COVID-19 patient experiences in pre-hospital pathways: a processual qualitative approach using the life-events calendar method highlights diagnostic delays and healthcare renunciation. (preprint)

Background During the height of the COVID-19 pandemic in 2020, 11% of patients who were hospitalized in France were immediately admitted to intensive care. We aimed to identify and characterize the different types of primary care pathways of patients hospitalized for COVID-19 using patients’ self-reported experiences. Method We conducted a qualitative study using biographic interviews of patients who were hospitalized for COVID-19 between September 2020 and December 2021 in the infectious disease departments in Marseille and Nice. The biographical interviews used a life-events calendar approach to understand the sequences of clinical and care events prior to hospitalization. Results 31 pathways were described. Short care pathways (i.e., admission to hospital ≤ 3 days after symptom onset) were more likely to be reported by older patients and those with comorbidities. These pathways were characterized by closer GP surveillance and by sudden symptom onset and rapid progression of the disease. Long care pathways (i.e., >10 days after system onset) were reported more by younger patients with no comorbidities. Multiple tests and medical consultations returning false-negatives had led this population to doubt they had COVID-19. They were more likely to present severe symptoms requiring intensive care. The study revealed key importance of patients’ loved ones in the process of their hospitalization. Conclusion This study highlights that primary care management of COVID-19 patients needing hospitalization in France was particularly slow and detrimental to their health. It also underlines the need to improve the identification and monitoring of patients at risk of complications.

Early Treatment with Hydroxychloroquine and Azithromycin: A Real-Life Monocentric Retrospective Cohort Study of 30,423 COVID-19 Patients (preprint)

Objective To estimate the comparative effectiveness of combination therapy with hydroxychloroquine (HCQ) and azithromycin for coronavirus disease 2019 (COVID-19)-related death based on a large monocentric cohort independent of investigators putative biases in a real-world setting. Design Retrospective monocentric cohort study, with comprehensive data collection authenticated by an external bailiff and death reports from a national database (French National Death Registry). Setting Institut Hospitalo-Universitaire Mediterranee Infection Center in Marseille, France. Participants All adults older than 18 years with PCR-proven COVID-19 who were treated directly in our centre between 2 March 2020 and 31 December 2021 and did not refuse the use of their data. Interventions HCQ and azithromycin (HCQ-AZ) as a reference treatment were compared to other regimens containing HCQ, ivermectin and azithromycin alone, combined, or none of these three drugs. The effect of vaccination was also evaluated. Main outcome measures 6-week all-cause mortality. Multivariable logistic regression estimated treatment effectiveness with adjustments for age, sex, comorbidities, vaccination, period of infection or virus variant, and outpatient or inpatient care. Results Total 30,423 COVID-19 patients were analysed (86 refused the analysis of their data) including 30,202 with available treatment data, and 535 died (1.77%). All-cause mortality was very low among patients < 50 years (8/15,925 (0.05%)) and among outpatients treated with HCQ-AZ (21 deaths out of 21,135 (0.1%), never exceeding 0.2% regardless of epidemic period). HCQ-AZ treatment was associated with a significantly lower mortality rate than no HCQ-AZ after adjustment for sex, age, period and patient care setting (adjusted OR (aOR) 95% confidence interval (CI) 0.55, 0.45-0.68). The effect was greater among outpatients (71% death protection rate) than among inpatients (45%). In a subset of 16,063 patients with available comorbidities and vaccinations status, obesity (2.01, 1.23-3.29), chronic respiratory disease (2.93, 1.29-6.64), and immunodeficiency (4.01, 1.69-9.50), on the one hand, and vaccination (0.29, 0.12-0.67) and HCQ-AZ treatment (0.47, 0.29-0.76), on the other hand, were independent factors associated with mortality. HCQ, alone or in any association, was associated with significant protection from death among outpatients (0.41, 0.21-0.79) and inpatients (0.59, 0.47-0.73). Conclusions HCQ prescribed early or late protects in part from COVID-19-related death. During pandemic health crises, financial stakes are enormous. Authentication of the data by an independent external judicial officer should be required. Public sharing of anonymized databases, ensuring their verifiability, should be mandatory in this context to avoid fake publications.

Cardiovascular Safety of Hydroxychloroquine-Azithromycin in 424 COVID-19 Patients (preprint)

Background Hydroxychloroquine (HCQ) combined with azithromycin (AZM) has been widely administered to patients with COVID-19 despite scientific controversies. In particular the potential to prolong cardiac repolarisation by using this combination has been discussed. Materials and methods We report a pragmatic and simple safety approach which we implemented in the first patients treated for COVID-19 in our center early 2020. Treatment contraindications were the presence of severe structural or electrical heart disease, baseline corrected QT interval (QTc) >500 ms, hypokalaemia, or other drugs prolonging QTc that could not be interrupted. Electrocardiogram and QTc was evaluated at admission and re-evaluated after 48 hours of the initial prescription. Results Among 424 consecutive adults (mean age 46.3 ± 16.1 years; 216 women). Patients were followed in conventional wards (21.5%) or in a day-care unit (78.5%). A total of 11 patients (2.6%) had contraindications to HCQ-AZ combination. In the remaining 413 treated patients, there were no arrhythmic events in any patient during the 10-day treatment regimen. QTc was slightly but statistically significantly prolonged by 3.75 ± 25.4 ms after two days (p=0.003). Ten patients (2.4%) developed QTc prolongation >60 ms, and none had QTc >500 ms. Conclusions This report do not aim to contribute to knowledge of the efficacy of treating COVID-19 with HCQ-AZ. However, a simple initial assessment of patient medical history, ECG and kalaemia identifies contraindicated patients and enables the safe treatment by HCQ-AZ of COVID-19 patients. QT-prolonging anti-infective drugs can be used safely in acute life-threatening infections, provided that a strict protocol and close collaboration between infectious disease specialists and rhythmologists are followed.

Culture and identification of a Deltamicron SARS-CoV-2 in a three cases cluster in southern France (preprint)

Multiple SARS-CoV-2 variants have successively, or concommitantly spread worldwide since summer 2020. A few co-infections with different variants were reported and genetic recombinations, common among coronaviruses, were reported or suspected based on co-detection of signature mutations of different variants in a given genome. Here were report three infections in southern France with a Delta 21J/AY.4-Omicron 21K/BA.1 Deltamicron recombinant. The hybrid genome harbors signature mutations of the two lineages, supported by a mean sequencing depth of 1,163-1,421 reads and mean nucleotide diversity of 0.1-0.6%. It is composed of the near full-length spike gene (from codons 156-179) of an Omicron 21K/BA.1 variant in a Delta 21J/AY.4 lineage backbone. It is similar to those reported for 15 other patients sampled since January 2022 in Europe. Importantly, we cultured an isolate of this recombinant and sequenced its genome. It was observed by scanning electron microscopy. As it is misidentified with current variant screening qPCR, we designed and implemented for routine diagnosis a specific duplex qPCR. Finally, structural analysis of the recombinant spike suggested its hybrid content could optimize viral binding to the host cell membrane. These findings prompt further studies of the virological, epidemiological, and clinical features of this recombinant.

First cases of infection with the 21L/BA.2 Omicron variant in Marseille, France (preprint)

The SARS-CoV-2 21K/BA.1, 21L/BA.2, and BA.3 Omicron variants have recently emerged worldwide. To date, the 21L/BA.2 Omicron variant has remained very minority globally but became predominant in Denmark instead of the 21K/BA.1 variant. Here we describe the first cases diagnosed with this variant in south-eastern France. We identified thirteen cases using variant-specific qPCR and next-generation sequencing between 28/11/2021 and 31/01/2022, the first two cases being diagnosed in travellers returning from Tanzania. Overall, viral genomes displayed a mean (+/-standard deviation) number of 65.9+/-2.5 (range, 61-69) nucleotide substitutions and 31.0+/-8.3 (27-50) nucleotide deletions, resulting in 49.6+/-2.2 (45-52) amino acid substitutions (including 28 in the spike protein) and 12.4+/-1.1 (12-15) amino acid deletions. Phylogeny showed the distribution in three different clusters of these genomes, which were most closely related to genomes from England and South Africa, from Singapore and Nepal, or from France and Denmark. Structural predictions pointed out a significant enlargement and flattening of the 21L/BA.2 N-terminal domain surface compared with that of the 21K/BA.2 Omicron variant, which may facilitate initial viral interactions with lipid rafts. Close surveillance is needed at global, country and center scales to monitor the incidence and clinical outcome of the 21L/BA.2 Omicron variant.

The emergence, spread and vanishing of a French SARS-CoV-2 variant exemplifies the fate of RNA virus epidemics and obeys the Black Queen rule (preprint)

Summary The nature and dynamics of mutations associated with the emergence, spread and vanishing of SARS-CoV-2 variants causing successive waves are complex 1-5 . We determined the kinetics of the most common French variant (“Marseille-4”) for 10 months since its onset in July 2020 5 . Here, we analysed and classified into subvariants and lineages 7,453 genomes obtained by next-generation sequencing. We identified two subvariants, Marseille-4A, which contains 22 different lineages of at least 50 genomes, and Marseille-4B. Their average lifetime was 4.1±1.4 months, during which 4.1±2.6 mutations accumulated. Growth rate was 0.079±0.045, varying from 0.010 to 0.173. All the lineages exhibited a “gamma” distribution. Several beneficial mutations at unpredicted sites initiated a new outbreak, while the accumulation of other mutations resulted in more viral heterogenicity, increased diversity and vanishing of the lineages. Marseille-4B emerged when the other Marseille-4 lineages vanished. Its ORF8 gene was knocked out by a stop codon, as reported in several mink lineages and in the alpha variant. This subvariant was associated with increased hospitalization and death rates, suggesting that ORF8 is a nonvirulence gene. We speculate that the observed heterogenicity of a lineage may predict the end of the outbreak.

Exploratory Study on Application of MALDI-TOF-MS to Detect SARS-CoV-2 Infection in Human Saliva (preprint)

SARS-CoV-2 caused a large outbreak since its emergence in December 2019. The COVID-19 diagnosis became a priority to isolate and treat infected individuals in order to break the contamination chain. Currently, the reference test for COVID-19 diagnosis is the molecular detection (RT-qPCR) of the virus from nasopharyngeal swab (NPS) samples. Although this sensitive and specific test remains the gold standard, it has several limitations, such as the invasive collection method, the relative high cost and the duration of the test. Moreover, the material shortage to perform tests due to the discrepancy between the high demand for tests and the production capacities puts additional constraints on RT-qPCR. Here, we propose a PCR-free method for diagnosing SARS-CoV-2 based on Matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) profiling and machine learning (ML) models from salivary samples. Kinetic saliva samples were collected at enrollment and ten and thirty days later (D0, D10 and D30), to assess the classification performance of the ML models compared to the molecular tests performed on NPS specimens. Spectra were generated using an optimized protocol of saliva collection and successive quality control steps were developed to ensure the reliability of spectra. A total of 360 averaged spectra were included in the study. At D0, the comparison of MS spectra from SARS-CoV-2 positive patients (n=105) with healthy healthcare controls (n=51) revealed nine peaks that significantly distinguished the two groups. Among the five ML models tested, Support Vector Machine with Linear Kernel (SVM-LK) provided the best performance on the training dataset (accuracy = 85.2 %, sensitivity = 85.1 %, specificity = 85.3 %, F1-Score = 85.1 %). The application of the SVM-LK model on independent datasets confirmed it performances with 88.9% and 80.8% of correct classification for samples collected at D0 and D30, respectively. Conversely, at D10, the proportion of correct classification fallen to 64.3%. The analysis of saliva samples by MALDI-TOF MS and ML appears as an interesting supplementary tool for COVID-19 diagnosis, despite the mitigated results obtained for convalescent patients (D10).

Analysis of SARS-CoV-2 variants from 24,181 patients exemplifies the role of globalisation and zoonosis in pandemics (preprint)

After the end of the first epidemic episode of SARS-CoV-2 infections, as cases began to rise again during the summer of 2020, we at IHU Mediterranee Infection in Marseille, France, intensified the genomic surveillance of SARS-CoV-2, and described the first viral variants. In this study, we compared the incidence curves of SARS-CoV-2-associated deaths in different countries and reported the classification of SARS-CoV-2 variants detected in our institute, as well as the kinetics and sources of the infections. We used mortality collected from a COVID-19 data repository for 221 countries. Viral variants were defined based on [≥]5 hallmark mutations shared by [≥]30 genomes. SARS-CoV-2 genotype was determined for 24,181 patients using next-generation genome and gene sequencing (in 47% and 11% of cases, respectively) or variant-specific qPCR (in 42% of cases). Sixteen variants were identified by analysing viral genomes from 9,788 SARS-CoV-2-diagnosed patients. Our data show that since the first SARS-CoV-2 epidemic episode in Marseille, importation through travel from abroad was documented for seven of the new variants. In addition, for the B.1.160 variant of Pangolin classification (a.k.a. Marseille-4), we suspect transmission from mink farms. In conclusion, we observed that the successive epidemic peaks of SARS-CoV-2 infections are not linked to rebounds of viral genotypes that are already present but to newly-introduced variants. We thus suggest that border control is the best mean of combating this type of introduction, and that intensive control of mink farms is also necessary to prevent the emergence of new variants generated in this animal reservoir.

Mouth Washing Impaired SARS-CoV-2 Detection in Saliva (preprint)

Background. A previous study demonstrated the performance of the Salivette® (SARSTEDT, Numbrecht, Germany) as a homogeneous saliva collection system to diagnose COVID-19 by RT-qPCR, notably for symptomatic and asymptomatic patients. However, for convalescent patients, the corroboration of molecular detection of SARS-CoV-2 in paired nasopharyngeal swabs (NPS) and saliva samples was unsatisfactory. Objectives. The aim of the present work was to assess the concordance level of SARS-CoV-2 detection between paired sampling of NPSs and saliva collected with Salivette® at two time points, with ten days of interval. Results. A total of 319 paired samples from 145 outpatients (OP) and 51 healthcare workers (HW) were collected. Due to significant waiting rate at hospital, most of the patients ate and/or drank in waiting their turn. Consequently, a mouth washing was systematically proposed prior saliva collection. None of the HW were diagnosed SARS-CoV-2 positive using NPS or saliva specimens at both time points (n=95) by RT-qPCR. The virus was detected in 56.3% (n=126/224) of the NPS samples from OP, but solely 26.8% (n=60/224) of the paired saliva specimens. The detection of the internal cellular control, the human RNase P, in more than 98% of the saliva samples, underlined that the low sensitivity of saliva specimens (45.2%) for SARS-CoV-2 detection was not attributed to an improper saliva sample storing or RNA extraction. Conclusions. Then, the mouth washing decreased viral load of buccal cavity conducting to impairment of SARS-CoV-2 detection. Viral loads in saliva neo-produced appeared insufficient for molecular detection of SARS-CoV-2. At the time that saliva tests could be a rapid, simple and noninvasive strategy to assess on large scale schooled children in France, the determination of the performance of saliva collection become imperative to standardize procedures.

The Need for Early Management in Patients With COVID-19 (preprint)

In March 2020, the IHU Méditerranée Infection set up a screening and treatment center for patients with COVID-19, a system that has been ultimately recommended by French public health authorities. The recent publication of the profiles of patients hospitalized in France published by the Directorate for Research, Studies, Evaluation and Statistics gives us the opportunity to measure the impact of this multidisciplinary early management system coupled with screening on mortality at 90 days. Analysis of the data shows that the system established at IHU-MI was associated with lower mortality, taking age and sex into account. Regarding the age-standardized mortality rate, mortality rates were lower than national data regardless of the period of the epidemic. Early management seems to have significantly decreased the mortality rate in the under-60 age group, suggesting the importance of early management, regardless of age. In addition, these patients had pejorative clinical criteria (high NEWS-2 score, ICU visits, oxygen saturation below 95%) requiring hospitalization, and co-morbidities that are now known to be aggravating factors [7]. This reinforces the need to care for all individuals, regardless of age. Early medical care, as part of a system integrating a screening center and a day hospital, may explain the lower mortality rates.

Expression of ACE2 receptor, soluble ACE2, Angiotensin I, Angiotensin II and Angiotensin (1-7), is modulated in COVID-19 patients (preprint)

Although SARS-CoV-2 is primarily a pulmonary-tropic virus, it is nonetheless responsible for multi-organ failure in patients with severe forms of COVID-19, particularly those with hypertension or cardiovascular disease. Infection requires virus binding to the angiotensin I converting enzyme 2 (ACE2) monocarboxypeptidase, a regulator of blood pressure homeostasis through its ability to catalyze the proteolysis of Angiotensin II (AngII) into Ang(1-7). Although assumed, it had not been proven so far whether the SARS-CoV-2 replication in COVID-19 patients could modulate the expression of the ACE2 receptor and/or the AngII plasma levels. We demonstrate here, that in COVID-19 patients the ACE2 mRNA expression is markedly reduced in circulating blood cells. This ACE2 gene dysregulation mainly affects the monocytes which also show a lower expression of membrane ACE2 protein. Moreover, a significant decrease in soluble ACE2 plasma levels is observed in COVID-19 patients, whereas the concentration of sACE2 returns to normal levels in patients recovered from COVID-19. In the plasma of COVID-19 patients, we also found an increase in AngI and AngII. On the other hand, the plasma levels of Ang(1-7) remains almost stable in COVID-19 patients. Despite the Ang(1-7) presence in the plasma of COVID-19 patients it seems insufficient to prevent the effects of massive AngII accumulation. These are the first direct evidence that the SARS-CoV-2 may affect the expression of blood pressure regulators with possible harmful consequences on COVID-19 outcome.

Introduction into the Marseille geographical area of a mild SARS-CoV-2 variant originating from sub-Saharan Africa (preprint)

BACKGROUNDIn Marseille, France, the COVID-19 incidence evolved unusually with several successive epidemic episodes. The second outbreak started in July, was associated with North Africa, and involved travelers and an outbreak on passenger ships. This suggested the involvement of a new viral variant. METHODSWe sequenced the genomes from 916 SARS-CoV-2 strains from COVID-19 patients in our institute. The patients demographic and clinical features were compared according to the infecting viral variant. RESULTSFrom June 26th to August 14th, we identified a new viral variant (Marseille-1). Based on genome sequences (n=89) or specific qPCR (n=53), 142 patients infected with this variant were detected. It is characterized by a combination of 10 mutations located in the nsp2, nsp3, nsp12, S, ORF3a, ORF8 and N/ORF14 genes. We identified Senegal and Gambia, where the virus had been transferred from China and Europe in February-April as the sources of the Marseille-1 variant, which then most likely reached Marseille through Maghreb when French borders reopened. In France, this variant apparently remained almost limited to Marseille. In addition, it was significantly associated with a milder disease compared to clade 20A ancestor strains. CONCLUSIONOur results demonstrate that SARS-CoV-2 can genetically diversify rapidly, its variants can diffuse internationally and cause successive outbreaks.

Saliva, a relevant alternative sample for SARS-CoV2 detection (preprint)

Background: Currently, COVID-19 diagnosis relies on quantitative reverse-transcriptase polymerase chain reaction (RT-qPCR) from nasopharyngeal swab (NPS) specimens, but NPSs present several limitations. The simplicity, low invasive and possibility of self-collection of saliva imposed this specimen as a relevant alternative for SARS-CoV-2 detection. However, the discrepancy of saliva test results compared to NPSs made of its use controversial. Here, we proposed to assess Salivettes, as a standardized saliva collection device, and to compare SARS-CoV-2 positivity on paired NPS and saliva specimens. Methods: A total of 303 individuals randomly selected among those investigated for SARS-CoV-2 were enrolled, including 30 (9.9%) patients previously positively tested using NPS (follow-up group), 90 (29.7%) mildly symptomatic and 183 (60.4%) asymptomatic. Results: The RT-qPCR revealed a positive rate of 11.6% (n=35) and 17.2% (n=52) for NPSs and saliva samples, respectively. The sensitivity and specificity of saliva samples were 82.9% and 91.4%, respectively, using NPS as reference. The highest proportion of discordant results concerned the follow-up group (33.3%). Although in the symptomatic and asymptomatic groups the agreement exceeded 90.0%, 17 individuals were detected positive only in saliva samples, with consistent medical arguments. Conclusion: Saliva collected with Salivette demonstrated more sensitive for detecting symptomatic and pre-symptomatic infections.

An ultra-sensitive, ultra-fast whole blood monocyte CD169 assay for COVID-19 screening (preprint)

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.

18F-FDG brain PET metabolism in post-SARS-CoV-2 infection: substrate for persistent/delayed disorders? (preprint)

Purpose: Several brain complications of SARS-CoV-2 infection have been reported. It has been moreover speculated that this neurotropism could potentially cause a delayed outbreak of neuropsychiatric and neurodegenerative diseases of neuroinflammatory origin. A propagation mechanism has been proposed across the cribriform plate of the ethmoid bone, from the nose to the olfactory epithelium, and possibly afterwards to other limbic structures, and deeper parts of the brain including the brainstem. Methods: : Review of clinical examination, and whole-brain voxel-based analysis of 18 F-FDG PET metabolism in comparison to healthy subjects (p-voxel<0.001, p-cluster<0.05), of two patients with confirmed diagnosis of SARS-CoV-2 pneumonia explored at the post-viral stage of the disease. Results: : Hypometabolism of the olfactory/rectus gyrus was found on the two patients, especially one with 4 weeks prolonged anosmia. Additional hypometabolisms were found within bilateral amygdala, hippocampus, cingulate cortex, thalamus, pons and medulla brainstem in the other patient who complained of delayed onset of an atypical painful syndrome. Conclusion: These preliminary findings reinforce the hypotheses of SARS-CoV-2 neurotropism through the olfactory bulb, and the possible extension of this impairment to other limbic structures and to the brainstem. 18 F-FDG PET hypometabolism could constitute a cerebral quantitative biomarker of this involvement. Post-viral cohort studies are required to specify the exact relationship between limbic/brainstem hypometabolisms and the possible persistent disorders, especially involving cognitive or emotion disturbances, residual respiratory symptoms or painful complaints.

Evaluating the serological status of COVID-19 patients using an indirect immunofluorescent assay, France. (preprint)

An indirect immunofluorescent assay was developed in order to assess the serological status of 888 RT-PCR-confirmed COVID-19 patients (1,302 serum samples) and controls in Marseille, France. Incorporating an inactivated clinical SARS CoV-2 isolate as the antigen, the specificity of the assay was measured as 100% for IgA titre [≥] 1:200; 98.6% for IgM titre [≥] 1:200; and 96.3% for IgG titre [≥] 1:100 after testing a series of negative controls as well as 150 serums collected from patients with non-SARS-CoV-2 Coronavirus infection, non-Coronavirus pneumonia and infections known to elicit false-positive serology. Seroprevalence was then measured at 3% before a five-day evolution up to 47% after more than 15 days of evolution. We observed that the seroprevalence as well as the titre of specific antibodies were both significantly higher in patients with a poor clinical outcome than in patients with a favourable evolution. These data, which have to be integrated into the ongoing understanding of the immunological phase of the infection, suggest that serotherapy may not be a therapeutic option in patients with severe COVID-19 infection. The IFA assay reported here is useful for monitoring SARS-CoV-2 exposure at the individual and population levels.