Interlaboratory reproducibility of Etest amphotericin B and caspofungin yeast susceptibility testing and comparison with the CLSI method.

This study aimed to assess the interlaboratory reproducibility at four university hospital laboratories in the southeast region of France of the Etest technique for the determination of caspofungin (CAS) and amphotericin B (AMB) MICs and to compare it to the CLSI broth microdilution reference method. Consecutive clinical yeast isolates (n = 198) were included in the study. AMB and CAS MICs were read at 24 and 48 h. Interlaboratory reproducibility was estimated by using (i) an intraclass correlation coefficient (ICC), (ii) essential agreement (EA), and (iii) categorical agreement (CA). For Etest interlaboratory reproducibility for CAS, ICCs were 0.80 (95% confidence interval [CI], 0.76 to 0.84) and 0.81 (95% CI, 0.77 to 0.85) at 24 and 48 h, respectively. For AMB, the ICCs were 0.51 (95% CI, 0.43 to 0.58) and 0.69 (95% CI, 0.63 to 0.74) at 24 and 48 h, respectively. At 48 h, the between-center EAs ranged from 94.4 to 99.0% for both antifungals. For the comparison of the CLSI method and the Etest, the between-technique ICCs were 0.69 (95% CI, 0.63 to 0.74) and 0.62 (95% CI, 0.55 to 0.68) for CAS and AMB, respectively. The EAs ranged from 76.5 to 98.5% for CAS and from 90.3 to 97.4% for AMB according to the centers. CAs ranged from 87.9% to 91.4%, with four very major errors for 2 strains (1 Candida albicans strain and 1 Candida krusei strain), for CAS and from 97.5 to 99.5%, with four major errors, for AMB. In conclusion, the Etest showed a good interlaboratory reproducibility and a good correlation with the CLSI technique. It is well suited for the routine clinical laboratory and can thus be used to monitor clinical yeast isolates' in vitro susceptibilities in this setting.

Platelia Aspergillus assay for diagnosis of disseminated histoplasmosis.

The Platelia Aspergillus assay tested positive in 8 of 11 patients with disseminated histoplasmosis. While other available methods for diagnosis have several drawbacks, this cross-reactivity is particularly valuable in the perspective of practitioners outside the USA who cannot use the test detecting antigen of Histoplasma capsulatum.

Results obtained with various antifungal susceptibility testing methods do not predict early clinical outcome in patients with cryptococcosis.

The in vitro susceptibilities of Cryptococcus neoformans isolates from consecutive human immunodeficiency virus-positive and -negative patients to the antifungal agents fluconazole, amphotericin B, and flucytosine were determined by different techniques, including the CLSI method, Etest, and broth microdilution in yeast nitrogen base (YNB) medium, during a multicenter prospective study in France. The relationship between the in vitro data and the clinical outcome 2 weeks after the initiation of antifungal therapy was assessed. In addition, the correlation between the strain serotype and the in vitro activities of the antifungals was determined, and the susceptibility results obtained with the different techniques were also compared. Thirty-seven patients received a combination of amphotericin B with flucytosine as first-line therapy, 22 were treated with amphotericin B alone, and 15 received fluconazole alone. Whatever the antifungal tested, there was no trend toward higher MICs for strains isolated from patients who failed to respond to a given therapy compared to those from patients who did not with either the CLSI method, Etest, or broth microdilution in YNB medium. The MICs obtained by the CLSI or Etest method were significantly lower for serotype D strains than for serotype A strains for both fluconazole and amphotericin B, while flucytosine MICs were not different according to serotype. These findings suggest that the in vitro antifungal susceptibility of C. neoformans, as determined with the techniques used, is not able to predict the early clinical outcome in patients with cryptococcosis.

Long-term epidemiological survey of Candida species: comparison of isolates found in an intensive care unit and in conventional wards.

The aim of this study was to compare the distribution of Candida species in patients hospitalized in an intensive care unit (ICU) and in conventional wards. A retrospective analysis was performed covering an 18-year period in a 700-bed teaching hospital. Various body sites were investigated in all patients admitted during the study and isolates were identified by microscopic and macroscopic morphology, and by commercially available kits. The susceptibility of strains to amphotericin B and flucytosine was assessed by the ATB-fungus system, itraconazole and fluconazole by Etest. No difference was observed between the distribution of Candida species in ICU and in conventional wards. Candida albicans represented about 70% of isolates and Candida glabrata was the second most common species involved in infection or colonization. The small number of C. glabrata resistant to fluconazole suggested this antifungal agent as suitable empirical treatment for non-immunocompromized patients in whom a fungal infection was suspected.

Sterol and fatty acid composition of Candida lusitaniae clinical isolates.

The sterol and fatty acid compositions of four amphotericin B-resistant and of two amphotericin B-susceptible Candida lusitaniae clinical isolates were determined. A flow cytofluorometric susceptibility test (FCST) with a membrane potential-sensitive cationic dye was used as a complement to the conventional method for selecting the isolates. Compared to susceptible isolates, resistant ones showed a greatly reduced ergosterol content and changes in sterol composition, consistent with a defect in Delta8-->7 isomerase. Within each group, no correlation between the sterol or fatty acid pattern or composition and both the degree of in vitro susceptibility and FCST MIC was found.

Improved detection of amphotericin B-resistant isolates of Candida lusitaniae by Etest.

Both intrinsic and acquired resistance to amphotericin B have been documented for Candida lusitaniae. Amphotericin B remains the drug of choice for many critical fungal infections, and the detection of resistance is essential to monitor treatment effectively. The limitations of the National Committee for Clinical Laboratory Standards (NCCLS) reference methodology for detection of amphotericin B resistance are well documented, and several alternative methods have been proposed. Etest assays with RPMI and antibiotic medium 3 (AM3) agar were compared to the NCCLS M27-A broth macrodilution method using AM3 for amphotericin B resistance testing with 49 clinical isolates of C. lusitaniae. The panel included nine isolates with known or presumed resistance to amphotericin B on the basis of in vivo and/or in vitro data. The distribution of amphotericin B MICs by Etest with RPMI ranged from 0. 032 to 16 microg/ml and was bimodal. All of the putatively resistant isolates were inhibited by amphotericin B at >/=0.38 microg/ml and could be categorized as resistant using this breakpoint. Etest with AM3 yielded a broader amphotericin B MIC range (0.047 to 32 microg/ml), and there were six putatively resistant isolates for which MICs were >1 microg/ml. The separation of putatively susceptible and resistant isolates was less obvious. Broth macrodilution with AM3 generated a unimodal distribution of MICs (ranging from 0.032 to 2 microg/ml) and failed to discriminate most of the putatively resistant isolates at both 24 and 48 h. Etest using RPMI and, to a lesser extent, using AM3 provided better discrimination between amphotericin B-resistant and -susceptible isolates of C. lusitaniae.

Comparative evaluation of a commercial system for identification of Candida lusitaniae.

The ability of the API Candida system (bioMérieux, France) to identify Candida lusitaniae isolates was evaluated in comparison to the Auxacolor and ID 32C systems using 52 clinical isolates previously identified on the basis of their morphology and their biochemical reactions in the Auxacolor and ID 32C systems. The API Candida system failed to definitively identify most of the strains tested within 24 h. No beta-maltosidase activity was detected in 28 strains, and supplementary tests were required to discriminate Candida lusitaniae, Candida famata and Candida guilliermondii. The API Candida system is not suitable for identification of Candida lusitaniae. In comparison, the Auxacolor system is easy to use and interpret, allowing rapid identification of this species; however, the ID 32C system is required for identification of atypical strains.

Evaluation of the E test for antifungal susceptibility testing of Candida glabrata.

The E test was compared to the reference NCCLS broth macrodilution method for susceptibility testing of Candida (Torulopsis) glabrata. The MICs of amphotericin B, flucytosine, fluconazole and itraconazole were determined using the appropriate culture media (RPMI 1640 agar with 2% glucose, Casitone agar or Antibiotic Medium 3 agar) according to the drug tested. Agreement between the two methods was within plus/minus two dilutions for 77-100% of test results, according to the drug/medium combination. The study revealed problems in determining the MICs of azoles using the E test, and confirmed the suitability of Casitone agar for susceptibility testing of fluconazole even if results were read within 24 h.

Do the components of heat and moisture exchanger filters affect their humidifying efficacy and the incidence of nosocomial pneumonia?

OBJECTIVES: To compare the efficiency of two heat and moisture exchange filters (HMEFs) of different compositions of the humidifying capacity and the rate of bronchial colonization and ventilator-associated pneumonia in patients in the intensive care unit (ICU). DESIGN: Prospective, randomized study. SETTING: ICU of a university hospital. PATIENTS: All patients who required mechanical ventilation for 24 hrs or more during the study period. INTERVENTIONS: At admission to the ICU, patients were randomly assigned to one of two groups. In one group, the patients were ventilated with Humid-Vent Filter Light HMEF. The condensation surface was made of paper impregnated with CaCl2. The filter membrane was made of polypropylene. In the other group, the patients were ventilated with the Clear ThermAl HMEF (Intersurgical, France). The condensation surface was made of plastic foam impregnated with AlCl2. The filter membrane was made of two polymer fibers (modacrylic and polypropylene). In both groups, HMEFs were changed daily. MEASUREMENTS AND MAIN RESULTS: Seventy-seven patients were ventilated for 19+/-7 days with the Humid-Vent Filter Light HMEF and 63 patients for 17+/-6 days with the Clear ThermAl HMEF. Patients ventilated with the Humid-Vent Filter Light underwent 8.7+/-3.7 tracheal aspirations and 1.2+/-2.0 instillations per day and those with the Clear ThermAl, 8.2+/-3.9 and 1.5+/-2.4 per day, respectively (NS). The abundance of tracheal secretions and the presence of blood and viscosity, as evaluated by semiquantitative scales, were similar in both groups. One episode of tracheal tube occlusion was observed with the Humid-Vent Filter Light HMEF and none with the other HMEF (NS). Tracheal colonization was observed at a rate of 91% with the Humid-Vent Filter Light and 97% with the Clear ThermAl (NS). The rate of ventilator-associated pneumonia was similar in both groups (35%). Bacteria responsible for tracheal colonization and pneumonia were similar in both groups. CONCLUSIONS: Despite differences in their components, the two HMEFs that were tested achieved similar performances in terms of humidification and heating of inspired gases. Only one episode of endotracheal tube occlusion was detected, and very few patients (three in each group) had to be switched to an active heated humidifier. No difference was observed either in the rate of tracheal colonization or of ventilator-associated pneumonia. These data show that the Humid-Vent Filter Light and the Clear ThermAl HMEFs are suited for use with ICU patients.

Amphotericin B susceptibility testing of Candida lusitaniae isolates by flow cytofluorometry: comparison with the Etest and the NCCLS broth macrodilution method.

A flow cytofluorometric susceptibility test (FCST) was used for rapid determination of the susceptibility of Candida lusitaniae isolates to amphotericin B. The test is based on the decrease in fluorescence intensity of cells stained with 3,3'-dipentyloxacarbocyanine iodide (DiOC5(3)), a membrane potential-sensitive cationic dye, after drug treatment. A total of 58 C. lusitaniae clinical isolates including strains known to be amphotericin B-resistant on the basis of in-vivo and/or in-vitro data were tested. MICs were determined concurrently by the NCCLS broth macrodilution method and the Etest, both with antibiotic medium 3. Regression analysis demonstrated that the data from the FCST and the Etest were better correlated (r = 0.93, n = 59, P < 0.001) than those from the FCST and the NCCLS method (r = 0.63, n = 59, P < 0.001). The FCST readily identified a series of putatively susceptible and resistant isolates. Our study points out the advantages of the flow cytometry approach in antifungal susceptibility testing of yeasts, since speed remains a major problem in conventional tests.

In-vitro susceptibility pattern of Candida lusitaniae and evaluation of the Etest method.

The antifungal susceptibility of 35 Candida lusitaniae isolates was determined in vitro by the National Committee for Clinical Laboratory Standards (NCCLS) M27-P macrodilution methodology. All the isolates were susceptible to ketoconazole, itraconazole and fluconazole. Of the 35 isolates, eight (23%) were resistant to flucytosine. For amphotericin B, M27-P yielded a narrow range of MICs (0.06-0.5 mg/L). We therefore investigated the activity of this drug by reading MICs at 72 h and by using alternative media, namely casitone complex medium (CCM) and antibiotic medium 3 (M-3). Reading at 72 h did not generate reproducible results. CCM and M-3 provided better discrimination between the isolates but did not change the rank order of the MICs. We thus concluded that all the isolates were susceptible to amphotericin B. We also conducted an evaluation with the Etest method according to the manufacturer's instructions with RPMI 1640 agar, CCM and the alternative semi-synthetic medium (SSM). For RPMI 1640, agreements +/-2 dilutions were 58% for amphotericin B, 92% for flucytosine, 57% for ketoconazole, 92% for fluconazole and 74% for itraconazole. CCM did not improve the agreement rates between the two methods but it led to better growth of all the isolates. The suitability of SSM was pointed out with itraconazole. The poor agreement rates for amphotericin B and ketoconazole call for further evaluation of the Etest method to assess several drug-organism combinations.

[Vaginal ecology, climate, landscapes and populations (xenoecies and facies)]. / L'écologie vaginale, climat, paysages et peuplements (xénoecies et faciès).

Brief study of vaginal populations, the human vagina being considered as a biotopic cavity. Allusion to dynamic aspects ("vaginal climate", "landscapes") and to various bacterial populations. Introduction of the concept of xenoecies and of facies. This study is preceded by essential definitions of terms widely used in ecology.