Evaluation of the LDBio Aspergillus ICT lateral flow assay for serodiagnosis of allergic bronchopulmonary aspergillosis.

BACKGROUND: Early recognition and diagnosis of allergic bronchopulmonary aspergillosis (ABPA) is critical to improve patient symptoms, and antifungal therapy may prevent or delay progression of bronchiectasis and development of chronic pulmonary aspergillosis. OBJECTIVE: A recently commercialized lateral flow assay (Aspergillus ICT) (LDBio Diagnostics, Lyons, France) detects Aspergillus-specific antibodies in <30 minutes, requiring minimal laboratory equipment. We evaluated this assay for diagnosis of ABPA compared to diseased (asthma and/or bronchiectasis) controls. METHODS: ABPA and control sera collected at the National Aspergillosis Centre (Manchester, UK) and/or from the Manchester Allergy, Respiratory and Thoracic Surgery research biobank were evaluated using the Aspergillus ICT assay. Results were read both visually and digitally (using a lateral flow reader). Serological Aspergillus-specific IgG and IgE, and total IgE titres were measured by ImmunoCAP. RESULTS: For 106 cases of ABPA versus all diseased controls, sensitivity and specificity for the Aspergillus ICT were 90.6% and 87.2%, respectively. Sensitivity for 'proven' ABPA alone (n = 96) was 89.8%, and 94.4% for 'presumed' ABPA (n = 18). 'Asthma only' controls (no bronchiectasis) and 'bronchiectasis controls' exhibited 91.4% and 81.7% specificity, respectively. Comparison of Aspergillus ICT result with Aspergillus-specific IgG and IgE titres showed no evident immunoglobulin isotype bias. Digital measurements displayed no correlation between ImmunoCAP Aspergillus-specific IgE level and ICT test line intensity. CONCLUSIONS: The Aspergillus ICT assay exhibits good sensitivity for ABPA serological screening. It is easy to perform and interpret, using minimal equipment and resources; and provides a valuable simple screening resource to rapidly distinguish more serious respiratory conditions from Aspergillus sensitization alone.

Chronic pulmonary aspergillosis commonly complicates treated pulmonary tuberculosis with residual cavitation.

Chronic pulmonary aspergillosis (CPA) complicates treated pulmonary tuberculosis (TB), with high 5-year mortality. We measured CPA prevalence in this group.398 Ugandans with treated pulmonary TB underwent clinical assessment, chest radiography and Aspergillus-specific IgG measurement. 285 were resurveyed 2 years later, including computed tomography of the thorax in 73 with suspected CPA. CPA was diagnosed in patients without active TB who had raised Aspergillus-specific IgG, radiological features of CPA and chronic cough or haemoptysis.Author-defined CPA was present in 14 (4.9%, 95% CI 2.8-7.9%) resurvey patients. CPA was significantly more common in those with chest radiography cavitation (26% versus 0.8%; p<0.001), but possibly less frequent in HIV co-infected patients (3% versus 6.7%; p=0.177) The annual rate of new CPA development between surveys was 6.5% in those with chest radiography cavitation and 0.2% in those without (p<0.001). Absence of cavitation and pleural thickening on chest radiography had 100% negative predictive value for CPA. The combination of raised Aspergillus-specific IgG, chronic cough or haemoptysis and chest radiography cavitation had 85.7% sensitivity and 99.6% specificity for CPA diagnosis.CPA commonly complicates treated pulmonary TB with residual chest radiography cavitation. Chest radiography alone can exclude CPA. Addition of serology can diagnose CPA with reasonable accuracy.

Siemens Immulite Aspergillus-specific IgG assay for chronic pulmonary aspergillosis diagnosis.

Chronic pulmonary aspergillosis (CPA) complicates underlying lung disease, including treated tuberculosis. Measurement of Aspergillus-specific immunoglobulin G (IgG) is a key diagnostic step. Cutoffs have been proposed based on receiver operating characteristic (ROC) curve analyses comparing CPA cases to healthy controls, but performance in at-risk populations with underlying lung disease is unclear. We evaluated optimal cutoffs for the Siemens Immulite Aspergillus-specific IgG assay for CPA diagnosis in relation to large groups of healthy and diseased controls with treated pulmonary tuberculosis. Sera from 241 patients with CPA attending the UK National Aspergillosis Centre, 299 Ugandan blood donors (healthy controls), and 398 Ugandans with treated pulmonary tuberculosis (diseased controls) were tested. Radiological screening removed potential CPA cases from diseased controls (234 screened diseased controls). ROC curve analyses were performed and optimal cutoffs identified by Youden J statistic. CPA versus control ROC area under curve (AUC) results were: healthy controls 0.984 (95% confidence interval 0.972-0.997), diseased controls 0.972 (0.959-0.985), screened diseased controls 0.979 (0.967-0.992). Optimal cutoffs were: healthy controls 15 mg/l (94.6% sensitivity, 98% specificity), unscreened diseased controls 15 mg/l (94.6% sensitivity, 94.5% specificity), screened diseased controls 25 mg/l (92.9% sensitivity, 98.7% specificity). Results were similar in healthy and diseased controls. We advocate a cutoff of 20 mg/l as this is the midpoint of the range of optimal cutoffs. Cutoffs calculated in relation to healthy controls for other assays are likely to remain valid for use in a treated tuberculosis population.

Case Definition of Chronic Pulmonary Aspergillosis in Resource-Constrained Settings.

Chronic pulmonary aspergillosis (CPA) is a recognized complication of pulmonary tuberculosis (TB). In 2015, the World Health Organization reported 2.2 million new cases of nonbacteriologically confirmed pulmonary TB; some of these patients probably had undiagnosed CPA. In October 2016, the Global Action Fund for Fungal Infections convened an international expert panel to develop a case definition of CPA for resource-constrained settings. This panel defined CPA as illness for >3 months and all of the following: 1) weight loss, persistent cough, and/or hemoptysis; 2) chest images showing progressive cavitary infiltrates and/or a fungal ball and/or pericavitary fibrosis or infiltrates or pleural thickening; and 3) a positive Aspergillus IgG assay result or other evidence of Aspergillus infection. The proposed definition will facilitate advancements in research, practice, and policy in lower- and middle-income countries as well as in resource-constrained settings.

Receiver operating characteristic curve analysis of four Aspergillus-specific IgG assays for the diagnosis of chronic pulmonary aspergillosis.

Measurement of Aspergillus-specific IgG is central to the diagnosis of chronic pulmonary aspergillosis (CPA), but manufacturers' guidance on test interpretation is based on unpublished data. We performed the first receiver operating characteristic (ROC) area under the curve (AUC) analysis to identify optimal cut-offs for this test in relation to European controls. Aspergillus-specific IgG levels were measured in sera from British adults with CPA and European healthy controls by ImmunoCAP, Immulite, Serion and Bio-Rad assays. ROC AUC analysis was performed to identify optimal cut-offs. ROC AUC results were; Bio-Rad 0.955, Immulite 0.948, ImmunoCAP 0.956 and Serion 0.944. Optimal diagnostic cut-offs were 1.5 AU/mL for Bio-Rad (93% sensitive, 98% specific), 25 mg/L for Immulite (93% sensitive, 99% specific), 50 mg/L for ImmunoCAP (84% sensitive, 96% specific) and 50 U/mL for Serion (84% sensitive, 91% specific). These cut-offs differ from manufacturers' guidance and from those previously calculated in relation to Ugandan controls.

Elevated Aspergillus-specific antibody levels among HIV infected Ugandans with pulmonary tuberculosis.

BACKGROUND: The incidence of tuberculosis (TB) is high among human immunodeficiency virus (HIV) infected Ugandans. Recent evidence suggests that Chronic Pulmonary Aspergillosis and Aspergillus sensitisation might be responsible for significant mortality in patients treated for tuberculosis in Uganda. METHODS: We retrieved and tested paired serum aliquots for 101 HIV-TB co-infected patients at the beginning and week 24 of TB treatment. We tested samples for Aspergillus-specific immunoglobulin G (IgG) and immunoglobulin E (IgE) using ImmunoCAP®; and Aspergillus-specific IgG and total serum IgE using Immulite® immunoassays. We compared antibody levels between baseline and week 24, relating them to selected baseline characteristics. RESULTS: 10% of the patients had elevated Aspergillus-specific IgE (Aspergillus sensitization) and Aspergillus-specific IgG antibodies were elevated in 9% of the patients at the end of TB treatment. There was a significant fall in the Aspergillus-specific IgG antibody levels between baseline and week 24 (P = 0.02). Patients with cluster of differentiation 4 (CD4) T-cell count <100 cells/µl and those who were not on anti-retroviral therapy at baseline had more elevated Aspergillus-specific IgG antibodies (P = 0.01, P = 0.03). The ImmunoCAP® Aspergillus-specific IgG antibody titres were higher at week 24 than baseline with more positives at week 24; even though the difference in means was small. However, this difference was statistically significant (P = 0.02). Pulmonary infiltrates were the commonest x-ray abnormality and only 5% of the patients had pulmonary cavities on chest x-ray at week 24. CONCLUSION: These results suggest that Aspergillus infection may complicate active pulmonary TB and further studies including fungal culture and thoracic imaging may now be indicated to measure the prevalence of pulmonary aspergillosis complicating tuberculosis. TRIAL REGISTRATION: The SOUTH trial was registered prospectively. ClinicalTrials.gov Identifier: NCT01782950 ; Registration date: 4th February 2013; Last verified: 13th April 2015.

Aspergillus serology: Have we arrived yet?

Aspergillosis presents in various clinical forms, among them chronic pulmonary aspergillosis, which is a spectrum of disease entities including aspergilloma, chronic cavitary pulmonary aspergillosis, and chronic fibrosing pulmonary aspergillosis. Aspergillus also contributes to fungal allergy and sensitization. Analysis of the immune response to Aspergillus and its antigens is an integral part of the diagnosis of these diseases. Over the past half century, the techniques used to determine antibody titers have evolved from testing for precipitating and agglutinating antibodies by agar gel double diffusion and immunolectrophoresis to enzyme-linked immunosorbent assays using recombinant proteins as capture antigens. A resurgence of interest in the detection of immunoglobulins, primarily Aspergillus-specific IgG, has hinted at the possibility of distinguishing between colonization and invasion in immunocompromised patients with invasive aspergillosis. Even though there appears to be a greater degree of discrimination between the clinical forms of aspergillosis there is still a long way to travel. This review presents illustrative examples of where new diagnostic platforms and technologies have been applied to this intriguing spectrum of diseases.

Comparison of six Aspergillus-specific IgG assays for the diagnosis of chronic pulmonary aspergillosis (CPA).

OBJECTIVES: Chronic pulmonary aspergillosis (CPA) is estimated to affect 3 million persons worldwide. Aspergillus-specific IgG is a key component in CPA diagnosis. We aimed to establish the optimal diagnostic cut offs for CPA and the comparative performance of six assays in this context. METHODS: Sera from 241 patients with CPA and 100 healthy blood donors were tested using five Aspergillus-specific IgG assays plus precipitin testing using Microgen Aspergillus antigens. RESULTS: Receiver operating characteristic (ROC) curve area under the curve (AUC) results were as follows: ThermoFisher Scientific ImmunoCAP 0.996 (95% confidence interval 0.992-1), Siemens Immulite 0.991 (0.982-1), Serion 0.973 (0.960-0.987), Dynamiker 0.918 (0.89-0.946) and Genesis 0.902 (0.871-0.933). Optimal CPA diagnostic cut-offs were; ImmunoCAP 20 mg/L (96% sensitivity, 98% specificity), Immulite 10 mg/L (96% sensitivity, 98% specificity), Serion 35 U/ml (90% sensitivity, 98% specificity), Dynamiker 65 AU/ml (77% sensitivity, 97% specificity) and Genesis 20 U/ml (75% sensitivity, 99% specificity). The precipitin test was 59% sensitive and 100% specific. CONCLUSIONS: ImmunoCAP and Immulite were statistically significantly superior to the other assays. Precipitins testing performed poorly. The currently accepted ImmunoCAP cut-off of 40 mg/L appears sub-optimal for CPA diagnosis and may require revision in this context.

Antibody testing in aspergillosis--quo vadis?

Humans are constantly exposed to airborne Aspergillus spores. Most develop Aspergillus-specific antibodies by adulthood. Persons with chronic lung disease or Aspergillus airway colonization often have raised levels of Aspergillus-specific immunoglobululin G (IgG). It is not known whether this signifies an increased risk of future aspergillosis.Chronic and allergic forms of pulmonary aspergillosis are estimated to affect over three million people worldwide. Antibody testing is central to diagnosis of these conditions, with raised Aspergillus-specific IgG in chronic pulmonary aspergillosis and raised Aspergillus-specific IgE in allergic aspergillosis. Antibody levels are also used to monitor treatment response in these syndromes. Acute invasive disease is less common. There is a more limited role for antibody testing in this setting as immunosuppression often results in delayed or absent antibody response.Many methods exist to detect Aspergillus-specific antibodies, but there are limited published data regarding comparative efficacy and reproducibility. We discuss the comparative merits of the available tests in the various clinical settings and their suitability for use in the resource-poor settings where the majority of cases of aspergillosis are thought to occur. We summarize the gaps in existing knowledge and opportunities for further study that could allow optimal use of antibody testing in this field.