Multilaboratory study of epidemiological cutoff values for detection of resistance in eight Candida species to fluconazole, posaconazole, and voriconazole.

Although epidemiological cutoff values (ECVs) have been established for Candida spp. and the triazoles, they are based on MIC data from a single laboratory. We have established ECVs for eight Candida species and fluconazole, posaconazole, and voriconazole based on wild-type (WT) MIC distributions for isolates of C. albicans (n=11,241 isolates), C. glabrata (7,538), C. parapsilosis (6,023), C. tropicalis (3,748), C. krusei (1,073), C. lusitaniae (574), C. guilliermondii (373), and C. dubliniensis (162). The 24-h CLSI broth microdilution MICs were collated from multiple laboratories (in Canada, Brazil, Europe, Mexico, Peru, and the United States). The ECVs for distributions originating from ≥6 laboratories, which included ≥95% of the modeled WT population, for fluconazole, posaconazole, and voriconazole were, respectively, 0.5, 0.06 and 0.03 µg/ml for C. albicans, 0.5, 0.25, and 0.03 µg/ml for C. dubliniensis, 8, 1, and 0.25 µg/ml for C. glabrata, 8, 0.5, and 0.12 µg/ml for C. guilliermondii, 32, 0.5, and 0.25 µg/ml for C. krusei, 1, 0.06, and 0.06 µg/ml for C. lusitaniae, 1, 0.25, and 0.03 µg/ml for C. parapsilosis, and 1, 0.12, and 0.06 µg/ml for C. tropicalis. The low number of MICs (<100) for other less prevalent species (C. famata, C. kefyr, C. orthopsilosis, C. rugosa) precluded ECV definition, but their MIC distributions are documented. Evaluation of our ECVs for some species/agent combinations using published individual MICs for 136 isolates (harboring mutations in or upregulation of ERG11, MDR1, CDR1, or CDR2) and 64 WT isolates indicated that our ECVs may be useful in distinguishing WT from non-WT isolates.

Multicenter study of anidulafungin and micafungin MIC distributions and epidemiological cutoff values for eight Candida species and the CLSI M27-A3 broth microdilution method.

Since epidemiological cutoff values (ECVs) using CLSI MICs from multiple laboratories are not available for Candida spp. and the echinocandins, we established ECVs for anidulafungin and micafungin on the basis of wild-type (WT) MIC distributions (for organisms in a species-drug combination with no detectable acquired resistance mechanisms) for 8,210 Candida albicans, 3,102 C. glabrata, 3,976 C. parapsilosis, 2,042 C. tropicalis, 617 C. krusei, 258 C. lusitaniae, 234 C. guilliermondii, and 131 C. dubliniensis isolates. CLSI broth microdilution MIC data gathered from 15 different laboratories in Canada, Europe, Mexico, Peru, and the United States were aggregated to statistically define ECVs. ECVs encompassing 97.5% of the statistically modeled population for anidulafungin and micafungin were, respectively, 0.12 and 0.03 µg/ml for C. albicans, 0.12 and 0.03 µg/ml for C. glabrata, 8 and 4 µg/ml for C. parapsilosis, 0.12 and 0.06 µg/ml for C. tropicalis, 0.25 and 0.25 µg/ml for C. krusei, 1 and 0.5 µg/ml for C. lusitaniae, 8 and 2 µg/ml for C. guilliermondii, and 0.12 and 0.12 µg/ml for C. dubliniensis. Previously reported single and multicenter ECVs defined in the present study were quite similar or within 1 2-fold dilution of each other. For a collection of 230 WT isolates (no fks mutations) and 51 isolates with fks mutations, the species-specific ECVs for anidulafungin and micafungin correctly classified 47 (92.2%) and 51 (100%) of the fks mutants, respectively, as non-WT strains. These ECVs may aid in detecting non-WT isolates with reduced susceptibility to anidulafungin and micafungin due to fks mutations.

Interlaboratory variability of Caspofungin MICs for Candida spp. Using CLSI and EUCAST methods: should the clinical laboratory be testing this agent?

Although Clinical and Laboratory Standards Institute (CLSI) clinical breakpoints (CBPs) are available for interpreting echinocandin MICs for Candida spp., epidemiologic cutoff values (ECVs) based on collective MIC data from multiple laboratories have not been defined. While collating CLSI caspofungin MICs for 145 to 11,550 Candida isolates from 17 laboratories (Brazil, Canada, Europe, Mexico, Peru, and the United States), we observed an extraordinary amount of modal variability (wide ranges) among laboratories as well as truncated and bimodal MIC distributions. The species-specific modes across different laboratories ranged from 0.016 to 0.5 µg/ml for C. albicans and C. tropicalis, 0.031 to 0.5 µg/ml for C. glabrata, and 0.063 to 1 µg/ml for C. krusei. Variability was also similar among MIC distributions for C. dubliniensis and C. lusitaniae. The exceptions were C. parapsilosis and C. guilliermondii MIC distributions, where most modes were within one 2-fold dilution of each other. These findings were consistent with available data from the European Committee on Antimicrobial Susceptibility Testing (EUCAST) (403 to 2,556 MICs) for C. albicans, C. glabrata, C. krusei, and C. tropicalis. Although many factors (caspofungin powder source, stock solution solvent, powder storage time length and temperature, and MIC determination testing parameters) were examined as a potential cause of such unprecedented variability, a single specific cause was not identified. Therefore, it seems highly likely that the use of the CLSI species-specific caspofungin CBPs could lead to reporting an excessive number of wild-type (WT) isolates (e.g., C. glabrata and C. krusei) as either non-WT or resistant isolates. Until this problem is resolved, routine testing or reporting of CLSI caspofungin MICs for Candida is not recommended; micafungin or anidulafungin data could be used instead.

Endemic human blastomycosis in Quebec, Canada, 1988-2011.

Blastomycosis is a systemic fungal infection found in various parts of the world. A review of literature for Quebec, Canada revealed only few case reports with the most recent one dating back to 1993. However, whether Quebec represents an important endemic region for blastomycosis in North America is unknown. In this work we reviewed 158 cases of human blastomycosis documented in Quebec during 1988-2011 using microbiological records available from the provincial public health laboratory. The estimated annual incidence of blastomycosis in the province is was ~0·133 cases per 100 000 individuals with the highest rates of 0·79 and 0·46 cases per 100 000 recorded in South-eastern and South-western Quebec. Moreover, the annual incidence rate significantly increased over the past 20 years. This study for the first time establishes Quebec as an important endemic region for Blastomyces dermatitidis.

Cryptococcus neoformans-Cryptococcus gattii species complex: an international study of wild-type susceptibility endpoint distributions and epidemiological cutoff values for fluconazole, itraconazole, posaconazole, and voriconazole.

Epidemiological cutoff values (ECVs) for the Cryptococcus neoformans-Cryptococcus gattii species complex versus fluconazole, itraconazole, posaconazole, and voriconazole are not available. We established ECVs for these species and agents based on wild-type (WT) MIC distributions. A total of 2,985 to 5,733 CLSI MICs for C. neoformans (including isolates of molecular type VNI [MICs for 759 to 1,137 isolates] and VNII, VNIII, and VNIV [MICs for 24 to 57 isolates]) and 705 to 975 MICs for C. gattii (including 42 to 260 for VGI, VGII, VGIII, and VGIV isolates) were gathered in 15 to 24 laboratories (Europe, United States, Argentina, Australia, Brazil, Canada, Cuba, India, Mexico, and South Africa) and were aggregated for analysis. Additionally, 220 to 359 MICs measured using CLSI yeast nitrogen base (YNB) medium instead of CLSI RPMI medium for C. neoformans were evaluated. CLSI RPMI medium ECVs for distributions originating from at least three laboratories, which included ≥95% of the modeled WT population, were as follows: fluconazole, 8 µg/ml (VNI, C. gattii nontyped, VGI, VGIIa, and VGIII), 16 µg/ml (C. neoformans nontyped, VNIII, and VGIV), and 32 µg/ml (VGII); itraconazole, 0.25 µg/ml (VNI), 0.5 µg/ml (C. neoformans and C. gattii nontyped and VGI to VGIII), and 1 µg/ml (VGIV); posaconazole, 0.25 µg/ml (C. neoformans nontyped and VNI) and 0.5 µg/ml (C. gattii nontyped and VGI); and voriconazole, 0.12 µg/ml (VNIV), 0.25 µg/ml (C. neoformans and C. gattii nontyped, VNI, VNIII, VGII, and VGIIa,), and 0.5 µg/ml (VGI). The number of laboratories contributing data for other molecular types was too low to ascertain that the differences were due to factors other than assay variation. In the absence of clinical breakpoints, our ECVs may aid in the detection of isolates with acquired resistance mechanisms and should be listed in the revised CLSI M27-A3 and CLSI M27-S3 documents.

Impact of endpoint definition on the outcome of antifungal susceptibility tests with Candida species: 24- versus 48-h incubation and 50 versus 80% reduction in growth.

The growth inhibition patterns of 764 clinical yeast isolates, in response to amphotericin B, flucytosine, itraconazole and fluconazole, were studied in order to determine the frequency of trailing growth and any impact this, as well as 24- or 48-h incubation periods, may have on minimum inhibitory concentration (MIC) results. A broth microdilution method following National Committee for Clinical Laboratory Standard No. M27A recommendations was used. Trailing growth was observed mainly with azoles. Furthermore, over 98% of isolates exhibiting a trailing effect at 24 h with fluconazole and itraconazole were either Candida albicans or Candida tropicalis. When comparing 24- and 48-h IC50 values, discrepancies were observed with itraconazole and fluconazole, respectively, in 18 and 11% of C. albicans and 24 and 30% of C. tropicalis. When comparing IC50 and IC80 values at 24 h, discrepancies were again essentially seen with itraconazole and fluconazole, respectively, in 11 and 10% of C. albicans, and 17 and 27% of C. tropicalis. In susceptibility tests performed with a microdilution method and read spectrophotometrically, 48-h IC80 values result in an unlikely high number of resistant isolates, indicating that a 24-h incubation and a 50% reduction in optical density may correlate better with clinical outcome.

Prevalence and antifungal susceptibility of 442 Candida isolates from blood and other normally sterile sites: results of a 2-year (1996 to 1998) multicenter surveillance study in Quebec, Canada.

During a 2-year surveillance program (1996 to 1998) in Quebec, Canada, 442 strains of Candida species were isolated from 415 patients in 51 hospitals. The distribution of species was as follows: Candida albicans, 54%; C. glabrata, 15%; C. parapsilosis, 12%; C. tropicalis, 9%; C. lusitaniae, 3%; C. krusei, 3%; and Candida spp., 3%. These data, compared to those of a 1985 survey, indicate variations in species distribution, with the proportions of C. glabrata and C. parapsilosis increasing by 9 and 4%, respectively, and those of C. albicans and C. tropicalis decreasing by 10 and 7%, respectively. However, these differences are statistically significant for C. glabrata and C. tropicalis only. MICs of amphotericin B were > or =4 microg/ml for 3% of isolates, all of which were non-C. albicans species. Three percent of C. albicans isolates were resistant to flucytosine (> or =32 microg/ml). Resistance to itraconazole (> or =1 microg/ml) and fluconazole (> or =64 microg/ml) was observed, respectively, in 1 and 1% of C. albicans, 14 and 9% of C. glabrata, 5 and 0% of C. tropicalis, and 0% of C. parapsilosis and C. lusitaniae isolates. Clinical data were obtained for 343 patients. The overall crude mortality rate was 38%, reflecting the multiple serious underlying illnesses found in these patients. Bloodstream infections were documented for 249 patients (73%). Overall, systemic triazoles had been administered to 10% of patients before the onset of candidiasis. The frequency of isolation of non-C. albicans species was significantly higher in this group of patients. Overall, only two C. albicans isolates were found to be resistant to fluconazole. These were obtained from an AIDS patient and a leukemia patient, both of whom had a history of previous exposure to fluconazole. At present, it appears that resistance to fluconazole in Quebec is rare and is restricted to patients with prior prolonged azole treatment.

Invasive fungal infections in Canada from 1992 to 1994.

PURPOSE: To describe the frequency, characteristics and impact of invasive fungal infection in Canada. METHODS: Nominal case reporting with standardized data collection from selected sites across Canada. Cases were found primarily through laboratory review with supplementation by record review and clinical surveillance at some sites. RESULTS: The frequency of invasive fungal infection varied from 3.54 to 6.64/100,000 population per year. Candida species were responsible for 66% of all reports; 80% of candidal infections were bloodstream isolates. Crytococcus neoformans, Aspergillus species and Histoplasma capsulatum each accounted for 5% to 10% of cases, and all other organisms less than 5% each. Human immunodeficiency virus infection was an important comorbidity for cryptococcus and histoplasma infections, and was associated with increased mortality for only histoplasma infections. Geographical variation of histoplasma, blastomyces and coccidioidomyces infection was confirmed. Case fatality was high for all invasive fungal infections, except coccidioidomycosis, blastomycosis and sporotrichosis. CONCLUSIONS: Candida species infections are the major pathogens in invasive fungal infections in Canada; all other species occur relatively infrequently. The potential for therapeutic intervention to limit mortality requires further assessment.

Effect of media on growth rate and susceptibility testing of Cryptococcus neoformans.

The effect of four media or media combinations on the growth and the in vitro susceptibility to amphotericin B, 5-fluorocytosine, fluconazole and itraconazole of 51 isolates of Cr. neoformans was studied. The drug concentration producing a 50% decrease in OD405 (IC50) was determined in microtiter format. The following MOPS buffered media or media combinations (antifungal drug diluent/inoculum diluent) were used: RPMI 1640 (RPMI)/RPMI, Yeast nitrogen base (YNB)/YNB, RPMI/YNB and RPMI/High resolution broth. Amphotericin B was further tested in M3 broth in the microdilution format and on RPMI agar with the E-test. Growth rates (OD405) were higher in YNB, RPMI/YNB, RPMI/HR and M3 as compared to RPMI alone. After 48 h of incubation, IC50 values obtained with RPMI alone and with RPMI/YNB correlated within two dilutions for 98, 95, 95 and 100% of test strains with amphotericin B, 5-fluorocytosine,fluconazole and itraconazole respectively. The E-test provided the widest range of IC values with amphotericin B. Growth of Cr. neoformans can be enhanced in susceptibility tests using microtiter plates with drugs diluted in RPMI by preparing the inoculum in YNB. However, when using spectrophotometric endpoint determinations, sufficient growth is obtainable with most isolates for test interpretation at 48 h in RPMI alone.

Ketoconazole and itraconazole susceptibility of Candida albicans isolated from patients infected with HIV.

Two hundred and fifty-five isolates of Candida albicans were collected from 93 patients infected with HIV during the course of a clinical trial comparing ketoconazole with itraconazole for the treatment of oropharyngeal or oesophageal candidosis. The susceptibility of the isolates to both drugs was determined by incubating 0.5-2.0 x 10(4) CFU/mL yeast in 0.25-16 mg/L drug in RPMI1640 buffered with MOPS for 24 h at 35 degrees C in 5%CO2 in microtitre trays. Each plate was agitated before reading the optical density. The IC90, IC80 and IC50 were defined as the lowest drug concentration to reduce the optical density to > or = 90%, 80% and 50% of the growth control respectively. IC90S > 0.25 mg/L of ketoconazole and/or itraconazole were found for 42 isolates recovered from 21 patients, 12 of whom had responded to treatment. IC80S > 0.25 mg/L were found for only six of these isolates which had been recovered from three patients, two of whom responded well to treatment. These results indicate that neither the IC90 nor the IC80 are useful in predicting clinical resistance. None of the isolates exhibited IC50 > 0.25 mg/L to either drug which is consistent with the low incidence of resistance reported for these antifungal agents.

Infection due to Rhizomucor pusillus: report of four cases in patients with leukemia and review.

Rhizomucor pusillus, a thermophilic fungus of the order Mucorales, is a rare cause of human infection. A search of the literature has produced only seven reports describing nine cases of infection caused by this organism. Recently, over a period of 17 months, four cases of R. pusillus infection in patients with leukemia were diagnosed: a cluster of three cases in a Montreal hospital and one isolated case from Quebec City. All four cases were proven both by histopathologic examination and by culture of tissues. In three cases, pulmonary involvement was confirmed following lung surgery, and in one case, disseminated infection was observed at autopsy. All patients received amphotericin B, and two underwent surgical debridement; however, none of the patients survived.

Blastomycosis in Quebec (1981-90): Report of 23 cases and review of published cases from Quebec.

Twenty-three cases of blastomycosis were reported in a survey conducted in the province of Quebec from 1981-90. Thirteen patients resided south of the St Lawrence River and the other 10, north. Two small geographical clusters were apparent in and around the cities of Sherbrooke and Quebec. The male to female ratio was 1.6:1 and the median age was 47 years (range 26 to 77). Lung involvement was observed in 19 cases and was the only site involved in 11. Cutaneous manifestations were reported in 11 cases while bone infection (three cases) and central nervous system (CNS) infections were also noted. Diagnosis was confirmed by culture in 21 cases and by histopathology in two cases. Of the 21 culture-positive cases, 12 strains of Blastomyces dermatitidis were isolated from lungs, nine from skin, and one each from bone and brain. Serodiagnostic tests by immunodiffusion or complement fixation were positive for only one of the 10 patients known to have been tested. Ten patients were treated with amphotericin B, 11 with ketoconazole, one with fluconazole and eight underwent surgery. While amphotericin B was used in eight of the 10 earliest treated cases, ketoconazole was administered in 10 of the 13 more recent cases. Of the patients for whom follow-up data have been obtained, 21 are reported cured (one of whom was not treated) and one patient died of another cause. This survey confirms that blastomycosis is a rare disease in this endemic area and that patterns of therapy are changing.

Graft-related endocarditis caused by Neosartorya fischeri var. spinosa.

The first case of endocarditis caused by Neosartorya fischeri var. spinosa is reported. The patient was a child who received a calf pericardium graft after removal of a previously inserted Dacron graft associated with deterioration of adjacent tissue. Copious vegetations removed from the heart were found to be composed of septate hyaline fungal filaments. The fungus was recognized in culture by its bivalved, winged, spiny ascospores, its Aspergillus fischerianus anamorph, and its thermotolerance.

Evaluation of the MicroScan Rapid Yeast Identification panel.

The MicroScan Rapid Yeast Identification (RYI) panel is a 4-h microdilution system for identification of clinical yeastlike isolates. Its accuracy was evaluated by using 357 isolates encompassing 11 genera and 30 species. The RYI panel identifications were compared with those obtained by the API 20C system assisted with morphological characterization on cornmeal-Tween 80 agar. The panels were read both visually and with the AutoScan-4, a computer-controlled microplate reader. Both the RYI panel and the API 20C system correctly identified 78% of the strains within 4 and 72 h, respectively, with no additional tests. Supplementary tests recommended by the manufacturers made it possible to identify up to 96.6% (AutoScan-4) and 98.9% (API 20C) of the strains. The accuracy of the RYI panel was 99.5% with common strains and 92.1% with less common strains. The RYI panel misidentified 10 or 12 strains and failed to identify 2 or 3 strains, depending on whether it was read with the AutoScan-4 or visually. Errors occurred with one strain of Torulopsis glabrata and the less common yeasts T. candida, Candida lusitaniae, C. lambica, C. rugosa, C. stellatoidea, Cryptococcus albidus, C. laurentii, and C. uniguttulatus. Overall, the RYI panel appears to be a reliable system for identification of the more common clinical yeast isolates.

Effects of pentamidine alone and in combination with ketoconazole or itraconazole on the growth of Candida albicans.

The in vitro interaction of pentamidine with ketoconazole and with itraconazole was studied with 10 strains of Candida albicans isolated from acquired immunodeficiency syndrome patients and one azole-resistant strain. Although growth curves indicated that concentrations of 1 microgram or more of pentamidine per ml significantly inhibited the growth of C. albicans, MICs and minimum fungicidal concentrations (MFCs) were greater than or equal to 10 micrograms/ml. Combinations of ketoconazole and pentamidine did not appear to have any significant effect on MICs or MFCs with most strains. However, the azole-resistant strain exhibited a 2-log decrease in MIC when exposed to ketoconazole combined with 1.0 microgram or more of pentamidine per ml. Similar results were obtained with itraconazole. An Eagle, or paradoxical, effect was observed with four strains exposed to itraconazole alone and in combination with 0.01 and 0.1 microgram of pentamidine per ml. This effect was not seen when concentrations of pentamidine reached 1.0 microgram/ml. Although no fungicidal effect was observed with any of these drugs alone, itraconazole combined with 10 micrograms of pentamidine per ml was fungicidal for eight strains. No signs of antagonism between pentamidine and these two antifungal agents were observed.

Collaborative evaluation of antigen detection by a commercial latex agglutination test and enzyme immunoassay in the diagnosis of invasive candidiasis.

The Cand-Tec Candida detection system and enzyme immunoassay for serum mannan were retrospectively compared in a controlled collaborative evaluation of antigen detection in 32 patients with candidiasis proven by biopsy or culture from a normally sterile site and with sera drawn within 7 days of inclusion. With a threshold titer of 1/8, which excluded false-positive results in 17 hospitalized patients without candidiasis, sensitivities for all 32 patients with candidiasis were 44% for the Cand-Tec assay and 17% for the enzyme immunoassay. Both assays provided greater sensitivity when sera were drawn within 24 h of inclusion in the study and in the category of patients with invasive candidiasis (57% by Cand-Tec and 33% by enzyme immunoassay). The Cand-Tec assay gave false-positive results (titer, greater than or equal to 1/8) in 4 of 6 patients with transient candidemia, in 1 of 20 otherwise healthy patients with rheumatoid factor, and in 1 patient with a positive cryptococcal latex agglutination test. Three serum specimens from 3 of 32 patients with candidiasis contained rheumatoid factor and gave titers of greater than or equal to 1/8 by the Cand-Tec assay. Detection of serum mannan by enzyme immunoassay was less sensitive but more specific than the Cand-Tec Candida detection system for the diagnosis of invasive candidiasis.

Performance characteristics of two bioassays and high-performance liquid chromatography for determination of flucytosine in serum.

We compared the accuracy and precision of two microbiological methods and one high-pressure liquid chromatography (HPLC) procedure used to measure the concentrations of flucytosine in serum. On the basis of an analysis of six standards, all methods were judged reliable within acceptable limits for clinical use. With the biological methods, a slight loss of linearity was observed in the 75- to 100-micrograms/ml range. Compared with the bioassays, the HPLC method did not present linearity problems and was more precise and accurate in the critical zone of 100 micrograms/ml. On average, results obtained with patient sera containing 50 to 100 micrograms of flucytosine per ml were 10.6% higher with the HPLC method than with the bioassays. Standards for the biological assays may be prepared in serum or water.