渡辺 哲

Invasive aspergillosis is a life-threatening mycosis caused by the pathogenic fungus Aspergillus. The predominant causal species is Aspergillus fumigatus, and azole drugs are the treatment of choice. Azole drugs approved for clinical use include itraconazole, voriconazole, posaconazole, and the recently added isavuconazole. However, epidemiological research has indicated that the prevalence of azole-resistant A. fumigatus isolates has increased significantly over the last decade. What is worse is that azole-resistant strains are likely to have emerged not only in response to long-term drug treatment but also because of exposure to azole fungicides in the environment. Resistance mechanisms include amino acid substitutions in the target Cyp51A protein, tandem repeat sequence insertions at the cyp51A promoter, and overexpression of the ABC transporter Cdr1B. Environmental azole-resistant strains harboring the association of a tandem repeat sequence and punctual mutation of the Cyp51A gene (TR34/L98H and TR46/Y121F/1-289A) have become widely disseminated across the world within a short time period. The epidemiological data also suggests that the number of Aspergillus spp. other than A. fumigatus isolated has risen. Some non-fumigatus species intrinsically show low susceptibility to azole drugs, imposing the need for accurate identification, and drug susceptibility testing in most clinical cases. Currently, our knowledge of azole resistance mechanisms in non-fumigatus Aspergillus species such as A. flavus, A. niger, A. tubingensis, A. terreus, A. fischeri, A. lentulus, A. udagawae, and A. calidoustus is limited. In this review, we present recent advances in our understanding of azole resistance mechanisms particularly in A. fumigatus. We then provide an overview of the genome sequences of non-fumigatus species, focusing on the proteins related to azole resistance mechanisms.

Multi-azole resistant Aspergillus fumigatus carrying TR46/Y121F/T289A was isolated from a patient in Japan in Dec 2013. This strain grouped into the same Glade of the ones which were clinically isolated in France and Germany. A. fumigatus harboring this mutation could be rapidly diffused outside the Eurasian continent. (C) 2016 Japanese Society of Chemotherapy and The Japanese Association for Infectious Diseases. Published by Elsevier Ltd. All rights reserved.

真菌の遺伝子学的分類法の発達により,これまで同種とされてきた菌種の中から,近年新しい菌種が報告されている。これら今まで知られていなかった菌種(隠蔽種)の中には抗真菌薬に対する薬剤感受性などが近縁種とは異なるものが存在する。各々の菌種による感染症の病態や治療反応性などについては報告症例が少数であり不明な点が多く,今後の研究課題である。(著者抄録)

輸入真菌症は健常人でも感染するその高度病原性が特徴であるが,同時に通常の感染症に比べると,さまざま病型,病態を呈することが知られており,診断の障害となっている。このため他の疾患と誤認され,治療により悪化するケースも懸念される。一般的な病態に関する理解に加えて,どのような病態を取りえるかをあらかじめ熟知し,必要な鑑別診断を心がけることが日常臨床では重要である。(著者抄録)

We developed new cycling probe-based real-time PCR and nested real-time PCR assays for the detection of Histoplasma capsulatum that were designed to detect the gene encoding N-acetylated alpha-linked acidic dipeptidase (NAALADase), which we previously identified as an H. capsulatum antigen reacting with sera from patients with histoplasmosis. Both assays specifically detected the DNAs of all H. capsulatum strains but not those of other fungi or human DNA. The limited of detection (LOD) of the real-time PCR assay was 10 DNA copies when using 10-fold serial dilutions of the standard plasmid DNA and 50 DNA copies when using human serum spiked with standard plasmid DNA. The nested real-time PCR improved the LOD to 5 DNA copies when using human serum spiked with standard plasmid DNA, which represents a 10-fold higher than that observed with the real-time PCR assay. To assess the ability of the two assays to diagnose histoplasmosis, we analyzed a small number of clinical specimens collected from five patients with histoplasmosis, such as sera (n = 4), formalin-fixed paraffin-embedded (FFPE) tissue (n = 4), and bronchoalveolar lavage fluid (BALF) (n = 1). Although clinical sensitivity of the real-time PCR assay was insufficiently sensitive (33%), the nested real-time PCR assay increased the clinical sensitivity (77%), suggesting it has a potential to be a useful method for detecting H. capsulatum DNA in clinical specimens.

代表的な呼吸器真菌症としてアスペルギルス症、ムーコル症、クリプトコックス症がある。原因菌はいずれも自然環境内に遍在する真菌であり、通常は経気道的に感染を起こす。ほとんどが高度免疫低下状態の患者が罹患する疾患であるが、クリプトコックス症のみは健常者でも罹患しうる国内唯一の肺真菌症である。近年の社会情勢、医療の発達、診断技術の進歩に伴い原因菌の多様化がみられる。菌種により感受性薬剤が異なるため、適切な治療薬選択のために可能な限り菌種同定に努める必要がある。(著者抄録)

A 16-year-old boy with chronic granulomatous disease presented with pneumonia and rib osteomyelitis. Emericella nidulans var. echinulata was isolated from his sputum. After starting voriconazole, Rasamsonia piperina was isolated from the rib swelling. A combination therapy of voriconazole and micafungin effectively eradicated this invasive mixed-mold infection. In immunocompromised patients, a precise pathogenic diagnosis is clinically useful for administration of an appropriate treatment regimen.

輸入真菌症の原因菌は感染力の強いものが多く、健常者でも発症する。診断上、最も重要なのは詳細な旅行歴の聴取である。肺病変以外の病型もとりうるため、疑わしい旅行歴があれば、どの診療科でも鑑別に挙げる必要がある。(著者抄録)

Aspergillus fumigatus is one of the major fungal pathogens which cause pulmonary aspergillosis [1]. Its conidia are taken into human airway from the environment, and adhere to lung epithelial cells in the first process of infection. Some virulence factors should be produced by A. fumigatus and facilitate the infection, but such substances have not confirmed yet. Gliotoxin, one of the secondary metabolites of this fungus, has been recognized as a candidate of virulence factor and has been investigated thoroughly. This molecule has been shown to have cytotoxicity to various cell lines such as human alveolar epithelial cells [2] and human neutrophils [3]. But the impact of gliotoxin in the first phase of the infection remains unclear. This study aimes to investigate cytotoxic effect of gliotoxin on epithelial cells and elucidate the effect of gliotoxin to the adhesion of conidia to them. We observed the surface of A549 lung epithelial cells by scanning electron microscope (SEM) and evaluated the adhesion rate of conidia of A. fumigatus Af293 to A549 cells, after exposure of gliotoxin. One hour exposure of gliotoxin led to cell shrinking. By the observation with SEM, microvilli decreasing or shortening were seen on the surface of A549 cells by gliotoxin. The adhesion rate of Af293 conidia to A549 cells tended to increase after exposure of gliotoxin at a concentration of 100 ng/ml, compared with control. From these results, we found that gliotoxin gives morphologic changes of lung epithelial cells and increases the adhesion of conidia to them. This may open a new approach to unravel the infection mechanism.

肺ノカルジア症は免疫低下宿主の増加を背景に報告症例数が増加している。しかしながら特異的な診断マーカーに乏しく、症状も非特異的であるため診断にはしばしば困難を伴う。培養検査のみが確定診断の根拠となる。中枢神経系への播種をしばしば起こすため、確定診断例は必ず画像検索を行うことが推奨される。また、菌種により大きく薬剤感受性が異なることに留意する。治療薬の選択肢は少なく、また治療期間も長期にわたる。(著者抄録)

We investigated the inhibitory effects of antibacterial, biocidal, and antifungal agents against Fusarium spp. Seven Fusarium spp: four F. falciforme (Fusarium solani species complex), one Fusarium spp, one Fusarium spp. (Fusarium incarnatum-equiseti species complex), and one F. napiforme (Gibberella fujikuroi species complex), isolated from eyes with fungal keratitis were used in this study. Their susceptibility to antibacterial agents: flomoxef, imipenem, gatifloxacin, levofloxacin, moxifloxacin, gentamicin, tobramycin, and Tobracin (R) (contained 3,000 mu g/ml of tobramycin and 25 mu g/ml of benzalkonium chloride (BAK), a biocidal agent: BAK, and antifungal agents: amphotericin B, pimaricin (natamycin), fluconazole, itraconazole, miconazole, voriconazole, and micafungin, was determined by broth microdilution tests. The half-maximal inhibitory concentration (IC50), 100% inhibitory concentration (IC100), and minimum inhibitory concentration (MIC) against the Fusarium isolates were determined. BAK had the highest activity against the Fusarium spp. except for the antifungal agents. Three fluoroquinolones and two aminoglycosides had inhibitory effects against the Fusarium spp. at relatively high concentrations. Tobracin (R) had a higher inhibitory effect against Fusarium spp. than tobramycin alone. Amphotericin B had the highest inhibitory effect against the Fusarium spp, although it had different degrees of activity against each isolate. Our findings showed that fluoroquinolones, aminoglycosides, and BAK had some degree of inhibitory effect against the seven Fusarium isolates, although these agents had considerably lower effect than amphotericin B. However, the inhibitory effects of amphotericin B against the Fusarium spp. varied for the different isolates. Further studies for more effective medications against Fusarium, such as different combinations of antibacterial, biocidal, and antifungal agents are needed.

肺アスペルギルス症やムーコル症などの深在性真菌症が、原則として免疫抑制患者のみに発症することとは対照的に、地域流行型真菌症(いわゆる輸入真菌症)の中には一般旅行者も含めた健常者にも感染が成立するものがある。臨床症状は非特異的なものが多いため、主治医が本疾患群を想定していなければ、診断に至らない可能性もある。確定診断のために最も重要なことは、詳細な渡航歴の聴取である。血清診断は感度、特異度ともに十分とは言えず、補助診断にとどまる例もある。確定診断のためには病変部の病理検査が極めて有用である。一般医療機関での培養検査は行うべきではない。治療は長期にわたる抗真菌薬投与が必要となる。(著者抄録)

The incidence of Aspergillus infection has been increasing in the past few years. Also, new Aspergillus fumigatus-related species, namely Aspergillus lentulus, Aspergillus udagawae, and Aspergillus viridinutans, were shown to infect humans. These fungi exhibit marked morphological similarities to A. fumigatus, albeit with different clinical courses and antifungal drug susceptibilities. The present study used liquid chromatography/time-of-flight mass spectrometry to identify the secondary metabolites secreted as virulence factors by these Aspergillus species and compared their antifungal susceptibility. The metabolite profiles varied widely among A. fumigatus, A. lentulus, A. udagawae, and A. viridinutans, producing 27, 13, 8, and 11 substances, respectively. Among the mycotoxins, fumifungin, fumiquinazoline A/B and D, fumitremorgin B, gliotoxin, sphingofungins, pseurotins, and verruculogen were only found in A. fumigatus, whereas auranthine was only found in A. lentulus. The amount of gliotoxin, one of the most abundant mycotoxins in A. fumigatus, was negligible in these related species. In addition, they had decreased susceptibility to antifungal agents such as itraconazole and voriconazole, even though metabolites that were shared in the isolates showing higher minimum inhibitory concentrations than epidemiological cutoff values were not detected. These strikingly different secondary metabolite profiles may lead to the development of more discriminative identification protocols for such closely related Aspergillus species as well as improved treatment outcomes. (C) 2015, Japanese Society of Chemotherapy and The Japanese Association for Infectious Diseases. Published by Elsevier Ltd. All rights reserved.

Aspergillus fumigatus is the Aspergillus species most commonly associated with aspergillosis. Of the various presentations of aspergillosis, one of the most frequently observed in cases involving A. fumigatus pulmonary infections is aspergilloma (PA). In such infections one finds a fungus ball composed of fungal hyphae, inflammatory cells, fibrin, mucus, and tissue debris. Chronic necrotizing pulmonary aspergillosis (CNPA), also known as semi-invasive or invasive aspergillosis, is locally invasive and predominantly seen in patients with mild immunodeficiency or with a chronic lung disease. In the present study, with the aid of a next-generation sequencer, we conducted whole genome sequence (WGS) analyses of 17 strains isolated from patients in Japan with PA and CNPA. A total of 99,088 SNPs were identified by mapping the reads to A. fumigatus genome reference strain Af293, and according to genome-wide phylogenetic analysis, there were no correlations between the whole genome sequence typing results and pathologic conditions of patients. Here, we conducted the first multi-genome WGS study to focus on the A. fumigatus strains isolated from patients with PA and CNPA, and comprehensively characterized genetic variations of strains. WGS approach will help in better understanding of molecular mechanisms of aspergillosis cases caused by A. fumigatus.

肺ノカルジア症の臨床的な特徴に関する報告は限られている。2003〜2014年に自施設で経験した肺ノカルジア症12例を、後方視的に検討した。平均年齢は69歳、男性10例。経過中に8例の混合感染を認めた。ST合剤を投与した9例中5例は、副作用のため他薬へ変更した。観察期間内に本症による喀血死1例を認め、5例が他病死した。結論として、肺ノカルジア症には混合感染がまれではなく、ST合剤は副作用の頻度が高い。本症の予後については、合併症の影響が無視できない。(著者抄録)

＜Key Points＞(1)輸入真菌症は本来わが国内に棲息していない真菌による感染症である。(2)各疾患の流行地への旅行歴、生活歴が診断にきわめて重要である。(3)免疫不全宿主では重症化しやすいが、健常人でも感染が成立する疾患が多い。(4)中枢神経病変を合併した場合の予後はきわめて不良である。(著者抄録)

The emergence of azole-resistant strains of Aspergillus fumigatus during treatment for aspergillosis occurs by a mutation selection process. Understanding how antifungal resistance mechanisms evolve in the host environment during infection is of great clinical importance and biological interest. Here, we used next-generation sequencing (NGS) to identify mutations that arose during infection by A. fumigatus strains sequentially isolated from two patients, one with invasive pulmonary aspergillosis (IPA) (five isolations) and the other with aspergilloma (three isolations). The serial isolates had identical microsatellite types, but their growth rates and conidia production levels were dissimilar. A whole-genome comparison showed that three of the five isolates from the IPA patient carried a mutation, while 22 mutations, including six nonsynonymous ones, were found among three isolates from the aspergilloma patient. One aspergilloma isolate carried the cyp51A mutation P216L, which is reported to confer azole resistance, and it displayed an MIC indicating resistance to itraconazole. This isolate harbored five other nonsynonymous mutations, some of which were found in the afyap1 and aldA genes. We further identified a large deletion in the aspergilloma isolate in a region containing 11 genes. This finding suggested the possibility that genomic deletions can occur during chronic infection with A. fumigatus. Overall, our results revealed dynamic alterations that occur in the A. fumigatus genome within its host during infection and treatment.

マルネッフェイ型ペニシリウム症は主に東南アジアを中心に風土病として流行している真菌症の1種である。主に胞子の吸入により経気道的に感染し、AIDSなどの細胞性免疫の低下を基礎疾患として、細網内皮系など全身の臓器に播種する。ペニシリウム症という言葉から受ける印象とは裏腹に、極めて病原性の高い真菌で致命率も高いことから、診断・治療には特段の注意が必要である。わが国では輸入真菌症として稀に症例が認められていたが、近年、毎年患者が確認されるようになり明らかな増加傾向にある。近年、研究が進むにつれて、明らかな全身的免疫障害がなくても感染する例が知られるようになり、これまでの知識が改められつつある。正しい診断、治療はもちろん、検査中の感染事故を防止する観点からも正確な知識をもつことが重要である。(著者抄録)