scholarly journals Influence of Shaking on Antifungal Susceptibility Testing of Cryptococcus neoformans: a Comparison of the NCCLS Standard M27A Medium, Buffered Yeast Nitrogen Base, and RPMI–2% Glucose

2000 ◽  
Vol 44 (2) ◽  
pp. 400-404 ◽  
Author(s):  
Juan L. Rodríguez-Tudela ◽  
Francisco Martín-Díez ◽  
Manuel Cuenca-Estrella ◽  
Laura Rodero ◽  
Yolanda Carpintero ◽  
...  
Keyword(s):  
Cryptococcus Neoformans ◽  
Clinical Laboratory ◽  
Culture Media ◽  
Antifungal Susceptibility ◽  
Optimal Growth ◽  
Inoculum Size ◽  
National Committee ◽  
Yeast Nitrogen Base ◽  
Nitrogen Base ◽  
Antifungal Susceptibility Test

ABSTRACT Cryptococcus neoformans is a nonfermentative yeast that requires oxygen for growth. The shaking of culture media achieves good oxygenation, promoting the growth of cryptococci. In this study, three test media (RPMI 1640, RPMI 1640–2% glucose, and buffered yeast nitrogen base [BYNB]) recommended in the National Committee for Clinical Laboratory Standards M27A standard were examined. Growth abilities and minimum inhibitory concentrations (MICs) in microplates incubated at 35°C for 48 h were determined. The results indicated that shaking and an inoculum size of 105 CFU/ml yielded optimal growth of this yeast. Compared to RPMI 1640, supplementation of RPMI 1640 with 2% glucose did not significantly improve growth of C. neoformans and resulted in an 8.7-h delay of exponential growth. Cryptococcal growth in RPMI 1640 at 24 h was notably better than that in RPMI–2% glucose, although by 48 h the growths were comparable. The MIC range of amphotericin B observed for the C. neoformans strains grown in RPMI 1640 with or without glucose was too narrow to allow the separation of susceptible and resistant strains based on clinical outcome. The widest ranges of MICs of flucytosine and fluconazole were obtained with BYNB. This work demonstrates the need for a new antifungal susceptibility test for C. neoformans.

scholarly journals Fluconazole Susceptibility Testing of Cryptococcus neoformans: Comparison of Two Broth Microdilution Methods and Clinical Correlates among Isolates from Ugandan AIDS Patients

1998 ◽  
Vol 36 (10) ◽  
pp. 2874-2876 ◽  
Author(s):  
C. J. Jessup ◽  
M. A. Pfaller ◽  
S. A. Messer ◽  
J. Zhang ◽  
M. Tumberland ◽  
...  
Keyword(s):  
Cryptococcus Neoformans ◽  
Clinical Laboratory ◽  
Reference Method ◽  
National Committee ◽  
Yeast Nitrogen Base ◽  
Broth Microdilution ◽  
Aids Patients ◽  
In Vitro Susceptibility ◽  
Nitrogen Base ◽  
Broth Microdilution Method

We compared the yeast nitrogen base (YNB) broth microdilution method with the National Committee for Clinical Laboratory Standards (NCCLS) M27-A microdilution reference method for measuring the in vitro susceptibility of Cryptococcus neoformans isolates to fluconazole. A total of 149 isolates of C. neoformans var.neoformans from Ugandan AIDS patients was tested by both methods. An overall agreement of 88% between the two microdilution methods was observed. All isolates grew well in both RPMI 1640 and YNB media, and MICs could be read after 48 h of incubation by both methods. The range of fluconazole MICs obtained with the YNB method was broader than that obtained with the NCCLS method. The extended range of MICs provided by the YNB method may be of clinical value, as it appears that the clinical outcome may be better among patients infected with strains inhibited by lower concentrations of fluconazole as determined by the YNB method. The YNB method appears to be a viable option for testing C. neoformans against fluconazole.

scholarly journals Antifungal susceptibility testing.

1993 ◽  
Vol 6 (4) ◽  
pp. 367-381 ◽  
Author(s):  
J H Rex ◽  
M A Pfaller ◽  
M G Rinaldi ◽  
A Polak ◽  
J N Galgiani
Keyword(s):  
Susceptibility Testing ◽  
Incubation Temperature ◽  
Clinical Laboratory ◽  
Antifungal Susceptibility ◽  
Inoculum Size ◽  
Antifungal Susceptibility Testing ◽  
National Committee ◽  
Candida Spp ◽  
Interlaboratory Variability ◽  
Future Work

Unlike antibacterial susceptibility testing, reliable antifungal susceptibility testing is still largely in its infancy. Many methods have been described, but they produce widely discrepant results unless such factors as pH, inoculum size, medium formulation, incubation time, and incubation temperature are carefully controlled. Even when laboratories agree upon a common method, interlaboratory agreement may be poor. As a result of numerous collaborative projects carried out both independently and under the aegis of the Subcommittee on Antifungal Susceptibility Testing of the National Committee for Clinical Laboratory Standards, the effects of varying these factors have been extensively studied and a standard method which minimizes interlaboratory variability during the testing of Candida spp. and Cryptococcus neoformans has been proposed. This review summarizes this work, reviews the strengths and weaknesses of the proposed susceptibility testing standard, and identifies directions for future work.

scholarly journals Evaluation of a Colorimetric Antifungal Susceptibility Test by Using 2,3-Diphenyl-5-Thienyl-(2)-Tetrazolium Chloride

2004 ◽  
Vol 48 (11) ◽  
pp. 4457-4459 ◽  
Author(s):  
Jeong Hwan Shin ◽  
Jae-Cheol Choi ◽  
Jeong Nyeo Lee ◽  
Hyung Hoi Kim ◽  
Eun Yup Lee ◽  
...  
Keyword(s):  
Clinical Laboratory ◽  
Colorimetric Assay ◽  
Antifungal Susceptibility ◽  
Susceptibility Test ◽  
National Committee ◽  
Tetrazolium Chloride ◽  
Antifungal Susceptibility Test

ABSTRACT A colorimetric antifungal susceptibility test was performed using 2,3-diphenyl-5-thienyl-(2)-tetrazolium chloride. Among 24 strains of Candida species, no trailing growth was found. In 22 and 20 strains, the MICs obtained in the colorimetric assay were within two dilutions of those obtained by the National Committee for Clinical Laboratory Standards method for ketoconazole and itraconazole, respectively.

scholarly journals Determination of Antifungal MICs by a Rapid Susceptibility Assay

2000 ◽  
Vol 38 (1) ◽  
pp. 333-340
Author(s):  
Marcia H. Riesselman ◽  
Kevin C. Hazen ◽  
Jim E. Cutler
Keyword(s):  
Clinical Laboratory ◽  
Antifungal Susceptibility ◽  
Colorimetric Method ◽  
Inoculum Size ◽  
Antifungal Drugs ◽  
Therapeutic Drug ◽  
National Committee ◽  
Substrate Uptake ◽  
Microtiter Assay ◽  
Susceptibility Assay

ABSTRACT A novel microtiter assay for antifungal susceptibility testing was developed. This method has several potential advantages over the M27-A assay of the National Committee for Clinical Laboratory Standards. These include provision of MIC results within 6 to 19 h, graphical display of data, and the availability of objective quantitative endpoints. We refer to the method as the rapid susceptibility assay (RSA). RSA is based on substrate utilization by fungi in the presence of antifungal drugs. Substrate uptake is determined by a colorimetric method, which can be scored by analysis of data obtained from a microplate reader. Variables evaluated in the development of the RSA included inoculum size, incubation period, and efficacy with different classes of antifungal drugs and different yeast isolates. With the rapidly available and quantitative endpoints of the RSA, correlation of MICs and therapeutic drug doses can be evaluated more successfully than they can be evaluated by existing assays.

scholarly journals Comparison of the in vitro activities of the echinocandin LY303366, the pneumocandin MK-0991, and fluconazole against Candida species and Cryptococcus neoformans.

1997 ◽  
Vol 41 (9) ◽  
pp. 1957-1960 ◽  
Author(s):  
T V Krishnarao ◽  
J N Galgiani
Keyword(s):  
Cryptococcus Neoformans ◽  
Candida Species ◽  
Clinical Laboratory ◽  
Inoculum Size ◽  
National Committee ◽  
Broth Microdilution ◽  
Test Conditions ◽  
Broth Macrodilution ◽  
Glucan Synthesis

Two new glucan synthesis inhibitors, the echinocandin LY303366 and the pneumocandin MK-0991 (formerly L-743,872), were studied for their antifungal activities in vitro in relation to each other and in relation to the activity of the triazole fluconazole. Systematic analysis of broth macrodilution testing by varying the starting inoculum size, medium composition, medium pH, temperature of incubation, length of incubation, or selection of endpoints failed to identify significant differences in antifungal activity for either LY303366 or MK-0991 in comparison to the activity under standard test conditions specified for other antifungal agents in National Committee for Clinical Laboratory Standards (NCCLS) document M27A. Under standardized conditions, both drugs exhibited prominent activity against Candida species including Candida glabrata and Candida krusei but showed little activity against Cryptococcus neoformans. This spectrum of activity differed from that of fluconazole, which exhibited marginal activity against C. glabrata and C. krusei but prominent activity against other Candida species and C. neoformans. For individual strains, broth microdilution MICs of LY303366 and MK-0991 were similar to but frequently higher than broth macrodilution results. In contrast, fluconazole broth microdilution MICs were often lower than broth microdilution results. We conclude that the test conditions specified in NCCLS document M27A are applicable to these two new glucan synthesis inhibitors and that systematic differences between broth microdilution procedures and the broth macrodilution reference standard will need to be addressed before the two test methods can be used interchangeably.

scholarly journals Interactions of Posaconazole and Flucytosine against Cryptococcus neoformans

2001 ◽  
Vol 45 (5) ◽  
pp. 1355-1359 ◽  
Author(s):  
Francesco Barchiesi ◽  
Anna Maria Schimizzi ◽  
Laura K. Najvar ◽  
Rosie Bocanegra ◽  
Francesca Caselli ◽  
...  
Keyword(s):  
Combination Therapy ◽  
Cryptococcus Neoformans ◽  
Clinical Laboratory ◽  
Combined Therapy ◽  
Antifungal Susceptibility ◽  
National Committee ◽  
Treatment Groups ◽  
Beneficial Effects

ABSTRACT A checkerboard methodology, based on standardized methods proposed by the National Committee for Clinical Laboratory Standards for broth microdilution antifungal susceptibility testing, was applied to study the in vitro interactions of flucytosine (FC) and posaconazole (SCH 56592) (FC-SCH) against 15 isolates of Cryptococcus neoformans. Synergy, defined as a fractional inhibitory concentration (FIC) index of <0.50, was observed for 33% of the isolates tested. When synergy was not achieved, there was still a decrease in the MIC of one or both drugs when they were used in combination. Antagonism, defined as a FIC of >4.0, was not observed. The in vitro efficacy of combined therapy was confirmed by quantitative determination of the CFU of C. neoformans 486, an isolate against which the FC-SCH association yielded a synergistic interaction. To investigate the potential beneficial effects of this combination therapy in vivo, we established two experimental murine models of cryptococcosis by intracranial or intravenous injection of cells ofC. neoformans 486. At 1 day postinfection, the mice were randomized into different treatment groups. One group each received each drug alone, and one group received the drugs in combination. While combination therapy was not found to be significantly more effective than each single drug in terms of survival, tissue burden experiments confirmed the potentiation of antifungal activity with the combination. Our study demonstrates that SCH and FC combined are significantly more active than either drug alone against C. neoformans in vitro as well in vivo. These findings suggest that this therapeutic approach could be useful in the treatment of cryptococcal infections.

scholarly journals Pentamidine Is Active In Vitro against Fusarium Species

2003 ◽  
Vol 47 (10) ◽  
pp. 3252-3259 ◽  
Author(s):  
Michail S. Lionakis ◽  
Russell E. Lewis ◽  
George Samonis ◽  
Dimitrios P. Kontoyiannis
Keyword(s):  
Clinical Laboratory ◽  
Fusarium Species ◽  
Tissue Perfusion ◽  
In Vivo Studies ◽  
National Committee ◽  
Yeast Nitrogen Base ◽  
Nitrogen Base ◽  
Minimum Fungicidal Concentration

ABSTRACT Fusariosis is an emerging opportunistic mycosis against which currently used antifungals have limited activity. Here, we investigated the in vitro activities of pentamidine (PNT) against 10 clinical isolates of Fusarium species (five Fusarium solani isolates and five non-F. solani isolates) by using the National Committee for Clinical Laboratory Standards microdilution method in three different media (RPMI, RPMI-2, and a yeast nitrogen base medium), disk diffusion testing, and viability dye staining. PNT had significant activities against all 10 Fusarium isolates. Non-F. solani isolates were more susceptible than F. solani isolates (P < 0.05). Additionally, PNT was fungicidal against all non-F. solani isolates, whereas it had fungistatic effects against four of the five F. solani isolates. PNT also exhibited greater activity against conidial than against hyphal development of the fungus. This fungicidal activity against non-F. solani Fusarium isolates was confirmed microscopically after staining of PNT-treated Fusarium oxysporum hyphae with the fluorescent viability dyes 5,(6)-carboxyfluorescein diacetate (CFDA) and bis-(1,3-dibutylbarbituric acid) trimethine oxonol (DiBAC). The MICs at which 50% of the isolates were inhibited (2 μg/ml for non-F. solani isolates and 4 μg/ml for F. solani isolates) and the minimum fungicidal concentration at which 50% of the isolates were killed (8 μg/ml for non-F. solani isolates) were much lower than the PNT tissue concentrations previously reported in humans using conventional daily intravenous PNT dosing. Finally, PNT was more active against Fusarium isolates in a hypoxic environment of in vitro growth (P < 0.05). This finding may be clinically significant, because Fusarium, an angiotropic mold, causes tissue infarcts with resultant low tissue perfusion. Our findings suggest that PNT may have a role in the management of Fusarium infections. Future in vivo studies are needed to verify these in vitro findings.

scholarly journals Detection of Resistance to Amphotericin B amongCryptococcus neoformans Clinical Isolates: Performances of Three Different Media Assessed by Using E-Test and National Committee for Clinical Laboratory Standards M27-A Methodologies

1998 ◽  
Vol 36 (10) ◽  
pp. 2817-2822 ◽  
Author(s):  
M. Lozano-Chiu ◽  
V. L. Paetznick ◽  
M. A. Ghannoum ◽  
J. H. Rex
Keyword(s):  
Amphotericin B ◽  
Susceptibility Testing ◽  
Clinical Laboratory ◽  
Diffusion Method ◽  
National Committee ◽  
Yeast Nitrogen Base ◽  
Nitrogen Base ◽  
Agar Diffusion ◽  
Agar Diffusion Method

Although reliable detection of resistance in vitro is critical to the overall performance of any susceptibility testing method, the recently released National Committee for Clinical Laboratory Standards M27-A methodology for susceptibility testing of yeasts discriminates poorly between resistant and susceptible isolates ofCandida spp. We have previously shown that both substitution of antibiotic medium 3 for RPMI 1640 medium in the microdilution variant of the M27-A method and use of the E-test agar diffusion methodology permit detection of amphotericin B-resistantCandida isolates. To determine the relevance of these observations to Cryptococcus neoformans, we have evaluated the performances of both the M27-A and the E-test methodologies with this yeast using three different media (RPMI 1640 medium, antibiotic medium 3, and yeast nitrogen base). As with Candida, we found that only antibiotic medium 3 permitted consistent detection of resistant isolates when testing was performed in broth by the M27-A method. When testing was performed by the E-test agar diffusion method, both RPMI 1640 medium and antibiotic medium 3 agar permitted ready detection of the resistant isolates. Reading of the results after 48 h of incubation was required for testing in broth by the M27-A method, while the MIC could be determined after either 48 or 72 h when the agar diffusion method was used.

scholarly journals Microdilution antifungal susceptibility testing of Candida albicans and Cryptococcus neoformans with and without agitation: an eight-center collaborative study.

1996 ◽  
Vol 40 (10) ◽  
pp. 2387-2391 ◽  
Author(s):  
E J Anaissie ◽  
V L Paetznick ◽  
L G Ensign ◽  
A Espinel-Ingroff ◽  
J N Galgiani ◽  
...  
Keyword(s):  
Candida Albicans ◽  
Cryptococcus Neoformans ◽  
Amphotericin B ◽  
Clinical Laboratory ◽  
Antifungal Susceptibility ◽  
Collaborative Study ◽  
Reference Method ◽  
Growth Patterns ◽  
National Committee ◽  
Resistant Strains

The growth patterns observed in the trailing wells when fluconazole is being tested may give rise to readings that suggest resistance or increased MICs for known susceptible strains. We conducted a multicenter study to evaluate the intralaboratory and interlaboratory reproducibilities of a method that uses agitation to disperse these types of growth. Ten strains of Candida albicans and five strains of Cryptococcus neoformans were tested against fluconazole, flucytosine, and amphotericin B by using a microdilution adaptation of the proposed reference method of the National Committee for Clinical Laboratory Standards for yeasts (M27-T). The endpoint criterion used before agitation was consistent with the M27-T recommendation, while a criterion of 50% or more reduction of growth compared with the control was used after agitation. The results of this study showed that use of agitation and the modified endpoint criterion both improved intralaboratory and inter-laboratory agreement and increased the frequency of interpretable MICs. The MICs obtained by this method were comparable to those obtained by the broth macrodilution M27-T method. Like M27-T, this method was not able to definitely distinguish amphotericin B-susceptible from -resistant strains, although the MICs for the resistant strains were consistently higher than those for the susceptible ones. The findings imply that agitation should be seriously considered when antifungal agents, particularly fluconazole, are tested in a microdilution format.

scholarly journals Clinical Correlates of Antifungal Macrodilution Susceptibility Test Results for Non-AIDS Patients with Severe CandidaInfections Treated with Fluconazole

2000 ◽  
Vol 44 (10) ◽  
pp. 2715-2718 ◽  
Author(s):  
Sai-Cheong Lee ◽  
Chang-Phone Fung ◽  
Jen-Seng Huang ◽  
Chi-Jen Tsai ◽  
Kuo-Su Chen ◽  
...  
Keyword(s):  
Bacterial Infections ◽  
Clinical Laboratory ◽  
Antifungal Susceptibility ◽  
Susceptibility Test ◽  
National Committee ◽  
Test Results ◽  
Aids Patients ◽  
Clinical Correlates ◽  
Antifungal Susceptibility Test ◽  
Dose Dependent

ABSTRACT Although the clinical correlates of the reference antifungal susceptibility test results in hematogenous and deep-seatedCandida infection are still controversial, we evaluated the clinical correlates of this test in deep-seatedCandida infections in non-AIDS patients. Thirty-two non-AIDS patients with hematogenous or deep-seated Candidainfections were treated with intravenous fluconazole (400 mg a day), and the clinical outcomes were evaluated. Coexisting bacterial infections were treated with appropriate antibiotics, superinfection or reinfection was excluded, inadequate fluconazole therapy was avoided, and essential surgical intervention was performed. The MICs of fluconazole for these 32 Candida isolates were determined according to the M27-A procedure approved by the National Committee on Clinical Laboratory Standards. MICs were interpreted as susceptible (≤8 μg/ml), dose-dependent susceptible (16 to 32 μg/ml), and resistant (≥64 μg/ml) according to the criteria of the M27-A standard. The success rates were 79% (19 of 24; 95% confidence interval [CI], 59 to 93%) in the susceptible category, 66% (4 of 6; 95% CI, 19 to 95%) in the dose-dependent susceptible category, and 0% (0 of 2; 95% CI, 0 to 84%) in the resistant category. We conclude that the clinical correlation of the reference antifungal susceptibility test results is high in hematogenous and deep-seatedCandida infections.

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