The cell wall of A. fumigatus is a unique structure which does not exist for human cells. It enables the fungus to resist against external aggressions, but, at the same time, it is its Achilles' heel since it is a major drug target as shown by the commercial launch of echinocandins that block cell wall biosynthesis. Polysaccharides represent the major part of the fungal cell wall and are responsible for its rigidity and plasticity. Five structural polysaccharides are present in the cell wall of A. fumigatus mycelium and conidia: β1-3)glucan, chitin, galactomannan, β1-3)glucan and β1-3/1-4)glucan. β1-3)glucan is highly branched with β1-6) linkages constituting a three-dimensional network with a large number of side-chains and ramifications. Other polysaccharides such as chitin, galactomannan and β1-3/1-4)glucan are cross-linked to the branched β1-3/1-6)glucan network. β(1-3)glucans are synthesized by a plasma membrane-bound β(1-3) glucan synthase complex which uses UDP-glucose as a substrate, and extrudes linear β(1-3)glucan chains through the membrane into the periplasmic space. A single catalytic FKS1 subunit is present in the genome of A. fumigatus and is essential. The regulation of the glucan synthase of A. fumigatus and the enzymes responsible for remodeling β(1-3) glucan remains however poorly understood.The chitin synthases that are responsible for the synthesis of linear chains of β(1-4) N-acetylgucosamine from the substrate UDP-N-acetylglucosamine are also a family of integral membrane proteins. Eight genes were found in A. fumigatus. The significance of each of these genes not understood but none of the CHS genes of A. fumigatus is essential.Three glucan synthase genes have been identified and none of the A. fumigatus genes is essential. Orthologs of most yeast mannosyltransferase genes can be found in the genome of A. fumigatus. How orthologs of the yeast mannosyltransferases synthesise a mannan chain different in A. fumigatus from S. cerevisiae remains a mystery. The addition of galactofuranose residues to the mannan core to form side-chains is also not understood. The current knowledge of the function of these cell wall enzymes and the role of the different polysaccharides in the cell wall organisation will be discussed in my talk with special emphasis on the use of cell wall as a drug target.
Cryptococcus neoformans is the most frequent cause of fungal meningoencephalitis in immunocompromised patients. Because of its minimal side effects and ready penetration into the central nervous system, fluconazole (triazole), has been the most widely used for the treatment as well as for the long term maintenance therapy for cryptococcosis. As with other fungi, fluconazole resistance associated with mutation or overexpression of P-450 lanosterol 14a-demethylase (ERG11) or constitutive upregulation of multidrug transporters have been reported in C. neoformans. In addition, we have reported a unique pattern of resistance to triazoles termed heteroresistance in two strains of C. neoformans isolated from patients in 1998. Heteroresistant strains produce cultures with heterogeneous compositions in which most of the cells are susceptible but cells with highly resistant to fluconazole (MICs, ≥ 64 ug/ml) are recovered at a variable frequency. Highly resistant population can be obtained in homogeneity by subculturing resistant clones on media with drug but homogeneous population of sensitive culture can never be obtained from heteroresistant strains. Furthermore, the highly resistant culture derived from a resistant subpopulation is lost during repeated transfer on drug free media without loosing the basal subpopulation of resistant cells. In order to determine the intrinsic nature of heteroresistance, we screened over 100 Cryptococcus strains isolated at least 10 to 20 years prior to the birth of azole drugs. Surprisingly, considerable number of the strains isolated between 1960 to 1973 manifested heteroresistance to fluconazole indicating that heteroresistance in C. neoformans is intrinsic.
Invasive aspergillosis presents a formidable problem for both diagnosis and therapy. Therefore, prevention is a very important strategy in controlling this disease. Currently, the environmental mycology of most cases of invasive aspergillosis is poorly defined. However, the development of molecular techniques more directly applicable to Aspergillus spp. may help resolve some of these difficulties. Preventing invasive aspergillosis demands a clear understanding of the environmental sources of Aspergillus spp. and how this mould is transmitted to patients before, during and after hospitalization for transplantation. Exposure to Aspergillus in hospital, especially during heavy construction or demolition, has been well documented. There appears to be a strong correlation between outbreaks of invasive aspergillosis and hospital building work. However, prevention of aspergillosis is relatively difficult. The HEPA filter appears to be the only effective means of decreasing the incidence of aspergillus infection. The principles of environmental control of nosocomial aspergillosis are complex given that even HEPA units are not completely effective in preventing disease. Alternatively, a systematic program of longitudinal patient and environmental surveillance may predict cases of invasive aspergillosis. Indeed, there appears to be a correlation between the recovery of Aspergillus spp. from the nose and mouth of patients in an open hematology ward and an elevated number of conidia in the air. The relationship between aspergillosis in predisposed patients and building work is also complex. Whether or not this activity is complicated by an outbreak of infection in the susceptible patients nearby, or is a risk related directly to the amount of disruption or some other factor, is unknown. Aspergillus spp. have a major reservoir in organic debris, dust and building material. Susceptible patients should not be treated in areas where there is construction or demolition activity. Although outbreaks of invasive aspergillosis have been associated with construction within or around a hospital, the precise source of the fungus is very difficult to trace. New data suggest that patients may be exposed to Aspergillus conidia in water supplies in hospitals and as a result of showering in patient bedroom facilities. Increasingly, cases of aspergillosis are being reported many months after transplantation and discharge from hospital. This scenario raises the question of community acquired disease following exposure to Aspergillus in the home, the workplace or the outdoor. Returning to work after transplantation is a much-discussed topic today. Returning to work should not endanger their health. This means that occupational risks such as occupational exposure to Aspergillus spores must be evaluated. The necessity of immunosuppressive therapy or the treatment for chronic graft versus host disease after transplantation elevates the aspergillosis risk, especially 1-6 months after transplantation. The risk of acquiring aspergillosis at work exists, but is not easily quantifiable. Nevertheless, the risk should be minimized during the period of vulnerability by preventive measures such as restriction of certain activities, changing work methods and reorganizing the work day to adapt to the risk, and wearing personal protective equipment, as well as attention to information about aspergillosis risk and about the likelihood of exposure in the patient's professional and leisure activities. From an epidemiological point of view, molecular study of moulds either isolated from patients or the environment will increase our understanding of the acquisition and route of infection. Various molecular techniques are available to genotype moulds. Discriminative typing methods are now available.
Cryptococcosis remains a worldwide invasive mycoses in immunocompromised hosts with significant morbidity and mortality. This presentation will focus on the recent IDSA guidelines as they are being adjusted. There will be discussions of management of cryptococcal meningoencephalitis in 3 groups:HIV-infected; transplant recipients; non-HIV, non-transplant hosts. There will be emphasis on management of complications such as increased intracranial pressure, IRIS and cryptococcomas. There will also be specific recommendations for unique populations such as children, pregnancy, and those in resource-limited environments. Recommendations for management will include other sites of infection such as the lungs and differences between C.neoformans and C. gattii infections. Important principles will be identified such as:(1) Induction therapy for meningitis using fungicidal regimens with amphotericin B plus flucytosine followed by consolidation and suppressive regimens using fluconazole. (2) Importance of early recognition and treatment of intracranial pressure and understanding/management of IRIS. (3)Use of lipid amphotericin B regimens in renally impaired patients. It is clear that cryptococcosis remains a challenging management issue with no new drugs or recent definitive studies. However, if diagnosis is made early, institution of guidelines and its principles considered and control of underlying disease available, cryptococcosis can be managed successfully in the vast majority of patients.
The IDSA guidelines review management of aspergilloma, invasive, chronic pulmonary and allergic forms of aspergillosis. Voriconazole is recommended for the primary treatment of invasive aspergillosis in most patients. Liposomal amphotericin B therapy could be considered as alternative primary therapy in some patients. For salvage therapy, agents include liposomal amphotericin, posaconazole, itraconazole or caspofungin. In patients whose infection is refractory to voriconazole, there are few data. The role of combination therapy in treatment of IA is uncertain and warrants a prospective controlled clinical trial. Management of breakthrough invasive aspergillosis in the setting of azole prophylaxis or suppressive therapy is not defined by clinical trial data. Antifungal prophylaxis with posaconazole can be recommended in the subgroup of HSCT recipients with graft versus host disease at high risk for IA and in neutropenic patients with acute leukemia or myelodysplastic syndrome who are at high risk for IA. Surgical resection unilateral pulmonary infection, pulmonary lesions contiguous with the heart or great vessels, invasion of the chest wall, osteomyelitis, and endocarditis. Recovery from neutropenia in a persistently neutropenic host or reduction of corticosteroids in a patient receiving high dose glucocorticosteroids is paramount for improved outcome in invasive aspergillosis. Single pulmonary aspergillomas may be best managed by surgical resection, whereas chronic pulmonary aspergillosis requires long-term medical therapy. The management of ABPA involves the administration of itraconazole and corticosteroids.
We have a little bit different choices in antifungal agents from the United States or other countries. The first edition of Japanaese practical guideline for fungal infectious diseases was published in 2003, when micafungin has been released. Amphotericin B, flucytosine, intravenous and oral fluconazole, itraconazole capsule, miconazole were antifungals that have been available before micafungin release. Since then, voriconazole, intravenous itraconazole and liposomal amphotericin B become available. Primary choice of antifungal in some situations such as invasive aspergillosis or prophylaxis changed accordingly. Another aspect of our guideline is a flowchart that connected diagnosis to antifungal choices. These charts were proposed according to each field of medical specialty. Since a guideline in Japan is not only for infectious diseases specialist but for all doctors including surgeons who might treat patients.
The treatment of candidiasis has evolved significantly over the past decade. The recognition of the influence of invasive Candida infections on morbidity and mortality, an abundance of recent in vitro and clinical data, and the availability of new antifungal agents with significant activity versus most Candida spp. have had a major influence on the therapeutic approach to this increasingly common disorder. Because of many significant changes in therapy since the publication of the last IDSA Candidiasis Treatment Guidelines in 2004, a revised version has recently been accepted by IDSA for publication in the near future. The most significant changes in these new guidelines relate to the importance of the echinocandins as a primary therapy for many forms of invasive candidiasis. For example, an echinocandin is favored for patients with proven or suspected candidemia among patients who are moderately to severely ill; de-escalation to fluconazole, when appropriate, is favored among patients who are culture-negative and improving clinically. The new guidelines emphasize the use of echinocandins for candidemia due to C. glabrata, and favor fluconazole or amphotericin B for infections due to C. parapsilosis. In neonates, fluconazole is favored for most Candida infections, but growing experience with the echinocandins suggests an important role for these agents in this population. For less common conditions such as endocarditis, hepatosplenic candidiasis, osteomyelitis, endophthalmitis, and central nervous system candidiasis, there have been few changes changed with regard to new therapies due to little or no new treatment data. The role of voriconazole for treatment of candidiasis is very limited due to the lack of significant benefit over fluconazole.
【はじめに】救急・集中治療領域の菌血症の起炎菌として，Candida 属の検出は多い。Candida glabrata（CG）の血流感染が主要臓器に与える影響を，動物研究で評価した。【方法と結果】8-12週齡の雄性Bulb-CマウスにCG 108/kgを生理的食塩水1 mLに撹拌し尾静脈内投与すると，2日死亡率は抗真菌薬を使用しない状況で約40%だった。CG投与後24時間で，全身潅流の後に肺，右心房，大動脈，腎臓を摘出し，転写因子nuclear factor-κB（NF-κB）活性をウエスタンブロッド法で解析した結果，対照群に比較して，肺で約7.2倍に，右心房で約4.5倍に，大動脈で約2.1倍に，腎臓で約4.3倍に，NF-κB活性が高められていた。肺に経気道的にToll-like受容体(TLR)2，TLR4，およびDectin-1の siRNAを遺伝子導入し，各受容体発現を肺選択的にノックアウトさせた系では，各々CGによる肺のNF-κB活性は対照群の約5倍に留まった。これに対して，tumor necrosis factor（TNF）受容体1をsiRNAで肺選択的にノックアウトさせると，CGによる肺のNF-κB活性は対照群の約3倍に減じた。CG投与後24時間の血漿TNF-α濃度は，700 pg/mLレベルに高められていた。【結語】CGは血流感染となることで，主要臓器の炎症強度を高めた。臓器に存在するTLRやDectin-1などはCGの炎症効果発現に必要である。しかし，臓器炎症の進展には，血中で高められたTNF-αなどの炎症性サイトカインが臓器の炎症性受容体のリガンドとして作用することが強く関与すると評価された。【文献】1. Matsuda N, et al. Nuclear Factor-κB decoy oligonucleotides prevent acute lung injury in mice with cecal ligation and puncture-induced sepsis. Mol Pharmacol 2005;67:1018-25.2. Matsuda N, et al. Silencing of caspase-8 and caspase-3 by RNA interference prevents vascular endothelial cell injury in mice with endotoxic shock. Cardiovasc Res 2007;76:132-40.
non-albicansは単一の性質をもつ菌種ではなく雑多なグループであり、かつて行われていたCandida albicansの簡易同定試験（germ-tubeや厚膜胞子形成）が陰性の株をnon-albicansと総称していた名残りにすぎない。正確に菌種の同定のできる現在、”non-albicans”とまとめることは、いわば”グラム陰性桿菌”とまとめて考えるのと同様の危険性をはらむ。カンジダ属の抗真菌薬感受性が菌種に依存することはよく知られた事実であり、フルコナゾール感受性という点では、感受性菌種であるC. albicans, C. parapsilosis, C. tropicalisと抵抗性菌種であるC. glabrata, C. kruseiに大別される。キャンディン感受性という点ではC. parapsilosis, C. guillermondiiではMICが他菌種に比べて有意に高い。キャンディン感受性のブレイクポイントやMICの高い菌種で治療成功率に差が現れるかは明らかではない。カンジダ症治療薬の比較試験においても全体としては良好な成績が得られてはいるが、複数の報告からはC. parapsilosisにおいて菌陰性化が遅延している。我が国のカンジダ血流感染症ではC. parapsilosisの割合が高いことから、治療成功率の差がわずかでもあれば実数の差は大きくなる可能性がある。さらに、キャンディンの選択において以下の点も注意に値する。 1）カンジダ属の複数菌種においてキャンディン耐性獲得株の報告は徐々に増加しており、いずれも治療中に耐性株が検出されたとされている。 2）キャンディンには点滴薬しかないため、静脈ライン留置期間および入院期間延長に伴う院内感染症のリスクが高まる。 したがって、non-albicansであることのみを根拠にキャンディンを選択すること、あるいは菌種判明後もそのまま継続することのデメリットを認識することは予後改善の最大化に必須であろう。
Mucor is a mold existed in nature. Mucor infections of humans, even in immunocompomised hosts, are rare. Clinical manifestations of mucormycosis are nonspecific and diagnosis is based on microscopic examination and culture of biopst specimens. Serologic test or molecular methods of speciation are used only as research tools We investigated medical records especially for underlying diseases, clinical findings, treatment, prognosis of patients diagnosed with rhinocerebral mucormycosis retrospectively in Asan medical Center from 1996 till 2007. Thirteen patients of rhinocerebral mucormycosis were enrolled, 6 male, 7 female. The underlying diseases were diabetes mellitus in 8 patients, acute leukemia 2 patients, Kidney transplantation 2 cases, myelodysplastic syndrome I patient. Six patients complained of nasal symptoms including stuffy nose, rhinorhhea, 5 patients complained of ophthalmic symptoms such as decreased visual acuity, diplopia, ophthalmic pain and 2 patients hard palate ulcer. Treatments were surgical debridement and antifungal agents in 10 patients, surgical debridement in 2 patients, antifungal agent in one patient. The mortality was 23% (3/13; two patients with kidney transplantation, one patient with acute leukemia) In summary mucormycosis should be considered in uncontrolled DM, immunocompromised host. The combined modality of early surgical debridement and antifungal agents were used for better treatment of rhinocerebral mucormycosis.
In Thailand, invasive aspergillosis and candidiasis are the most common opportunistic fungal infections whereas disseminated penicilliosis and histoplasmosis are the most common endemic mycoses. Definite diagnosis of invasive aspergillosis is problematic due to poor host conditions, difficulty on performing invasive procedures, and unavailability of diagnostic tests. Mortality of patients with invasive aspergillosis is relatively high. Primay antifungal therapy still relies on conventional amphotericin B since lipid amphotericin B, echinocandins, and voriconazole are expensive. Several studies have been done from Thailand on epidemiology, clinical manifestations, diagnosis, treatment, and prevention of penicilliosis; many of them have been incorporated into the standard practice.
Fungal infections are increasing in frequency and complexity. The number of reported candidaemia cases has been rising in the UK from 1398 cases in 2003 to 1873 cases in 2006. Invasive aspergillosis has increased in incidence so that in Europe it is found at autopsy in 4% of all patients dying in European teaching hospitals. Other figures from Japan and USA support this upward trend. There is a high frequency (∼4%) in medical ICU patients, of whom > 40% are COPD patients. More than 500 cases of IA have been described postoperatively in non-immunocompromised patients. Overall risk estimates by underlying condition are shown in the table below, assuming no prophylaxis. Poor diagnostic performance of current methods accounts for some underdiagnosis AmBisome (liposomal amphotericin B) is useful for the management of candidaemia, cryptococcal meningitis invasive aspergillosis and zygomycosis. The dose of 3mg/Kg appears to be the optimal dose for all or almost all cases, with the possible exception of zygomycosis in which higher doses are often used. While voriconazole remains the drug of first choice for invasive aspergillosis, drug interactions, organ dysfunction, azole resistance and breakthrough infection are reasons to consider AmBisome. The echinocandins have no useful activity for cryptococcal infection or zygomycosis, and so amphotericin B is first-line therapy in almost all these cases.
Fifty years ago, no effective treatment of invasive fungal infections was known. In the absence of treatment, diagnosis was given little importance and often made at autopsy. Introduction of amphotericin B into clinical practice in the 1950's, followed by flucytosine changed everything. Despite its toxicity, amphotericin B deoxycholate proved to be a potent, broad spectrum antifungal. In the next decade, intravenous miconazole and oral clotrimazole were introduced but seemed to lack promise. It was only in the 1980's when ketoconazole and then itraconazole and fluconazole were introduced that the potential of oral, well tolerated broad spectrum antifungal agents was realized. In the 1990's lipid formulations of amphotericin B and voriconazole came into clinical trials and were significant additions. The first echinocandin, caspofungin, was introduced in the 1990's. Commercial success prompted development of two very similar echinocandins, micafungin and anidulafungin. Now, this cornucopia of new antifungals appears to be at an end, with the newer azoles, echinocandins and a monoclonal antibody (Mycograb) now in development appearing to offer no major advantages. Trials in which existing drugs are used in combination may expand our therapeutic arsenal a little. Perhaps the next leap forward will be in better diagnostic tests so that empirical therapy will be more and more replaced by treatment of diagnosed infection.
Invasive candidiasis (IC) is an important healthcare-associated infection that continues to impact hospitalized and chronically ill patients in much of the developed world. Because of concerns regarding the changing epidemiology of these infections and the shift away from traditionally fluconazole-susceptible isolates such as Candida albicans and Candida parapsilosis, there is a need for newer antifungal agents that are both safe and offer improved efficacy than fluconazole and amphotericin B. In recently published studies comparing an echinocandin to a standard agent (amphotericin B, a lipid formulation of amphotericin B, or fluconazole), the echinocandins have been consistently efficacious and safe. Because of this outstanding safety profile and consistent efficacy (70-75% successfully treated patients), these agents have become important choices for first-line therapy for proven and suspected IC. For IC, many clinicians have adopted the approach of initiating therapy with an echinocandin, then 'de-escalating' to fluconazole or voriconazole when the patient has become clinically stable, has cleared the bloodstream of Candida and has an isolate that is predictably susceptible to an azole. There is much less experience with echinocandins in rarer forms of IC such as endocarditis, endophthalmitis and osteomyelitis, although anecdotal reports suggest that these agents perform well in this setting. There is a growing body of data among neonates and infants suggesting both the safety and efficacy of the echinocandins in this population. Echinocandins should generally be avoided in a setting of CNS candidiasis due to poor penetration into the CNS. Because of consistently increased MICs to these agents, first-line use of echinocandins for the treatment of Candida parapsilosis infections should be approached cautiously.
Cryptococcus neoformans is an environmental fungal pathogen that can cause devastating infection in the central nervous system. The important issues in the pathogenesis of cryptococcal brain infection is how C. neoformans enters the brain and then adopt to the brain oxygen level which is significantly lower than the optimum level (21%) for the growth of the fungus. In order to address the mechanism by which C. neoformans crosses the blood-brain barrier (BBB), we used an in vitro model of the human BBB. We demonstrated that C. neoformans invades the brain by transcellular crossing of the endothelium of the BBB. To investigate the genetic basis for the crossing of BBB and adaptation to the low oxygen conditions in the brain, we performed genetic screening of mutant strains that showed defects in crossing the BBB and/or growth under low oxygen conditions. Molecular dissecting of the mutated genes and the virulence of the mutants in experimental animas showed that the ability to grow under low oxygen conditions is an important pathobiological factor of C. neoformans.