Use of a newly developed lateral-flow device (Afu-LFD) for urine-based detection of invasive aspergillosis in Haemato-oncology and post-haematopoietic Stem Cell Transplant (HSCT) patients: A proof of concept study.
Aims of the study
To determine the usefulness of a newly developed LFD for urine-based diagnosis of pulmonary IA in children and young people aged < 18 years with an underlying malignancy or undergoing HSCT.
Paediatric patients (<18 years of age) with an underlying malignancy including ALL (induction, consolidation and delayed intensification), relapsed ALL, AML, relapsed AML or post-HSCT who are admitted at any of the participating centres with:
- A signed informed consent form AND Febrile neutropenia lasting ≥ 48h
- Diagnosis of probable/proven IA
- Note: To have previously presented an episode of FN with no diagnosis of probable or proven IFD would not be an exclusion criteria.
- Patients ≥ 18 years of age.
The following data will be collected
Urine samples (volume of 20ml) will be collected, labelled and stored at -20oC, starting on day 0 as defined in the protocol. As much as possible, samples will be taken by clean catch from the first urine in the morning. Samples will be repeated twice a week until end of febrile neutropenia or at discharge for participants with a diagnosis of probable/proven IA. Samples will be shipped in seal tight sample bags, with absorbent material and in dry ice. Samples will be sent in one batch once the target of 30 patients has been reached.
Urine samples stored at -20oC will be thawed, vortexed briefly, and then centrifuged for 10 min at 16,000 g. Five hundred-l of urine sample will be added to an Amicon Ultra-0.5 centrifugal (10kDa cut-off) filter unit, and concentrated 10-fold by centrifugation for 5 min at 14,000 g, followed by buffer exchange with PBS for 8 min at 14,000 g. The concentrated sample, recovered by centrifugation at 1000 g for 2 min, will then be heated at 100oC for 10 min. The cooled sample will be mixed 1:1 (vol:vol) with PBST, and the resultant 100 l volume then added to an Afu-LFD device. The test result will be recorded after 15 min as negative (single internal control (C) line only) or positive (both control (C) and test (T) lines visible) for PD7 protein biomarker.
Diagnostic performance testing
Aspergillus LFD urine test results will be compared to the final patient diagnosis, adjusted based on the reviewed European Organization for Research and Treatment of Cancer/Invasive Fungal Infections Cooperative Group and National Institute of Allergy and Infectious Diseases Mycoses Study Group (EORTC/MSG) definitions7. Proven IA or other IFDs requires documentation of the causative organism by culture and histopathology from invasive tissue samples (e.g. lung biopsy). IA will be considered “probable” with documentation of positive radiological changes on the HRCT-chest and any of the following: (1) culture of Aspergillus species from sputum and/or BAL fluid; (2) documented positivity of BAL GM EIA with indices ≥0.7; and/or (3) documented positivity of serum GM EIA with indices ≥0.5. Patients with clinical criteria in whom IA is suspected but not confirmed by at least 1 of the above mycological criteria will be labelled to have “possible” IA (see variables list in appendix).