Comparative genomic analysis of clinical Candida glabrata isolates identifies multiple polymorphic loci that can improve existing multilocus sequence typing strategy.

Candida glabrata is the second leading cause of candidemia in many countries and is one of the most concerning yeast species of nosocomial importance due to its increasing rate of antifungal drug resistance and emerging multidrug-resistant isolates. Application of multilocus sequence typing (MLST) to clinical C. glabrata isolates revealed an association of certain sequence types (STs) with drug resistance and mortality. The current C. glabrata MLST scheme is based on single nucleotide polymorphisms (SNPs) at six loci and is therefore relatively laborious and costly. Furthermore, only a few high-quality C. glabrata reference genomes are available, limiting rapid analysis of clinical isolates by whole genome sequencing. In this study we provide long-read based assemblies for seven additional clinical strains belonging to three different STs and use this information to simplify the C. glabrata MLST scheme. Specifically, a comparison of these genomes identified highly polymorphic loci (HPL) defined by frequent insertions and deletions (indels), two of which proved to be highly resolutive for ST. When challenged with 53 additional isolates, a combination of TRP1 (a component of the current MLST scheme) with either of the two HPL fully recapitulated ST identification. Therefore, our comparative genomic analysis identified a new typing approach combining SNPs and indels and based on only two loci, thus significantly simplifying ST identification in C. glabrata. Because typing tools are instrumental in addressing numerous clinical and biological questions, our new MLST scheme can be used for high throughput typing of C. glabrata in clinical and research settings.

Aspergillus fumigatus and aspergillosis: From basics to clinics.

The airborne fungus Aspergillus fumigatus poses a serious health threat to humans by causing numerous invasive infections and a notable mortality in humans, especially in immunocompromised patients. Mould-active azoles are the frontline therapeutics employed to treat aspergillosis. The global emergence of azole-resistant A. fumigatus isolates in clinic and environment, however, notoriously limits the therapeutic options of mould-active antifungals and potentially can be attributed to a mortality rate reaching up to 100 %. Although specific mutations in CYP 51A are the main cause of azole resistance, there is a new wave of azole-resistant isolates with wild-type CYP 51A genotype challenging the efficacy of the current diagnostic tools. Therefore, applications of whole-genome sequencing are increasingly gaining popularity to overcome such challenges. Prominent echinocandin tolerance, as well as liver and kidney toxicity posed by amphotericin B, necessitate a continuous quest for novel antifungal drugs to combat emerging azole-resistant A. fumigatus isolates. Animal models and the tools used for genetic engineering require further refinement to facilitate a better understanding about the resistance mechanisms, virulence, and immune reactions orchestrated against A. fumigatus. This review paper comprehensively discusses the current clinical challenges caused by A. fumigatus and provides insights on how to address them.

Multicenter study of epidemiological cutoff values and detection of resistance in Candida spp. to anidulafungin, caspofungin, and micafungin using the Sensititre YeastOne colorimetric method.

Neither breakpoints (BPs) nor epidemiological cutoff values (ECVs) have been established for Candida spp. with anidulafungin, caspofungin, and micafungin when using the Sensititre YeastOne (SYO) broth dilution colorimetric method. In addition, reference caspofungin MICs have so far proven to be unreliable. Candida species wild-type (WT) MIC distributions (for microorganisms in a species/drug combination with no detectable phenotypic resistance) were established for 6,007 Candida albicans, 186 C. dubliniensis, 3,188 C. glabrata complex, 119 C. guilliermondii, 493 C. krusei, 205 C. lusitaniae, 3,136 C. parapsilosis complex, and 1,016 C. tropicalis isolates. SYO MIC data gathered from 38 laboratories in Australia, Canada, Europe, Mexico, New Zealand, South Africa, and the United States were pooled to statistically define SYO ECVs. ECVs for anidulafungin, caspofungin, and micafungin encompassing ≥97.5% of the statistically modeled population were, respectively, 0.12, 0.25, and 0.06 µg/ml for C. albicans, 0.12, 0.25, and 0.03 µg/ml for C. glabrata complex, 4, 2, and 4 µg/ml for C. parapsilosis complex, 0.5, 0.25, and 0.06 µg/ml for C. tropicalis, 0.25, 1, and 0.25 µg/ml for C. krusei, 0.25, 1, and 0.12 µg/ml for C. lusitaniae, 4, 2, and 2 µg/ml for C. guilliermondii, and 0.25, 0.25, and 0.12 µg/ml for C. dubliniensis. Species-specific SYO ECVs for anidulafungin, caspofungin, and micafungin correctly classified 72 (88.9%), 74 (91.4%), 76 (93.8%), respectively, of 81 Candida isolates with identified fks mutations. SYO ECVs may aid in detecting non-WT isolates with reduced susceptibility to anidulafungin, micafungin, and especially caspofungin, since testing the susceptibilities of Candida spp. to caspofungin by reference methodologies is not recommended.

Different risk factors for candidemia occur for Candida species belonging to the C. parapsilosis complex.

Previous studies have demonstrated reduced virulence in the species that comprise the Candida parapsilosis complex. We investigated a cohort of 93 patients with candidemia caused by this complex. Most infections were caused by C. parapsilosis (80.6%), followed by C. orthopsilosis (18.3%) and C. metapsilosis (1.1%). Renal failure (P < 0.001) and chronic liver diseases (P = 0.019) were more frequently encountered with infections caused by the C. orthopsilosis group, suggesting an association with patients who had a greater state of immune suppression in comparison with infections caused by C. parapsilosis sensu stricto.

Epidemiology and molecular characterization of bacteremia due to carbapenem-resistant Klebsiella pneumoniae in transplant recipients.

We conducted a retrospective study of 17 transplant recipients with carbapenem-resistant Klebsiella pneumoniae bacteremia, and described epidemiology, clinical characteristics and strain genotypes. Eighty-eight percent (15/17) of patients were liver or intestinal transplant recipients. Outcomes were death due to septic shock (18%), cure (24%) and persistent (>7 days) or recurrent bacteremia (29% each). Thirty- and 90-day mortality was 18% and 47%, respectively. Patients who were cured received at least one active antimicrobial agent and underwent source control interventions. Forty-one percent (7/17) of patients had intra-abdominal infections; all except one developed persistent/recurrent bacteremia despite drainage. Two patients tolerated persistent bacteremia for >300 days. All patients except one were infected with sequence type 258 (ST258), K. pneumoniae carbapenemase (KPC)-2-producing strains harboring a mutant ompK35 porin gene; the exception was infected with an ST37, KPC-3-producing strain. Seventy-one percent (12/17) of patients were infected with ST258 ompK36 mutant strains. In two patients, persistent bacteremia was caused by two strains with different ompK36 genotypes. Three ompK36 mutations were associated with significantly higher carbapenem minimum inhibitory concentrations than wild-type ompK36. Pulse-field gel electrophoresis identified a single ST258 lineage; serial strains from individual patients were indistinguishable. In conclusion, KPC-K. pneumoniae bacteremia exhibited highly diverse clinical courses following transplantation, and was caused by clonal ST258 strains with different ompK36 genotypes.

Isavuconazole activity against Aspergillus lentulus, Neosartorya udagawae, and Cryptococcus gattii, emerging fungal pathogens with reduced azole susceptibility.

Isavuconazole is an extended-spectrum triazole with in vitro activity against a wide variety of fungal pathogens. Clinical isolates of molds Aspergillus lentulus and Neosartorya udagawae and yeast Cryptococcus gattii VGII (implicated in the outbreak in the Pacific Northwest, North America) exhibit reduced susceptibilities to several azoles but higher susceptibilities to isavuconazole.

Clinical breakpoints for the echinocandins and Candida revisited: integration of molecular, clinical, and microbiological data to arrive at species-specific interpretive criteria.

The CLSI established clinical breakpoints (CBPs) for caspofungin (CSF), micafungin (MCF) and anidulafungin (ANF) versus Candida. The same CBP (susceptible (S): MIC ≤ 2 mcg/ml; non-S: MIC > 2 mcg/ml) was applied to all echinocandins and species. More data now allow reassessment of these CBPs. We examined cases of echinocandin failure where both MICs and fks mutations were assessed; wild type (WT) MICs and epidemiological cutoff values (ECVs) for a large Candida collection; molecular analysis of fks hotspots for Candida with known MICs; and pharmacokinetic and pharmacodynamic (PK/PD) data. We applied these findings to propose new species-specific CBPs for echinocandins and Candida. Of 18 candidiasis cases refractory to echinocandins and with fks mutations, 28% (CSF), 58% (ANF) and 66% (MCF) had MICs in the S category using CBP of ≤ 2 mcg/ml, while 0-8% would be S using CBP of ≤ 0.25 mcg/ml. WT MIC distributions revealed ECV ranges of 0.03-0.25 mcg/ml for all major species except C. parapsilosis (1-4 mcg/ml) and C. guilliermondii (4-16 mcg/ml). Among Candida tested for fks mutations, only 15.7-45.1% of 51 mutants were detected using the CBP for NS of >2 mcg/ml. In contrast, a cutoff of >0.25 mcg/ml for C. albicans, C. tropicalis, C. krusei, and C. dubliniensis detected 85.6% (MCF) to 95.2% (CSF) of 21 mutant strains. Likewise, a cutoff of >0.12 mcg/ml for ANF and CSF and of >0.06 mcg/ml for MCF detected 93% (ANF) to 97% (CSF, MCF) of 30 mutant strains of C. glabrata. These data, combined with PK/PD considerations, support CBPs of ≤ 0.25 mcg/ml (S), 0.5 mcg/ml (I), ≥ 1 (R) for CSF/MCF/ANF and C. albicans, C. tropicalis and C. krusei and ≤ 2 mcg/ml (S), 4 mcg/ml (I), and ≥ 8 mcg/ml (R) for these agents and C. parapsilosis. The CBPs for ANF and CSF and C. glabrata are ≤ 0.12 mcg/ml (S), 0.25 mcg/ml (I), and ≥ 0.5 mcg/ml (R), whereas those for MCF are ≤ 0.06 mcg/ml (S), 0.12 mcg/ml (I), and ≥ 0.25 mcg/ml (R). New, species-specific CBPs for Candida and the echinocandins are more sensitive to detect emerging resistance associated with fks mutations, and better able to predict risk for clinical failure.

Disseminated Candidiasis caused by Candida albicans with amino acid substitutions in Fks1 at position Ser645 cannot be successfully treated with micafungin.

The clinical utility of the echinocandins is potentially compromised by the emergence of drug resistance. We investigated whether Candida albicans with amino acid substitutions at position Ser645 in Fks1 can be treated with either a conventional or an elevated dosage of micafungin. We studied Candida albicans (wild-type SC5314; MIC, 0.06 mg/liter) and four fks1 mutants (one FKS1/fks1 heterozygote mutant [MIC, 0.5 mg/liter] and three fks1/fks1 homozygous mutants [MICs for all, 2 mg/liter]) with a variety of amino acid substitutions at Ser645. The pharmacokinetic and pharmacodynamic relationships were characterized in a persistently neutropenic murine model of disseminated candidiasis. A mathematical model was fitted to all pharmacokinetic and pharmacodynamic data. This mathematical model was then used to "humanize" the murine pharmacokinetics, and the predicted antifungal effect was determined. The estimated maximal rate of growth and ultimate fungal densities in the kidney for each of the strains were similar. The administration of micafungin at 1 mg/kg of body weight to the wild type resulted in moderate antifungal activity, whereas the administration of 5 and 20 mg/kg resulted in rapid fungicidal activity. In contrast, the FKS1/fks heterozygote was killed only with 20 mg/kg, and the homozygous fks1 mutants failed to respond to any dosage. The bridging study revealed that human dosages of 100 and 400 mg/day were active only against the wild type, with no activity against either the heterozygote or the homozygote mutants. Ser645 Fks1 Candida albicans mutants cannot be treated with either conventional or elevated dosages of micafungin and should be deemed resistant.

Pharmacodynamics of echinocandins against Candida glabrata: requirement for dosage escalation to achieve maximal antifungal activity in neutropenic hosts.

Candida glabrata is a leading cause of disseminated candidiasis. The echinocandins are increasingly used as first-line agents for the treatment of patients with this syndrome, although the optimal regimen for the treatment of invasive Candida glabrata infections in neutropenic patients is not known. We studied the pharmacokinetics (PK) and pharmacodynamics (PD) of micafungin, anidulafungin, and caspofungin in a neutropenic murine model of disseminated Candida glabrata infection to gain further insight into optimal therapeutic options for patients with this syndrome. A mathematical model was fitted to the data and used to bridge the experimental results to humans. The intravenous inoculation of Candida glabrata in mice was followed by logarithmic growth throughout the experimental period (101 h). A dose-dependent decline in fungal burden was observed following the administration of 0.1 to 20 mg/kg of body weight every 24 h for all three agents. The exposure-response relationships for each drug partitioned into distinct fungistatic and fungicidal components of activity. Surprisingly, the average human drug exposures following currently licensed regimens were predicted to result in a fungistatic antifungal effect. Higher human dosages of all three echinocandins are required to induce fungicidal effects in neutropenic hosts.

Use of a high-resolution melt assay to characterize codon 54 of the cyp51A gene of Aspergillus fumigatus on a Rotor-Gene 6000 instrument.

A high-resolution melt (HRM) assay using a Rotor-Gene 6000 instrument was developed to characterize the codon for glycine 54 in the cyp51A genes from 13 reference isolates and 12 clinical isolates of Aspergillus fumigatus. Mutations in this codon confer reduced susceptibility to itraconazole and posaconazole. The assay is simple to perform, and a result of "wild type" or "mutant" is available after approximately 1 h following DNA extraction using commercially available reagents and conventional primers.