Yasunori Muraosa's research while affiliated with The National Institute of Infectious Diseases and other places

Hyphal pellet formation by Aspergillus species in liquid cultures is one of the main obstacles to high-throughput anti-Aspergillus reagent screening. We previously constructed a hyphal dispersion mutant of Aspergillus fumigatus by disrupting the genes encoding the primary cell wall α-1,3-glucan synthase Ags1 and putative galactosaminogalactan synthase Gtb3 (Δags1Δgtb3). Mycelial growth of the mutant in liquid cultures monitored by optical density was reproducible, and the dose-response of hyphal growth to antifungal agents has been quantified by optical density. However, Δags1Δgtb3 still forms hyphal pellets in some rich growth media. Here, we constructed a disruptant lacking all three α-1,3-glucan synthases and galactosaminogalactan synthase (Δags1Δags2Δags3Δgtb3), and confirmed that its hyphae were dispersed in all the media tested. We established an automatic method to monitor hyphal growth of the mutant in a 24-well plate shaken with a real-time plate reader. Dose-dependent growth suppression and unique growth responses to antifungal agents (voriconazole, amphotericin B, and micafungin) were clearly observed. A 96-well plate was also found to be useful for the evaluation of mycelial growth by optical density. Our method is potentially applicable to high-throughput screening for anti-Aspergillus agents.

Although chitin in fungal cell walls is associated with allergic airway inflammation, the precise mechanism underlying this association has yet to be elucidated. Here, we investigated the involvement of fungal chitin-binding protein and chitin in allergic airway inflammation. Recombinant Aspergillus fumigatus LdpA (rLdpA) expressed in Pichia pastoris was shown to be an O -linked glycoprotein containing terminal α-mannose residues recognized by the host C-type lectin receptor, Dectin-2. Chitin particles were shown to induce acute neutrophilic airway inflammation mediated release of interleukin-1α (IL-1α) associated with cell death. Furthermore, rLdpA–Dectin-2 interaction was shown to promote phagocytosis of rLdpA–chitin complex and activation of mouse bone marrow-derived dendritic cells (BMDCs). Moreover, we showed that rLdpA potently induced T helper 2 (Th2)-driven allergic airway inflammation synergistically with chitin, and Dectin-2 deficiency attenuated the rLdpA–chitin complex-induced immune response in vivo . In addition, we showed that serum LdpA-specific immunoglobulin levels were elevated in patients with pulmonary aspergillosis.

Poster session 3, September 23, 2022, 12:30 PM - 1:30 PM Objectives Filamentous fungi generally form hyphal pellets in liquid culture. This property prevents filamentous fungi from applying the growth monitoring methods used for unicellular organisms such as yeast and bacteria. We have analyzed the biological functions of cell wall polysaccharide α-1,3-glucan and extracellular polysaccharide galactosaminogalactan (GAG) in Aspergillus species, and revealed that both α-1,3-glucan and GAG contribute to the pellet formation. Here we constructed the double disruption mutant of α-1,3-glucan and GAG biosynthesis-related genes (Δags1Δgtb3) in Aspergillus fumigatus AfS35 strain, and used the mutant for quantitative monitoring of the mycelial growth by optical density. Methods To measure the optical density of conidia and mycelia in shake-flask culture, conidia (final concentration, 1.0 × 10⁷/ml) of AfS35 or Δags1Δgtb3 strain were inoculated into 50 ml of Aspergillus minimal medium (AMM) medium in a 200-ml Erlenmeyer flask and rotated at 160 rpm at 37°C. At each sampling point, the culture (2 ml) was withdrawn, and 100 μL of the culture was mixed with 100 μL of 4% paraformaldehyde solution in a 96-well plate. OD₆₀₀ was measured in a microplate reader. To evaluate the susceptibility of the Δags1Δgtb3 strain to antifungal agents, conidia (final concentration, 5.0 × 10⁶/ml) were inoculated into 500 μL of RPMI medium containing an antifungal agent (voriconazole, itraconazole, amphotericin B, flucytosine, or micafungin) in a 48-well plate and rotated at 300 rpm using a microplate mixer at 35°C for 15 h, and OD₆₀₀ was measured in triplicate. Results In AMM flask culture, pellets became visible in AfS35 from 9 h, and their size increased with time, whereas the Δags1Δgtb3 hyphae were continuously dispersed (Fig. 1a). The OD₆₀₀ measurements of AfS35 suggested no correlation between apparent AfS35 growth and OD₆₀₀ (Fig. 1b). In the Δags1Δgtb3 strain, the first and third quartiles were 0.633 and 0.595 at 15 h (Fig. 1b), suggesting that the measurement of OD₆₀₀ is suitable for monitoring the growth of the mutant. As an application of the monitoring method, the Δags1Δgtb3 strain was grown in an RPMI medium containing the indicating antifungal agent for 15 h in a 48-well plate (Fig. 2). Growth was repressed in the presence of antifungal agents tested, except for micafungin (Fig. 2). Growth was completely inhibited at 2 μg/ml of voriconazole, 1 μg/ml of itraconazole, 0.5 μg/ml of amphotericin B, and 256 μg/ml of flucytosine (Fig. 2), which was in agreement with MICs determined by CLSI M38-A2. Conclusion We established a convenient strategy to monitor A. fumigatus hyphal growth. Our method can be directly applied to screening for novel antifungals against Aspergillus species.

Purpose To evaluate an outbreak of endophthalmitis caused by Fusarium oxysporum after cataract surgery. Methods In the present study, we conducted a retrospective review of the medical records of cases of endophthalmitis that developed after cataract surgery. All eyes underwent phacoemulsification and intraocular lens implantation (PEA + IOL) at a single eye clinic on the same date. Symptoms of endophthalmitis occurred 21.5 ± 3.4 days after the cataract surgery. Results Nine eyes of 9 patients with fungal endophthalmitis (5 males and 4 females) were enrolled in the current study. The mean age of the patients was 63.4 ± 8.5 years. Soon after the diagnosis of endophthalmitis, pars plana vitrectomy (PPV) had been performed in all the eyes. However, because there was no response to the first PPV plus antibacterial drug therapy, we performed repeat PPV for all the eyes, combined with IOL removal and antifungal therapy (natamycin eye drops plus oral voriconazole or fosfluconazole). After the antifungal drug therapy, no recurrence of endophthalmitis was observed in any of the operated eyes, and good visual outcomes were obtained. Fusarium oxysporum was identified by culture and sequencing analysis. Conclusion Early diagnosis and appropriate, adequate treatment are needed for successful management of fungal endophthalmitis.

Aspergillus is a widespread fungus in the environment, usually invades through the respiratory tract. Invasive aspergillosis is a fatal disseminated infection in immunocompromised hosts. Appendicitis occurs scarcely in patients with leukemia. We report a case of Aspergillus appendicitis that underwent an urgent appendectomy. An 11-year-old boy received the diagnosis of acute myeloid leukemia, because of the bone pain and results of the bone marrow study. He obtained a complete remission after cancer chemotherapy and received peripheral blood stem cell transplantation from a histocompatible sibling. Leukemia relapsed 5 months post-transplant. Induction therapy with etoposide, cytarabine and mitoxantrone was started on Candida prophylaxis. Fifteen days after the end of chemotherapy, he presented with febrile neutropenia and abdominal pain, that did not respond to broad-spectrum antibiotics. Serum levels of C-reactive protein, β-D-glucan and procalcitonin were unremarkable. Computed tomography scan revealed a swollen appendix and the adjacent tissue inflammation. An urgent appendectomy led to a tentative diagnosis of Aspergillus appendicitis based on the histopathological findings of many fungal hyphal forms. Panfungal polymerase chain reaction using DNA extracted from the lesion determined the pathogen of Aspergillus niger. There was no evidence of invasive aspergillosis. During the prolonged anti-fungal therapy, he achieved a remission of leukemia and underwent the second hematopoietic cell transplantation. To our knowledge, Aspergillus appendicitis was reported to occur in 5 leukemia patients. Four of them survived after appendectomy and one died from intestinal perforation. Early surgical intervention is mandatory for a cure of Aspergillus appendicitis in neutropenic patients on Candida prophylaxis.

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.

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.

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.

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.