Analysis of sars-cov-2 nucleocapsid protein in upper respiratory tract, blood and urine as a diagnostic and prognostic marker for covid-19 in hospitalized patients

Signalée pour la première fois en décembre 2019, la maladie à coronavirus 2019 (COVID-19) est devenue une préoccupation majeure de santé publique dans le monde entier. Le système de santé a très rapidement été mis à mal. La prise en charge clinique de la COVID-19 a reposé initialement sur la prévention de la transmission par l’identification des cas par les tests viraux, combinés à la vaccination à partir de 2021, et les soins de soutien. Dans la première phase de la pandémie les plateformes de biologie moléculaire ont vite été dépassées. La mise en œuvre de tests de diagnostic rapide (TDR) basés sur la détection de la protéine de nucléocapside ou de la protéine spike du SARS-CoV-2 a amélioré l'efficacité des stratégies de test à grande échelle.De plus, le SARS-CoV-2 a remis en question les connaissances médicales sur les infections respiratoires virales. L'une des principales caractéristiques de l'infection par le SARS-CoV-2 est la diversité de son expression clinique. La présence de composants du SARS-CoV-2 n’est pas limitée aux voies respiratoires et a été détectée dans de nombreux organes extra-pulmonaires et divers liquides biologiques. La présence d’antigènes viraux (notamment la nucléocapside) en extra-pulmonaire est tout particulièrement retrouvée chez les patients immunodéprimés ou présentant une forme grave de COVID-19. A l’inverse, chez les patients avec une forme modérée de COVID-19, la présence d’antigène viral est limitée aux voies respiratoires.Dans ce travail de thèse nous avons : i) analysé les performances diagnostiques du 1er TDR antigénique ciblant la protéine de nucléocapside mis sur le marché en Europe au tout début de la pandémie, ii) Étudier la cinétique de la protéine de la nucléocapside dans les voies respiratoires, le sang et l’urine, comparativement à la détection de l’ARN SARS-CoV-2 et de la réponse sérologique iii) Mis en évidence la dissémination de la protéine de la nucléocapside dans le sang et l’urine et son rôle potentiel de marqueur de sévérité de la COVID-19 chez le patient hospitalisé.La protéine de la nucléocapside est un bon marqueur diagnostique. Dans une première étude nous montrons que le TDR antigénique de première génération Coris Respi-Strip possédait des performances diagnostiques très en retrait par rapport à la PCR. Néanmoins, lorsqu’il était combiné à la détection d’anticorps par TDR ou test ELISA, les performances obtenues faisaient de ce test un outil intéressant. Les progrès des TDR de 2e et 3e génération ont permis de répondre aux exigences de performances de l’OMS définies par une sensibilité ≥ 80% et une spécificité ≥ 97% par rapport aux tests de RT-PCR, rendant inutile le diagnostic sérologique.Des études ont montré que la protéine de nucléocapside était également présente dans le compartiment circulant chez la plupart des sujets infectés, constituant un marqueur diagnostique alternatif et un marqueur de sévérité potentiel. Dans le cadre d’une étude cas contrôle réalisée au CHU de Montpellier (CoroTri, PHRC inter-régional) nous avons recherché la présence de la protéine de nucléocapside du SARS-CoV-2 dans les urines des patients hospitalisés pour Covid-19 et évalué sa valeur comme marqueur de sévérité. Nous observons que la protéine virale est présente en forte concentration dans l’urine et que les personnes nécessitant un recours aux soins intensifs présentaient une antigénurie N supérieure à celle des patients avec une forme modérée de COVID-19. Nos résultats suggèrent que la valeur pronostique de l’antigénurie est supérieure à celle de l’antigénémie. La présence de la protéine N dans le sang et l’urine semble refléter une infection disséminée du SARS-CoV-2 associée à un mauvais pronostic. Le développement d’un score se basant sur différents marqueurs précoces de gravité incluant la protéine N dans les urines pourrait permettre de mieux prédire la sévérité des formes de COVID-19.First reported in December 2019, coronavirus disease 2019 (COVID-19) has become a major public health concern worldwide. The health-care system was very quickly challenged. Clinical management of COVID-19 initially relied on prevention of transmission through case identification by viral testing combined with vaccination starting in 2021, and supportive care. In the first phase of the pandemic, the molecular biology platforms were quickly overwhelmed. The implementation of rapid diagnostic tests (RDTs) based on the detection of the nucleocapsid or spike protein of SARS-CoV-2 has improved the efficiency of large-scale testing strategies.Furthermore, SARS-CoV-2 has challenged the medical understanding of viral respiratory infections. One of the main characteristics of SARS-CoV-2 infection is the diversity of its clinical expression. The presence of SARS-CoV-2 components is not limited to the respiratory tract and has been detected in many extra-pulmonary organs and various biological fluids. The presence of viral antigens (notably the nucleocapsid) in extra-pulmonary organs is particularly found in immunocompromised patients or in patients with a severe form of COVID-19. However, in patients with a moderate form of COVID-19, the presence of viral antigen is limited to the respiratory tract.In this thesis work we have: i) Analyzed the diagnostic performance of the 1st antigenic RDT test targeting the nucleocapsid protein marketed in Europe at the beginning of the pandemic, ii) Studied the kinetics of the nucleocapsid protein in the respiratory tract, blood and urine, (iii) Demonstrated the dissemination of the nucleocapsid protein in blood and urine and its potential role as a marker of the severity of COVID-19 in hospitalized patients.Nucleocapsid protein is a good diagnostic marker. In a first study we show that the first generation antigenic RDT Coris Respi-Strip had a much lower diagnostic performance than the PCR. Nevertheless, when combined with antibody detection by RDT or ELISA, the performances obtained made this test an interesting tool. Advances in 2nd and 3rd generation RDTs have made it possible to have WHO performance requirements defined by a sensitivity ≥ 80% and a specificity ≥ 97% compared to RT-PCR tests, making serological diagnosis unnecessary.Studies have shown that the nucleocapsid protein is also present in the circulating compartment in most infected peoples, constituting an alternative diagnostic marker and a potential severity marker. In a case-control study conducted at the University Hospital of Montpellier (CoroTri, inter-regional PHRC), we investigated the presence of SARS-CoV-2 nucleoprotein in the urine of patients hospitalized for Covid-19 and evaluated its value as a marker of severity. We observe that the viral protein is present in high concentration in urine and that people requiring intensive care had a higher nucleocapsid antigenuria than patients with a moderate form of COVID-19. Our results suggest that the prognostic value of antigenuria is greater than antigenemia. The presence of N protein in blood and urine seems to reflect a disseminated infection of SARS-CoV-2 associated with a worse prognosis. The development of a score based on different early markers of severity including the N protein in urine could better predict the severity of COVID-19

Th17 CD4+ T-Cell as a Preferential Target for HIV Reservoirs

International audienceAmong CD4+ T-cells, T helper 17 (Th17) cells play a sentinel role in the defense against bacterial/fungal pathogens at mucosal barriers. However, Th17 cells are also highly susceptible to HIV-1 infection and are rapidly depleted from gut mucosal sites, causing an imbalance of the Th17/Treg ratio and impairing cytokines production. Consequently, damage to the gut mucosal barrier leads to an enhanced microbial translocation and systemic inflammation, a hallmark of HIV-1 disease progression. Th17 cells’ expression of mucosal homing receptors (CCR6 and α4β7), as well as HIV receptors and co-receptors (CD4, α4β7, CCR5, and CXCR4), contributes to susceptibility to HIV infection. The up-regulation of numerous intracellular factors facilitating HIV production, alongside the downregulation of factors inhibiting HIV, helps to explain the frequency of HIV DNA within Th17 cells. Th17 cells harbor long-lived viral reservoirs in people living with HIV (PLWH) receiving antiretroviral therapy (ART). Moreover, cell longevity and the proliferation of a fraction of Th17 CD4 T cells allow HIV reservoirs to be maintained in ART patients

Antibody response after first and second BNT162b2 vaccination to predict the need for subsequent injections in nursing home residents

International audienceAbstract We explored antibody response after first and second BNT162b2 vaccinations, to predict the need for subsequent injections in nursing home (NH) residents. 369 NH residents were tested for IgG against SARS-CoV-2 Receptor-Binding Domain (RBD-IgG) and nucleoprotein-IgG (SARS-CoV-2 IgG II Quant and SARS-CoV-2 IgG Alinity assays, Abbott Diagnostics). In NH residents with prior SARS-CoV-2 infection, the first dose elicited high RBD-IgG levels (≥ 4160 AU/mL) in 99/129 cases (76.9%), with no additional antibody gain after the second dose in 74 cases (74.7%). However, a low RBD-IgG level (< 1050 AU/mL) was observed in 28 (21.7%) residents. The persistence of nucleoprotein-IgG and a longer interval between infection and the first dose were associated with a higher RBD-IgG response (p < 0.0001 and p = 0.0013, respectively). RBD-IgG below 50 AU/mL after the first dose predicted failure to reach the antibody concentration associated with a neutralizing effect after the second dose (≥ 1050 AU/mL). The BNT162b2 vaccine elicited a strong humoral response after the first dose in a majority of NH residents with prior SARS-CoV-2 infection. However, about one quarter of these residents require a second injection. Consideration should be given to immunological monitoring in NH residents to optimize the vaccine response in this vulnerable population

Severe Acute Respiratory Syndrome Coronavirus 2 Nucleocapsid Antigen in Urine of Hospitalized Patients With Coronavirus Disease 2019

International audienceAbstract Background Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) nucleocapsid antigen (N-Ag) can be detected in the blood of patients with coronavirus disease 2019 (COVID-19). We used a highly sensitive and specific assay to explore the presence of N-Ag in urine during the course of COVID-19 and its relationship with the severity of disease. Methods We studied urinary and plasma N-Ag using a highly sensitive immunoassay in 82 patients with SARS-CoV-2 infection proved by polymerase chain reaction. Results In the first and second weeks of COVID-19, hospitalized patients tested positive for urinary N-Ag (81.25% and 71.79%, respectively) and plasma N-Ag (93.75% and 94.87%, respectively). High urinary N-Ag levels were associated with the absence of SARS-CoV-2 nucleocapsid antibodies, admission in intensive care units, high C-reactive protein levels, lymphopenia, eosinopenia, and high lactate dehydrogenase levels. Higher accuracy was observed for urinary N-Ag as a predictor of severe COVID-19 than for plasma N-Ag. Conclusions Our study demonstrates that N-Ag is present in the urine of patients hospitalized in the early phase of COVID-19. As a direct marker of SARS-CoV-2, urinary N-Ag reflects the dissemination of viral compounds in the body. Urinary N-Ag may be a useful marker for disease severity in SARS-CoV-2 infections

Diagnosis value of SARS‐CoV‐2 antigen/antibody combined testing using rapid diagnostic tests at hospital admission

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Genetic Background of the Sickle Cell Disease Pediatric Population of Dakar, Senegal, and Characterization of a Novel Frameshift β -Thalassemia Mutation [ HBB : c.265_266del; p.Leu89Glufs*2]

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Erratum to: Monocyte CD169 Expression as a Biomarker in the Early Diagnosis of Coronavirus Disease 2019

International audienceAbstract We assessed the expression of CD169, a type I interferon-inducible receptor, on monocytes (monocyte CD169 [mCD169]) in 53 adult patients admitted to the hospital during the coronavirus disease 2019 (COVID-19) outbreak for a suspicion of severe acute respiratory syndrome coronavirus 2 infection. Monocyte CD169 was strongly overexpressed in 30 of 32 (93.7%) confirmed COVID-19 cases, compared with 3 of 21 (14.3%) patients in whom the diagnosis of COVID-19 was finally ruled out. Monocyte CD169 was associated with the plasma interferon-alpha level and thrombocytopenia. Monocyte CD169 testing may be helpful for the rapid triage of suspected COVID-19 patients during an outbreak

Monocyte CD169 expression as a biomarker in the early diagnosis of COVID-19

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