亀井 克彦

Invasive fungal disease (IFD) in patients with haematological disorders is a fatal disease, making rapid identification and treatment crucial. However, the identification of the causative fungus is often difficult, sometimes even impossible. There have been few reports concerning the causative species of IFD. This study aimed to investigate the epidemiology and causative organism of IFD in patients with haematological diseases in Japan. We analyzed the IFD cases among the patients with haematological malignancies identified at the Medical Mycological Research Center, Chiba University, between 2013 and 2019. The most common underlying disease was acute myeloid leukaemia (34.3%). Forty-six point one percent of IFD patients received haematopoietic stem cell transplantation (HSCT). The major pathogens were Aspergillus, Candida, and Fusarium. Aspergillus fumigatus was the most common Aspergillus species, and Candida fermentati and Fusarium petroliphilum were the most common Candida and Fusarium species, respectively, in this analysis. Furthermore, various cryptic species and non-albicans Candida were identified. The drug susceptibility of such relatively rare strains suggests that analysis of the causative fungi should provide valuable information for therapeutic options. Therefore, our study indicated that it is clinically significant to identify the organism in as much detail as possible.

＜文献概要＞82歳,男性.2015年12月に右第3指に棘が刺さり,近医で切開,異物除去と後に縫合術を受けた.2016年2月頃右前腕にびらんが出現し,同部位に結節が多発してきたため当科を受診した.初診時,右前腕に3ヶ所の皮膚結節があった.結節を生検したところ,真皮内に好中球,形質細胞,多核巨細胞を含む膿瘍がみられた.組織培養の結果,菌の形態はAspergillus nigerに合致したが,遺伝子解析の結果,A. nigerの関連種であるA. tubingensisと判明した.画像検査上,他臓器にアスペルギルス症を示唆する所見はなく,A. tubingensisによる皮膚アスペルギルス症と診断した.イトラコナゾール200mg/日の3ヵ月間内服で治癒した.真菌の遺伝子検査が発達し,「関連種(隠蔽種)」と呼ばれる,形態学的に同一視されてきた別菌種の存在が明らかになった.関連種によっては薬剤感受性が異なり,早期鑑別が適切な抗真菌薬治療に役立つ可能性がある.真菌の分子生物学的検査に関心を持って症例を蓄積・検討していくことは臨床的に有益だと考える.

Schizophyllum commune is a causative agent of allergic bronchopulmonary mycosis, allergic fungal rhinosinusitis, and basidiomycosis. Diagnosis of these diseases remains difficult because no commercially available tool exists to identify the pathogen. Unique volatile organic compounds produced by a pathogen might be useful for non-invasive diagnosis. Here, we explored microbial volatile organic compounds produced by S. commune. Volatile sulfur compounds, dimethyl disulfide (48 of 49 strains) and methyl ethyl disulfide (49 of 49 strains), diethyl disulfide (34 of 49 strains), dimethyl trisulfide (40 of 49 strains), and dimethyl tetrasulfide (32 of 49 strains) were detected from headspace air in S. commune cultured vials. Every S. commune strain produced at least one volatile sulfur compound analyzed in this study. Those volatile sulfur compounds were not detected from the cultures of Aspergillus spp. (A. fumigatus, A. flavus, A. niger, and A. terreus), which are other major causative agents of allergic bronchopulmonary mycosis. The last, we examined H2S detection using lead acetate paper. Headspace air from S. commune rapidly turned the lead acetate paper black. These results suggest that those volatile sulfur compounds are potent targets for the diagnosis of S. commune and infectious diseases.

Chrysosporium zonatum is a soil-dwelling fungus that rarely causes pulmonary infections, and a small number of cases have been reported to date. A 74-year-old man, who had previously been treated for tuberculosis, presented with symptoms of low-grade fever, anorexia, cough, and bloody sputum. Chest computed tomography (CT) showed a thick-walled cavitary lesion in the right upper lobe, in which there was a suspected mycotic mass. Initially, the patient was suspected to have chronic aspergillosis due to positive serum anti-Aspergillus antibodies. However, bronchoscopic culture revealed the growth of C. zonatum. Symptoms and imaging findings improved with administration of voriconazole for 18 months. Infection by C. zonatum is very rare and is difficult to differentiate from aspergillosis by clinical features. Clinicians should be aware of the possibility of coinfection with C. zonatum and Aspergillus sp. Voriconazole may be an effective treatment option.

Introduction: Infections with Candida glabrata have recently gained worldwide attention owing to its association with long hospitalizations and high mortality rates. This problem is highlighted when the infection is associated with echinocandin resistance, which is used for first-line therapy. Echinocandin resistance is exclusively attributed to functional mutations in FKS genes, and especially in hot spot (HS) regions. Unfortunately, few studies have focused on the rapid identification of FKS mutations associated with echinocandin resistance in C. glabrata. This study was intended to evaluate and validate the use of Surveyor nuclease assay (SN) for detection of FKS gene mutations. Methods: SN was evaluated against three segments of FKS1 and FKS2 genes including whole gene, regions including all HSs, and the region including only HS1. Results: Our results showed that SN results are basically dependent on the type of gene as well as the segment type. Interestingly, SN can detect mutations in the region containing HS1 in both FKS1 and FKS2 genes. Furthermore, SN can detect mutations in the segment containing all HS regions for FKS1 but not FKS2. SN was unable to detect mutations in the whole FKS1 and FKS2 genes. Conclusions: As far as we know, this is the first study to validate SN for rapid identification of FKS gene mutations. This assay could be used as a sample for rapid identification of mutations associated with HS1 region in FKS genes, which have a predominant role for echinocandin resistance induction in C. glabrata.

Triazole-resistant Aspergillus fumigatus is a global health concern. In general, each triazole resistance pattern caused by the specified amino acid substitution of Cyp51A has a typical pattern depending on the mutation site. We evaluated the contribution of both Cyp51A and Hmg1 mutations to atypical triazole resistance in A. fumigatus. We used clinical triazole-resistant A. fumigatus strains collected in Japan and investigated the sequences of cyp51A and hmg1 genes. To delineate the association between the hmg1 mutation and atypical triazole resistance, the mutant hmg1 alleles in clinical multi-azole resistant strains were replaced with the wild-type hmg1 allele by CRISPR/Cas9 system. In our study, the combination of Cyp51A mutation and Hmg1 mutation was shown to additively contribute to triazole resistance. We also demonstrated that the triazole resistance conferred by the Hmg1 mutation showed a different pattern depending on the mutation site, similar to the Cyp51A mutation. Our results indicate that focusing on the phenotypes of multiple genes is essential to clarify the overall picture of the triazole resistance mechanism of A. fumigatus. LAY SUMMARY: The number of triazole-resistant Aspergillus fumigatus is increasing. We confirmed thatmutation in a hydroxymethylglutaryl-CoA reductase (Hmg1) in the fungus contributesto the resistance separately from Cyp51A mutation, and that susceptibility patterns aredifferent based on mutation site.

Filamentous fungi produce various bioactive compounds that are biosynthesized by sets of proteins encoded in biosynthesis gene clusters (BGCs). For an unknown reason, many BGCs are transcriptionally silent in laboratory conditions, which has hampered the discovery of novel fungal compounds. The transcriptional reactiveness of fungal secondary metabolism is not fully understood. To gain the comprehensive view, we conducted comparative genomic and transcriptomic analyses of nine closely-related species of <italic>Aspergillus</italic> section <italic>Fumigati</italic> (<italic>A. fumigatus, A. fumigatiaffinis, A. novofumigatus, A. thermomutatus, A. viridinutans, A. pseudoviridinutans, A. lentulus, A. udagawae</italic>, and <italic>Neosartorya fischeri</italic>). For expanding our knowledge, we newly sequenced genomes of <italic>A. viridinutans</italic> and <italic>A. pseudoviridinutans</italic>, and reassembled and reannotated the previously released genomes of <italic>A. lentulus</italic> and <italic>A. udagawae</italic>. Between 34 and 84 secondary metabolite (SM) backbone genes were identified in the genomes of these nine respective species, with 8.7–51.2% being unique to the species. A total of 247 SM backbone gene types were identified in the nine fungi. Ten BGCs are shared by all nine species. Transcriptomic analysis using <italic>A. fumigatus, A. lentulus, A. udagawae, A. viridinutans</italic>, and <italic>N. fischeri</italic> was conducted to compare expression levels of all SM backbone genes in four different culture conditions; 32–83% of SM backbone genes in these species were not expressed in the tested conditions, which reconfirmed that large part of fungal SM genes are hard to be expressed. The species-unique SM genes of the five species were expressed with lower frequency (18.8% in total) than the SM genes that are conserved in all five species (56%). These results suggest that the expression tendency of BGCs is correlated with their interspecies distribution pattern. Our findings increase understanding of the evolutionary processes associated with the regulation of fungal secondary metabolism.

The prevalence of azole-resistant Aspergillus fumigatus (ARAF) among chronic pulmonary aspergillosis (CPA) patients treated with azoles in Japan is unknown. The aim of this study was to determine the detection rate of ARAF in isolates from CPA patients who were treated with azoles for varying durations. The potential mechanism of acquiring resistance was examined by sequencing cyp51A and hmg1, two genes associated with ARAF. A. fumigatus isolates (n = 120) were collected from CPA patients (n = 104) between February 2012 and February 2019, at National Hospital Organization Tokyo National Hospital. The isolates were tested for susceptibility to the azole drugs itraconazole (ITCZ) and voriconazole (VRCZ). The detection rate of ARAF among all isolates was 8.3% (n = 10). Of the 10 resistant isolates, eight were ITCZ-resistant and five were VRCZ-resistant. Among 47 isolates obtained from 36 CPA patients who were treated with ITCZ (for an average of 256 days) and/or VRCZ (for an average of 29 days), the resistance rates were 17.0% and 10.6%, respectively. In addition, 46.2% of 13 isolates obtained from CPA patients with ongoing azole treatment at the time of antifungal therapy failure were resistant to azoles. Among the 10 ARAF isolates, a point mutation was detected in cyp51A in seven isolates and in hmg1 in two isolates. ARAF was detected at a high rate in CPA patients, particularly in those with ongoing long-term azole treatment, at the time of azole antifungal therapy failure.

67歳男性。発熱で受診し、CTで右肺上葉に浸潤影を認め、抗菌化学療法を行ったが、改善に乏しかった。気管支洗浄液でAspergillus nigerを検出し、慢性進行性肺アスペルギルス症と診断した。ボリコナゾール(voriconazole:VRCZ)で改善せず、リポソーマルアムホテリシンB(liposomal amphotericin B:L-AMB)へ変更、ミカファンギン(micafungin:MCFG)も併用したものの治療に難渋した。菌種の同定を専門施設に依頼し、A.tubingensisと同定された。感受性検査の結果を基に、L-AMB、カサポファンギン(caspofungin:CPFG)、フルシトシン(flucytosin:5-FC)の3剤併用療法で約3ヵ月間の治療を継続し改善を得た。治療に難渋する肺アスペルギルス症では薬剤感受性検査は有用な手段となり得る。(著者抄録)

This study was designed to analyze the interaction of 21 antifungal combinations consisting of seven major antifungal agents against 11 echinocandin- susceptible and six-resistant C. glabrata isolates. The combinations were divided into five major groups and were evaluated by checkerboard, disc diffusion, and time-killing assays. Synergy based on the fractional inhibitory concentration index of ≤0.50 was observed in 17.65-29.41% of the cases for caspofungin combinations with azoles or amphotericin B. Amphotericin B combination with azoles induced synergistic interaction in a range of 11.76-29.41%. Azole combinations and 5-flucytosine combinations with azoles or amphotericin B did not show synergistic interactions. None of the 21 combinations showed antagonistic interactions. Interestingly, 90% of the detected synergism was among the echinocandin-resistant isolates. Disk diffusion assays showed that the inhibition zones produced by antifungal combinations were equal to or greater than those produced by single drugs. The time-killing assay showed the synergistic action of caspofungin combination with fluconazole, voriconazole, and posaconazole, and the amphotericin B-5-flucytosine combination. Furthermore, for the first time, this assay confirmed the fungicidal activity of caspofungin-voriconazole and amphotericin B-5-flucytosine combinations. The combination interactions ranged from synergism to indifference and, most importantly, no antagonism was reported and most of the synergistic action was among echinocandin-resistant isolates.

A 53-year-old man was diagnosed with acute myeloid leukemia, which was refractory to chemotherapies. Systemic papules appeared afterward. The skin biopsies revealed filamentous fungal infection including fusariosis. Despite antifungal therapy, the infection did not resolve, because neutropenia persisted with the leukemia. He underwent hematopoietic stem cell transplantation (HSCT) to overcome the leukemia and restore normal hematopoiesis but died from fusariosis just before engraftment. Fusarium fujikuroi species complex was detected in blood cultures with poor antifungal susceptibility. Because restoring normal hematopoiesis is important in the treatment of fusariosis, HSCT might be considered for patients with persistent pancytopenia.

症例は22歳男性。20XX-3年から10ヵ月間米国オレゴン州に留学していた。胸部CTで左肺上葉に空洞を伴う結節影を指摘され、20XX年6月に埼玉県立循環器・呼吸器病センターを受診した。自覚症状はなく血清クリプトコッカス抗原は陰性だった。診断目的で同年8月に胸腔鏡下肺生検で採取した肺組織からCryptococcus sp.が培養され、遺伝子解析にてCryptococcus gattii(分子型VG IIc)と判明した。抗真菌薬に対する最小発育阻止濃度の結果を確認した後にイトラコナゾール(itraconazole:ITCZ)を6ヵ月間投与し、以後4年間再発していない。(著者抄録)

Infections caused by Candida glabrata have caused worldwide concern, especially when they are associated with increasing echinocandin and azole resistance. In this study, we analyzed the molecular mechanisms of azole and echinocandin resistance in C. glabrata isolates obtained from hospitalized patients in Japan from 1997 to 2019. All isolates were checked phenotypically for resistance and genotypically for mutations in PDR1, ERG11, hot spot 1 (HS1), HS2, and HS3 of FKS1, and HS1 and HS2 of FKS2, and all isolates were genotyped by multilocus sequence typing (MLST). Interestingly, 32.6% of the isolates were resistant to caspofungin, and 4.7% were resistant to micafungin. The isolates showed low rates of resistance to azoles, ranging from 2.3% to 9.3%, and only 4.7% of the isolates were non-wild type for flucytosine susceptibility. For the first time in Japan, 4.7% of the isolates were identified as multidrug-resistant strains. Nonsynonymous mutations in PDR1, including two novel mutations associated with azole resistance, were identified in 39.5% of the isolates, and a single nonsynonymous mutation was identified in ERG11 Nine isolates from the same patient harbored nonsynonymous mutations in HS1 of FKS2, and a single isolate harbored a single nonsynonymous mutation in HS1 of FKS1 MLST genotyping revealed 13 different sequence types (STs), with 3 new STs, and ST7 was the most prevalent among the patients (35%) and was associated with high resistance rates. Our results are of crucial clinical concern, since understanding the molecular mechanisms underlying fungal resistance is imperative for guiding specific therapy for efficient patient treatment and promoting strategies to prevent epidemic spread.

Biofilm is a complex structure consisting of microorganisms such as bacteria, fungi and an extracellular matrix (ECM). Biofilms are involved in most microbial infections and show persistent resistance to antibiotic treatment and immune response. Both Aspergillus fumigatus and Streptococcus pneumoniae are colonizers that can form biofilms in the respiratory tract. These pathogens have been simultaneously isolated from the same patient, but their interaction is poorly understood. We observed morphological changes in single- and mixed-species biofilms prepared for confocal laser scanning microscopy and scanning electron microscopy (SEM). Pneumococci suppressed the development of a fungal biofilm, and it even disrupted a preformed fungal biofilm. When a preformed fungal biofilm was treated with pneumococci, the mycelial network was fragmented, and only bacteria could develop. SEM revealed that the fragmented mycelium was further disrupted into fine filaments as treatment time progressed, and that the ECM of the preformed fungal biofilm had disappeared. The pneumococcal culture supernatant contained mycelial fragmentation activity that was heat-sensitive. The culture supernatant of a mutant pneumococcal strain deficient in pneumolysin (Δply) also exhibited the mycelial fragmentation activity. Enolase and lactate oxidase, which are involved in glycolysis and hydrogen peroxide production, were identified in the culture supernatant of the Δply mutant. Neither the wild type nor the mutant strain could fragment the mycelium in the presence of catalase. These data suggest that hydrogen peroxide could fragment the mycelium and would terminate the co-existence of A. fumigatus and S. pneumoniae in biofilm.

Aspergillus empyema is treated with either systemic administration of antifungal drugs or surgery, but the mortality rate is very high. Here, we report a case of Aspergillus empyema successfully treated using combined intrathoracic and intravenous administration of voriconazole (VRCZ). Treatment success was achieved by monitoring VRCZ plasma trough concentration. The patient was a 71-year-old Japanese woman diagnosed with Aspergillus empyema whom we started on intravenous administration of VRCZ. Although penetration of VRCZ into the pleural effusion was confirmed, the level was below 1 μg/mL, which is the minimum inhibitory concentration for Aspergillus fumigatus determined by antifungal susceptibility testing in pleural effusion culture. Therefore, we initiated combination therapy with intrathoracic and intravenous administration of VRCZ. VRCZ 200 mg was first dissolved in 50-100 mL of saline and administered into the thoracic cavity via a chest tube. The chest tube was clamped for 5-6 h, and then VRCZ solution was excreted though the chest tube. When a single dose of the VRCZ was administered into the intrathoracic space, the plasma concentration before intravenous administration increased from 1.45 μg/mL on day 27 to 1.53 μg/mL on day 28. Although intravenous administration was continued, the VRCZ plasma trough concentration decreased to 1.36 μg/mL on day 29. We therefore decided on an intrathoracic administration schedule of 2-3 times a week. Intrathoracic administration was performed 14 times in total until fenestration surgery on day 64. Our case suggests that combined intrathoracic and intravenous administration of VRCZ may be a valid treatment option for Aspergillus empyema.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Background: Invasive fusariosis (IF) is a frequently fatal disease as there are few antifungals to treat it, making the prevention of IF crucial. However, fusarium infections have not been as thoroughly studied as other common pathogenic fungi such as Aspergillus or Candida. Aim: To investigate the epidemiology of IF in patients with haematological diseases in Japan and to elucidate the infectious route of fusarium infection. Methods: We retrospectively analysed 29 IF cases in patients with haematological diseases from 2009 to 2019 in Japan. To discover the infectious source of IF, we performed an indoor environment survey targeted at indoor air and drain outlets in medical institutions and residences using culture-based and metagenomic methods. Finally, we performed aerosol- and droplet-mediated dispersion studies. Findings: The epidemiological study showed that the primary pathogen of IF was Fusarium solani species complex (FSSC), and the most common species was Fusarium petroliphilum. Most patients were likely to develop IF during hospitalization. A fusarium culture was positive in 26 of 72 drain samples. Few fusarium were detected from air samples; by contrast, 29 of 108 isolates from the drain outlets were identified as fusarium. Furthermore, similar results were obtained in the metagenomic analysis. Interestingly, species belonging to FSSC were isolated from indoor drain outlets, which was similar to those of the IF patients. In the droplet-mediated dispersion study, eight to 17 colonies of fusarium were isolated. Conclusion: Our study indicates that causative Fusarium spp. could inhabit drain outlets in hospitals or residences, and droplet-mediated fusarium dispersion is a potential cause of IF.

今回我々はSchizophyllum communeによるアレルギー性気管支肺真菌症の1症例を経験したので報告する。症例は60代男性,現病歴は糖尿病,前立腺肥大。発熱,咳嗽のため受診,胸部単純X線写真にて右上肺野に浸潤影が認められ,抗菌薬が処方されたが改善しないため,呼吸器内科に紹介された。気管支鏡にて粘液栓が認められ,グラム染色では糸状真菌を確認した。培養では白い綿毛状のコロニーが発育,質量分析装置,遺伝子解析によりS.communeと同定された。また血清学的検査にてELISA法によるS.communeに対するIgG抗体,IgE抗体ともに強陽性であり,S.communeによるABPMと診断した。itraconazole(ITCZ)とステロイドによる治療を6ヵ月間行い軽快した。(著者抄録)

Introduction: Cunninghamella bertholletiae although rarely causing mucormycosis, is responsible for the highest mortality among mucormycetes. The diagnosis of mucormycosis is challenged by the absence of specific biomarkers. Herein, we report a fatal case of C. bertholletiae infection and detection of its DNA in the serum by polymerase chain reaction (PCR). Presentation of case: A 23-year-old male with refractory osteosarcoma was admitted with multiple lung metastases. He was on oral voriconazole prophylaxis after pulmonary aspergillosis. He suffered from fever during temporary neutropenia following chemotherapy and showed several neurological and respiratory symptoms. Despite liposomal-amphotericin B administration, the symptoms rapidly progressed, and he died five days after the onset of neurological symptoms.We retrospectively evaluated the filamentous fungus isolated after his death from gastric juices. Based on the sequence of the internal transcribed spacer (ITS) region we identified the fungal isolate as C. bertholletiae. A 146-bp portion of the D1/D2 region was quantified by quantitative-PCR using DNA extracted from the serum. C. bertholletiae DNA load in the serum was 18.0 copies/μL on the day of onset of neurological symptoms, with the highest (101.0 copies/μL) on the day of his death. Discussion: Detection of circulating DNA of mucormycetes in the blood would greatly enhance the diagnosis of mucormycosis. Rapid diagnosis might alleviate mortality due to mucormycosis. Conclusion: The present case-report suggests that the quantification of C. bertholletiae DNA in the serum could be useful for the diagnosis and evaluation of mucormycosis pathogenesis in patients.

細菌や真菌には、呼吸器を主な感染の場とするものがあり、バイオフィルムを形成することで化学療法や宿主防御機構に対する抵抗性が増強する。我々は、呼吸器感染症の主要な病原体であるStreptococcus pneumoniaeとAspergillus fumigatusの混合バイオフィルムを解析し、S.pneumoniaeがA.fumigatusの菌糸体を分断することを見出した。菌糸体分断活性はS.pneumoniaeの培養上清中にも存在し、培養上清の分画により過酸化水素を生成する乳酸オキシダーゼを同定した。A.fumigatusのバイオフィルムを乳酸オキシダーゼとその基質であるL-乳酸ナトリウムを加えた培地で培養すると菌糸体が分断されたが、菌糸体分断はカタラーゼ存在下では阻害された。菌糸体分断には、S.pneumoniaeが産生する過酸化水素と、S.pneumoniaeの自己溶菌により遊離した酵素の反応で生じる過酸化水素の両方が関与していると考えられる。(著者抄録)

症例は18歳男性で，2歳時に慢性肉芽腫症と診断された。適切なドナーがなく，強度減弱前処置の下，母親からHLA半合致骨髄移植を施行した。移植後17日目に生着したが，キメリズムが低下し，96日目にドナーリンパ球輸注を施行した。120日目に急性移植片対宿主病を発症し，ステロイド投与を開始した。173日目に痙攣出現し，脳・肺・腎・前立腺に膿瘍を認めた。脳・前立腺・腎の膿瘍穿刺液からAspergillus siamensisが検出され，播種性アスペルギルス症と診断した。A. siamensis感染症はこれまで報告はなかったが，各種抗真菌薬を併用するも増悪し，239日目に永眠された。移植後の播種性アスペルギルス症は致死的であり，各種抗原をモニタリングしつつ，感染徴候を認めた場合には速やかに適切な検体を採取し，菌種・感受性に則した薬剤の選択が求められる。

Species of Aspergillus section Nigri are generally identified by molecular genetics approaches, whereas in clinical practice, they are classified as A. niger by their morphological characteristics. This study aimed to investigate whether the species of Aspergillus section Nigri isolated from the respiratory tract vary depending on clinical diagnosis. Forty-four Aspergillus section Nigri isolates isolated from the lower respiratory tracts of 43 patients were collected from February 2012 to January 2017 at the National Hospital Organization (NHO) Tokyo National Hospital. Species identification was carried out based on β-tubulin gene analysis. Drug susceptibility tests were performed according to the Clinical and Laboratory Standards Institute (CLSI) M38 3rd edition, and the clinical characteristics were retrospectively reviewed. A. welwitschiae was isolated most frequently, followed by A. tubingensis. More than half of the A. tubingensis isolates exhibited low susceptibility to azoles in contrast to only one A. welwitschiae isolate. Approximately three quarters of the patients from whom A. welwitschiae was isolated were diagnosed with colonization, whereas more than half the patients from whom A. tubingensis was isolated were diagnosed with chronic pulmonary aspergillosis (CPA). More attention needs to be given to the drug choice for patients with CPA with Aspergillus section Nigri infection because A. tubingensis, which was found to be frequently azole-resistant, was the most prevalent in these patients.

Azole-resistant Aspergillus fumigatus containing unique mutation(s) of cyp51A with tandem repeats in the promoter region has emerged and has become dispersed in environments worldwide. For this study, we designed primers and cycling probes to detect mutations associated with tandem repeats. Substitutions at the 293rd nucleotide (leucine or histidine at the 98th amino acid residue), at the 362nd nucleotide (tyrosine or phenylalanine at the 121st amino acid residue), and at the 865th nucleotide (threonine or alanine at the 289th amino acid residue) in cyp51A were detected using these primers and probes. These results suggest that the primer and probe sets are helpful in detecting these mutations and in differentiating the types of tandem repeats in cyp51A.

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.