Environmental Clonal Spread of Azole-Resistant Candida parapsilosis with Erg11-Y132F Mutation Causing a Large Candidemia Outbreak in a Brazilian Cancer Referral Center

Clonal outbreaks due to azole-resistant Candida parapsilosis (ARCP) isolates have been reported in numerous studies, but the environmental niche of such isolates has yet to be defined. Herein, we aimed to identify the environmental niche of ARCP isolates causing unremitting clonal outbreaks in an adult ICU from a Brazilian cancer referral center. C. parapsilosis sensu stricto isolates recovered from blood cultures, pericatheter skins, healthcare workers (HCW), and nosocomial surfaces were genotyped by multilocus microsatellite typing (MLMT). Antifungal susceptibility testing was performed by the EUCAST (European Committee for Antimicrobial Susceptibility Testing) broth microdilution reference method and ERG11 was sequenced to determine the azole resistance mechanism. Approximately 68% of isolates were fluconazole-resistant (76/112), including pericatheter skins (3/3, 100%), blood cultures (63/70, 90%), nosocomial surfaces (6/11, 54.5%), and HCW’s hands (4/28, 14.2%). MLMT revealed five clusters: the major cluster contained 88.2% of ARCP isolates (67/76) collected from blood (57/70), bed (2/2), pericatheter skin (2/3), from carts (3/7), and HCW’s hands (3/27). ARCP isolates were associated with a higher 30 day crude mortality rate (63.8%) than non-ARCP ones (20%, p = 0.008), and resisted two environmental decontamination attempts using quaternary ammonium. This study for the first time identified ARCP isolates harboring the Erg11-Y132F mutation from nosocomial surfaces and HCW’s hands, which were genetically identical to ARCP blood isolates. Therefore, it is likely that persisting clonal outbreak due to ARCP isolates was fueled by environmental sources. The resistance of Y132F ARCP isolates to disinfectants, and their potential association with a high mortality rate, warrant vigilant source control using effective environmental decontamination.

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Use of spectrophotometric reading for in vitro antifungal susceptibility testing ofAspergillusspp.

Canadian Journal of Microbiology ◽

10.1139/w99-075 ◽

1999 ◽

Vol 45 (10) ◽

pp. 871-874 ◽

Cited By ~ 7

Author(s):

Eric Dannaoui ◽

Florence Persat ◽

Marie-France Monier ◽

Elisabeth Borel ◽

Marie-Antoinette Piens ◽

...

Keyword(s):

Amphotericin B ◽

Susceptibility Testing ◽

Clinical Laboratory ◽

Antifungal Susceptibility ◽

Reference Method ◽

Antifungal Susceptibility Testing ◽

Aspergillus Species ◽

National Committee ◽

Broth Microdilution Method

A comparative study of visual and spectrophotometric MIC endpoint determinations for antifungal susceptibility testing of Aspergillus species was performed. A broth microdilution method adapted from the National Committee for Clinical Laboratory Standards (NCCLS) was used for susceptibility testing of 180 clinical isolates of Aspergillus species against amphotericin B and itraconazole. MICs were determined visually and spectrophotometrically at 490 nm after 24, 48, and 72h of incubation, and MIC pairs were compared. The agreement between the two methods was 99% for amphotericin B and ranged from 95 to 98% for itraconazole. It is concluded that spectrophotometric MIC endpoint determination is a valuable alternative to the visual reference method for susceptibility testing of Aspergillus species.Key words: antifungal, susceptibility testing, Aspergillus, spectrophotometric reading.

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In Vitro Interaction between Isavuconazole and Tacrolimus, Cyclosporin A, or Sirolimus against Aspergillus Species

Journal of Fungi ◽

10.3390/jof6030103 ◽

2020 ◽

Vol 6 (3) ◽

pp. 103 ◽

Cited By ~ 3

Author(s):

Patrick Schwarz ◽

Eric Dannaoui

Keyword(s):

Cyclosporin A ◽

Aspergillus Terreus ◽

Susceptibility Testing ◽

Inhibitory Concentration ◽

Antifungal Susceptibility ◽

Reference Method ◽

Common Species ◽

Minimal Inhibitory Concentrations ◽

In Vitro Interaction

The interaction of isavuconazole with immunosuppressors (tacrolimus, cyclosporin A, or sirolimus) against 30 Aspergillus isolates belonging to the most common species responsible for invasive aspergillosis in humans (Aspergillus flavus, Aspergillus fumigatus, Aspergillus nidulans, Aspergillus niger, and Aspergillus terreus) was evaluated in vitro by a microdilution checkerboard technique based on the EUCAST reference method for antifungal susceptibility testing. The interpretation of the results was performed based on the fractional inhibitory concentration index. The combination of isavuconazole with tacrolimus, cyclosporin A, or sirolimus, was synergistic for 56, 20, or 10% of the isolates, respectively. Interestingly synergy of the combination of isavuconazole with tacrolimus was also achieved for the majority of azole-resistant isolates of A. fumigatus, and for all A. niger isolates with isavuconazole minimal inhibitory concentrations ≥ 8 µg/mL. Antagonistic interactions were never observed for any combination tested.

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Rapid Detection of Echinocandins Resistance by MALDI-TOF MS in Candida parapsilosis Complex

Microorganisms ◽

10.3390/microorganisms8010109 ◽

2020 ◽

Vol 8 (1) ◽

pp. 109

Author(s):

Ana Emília M. Roberto ◽

Danilo E. Xavier ◽

Esteban E. Vidal ◽

Cláudia Fernanda de L. Vidal ◽

Rejane P. Neves ◽

...

Keyword(s):

Mass Spectrometry ◽

Incubation Time ◽

Candida Parapsilosis ◽

Clinical Laboratory ◽

Antifungal Susceptibility ◽

Reference Method ◽

Sensu Stricto ◽

Maldi Tof ◽

Separation Parameter ◽

Candida Parapsilosis Complex

Mass spectrometry by matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) was used to identify and differentiate the pattern of susceptibility of clinical isolates of Candida parapsilosis complex. 17 C. parapsilosis sensu stricto, 2 C. orthopsilosis, and 1 C. metapsilosis strains were obtained from blood cultures, and three different inocula (103, 105, and 107 CFU/mL) were evaluated against three echinocandins at concentrations ranging from 0.03 to 16 µg/mL after incubation of 1 h, 2 h, and 3 h. Drug-free control was used. The spectra obtained at these concentrations were applied to generate composite correlation index (CCI) matrices for each yeast individually. After cross correlations and autocorrelations of each spectra with null (zero) and maximal (16) concentrations, the CCI was used as separation parameter among spectra. Incubation time and inoculum were critical factors to reach higher precision and reliability of this trial. With an incubation time of 3 h and inoculum of 107 CFU/mL, it was possible to determine the breakpoint of the clinical yeasts by MALDI-TOF that presented high agreement with the clinical laboratory standard institute (CLSI) reference method. Herein, we show that mass spectrometry using the MALDI-TOF technique is powerful when it exploits antifungal susceptibility testing assays.

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Colistin and Isavuconazole Interact Synergistically In Vitro against Aspergillus nidulans and Aspergillus niger

Microorganisms ◽

10.3390/microorganisms8091447 ◽

2020 ◽

Vol 8 (9) ◽

pp. 1447

Author(s):

Patrick Schwarz ◽

Elie Djenontin ◽

Eric Dannaoui

Keyword(s):

Aspergillus Niger ◽

Aspergillus Nidulans ◽

Susceptibility Testing ◽

Antifungal Susceptibility ◽

Reference Method ◽

Important Species ◽

Agar Diffusion ◽

Agar Diffusion Assay ◽

Diffusion Assay

The in vitro interactions of isavuconazole in combination with colistin were evaluated against 55 clinical Aspergillus species isolates belonging to the five most important species (Aspergillus flavus, Aspergillus fumigatus, Aspergillus nidulans, Aspergillus niger, and Aspergillus terreus) responsible for human aspergillosis by a microdilution checkerboard technique based on the European Committee on Antimicrobial Susceptibility Testing (EUCAST) reference method for antifungal susceptibility testing. Selected isolates (A. nidulans, n = 10; A. niger, n = 15) were additionally evaluated by an agar diffusion assay using isavuconazole gradient concentration strips with or without colistin incorporated Roswell Parc Memorial Institute (RPMI) agar. Interpretation of the checkerboard results was done by the fractional inhibitory concentration index. Using the checkerboard method, combination isavuconazole–colistin was synergistic for 100% of the 15 A. nidulans isolates and for 60% of the 20 A. niger isolates. No interactions were found for any of the other isolates. By agar diffusion assay, minimal inhibitory concentrations (MICs) in combination decreased compared to isavuconazole alone for 92% of the isolates. No interactions were found for any A. nidulans isolates, but synergy was observed for 40% of the A. niger isolates. A poor essential agreement of EUCAST and gradient concentration strip MICs at ± 2 log2 dilutions with 0% was obtained. Antagonistic interactions were never observed regardless of the technique used.

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Identification and broth-microdilution antifungal susceptibility testing of yeast directly from automated blood cultures

Future Microbiology ◽

10.2217/fmb-2020-0077 ◽

2020 ◽

Vol 15 (15) ◽

pp. 1453-1464

Author(s):

Sourav Das ◽

Yamini Tawde ◽

Shreya Singh ◽

Arunaloke Chakrabarti ◽

Pallab Ray ◽

...

Keyword(s):

Susceptibility Testing ◽

Antifungal Susceptibility ◽

High Sensitivity ◽

Turnaround Time ◽

Blood Cultures ◽

Species Level ◽

Robust Method ◽

Broth Microdilution ◽

Level Identification ◽

Tof Ms

Aim: To standardize MALDI-TOF-MS based identification and antifungal susceptibility (AFST) for yeasts directly from automated blood cultures to reduce turnaround time. Materials & methods: Direct-ID after lysis-centrifugation (0.5% SDS) standardized in 40 and validated in 250 yeast positive samples. Direct-AFST was standardized with fluconazole (28 samples) and evaluated (70 samples) for seven antifungals. Results: Direct-ID had a high sensitivity (97.2%) and specificity (94.3%). Correct species-level identification showed 100% in C. tropicalis, C. krusei, C. parapsilosis. Direct-AFST had a 100% categorical agreement with culture-AFST for posaconazole, anidulafungin and >90% categorical agreement for amphotericin B, voriconazole and fluconazole. Conclusion: Direct-ID and subsequent direct-AFST is a rapid and robust method to reduce the turnaround time for the diagnosis of invasive candidiasis.

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Comparison of the Sensititre YeastOne and CLSI M38-A2 Microdilution Methods in Determining the Activity of Amphotericin B, Itraconazole, Voriconazole, and Posaconazole against Aspergillus Species

Journal of Clinical Microbiology ◽

10.1128/jcm.00780-18 ◽

2018 ◽

Vol 56 (10) ◽

Cited By ~ 7

Author(s):

Hsuan-Chen Wang ◽

Ming-I Hsieh ◽

Pui-Ching Choi ◽

Chi-Jung Wu

Keyword(s):

Aspergillus Fumigatus ◽

Amphotericin B ◽

Antifungal Agents ◽

Susceptibility Testing ◽

Geometric Mean ◽

Antifungal Susceptibility ◽

Reference Method ◽

Aspergillus Species ◽

Broth Microdilution ◽

Content Type

ABSTRACT This study compared the YeastOne and reference CLSI M38-A2 broth microdilution methods for antifungal susceptibility testing of Aspergillus species. The MICs of antifungal agents were determined for 100 Aspergillus isolates, including 54 Aspergillus fumigatus (24 TR34/L98H isolates), 23 A. flavus, 13 A. terreus, and 10 A. niger isolates. The overall agreement (within 2 2-fold dilutions) between the two methods was 100%, 95%, 92%, and 90% for voriconazole, posaconazole, itraconazole, and amphotericin B, respectively. The voriconazole geometric mean (GM) MICs were nearly identical for all isolates using both methods, whereas the itraconazole and posaconazole GM MICs obtained using the YeastOne method were approximately 1 dilution lower than those obtained using the reference method. In contrast, the amphotericin B GM MIC obtained using the YeastOne method was 3.3-fold higher than that observed using the reference method. For the 24 A. fumigatus TR34/L98H isolates assayed, the categorical agreement (classified according to the CLSI epidemiological cutoff values) was 100%, 87.5%, and 83.3% for itraconazole, voriconazole, and posaconazole, respectively. For four A. niger isolates, the itraconazole MICs were >8 μg/ml using the M38-A2 method due to trailing growth, whereas the corresponding itraconazole MICs obtained using the YeastOne method were all ≤0.25 μg/ml without trailing growth. These data suggest that the YeastOne method can be used as an alternative for azole susceptibility testing of Aspergillus species and for detecting the A. fumigatus TR34/L98H isolates but that this method fails to detect A. niger isolates exhibiting trailing growth with itraconazole. Additionally, for isolates with azole MICs that approach or that are at susceptibility breakpoints or with high amphotericin B MICs detected using the YeastOne method, further MIC confirmation using the reference CLSI method is needed.

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Clonal Candidemia Outbreak by Candida parapsilosis Carrying Y132F in Turkey: Evolution of a Persisting Challenge

Frontiers in Cellular and Infection Microbiology ◽

10.3389/fcimb.2021.676177 ◽

2021 ◽

Vol 11 ◽

Author(s):

Amir Arastehfar ◽

Suleyha Hilmioğlu-Polat ◽

Farnaz Daneshnia ◽

Weihua Pan ◽

Ahmed Hafez ◽

...

Keyword(s):

Candida Parapsilosis ◽

Control Strategies ◽

Antifungal Susceptibility ◽

Therapeutic Failure ◽

University Hospital ◽

Antifungal Stewardship ◽

Major Health ◽

Major Cluster ◽

Echinocandin Resistance ◽

Clinical Profiles

As the second leading etiological agent of candidemia in Turkey and the cause of severe fluconazole-non-susceptible (FNS) clonal outbreaks, Candida parapsilosis emerged as a major health threat at Ege University Hospital (EUH). Evaluation of microbiological and pertinent clinical profiles of candidemia patients due to C. parapsilosis in EUH in 2019–2020. Candida parapsilosis isolates were collected from blood samples and identified by sequencing internal transcribed spacer ribosomal DNA. Antifungal susceptibility testing was performed in accordance with CLSI M60 protocol and ERG11 and HS1/HS2-FKS1 were sequenced to explore the fluconazole and echinocandin resistance, respectively. Isolates were typed using a multilocus microsatellite typing assay. Relevant clinical data were obtained for patients recruited in the current study. FNS C. parapsilosis isolates were recovered from 53% of the patients admitted to EUH in 2019–2020. Y132F was the most frequent mutation in Erg11. All patients infected with C. parapsilosis isolates carrying Y132F, who received fluconazole showed therapeutic failure and significantly had a higher mortality than those infected with other FNS and susceptible isolates (50% vs. 16.1%). All isolates carrying Y132F grouped into one major cluster and mainly recovered from patients admitted to chest diseases and pediatric surgery wards. The unprecedented increase in the number of Y132F C. parapsilosis, which corresponded with increased rates of fluconazole therapeutic failure and mortality, is worrisome and highlights the urgency for strict infection control strategies, antifungal stewardship, and environmental screening in EUH.

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Antifungal susceptibility testing identifies the abdominal cavity as a source of Candida glabrata resistant isolates

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.01249-21 ◽

2021 ◽

Author(s):

Judith Díaz-García ◽

Aina Mesquida ◽

Ana Gómez ◽

Marina Machado ◽

Pablo Martín-Rabadán ◽

...

Keyword(s):

Candida Glabrata ◽

Susceptibility Testing ◽

Abdominal Cavity ◽

Antifungal Susceptibility ◽

Treatment Resistance ◽

Resistance Rate ◽

Blood Cultures ◽

Wild Type ◽

Echinocandin Resistance ◽

Resistance Rates

To identify unrecognized niches of resistant Candida isolates and compartmentalization we retrospectively studied the antifungal susceptibility of 1,103 Candida spp. isolates from blood cultures, non-blood sterile samples, and non-sterile samples. Antifungal susceptibility was assessed by EUCAST E.Def 7.3.2; sequencing and genotyping of the FKS1-2 and ERG11 genes were carried out for non-wild type isolates. Resistance compartmentalization (presence of resistant and susceptible isogenic isolates in different anatomical sites of a given patient) was studied. Clinical charts of patients carrying non-wild type isolates were reviewed. Most isolates (63%) were Candida albicans , regardless the clinical source; Candida glabrata (27%) was the second most frequently found species in abdominal cavity samples. Fluconazole and echinocandin resistance rates were 1.5% and 1.3%, respectively, and highest in C. glabrata . We found 22 genotypes among non-wild type isolates, none of them widespread across the hospital. Fluconazole/echinocandin resistance rates of isolates from abdominal cavity (3.2%/3.2%) were significantly higher than those from blood cultures (0.7%/1.3%) ( P <0.05). Overall, fifteen patients with different forms of candidiasis were infected by resistant isolates, 80% of whom had received antifungals before or at the time of isolate collection; resistance compartmentalization was found in six patients, mainly due to C. glabrata . The highest antifungal resistance rate was detected in isolates from the abdominal cavity, mostly C. glabrata . Resistance was not caused by the spread of resistant clones, but because of antifungal treatment. Resistance compartmentalization illustrates how resistance might be overlooked if susceptibility testing is restricted to bloodstream isolates.

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Molecular differentiation and antifungal susceptibilities of Candida parapsilosis isolated from patients with bloodstream infections

Journal of Medical Microbiology ◽

10.1099/jmm.0.004242-0 ◽

2009 ◽

Vol 58 (2) ◽

pp. 185-191 ◽

Cited By ~ 57

Author(s):

Sun Tee Tay ◽

Shiang Ling Na ◽

Jennifer Chong

Keyword(s):

Candida Parapsilosis ◽

Susceptibility Testing ◽

Rapd Analysis ◽

Antifungal Susceptibility ◽

Bloodstream Infections ◽

Candida Metapsilosis ◽

Significant Difference ◽

Its Gene ◽

Candida Orthopsilosis ◽

Susceptibility Patterns

The genetic heterogeneity and antifungal susceptibility patterns of Candida parapsilosis isolated from blood cultures of patients were investigated in this study. Randomly amplified polymorphic DNA (RAPD) analysis generated 5 unique profiles from 42 isolates. Based on the major DNA fragments of the RAPD profiles, the isolates were identified as RAPD type P1 (29 isolates), P2 (6 isolates), P3 (4 isolates), P4 (2 isolates) and P5 (1 isolate). Sequence analysis of the internal transcribed spacer (ITS) gene of the isolates identified RAPD type P1 as C. parapsilosis, P2 and P3 as Candida orthopsilosis, P4 as Candida metapsilosis, and P5 as Lodderomyces elongisporus. Nucleotide variations in ITS gene sequences of C. orthopsilosis and C. metapsilosis were detected. Antifungal susceptibility testing using Etests showed that all isolates tested in this study were susceptible to amphotericin B, fluconazole, ketoconazole, itraconazole and voriconazole. C. parapsilosis isolates exhibited higher MIC50 values than those of C. orthopsilosis for all of the drugs tested in this study; however, no significant difference in the MICs for these two Candida species was observed. The fact that C. orthopsilosis and C. metapsilosis were responsible for 23.8 and 4.8 % of the cases attributed to C. parapsilosis bloodstream infections, respectively, indicates the clinical relevance of these newly described yeasts. Further investigations of the ecological niche, mode of transmission and virulence of these species are thus essential.

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Comparison of Two Commercial Colorimetric Broth Microdilution Tests for Candida Susceptibility Testing: Sensititre YeastOne versus MICRONAUT-AM

Journal of Fungi ◽

10.3390/jof7050356 ◽

2021 ◽

Vol 7 (5) ◽

pp. 356

Author(s):

Sophie Philips ◽

Frederik Van Van Hoecke ◽

Emmanuel De De Laere ◽

Steven Vervaeke ◽

Roos De De Smedt ◽

...

Keyword(s):

Amphotericin B ◽

Antifungal Agents ◽

Susceptibility Testing ◽

Antifungal Susceptibility ◽

Reference Method ◽

Daily Practice ◽

Broth Microdilution ◽

Minimal Inhibitory Concentrations ◽

Clinical Breakpoints ◽

Susceptibility Tests

Two colorimetric broth microdilution antifungal susceptibility tests were compared, Sensititre YeastOne and MICRONAUT-AM for nine antifungal agents. One hundred clinical Candida isolates were tested, representing a realistic population for susceptibility testing in daily practice. The reproducibility characteristics were comparable. Only for fluconazole, caspofungin, 5-flucytosine and amphotericin B, an essential agreement of ≥90% could be demonstrated. Sensititre minimal inhibitory concentrations (MICs) were systematically higher than MICRONAUT MICs for all antifungals, except for itraconazole. CLSI clinical breakpoints (CBPs) and epidemiological cut-off values (ECVs) were used for Sensititre MICs while for MICRONAUT the EUCAST CBPs and ECVs were used. Only fluconazole, micafungin, and amphotericin B had a categorical agreement of ≥90%. For fluconazole, micafungin, and amphotericin B the susceptibility proportions were comparable. Susceptibility proportion of posaconazole and voriconazole was higher using the MICRONAUT system. For itraconazole and anidulafungin, the susceptibility proportion was higher using Sensititre. It was not possible to determine the true MIC values or the correctness of a S/I/R result since both commercial systems were validated against a different reference method. These findings show that there is a significant variability in susceptibility pattern and consequently on use of antifungals in daily practice, depending on the choice of commercial system.

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