DEVELOPMENT OF NOVEL METHODS FOR THE DIAGNOSIS OF INVASIVE PULMONARY ASPERGILLOSIS

Abstract

Background: Invasive pulmonary aspergillosis (IPA) is a common cause of mortality in haemato-oncology patients and early diagnosis is vital for improving outcomes. Since lung biopsy in this acute setting is rarely performed due to the associated risks, an empirical strategy remains the standard of care in many haematology units, but leads to overtreatment with antifungal drugs, which have significant side-effects. This project has developed novel approaches for detecting IPA, allowing early and specific treatment of genuine fungal infection. Methods: A combination marker approach involving a new Aspergillus qPCR assay, an EORTC/MSG-endorsed GM ELISA and an Aspergillus LFD, was used to establish a robust diagnosis of IPA from clinical broncho-alveolar lavage (BAL) fluid samples. The inflammatory cytokine profile associated with IPA biomarker positive BAL fluid was also evaluated. Finally, antigen and qPCR detection were combined in a proximity ligation assay (PLA), to demonstrate proof-of-principle for a diagnostic assay for the earliest possible detection of fungal infections. Results: A dual testing approach involving a novel MIQE-compliant Aspergillus qPCR assay and an Aspergillus LFD showed a sensitivity and specificity of 100% and 94%, respectively in BAL fluid, unlike in blood where this approach was not sensitive. Results confirmed previously published concerns over the repeatability of GM in serum, whereas BAL GM results appear stable. Biomarker detection results in exhaled breath condensate did not correlate well with results in BAL fluid samples. Respiratory samples did not identify a distinct inflammatory marker profile in IPA. Finally, antibodies raised against JF5 mannoprotein were used to develop a PLA test to detect active growth of Aspergillus. Conclusions: The optimised qPCR is a very sensitive and highly specific aid in IPA diagnosis. A combination biomarker approach could be incorporated into a diagnostic-driven approach to patient management to direct antifungal treatment to patients with evidence of invasive fungal disease.