Mistaken Identity: Neosartorya pseudofischeri and Its Anamorph Masquerading as Aspergillus fumigatus
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微生物临床杂志 2005年第12期
Program in Infectious Diseases, Fred Hutchinson Cancer Research Center, Seattle, Washington
Centers for Disease Control and Prevention, Atlanta, Georgia
City of Hope Medical Center, Duarte, California
Fungus Testing Laboratory, University of Texas Health Science Center at San Antonio, San Antonio, Texas
Departments of Medicine and Microbiology, University of Washington, Seattle, Washington
ABSTRACT
Invasive fungal infections caused by Neosartorya pseudofischeri S. W. Peterson [anamorph Aspergillus thermomutatus (Paden) S. W. Peterson] are extremely rare. Phenotypically, the anamorphic state of N. pseudofischeri resembles Aspergillus fumigatus, the predominant agent of invasive aspergillosis in immunocompromised hosts. We report the recovery of three clinical isolates of N. pseudofischeri, all initially misidentified by morphological characteristics as A. fumigatus. All three isolates were correctly identified by sequencing portions of the -tubulin and the rodlet A genes. Only one of the three isolates produced the confirmatory fruiting bodies and was thus classified as N. pseudofischeri; the other isolates did not produce asci and were therefore identified as A. thermomutatus. All three isolates had higher MICs to voriconazole in vitro compared to A. fumigatus Af293. This report emphasizes that phenotypic identification of filamentous fungi may not identify morphologically similar, but genetically distinct, members of the genus Aspergillus section Fumigati. Accurate identification of these organisms may be clinically meaningful, given their potential differences in antifungal susceptibilities.
INTRODUCTION
Aspergillus fumigatus, the principal etiological agent of invasive aspergillosis, belongs to the genus Aspergillus section Fumigati and is identified in the laboratory predominantly by morphological features. Neosartorya fischeri and Neosartorya pseudofischeri also belong to section Fumigati, and their asexual (conidial) state closely overlaps that of A. fumigatus; thus, these two fungi appear morphologically very similar to A. fumigatus. Despite phenotypic similarity with A. fumigatus, N. fischeri and N. pseudofischeri have seldom been reported as etiologic agents of human aspergillosis. Phenotypically, Neosartorya fischeri (Wehmer, Malloch and Cain 1972) can be differentiated from the closely related N. pseudofischeri only by electron microscope analysis of the ascospore structure.
To date there are only seven reported cases in which N. pseudofischeri has been recovered from invasive fungal infections (Table 1). The anamorphic (asexual) form of N. pseudofischeri, Aspergillus thermomutatus, grows as whitish, fast-growing, slowly sporulating colonies (producing conidiophores with conidia [sporulating] only after prolonged incubation on laboratory medium). Recently, several slowly sporulating Aspergillus isolates have been identified as members of a new species, Aspergillus lentulus (sp. nov. S. A. Balajee and K. A. Marr [1]). As part of a screening study to identify other A. lentulus isolates among culture collections in the United States, we recovered three poorly sporulating isolates that were phenotypically identified as A. fumigatus but were not A. lentulus by sequence typing of -tubulin (benA) and rodlet A (rodA) gene regions. Data presented within demonstrate that these isolates are N. pseudofischeri and its anamorph, A. thermomutatus.
MATERIALS AND METHODS
Isolates. Isolate FH274 was received from the Fungus Reference and Molecular Subtyping Unit at the Centers for Disease Control and Prevention (CDC) and was originally recovered from a patient 3 months after receipt of hematopoietic stem cell transplantation. Biopsy of a right ear wound showed branching septate hyphae by KOH stain and grew what was first identified by the local laboratory as Aspergillus versicolor. The patient was treated successfully with amphotericin B lipid complex (ABLC), followed by voriconazole (VRZ) and caspofungin (CAS); the patient died 1 month later due to progressive leukemia. This isolate was sent to the CDC, where the fungus was reidentified by morphology as A. fumigatus. At that time, no asci or ascopores were found after incubation on several media for 4 weeks. Isolates FH240 and FH242 were originally obtained from the sputum of cystic fibrosis patients in Montana and Texas, respectively. Both isolates were initially identified as A. fumigatus by referring institutions submitting the isolates to the Fungus Testing Laboratory, University of Texas Health Science Center at San Antonio (UT), for antifungal susceptibility testing. Reidentification was not performed by UT. Aspergillus fumigatus isolate Af293 was provided by David Denning and was confirmed to be A. fumigatus Fresenius by the National Collection of Pathogenic Fungi (NCPF 7367) at the Mycology Reference Laboratory, Bristol, United Kingdom, and by the Centraal Bureau voor Schimmelcultures (CBS 101355), Baarn, The Netherlands (11).
Media and antifungals. Potato dextrose agar (PDA; Becton Dickinson, Sparks, MD), Czapek-dox (CZD; Becton Dickinson, Sparks, MD) supplemented with 20% dextrose, malt extract agar (MEA; Becton Dickinson, Sparks, MD), and Sabouraud dextrose agar (SDA; Becton Dickinson, Sparks, MD) were used in the study. The antifungal agents amphotericin B (AMB; Bristol-Myers Squibb Pharmaceutical Research Institute, Wallingford, CT), itraconazole (ITZ; Ortho Biotech, Bridgewater, NJ), and VRZ (Pfizer Pharmaceuticals, New York, NY) were dissolved in dimethyl sulfoxide, and CAS (Merck & Co. Inc., Rahway, NJ) was dissolved in distilled water. Further dilutions were made in RPMI 1640 with L-glutamine, without bicarbonate, buffered with 0.165 morpholinepropanesulfonic acid to pH 7.0 (RPMI; Sigma Chemical Co., St. Louis, MO), as outlined in CLSI (formerly NCCLS) document M38A (9).
Molecular typing. Genomic DNA was extracted from hyphal mats of isolates Af293, FH240, FH242, and FH274 grown in SDB for 3 days as previously described (1). In brief, hyphal cells were treated with lyticase (10 U/μl; Sigma Chemical Co., St. Louis, MO) for 1 h at 37°C and then incubated in proteinase K (10 μg/ml; Sigma) and 0.5% sodium dodecyl sulfate (Sigma) for 2 h at 60°C. This suspension was then subjected to three cycles of freeze-thaw in liquid nitrogen alternating with vortexing 1 min with 0.2 g sterile glass beads (Sigma). Genomic DNA was isolated with the DNeasy tissue kit (69504; QIAGEN, Hilden, Germany) according to the manufacturer's instructions.
PCR primers were designed to amplify -tubulin (benA-F, 5'-AATTGGTGCCGCTTTCTGG-3', and R, 5'-AGTTGTCGGGACGGAATAG-3') and rodlet A (rodA-F, 5'-GCTGGCAATGGTGTTGGCAA-3', and R, 5'-AGGGCAATGCAAGGAAGACC-3') regions as previously described (1). PCR amplification was performed with 2 to 4 μl of genomic DNA as template in a total reaction volume of 50 μl consisting of PCR buffer (20 mM Tris-HCl, pH 8.4, 50 mM KCl), 0.2 mM (each) dATP, dGTP, dCTP, and dTTP, 1.2 mM MgSO4, 0.2 pmol (each) primer, and 1 U of Pfx polymerase (Invitrogen-BRL Life Technologies, Carlsbad, CA) and 1x Pfx enhancer (Invitrogen). Thirty cycles of amplification were performed in a GeneAmp PCR system 9700 thermocycler (PE-Applied Biosystems) after initial denaturation of DNA at 94°C for 3 min. Thirty cycles consisted of a denaturation step at 94°C for 15 seconds, an annealing step at 55°C for 30 seconds, and an extension step at 68°C for 30 seconds, with a final extension at 68°C for 3 min following the last cycle.
Amplicons were purified using the QIAquick PCR purification kit (catalog no. 28104) and directly sequenced on a Perkin-Elmer/ABI model 373 DNA sequencer with protocols supplied by the manufacturer. The resultant nucleotide sequences were edited using the Sequencher program, and each set of homologous gene sequences was aligned using ClustalW (17). Sequences were compared to the other available sequences in GenBank using the BLAST program of the National Center for Biotechnology Information.
Phenotypic analysis. FH274, FH240, and FH242 were grown on PDA, MEA, and CZD at two temperatures, 25°C and 37°C, for 7 to 21 days with periodic microscopic examination. When asci were produced, specimens prepared with lactophenol cotton blue were examined by differential interference contrast microscopy (magnification, 100x) and images were photographed.
Antifungal susceptibility testing. Susceptibilities of the isolates to AMB, ITZ, VRZ, and CAS were assayed by the CLSI M38A broth microdilution method, as previously published (1). Isolate FH240 was grown on CZD at 37°C to maximize conidial harvest, and the conidia from all three isolates tested were counted with a hemocytometer and adjusted to a concentration of 106 CFU/ml. As per the CLSI recommendations (9), MICs for ITZ, VRZ, and AMB were defined as the lowest concentration of the drug that resulted in 100% growth reduction when compared to the drug-free control. For CAS, the minimal effective concentration (MEC) was defined as the minimum concentration of drug that produced morphological alterations, such as abnormal hyphal growth with highly branched tips, swollen germ tubes, and distended balloon-like hyphae, when observed under the light microscope (7). Susceptibilities were determined by duplicate measures in three different experiments. MICs for FH240 and FH242 were confirmed at the Fungus Testing Laboratory, Department of Pathology, University of Texas Health Science Center at San Antonio.
RESULTS
A large number of slowly sporulating isolates of A. fumigatus from various culture collections in the United States were screened for the presence of A. lentulus. As part of this project, we sequenced the benA and the rodA genes of the slowly sporulating isolates FH274, FH240, and FH242. Results showed that FH274 and FH240 were 100% homologous and FH242 was 99% homologous to the sequence of N. pseudofischeri (GenBank accession no. AF057325). Similarly, rodA sequences of FH274, FH240, and FH242 were 99% homologous to the sequence of N. pseudofischeri (GenBank accession no. AF057345). Sequences of all three isolates were only 90% homologous to the benA sequence and 88% homologous to the rodA sequence of A. fumigatus isolate Af293 (data not shown), and the sequences were 91% homologous to the benA sequence and 89% homologous to the rodA sequence of A. lentulus isolate FH5 (data not shown).
All three isolates were initially identified as A. fumigatus by the CDC and UT by phenotypic characteristics. Since molecular data suggested that these isolates were not A. fumigatus but N. pseudofischeri, we sought to corroborate the molecular findings by phenotype analyses. All isolates were grown on PDA, CZD, and MEA at 25°C and 37°C. At the higher temperature, the isolates were fast growing on all three media and appeared as whitish velvety colonies with sparse conidiation on CZD and PDA and without conidiation on MEA. Microscopic examination revealed scant conidiophores that were smooth and hyaline, terminating in subglobose vesicles with a single series of ampulliform phialides bearing dull green globose conidia (Fig. 1a). At 25°C, FH240 and FH242 appeared as fast-growing colonies (on all three media) with absolutely no conidiation. However, FH274 appeared granular on PDA, with ascogonial initials produced within 7 days. By the 10th day, ascomata were produced and eight-spored asci were seen microscopically (Fig. 1b). The ascospores were hyaline, one-celled, and lenticular with two closely oppressed equatorial crests extending beyond the spore body (Fig. 1c). Hence, isolate FH274 was identified as N. pseudofischeri. Both FH240 and FH242 failed to produce the confirmatory asci and ascospores in PDA, CZD, and MEA at both 25°C and 37°C and were classified as A. thermomutatus. All three isolates were able to grow profusely at 45°C and exhibited limited growth at 48°C.
It is being increasingly appreciated that members of the Aspergillus species may have variable antifungal susceptibility patterns, a case in point being A. terreus, which has high MICs of AMB in vitro (18) and is associated with high rates of treatment failure (13, 18). Hence, we evaluated the antifungal susceptibilities of the three isolates and found that MICs were variable, although each of the isolates appeared to have relatively high MICs of the azoles, VRZ and ITZ (Table 2). One isolate (FH240) had a relatively high MIC of AMB; all CAS MECs were low. Aspergillus fumigatus Af293 was susceptible to all the drugs tested.
DISCUSSION
The present study brings into focus the limitations of phenotypic methods of identification of filamentous fungi, since all three N. pseudofischeri/A. thermomutatus isolates were initially misidentified as A. fumigatus by morphological typing. Limitations of traditional fungal species characterization by phenotype are being increasingly documented, and molecular phylogenetics-based methods appear to be a more reliable and robust alternative to discriminate fungal species (3, 6, 12, 16). For example, in the human pathogenic fungus Histoplasma capsulatum, eight genetically isolated groups have been distinguished by molecular methods with biologically relevant features, such as geographic distribution and pathogenicity characteristic of each group (5). Similarly, cryptic species were revealed in several medically important fungi, including Coccidioides immitis, Candida albicans, Cryptococcus neoformans, and A. flavus (15), and recently isolates belonging to the morphologically indistinguishable Aspergillus niger aggregate have been divided into two species, A. niger and A. tubingensis, based on molecular differences (12). We recently used multilocus sequence typing to identify a new species in the section Fumigati, A. lentulus (1). Thus, it appears that traditional phenotypic methods alone may be inadequate for fungal speciation, and an integration of molecular speciation methods with available classical techniques appears to be warranted to accurately characterize fungi, especially ascomycetes.
Consistent with the ambiguity of morphological methods of fungal characterization, two of three isolates (identified as N. pseudofischeri by molecular methods) did not produce any ascoma. Although the anamorphic states of N. pseudofischeri and A. fumigatus have overlapping phenotypic characteristics, such as conidiophore morphology and thermotolerance, it is thought that N. pseudofischeri can be clearly differentiated from A. fumigatus by the fact that N. pseudofischeri produces ascoma on MEA or CZD, whereas A. fumigatus does not (10). Some fungal isolates may not produce fruiting bodies in the laboratory, possibly due to repeated subculturing on rich medium. Reliance on such unstable phenotypic characteristics for speciation of clinical isolates may lead to misidentification of the etiological agent.
In contrast to A. fumigatus, all three isolates of N. pseudofischeri/A. thermomutatus recovered in the current study had elevated MICs of VRZ. Low susceptibility of these isolates to VRZ is an important observation and if validated in vivo may have significant implications in clinical practice. All three isolates were extremely susceptible to the echinocandin CAS, moderately susceptible to ITZ, and had variable MICs of AMB. Whether differences in susceptibility are inherent to the species or are acquired traits is not clear but, previously, a clinical isolate of N. pseudofischeri was shown to be very sensitive to AMB, ITZ, ketoconazole, and flucytosine (14).
In the present study, N. pseudofischeri was not isolated as a cause of invasive pulmonary aspergillosis but was recovered from sputum of cystic fibrosis patients and as a cause of invasive otitis. However, N. pseudofischeri clearly is able to cause infection in the right environment, as can be seen from its isolation in the past from cases of peritonitis, invasive pulmonary aspergillosis, and graft-related endocarditis (Table 1). Our investigation has also uncovered the possibility that the difficulty in distinguishing A. fumigatus from N. pseudofischeri may underestimate the frequency of disease caused by N. pseudofischeri. Detailed molecular screening and typing studies may be needed to assess the true rates of recovery of N. pseudofischeri as a cause of human disease. Correct identification of N. pseudofischeri may be important if in vitro susceptibility results predict clinical outcomes in in vivo models of infection.
ACKNOWLEDGMENTS
We thank S. W. Peterson, U.S. Department of Agriculture, for helpful advice on N. pseudofischeri classification.
Financial support for this study was provided by an R21 grant (AI 055928) from the National Institutes of Health.
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Centers for Disease Control and Prevention, Atlanta, Georgia
City of Hope Medical Center, Duarte, California
Fungus Testing Laboratory, University of Texas Health Science Center at San Antonio, San Antonio, Texas
Departments of Medicine and Microbiology, University of Washington, Seattle, Washington
ABSTRACT
Invasive fungal infections caused by Neosartorya pseudofischeri S. W. Peterson [anamorph Aspergillus thermomutatus (Paden) S. W. Peterson] are extremely rare. Phenotypically, the anamorphic state of N. pseudofischeri resembles Aspergillus fumigatus, the predominant agent of invasive aspergillosis in immunocompromised hosts. We report the recovery of three clinical isolates of N. pseudofischeri, all initially misidentified by morphological characteristics as A. fumigatus. All three isolates were correctly identified by sequencing portions of the -tubulin and the rodlet A genes. Only one of the three isolates produced the confirmatory fruiting bodies and was thus classified as N. pseudofischeri; the other isolates did not produce asci and were therefore identified as A. thermomutatus. All three isolates had higher MICs to voriconazole in vitro compared to A. fumigatus Af293. This report emphasizes that phenotypic identification of filamentous fungi may not identify morphologically similar, but genetically distinct, members of the genus Aspergillus section Fumigati. Accurate identification of these organisms may be clinically meaningful, given their potential differences in antifungal susceptibilities.
INTRODUCTION
Aspergillus fumigatus, the principal etiological agent of invasive aspergillosis, belongs to the genus Aspergillus section Fumigati and is identified in the laboratory predominantly by morphological features. Neosartorya fischeri and Neosartorya pseudofischeri also belong to section Fumigati, and their asexual (conidial) state closely overlaps that of A. fumigatus; thus, these two fungi appear morphologically very similar to A. fumigatus. Despite phenotypic similarity with A. fumigatus, N. fischeri and N. pseudofischeri have seldom been reported as etiologic agents of human aspergillosis. Phenotypically, Neosartorya fischeri (Wehmer, Malloch and Cain 1972) can be differentiated from the closely related N. pseudofischeri only by electron microscope analysis of the ascospore structure.
To date there are only seven reported cases in which N. pseudofischeri has been recovered from invasive fungal infections (Table 1). The anamorphic (asexual) form of N. pseudofischeri, Aspergillus thermomutatus, grows as whitish, fast-growing, slowly sporulating colonies (producing conidiophores with conidia [sporulating] only after prolonged incubation on laboratory medium). Recently, several slowly sporulating Aspergillus isolates have been identified as members of a new species, Aspergillus lentulus (sp. nov. S. A. Balajee and K. A. Marr [1]). As part of a screening study to identify other A. lentulus isolates among culture collections in the United States, we recovered three poorly sporulating isolates that were phenotypically identified as A. fumigatus but were not A. lentulus by sequence typing of -tubulin (benA) and rodlet A (rodA) gene regions. Data presented within demonstrate that these isolates are N. pseudofischeri and its anamorph, A. thermomutatus.
MATERIALS AND METHODS
Isolates. Isolate FH274 was received from the Fungus Reference and Molecular Subtyping Unit at the Centers for Disease Control and Prevention (CDC) and was originally recovered from a patient 3 months after receipt of hematopoietic stem cell transplantation. Biopsy of a right ear wound showed branching septate hyphae by KOH stain and grew what was first identified by the local laboratory as Aspergillus versicolor. The patient was treated successfully with amphotericin B lipid complex (ABLC), followed by voriconazole (VRZ) and caspofungin (CAS); the patient died 1 month later due to progressive leukemia. This isolate was sent to the CDC, where the fungus was reidentified by morphology as A. fumigatus. At that time, no asci or ascopores were found after incubation on several media for 4 weeks. Isolates FH240 and FH242 were originally obtained from the sputum of cystic fibrosis patients in Montana and Texas, respectively. Both isolates were initially identified as A. fumigatus by referring institutions submitting the isolates to the Fungus Testing Laboratory, University of Texas Health Science Center at San Antonio (UT), for antifungal susceptibility testing. Reidentification was not performed by UT. Aspergillus fumigatus isolate Af293 was provided by David Denning and was confirmed to be A. fumigatus Fresenius by the National Collection of Pathogenic Fungi (NCPF 7367) at the Mycology Reference Laboratory, Bristol, United Kingdom, and by the Centraal Bureau voor Schimmelcultures (CBS 101355), Baarn, The Netherlands (11).
Media and antifungals. Potato dextrose agar (PDA; Becton Dickinson, Sparks, MD), Czapek-dox (CZD; Becton Dickinson, Sparks, MD) supplemented with 20% dextrose, malt extract agar (MEA; Becton Dickinson, Sparks, MD), and Sabouraud dextrose agar (SDA; Becton Dickinson, Sparks, MD) were used in the study. The antifungal agents amphotericin B (AMB; Bristol-Myers Squibb Pharmaceutical Research Institute, Wallingford, CT), itraconazole (ITZ; Ortho Biotech, Bridgewater, NJ), and VRZ (Pfizer Pharmaceuticals, New York, NY) were dissolved in dimethyl sulfoxide, and CAS (Merck & Co. Inc., Rahway, NJ) was dissolved in distilled water. Further dilutions were made in RPMI 1640 with L-glutamine, without bicarbonate, buffered with 0.165 morpholinepropanesulfonic acid to pH 7.0 (RPMI; Sigma Chemical Co., St. Louis, MO), as outlined in CLSI (formerly NCCLS) document M38A (9).
Molecular typing. Genomic DNA was extracted from hyphal mats of isolates Af293, FH240, FH242, and FH274 grown in SDB for 3 days as previously described (1). In brief, hyphal cells were treated with lyticase (10 U/μl; Sigma Chemical Co., St. Louis, MO) for 1 h at 37°C and then incubated in proteinase K (10 μg/ml; Sigma) and 0.5% sodium dodecyl sulfate (Sigma) for 2 h at 60°C. This suspension was then subjected to three cycles of freeze-thaw in liquid nitrogen alternating with vortexing 1 min with 0.2 g sterile glass beads (Sigma). Genomic DNA was isolated with the DNeasy tissue kit (69504; QIAGEN, Hilden, Germany) according to the manufacturer's instructions.
PCR primers were designed to amplify -tubulin (benA-F, 5'-AATTGGTGCCGCTTTCTGG-3', and R, 5'-AGTTGTCGGGACGGAATAG-3') and rodlet A (rodA-F, 5'-GCTGGCAATGGTGTTGGCAA-3', and R, 5'-AGGGCAATGCAAGGAAGACC-3') regions as previously described (1). PCR amplification was performed with 2 to 4 μl of genomic DNA as template in a total reaction volume of 50 μl consisting of PCR buffer (20 mM Tris-HCl, pH 8.4, 50 mM KCl), 0.2 mM (each) dATP, dGTP, dCTP, and dTTP, 1.2 mM MgSO4, 0.2 pmol (each) primer, and 1 U of Pfx polymerase (Invitrogen-BRL Life Technologies, Carlsbad, CA) and 1x Pfx enhancer (Invitrogen). Thirty cycles of amplification were performed in a GeneAmp PCR system 9700 thermocycler (PE-Applied Biosystems) after initial denaturation of DNA at 94°C for 3 min. Thirty cycles consisted of a denaturation step at 94°C for 15 seconds, an annealing step at 55°C for 30 seconds, and an extension step at 68°C for 30 seconds, with a final extension at 68°C for 3 min following the last cycle.
Amplicons were purified using the QIAquick PCR purification kit (catalog no. 28104) and directly sequenced on a Perkin-Elmer/ABI model 373 DNA sequencer with protocols supplied by the manufacturer. The resultant nucleotide sequences were edited using the Sequencher program, and each set of homologous gene sequences was aligned using ClustalW (17). Sequences were compared to the other available sequences in GenBank using the BLAST program of the National Center for Biotechnology Information.
Phenotypic analysis. FH274, FH240, and FH242 were grown on PDA, MEA, and CZD at two temperatures, 25°C and 37°C, for 7 to 21 days with periodic microscopic examination. When asci were produced, specimens prepared with lactophenol cotton blue were examined by differential interference contrast microscopy (magnification, 100x) and images were photographed.
Antifungal susceptibility testing. Susceptibilities of the isolates to AMB, ITZ, VRZ, and CAS were assayed by the CLSI M38A broth microdilution method, as previously published (1). Isolate FH240 was grown on CZD at 37°C to maximize conidial harvest, and the conidia from all three isolates tested were counted with a hemocytometer and adjusted to a concentration of 106 CFU/ml. As per the CLSI recommendations (9), MICs for ITZ, VRZ, and AMB were defined as the lowest concentration of the drug that resulted in 100% growth reduction when compared to the drug-free control. For CAS, the minimal effective concentration (MEC) was defined as the minimum concentration of drug that produced morphological alterations, such as abnormal hyphal growth with highly branched tips, swollen germ tubes, and distended balloon-like hyphae, when observed under the light microscope (7). Susceptibilities were determined by duplicate measures in three different experiments. MICs for FH240 and FH242 were confirmed at the Fungus Testing Laboratory, Department of Pathology, University of Texas Health Science Center at San Antonio.
RESULTS
A large number of slowly sporulating isolates of A. fumigatus from various culture collections in the United States were screened for the presence of A. lentulus. As part of this project, we sequenced the benA and the rodA genes of the slowly sporulating isolates FH274, FH240, and FH242. Results showed that FH274 and FH240 were 100% homologous and FH242 was 99% homologous to the sequence of N. pseudofischeri (GenBank accession no. AF057325). Similarly, rodA sequences of FH274, FH240, and FH242 were 99% homologous to the sequence of N. pseudofischeri (GenBank accession no. AF057345). Sequences of all three isolates were only 90% homologous to the benA sequence and 88% homologous to the rodA sequence of A. fumigatus isolate Af293 (data not shown), and the sequences were 91% homologous to the benA sequence and 89% homologous to the rodA sequence of A. lentulus isolate FH5 (data not shown).
All three isolates were initially identified as A. fumigatus by the CDC and UT by phenotypic characteristics. Since molecular data suggested that these isolates were not A. fumigatus but N. pseudofischeri, we sought to corroborate the molecular findings by phenotype analyses. All isolates were grown on PDA, CZD, and MEA at 25°C and 37°C. At the higher temperature, the isolates were fast growing on all three media and appeared as whitish velvety colonies with sparse conidiation on CZD and PDA and without conidiation on MEA. Microscopic examination revealed scant conidiophores that were smooth and hyaline, terminating in subglobose vesicles with a single series of ampulliform phialides bearing dull green globose conidia (Fig. 1a). At 25°C, FH240 and FH242 appeared as fast-growing colonies (on all three media) with absolutely no conidiation. However, FH274 appeared granular on PDA, with ascogonial initials produced within 7 days. By the 10th day, ascomata were produced and eight-spored asci were seen microscopically (Fig. 1b). The ascospores were hyaline, one-celled, and lenticular with two closely oppressed equatorial crests extending beyond the spore body (Fig. 1c). Hence, isolate FH274 was identified as N. pseudofischeri. Both FH240 and FH242 failed to produce the confirmatory asci and ascospores in PDA, CZD, and MEA at both 25°C and 37°C and were classified as A. thermomutatus. All three isolates were able to grow profusely at 45°C and exhibited limited growth at 48°C.
It is being increasingly appreciated that members of the Aspergillus species may have variable antifungal susceptibility patterns, a case in point being A. terreus, which has high MICs of AMB in vitro (18) and is associated with high rates of treatment failure (13, 18). Hence, we evaluated the antifungal susceptibilities of the three isolates and found that MICs were variable, although each of the isolates appeared to have relatively high MICs of the azoles, VRZ and ITZ (Table 2). One isolate (FH240) had a relatively high MIC of AMB; all CAS MECs were low. Aspergillus fumigatus Af293 was susceptible to all the drugs tested.
DISCUSSION
The present study brings into focus the limitations of phenotypic methods of identification of filamentous fungi, since all three N. pseudofischeri/A. thermomutatus isolates were initially misidentified as A. fumigatus by morphological typing. Limitations of traditional fungal species characterization by phenotype are being increasingly documented, and molecular phylogenetics-based methods appear to be a more reliable and robust alternative to discriminate fungal species (3, 6, 12, 16). For example, in the human pathogenic fungus Histoplasma capsulatum, eight genetically isolated groups have been distinguished by molecular methods with biologically relevant features, such as geographic distribution and pathogenicity characteristic of each group (5). Similarly, cryptic species were revealed in several medically important fungi, including Coccidioides immitis, Candida albicans, Cryptococcus neoformans, and A. flavus (15), and recently isolates belonging to the morphologically indistinguishable Aspergillus niger aggregate have been divided into two species, A. niger and A. tubingensis, based on molecular differences (12). We recently used multilocus sequence typing to identify a new species in the section Fumigati, A. lentulus (1). Thus, it appears that traditional phenotypic methods alone may be inadequate for fungal speciation, and an integration of molecular speciation methods with available classical techniques appears to be warranted to accurately characterize fungi, especially ascomycetes.
Consistent with the ambiguity of morphological methods of fungal characterization, two of three isolates (identified as N. pseudofischeri by molecular methods) did not produce any ascoma. Although the anamorphic states of N. pseudofischeri and A. fumigatus have overlapping phenotypic characteristics, such as conidiophore morphology and thermotolerance, it is thought that N. pseudofischeri can be clearly differentiated from A. fumigatus by the fact that N. pseudofischeri produces ascoma on MEA or CZD, whereas A. fumigatus does not (10). Some fungal isolates may not produce fruiting bodies in the laboratory, possibly due to repeated subculturing on rich medium. Reliance on such unstable phenotypic characteristics for speciation of clinical isolates may lead to misidentification of the etiological agent.
In contrast to A. fumigatus, all three isolates of N. pseudofischeri/A. thermomutatus recovered in the current study had elevated MICs of VRZ. Low susceptibility of these isolates to VRZ is an important observation and if validated in vivo may have significant implications in clinical practice. All three isolates were extremely susceptible to the echinocandin CAS, moderately susceptible to ITZ, and had variable MICs of AMB. Whether differences in susceptibility are inherent to the species or are acquired traits is not clear but, previously, a clinical isolate of N. pseudofischeri was shown to be very sensitive to AMB, ITZ, ketoconazole, and flucytosine (14).
In the present study, N. pseudofischeri was not isolated as a cause of invasive pulmonary aspergillosis but was recovered from sputum of cystic fibrosis patients and as a cause of invasive otitis. However, N. pseudofischeri clearly is able to cause infection in the right environment, as can be seen from its isolation in the past from cases of peritonitis, invasive pulmonary aspergillosis, and graft-related endocarditis (Table 1). Our investigation has also uncovered the possibility that the difficulty in distinguishing A. fumigatus from N. pseudofischeri may underestimate the frequency of disease caused by N. pseudofischeri. Detailed molecular screening and typing studies may be needed to assess the true rates of recovery of N. pseudofischeri as a cause of human disease. Correct identification of N. pseudofischeri may be important if in vitro susceptibility results predict clinical outcomes in in vivo models of infection.
ACKNOWLEDGMENTS
We thank S. W. Peterson, U.S. Department of Agriculture, for helpful advice on N. pseudofischeri classification.
Financial support for this study was provided by an R21 grant (AI 055928) from the National Institutes of Health.
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