David S. Perlin

Multilocus Sequence Typing Is a Reliable Alternative Method to DNA Fingerprinting for Discriminating among Strains of Candida albicans

ABSTRACT Multilocus sequence typing (MLST) has emerged as a powerful new DNA-typing tool for the evaluation of intraspecies genetic relatedness. This method relies on DNA sequence analysis of nucleotide polymorphisms in housekeeping genes and has shown a high degree of intraspecies discriminatory power for bacterial and fungal pathogens. However, the results of the MLST scheme for Candida albicans have heretofore never been formally compared to those of other established typing techniques. To assess the value of MLST relative to those of other DNA fingerprinting tools for discriminating among strains of C. albicans, we applied it to a previously well-characterized set of 29 C. albicans isolates evaluated by the random amplified polymorphic DNA (RAPD), multilocus enzyme electrophoresis (MLEE), and Ca3 Southern hybridization probe techniques. MLST identified three clusters of genetically related isolates, with 82.3% direct concordance with MLEE, 82.7% with RAPD analysis, and 86.2% with the Ca3 Southern hybridization technique. When MLST was applied to a subset of 22 isolates of unrelated origins, it identified 21 independent diploid sequence types (DSTs), resulting in a discriminatory power of 99.6%. These DSTs were 96.9, 99.6, and 99.6% concordant with the genotypes identified by RAPD analysis, MLEE, and Ca3 Southern hybridization, respectively. These results demonstrate that MLST is a highly effective technique that performs at least comparably to other established DNA fingerprinting techniques.

Frequency and evolution of azole resistance in Aspergillus fumigatus associated with treatment failure

An increase in the frequency of azole-resistant Aspergillus fumigatus has emerged.

Increasing Echinocandin Resistance in Candida glabrata: Clinical Failure Correlates With Presence of FKS Mutations and Elevated Minimum Inhibitory Concentrations

BACKGROUND Fluconazole (FLC) resistance is common in C. glabrata and echinocandins are often used as first-line therapy. Resistance to echinocandin therapy has been associated with FKS1 and FKS2 gene alterations. METHODS We reviewed records of all patients with C. glabrata bloodstream infection at Duke Hospital over the past decade (2001-2010) and correlated treatment outcome with minimum inhibitory concentration (MIC) results and the presence of FKS gene mutations. For each isolate, MICs to FLC and echinocandins (anidulafungin, caspofungin, and micafungin) and FKS1 and FKS2 gene sequences were determined. RESULTS Two hundred ninety-three episodes (313 isolates) of C. glabrata bloodstream infection were analyzed. Resistance to echinocandins increased from 4.9% to 12.3% and to FLC from 18% to 30% between 2001 and 2010, respectively. Among the 78 FLC resistant isolates, 14.1% were resistant to 1 or more echinocandin. Twenty-five (7.9%) isolates harbored a FKS mutation. The predictor of a FKS mutant strain was prior echinocandin therapy (stepwise multivariable analysis, odds ratio, 19.647 [95% confidence interval, 7.19-58.1]). Eighty percent (8/10) of patients infected with FKS mutants demonstrating intermediate or resistant MICs to an echinocandin and treated with an echinocandin failed to respond or responded initially but experienced a recurrence. CONCLUSIONS Echinocandin resistance is increasing, including among FLC-resistant isolates. The new Clinical and Laboratory Standards Institute clinical breakpoints differentiate wild-type from C. glabrata strains bearing clinically significant FKS1/FKS2 mutations. These observations underscore the importance of knowing the local epidemiology and resistance patterns for Candida within institutions and susceptibility testing of echinocandins for C. glabrata to guide therapeutic decision making.

Specific Substitutions in the Echinocandin Target Fks1p Account for Reduced Susceptibility of Rare Laboratory and Clinical Candida sp. Isolates

ABSTRACT An association between reduced susceptibility to echinocandins and changes in the 1,3-β-d-glucan synthase (GS) subunit Fks1p was investigated. Specific mutations in fks1 genes from Saccharomyces cerevisiae and Candida albicans mutants are described that are necessary and sufficient for reduced susceptibility to the echinocandin drug caspofungin. One group of amino acid changes in ScFks1p, ScFks2p, and CaFks1p defines a conserved region (Phe 641 to Asp 648 of CaFks1p) in the Fks1 family of proteins. The relationship between several of these fks1 mutations and the phenotype of reduced caspofungin susceptibility was confirmed using site-directed mutagenesis or integrative transformation. Glucan synthase activity from these mutants was less susceptible to caspofungin inhibition, and heterozygous and homozygous Cafks1 C. albicans mutants could be distinguished based on the shape of inhibition curves. The C. albicans mutants were less susceptible to caspofungin than wild-type strains in a murine model of disseminated candidiasis. Five Candida isolates with reduced susceptibility to caspofungin were recovered from three patients enrolled in a clinical trial. Four C. albicans strains showed amino acid changes at Ser 645 of CaFks1p, while a single Candida krusei isolate had a deduced R1361G substitution. The clinical C. albicans mutants were less susceptible to caspofungin in the disseminated candidiasis model, and GS inhibition profiles and DNA sequence analyses were consistent with a homozygous fks1 mutation. Our results indicate that substitutions in the Fks1p subunit of GS are sufficient to confer reduced susceptibility to echinocandins in S. cerevisiae and the pathogens C. albicans and C. krusei.

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.

A Naturally Occurring Proline-to-Alanine Amino Acid Change in Fks1p in Candida parapsilosis, Candida orthopsilosis, and Candida metapsilosis Accounts for Reduced Echinocandin Susceptibility

ABSTRACT Candida parapsilosis has emerged as a common cause of invasive fungal infection, especially in Latin America and in the neonatal setting. C. parapsilosis is part of a closely related group of organisms that includes the species Candida orthopsilosis and Candida metapsilosis. All three species show elevated MICs for the new echinocandin class drugs caspofungin, micafungin, and anidulafungin relative to other Candida species. Despite potential impacts on therapy, the mechanism behind this reduced echinocandin susceptibility has not been determined. In this report, we investigated the role of a naturally occurring Pro-to-Ala substitution at amino acid position 660 (P660A), immediately distal to the highly conserved hot spot 1 region of Fks1p, in the reduced-echinocandin-susceptibility phenotype. Kinetic inhibition studies demonstrated that glucan synthase from the C. parapsilosis group was 1 to 2 logs less sensitive to echinocandin drugs than the reference enzyme from C. albicans. Furthermore, clinical isolates of C. albicans and C. glabrata which harbor mutations at this equivalent position also showed comparable 2-log decreases in target enzyme sensitivity, which correlated with increased MICs. These mutations also resulted in 2.4- to 18.8-fold-reduced Vmax values relative to those for the wild-type enzyme, consistent with kinetic parameters obtained for C. parapsilosis group enzymes. Finally, the importance of the P660A substitution for intrinsic resistance was confirmed by engineering an equivalent P647A mutation into Fks1p of Saccharomyces cerevisiae. The mutant glucan synthase displayed characteristic 2-log decreases in sensitivity to the echinocandin drugs. Overall, these data firmly indicate that a naturally occurring P660A substitution in Fks1p from the C. parapsilosis group accounts for the reduced susceptibility phenotype.

High-frequency Triazole Resistance Found In Nonculturable Aspergillus fumigatus from Lungs of Patients with Chronic Fungal Disease

BACKGROUND Oral triazole therapy is well established for the treatment of invasive (IPA), allergic (ABPA), and chronic pulmonary (CPA) aspergillosis, and is often long-term. Triazole resistance rates are rising internationally. Microbiological diagnosis of aspergillosis is limited by poor culture yield, leading to uncertainty about the frequency of triazole resistance. METHODS Using an ultrasensitive real-time polymerase chain reaction (PCR) assay for Aspergillus spp., we assessed respiratory fungal load in bronchoalveolar lavage (BAL) and sputum specimens. In a subset of PCR-positive, culture negative samples, we further amplified the CYP51A gene to detect key single-nucleotide polymorphisms (SNPs) associated with triazole resistance. RESULTS Aspergillus DNA was detected in BAL from normal volunteers (4/11, 36.4%) and patients with culture or microscopy confirmed IPA (21/22, 95%). Aspergillus DNA was detected in sputum in 15 of 19 (78.9%) and 30 of 42 (71.4%) patients with ABPA and CPA, compared with 0% and 16.7% by culture, respectively. In culture-negative, PCR-positive samples, we detected triazole-resistance mutations (L98H with tandem repeat [TR] and M220) within the drug target CYP51A in 55.1% of samples. Six of 8 (75%) of those with ABPA and 12 of 24 (50%) with CPA had resistance markers present, some without prior triazole treatment, and in most despite adequate plasma drug concentrations around the time of sampling. CONCLUSIONS The very low organism burdens of fungi causing infection have previously prevented direct culture and detection of antifungal resistance in clinical samples. These findings have major implications for the sustainability of triazoles for human antifungal therapy.

Multiple Resistance Mechanisms among Aspergillus fumigatus Mutants with High-Level Resistance to Itraconazole

ABSTRACT A collection of Aspergillus fumigatus mutants highly resistant to itraconazole (RIT) at 100 μg ml−1 were selected in vitro (following UV irradiation as a preliminary step) to investigate mechanisms of drug resistance in this clinically important pathogen. Eight of the RIT mutants were found to have a mutation at Gly54 (G54E, -K, or -R) in the azole target gene CYP51A. Primers designed for highly conserved regions of multidrug resistance (MDR) pumps were used in reverse transcriptase PCR amplification reactions to identify novel genes encoding potential MDR efflux pumps in A. fumigatus. Two genes, AfuMDR3 and AfuMDR4, showed prominent changes in expression levels in many RIT mutants and were characterized in more detail. Analysis of the deduced amino acid sequence encoded by AfuMDR3 revealed high similarity to major facilitator superfamily transporters, while AfuMDR4 was a typical member of the ATP-binding cassette superfamily. Real-time quantitative PCR with molecular beacon probes was used to assess expression levels of AfuMDR3 and AfuMDR4. Most RIT mutants showed either constitutive high-level expression of both genes or induction of expression upon exposure to itraconazole. Our results suggest that overexpression of one or both of these newly identified drug efflux pump genes of A. fumigatus and/or selection of drug target site mutations are linked to high-level itraconazole resistance and are mechanistic considerations for the emergence of clinical resistance to itraconazole.

Effect of Candida glabrata FKS1 and FKS2 Mutations on Echinocandin Sensitivity and Kinetics of 1,3-β-d-Glucan Synthase: Implication for the Existing Susceptibility Breakpoint

ABSTRACT Thirteen Candida glabrata strains harboring a range of mutations in hot spot regions of FKS1 and FKS2 were studied. The mutations were linked to an echinocandin reduced susceptibility phenotype. Sequence alignments showed that 11 out of the 13 mutants harbored a mutation in FKS1 or FKS2 not previously implicated in echinocandin reduced susceptibility in C. glabrata. A detailed kinetic characterization demonstrated that amino acid substitutions in Fks1p and Fks2p reduced drug sensitivity in mutant 1,3-β-d-glucan synthase by 2 to 3 log orders relative to that in wild-type enzyme. These mutations were also found to reduce the catalytic efficiency of the enzyme (Vmax) and to influence the relative expression of FKS genes. In view of the association of FKS mutations and reduced susceptibility of 1,3-β-d-glucan synthase, an evaluation of the new CLSI echinocandin susceptibility breakpoint was conducted. Only 3 of 13 resistant fks mutants (23%) were considered anidulafungin or micafungin nonsusceptible (MIC > 2 μg/ml) by this criterion. In contrast, most fks mutants (92%) exceeded a MIC of >2 μg/ml with caspofungin. However, when MIC determinations were performed in the presence of 50% serum, all C. glabrata fks mutants showed MICs of ≥2 μg/ml for the three echinocandin drugs. As has been observed with Candida albicans, the kinetic inhibition parameter 50% inhibitory concentration may be a better predictor of FKS-mediated resistance. Finally, the close association between FKS1/FKS2 hot spot mutations provides a basis for understanding echinocandin resistance in C. glabrata.

Correlating Echinocandin MIC and Kinetic Inhibition of fks1 Mutant Glucan Synthases for Candida albicans: Implications for Interpretive Breakpoints

ABSTRACT A detailed kinetic characterization of echinocandin inhibition was performed for mutant 1,3-β-d-glucan synthase enzymes from clinical isolates of Candida albicans with nine different FKS1 mutations resulting in high MICs. Among 14 mutant Fks1p enzymes studied, the kinetic parameters 50% inhibitory concentration and Ki increased 50-fold to several thousandfold relative to those for the wild type. Enzymes with mutations at Ser645 (S645P, S645Y, and S645F) within hot spot 1 showed the most prominent decrease in sensitivity, while those with mutations at the N- and C-terminal ends of hot spot 1 generally retained greater sensitivity to all three drugs. Kinetic inhibitions by caspofungin, micafungin, and anidulafungin were comparable among the fks1 mutant enzymes, although absolute values did vary with specific mutations. Amino acid substitutions in Fks1p did not alter Km values, although some mutations decreased the Vmax. Given the association of FKS1 mutations with clinical resistance, an evaluation of the kinetic parameters for the inhibition of mutant 1,3-β-d-glucan synthase as a function of the MIC enabled an independent evaluation of the recently adopted susceptibility breakpoint for echinocandin drugs. Overall, a breakpoint MIC of ≥2 μg/ml for caspofungin captured nearly 100% of fks1 C. albicans strains when a kinetic inhibition rise threshold of ≤50-fold for the Ki was used as a measure of susceptibility. A similar MIC breakpoint for micafungin and anidulafungin was less inclusive, and a projected MIC of ≥0.5 μg/ml was required for >95% coverage of clinical isolates. However, when MIC determinations were performed in the presence of 50% serum, all fks1 mutants showed MIC values of ≥2 μg/ml for the three echinocandin drugs. The 1,3-β-d-glucan synthase kinetic inhibition data support the proposed susceptibility breakpoint for caspofungin in C. albicans, but a lower susceptibility breakpoint (≤0.5 μg/ml) may be more appropriate for anidulafungin and micafungin. Overall, the data indicate that MIC testing with caspofungin may serve as a surrogate marker for resistance among the class of echinocandin drugs.