From an ecological point of view, pathogens and autochthonous commensals are species that enhance their evolutionary fitness by using an animal host in any part of their life cycle. Conversely, growth on or in humans is evolutionary spill-over for opportunists and allochthonous commensals, decreasing the species' fitness. Strategic use of animals includes decrease of virulence over evolutionary time, such as to guarantee optimal condition of the host for dispersal and carriage to nutrient sources. Pathogens are able to overcome the actions of innate immune cells, and are limited or dormant with functioning acquired cellular immunity. Opportunists are poorly adapted, and hence are more virulent and less specific in their pathology. The great majority of opportunistic infections is quickly resolved by phagocytic action, but occasionally chronic infections are noted, where acquired immunity nevertheless remains insignificant. The number of opportunistic species is very small compared to the number of extant fungi, and therefore strategic and coincidental virulence factors must be of a quite specific nature. We aim to understand the role of such factors by revealing counterparts in the natural ecological niches of recurrent opportunists, such as Scedosporium species and black yeasts with their relatives. As an example, a possible clue to the high frequency of Asian cerebral infections by Chaetothyrialean fungi may their ablity of alkylbenzene assimilation.
Aspergillus species such as A. fumigatus and A. flavus are now the major cause of aspergillosis, which leads to a high mortality in immunocompromised hosts, especially in hematology patients. For the treatment of invasive aspergillosis (IA), amphotericin B and itraconazole have been the drugs of choice although amphotericin B therapy is limited by toxicity. Itraconazole is a triazole antifungal drug with good in vitro activity against Aspergillus spp. and has less toxicity. These two antifungal agents have been widely used to treat IA in China during the last decade. However, the extensive use of these compounds has been associated with the resistance. The earliest such findings were amongst Candida species. Recently, A. fumigatus isolates with in vitro resistance to itraconazole have been described. Also, an A. flavus isolate was found to be resistant to amphotericin B in vitro. We report the acquired itraconazole resistance in an A. fumigatus isolated from an aspergilloma patient treated with itraconazole in 2005: six A. fumigatus isolates were recovered at different times from the patient's sputum. After careful evaluation for antifungal susceptibility in vitro, the serial isolates were typed by RAPD with five different primers to confirm the identity. The interesting finding was that the itraconazole MIC against the pre-treatment isolate was 0.25 mg/L, while the second isolate, recovered after 6 months of itraconazole therapy, had an itraconazole MIC of >16 mg/L. The third strain was isolated two months after the therapy was discontinued and the itraconazole MIC was 0.5 mg/L. The MIC against the last three isolates acquired from the restoration of itraconazole therapy of 4 to 7 month was >16 mg/L. The molecular mechanisms of Aspergillus species resistance to these antifungals was studied through mutation detection of the target gene of the azoles ERG11 and the expression changes of pump genes such as MDR3. Cross resistance among the antifungal agents was detected and the role of ERG3 was studied. Our study revealed that the acquired itraconazole resistance of A. fumigatus may result from mutations in ERG11 gene.
Onychomycosis is a common disease which can affect anyone - men and women, old and young and accounts for approximately 23% of diagnosed foot diseases1. In addition, according to community studies have generally estimated a prevalence of approximately 3-5% in the general population. The majority of onychomycosis infections are associated with current or past dermatophyte infection of the feet2. Toe nails were affected 8 times more frequently than finger nails and nails of big toes and thumbs were affected in 89.1% and 69.5% of the patients respectively3. The main types are distal and lateral onychomycosis, proximal subungual onychomycosis, superficial onychomycosis and total dystrophic onychomycosis. In addition, patients may show different combinations of these patterns. Concerning a treatment, it can be categorized into three board groups: oral treatment, topical treatment and surgical/chemical avulsion of the nail plate. However, there are some specific difficulties and it is a source of frustration for many dermatologists. As a consequence, for the first time, possibility to establish international guidelines for the treatment of onychomycosis is discussed by the Taskforce on Onychomycosis Education(TOE). TOE is the first international board of key opinion leaders on onychomycosis, it was established with 13 international opinion leaders specialized in the field. One of the purposes of this board is that to establish international guidelines for treatment of onychomycosis. As a results, the latest consensus guidelines, primarily discuss therapeutic recommendations4, which is published on JEADV in 2005, was discussed during the 2nd international TOE meeting. This practical guideline for treatment of onychomycosis will certainly be helpful for both physicians and patients. However, there is no guideline for Asia country yet; therefore, it is time to generate an Asia of credible guidelines made by experts, in which all published guidelines from other countries will also be taken into account. It provides a guide to identify and effectively manage patients with onychomycosis.
References: 1. Roseeuw D. Achilles foot screening project : preliminary results of patients screened by dermatologists. J Eur Acad Dermtol Venereol 1999; 12: S6-9 2. Williams HC. The epidemiology of onychomycosis in Britain. Br J Dermatol 1993; 129: 101-9 3. Lee JM, Shin DH, Choi JS, Kim KH. Analysis of treatment of patients with tinea unguium and assessment of the real Effectiveness of antifungal agents and patient compliance. Korean J Dermatol 1998 ; 36(5) : 772-779 4. Lecha M, Effendy I, Feuilhade de Chauvin M, et al. Treatment options-development of consensus guidelines. J Eur Dermatol Venereol 2005 ; 19 (Suppl I) 25-33
In recent years fungi are flourishing in immunocompromised patients of tertiary care centers. The data on the burden of opportunistic mycoses in India is not clear though the climate in this country is well suited for wide variety of fungal infections. There are very few good diagnostic mycology laboratories and clinicians are still not aware of the emerging trends. Within the limited data available, it is found that the global change in Mycotic infections has been reflected on India as well, with increased incidence of invasive candidiasis, aspergillosis, and zygomycosis. The emergence of fungal sinusitis, penicilliosis marneffei and zygomycosis due to Apophysomyces elegans is quite unique in Indian scenario. Invasive candidiasis is most common opportunistic mycosis. In our center where regular medical autopsies are performed and systematic data is available, a 11-fold increase in candidemia was reported in second half of 1980s. A further 18-fold increase was observed in 1995 as compared to 1991. During second half of last decade, over 500 cases of candidemia were observed annually in 1400-bedded hospital. With the judicious use of antifungal prophylaxis, the incidence has come down to 200-300 cases annually at present. Along with the change in spectrum of Candida species globally, India observed similar change in scenario in recent years. However, the higher prevalence of candidemia due to Candida tropicalis in contrast to C. glabrata or C. parapsilosis among non-C. albicans, Candida species is unique in this part of the world. The reason is still not clear. Besides, an outbreak due to Pichia anomala (anamorph C. pelliculosa) affecting 379 neonates and children (4.2% of all admission) over 23 months period in our Institute is interesting and deserves attention. Recent molecular study has proved it as a point-source outbreak. Invasive aspergillosis is the second contender after candidiasis. Though due to difficulty in antemortem diagnosis the exact prevalence of this disease is not known, high prevalence is expected in Indian Hospitals where construction activities continue in hospital vicinity without proper impervious barrier. High incidence of Aspergillus fungal sinusitis and outbreak of Aspergillus endophthalmitis are the other challenges. Cryptococcus neoformans and Penicillium marneffei are thriving with high frequency of patients with AIDS in India. We observed a 15-fold increase of cryptococcosis cases compared to pre-AIDS period. The other opportunistic mycosis, invasive zygomycosis is an important concern as world's highest number of this disease is reported from India. The infection is commonly observed in patients with uncontrolled diabetes mellitus. A new clinical entity of renal zygomycosis and higher prevalence of Apophysomyces elegans infection in India require special attention. Other rare agents like species under Fusarium, Scedosporium, Trichosporon etc. cannot be ignored as they produce resistant opportunistic mycoses. Thus there is need of good diagnostic mycology laboratories, rapid diagnosis, and refinement of antifungal strategies in India.
シンポジウム I : International Symposium
International cross-talk on moving dimension of medical mycology
During the last two decades, numerous scientists have focused on molecular studies of fungal pathogenesis and development of antifungal drugs, yet many problems haven't been resolved due to technical difficulties in studying specific fungi. With the completion of genome sequences of the major fungal pathogens, knowledge gained from study of a specific fungus can immediately be applied to other fungi, using genomics as a platform. Of several dozen fungal species causing disease in humans, Candida glabrata is the most amenable species for molecular biology. For this reason, C. glabrata is a good resource for basic studies that may be applicable to many pathogenic fungi. The C. glabrata phenome project consists of an effort to construct a library of strains bearing mutations in single genes. Essential genes will have a regulatable promoter inserted at the 5' position, whereas all dispensable genes will be knocked out. The first aim of this project is prioritization of drug targets amongst the 5,300 C. glabrata genes. Genes representing potential new antifungal drug targets must be essential for growth, and highly conserved in other pathogenic fungi to ensure drugs will have a broad spectrum, but should not be conserved in the human genome. Consequently, a few dozen genes have been identified as the high priority candidates. In this talk we present the strategy of the prioritization and challenge of the fusion study of in silico and experimental research to develop new anti fungal drugs.
The human pathogenic fungus Candida albicans can cause a wide range of infections including oral thrush and life threatening systemic candidosis. However, the mechanisms by which C. albicans invades and persists within mucosal epithelium or organs such as the liver are not clear. We characterized the cellular and molecular mechanisms of epithelial-fungus interactions and liver invasion using reconstituted human oral epithelium (RHE), infected organs from animals and clinical samples. We observed that hyphal formation facilitates epithelial invasion via both active (physical penetration) and passive (induced endocytosis) processes. To identify C. albicans genes that are expressed during invasion of epithelial cells and liver tissue, we used genome-wide transcriptional profiling in vivo and ex vivo. By analysing the different phases of infection from attachment to tissue penetration in a time course experiment and in patient samples, and by comparing the profiles of an invasive with those of a non-invasive strain, we identified genes and transcriptional patterns which are associated with the invasion process. This includes genes involved in metabolism, stress, nutrient uptake, as well as transcriptional programmes regulating morphology and environmental sensing. We identified several novel infection-associated genes with unknown function. One gene, up regulated in both RHE infection and patients, named EED1, was essential for maintenance of hyphal elongation. Mutants lacking EED1 showed transient cell elongation on epithelial tissue, which enabled only superficial invasion of epithelial cells. Once inside an epithelial cell, Δeed1 cells could proliferate as yeasts or pseudohyphae but remained trapped intracellularly. Our results suggest that the adaptive response and morphology of C. albicans play specific roles for host-fungal interactions during mucosal and systemic infections.
GPI-anchored mannoproteins are one of the major cell wall components of eukaryotic microorganisms, including yeast and fungi. Some of GPI-anchored proteins are localized at the plasma membrane, but the others are further processed at the plasma membrane and are covalently linked to beta-1,6-glucan of the cell wall through the GPI portion. The genes and enzymes responsible for their biosynthesis and its cell wall assembly are potential targets of anti-fungal reagents. We identified GWT1 as a target of new anti-fungal drug candidates and elucidated its function to be involved in the acylation of inositol ring. We also found a new function of GPI7, which is involved in transfer of ethanolamine phosphate to Man2 of GPI. Our results indicate that the localization of GPI-anchored endoglucanase Egt2p is displaced from the septal region to the cell cortex at the restrictive temperature in gpi7Δ cells, suggesting that GPI7 is involved in the separation of mother and daughter cells and its defective phenotype is a good marker to select a new inhibitor towards Gpi7 function. We have recently reported that PER1 is involved in lipid remodeling of GPI-anchored proteins, indicating that Per1p has a GPI-phospholipase A2 activity to eliminate unsaturated fatty acyl chain at the sn-2 position of PI moiety. We further found that human PERLD1, which is now known as an oncogene, is a functional homologue of yeast PER1, indicating that this is a potential target for new anti-cancer drugs.
TAC1 (for Transcriptional Activator of CDR genes) is needed for upregulation of the ABC-transporters CDR1/CDR2, which mediate azole resistance (AR) in Candida albicans. While wild-type TAC1 drives high expression of CDR1/2 in response to inducers, TAC1 can be hyperactive (HYP) by gain-of-function (GOF) mutations responsible for constitutive high expression of CDR1/2. High AR levels are achieved when C. albicans carries HYP alleles as a consequence of loss of heterozygosity (LOH) at the TAC1 locus on Chr. 5, which is next to the mating-type like (MTL) locus. Both are located on Chr. 5 left arm (5L) with ERG11 (target of azoles). A detailed genotypic evolution in 5 groups of related isolates containing azole-susceptible and -resistant strains was undertaken by analysis of TAC1 and ERG11 alleles and Chr. 5 alterations. ERG11 alleles contained known mutations and 17 new TAC1 alleles were isolated, including seven HYP alleles with 5 separate new GOF mutations. SNP analysis of Chr. 5 revealed that AR strains acquired TAC1 HYP alleles and, in most cases, ERG11 mutant alleles by LOH events not systematically including the MTL locus. TAC1 LOH resulted mostly from mitotic recombination of 5L. Comparative genome hybridization and karyotype analysis revealed the presence of isochromosome i(5L) in two AR strains. i(5L) leads to increased copy number of AR genes present on 5L, among them TAC1 and ERG11. Our work shows that AR was not only due to the presence of specific mutation(s) in AR genes (at least ERG11 and TAC1) but also to their increase in copy number by LOH and to the addition of extra Chr.5 copies. The development of AR (that looks here only at 2 different mechanisms) in C. albicans is therefore a powerful instrument for generating genetic diversity. The existence of other resistance mechanisms in C. albicans is still able to elevate the complexity of this phenomenon.
Clinical isolates of Candida species with reduced susceptibility are rare. We have investigated the MCF resistance mechanism in C. glabrata clinical isolates and laboratory mutants. DNA sequence analysis of the ORFs encoding the β-1,3-glucan synthase catalytic subunits from sensitive and resistant clinical strains identified nucleotide changes that were predicted to cause a S629P amino acid change in the echinocandin resistance 'hot spot' of FKS2, and to introduce a premature stop codon in FKS1. As C. glabrata is haploid, one mutation in each of the two FKS genes may be required to confer full resistance to MCF. This hypothesis was tested by introducing the FKS2 S629P mutation in a sensitive strain using site-directed mutagenesis. The mutant had intermediate resistance to MCF, and developed elevated resistance when exposed to MCF. All resistant variants derived from the mutant showed the same MIC as the resistant clinical isolates, and each had either a predicted single amino acid change or a premature stop codon in FKS1. These findings indicate that functional homozygosity in glucan synthase subunits is required to confer clinically significant MCF resistance on C. glabrata.