0.6 versus 6.4 +/- 0.4 for rabbits immunized with C-Ag versus 2.4 +/- 0.3 for all other regimens combined). Yeast-i.v. antibodies were highly sensitive for the detection of antigenuria in patients with histoplasmosis, as shown by the following results: 12/12 patients compared to 10/12, 6/12, 3/12, and 3/12, respectively, for antibodies from rabbits immunized with (i) C-Ag; (ii) whole, killed yeast-phase cells administered subcutaneously and intramuscularly; (iii) yeast-phase culture filtrates; and (iv) HPA-positive urine. Rabbits immunized using the yeast-i.v. regimen also gave higher peak antibody titers than rabbits immunized by any other regimen (P < 0.03), and their antibodies were most comparable in reactivity to antibodies produced for use in the standard HPA-EIA test (P < 0.001). Therefore, rabbits immunized using the yeast-i.v. regimen produced the most sensitive antibodies with the highest titers for detection of C-Ag and antigenuria in histoplasmosis patients.

Assessment of ribosomal large-subunit D1-D2, internal transcribed spacer 1, and internal transcribed spacer 2 regions as targets for molecular identification of medically important Aspergillus species.

Molecular approaches are now being developed to provide a more rapid and objective identification of fungi compared to traditional phenotypic methods. Ribosomal targets, especially the large-subunit RNA gene (D1-D2 region) and internal transcribed spacers 1 and 2 (ITS1 and ITS2 regions), have shown particular promise for the molecular identification of some fungi. We therefore conducted an assessment of these regions for the identification of 13 medically important Aspergillus species: Aspergillus candidus, Aspergillus (Eurotium) chevalieri, Aspergillus (Fennellia) flavipes, Aspergillus flavus, Aspergillus fumigatus, Aspergillus granulosus, Aspergillus (Emericella) nidulans, Aspergillus niger, Aspergillus restrictus, Aspergillus sydowii, Aspergillus terreus, Aspergillus ustus, and Aspergillus versicolor. The length of ribosomal regions could not be reliably used to differentiate among all Aspergillus species examined. DNA alignment and pairwise nucleotide comparisons demonstrated 91.9 to 99.6% interspecies sequence identities in the D1-D2 region, 57.4 to 98.1% in the ITS1 region, and 75.6 to 98.3% in the ITS2 region. Comparative analysis using GenBank reference data showed that 10 of the 13 species examined exhibited a < or = 1-nucleotide divergence in the D1-D2 region from closely related but different species. In contrast, only 5 of the species examined exhibited a < or = 1-nucleotide divergence from sibling species in their ITS1 or ITS2 sequences. Although the GenBank database currently lacks ITS sequence entries for some species, and major improvement in the quality and accuracy of GenBank entries is needed, current identification of medically important Aspergillus species using GenBank reference data seems more reliable using ITS query sequences than D1-D2 sequences, especially for the identification of closely related species.

Competitive binding inhibition enzyme-linked immunosorbent assay that uses the secreted aspartyl proteinase of Candida albicans as an antigenic marker for diagnosis of disseminated candidiasis.

The secreted aspartyl proteinases (Saps) of Candida albicans have been implicated as virulence factors associated with adherence and tissue invasion. The potential use of proteinases as markers of invasive candidiasis led us to develop a competitive binding inhibition enzyme-linked immunosorbent assay (ELISA) to detect Sap in clinical specimens. Daily serum and urine specimens were collected from rabbits that had been immunosuppressed with cyclophosphamide and cortisone acetate and infected intravenously with 10(7) C. albicans blastoconidia. Disseminated infection was confirmed by organ culture and histopathology. Although ELISA inhibition was observed when serum specimens from these rabbits were used, more significant inhibition, which correlated with disease progression, occurred when urine specimens were used. Urine collected as early as 1 day after infection resulted in significant ELISA inhibition (mean inhibition +/- standard error [SE] compared with preinfection control urine, 15.7% +/- 2.7% [P < 0.01]), and inhibition increased on days 2 through 5 (29.4% +/- 4.8% to 44.5% +/- 3.5% [P < 0.001]). Urine specimens from immunosuppressed rabbits infected intravenously with Candida tropicalis, Candida parapsilosis, Candida krusei, Cryptococcus neoformans, Aspergillus fumigatus, or Staphylococcus aureus were negative in the assay despite culture-proven dissemination. Nonimmunosuppressed rabbits receiving oral tetracycline and gentamicin treatment were given 2 x 10(8) C. albicans blastoconidia orally or intraurethrally to establish colonization of the gastrointestinal tract or bladder, respectively, without systemic dissemination; urine specimens from these rabbits also gave negative ELISA results. Dissemination to the kidney and spleen occurred in one rabbit challenged by intragastric inoculation, and urine from this rabbit demonstrated significant inhibition in the ELISA (mean inhibition +/- SE by day 3 after infection, 32.9% +/- 2.7% [P < 0.001]). The overall test sensitivity was 83%, the specificity was 92%, the positive predictive value was 84%, the negative predictive value was 91%, and the efficiency was 89% (166 urine samples from 33 rabbits tested). The specificity, positive predictive value, and efficiency could be increased to 97, 95, and 92%, respectively, if at least two positive test results were required for a true positive designation. The ELISA was sensitive and specific for the detection of Sap in urine specimens from rabbits with disseminated C. albicans infection, discriminated between colonization and invasive disease, reflected disease progression and severity, and has the potential to be a noninvasive means to diagnose disseminated candidiasis.

Resolution of discrepant results for Candida species identification by using DNA probes.

Candida species bloodstream isolates were collected from institutions participating in an active, population-based surveillance for candidemia. Species identifications were performed locally and then confirmed at the Centers for Disease Control and Prevention (CDC) by phenotype-based methods. Discrepancies in species identification between the referring institution and the CDC were noted for 43 of 935 isolates (4.6%). A DNA probe-based species identification system (PCR-enzyme immunoassay [EIA]) was then used to resolve these discrepancies. The PCR-EIA result was identical to the CDC phenotypic identification method for 98% of the isolates tested. The most frequently misidentified species was Candida glabrata (37% of all discrepant identifications). Such misidentifications could lead to the administration of inappropriate therapy given the propensity of C. glabrata to develop resistance to azole antifungal drugs.

Rapid differentiation of Aspergillus species from other medically important opportunistic molds and yeasts by PCR-enzyme immunoassay.

We developed a PCR-based assay to differentiate medically important species of Aspergillus from one another and from other opportunistic molds and yeasts by employing universal, fungus-specific primers and DNA probes in an enzyme immunoassay format (PCR-EIA). Oligonucleotide probes, directed to the internal transcribed spacer 2 region of ribosomal DNA from Aspergillus flavus, Aspergillus fumigatus, Aspergillus nidulans, Aspergillus niger, Aspergillus terreus, Aspergillus ustus, and Aspergillus versicolor, differentiated 41 isolates (3 to 9 each of the respective species; P < 0.001) in a PCR-EIA detection matrix and gave no false-positive reactions with 33 species of Acremonium, Exophiala, Candida, Fusarium, Mucor, Paecilomyces, Penicillium, Rhizopus, Scedosporium, Sporothrix, or other aspergilli tested. A single DNA probe to detect all seven of the most medically important Aspergillus species (A. flavus, A. fumigatus, A. nidulans, A. niger, A. terreus, A. ustus, and A. versicolor) was also designed. Identification of Aspergillus species was accomplished within a single day by the PCR-EIA, and as little as 0.5 pg of fungal DNA could be detected by this system. In addition, fungal DNA extracted from tissues of experimentally infected rabbits was successfully amplified and identified using the PCR-EIA system. This method is simple, rapid, and sensitive for the identification of medically important Aspergillus species and for their differentiation from other opportunistic fungi.