key: cord-0818720-sgop2bpf authors: Michel, M.; Malergue, F.; Ait-Belkacem, I.; Bourgoin, P.; Morange, P.-E.; Arnoux, I.; Miloud, T.; Million, M.; Tissot-Dupont, H.; Mege, J.-L.; Busnel, J.-M.; Vitte, J. title: An ultra-sensitive, ultra-fast whole blood monocyte CD169 assay for COVID-19 screening date: 2020-10-26 journal: nan DOI: 10.1101/2020.10.22.20215749 sha: 74e2dcf04627ac0a2a51f4e54db21f9e3af88cf5 doc_id: 818720 cord_uid: sgop2bpf CoVID-19 is an unprecedented epidemic, globally challenging health systems, societies, and economy. Its diagnosis relies on molecular methods, with drawbacks revealed by current use as mass screening. Monocyte CD169 upregulation has been reported as a marker of viral infections, we evaluated a flow cytometry three-color rapid assay of whole blood monocyte CD169 for CoVID-19 screening. Outpatients (n=177) with confirmed CoVID-19 infection, comprising 80 early-stage ([≤]14 days after symptom onset), 71 late-stage ([≥]15 days), and 26 asymptomatic patients received whole blood CD169 testing in parallel with SARS-CoV-2 RT-PCR. Upregulation of monocyte CD169 without polymorphonuclear neutrophil CD64 changes was the primary endpoint. Sensitivity was 98% and 100% in early-stage and asymptomatic patients respectively, specificity was 50% and 84%. Rapid whole blood monocyte CD169 evaluation was highly sensitive when compared with RT-PCR, especially in early-stage, asymptomatic patients whose RT-PCR tests were not yet positive. Diagnostic accuracy, easy finger prick sampling and minimal time-to-result (15-30 minutes) rank whole blood monocyte CD169 upregulation as a potential screening and diagnostic support for CoVID-19. Secondary endpoints were neutrophil CD64 upregulation as a marker of bacterial infections and monocyte HLA-DR downregulation as a surrogate of immune fitness, both assisting with adequate and rapid management of non-CoVID cases. In this context, harnessing immune markers of leukocyte activation is a promising tool. Indeed, leukocytes 23 detect and rapidly respond to infection with secreted and surface activation molecules. We and others 24 have previously reported that acute viral infections induce the appearance of CD169 (Siglec- 1, 25 sialoadhesin) at the surface of blood monocytes (6, 7). Monocyte CD169 expression is upregulated by type 26 1 interferons (8), produced by locally attacked tissues, and is found in all circulating blood monocytes, 27 allowing its detection in minimal blood volumes such as a drop of blood at the fingertip. CD169 28 upregulation has been found in patients with HIV (9), EBV (10), RSV (11), CMV (12), Dengue (13, 14), Zika 29 (15), noroviruses (16), Lassa and Marburg (17). Transcriptomic and mass cytometry studies have identified 30 CD169 as a relevant biomarker for 19 ). The first evaluation by flow cytometry on patients 31 not only confirmed CD169 as a SARS-CoV-2 infection marker, but also showed that its expression was 32 much higher than for any other virus tested so far (20, 21). 33 Having developed a rapid (15 min) and affordable assay to measure monocyte CD169 upregulation in a 34 few microliters of blood (22), we set out to assess its diagnostic efficacy in a large cohort of CoVID-19-35 confirmed patients, with SARS-CoV-2 RT-PCR as the reference method. This assay evaluated in parallel 36 two other immune markers: upregulation of CD64 on polymorphonuclear neutrophils, which is widely 37 used as an indicator of bacterial infection (23), and expression of HLA-DR on monocytes, which reflects 38 the general state of the immune system (24): increased when activated by a pathogen (viral or bacterial), 39 and decreased if the immune system is "exhausted" by a severe infection (e.g. sepsis). 40 All rights reserved. No reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. On the basis of the clinical and laboratory results, the 177 RT-PCR-confirmed SARS-CoV-2 patients were 42 sorted into 3 groups: patients who presented at an early stage of the disease (less than 14 days after 43 symptoms onset, n=80, group 1), those at a later stage of the disease (with a median of 19 days after 44 symptoms onset (range 14-48), n=71, group 2), and asymptomatic patients (n=26, group 3). In each group, 45 patients were further separated according to the concomitant RT-PCR results (Figure 1) In the first group of CoVID-19 patients (early stage), the CD169 index was higher than the 3.5 threshold in 52 80% of patients (64/80) (Figure 1A) , while concomitant RT-PCR detected the virus (new cases), or 53 confirmed it (re-tested cases), in 65% of patients (52/80). Among the 16 CD169-negative early-stage 54 patients, 15 also had a negative concomitant RT-PCR. Sensitivity was 98%, with one patient exhibited a 55 CD169 index below the threshold but a positive nasopharyngeal RT-PCR in the swab. Review of laboratory 56 data for this patient showed very low and decreasing RNA quantities (cycle threshold (Ct) at 34.5 and 33.5, 57 respectively 24 and 48 hours earlier), suggesting a near complete viral clearance. In line with this 58 observation, the 15 other CD169 negative samples had been collected 6 to 14 days after the onset of 59 symptoms, and the corresponding RT-PCR were also negative. Thus, sensitivity of monocyte CD169 was 60 98% when compared to RT-PCR. 61 All rights reserved. No reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. In the second group (late stage), CD169 and RT-PCR were positive in only 7% of patients (5/71), in which 62 the RT-PCR were negative (Figure 1B) , indicating that CD169 expression returns to baseline levels upon 63 viral clearance. 64 In the third group (asymptomatic cases), significant CD169 upregulation was detected in 46% of patients 65 (12/26), at levels similar to those of group 1 comprising recent infections ( Figure 1C) , while concomitant 66 RT-PCR was positive in 35% of patients (9/26). All nine PCR positive cases were detected unambiguously. 67 It is remarkable that almost half of asymptomatic patients, who made up 15% of the study cohort, 68 expressed CD169 at the same level as patients experiencing symptoms. Indeed, the area under the curve 69 (AUC) and overall performances, as displayed in Figure 1D There was only a weak correlation between CD169 level and RT-PCR Ct ( Figure 2) . Still, the few CD169 77 negative patients were found among the weakest RT-PCR Ct. 78 Neutrophil CD64 expression, a marker of bacterial infections, was unchanged in 75% of the cases and 79 weakly upregulated in 25% of the cases (45/177), showing no significant differences between groups (23, 80 27, and 31% respectively). Within this cohort of outpatients presenting with mild disease, HLA-DR was 81 expressed at normal or slightly increased levels, an expected finding as opposed to the decrease usually 82 observed in severe cases(25). (Figure 3) . 83 All rights reserved. No reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted October 26, 2020. ; https://doi.org/10.1101/2020.10.22.20215749 doi: medRxiv preprint The sensitivity of monocyte CD169 upregulation was almost equivalent to RT-PCR for early-stage patients 84 and asymptomatic patients. There was a high percentage (80%) of positive CD169 results in early-stage 85 patients, peaking at 98.5% during the first week. As exemplified by these cases, false negative RT-PCR has 86 a frequent occurrence, in agreement with the commonly described false negative rates ranging from 10 87 to 30%(5). 88 Considering the main screening target, i.e., recent symptomatic cases (within 7 days) with a positive RT-89 PCR the same day (n=49) or less than 48 h prior to or following CD169 assessment (n=54), 98. Affordable reagents and lighter logistics. 5. Not using a closed system, as the assay is supported by most 103 flow cytometers equipping clinical laboratories. 104 Screening for monocyte CD169 upregulation could alleviate the load on specialized RT-PCR services and 105 reduce overall costs, while making the sample collection step easier for the greatest number of people. 106 All rights reserved. No reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted October 26, 2020. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted October 26, 2020. Coulter Life Sciences). The cocktail was then pre-mixed with 0.5 mL of Versalyse RBC lysing solution, and 149 10 µL of blood were added in the reaction tube. The mixture was finally mixed manually. After 10 minutes 150 All rights reserved. No reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted October 26, 2020. Wallis tests as appropriate. A two-sided p-value < 0.05 was considered statistically significant. 162 All rights reserved. No reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted October 26, 2020. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. 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