Inherited CARD9 Deficiency Due to a Founder Effect in East Asia.

Autosomal recessive CARD9 deficiency can underly deep and superficial fungal diseases. We identified two Japanese patients, suffering from superficial and invasive Candida albicans diseases, carrying biallelic variants of CARD9. Both patients, in addition to another Japanese and two Korean patients who were previously reported, carried the c.820dup CARD9 variant, either in the homozygous (two patients) or heterozygous (three patients) state. The other CARD9 alleles were c.104G > A, c.1534C > T and c.1558del. The c.820dup CARD9 variant has thus been reported, in the homozygous or heterozygous state, in patients originating from China, Japan, or South Korea. The Japanese, Korean, and Chinese patients share a 10 Kb haplotype encompassing the c.820dup CARD9 variant. This variant thus originates from a common ancestor, estimated to have lived less than 4,000 years ago. While phaeohyphomycosis caused by Phialophora spp. was common in the Chinese patients, none of the five patients in our study displayed Phialophora spp.-induced disease. This difference between Chinese and our patients probably results from environmental factors. (161/250).

Hmg1 mutations in Aspergillus fumigatus and their contribution to triazole susceptibility.

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.

Evaluation of Surveyor nuclease for rapid identification of FKS genes mutations in Candida glabrata.

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.

Genetic differences between Japan and other countries in cyp51A polymorphisms of Aspergillus fumigatus.

BACKGROUND: Mutations in cyp51A gene are known as main mechanisms of azole resistance in Aspergillus fumigatus, whereas azole-susceptible strains also carry cyp51A mutations (polymorphisms). The polymorphisms found in Europe mainly consist of two combinations of mutations, that is combinations of five single-nucleotide polymorphisms (SNPs) of cyp51A, referred to as cyp51A-5SNPs, and combinations of three SNPs of cyp51A, referred to as cyp51A-3SNPs. Few studies have compared the distributions of cyp51A polymorphisms between different regions. OBJECTIVES: The aim of this study was to investigate the regional differences of cyp51A polymorphisms. METHODS: We compared the proportions of cyp51A polymorphisms in clinical and environmental strains isolated in various countries, and analysed the strains phylogenetically using short tandem repeats (STRs) and whole-genome sequence (WGS). RESULTS: Among the Japanese strains, 15 out of 98 (15.3%) clinical strains and 8 out of 95 (8.4%) environmental strains had cyp51A polymorphisms. A mutation of cyp51AN248K was the most prevalent polymorphism in both clinical (n = 14, 14.3%) and environmental strains (n = 3, 3.2%). Only one environmental strain harboured cyp51A-5SNPs, which was reported to be the most prevalent in Europe. For phylogenetic analyses using STRs and WGS, 183 and 134 strains, respectively, were employed. They showed that most of the strains with cyp51AN248K clustered in the clades different from those of the strains with cyp51A-5SNPs and cyp51A-3SNPs as well as from those with TR34 /L98H mutations. CONCLUSIONS: This study suggests that there are genetic differences between cyp51A polymorphisms of A. fumigatus in Japan and Europe.

A Simple Method To Detect Point Mutations in Aspergillus fumigatus cyp51A Gene Using a Surveyor Nuclease Assay.

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.

Genetic Basis of Azole and Echinocandin Resistance in Clinical Candida glabrata in Japan.

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.

[A Case of Pulmonary Enteric Adenocarcinoma with Soleus Muscle Metastasis].

Pulmonary enteric adenocarcinoma is a unique pulmonary adenocarcinoma subtype and has histopathological findings that are similar to those of colorectal adenocarcinoma. A man in his 50s visited our hospital because of discomfort in his right lower leg for the last 9 months. Imaging studies revealed a mass in his right soleus muscle, and needle biopsy was performed. Histological findings revealed adenocarcinoma, and immunohistochemical staining showed that the tumor cells were positive for CK20 and CDX-2. The tumor was first suspected to be metastasis of gastrointestinal malignant tumors. FDG-PET/CT showed increased FDG uptake in the right soleus muscle mass and presented with increased FDG uptake in a right upper lobe mass and right mediastinum lymphadenopathy. There were no findings in other organs. Scraping cytology of a transbronchial biopsy indicated adenocarcinoma. Upper and lower gastrointestinal endoscopy showed no findings of malignancy. He was finally diagnosed with pulmonary enteric adenocarcinoma(cT3N2M1b, Stage ⅣA). Treatment with cisplatin(CDDP), pemetrexed( PEM), and bevacizumab(BEV) was initiated. After 4 courses of the regimen, the tumor was partially reduced, and the patient showed stable disease(SD).

In vivo efficacy of pitavastatin combined with itraconazole against Aspergillus fumigatus in silkworm models.

Azole resistance in Aspergillus fumigatus is a worldwide concern and new antifungal drugs are required to overcome this problem. Statin, a 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase inhibitor, has been reported to suppress the growth of A. fumigatus, but little is known about its in vivo antifungal effect against A. fumigatus. In this study, we evaluated the in vivo efficacy of pitavastatin (PIT) combined with itraconazole (ITC) against azole-susceptible and azole-resistant strains with silkworm models. Prolongation of survival was confirmed in the combination-therapy (PIT and ITC) group compared to the no-treatment group in both azole-susceptible and azole-resistant strain models. Furthermore, when the azole-susceptible strain was used, the combination-therapy resulted in a higher survival rate than with ITC alone. Histopathological analysis of the silkworms revealed a reduction of the hyphal amount in both azole-susceptible and azole-resistant strain models. Quantitative evaluation of fungal DNA by qPCR in azole-susceptible strain models clarified the reduction of fungal burden in the combination-therapy group compared with the no-treatment group and ITC-alone group. These results indicate that the efficacy of PIT was enhanced when combined with ITC in vivo. As opposed to most statins, PIT has little drug-drug interaction with azoles in humans and can be used safely with ITC. This combination therapy may be a promising option as an effective treatment in clinical settings in the future. IMPORTANCE Azole resistance among A. fumigatus isolates has recently been increasingly recognized as a cause of treatment failure, and alternative antifungal therapies are required to overcome this problem. Our study shows the in vivo efficacy of PIT combined with ITC against A. fumigatus using silkworm models by several methods including evaluation of survival rates, histopathological analysis, and assessment of fungal burden. Contrary to most statins, PIT can be safely administered with azoles because of less drug-drug interactions, so this study should help us to verify how to make use of the drug in clinical settings in the future.

Lymphadenitis caused by Purpureocillium lilacinum in a patient with CARD9 deficiency.

We hereby make the first report of a case of mycosis caused by Purpureocillium lilacinum in CARD9 deficiency. A 40-year-old woman complained of lymph node swellings in the left cervical area. She also had chronic mucocutaneous candidiasis (CMC), and was found to have CARD9 deficiency. Lymphadenitis by P. lilacinum was confirmed. The diagnosis was difficult, as culturing the biopsy specimen at a cautiously selected temperature (25 °C) and genetic analysis were both required. Oral administration of voriconazole improved her lymphadenopathy.

In Vitro Characterization of Twenty-One Antifungal Combinations against Echinocandin-Resistant and -Susceptible Candida glabrata.

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.