Program and Abstracts of Annual Meeting of the Japanese Society for Medical Mycology

Members of Cryptococcus neoformans (C.n. ) species complex cause life-threatening infections. Its taxonomy is an open question, with 2 anamorph species originally described: C. neoformans & C. gattii. Mating experiments linked them to the teleomorph genus Filobasidiella, establishing 2 varieties: C.n. var. neoformans = F.n. var. neoformans (serotypes A, D & AD) & C.n. var. gattii = F.n. var. bacillispora (serotypes B & C). C.n. var. grubii, was established as a separate variety causing most infections. Molecular data suggested C.n. var. gattii as a new species, C. bacillisporus, recently renamed as C. gattii. PCR-fingerprinting, AFLP, URA5, PLB1 & ACT1 RFLP analysis, sequencing (ITS, URA5, PLB1 & ACT1) & microsatellite analysis have been used to study the genetic diversity of the C.n. species complex. PCR-fingerprinting, AFLP & RFLP analysis divided more than 1000 global isolates into 8 molecular types. VNI/AFLP1 & VNII/AFLP1A = C.n. var. grubii, serotype A; VNIII/AFLP3 = hybrid between C.n. var. grubii & C.n. var. neoformans, serotype AD; VNIV/AFLP2 = C.n. var. neoformans, serotype D; & VGI/AFLP4, VGII/AFLP6, VGIII/AFLP5 & VGIV/AFLP7 = C. gattii, serotypes B & C. AD hybrid isolates (VNIII/AFLP3) revealed patterns that correspond to a number of molecular types, suggesting recombination events leading to diploid or aneuploid strains. VNI & VGI are the predominant genotypes worldwide. Similar regional profiles of tree-derived & clinical isolates support an epidemiological link. The Vancouver Island outbreak is caused by the rare genotype VGII/AFLP6, which seems to be emerging in temperate climates (Greece & Colombia). Whole genome sequence analysis of the strains H99 (C.n. var. grubii ), B3501 & JEC21 (C.n. var. neoformans ) & WM276 (C. gattii ) has shown different levels of microsatellite abundance & polymorphism per variety/species & large variations in the flanking regions. The genotypic variation found among the 8 molecular types lies within a comparable range of that found in established fungal species, suggesting further speciation of the C.n. species complex. The specificity of the microsatellite flanking regions to a certain genotype is adding evidence to the existence of separate species.

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Reduced intensity stem-cell transplantation (RIST) has been developed as a novel curative option for advanced hematologic diseases. Its minimal toxicity has made transplantation possible for patients of advanced age or having organ dysfunction. Young patients without comorbidity can undergo RIST in the outpatient setting. Fungal infection remains an important complication in RIST. Since the prognosis of fungal infection is poor, prophylaxis is critical for its management; given recent progression in RIST management, the strategy of infectious prophylaxis has also changed. Equipment in the hospital is important for fungal infection; however, the median day of the development of fungal infection is day 100, the stage at which most of patients are followed as outpatients. The focus of fungal management after RIST is oral antifungals rather than in-hospital equipment. Various antifungals have recently been developed and applied to clinical use. A major change in antifungal management will probably occur in several years.

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The most common cause of high-level azole resistance in clinical isolates of Candida albicans is over-expression of drug efflux proteins, in particular, the ABC-type membrane transporters Cdr1p and Cdr2p. We examined the function of individual efflux proteins by expressing them in the genetically tractable yeast Saccharomyces cerevisiae. Individual CDR1 and CDR2 alleles were cloned and hyper-expressed in an S. cerevisiae expression system (host strain AD1-8u^- and transformation cassette constructed in plasmid pABC3) that correctly trafficked the hyper-expressed efflux pump protein to the host yeast plasma membrane.
Recombinant S. cerevisiae strains were constructed that expressed variants of one allele (A) of the C. albicans ATCC 10261 CDR1 gene generated by low-fidelity PCR mutagenesis. Specific regions of the CDR1 gene, such as the Nucleotide Binding Domain (NBD) were deleted and replaced by mutagenised PCR-generated fragments. One recombinant had a reduced fluconazole Minimum Inhibitory Concentration (MIC) of 75 μg/ml relative to the parental A allele strain (200 μg/ml), reduced rhodamine 6G (R6G) efflux, and contained two unique amino acid substitutions (F371S and R420S) in NBD1. Comparative SDS-PAGE analysis demonstrated reduced amounts of Cdr1p in the plasma membrane of this strain, possibly due to instability of the mutant polypeptide. In another mutant CDR1 allele strain, a L1021S mutation, immediately N-terminal of the NBD2 Walker B motif, conferred reduced pump activity without reducing Cdr1p expression in the plasma membrane. The hyper-expression system was also used to investigate natural allelic variation in both CDR1 and CDR2 in eight strains. Whereas only one strain had non-synonymous single nucleotide polymorphisms (SNPs) within the CDR1 gene, that gave minor functional change, the CDR2 alleles of seven strains contained both intra- and inter-strain non-synonymous SNPs. Functional variation between CDR2 alleles was detected, and confirmed by site-directed mutagenesis of individual SNPs.
The use of a heterologous hyper-expression system has facilitated analysis of allelic variation and identified amino acid residues important for function in the C. albicans drug efflux genes CDR1 and CDR2.

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Since the 1980s, the necessity for a clinically significant drug susceptibility testing method with a high level of reproducibility has increasingly been recognized due to the increased incidence of deep mycosis, a wave of novel antifungal agents introduced to the clinical setting, and the emergence of resistant fungi to antifungal agents (particularly, azole-resitant Candida spp.).Studies on susceptibility testing methods for yeasts that have been vigorously carried on primarily by the National Committee for Clinical Laboratory Standards (NCCLS) in the US came to fruition in the form of the standard method called NCCLS M27. The reproducibility of this method, as well as a favorable correlation between in vitro susceptibility and in vivo clinical effects for Candida spp. and azole antifungal agents, particularly fluconazole (FLCZ), have been confirmed. However, concerning other pathogenic fungi and antifungal agents, there are many issues to be solved in the future, since an in vitro-in vivo correlation has hardly been obtained, and testing methods for candin agents have never been standardized before.In Japan, the Standardization Committee of this Society started working on the establishment of a standard method for commercially available agents in Japan around 1992 when the guidelines for M27-P, the first NCCLS method, were released. The method of this Society was established in compliance with the M27-P method in terms of test media and other basic testing conditions. However, the M27-P method only described a macro-dilution technique, whereas this institute employed a micro-dilution technique that was announced in 1995, and is still available today. The method using such a technique was subsequently commercialized as test kits covered by medical insurance. Meanwhile, the NCCLS also standardized the micro-dilution technique, and this format predominates currently.In this seminar, we will explain the current status of drug susceptibility testing methods for yeasts and related tasks based on the results obtained by the Japan Antifungal Surveillance Program that were implemented by our research group and the discussion on the published literature, and urgent amendments to the assessment criteria of MIC end points used by this Society will be proposed. Therefore, we would like to obtain our Society Members' views on this issue.

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