key: cord-0730115-2u91umk6 authors: Hoenigl, Martin; Egger, Matthias; Boyer, Johannes; Schulz, Eduard; Prattes, Juergen; Jenks, Jeffrey D. title: Serum Lateral Flow assay with digital reader for the diagnosis of invasive pulmonary aspergillosis: A two‐centre mixed cohort study date: 2021-07-24 journal: Mycoses DOI: 10.1111/myc.13352 sha: 4ca07c22c4804c8064dae6e2da8fdc2698e17771 doc_id: 730115 cord_uid: 2u91umk6 BACKGROUND: Detection of galactomannan (GM) from bronchoalveolar lavage fluid (BALF) or serum is broadly used for diagnosis of invasive aspergillosis (IA), although the sensitivity of GM from serum is lower in non‐neutropenic patients. We evaluated the Aspergillus galactomannan Lateral Flow assay (LFA) with digital readout from serum in a mixed cohort of patients. METHODS: We performed a retrospective two‐centre study evaluating the LFA from serum of patients with clinical suspicion of IA obtained between 2015 and 2021 at the University of California San Diego and the Medical University of Graz. The sensitivity and specificity was calculated for proven/probable aspergillosis versus no aspergillosis. Correlation with same‐sample GM was calculated using Spearman correlation analysis and kappa statistics. RESULTS: In total, 122 serum samples from 122 patients were analysed, including proven IA (n = 1), probable IA or coronavirus‐associated pulmonary aspergillosis (CAPA) (n = 27), and no IA/CAPA/non‐classifiable (n = 94). At a 0.5 ODI cut‐off, the sensitivity and specificity of the LFA was 78.6% and 80.5%. Spearman correlation analysis showed a strong correlation between serum LFA ODI and serum GM ODI (ρ 0.459, p < .0001). Kappa was 0.611 when both LFA and GM were used with a 0.5 ODI cut‐off, showing substantial agreement (p < .001). DISCUSSION: The LFA with digital read out from serum showed good performance for the diagnosis of probable/proven aspergillosis, with substantial agreement to GM from serum. Like the LFA from BALF, the LFA from serum may serve as a more rapid test compared to conventional GM, particularly in settings where GM is not readily available. Invasive aspergillosis (IA) continues to emerge as a major cause of morbidity and mortality, including among non-traditional risk groups such as critically ill patients in the intensive care unit (ICU), 1 including those with severe coronavirus disease 2019 (COVID- 19) infection. 2 Although microscopy and culture are the gold standard for the diagnosis of IA, sensitivity is limited 3 and therefore the detection of the fungal cell wall component galactomannan (GM) from bronchoalveolar lavage fluid (BALF) or serum 4 is commonly used (sensitivity 78% and specificity 85% at 0.5 cut-off, sensitivity 71% and specificity 90% at 1.0 cut-off 4 ), although the sensitivity of GM from serum is low in non-neutropenic patients and those with SARS-CoV-2 associated pulmonary aspergillosis (CAPA). 2, 5, 6 Furthermore, the sensitivity of GM from blood declines in those on antifungal prophylaxis or treatment. 5, 7, 8 Other molecular tests such as polymerase chain reaction lack standardisation, have variable diagnostic performance across studies and settings, with declining performance in those on mould-active prophylaxis. 9 Thus, there is a need for improved diagnostics from blood for IA. The performance of the IMMY sōna Aspergillus GM Lateral Flow assay (LFA; IMMY) with manual readout in BALF demonstrated a sensitivity and specificity of 77% and 81%, respectively. [10] [11] [12] [13] A recent multicentre study in a mixed cohort of patients found a sensitivity of 74% and specificity of 83% with digital readout from BALF at an optical density cut-off (ODI) of 1.5, with stable test performance across centres and patient groups. 14 In serum, the LFA with digital readout was evaluated in three studies focusing exclusively on patients with haematologic malignancy, with one study reporting a sensitivity of 49% and a specificity of 95%, 15 and two reporting higher sensitivities of 96.9% 16 and 90.9% 17 with specificities between 90% and 96% at an ODI of ≥0.5. However, evaluation of the LFA test with digital readout from serum outside the haematologic malignancy setting is lacking. We performed a retrospective two-centre study evaluating the LFA with digital readout from serum in a mixed cohort of patients. Patients who did not fall into either of these categories but who had clinical, radiological and mycological evidence of IA were categorised as non-classifiable. GM (Platelia Aspergillus Ag ELISA; Bio-Rad Laboratories) was routinely and prospectively performed in the majority of samples at each participating centre before samples were stored at −70°C for up to 5 years. Between January 2020 and April 2021 these serum samples where thawed, vortexed, and tested with the LFA according to the manufacturer's instructions, as previously described. 14 Test lines intensities were first read by naked eye by two investigators blinded to disease classification and then by an automated cube reader that was included with the test kits and displayed in ODI. 14 Statistical analyses were performed using SPSS 25 (SPSS Inc.). For continuous data, including LFA and GM ODIs, receiver operating characteristic curve analyses were performed and area under the curve (AUC) values were presented including 95% confidence intervals (95% CI) for the outcomes proven/probable aspergillosis (vs. no aspergillosis) in the overall study cohort and sub-cohorts. LFA ODI cut-offs of 0.5 ODI and 1.0 ODI were compared by calculating sensitivity and specificity for aspergillosis (ie fulfilling criteria of probable or proven) versus no aspergillosis (exclusion of cases that were non-classifiable). Correlation between serum GM and LFA ODIs was calculated using Spearman rho correlation analysis due to the nonnormal distributions as well as Kappa statistics. Two-sided p < 0.05 was taken as cut-off for statistical significance. The study protocol and all study-related procedures were approved by the Human Research Protections Program at UCSD (IRB project no. 171104), and the Medical University of Graz (EC no. 23-343). Of 122 serum samples, 50 were from the UCSD and 72 from the Medical University of Graz. The LFA produced valid results in all samples. One patient fulfilled criteria of proven IA, 27 probable IA or CAPA, and 87 did not fulfil criteria for IA or CAPA. Seven patients were non-classifiable. Figure 2 ). Kappa was 0.611 when both LFA and GM were used with a 0.5 ODI cut-off, showing substantial agreement (p < .001). Thirty-eight per cent (46/122) were receiving mould-active antifungal prophylaxis or treatment at the time of serum sampling but this did not impact discriminatory power of the LFA (AUC 0.900, 95%CI 0.795-1.000, n = 13 with probable/proven disease). We evaluated the LFA from serum in a mixed patient cohort and found that when used with the automated reader the LFA from serum showed a strong correlation with serum GM and a good performance for diagnosing probable/proven aspergillosis. At a 0.5 ODI cut-off the overall sensitivity and specificity was 78.6% and 80.5%, respectively, in differentiating probable/proven disease versus no aspergillosis with similar performance in patients without haematological malignancies (eg, 83.3% sensitivity and 75.9% specificity in those with other established risk factors for IA but without haematologic malignancy), which represented 55% of our study cohort. Previously the LFA with automated read out was evaluated exclusively in serum samples from patients with haematological malignancies, where two studies found higher sensitivity and specificity 16, 17 and one a lower sensitivity, compared to our findings. 14 The serum LFA has several advantages over GM ELISA testing, including that it allows for single sample testing, requires minimal laboratory equipment so can potentially be performed at a lower cost than ELISA testing, and has shorter turn-around times, making it an attractive option for smaller centres that don't test GM in house. It has also shown promise in combination with other diagnostic tests and immunological markers. The results of the LFA from serum at a 0.5 ODI cut-off in our mixed cohort are slightly superior to those of a recent multicentre study evaluating the LFA from BALF in a mixed cohort of patients, where the sensitivity was 74% and specificity 83% at an ODI of 1.5. 10 In contrast to BALF, where a higher ODI cut-off may be warranted to increase test performance, a 0.5 ODI cut-off may be optimal in serum samples, mirroring similar findings for GM. This is not surprising given the strong correlation and substantial agreement between serum LFA ODI and serum GM ODI found in our study and other studies before. 10, 15 Finally, we found the automated reader increased the reliability and test performance compared to manual read. Our study has several limitations including the low number of proven cases of aspergillosis. In addition, this study was performed with banked serum samples that were tested after they were stored and frozen. The authors wish to acknowledge Marina Linhofers support as research coordinator. Aspergillus GM LFAs were provided by IMMY, Norman, Oklahoma, USA. They had no role in the study design, data collection, analysis, interpretation, decision to publish, in the writing of the manuscript, or in the decision to submit the manuscript for publication. MH received research funding from Gilead, Astellas, Scynexis, Data is available on request. EORTC/MSGERC definitions of invasive fungal diseases: summary of activities of the Intensive Care Unit Working Group COVID-19 Associated Pulmonary Aspergillosis (CAPA)-from immunology to treatment Diagnosis of invasive aspergillosis in hematological malignancy patients: performance of cytokines, Asp LFD, and Aspergillus PCR in same day blood and bronchoalveolar lavage samples Galactomannan detection for invasive aspergillosis in immunocompromised patients Invasive aspergillosis in critically ill patients: review of definitions and diagnostic approaches Defining and managing COVID-19-associated pulmonary aspergillosis: the 2020 ECMM/ ISHAM consensus criteria for research and clinical guidance Antifungal therapy decreases sensitivity of the Aspergillus galactomannan enzyme immunoassay Galactomannan testing and Aspergillus PCR in same-day bronchoalveolar lavage and blood samples for diagnosis of invasive aspergillosis Blood Aspergillus PCR: the good, the bad, and the ugly Point-of-care diagnostics for invasive aspergillosis: nearing the finish line Lateral flow assays for diagnosing invasive pulmonary aspergillosis in adult hematology patients: a comparative multicenter study Bronchoalveolar lavage Aspergillus galactomannan lateral flow assay versus Aspergillus-specific lateral flow device test for diagnosis of invasive pulmonary aspergillosis in patients with hematological malignancies Pointof-care diagnosis of invasive aspergillosis in non-neutropenic patients: Aspergillus Galactomannan Lateral Flow Assay versus Aspergillus-specific Lateral Flow Device test in bronchoalveolar lavage Performance of the bronchoalveolar lavage fluid Aspergillus Galactomannan Lateral Flow Assay with cube reader for diagnosis of invasive pulmonary aspergillosis: a multicenter cohort study Prospective evaluation of the turbidimetric β-D-glucan assay and two lateral flow assays on serum in invasive aspergillosis Evaluation of the performance of the IMMY sona Aspergillus galactomannan lateral flow assay when testing serum to aid in diagnosis of invasive aspergillosis Serum Aspergillus galactomannan lateral flow assay for the diagnosis of invasive aspergillosis: a single-centre study Revision and update of the consensus definitions of invasive fungal disease from the European Organization for Research and Treatment of Cancer and the Mycoses Study Group Education and Research Consortium COVID-19-associated pulmonary aspergillosis Invasive fungal disease complicating COVID-19: when it rains it pours ECMM/ISHAM recommendations for clinical management of COVID-19 associated mucormycosis in low-and middle-income countries The emergence of COVID-19 associated mucormycosis: analysis of cases from 18 countries COVID-19-Associated Candidiasis (CAC): an underestimated complication in the absence of immunological predispositions? Accuracy of galactomannan testing on tracheal aspirates in COVID-19-associated pulmonary aspergillosis Diagnosis and treatment of COVID-19 associated pulmonary apergillosis in critically ill patients: results from a European confederation of medical mycology registry Serum Lateral Flow assay with digital reader for the diagnosis of invasive pulmonary aspergillosis: A two-centre mixed cohort study