Evaluation of direct antifungal susceptibility testing methods of Candida spp. from positive blood culture bottles.

BACKGROUND: Blood culture is the gold standard diagnostic method in candidemia in spite of long result time and low sensitivity rate. Early diagnosis is crucial for management of candidemia because a delay in treatment is related with increased mortality. We aimed to evaluate the direct applicability of antifungal susceptibility testing methods from positive blood culture bottles to save at least 24 hours. METHODS: Blood culture bottles were inoculated with 62 Candida isolates. Etest and broth microdilution (BMD) methods for six antifungals, disk diffusion (DD) method for two antifungals were performed, both directly from bottles and standardly. RESULTS: Essential agreements between direct and standard Etest methods were 87.1% for caspofungin and >90% for other antifungals, but the agreements of them with reference BMD were relatively low. Essential agreement between direct and standard BMD was >93%. Correlation between direct and standard DD methods was very high, negative correlations were observed between reference BMD and DD methods. CONCLUSION: BMD is a reference method to evaluate the antifungal susceptibility, direct application of BMD might provide reliable results at least 24 hours earlier. Direct DD method may be a qualitative alternative. Direct susceptibility testing methods may be very useful to initiating the appropriate treatment on time.

Evaluation of three different bottles in BACTEC 9240 automated blood culture system and direct identification of Candida species to shorten the turnaround time of blood culture.

PURPOSE: Candida spp. are the most common causes of fungemia. Rapid and accurate diagnostic methods are very important for appropriate management of candidemia. At present, blood culture is the essential diagnostic test despite having a long detection time and low sensitivity rate. We aimed to investigate the ways to shorten the turnaround time from blood culture collection to final identification in candidemia. METHODOLOGY: Sixty clinical bloodstream isolates of Candida were included, and Plus Aerobic/F, Peds Plus/F and Mycosis IC/Fbottles were used with a BACTEC 9240 blood culture instrument. Germ tube production, carbohydrate assimilation (API 20C AUX) and peptide nucleic acid fluorescent in situ hybridization yeast traffic light tests were performed directly from positive-signalled bottles. RESULTS: Time to positivity of blood cultures was affected by species of Candida, fungal load and bottle type. Candidatropicalis had the shortest and Candidaglabrata had the longest time to positivity. Mycosis IC/F culture bottle had a significant superiority in the isolation of yeasts, especially for C. glabrata and if there was a low fungal load in the bottle. Direct germ tube test had 90 % sensitivity and 97.6 % specificity for Candidaalbicans in two hours after signalling. The compliance between direct and classical assimilation tests was 98.3 %. Sensitivity and specificity of peptide nucleic acid fluorescent in situ hybridization were 100 %. CONCLUSION: We think that it is possible to shorten the turnaround time for the identification of Candida in blood culture even with currently available methods.

Time-Kill Kinetics and In Vitro Antifungal Susceptibility of Non-fumigatus Aspergillus Species Isolated from Patients with Ocular Mycoses.

Aspergillus species can cause ocular morbidity and blindness, and thus, appropriate antifungal therapy is needed. We investigated the in vitro activity of itraconazole, voriconazole, posaconazole, caspofungin, anidulafungin, and amphotericin B against 14 Aspergillus isolates obtained from patients with ocular mycoses, using the CLSI reference broth microdilution methodology. In addition, time-kill assays were performed, exposing each isolate separately to 1-, 4-, and 16-fold concentrations above the minimum inhibitory concentration (MIC) of each antifungal agent. A sigmoid maximum-effect (E max) model was used to fit the time-kill curve data. The drug effect was further evaluated by measuring an increase/decrease in the killing rate of the tested isolates. The MICs of amphotericin B, itraconazole, voriconazole, and posaconazole were 0.5-1.0, 1.0, 0.5-1.0, and 0.25 µg/ml for A. brasiliensis, A. niger, and A. tubingensis isolates, respectively, and 2.0-4.0, 0.5, 1.0 for A. flavus, and 0.12-0.25 µg/ml for A. nomius isolates, respectively. A. calidoustus had the highest MIC range for the azoles (4.0-16.0 µg/ml) among all isolates tested. The minimum effective concentrations of caspofungin and anidulafungin were ≤0.03-0.5 µg/ml and ≤0.03 µg/ml for all isolates, respectively. Posaconazole demonstrated maximal killing rates (E(max) = 0.63 h(-1), r(2) = 0.71) against 14 ocular Aspergillus isolates, followed by amphotericin B (E(max) = 0.39 h(-1), r(2) = 0.87), voriconazole (E(max) = 0.35 h(-1), r(2) = 0.098), and itraconazole (E(max) = 0.01 h(-1), r(2) = 0.98). Overall, the antifungal susceptibility of the non-fumigatus Aspergillus isolates tested was species and antifungal agent dependent. Analysis of the kinetic growth assays, along with consideration of the killing rates, revealed that posaconazole was the most effective antifungal against all of the isolates.