There are several different types of repeated sequences in the genome of Candida albicans, including the MRS (Major repeated sequence). In 2004, the whole genome sequence of C. albicans was published. Assembly of the sequences to chromosomal length contigs was not achieved, mainly due to interruption of the sequences by MRS. However, MRS including Ca3, 27A and RPS have been playing important roles in a number of epidemiological research studies and basic biological investigations into C. albicans chromosome loss events and associated phenotypic changes. Here we summarize structural analyses from subrepeat sequences to the chromosome level, and functional analyses of MRS.

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Serological surrogate tests for invasive fungal infection have been used practically in Japan. The Aspergillus galactomannan antigen detection kit by enzyme linked immunosorbent assay is the most reliable test for making a diagnosis of invasive aspergillosis. But in recent years, occurrences of false positive and negative results have also been reported by several investigators. Therefore, evaluation of the results of this assay should be done carefully in the clinical stage. Moreover, some other methods to detect the Aspergillus antigen or anti Aspergillus antibody in serum have also been reported.
The problem of false positive results due to the frequent occurrence of non-specific reaction in the alkaline treatment, chromogenic automated kinetic assay to mesure (1→3)-β-D-glucan had been noticed in Japan. But this important problem was resolved by improvement of the pretreatment reagent in this kit in July 2005. In this manuscript, I describe recent trends of serological surrogate tests for Aspergillus infection and the process of improvement of the (1→3)-β-D-glucan measurement kit.

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Hematopoietic stem cell transplantation has been established as a curative treatment for advanced hematologic malignancies. Transplantation with a reduced-intensity conditioning regimen has been developed, and the minimal toxicity of reduced-intensity stem cell transplantation (RIST) has made this procedure available for patients of advanced age or with organ dysfunction. The response of malignant lymphoma and some solid tumors to RIST has been observed. RIST with unrelated donors and umbilical cord blood has been studied. Fungal infection is an important complication of RIST. Since the prognosis of fungal infection is poor, the management has been focused on its prophylaxis. 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, when most patients are followed as outpatients. The focus of fungal management after RIST is oral antifungal agents rather than in-hospital equipment. Various antifungal agents have recently been developed and applied for clinical use, and many of these have been developed simultaneously for the first time. A major change in antifungal management will probably occur in the next several years.

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Micafungin, a new class of the antifungal agent “echinocandin” released in 2002, and voriconazole, a new triazole antifungal agent released in 2005 in Japan have in vitro activities against Aspergillus spp. Results of large-scale clinical trials in Europe and the United States showed voriconazole to have superior efficacy against invasive pulmonary aspergillosis in comparison with conventional amphotericin B, and caspofungin, a member of the echinocandins, was effective as an empirical antifungal therapy in patients with persistent fever and neutropenia. In this way, choices of therapeutic medicine for aspergillosis are increasing more and more, and it is expected that the method of treatment will change greatly in future. On the other hand, we need to establish a new standard therapy for aspergillosis to avoid the clinical disruption caused by the variety of pharmaceutical choice caused. In this report, we describe the role of new antifungal agents for non-fumigatus Aspergillus infections, and the breakthrough in counteracting fungal infection using these new drugs.

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Recent situation of HIV-related mycosis was discussed in this paper, with the analysis of 1) annual report of HIV trends in Japan by the AIDS epidemiology committee, 2) report of HIV-related opportunistic infections (OIs) collected by the AIDS-OIs research group funded by the Ministry of Health, Labour and Welfare, and 3) 17 cases of HIV-related aspergillosis collected by the author. Annual AIDS cases were increasing, and their major diseases were included with the following mycosis: pneumosystis pneumonia 35.7%, candidiasis 19.1%, and cryptococcosis 2.4%. There were two foreigner's cases of histoplasmosis and no coccidioidosis. Candidiasis was likely to be shown in Japanese patients and cryptococcosis was in foreigners. Outcome of cryptococcosis was very poor as 32.7% of patients died.
There were 17 HIV-related aspergillosis, which consisted of 13 cases of lung diseases, 2 of brain lesions, and one each of sinus and stomach disease. Remarkable risk factor of HIV-related aspergillosis was decrease of CD4 cell count less than 10/μl, in addition to the usual risk factors of aspergillosis. Outcome of aspergillosis was very poor, as all treated cases died except one recent case treated with voriconazole.

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Solid organ transplantation is becoming increasingly common in Japan. Despite invasive fungal infections being less common than bacterial and viral infections, fungal infections still result in a higher mortality rate. Empiric and pre-emptive therapy plays an important role in management of invasive fungal infections, because successful treatment is difficult after a definite diagnosis of an invasive disease, especially invasive aspergillosis. Given this situation, to improve outcome, high risk patients need to be identified and antifungal prophylaxis is mandatory in preventing the development of the disease. However, antifungal prophylaxis for solid organ transplant recipients remains controversial. New antifungal agents might change the choice of fungal prevention and treatment.

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We studied the clinical features of 59 chronic pulmonary aspergillosis cases (aspergilloma, chronic necrotizing pulmonary aspergillosis) which we experienced in our hospital. To diagnose this disease, X-rays, sputum culture and serologic tests were mainly examined, X-ray findings were a fungus ball type in 47% of cases and thickened wall of a cavity type in 32%. Positive sputum culture found was A. fumigatus 78%, A. niger 13% and A. flavus 2%. Positive rates of serologic tests showed precipitating antibody 81% and antigen 11%; 39% of β-D glucan exceeded the reference value. As clinical symptoms, bloody sputum and hemoptysis were found at high frequency. Antifungal agents were administered intravenously or topically for treatment, primarily AMPH-B, ITCZ and MCFG. As adjuvant therapy, we administered Ulinastatin which is an elastase inhibitor for use aginst hemoptysis, and we performed steroid combination for cases considered to be associated with allergy. In all of 6 cases of chronic necrotizing pulmonary aspergillosis which were administered MCFG, X-ray findings improved.
A pathogenic factor, elastase was isolated from Aspergillus spp., and we also found the elastase inhibitor from this series. Five of 12 strains of A. fumigatus, and one of 2 strains of A. flavus expressed elastase inhibitory activity when we screened for the culture supernatant of various Aspergillus spp. of a clinical isolate. Elastase inhibitory activity from A. niger was very weak. Culture supernatants from 5 strains of A. fumigatus and one strain of A. flavus were stable for a fever, and human leucocyte elastase was inhibited, but these did not inhibit porcine pancreas elastase. We are aiming at clinical application and plan to continue further study.

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Candida albicans mannan consists of the α-1,6-linked backbone moiety and the α-1,2- and α-1,3-linked side chains. It also contains α-1,6-branched mannose units, β-1,2-linked mannose units, and phosphate groups. The cell wall mannans of the genus Candida possess three types of β-1,2 linked mannose units. One is linked via the phosphodiester linkage, the second type is connected to an α-1,2-linked mannose unit, and the third type is attached to an α-1,3-linked mannose unit. These β-1,2-linked mannose units showed a strong antigenicity and produce the characteristic NMR chemical shifts. Using two-dimensional NMR techniques, we will practically determine the structure of these polysaccharides in a nondestructive manner.

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1,3-β-glucans is a major cell wall component in fungi. Receptor molecules relating to innate immunity may recognize such cell wall products, and affect host defense systems. A β-glucan receptor, dectin-1, is a C-type lectin and may contribute to the innate immune responses. To examine the role of dectin-1 in recognition of 1,3-β-glucans and subsequent activation of intracellular signaling, the molecular characteristics of a carbohydrate recognition domain (CRD) of dectin-1 were investigated. The binding ability to β-glucans was abolished by mutating two amino acid residues, Trp221 and His223, on the CRD. Dectin-1 increased TLR2-mediated NF-κB activation in response to zymosan. However, dectin-1 alone could not affect the activation pathway for NF-κB, nor did co-expression of dectin-1 mutant and TLR2 increase the NF-κB activation. These results suggest that dectin-1 may have a co-stimulatory effect on leukocyte activation in response to fungal infection.

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Neutrophils are believed to be the first line of defense against invading microorganisms, but in vivo roles of reactive oxygens produced by neutrophils are not well known. Myeloperoxidase (MPO) catalyzes reaction of hydrogen peroxide with chloride ion to produce hypochlorous acid that is used for microbial killing by phagocytic cells. To define the in vivo role of MPO, we generated mice having no peroxidase activity in their neutrophils or monocytes. MPO-deficient (MPO-KO) mice showed severely reduced cytotoxicity to Candida albicans, Aspergillus fumigatus, Cryptococcus neoformans, and other microorganisms, demonstrating that an MPO-dependent oxidative system is important for host defense against fungi. However, the significance of MPO compared to the NADPH-oxidase is still unclear because individuals with MPO deficiency are usually healthy in contrast to patients with chronic granulomatous disease (CGD) who present clinical symptoms early in life. To better understand the contributions of MPO and NADPH-oxidase to antifungal defense mechanisms, we compared the susceptibility of MPO-KO mice and CGD mice to infections by C. albicans. Interestingly, at the highest dose, the mortality of MPO-KO mice was comparable to CGD mice, but was the same as normal mice at the lowest dose. These results suggest that MPO and NADPH-oxidase are equally important for early host defense against a large inocula of Candida. Our present results suggest that MPO-deficient individuals could exhibit similar problems as CGD patients if exposed to a large number of microorganisms.

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Cryptococcus neoformans is an opportunistic fungal infectious pathogen in immunocompromised patients with acquired immunodeficiency syndrome and hematological malignancies. Recently, innate immune cells, such as NK, NKT and γδT cells, have been found critical for determining the quality of acquired immunity by affecting the direction of Th1-Th2 balance. Th1-type immune response is important for the host defense against C. neoformans, and innate immunity may involve this process. In the present review, the accumulated knowledge including our own data on the role of innate immune lymphocytes in the host defense to this fungal pathogen are summarized, focusing on NKT and γδT cells.

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To evaluate the currently used Japanese Society for Medical Mycology (JSMM) method for testing the azole susceptibility of yeasts, the activities of fluconazole and itraconazole were tested against recently collected clinical isolates of Candida spp. (n=946) and compared with the National Committee for Clinical Laboratory Standards (NCCLS) M27-A2 microdilution reference method. Favorable correlation with the M27-A2 method was not seen for isolates of C. albicans, C. tropicalis or other Candida spp., particularly their trailing-growth isolates. However, the degree of correlation and agreement of MIC values were markedly improved when testing was performed by the modified JSMM method in which the end-point to be read was changed from IC₈₀ (for the current JSMM method) to IC₅₀. These results suggest that there is an urgent need to revise the current JSMM method.

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A novel elastase inhibitor from Aspergillus flavus (AFLEI) was isolated, and biochemical properties of AFLEI were examined. Column chromatography using diethylaminoethyl (DE) 52-Cellulose and Sephadex G-75 was used to purify the inhibitor. The final preparation was found to be homogeneous as indicated by a single band after disc polyacrylamide gel (PAGE) and isoelectric focusing electrophoreses. AFLEI had a molecular weight of 7,525.8 as determined by TOF-MS (time of flight mass spectrometry). The elastolytic activity of elastases from A. flavus, A. fumigatus and human leukocytes were inhibited by AFLEI. However, this activity from porcine pancreas elastase, trypsin, chymotrypsin, thrombin, and Ac₁-Proteinase from snake venom was not affected by AFLEI. The fibrinogenase activity of the elastase from A. flavus was inhibited by AFLEI. AFLEI was inhibited by α₂-macroglobulin. However, ethylenediaminetetraacetic acid (EDTA-2Na), benzamidine, chymostatin, tosyl phenylalanine chloromethyl ketone (TPCK) and dithiothreitol (DTT) did not show any inhibitory effect on the elastase inhibitory activity of AFLEI.

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Early identification of Candida isolates to the species level is necessary for effective antifungal therapy, and can also facilitate control of hospital infections. Phenotype-based methods for identifying Candida species are often difficult and time-consuming. Molecular biological techniques provide a useful alternative approach. In the present study, the ITS1-5.8S-ITS2 regions of fungal rRNA genes were amplified with universal primers in 20 standard strains. Digestion of the PCR products with one restriction enzyme, MspI, allowed discrimination of medically important Candida species, including C. albicans, C. glabrata, C. parapsilosis, C. tropicalis, C. krusei, and C. guilliermondii. Using this method, we successfully identified 137 clinical isolates of Candida. Among them, C. albicans was identified as the most common species, followed by C. parapsilosis, C. tropicalis, C. glabrata, C. krusei, and C. guilliermondii. This method is a simple, rapid, and cost-effective method for differentiation between species that is applicable in clinical laboratories.

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The production of chlamydospores is a diagnostic tool used to identify Candida albicans; these structures also represent a model for morphogenetic research. The time required to produce them with standard methods is 48-72 hours in rice meal agar and tensoactive agents. This time can be shorted using liquid media such as cornmeal broth (CMB) and dairy supplements.
Five media were tested: CMB plus 1% Tween-80, CMB plus 5% milk, CMB plus 5% milk serum, milk serum, and milk serum plus 1% Tween-80, under different incubation conditions: at 28°C and 37°C in a metabolic bath stirring at 150rpm, and at 28°C in a culture stove. The reading time points were established at 8 and 16 hours. The best results were obtained at 16 hours with CMB plus 5% milk under incubation at 28°C and stirring at 150 rpm. The next most efficient methods were CMB plus 5% milk serum and CMB plus 1% Tween-80, under the same incubation conditions. The other media were ineffective in producing chlamydospores. The absence of stirring at 28°C prevented the formation of chlamydospores within the set time points, and incubation at 37°C decreased their production.
This paper reports that the time to form C. albicans chlamydospores can be reduced.

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The conidia of filamentous fungi can be easily blown into the air and tend to be contaminants in the laboratory environment.
We developed a new “safety culture tube for fungi” to prevent biohazards and a procedure for collecting conidia for passage or fixing strains was proposed.

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