Etest ECVs/ECOFFs for Detection of Resistance in Prevalent and Three Nonprevalent Candida spp. to Triazoles and Amphotericin B and Aspergillus spp. to Caspofungin: Further Assessment of Modal Variability.

Susceptibility testing is an important tool in the clinical setting; its utility is based on the availability of categorical endpoints, breakpoints (BPs), or epidemiological cutoff values (ECVs/ECOFFs). CLSI and EUCAST have developed antifungal susceptibility testing, BPs, and ECVs for some fungal species. Although the concentration gradient strip bioMérieux Etest is useful for routine testing in the clinical laboratory, ECVs are not available for all agent/species; the lack of clinical data precludes development of BPs. We reevaluated and consolidated Etest data points from three previous studies and included new data. We defined ECOFFinder Etest ECVs for three sets of species-agent combinations: fluconazole, posaconazole, and voriconazole and 9 Candida spp.; amphotericin B and 3 nonprevalent Candida spp.; and caspofungin and 4 Aspergillus spp. The total of Etest MICs from 23 laboratories (Europe, the Americas, and South Africa) included (antifungal agent dependent): 17,242 Candida albicans, 244 C. dubliniensis, 5,129 C. glabrata species complex (SC), 275 C. guilliermondii (Meyerozyma guilliermondii), 1,133 C. krusei (Pichia kudriavzevii), 933 C. kefyr (Kluyveromyces marxianus), 519 C. lusitaniae (Clavispora lusitaniae), 2,947 C. parapsilosis SC, 2,214 C. tropicalis, 3,212 Aspergillus fumigatus, 232 A. flavus, 181 A. niger, and 267 A. terreus SC isolates. Triazole MICs for 66 confirmed non-wild-type (non-WT) Candida isolates were available (ERG11 point mutations). Distributions fulfilling CLSI ECV criteria were pooled, and ECOFFinder Etest ECVs were established for triazoles (9 Candida spp.), amphotericin B (3 less-prevalent Candida spp.), and caspofungin (4 Aspergillus spp.). Etest fluconazole ECVs could be good detectors of Candida non-WT isolates (59/61 non-WT, 4 of 6 species).

Blood and tissue distribution of posaconazole in a rat model of invasive pulmonary aspergillosis.

Posaconazole is the recommended prophylactic agent in patients at high risk of invasive fungal infection, since adequate drug levels seem to be reached in target sites despite the relatively low levels detected in blood. The objective of this study is to obtain pharmacokinetic (PK) information associated to blood and tissue distribution of posaconazole in an animal model of invasive pulmonary aspergillosis. The PK parameters in lung samples were systematically higher than in serum. After multiple-dose administration of posaconazole, a significant accumulation of the drug was evident in lung tissue. The PK behavior of posaconazole in this particular experimental model is similar to that observed in humans. Thus, we believe this model could be a valid tool to evaluate posaconazole exposure-response relationship.

Rapid development of Candida krusei echinocandin resistance during caspofungin therapy.

In invasive candidiasis, there has been an epidemiological shift from Candida albicans to non-albicans species infections, including infections with C. glabrata, C. parapsilosis, C. tropicalis, and C. krusei. Although the prevalence of C. krusei remains low among yeast infections, its intrinsic resistance to fluconazole raises epidemiological and therapeutic concerns. Echinocandins have in vitro activity against most Candida spp. and are the first-line agents in the treatment of candidemia. Although resistance to echinocandin drugs is still rare, individual cases of C. krusei resistance have been reported in recent years, especially with strains that have been under selective pressure. A total of 15 C. krusei strains, isolated from the blood, urine, and soft tissue of an acute lymphocytic leukemia patient, were analyzed. Strains developed echinocandin resistance during 10 days of caspofungin therapy. The molecular epidemiology of the isolates was investigated using two different typing methods: PCR-based amplification of the species-specific repetitive polymorphic CKRS-1 sequence and multilocus sequence typing. All isolates were genetically related, and the mechanism involved in decreased echinocandin susceptibility was characterized. Clinical resistance was associated with an increase in echinocandin MICs in vitro and was related to three different mutations in hot spot 1 of the target enzyme Fks1p. Molecular evidence of the rapid acquisition of resistance by different mutations in FKS1 highlights the need to monitor the development of resistance in C. krusei infections treated with echinocandin drugs.

In vivo efficacy of voriconazole and posaconazole therapy in a novel invertebrate model of Aspergillus fumigatus infection.

The emergence of azole resistance in Aspergillus fumigatus is a clinically important issue in the management of invasive aspergillosis as it could limit therapeutic options. Accurate measurement of in vitro antifungal activity in terms of minimum inhibitory concentration (MIC) is considered of clinical relevance and often gives useful therapeutic information for physicians. However, the lack of in vitro-in vivo correlation is frequent and the observed in vitro phenotype does not always correlate with the in vivo response. In this regard, a wild-type strain and five A. fumigatus cyp51A mutated strains showing different azole susceptibility profiles were used to investigate whether the greater wax moth (Galleria mellonella) is an alternative model to assess the in vivo efficacy of voriconazole and posaconazole. Administration of both azoles improved the survival of larvae infected with susceptible strains. However, those larvae infected with resistant strains did not respond to treatment. The phenotype observed in vitro was found to correlate with the efficacy observed in vivo. Moreover, using this in vivo model, the pharmacodynamic target predicting therapeutic success (AUC(0-24)/MIC) was in the same range as previously described, allowing the use of the G. mellonella model to predict the azole susceptibility profile of A. fumigatus strains.

An invertebrate model to evaluate virulence in Aspergillus fumigatus: the role of azole resistance.

The impact of different mutations in the Aspergillus fumigatus ergosterol biosynthesis pathway on pathogenesis has been evaluated using a simple invertebrate mini host, the caterpillar Galleria mellonella. A set of strains that includes clinical isolates and isogenic mutants with mutations at the cyp51A gene conferring azole resistance were studied. All strains demonstrated a similar in vitro growth pattern and are equally virulent against the insect larvae. These results suggest that in A. fumigatus acquisition of this particular azole-resistance mechanism would not imply any significant change in virulence. G. mellonella may provide a convenient and inexpensive model for the in vivo prescreening of mutants of A. fumigatus, contributing to the generation of a hypotheses that can be further tested in refined experiments in mammalian models.

Candida tropicalis antifungal cross-resistance is related to different azole target (Erg11p) modifications.

Candida tropicalis ranks between third and fourth among Candida species most commonly isolated from clinical specimens. Invasive candidiasis and candidemia are treated with amphotericin B or echinocandins as first-line therapy, with extended-spectrum triazoles as acceptable alternatives. Candida tropicalis is usually susceptible to all antifungal agents, although several azole drug-resistant clinical isolates are being reported. However, C. tropicalis resistant to amphotericin B is uncommon, and only a few strains have reliably demonstrated a high level of resistance to this agent. The resistance mechanisms operating in C. tropicalis strains isolated from clinical samples showing resistance to azole drugs alone or with amphotericin B cross-resistance were elucidated. Antifungal drug resistance was related to mutations of the azole target (Erg11p) with or without alterations of the ergosterol biosynthesis pathway. The antifungal drug resistance shown in vitro correlated very well with the results obtained in vivo using the model host Galleria mellonella. Using this panel of strains, the G. mellonella model system was validated as a simple, nonmammalian minihost model that can be used to study in vitro-in vivo correlation of antifungals in C. tropicalis. The development in C. tropicalis of antifungal drug resistance with different mechanisms during antifungal treatment has potential clinical impact and deserves specific prospective studies.

HPLC/UV or bioassay: two valid methods for posaconazole quantification in human serum samples.

In this paper we report on the development and validation of two different methods for posaconazole quantification from serum samples, HPLC/UV and bioassay. Both methods have been validated according to international guidelines and were also applied to the analysis of 61 trough serum samples from treated patients. A good correlation between both methods was observed. The HPLC method, more laborious and expensive, was demonstrated to be more accurate, precise and faster (analytical range 0.125-16 µg/mL, accuracy between -2.48 and 3.70% and precision between 2.77 and 5.93%, with an analytical run time of 11 min), making it a valuable tool for reference laboratories that centralize high numbers of samples. The microbiological method, however, is simple and offers sufficient precision and accuracy (analytical range 0.125-16 µg/mL, accuracy between -8.10 and 3.77% and a precision between 4.52 and 10.07%), to be used to monitor posaconazole. It may be a valid alternative to chromatographic methods in clinical laboratories without specialized facilities.