亀井 克彦

The increasing incidence of infectious diseases caused by fungi in immunocompromised patients has encouraged researchers to develop rapid and accurate diagnosis methods. Identification of the causative fungal species is critical in deciding the appropriate treatment, but it is not easy to get satisfactory results due to the difficulty of fungal cultivation and morphological identification from clinical samples. In this study, we established a microarray system that can identify 42 species from 24 genera of clinically important fungal pathogens by using a chemical color reaction in the detection process. The array uses the internal transcribed spacer region of the rRNA gene for identification of fungal DNA at the species level. The specificity of this array was tested against a total of 355 target and nontarget fungal species. The fungal detection was succeeded directly from 10(3) CFU/ml for whole blood samples, and 50 fg DNA per 1 ml of serum samples indicating that the array system we established is sensitive to identify infecting fungi from clinical sample. Furthermore, we conducted isothermal amplification in place of PCR amplification and labeling. The successful identification with PCR-amplified as well as isothermally amplified target genes demonstrated that our microarray system is an efficient and robust method for identifying a variety of fungal species in a sample.

Introduction: During the last decades, Fusarium spp. has been reported as a significant cause of disease in humans, especially in immunocompromised patients, who have high risk of invasive life-threatening disease. Fusarium species usually reported as cause of human disease are F. solani, F. oxysporum and F. verticillioides. Case description: We describe the second case in the literature of disseminated fusariosis caused by Fusarium napiforme, that occurred in a 60-year-old woman with multiple myeloma after subsequent cycles of chemotherapy. Discussion and Evaluation: We identified the F. napiforme not only by standard morphologic criteria by macroscopic and microscopic characteristics, but also confirmed by molecular biology methods, including sequencing. The antifungal susceptibility of the F. napiforme isolates were tested to seven antifungal drugs; the azoles were the most active drug against all the isolates tested. Conclusions: Fusarium spp. are of relevance in medical mycology, and their profiles of low susceptibility to antifungal drugs highlight the importance for faster and more accurate diagnostic tests, what can contribute to an earlier and precise diagnosis and treatment.

症例は38歳、女性。乾性咳嗽で受診。胸部X線写真では左中肺野に浸潤影、胸部CTでは左舌区に粘液栓で充満し拡張した気管支による棍棒状陰影を認めた。気管支鏡では左B5入口部の狭窄と粘液栓を認め、血清IgE高値、アスペルギルス特異的IgE高値よりアレルギー性気管支肺アスペルギルス症が疑われた。しかし気管支洗浄液からCurvularia lunataが検出され、本真菌によるアレルギー性気管支肺真菌症と診断した。ステロイド内服治療のみでは効果なくボリコナゾールの併用にて改善を認めた。(著者抄録)

Exophiala dermatitidis pneumonia is extremely rare. Here we report a case of E. dermatitidis pneumonia successfully treated with long-term itraconazole therapy. A 63-year-old woman without a remarkable medical history developed a dry and chest pain. Chest radiographs revealed consolidation in the middle lobe of the lung. Cytologic examination by bronchoscopy showed filamentous fungi and E. dermatitidis was detected in the bronchoalveolar lavage fluid. After 5 months of itraconazole therapy, her symptoms improved and the area of consolidation diminished. Two weeks after discontinuing the itraconazole therapy, the area of consolidation reappeared. Itraconazole therapy was restarted and continued for 7 months. The abnormal shadow observed on the chest X-ray gradually diminished. Over a 27-month follow-up with periodic examination, there was no relapse and the patient had a favorable clinical course. (C) 2014, Japanese Society of Chemotherapy and The Japanese Association for Infectious Diseases. Published by Elsevier Ltd. All rights reserved.

Gliotoxin is an important virulence factor of Aspergillus fumigatus. Although GliA putatively belongs to the major facilitator superfamily in the gliotoxin biosynthesis cluster, its roles remain unclear. To determine the function of GliA, we disrupted gliA in A. fumigatus. gliA disruption increased the susceptibility of A. fumigatus to gliotoxin. The gliT and gliA double-disrupted mutant had even higher susceptibility to gliotoxin than each individual disruptant. The extracellular release of gliotoxin was greatly decreased in the gliA disruptant. Mice infected with the gliA disruptant of A. fumigatus showed higher survival rates than those infected with the parent strain. These results strongly indicate that GliA, in addition to GliT, plays a significant role in the tolerance to gliotoxin and protection from extracellular gliotoxin in A. fumigatus by exporting the toxin. This also allows the fungus to evade the harmful effect of its own gliotoxin production. Moreover, GliA contributes to the virulence of A. fumigatus through gliotoxin secretion.

In the present study, we developed a new real-time PCR system based on the cycling probe technology (CPT), which is composed of two single tube real-time PCR assays: the Fusarium genus-specific assay and the Fusanum solani species complex (FSSC)-specific assay with primers targeting the 28s ribosomal RNA gene. The Fusanum genus-specific assay was shown to be highly specific, detecting all reference Fusarium strains with no cross-reaction with other reference fungal strains, such as Aspergillus spp. and human DNA. The FSSC-specific assay also reacted very specifically with FSSC, except for a cross-reaction with Fusarium lunatum. To validate the real-time PCR system, we tested 87 clinical isolates of Fusarium spp. Identification results from the real-time PCR system were found to be 100% concordant with those from DNA sequencing of EF-1 alpha gene. The sensitivity testing also demonstrated high sensitivity, enabling detection of one copy of standard DNA with good reproducibility. Furthermore, both assays were shown to be extremely sensitive even when fungal cells were mixed with human cells, detecting 3 germinated conidia spiked in 3 mL of human blood. To apply our new real-time PCR system to the molecular diagnosis of fusariosis, we evaluated its efficacy using a mouse model of invasive F. solani infection. Plasma and whole blood samples of infected mice were tested. using the real-time PCR system. The sensitivity of the real-time PCR system was found to be 100% (n =4) in plasma samples. In contrast, no amplification signal was detected in whole blood samples. This system could provide a rapid and precise diagnostic tool for early diagnosis, which is necessary for appropriate treatment and improvement of prognosis of disseminated fusariosis. (C) 2014 Elsevier GmbH. All rights reserved.

Azole resistance among clinical isolates of Aspergillus fumigatus is becoming a serious problem in Europe, but the status in Japan is not yet known in detail. The aim of this study was to determine the present status of azole resistance in A. fumigatus in Japan. We employed 171 clinical isolates of A. fumigatus sensu stricto collected from 1987 to 2008 at the Medical Mycology Research Center, Chiba University, Japan for azole resistance determination. Identification of all isolates were re-examined both from the aspect of morphology and molecular phylogeny. The antifungal susceptibility of these isolates was tested based on the CLSI M38-A2 broth microdilution method. In our collection, only 1 (0.6%) and 2 isolates (1.2%) showed elevated MIC to voriconazole and itraconazole, respectively. Our study disclosed that the frequency of azole resistance in A. fumigatus still remains low in this collection. (C) 2014, Japanese Society of Chemotherapy and The Japanese Association for Infectious Diseases. Published by Elsevier Ltd. All rights reserved.

Biofilm production by microorganisms is critical for their pathogenicity. Serum promotes biofilm production by Aspergillus fumigatus; however, its effects on other Aspergillus spp. have not been reported. We analyzed biofilm formation by five Aspergillus spp., i.e., A. fumigatus, A. flavus, A. nidulans, A. niger, and A. terreus, and examined the effects of serum/serum proteins such as fetal bovine serum (FBS), fetuin A, and bovine serum albumin (BSA) on hyphal growth, hyphal branching, and extracellular matrix (ECM) formation. The antifungal susceptibility of A. fumigatus isolates that formed biofilms was also examined. All serum/serum proteins promoted the growth of all these fungal species; growth promotion was most evident with FBS, followed by fetuin A and BSA. This effect was most evident in case of A. fumigatus and least evident in case of A. terreus. Electron microscopy showed thick ECM layers surrounding fungal cell walls after culture with FBS, particularly in A. fumigatus. An increase in hyphal branching caused by fetuin A was the highest in case of A. fumigatus and A. nidulans. Biofilm-forming A. fumigatus showed resistance to most antifungal agents, although a synergism of micafungin and amphotericin B was suggested. Our results indicate that serum promotes biofilm formation, including thick ECM, by many Aspergillus spp., particularly A. fumigatus, and that this may be closely related to its virulence.

79歳,男性.約15年前より両上肢,手背にそう痒を伴う紅斑,乳頭状結節が出現した.ステロイド剤を外用したが改善しないため,紹介され受診した.右手背から前腕伸側に表面凹凸,虫食い状の硬い紅色局面を認め,乳頭状に隆起した小結節が多発していた.左前腕伸側に落屑を付着する紅斑を認めた.血中β-Dグルカンは,546.6pg/mlと高値であった.右手背の乳頭状結節および右前腕の局面の2ヶ所から生検し,真皮内に肉芽腫および多数の細胞浸潤を認め,Grocott染色陽性の菌糸を多数認めた.同部位の真菌培養では,灰黒色のコロニーを形成し,スライド培養では洋梨状で石垣状の多細胞からなる分生子が確認された.菌学的性状および遺伝子解析によりAlternaria alternataと同定し,深在性皮膚アルテルナリア症と診断した.イトラコナゾール200mg/日の内服を開始し,約半年で皮疹はほぼ平坦化した.自験例では,免疫不全を引き起こす合併症はないが,不適切なステロイド剤の長期外用により深在性皮膚真菌症の症状が遷延,増悪したと考えられた.(著者抄録)

Scedosporium prolificans (S. prolificans) is a type of mold, which rarely affects immunocompromised people. We treated a 71-year-old woman with acute myeloid leukemia (AML-M5a) with low-dose cytarabine, acralubicin, and filgrastim as the induction therapy. On day 7 after the initiation of chemotherapy, she became febrile and agranulocytic, and developed anal pain ; therefore, we discontinued the chemotherapy on day 8. Broad-spectrum antibiotics, micafungin, and then liposomal amphotericin B were ineffective. The serum concentration of β-D-glucan was 525 pg/mL. She died of multiple organ failure on day 17. S. prolificans was detected from the blood culture on day 13. Physicians should consider Scedosporium spp. infection when principal antifungal agents are ineffective and fungal infection is strongly suspected.

This report describes a case of Cryptococcus gattii VGIIb infection of the pulmonary and central nervous systems in an immunocompetent Japanese man with a travel history, and it hypothesizes the place where he was infected with C. gattii using the genotype information.

A 71-year-old man with interstitial pneumonia was hospitalized due to a pulmonary infection. He had been living in Thailand and had returned to Japan three months earlier. Antibiotic therapy initially cleared the infection; however, the patient's condition relapsed. Pseudomonas aeruginosa and Penicillium sp. were both detected in sputum and bronchial lavage fluid cultures and Penicillium sp. was identified to be P. marneffei. The infiltration observed on chest radiographs improved following treatment with itraconazole and tazobactam/piperacillin, and no relapse occurred. We herein report the first case of a non-HIV patient with P. marneffei infection in Japan.

Mucormycosis is an increasingly important cause of morbidity and mortality for patients with hematological malignancies. The diagnosis of mucormycosis usually requires mycological evidence through tissue biopsy or autopsy because the signs and symptoms are nonspecific and there are currently no biomarkers to identify the disease. We herein present two autopsied cases of acute myeloid leukemia with prolonged neutropenia who developed invasive mucormycosis accompanied by pulmonary artery embolism. Our cases were featured by unexplained fever and rapidly progressive dyspnea. Computed tomography scan detected nodular lesions or nonspecific consolidations in the lungs. Cultures, cytological study, and serum fungal markers consistently gave negative results. Autopsy revealed embolism of the pulmonary artery which consisted of fibrin clots by filamentous fungi. Genomic DNA was extracted from the paraffin-embedded clots and was applied to polymerase chain reaction amplification, leading to the diagnosis of infection by Rhizopus microsporus. We should carefully search for life-threatening pulmonary embolism when patients with hematological malignancies develop pulmonary mucormycosis.

The performance of a visual slide-based DNA microarray for the identification of non-albicans Candida spp. was evaluated. Among 167 isolates that had previously been identified by Vitek 2, the agreement between DNA microarray and sequencing results was 97.6%. This DNA microarray platform showed excellent performance.

76歳,男性.初診日1ヵ月前に屋外で転倒し左母指を受傷した.左母指の基部に辺縁が周堤状に隆起し,中央に潰瘍を伴う結節を認めた.潰瘍部を生検したところ,真皮間質にPAS染色陽性の淡紅色の円形構造物を多数認め,真菌培養によりCryptococcus neoformansを同定し,皮膚クリプトコッカス症と診断した.イトラコナゾール内服にて消長を繰り返しつつ徐々に軽快した.外傷を契機に発症したため,一見原発性を疑うが,胸部X線にて左下肺に陰影を認めたこと,単発性でなく外傷部以外にも紅斑局面を生じ,そこからも同じ菌が検出されたこと,病理組織像にて炎症細胞浸潤に乏しく,肉芽腫形成のないゼラチン状と称される所見であることなどより続発性と考えた.(著者抄録)

症例は50歳、男性。近医で慢性糸球体腎炎(人工透析)と間質性肺炎にて通院中であった。その後、間質性肺炎急性増悪にて同院より紹介された。入院直後からICU管理となり、人工呼吸管理、各種抗菌薬治療、ステロイドパルス療法、エンドキサンパルス療法を施行したが、肺炎像は増悪傾向を示した。その後、左中肺野に空洞性陰影を認め、アムホテリシンBを併用したが、第10病日に左の緊張性気胸を併発し永眠された。剖検にて左S3に穿破した空洞病変を認め、特徴的な病理所見から侵襲性肺ムーコル症と診断した。本症において、人工呼吸管理中に気胸を併発した場合、報告例は少ないが致死的となる可能性が高く、文献的考察を加え報告する。(著者抄録)

Although Cryptococcus gattii can cause life-threatening complications, putative virulence factors of C. gattii remain controversial. Therefore, we conducted the present study to elucidate the virulence factors of the yeast and found that the mortality rate of mice infected with C. gattii R265 was significantly higher than that of those infected with C. gattii 5815; however, no difference was found in the mortality rates between mice infected with C. gattii R265 and Cryptococcus neoformans H99. In contrast, we found a significant difference in histopathological findings of the lungs between mice infected with C. gattii R265 and C. neoformans H99. The former showed alveolar expansion due to yeast proliferation with much lesser macrophage response, whereas the latter showed numerous nodules in the alveolar space consisting of macrophages and multinucleated giant cells. Furthermore, alveolar expansion was more enhanced in mice infected with C. gattii R265 than in those infected with C. gattii 5815. Our study confirmed that there is a different pathophysiology leading to death during C. gattii and C. neoformans infections. The result can provide two characteristics of C. gattii: one includes some mechanisms to escape from host recognition via macrophage and another includes a high performance of pulmonary structural alteration. These characteristics may be associated with the high virulence of C. gattii.

症例は35歳、男性。2008年11月から2011年10月まで仕事のため米国アリゾナ州ツーソンに滞在。帰国後の検診にて胸部異常陰影(右下葉の結節影)を指摘された。気管支鏡検査を施行し、経気管支肺生検組織にて形態学的にコクシジオイデス症と診断。国立感染症研究所に移送した気管支肺胞洗浄液の培養から真菌コロニーを認め、その遺伝子解析の結果Coccidioides posadasiiと同定された。気管支鏡を用いて肺コクシジオイデス症と診断された症例は少ないため報告する。(著者抄録)

The fungal high osmolarity glycerol (HOG) pathway is composed of a two-component system (TCS) and Hog1-type mitogen-activated protein kinase (MAPK) cascade. A group III (Nik1-type) histidine kinase plays a major role in the HOG pathway of several filamentous fungi. In this study, we characterized a group III histidine kinase, NikA/TcsC, in the life-threatening pathogenic fungus, Aspergillus fumigatus. A deletion mutant of nikA showed low conidia production, abnormal hyphae, marked sensitivity to high osmolarity stresses, and resistance to cell wall perturbing reagents such as congo red and calcofluor white, as well as to fungicides such as fludioxonil, iprodione, and pyrrolnitrin. None of these phenotypes were observed in mutants of the SskA response regulator and SakA MAPK, which were thought to be downstream components of NikA. In contrast, in response to fludioxonil treatment, NikA was implicated in the phosphorylation of SakA MAPK and the transcriptional upregulation of catA, dprA, and dprB, which are regulated under the control of SakA. We then tested the idea that not only NikA, but also the other 13 histidine kinases play certain roles in the regulation of the HOG pathway. Interestingly, the expression of fos1, phkA, phkB, fhk5, and fhk6 increased by osmotic shock or fludioxonil treatment in a SakA-dependent manner. However, deletion mutants of the histidine kinases showed no significant defects in growth under the tested conditions. Collectively, although the signal transduction network related to NikA seems complicated, NikA plays a crucial role in several aspects of A. fumigatus physiology and, to a certain extent, modulates the HOG pathway.

Abstract Background Idiopathic pulmonary arterial hypertension (IPAH) continues to be one of the most serious intractable diseases that might start with activation of several triggers representing the genetic susceptibility of a patient. To elucidate what essentially contributes to the onset and progression of IPAH, we investigated factors playing an important role in IPAH by searching discrepant or controversial expression patterns between our murine model and those previously published for human IPAH. We employed the mouse model, which induced muscularization of pulmonary artery leading to hypertension by repeated intratracheal injection of Stachybotrys chartarum, a member of nonpathogenic and ubiquitous fungus in our envelopment. Methods Microarray assays with ontology and pathway analyses were performed with the lungs of mice. A comparison was made of the expression patterns of biological pathways between our model and those published for IPAH. Results Some pathways in our model showed the same expression patterns in IPAH, which included bone morphogenetic protein (BMP) signaling with down-regulation of BMP receptor type 2, activin-like kinase type 1, and endoglin. On the other hand, both Wnt/planar cell polarity (PCP) signaling and its downstream Rho/ROCK signaling were found alone to be activated in IPAH and not in our model. Conclusions Activation of Wnt/PCP signaling, in upstream positions of the pathway, found alone in lungs from end stage IPAH may play essential roles in the pathogenesis of the disease.

A man was admitted to our hospital with shortness of breath. He was involved in making wood chips from contaminated debris created by the tsunami that occurred after the Great East Japan Earthquake. Fungi detected at his home and workplace were possible inducers of hypersensitivity pneumonitis, but the absence of precipitating antibodies countered this diagnosis. His rapid and progressive clinical course and surgical lung biopsy and bronchoalveolar lavage findings suggested acute interstitial pneumonia. Electron probe X-ray microanalysis revealed the deposition of excessive exogenous substances in bronchiolar regions. Inhalation of harmful materials was suspected to be the cause of acute lung injury.

東日本大震災において津波肺からScedosporium属が分離された3症例を経験した。3症例とも津波による溺水後に病院に搬送された。症例1は,重症肺炎を発症した33歳の女性。喀痰からS.apiospermum(Pseudallescheria apiosperma)とS.prolificansが分離された。症例2は血痰と胸痛が出現した68歳の女性。喀痰からS.aurantiacumが分離された。症例3は,両肺野に浸潤影を認めた59歳の女性。気管支肺胞洗浄液からS.apiospermum(P.apiosperma)が分離された。血中(1→3)-β-D-グルカン濃度(pg/ml)は,それぞれ48.4,47.8,94.5と高値であった。スマトラ島沖地震後の溺水患者からScedosporium属が分離された報告を踏まえ,津波肺や溺水に伴う感染症では,スケドスポリウム症も念頭に置くことが重要である。(著者抄録)

Aspergillus udagawae and A. fumigatus share similar morphological features but they differ genetically. There is also an important clinical distinction as A. udagawae is less sensitive to amphotericin B than A. fumigatus. We encountered a rare case of bronchial infection due to A. udagawae that was successfully treated with voriconazole. An 82-year-old woman with diabetes mellitus complained of bloody sputum. Bronchoscopy revealed a white plugged region at the origin of the right bronchi B5. Cytological study revealed a clot composed of filamentous fungi and Aspergillus spp. was detected by culture. Molecular analysis revealed that the causative agent was A. udagawae, and voriconazole was used for the treatment. In comparison to A. fumigatus, the A. udagawae strain isolated in this case was less sensitive to amphotericin B, less virulent in immunosuppressed mice, and more sensitive to hydrogen peroxide, features that are almost identical to those of the previously reported isolates of the fungus. We should be aware of the emergence of new Aspergillus species that might pose a clinical threat.

In the respiratory field, chronic pulmonary aspergillosis, such as chronic necrotizing pulmonary aspergillosis (CNPA) or aspergilloma, is important. We examined the efficacy and safety of short- and long-term itraconazole (ITCZ) administration, involving a switch from injection to an oral preparation, in patients with CNPA. In all hospitals participating in this study, the protocol was approved by the ethics review board. This study started after UMIN registration (UMIN000001727). Subjects enrolled in this study were patients who were clinically or definitively diagnosed with CNPA in the respiratory field, according to the diagnostic criteria of the Japanese "Guidelines for management of deep-seated mycosis 2007," in 16 hospitals that participated in this study between May 2008 and March 2011. Treatment was started with ITCZ injection. Subsequently, the agent was switched to an oral preparation. Efficacy was evaluated with major items (clinical symptoms, fever, imaging findings) and minor items (nutritional status, inflammatory markers). Twenty-nine patients were enrolled; safety was evaluated in 24 and efficacy in 23. Of the 23 patients, 10 (43.5 %) responded. With respect to the administration period, the response rates in 8 patients treated for a short period and 15 treated for a long period were 25.0 % and 53.3 %, respectively. Trough blood concentration of ITCZ reached a level at which ITCZ may be effective for aspergillosis at 3 days after the start of ITCZ injection therapy. After changing to high-dose capsules, its level was also maintained. Adverse events such as liver dysfunction and heart failure were observed in 9 of the 24 patients. Furthermore, 6 patients died. However, there was no relationship between these events and ITCZ. Step-down therapy from ITCZ injection to oral administration may be a useful treatment option in CNPA patients requiring long-term treatment.

64歳女性。半年前より右頬部にそう痒を伴う皮疹が出現し、クロベタゾールプロピオン酸エステルを外用するも改善せず、著者らの施設へ受診となった。所見では右頬部に鱗屑と痂皮が付着し、隆起を伴う境界明瞭な浸潤性の紅斑局面が認められた。また、病理組織学的にはPAS染色やグロコット染色にて多数の菌糸がみられ、真菌培養では黒色真菌が検出された。以上、これらを踏まえ、遺伝子検査を行った結果、本症例はOchroconis humicola感染症と診断され、真菌による内臓病変がないことからイトラコナゾールの内服を開始した。だが、胃部不快感が出現したためテルビナフィンに変更したところ、1ヵ月で皮疹は徐々に改善した。その後、8ヵ月間はテルビナフィンの内服が継続されたが、内服終了後10ヵ月経過した現在、再燃はみられていない。

症例は42歳男性。米国から帰国後、健康診断の胸部X線にて左肺野の結節影を指摘された。1年後の胸部X線にてさらに左肺門リンパ節の腫脹を指摘され、埼玉県立循環器・呼吸器病センターを受診した。胸部CT検査では左肺の結節と左肺門・縦隔リンパ節の腫脹を認めた。結節に対し胸腔鏡下肺生検を施行した。酵母様真菌を認め、肺ヒストプラズマ症を疑った。血清抗ヒストプラズマ抗体陽性、摘出肺のpolymerase chain reaction法によりHistoplasma capsulatumの遺伝子配列と一致したことから、肺ヒストプラズマ症と診断した。縦隔リンパ節は経過、画像所見よりヒストプラズマ症による縦隔肉芽腫と考えられた。フルコナゾールの投与を開始し半年後には縦隔リンパ節の縮小を認めた。これまで我が国では、縦隔肉芽腫を合併した肺ヒストプラズマ症の報告がないため報告する。(著者抄録)

50歳代男。右下顎歯のぐらつきを自覚した。抜歯したが、創傷治癒不全および口内痛による食事摂取困難のために歯科へ入院した。このとき低Na血症が指摘され、補液によって改善した。再び食事摂取不能となり入院となった。口腔内びらん性病変の病理学的所見、血清学的所見よりパラコクシジオイデス症と診断した。血中コルチゾール低下、血中ACTH著増を認め、副腎機能不全による全身衰弱、低Na血症、食欲低下と診断した。貧血は小球性だが、フェリチンが増加していることおよび経口摂取が不良だったことより、慢性炎症と低栄養が原因と考えた。低Na血症に対して、Na負荷による補正を行った。全身衰弱や食欲低下といった症状はプレドニゾロン内服の開始ですみやかに改善した。入院22日目に軽快退院した。退院半年後、イトラコナゾール内服を継続して順調に軽快している。

Interstitial lung diseases (ILDs) represent a large group of different diseases, with a large part comprising idiopathic interstitial pneumonias. Differentiating hypersensitivity pneumonitis (HP), especially its chronic form and other ILDs, is difficult because of similarities in radiological manifestation and clinical course, and the difficulty of identifying causative antigens. We recently experienced a patient with Cladosporium-induced chronic HP that developed in a household environment, but the cause had been misdiagnosed as idiopathic interstitial pneumonia for several years. This case highlighted the need for measures differentiating HP from idiopathic interstitial pneumonia. In this study, we examined fungal exposure in ILDs using an antibody titer in serum to identify possible fungus-related HP. We measured the antibody titer to Cladosporium spp. in 34 patients with various ILDs, 17 patients with bronchial asthma, and 21 control subjects using an immunofluorescence assay. ILDs included HP (5 patients), idiopathic interstitial pneumonias (21 patients), and ILDs with collagen vascular diseases (8 patients). Results showed a significantly higher tendency for high anti-Cladosporium antibody titers in ILD groups (12 patients out of 34 patients), compared to patients with bronchial asthma (0/17) or control subjects (0/21). This increase in antibody titers was observed not only in patients with HP, but also in those with idiopathic interstitial pneumonias and those exhibiting collagen vascular diseases with ILDs. This report highlights the pathogenic role of fungal antigens in various ILDs. In conclusion, fungi commonly observed in our living environment such as Cladosporium could be involved in the development of ILDs.

86歳，男性．23年間関節リウマチにて加療され，8年前よりリウマチ性多発筋痛症の診断にてプレドニゾロン内服治療を開始し10mg/日で内服継続中である．約2か月前より右手背に腫瘤を生じ徐々に多発してきたため当科を受診した．右手背に母指頭大から鳩卵大のやや柔らかい膿瘍が多発しており，一部排膿していた．MRI所見にて骨破壊像を認めた．皮膚病理組織像ではPAS染色にて囊腫壁内にさまざまな形の淡褐色の菌糸と酵母様細胞を認め，硬壁細胞(sclerotic cell)や顆粒はみられなかった．以上より黒色菌糸症(phaeohyphomycosis)と診断した．また培養形態と塩基配列から本菌をExophiala jeanselmeiと同定した．骨破壊像があり外科的治療で根治が難しいと判断し手術を行わず排膿とイトラコナゾール内服を開始した．一時結節の新生を認めたため温熱療法を併用し病変の縮小を認めた．比較的まれな黒色菌糸症を経験したので報告する．

Aspergillus fumigatus is an all-important pathogenic fungus and is known for its angiotropism. When it invades human organs, A. fumigatus makes direct contact with blood and its components by causing inflammation and invading vascular structures. To learn the effect of its contact with blood on the development of infection, we examined the effect of serum on A. fumigatus growth. In Dulbecco's modified Eagle's medium containing 10% fetal bovine serum, hyphal tip growth was accelerated, forming a thickened and well-networked biofilm associated with extracellular matrix, and fetuin A was identified as the active component in the serum that accelerates fungal growth leading to formation of a community. These results suggest that fetuin A is a novel accelerator of the growth of A. fumigatus and that it participates in the formation of thick biofilm. (C) 2012 Elsevier GmbH. All rights reserved.

パラコクシジオイデス症は南米諸国にみられる真菌症であり、肺や皮膚粘膜に病変を作る。日本からの報告は南米長期滞在者に限られており、多くは難治性の口腔内潰瘍をきっかけに診断される。パラコクシジオイデス症の潰瘍性病変は口腔内悪性腫瘍と肉眼的に酷似している。南米での生活歴があり組織で肉芽腫性病変を認めた場合には、本症例も鑑別に挙げる必要がある。今回われわれは、口腔内潰瘍と肺病変を伴ったブラジル人男性のパラコクシジオイデス症例を経験したので報告する。(著者抄録)

Trichophyton tonsurans has been isolated among judo practitioners, wrestlers, and sumo wrestlers during an epidemic of tinea corporis and tinea capitis in Japan. A previous study using restriction fragment length polymorphism (RFLP) analysis of the non-transcribed spacer (NTS) region of the ribosomal RNA gene revealed different sources for the causative fungus in epidemics among judo practitioners and among wrestlers. Many different fungal strains have since been isolated from practitioners of these sports. The present study evaluated fungal characteristics of strains newly isolated between July 2006 and December 2010 using this molecular method. PCR-RFLP analysis using Mval and AvaI was performed on 263 strains, composed of 186 isolates from judo practitioners, 32 from wrestlers, 30 from sumo wrestlers, 5 from other sports, 7 from family members or friends of the sports practitioner patients, and 3 from sporadic (non-epidemic) cases. Four molecular types, NTS I, II, III, and VII were detected. Of these, NTS I was the most predominant, occurring in 243 of 263 strains (92.4%). All of the 30 strains isolated from sumo wrestlers were classified as NTS I, suggesting that the epidemic among sumo wrestlers originated from an earlier epidemic among judo practitioners. Thirteen strains were classified as NTS II; all were related to wrestling and were isolated mainly from the Chubu and Kansai areas in the central part of Honshu island. NTS III was detected in 6 strains, and one strain classified as NTS VII was isolated from a sporadic case of tinea capitis in a Peruvian immigrant. The minimum inhibitory concentrations (MICs) of terbinafine, itraconazole, fluconazole, and griseofulvin on 10 strains of NTS I and NTS II and 4 strains of NTS III were examined; there were no differences in MIC between these molecular types.

Reference methods for antifungal susceptibility tests recommend the use of conidia as inoculum. However, some isolates produce few conidia, while the invasive form of filamentous fungi in general is hyphae making susceptibility tests infeaseble. These facts suggest that other than conidia broth dilution method is required for susceptibility tests. The aim of this study was to clarify if the hyphal growth inhibition rate could be used as a method of determining the antifungal susceptibility of genus Microsporum. For this reason, a method which traces hyphal tips automatically and measures their growth rate was standardized for Microsporum spp. Control growth curves and test growth curves obtained by real-time observation of the hyphae groups responses to different concentrations of terbinafine, griseofulvin, and ciclopiroxolamine were used to compare with minimum inhibitory concentrations (MICs) obtained by conidia broth microdilution method. A visible reduction in the growth inhibition rate was observed when hyphal activity was evaluated using the third or fourth serial two-fold dilution below the MIC determined by broth microdilution for terbinafine and ciclopiroxolamine. For griseofulvin, this reduction occurred after the fifth dilution below the MIC. This study highlights the importance of the inoculum type used to determine the in vitro susceptibility of Microsporum strains. We conclude that measurement of hyphal growth inhibition, despite being time consuming, could be a suitable method for evaluating antifungal susceptibility, particularly for fungi as Microsporum spp. that produce a small (or not at all) number of conidia.

A 65-year-old Chinese man with diabetes mellitus was admitted to our hospital complaining of bloody sputum, fever, and dyspnea. Despite antibiotic treatment, his condition deteriorated, necessitating mechanical ventilation. Diffuse alveolar hemorrhage was suspected, and steroid therapy was initiated. Although his condition improved and he was extubated, the fever recurred twice, and on both occasions blood cultures yielded yeasts. The yeasts were misidentified as Cryptococcus humicola with a commercially available phenotype test (API ID32C), which did not match the clinical profile, and molecular identification was then performed. The isolates were identified as Candida intermedia by molecular phylogenetic analyses of the chromosomal regions coding for the D1/D2 domain of the large-subunit 26S rRNA gene. The patient responded well to several antifungal agents and was discharged on the 34th hospital day. To our knowledge, this is the first case of C. intermedia infection reported in Japan, and the tenth case reported in the international medical literature.

症例は気管支喘息に罹患している53歳男性である。胸部腫瘤影を指摘されて当センターを受診した。気管支鏡検査により好酸球、シャルコーライデン結晶、真菌を有する粘液栓と気管支壁への好酸球浸潤、及びスエヒロタケが証明され、喀痰からアスペルギルスが培養された。アスペルギルス特異的IgE、アスペルギルスとスエヒロタケに対する沈降抗体が陽性だったため、両者によるアレルギー性気管支肺真菌症と診断した。フルチカゾン/サルメテロール配合剤、イトラコナゾールを投与し、自覚症状、画像所見の改善を認めた。(著者抄録)

Stachybotrys chartarum, a ubiquitous fungus in our environment, has been suspected of causing respiratory symptoms in humans, such as acute infant pulmonary hemorrhage and asthma. We previously established a mouse model in which repeated inhalation of Stachybotrys chartarum spores caused pulmonary hypertension. To further investigate the model, particularly in the pulmonary circulation, mice were intra-tracheally injected with spores, 18 times over 12 weeks. Severe muscularization was observed in the small- to medium-sized pulmonary arteries. Bronchoalveolar lavage fluid revealed an increase in eosinophils accompanied by high concentrations of Th2-associated cytokines, IL-4, IL-5, but not Th1-associated IFN-gamma. The remodeling was temporary, resolving after cessation of spore inhalation. Chronic inhibition of the RhoA/Rho-kinase pathway by fasudil attenuated pulmonary arterial remodeling. These data suggest that Stachybotrys-mediated remodeling is caused by Th2-associated inflammation and can be resolved by Rho-kinase inhibition, either through direct effects on smooth muscle hypertrophy or through indirect effects on vascular inflammation. These data also show that extensive pulmonary vascular remodeling, often thought of as a fixed lesion, will spontaneously resolve in the absence of underlying molecular etiology.

クロモブラストミコーシス(CBM)の主要な原因菌種Fonsecaea属は、近年分子系統解析により再検討され、F.pedorosoi、F.monophora、F.nubicaの3菌種が提唱されている。今回、その原因菌の形態学的同定が困難であったが、系統解析によりF.monophoraと同定することができた慢性重症型CBMの症例を報告する。患者は55歳のフィリピン出身の男性。ココナッツ農園で働いていた1973年ごろ、左下腿に小紅斑が出現。1999年に下腿部から大腿部にかけて皮疹が多発し、同年から2005年まで母国で治療し略治。2005年来日後、下腿の皮疹が再燃し、イトラコナゾールの内服を再開したが悪化してきたため当科を受診。2008年10月初診時、左下腿部に瘢痕型病変と周囲に腫瘤型病変を伴っていた。その病変の上部から大腿にかけて、多数の斑状瘢痕を認めた。腫瘤性病変からmuriform cellを確認、黒色真菌を分離した。集落は、はじめ炭粉状、後に短絨毛様となった。本分離株は顕微鏡所見による菌種同定が困難なため、リボソームRNA遺伝子internal transcribed spacer領域の塩基配列を解析し、F.monophora(DDBJ accession number AB566420)と同定した。治療はボリコナゾール内服とカイロによる温熱療法を併用し、6ヵ月後には腫瘤性病変は消退し、1年6ヵ月で瘢痕様紅斑を残し内服治療を終了した。瘢痕型は薬剤の到達が難しいため再発しやすく、治癒判定も難しいため、今後も経過観察が必要である。(著者抄録)

Previously, we reported that epigallocatechin 3-O-gallate (EGCg) has growth-inhibitory effect on clinical isolates of Candida species. In this study, we investigated the antifungal activity of EGCg and antifungal agents against thirty-five of dermatophytes clinically isolated by the international guidelines (M38-A2). All isolates exhibited good susceptibility to EGCg (MIC(50), 2-4 mu g/mL, MIC(90), 4-8 mu g/mL, and geometric mean (GM) MICs, 3.36-4 mu g/mL) than those of fluconazole (MIC(50), 2-16 mu g/mL, MIC(90), 4-32 mu g/mL, and GM MICs, 3.45-25.8 mu g/mL) and flucytosin (MIC(50), MIC(90), and GM MICs, > 64 mu g/mL), although they were less susceptible to other antifungal agents, such as amphotericin B, itraconazole, and miconazole. These activities of EGCg were approximately 4-fold higher than those of fluconazole, and were 4 to 16-fold higher than flucytoin. This result indicates that EGCg can inhibit pathogenic dermatophyte species. Therefore, we suggest that EGCg may be effectively used solely as a possible agent or combined with other antifungal agents for antifungal therapy in dermatophytosis.

Background: Group 1 introns (ribozymes) are among the most ancient and have the broadest phylogenetic distribution among the known self-splicing ribozymes. Fungi are known to be rich in rDNA group 1 introns. In the present study, five sequences of the 28S ribosomal RNA gene (rDNA) regions of pathogenic dematiaceous Phialophora verrucosa were analyzed using PCR by site-specific primers and were found to have three insertions, termed intron-F, G and H, at three positions of the gene. We investigated the distribution of group 1 introns in this fungus by surveying 34 strains of P. verrucosa and seven strains of Phialophora americana as the allied species. Results: Intron-F's (inserted at L798 position) were found in 88% of P. verrucosa strains, while intron-G's (inserted at L1921) at 12% and intron-H's (inserted at L2563) at 18%. There was some correlation between intron distribution and geographic location. In addition, we confirmed that the three kinds of introns are group 1 introns from results of BLAST search, alignment analysis and Reverse Transcriptase-Polymerase Chain Reaction (RT-PCR). Prediction of secondary structures and phylogenetic analysis of intron sequences identified introns-F and G as belonging to subgroup IC1. In addition, intron-H was identified as IE. Conclusion: The three intron insertions and their insertion position in the 28S rDNA allowed the characterization of the clinical and environmental isolates of P. verrucosa and P. americana into five genotypes. All subgroups of introns-F and G and intron-H were characterized and observed for the first time in both species.

症例は71歳男性。2008年5月に発熱、咳嗽を主訴に紹介受診した。胸部単純写真にて左肺に空洞形成と内部の菌塊と思われる構造を認め、気管支肺胞洗浄液及びCTガイド下生検検体よりScedosporium apiospermumが検出されたことから肺スケドスポリウム症と診断した。ボリコナゾール(VRCZ)200mg/日内服による治療を2ヵ月間行なったが症状やレントゲン陰影の改善はみられなかった。同薬剤の血中濃度はピーク値2.15μg/ml、トラフ値0.72μg/mlであったため、血中濃度が十分でないと判断し、400mg/日に増量したところ、血中濃度はピーク値5.11μg/ml、トラフ値3.13μg/mlまで上昇した。投与量増量後より症状及びレントゲン陰影の明らかな改善がみられ、6ヵ月間の内服で治癒した。難治性の肺スケドスポリウム症にはVRCZの血中濃度測定が有効と考え報告する。(著者抄録)

Fonsecaea species are major etiologic agents of Chromoblastomycosis(CBM). By genetic analysis, the genus Fonsecaea has recently been revised and classified into F. pedorosoi, F. monophora and F. nubica. Here we report a severe chronic case of CBM caused by F. monophora. A 55-year-old Filipino male developed progressive skin lesions on the left lateral ankle in 1973, when he worked at a coconut plantation in the Philippines. In 1999, he received medical treatments for enlarged, multiple lesions on the left lower limb. When he moved to Japan in 2005, the lesions were remarkably improved and he discontinued taking the medicine. On our first examination in October 2008, a large, reddish, cicatricial plaque was observed on the left lower aspect of his leg. Several tumorous lesions surrounded the plaque, indicating that the therapies performed before had been insufficient. In addition, there were many patchy scars scattered on the thigh and the upper part of the lower leg. The diagnosis of CBM was made by the presence of muriform cells. Black, pulverulent colonies were yielded in culture of skin scrapings and tissues. Although the fungus could not be identified by microscopic morphology, r-RNA ITS sequence analysis enabled identification of Fonsecaea monophora. The patient responded well to oral voriconazole combined with local thermotherapy using pocket warmers. The tumoral masses subsided in 6 months, leaving pink scars with negative fungal culture. Voriconazole treatment was continued for 18 months. It seems that drugs are insufficiently delivered in the cicatricial lesions because of the paucity of blood flow, suggesting that a long-term follow-up is necessary for such a case.