Chromatin profiling reveals heterogeneity in clinical isolates of the human pathogen Aspergillus fumigatus.

Invasive Pulmonary Aspergillosis, which is caused by the filamentous fungus Aspergillus fumigatus, is a life-threatening infection for immunosuppressed patients. Chromatin structure regulation is important for genome stability maintenance and has the potential to drive genome rearrangements and affect virulence and pathogenesis of pathogens. Here, we performed the first A. fumigatus global chromatin profiling of two histone modifications, H3K4me3 and H3K9me3, focusing on the two most investigated A. fumigatus clinical isolates, Af293 and CEA17. In eukaryotes, H3K4me3 is associated with active transcription, while H3K9me3 often marks silent genes, DNA repeats, and transposons. We found that H3K4me3 deposition is similar between the two isolates, while H3K9me3 is more variable and does not always represent transcriptional silencing. Our work uncovered striking differences in the number, locations, and expression of transposable elements between Af293 and CEA17, and the differences are correlated with H3K9me3 modifications and higher genomic variations among strains of Af293 background. Moreover, we further showed that the Af293 strains from different laboratories actually differ in their genome contents and found a frequently lost region in chromosome VIII. For one such Af293 variant, we identified the chromosomal changes and demonstrated their impacts on its secondary metabolites production, growth and virulence. Overall, our findings not only emphasize the influence of genome heterogeneity on A. fumigatus fitness, but also caution about unnoticed chromosomal variations among common laboratory strains.

Pulmonary Aspergillosis in People with Cystic Fibrosis.

In the last decade, fungal respiratory diseases have been increasingly investigated for their impact on the clinical course of people with cystic fibrosis (CF), with a particular focus on infections caused by Aspergillus spp. The most common organisms from this genus detected from respiratory cultures are Aspergillus fumigatus and Aspergillus terreus, followed by Aspergillus flavus, Aspergillus niger, and Aspergillus nidulans. These species have been identified to be both chronic colonizers and sources of active infection and may negatively impact lung function in people with CF. This review article discusses definitions of aspergillosis, challenges in clinical practice, and current literature available for laboratory findings, clinical diagnosis, and treatment options for pulmonary diseases caused by Aspergillus spp. in people with CF.

Antifungal Resistance in Pulmonary Aspergillosis.

Aspergilli may cause various pulmonary diseases in humans, including allergic bronchopulmonary aspergillosis (ABPA), chronic pulmonary aspergillosis (CPA), and acute invasive pulmonary aspergillosis (IPA). In addition, chronic colonization may occur in cystic fibrosis (CF). Aspergillus fumigatus represents the main pathogen, which may employ different morphotypes, for example, conidia, hyphal growth, and asexual sporulation, in the various Aspergillus diseases. These morphotypes determine the ease by which A. fumigatus can adapt to stress by antifungal drug exposure, usually resulting in one or more resistance mutations. Key factors that enable the emergence of resistance include genetic variation and selection. The ability to create genetic variation depends on the reproduction mode, including, sexual, parasexual, and asexual, and the population size. These reproduction cycles may take place in the host and/or in the environment, usually when specific conditions are present. Environmental resistance is commonly characterized by tandem repeat (TR)-mediated mutations, while in-host resistance selection results in single-resistance mutations. Reported cases from the literature indicate that environmental resistance mutations are almost exclusively present in patients with IA indicating that the risk for in-host resistance selection is very low. In aspergilloma, single-point mutations are the dominant resistance genotype, while in other chronic Aspergillus diseases, for example, ABPA, CPA, and CF, both TR-mediated and single-resistance mutations are reported. Insights into the pathogenesis of resistance selection in various Aspergillus diseases may help to improve diagnostic and therapeutic strategies.

Clinical epidemiology of pulmonary aspergillosis in hospitalized patients and contribution of Cyp51A, Yap1, and Cdr1B mutations to voriconazole resistance in etiologic Aspergillus species.

Pulmonary aspergillosis is a life-threatening fungal infection with worldwide distribution. In the present study, clinical epidemiology of pulmonary aspergillosis and antifungal susceptibility of etiologic Aspergillus species were evaluated in one-hundred fifty patients with special focus on the frequency of voriconazole resistance. All the cases were confirmed by the clinical pictures, laboratory findings, and isolation of etiologic Aspergillus species which belonged to two major species, i.e., A. flavus and A. fumigatus. Seventeen isolates displayed voriconazole MIC greater than or equal to the epidemiological cutoff value. Expression of cyp51A, Cdr1B, and Yap1 genes was analyzed in voriconazole-intermediate/resistant isolates. In A. flavus, Cyp51A protein sequencing showed the substitutions T335A and D282E. In the Yap1 gene, A78C replacement led to Q26H amino acid substitution that was not reported previously in A. flavus resistant to voriconazole. No mutations associated with voriconazole resistance were found in the three genes of A. fumigatus. The expression of Yap1 was higher than that of two other genes in both A. flavus and A. fumigatus. Overall, voriconazole-resistant strains of both A. fumigatus and A. flavus demonstrated overexpression of Cdr1B, Cyp51A, and Yap1 genes compared to voriconazole-susceptible strains. Although there are still ambiguous points about the mechanisms of azole resistance, our results showed that mutations were not present in majority of resistant and intermediate isolates, while all of these isolates showed overexpression in all three genes studied. As a conclusion, it seems that the main reason of the emergence of mutation in voriconazole-resistant isolates of A. flavus and A. fumigatus is previous or prolonged exposure to azoles.

Identification of distinct immunophenotypes in chronic pulmonary aspergillosis using cluster analysis.

BACKGROUND: Whether chronic pulmonary aspergillosis (CPA) has different immunophenotypes remains unknown. OBJECTIVE: To identify different CPA immunophenotypes using cluster analysis. METHODS: We used a subject-centred multivariate clustering approach without prior assumptions to identify CPA phenotypes. We retrospectively included the data of treatment-naïve subjects with CPA and excluded subjects with asthma and allergic bronchopulmonary aspergillosis (ABPA). We performed a scalable two-step cluster analysis using the log-likelihood distance measures to identify CPA phenotypes based on the blood immunological profile (total IgE, eosinophil count and Aspergillus-specific IgE and IgG). RESULTS: We included 351 CPA subjects and found two clusters. Cluster 2 (n = 118) had significantly higher serum total IgE, peripheral blood eosinophil count, and serum A. fumigatus-specific IgE and IgG than cluster 1 (n = 233). Cluster 2 subjects had a lower FEV1:FVC ratio on spirometry and were more likely to have a fungal ball (88 [74.6%] vs. 145 (62.2%), p = .023) on the CT thorax than cluster 1. After treatment discontinuation, cluster 2 had a longer median (interquartile range) time to relapse than cluster 1 (11.5 [7.3-27.4] vs. 4 [1.1-8.9] months, p = .005). CONCLUSION: We identified two distinct CPA phenotypes, type-2 dominant and non-type-2, with different clinical and radiological findings and treatment outcomes. Future studies should confirm our findings and investigate different treatment strategies based on CPA phenotypes.

Non-fumigatus Aspergillus Infection Associated with a Negative Aspergillus Precipitin Test in Patients with Chronic Pulmonary Aspergillosis.

Aspergillus antibody testing is key for the clinical diagnosis of chronic pulmonary aspergillosis (CPA) with high sensitivity. However, false-negative results in patients with CPA might be obtained, depending on the Aspergillus species. The aim of this study was to investigate which factors are associated with false-negative results in Aspergillus precipitin tests and whether the sensitivity of precipitin tests in CPA is influenced by Aspergillus fumigatus and non-fumigatus Aspergillus species. Between February 2012 and December 2020, 116 consecutive antifungal treatment-naive patients with CPA were identified and included in this retrospective chart review. Aspergillus species isolated from the respiratory tract of patients were identified by DNA sequencing. Characteristics of patients with positive and negative results for Aspergillus precipitin tests were compared. The sensitivity of the Aspergillus precipitin tests was compared between patients with A. fumigatus-associated CPA and non-fumigatus Aspergillus-associated CPA. A non-fumigatus Aspergillus species was the only factor significantly associated with negative Aspergillus precipitin test results in patients with CPA in the multivariate analysis (hazard ratio, 8.3; 95% confidence interval, 3.2 to 22.1; P < 0.0001). The positivity of the Aspergillus precipitin test for patients with non-fumigatus Aspergillus-associated CPA was lower than that for patients with A. fumigatus-associated CPA (84.8% versus 37.9%; P < 0.0001). These results revealed that the presence of non-fumigatus Aspergillus-associated CPA should be considered with a negative Aspergillus precipitin test; this finding may prevent diagnostic delay or misdiagnosis for CPA.

Can bronchial secretion cultures identify the etiologic agent of COVID-19-associated pulmonary aspergillosis in ICU patients? Comparison with a species-specific Aspergillus PCR in serum.

Aspergillus spp. isolated from non-BAL cultures of coronavirus disease 2019 (COVID-19)-associated pulmonary aspergillosis (CAPA) patients may reflect colonization rather than infection. Sera (n = 181) from 49 adult ICU CAPA patients (24 probable and 25 possible CAPA) with bronchial secretions (BS) culture positive for Aspergillus spp. were collected and tested for Aspergillus DNA detection by species-specific real-time PCR. Overall, 30/49 (61%) patients were PCR positive. BS culture/serum PCR agreement was moderate (21/30; 70%). Based on serum PCR positive patients, all CAPAs were due to A. fumigatus (80%), A. flavus (10%), and A. terreus (10%). No A. niger/A. nidulans or mixed infections were found despite positive BS cultures.

Discordant results were observed between bronchial secretion cultures and species-specific serum PCR (30%) with A. fumigatus being by far the most common etiological agent of CAPA (80%). No A. niger/A. nidulans or mixed infections were found despite positive cultures.

Chronic pulmonary aspergillosis is common among patients with presumed tuberculosis relapse in Ghana.

Chronic pulmonary aspergillosis (CPA) may mimic pulmonary tuberculosis (PTB). The two diseases are clinically indistinguishable and may result in CPA misdiagnosed as PTB or vice versa. Although PTB is largely recognised as a differential diagnosis of CPA and often ruled out prior to CPA diagnosis, the reverse is uncommon. The aim of this study was to determine the proportion of CPA cases among patients being assessed for PTB. A cross-sectional survey was conducted among consecutive patients referred for GeneXpert Mycobacterium tuberculosis test for the diagnosis of PTB at the Korle-Bu Teaching Hospital, Accra, Ghana. Patients' demographics, clinical and socioeconomic details were obtained using a structured questionnaire. Blood was collected for Aspergillus and HIV serology, and sputum samples obtained for Aspergillus culture. Chest radiograph was obtained, and computed tomography scan was also done for patients with positive Aspergillus serology or cavitation. CPA was defined using an algorithm developed by the Global Action for Fungal Infections (GAFFI) international expert panel. A total of 154 patients were included in the analysis, of whom 134 (87%) did not have a prior PTB diagnosis. There were 41 (26.6%) GeneXpert positive cases. CPA prevalence was 9.7% overall, but 50% in patients with a prior history of PTB and 3.7% in those without previous PTB. Although CPA is rarely considered as a differential diagnosis of PTB in Ghana, our findings show that CPA may affect half of patients being assessed for PTB relapse. Efforts to diagnose CPA should be prioritised in this patient group.

Chronic pulmonary aspergillosis (CPA) may be misdiagnosed as pulmonary tuberculosis (PTB), or vice versa due to clinical similarities. Screening for CPA among patients undergoing investigation for relapsed PTB and new PTB revealed that half and about four in 100 patients, respectively, had CPA.

Aspergillus fumigatus calcium-responsive transcription factors regulate cell wall architecture promoting stress tolerance, virulence and caspofungin resistance.

Aspergillus fumigatus causes invasive aspergillosis, the most common life-threatening fungal disease of immuno-compromised humans. The treatment of disseminated infections with antifungal drugs, including echinocandin cell wall biosynthesis inhibitors, is increasingly challenging due to the rise of drug-resistant pathogens. The fungal calcium responsive calcineurin-CrzA pathway influences cell morphology, cell wall composition, virulence, and echinocandin resistance. A screen of 395 A. fumigatus transcription factor mutants identified nine transcription factors important to calcium stress tolerance, including CrzA and ZipD. Here, comparative transcriptomics revealed CrzA and ZipD regulated the expression of shared and unique gene networks, suggesting they participate in both converged and distinct stress response mechanisms. CrzA and ZipD additively promoted calcium stress tolerance. However, ZipD also regulated cell wall organization, osmotic stress tolerance and echinocandin resistance. The absence of ZipD in A. fumigatus caused a significant virulence reduction in immunodeficient and immunocompetent mice. The ΔzipD mutant displayed altered cell wall organization and composition, while being more susceptible to macrophage killing and eliciting an increased pro-inflammatory cytokine response. A higher number of neutrophils, macrophages and activated macrophages were found in ΔzipD infected mice lungs. Collectively, this shows that ZipD-mediated regulation of the fungal cell wall contributes to the evasion of pro-inflammatory responses and tolerance of echinocandin antifungals, and in turn promoting virulence and complicating treatment options.

Novel acute hypersensitivity pneumonitis model induced by airway mycosis and high dose lipopolysaccharide.

BACKGROUND: Inhalation of fungal spores is a strong risk factor for severe asthma and experimentally leads to development of airway mycosis and asthma-like disease in mice. However, in addition to fungal spores, humans are simultaneously exposed to other inflammatory agents such as lipopolysaccharide (LPS), with uncertain relevance to disease expression. To determine how high dose inhalation of LPS influences the expression of allergic airway disease induced by the allergenic mold Aspergillus niger (A. niger). METHODS: C57BL/6J mice were intranasally challenged with the viable spores of A. niger with and without 1 µg of LPS over two weeks. Changes in airway hyperreactivity, airway and lung inflammatory cell recruitment, antigen-specific immunoglobulins, and histopathology were determined. RESULTS: In comparison to mice challenged only with A. niger, addition of LPS (1 µg) to A. niger abrogated airway hyperresponsiveness and strongly attenuated airway eosinophilia, PAS+ goblet cells and TH2 responses while enhancing TH1 and TH17 cell recruitment to lung. Addition of LPS resulted in more severe, diffuse lung inflammation with scattered, loosely-formed parenchymal granulomas, but failed to alter fungus-induced IgE and IgG antibodies. CONCLUSIONS: In contrast to the strongly allergic lung phenotype induced by fungal spores alone, addition of a relatively high dose of LPS abrogates asthma-like features, replacing them with a phenotype more consistent with acute hypersensitivity pneumonitis (HP). These findings extend the already established link between airway mycosis and asthma to HP and describe a robust model for further dissecting the pathophysiology of HP.

High detection rate of azole-resistant Aspergillus fumigatus after treatment with azole antifungal drugs among patients with chronic pulmonary aspergillosis in a single hospital setting with low azole resistance.

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.

Aspergillus fumigatus can acquire azole resistance during long-term treatment with azole drugs in patients with chronic pulmonary aspergillosis (CPA). The aim of this study was to determine the detection rate of azole-resistant A. fumigatus (ARAF) in isolates from CPA patients who had been treated with azoles. In addition, a 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). The isolates were tested for susceptibility to the azole drugs itraconazole (ITCZ) and voriconazole (VRCZ). The detection rate of ARAF from all isolates was 8.3% (n = 10). Greater than 10% of the 47 isolates obtained from 36 CPA patients who had been treated with azoles exhibited resistance. Furthermore, 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 undergoing long-term azole treatment at the time of antifungal therapy failure.

Clinical and experimental phenotype of azole-resistant Aspergillus fumigatus with a HapE splice site mutation: a case report.

BACKGROUND: The recent increase in cases of azole-resistant Aspergillus fumigatus (ARAf) infections is a major clinical concern owing to its treatment limitations. Patient-derived ARAf occurs after prolonged azole treatment in patients with aspergillosis and involves various cyp51A point mutations or non-cyp51A mutations. The prognosis of patients with chronic pulmonary aspergillosis (CPA) with patient-derived ARAf infection remains unclear. In this study, we reported the case of a patient with ARAf due to HapE mutation, as well as the virulence of the isolate. CASE PRESENTATION: A 37-year-old male was presented with productive cough and low-grade fever. The patient was diagnosed with CPA based on the chronic course, presence of a fungus ball in the upper left lobe on chest computed tomography (CT), positivity for Aspergillus-precipitating antibody and denial of other diseases. The patient underwent left upper lobe and left S6 segment resection surgery because of repeated haemoptysis during voriconazole (VRC) treatment. The patient was postoperatively treated with VRC for 6 months. Since then, the patient was followed up without antifungal treatment but relapsed 4 years later, and VRC treatment was reinitiated. Although an azole-resistant isolate was isolated after VRC treatment, the patient did not show any disease progression in either respiratory symptoms or radiological findings. The ARAf isolated from this patient showed slow growth, decreased biomass and biofilm formation in vitro, and decreased virulence in the Galleria mellonella infection model compared with its parental strain. These phenotypes could be caused by the HapE splice site mutation. CONCLUSIONS: This is the first to report a case demonstrating the clinical manifestation of a CPA patient infected with ARAf with a HapE splice site mutation, which was consistent with the in vitro and in vivo attenuated virulence of the ARAf isolate. These results imply that not all the ARAf infections in immunocompetent patients require antifungal treatment. Further studies on the virulence of non-cyp51A mutations in ARAf are warranted.

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.

Proven Aspergillus flavus pulmonary aspergillosis in a COVID-19 patient: A case report and review of the literature.

Severe COVID-19 patients complicated with aspergillosis are increasingly reported. We present a histopathological proven case of fatal COVID-19-associated pulmonary aspergillosis (CAPA), due to Aspergillus flavus. This report and existing published literature indicate diagnostic challenges and poor outcomes of CAPA in ICU patients.

Diagnostic Value of Galactomannan in Bronchoalveolar Lavage Fluid for Chronic Respiratory Disease with Pulmonary Aspergillosis.

The objective of this study was to explore the diagnostic value of the bronchoalveolar lavage fluid galactomannan (BALF GM) test for chronic respiratory disease with pulmonary aspergillosis and to establish the optimal cutoff value. Samples from a total of 309 chronic respiratory disease patients seen at the respiratory medicine department of Peking University Shenzhen Hospital from September 2016 to September 2019 were analyzed. According to the diagnostic criteria, we divided the patients into a case group (n = 79, comprising 25 proven cases and 54 probable cases) and a control group (n = 230). Bronchoalveolar lavage fluid was collected, and the BALF GM test results were analyzed. A nonparametric rank sum test showed that the mean rank of the case group was 255.30, which was higher than that of the control group (120.55). The Z-value was -11.567 (P = 0.000), indicating that the general distributions of BALF GM differed between the two groups. A BALF GM cutoff value of 0.88 showed the highest diagnostic efficacy for pulmonary aspergillosis. The sensitivity, specificity, positive predictive value, and negative predictive value were 77.2%, 93%, 79.2%, and 92.2%, respectively. As the cutoff value increased, the specificity and sensitivity of the BALF GM test increased and decreased, respectively. The BALF GM test can be used confirm the diagnosis of patients with pulmonary aspergillosis and chronic respiratory disease. The optimum BALF GM cutoff value is 0.88.

Clinical relevance of Aspergillus fumigatus sensitization in cystic fibrosis.

RATIONALE: The clinical relevance of sensitization to Aspergillus (A) fumigatus in cystic fibrosis (CF) is unclear. Some researchers propose that specific A fumigatus IgE is an innocent bystander, whereas others describe it as the major cause of TH-2-driven asthma-like disease. OBJECTIVES: Lung function parameters in mild CF patients may be different in patients with and without A fumigatus sensitization. We aimed to ascertain whether allergen exposure to A fumigatus by bronchial allergen provocation (BAP) induces TH-2 inflammation comparable to an asthma-like disease. METHODS: A total of 35 patients, aged 14.8 ± 8.5 years, and 20 healthy controls were investigated prospectively. The patients were divided into two groups: group 1 (n = 18): specific (s)IgE negative, and group 2 (n = 17): sIgE positive (≥0.7 KU/L) for A fumigatus. Lung function, exhaled NO, and induced sputum were analysed. All sensitized patients with an FEV1 > 75% (n = 13) underwent BAP with A fumigatus, and cell counts, and the expression of IL-5, IL-13, INF-γ, and IL-8 as well as transcription factors T-bet, GATA-3, and FoxP3, were measured. RESULTS: Lung function parameters decreased significantly compared to controls, but not within the CF patient group. After BAP, 8 of 13 patients (61%) had a significant asthmatic response and increased eNO 24 hours later. In addition, marked TH-2-mediated inflammation involving eosinophils, IL-5, IL-13, and FoxP3 became apparent in induced sputum cells. CONCLUSION: Our study demonstrated the clinical relevance of A fumigatus for the majority of sensitized CF patients. A distinct IgE/TH-2-dominated inflammation was found in induced sputum after A fumigatus exposure.

Species identification, antifungal susceptibility, and clinical feature association of Aspergillus section Nigri isolates from the lower respiratory tract.

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 ß-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.

In vitro and in vivo characterization of two nonsporulating Aspergillus fumigatus clinical isolates from immunocompetent patients.

Aspergillus fumigatus is a pathogenic fungus responsible for invasive aspergillosis (IA). Typically, it can produce abundant conidia to survive and spread. The infection by A. fumigatus usually occurs in immunocompromised patients due to failed clearance of inhaled conidia. However, the incidence of aspergillosis in immunocompetent hosts has been increasing, the pathogenesis of which is still unknown. Our team previously obtained two clinical nonsporulating A. fumigatus isolates from non-immunocompromised patients, which only have the form of hyphae. This present study demonstrated the in vitro and in vivo characteristics of the two nonsporulating A. fumigatus isolates and verified that their conidiation defects are associated to abolished expression of the sporulation-related central regulatory pathway brlA gene. In addition, we confirmed the mutation site of brlA gene (c.657_660delTCCT) contributes to the nonsporulating phenotype in one clinical isolate. Plate assay showed that the two nonsporulating isolates have a similar resistance to antifungal drugs, cell wall disturbing substances, and oxidative stress compared with the wild-type reference Af293. Most important of all, we employed an immunocompetent mouse model to mimic the pathogenesis of pulmonary aspergillosis in non-immunocompromised patients. It revealed that the hyphae of two nonsporulating isolates and Af293 have similar virulence in immunocompetent hosts. Interestingly, the hyphae fragments of Af293 but not conidia are able to induce invasive aspergillosis in immunocompetent mice. In conclusion, our study indicate that the form of hyphae may play a dominant causative role in pulmonary aspergillosis of immunocompetent hosts rather than conidia.

Azole resistance among clinical isolates of Aspergillus fumigatus in Lima-Peru.

Azole resistance among Aspergillus fumigatus isolates, which is mainly related to mutations in the cyp51A gene, is a concern because it is rising, worldwide disseminated, and associated with treatment failure and death. Data on azole resistance of aspergillus from Latin American countries is very scarce and do not exist for Peru. Two hundred and seven Aspergillus clinical isolates collected prospectively underwent mycology and molecular testing for specie identification, and 143 isolates were confirmed as A. fumigatus sensu stricto (AFSS). All AFSS were tested for in vitro azole susceptibility, and resistant isolates underwent PCR amplification and sequencing of the whole cyp51A gene and its promoter. The in vitro susceptibility showed a minimal inhibitory concentration (MIC) range, MIC50 and MIC90 of 0.125 to >16, 0.25, and 0.5 µg/ml for itraconazole; 0.25 to 2, 0.5, and 0.5 µg/ml for voriconazole; and 0.003 to 1, 0.06, and 0.125 µg/ml for posaconazole. Three isolates (2%) showed resistance to itraconazole and exhibited different mutations of the cyp51A gene. One isolate harbored the mutation M220K, while a second one exhibited the G54 mutation plus a modification in the cyp51A gene promoter. The third isolate, from an azole naive patient, presented an integration of a 34-bp tandem repeat (TR34) in the promoter region of the gene and a substitution of leucine 98 by histidine (L98H). The three source patients had a diagnosis or suspicion of chronic pulmonary aspergillosis.

Differential diagnosis of atypical encephalopathy in critical care: a case report.

BACKGROUND: A lower level of consciousness is a common presentation in critical care, with many different causes and contributory factors, of which more than one may be present concurrently. CASE PRESENTATION: We described a woman with poorly controlled diabetes and steroid-dependent asthma who presented in a deep coma. She was found to have Streptococcus intermedius bacteremia and pyogenic ventriculitis that originated from right middle lobe pneumonia. Also, multiple small parenchymal lesions were observed on brain magnetic resonance imaging and increased protein concentration was noted in cerebral spinal fluid. Initially, her coma was thought to be due to diabetic ketoacidosis and septic encephalopathy. However, her lowered level of consciousness was disproportionate to either diabetic ketoacidosis or septic encephalopathy, and her clinical course was not as expected for these two conditions. Treatment with antibiotic, corticosteroid and antihelminthic drugs was administered resulting in improving consciousness. The Streptococcus intermedius pneumonia progressed to form a large cavity that needed an early surgical lobectomy and resulted in the unexpected diagnosis of chronic cavitary pulmonary aspergillosus. CONCLUSIONS: In critical care, a lowered level of consciousness may have many etiologies, and critical care clinicians should be familiar with the signs and symptoms of all possible causes to enable prompt diagnosis and appropriate treatment.