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ABSTRACTIntroductionThe epidemiology of invasive Candidainfections is evolving. Infections caused by non-albicans Candidaspp. are increasing; however, the antifungal pipeline is more promising than ever and is enriched with repurposed drugs and agents that have new mechanisms of action. Despite progress, unmet needs in the treatment of invasive candidiasis remain, and there are still too few antifungals that can be administered orally or that have CNS penetration.Areas coveredThe authors shed light on those antifungal agents active against Candidathat are in early- and late-stage clinical development. Mechanisms of action and key pharmacokinetic and pharmacodynamic properties are discussed. Insights are offered on the potential future roles of the investigational agents MAT-2203, oteseconazole, ATI-2307, VL-2397, NP-339, and the repurposed drug miltefosine.Expert opinionIbrexafungerp and fosmanogepix have novel mechanisms of action and will provide effective options for the treatment of Candidainfections (including those caused by multiresistant Candidaspp). Rezafungin, an echinocandin with an extended half-life allowing for once weekly administration, will be particularly valuable for outpatient treatment and prophylaxis. Despite this, there is an urgent need to garner clinical data on investigational drugs, especially in the current rise of azole-resistant and multidrug-resistant Candidaspp.

The discovery of antibiotics more than 80 years ago has led to considerable improvements in human and animal health. Although antibiotic resistance in environmental bacteria is ancient, resistance in human pathogens is thought to be a modern phenomenon that is driven by the clinical use of antibiotics1. Here we show that particular lineages of methicillin-resistant Staphylococcus aureus—a notorious human pathogen—appeared in European hedgehogs in the pre-antibiotic era. Subsequently, these lineages spread within the local hedgehog populations and between hedgehogs and secondary hosts, including livestock and humans. We also demonstrate that the hedgehog dermatophyte Trichophyton erinaceiproduces two β-lactam antibiotics that provide a natural selective environment in which methicillin-resistant S. aureusisolates have an advantage over susceptible isolates. Together, these results suggest that methicillin resistance emerged in the pre-antibiotic era as a co-evolutionary adaptation of S. aureusto the colonization of dermatophyte-infected hedgehogs. The evolution of clinically relevant antibiotic-resistance genes in wild animals and the connectivity of natural, agricultural and human ecosystems demonstrate that the use of a One Health approach is critical for our understanding and management of antibiotic resistance, which is one of the biggest threats to global health, food security and development.

HIV pseudotypes appear to be able to infect both CD4-positive and CD4-negative cells. Pseudotypes are genetically mixed viruses. Pseudotypes may occur when two viruses infect the same cell. CD4 cells are components of the immune system. An HIV-HTLV-I pseudotype was prepared. Both CD4-positive and CD4-negative cells were infected by the pseudotype. Generally, CD4-negative cells are not infected by HIV. An anti-HTLV-I serum effectively stopped infection of CD4-negative cells by the pseudotype. An anti-HIV serum did not prevent infection of CD4-negative cells. Lectins and anti-carbohydrate monoclonal antibodies prevented CD4-negative cell infection. This suggests that the infective ability of HIV may be increased in cases of coinfection with human T-cell lymphotropic virus type I (HTLV-I). Many HIV-positive patients are also infected with HTLV-I.

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The virulence of isolates among different Candidaspecies causing candidemia may play a role in the prognosis of the patients. Furthermore, the potential relationship between genotype and virulence is still unclear and need to be further studied.

Rhino-orbital-cerebral mucormycosis (ROCM) is a rare fulminant opportunistic fungal infection that despite relevant treatment has high mortality. We present a case of a 3-year-old girl with acute lymphoblastic leukemia and ROCM, who was treated successfully with excessive surgery, systemic antifungal treatment with amphotericin B (AmB), posaconazole, and terbinafine as well as hyperbaric oxygen. Surgery included, beside extracranial and intracranial removal of infected areas, endoscopic sinus and skull base surgery with local AmB installation and in addition placement of an Ommaya reservoir for 114 intrathecal administrations of AmB. In addition, we review the literature of ROCM in pediatric patients with hematological diseases.

ABSTRACTASP2397 is a new compound with a novel and as-yet-unknown target different from that of licensed antifungal agents. It has activity against Aspergillusand Candida glabrata. We compared its in vitroactivity against wild-type and azole-resistant A. fumigatusand A. terreusisolates with that of amphotericin B, itraconazole, posaconazole, and voriconazole. Thirty-four isolates, including 4 wild-type A. fumigatusisolates, 24 A. fumigatusisolates with alterations in CYP51A TR/L98H (5 isolates), M220 (9 isolates), G54 (9 isolates), and HapE (1 isolate), and A. terreusisolates (2 wild-type isolates and 1 isolate with an M217I CYP51A alteration), were analyzed. EUCAST E.Def 9.2 and CLSI M38-A2 MIC susceptibility testing was performed. ASP2397 MIC50values (in milligrams per liter, with MIC ranges in parentheses) determined by EUCAST and CLSI were 0.5 (0.25 to 1) and 0.25 (0.06 to 0.25) against A. fumigatusCYP51A wild-type isolates and were similarly 0.5 (0.125 to >4) and 0.125 (0.06 to >4) against azole-resistant A. fumigatusisolates, respectively. These values were comparable to those for amphotericin B, which were 0.25 (0.125 to 0.5) and 0.25 (0.125 to 0.25) against wild-type isolates and 0.25 (0.125 to 1) and 0.25 (0.125 to 1) against isolates with azole resistance mechanisms, respectively. In contrast, MICs for the azole compounds were elevated and highest for itraconazole: >4 (1 to >4) and 4 (0.5 to >4) against isolates with azole resistance mechanisms compared to 0.125 (0.125 to 0.25) and 0.125 (0.06 to 0.25) against wild-type isolates, respectively. ASP2397 was active against A. terreusCYP51A wild-type isolates (MIC 0.5 to 1), whereas MICs of both azole and ASP2397 were elevated for the mutant isolate. ASP2397 displayed in vitroactivity against A. fumigatusand A. terreusisolates which was independent of the presence or absence of azole target gene resistance mutations in A. fumigatus. The findings are promising at a time when azole-resistant A. fumigatusis emerging globally.

ABSTRACTThe in vitroactivity of isavuconazole against Mucoralesisolates measured by EUCAST E.Def 9.2 and CLSI M38-A2 methodologies was investigated in comparison with those of amphotericin B, posaconazole, and voriconazole. Seventy-two isolates were included: 12 of Lichtheimia corymbifera, 5 of Lichtheimia ramosa, 5 of group I and 9 of group II of Mucor circinelloides, 9 of Rhizomucor pusillus, 26 of Rhizopus microsporus, and 6 of Rhizopus oryzae. Species identification was confirmed by internal transcribed spacer (ITS) sequencing. EUCAST MICs were read on day 1 (EUCAST-d1) and day 2 (EUCAST-d2), and CLSI MICs were read on day 2 (CLSI-d2). Isavuconazole MIC50s (range) (mg/liter) by EUCAST-d1, CLSI-d2, and EUCAST-d2 were 1 (0.125 to 16), 1 (0.125 to 2), and 4 (0.5 to >16), respectively, across all isolates. The similar values for comparator drugs were as follows: posaconazole, 0.25 (≤0.03 to >16), 0.25 (0.06 to >16), and 1 (0.06 to >16); amphotericin, 0.06 (≤0.03 to 0.5), 0.06 (≤0.03 to 0.25), and 0.125 (≤0.03 to 1); voriconazole, 16 (2 to >16), 8 (1 to >16), and >16 (8 to >16), respectively. Isavuconazole activity varied by species: Lichtheimia corymbifera, 1 (0.5 to 2), 1 (1 to 2), and 2 (1 to 4); Lichtheimia ramosa, 0.25 (0.125 to 0.5), 1 (0.5 to 2), and 2 (0.5 to 4); Rhizomucor pusillus, 0.5 (0.5 to 1), 1 (0.125 to 1), and 2 (1 to 2); Rhizopus microsporus, 1 (0.5 to 4), 0.5 (0.125 to 1), and 4 (1 to 8); and Rhizopus oryzae, 1 (0.5 to 4), 1 (0.125 to 2), and 4 (0.5 to 8), respectively, were more susceptible than Mucor circinelloides: group I, 8 (4 to 8), 4 (2 to 4), and 16 (2 to 16), respectively, and group II, 8 (1 to 16), 8 (1 to 8), and 16 (4 to >16), respectively. This was also observed for posaconazole. The essential agreement was best between EUCAST-d1 and CLSI-d2 (75% to 83%). Isavuconazole displayed in vitroactivity against Mucoralesisolates with the exception of Mucor circinelloides. The MICs were in general 1 to 3 steps higher than those for posaconazole. However, in the clinical setting this may be compensated for by the higher exposure at standard dosing.

ABSTRACTThe EUCAST E.DEF9.1 standard recommends standardization of the inoculum concentration by conidium counting using a hemocytometer rather than a spectrophotometer. In this study, we investigated whether the choice of these methods influenced isavuconazole MICs. A blinded collection of 30 molecularly characterized azole-resistant isolates and 10 wild-type Aspergillus fumigatusisolates was shared with four different laboratories. Additionally, each laboratory selected approximately 100 A. fumigatusisolates and 50 isolates each of A. flavus, A. nidulans, A. niger, and A. terreus(1,237 isolates in total). Three laboratories (laboratories 1 to 3) used conidium counting. One laboratory standardized the inoculum using a spectrophotometer (that is, by use of the optical density [OD]) and is referred to as the OD laboratory. Correlation coefficients, intraclass correlation coefficients, and essential agreement were calculated, and 2-log-unit differences were assessed (paired ttest). The MIC range for the blinded collection was 0.25 to 16 mg/liter, and a 1-dilution-step difference between the MIC50and MIC90across the four laboratories was detected and a 2-dilution-step difference between the modal MICs was detected. Compared to the results for laboratories 1 and 2, a significant correlation was found for the OD laboratory MIC data (correlation coefficients, 0.85 and 0.93, respectively; intraclass correlation coefficients, 0.88 and 0.96, respectively). The number of mutant isolates whose MICs overlapped those of the wild-type isolates was the lowest for the OD laboratory (14/30 [46.7%] mutant isolates), whereas the numbers were 18/30 (60%) isolates for laboratory 1, 17/30 (56.7%) isolates for laboratory 2, and 21/30 (70%) isolates for laboratory 3. For the A. flavus, A. fumigatus, A. nidulans, A. niger, and A. terreusisolates, comparative analysis again defined the MIC distributions from the OD laboratory to be in excellent agreement with those from laboratories 1 and 2 across all five Aspergillusspp. The findings suggest that EUCAST testing using OD determination is an appropriate alternative for standardization of Aspergillusinoculum concentrations.

ABSTRACTIsavuconazole is a novel expanded-spectrum triazole, which has recently been approved by the FDA as an orphan drug to treat invasive aspergillosis and is currently being studied in phase III clinical trials for invasive candidiasis. The susceptibility of relatively few clinical isolates has been reported. In this study, the isavuconazole susceptibilities of 1,237 Aspergillusand 2,010 Candidageographically diverse clinical isolates were determined by EUCAST methodology at four European mycology laboratories, producing the largest multicenter data set thus far for this compound. In addition, a blinded collection of 30 cyp51Amutant Aspergillus fumigatusclinical isolates and 10 wild-type isolates was tested. From these two data sets, the following preliminary epidemiological cutoff (ECOFF) values were suggested: 2 mg/liter for Aspergillus fumigatus, Aspergillus terreus, and Aspergillus flavus; 4 mg/liter for Aspergillus niger; 0.25 mg/liter for Aspergillus nidulans; and 0.03 mg/liter for Candida albicans, Candida parapsilosis, and Candida tropicalis. Unfortunately, ECOFFs could not be determined for Candida glabrataor Candida kruseidue to an unexplained interlaboratory MIC variation. For the blinded collection of A. fumigatusisolates, all MICs were ≤2 mg/liter for wild-type isolates. Differential isavuconazole MICs were observed for triazole-resistant A. fumigatusisolates with different cyp51Aalterations: TR34/L98H mutants had elevated isavuconazole MICs, whereas isolates with G54 and M220 alterations had MICs in the wild-type range, suggesting that the efficacy of isavuconazole may not be affected by these alterations. This study will be an aid in interpreting isavuconazole MICs for clinical care and an important step in the future process of setting official clinical breakpoints.

ABSTRACTFKSmutant isolates associated with breakthrough or failure cases are emerging in clinical settings. Discrimination of these from wild-type (wt) isolates in a routine laboratory setting is complicated. We evaluated the ability of caspofungin MIC determination using the new Vitek 2 AST-Y06 yeast susceptibility card to correctly identify the fksmutants from wt isolates and compared the performance to those of the CLSI and EUCAST reference methods. A collection of 98 Candidaisolates, including 31 fkshot spot mutants, were included. Performance was evaluated using the FKSgenotype as the “gold standard” and compared to those of the CLSI and EUCAST methodologies. The categorical agreement for Vitek 2 was 93.9%, compared to 88.4% for the CLSI method and 98.7% for the EUCAST method. Vitek 2 misclassified 19.4% (6/31) of the fksmutant isolates as susceptible, in contrast to <4% for each of the reference methods. The overall essential agreement between the CLSI method and Vitek 2 MICs was 92.6% (88/95) but was substantially lower for fksmutant isolates (78.6% [22/28]). Correct discrimination between susceptible and intermediate Candida glabrataisolates was not possible, as the revised species-specific susceptibility breakpoint was not included in the Vitek 2 detection range (MIC of ≤0.250 to ≥4 mg/liter). In conclusion, the Vitek 2 allowed correct categorization of all wt isolates as susceptible. However, despite an acceptable categorical agreement, it failed to reliably classify isolates harboring fkshot spot mutations as intermediate or resistant, which was in part due to the fact that the detection range did not span the susceptibility breakpoint for C. glabrata.

ABSTRACTThree Candida tropicalisisolates were obtained from a patient with acute lymphoblastic leukemia. The first isolate was susceptible to all drug classes, while isolates 2 and 3, obtained after 8 and 8.5 weeks of caspofungin treatment, respectively, were resistant to the three echinocandins. Multilocus sequence genotyping suggested a clonal relation among all isolates. FKS1sequencing revealed a stepwise development of a heterozygous and finally a homozygous mutation, leading to S80S/P and S80P amino acid substitutions.

ABSTRACTNinety-six strains of Candida, including 29 resistant and 67 susceptible isolates with mutations in the FKS1and FKS2genes were tested by the European Committee on Antibiotic Susceptibility Testing EDef 7.1 and 7.2 methodologies to determine the impact on the MIC when water was replaced with dimethyl sulfoxide (DMSO) as the solvent for caspofungin and micafungin. The MICs were significantly lower and the MIC ranges were narrower when DMSO was used as the solvent. The use of DMSO may help to better discriminate between susceptible and resistant populations.

ABSTRACTThe purpose of this study was to evaluate the performance of caspofungin Etest and the recently revised CLSI breakpoints. A total of 497 blood isolates, of which 496 were wild-type isolates, were included. A total of 65/496 susceptible isolates (13.1%) were misclassified as intermediate (I) or resistant (R). Such misclassifications were most commonly observed for Candida krusei(73.1%) and Candida glabrata(33.1%). The revised breakpoints cannot be safely adopted for these two species.

Recent advances in the molecular diagnostics of dermatophytosis may improve speed, specificities and sensitivities. This review provides an update on the current available molecular techniques for the diagnosis of dermatophytosis.

ABSTRACTWe recently observed that the micafungin MICs for some Candida glabrata fkshot spot mutant isolates are less elevated than those for the other echinocandins, suggesting that the efficacy of micafungin may be differentially dependent on such mutations. Three clinical C. glabrataisolates with or without (S3) fkshot spot mutations R83 (Fks2p-S663F) and RR24 (Fks1p-S629P) and low, medium, and high echinocandin MICs, respectively, were evaluated to assess the in vivoefficacy in an immunocompetent mouse model using three doses of each echinocandin. Drug concentrations were determined in plasma and kidneys by high-performance liquid chromatography (HPLC). A pharmacokinetic-pharmacodynamic mathematical model was used to define the area under the concentration-time curve (AUC) that produced half- and near-maximal activity. Micafungin was equally efficacious against the S3 and R83 isolates. The estimates for the AUCs of each echinocandin that induced half-maximal effect (E50s) were 194.2 and 53.99 mg · h/liter, respectively. In contrast, the maximum effect (Emax) for caspofungin was higher against S3 than R83, but the estimates for E50were similar (187.1 and 203.5 mg · h/liter, respectively). Anidulafungin failed to induce a ≥1-log reduction for any of the isolates (AUC range, 139 to 557 mg · h/liter). None of the echinocandins were efficacious in mice challenged with the RR24 isolate despite lower virulence (reduced maximal growth, prolonged lag phase, and lower kidney burden). The AUC associated with half-maximal effect was higher than the average human exposure for all drug-dose-bug combinations except micafungin and the R83 isolate. In conclusion, differences in micafungin MICs are associated with differential antifungal activities in the animal model. This study may have implications for clinical practice and echinocandin breakpoint determination, and further studies are warranted.

We recently observed that the micafungin MICs for some Candida glabrata fks hot spot mutant isolates are less elevated than those for the other echinocandins, suggesting that the efficacy of micafungin may be differentially dependent on such mutations. Three clinical C. glabrata isolates with or without (S3) fks hot spot mutations R83 (Fks2p-S663F) and RR24 (Fks1p-S629P) and low, medium, and high echinocandin MICs, respectively, were evaluated to assess the in vivo efficacy in an immunocompetent mouse model using three doses of each echinocandin. Drug concentrations were determined in plasma and kidneys by high-performance liquid chromatography (HPLC). A pharmacokinetic-pharmacodynamic mathematical model was used to define the area under the concentration-time curve (AUC) that produced half- and near-maximal activity. Micafungin was equally efficacious against the S3 and R83 isolates. The estimates for the AUCs of each echinocandin that induced half-maximal effect (E50s) were 194.2 and 53.99 mg · h/liter, respectively. In contrast, the maximum effect (Emax) for caspofungin was higher against S3 than R83, but the estimates for E50were similar (187.1 and 203.5 mg · h/liter, respectively). Anidulafungin failed to induce a ≥1-log reduction for any of the isolates (AUC range, 139 to 557 mg · h/liter). None of the echinocandins were efficacious in mice challenged with the RR24 isolate despite lower virulence (reduced maximal growth, prolonged lag phase, and lower kidney burden). The AUC associated with half-maximal effect was higher than the average human exposure for all drug-dose-bug combinations except micafungin and the R83 isolate. In conclusion, differences in micafungin MICs are associated with differential antifungal activities in the animal model. This study may have implications for clinical practice and echinocandin breakpoint determination, and further studies are warranted.

ABSTRACTThis study investigated microbiological, clinical, and management issues and outcomes for Danish fungemia patients. Isolates and clinical information were collected at six centers. A total of 334 isolates, 316 episodes, and 305 patients were included, corresponding to 2/3 of the national episodes. Blood culture positivity varied by system, species, and procedure. Thus, cases with concomitant bacteremia were reported less commonly by BacT/Alert than by the Bactec system (9% [11/124 cases] versus 28% [53/192 cases]; P< 0.0001), and cultures with Candida glabrataor those drawn via arterial lines needed longer incubation. Species distribution varied by age, prior antifungal treatment (57% occurrence of C. glabrata, Saccharomyces cerevisiae, or C. kruseiin patients with prior antifungal treatment versus 28% occurrence in those without it; P= 0.007), and clinical specialty (61% occurrence of C. glabrataor C. kruseiin hematology wards versus 27% occurrence in other wards; P= 0.002). Colonization samples were not predictive for the invasive species in 11/100 cases. Fifty-six percent of the patients had undergone surgery, 51% were intensive care unit (ICU) patients, and 33% had malignant disease. Mortality increased by age (P= 0.009) and varied by species (36% for C. krusei, 25% for C. parapsilosis, and 14% for other Candidaspecies), severity of underlying disease (47% for ICU patients versus 24% for others; P= 0.0001), and choice but not timing of initial therapy (12% versus 48% for patients with C. glabratainfection receiving caspofungin versus fluconazole; P= 0.023). The initial antifungal agent was deemed suboptimal upon species identification in 15% of the cases, which would have been 6.5% if current guidelines had been followed. A large proportion of Danish fungemia patients were severely ill and received suboptimal initial antifungal treatment. Optimization of diagnosis and therapy is possible.

ABSTRACTThe susceptibilities of Candidaisolates to the echinocandins anidulafungin, caspofungin, and micafungin were determined by using the recently revised CLSI breakpoints and Etest on 238 clinical bloodstream Candidaisolates collected between September 2005 and August 2006. The isolates represent approximately 95% of all non-albicans Candidabloodstream infections and one-third of Candida albicansbloodstream infections during this 1-year period in Sweden. The collection included 81 C. albicans, 81 C. glabrata, 36 C. parapsilosis, 14 C. dubliniensis, 8 C. tropicalis, 8 C. lusitaniae, 5 C. krusei, 2 C. guilliermondiiand 2 C. inconspicuaisolates as well as 1 C. pelliculosaisolate. The MICs were largely consistent with the global epidemiology of bloodstream Candidaisolates. All C. albicansand C. glabrataisolates were susceptible to all 3 echinocandins (MIC = 0.016 µg/ml in all instances). Resistance (MIC = 8 µg/ml) to anidulafungin alone was observed for 4 (11.1%) C. parapsilosisisolates and for 1/2 C. guilliermondiiisolates. Intermediate susceptibility to caspofungin alone was observed for 2/5 C. kruseiisolates. One of the eight C. tropicalisisolates was classified as being intermediately susceptible to micafungin (MIC, 0.5 µg/ml) and as being resistant to anidulafungin and caspofungin (MIC = 1 µg/ml). This isolate harbored a heterozygous FKS1hot spot mutation (S80P) known to confer echinocandin resistance. This first study to apply the revised CLSI breakpoints for Etest endpoints showed that the breakpoints worked successfully in detecting an isolate with a hot spot mutation. Acquired echinocandin resistance is rare in Sweden. Echinocandin MICs against C. parapsilosisand C. guilliermondiiwere lowest for micafungin.

Respiratory tract colonization by molds in patients with cystic fibrosis (CF) were analyzed, with particular focus on the frequency, genotype, and underlying mechanism of azole resistance among Aspergillus fumigatus isolates. Clinical and demographic data were also analyzed. A total of 3,336 respiratory samples from 287 CF patients were collected during two 6-month periods in 2007 and 2009. Azole resistance was detected using an itraconazole screening agar (4 mg/liter) and the EUCAST method. cyp51A gene sequencing and microsatellite genotyping were performed for isolates from patients harboring azole-resistant A. fumigatus. Aspergillus spp. were present in 145 patients (51%), of whom 63 (22%) were persistently colonized. Twelve patients (4%) harbored other molds. Persistently colonized patients were older, provided more samples, and more often had a chronic bacterial infection. Six of 133 patients (4.5%) harbored azole-nonsusceptible or -resistant A. fumigatus isolates, and five of those six patients had isolates with Cyp51A alterations (M220K, tandem repeat [TR]/L98H, TR/L98H-S297T-F495I, M220I-V101F, and Y431C). All six patients were previously exposed to azoles. Genotyping revealed (i) microevolution for A. fumigatus isolates received consecutively over the 2-year period, (ii) susceptible and resistant isolates (not involving TR/L98H isolates) with identical or very closely related genotypes (two patients), and (iii) two related susceptible isolates and a third unrelated resistant isolate with a unique genotype and the TR/L98H resistance combination (one patient). Aspergilli were frequently found in Danish CF patients, with 4.5% of the A. fumigatus isolates being azole nonsusceptible or resistant. Genotyping suggested selection of resistance in the patient as well as resistance being achieved in the environment.

ABSTRACTRespiratory tract colonization by molds in patients with cystic fibrosis (CF) were analyzed, with particular focus on the frequency, genotype, and underlying mechanism of azole resistance among Aspergillus fumigatusisolates. Clinical and demographic data were also analyzed. A total of 3,336 respiratory samples from 287 CF patients were collected during two 6-month periods in 2007 and 2009. Azole resistance was detected using an itraconazole screening agar (4 mg/liter) and the EUCAST method. cyp51Agene sequencing and microsatellite genotyping were performed for isolates from patients harboring azole-resistant A. fumigatus. Aspergillusspp. were present in 145 patients (51%), of whom 63 (22%) were persistently colonized. Twelve patients (4%) harbored other molds. Persistently colonized patients were older, provided more samples, and more often had a chronic bacterial infection. Six of 133 patients (4.5%) harbored azole-nonsusceptible or -resistant A. fumigatusisolates, and five of those six patients had isolates with Cyp51A alterations (M220K, tandem repeat [TR]/L98H, TR/L98H-S297T-F495I, M220I-V101F, and Y431C). All six patients were previously exposed to azoles. Genotyping revealed (i) microevolution for A. fumigatusisolates received consecutively over the 2-year period, (ii) susceptible and resistant isolates (not involving TR/L98H isolates) with identical or very closely related genotypes (two patients), and (iii) two related susceptible isolates and a third unrelated resistant isolate with a unique genotype and the TR/L98H resistance combination (one patient). Aspergilli were frequently found in Danish CF patients, with 4.5% of the A. fumigatusisolates being azole nonsusceptible or resistant. Genotyping suggested selection of resistance in the patient as well as resistance being achieved in the environment.

ABSTRACTThe MICs of echinocandins against Candidaisolates with fksmutations are higher than those for wild-type (WT) isolates. However, the MIC ranges for susceptible and mutant populations overlap or are poorly separated. It was recently reported that a greater separation could be achieved in the presence of serum. To more fully explore this possibility, we compared the performances of the reference microdilution methods by using standard and bovine serum albumin (BSA)-supplemented growth medium. Anidulafungin, caspofungin, and micafungin MICs were determined according to EUCAST and CLSI methods and with 50% BSA in the medium for 93 clinical isolates, including Candida albicans(20/10 [number of isolates/number of mutants]), C. glabrata(19/10), C. dubliniensis(2/1), C. krusei(16/3), C. parapsilosis(19), and C. tropicalis(19/4) isolates. Stability of the plates was tested after storage at −80°C for 2 and 6 months, and the performance of two different lots of caspofungin was investigated. The addition of BSA to the medium resulted in higher MICs (1 to 9 2-fold dilution steps) for all isolates and compounds. The increases were greatest for anidulafungin and micafungin and, among WT isolates, for C. parapsilosis. The number of very major errors (VMEs) was reduced (24% [20/84 isolates] versus ≤7% [6/84 isolates]) using BSA-supplemented EUCAST medium but not using BSA-supplemented CLSI medium (6% versus 9%). MIC results were unchanged after 6 months of storage of test plates. The two lots of caspofungin yielded identical results. Addition of BSA to the EUCAST medium increases the ability to differentiate between WT isolates and isolates harboring resistance mutations.

ABSTRACTDisk diffusion testing has recently been standardized by the CLSI, and susceptibility breakpoints have been established for several antifungal compounds. For caspofungin, 5-μg disks are approved, and for micafungin, 10-μg disks are under evaluation. We evaluated the performances of caspofungin and micafungin disk testing using a panel of Candidaisolates with and without known FKSechinocandin resistance mechanisms. Disk diffusion and microdilution assays were performed strictly according to CLSI documents M44-A2 and M27-A3. Eighty-nine clinical Candidaisolates were included: Candida albicans(20 isolates/10 mutants), C. glabrata(19 isolates/10 mutants), C. dubliniensis(2 isolates/1 mutant), C. krusei(16 isolates/3 mutants), C. parapsilosis(14 isolates/0 mutants), and C. tropicalis(18 isolates/4 mutants). Quality control strains were C. parapsilosisATCC 22019 and C. kruseiATCC 6258. The correlations between zone diameters and MIC results were good for both compounds, with identical susceptibility classifications for 93.3% of the isolates by applying the current CLSI breakpoints. However, the numbers of fkshot spot mutant isolates misclassified as being susceptible (S) (very major errors [VMEs]) were high (61% for caspofungin [S, ≥11 mm] and 93% for micafungin [S, ≥14 mm]). Changing the disk diffusion breakpoint to S at ≥22 mm significantly improved the discrimination. For caspofungin, 1 VME was detected (a C. tropicalisisolate with an F76S substitution) (3.5%), and for micafungin, 10 VMEs were detected, the majority of which were for C. glabrata(8/10). The broadest separation between zone diameter ranges for wild-type (WT) and mutant isolates was seen for caspofungin (6 to 12 mm versus −4 to 7 mm). In conclusion, caspofungin disk diffusion testing with a modified breakpoint led to excellent separation between WT and mutant isolates for all Candidaspecies.

ABSTRACTCandida palmioleophilahas previously been misidentified as C. famataor C. guilliermondii. We have investigated traditional and modern identification methods for the identification of this and related species. Forty-one clinical isolates previously identified as C. famataor C. guilliermondiiand 8 reference strains were included. Color development on CHROMagar, growth temperature ranges, micromorphologies, carbon assimilation (ID32C), matrix-assisted laser desorption ionization–time of flight mass spectrometry (MALDI-TOF MS) profiles, and susceptibility profiles (mica- and anidulafungin and itra-, vori-, posa-, and fluconazole MICs were determined by EUCAST method EDef 7.1, and caspofungin MICs were determined by Etest) were determined, and results were compared to those of molecular identification (ITS1 and ITS2 sequencing). The following five different species were identified among the clinical isolates by sequencing, but no C. famataisolates were found: C. guilliermondii(22 isolates), C. palmioleophila(8 isolates), C. fermentati(6 isolates), C. lusitaniae(3 isolates), and C. intermedia(2 isolates). C. palmioleophiladeveloped a distinct scintillating color of turquoise to rose, grew at 40°C, and failed to produce pseudohyphae within 14 days. The ID32C profile for 7/9 C. palmioleophilaisolates was 5367352315, and all were unable to hydrolyze esculin (Esc). The six related species were well discriminated by MALDI-TOF MS. The susceptibility pattern for C. palmioleophilawas unique, as the echinocandin MICs were low (range, 0.008 to 0.125 µg/ml) and fluconazole MICs were high (range, 8 to >16 µg/ml). Correct identification of C. palmioleophilais important due to its unique susceptibility profile. Identification is possible yet laborious with conventional techniques, whereas MALDI-TOF MS easily separated the related species.

ABSTRACTA 6-year nationwide study of fungemia in Denmark was performed using data from an active fungemia surveillance program and from laboratory information systems in nonparticipating regions. A total of 2,820 episodes of fungemia were recorded. The incidence increased from 2004 to 2007 (7.7 to 9.6/100,000) and decreased slightly from 2008 to 2009 (8.7 to 8.6/100,000). The highest incidences were seen at the extremes of age (i.e., 11.3 and 37.1/100,000 for those <1 and 70 to 79 years old, respectively). The rate was higher for males than for females (10.1 versus 7.6/100,000, P= 0.003), with the largest difference observed for patients >50 years of age. The species distribution varied significantly by both age and gender. Candidaspecies accounted for 98% of the pathogens, and C. albicanswas predominant, although the proportion decreased (64.4% to 53.2%, P< 0.0001). C. glabrataranked second, and the proportion increased (16.5% to 25.9%, P= 0.003). C. glabratawas more common in adults and females than in children and males, whereas C. tropicaliswas more common in males (P= 0.020). C. kruseiwas a rare isolate (4.1%) except at one university hospital. Acquired resistance to amphotericin and echinocandins was rare. However, resistance to fluconazole (MIC of >4 µg/ml) occurred in C. albicans(7/1,183 [0.6%]), C. dubliniensis(2/65 [3.1%]), C. parapsilosis(5/83 [6.0%]), and C. tropicalis(7/104 [6.7%]). Overall, 70.8% of fungemia isolates were fully fluconazole susceptible, but the proportion decreased (79.7% to 68.9%, P= 0.02). The study confirmed an incidence rate of fungemia in Denmark three times higher than those in other Nordic countries and identified marked differences related to age and gender. Decreased susceptibility to fluconazole was frequent and increasing.

ABSTRACTA single mechanism of azole resistance was shown to predominate in clinical and environmental Aspergillus fumigatusisolates from the Netherlands, and a link to the use of azoles in the environment was suggested. To explore the prevalence of azole-resistant A. fumigatusand other aspergilli in the environment in other European countries, we collected samples from the surroundings of hospitals in Copenhagen, Innsbruck, and Madrid, flowerbeds in an amusement park in Copenhagen, and compost bags purchased in Austria, Denmark, and Spain and screened for azole resistance using multidish agars with itraconazole, voriconazole, and posaconazole. EUCAST method E.DEF 9.1 was used to confirm azole resistance. The promoter and entire coding sequence of the cyp51Agene were sequenced to identify azole-resistant A. fumigatusisolates. A. fumigatuswas recovered in 144 out of 185 samples (77.8%). Four A. fumigatusisolates from four Danish soil samples displayed elevated azole MICs (8%), and all harbored the same TR/L98H mutation of cyp51A. One A. lentulusisolate with voriconazole MIC of 4 mg/liter was detected in Spain. No azole-resistant aspergilli were detected in compost. Finally, A. terreuswas present in seven samples from Austria. Multi-azole-resistant A. fumigatusis present in the environment in Denmark. The resistance mechanism is identical to that of environmental isolates in the Netherlands. No link to commercial compost could be detected. In Spain and Austria, only Aspergillusspecies with intrinsic resistance to either azoles or amphotericin B were found.

This review covers candidaemia in numbers, susceptibility issues, host groups, risk factors and outcome.

ABSTRACTThis study compared nine susceptibility testing methods and 12 endpoints for anidulafungin, caspofungin, and micafungin with the same collection of blinded FKShot spot mutant (n= 29) and wild-type isolates (n= 94). The susceptibility tests included EUCAST Edef 7.1, agar dilution, Etest, and disk diffusion with RPMI-1640 plus 2% glucose (2G) and IsoSensitest-2G media and CLSI M27A-3. Microdilution plates were read after 24 and 48 h. The following test parameters were evaluated: fkshot spot mutants overlapping the wild-type distribution, distance between the two populations, number of very major errors (VMEs; fksmutants misclassified as susceptible), and major errors (MEs; wild-type isolates classified as resistant) using a wild-type-upper-limit value (WT-UL) (two twofold-dilutions higher than the MIC50) as the susceptibility breakpoint. The methods with the lowest number of errors (given as VMEs/MEs) across the three echinocandins were CLSI (12%/1%), agar dilution with RPMI-2G medium (14%/0%), and Etest with RPMI-2G medium (8%/3%). The fewest errors overall were observed for anidulafungin (4%/1% for EUCAST, 4%/3% for CLSI, and 3%/9% for Etest with RPMI-2G). For micafungin, VME rates of 10 to 71% were observed. For caspofungin, agar dilution with either medium was superior (VMEs/MEs of 0%/1%), while CLSI, EUCAST with IsoSensitest-2G medium, and Etest were less optimal (VMEs of 7%, 10%, and 10%, respectively). Applying the CLSI breakpoint (S ≤ 2 μg/ml) for CLSI results, 89.2% fkshot spot mutants were classified as anidulafungin susceptible, 60.7% as caspofungin susceptible, and 92.9% as micafungin susceptible. In conclusion, no test was perfect, but anidulafungin susceptibility testing using the WT-UL to define susceptibility reliably identified fkshot spot mutants.

ABSTRACTCaspofungin is used for the treatment of acute invasive candidiasis and as salvage treatment for invasive aspergillosis. We report characteristics of isolates of Candida albicansand Aspergillus fumigatusdetected in a patient with breakthrough infection complicating severe gastrointestinal surgery and evaluate the capability of susceptibility methods to identify candin resistance. The susceptibility of C. albicansto caspofungin and anidulafungin was investigated by Etest, microdilution (European Committee on Antibiotic Susceptibility Testing [EUCAST] and CLSI), disk diffusion, agar dilution, and FKS1sequencing and in a mouse model. Tissue was examined by immunohistochemistry, PCR, and sequencing for the presence of A. fumigatusand resistance mutations. The MICs for the C. albicansisolate were as follows: >32 μg/ml caspofungin and 0.5 μg/ml anidulafungin by Etest, 2 μg/ml caspofungin and 0.125 μg/ml anidulafungin by EUCAST methods, and 1 μg/ml caspofungin and 0.5 μg/ml anidulafungin by CLSI methods. Sequencing of the FKS1gene revealed a mutation leading to an S645P substitution. Caspofungin and anidulafungin failed to reduce kidney CFU counts in animals inoculated with this isolate (P> 0.05 compared to untreated control animals), while both candins completely sterilized the kidneys in animals infected with a control isolate. Disk diffusion and agar dilution methods clearly separated the two isolates. Immunohistochemistry and sequencing confirmed the presence of A. fumigatuswithout FSK1resistance mutations in liver and lung tissues. Breakthrough disseminated aspergillosis and candidiasis developed despite an absence of characteristic FKS1resistance mutations in the Aspergillusisolates. EUCAST and CLSI methodology did not separate the candin-resistant clinical isolate from the sensitive control isolate as well as did the Etest and agar methods.

ABSTRACTTwo clinical isolates of Aspergillus fumigatus, designated AT and DK, were recently obtained from patients failing caspofungin and itraconazole therapy, respectively. The isolates were tested by microdilution for susceptibility to itraconazole, voriconazole, posaconazole, ravuconazole, and caspofungin and by Etest for susceptibility to amphotericin B and caspofungin. Susceptibility testing documented that the DK isolate was azole resistant (itraconazole and posaconazole MICs, >4 μg/ml; voriconazole MIC, 2 μg/ml; ravuconazole MIC, 4 μg/ml), and the resistance was confirmed in a hematogenous mouse model, with mortality and the galactomannan index as the primary and secondary end points. Sequencing of the cyp51Agene revealed the M220K mutation, conferring multiazole resistance. The Etest, but not microdilution, suggested that the AT isolate was resistant to caspofungin (MIC, >32 μg/ml). In the animal model, this isolate showed reduced susceptibility to caspofungin. Sequencing of the FKS1gene revealed no mutations; the enzyme retained full sensitivity in vitro; and investigation of the polysaccharide composition showed that the β-(1,3)-glucan proportion was unchanged. However, gene expression profiling by Northern blotting and real-time PCR demonstrated that the FKSgene was expressed at a higher level in the AT isolate than in the susceptible control isolate. To our knowledge, this is the first report to document the presence of multiazole-resistant clinical isolates in Denmark and to demonstrate reduced susceptibility to caspofungin in a clinical A. fumigatusisolate with increased expression of the FKSgene. Further research to determine the prevalence of resistance in A. fumigatusworldwide, and to develop easier and reliable tools for the identification of such isolates in routine laboratories, is warranted.

ABSTRACTWe developed the simple, rapid (1 h), and accurate PNA FISHFlowmethod for the identification of Candida albicans. The method exploits unique in solution in situ hybridization conditions under which the cells are simultaneously fixed and hybridized. This method facilitates the accurate identification of clinical yeast isolates using two scoring techniques: flow cytometry and fluorescence microscopy.

A rapid two-step DNA extraction method and a multiplex PCR for the detection of dermatophytes in general and Trichophyton rubrum specifically were developed and evaluated with DNA extracted from pure cultures and from clinically diseased nails. DNA from the following dermatophytes was used: Epidermophyton floccosum, Microsporum audouinii, Microsporum canis, Microsporum gypseum, Microsporum nanum, Trichophyton mentagrophytes, Trichophyton rubrum, Trichophyton schoenleinii, Trichophyton soudanense, Trichophyton terrestre, Trichophyton tonsurans, Trichophyton verrucosum, and Trichophyton violaceum. Human DNA and DNA from the following nondermatophyte fungi were included as controls: Alternaria, Aspergillus niger, Candida albicans, Candida glabrata, Candida krusei, Malassezia furfur, Saccharomyces cerevisiae, and Scopulariopsis brevicaulis. A total of 118 nail samples received for routine microscopy and culture for dermatophytes were subsequently tested by the two PCRs separately and in a multiplex format. Using DNA extracted from pure cultures and the pan-dermatophyte PCR, the T. rubrum-specific PCR sequentially and in a multiplex format correctly detected all dermatophytes and additionally correctly identified T. rubrum. Comparison of the traditional diagnostic evaluation (microscopy and culture) of nail samples with PCR on DNA directly extracted from the nails showed excellent agreement between PCR and microscopy, but the number of samples with dermatophyte species identification was increased considerably from 22.9% to 41.5%, mainly due to the identification of T. rubrum by PCR in microscopy-positive but culture-negative samples. In conclusion, this 5-hour diagnostic test was shown to increase not only the speed but also the sensitivity of investigation for nail dermatophytosis.

ABSTRACTThe aim of this study was to present the first set of comprehensive data on fungemia in Denmark including the distribution of species and range of susceptibility to major antifungal compounds based on a seminational surveillance study initiated in 2003. The catchment area of the participating hospitals had a population of 2.8 million, or 53% of the Danish population. A total of 303 episodes of fungemia were registered (annual rate, 11 of 100,000 people or 0.49 of 1,000 hospital discharges). Candidaspecies accounted for 97.4% of the fungal pathogens. C. albicanswas the predominant species (63%), but the proportion varied from 57% to 72% among participating departments of clinical microbiology. C. glabratawas the second most frequent species (20%; range, 8% to 32%). C. kruseiwas a rare isolate (3%) and occurred only at two of the participating hospitals. Retrospective data retrieved from the Danish laboratory systems documented a continuous increase of candidemia cases since the early 1990s. For the 272 susceptibility-tested isolates, MICs of amphotericin B and caspofungin were within the limits expected for the species or genus. However, decreased azole susceptibility, defined as a fluconazole MIC of >8 µg/ml and/or itraconazole MIC of >0.125 µg/ml, was detected for 11 Candidaisolates that were neither C. glabratanor C. krusei. Including intrinsically resistant fungi, we detected decreased susceptibility to fluconazole and/or itraconazole in 87 (32%) current Danish bloodstream fungal isolates. We showed a continuous increase of fungemia in Denmark and an annual rate in 2003 to 2004 higher than in most other countries. The proportion of bloodstream fungal isolates with reduced susceptibility to fluconazole and/or itraconazole was also notably high.

In order to elucidate the time when HIV was introduced into a population of patients with acute hepatitis B, serum samples collected in the period 1975-1984 from 331 patients with hepatitis B were analysed for the presence of antibody to HIV (anti-HIV). Anti-HIV was not detected in any of the serum samples from 97 females. 5/234 serum samples from males (2%) were repeatedly positive. Anti-HIV was first demonstrated in 1978, 3 years before the first patients with AIDS were recognized in Denmark. None of the 4 Danish patients with anti-HIV developed AIDS during a follow-up period of 1-7 years. However, at the time of follow-up in 1985 3 had decreased cell mediated immunity. The hepatitis B infection had an uncomplicated course in 4/5 patients with anti-HIV. One patient had a protracted delta hepatitis and was a HBsAg carrier before as well as after the acute hepatitis. Thus, the HIV infection did not cause any complicated course in this study.

Serum neutralization was measured in 72 sera collected during a 5.5-year period from 10 HIV infected individuals. Neutralizing antibodies (NA) were present in all sera. NA titers ranging from >40 to >640 were detected in sera from 4 patients, who all remained healthy and further an increase with time of NA was observed in these 4 patients. Progression to disease was observed in 3 persons with NA titers ≤40 who also lacked or lost anti-gag antibodies. Two of these patients were HIV antigenaemic prior to development of disease, whereas antigen was not detected in the remaining 7 healthy persons. A weak positive correlation (R(S) = +0.643, p<0.001) was found between titers of NA and whole virus antibody (WVA), with the ratios between titers (NA titer/WVA titer) varying a 100-fold. The results suggest that the presence of NA in some cases might be related to a healthy carrier state and that a combination of low titer NA with decline of anti-gag antibodies and/or HIV an-tigenaemia is associated with progression to clinical disease.

97 sera collected during a 10-year period from 10 HIV-1 infected individuals were tested for neutralizing capacity against a virus isolate FICPH-22 obtained from a Danish AIDS patient, and the laboratory strain HTLV-IIIB. Three patterns of serum neutralizing activity were demonstrated: (a) patients developing high neutralizing activity against both HIV strains; (b) patients developing high neutralizing activity against the Danish virus isolate; and (c) patients developing only low titers of neutralizing antibodies (NA) against both HIV strains. The HTLV-IIIB strain was less sensitive to serum neutralization than the FICPH-22 isolate and the appearance of NA against HTLV-IIIB was typically lacking several years behind that against FICPH-22 indicating a broadening of the NA response over time. No difference in clinical outcome was observed comparing patients reaching high titers of NA and patients with low titers. Development of AIDS among patients reaching high titers of NA was preceded by a decline in NA titers, indicating an association of high titers of NA with the healthy carrier state and of declining or low titers of NA with disease progression. The majority of the neutralizing activity was mediated by IgG, but some neutralizing activity was demonstrated in the IgG depleted serum, indicating the presence of additional neutralizing substances in serum.

The objective of the study was to evaluate the yield of a blood culture based on inoculating 40 ml of blood drawn from 1 venipuncture. During a 3-year period (1990-1992) 1351 (19.3%) of 6994 blood culture sets were positive. The increased yield of true bacteremic events using 40 ml instead of 30 ml of blood per blood culture was estimated to be 4,2%. Contaminants were isolated in 5% of the blood culture sets, with coagulase negative staphylococci being the most frequently isolated contaminants (3.2%, 1.3% of the culture bottles). In most of cases, contaminants were only isolated in 1 or 2 of the 4 bottles of the set. Furthermore, a positive correlation was observed between the number of positive culture bottles and the recovery of a clinically-significant microorganism. Neither the frequency nor the interpretations of positive blood culture events with microorganisms of questionable significance were major obstacles. In conclusion, the spectrum and yield of microorganisms drawing 40 ml of blood from one venipuncture into 4 culture bottles was satisfactory. The method bears obvious advantages from a clinical point of view, since usually only 1 venipuncture is needed before institution of antibiotic treatment

ABSTRACTEchinocandins are the preferred therapy for invasive infections due to Candida krusei. We present here a case of clinical failure involving C. kruseiwith a characteristic FKS1hot spot mutation not previously reported in C. kruseithat was isolated after 14 days of treatment. Anidulafungin MICs were elevated by ≥5 dilution steps above the clinical breakpoint but by only 1 step for a Candida albicansisolate harboring the corresponding mutation, suggesting a notable species-specific difference in the MIC increase conferred by this mutation.

ABSTRACTA multicenter study was conducted to assess the accuracy of the ISO standard 20776-1 and the serial 2-fold dilution procedures for antifungal susceptibility testing. Fluconazole trays can be accurately prepared by following ISO and serial dilution schemes. However, itraconazole trays showed a significant lack of reproducibility that was independent of which method was followed.

Echinocandins have been used as primary therapy of invasive aspergillosis (IA), with suboptimal results at standard dosing. Here, we explored the efficacy of dose escalation in a validated in vitropharmacokinetic/pharmacodynamic (PK/PD) model.

Posaconazole is more active than fluconazole against Candida albicansin vitroand is approved for the treatment of oropharyngeal candidiasis but not for that of invasive candidiasis (IC). Here, we explored the efficacy of posaconazole against C. albicansin an in vitropharmacokinetic/pharmacodynamic (PK/PD) model of IC and determined the probability of pharmacodynamic target attainment for the oral solution and intravenous (i.v.)/tablet formulations.

Olorofim is a novel antifungal drug in phase 2 trials. It has shown promising in vitroactivity against various molds, except for Mucorales. Initially, we observed a broad range of EUCAST MICs for Aspergillus fumigatus. Here, we explored the MIC variability in more detail and prospectively investigated the susceptibility of contemporary clinical mold isolates, as population data are needed for future epidemiological cutoff (ECOFF) settings. Fifteen A. fumigatusisolates previously found with low/medium/high MICs (≤0.

Fosmanogepix is a novel prodrug in a new class of antifungal agents. Manogepix is the active moiety. We evaluated the CLSI and EUCAST MICs of manogepix and eight comparators against Candida auris. CLSI M27-A3 susceptibility testing of manogepix was performed for 122 C. aurisisolates and compared to CLSI and EUCAST MICs for manogepix and eight comparators. Differences and agreement were calculated for each compound. Wild-type upper limits (WT-ULs; the upper MIC where the wild-type distribution ends) for manogepix and correlations with other drugs’ MICs were determined.

Manogepix (APX001A) is the active moiety of the drug candidate fosmanogepix (APX001), currently in clinical development for the treatment of invasive fungal infections. We compared manogepix EUCAST minimum effective concentrations (MECs) to MICs of five comparators and CLSI MECs and MICs by a colorimetric method against contemporary molds. EUCAST susceptibility testing was performed for 161 isolates. Interlaboratory and intermethod reproducibility were determined by comparison with published manogepix MECs.

Manogepix (APX001A) is the active moiety of the novel drug candidate fosmanogepix (APX001). We previously reported the broad-spectrum activity of manogepix but also observed a correlation between increased manogepix and fluconazole MICs. Here, we extended this study and included isolates with acquired fluconazole resistance. Isolates (n= 835) were identified using CHROMagar, matrix-assisted laser desorption ionization–time of flight mass spectrometry (MALDI-TOF MS), and, when needed, internal transcribed spacer (ITS) sequencing.

Rezafungin (formerly CD101) is a novel echinocandin in clinical development. EUCAST epidemiological cutoff values (ECOFFs) have not yet been established. We determined the in vitroactivity of rezafungin and comparators against 1,293 Nordic yeast isolates and 122 Indian Candida aurisisolates and established single-center wild-type upper limits (WT-UL). The isolates (19 Candidaspp. and 13 other yeast species) were identified using Chromagar; matrix-assisted laser desorption ionization–time of flight (MALDI-TOF); and, when needed, internal transcribed spacer sequencing.