In addition to serial screening for GM antigenemia and radiological surveillance, PCR-ELISA may improve the rates of early diagnosis of IA and the management of patients with hematological malignancies.

Inactivation of the FCY2 (cytosine permease), FCY1 (cytosine deaminase), and FUR1 (uracil phosphoribosyltransferase) genes in Candida lusitaniae produced two patterns of resistance to flucytosine. Mutant fur1 demonstrated resistance to 5-fluorouracil, whereas mutants fcy1 and fcy2 demonstrated fluconazole resistance in the presence of subinhibitory flucytosine concentrations.Flucytosine (5FC) is one of the oldest antifungal agents. When this drug is taken up and converted to 5-fluorouracil (5FU) by fungal cells, it inhibits DNA replication and protein synthesis (11). However, it should be administered in combination with amphotericin B or azole antifungal agents, such as fluconazole (FLC), because the frequency of resistance development precludes its use as a single agent (10). In a previous work, we demonstrated that 5FC resistance in four Candida lusitaniae clinical isolates was due to a defect of purine-cytosine permease and that these isolates were specifically crossresistant to FLC when both the antifungals 5FC and FLC were used in combination (8). More recently, we provided molecular evidence that inactivation of the FCY2 gene in C. lusitaniae promotes cross-resistance to the antifungal combination 5FC-FLC (3). The goal of this study was to determine the precise contribution of the two other main genes possibly involved in 5FC resistance, FCY1 and FUR1, to the 5FC-FLC cross-resistance phenotype.Cloning and disruption of FCY1, FUR1, and FCY2 genes.

The aim of this work was to elucidate the molecular mechanisms of flucytosine (5FC) resistance and 5FC/fluconazole (FLC) cross-resistance in 11 genetically and epidemiologically unrelated clinical isolates of Candida lusitaniae. We first showed that the levels of transcription of the FCY2 gene encoding purine-cytosine permease (PCP) in the isolates were similar to that in the wild-type strain, 6936. Nucleotide sequencing of the FCY2 alleles revealed that 5FC and 5FC/FLC resistance could be correlated with a cytosine-to-thymine substitution at nucleotide 505 in the fcy2 genes of seven clinical isolates, resulting in a nonsense mutation and in a putative nonfunctional truncated PCP of 168 amino acids. Reintroducing a FCY2 wild-type allele at the fcy2 locus of a ura3 auxotrophic strain derived from the clinical isolate CL38 fcy2(C505T) restored levels of susceptibility to antifungals comparable to those of the wild-type strains. In the remaining four isolates, a polymorphic nucleotide was found in FCY1 where the nucleotide substitution T26C resulted in the amino acid replacement M9T in cytosine deaminase. Introducing this mutated allele into a 5FC-and 5FC/FLC-resistant fcy1⌬ strain failed to restore antifungal susceptibility, while susceptibility was obtained by introducing a wild-type FCY1 allele. We thus found a correlation between the fcy1 T26C mutation and both 5FC and 5FC/FLC resistances. We demonstrated that only two genetic events occurred in 11 unrelated clinical isolates of C. lusitaniae to support 5FC and 5FC/FLC resistance: either the nonsense mutation C505T in the fcy2 gene or the missense mutation T26C in the fcy1 gene.Treatment of fungal infections is challenged by a limited number of available antifungal agents and by the emergence of antifungal resistance. Therefore, during the last few years, research has focused on elucidating the molecular mechanisms of antifungal resistance (4, 28). The haploid opportunistic yeast Candida lusitaniae (teleomorph, Clavispora lusitaniae) is a good model for studying antifungal resistance. Although much less common than other Candida species, this yeast is characterized by its propensity to develop resistance to antifungal agents during treatment, mainly to amphotericin B, but also to azole drugs and to flucytosine (5FC) (11,16,17,22,26).5FC is a fluorinated pyrimidine that is used in combination with other antifungals to treat human cryptococcosis and sometimes candidiasis (3, 33). 5FC is actively transported into the cell through the action of purine-cytosine permease (PCP), encoded by the FCY2 gene. Once inside the cell, 5FC is rapidly converted to 5-fluorouracil (5FU) by cytosine deaminase (encoded by the FCY1 gene) and to 5-fluorouridine monophosphate (5FUMP) by uracil phosphoribosyltransferase (UPRTase; encoded by the FUR1 gene). 5FUMP is then converted to 5-fluorouridine triphosphate, which, when incorporated into fungal RNA instead of uridylic acid, disrupts protein synthesis. 5FUMP is also converted to 5-fluorodeoxyuridine monophosphate, which is a potent i...