Aspergillus Fumigatus Antigens Research Articles

Long-term use of omalizumab in patients with allergic bronchopulmonary aspergillosis: a tertiary-level care center experience

Introduction Allergic bronchopulmonary aspergillosis (ABPA) is a lung disease caused by a hypersensitivity reaction to antigens of Aspergillus fumigatus. Objective The aim of this study was to evaluate the long-term clinical outcomes of omalizumab use in patients with ABPA. Methods In this retrospective study, 12 patients diagnosed with ABPA and receiving omalizumab for at least 2 years, and 32 patients diagnosed with severe allergic asthma and receiving omalizumab for at least 2 years (control group) were evaluated. Results Evaluation was made of a total of 44 participants, comprising 11 (25%) males and 33 (75%) females, who received omalizumab for at least 2 years with the diagnosis of the control group (n = 32) and ABPA (n = 12). The increase in asthma control test (ACT) score after omalizumab was significant at 12 months and at 24 months in patients with ABPA. After omalizumab, the use of oral corticosteroid (OCS), the annual number of asthma attacks and hospitalizations were significantly decreased at 12 months and at 24 months in patients with ABPA. The increase in forced expiratory volume in 1 s (FEV1) (%) and ACT score after omalizumab were significant at 12 months and at 24 months in the control group. After omalizumab, the use of OCS, annual number of asthma attacks and hospitalizations were significantly decreased at 12 months and at 24 months in the control group. Conclusion Long-term omalizumab use in patients with ABPA seems to be an effective treatment for improving pulmonary function and reducing asthma exacerbations and hospitalizations.

Proportion of Allergic Bronchopulmonary Aspergillosis Presenting as Difficult-to-control Asthma in Patients Attending a Tertiary Care Centre using the Modified ISHAM Criteria: A Cross-sectional Study

Introduction: Difficult-to-control asthmatics, as defined by the Global Initiative for Management of Asthma (GINA), belong to a subset of patients whose symptoms remain uncontrolled despite adhering to maximal optimised therapy. Complex hypersensitivity reactions in response to airway colonization with Aspergillus fumigatus, which occurs in patients with asthma or cystic fibrosis, are established factors for a poor response to treatment and frequent exacerbations. Only limited data related to Allergic Bronchopulmonary Aspergillosis (ABPA) is available from India, particularly from Kerala. Aim: To assess the occurrence of ABPA in patients with difficultto-control asthma using the modified ISHAM criteria. Materials and Methods: This cross-sectional analysis was conducted in the Department of Pulmonary Medicine, Institute of Chest Diseases, Government Medical College, Kozhikode, Kerala, India from February 2019 to July 2020. The study population comprised asthmatics attending the Pulmonary Medicine Outpatient Services who were on regular medications, including optimal doses of inhaled corticosteroid and longacting beta-agonist combinations. Patients with two or more exacerbations per year requiring systemic steroids for symptom control and a positive skin test for Aspergillus fumigatus antigen were further analysed using the modified International Society for Human and Animal Mycology (IHAM) criteria to determine the proportion of difficult-to-control asthmatics with ABPA. A total of 185 subjects were enrolled. Twelve patients opted out of the study, and the remaining 173 patients were screened using the modified ISHAM criteria. Statistical analysis was performed using Stastistical Packages of Social Sciences (SPSS) software version 21.0. Continuous parameters were expressed as mean and median, while categorical parameters were measured as frequency and percentages. Results: It was observed that 104 (60.1%) patients belonged to the age group between 41-60 years, while approximately 60 patients (34.7%) were below 40 years of age. Among the 173 patients, 86 (49.7%) tested positive for Aspergillus fumigatus antigen. Applying the ISHAM criteria, it was found that only 17 (9.8%) of these patients satisfied the criteria for co-existent ABPA. A total of 101 patients (58.4%) required at least one hospital admission, while 4 (2.3%) patients required more than three hospital admissions per year. Total 21 (12.1%) patients had IgE specific to Aspergillus fumigatus, while total IgE levels were elevated in 46 (26.6%) cases. Thirty-six cases (20.8%) had a high peripheral eosinophil count. Conclusion: This study suggests the possibility of the treating physician overlooking 10% of asthmatics in this region who are being managed as difficult-to-control asthma, but who have co-existent ABPA. This subset should be identified early in the course and managed separately for better treatment response.

COVID-19-Associated Rhino-Mucormycosis and Pulmonary Aspergillosis Infection

In the current pandemic, the incidence of fungal infections-associated COVID-19 is surging. Diabetes mellitus, overzealous steroid, and antibiotic use for COVID-19 management may cause or exacerbate the fungal disease. In paranasal sinus (PNS), ethmoids followed by the maxillary sinus are commonly involved with the risk of intra-orbital or/and intracranial involvement is quite common. In lungs, COVID-19 infection has higher mortality rate, if there is an associated fungal infection. Aspergillus fumigates is the most common fungus that cause lung infection and present as discrete lesion different from COVID-19 manifestation. Two cases of COVID-associated fungal infections, i.e., rhino-mucormycosis and pulmonary aspergillosis are described here. In the first case, computed tomography (CT) of PNS was done for the complaints of mild right-sided facial swelling and mild restriction of the eye globe on day 27 of illness. CT showed hyperdense content involving the right-sided nasal sinus with the remodeling of bones with erosion and thinning of the inferior and medial orbital wall with the extension of soft tissue into the extraorbital space. The patient was treated with Amphotericin B and posaconazole oral suspension as the first-line antifungal monotherapy. In the second case, on complaints of cough with expectoration on the 20th day of illness, X-ray chest and CT chest were done which confirmed a thick-walled cavity in the right lung and other post COVID features. The tracheal aspirate culture was suggestive of Aspergillus fumigatus and Aspergillus antigen galactomannan was found positive in the fluid. The patient was treated for the same with antifungal therapy. Hence it is important to pay attention to the high probability of fungal infections in COVID-19 patients. The association of coronavirus with mucormycosis of the PNSs and aspergillosis of the lung must be given outmost consideration. Noncontrast CT of the PNSs is usually the first investigation of choice for PNS involvement, and CT chest help in the diagnosis of pulmonary fungal infection. Uncontrolled diabetes and use of steroids are two of the main factors for aggravating factors. Both early surgical intervention and anti-fungal treatment should be sought for the management.

COVID-19 patients share common, corticosteroid-independent features of impaired host immunity to pathogenic molds.

Patients suffering from coronavirus disease-2019 (COVID-19) are susceptible to deadly secondary fungal infections such as COVID-19-associated pulmonary aspergillosis and COVID-19-associated mucormycosis. Despite this clinical observation, direct experimental evidence for severe acute respiratory syndrome coronavirus type 2 (SARS-CoV-2)-driven alterations of antifungal immunity is scarce. Using an ex-vivo whole blood stimulation assay, we challenged blood from twelve COVID-19 patients with Aspergillus fumigatus and Rhizopus arrhizus antigens and studied the expression of activation, maturation, and exhaustion markers, as well as cytokine secretion. Compared to healthy controls, T-helper cells from COVID-19 patients displayed increased expression levels of the exhaustion marker PD-1 and weakened A. fumigatus- and R. arrhizus-induced activation. While baseline secretion of proinflammatory cytokines was massively elevated, whole blood from COVID-19 patients elicited diminished release of T-cellular (e.g., IFN-γ, IL-2) and innate immune cell-derived (e.g., CXCL9, CXCL10) cytokines in response to A. fumigatus and R. arrhizus antigens. Additionally, samples from COVID-19 patients showed deficient granulocyte activation by mold antigens and reduced fungal killing capacity of neutrophils. These features of weakened anti-mold immune responses were largely decoupled from COVID-19 severity, the time elapsed since diagnosis of COVID-19, and recent corticosteroid uptake, suggesting that impaired anti-mold defense is a common denominator of the underlying SARS-CoV-2 infection. Taken together, these results expand our understanding of the immune predisposition to post-viral mold infections and could inform future studies of immunotherapeutic strategies to prevent and treat fungal superinfections in COVID-19 patients.

Improved IgG antibody diagnostics of hypersensitivity pneumonitis and pulmonary mycoses by means of newly evaluated serum antibody ranges and frequencies using IgG ImmunoCAP™

Summary Background The ranges of most human IgG antibodies against avian, microbial, and chemical antigens between their cut off values and their maximum values detected by IgG ImmunoCAP™ (Thermo Fisher Diagnostics, Freiburg, Germany) are not well known in pulmonary immune-mediated disorders. In addition, for many antigens it is not yet known how frequently their IgG antibodies appear in patients with these lung diseases. Therefore, we evaluated ranges and frequencies of these IgG antibodies. Methods The sera of 47,200 patients with suspected hypersensitivity pneumonitis (HP, extrinsic allergic alveolitis) or bronchopulmonary mycoses (mainly allergic bronchopulmonary aspergillosis [ABPA]), which were examined for 32 various IgG antibodies against birds, bacteria, molds, yeasts, and chemicals using the IgG ImmunoCAP™ assay, were evaluated retrospectively. Results We found a large spreading of the specific IgG ranges with maximum values from 26 mgA/l for Rhizopus nigricans up to 4640 mgA/l for pigeon. When the maximum values in the literature are also taken into account, the ranges of avian antibodies reach values up to 7280 mgA/l, the ranges of molds and yeasts up to 1707 mgA/l, of bacterial thermoactinomycetes up to 206 mgA/l, and of chemicals up to 139 mgA/l. The evaluated antibody ranges of the individual antigens can be used to decide whether a detected IgG antibody value is weakly, moderately or strongly positive. According to consistent evidence from numerous studies, a strongly positive antibody indicates HP or pulmonary mycosis more likely than a weakly positive antibody. It was found that the antigens of the highest maximum antibody levels—pigeon, budgerigar, parrot, Aspergillus fumigatus—are the causative agents of the most common immune-mediated lung diseases: bird breeder’s lung and pulmonary aspergillosis. Evaluation of the frequencies of eight major IgG antibodies of HP revealed the following rates: pigeon 28%, Aspergillus fumigatus 25%, budgerigar 23%, Penicillium chrysogenum 11%, Saccharopolyspora rectivirgula 7%, Acremonium kiliense 6%, Aureobasidium pullulans 5%, and Thermoactinomyces vulgaris 2%. Conclusion This study is the first to evaluate the ranges not only of avian and Aspergillus fumigatus antigens, as has been done up to now, but also of antibodies against 24 other environmental antigens. Quantification of IgG antibodies regarding their specific ranges can help to improve the serodiagnostics of immune-mediated lung diseases. In the lower ranges IgG antibodies are mainly physiological, while higher ranges correlate with the mentioned diseases as HP and ABPA/allergic bronchopulmonary mycoses (ABPM). The determined frequencies of the eight HP antibodies can be helpful in establishing HP screening tests.

Development of a Simple and Robust Whole Blood Assay with Dual Co-Stimulation to Quantify the Release of T-Cellular Signature Cytokines in Response to Aspergillus fumigatus Antigens.

Deeper understanding of mold-induced cytokine signatures could promote advances in the diagnosis and treatment of invasive mycoses and mold-associated hypersensitivity syndromes. Currently, most T-cellular immunoassays in medical mycology require the isolation of mononuclear cells and have limited robustness and practicability, hampering their broader applicability in clinical practice. Therefore, we developed a simple, cost-efficient whole blood (WB) assay with dual α-CD28 and α-CD49d co-stimulation to quantify cytokine secretion in response to Aspergillus fumigatus antigens. Dual co-stimulation strongly enhanced A. fumigatus-induced release of T-cellular signature cytokines detectable by enzyme-linked immunosorbent assay (ELISA) or a multiplex cytokine assay. Furthermore, T-cell-dependent activation and cytokine response of innate immune cells was captured by the assay. The protocol consistently showed little technical variation and high robustness to pre-analytic delays of up to 8 h. Stimulation with an A. fumigatus lysate elicited at least 7-fold greater median concentrations of key T-helper cell signature cytokines, including IL-17 and the type 2 T-helper cell cytokines IL-4 and IL-5 in WB samples from patients with Aspergillus-associated lung pathologies versus patients with non-mold-related lung diseases, suggesting high discriminatory power of the assay. These results position WB-ELISA with dual co-stimulation as a simple, accurate, and robust immunoassay for translational applications, encouraging further evaluation as a platform to monitor host immunity to opportunistic pathogens.

COVID-19 Impairs Immune Response to Candida albicans.

Infection with SARS-CoV-2 can lead to Coronavirus disease-2019 (COVID-19) and result in severe acute respiratory distress syndrome (ARDS). Recent reports indicate an increased rate of fungal coinfections during COVID-19. With incomplete understanding of the pathogenesis and without any causative therapy available, secondary infections may be detrimental to the prognosis. We monitored 11 COVID-19 patients with ARDS for their immune phenotype, plasma cytokines, and clinical parameters on the day of ICU admission and on day 4 and day 7 of their ICU stay. Whole blood stimulation assays with lipopolysaccharide (LPS), heat-killed Listeria monocytogenes (HKLM), Aspergillus fumigatus, and Candida albicans were used to mimic secondary infections, and changes in immune phenotype and cytokine release were assessed. COVID-19 patients displayed an immune phenotype characterized by increased HLA-DR+CD38+ and PD-1+ CD4+ and CD8+ T cells, and elevated CD8+CD244+ lymphocytes, compared to healthy controls. Monocyte activation markers and cytokines IL-6, IL-8, TNF, IL-10, and sIL2Rα were elevated, corresponding to monocyte activation syndrome, while IL-1β levels were low. LPS, HKLM and Aspergillus fumigatus antigen stimulation provoked an immune response that did not differ between COVID-19 patients and healthy controls, while COVID-19 patients showed an attenuated monocyte CD80 upregulation and abrogated release of IL-6, TNF, IL-1α, and IL-1β toward Candida albicans. This study adds further detail to the characterization of the immune response in critically ill COVID-19 patients and hints at an increased susceptibility for Candida albicans infection.

Role of recombinant Aspergillus fumigatus antigens in diagnosing Aspergillus sensitisation among asthmatics.

The diagnosis of Aspergillus-sensitised asthma (ASA) and allergic bronchopulmonary aspergillosis (ABPA) is made using IgE against crude antigens of Afumigatus (cAsp). However, the IgE against cAsp has limitations due to cross-reactivity with other fungi. To evaluate the utility of recombinant Afumigatus (rAsp) antigens in detecting ASA and their role in differentiating true from cross-sensitisation. We performed IgE against rAsp (f 1, f 2, f 3, f 4 and f 6), cAsp and other fungal (Alternaria, Candida, Cladosporium, Malassezia and Trichophyton) antigens in subjects with Afumigatus-unsensitised asthma (Af-UA [n=51]), ASA (n=71) and ABPA (n=123). The diagnoses were made using cAsp-IgE and compared using rAsp-IgE. Subjects with elevated cAsp-IgE, but negative rAsp f 1 and f 2, were presumed to lack true Afumigatus sensitisation. The prevalence of any rAsp antigen positivity (cut-off, 0.35kUA/L) varied from 2%-22%, 32%-73% and 84%-98% for Af-UA, ASA and ABPA, respectively. The prevalence of sensitisation to other fungi ranged from 29%-65%, 59%-85% and 87%-95%, respectively, among subjects with Af-UA, ASA and ABPA. Nineteen subjects of ASA and one subject with ABPA were positive with cAsp-IgE but negative for rAsp f 1 and f 2 and were also cross-sensitised to at least one of the other fungi. Five subjects of Af-UA (cAsp-IgE negative) were rAsp f 1 or f 2 positive. Crude Aspergillus antigens may misclassify Aspergillus sensitisation among asthmatics. IgE against rAsp antigens (f 1 and f 2) potentially detect true Aspergillus sensitisation and could be used for this purpose.

The utility of the basophil activation test in differentiating asthmatic subjects with and without allergic bronchopulmonary aspergillosis.

Asthma with Aspergillus sensitisation (ASA) and allergic bronchopulmonary aspergillosis (ABPA) are distinct clinical conditions, with different management strategies. To determine the utility of the basophil activation test (BAT) in differentiating between asthmatic subjects with and without ABPA. We performed the BAT using flow cytometry for the basophil activation markers CD63, CD193, and CD203c in consecutive subjects with unsensitised asthma (UA), ASA and ABPA. The BAT was performed unstimulated (baseline) and after stimulation with peanut and Aspergillus fumigatus antigens. Stimulation indices (SI) for CD63, CD193 and CD203c were compared between the study groups. We enrolled 82 (UA [n=25], ASA [n=25], ABPA [n=32]) subjects. Only those subjects without peanut sensitisation on serological testing were included in further analyses (n=50). The receiver operating characteristic analysis of SI for CD63, CD193 and CD203c for the diagnosis of ABPA vs other asthmatics (UA and ASA) showed an area under the curve of 0.577, 0.317 and 0.625, respectively. The SI CD203c at a cut-off of 1.2 (sensitivity 50.0%, specificity 88.9%) and the SI CD63 at a cut-off of 1.3 (sensitivity 42.9%, specificity 91.7%) also had limited utility for the diagnosis of ABPA. The BAT cannot be used as a diagnostic test in distinguishing ABPA complicating asthma from ASA and UA.

A rare case report of allergic bronchopulmonary aspergillosis with active pulmonary tuberculosis

Allergic bronchopulmonary aspergillosis (ABPA) is a disorder caused by a hypersensitivity reaction to antigens of Aspergillus fumigatus and is commonly seen in patients suffering from bronchial asthma or cystic fibrosis. ABPA in patients with active pulmonary tuberculosis a rare finding because tuberculosis, being very common in this part of the world, often masks the clinical features of ABPA. A high index of suspicion is thus necessary to confirm the presence of the latter in patients with atypical presentations. We wish to report two such cases, in which ABPA was diagnosed while the patient was receiving anti-tubercular therapy.

Diagnostic Cutoffs and Clinical Utility of Recombinant Aspergillus fumigatus Antigens in the Diagnosis of Allergic Bronchopulmonary Aspergillosis

The clinical utility of IgE against recombinant Aspergillus fumigatus (rAsp)-specific antigens in allergic bronchopulmonary aspergillosis (ABPA) remains unclear. To identify the optimal diagnostic cutoffs of rAsp-specific IgE in differentiating ABPA from A fumigatus-sensitized asthma (ASA), and define their utility in the diagnosis of ABPA. We enrolled consecutive subjects with ASA and ABPA. IgE against rAsp f1, f2, f3, f4, and f6 was assayed in all the subjects. We evaluated 3 fixed cutoffs (0.35, 0.5, and 1.0 kUA/L) for their diagnostic performance in the entire cohort. We also divided the study population into derivation and validation cohorts. Cutoffs for rAsp-specific IgE were obtained using the receiver-operating characteristic analysis in the derivation cohort. We then evaluated the diagnostic performance of these cutoffs in the validation cohort. We further correlated rAsp-specific IgE levels in ABPA with asthma control, spirometry, imaging, and immunologic markers. We included 194 subjects (123 ABPA and 71 ASA). The statistically derived cutoffs proved superior to fixed cutoffs. IgE against rAsp f1 yielded the best combination of sensitivity (89%) and specificity (100%). The sensitivity and specificity of IgE against either rAsp f1 (cutoff, 4.465 kUA/L) or f2 (cutoff, 1.300 kUA/L) for diagnosing ABPA were 100% and 81%, respectively. The correlation between rAsp-specific IgE and most clinical parameters of ABPA was weak. IgE against rAsp f1 and f2 (using receiver-operating characteristic-derived cutoffs) were found to be the most useful in differentiating ABPA from ASA. Because this study was conducted at a single center, our results require further validation.

Update of reference values for IgG antibodies against typical antigens of hypersensitivity pneumonitis

Specific (s)IgG antibodies against environmental and occupational antigens, especially from bacteria, moulds, yeasts, birds and chemicals play an important role for hypersensitivity pneumonitis (HP). An increased serum level of sIgG is one criterion in the diagnostic procedure of HP and crucial for the detection of the triggering antigen for successful avoidance of further exposure. In contrast to specific IgE, sIgG concentrations in healthy individuals vary greatly depending on the antigen, which makes it difficult to differentiate from patients with HP. The aim of this study is to update or establish sIgG-reference values for important HP antigens in a healthy blood donor group. Therefore a study including six clinical centres in Germany was conducted to collect sera from 121 subjects without any signs of HP and without obvious exposure to potential HP antigens. Specific IgG to 32 typical HP antigens were quantified by ImmunoCAP (ThermoFisher Scientific; Phadia, Uppsala, Sweden). For validation selected measurements were repeated, total IgG was determined, sera were tested for unspecific binding with the human serum albumin ImmunoCAP Ro401, and influence of potential confounders was analysed. Statistical distribution of the antigen-specific IgG values was evaluated and the nonparametric method of percentile calculation was applied. The levels of IgG antibodies to the different antigens varied considerably in the study group from <0.02 to 726 mgA/L. Low sIgG levels were found against the chemicals and the highest levels to fungal antigens, especially to Aspergillus fumigatus and Botrytis cinerea. For three isocyanates, three acid anhydrides, Trichosporon pullulans and Acremonium kiliense reference values were proposed for the first time. For several avian antigens, moulds, and bacteria pre-existing reference values nearly could be confirmed without significant deviations, but already the 90% quantile for sIgG against Penicillium chrysogenum, Aspergillus fumigatus and pigeon antigen (Ge91) clearly exceeded the pre-existing values. In contrast, the 97.5% quantile value for Candida albicans was nearly half of the pre-existing cut-off value. In most cases specific IgG values were not significantly influenced by smoking and gender and most of them were unaffected by age. For implementation of these sIgG reference values into the routine diagnosis of HP, we provide an online available calculator to rank measured sIgG concentrations to the 32 different ImmunoCAP antigens.

Fungus Among Us: The Frenemies Within

A recent study shows that the commensal fungus Candida albicans is an inducer of differentiation of human CD4+ Th17 cells that harbor heterologous specificity for other fungi, which may explain evolutionary benefits of C. albicans as a commensal microbe (Bacher et al. Cell 2019;176;1340-1355). However, Th17 cells that are crossreactive to Aspergillus fumigatus antigens can also drive exaggerated airway inflammation in humans.

Diagnosing Allergic Bronchopulmonary Aspergillosis: A Review.

Dr. Hinson and his colleagues first described allergic bronchopulmonary aspergillosis (ABPA) in 1952. Later in 1977, Rosenberg proposed a diagnostic criteria for ABPA that even today remains widely acknowledged. Despite these steps taken, there still isn't a standardized diagnostic criteria set for ABPA although many have been proposed by various physicians over the years. ABPA is a condition caused by hypersensitivity to Aspergillus fumigatus antigens. It is seen most commonly in patients with either asthma or cystic fibrosis. In susceptible hosts, repeated inhalation of Aspergillus spores can cause an allergic response. Although a standardized diagnostic criteria is re­quired, there is no single test that establishes the diagnosis oth­er than a demonstration of central bronchiectasis (CB) with nor­mal tapering bronchi, a feature that is still considered pathognomonic of ABPA. Because of lack of standardized diagnostic criteria and screening, even today ABPA is under diagnosed and often times treatment for it is delayed. This can lead to complications in patients like pulmonary fibrosis, bronchiectasis with chronic sputum production, and increasingly severe persistent asthma with loss of lung function. For this alone, it becomes imperative that the diagnostic criteria guidelines need to be reviewed and standardized preferably with the help of larger research studies. In the following review article, we address the epidemiology, pathophysiology, and the current cumulative view regarding the diagnosis of ABPA.

A novel monoclonal antibody against enolase antigen of Aspergillus fumigatus protects experimental aspergillosis in mice.

Aspergillus fumigatus is one of the most common opportunistic fungal pathogens responsible for a variety of diseases in human, from allergic bronchopulmonary aspergillosis to chronic pulmonary aspergillosis, mostly in immunocompromized patients. In this study, one monoclonal antibody MAb R-5 (IgM) raised against enolase cell surface protein of A. fumigatus exhibited significant inhibition of spore germination in A. fumigatus (88.3%), Aspergillus flavus (57.4%) and Aspergillus niger (30.6%). The MAb R-5 also showed in vitro fungicidal activity against these species as follows: A. fumigatus (24.1%), A. flavus (13.3%) and A. niger (8.8%). These findings were supported by the indirect immunofluorescence microscopy, where the antibody showed binding with germinated spores and hyphae of A. fumigatus as well as A. flavus and A. niger.In vivo protective effect of MAb R-5 was evaluated in BALB/c mice challenged intravenously with A. fumigatus spores, where a significant reduction in CFU (85.9%) was observed in kidney tissue. The mean survival time of mice treated with MAb R-5 (18.5 days) was also enhanced compared to control (6.5 days). These results indicate that MAb R-5 could be valuable in diagnosis as well as in the treatment of broad range of Aspergillus infections.

Development of Aspergillus fumigatus-induced chronic atopic dermatitis mouse model

Purpose: Atopic dermatitis (AD) is the most common chronic and relapsing inflammatory skin disease with skin barrier defects and altered immune responses. Chronic inflammation leads to irreversible fibrosis in the skin and there is no treatment to completely abolish the inflammation and fibrosis. To prevent or treat the chronic process of AD, it is necessary to develop a murine model of AD that reflects the chronic process to identify the mechanism. The aims of this study were to develop a chronic AD model with a crude extract Aspergillus fumigatus (Af) antigen. Methods: We applied Af extract (40 μg) epicutaneously to the dorsal skin of BALB/c mice for 5 consecutive days per week during a period of 5 weeks for a chronic AD model, and 5 consecutive days repeatedly with 2 weeks interval for an acute AD model. Results: The clinical score and transepidermal water loss were more increased in the chronic AD model than in the acute AD model. Histologic findings showed that more increased epidermal thickness, neutrophil infiltration and hyperkeratosis in the chronic model than in the acute model. Skin fibrosis was more prominent in the chronic model than in the acute model. The mRNA expression lev els of transforming growth factor (TGF)-β, thymic stromal lymphopoietin, and interleukin-33 were increased in the skin of the chronic model compared to the acute model. The levels of total IgE, Af-specific IgE, IgG1, and IgG2a were significantly increased in the chronic model compared to controls. Conclusion: The Af-induced chronic AD model showed prominent fibrosis and increased TGF-β expression in the skin, which sug gests that these models may be useful in the research for the mechanism of the chronic process in AD. (Allergy Asthma Respir Dis 2019;7:150-157)

A cross-sectional study of skin prick test to Aspergillus fumigatus antigen in asthmatic patients seen at a tertiary healthcare center

INTRODUCTION: Asthma is a significant public health problem. The severity of asthma varies from patient to patient and the reasons for this are not fully understood. Atopy is known to play an important part in the pathogenesis of asthma. Sensitization to aeroallergens like house dust mite, animal dander from pets and environmental fungi are evaluated in asthmatics. Severe asthma seems to be associated to environmental fungi with sensitization especially to Aspergillus species. AIMS AND OBJECTIVE: The study aimed at determining the prevalence of Cutaneous Sensitization to Aspergillus species by Skin Prick Test (SPT) in moderate- severe asthmatics. MATERIAL AND METHODS: This study was done on 205 clinically diagnosed asthmatic patients, between September 2012 and August 2013. SPT and spirometry was done in all subjects along with a detailed history. RESULTS: The prevalence rate of Aspergillus sensitized patients is 59.5%. Observations of this study also suggest that the severity of asthma is more in Aspergillus sensitized patients when compared to non-sensitized patients and the duration of asthma was more in Non sensitized. There was no significant association between AEC, total IgE levels and Aspergillus species culture positivity in Aspergillus sensitized patients. Aspergillus terreus was the predominant fungal isolate from both SPT positive and negative patient. There was no significant correlation of fungal culture with SPT. CONCLUSIONS: High levels of Aspergillus sensitization is seen in south Indian subjects and is associated with greater severity and shorter duration of asthma.

FACS isolation of live mouse eosinophils at high purity via a protocol that does not target Siglec F

Flow cytometry protocols designed to identify mouse eosinophils typically target Siglec F, an α-2,3-sialic acid binding transmembrane protein expressed universally on cells of this lineage. While a convenient target, antibody-mediated ligation of Siglec F induces eosinophil apoptosis, which limits its usefulness for isolations that are to be followed by functional and/or gene expression studies. We present here a method for FACS isolation which does not target Siglec F and likewise utilizes no antibodies targeting IL5Rα (CD125) or CCR3. Single cell suspensions are prepared from lungs of mice that were sensitized and challenged with Aspergillus fumigatus antigens; eosinophils were identified and isolated by FACS as live SSChi/FSChi CD11c−Gr1−/loMHCII− cells. This strategy was also effective for eosinophil isolation from the lungs of IL5tg mice. Purity by visual inspection of stained cytospin preparations and by Siglec F-diagnostic flow cytometry was 98–99% and 97–99%, respectively. Eosinophils isolated by this method (yield, ~4×106/mouse) generated high-quality RNA suitable for gene expression analysis.

Prospective Evaluation of a New Aspergillus IgG Enzyme Immunoassay Kit for Diagnosis of Chronic and Allergic Pulmonary Aspergillosis

Anti-Aspergillus IgG antibodies are important biomarkers for the diagnosis of chronic pulmonary aspergillosis (CPA) and allergic bronchopulmonary aspergillosis (ABPA). We compared the performance of a new commercial enzyme immunoassay (EIA) (Bordier Affinity Products) with that of the Bio-Rad and Virion\Serion EIAs. This assay is novel in its association of two recombinant antigens with somatic and metabolic antigens of Aspergillus fumigatus In a prospective multicenter study, 436 serum samples from 147 patients diagnosed with CPA (136 samples/104 patients) or ABPA (94 samples/43 patients) and from 205 controls (206 samples) were tested. We obtained sensitivities of 97%, 91.7%, and 86.1%, and specificities of 90.3%, 91.3%, and 81.5% for the Bordier, Bio-Rad, and Virion\Serion tests, respectively. The Bordier kit was more sensitive than the Bio-Rad kit (P < 0.01), which was itself more sensitive than the Virion\Serion kit (P = 0.04). The Bordier and Bio-Rad kits had similar specificity (P = 0.8), both higher than that of the Virion\Serion kit (P = 0.02). The area under the receiver operating characteristic (ROC) curves confirmed the superiority of the Bordier kit over the Bio-Rad and the Virion\Serion kits (0.977, 0.951, and 0.897, respectively; P < 0.01 for each comparison). In a subset analysis of 279 serum samples tested with the Bordier and Bio-Rad kits and an in-house immunoprecipitin assay (IPD), the Bordier kit had the highest sensitivity (97.7%), but the IPD tended to be more specific (71.2 and 84.7%, respectively; P = 0.10). The use of recombinant, somatic, and metabolic antigens in a single EIA improved the balance of sensitivity and specificity, resulting in an assay highly suitable for use in the diagnosis of chronic and allergic aspergillosis.

Allergic Bronchopulmonary Aspergillosis: Diagnostic and Treatment Challenges

Allergic bronchopulmonary aspergillosis (ABPA) is a pulmonary disorder, occurring mostly in asthmatic and cystic fibrosis patients, caused by an abnormal T-helper 2 lymphocyte response of the host to Aspergillus fumigatus antigens. ABPA diagnosis is defined by clinical, laboratory and radiological criteria including active asthma, immediate skin reactivity to A. fumigatus antigens, total serum IgE levels>1000 IU/mL, fleeting pulmonary parenchymal opacities and central bronchiectases that represent an irreversible complication of ABPA. Despite advances in our understanding of the role of the allergic response in the pathophysiology of ABPA, pathogenesis of the disease is still not completely clear. In addition, the absence of consensus regarding its prevalence, diagnostic criteria and staging limits the possibility of diagnosing the disease at early stages. This may delay the administration of a therapy that can potentially prevent permanent lung damage. Long-term management is still poorly studied. Present primary therapies, based on clinical experience, are not yet standardized. These consist in oral corticosteroids, which control acute symptoms by mitigating the allergic inflammatory response, azoles and, more recently, anti-IgE antibodies. The latter two are used as a steroid-sparing agent to prolong the remission stage of the disease. Anti-IgE antibodies also have immunomodulatory properties. At present, the only way to bypass these limits and allow for an early diagnosis, is to assume ABPA in all patients with difficult-to-control asthma or cystic fibrosis. They should then be screened for sensitization to A. fumigatus antigens and, if positive, monitored more closely. Future controlled studies are needed to standardize present therapy, standardize cut-off values of various investigations, define the role of different novel immunomodulatory therapies, define the role of novel assays (such as recombinant A. fumigatus antigens and CCL17) and confirm new diagnostic and staging criteria.