Fungal metabolite gliotoxin blocks mast cell activation by a calcium- and superoxide-dependent mechanism: implications for immunosuppressive activities

Fungal secondary metabolites such as gliotoxin, an epipolythiodioxopiperazine toxin produced by pathogenic fungi like Candida and Aspergillus, possess immunosuppressive activities and have been thought to contribute to pathology of fungal infections in animals and humans. Since recent studies show that mast cell plays a crucial role in the front of host defense, we examined whether fungal secondary metabolites affected mast cell activation. We found that gliotoxin had suppressive effects on FcepsilonRI-dependent or -independent mast cell activation, including degranulation, leukotriene C4 secretion, and TNF-alpha and IL-13 production. Gliotoxin also suppressed intracellular Ca2+ rise through store-operated Ca2+ channels with a minimal effect on depletion of internal Ca2+ stores. Finally, gliotoxin induced intracellular production of superoxide possibly through a thiol redox cycling, which appeared to mediate suppressive effects on mast cell activation. These findings suggest that suppression of mast cell activation might contribute to the establishment of infections with gliotoxin-producing fungi.     

Evaluation of Luminex xTAG fungal analyte-specific reagents for rapid identification of clinically relevant fungi

Invasive fungal infections (IFI) remain a serious threat to immunocompromised hosts. Current diagnostic methods, including fungal culture and antigen detection, are slow and often lack specificity. Rapid diagnostic tools with increased sensitivity and specificity could improve the care of patients with IFI. Recently, Luminex Molecular Diagnostics (Toronto, Canada) developed 23 analyte-specific reagents (ASRs) for the detection of the most common clinically relevant fungi. This study's objective was to evaluate the sensitivity and specificity of a subset of these ASRs for fungal isolates and clinical specimens. Previously characterized fungal and bacterial isolates (n = 110), blood culture specimens (n = 34), and respiratory specimens (n = 44) were tested using either a Candida 7-plex panel (Candida albicans, Candida glabrata, Candida tropicalis, Candida parapsilosis, Candida lusitaniae, Candida guilliermondii, and Candida krusei) or a mold 11-plex panel (Aspergillus fumigatus, Aspergillus flavus, Aspergillus niger, Aspergillus terreus, Scedosporium prolificans, Scedosporium apiospermum, Fusarium oxysporum/Fusarium solani, Rhizopus arrhizus, Rhizopus microsporus, Mucor indicus, and Cunninghamella bertholletiae). The Candida 7-plex panel correctly identified all Candida isolates as confirmed by fungal culture and biochemical tests, for a sensitivity and specificity of 100%. The mold 11-plex panel correctly identified all mold isolates tested except for A. niger. Fungal isolates of Rhizopus and Mucor species were not detected, either, although they could represent species other than those targeted by the ASRs. Further evaluation will be necessary to confirm the sensitivities of some of the mold ASRs. Implementation of these ASRs will allow same-day detection of fungal DNA in clinical specimens.     

Temperature and aflatoxin production by Aspergillus flavus and A. parasiticus strains from Nigerian groundnuts

The ability of four strains of Aspergillus parasiticus (IMI 301,001) and two strains of Aspergillus flavus (IMI 300,998) to produce aflatoxins on Nigerian groundnuts under varying conditions of temperature was studied. While all the A. parasiticus strains produced the four major aflatoxins (B1, B2, G1, G2), only aflatoxin B1, and B2 were produced by the A. flavus strains used. The optimum temperature for aflatoxin production by both the fungal species was 30 degrees C with no toxin production at 10 degrees C.     

Simultaneous detection and identification of Aspergillus and mucorales species in tissues collected from patients with fungal rhinosinusitis

Rapid detection and differentiation of Aspergillus and Mucorales species in fungal rhinosinusitis diagnosis are desirable, since the clinical management and prognosis associated with the two taxa are fundamentally different. We describe an assay based on a combination of broad-range PCR amplification and reverse line blot hybridization (PCR/RLB) to detect and differentiate the pathogens causing fungal rhinosinusitis, which include five Aspergillus species (A. fumigatus, A. flavus, A. niger, A. terreus, and A. nidulans) and seven Mucorales species (Mucor heimalis, Mucor racemosus, Mucor cercinelloidea, Rhizopus arrhizus, Rhizopus microsporus, Rhizomucor pusillus, and Absidia corymbifera). The assay was validated with 98 well-characterized clinical isolates and 41 clinical tissue specimens. PCR/RLB showed high sensitivity and specificity, with 100% correct identifications of 98 clinical isolates and no cross-hybridization between the species-specific probes. Results for five control isolates, Candida albicans, Fusarium solani, Scedosporium apiospermum, Penicillium marneffei, and Exophiala verrucosa, were negative as judged by PCR/RLB. The analytical sensitivity of PCR/RLB was found to be 1.8 × 10(-3) ng/μl by 10-fold serial dilution of Aspergillus genomic DNA. The assay identified 35 of 41 (85.4%) clinical specimens, exhibiting a higher sensitivity than fungal culture (22 of 41; 53.7%) and direct sequencing (18 of 41; 43.9%). PCR/RLB similarly showed high specificity, with correct identification 16 of 18 specimens detected by internal transcribed spacer (ITS) sequencing and 16 of 22 detected by fungal culture, but it also has the additional advantage of being able to detect mixed infection in a single clinical specimen. The PCR/RLB assay thus provides a rapid and reliable option for laboratory diagnosis of fungal rhinosinusitis.     

黏蛋白5B在鉴别真菌性鼻窦炎组织中曲霉菌和毛霉菌的意义

目的 探讨黏蛋白抗体在真菌性鼻窦炎活检组织中的曲霉菌和毛霉菌鉴别中的意义.方法 选取北京同仁医院病理科2001-2006年间66例真菌性鼻窦炎活检标本存档蜡块,其中真菌球29例,变应性真菌性鼻窦炎12例,慢性侵袭性真菌性鼻窦炎24例,急性侵袭性真菌性鼻窦炎1例;确认曲霉菌属44例(烟曲霉菌31例,黄曲霉菌7例,土曲霉菌6例),毛霉科真菌22例(毛霉菌14例,根霉菌8例).用黏蛋白(MUC)2、MUC5AC和MUC5B抗体以免疫组织化学方法标记真菌,并与过碘酸-雪夫(PAS)和Grocott环六亚甲基四胺银(GMS)特殊染色结果进行对比.结果 MUC5B免疫组织化学结果显示烟曲霉菌、黄曲霉菌和土曲霉菌总阳性率为90.9%,毛霉菌和根霉菌总阳性率为4.5%,二者差异有统计学意义(P＜0.001);MUC2和MUC5AC均为阴性;PAS和GMS染色上述真菌均为阳性.结论 MUC5B抗体可作为检测组织内真菌的有效指标,应用MUC5B抗体可以对真菌性鼻窦炎中曲霉菌属和毛霉科真菌进行鉴别.     

Toxicity and mutagenicity of anthraquinones from Aspergillus chevalieri.

More than 100 strains of the Aspergillus glaucus group were cultivated on synthetic media for 11 days at 28 degrees C. Organic extracts of fungal material were screened by thin-layer chromatography (TLC) for the mycotoxins aflatoxins B1,2 and G1,2, sterigmatocystin, ochratoxin A, gliotoxin, patulin, and xanthocillin X. None of these toxins were produced in detectable amounts under experimental conditions. Nevertheless, organic extracts exhibited high toxicity after intraperitoneal (i.p.) administration in mice. Aspergillus chevalieri strain ZT 8268 was selected for further investigation of its toxic metabolites. The main toxic action was attributed to the four anthraquinone derivatives, physicion, physcionanthrone B, physciondianthrone, and erythroglaucin, which were isolated and identified. No toxic effects were found after oral administration. Using the Salmonella/mammalian microsome test, mutagenic activity (frame-shift) was detected in strain TA 1537 in the presence of S-9 liver microsome preparation.     

Determination of MICs of aminocandin for Candida spp. and filamentous fungi

Candida and Aspergillus spp., as well as other filamentous molds, have increasingly been reported as the causes of severe invasive fungal infections. We evaluated the new echinocandin aminocandin (AMN) for its antifungal activities against a range of fungal pathogens by determination of the MICs for the organisms. The MICs of the comparator drugs amphotericin B, caspofungin, micafungin, and voriconazole were also determined. The MICs of AMN for 25 strains each of non-Candida albicans Candida spp. (including Candida parapsilosis, Candida krusei, Candida guilliermondii, and Candida tropicalis), Aspergillus fumigatus, Scedosporium spp., Fusarium spp., and zygomycetes (including Absidia, Mucor, and Rhizopus spp.) were determined by using the Clinical and Laboratory Standards Institute M27-A2 and M38-A methodologies for yeasts and filamentous molds, respectively. The MIC ranges of AMN for all yeasts were similar (0.03 to 4.0 microg/ml), while the MIC ranges of AMN for filamentous fungi were species specific. AMN demonstrated potent antifungal activity against A. fumigatus, limited activity against Scedosporium spp., and no activity against zygomycetes or Fusarium spp. Our data showed that AMN demonstrated potent antifungal activities against all of the yeasts and Aspergillus isolates tested, suggesting that AMN could be an important addition to our arsenal of antifungals for the treatment of invasive fungal disease.     

Airborne fungal spores at the Belgrad forest near the city of Istanbul (Turkey) in the year 2001 and their relation to allergic diseases

The aim of this study was to investigate the relationship between airborne fungal spores and allergic diseases; for this reason, the airborne fungal spores that were obtained from five different locations of Belgrad Forest were isolated, determined and studied quantitatively.Totally 120 samples were examined by using the Petri Plate Gravitational Method, and fungi obtained from these samples were isolated and 600 colonies were counted. By identification of these isolations, 13 genera (Mucor, Rhizopus, Absidia, Aspergillus, Penicillium, Trichoderma, Trichothecium, Stemphylium, Cladosporium, Alternaria, Ulocladium, Aureobasidium and Fusarium), 25 species and 10 different sterile fungi were determined. The identifications of these fungi were made according to their microscopic, macromorphological features and references.     

Comparison of six DNA extraction methods for recovery of fungal DNA as assessed by quantitative PCR

The detection of fungal pathogens in clinical samples by PCR requires the use of extraction methods that efficiently lyse fungal cells and recover DNA suitable for amplification. We used quantitative PCR assays to measure the recovery of DNA from two important fungal pathogens subjected to six DNA extraction methods. Aspergillus fumigatus conidia or Candida albicans yeast cells were added to bronchoalveolar lavage fluid and subjected to DNA extraction in order to assess the recovery of DNA from a defined number of fungal propagules. In order to simulate hyphal growth in tissue, Aspergillus fumigatus conidia were allowed to form mycelia in tissue culture media and then harvested for DNA extraction. Differences among the DNA yields from the six extraction methods were highly significant (P<0.0001) in each of the three experimental systems. An extraction method based on enzymatic lysis of fungal cell walls (yeast cell lysis plus the use of GNOME kits) produced high levels of fungal DNA with Candida albicans but low levels of fungal DNA with Aspergillus fumigatus conidia or hyphae. Extraction methods employing mechanical agitation with beads produced the highest yields with Aspergillus hyphae. The Master Pure yeast method produced high levels of DNA from C. albicans but only moderate yields from A. fumigatus. A reagent from one extraction method was contaminated with fungal DNA, including DNA from Aspergillus and Candida species. In conclusion, the six extraction methods produce markedly differing yields of fungal DNA and thus can significantly affect the results of fungal PCR assays. No single extraction method was optimal for all organisms.     

Species identification of Aspergillus, Fusarium and Mucorales with direct surface analysis by matrix-assisted laser desorption ionization time-of-flight mass spectrometry

Accurate species discrimination of filamentous fungi is essential, because some species have specific antifungal susceptibility patterns, and misidentification may result in inappropriate therapy. We evaluated matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) for species identification through direct surface analysis of the fungal culture. By use of culture collection strains representing 55 species of Aspergillus, Fusarium and Mucorales, a reference database was established for MALDI-TOF MS-based species identification according to the manufacturer's recommendations for microflex measurements and MALDI BioTyper 2.0 software. The profiles of young and mature colonies were analysed for each of the reference strains, and species-specific spectral fingerprints were obtained. To evaluate the database, 103 blind-coded fungal isolates collected in the routine clinical microbiology laboratory were tested. As a reference method for species designation, multilocus sequencing was used. Eighty-five isolates were unequivocally identified to the species level (≥99% sequence similarity); 18 isolates producing ambiguous results at this threshold were initially rated as identified to the genus level only. Further molecular analysis definitively assigned these isolates to the species Aspergillus oryzae (17 isolates) and Aspergillus flavus (one isolate), concordant with the MALDI-TOF MS results. Excluding nine isolates that belong to the fungal species not included in our reference database, 91 (96.8%) of 94 isolates were identified by MALDI-TOF MS to the species level, in agreement with the results of the reference method; three isolates were identified to the genus level. In conclusion, MALDI-TOF MS is suitable for the routine identification of filamentous fungi in a medical microbiology laboratory.     

Simultaneous detection and identification of Candida, Aspergillus, and Cryptococcus species by reverse line blot hybridization

We report on a reverse line blot (RLB) assay, utilizing fungal species-specific oligonucleotide probes to hybridize with internal transcribed spacer 2 region sequences amplified using a nested panfungal PCR. Reference and clinical strains of 16 Candida species (116 strains), Cryptococcus neoformans (five strains of Cryptococcus neoformans var. neoformans, five strains of Cryptococcus neoformans var. grubii, and six strains of Cryptococcus gatti), and five Aspergillus species (68 strains) were all correctly identified by the RLB assay. Additional fungal species (16 species and 26 strains) not represented on the assay did not exhibit cross-hybridization with the oligonucleotide probes. In simulated clinical specimens, the sensitivity of the assay for Candida spp. and Aspergillus spp. was 10(0.5) cells/ml and 10(2) conidia/ml, respectively. This assay allows sensitive and specific simultaneous detection and identification of a broad range of fungal pathogens.     

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.     

Molecular identification of zygomycetes from culture and experimentally infected tissues

Mucormycosis is an emerging infection associated with a high mortality rate. Identification of the causative agents remains difficult and time-consuming by standard mycological procedures. In this study, internal transcribed spacer (ITS) sequencing was validated as a reliable technique for identification of Zygomycetes to the species level. Furthermore, species identification directly from infected tissues was evaluated in experimentally infected mice. Fifty-four Zygomycetes strains belonging to 16 species, including the most common pathogenic species of Rhizopus spp., Absidia spp., Mucor spp., and Rhizomucor spp., were used to assess intra- and interspecies variability. Ribosomal DNA including the complete ITS1-5.8S-ITS2 region was amplified with fungal universal primers, sequenced, and compared. Overall, for a given species, sequence similarities between isolates were >98%. In contrast, ITS sequences were very different between species, allowing an accurate identification of Zygomycetes to the species level in most cases. Six species (Rhizopus oryzae, Rhizopus microsporus, Rhizomucor pusillus, Mucor circinelloides, and Mucor indicus) were also used to induce disseminated mucormycosis in mice and to demonstrate that DNA extraction, amplification of fungal DNA, sequencing, and molecular identification were possible directly from frozen tissues.     

Production of melanin pigments in saprophytic fungi in vitro and during infection

Melanins are one of the great natural pigments produced by a wide variety of fungal species that promote fitness and cell survival in diverse hostile environments, including during mammalian infection. In this study, we sought to demonstrate the production of melanin in the conidia and hyphae of saprophytic fungi, including dematiaceous and hyaline fungi. We showed that a melanin‐specific monoclonal antibody (MAb) avidly labeled the cell walls of hyphae and conidia, consistent with the presence of melanin in these structures, in 14 diverse fungal species. The conidia of saprophytic fungi were treated with proteolytic enzymes, denaturant, and concentrated hot acid to yield dark particles, which were shown to be stable free radicals, consistent with their identification as melanins. Samples obtained from patients with fungal keratitis due to Fusarium falciforme, Aspergillus fumigatus, Aspergillus flavus, Curvularia lunata, Exserohilum rostratum, or Fonsecaea pedrosoi were found to be intensely labeled by the melanin‐specific MAb at the fungal hyphal cell walls. These results support the hypothesis that melanin is a common component that promotes survival under harsh conditions and facilitates fungal virulence. Increased understanding of the processes of melanization and the development of methods to interfere with pigment formation may lead to novel approaches to combat these complex pathogens that are associated with high rates of morbidity and mortality.     

Mycoflora of air-conditioners dust from Riyadh, Saudi Arabia

Using the hair baiting technique, 6 genera and 14 species were collected on Sabouraud's dextrose agar from 37 dust samples from air-conditioners. The most common fungi were Chrysosporium tropicum, C. indicum, C. keratinophilum, Aspergillus flavus followed by Acremonium strictum and Scopulariopsis brevicaulis. Using the dilution-plate method, 26 genera and 52 species were collected from 37 dust samples on glucose-(23 genera and 45 species) and cellulose-(18 genera and 34 species) Czapek's agar at 28 degrees C. The most prevalent species were Aspergillus niger, A. flavus, Penicillium chrysogenum, Stachybotrys chartarum, Ulocladium atrum, Mucor racemosus and Fusarium solani and A. niger, A. flavus, Trichoderma viride, P. chrysogenum, Ulocladium atrum, Chaetomium globosum, C. spirale, Stachybotrys chartarum and Mucor racemosus on the two media, respectively.     

Production of mycotoxins by Aspergillus lentulus and other medically important and closely related species in section Fumigati.

The production of mycotoxins and other secondary metabolites have been studied by LC-DAD-MS from six species in Aspergillus section Fumigati. This includes the three new species Aspergillus lentulus, A. novofumigatus and A. fumigatiaffinis as well as A. fumigatus, Neosartoria fisheri and N. pseudofisheri. A major finding was detection of gliotoxin from N. pseudofisheri, a species not previously reported to produce this mycotoxin. Gliotoxin was also detected from A. fumigatus together with fumagillin, fumigaclavine C, fumitremorgin C, fumiquinazolines, trypacidin, methyl-sulochrin, TR-2, verruculogen, helvolic acid and pyripyropenes. Major compounds from A. lentulus were cyclopiazonic acid, terrein, neosartorin, auranthine and pyripyropenes A, E and O. Thus in the present study A. fumigatus and A. lentulus did not produce any of the same metabolites except for pyripyropenes. The fact that A. lentulus apparently does not produce gliotoxin supports the idea that other compounds than gliotoxin might play an important role in the effective invasiveness of A. lentulus. An overall comparison of secondary metabolite production by strains of the six species was achieved by analysis of fungal extracts by direct injection mass spectrometry and cluster analysis. Separate groupings were seen for all the six species even though only one isolate was included in this study for the two species A. novofumigatus and A. fumigatiaffinis.     

Disseminated coccidioidomycosis detected by percutaneous liver biopsy in a liver transplant recipient

The authors report the first case, to their knowledge, of disseminated coccidioidomycosis occurring in a liver transplant recipient. The case is also interesting in that the diagnosis of disseminated coccidioidomycosis was made fortuitously, only after finding the characteristic endosporulating spherules on a percutaneous liver biopsy. In addition, the authors reviewed the literature on post-transplant infection with particular emphasis on fungal pathogens. All studies concurred that Candida species was the most prevalent infecting fungal organism when both localized and disseminated forms of infection are included. Aspergillus was the second most common offender, and disseminated infection was associated with a very grave prognosis for the transplant recipient. Rare infections with Mucor and Cryptococcus neoformans are described in the literature.     

Mycosis in the ear, nose and throat]

Recent trends of fungal infections of the ear, nose and throat were introduced from the viewpoint of otolaryngologic practice. Aspergillus terreus was the most common pathogen of otomycosis followed by A. niger and A. flavus. Lanoconazole showed the most effective antifungal function for these Aspergillus species by drug sensitivity test. Biological differences between clinical and soil-borne strains of A. terreus were evaluated. The clinical strains showed slower growth-rate on malt extract agar and different patterns of fingerprinting by Random Amplified Polymorphic DNA. Aspergillosis is the most common fungal disease in the paranasal sinuses. Unilateral opacity of the maxillary sinus which contains flecks of calcification was specifically found by CT-study. Surgical removal of the fungus ball and establishment of a drainage route to the nasal passage by endoscopic sinus surgery are effective to manage aspergillomas in paranasal sinuses. Although candidosis is a common and mild infection in the oral mucosa, underlying problems related to immunodeficiency syndrome must be evaluated.     

Limitations of monoclonal antibodies for monitoring of fungal aerosols using Penicillium brevicompactum as a model fungus

Molds are ubiquitous in every environment and many species have been recently associated with an increase in opportunistic infections in immunocompromised patients or the exacerbation of asthmatic episodes in allergic patients. The degree of environmental contamination with fungi thus needs to be monitored and in this study we report the development of a monoclonal antibody (mAb)-mediated enzyme-linked immunosorbent assay (ELISA) for the detection of spores of Penicillium brevicompactum in experimental model aerosols. In addition, we have investigated the influence of different parameters of air sampling and sample recovery on ELISA performance. MAbs were produced with standard hybridoma techniques and cross-reactivities were determined against spores of 53 fungal species by indirect ELISA. Standardized experimental fungal aerosols were collected with the Button Personal Inhalable Aerosol Sampler onto polycarbonate or polytetrafluoroethylene filters (PTFE) and the effects of different extraction buffers and filter agitation methods during sample processing on spore recovery and ELISA detection were investigated. Five mAbs were produced and all of them cross-reacted with several of 31 related Aspergillus, Penicillium and Eurotium species. However, cross-reactivities with 21 non-related fungi were rare.Spores were recovered in much higher numbers from polycarbonate filters (PFs) than from polytetrafluoroethylene filters. Optical densities (ODs) in ELISA were higher for spores collected into carbonate coating buffer (CCB) than phosphate-buffered saline (PBS). Filter bath sonication following filter vortexing had no positive effects on ELISA sensitivity. The cross-reactivity patterns of mAbs suggest that Aspergillus and Penicillium species share multiple antigens. Quantitative ELISA results for fungal aerosols were found to be influenced by differential sample processing and thus method standardization will be essential to maintain the comparability of immunometric monitoring results.     

Antifungal susceptibility and phylogeny of opportunistic members of the order mucorales

The in vitro susceptibilities of 66 molecularly identified strains of the Mucorales to eight antifungals (amphotericin B, terbinafine, itraconazole, posaconazole, voriconazole, caspofungin, micafungin, and 5-fluorocytosine) were tested. Molecular phylogeny was reconstructed based on the nuclear ribosomal large subunit to reveal taxon-specific susceptibility profiles. The impressive phylogenetic diversity of the Mucorales was reflected in susceptibilities differing at family, genus, and species levels. Amphotericin B was the most active drug, though somewhat less against Rhizopus and Cunninghamella species. Posaconazole was the second most effective antifungal agent but showed reduced activity in Mucor and Cunninghamella strains, while voriconazole lacked in vitro activity for most strains. Genera attributed to the Mucoraceae exhibited a wide range of MICs for posaconazole, itraconazole, and terbinafine and included resistant strains. Cunninghamella also comprised strains resistant to all azoles tested but was fully susceptible to terbinafine. In contrast, the Lichtheimiaceae completely lacked strains with reduced susceptibility for these antifungals. Syncephalastrum species exhibited susceptibility profiles similar to those of the Lichtheimiaceae. Mucor species were more resistant to azoles than Rhizopus species. Species-specific responses were obtained for terbinafine where only Rhizopus arrhizus and Mucor circinelloides were resistant. Complete or vast resistance was observed for 5-fluorocytosine, caspofungin, and micafungin. Intraspecific variability of in vitro susceptibility was found in all genera tested but was especially high in Mucor and Rhizopus for azoles and terbinafine. Accurate molecular identification of etiologic agents is compulsory to predict therapy outcome. For species of critical genera such as Mucor and Rhizopus, exhibiting high intraspecific variation, susceptibility testing before the onset of therapy is recommended.