Magnesium sensing via LFA-1 regulates CD8+ T cell effector function.

The relevance of extracellular magnesium in cellular immunity remains largely unknown. Here, we show that the co-stimulatory cell-surface molecule LFA-1 requires magnesium to adopt its active conformation on CD8+ T cells, thereby augmenting calcium flux, signal transduction, metabolic reprogramming, immune synapse formation, and, as a consequence, specific cytotoxicity. Accordingly, magnesium-sufficiency sensed via LFA-1 translated to the superior performance of pathogen- and tumor-specific T cells, enhanced effectiveness of bi-specific T cell engaging antibodies, and improved CAR T cell function. Clinically, low serum magnesium levels were associated with more rapid disease progression and shorter overall survival in CAR T cell and immune checkpoint antibody-treated patients. LFA-1 thus directly incorporates information on the composition of the microenvironment as a determinant of outside-in signaling activity. These findings conceptually link co-stimulation and nutrient sensing and point to the magnesium-LFA-1 axis as a therapeutically amenable biologic system.

Diapedesis-Induced Integrin Signaling via LFA-1 Facilitates Tissue Immunity by Inducing Intrinsic Complement C3 Expression in Immune Cells.

Intrinsic complement C3 activity is integral to human T helper type 1 (Th1) and cytotoxic T cell responses. Increased or decreased intracellular C3 results in autoimmunity and infections, respectively. The mechanisms regulating intracellular C3 expression remain undefined. We identified complement, including C3, as among the most significantly enriched biological pathway in tissue-occupying cells. We generated C3-reporter mice and confirmed that C3 expression was a defining feature of tissue-immune cells, including T cells and monocytes, occurred during transendothelial diapedesis, and depended on integrin lymphocyte-function-associated antigen 1 (LFA-1) signals. Immune cells from patients with leukocyte adhesion deficiency type 1 (LAD-1) had reduced C3 transcripts and diminished effector activities, which could be rescued proportionally by intracellular C3 provision. Conversely, increased C3 expression by T cells from arthritis patients correlated with disease severity. Our study defines integrins as key controllers of intracellular complement, demonstrates that perturbations in the LFA-1-C3-axis contribute to primary immunodeficiency, and identifies intracellular C3 as biomarker of severity in autoimmunity.

Eomes-Dependent Loss of the Co-activating Receptor CD226 Restrains CD8+ T Cell Anti-tumor Functions and Limits the Efficacy of Cancer Immunotherapy.

CD8+ T cells within the tumor microenvironment (TME) are exposed to various signals that ultimately determine functional outcomes. Here, we examined the role of the co-activating receptor CD226 (DNAM-1) in CD8+ T cell function. The absence of CD226 expression identified a subset of dysfunctional CD8+ T cells present in peripheral blood of healthy individuals. These cells exhibited reduced LFA-1 activation, altered TCR signaling, and a distinct transcriptomic program upon stimulation. CD226neg CD8+ T cells accumulated in human and mouse tumors of diverse origin through an antigen-specific mechanism involving the transcriptional regulator Eomesodermin (Eomes). Despite similar expression of co-inhibitory receptors, CD8+ tumor-infiltrating lymphocyte failed to respond to anti-PD-1 in the absence of CD226. Immune checkpoint blockade efficacy was hampered in Cd226-/- mice. Anti-CD137 (4-1BB) agonists also stimulated Eomes-dependent CD226 loss that limited the anti-tumor efficacy of this treatment. Thus, CD226 loss restrains CD8+ T cell function and limits the efficacy of cancer immunotherapy.

How integrin phosphorylations regulate cell adhesion and signaling.

Cell adhesion is essential for the formation of organs, cellular migration, and interaction with target cells and the extracellular matrix. Integrins are large protein α/ß-chain heterodimers and form a major family of cell adhesion molecules. Recent research has dramatically increased our knowledge of how integrin phosphorylations regulate integrin activity. Phosphorylations determine the signaling complexes formed on the cytoplasmic tails, regulating downstream signaling. α-Chain phosphorylation is necessary for inducing ß-chain phosphorylation in LFA-1, and the crosstalk from one integrin to another activating or inactivating its function is in part mediated by phosphorylation of ß-chains. The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) virus receptor angiotensin-converting enzyme 2 (ACE2) and possible integrin coreceptors may crosstalk and induce a phosphorylation switch and autophagy.

Extracellular ISG15 Signals Cytokine Secretion through the LFA-1 Integrin Receptor.

ISG15 is a ubiquitin-like protein that functions in innate immunity both as an intracellular protein modifier and as an extracellular signaling molecule that stimulates IFN-γ secretion. The extracellular function, important for resistance to mycobacterial disease, has remained biochemically uncharacterized. We have established an NK-92 cell-based assay for IFN-γ release, identified residues critical for ISG15 signaling, and identified the cell surface receptor as LFA-1 (CD11a/CD18; αLß2 integrin). LFA-1 inhibition blocked IFN-γ secretion, splenocytes from CD11a-/- mice did not respond to ISG15, and ISG15 bound directly to the αI domain of CD11a in vitro. ISG15 also enhanced secretion of IL-10, indicating a broader role for ISG15 in cytokine signaling. ISG15 engagement of LFA-1 led to the activation of SRC family kinases (SFKs) and SFK inhibition blocked cytokine secretion. These findings establish the molecular basis of the extracellular function of ISG15 and the initial outside-in signaling events that drive ISG15-dependent cytokine secretion.

Carba PBP: a novel penicillin-binding protein-based lateral flow assay for rapid phenotypic detection of carbapenemase-producing Enterobacterales.

Rapid phenotypic detection assays, including Carba NP and its variants, are widely applied for clinical diagnosis of carbapenemase-producing Enterobacterales (CPE). However, these tests are based on the acidification of the pH indicator during carbapenem hydrolysis, which limits test sensitivity and speed, especially for the detection of CPE producing low-activity carbapenem (e.g., OXA-48 variants). Herein, we developed a novel rapid and sensitive CPE detection method (Carba PBP) that could measure substrate (meropenem) consumption based on penicillin-binding protein (PBP). Meropenem-specific PBP was used to develop a competitive lateral flow assay (LFA) for meropenem identification. For the detection of carbapenemase activity, meropenem concentration was optimized using a checkerboard assay. The performance of Carba PBP was evaluated and compared with that of Carba NP using a panel of 94 clinical strains characterized by whole-genome sequencing and carbapenem susceptibility test. The limit of detection of PBP-based LFA for meropenem identification was 7 ng mL-1. Using 10 ng mL-1 meropenem as the substrate, Carba PBP and Carba NP could detect 10 ng mL-1 carbapenemase within 25 min and 1,280 ng mL-1 CPE in 2 h, respectively. The sensitivity and specificity were 100% (75/75) and 100% (19/19) for Carba PBP and 85.3% (64/75) and 100% (19/19) for Carba NP, respectively. When compared with Carba NP, Carba PBP showed superior performance in detecting all the tested CPE strains (including OXA-48-like variants) within 25 min and presented two orders of magnitude higher analytical sensitivity, demonstrating potential for clinical diagnosis of CPE. IMPORTANCE This study successfully achieved the goal of carbapenemase activity detection with both high sensitivity and convenience, offering a convenient lateral flow assay for clinical diagnosis of carbapenemase-producing Enterobacterales.

LFA-1 in T cell priming, differentiation, and effector functions.

The integrin LFA-1 is crucial for T cell entry into mammalian lymph nodes and tissues, and for promoting interactions with antigen-presenting cells (APCs). However, it is increasingly evident that LFA-1 has additional key roles beyond the mere support of adhesion between T cells, the endothelium, and/or APCs. These include roles in homotypic T cell-T cell (T-T) communication, the induction of intracellular complement activity underlying Th1 effector cell polarization, and the support of long-lasting T cell memory. Here, we briefly summarize current knowledge of LFA-1 biology, discuss novel cytoskeletal regulators of LFA-1 functions, and review new aspects of LFA-1 mechanobiology that are relevant to its function in immunological synapses and in specific pathologies arising from LFA-1 dysregulation.

Integrin activation enables rapid detection of functional Vδ1+ and Vδ2+ γδ T cells.

Conformational change of the ß2 integrin lymphocyte function-associated antigen 1 (LFA-1) is an early marker of T cell activation. A protocol using the mAb clone m24 recognizing the active, extended high-affinity conformation has been previously described for the assessment of functional CD4+ and CD8+ T cells in response to MHC-peptide stimulation. We investigated the applicability of the m24 mAb to detect the activation of γδ T cells in response to different soluble and immobilized stimuli. m24 mAb staining was associated with the expression of cytokines and was detectable as early as 10 min after stimulation, but with different kinetics depending on the nature of the stimulus. Hence, we conclude that this assay is suitable for the detection of functional γδ T cells and allows the assessment of activation more rapidly than alternative methods such as cytokine detection. Intracellular staining, protein trafficking inhibitors, or prior knowledge of the stimulating moiety recognized are no longer required for monitoring γδ T cell activation.

Development of a rapid lateral flow assay for detection of anti-coccidioidal antibodies.

Coccidioides spp. are dimorphic fungi that are capable of infecting human and non-human mammals and can cause diverse manifestations of coccidioidomycosis or Valley fever (VF). In combination with clinical symptoms and radiographic findings, antibody-based diagnostic tests are often used to diagnose and monitor patients with VF. Chitinase 1 (CTS1) has previously been identified as the seroreactive antigen used in these diagnostic assays to detect anticoccidial IgG. Here, an indirect enzyme-linked immunosorbent assay to detect IgG to CTS1 demonstrated 165 of 178 (92.7%) patients with a positive result by immunodiffusion (ID) and/or complement fixation (CF) had antibodies to the single antigen CTS1. We then developed a rapid antibody lateral flow assay (LFA) to detect anti-CTS1 antibodies. Out of 143 samples tested, the LFA showed 92.9% positive percent agreement [95% confidence interval (CI), 84.3%-96.9%] and 97.7% negative percent agreement (95% CI, 87.9%-99.6%) with ID and CF assays. Serum or plasma from canines, macaques, and dolphins was also tested by the CTS1 LFA. Test line densities of the CTS1 LFA correlated in a linear manner with the reported CF and ID titers for human and non-human samples, respectively. This 10-min point-of-care test for the rapid detection of anti-coccidioidal antibodies could help to inform healthcare providers in real-time, potentially improving the efficiency of healthcare delivery.

Magnesium enhances the graft-versus-tumor effect of donor lymphocytic infusion on hematologic malignancies.

Donor lymphocyte infusion (DLI) cures relapsed hematologic malignancies after allogeneic hematopoietic stem cell transplantation through the graft-versus-tumor (GVT) effect. Although the important role of magnesium in enhancing immunity has been mentioned in studies, limited clinical data have explored how magnesium affects the efficacy of DLI. Besides, although laboratory data demonstrate that magnesium can enhance CD8+ T cells effector function, whether magnesium regulates the tumor killing effect of peripheral blood mononuclear cells (PBMCs) remains to be explored. Here, for the retrospective study, we collected clinical data of relapsed patients receiving DLI and explored the relationship between different serum magnesium levels and patient outcomes. For in vitro studies, we investigated the effect of magnesium on the cytotoxicity of DLI cells which were PBMCs and preliminarily explored the mechanism. Eighty-one patients were enrolled in this study. It was found that the high post-DLI magnesium level was significantly associated with a higher incidence of complete remission (CR) or partial remission (CR/PR) and a higher possibility of survival. The magnesium level after DLI was an independent risk factor of overall survival. In vitro studies proved that increased magnesium enhanced the cytotoxic function of PBMCs on hematologic malignancies. Besides, magnesium modulated LFA-1 headpiece opening. When blocking the integrin-ligand interaction between LFA-1 and ICAM-1, the regulation effect of magnesium on PBMCs was weakened. Therefore, it was possible that magnesium regulated PBMCs effector function by stimulating LFA-1. These results show that serum magnesium levels affect immunological responses mediated by donor lymphocytes in hematologic malignancies.

LFA-1 knockout inhibited the tumor growth and is correlated with treg cells.

Cancer immunotherapy has been proven to be clinically effective in multiple types of cancers. Lymphocyte function-associated antigen 1 (LFA-1), a member of the integrin family of adhesion molecules, is expressed mainly on αß T cells. LFA-1 is associated with tumor immune responses, but its exact mechanism remains unknown. Here, two kinds of mice tumor model of LFA-1 knockout (LFA-1-/-) mice bearing subcutaneous tumor and Apc Min/+;LFA-1-/- mice were used to confirm that LFA-1 knockout resulted in inhibition of tumor growth. Furthermore, it also demonstrated that the numbers of regulatory T cells (Treg cells) in the spleen, blood, mesenteric lymph nodes were decreased in LFA-1-/- mice, and the numbers of Treg cells in mesenteric lymph nodes were also decreased in Apc Min/+;LFA-1-/- mice compared with Apc Min/+ mice. LFA-1 inhibitor (BIRT377) was administered to subcutaneous tumor-bearing LFA-1+/+ mice, and the results showed that the tumor growth was inhibited and the number of Treg cells was reduced. The analysis of TIMER tumor database indicated that LFA-1 expression is positively associated with Treg cells and TNM stage. Conclusively, this suggests that LFA-1 knockout would inhibit tumor growth and is correlated with Treg cells. LFA-1 may be one potential target for cancer immunotherapy. Video Abstract.

A review of rapid food safety testing: using lateral flow assay platform to detect foodborne pathogens.

The detrimental impact of foodborne pathogens on human health makes food safety a major concern at all levels of production. Conventional methods to detect foodborne pathogens, such as live culture, high-performance liquid chromatography, and molecular techniques, are relatively tedious, time-consuming, laborious, and expensive, which hinders their use for on-site applications. Recurrent outbreaks of foodborne illness have heightened the demand for rapid and simple technologies for detection of foodborne pathogens. Recently, Lateral flow assays (LFA) have drawn attention because of their ability to detect pathogens rapidly, cheaply, and on-site. Here, we reviewed the latest developments in LFAs to detect various foodborne pathogens in food samples, giving special attention to how reporters and labels have improved LFA performance. We also discussed different approaches to improve LFA sensitivity and specificity. Most importantly, due to the lack of studies on LFAs for the detection of viral foodborne pathogens in food samples, we summarized our recent research on developing LFAs for the detection of viral foodborne pathogens. Finally, we highlighted the main challenges for further development of LFA platforms. In summary, with continuing improvements, LFAs may soon offer excellent performance at point-of-care that is competitive with laboratory techniques while retaining a rapid format.

Self-directed molecular diagnostics (SdMDx) system for COVID-19 via one-pot processing.

Rapid, sensitive, and specific detection of the severe acute respiratory syndrome coronavirus (SARS-CoV)- 2 during early infection is pivotal in controlling the spread and pathological progression of Coronavirus Disease 2019 (COVID-19). Thus, highly accurate, affordable, and scalable point-of-care (POC) diagnostic technologies are necessary. Herein, we developed a rapid and efficient self-directed molecular diagnostic (SdMDx) system for SARS-CoV-2. This system combines the sample preparation step, including virus enrichment and extraction processes, which involve dimethyl suberimidate dihydrochloride and diatomaceous earth functionalized with 3-aminopropyl(diethoxy)methylsilane, and the detection step using loop-mediated isothermal amplification-lateral flow assay (LAMP-LFA). Using the SdMDx system, SARS-CoV-2 could be detected within 47 min by hand without the need for any larger instruments. The SdMDx system enabled detection as low as 0.05 PFU in the culture fluid of SARS-CoV-2-infected VeroE6 cells. We validated the accuracy of the SdMDx system on 38 clinical nasopharyngeal specimens. The clinical utility of the SdMDx system for targeting the S gene of SARS-CoV-2 showed 94.4% sensitivity and 100% specificity. This system is more sensitive than antigen and antibody assays, and it minimizes the use of complicated processes and reduces contamination risks. Accordingly, we demonstrated that the SdMDx system enables a rapid, accurate, simple, efficient, and inexpensive detection of SARS-CoV-2 at home, in emergency facilities, and in low-resource sites as a pre-screening platform and POC testing through self-operation and self-diagnosis.

Fluorophore-encapsulated nanobeads for on-site, rapid, and sensitive lateral flow assay.

Since December 2019, the rapid and sensitive detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has become a priority for public health. Although the lateral flow assay (LFA) sensor has emerged as a rapid and on-site SARS-CoV-2 detection technique, the conventional approach of using gold nanoparticles for the signaling probe had limitations in increasing the sensitivity of the sensor. Herein, our newly suggested methodology to improve the performance of the LFA system could amplify the sensor signal with a facile fabrication method by concentrating fluorescent organic molecules. A large Stokes shift fluorophore (single benzene) was encapsulated into polystyrene nanobeads to enhance the fluorescence intensity of the probe for LFA sensor, which was detected on the test line with a longpass filter under ultraviolet light irradiation. This approach provides comparatively high sensitivity with the limit of detection of 1 ng mL-1 for the SARS-CoV-2 spike protein and a fast detection process, which takes less than 20 min. Furthermore, our sensor showed higher performance than gold nanoparticle-based commercial rapid diagnostics test kits in clinical tests, proving that this approach is more suitable and reliable for the sensitive and rapid detection of viruses, bacteria, and other hazardous materials.

Proteomic analysis in primary T cells reveals IL-7 alters T cell receptor thresholding via CYTIP/cytohesin/LFA-1 localisation and activation.

The ability of the cellular immune system to discriminate self from foreign antigens depends on the appropriate calibration of the T cell receptor (TCR) signalling threshold. The lymphocyte homeostatic cytokine interleukin 7 (IL-7) is known to affect TCR thresholding, but the molecular mechanism is not fully elucidated. A better understanding of this process is highly relevant in the context of autoimmune disease therapy and cancer immunotherapy. We sought to characterise the early signalling events attributable to IL-7 priming; in particular, the altered phosphorylation of signal transduction proteins and their molecular localisation to the TCR. By integrating high-resolution proximity- phospho-proteomic and imaging approaches using primary T cells, rather than engineered cell lines or an in vitro expanded T cell population, we uncovered transduction events previously not linked to IL-7. We show that IL-7 leads to dephosphorylation of cytohesin interacting protein (CYTIP) at a hitherto undescribed phosphorylation site (pThr280) and alters the co-localisation of cytohesin-1 with the TCR and LFA-1 integrin. These results show that IL-7, acting via CYTIP and cytohesin-1, may impact TCR activation thresholds by enhancing the co-clustering of TCR and LFA-1 integrin.

Comparative assessment of homemade ELISA and lateral flow assay (LFA)in the rapid, specific and sensitive detection of SARS-CoV-2 anti-nucleocapsid protein in sera of Egyptian patients.

Several diagnostic measures have been employed to precisely detect the SARS-CoV-2 viral infection using viral antigens, nucleic acids, and other serological approaches. The sensitivity and specificity of the serological tests remain a challenging need. Here, we describe the detection of human anti-SARS-CoV-2 IgG and IgM antibodies qualitatively through two optimized in-house ELISA and lateral flow immunoassay. Both approaches are based on the prokaryotic expression of 50 kDa SARS-CoV-2 recombinant nucleocapsid protein. This SARS-CoV-2rN-6×His was used either to coat ELISA plates or to be conjugated to gold nanoparticles followed by colorimetric detection of bound human IgG or IgM. In the LFA, we show the optimization of nanoparticle size, protein-binding capacity, membrane treatment, and finally testing the potential capacity of using either the optimized ELISA or LFA in detecting antibodies raised against viral infection. Assessment of both methods was carried out using human sera-positive and negative SARS-CoV-2 antibodies. The ELISA and LFA tests showed 86%, 96.5% sensitivity, 92%, 93.75% specificity, 97%, 98.2% PPV, and 64%, 88.2% NPV, respectively. In conclusion, both approaches were able to successfully detect human antibodies against SARS-CoV-2 nucleocapsid protein. The importance of both protocols cannot be overstated in the detection and diagnosis of viral infections, especially in developing countries.

Implementation of Lateral Flow Assays for the Diagnosis of Invasive Aspergillosis in European Hospitals: A Survey from Belgium and a Literature Review of Test Performances in Different Patient Populations.

OBJECTIVES: Diagnosis of invasive aspergillosis is based on a combination of criteria, of which the detection of Aspergillus galactomannan (GM) often is decisive. To date, the most commonly used method to determine GM is an enzyme-linked immune assay (EIA). But since a few years lateral flow assays (LFAs) were introduced, providing the possibility for rapid single sample testing. More and more LFAs are entering the market, but, although often being equated, all use their own antibodies, procedures and interpretation criteria. A recent European survey revealed that about 24-33% of laboratories implemented a lateral flow assay on-site. METHODS: We conducted a survey at 81 Belgian hospital laboratories regarding the implementation of LFAs in their centre. In addition, we performed an extensive review of all publicly available studies on the performance of lateral flow assays to diagnose invasive aspergillosis. RESULTS: Response rate to the survey was 69%. Of the 56 responding hospital laboratories, 6 (11%) used an LFA. The Soña Aspergillus galactomannan LFA (IMMY, Norman, Oklahoma, USA) was used in 4/6 centres, while two centres used the QuicGM (Dynamiker, Tianjin, China) and one centre used the FungiXpert Aspergillus Galactomannan Detection K-set LFA (Genobio [Era Biology Technology], Tianjin, China). One centre used 2 distinct LFAs. In 3/6 centres, the sample is sent to another lab for confirmation with GM-EIA when the LFA result is positive and in 2/6 when the LFA results is negative. In one centre, a confirmatory GM-EIA is always performed in house. In three centres the LFA result is used as a complete substitute for GM-EIA. Available LFA performance studies are very diverse and results vary in function of the study population and type of LFA. Apart from the IMMY and OLM LFA, only very limited performance data are available. From two out of three LFAs used in Belgium, no clinical performance studies are published in literature. CONCLUSIONS: A large variety of LFAs are used in Belgian Hospitals, some of which no clinical validation studies are published. These results do likely have implications for other parts of Europe and for the rest of the world as well. Due to the variable performance of LFA tests and the limited validation data available, each laboratory must check the available performance information of the specific test considered for implementation. In addition, laboratories should perform an implementation verification study.

The Role of LFA-1 for the Differentiation and Function of Regulatory T Cells-Lessons Learned from Different Transgenic Mouse Models.

Regulatory T cells (Treg) are essential for the maintenance of peripheral tolerance. Treg dysfunction results in diverse inflammatory and autoimmune diseases with life-threatening consequences. ß2-integrins (CD11a-d/CD18) play important roles in the migration of leukocytes into inflamed tissues and cell signaling. Of all ß2-integrins, T cells, including Treg, only express CD11a/CD18, termed lymphocyte function-associated antigen 1 (LFA-1), on their surface. In humans, loss-of-function mutations in the common subunit CD18 result in leukocyte adhesion deficiency type-1 (LAD-1). Clinical symptoms vary depending on the extent of residual ß2-integrin function, and patients may experience leukocytosis and recurrent infections. Some patients can develop autoimmune diseases, but the immune processes underlying the paradoxical situation of immune deficiency and autoimmunity have been scarcely investigated. To understand this complex phenotype, different transgenic mouse strains with a constitutive knockout of ß2-integrins have been established. However, since a constitutive knockout affects all leukocytes and may limit the validity of studies focusing on their cell type-specific role, we established a Treg-specific CD18-floxed mouse strain. This mini-review aims to delineate the role of LFA-1 for the induction, maintenance, and regulatory function of Treg in vitro and in vivo as deduced from observations using the various ß2-integrin-deficient mouse models.

Lymphocytes perform reverse adhesive haptotaxis mediated by LFA-1 integrins.

Cell guidance by anchored molecules, or haptotaxis, is crucial in development, immunology and cancer. Adhesive haptotaxis, or guidance by adhesion molecules, is well established for mesenchymal cells such as fibroblasts, whereas its existence remains unreported for amoeboid cells that require less or no adhesion in order to migrate. We show that, in vitro, amoeboid human T lymphocytes develop adhesive haptotaxis mediated by densities of integrin ligands expressed by high endothelial venules. Moreover, lymphocytes orient towards increasing adhesion with VLA-4 integrins (also known as integrin α4ß1), like all mesenchymal cells, but towards decreasing adhesion with LFA-1 integrins (also known as integrin αLß4), which has not previously been observed. This counterintuitive 'reverse haptotaxis' cannot be explained by existing mechanisms of mesenchymal haptotaxis involving either competitive anchoring of cell edges under tension or differential integrin-activated growth of lamellipodia, because they both favor orientation towards increasing adhesion. The mechanisms and functions of amoeboid adhesive haptotaxis remain unclear; however, multidirectional integrin-mediated haptotaxis might operate around transmigration ports on endothelia, stromal cells in lymph nodes, and inflamed tissue where integrin ligands are spatially modulated.

Aspergillus Lateral Flow Assay with Digital Reader for the Diagnosis of COVID-19-Associated Pulmonary Aspergillosis (CAPA): a Multicenter Study.

This multicenter study evaluated the IMMY Aspergillus Galactomannan Lateral Flow Assay (LFA) with automated reader for diagnosis of pulmonary aspergillosis in patients with COVID-19-associated acute respiratory failure (ARF) requiring intensive care unit (ICU) admission between 03/2020 and 04/2021. A total of 196 respiratory samples and 148 serum samples (n = 344) from 238 patients were retrospectively included, with a maximum of one of each sample type per patient. Cases were retrospectively classified for COVID-19-associated pulmonary aspergillosis (CAPA) status following the 2020 consensus criteria, with the exclusion of LFA results as a mycological criterion. At the 1.0 cutoff, sensitivity of LFA for CAPA (proven/probable/possible) was 52%, 80% and 81%, and specificity was 98%, 88% and 67%, for bronchoalveolar lavage fluid (BALF), nondirected bronchoalveolar lavage (NBL), and tracheal aspiration (TA), respectively. At the 0.5 manufacturer's cutoff, sensitivity was 72%, 90% and 100%, and specificity was 79%, 83% and 44%, for BALF, NBL and TA, respectively. When combining all respiratory samples, the receiver operating characteristic (ROC) area under the curve (AUC) was 0.823, versus 0.754, 0.890 and 0.814 for BALF, NBL and TA, respectively. Sensitivity and specificity of serum LFA were 20% and 93%, respectively, at the 0.5 ODI cutoff. Overall, the Aspergillus Galactomannan LFA showed good performances for CAPA diagnosis, when used from respiratory samples at the 1.0 cutoff, while sensitivity from serum was limited, linked to weak invasiveness during CAPA. As some false-positive results can occur, isolated results slightly above the recommended cutoff should lead to further mycological investigations.