Clinical Isolates Of Filamentous Fungi Research Articles

S4.1d In vitro activity of eight antifungals against 3343 filamentous pathogenic fungi collected during 18 years at the French National Reference Center for invasive mycosis and antifungals

S4.1 Treatment of rare mold infections in 2021: the role of new and old antifungals, September 22, 2022, 10:30 AM - 12:00 PM Background & ObjectivesThe NRCMA oversees the surveillance of invasive fungal diseases in France. As part of our expertise, we perform antifungal susceptibility testing based on European Committee on Antimicrobial Susceptibility Testing (EUCAST) methodology on all isolates. The antifungal profiles help us monitor the emergence of resistant isolates, determine the susceptibility pattern of wild-type strains to new antifungals, and in specific cases, determine the relationship between clinical failure and selection/emergence of a less susceptible isolate.We review the Minimum inhibitory concentrations (MIC) distribution of eight antifungals on clinical isolates of filamentous fungi identified at the NRCMA from 2003 to 2021.MethodsSpecies identification was performed by a combination of morphological features and multilocus sequencing. Only strains that produced enough conidia or spores were tested. In vitro susceptibility testing was performed according to the EUCAST procedure. Eight antifungal agents were used: triazoles [itraconazole, voriconazole, posaconazole, isavuconazole (since 2015)], echinocandins (caspofungin, micafungin), amphothericin B, and terbinafine. The concentrations inhibiting 50% (MIC50) and 90% (MIC90) of the isolates were determined for species with at least 5 and 10 isolates, respectively. For Aspergillus fumigatus isolates exhibiting high azole MICs, we sequenced the cyp51A gene for mutation screening.ResultsMICs were obtained for 3343 pathogenic strains. We identified Aspergillus spp. including cryptic species (32%), Fusarium spp. (21%), Mucorales (18%), phaeohyphomycetes (10%), i.e., Alternaria spp., Curvularia spp., Fonsecaea, Exophiala, Phaeoacremonium, rare hyphomycetes (9%), i.e., Rasamsonia, Paecilomyces, Trichoderma, Scopulariopsis. Scedosporium spp. (7%) and emergent pathogens such as Nannizziopsis obscura (1%), dimorphic fungi (1%), or to a less extent basidiomycetous molds. Examples of MIC results are presented in Table 1.For the nine genera of the order Mucorales, amphotericin B (AmB) exhibited low MICs except for Cunninghamella species and Saksenaea vasiformis. Posaconazole had variable activity depending on the species (high MIC values were observed for Mucor species) while voriconazole and echinocandins had none.In section Fumigati, amphotericin B MIC values were high due to the intrinsic resistance of cryptic species Aspergillus lentulus and A. udagawae. Other cryptic species were identified such as A. hiratsukae isolates which exhibited low MICs to all antifungals.Members of Aspergillus section Nidulantes such as A. nidulans and A. quadrilineatus showed variable MICs to caspofungin and amphotericin B.Among melanized fungi, Alternaria infectoria and A. alternata species group displayed low MICs of all azoles with the exception of voriconazole.Four Exophiala species were analyzed and had similar azole MICs, E. spinifera being the species with the lowest values.Multi-drug resistant profiles were observed especially in species belonging to Microascales such as Lomentospora prolificans, Microascus cirrosus, and Scopulariopsis brevicaulis. Members of Fusarium species complexes had predictably high MIC values to all antifungals including isavuconazole. Only Fusarium dimerum species complex had low MICs to amphotericin B.Further distribution analysis is ongoing. These large datasets provide a baseline for monitoring the emergence of antifungal resistance in our country and supports the fact that MIC determination should be performed for rare/emergent species and also in case of infections due to cryptic species, which would benefit patient management.

Comparative activity of posaconazole and systemic azole agents against clinical isolates of filamentous fungi from a global surveillance programme.

ObjectivesThe activity of mould-active azoles was evaluated against 397 filamentous fungi causing invasive mould infections (IMI) worldwide. In addition, a tentative posaconazole epidemiological cut-off value (ECV) against Aspergillus fumigatus was investigated.MethodsIsolates were susceptibility tested by the CLSI reference broth microdilution methods. Species identification was confirmed by MALDI-TOF and/or sequencing analysis.ResultsAspergillus spp. (81.9%) remained the most common organism causing IMI worldwide; approximately two-thirds of Aspergillus spp. recovered were A. fumigatus. In general, more than 90% of 220 A. fumigatus isolates were wild type (WT) to all mould-active azoles, except itraconazole (84.5% WT). The voriconazole non-susceptible (NS) A. fumigatus rate was 7.7% overall and was higher in Europe (12.9%) than in the other regions (0%–5.8%). Posaconazole (MIC50/MIC90, 0.25/0.5 mg/L) showed similar or slightly higher activity than voriconazole (MIC50/MIC90, 0.5/0.5 mg/L) and isavuconazole (MIC50/MIC90, 0.5/1 mg/L) against A. fumigatus. The mould-active azoles displayed similar activity against non-fumigatus Aspergillus (WT rates >93%), but differences were observed among the main species/sections. Posaconazole, voriconazole, and isavuconazole inhibited at their respective ECVs 100%, 97.0%, and 100% of A. section Nigri; 100%, 100%, and 93.8% of A. section Terrei; and 97.3%, 100%, and 100% of A. section Flavi isolates. Posaconazole displayed potency greater than or equal to the other azoles against the Mucorales group and Scedosporium spp.ConclusionsPosaconazole and other mould-active azoles showed good activity against Aspergillus spp. causing IMI, but clinicians should be aware of regional rates of voriconazole-NS A. fumigatus.

Evaluation of Vitek MS in identification of clinical filamentous fungi isolates

Objective To evaluate the performance of Vitek MS, an automated mass spectrometry microbial identification system, in identification of clinical filamentous fungi isolates, which provide a new insight into filamentous fungi identification strategy. Methods Twenty-five isolates of filamentous fungi genetically confirmed by molecular sequencing method were inoculated in different culture conditions to analyze the potential influences of culture conditions on the identification of filamentous fungi with Vitek MS. Further evaluation of the performance of Vitek MS in filamentous fungi identification was carried out with 207 clinical isolates of filamentous fungi and the comparison of the results with those of morphology and sequencing analysis. Results The performance of Vitek MS in filamentous fungi identification was not significantly influenced by culture conditions. Vitek MS successfully identified 87.9% (182/207) of all tested filamentous fungi. Lacking of reference MS spectra in Vitek database was the main reason that the other isolates could not be identified. Vitek MS was superior to the traditional morphological method in identification of filamentous fungi especially non-Aspergillus molds at species level. Conclusion Vitek MS is an efficient approach for clinical filamentous fungi identification. Continuous enrichment of the species coverage in database will be of great importance for improving the performance of Vitek MS in identification of filamentous fungi. Key words: Vitek MS; Filamentous fungi; Identification

Comparison of E Test and Disc Diffusion Methods for Susceptibility Testing of Filamentous Fungi; Experience of a Routine Lab

Background: Testing for antifungal susceptibility to newly introduced drugs is standardized through the clinical laboratory standard institute (CLSI) broth micro-dilution method for testing of molds (M38-A2), yet is difficult to use routinely. Objectives: To compare two agar-based diffusion methods on two types of media for fungal drug sensitivity testing. Materials and Methods: The E-test method and the disc diffusion method were used on non-supplemented Muller Hinton agar (MHA) and RPMI for evaluating the in-vitro susceptibility of 48 clinical isolates of filamentous fungi to amphotericin B, itraconazole, voriconazole, and caspofungin. Results: Categorical agreement between E-test and disc diffusion method for itraconazole and voriconazole was 100%, yet for amphotericin B on MHA agar was 66.67%, and on RPMI, it was 47.92%. The correlation coefficient (R) between the inhibition zone diameters when using MHA and RPMI for itraconazole, voriconazole, amphotericin B and caspofungin was +0.745, +0.901 +0.649, and +0.409, respectively. Conclusions: Routine antifungal susceptibility testing using disk diffusion can be implemented in routine microbiology work up in limited resources.

Capability of internal transcribed spacer (ITS) region sequencing analysis to identify clinical isolates of filamentous fungi

Objective To evaluate the capability of internal transcribed spacer (ITS) region sequencing analysis to identify clinical isolates of filamentous fungi. Methods A total of 267 filamentous fungi isolates collected from clinical specimens were analyzed by ITS region sequencing analysis. Alignment of acquired sequences with known sequences in GenBank and MycoBank was conducted to identify the species of those isolates. Results ITS sequences of the 267 isolates were amplified successfully. Among these isolates, 53.9% (144/267) were identified to species level and 44.2% (118/267) to genus level. Only five isolates were failed to be identified at genus level as they shared >95% homology in ITS sequence with multiple genera. Conclusion ITS region sequencing analysis is preferred for identification of clinical isolates of filamentous fungi at genus level for its high universality and great capability. When species-level identification is required, some informative DNA markers besides ITS region should be included accordingly. Key words: ITS; Sequencing analysis; Filamentous fungi; Identification

Evaluation of Etest Performed in Mueller–Hinton Agar Supplemented with Glucose for Antifungal Susceptibility Testing of Clinical Isolates of Filamentous Fungi

Although reference broth microdilution protocol is currently available for filamentous fungi antifungal susceptibility testing (AFST), simpler alternatives as Etest(®) tend to be favoured in clinical routine, making their validation of utmost importance. In this study, Etest(®) method using 2% glucose supplemented Muller-Hinton agar was compared to the Clinical and Laboratory Standards Institute (CLSI) M38-A2 protocol for filamentous fungi AFST. The echinocandins, caspofungin and anidulafungin, the azoles voriconazole and posaconazole, and the polyene amphotericin B were tested against 48 Aspergillus spp., seven Fusarium spp., one Beauveria bassiana and three Paecilomyces lilacinus isolates. The majority of the isolates were susceptible to the antifungals tested, and the overall level of agreement between the CLSI and Etest methods was 71.9% for one dilution and 99.7% when using two dilutions. Since interpretative breakpoints for filamentous fungi employing the CLSI or Etest methods are not available yet, the established epidemiological cut-off values for Aspergillus spp. were used to distinguish wild-type isolates from those with acquired resistance mechanisms. Forty-five Aspergillus strains did not evidence resistance mutations.

In Vitro Susceptibility Testing of Aspergillus and Non-Aspergillus Filamentous Moulds to Antifungal Agents: Evaluation of Three Different Methods

Aim: The aim of this study was to evaluate the suitability of Sensititre YeastOne (SYO) method for susceptibility profiles’ determination of non- Aspergillus moulds and of E-test for Aspergillus spp. and non- Aspergillus moulds, in comparison with the M38-A reference broth microdilution (BMD) method. Material and Methods: A total of 33 clinical isolates of filamentous fungi were tested. Results: The agreement between E-test and BMD at ± 2 dilutions was 82.4%, 83.3% and 82.4% for amphotericin B, itraconazole and voriconazole, respectively. The agreement between SYO and BMD at ± 2 dilutions was 76.5%, 66.7% and 88.2% for amphotericin B, itraconazole and voriconazole, respectively. The majority of differing results are due to higher MICs with the reference method. In particular, SYO was unable to detect some of the potentially amphotericin B resistant strains. We found that both E-test and SYO method were reproducible and served as suitable methods for antifungal susceptibility testing of moulds. Conclusion: In conclusion, both E-test and SYO method are promising, but require further investigation to identify the optimum conditions for their use in testing of susceptibility profiles of filamentous fungi to antifungal agents.

Susceptibility of 100 filamentous fungi: comparison of two diffusion methods, Neo-Sensitabs and E-test, for amphotericin B, caspofungin, itraconazole, voriconazole and posaconazole

We compared the E-test method to that of the Neo-Sensitabs tablet diffusion assay for evaluating the in vitro susceptibility of 100 clinical isolates of filamentous fungi (Aspergillus spp., Fusarium spp., Scedosporium spp., zygomycetes and other molds) to amphotericin B, itraconazole, voriconazole, caspofungin, and posaconazole. We determined the categorical agreement level between E-test minimum inhibitory concentrations (MIC) and tablet end-points, as opposed to the following disagreement parameters: very major error - resistant parameter (R) in E-test and susceptible (S) in tablet; major error - S by E-test and R by tablet; minor error - shifts between S and susceptible dose-dependent (S-DD) or S-DD and R. We also performed linear regression analyses and computed Pearson's correlation coefficients (R values) between the log transforms of MICs and the inhibition zone diameters of the five studied antifungal agents. For itraconazole we obtained 97% categorical agreement and R = -0.727. Categorical agreement for caspofungin and voriconazole was 96% and R =-0.821 and R = -0.789, respectively. For posaconazole the categorical agreement was 94% and R =-0.743. Amphotericin B exhibited a lower degree of agreement (76%, R = -0.672), especially in studies of Aspergillus spp. Our results suggest a potential value of the Neo-Sensitabs assay for in vitro susceptibility testing of molds to itraconazole, voriconazole, caspofungin and posaconazole, while amphotericin B exhibited an overall lower degree of agreement.

Antifungal activity of a new triazole, voriconazole (UK-109,496), compared with three other antifungal agents tested against clinical isolates of filamentous fungi

Antifungal activity of a new triazole, voriconazole (UK-109,496), compared with three other antifungal agents tested against clinical isolates of filamentous fungi

Activity of BAL 4815 against filamentous fungi

BAL 4815 is a new antifungal drug and it is the active component of the antifungal triazole BAL 8557 (the water-soluble precursor). We studied the in vitro fungistatic and fungicidal activities of BAL 4815 against 103 clinical isolates of filamentous fungi, including 51 isolates of Aspergillus spp. and 52 isolates of non-Aspergillus filamentous fungi. We evaluated the in vitro activity of BAL 4815 against 51 isolates of Aspergillus spp., 20 isolates of dematiaceous fungi, 18 isolates of hyaline Hyphomycetes and 14 isolates of Zygomycetes. MICs were determined following the CLSI M38-A broth microdilution method, using RPMI 1640 medium buffered to pH 7.0 with MOPS. Microdilution plates were incubated at 35 degrees C and read at 24 and 48 h (Mucorales were read at 24 h). Minimal fungicidal concentrations were also determined. For all isolates, geometric mean MICs, MIC(50)s, MIC(90)s and MIC ranges (mg/L) were: Aspergillus spp., 1.67, 2, 4 and 0.5-4; dematiaceous fungi, 1.62, 1, >8 and 0.03 to >8; hyaline Hyphomycetes, 2.41, 2, >8 and 0.03 to >8; and Zygomycetes, 6.81, 8, >8 and 0.03 to >8. Differences in susceptibility between genera were noted. Scedosporium prolificans, Fusarium spp., Mucor spp. and Rhizopus spp. (MIC(90) > 8 mg/L) were less susceptible than Aspergillus spp. (MIC(90) = 4 mg/L). BAL 4815 has excellent in vitro activity against Aspergillus spp. and variable activity against other filamentous fungi.

Evaluation of Disk Diffusion and Etest Compared to Broth Microdilution for Antifungal Susceptibility Testing of Posaconazole against Clinical Isolates of Filamentous Fungi

We performed Etest, disk diffusion, and broth microdilution susceptibility testing of posaconazole against 146 clinical isolates of filamentous fungi. By using provisional breakpoints for comparison purposes only, categorical agreement between the results of the agar-based methods and those of broth microdilution were 96 to 98%, with no very major errors. These agar-based methods hold promise as simple and reliable methods for determining the posaconazole susceptibilities of filamentous fungi.

Estudio de la sensibilidad in vitro de aislamientos clínicos de mohos y levaduras a itraconazol y voriconazol

The interest on the in vitro susceptibility to itraconazole has recently increased due the availability of the intravenous formulation. In this study, comparative MICs of this antifungal with voriconazole were carried out in 62 clinical isolates of filamentous fungi and 100 yeasts isolates using the NCCLS microbroth methods described in M38-A and M27-A2 documents. A MIC90 of 0.125 micrograms per ml was observed for itraconazole and voriconazole against Aspergillus fumigatus. Higher susceptibility to itraconazole was found for the filamentous form of Sporotrhix schenckii (p = 0.001). Voriconazole was more effective against Scedosporium apiospermium while Scedosporium prolificans isolates were resistant to both azoles. Some isolates of Rhizopus stolonifer were susceptible to itraconazole and resistant to voriconazole, but without statistical significance. Susceptibility of nine species of Candida was similar for both triazoles used in this study. However, Candida glabrata was more susceptible to voriconazole. Some fluconazole-resistant Candida albicans isolates were susceptible to itraconazole and / or voriconazole. Cryptococcus neoformans was more susceptible to itraconazole than to voriconazole. Itraconazole and voriconazole showed very close in vitro activity against the tested fungal isolated, except against S. schenckii. In spite of this, there were some differences in susceptibility among isolates within the same fungal species.

Activities of caspofungin, itraconazole, posaconazole, ravuconazole, voriconazole, and amphotericin B against 448 recent clinical isolates of filamentous fungi.

We examined the in vitro activity of caspofungin, posaconazole, voriconazole, ravuconazole, itraconazole, and amphotericin B against 448 recent clinical mold isolates. The endpoint for reading caspofungin was the minimum effective concentration (MEC). Among the triazoles, posaconazole was most active, inhibiting 95% of isolates at <or=1 microg/ml, followed by ravuconazole (91%), voriconazole (90%), and itraconazole (79%). Caspofungin and amphotericin B inhibited 93% and 89% of isolates at <or=1 microg/ml, respectively, with caspofungin demonstrating an MEC 90 of 0.12 microg/ml. All three new triazoles and caspofungin inhibited >95% of Aspergillus spp. at <or=1 microg/ml compared to 83% for itraconazole and 91% for amphotericin B. Amphotericin B inhibited only 38% of Aspergillus terreus isolates at <or=1 microg/ml, whereas the three new triazoles and caspofungin inhibited all A. terreus at <or=0.5 microg/ml. The new triazoles and caspofungin have excellent in vitro activity against a very large collection of recent clinical isolates of Aspergillus spp., and some in vitro activity against selected other filamentous fungi.

Antifungal activity of a new triazole, voriconazole (UK-109,496), compared with three other antifungal agents tested against clinical isolates of filamentous fungi

Antifungal activity of a new triazole, voriconazole (UK-109,496), compared with three other antifungal agents tested against clinical isolates of filamentous fungi

In Vitro Activity of Two Echinocandin Derivatives, LY303366 and MK-0991 (L-743,792), Against Clinical Isolates of Aspergillus, Fusarium, Rhizopus, and Other Filamentous Fungi

LY303366 and MK-0991 (previously L-743,792) are new echinocandin derivatives with excellent broad-spectrum antifungal activity. We investigated the in vitro activity of LY303366, MK-0991, itraconazole, amphotericin B, and 5-flucytosine against 51 clinical isolates of filamentous fungi, including Aspergillus flavus (10), A. fumigatus (12), Fusarium spp. (13), Rhizopus spp. (6), Pseudallescheria boydii (5), and one isolate each of Acremonium spp., A. niger, A. terreus, Paecilomyces spp., and Trichoderma spp. In vitro susceptibility testing was performed using a microdilution broth method performed according to National Committee for Clinical Laboratory Standards guidelines. LY303366 was two- to fourfold more active than MK-0991 against A. flavus, A. fumigatus, and Trichoderma spp. Both LY303366 and MK-0991 were considerably more active (MIC 90 of 0.03–0.12 μg/mL) than itraconazole, amphotericin B, and 5-flucytosine against Aspergillus spp., but were less active than itraconazole and amphotericin B against Rhizopus spp. MK-0991 was more active than either LY303366 or itraconazole against Acremonium spp., Paecilomyces spp., and P. boydii. These data demonstrate promising activity of both LY303366 and MK-0991 against Aspergillus spp. and other species of filamentous fungi that are likely to be encountered clinically. Further in vitro and in vivo investigation is indicated.

In vitro activity of a new triazole antifungal agent, Sch 56592, against clinical isolates of filamentous fungi.

Sch 56592 is a new triazole derivative that possesses potent, broad-spectrum antifungal activity. We evaluated the in vitro activity of Sch 56592 compared with that of itraconazole, amphotericin B and 5-fluorocytosine against 51 clinical isolates of filamentous fungi, including Aspergillus flavus (10), A. fumigatus (12), Fusarium spp. (13), Rhizopus spp. (6), Pseudallescheria boydii (5), and one isolate each of Acremonium spp., A. niger, A. terreus, Paecilomyces spp., and Trichoderma spp. In vitro susceptibility testing was performed using the microdilution broth method outlined in the NCCLS 27-A document. Sch 56592 was highly active against A. flavus (MIC90, 0.25 micrograms/ml), A. fumigatus (MIC90, 0.12 micrograms/ml), P. boydii (MIC50, 1 microgram/ml) and Rhizopus spp (MIC50, 1 microgram/ml). By comparison with itraconazole, Sch 56592 was four-to eight-fold more active against isolates of Aspergillus and both compounds showed equipotent in vitro activity against P. boydii and Rhizopus spp. Sch 56592 was four- to 16-fold more active than amphotericin B against Aspergillus spp. and P. boydii and both antifungal drugs displayed similar activity against Rhizopus spp. Overall, Sch 56592 showed good in vitro activity against all isolates tested (MIC, < or = 2 micrograms/ml) except isolates of Fusarium (MIC range, (1-->4 micrograms/ml). On the basis of these data Sch 56592 has promising activity against Aspergillus spp. and other species of filamentous fungi that are likely to be encountered clinically. Additional in vitro and in vivo studies are warranted.

Antifungal activity of a new triazole, voriconazole (UK-109,496), compared with three other antifungal agents tested against clinical isolates of filamentous fungi

Voriconazole is a new triazole antifungal agent with potent activity against yeast and moulds. We investigated the in vitro activity of voriconazole, itraconazole, amphotericin B and 5-flucytosine against 51 clinical isolates of filamentous fungi. Overall, voriconazole was active (MIC50, 0.5 mg l(-1) and MIC90, 8 mg l(-1)) against these mould isolates. Voriconazole was most active against P. boydii (MIC50, 0.12 mg l(-1)) and Aspergillus spp. (MIC90, 0.5 mg l(-1)) and least active against Fusarium spp. (MIC90, 8 mg l(-1)) and Rhizopus spp. (MIC50, 8 mg l(-1)). Voriconazole was more active than amphotericin B against Aspergillus spp. and P. boydii. By comparison with itraconazole, voriconazole was more active against all isolates except Rhizopus spp. Based on these results, voriconazole has promising activity against commonly encountered isolates of filamentous fungi and its clinical usefulness should be established by further studies.