Single-Center Evaluation of an Agar-Based Screening for Azole Resistance in Aspergillus fumigatus by Using VIPcheck.

Antifungal susceptibility testing is an essential tool for guiding therapy, although EUCAST and CLSI reference methods are often available only in specialized centers. We studied the performance of an agar-based screening method for the detection of azole resistance in Aspergillus fumigatus cultures. The VIPcheck consists of four wells containing voriconazole, itraconazole, posaconazole, or a growth control. Ninety-six A. fumigatus isolates were used. Thirty-three isolates harbored a known resistance mechanism: TR34/L98H (11 isolates), TR46/Y121F/T289A (6 isolates), TR53 (2 isolates), and 14 isolates with other cyp51A gene point mutations. Eighteen resistant isolates had no cyp51A-mediated azole resistance. Forty-five isolates had a wild-type (WT) azole phenotype. Four technicians and two inexperienced interns, blinded to the genotype/phenotype, read the plates visually after 24 h and 48 h and documented minimal growth, uninhibited growth, and no growth. The performance was compared to the EUCAST method. After 24 h of incubation, the mean sensitivity and specificity were 0.54 and 1.00, respectively, with uninhibited growth as the threshold. After 48 h of incubation, the performance mean sensitivity and specificity were 0.98 and 0.93, respectively, with minimal growth. The performance was not affected by observer experience in mycology. The interclass correlation coefficient was 0.87 after 24 h and 0.85 after 48 h. VIPcheck enabled the selection of azole-resistant A. fumigatus colonies, with a mean sensitivity and specificity of 0.98 and 0.93, respectively. Uninhibited growth on any azole-containing well after 24 h and minimal growth after 48 h were indicative of resistance. These results indicate that the VIPcheck is an easy-to-use tool for azole resistance screening and the selection of colonies that require MIC testing.

In vitro activity of the novel antifungal compound F901318 against difficult-to-treat Aspergillus isolates.

Background: F901318 is a new antifungal agent with a novel mechanism of action with activity against Aspergillus species. We investigated the in vitro activity of F901318 against a collection of Aspergillus isolates. Methods: A total of 213 Aspergillus isolates were used in this study. A total of 143 Aspergillus fumigatus sensu stricto isolates were used, of which 133 were azole resistant [25 TR34/L98H; 25 TR46/Y121F/T289A; 33 A. fumigatus with cyp51A-associated point mutations (25 G54, 1 G432 and 7 M220); and 50 azole-resistant A. fumigatus without known resistance mechanisms]. Ten azole-susceptible A. fumigatus isolates were used as WT controls. The in vitro activity was also determined against Aspergillus calidoustus (25 isolates), Aspergillus flavus (10), Aspergillus nidulans (10) and Aspergillus tubingensis (25). F901318 activity was compared with that of itraconazole, voriconazole, posaconazole, isavuconazole, amphotericin B and anidulafungin. Minimum effective concentrations and MICs were determined using the EUCAST broth microdilution method. Results: F901318 was active against all tested isolates: A. fumigatus WT, MIC90 0.125 mg/L (range 0.031-0.125); TR34/L98H,TR46/Y121F/T289A and azole resistant without known resistance mechanisms, MIC90 0.125 mg/L (range 0.031-0.25); A. fumigatus with cyp51A-associated point mutations, MIC90 0.062 mg/L (range 0.015-0.125); and other species, A. calidoustus MIC90 0.5 mg/L (range 0.125-0.5), A. flavus MIC90 0.062 mg/L (range 0.015-0.62), A. nidulans MIC90 0.125 mg/L (range 0.062-0.25) and A. tubingensis MIC90 0.062 mg/L (range 0.015-0.25). Conclusions: F901318 showed potent and consistent in vitro activity against difficult-to-treat Aspergillus spp. with intrinsic and acquired antifungal resistance due to known and unknown resistance mechanisms, suggesting no significant implications of azole resistance mechanisms for the mode of action of F901318.

Combination of Amphotericin B and Flucytosine against Neurotropic Species of Melanized Fungi Causing Primary Cerebral Phaeohyphomycosis.

Primary central nervous system phaeohyphomycosis is a fatal fungal infection due mainly to the neurotropic melanized fungiCladophialophora bantiana,Rhinocladiella mackenziei, andExophiala dermatitidis.Despite the combination of surgery with antifungal treatment, the prognosis continues to be poor, with mortality rates ranging from 50 to 70%. Therefore, a search for a more-appropriate therapeutic approach is urgently needed. Ourin vitrostudies showed that with the combination of amphotericin B and flucytosine against these species, the median fractional inhibitory concentration (FIC) indices for strains ranged from 0.25 to 0.38, indicating synergy. By use of Bliss independence analysis, a significant degree of synergy was confirmed for all strains, with the sum ΔE ranging from 90.2 to 698.61%. No antagonism was observed. These results indicate that amphotericin B, in combination with flucytosine, may have a role in the treatment of primary cerebral infections caused by melanized fungi belonging to the orderChaetothyriales Furtherin vivostudies and clinical investigations to elucidate and confirm these observations are warranted.

Emergence of fusarioses in a university hospital in Turkey during a 20-year period.

Fusarium species have started appearing increasingly as the main cause of infections, particularly in immunocompromised patients. In this study, we aimed to present the first epidemiological data from Turkey, analyze fusariosis cases that have been monitored in a university hospital during the past 20 years, identify the responsible Fusarium species, and determine antifungal susceptibilities. A total of 47 cases of fusariosis was included in the study. Fusarium isolates were identified by multilocus sequence typing (MLST). Antifungal susceptibility was tested by the broth microdilution method according to the Clinical and Laboratory Standards Institute (CLSI) methodology. Of the Fusarium infections, 23.4 % were superficial, 44.7 % were locally invasive, and 31.9 % were disseminated. A significant increase was observed over the years. The Fusarium fujikuroi species complex (FFSC) proved to be the most frequent agent group (17 cases; 51.5 %), followed by the Fusarium solani species complex (FSSC) (14 cases; 42.4 %), the Fusarium dimerum species complex (FDSC), and the Fusarium oxysporum species complexes (FOSC) (one case each). Amphotericin B had the highest in vitro activity against all species. Voriconazole and posaconazole showed interspecies variability across and within Fusarium species complexes. In conclusion, our data support the fact that regional differences exist in the distribution of the Fusarium species and that species-specific differences are observed in antifungal susceptibility patterns. The monitoring of local epidemiological data by determining fungal identity and susceptibility are of importance in guiding the clinical follow-up of patients.

Antifungal susceptibility patterns of opportunistic fungi in the genera Verruconis and Ochroconis.

Species of Verruconis and species of Ochroconis are dematiaceous fungi generally found in the environment but having the ability to infect humans, dogs, cats, poultry, and fish. This study presents the antifungal susceptibility patterns of these fungi at the species level. Forty strains originating from clinical and environmental sources were phylogenetically identified at the species level by using sequences of the ribosomal DNA internal transcribed spacer (rDNA ITS). In vitro antifungal susceptibility testing was performed against eight antifungals, using the Clinical and Laboratory Standards Institute (CLSI) broth microdilution method. The geometric mean MICs for amphotericin B (AMB), flucytosine (5FC), fluconazole (FLC), itraconazole (ITC), voriconazole (VRC), and posaconazole (POS) and minimum effective concentrations (MECs) for caspofungin (CAS) and anidulafungin (AFG) across the Ochroconis and Verruconis species were as follows, in increasing order. For Verruconis species, the values (µg/ml) were as follows: AFG, 0.04; POS, 0.25; ITC, 0.37; AMB, 0.50; CAS, 0.65; VRC, 0.96; 5FC, 10.45; and FLC, 47.25. For Ochroconis species, the values (µg/ml) were as follows: AFG, 0.06; POS, 0.11; CAS, 0.67; VRC, 2.76; ITC, 3.94; AMB, 5.68; 5FC, 34.48; and FLC, 61.33. Antifungal susceptibility of Ochroconis and Verruconis was linked with phylogenetic distance and thermotolerance. Echinocandins and POS showed the greatest in vitro activity, providing possible treatment options for Ochroconis and Verruconis infections.

Absence of microbial adaptation to taurolidine in patients on home parenteral nutrition who develop catheter related bloodstream infections and use taurolidine locks.

BACKGROUND & AIMS: Some home parenteral nutrition (HPN) patients develop catheter related bloodstream infections (CRBSI) despite using an anti-microbial catheter lock solution taurolidine. The aim of this study was to assess whether long-term use of taurolidine leads to selective growth of microorganisms with increased taurolidine minimum inhibitory concentrations (MICs). METHODS: Bloodstream infections among 158 HPN patients with long-term taurolidine catheter locking were analyzed retrospectively. CRBSI-diagnosis was based on clinical symptoms, culture results, and absence of other sources of infections. CRBSIs were classified as definitive, probable or possible and exit site/tunnel/port or luminal infections. MICs were determined by broth microdilution. RESULTS: Between January 2009 and April 2011, 14 patients developed at least one luminal CRBSI episode during long-term taurolidine catheter locking (median (range) = 451 (78-1394) days). Coagulase-negative Staphylococcus species or Staphylococcus aureus predominated among CRBSI-causing Gram-positive bacteria. Taurolidine MICs were 512 mg/l or less in 50% of these isolates (MIC50). Taurolidine MIC50 for Klebsiella pneumoniae and Escherichia coli, the most common CRBSI-causing Gram-negative bacteria, were 256 and 512 mg/l, respectively. Taurolidine MIC50 among CRBSI-causing Candida albicans were 2048 mg/l. CONCLUSION: Adaptation of microorganisms to taurolidine has not yet emerged as a factor in the pathogenesis of CRBSI in HPN patients with long-term taurolidine catheter locking.

A colorimetric and spectrophotometric method for in vitro susceptibility testing of Aspergillus species against caspofungin.

The in vitro susceptibility of 45 Aspergillus fumigatus, Aspergillus flavus and Aspergillus terreus isolates against caspofungin (CAS) was assessed by the CLSI reference method with spectrophotometric reading and by a colorimetric method that employed the dye MTT. Perfect agreement was found between the minimal effective concentrations (MECs) and the MIC-2 values (50% growth reduction) obtained by the MTT method. The agreement found between the MICs obtained by the CLSI and the MTT method was dependent on the MIC endpoint used, the antifungal agent tested, and the species investigated.