Susceptibility testing of fungi: current status of the standardization process

The technical parameters for antifungal susceptibility testing with Candida species were reexamined to determine the optimal conditions for testing with semiautomated preparations of broth microdilution cultures, automated spectrophotometric readings of the cultures, and dose-response and endpoint determinations by means of a computer spreadsheet. Tests were based on proposed standard method M27P of the National Committee for Clinical Laboratory Standards for antifungal agents. RPMI 1640 broth with extra glucose to a final concentration of 2% gave higher and more reproducible drug-free control readings without affecting susceptibility endpoint readings. An inoculum of 8 ؋ 10 4 yeasts per ml prepared from a carbon-limiting broth culture without further standardization was found to give optimal control readings after 48 h of incubation at 37؇C. For flucytosine, fluconazole, itraconazole, and ketoconazole, endpoints based on 50% growth inhibition (50% inhibitory concentration) gave the minimum variation with inoculum size and the fewest endpoint differences with RPMI 1640 medium obtained from two different suppliers. The 50% inhibitory concentration was also the optimal endpoint for fluconazole and ketoconazole susceptibilities in comparison with broth macrodilution MICs determined by the method of the National Committee for Clinical Laboratory Standards. Intralaboratory reproducibility was determined by retrospective analysis of replicate results for isolates retested at random over a 2-year period. This approach showed less favorable reproducibility than has been reported from purpose-designed, prospective antifungal susceptibility studies, but it may better reflect real-life test reproducibility. Susceptibility data for 616 clinical isolates of yeasts, representing 16 Candida and Saccharomyces spp., confirmed the tendency of Candida lusitaniae isolates to show relatively low susceptibilities to amphotericin B, the tendency of Candida krusei isolates to show low flucytosine and fluconazole susceptibilities, and the presence of some isolates in the species Candida albicans, Candida glabrata, and Candida tropicalis with low susceptibilities to azole derivative antifungal agents. The study demonstrates the value of automation and standardization in all stages of yeast susceptibility testing, from plate preparation to data analysis.Interest in antifungal susceptibility testing has increased in recent years, as witnessed by the growing number of published articles in this field. Most investigations have focused on tests with fluconazole against pathogenic yeasts, particularly since reports have appeared suggesting that Candida isolates with reduced fluconazole susceptibilities are being encountered clinically after long-term fluconazole treatment (4,5,19,24,27,31,32,34,35,41). Current methods for antifungal susceptibility testing need to conform to three considerations, which overlap mutually. They should give results that correspond to the clinical outcome of antifungal treatment, that show some correlation to those ...

supporting

confidence: 65%

Antifungal susceptibility testing of yeasts: evaluation of technical variables for test automation

Odds

Vranckx

Woestenborghs

1995

“…Because of a lack of standardized tests, the comparisons of MICs from laboratory to laboratory and from study to study should be interpreted with caution. Moreover, no relationship has been established between in vitro results and patients' responses (13).…”

Section: Discussionmentioning

confidence: 99%

Fluconazole penetration in cerebral parenchyma in humans at steady state

Thaler

Bernard

Tod

et al. 1995

We studied fluconazole penetration in the brain in five patients who had a deep cerebral tumor whose removal required the excision of healthy brain tissue. Plasma and brain samples were simultaneously obtained after oral ingestion of 400 mg of fluconazole daily for 4 days (90% of steady state). Fluconazole penetration in healthy cerebral parenchyma was determined. Plasma and brain samples were assayed by high-pressure liquid chromatography. Concentrations in plasma and brain tissue were 13.5 ؎ 5.5 g/ml and 17.6 ؎ 6.6 g/g, respectively. The average ratio of concentrations in the brain and plasma (four patients) was 1.33 (range, 0.70 to 2.39). Despite the lack of data concerning the penetration of fluconazole in brain abscesses, these results should permit the use of a daily dose of 400 mg of fluconazole in prospective clinical studies that evaluate the effectiveness of this drug in the treatment of brain abscesses due to susceptible species of fungi.Fluconazole, a triazole compound, is a systemic antifungal synthetic agent. Its fungistatic action is the inhibition of fungal cytochrome P-450 sterol C-14 alpha demethylation (18). During recent years, an increase in the rate of central nervous system fungal infections mainly due to an increased prevalence of immunosuppressed states (e.g., AIDS, malignancies, and organ transplantation) has been reported. Treatment by fluconazole of invasive infections due to various fungi has been a significant advance in antimicrobial chemotherapy (1,19,21). Its efficacy in treating cryptococcal and coccidioidal meningitis (2,3,5,14,22,23) is well established in clinical studies.In acute AIDS-associated cryptococcal meningitis fluconazole was shown to be as effective as amphotericin B (23) and more so in preventing relapse (3,22). However, use of fluconazole for treatment of this disease remains controversial (6,20,24), and the optimal dose remains uncertain. The incidence of cryptococcal intracerebral lesions is unknown and probably underestimated (6,8,11,27). Inadequate control of these lesions is perhaps one of the risk factors for failure or relapse of cryptococcal meningitis. Actually, therapeutic failures and relapses may occur when using amphotericin B or fluconazole (400 mg/day), and higher doses of fluconazole as salvage therapy have been proposed (2).While the penetration of fluconazole in human cerebrospinal fluid is known to be good (60 to 80% of levels in serum) (10, 25), data regarding its penetration in brain tissue other than in animals are lacking (17,26). The objective of this research was to study the penetration of fluconazole in human brain tissue and to determine the ratio of concentrations in the brain and plasma at steady state. MATERIALS AND METHODSObtaining brain tissue samples from patients with cryptococcal meningitis is not acceptable in light of current diagnostic and therapeutic strategies. Obtaining tissue samples from healthy volunteers is not conceivable. We therefore decided to study the penetration of fluconazole in the healthy cerebral parenchyma...

“…More recently, resistance to the triazole FZ has been described in C. albicans strains isolated from the oral cavities of AIDS patients in different parts of the world (2, 5-8, 15, 18, 22, 24, 25, 28, 29, 32, 34, 36). The absence in most cases of comparative susceptibility data for alternative azole antifungal agents in these isolates still leaves open the question of whether cross-resistance to azole drugs in C. albicans is a general phenomenon.All of the azole antifungal agents share certain properties that makes in vitro susceptibility testing difficult, mainly partial inhibition of fungal growth that makes MIC endpoint determinations both very difficult and subjective (12,29,35). In the proposed standard of the National Committee for Clinical Laboratory Standards (NCCLS) (23), endpoints for azoles are determined visually at 48 h and the MIC is defined as the lowest drug concentration that reduces growth by 80% relative to the growth of the control (MIC 80%).…”

mentioning

confidence: 99%

“…All of the azole antifungal agents share certain properties that makes in vitro susceptibility testing difficult, mainly partial inhibition of fungal growth that makes MIC endpoint determinations both very difficult and subjective (12,29,35). In the proposed standard of the National Committee for Clinical Laboratory Standards (NCCLS) (23), endpoints for azoles are determined visually at 48 h and the MIC is defined as the lowest drug concentration that reduces growth by 80% relative to the growth of the control (MIC 80%).…”

mentioning

confidence: 99%

Patterns of in vitro activity of itraconazole and imidazole antifungal agents against Candida albicans with decreased susceptibility to fluconazole from Spain

Martı́nez-Suárez

Rodríguez-Tudela

1995

Two groups of recent clinical isolates of Candida albicans consisting of 101 isolates for which fluconazole MICs were <0.5 g/ml (n ‫؍‬ 50) and >4.0 g/ml (n ‫؍‬ 51), respectively, were compared for their susceptibilities to fluconazole, clotrimazole, miconazole, ketoconazole, and itraconazole. Susceptibility tests were performed by a photometer-read broth microdilution method with an improved RPMI 1640 medium supplemented with 18 g of glucose per liter (RPMI-2% glucose; J. L. Rodríguez-Tudela and J. V. Martínez-Suárez, Antimicrob. Agents Chemother. 38:45-48, 1994). Preparation of drugs, basal medium, and inocula was done by the recommendations of the National Committee for Clinical Laboratory Standards. The MIC endpoint was calculated objectively from the turbidimetric data read at 24 h as the lowest drug concentration at which growth was just equal to or less than 20% of that in the positive control well (MIC 80%). In vitro susceptibility testing separated azole-susceptible strains from the strains with decreased susceptibilities to azoles if wide ranges of concentrations (20 doubling dilutions) were used for ketoconazole, miconazole, and clotrimazole. By comparison with isolates for which fluconazole MICs were <0.5 g/ml, those isolates for which fluconazole MICs were >4.0 g/ml were in general less susceptible to other azole drugs, but different patterns of decreased susceptibility were found, including uniform increases in the MICs of all azole derivatives, higher MICs of several azoles but not others, and elevated MICs of fluconazole only. On the other hand, decreased susceptibility to any other azole drug was never found among strains for which MICs of fluconazole were lower. Secondary resistance to azole antifungal agents in Candida albicans first appeared in the 1980s in patients with chronic mucocutaneous candidiasis treated with KZ for very long periods of time (25,33,36). The isolates involved had markedly reduced susceptibilities to KZ and cross-resistance to other azole drugs. More recently, resistance to the triazole FZ has been described in C. albicans strains isolated from the oral cavities of AIDS patients in different parts of the world (2, 5-8, 15, 18, 22, 24, 25, 28, 29, 32, 34, 36). The absence in most cases of comparative susceptibility data for alternative azole antifungal agents in these isolates still leaves open the question of whether cross-resistance to azole drugs in C. albicans is a general phenomenon.All of the azole antifungal agents share certain properties that makes in vitro susceptibility testing difficult, mainly partial inhibition of fungal growth that makes MIC endpoint determinations both very difficult and subjective (12,29,35). In the proposed standard of the National Committee for Clinical Laboratory Standards (NCCLS) (23), endpoints for azoles are determined visually at 48 h and the MIC is defined as the lowest drug concentration that reduces growth by 80% relative to the growth of the control (MIC 80%). We have previously shown that increased growth of C. albicans in RP...