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.
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 5-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 pairs of the major systematic antimycotic drugs, amphotericin B (AmphB), 5-fluorocytosine (5-FC) and ketoconazole (Ktz) were administered to mice with experimental candidiasis, cryptococcosis and aspergillosis at a variety of combination ratios. The 3 mycoses were produced with 3 strains each of Candida albicans, Cryptococcusneoformans, and Aspergillus jumigatus, respectively, which were preselected to represent 3 different degrees of 5-FC sensitivity (‘normally sensitive’, ‘moderately resistant’, and ‘definitely resistant’). The life-prolonging effect of the combinations was compared with the effect of each partner administered alone at the same and at the double dosage. Using the U test of Mann and Whitney and setting limits which on the whole were more rigorous than those of the isobole methods commonly applied to the study of drug interactions, the effects of the concentrations were classified as ‘synergistic’, ‘additive’, ‘indifferent’ or ‘antagonistic’. The combination AmphB plus 5-FC was definitely synergistic or definitely additive in all 3 candidiasis models, the most pronounced synergism occurring in the infection with the ‘definitely 5-FC-resistant’ C. albicans strain; in cryptococcosis produced by any of the 3 C. neoformans strains the effect was definitely additive, but only slightly additive or indifferent in the 3 aspergillosis models. The combination AmphBplus Ktz was slightly synergistic in candidiasis produced by one C. albicans strain, but definitely antagonistic in this mycosis produced by the remaining 2 strains of the same species; the combination was definitely additive or, even, slightly synergistic in the 3 cryptococcus models, but, again, antagonistic in aspergillosis produced by all 3 strains of A. fumigatus. 5-FC plus Ktz was additive or indifferent in the 3 candidiasis models, but throughout indifferent in cryptococcosis and aspergillosis.
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.Wangiella dermatitidis is a polymorphic, dematiaceous fungal zoopathogen which causes phaeohyphomycosis [3,9]. In a recent, thorough review of the human infections produced by this fungus, Matsumoto et al. clarified the literature and documented the significance of the fungus as a neurotropic pathogen [8]. They calculated a mortality rate of nearly 50% from all reported cases and 100% when the central nervous system was involved. This fungus is joined by Dactylaria constricta [1], Curvularia pallescens [7], Fonsecaea pedrosoi [9], Xylohypha bantiana [10], Bipolaris hawaiiensis [12] and Bipolaris spicifera [12] as dematiaceous fungi which produce brain infections in humans.Cryptococcus neoformans, although not considered to be dematiaceous, does produce melanin and is similarly neurotropic in man and other animals [6]. The melanin
Combination pairs of 5-fluorocytosine (5-FC) + itraconazole (Itra), 5-FC + fluconazole (Flue), 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 + Flue was synergistic, but indifferent in cryptococcosis and aspergillosis.
5-Fluorouracil and 5-fluorodeoxyuridine monophosphate levels were estimated in 75 isolates of Candida albicans to determine whether 5-fluorocytosine susceptibility could be ideally correlated with the intrafungal formation of both 5-fluorodeoxyuridine monophosphate and 5-fluorouridine triphosphate or a reciprocal formation of the two metabolites to prove the mechanism of 5-fluorocytosine activity. Using the results of four in vitro susceptibility tests, we separated isolates of C. albicans into susceptibility groups. For most strains, there was a positive correlation between the degree of 5-fluorocytosine susceptibility and the inhibition of biosynthesis of both RNA and DNA, incorporation of 5-fluorouracil into RNA, inhibition of ribosomal protein synthesis, and levels of 5-fluorodeoxyuridine monophosphate. However, in some strains with a similar degree of 5-fluorocytosine resistance, either reduced incorporation of 5-fluorouracil or reduced 5-fluorodeoxyuridine monophosphate levels occurred, suggesting that these two mechanisms are not necessarily linked to each other and that both may be responsible for 5-fluorocytosine activity.The mechanism of action of the antifungal drug 5-fluorocytosine (5-FC) has been investigated in yeasts (7,8,12), aspergilli (12), and dematiaceous fungi (17). It has been suggested that the activity of 5-FC after uptake by the fungi and intracellular deamination is a consequence of intrafungal formation of two metabolites, 5-fluorodeoxyuridine monophosphate (FdUMP) and 5-fluorouridine triphosphate (FURTP) (11,15).FdUMP is a potent inhibitor of thymidylate synthetase (5), causing an impairment of DNA synthesis in bacteria and tumor cells. Furthermore, in one 5-FC-susceptible strain of Candida albicans, in the presence of 5-FC, FdUMP was formed, followed by a decrease in the thymidylate synthetase activity (2). Moreover, the addition of 5-FC to both hyphal and yeast phases of C. albicans cultures resulted in an immediate inhibition in the increase of DNA (12).FURTP is incorporated into fungal RNA in place of uridylic acid (11, 15), which alters amino acylation of tRNA in vitro (4) and is believed to cause unbalanced internal pool and disturbed synthesis of proteins and carbohydrates (6, 13, 14). There is a statistical correlation between incorporation of the fluorinated metabolite into RNA and the antifungal activity of 5-FC, although there are considerable individual variations in incorporation in mutants with similar 5-FC susceptibility (1, 7, 10). Whether these two suggested mechanisms of 5-FC activity (FdUMP and FURTP) are linked or independent of each other is still unknown. The formation of FdUMP and decreased thymidylate activity may explain the antifungal 5-FC activity, the incorporation of FURTP in RNA being only incidental, or the converse. The purpose of this study was to determine on which of these mechanisms the activity of 5-FC is based.
Candida albicans isolates from 402 patients with no prior history of treatment with 5-fluorocytosine were collected at five medical centers from different areas of the United States. Isolates could be separated into four groups based on their minimum inhibitory concentrations (MICs) to 5-fluorocytosine. Group I isolates (60%) had MICs less than or equal to 12.5 micrograms/ml after 7 days, whereas groups II (22%), III (14%), and IV (4%) demonstrated MICs greater than 12.5 micrograms/ml on days 7, 2, and 1, respectively. Serotypes A and B accounted for 50.7 and 49.3%, respectively, of the 398 isolates typed. Serotype B was less prevalent in group I (26%), but predominated in the more resistant groups, groups II (85%), III (86%), and IV (53%). The common practice of identifying as "resistant" those isolates with MICs greater than 12.5 micrograms/ml after 48 h of incubation would yield a resistance rate in the United States of 11.5 to 15.5% in four centers and 35% in the fifth. Although serotype B and small agar disk diffusion zone sizes correlated with poor 5-fluorocytosine susceptibility, their ability to predict tube dilution MICs was limited. The true predictive value of such tests awaits correlation with in vivo studies.
The in vitro susceptibility of Candida albicans isolates to flucytosine was compared to therapeutic effect in experimental murine candidiasis (candidosis). Four groups of 10 isolates were chosen, based upon their broth dilution minimal inhibitory concentrations (MICs), from a group of 402 isolates from patients without prior flucytosine therapy. Group I MICs were less than 12.5 micrograms/ml after seven days, whereas group II, III, and IV MICs exceeded 12.5 micrograms/ml on days 7, 2, and 1, respectively. Pilot experiments selected challenge inocula of similar virulence. Mice were infected intravenously and given various flucytosine doses. Significant prolongation of survival correlated with MICs and with agar disk-diffusion zone diameters (P less than 0.05). In vivo response to therapy was more favorable for group I isolates compared with group IV isolates (P less than 0.01). The present study demonstrates in this animal model that in vitro susceptibility does correlate with in vivo response to therapy, although exceptions occur with individual isolates.
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