The inhibitory activities of 2 new orally absorbed antifungal imidazole derivatives, BAY n 7133 and BAY l 9139, were compared in vitro with those of ketoconazole and miconazole. Clinical isolates of pathogenic fungi tested included 35 yeasts, 62 dimorphic fungal pathogens, 37 filamentous fungi and 31 dermatophytes. LY 121019, a semisynthetic analog of echinocandin B, was included in tests with the pathogenic yeasts. Both BAY n 7133 and BAY l 9139 were found to be broad spectrum antifungal agents. The spectra of these newer compounds were comparable to those of ketoconazole and miconazole; however only BAY n 7133 resembled these latter 2 imidazoles quantitatively in terms of the degree of antifungal activity as indicated by measurable MICs. In contrast, the spectrum of LY 121019 appeared to be confined only to isolates of Candida.

An evaluation of broth dilution antifungal susceptibility tests was performed by determining both the micro- and macrodilution MICs of amphotericin B, flucytosine, fluconazole, ketoconazole, and cilofungin against 38 isolates of Candida albicans, Candida lusitaniae, Candida parapsilosis, Candida tropicalis, Cryptococcus neoformans, and Torulopsis glabrata. The following preliminary antifungal working group recommendations of the National Committee for Clinical Laboratory Standards for broth macrodilution tests with antifungal agents were used: inocula standardized to 1 x 10(4) to 5 x 10(4) CFU/ml with a spectrophotometer, RPMI 1640 medium buffered with morpholinopropanesulfonic acid (pH 7.0), incubation at 35 degrees C for 24 to 48 h, and an additive drug dilution procedure. Broth microdilution MICs were higher (two or more dilutions) than broth macrodilution MICs for all isolates tested with amphotericin B and for most isolates tested with ketoconazole, fluconazole, and cilofungin. MICs of flucytosine were the same by both techniques or lower by the broth microdilution test except in tests with C. neoformans. However, the only statistically significant differences between the two tests were observed with amphotericin B against all isolates (P = 0.01 to 0.07), ketoconazole against C. neoformans (P = 0.01 to 0.02), and cilofungin against C. albicans (P = 0.05 to 0.14). Tests performed with less dense inocula (1 x 10(3) to 5 x 10(3] produced similar results.

Cilofungin (LY 121019) is a semi-biosynthetic lipopeptide agent which is active both in vitro and in vivo against isolates of Candida species. The purpose of this study was to demonstrate the usefulness of experimental 10- and 20-micrograms cilofungin disks in predicting probable in vitro susceptibility to this drug. Fifty-two isolates of pathogenic yeasts, which included 34 isolates of Candida albicans, 10 isolates of Candida tropicalis and eight isolates of Candida glabrata, were tested. Both agar dilution minimal inhibitory concentration (MIC) tests and matched agar disk diffusion tests were performed using 14-h-old broth cultures grown in single strength yeast nitrogen base (YNB) supplemented with asparagine and dextrose. Both tests were incubated at 35 degrees C and the results were read after 24 h. Regression analysis was used to measure the degree of correlation between MIC values and matched averaged zones of inhibition, and demonstrated that both the 10-micrograms (r = 0.9278) and 20-micrograms (r = 0.9082) disks can predict probable MIC values.

Fifteen isolates, each of serotypes A and B of Candida albicans, were tested in vitro against flucytosine, amphotericin B, and cilofungin. All 15 serotype A isolates were susceptible to all three drugs. Three serotype B isolates were resistant to flucytosine. Two isolates, one serotype A and one serotype B, had elevated amphotericin B and cilofungin MICs of 5-mu-g/ml.

SummaryGuidelines for the treatment of Infections caused by unusual opportunistic fungi are limited and available in vitro data are scanty. In vitro susceptibility tests, employing an agar dilution procedure, were performed with amphotericin B (AMB), natamycin (NTC), itraconazole (ICZ), and ketoconazole (KTZ). Two media were used: Kimmig's agar (KA) and Yeast Morphology Agar (YMA). Fungi tested included isolates (n) of A cremonium spp. (10), Cunninghamella spp. (6), Fusarium spp. (18), Pseudallescheria boydii (14), and Trichosporon beigelii (5). All Acremonium and Cunninghamella isolates were susceptible to NTC (MIC ≤4 μg/ml) but many appeared to be resistant to AMB, (MIC ≧32 μg/m1), KTZ and ICZ (MIC ≧128 μg/ml). Most isolates of Fusarium spp. were susceptible to both AMB and NTC (MIC90 = 4 μg/ml); one isolate was cross‐resistant to both polyenes (MIC > 32 μg/ml ). Only two of 18 Fusarium isolates appeared susceptible to the imidazoles (MIC ≤4 μg/ml); the remaining isolates exhibited high MICs (≧64 μg/ ml). All 14 isolates of P. boydii were susceptible to NTC (MIC ≤ μg/ml) but four appeared to be resistant to AMB (MIC 32 μg/ ml). Most isolates of P. boydii were susceptible to both KTZ (MIC ≤4 μg/ml) and ICZ (MIC ≤ 16 μg/ml) but two isolates appeared to be resistant (MIC ≧ 16 μg/ml). AMB was the most active compound against T. beigelii (MIC90 = 0.25 μg/ml) while KTZ was the least active (MIC90 = 64 μg/ml). Sixty‐one MICs were determined for the four drugs on both KA and YMA. The best inter‐test agreement (equal or ± 1 dilution) between paired KA and YMA MICs was observed in tests with NTC (93 %) while the poorest was observed in tests with ICZ (79 %). Four‐fold or greater differences in paired MICs were observed in two tests each with AMB and ICZ.

The evaluation of any antifungal agent involves the determination of its in vitro and in vivo activity against pathogenic and/or opportunistic fungi. The in vitro evaluation is followed by an in vivo evaluation in animal models, and clinical trials in humans. From the first report of the efficacy of the iodides for the treatment of sporotrichosis (1903) until the introduction of the imidazoles (azoles, 1960s), the number of antifungal agents available was very limited, including griseofulvin (1939), nystatin (1950), amphotericin B (1956), and flucytosine (1964). This paper briefly reviews the status of the antifungal agents currently used, and gives a more in depth evaluation of progress during recent years in the search for new antifungal drugs. Efforts to improve the efficacy of the current antifungal agents are also reviewed.

Oxiconazole nitrate was compared in vitro with ketoconazole and econazole nitrate in tests with 96 dermatophytes, 18 isolates of Malassezia furfur, and seven isolates of Exophiala werneckii. An agar dilution procedure was used employing either Kimmig's agar or Sabouraud's dextrose agar supplemented with Olive oil and Tween 80. Econazole was the more active compound in tests with Microsporum species (41 isolates) and most isolates of Trichophyton species (45 isolates). Oxiconazole was the more active compound in tests with T. tonsurans and T. rubrum. However, differences between results for oxiconazole and econazole and the dermatophytes were only marginal. Ketoconazole was the most active compound in tests with M. furfur; some cross resistance on the part of several isolates of M. furfur between oxiconazole and econazole was noted. All three compounds were active against E. werneckii with MIC90 values of either 0.25 or 0.5 microgram/ml. Epidermophyton floccosum was the most susceptible of all organisms tested with all MIC values for all three drugs being less than or equal to 0.063 microgram/ml, the lowest concentration tested.

A multicenter prospective randomized trial of four versus six weeks of amphotericin B, 0.3 mg/kg per day, plus flucytosine, 150 mg/kg per day, was performed with 194 patients with cryptococcal meningitis. One or more toxic drug reactions developed in 103 patients: azotemia (51), renal tubular acidosis (two), leukopenia (30), thrombocytopenia (22), diarrhea (26), nausea/vomiting (10), and hepatitis (13). The four- and six-week regimens were complicated by toxicity in 44 percent and 43 percent of cases, respectively. Toxicity appeared during the first two weeks of therapy in 56 percent and during the first four weeks in 87 percent. Azotemia did not occur more frequently in renal transplant recipients or diabetic patients. Cytopenias did not appear more often in patients with hematologic malignancies or those receiving immunosuppressive therapies. Toxic reactions that contributed to death developed in five patients (two with azotemia, one with pancytopenia, one with hepatitis, one with ileus). Amphotericin B- induced azotemia was not a significant risk factor for the subsequent development of bone marrow, gastrointestinal, or hepatic toxicity attributable to flucytosine. Flucytosine toxocity was associated with peak serum flucytosine levels of 100 μg/ml or more during two or more weeks of therapy (p = 0.005). Peak 5-fluorouracil levels were not predictive of toxicity. An initial dose of flucytosine is recommended based on the creatinine clearance: 150 mg/kg per day at a creatinine clearance above 50 ml/minute, 75 mg/kg per day at a creatinine clearance of 26 to 50 ml/minute, and 37 mg/kg per day at a creatinine clearance of 13 to 25 ml/minute. The serum creatinine leve should be monitored twice weekly and the creatinine clearance weekly during therapy in order to anticipate changes in serum flucytosine concentration. In addition, it is recommended that the serum flucytosine level be determined two hours after an oral dose once a week, and that the dose be adjusted to maintain a level of 50 to 100 μg/ml.

Three hundred twenty-three clinical isolates of Cryptococcus neoformans of diverse geographic origins were biochemically serogrouped using glycine-cycloheximide-phenol red agar (GCP), the same medium less cycloheximide (GOP), and glycine-L-canavanine bromothymol blue agar (CGB). Twenty isolates gave positive reactions on all three media typical of the B and C serotypes. Three were from the Peoples' Republic of China; three each were from Michigan (two patients) and Louisiana; two each were from California, Georgia, and Virginia; and one each was from Alabama, Florida, North Carolina, Oklahoma, and Tennessee. Two hundred seventy-six isolates were identified as belonging to the A/D serogroup; 272 were of American origin and four were from China. Twenty-seven isolates were biochemically ungroupable. Evaluations of the reactions on all three media were open to subjective interpretations. Utilization of glycine was the most frequent atypical variable; 36 of 276 (13%) A/D isolates utilized glycine while being inhibited by either GCP or CGB or both. Significant differences between A/D and B/C serogroups in terms of susceptibility to 5-fluorocytosine but not to amphotericin B were observed; B/C serogroup isolates appeared to be less susceptible to 5-fluorocytosine in vitro than were the A/D serogroup isolates. These results provided new evidence on the distribution of B/C serogroup isolates of C. neoformans in America and demonstrate the difficulties of using biochemical tests for serotyping purposes. They also offer a possible explanation for the apparent more refractory therapeutic responses of infections caused by B and C serotypes to conventional antifungal chemotherapy.

One hundred ninety-four patients with cryptococcal meningitis were enrolled in a multicenter, prospective, randomized clinical trial to compare the efficacy and toxicity of four as compared with six weeks of combination amphotericin B and flucytosine therapy. Among 91 patients who met preestablished criteria for randomization, cure or improvement was noted in 75 percent of those treated for four weeks and in 85 percent of those treated for six weeks. The estimated relapse rate for the four-week regimen was higher--27 as compared with 16 percent--whereas the incidence of toxic effects for the two regimens was similar--44 as compared with 43 percent. Among 23 transplant recipients, 4 of 5 treated for four weeks relapsed, leading to the decision to treat the rest of the group for six weeks. Only 3 of the 18 treated for six weeks relapsed. In a third group of 80 patients, the protocol was not followed during the initial four weeks, and these patients were not randomized. Thirty-eight died or relapsed. Multifactorial analysis of pretreatment factors for all 194 patients identified three significant predictors (P less than 0.05) of a favorable response: headache as a symptom, normal mental status, and a cerebrospinal fluid white-cell count above 20 per cubic millimeter. These and other findings in this study are consistent with the view that the four-week regimen should be reserved for patients who have meningitis without neurologic complications, underlying disease, or immunosuppressive therapy; a pretreatment cerebrospinal fluid white-cell count above 20 per cubic millimeter and a serum cryptococcal antigen titer below 1:32; and at four weeks of therapy, a negative cerebrospinal fluid India ink preparation and serum and cerebrospinal fluid cryptococcal-antigen titers below 1:8. Patients who do not meet these criteria should receive at least six weeks of therapy.

Nine strains of Clostridium difficile isolated from symptomatic and asymptomatic patients and four other species of clostridia were tested for relative hydrophobicity by determining the degree of adherence to polystyrene. Under three different conditions of growth all strains of Clostridium difficile had high rates of adherence, whereas the other clostridial species showed no pronounced adherence. Isolates of Clostridium difficile were also tested for their ability to adhere to human embryonic intestinal cells and adult colon cells. All strains adhered to both cell lines, although the percentages of organisms adhering varied. Adherence was greatest at pH 5.5-6.0 but was not significantly altered at a pH of 7.0-7.8 (p = 0.15, p = 0.20); it decreased significantly upon washing with 1% Tween 80 but not with 0.1% Tween 80. This capacity for adherence may play a part in the organism's colonization of the human intestinal tract.

Exoantigens from 10-day-old cultures of 100 isolates of pathogenic and saprophytic dematiaceous fungi were analyzed by the exoantigen test. Antisera to Cladosporium bantianum ATCC 10958, Fonsecaea pedrosoi CDC AMO-B06, and Phialophora verrucosa CDC AMO-C12 were prepared in New Zealand rabbits immunized with soluble antigens from 1-month-old cultures. Absorbed and nonabsorbed antisera and exoantigens from the same organisms were used as reference reagents. Serologic reactions were analyzed in terms of the presence or absence of lines of identity or nonidentity. These reactions allowed presumptive differentiation of C. bantianum, F. pedrosoi, and Phialophora verrucosa from other dematiaceous fungi, including Cladosporium spp. (28 isolates), Exophiala spp. (18 isolates), Fonsecaea spp. (17 isolates). Lecythophora hoffmannii (4 isolates), Phaeoannellomyces werneckii (3 isolates), Phialophora spp. (17 isolates), Wangiella dermatitidis (9 isolates), and Rhinocladiella spp. (4 isolates).

The independent University-based research scientist conducting research neither involving human subjects nor dealing with safety has been excluded from the drug regulatory affair process. This poses problems in terms of preclinical studies with potentially regulated “test articles”. These problems involve quality control, records and documentation of research data, confidentiality, investigator compliance and the need for external monitoring. Quality control can be provided by use of appropriate “control articles”. Guidelines are needed from sponsors of preclinical studies regarding documentation and retention of research data. Confidentiality is an ethical issue not subject to regulation and investigator compliance remains an unresolved problem. All of these problems suggest one ultimate final solution — the need for external monitoring of all preclinical studies with “test articles” which ultimately may be subject to regulation.

Ketoconazole was tested in vitro in three different media against 69 isolates of pathogenic fungi by using a macro-broth dilution procedure. The dimorphic systemic pathogens were highly susceptible, with most isolates of Blastomyces dermatitidis and Histoplasma capsulatum being inhibited and killed by concentrations less than or equal to 0.39 micrograms of ketoconazole/ml. Most isolates of Coccidioides immitis were also inhibited or killed by 0.39 micrograms of ketoconazole/ml; however, several were not killed by 100 micrograms/ml. Isolates of Cryptococcus neoformans and Sporothrix schenckii appeared to be less susceptible, with many isolates being resistant to less than or equal to 1.56 micrograms of ketoconazole/ml. There were 19 isolates of B. dermatitidis, C. immitis, and H. capsulatum recovered from 12 patients either during or following treatment with ketoconazole. Evidence for selection of secondary resistance to ketoconazole in these isolates was not observed. Results of these in vitro studies correlated poorly with the clinical responses to ketoconazole observed in the patients from whom the isolates were recovered.

The in vitro activity of R 51,211 (itraconazole, accepted generic name; Janssen Pharmaceutica, Beerse, Belgium), a new orally active triazole, was compared with those of two existing orally active azoles, ketoconazole and BAY n 7133, and a topical agent, Ro 14-4767/002. An agar dilution procedure (Kimmig agar) was performed with 148 isolates of pathogenic fungi. Incubation was at 30 degrees C from 48 h to 7 days. R 51,211 was dissolved in 0.2 N HCl in absolute ethanol, ketoconazole was dissolved in 0.2 N HCl alone, BAY n 7133 was dissolved in absolute ethanol, and Ro 14-4767/002 was dissolved in dimethyl sulfoxide. R 51,211 and Ro 14-4767/002 were the most active drugs against isolates of Histoplasma capsulatum, and R 51,211 showed the greatest activity in vitro against isolates of Blastomyces dermatitidis and Cryptococcus neoformans. Ro 14-4767/002 was the most active drug against 30 isolates of dermatophytes, followed by R 51,211, ketoconazole, and BAY n 7133. R 51,211 showed the best activity in vitro against 19 isolates of Aspergillus fumigatus and Aspergillus flavus, as well as 19 isolates of dematiaceous fungi. All four drugs had 90% MICs of greater than or equal to 16 micrograms/ml when tested with isolates of zygomycetous fungi.

Concentrated (25X) exoantigens of 105 isolates of pathogenic and saprophytic dematiaceous fungi and 3 isolates of Sporothrix schenckii were analyzed by the microimmunodiffusion method. The reagents used were nonadsorbed and adsorbed sera produced in New Zealand rabbits. One set of rabbits was immunized with soluble antigens of a 1-month-old culture of Exophiala jeanselmei (ATCC 34123), and the other set was immunized with soluble antigens from a culture of Wangiella dermatitidis (ATCC 28869). The reference antigens were 25X-concentrated exoantigens of the above cultures. This exoantigen test permitted the differentiation of E. jeanselmei and W. dermatitidis from one another as well as from other Exophiala species, Fonsecaea species, Phialophora species, Cladosporium species, Rhinocladiella species, and Sporothrix schenckii by presence or absence of lines of identity or of partial identity, or lines of nonidentity. Using adsorbed serum eliminated the problems with cross-reactivity seen with nonadsorbed serum. Thus, with an adsorbed serum as the reagent, it was possible to presumptively differentiate E. jeanselmei and W. dermatitidis from one another and from other dematiaceous fungi.

Four new antifungal agents were compared in vitro with miconazole and ketoconazole. The agents were BAY n 7133 and ICI 153,066, two orally active triazoles, and bifonazole (BAY h 4502) and Ro 14-4767/002, both topical agents. While all four were found to be broad spectrum antifungal agents they also demonstrated certain gaps in their spectra. In general, Ro 14-4767/002 was the most active agent tested whereas bifonazole and BAY n 7133 were the least active. Noteworthy activities included that of Ro 14-4767/002 against Candida albicans , the dermatophytes and Sporothrix schenckii and that of ICI 153,066 against Torulopsis glabrata .