Epidemiological and Molecular Evolution of the Hiv-1 Crf94: Birth of Crf132

Background: In 2018 we reported the emergence of the new HIV-1 recombinant CRF94-02BF2 involved in a large transmission cluster of 49 French MSM mostly infected in 2016-2017. This CRF94 raised concerns of enhanced virulence. Prevention actions were undertaken in the area and population affected. This study reported the molecular and epidemiological evolution of this CRF94 until June 2022. Method(s): In 2021-2022, French sequence databases were screened for patients infected with HIV-1 subtype CRF94 or similar strain. HIV subtyping was confirmed by phylogenetic analysis of genes encoding both protease and reverse transcriptase (1070bps), and integrase (696bps) using IQ-Tree. Five whole genomes, related but distinct from CRF94, were obtained with the DeepChek assay Whole Genome kits. Recombination breakpoints were estimated using RDP4 and SimPlot. Mann-Whitney and LogRank tests were used for statistical analyses to compare patients' characteristics. Result(s): In June 2022, 49 new HIV-1 sequences were collected: 14 clustered with the 49 previous CRF94, 32 formed a new cluster next to but distinct from CRF94, and 3 strains could not be classified. Analysis of 5 whole genomes from the new cluster revealed a new recombinant, the CRF132-94B, mainly consisting of CRF94 which recombined with subtype B in the POL and accessory genes. Vif gene changed from the F2 to the B subtype. Both CRF94 and 132 clusters involved >95% of MSM, mostly infected < 1 year before diagnosis. However, there were differences: 97% were diagnosed in 2013-2019 for CRF94 vs 90% in 2020-2022 for CRF132. At time of diagnosis, 33% of patients infected with CRF94 knew the Prep vs 95% for CRF132. In the cluster CRF94, patients were older (34 vs 30 years, p=0.02), had higher viral loads (5.42 vs 4.42 log10 copies/Ml;p< 0.001), a lower CD4 cell counts (358 vs 508 /mm3, p=0.002). On treatment, the patients with the CRF94 reached viremia < 50 copies/Ml significantly later than those infected with CRF132 (p=0.0002). The prevention activities targeting the CRF94 cluster could explained the few patients infected with this strain after 2018. The CRF132 is mainly located in another Paris region area, but no specific transmission place has been identified. Conclusion(s): After 2019, the CRF94 spread seems greatly slowed down but the very close CRF132-94B has given birth to a new highly active cluster in 2020- 2022, despite the COVID social-distancing and a strong knowledge of the Prep. CRF132 appears to be less virulent perhaps due to the Vif gene change. Identified breakpoints positions of the new HIV-1 CRF132-94B. GenBank accession numbers of the five references : ON901787 to ON901791.

Evolution of Clusters Transmission Patterns in France during the Last Decade

Background: The recent transmission clusters (RTCs) identified through phylogenetic approaches allow to describe the main transmission networks. This render possible to describe potential shifts among HIV transmission routes and populations and, in some cases, to specifically target prevention measures. Here we describe the evolution of RTCs over the last decade in a specialized laboratory serving centers from the entire French territory. Method(s): We extracted all the HIV reverse transcriptase sequences available between 01/01/2013 and 31/08/2022. The sequences dataset was studied overall and divided into three equal time periods: 2013-15, 2016-18, 2019-2021. The first sequences available for each patient were aligned and the trees were reconstructed by maximum likelihood using IQtree software. Clusters, defined by a maximum genetic distance < 4.5% and a branch support >90%, were extracted using ClusterPicker. Result(s): Overall, 8591 sequences were included. Among them, 950 RTCs were identified including 2492 sequences (29%) and 68 large RTCs ( >4 sequences) with 475 (5.6%) sequences. The mean duration of large RTCs (from the first to the last sequences) was 5.1 years [IQR: 4.1-7.1] and 34 were still active (including at least one sequence during the last year of the study period). 3640, 2897 and 2157 sequences were included for the 2013-15, 2016-18 and 2019-2021 periods, respectively. We identified 298 RTCs (19.5% of sequences), 249 (20.4%) and 226 (27.5%) among those periods, respectively. While the number of sequence pairs decreased from 2013-15 to 2019-21, the number of large RTCs increased steadily (see Table 1). During the period 2019-21, including the largest clusters, patients belonging to a RTC were more often male (68 vs 58%, p< 0.001) and younger (average age: 39 vs 44 years, p< 0.001) than non-RTC patients. This observation was even more marked for very large RTCs (see Table 2). It should be noted that the largest cluster (14 patients) was mainly composed of women and located in French overseas territories. Conclusion(s): This study shows an evolution of the structure of HIV sequence clusters over time with a decreasing number of small RTCs but an increasing number of large RTCs. These trends can be explained by a better global control of transmission, due in part to TasP, but not preventing some super-transmitters networks, despite PrEP use and not only including MSM is some settings. The COVID period does not seem to have strongly prevented such large transmission networks.

DUAL MONOCLONAL ANTIBODIES in COVID-19 PATIENTS: CLINICAL and VIROLOGICAL EFFICACY

Background: An emergency use authorization was issued in March 2021 for two combinations of monoclonal antibodies (MAbs) for SARS-CoV-2 infected patients at high risk of severe COVID-19. We performed a cohort study of patients receiving early treatment with Bamlanivimab/Etesevimab (B/E) or Casirivimab/Imdevimab (C/I) in a Paris university hospital. Methods: All patients receiving a MAbs therapy from March to July 2021 were included. Prescriptions were systematically advised by a multidisciplinary team. Both MAbs dual therapies were used up to May 12th, then only C/I due to local emergence of Delta variant. Nasopharyngeal swabs (NPS) were performed at diagnosis and 7 days after infusion. Additional NPS were collected for hospitalized patients at day 3 and during follow-up until negative RT-PCR or patients discharge. Viral sequencing was carried out and viral mutations were retained if present at more than 20% of viral subpopulations. Results: Overall, 66 patients (19 ambulatory) received a MAbs dual therapy for a documented SARS-CoV-2 asymptomatic infection or within 5 days after symptoms onset. Patients had a median age of 67 years [IQR=41-75], 53% were male, 30 (45%) were receiving immunosuppressive treatment (17 being solid organ recipients), 8 (12%) had chronic respiratory insufficiency, and 6 (9%) were receiving chemotherapy. Regarding variants, 82% were Alpha, 5% Delta and 13% other variants. 8 patients (12%) died (6 treated with B/E and two with C/I). Five deaths were related to COVID-19 worsening and three were unrelated. Among the surviving patients, 42 (64%) did not require any oxygen and 16 (24%) required low-flow oxygen. No severe adverse event related to MAbs occurred. A slower viral decay was observed among patients receiving B/E than C/I, with 17/29 and 5/13 having <30 Ct at day 7 post-infusion (p=0.3), respectively, and 9/14 and 1/8 at day 14 (p=0.03). Different Spike mutations emergence were observed including Q493R in 7 patients and E484K in 2 patients, all infected with an Alpha variant, and detected from 6 to 18 days after MAbs infusion. Among the 9 mutations, 8 occurred after B/E infusion and one Q493R occurred after C/I infusions. Conclusion: We described safety and efficacy of early MAbs therapies administration in a cohort of 66 patients at risk of severe COVID-19. Emergence of mutations were observed under both therapies, with increased frequency under B/E. Further studies including patients infected by Delta variant and receiving C/I infusion are ongoing.

PHYLODYNAMIC of SARS-CoV-2 TRANSMISSION in FRANCE: 2020

Background: In 2020, France reported 2.7 million cases of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), making it the second most affected European country by the COVID-19 pandemic after the United Kingdom. However, dynamics of SARS-CoV-2 transmissions within France or between France and other countries remains partially characterized. We propose an analysis of these dynamics on multiple scales, from the continents to the French administrative regions. Methods: We produced 736 SARS-CoV-2 sequences from Ile-de-France (Paris area, France) and analyzed them concomitantly with GISAID deposited sequences to elucidate the origins and spread of the virus from January 2020 to December 2020. A total of 4,571 worldwide sequences, including 1,652 French sequences, constituted the final dataset. All sequences were selected to be representative of each country temporal distribution of SARS-CoV-2 to the week resolution. We used a maximum likelihood phylogenetic framework to estimate the most probable temporal and geographic spread of SARS-CoV-2 within France and worldwide. Depending on the geographical focus (France, Europe or worldwide), we pruned the tree accordingly in 1,000 independent replicates. Results: Phylogenetic analysis revealed that, during the 1st French epidemic wave (from March to May), the majority of viruses introduced to France came from North America (USA) and Europe (Spain, Italy, ?). France regularly transmitted to neighboring European countries: Belgium, Germany, Italy and United Kingdom. Contrary to the 1st wave, inter-country transmission events were limited to neighboring countries and intercontinental transmission were almost absent during the French 2nd wave (from September to November). At the French regions-scale, we observed that Ile-de-France (IDF) was the main source of infections for all other French regions during the 1st epidemic wave, with a minor participation of Provence-Alpes-Côte d'Azur (PACA). For the 2nd epidemic wave, PACA was the main source of infections for all other French regions, with a lower participation of IDF and other regions. Conclusion: Overall, our findings allow a more comprehensive representation of SARS-CoV-2 transmission chains related to and within France and the global temporal distribution of those events, in link with control measures applied during the whole 2020 period. IDF and PACA were the main hubs of transmissions in France for the 1st and the 2nd epidemic waves, respectively.

SARS-CoV-2 N-ANTIGEN SERA LEVELS ARE ASSOCIATED with COVID-19 PROGNOSTIC VALUE

Background: Small studies have reported that high levels of free SARS-CoV-2 nucleocapsid-antigen in sera (N-antigenemia) was associated with prognostic. Here, we assessed the association between N-antigenemia levels and patient's outcome in a large cohort of hospitalized patients. Methods: We analysed data from patients with at least one sera sample that were included in the French COVID cohort between January, 25 2020 and September, 2 2020. N-antigenemia levels were determined with the COV-Quanto® assay (AAZ) with a limit of detection of 2.98 pg/mL. IgG (anti-N, anti-S and anti-RBD) levels were assessed using the V-PLEX panel assay (MSD). Patient's outcome was classified in three groups: death, recovery with ICU transfer (ICU) and recovery without ICU transfer (Hospital). Results: We included 1166 samples from 357 patients, with 66% of male and a median age at 63 years [IQR: 52-71]. A total of 82, 96 and 142 patients were in the Death, ICU and Hospital groups, respectively. The sensitivity of N-antigenemia was 79% (131/165) within the first 10 days SSO (since symptoms onset) and 62% (365/589) from 11 to 30 days SSO. Positivity rates were significantly different across severity groups from 0 to 15 days SSO: 95% (95/100), 64% (118/183), 79% (83/105) (p<0.001) for Death, ICU and Hospital groups, respectively. Among positive patients, a significant gradient was found in the levels of N-antigenemia according to disease severity, with median levels of 302, 134 and 89 in Death, ICU and hospital groups, respectively. Similar relationships were found when stratifying on the time SSO (see figure). Overall, 95, 80, 43 and 22% of N-antigenemia >10,000, >5,000, >1,000 and <1,000 pg/mL corresponded to patients who died. IgG antibodies titers were not correlated to severity and the presence of both sera N-antigen and anti-N IgG was observed for 42% (490/1166) samples. Conclusion: We observed, on a large prospective cohort, a strong relationship between N-antigenemia and COVID-19 severity. This new diagnostic tool should help to prognostic evaluation of COVID-19 patients. To our knowledge, COVID-19 is the first demonstration of the presence of free antigenemia in a viral pneumonia, and its association with prognostic.

Concordance de la détection des virus respiratoires sur écouvillonnage nasopharyngé et lavage broncho-alvéolaire chez l'adulte admis pour une suspicion de pneumonie

Introduction A ce jour, peu de données sont disponibles sur la concordance de la détection des virus respiratoires (en dehors du SARS-CoV2) entre le prélèvement nasopharyngé (NP) et le lavage broncho-alvéolaire (LBA) chez l'adulte. L'objectif de cette étude était de décrire la concordance de leurs résultats. Matériels et méthodes Une analyse rétrospective monocentrique a été réalisée à l'aide des données de 276 adultes suspects de pneumonie et testés par PCR multiplex dans le NP et le LBA à 24 heures d'intervalle. Résultats Les patients étaient majoritairement des hommes (65 %), avec un âge médian de 60 ans[IQR : 50,9-67,8]. 169 patients (61 %) ont été admis en réanimation Nous avons détecté au moins un virus respiratoire dans 95 NP (34 %) et dans 80 BAL (29 %). Comparé au LBA, le NP avait une sensibilité de 71,6 % et une spécificité de 93,4 % et un coefficient Kappa de 0,67. Le même agent pathogène ou combinaison d'agents pathogènes a été observé chez 84 % des patients positifs à la fois sur le NP et le LBA. La grippe B, le parainfluenza, les coronavirus HKU1, NL63, 229E présentaient la concordance la plus élevée (100 %) entre le NP et la LBA, tandis que le coronavirus OC43 et le rhinovirus présentaient la concordance la plus faible (33 % et 67 %, respectivement).Nous avons observé que les patients atteints d'une maladie respiratoire chronique ont une plus faible concordance entre le NP et LBA avec un OR ajusté à 0,5, IC 95 % (0,25-0,97), p = 0,043. Conclusion Dans ce travai, Il y a une bonne concordance entre le NP et le LBA dans la détection des virus respiratoires chez les patients consultant avec une suspicion de pneumonie. Néanmoins, ces données encouragent toujours à réaliser un LBA lorsque cela est possible afin d'obtenir un diagnostic étiologique plus précis. Aucun lien d'intérêt

Pulmonary bacterial infections in adult patients hospitalized for COVID-19 in standard wards.

OBJECTIVES: During the COVID-19 pandemic, antibiotic use was very common. However, bacterial co-/secondary infections with coronaviruses remain largely unknown in standard wards. We aimed to investigate the characteristics of pulmonary bacterial infections associated with COVID-19 in hospitalized patients. METHODS: A retrospective monocentric observational study was conducted in Bichat hospital, France, between February 26 and April 22, 2020. All patients hospitalized in standard wards with COVID-19 (positive nasopharyngeal PCR and/or typical aspect on CT-scan) and diagnosed with pulmonary bacterial infection (positive bacteriological samples) were included. Bacteriological and clinical data were collected from the microbiology laboratories and patient's medical records. RESULTS: Twenty-three bacteriological samples from 22 patients were positive out of 2075 screened samples (1.1%) from 784 patients (2.8%). Bacterial infection occurred within a median of 10 days after COVID-19 onset. Diagnosis of pulmonary bacterial infection was suspected on increase of oxygen requirements (20/22), productive cough or modification of sputum aspect (17/22), or fever (10/22). Positive samples included 13 sputum cultures, one FilmArray® assay on sputum samples, one bronchoalveolar lavage, six blood cultures, and two pneumococcal urinary antigen tests. The most frequent bacteria were Pseudomonas aeruginosa (6/23), Staphylococcus aureus (5/23), Streptococcus pneumoniae (4/23), Enterococcus faecalis (3/23), and Klebsiella aerogenes (3/23). No Legionella urinary antigen test was positive. Four out of 496 nasopharyngeal PCR tests (0.8%) were positive for intracellular bacteria (two Bordetella pertussis and two Mycoplasma pneumonia). CONCLUSIONS: Pulmonary bacterial secondary infections and co-infections with SARS-CoV-2 are uncommon. Antibiotic use should remain limited in the management of COVID-19.

Focus on Middle East respiratory syndrome coronavirus (MERS-CoV).

Since the first case of human infection by the Middle East respiratory syndrome coronavirus (MERS-CoV) in Saudi Arabia in June 2012, more than 2260 cases of confirmed MERS-CoV infection and 803 related deaths have been reported since the 16th of October 2018. The vast majority of these cases (71%) were reported in Saudi Arabia but the epidemic has now spread to 27 countries and has not ceased 6 years later, unlike SARS-CoV that disappeared a little less than 2 years after emerging. Due to the high fatality rate observed in MERS-CoV infected patients (36%), much effort has been put into understanding the origin and pathophysiology of this novel coronavirus to prevent it from becoming endemic in humans. This review focuses in particular on the origin, epidemiology and clinical manifestations of MERS-CoV, as well as the diagnosis and treatment of infected patients. The experience gained over recent years on how to manage the different risks related to this kind of epidemic will be key to being prepared for future outbreaks of communicable disease.

Émergence de variants E484K suite à une monothérapie bamlanivimab chez des patients COVID-19 à haut risque d’évolution vers une forme sévère

Introduction En France, une Autorisation d’Utilisation Temporaire de cohorte (ATUc) a été délivrée le 27/02/2021 pour l’utilisation du bamlanivimab en monothérapie dans le traitement précoce (<5jours du début des symptômes) de patients adultes avec un COVID-19 léger à modéré confirmé par PCR et à haut risque d’évolution vers une forme sévère. Les patients concernés étaient les patients de >80 ans ou les patients de <80 ans immunodéprimés (transplantation, chimiothérapie, traitement immunosuppresseur). Le risque d’émergence de variants potentiellement résistants à un monothérapie par anticorps monoclonal anti-Spike, en particulier les variants E484K, avait été pris en compte lors de l’ATUc. Cependant, le bénéfice potentiel de ces traitements chez les patients à haut risque a été considéré comme supérieur au risque. Ici nous décrivons 6 patients ayant reçu de ce traitement, leur évolution et l’émergence de mutations de résistance sous pression de sélection. Matériels et méthodes Il s’agit d’une étude unicentrique en centre hospitalier universitaire. Le bamlanivimab a été administré à une dose unique de 700mg en injection IV d’une heure chez 6 patients qui ont accepté d’utiliser le traitement dans le cadre de l’ATUc. Le suivi virologique des patients a consisté en un test RT-qPCR itératif réalisé le jour de la perfusion ou la veille, à J3±1, à J5±1, à J7±1 puis tous les 3jours jusqu’à ce que la PCR soit négative. La sélection de mutation de résistance a été vérifiée par séquençage du génome complet du SARS-CoV-2 chez tous les patients. Résultats Les six hommes traités avaient un âge médian de 65 ans (extrêmes 35-97), plus de 3 comorbidités à haut risque d’évolution vers une forme sévère et 5 étaient infectés par un variant UK (N501Y.V1, B.1.1.7). Le traitement a été administré dans les 4jours suivant l’apparition des symptômes (médiane 2jours). Les 6 patients ont eu une évolution clinique favorable, deux ont eu besoin d’oxygène au débit maximal de 4 L/min. Aucun patient n’a eu besoin d’oxygénothérapie haut débit, d’une ventilation non invasive ou d’une ventilation invasive. À J20 après l’administration, un seul présentait une PCR nasopharyngée négative et 5 présentaient l’apparition d’une mutation E484K à différents moments après l’administration (J6, J7, J12, J14 et J26). Conclusion Chez ces patients à très haut risque d’évolution vers une forme sévère, la sélection de la mutation E484K après administration de bamlanivimab en monothérapie était très fréquente et bien plus importante qu’observée dans les premiers essais cliniques. Ceci peut être expliqué en partie par l’infection par un variant UK pour 5/6 patients décrits ici. L’émergence de résistance lors des bithérapies devra être suivi attentivement, notamment avec l’émergence des nouveaux variants.

Detection of SARS-CoV-2 N-antigen in blood during acute COVID-19 provides a sensitive new marker and new testing alternatives

OBJECTIVES: Molecular assays on nasopharyngeal swabs remain the cornerstone of COVID-19 diagnostic. The high technicalities of nasopharyngeal sampling and molecular assays, as well as scarce resources of reagents, limit our testing capabilities. Several strategies failed, to date, to fully alleviate this testing process (e.g. saliva sampling or antigen testing on nasopharyngeal samples). We assessed the clinical performances of SARS-CoV-2 nucleocapsid antigen (N-antigen) ELISA detection in serum or plasma using the COVID-19 Quantigene R (AAZ, France) assay. METHODS: Performances were determined on 63 sera from 63 non-COVID patients and 227 serum samples (165 patients) from the French COVID and CoV-CONTACT cohorts with RT-PCR confirmed SARS-CoV-2 infection, including 142 serum (114 patients) obtained within 14 days after symptoms' onset. RESULTS: Specificity was 98.4% (95% confidence interval [CI], 95.3 to 100). Sensitivity was 79.3% overall (180/227, 95% CI, 74.0 to 84.6) and 93.0% (132/142, 95% CI, 88.7 to 97.2) within 14 days after symptoms onset. 91 included patients had a sera and nasopharyngeal swabs collected in the same 24 hours. Among those with high nasopharyngeal viral loads, i.e. Ct value below 30 and 33, only 1/50 and 4/67 tested negative for N-antigenemia, respectively. Among those with a negative nasopharyngeal RT-PCR, 8/12 presented positive N-antigenemia;the lower respiratory tract was explored for 6 of these 8 patients, showing positive RT-PCR in 5 cases. CONCLUSION: This is the first evaluation of a commercially available serum N-antigen detection assay. It presents a robust specificity and sensitivity within the first 14 days after symptoms onset. This approach provides a valuable new option for COVID-19 diagnosis, only requiring a blood draw and easily scalable in all clinical laboratories.

Co-infection et surinfection bactérienne chez les patients hospitalisés pour COVID-19: une étude rétrospective monocentrique

Introduction Durant la pandémie de COVID-19, la prescription d’antibiotique a été importante. Cependant, les surinfections et co-infections bactériennes associées aux coronavirus sont peu décrites, et concernent principalement les patients de soins intensifs. Matériels et méthodes Une étude observationnelle rétrospective monocentrique a été menée dans notre CHU du 26/02/2020 au 22/04/2020. Ont été inclus tous les patients hospitalisés pour une COVID-19 (PCR respiratoire positive à SARS-CoV-2 ou atteinte scannographique compatible) hors service de réanimation et présentant une infection bactérienne documentée par un prélèvement bactériologique positif parmi: prélèvement respiratoire (ECBC, aspiration bronchique, LBA), hémoculture, antigénurie légionnelle ou pneumocoque, ou PCR nasopharyngée (Bordetella pertussis, Mycoplasma pneumoniae et légionnelle). Les données bactériologiques ont été obtenues par les laboratoires de bactériologie et virologie et les données cliniques recueillies à partir du dossier médical des patients. Résultats Au total, 2710 prélèvements ont été réalisés chez 778 patients hospitalisés: 957 hémocultures, 169 ECBC, 3 aspirations bronchiques, 2 LBA, 537 PCR nasopharyngées, 517 antigénuries légionnelle et 525 antigénuries pneumocoque. Quarante et un prélèvements étaient positifs (1,5 %) dont quatorze ont été considérés comme une colonisation par les cliniciens. Vingt neuf prélèvements positifs (1,1 %) ont été considérés comme pathogènes chez 27 patients (3,5 %): 15 ECBC, 2 aspirations bronchiques, 1 LBA, 7 hémocultures, 2 antigénuries et 4 PCR nasopharyngées. Parmi les 27 patients, 18 (66,7 %) avaient des comorbidités dont 10 (37 %) une pathologie respiratoire chronique. Huit patients (29,6 %) étaient sous traitement immunosuppresseur au long cours et 15 (55,6 %) ont reçu des corticoïdes ou autres traitements immunomodulateurs pour la COVID-19. Le delais entre l’apparition des symptômes de COVID-19 et la survenue de la surinfection bactérienne était en moyenne de 11jours. Les arguments en faveur d’une surinfection étaient l’augmentation des besoins en oxygène (n=22, 81,5 %), la toux expectorante (n= 20, 74,0 %) ou la modification de la toux (n=5, 18,5 %) et la présence de fièvre (n= 5, 18,5 %). Un patient avait un tableau de sepsis. Les pathogènes les plus fréquemment retrouvés nétaient: Pseudomonas aeruginosa (n=8, 29,6 %), Staphylococcus aureus (n=5, 18,5 %), Streptococcus pneumoniae (n=4, 14,8 %) et Enterococcus faecalis (n=3, 11,1 %). Quatre patients (0,5 %) avaient une co-infection à bactérie intracellulaire: 2 à Bordetella pertussis et 2 à Mycoplasma pneumoniae. Aucune antigénurie légionnelle n’est revenue positive. Conclusion Le taux de surinfections et de co-infections bactériennes dans la COVID-19 semble faible. Ce faible taux est en faveur d’une utilisation limitée des antibiotiques dans la prise en charge de la COVID-19.