Aspergillus fumigatus Supernatants Disrupt Bronchial Epithelial Monolayers: Potential Role for Enhanced Invasion in Cystic Fibrosis.

Aspergillus fumigatus is the most commonly isolated fungus in chronic lung diseases, with a prevalence of up to 60% in cystic fibrosis patients. Despite this, the impact of A. fumigatus colonisation on lung epithelia has not been thoroughly explored. We investigated the influence of A. fumigatus supernatants and the secondary metabolite, gliotoxin, on human bronchial epithelial cells (HBE) and CF bronchial epithelial (CFBE) cells. CFBE (F508del CFBE41o-) and HBE (16HBE14o-) trans-epithelial electrical resistance (TEER) was measured following exposure to A. fumigatus reference and clinical isolates, a gliotoxin-deficient mutant (ΔgliG) and pure gliotoxin. The impact on tight junction (TJ) proteins, zonula occludens-1 (ZO-1) and junctional adhesion molecule-A (JAM-A) were determined by western blot analysis and confocal microscopy. A. fumigatus conidia and supernatants caused significant disruption to CFBE and HBE TJs within 24 h. Supernatants from later cultures (72 h) caused the greatest disruption while ΔgliG mutant supernatants caused no disruption to TJ integrity. The ZO-1 and JAM-A distribution in epithelial monolayers were altered by A. fumigatus supernatants but not by ΔgliG supernatants, suggesting that gliotoxin is involved in this process. The fact that ΔgliG conidia were still capable of disrupting epithelial monolayers indicates that direct cell-cell contact also plays a role, independently of gliotoxin production. Gliotoxin is capable of disrupting TJ integrity which has the potential to contribute to airway damage, and enhance microbial invasion and sensitisation in CF.

Population genomics confirms acquisition of drug-resistant Aspergillus fumigatus infection by humans from the environment.

Infections caused by the fungal pathogen Aspergillus fumigatus are increasingly resistant to first-line azole antifungal drugs. However, despite its clinical importance, little is known about how susceptible patients acquire infection from drug-resistant genotypes in the environment. Here, we present a population genomic analysis of 218 A. fumigatus isolates from across the UK and Ireland (comprising 153 clinical isolates from 143 patients and 65 environmental isolates). First, phylogenomic analysis shows strong genetic structuring into two clades (A and B) with little interclade recombination and the majority of environmental azole resistance found within clade A. Second, we show occurrences where azole-resistant isolates of near-identical genotypes were obtained from both environmental and clinical sources, indicating with high confidence the infection of patients with resistant isolates transmitted from the environment. Third, genome-wide scans identified selective sweeps across multiple regions indicating a polygenic basis to the trait in some genetic backgrounds. These signatures of positive selection are seen for loci containing the canonical genes encoding fungicide resistance in the ergosterol biosynthetic pathway, while other regions under selection have no defined function. Lastly, pan-genome analysis identified genes linked to azole resistance and previously unknown resistance mechanisms. Understanding the environmental drivers and genetic basis of evolving fungal drug resistance needs urgent attention, especially in light of increasing numbers of patients with severe viral respiratory tract infections who are susceptible to opportunistic fungal superinfections.

Prospective observational study of respiratory Aspergillus colonization or disease in patients with various stages of chronic obstructive pulmonary disease utilizing culture versus nonculture techniques.

Chronic obstructive pulmonary disease (COPD) patients have been recognized to be at increased risk of Aspergillus spp. colonization, which may progress to invasive pulmonary aspergillosis (IPA). The objective of this study was to determine the frequency of Aspergillus colonization, or disease, in a cohort of COPD patients. A prospective observational study was undertaken to determine Aspergillus colonization, or disease, in consecutive COPD patients undergoing bronchoscopy. Fungal culture as well as galactomannan antigen (GM) and Aspergillus nucleic acid detection (PCR) were performed on bronchoalveolar lavage fluid (BAL) samples. One hundred and fifty patients were recruited. One hundred and twelve (74.7%) were outpatients, 38 (25.33%) were inpatients, of whom 6 (4%) were in the intensive care unit. Most patients (N = 122, 81.3%) were either COPD GOLD (Global Initiative for Chronic Obstructive Lung Disease) stages 1 or 2. Nine (6%) patients were on systemic steroids, 64 (42.7%) on inhaled steroids, and 9 (6%) on both. Seventeen patients (11.3%) had at least one positive test for Aspergillus detection (culture ± galactomannan ± polymerase chain reaction [PCR]), 13 (76.4%) of whom were COPD GOLD stages 1 or 2.  Five patients had probable or putative IPA. Aspergillus sp. was detected in five patients (3.3%) by culture, but detection increased to 17 (11.3%) by the additional testing for GM or Aspergillus DNA. The frequency of Aspergillus detection in this cohort of COPD patients may reflect the predominance of early GOLD stages among the study population but deserves further investigation to determine its relevance as a predictive risk factor for IPA. LAY SUMMARY: COPD is a risk factor for Aspergillus spp. colonization. Bronchoalveolar lavage samples of 150 COPD patients were tested for presence of Aspergillus fumigatus, which was detected in five patients (3.3%) by culture, but detection of Aspergillus increased to 17 (11.3%) by additional GM and PCR testing.

CD3ε Expression Defines Functionally Distinct Subsets of Vδ1 T Cells in Patients With Human Immunodeficiency Virus Infection.

Human γδ T cells expressing the Vδ1 T cell receptor (TCR) recognize self and microbial antigens and stress-inducible molecules in a major histocompatibility complex-unrestricted manner and are an important source of innate interleukin (IL)-17. Vδ1 T cells are expanded in the circulation and intestines of patients with human immunodeficiency virus (HIV) infection. In this study, we show that patients with HIV have elevated frequencies, but not absolute numbers, of circulating Vδ1 T cells compared to control subjects. This increase was most striking in the patients with Candida albicans co-infection. Using flow cytometry and confocal microscopy, we identify two populations of Vδ1 T cells, based on low and high expression of the ε chain of the CD3 protein complex responsible for transducing TCR-mediated signals (denoted CD3εlo and CD3εhi Vδ1 T cells). Both Vδ1 T cell populations expressed the CD3 ζ-chain, also used for TCR signaling. Using lines of Vδ1 T cells generated from healthy donors, we show that CD3ε can be transiently downregulated by activation but that its expression is restored over time in culture in the presence of exogenous IL-2. Compared to CD3εhi Vδ1 T cells, CD3εlo Vδ1 T cells more frequently expressed terminally differentiated phenotypes and the negative regulator of T cell activation, programmed death-1 (PD-1), but not lymphocyte-activation gene 3, and upon stimulation in vitro, only the CD3εhi subset of Vδ1 T cells, produced IL-17. Thus, while HIV can infect and kill IL-17-producing CD4+ T cells, Vδ1 T cells are another source of IL-17, but many of them exist in a state of exhaustion, mediated either by the induction of PD-1 or by downregulation of CD3ε expression.

Post-influenzal triazole-resistant aspergillosis following allogeneic stem cell transplantation.

Influenza virus infection is now recognised as a risk factor for invasive pulmonary aspergillosis (IPA). Delays in diagnosis contribute to delayed commencement of antifungal therapy. In addition, the emergence of resistance to first-line triazole antifungal agents puts emphasis on early detection to prevent adverse outcomes. We present 2 allogeneic stem cell transplant patients who developed IPA due to triazole-resistant Aspergillus fumigatus following influenza infection. We underline the challenges faced in the management of these cases, the importance of early diagnosis and need for surveillance given the emergence of triazole resistance.

Intercountry Transfer of Triazole-Resistant Aspergillus fumigatus on Plant Bulbs.

We investigated whether plants imported to Ireland from the Netherlands might harbor triazole-resistant Aspergillus fumigatus. Samples of plant bulbs were positive for triazole-resistant A. fumigatus with CYP51A mutations. We hypothesize that this represents a route for intercountry transfer of an emerging resistance mechanism in a major opportunistic mold pathogen.

A prospective study of fungal biomarkers to improve management of invasive fungal diseases in a mixed specialty critical care unit.

PURPOSE: The diagnosis of invasive fungal diseases (IFD) in critical care patients (CrCP) is difficult. The study investigated the performance of a set of biomarkers for diagnosis of IFD in a mixed specialty critical care unit (CrCU). METHODS: A prospective observational study in patients receiving critical care for ≥7days was performed. Serum samples were tested for the presence of: (1-3) - ß-d-glucan (BDG), galactomannan (GM), and Aspergillus fumigatus DNA. GM antigen detection was also performed on bronchoalveolar lavage (BAL) samples. The patients were classified using published definitions for IFD and a diagnostic algorithm for invasive pulmonary aspergillosis. Performance parameters of the assays were determined. RESULTS: In patients with proven and probable IFD, the sensitivity, specificity, PPV and NPV of a single positive BDG were 63%, 83%, 65% and 83% respectively. Specificity increased to 86% with 2 consecutive positive results. The mean BDG value of patients with proven and probable IFD was significantly higher compared to those with fungal colonization and no IFD (p value<0.0001). CONCLUSION: New diagnostic criteria which incorporate these biomarkers, in particular BDG, and host factors unique to critical care patients should enhance diagnosis of IFD and positively impact antifungal stewardship programs.

The basophil surface marker CD203c identifies Aspergillus species sensitization in patients with cystic fibrosis.

BACKGROUND: Colonization by Aspergillus fumigatus in patients with cystic fibrosis (CF) can cause A fumigatus sensitization and/or allergic bronchopulmonary aspergillosis (ABPA), which affects pulmonary function and clinical outcomes. Recent studies show that specific allergens upregulate the surface-expressed basophil marker CD203c in sensitized subjects, a response that can be readily measured by using flow cytometry. OBJECTIVE: We sought to identify A fumigatus sensitization in patients with CF by using the basophil activation test (BAT). METHODS: Patients with CF attending Beaumont Hospital were screened for study inclusion. BAT was used to identify A fumigatus sensitization. Serologic (total and A fumigatus-specific IgE), pulmonary function, and body mass index measurements were performed. RESULTS: The BAT discriminates A fumigatus-sensitized from nonsensitized patients with CF. Persistent isolation of A fumigatus in sputum is a significant risk factor for A fumigatus sensitization. Levels of the A fumigatus-stimulated basophil activation marker CD203c inversely correlated with pulmonary function and body mass index in A fumigatus-sensitized but not nonsensitized patients with CF. Total and A fumigatus-specific IgE, but not IgG, levels are increased in A fumigatus-sensitized patients with CF and ABPA when compared with those in A fumigatus-sensitized and nonsensitized patients with CF without ABPA. Itraconazole treatment did not affect A fumigatus sensitization. CONCLUSION: Combining the BAT with routine serologic testing allows classification of patients with CF into 3 groups: nonsensitized, A fumigatus-sensitized, and ABPA. Accurate and prompt identification of A fumigatus-associated clinical status might allow early and targeted therapeutic intervention, potentially improving clinical outcomes.

Candida albicans stimulates IL-23 release by human dendritic cells and downstream IL-17 secretion by Vδ1 T cells.

γδ T cells expressing the Vδ1 TCR are expanded in patients with HIV infection. We show in this article that circulating Vδ1 T cell numbers are particularly high in patients with HIV and candidiasis, and that these cells expand and produce IL-17 in response to Candida albicans in vitro. Although C. albicans could directly stimulate IL-17 production by a subset of Vδ1 T cells, fungus-treated dendritic cells (DCs) were required to expand C. albicans-responsive Vδ1 T cells to generate sufficient numbers of cells to release IL-17 at levels detectable by ELISA. C. albicans induced the release of IL-1ß, IL-6, and IL-23 by DCs, but addition of these cytokines or supernatants of C. albicans-treated DCs to Vδ1 T cells was not sufficient to induce proliferation. We found that direct contact with DCs was required for Vδ1 T cell proliferation, whereas IL-23R-blocking studies showed that IL-23 was required for optimal C. albicans-induced IL-17 production. Because IL-17 affords protection against both HIV and C. albicans, and because Vδ1 T cells are not depleted by HIV, these cells are likely to be an important source of IL-17 in HIV-infected patients with candidiasis, in whom CD4(+) Th17 responses are impaired. These data show that C. albicans stimulates proliferation and IL-17 production by Vδ1 T cells by a mechanism that involves IL-23 release by DCs.

The effect of Aspergillus fumigatus infection on vitamin D receptor expression in cystic fibrosis.

RATIONALE: Aspergillus fumigatus (A. fumigatus) in cystic fibrosis (CF) is increasingly recognized. Although allergic bronchopulmonary aspergillosis (ABPA) leads to deterioration of pulmonary function, the effect of A. fumigatus colonization in the absence of ABPA remains unclear. OBJECTIVES: To address this, we examined individuals with CF with A. fumigatus who were ABPA negative to identify the effects of itraconazole therapy on Aspergillus-induced lung inflammation. METHODS: The effect of A. fumigatus on nuclear vitamin D receptor (VDR) expression was investigated using qRT-PCR and Western blotting. IL-5 and IL-13 levels were quantified by ELISA. The effect of itraconazole was assessed by a combination of high-resolution computed tomography, lung function test, and microbiological analysis. MEASUREMENTS AND MAIN RESULTS: We demonstrate that A. fumigatus down-regulates VDR in macrophages and airway epithelial cells and that the fungal metabolite gliotoxin (Gt) is the main causative agent. Gt overcame the positive effect of 1,25-OH vitamin D(3) on VDR expression in vitro, resulting in increased IL-5 and IL-13 production. In vivo, A. fumigatus positivity was associated with increased Gt in CF bronchoalveolar lavage fluid and increased bronchoalveolar lavage fluid levels of IL-5 and IL-13. After airway eradication of A. fumigatus with itraconazole, we observed decreased Gt, IL-5 and IL-13, improved respiratory symptoms, and diminished high-resolution computed tomography mosaic pattern consistent with sustained pulmonary function. CONCLUSIONS: This study provides a rationale for the therapeutic effect of itraconazole and implied that the therapeutic potential of vitamin D supplementation in preventing ABPA is only feasible with concurrent elimination of A. fumigatus to permit VDR expression and its positive functional consequences.