Annemarie Polak

Mode of Action of 5-Fluorocytosine and Mechanisms of Resistance

Mode of action of 5-fluorocytosine (5-FC) and mechanisms of resistance to the drug are discussed on the basis of experiments performed with Candida albicans ATCC 26790 and with 50 selected clinical isolates of C. albicans belonging to serological type A or B and representing various degrees and models of 5-FC resistance (sensitivity). Incorporation of 5-fluorouridylic acid into RNA appeared as a prerequisite to antifungal activity, although at a given incorporation rate, growth inhibition varied considerably from one strain to the other. The amino acid pool was unbalanced, and there was evidence for disturbance of protein synthesis. These dysfunctions of RNA probably account for growth inhibition and cell death, whereas up to the present, there was no proof of formation of 5-fluorodeoxyuridylic acid nor of subsequent inhibition of thymidylate synthetase. Incorporation of fluorinated pyrimidine into RNA was lower in normally sensitive type B strains than in normally sensitive ones of type A, whereas the frequency of 5-FC-resistant mutants was the same. The two serological types did not differ in the activity of cytosine permease nor in that of cytosine deaminase. Among 29 clinical isolates with 6-FC resistance (or impaired sensitivity) no instance of cytosine permease deficiency was found. Two isolates (belonging to the serological type A) were deficient in cytosine deaminase, whereas the majority was probably deficient in uridine monophosphate pyrophosphorylase or had a surplus of de novo synthesis of pyrimidines. Relative 5-FC resistance was more common than complete resistance.

Combination Therapy of Experimental Candidiasis, Cryptococcosis, Aspergillosis and Wangiellosis in Mice

Combination pairs of 5-fluorocytosine (5-FC) + itraconazole (Itra), 5-FC + fluconazole (Fluc), and amphotericin B (Amph B) + Itra were administered to mice with experimental candidiasis, cryptococcosis, aspergillosis and wangiellosis with a variety of combination ratios. The life-prolonging effect of the combinations was compared with the effect of each partner administered alone and with a double dosage. Using the U test of Mann and Whitney, the effects of the concentration were classified as synergistic, additive, indifferent or antagonistic; the degree of the interaction was compared with the known effect of Amph B and 5-FC combinations. The combination 5-FC + Itra was definitely synergistic or additive in candidiasis and aspergillosis. The most pronounced synergism occurred in the infection with a 5-FC-resistant strain of Candida albicans. The degree of synergism was the same as with 5-FC + Amph B. In cryptococcosis this combination was indifferent. The combination of 5-FC + Itra merits clinical investigation, especially in candidiasis and aspergillosis. Amph B + Itra was mostly indifferent and weakly antagonistic; the degree of antagonism was significantly weaker than the one observed with Amph B + ketoconazole (Keto). In candidiasis, 5-FC + Fluc was synergistic, but indifferent in cryptococcosis and aspergillosis.

Pathogenicity and virulence of wild-type and melanin-deficient Wangiella dermatitidis

Wild-type, dematiaceous Wangiella dermatitidis (DMD 368) and melanin-deficient mutant (Mel 3) strains derived therefrom were compared for pathogenic and virulent effects in Swiss albino mice following intravenous infection. Parameters examined were mouse survival and central nervous system signs of infection, time-course cultures of fungus from brains, lungs, livers, spleens and kidneys, and histopathology of brains. Over a range of concentrations, DMD 368 produced 100% mortality while one Mel 3 strain, DMD 369, produced no mortality by 21 days after inoculation. However, in chronic infections with DMD 369, mice developed ataxia and torticollis. These signs of disease were indistinguishable from those produced by low concentrations of DMD 368. The brain was the most severely affected organ where both DMD 368 and 369 grew exponentially. Histological responses to the two strains appeared to be indistinguishable. However, the mutant appeared not to form the invasive hyphal forms of growth associated with the acute, fatal infections caused by the wild type. Thus, although the absence of melanin was associated with decreased mortality in mice, the chronic neurological signs of mouse phaeohyphomycosis appeared to be unrelated to melanin.

Melanin as a virulence factor in pathogenic fungi.

The pigment melanin is found universally in nature and is attributed to a variety of functions. In some fungi it is thought to play a decisive role in the determination of virulence. This review examines the experimental evidence which has led to an understanding of the mechanisms by which melanin functions in pathogenic fungi, particularly in plant pathogens, in Cryptococcus neoformans and Wangiella dermatitidis.

Pharmacokinetic Studies on the Oral Antimycotic Agent 5-Fluorocytosine in Individuals with Normal and Impaired Kidney Function

Pharmacokinetic studies were performed on the systemic antimycotic agent 5-fluorocytosine (5-FC) by giving 2 g orally to 10 healthy adult volunteers and to 40 patients with renal impairment of various degree (serum creatinine concentration varying from 1.0 to 16.4 mg/l00 ml) including 5 nephrectomized or anuric patients. The microbiological method used for the measurement of the serum and urinary concentrations of the drug is described in detail. In 20 patients endogenous creatinine clearance was determined in addition to the serum creatinine concentration. The average biological half-life of 5-FC in serum was 2.89 h in the healthy individuals (range 2.36–3.99 h) but in the patients, it was significantly prolonged with increasing serum creatinine concentration and decreasing creatinine clearance. The average half-life in the five nephrectomized or anuric patients was 85.0 h (29.9–250 h). A linear correlation was found to exist between the elimination rate constant of the drug and creatinine clearance, and based on this, a 5-FC dosage schedule has been proposed for patients with impaired kidney function. 5-FC is eliminated almost exclusively by the kidneys but has no renal toxicity. Provided that dosage is adapted accordingly, the drug is appropriate for chemotherapeutic treatment of patients with renal impairment which is a relative contraindication to amphotericin B.

Combination therapy for systemic mycosis

ConclusionThe use of antifungals in combination can clearly improve efficacy, broaden spectrum and reduce the duration of therapy. The availability of more systemic antifungals may lead to an increase in the use of triple combinations, especially against fungal infection in immunosuppressed patients. 5-flucytosine plus amphotericin B is still the most active combination with proven clinical application in cryptococcosis and candidosis. Combinations of 5-flucytosine with ketoconazole or itraconazole have shown promising results and may well play a role in human chemotherapy.

In vitro and in vivo Drug Studies with Three Agents of Central Nervous System Phaeohyphomycosis

Amphotericin B (Amph B), 5-fluorocytosine (5-FC), ketoconazole (KTZ), fluconazole (FLZ), amorolfine (AMOR) and terbinafine (TER) were tested against 3 agents of central nervous system phaeohyphomycosis in vitro and in life-threatening infections in mice. The fungi studied were Cladosporium bantianum, Dactylaria constricta and Wangiella dermatitidis. The broadest protection against this group of fungi in mice was offered by 5-FC followed by Amph B and FLZ, then KTZ. AMOR and TER were inactive in vivo. The results of in vitro susceptibility testing had no predictive value. In contrast, the data obtained from the mouse models should be useful clinically.

Virulence of Candida albicans mutants.

Summary. Mutant strains of the fungal pathogen Candida albicans blocked in pyrimidine transport and salvage metabolism were tested for virulence in various animal models. The growth rate, germination and proteolytic enzyme production did not correlate with the virulence of the strains.

Antifungal activity in vitro of Ro 14-4767/002, a phenylpropyl-morpholine

Minimal inhibitory concentrations (MICs) of Ro 14-4767/002 for pathogenic yeasts, Aspergillus spp., dermatophytes and other filamentous fungi were determined in dilution tests under a variety of experimental conditions and, for the most of the species and a number of different isolates. Ro 14-4767/002 showed the highest effect against dermatophytes and Cryptococcus neoformans, followed by Candida spp., whereas its activity against Aspergillus spp. was weak. Its activity against most pathogens compared favourably with antifungals of the imidazole class. The activity of Ro 14-4767/002 not only differed between the species but there was also a significant intra-species variation. The MICs were influenced by the inoculum size, the incubation time, and by the composition of the medium. The activity of the compound was significantly higher on Casitone agar than on a chemically defined medium (Yeast Nitrogen Base + glucose). Ro 14-4767/002 was also found to exert fungicidal activity which was time- and concentration-dependent.

Dihydroxynaphthalene (DHN) Melanin and Its Relationship with Virulence in the Early Stages of Phaeohyphomycosis

Pathogenicity can be defined as “giving rise to, or producing disease.” Virulence is generally considered as the degree of pathogenicity. If a given microorganism possesses no degree of pathogenicity, then it would be considered both avirulent and nonpathogenic.