亀井 克彦

One of the main mechanisms of azole resistance of Aspergillus fumigatus is thought to be a reduction in the drug's affinity for the target molecule, Cyp51A, due to its amino acid mutation(s). It is known that the azole resistance pattern is closely related to the mutation site(s) of the molecule. In this study, we tried to develop a simple and rapid detection method for cyp51A mutations using the endonuclease Surveyor nuclease. The Surveyor nuclease assay was verified using several azole-resistant strains of A. fumigatus that possess point mutations in Cyp51A. For validation of the Surveyor nuclease assay, blind tests were conducted using 48 strains of A. fumigatus (17 azole-resistant and 31 azole-susceptible strains). The Surveyor nuclease assay could rapidly detect cyp51A mutations with one primer set. Also, all the tested strains harboring different cyp51A single point mutations could be clearly distinguished from the wild type. The Surveyor nuclease assay is a simple method that can detect cyp51A mutations rapidly.

We report a fatal case of invasive pulmonary aspergillosis caused by voriconazole-resistant Aspergillus tubingensis in a patient with solid tumor but without severe immunosuppression. A high index of suspicion for invasive pulmonary aspergillosis, coupled with molecular identification of species and resistance genes, may facilitate early initiation of appropriate treatment.

Histoplasmosis, a fungal infection caused by Histoplasma capsulatum, is poor prognosis once it disseminated, especially in immunocompromised patients. A 50-year-old Japanese-Brazilian male with multiple cervical lymphadenopathies was diagnosed as disseminated histoplasmosis and acquired immunodeficiency syndrome (AIDS). Anti-fungal therapy was initiated followed by anti-retroviral therapy (ART). He achieved long-term remission by treatment with voriconazole. Here we report a case of an AIDS patient with disseminated histoplasmosis who achieved long-term survival in non-endemic area.

The pathogenic fungus Aspergillus fumigatus contains galactomannans localized on the surface layer of its cell walls, which are involved in various biological processes. Galactomannans comprise α-(1→2)-/α-(1→6)-mannan and β-(1→5)-/β-(1→6)-galactofuranosyl chains. We previously revealed that GfsA is a β-galactofuranoside β-(1→5)-galactofuranosyltransferase involved in the biosynthesis of β-(1→5)-galactofuranosyl chains. In this study, we clarified the biosynthesis of β-(1→5)-galactofuranosyl chains in A. fumigatus Two paralogs exist within A. fumigatus: GfsB and GfsC. We show that GfsB and GfsC, in addition to GfsA, are β-galactofuranoside β-(1→5)-galactofuranosyltransferases by biochemical and genetic analyses. GfsA, GfsB, and GfsC can synthesize β-(1→5)-galactofuranosyl oligomers at up to lengths of 7, 3, and 5 galactofuranoses within an established in vitro highly efficient assay of galactofuranosyltransferase activity. Structural analyses of galactomannans extracted from ΔgfsB, ΔgfsC, ΔgfsAC, and ΔgfsABC strains revealed that GfsA and GfsC synthesized all β-(1→5)-galactofuranosyl residues of fungal-type and O-mannose-type galactomannans and that GfsB exhibited limited function in A. fumigatus The loss of β-(1→5)-galactofuranosyl residues decreased the hyphal growth rate and conidium formation ability and increased the abnormal hyphal branching structure and cell surface hydrophobicity, but this loss is dispensable for sensitivity to antifungal agents and virulence toward immunocompromised mice.IMPORTANCE β-(1→5)-Galactofuranosyl residues are widely distributed in the subphylum Pezizomycotina of the phylum Ascomycota. Pezizomycotina includes many plant and animal pathogens. Although the structure of β-(1→5)-galactofuranosyl residues of galactomannans in filamentous fungi was discovered long ago, it remains unclear which enzyme is responsible for biosynthesis of this glycan. Fungal cell wall formation processes are complicated, and information concerning glycosyltransferases is essential for understanding them. In this study, we showed that GfsA and GfsC are responsible for the biosynthesis of all β-(1→5)-galactofuranosyl residues of fungal-type and O-mannose-type galactomannans. The data presented here indicate that β-(1→5)-galactofuranosyl residues are involved in cell growth, conidiation, polarity, and cell surface hydrophobicity. Our new understanding of β-(1→5)-galactofuranosyl residue biosynthesis provides important novel insights into the formation of the complex cell wall structure and the virulence of the members of the subphylum Pezizomycotina.

BACKGROUND: Chronic rhinosinusitis with nasal polyps (CRSwNP) is a heterogeneous disease, and its pathogenesis remains controversial. This study aimed to examine the involvement of fungi in CRSwNP pathogenesis. METHODS: We enrolled 29 controls and 111 CRSwNP patients. We analyzed fungi in the nasal secretions, serum fungus-specific immunoglobulin E (IgE) levels, and nasal polyp (NP) IgE levels. Moreover, we evaluated the correlation between patients' IgE levels and computed tomography (CT) scores. RESULTS: There was no difference in fungal detection rate between CRSwNP patients with and without asthma. Specific IgEs against various antigens were highly detectable in NPs of CRSwNP patients. In CRSwNP patients, fungus-specific IgE levels in NPs were correlated with CT scores. Serum fungus-specific IgEs became undetectable after operation in more than half of the CRSwNP patients without asthma but not in those with asthma. Other serum airborne antigen-specific IgEs did not become undetectable after operation. CONCLUSIONS: Fungus-specific IgEs were highly detectable in NPs of CRSwNP patients, and NPs comprised a major region of specific IgE production. Fungi may therefore play an important role in CRSwNP pathogenesis by inducing Th2 immune responses, including IgE synthesis.

症例は46歳女性。中米を拠点として中南米で勤務している。健診で胸部異常陰影を指摘され、CT検査で右肺下葉に辺縁明瞭な2cmの小結節が認められた。現地でCTガイド下生検術を施行されるも悪性所見は認めず、抗酸菌、真菌の感染所見も認めなかった。精査目的で帰国後、前医で気管支鏡検査を施行されるも壊死のみで診断がつかず、肺生検目的で当科に紹介となった。胸腔鏡下右下葉部分切除術を施行し、術中迅速検査にて悪性所見は認めず、炎症性肉芽腫の診断であった。検体は黄白色調、凝固壊死を伴った類上皮肉芽腫であり、病理診断では抗酸菌染色は陰性、Grocott染色で類円形、楕円形の酵母様真菌が認められた。術後血清H.capsulatum陽性を確認し、肺ヒストプラズマ症と診断した。免疫正常者であり、無症状であることからIDSAガイドラインに則り、経過観察となった。術後20ヵ月現在、感染の再燃なく経過している。(著者抄録)

Aspergillosis is a rare fungal infection in newborns, and its morbidity and mortality are high. Voriconazole (VRCZ) is the first-line antifungal agent for invasive Aspergillus infection, but little data is available about its pharmacokinetics in infants. We report a case of a premature infant who developed ventriculitis due to Aspergillus fumigatus and received combination antifungal therapy including VRCZ. β-D glucan and Aspergillus antigen index were elevated in the cerebrospinal fluid (CSF). We titrated the dose of VRCZ by monitoring plasma and CSF concentrations. The CSF to plasma concentration ratio of VRCZ ranged from 0.47 to 1.36 (median 0.71). While VRCZ adequately penetrates the blood-brain barrier, its concentration is highly variable in infants.

Schizophyllum commune is a ubiquitous basidiomycetous fungus typically found across the world, which has been detected in indoor and outdoor air. Some studies indicated that sensitization to S. commune is correlated with asthma severity in patients. Patients with chronic severe or acute fatal asthma have neutrophil-dominant airway inflammation. We hypothesized that S. commune can exacerbate asthma. To test this hypothesis, we evaluated the direct immunomodulatory activities of S. commune in allergic airway inflammation induced by non-fungal sensitization. Ovalbumin (OVA)-induced asthma model mice were generated using wild-type (WT) and Il-17a-/-Il-17f-/- mice that were intratracheally exposed to S. commune, then immune responses in the lungs were assessed after 24 h. Intratracheal administration of S. commune in OVA-induced asthma model mice enhanced neutrophilic airway inflammation, increased the mRNA expression of CXCL1 and CXCL2 in the lungs, and provoked IL-17A, and IL-17F production in BAL fluid. In addition, neutrophilic airway inflammation was significantly inhibited in Il-17a-/-Il-17f-/- mice compared with those found in WT mice. We demonstrated that S. commune induces neutrophilic airway inflammation in OVA-induced asthma model mice, and IL-17A and IL-17F had central roles in this activity. As S. commune inhabits the general environment, including indoor and outdoor air, our results suggested that S. commune is a causative agent of asthma exacerbation. This study has provided clues regarding the mechanisms behind fungi and asthma exacerbation.

A 42-year-old man with asthma presented in 2007 with chest infiltration and productive cough. Pycnoporus sanguineus and Perenniporia tephropora were repeatedly isolated from sputum and bronchial washing fluids. Because we lacked immunologic evidence, we could not diagnose him with allergic bronchopulmonary mycosis (ABPM) due to these basidiomycetous fungi. At that time, serum-specific IgE and IgG against Schizophyllum commune findings were negative. Inhaled beclomethasone/salmeterol improved his condition. Seven years later, mucous plugs obtained via bronchoscopy at a relapse were compatible with allergic mucin. Because S. commune was isolated from mucous plugs and serum-specific IgG against S. commune turned positive, we diagnosed the patient with ABPM due to S. commune.

Trichophyton verrucosum(T.verrucosum)感染症は,主にウシからヒトへと接触感染する人畜共通感染症として知られ,ヒトに感染した場合には一時的に激しい皮膚症状を来すことがある。小膿疱が多発融合して隆起し,中心治癒傾向の乏しい浸潤性紅斑局面をとることもあり,診断に苦慮する例も多い。今回,診断が遅れた結果,ステロイド外用剤により難治となった,いわゆる生毛部急性深在性白癬と小児のケルスス禿瘡を経験したので報告する。症例1:50歳,男性。畜産農家。左前腕伸側に小膿疱を伴う暗赤色の紅斑性局面を認めた。痂皮のKOH標本にて分枝した糸状菌を認めたため,いわゆる生毛部急性深在性白癬と診断し,イトラコナゾール(以下ITCZ) 125mg/日の内服とネチコナゾール塩酸塩(以下NCZ)の外用を約9週間行い軽快した。症例2:7歳,男児。自宅の流し台の棚の開き戸で頭部を受傷し,生じた潰瘍は難治で排膿を認めた。母子ともにウシとの直接的な接触歴はないが,母親の勤務先には牛舎があった。病変部の毛髪のKOH標本にて菌糸,胞子を認めたため,ケルスス禿瘡と診断し,テルビナフィン塩酸塩60mg/日の内服,NCZの外用にて加療を約4週間継続したが,効果が乏しく,ITCZ 150mg/日の内服と白色ワセリン外用を約16週間継続し軽快した。形態学的特徴と遺伝子解析にて症例1,2ともに原因菌はT.verrucosumと同定した。(著者抄録)

An otherwise healthy 3-year-old girl presented with a several-month history of scaly lesions on her palms and soles. The lesions on the palms and right sole had been successfully treated with a steroid for pompholyx by a nearby dermatology clinic, but the lesion on the left sole persisted and spread to the back of the foot. On the initial visit, the patient exhibited an itchy and scaly erythematous left foot lesion. Direct microscopic examination of the scales revealed a considerable amount of fungal elements. A diagnosis of tinea pedis was made, and antifungal treatment with a neticonazole ointment was initiated. Complete cure was achieved after 4 weeks of treatment. The primary mycological cultures from the scales simultaneously revealed two types of colonies: a white powdery flat colony and a white downy elevated colony with a reddish-yellow bottom. Although the powdery colony was identified as Trichophyton mentagrophytes complex on slide culture, the downy colonies could not be identified based on cultural and morphological characteristics. The nucleotide sequences of the internal transcribed spacer region from both colonies showed an exact match, which eventually led to their identification as Trichophyton interdigitale. Further genotyping at three points in the non-transcribed spacer region in both colonies also showed the same NTS type of D2II. It is very rare for two morphologically different colonies to be isolated from the primary culture under the same conditions in tinea cases. Genetic tests are of extreme value to identify the strain in such cases.

Annulohypoxylon sp.による趾間黒癬(右第2・3・4趾間),足爪黒癬(右第4趾)の93歳,男性例を報告する。臨床像は炎症のない点状,線状,斑状の黒褐色斑で,Hortaea werneckiiによる報告例よりも黒色調が強かった。再発時の爪甲病変のダーマスコピーでは淡褐色の線状色素斑がモザイク状にみられた。趾間と爪甲の直接鏡検で多数の黒褐色菌糸を認めた。趾間鱗屑の培養では,コロニーは継代の度に黒色調から白色絨毛状になった。スライドカルチャーでは,菌糸のみで胞子,分生子の発育はなかった。Hortaea werneckiiや,ベネズエラ固有のStenella araguataは否定された。培養検体からのITS〜D1/D2領域の遺伝子解析により,Annulohypoxylon sp.と同定された。植物腐敗菌の菌種のヒトへの感染例はない。明らかに黒癬の臨床像を呈し,Annulohypoxylon sp.のみが分離培養できたのでコンタミネーションは否定した。居住環境からの感染と推察された。自験例はAnnulohypoxylon sp.による黒癬の世界第1例である。抗真菌剤の外用療法では趾間,爪甲ともに再燃がみられたので,この菌種による黒癬は難治な疾患として対応すべき可能性が示唆された。(著者抄録)

BACKGROUND: Botrytis species are well known fungal pathogens of various plants but have not been reported as human pathogens, except as allergenic precipitants of asthma and hypersensitivity pneumonitis. CASE PRESENTATION: The asymptomatic patient was referred because of a nodule revealed by chest X-ray. Computed tomography (CT) showed a cavitary nodule in the right upper lobe of the lung. He underwent wedge resection of the nodule, which revealed necrotizing granulomas and a fungus ball containing Y-shaped filamentous fungi, which was confirmed histopathologically. Culture of the specimen yielded white to grayish cotton-like colonies with black sclerotia. We performed multilocus gene sequence analyses including three single-copy nuclear DNA genes encoding glyceraldehyde-3-phosphate dehydrogenase, heat-shock protein 60, and DNA-dependent RNA polymerase subunit II. The analyses revealed that the isolate was most similar to Botrytis elliptica. To date, the pulmonary Botrytis sp. infection has not recurred after lung resection and the patient did not require any additional medication. CONCLUSIONS: We report the first case of an immunocompetent patient with pulmonary Botrytis sp. infection, which has not recurred after lung resection without any additional medication. Precise evaluation is necessary for the diagnosis of pulmonary Botrytis infection because it is indistinguishable from other filamentous fungi both radiologically and by histopathology. The etiology and pathophysiology of pulmonary Botrytis infection remains unclear. Further accumulation and analysis of Botrytis cases is warranted.

73歳男性。四肢の浮腫を主訴に前医を受診、胸部X線で心不全を指摘され、精査加療目的で当院の循環器内科へ紹介となった。入院時、胸部X線で両側胸水貯留と心拡大を認め、12誘導心電図では完全房室ブロックが認められた。右内頸静脈に中心静脈カテーテルを留置し、硝酸剤、降圧剤、利尿薬の投与後、完全房室ブロックに対し恒久式ペースメーカー植え込み術が施行された。だが、第3病日目に発熱を認めたため、ABPC/SBTが投与されたが、第8病日目の血液培養、第12病日目の抜去した中心静脈カテーテル先端からは酵母様真菌が認められた。そこで、第21病日目に一時的ペースメーカーの交換が行われるも、血液培養は陽性を持続し、F-FLCZやVCMの追加も効果なく、患者は第52病日目に死亡となった。その後、血液培養および遺伝子解析にてYarrowia lipolyticaが同定され、本症例は同菌によるカテーテル関連血流感染症と確定診断された。

Aspergillus fumigatus is the commonest cause of pulmonary aspergillosis; however, a recently developed molecular genetic technique identified A. lentulus as a sibling species. Most of the isolates were found in solid organ recipients, often associated with a fatal outcome. Moreover, there is concern that A. lentulus has low susceptibility to multiple antifungal agents. Herein, we report an adult immunocompromised patient with proven invasive pulmonary aspergillosis (IPA) caused by A. lentulus, which was identified through molecular genetic analysis. The patient was diagnosed with IPA by bronchoscopy 3 weeks after initiating systemic corticosteroid therapy for anti-neutrophil cytoplasmic antibody-associated vasculitis. The clinical course of IPA due to A. lentulus showed improvement after treatment with the antifungal agent voriconazole. In summary, we report an adult immunocompromised patient without a history of transplantation who was diagnosed with IPA due to A. lentulus successfully treated with voriconazole, and we also report the findings of a literature review on IPA caused by A. lentulus.

＜文献概要＞64歳,女性.初診の3年前に左2指爪基部の色調変化を自覚した.近医で爪白癬と診断され,抗真菌薬内服および外用による治療を受けたが改善しなかった.初診時,左2指爪甲は肥厚し,黒色-灰白色に混濁し,後爪部の腫脹・発赤を伴っていた.KOH直接検鏡法で菌糸が陽性,爪甲の真菌培養では2回の培養で同様の形態の菌が分離された.分離菌のコロニーは表面羊毛状で淡ピンク,裏面は黄白色調を示し,検鏡でFusarium属に特徴的な鎌状の大分生子を認めた.分離菌はリボソームRNA遺伝子のITS領域およびEF1-alpha遺伝子の塩基配列よりFusarium verticillioidesと同定した.現在ルリコナゾール爪外用液で治療し,爪囲炎は消失し,爪甲は平坦化,色調変化も改善傾向にある.自験例のような爪囲炎を伴う手爪1ヶ所の病変は,爪白癬として非典型的である.このような症例ではFusarium属をはじめとする非白癬性爪真菌症を疑う必要がある.

＜文献概要＞症例のポイント ・Sporothrix globosa(S. globosa)による播種型スポロトリコーシスを経験した.・免疫異常や基礎疾患のない患者に生じた播種型スポロトリコーシスの本邦邦文報告例はこれまでになく,まれな症例と思われた.

71歳男性、56歳女性、68歳男性、73歳男性のスポロトリコーシスを報告する。第3・4例は遺伝子解析でS.globosaと同定した。第4例はイトラコナゾール(ITCZ)が奏効せずヨウ化カリウム(KI)に変更後病変を全摘した。他はKIが奏功した。遺伝子解析から本邦での起因菌はほぼS.globosa(37℃で発育せずITCZ低感受性)でKIや温熱療法が適することが裏付けられた。皮膚科医にも最新の真菌学的知見や遺伝子学的検査を活用することが求められ始めている。(著者抄録)

Chronic granulomatous disease (CGD) is a type of primary immunodeficiency disease, which increases susceptibility to recurrent bacterial and fungal infections. Sputum and bronchoalveolar lavage fluid are often obtained using bronchoscopy from adult patients for pathogenic diagnosis, although this approach is much more invasive for infants. We report the case of a 2-month-old boy with CGD, in which gastric aspirate culture was used to diagnose fungal pneumonia. Rasamsonia piperina was isolated from the gastric aspirate, and the patient was successfully treated with micafungin based on the drug susceptibility test results for the fungal isolate. The acid tolerance test revealed that R. piperina could grow at pH 2, indicating high acid resistance. Although we can only report our experience with a single case, gastric aspirate culture may be a useful tool for detecting fungal respiratory pathogens in children with primary immunodeficiency. Detecting these pathogens may help improve outcomes, as early diagnosis and appropriate treatment are extremely important for immunocompromised patients with respiratory infections.

RATIONALE: Histoplasmosis occurs most commonly in Northern and Central America and Southeast Asia. Increased international travel in Japan has led to a few annual reports of imported histoplasmosis. Healed sites of histoplasmosis lung infection may remain as nodules and are often accompanied by calcification. Previous studies in endemic areas supported the hypothesis that new infection/reinfection, rather than reactivation, is the main etiology of symptomatic histoplasmosis. No previous reports have presented clinical evidence of reactivation. PATIENT CONCERNS: An 83-year-old Japanese man was hospitalized with general fatigue and high fever. He had been treated with prednisolone at 13 mg/d for 7 years because of an eczematous skin disease. He had a history of travel to Los Angeles, Egypt, and Malaysia 10 to 15 years prior to admission. Five years earlier, computed tomography (CT) identified a solitary calcified nodule in the left lingual lung segment. The nodule size remained unchanged throughout a 5-year observation period. Upon admission, his respiratory condition remained stable while breathing room air. CT revealed small, randomly distributed nodular shadows in the bilateral lungs, in addition to the solitary nodule. DIAGNOSIS: Disseminated histoplasmosis, based on fungal staining and cultures of autopsy specimens. INTERVENTIONS: The patient's fever continued despite several days of treatment with meropenem, minocycline, and micafungin. Although he refused bone marrow aspiration, isoniazid, rifampicin, ethambutol, and prednisolone were administered for a tentative diagnosis of miliary tuberculosis. OUTCOMES: His fever persisted, and a laboratory examination indicated severe thrombocytopenia with disseminated intravascular coagulation. He died on day 43 postadmission. During autopsy, the fungal burden was noted to be higher in the calcified nodule than in the disseminated nodules of the lung, suggesting a pathogenesis involving endogenous reactivation of the nodule and subsequent hematogenous and lymphatic spread. LESSONS: Physicians should consider histoplasmosis in patients with calcified nodules because the infection may reactivate during long-term corticosteroid therapy.

Aspergillus fumigatus, a filamentous fungus that is ubiquitous in the environment, causes several human pulmonary disorders, including chronic and acute invasive infections and allergic diseases. Lysin motif (LysM) is a small protein domain that binds chitin, a major component of fungal cell wall polysaccharides. Several secreted LysM-domain proteins without catalytic function (LysM effectors) have been identified. They act as virulence factors in plant pathogenic fungi by preventing the immune response induced by chitin; however, LysM proteins in mammalian pathogenic fungi remain largely unexplored. We describe two novel LysM-domain proteins, LdpA and LdpB, in A. fumigatus. Functional analyses of single and double knockouts revealed no significant effects on cell wall chitin content, cell wall integrity, fungal morphology and fungal growth. Fluorescent signals from LdpA-green fluorescent protein (GFP) and LdpB-GFP were observed in cell wall and extracellular matrix. In a mouse model of invasive pulmonary aspergillosis, survival did not differ between ΔldpA/B and wild-type infection; however, further studies are required to reveal their functions in fungal-host interactions.

Aspergillus flavus is an important zoonotic pathogen and a well-known aflatoxin producer. Aspergillus flavus strains that are prevalent in Japanese environments are reported to be non-aflatoxigenic, although their aflatoxin productivity, especially among clinical isolates, has not been thoroughly investigated to date. In this study, we sequenced the genomes of ten strains of A. flavus isolated in Japan and compared their sequences with each other as well as with those of Aspergillus oryzae RIB40 and A. flavus NRRL3357. The phylogenetic analysis based on identified SNPs indicated that five strains were closer to A. oryzae RIB40 than to A. flavus NRRL3357. In contrast, of those isolates that were closer to A. flavus NRRL3357 than to A. oryzae RIB40, three were found to possess either the entire or partial aflatoxin biosynthesis gene cluster of NRRL3357-type. Furthermore, two of the three actually produced either aflatoxin B1 or an intermediate of the reaction leading to aflatoxin formation. Three of the ten strains we isolated were identified to possess part of the aflatoxin gene cluster, while five others retained the A. oryzae RIB40-type cluster. The genome data thus obtained may be further explored and utilized for comparative analysis of aflatoxin production in environmental and clinical isolates of A. flavus.

Aspergillus fumigatus is an airborne fungal pathogen that causes severe infections with invasive growth in immunocompromised patients. Several mycoviruses have recently been isolated from A. fumigatus strains, but there are presently no reports of mycoviral-mediated reduction or elimination of fungal pathogenicity in vertebrate models. Here, we report the biological features of a novel mycovirus, A. fumigatus chrysovirus 41362 (AfuCV41362), isolated from the hypovirulent A. fumigatus strain IFM 41362. The AfuCV41362 genome is comprised of four dsRNAs, each of which contains a single ORF (ORF1-4). ORF1 encodes a protein with sequence similarity to RNA-dependent RNA polymerases of viruses in the family Chrysoviridae, while ORF3 encodes a putative capsid protein. Viral RNAs are expressed primarily during the germination stage, and RNA-seq analysis of virus-infected A. fumigatus at the germination stage suggested that the virus suppressed expression of several pathogenicity-associated host genes, including hypoxia adaptation and nitric oxide detoxification genes. In vitro functional analysis revealed that the virus-infected strain had reduced tolerance to environmental stressors. Virus-infected A. fumigatus strain IFM 41362 had reduced virulence in vivo compared to the virus-free strain in a mouse infection model. Furthermore, introduction of the mycovirus to a natively virus-free KU A. fumigatus strain induced virus-infected phenotypes. To identify mycovirus genes responsible for the reduced virulence of A. fumigatus, each viral ORF was ectopically expressed in the virus-free KU strain. Ectopic expression of the individual ORFs only nominally reduced virulence of the host fungus in a mouse infection model. However, we found that ORF3 and ORF4 reduced tolerance to environmental stresses in in vitro analysis. Based on these results, we suggest that the AfuCV41362 mycovirus ORF3 and ORF4 reduce fungal virulence by suppressing stress tolerance together with other viral genes, rather than alone.

マルネッフェイ型ペニシリウム症は,東南アジアを中心に分布する真菌Talaromyces marneffeiを原因とし,免疫機能が低下した易感染性宿主に発症することが多い.今回,われわれはHuman Immunodeficiency Virus(HIV)感染を背景とした輸入真菌症として本症を経験した.症例は20歳代ベトナム国籍の男性で,発熱・腹痛・皮疹にて近医を受診しHIVスクリーニング検査で陽性と判明した.さらに吐血・下血を認めたため当院へ紹介入院となった.来院時の上部消化管内視鏡検査で食道カンジダ症,出血性胃炎を認めた.血液培養が好気ボトルにて約48時間後に陽性となり,鏡検にて糸状菌を確認した.クロムアガーカンジダ培地にて28℃で3日間サブカルチャーしたところ,深紅色素の拡散を伴うアスペルギルス様の比較的大きな灰白色のコロニーを認めた.免疫血清学的検査でβ-D-グルカンが高値であったため真菌症を疑いアムホテリシンBリポソーム製剤(L-AMB)を2週間投与,その後イトラコナゾール(ITCZ)に変更し軽快した.退院後,β-tubulin遺伝子の塩基配列による検査でT.marneffeiと同定された.本症例はHIV感染により免疫力の低下が進行,全身性播種性マルネッフェイ型ペニシリウム症と考えられた.今後は海外交流の増加に伴い輸入真菌症に遭遇する可能性が高くなる.まれな輸入感染症を想定した診療・検査体制および院内感染制御対策が必要と考える.(著者抄録)

Kodamaea ohmeri is a relatively rare yeast isolated form clinical specimens, and it is known to be a causative fungus of severe invasive infectious diseases in immunocompromised hosts. Herein, we describe fungemia due to K. ohmeri in a patient with a severe extended burn. The isolate was obtained from not only blood specimens but also skin lesions. We should be aware of risk for fungemia including K. ohmeri in case of severe burn.

We designed primers and cycling probes to detect the tandem repeat (TR) of cyp51A promoter in Aspergillus fumigatus. A control-probe was designed to anneal to the outside of the TR region, whereas a TR-probe was designed to anneal to the inside of the TR region. For amplification and probe-hydrolysis detection, the CycleavePCR system was used. Although the difference between Ct values of the wild-type genome for the control-probe and the TR-probe was around -0.1, the difference between Ct values of TR-harboring strains was around 0.7. These data indicate that this is a simple method to detect TR in azole-resistant A. fumigatus.

Disseminated fusariosis is a fatal infection in immunocompromised hosts. However, the optimal antifungal treatment for disseminated fusariosis has not yet been established. We report a case of disseminated fusariosis after chemotherapy for acute lymphoblastic leukemia, presenting with multiple skin, lung and kidney lesions and cerebrospinal fluid invasion. The combination therapy of liposomal amphotericin B and caspofungin resolved disseminated fusariosis successfully.

Purpose of Review The purpose of this review is to give an overview of recent findings on antifungal resistance in Aspergillus fumigatus (the major causative agent of aspergillosis) and sibling Aspergillus species, which can be hidden agents of aspergillosis. Recent Findings Azole resistance by Cyp51A mutation in A. fumigatus is a growing problem worldwide. The resistance can occur in patients or in the environment. The former occurs by drug selection in the host, inducing mutations in Cyp51A. The latter is characterized by a tandem repeat in the promoter region of cyp51A gene and mutation(s) in Cyp51A. Environmental resistant strains are prevailing rapidly and globally. Moreover, efflux pump and biofilm formation are closely related with antifungal resistance of A. fumigatus. Finally, sibling species of Aspergillus are described with regard to antifungal resistance. Summary Environmental azole-resistant strains have newly emerged and been dispersed globally, and continuous survey and countermeasures are urgently needed against these strains. Although the contributions of Cyp51A and efflux pumps to antifungal resistance are becoming clear, other resistance mechanisms remain unclear. Further investigations including genome compari- sons will help to clarify the novel resistant mechanisms and to develop countermeasures or novel antifungal drugs against resistant strains of A. fumigatus and other Aspergillus species that have low susceptibility to antifungal therapeutics.

A pan-azole-resistant Aspergillus fumigatus strain with the cyp51A mutations Gly138Ser and Asn248Lys was isolated from a patient receiving long-term voriconazole treatment. PCR fragments containing cyp51A with the mutations were introduced along with the Cas9 protein and single guide RNA into the azole-resistant/susceptible strains.

Increasing of detection rate of antifungal-resistant fungi is a serious global concern in public health. As few epidemiological data on resistant fungi in Brazil have been available, we planned an international joint project with the University of Campinas, São Paulo, Brazil to research antifungal-resistant fungi. This project, supported by the SATREPS program of the Japanese government, started in fiscal 2017.

Invasive fungal infection (IFI) is a life-threating infectious disease in high-risk neonates. Strategies for the treatment and prevention of IFI in neonates in Japan remain unclear. We conducted a nationwide retrospective survey to determine IFI incidence between January 2014 and October 2015. Primary survey questionnaires were submitted to 309 medical facilities that regularly treat high-risk neonates. The questionnaire assessed IFI incidence during the study period, methods for preventing fungal infection in early delivery neonates, and methods for preventing mother-to-child fungal transmission. The secondary questionnaire was for facilities that had IFI cases and replied to the primary questionnaire. In total, 128 medical facilities (41.4%) completed the primary questionnaire, 17/128 facilities recorded 23 proven or probable IFI cases. Estimated annual IFI incidence was 0.33/1000 live births of hospitalized neonates. Patient data at IFI onset were available for all 23 patients. Birth weight was < 1000 g in 18 patients. Causative microorganisms were identified in 22 patients. Candida species (n = 21) were the most common pathogens, and one patient had mucormycosis. The mortality rate was 17.4%. Regarding neonatal fungal prophylaxis, 55/128 facilities (43.0%) reported administering therapy. The most frequently used prophylactic drugs were fluconazole, then micafungin. Fungal prophylaxis for mothers who showed fungal colonization was performed in 30/128 facilities (23.4%). Oxiconazole vaginal tablets were most commonly used as prophylaxis for high-risk mothers. In Japan, the diagnosis, treatment, and prevention of neonatal IFI varied. Continuous surveillance and treatment regimen for neonatal IFI are required to improve outcomes in high-risk neonates.

Disseminated fusariosis (DF) is a rare life threatening fungal infection in immunocompromised hosts. We herein report a case of a fatal DF mimicking varicella zoster virus (VZV) infection that was emerged from a localized genital infection during cord blood transplantation (CBT) in a patient with severe aplastic anemia (SAA). The patient developed an ulcer following small painful vesicles mimics herpes simplex virus infection (HSV) on the glans penis before CBT, but a Fusarium species was identified. Despite administration of voriconazole, liposomal amphotericin B and granulocyte transfusion, the lesion was extended to extensive skin looked like VZV infection and the patients died after CBT. Massive fusarium infiltration was detected in multiple organs at autopsy. A genetic analysis of the mold identified Fusarium solani after his death. It should be noted that in patients with fusarium infection, localized and disseminated lesions of fusarium infection sometimes mimic HSV and VZV infections, which hampers an early diagnosis.

小児がん患者では、真菌感染症が疑われる場合でも、真菌の分離同定はしばしば困難であり臨床経過により診断治療が行われることが多い。2004年1月から2014年12月までに当科で治療を受けた小児血液がん患者6人より分離同定された糸状菌2株、酵母5株に関し、薬剤感受性試験を行い、分離同定することの意義について後方視的に検討した。糸状菌はいずれも耳漏より検出された。1例では好中球抑制期間に外耳炎を繰り返し、Aspergillus terreusが同定された。薬剤感受性試験の結果よりミカファンギン(MCFG)、ボリコナゾール(VRCZ)を併用し造血幹細胞移植を行った。酵母はすべてカンジダで、血液より分離同定された。Candida tropicalis分離例は治療開始後にβ-Dグルカンの上昇、脾膿瘍の悪化を認めたが感受性試験にてMCFG感受性良好であることを確認し治療遂行できた。C.parapsilosis、C.glabrata分離例はいずれもMCFG投与下のブレイクスルー感染であった。MCFG感受性良好として知られているC.glabrataに関しては薬剤感受性試験の結果、キャンディン系薬剤に対するMICの上昇が確認された。近年米国でもキャンディン系耐性カンジダが問題となっており、今後小児がん患者においても、治療効果が思わしくない際には薬剤感受性試験を行うことが必要だろう。(著者抄録)

Allergic bronchopulmonary mycosis (ABPM) develops mainly in patients with asthma or cystic fibrosis via types I and III hypersensitivity reactions to filamentous fungi. Aspergillus spp., especially Aspergillus fumigatus, is the major causative fungus because of its small conidia, thermophilic hyphae, and ability to secrete serine proteases. The cardinal histological feature of ABPM is allergic (eosinophilic) mucin-harboring hyphae in the bronchi, for which the formation of extracellular DNA trap cell death (ETosis) of eosinophils induced by viable fungi is essential. Clinically, ABPM is characterized by peripheral blood eosinophilia, increased IgE levels in the serum, IgE and IgG antibodies specific for fungi, and characteristic radiographic findings; however, there are substantial differences in the clinical features of this disease between East and South Asian populations. Systemic corticosteroids and/or antifungal drugs effectively control acute diseases, but recurrences are quite common, and development of novel treatments are warranted to avoid adverse effects and emergence of drug-resistance due to prolonged treatment with corticosteroids and/or antifungal drugs.

We elucidated a unique feature of sulfur metabolism in Cryptococcus neoformans. C. neoformans produces cysteine solely by the O-acetylserine pathway that consists of serine-O-acetyl transferase and cysteine synthase. We designated the gene encoding the former enzyme CYS2 (locus tag CNE02740) and the latter enzyme CYS1 (locus tag CNL05880). The cys1Δmutant strain was found to be avirulent in a murine infection model. Methionine practically does not support growth of the cys1Δ strain, and cysteine does not serve as a methionine source, indicating that the transsulfuration pathway does not contribute to sulfur amino acid synthesis in C. neoformans. Among the genes encoding enzymes catalyzing the reactions from homoserine to methionine, the gene corresponding to the Saccharomyces cerevisiae MET17 encoding O-acetylhomoserine sulfhydrylase (Met17p) had remained to be identified in C. neoformans. By genetic analysis of Met- mutants obtained by Agrobacterium tumefaciens-mediated mutagenesis, we concluded that Cnc01220, most similar to Str2p (36% identity), cystathionine-γ-synthase, in the Saccharomyces genome, is the C. neoformans version of O-acetylhomoserine sulfhydrylase. We designated CNC01220 as MET17. The C. neoformans met3Δ mutant defective in the first step of the sulfate assimilation pathway, sulfate adenylyltransferase, barely uses methionine as a sulfur source, whereas it uses cysteine efficiently. The poor utilization of methionine by the met3Δ mutant is most probably due to the absence of the transsulfuration pathway, causing an incapability of C. neoformans to produce cysteine and hydrogen sulfide from methionine. When cysteine is used as a sulfur source, methionine is likely produced de novo by using hydrogen sulfide derived from cysteine via an unidentified pathway. Altogether, the unique features of sulfur amino acid metabolism in C. neoformans will make this fungus a valuable experimental system to develop anti-fungal agents and to investigate physiology of hydrogen sulfide.

A DNA microarray platform, based on the nucleotide sequences of the internal transcribed spacer regions (ITS1 and ITS2) of the rRNA gene, was developed to identify 32 fungal pathogens at the species level. The probe sequences were spotted onto polycarbonate slides with a mini-microarray printer, and after the hybridization, the results were visible with the naked eye. The performance of the microarray platform was evaluated against the commercial automated systems (Vitek 2 and BD Phoenix systems) and DNA sequencing (gold standard). A total of 461 blood culture bottles were tested: 127 positive for fungi, 302 positive for bacteria, and 32 that were negative. Once the microorganisms were identified by automated systems, fungal DNA was extracted directly from the blood culture bottles. The DNA products were tested using the microarray platform, and DNA sequencing was performed. The results of the microarray and DNA sequencing were concordant in 96.7% of cases, and the results from the automated systems and DNA sequencing were concordant in 98.4%. Of all the nucleotide sequences contained in the microarray platform, the microarray failed to identify four fungal isolates (one Candida parapsilosis, two Candida tropicalis, and one Cryptococcus neoformans). Of note, the microarray detected Candida krusei DNA in two blood cultures from the same patient, whereas the automated system was only positive for Enterococcus faecium. Our microarray system provided reliable and fast fungal identification compared to that from DNA sequencing and the automated systems. The simplicity of reading the results by the naked eye made this DNA platform a suitable method for fungal molecular diagnosis.

We report a very rare case of pulmonary chromomycosis caused by Scedosporium prolificans that developed after lung transplantation and was successfully treated with endobronchial topical amphotericin B instillation. The subject was a woman in her 50s with a history of bilateral lobar lung transplantation from living donors for idiopathic pulmonary hypertension. Eight years after the lung transplantation, chest radiography X-ray and computed tomography showed an abnormal shadow in the right lung. Bronchoscopic findings showed obstruction by a fungal component at the laterobasal bronchus B9. She was diagnosed with pulmonary chromomycosis after S. prolificans was detected in the bronchial aspirate. Systemic antifungal treatment with itraconazole was ineffective. Therefore, we administered topical amphotericin B weekly via endobronchial instillation and replaced oral itraconazole with voriconazole. The endobronchial procedure was safe and tolerable. Bronchial obstruction improved after three 3 instillations. We continued topical amphotericin B instillation once every 3 months for 2 years, and the abnormal shadow nearly disappeared. This case report describes infection by S. prolificans, which rarely becomes an etiologic agent in lung transplant patients, and shows that endobronchial topical amphotericin B instillation is a therapeutic option when systemic antifungal treatment is ineffective.

From 2006 to 2013, an increasing incidence of fusariosis was observed in the hematologic patients of our University Hospital. We suspected of an environmental source, and the indoor hospital air was investigated as a potential source of the fungemia. Air samplings were performed in the hematology and bone marrow transplant (BMT) wards using an air sampler with pre-defined air volumes. To study the molecular relationship among environmental and clinical isolates, 18 Fusarium spp. recovered from blood cultures were included in the study. DNA sequencing of a partial portion of TEF1α gene was performed for molecular identification. Molecular typing was carried out by multi-locus sequence typing (MLST) using a four-gene scheme: TEF1α, rDNA, RPB1 and RPB2. One hundred four isolates were recovered from the air of the hematology (n = 76) and the BMT (n = 28) wards. Fusarium isolates from the air were from five species complexes: Fusarium fujikuroi (FFSC, n = 56), Fusarium incarnatum-equiseti (FIESC, n = 24), Fusarium solani (FSSC, n = 13), Fusarium chlamydosporum (FCSC, n = 10), and Fusarium oxysporum (FOSC, n = 1). Fifteen Fusarium isolates recovered from blood belonged to FSSC, and three to FFSC. MLST identified the same sequence type (ST) in clinical and environmental isolates. ST1 was found in 5 isolates from blood and in 7 from the air, both identified as FSSC (Fusarium petroliphilum). STn1 was found in one isolate from blood and in one from the air, both identified as FFSC (Fusarium napiforme). F. napiforme was isolated from the air of the hospital room of the patient with fungemia due to F. napiforme. These findings suggested a possible clonal origin of the Fusarium spp. recovered from air and bloodcultures. In conclusion, our study found a diversity of Fusarium species in the air of our hospital, and a possible role of the air as source of systemic fusariosis in our immunocompromised patients.

Aspergillus fumigatus is an important fungal pathogen of humans. Inhaled conidia of A. fumigatus adhere to pulmonary epithelial cells, causing opportunistic infection. However, little is known about the molecular mechanism of the adherence of resting conidia. Fungal molecules adhesive to host cells are presumed to be displayed on the conidial surface during conidial formation as a result of changes in gene expression. Therefore, we exhaustively searched for adhesion molecules by comparing the phenotypes and the gene expression profiles of A. fumigatus strains that have conidia showing either high or low adherence to human pulmonary A549 cells. Morphological observation suggested that strains that produce conidia of reduced size, hydrophobicity, or number show decreased adherence to A549 cells. K-means cluster analyses of gene expression revealed 31 genes that were differentially expressed in the high-adherence strains during conidial formation. We knocked out three of these genes and showed that the conidia of AFUA_4G01030 (encoding a hypothetical protein) and AFUA_4G08805 (encoding a haemolysin-like protein) knockout strains had significantly reduced adherence to host cells. Furthermore, the conidia of these knockout strains had lower hydrophobicity and fewer surface spikes compared to the control strain. We suggest that the selectively expressed gene products, including those we identified experimentally, have composite synergistic roles in the adhesion of conidia to pulmonary epithelial cells.

Koji is a fermenting agent used in many traditional Japanese foods, and Aspergillus oryzae is the most frequently used microorganism in koji production. Few cases of hypersensitivity pneumonitis due to A. oryzae have been reported. However, physicians should recognize the disease because of the increasing globalization of food production.