key: cord-0861021-i6q5i6yi
authors: Pauline, Maisonnasse; Poynard, Thierry; Sakka, Mehdi; Akhavan, Sepideh; Marlin, Romain; Peta, Valentina; Deckmyn, Olivier; Ghedira, Nesrine Braham; Ngo, Yen; Rudler, Marika; van der Werf, Sylvie; Marot, Stephane; Thabut, Dominique; Sokol, Harry; Housset, Chantal; Combes, Alain; Le Grand, Roger; Cacoub, Patrice
title: Validation of the performance of A1HPV6, a triage blood test for the early diagnosis and prognosis of SARS-CoV-2 infection.
date: 2022-02-07
journal: Gastro hep advances
DOI: 10.1016/j.gastha.2021.12.009
sha: 54492de7dcf99a002d5efa2f5c95451a9da0b116
doc_id: 861021
cord_uid: i6q5i6yi

Background and aims Apolipoprotein A1 (A1) and haptoglobin (HP) serum levels are associated with the spread and severity of SARS-CoV-2 infection. We have constructed and validated a multivariable risk calculator (A1HPV6) integrating A1, HP, alpha2-macroglobulin, and gamma glutamyl transferase to improve the performances of virological biomarkers. Methods In a prospective observational study of hospitalized patients with non-severe SARS-CoV-2 infection, A1HPV6 was constructed in 127 patients and validated in 116. The specificity was assessed in 7,482 controls representing the general population. The primary diagnostic endpoint was the AUROC in patients with positive SARS-CoV-2 PCR. The primary prognostic endpoint was the age-sex adjusted risk of A1HPV6 to predict patients with WHO-stage>4 (W>4) severity. We assessed the kinetics of the A1HPV6 components in a non-human primate model (NHP), from baseline to 7 days (D7) after SARS-CoV-2 infection. Results The AUROC A1HPV6 was 0.99 (95%CI .97-.99) in the validation subset, which was not significantly different from the construction subset, 0.99 (.99-.99; P=.80), like for sensitivity 92% (85-96) vs. 94% (88-97; P=.29). A1HPV6 was associated with W>4, with significant Odds-Ratio of 1.3 (1.1-1.5; 0.002). In NHP, A1 levels decreased (P<0.01) at D2 and normalized at D4; HP levels increased at D2, and peaked at D4. In patients, A1 concentration was very low at D2 vs. controls (P<0.01) and increased at D14 (P<0.01) but was still lower than controls; HP increased at D2 and remained elevated at D14. Conclusion These results validate the diagnostic and prognostic performances of A1HPV6. Similar kinetics of apolipoprotein A1, haptoglobin and alpha2-macroglobulin were observed in NHP model. ClinicalTrials.gov number, NCT01927133.conclusion

Data transparency statement. 83 Data, analytic methods, and study materials of this non)intervantional study will be made 84 available to other researchers, at thierry@poynard.com. 85 86

The pandemic of the respiratory disease (COVID-19) associated with the novel 119 coronavirus (SARS-CoV-2) has highlighted the need for biomarkers that detect different risks 120

i.e. the risk of infection before the exposure, named "predisposing biomarkers", the prognostic 121 factors during the exposure, named "acute phase biomarker", and the risk of sequelae after 122 exposure, named "sequelae biomarker". 1-3 Among simple available blood biomarkers, two 123 proteins associated with cell repair, apolipoprotein-A1 (A1) and haptoglobin (HP) could be 124 accurate components of such multi-analyte risk markers. 125

Several prospective population-based studies have shown that a low level of A1 as well 126 as an associated low level of high-density lipoprotein cholesterol (HDL-C), were associated 127 with a significant risk of hospitalization 10 years later, confirming their importance as a 128 predisposing biomarker. 4-7 Several prospective studies in COVID-19 hospitalized patients have 129 shown that A1 alone or associated with HP, also had significant diagnostic and short term 130 prognostic value for the acute phase of infection. [8] [9] [10] [11] [12] Biologically, A1 interacts with lipid rafts 131 on cellular membranes that are enriched in immune cell receptors such as toll-like receptors on 132 macrophages, T-cell receptors, and B-cell receptors which may all modulate immune 133 responses. 9-13 HDL also has immunomodulatory, antithrombotic, and antioxidant effects that 134 could provide important clarify why genetically determined levels of HDL-cholesterol, but not 135 LDL-cholesterol, provide a protective effect against infectious disease. 4,12 136

The increase in serum HP levels associated with SARS-CoV-2 infection was expected 137 based on its well-known acute phase protein profile. 14-16 HP is the most abundant protein among 138 those modulated in SARS-CoV-2 infection. 17 Unlike A1, there is no association between serum 139 HP levels before SARS-CoV-2 exposure or genetic polymorphisms. 3,14 However, A1 and HP 140 interact during the acute phase response to infection. 14-16 A1 collaborates with HP to 141 downregulate hemoglobin-redox activity. 15, 16 It also facilitates the uptake of hemoglobin by 142 6 interacting with the HDL-scavenger receptor B type 1 (SR-B1) displayed on macrophages and 143 hepatocytes. 16 144

The interaction of the SARS-CoV-2 S1 spike protein with HDL cholesterol could also 145 participate in the transient decrease of A1 by its main transporter during infection. The SARS-146

CoV-2 S protein binds to cholesterol, followed by an enhanced attachment via SR-B1, which 147 facilitates the SARS-CoV-2 entry. SR-B1 and host cell entry are initiated through interactions 148 with the angiotensin-converting enzyme 2 (ACE2). 18 149

In a previous study we described the temporal association between A1 and HP in 150 patients with non-alcoholic fatty liver disease (NAFLD) with the incidence of SARS-CoV-2-151 infected cases in the USA. The significant association between serum A1 levels and SARS- The primary endpoint of this study was to assess the diagnostic value of A1HPV6, a 158 multi-analyte SARS-CoV-2 risk marker integrating A1 and HP, alpha2-macroglobulin (A2M) 159 a marker of liver fibrosis, and the prognostic value of gamma glutamyl transpeptidase (GGT) a 160 sensitive marker of liver injury. 3 We report the prospective construction of A1HPV6 during the 161 first wave of COVID-19, the "construction subset", and the internal validation in the 162 "validation-subset" during the following two waves. 163

The second aim was to assess the prognostic value of A1HPV6 to predict WHO-164 stage>4 (W>4) severity COVID-19 disease in patients hospitalized in an internal medicine 165 department, ( Table 1) Five subsets were analyzed, a "construction-subset", a "validation-subset", a "prognostic-178 subset", a "controls-subset", and a "kinetic-subset" combining all the positive SARS-CoV-2 179 PCR cases admitted in the internal medicine department, plus three severe cases with persistent 180 plasma infection followed in the intensive care unit (ICU), and controls from the general 181 population (Figure 1) . 182

The prospective observational study in COVID-19 patients was approved by CER-Sorbonne 184

University IRB, CER-2020-14, with signed informed consent. All of the previously published 185 patient analyses from retrospective databases were non-interventional studies, without 186 supplementary blood samples, and were exempt from IRB review (NCT01927133). This study 187 was performed according to the principles of the Declaration of Helsinki. All authors had access 188 to the study data and reviewed and approved the final manuscript. 189

The construction-subset was performed between January and June 2020 and the validation- The prognostic analysis was performed in all patients by combining the construction and 200 validation subsets, and called the "prognostic-subset" (Figure 1) . 201

The kinetics were analyzed in the "kinetics-subset", which included the prospective integrated 203 prognostic subset and retrospectively severe cases admitted directly in the ICU (ICU-patients). 204

In non-ICU-patients, all cases with at least 3 repeated samples were analyzed. The aim was to 205 describe the kinetics of A1HPV6 and its components, 8 and 14 days after hospital admission, 206 that is around 16 and 24 days post infection (Dpi). Only kinetics in NHP were able to assess 207 the kinetics during the first 14 Dpi. 208

The severe cases were identified as ICU patients as those with persistent SARS-CoV-2 viremia 209 and with at least nine samples repeated frozen plasma samples. The severe cases allowed us to 210 identify the risks of A1HPV6 false negatives and its components, such as organ failure and 211 aggressive treatment. 22 212

We previously collected five cohorts, called "specificity-cohorts" which were used to 214 retrospectively validate the specificity of A1 and haptoglobin in a large group of subjects 215 without COVID-19 or pneumonia (Supplementary Table S1 ). Specificity was assessed in the 216 group of healthy volunteers that was representative of the French population before 2019. 23 217

Measurements were all performed on fresh prospectively collected serum or plasma in the 219 biochemistry unit of the APHP-PSL hospital. A1, HP, A2M, GGT, ALT and bilirubin were 220 

Briefly, four female and three male cynomolgus macaques and two female rhesus macaques, 226 aged 3-6 years old, were originating from Mauritian and Chinese AAALAC certified breeding 227 centers, 9,25 respectively and with the related NHP references .described in Supplementary File 228

The criterion for main prognostic endpoint was the accurate prediction of severe stage WHO 5 231 to 8 ( Table 1) Table  249 1). Forty out of 156 patients (25.6%) were not eligible, due to the absence of contemporaneous 250 serum samples, vs. 9 out of 136 patients (6.6%) pre-included in the construction subset 251 (P<0.001) (Table S2) . variable did not change the prognostic significance of A1HPV6 odds ratio = 2.14 (1.28-3.57; 275 p=0.004) ( Table 2) . 276

A total of 222 sera were assessed in 59 patients, (Figure 2 

Three ICU patients with persistent SARS-CoV-2 plasma viremia for more than two months 289 who later to died had 9 aliquots available each, (Supplementary Table S2 ). The repeated 290 13 mean A1HPV6 values were not significantly different during follow-up compared to values at 291 admission (D0) (Figure 3. Panel A) . The mean A1 on D0 and D80, were below 1g/L1 D2 which 292 were extremely low compared to normal values (>1. 15 (Table S2) . 

We used the COVID-19 WHO severity staging system to compare the performance of A1HPV6 353

with other blood tests. The main limitations were the small sample size, the limited number of 354 events, and the short follow-up during hospitalization, which prevents a powerful multivariate 355 analysis. There were three significant differences, in the validation subset vs. the construction 356 subset: median age three-years younger, twice more patients from Asian and Subsaharan origin, 357 and twice less high flow or ventilation. However, the A1HPV6 score already included age as a 358 covariable, and at the inclusion there were three significant differences, in the validation vs. 359 construction subset: median age three-years younger, twice more patients from Asian and 360 Subsaharan origin, and twice less high flow or ventilation. The A1HPV6 score already included 361 age as a covariable. One regression analysis included the subset as an independent variable 362 (validation vs. construction 2.07 (1.24-3.48;P=0.006), without a significant change in the 363 prognostic performance of A1HPV6. A second regression analysis included geographical 364 origin as an independent variable which also did not change the prognostic significance of 365 A1HPV6 odds ratio = 2.14 (1.28-3.57; p=0.004) ( Table 2) 

The binding of haptoglobin to apolipoprotein AI: 479 influence of hemoglobin and concanavalin A

Lipid-free apolipoprotein A-I exerts an antioxidative role 482 against cell-free hemoglobin

Circulating Exosomes are strongly involved in 485 SARS-CoV-2 Infection

HDL-scavenger receptor B type 1 facilitates SARS-CoV-2 488 entry

SARS-CoV-2 infection in nonhuman primates 491 alters the composition and functional activity of the gut microbiota

Lipoprotein concentrations over time in the 498 intensive care unit COVID-19 patients: Results from the ApoCOVID study

Prevalence of liver fibrosis and risk factors in a general 505 population using non-invasive biomarkers (FibroTest)

COVA1-18 neutralizing antibody protects against 511 SARS-CoV-2 in three preclinical models

Inferred duration of infectious period of SARS-514

CoV-2: rapid scoping review and analysis of available evidence for asymptomatic and 515 symptomatic COVID-19 cases

Validation of liver fibrosis biomarker 521 (FibroTest) for assessing liver fibrosis progression: proof of concept and first application in a 522 large population

Prognosis of French COVID-19 patients with chronic 525 liver disease: A national retrospective cohort study for 2020