Fungal microbiota dysbiosis in IBD.

International audienceThe bacterial intestinal microbiota plays major roles in human physiology and IBDs. Although some data suggest a role of the fungal microbiota in IBD pathogenesis, the available data are scarce. The aim of our study was to characterise the faecal fungal microbiota in patients with IBD. Bacterial and fungal composition of the faecal microbiota of 235 patients with IBD and 38 healthy subjects (HS) was determined using 16S and ITS2 sequencing, respectively. The obtained sequences were analysed using the Qiime pipeline to assess composition and diversity. Bacterial and fungal taxa associated with clinical parameters were identified using multivariate association with linear models. Correlation between bacterial and fungal microbiota was investigated using Spearman's test and distance correlation. We observed that fungal microbiota is skewed in IBD, with an increased Basidiomycota/Ascomycota ratio, a decreased proportion of Saccharomyces cerevisiae and an increased proportion of Candida albicans compared with HS. We also identified disease-specific alterations in diversity, indicating that a Crohn's disease-specific gut environment may favour fungi at the expense of bacteria. The concomitant analysis of bacterial and fungal microbiota showed a dense and homogenous correlation network in HS but a dramatically unbalanced network in IBD, suggesting the existence of disease-specific inter-kingdom alterations. Besides bacterial dysbiosis, our study identifies a distinct fungal microbiota dysbiosis in IBD characterised by alterations in biodiversity and composition. Moreover, we unravel here disease-specific inter-kingdom network alterations in IBD, suggesting that, beyond bacteria, fungi might also play a role in IBD pathogenesis

Contribution of molecular biology tools in the study and surveillance of SARS-CoV-2 during the COVID-19 pandemic

Dès le début de la pandémie de COVID-19, des outils de biologie moléculaire permettant la détection et l’analyse du génome du SARS-CoV-2 ont été développés et déployés à une échelle sans précédent. Les techniques de PCR ont été utilisées pour diagnostiquer quotidiennement des millions de personnes, tandis que la démocratisation du séquençage de nouvelle génération (NGS) a permis de générer rapidement des centaines de milliers de génomes viraux. Ces outils ont été une source d’information majeure dans l’étude et la compréhension de la physiopathologie du SARS-CoV-2, mais également dans le suivi en temps réel de la dynamique de la pandémie et de l’évolution de ce virus. Nous avons entrepris cette thèse afin d’évaluer comment ces outils de biologie moléculaire peuvent contribuer à l’étude et la surveillance du SARS-CoV-2, dans le cadre de trois études répondants à différents objectifs. Avec l’étude PhyloCoV, nous avons tout d’abord apporté une description précise de l’évolution de la diversité virale observée au cours de l’année 2020 en région Ile-de-France, grâce au séquençage de 736 génomes du SARS-CoV-2 provenant de patients et de personnels soignant des CHU Pitié-Salpêtrière et Bichat Claude-Bernard. Cette étude nous a également permis de développer une méthode de clustering phylogénétique permettant la détection des transmissions intra-hospitalières de la COVID-19. Ensuite, dans le cadre d’une étude observationnelle au sein du GH Sorbonne Université, nous avons estimé la fréquence et la mortalité associées aux transmissions nosocomiales du SARS-CoV-2. Entre septembre et novembre 2020, 209 cas de COVID-19 nosocomial ont été identifiés, représentant 13,9% des patients hospitalisés infectés par le SARS-CoV-2 et associés à une mortalité de 31%. Le séquençage des génomes viraux a permis de réfuter près d’un tiers des transmissions nosocomiales et de révéler de précieuses informations sur les voies de transmission au sein des services hospitaliers. Finalement, avec l’étude COCOPREV, nous avons étudié l’évolution du génome du SARS-CoV-2 au cours de l’infection chez des patients traités par des anticorps monoclonaux, majoritairement immunodéprimés. Plusieurs mutations de résistance émergent dans la protéine SPIKE sous les trois traitements étudiés. Les patients traités avec du Tixagevimab/Cilgavimab présentent un risque 5 fois plus élevé de développer des mutations au niveau des résidus R346 et K444. A la différence des autres mutations retrouvées, les substitutions R346 et K444 ont plusieurs fois émergé dans différents variants grâce à leur impact sur le fitness du SARS-CoV-2. Elles ont finalement été sélectionnées par les variants BQ.1 et XBB, variants actuellement majoritaires. En conclusion, nous avons démontré l’utilité des outils de biologie moléculaire dans la surveillance de la transmission du SARS-CoV-2 dans les hôpitaux et dans la population en général, ainsi que dans l'identification de mutations de résistance émergentes chez des patients traités par des anticorps monoclonaux. Ces outils continueront très probablement d'être utilisés dans le cadre de la pandémie de COVID-19 mais également dans la surveillance de l’émergence et de la propagation d’autres virus.From the onset of the COVID-19 pandemic, molecular biology tools for detection and analysis of the SARS-CoV-2 genome were developed and deployed on an unprecedented scale. PCR techniques have been used to diagnose millions of people on a daily basis, while the democratization of next-generation sequencing (NGS) has enabled the rapid generation of hundreds of thousands of viral genomes. These tools have been a major source of information in the study and understanding of the pathophysiology of SARS-CoV-2, but also in the real-time monitoring of the dynamics of the pandemic and the evolution of this virus. We undertook this thesis to evaluate how these molecular biology tools can contribute to the study and surveillance of SARS-CoV-2, in three studies with different objectives. With the PhyloCoV study, we first provided a precise description of the evolution of viral diversity observed during the year 2020 in the Ile-de-France area, thanks to the sequencing of 736 SARS-CoV-2 genomes from patients and healthcare workers of the Pitié-Salpêtrière and Bichat Claude-Bernard University Hospital. This study also allowed us to develop a phylogenetic clustering method allowing the detection of intra-hospital transmission of COVID-19. Then, in the framework of an observational study within the GH Sorbonne Université, we estimated the frequency and mortality associated with nosocomial transmissions of SARS-CoV-2. Between September and November 2020, 209 cases of nosocomial COVID-19 were identified, representing 13.9% of hospitalized patients infected with SARS-CoV-2 and associated with a mortality of 31%. Sequencing of the viral genomes disproved nearly one third of nosocomial transmissions and revealed valuable information about transmission routes within hospital wards. Finally, with the COCOPREV study, we studied the evolution of the SARS-CoV-2 genome during infection in patients treated with monoclonal antibodies, mostly immunocompromised. Several resistance mutations emerge in the SPIKE protein under the three treatments studied. Patients treated with Tixagevimab/Cilgavimab had a 5-fold increased risk of developing mutations at residues R346 and K444. Unlike other mutations found, R346 and K444 substitutions have repeatedly emerged in different variants due to their impact on SARS-CoV-2 fitness. They were finally selected by the currently dominant BQ.1 and XBB variants. In conclusion, we have demonstrated the utility of molecular biology tools in monitoring SARS-CoV-2 transmission in hospitals and the general population, as well as in identifying emerging resistance mutations in patients treated with monoclonal antibodies. These tools will most likely continue to be used in the context of the COVID-19 pandemic but also in monitoring the emergence and spread of other viruses

Kaposi’s Sarcoma-Associated Herpesvirus, the Etiological Agent of All Epidemiological Forms of Kaposi’s Sarcoma

International audienceSimple SummaryKaposi’s sarcoma-associated herpesvirus (KSHV) is one of the seven oncogenic viruses currently recognized by the International Agency for Research on Cancer. Its presence for Kaposi’s sarcoma development is essential and knowledge on the oncogenic process has increased since its discovery in 1994. However, some uncertainties remain to be clarified, in particular on the exact routes of transmission and disparities in KSHV seroprevalence and the prevalence of Kaposi’s sarcoma worldwide. Here, we summarized the current data on the KSHV viral particle’s structure, its genome, the replication, its seroprevalence, the viral diversity and the lytic and latent oncogenesis proteins involved in Kaposi’s sarcoma. Lastly, we reported the environmental, immunological and viral factors possibly associated with KSHV transmission that could also play a role in the development of Kaposi’s sarcoma.AbstractKaposi’s sarcoma-associated herpesvirus (KSHV), also called human herpesvirus 8 (HHV-8), is an oncogenic virus belonging to the Herpesviridae family. The viral particle is composed of a double-stranded DNA harboring 90 open reading frames, incorporated in an icosahedral capsid and enveloped. The viral cycle is divided in the following two states: a short lytic phase, and a latency phase that leads to a persistent infection in target cells and the expression of a small number of genes, including LANA-1, v-FLIP and v-cyclin. The seroprevalence and risk factors of infection differ around the world, and saliva seems to play a major role in viral transmission. KSHV is found in all epidemiological forms of Kaposi’s sarcoma including classic, endemic, iatrogenic, epidemic and non-epidemic forms. In a Kaposi’s sarcoma lesion, KSHV is mainly in a latent state; however, a small proportion of viral particles (<5%) are in a replicative state and are reported to be potentially involved in the proliferation of neighboring cells, suggesting they have crucial roles in the process of tumorigenesis. KSHV encodes oncogenic proteins (LANA-1, v-FLIP, v-cyclin, v-GPCR, v-IL6, v-CCL, v-MIP, v-IRF, etc.) that can modulate cellular pathways in order to induce the characteristics found in all cancer, including the inhibition of apoptosis, cells’ proliferation stimulation, angiogenesis, inflammation and immune escape, and, therefore, are involved in the development of Kaposi’s sarcoma

Epidemiology and Characteristics of SARS-CoV-2 Variants of Concern: The Impacts of the Spike Mutations

SARS-CoV-2 expresses on its surface the Spike protein responsible for binding with the ACE2 receptor and which carries the majority of immunodominant epitopes. Mutations mainly affect this protein and can modify characteristics of the virus, giving each variant a unique profile concerning its transmissibility, virulence, and immune escape. The first lineage selected is the B.1 lineage characterized by the D614G substitution and from which all SARS-CoV-2 variants of concern have emerged. The first three variants of concern Alpha, Beta, and Gamma spread in early 2021: all shared the N501Y substitution. These variants were replaced by the Delta variant in summer 2021, carrying unique mutations like the L452R substitution and associated with higher virulence. It was in turn quickly replaced by the Omicron variant at the end of 2021, which has predominated since then, characterized by its large number of mutations. The successive appearance of variants of concern showed a dynamic evolution of SARS-CoV-2 through the selection and accumulation of mutations. This has not only allowed progressive improvement of the transmissibility of SARS-CoV-2, but has also participated in a better immune escape of the virus. This review brings together acquired knowledge about SARS-CoV-2 variants of concern and the impacts of the Spike mutations

Harnessing Redox Disruption to Treat Human Herpesvirus 8 (HHV-8) Related Malignancies

Reprogrammed metabolism is regarded as a hallmark of cancer and offers a selective advantage to tumor cells during carcinogenesis. The redox equilibrium is necessary for growth, spread and the antioxidant pathways are boosted following Reactive Oxygen Species (ROS) production to prevent cell damage in tumor cells. Human herpesvirus 8 (HHV-8), the etiologic agent of Kaposi sarcoma KS and primary effusion lymphoma (PEL), is an oncogenic virus that disrupts cell survival-related molecular signaling pathways leading to immune host evasion, cells growths, angiogenesis and inflammatory tumor-environment. We recently reported that primaquine diphosphate causes cell death by apoptosis in HHV-8 infected PEL cell lines in vivo and exhibits therapeutic anti-tumor activity in mice models and advanced KS. Our findings also suggest that the primaquine-induced apoptosis in PEL cells is mostly influenced by ROS production and targeting the redox balance could be a new approach to treat HHV-8 related diseases. In this review, we summarized the knowledge about the influence of ROS in cancer development; more specifically, the proof of evidence from our work and from the literature that redox pathways are important for the development of HHV-8 pathologies

Seroprevalence and molecular diversity of Human Herpesvirus 8 among people living with HIV in Brazzaville, Congo

International audienceHuman herpesvirus 8 (HHV8) is endemic in Africa, although studies of this infection are rare in Congo. We evaluated seroprevalence and HHV-8 diversity among people living with HIV. We included 353 patients receiving highly active antiretroviral therapy. Antibodies against HHV-8 latency-associated nuclear antigen were detected by indirect immunofluorescence. In HHV-8 positive patients, we performed HHV-8 quantification in blood and saliva by real-time PCR and typing by Sanger sequencing of K1 open reading frame. HHV-8 seroprevalence was 19%, being male (odd ratio [OR] = 1.741, [95% Confidence interval {CI}, 0.97–3.07]; p = 0.0581) and having multiple sex partners before HIV diagnosis (OR = 1.682, [CI 95%, 0.97–2.92]; p = 0.0629) tended to be associated with HHV-8 seropositivity. Of the 64 HHV-8 seropositive patients, HHV-8 DNA was detected in 10 (16%) in saliva, 6 (9%) in whole-blood and in 2 (3%) in both whole-blood and saliva. Three out of 6 HHV-8 strains were subtypes A5, 2 subtype B1 and 1 subtype C. HHV-8 seroprevalence was relatively low with more frequent carriage in men, associated with asymptomatic oral excretion and a predominance of subtype A5. These data tend to support the hypothesis of horizontal transmission in people living with HIV in Brazzaville

Decreased Sensitivity of Rapid Antigen Test Is Associated with a Lower Viral Load of Omicron than Delta SARS-CoV-2 Variant

International audienceLarge-scale screening for SARS-CoV-2 infection is an important tool for epidemic prevention and control. The appearance of new variants associated with specific mutations can call into question the effectiveness of rapid diagnostic tests (RDTs) deployed massively at national and international levels. We compared the clinical and virological characteristics of individuals infected by Delta or Omicron variants to assess which factors were associated with a reduced performance of RDT. A commercially available RDT as well as the evaluation of the viral load (VL) and the detection of replicate intermediates (RIs) were carried out retrospectively on positive SARS-CoV-2 nasopharyngeal specimens from health care workers of the Pitié-Salpêtrière Hospital infected by the Delta or Omicron variant between July 2021 and January 2022. Of the 205 samples analyzed (104 from individuals infected with Delta and 101 with Omicron), 176 were analyzed by RDT and 200 by RT-PCR for VL and RIs. The sensitivity of the TDR for Omicron was significantly lower than that observed for Delta (53.8% versus 74.7%, respectively, P < 0.01). Moreover, the Delta VL was significantly higher than that measured for Omicron (median Ct 21.2 versus 24.1, respectively, P < 0.01) and associated with the positivity of the RDT in multivariate analysis. We demonstrate a lower RDT sensitivity associated with a lower VL at the time of diagnosis on Omicron-infected individuals in comparison to those infected with the Delta variant. This RDT lower sensitivity should be taken into account in the large-scale screening strategy and in particular in case of strong suspicion of infection where testing should be repeated. IMPORTANCE Previous reports have shown a variability in the diagnostic performance of RDTs. In the era of SARS-CoV-2 variants and the use of RDT, mutation associated with these variants could affect the test performance. We evaluate the sensitivity of the RDT Panbio COVID-19 Ag (Abbott) with two variants of concern (VOC), the Delta and Omicron variants. In order to investigate whether clinical characteristics or virological characteristics can affect this sensitivity, we collected clinical information and performed a specific RT-PCR that detected the RIs as a marker of the viral replication and viral cycle stage. Our results showed that Omicron was less detected than the Delta variant. A lower viral load of Omicron variant in comparison to Delta variant explained this decreased sensitivity, even if they are at the same stage of the disease and the viral cycle and should be taken into account with the use of RDT as diagnostic tool

Primaquine as a Candidate for HHV-8-Associated Primary Effusion Lymphoma and Kaposi’s Sarcoma Treatment

International audienceHuman Herpesvirus 8 (HHV-8) is associated with three main severe orphan malignancies, Kaposi’s sarcoma (KS), multicentric Castleman’s disease (MCD), and primary effusion lymphoma (PEL), which present few therapeutic options. We identified the antimalarial primaquine diphosphate (PQ) as a promising therapeutic candidate for HHV-8-associated PEL and KS. Indeed, PQ strongly reduced cell viability through caspase-dependent apoptosis, specifically in HHV-8-infected PEL cells. Reactive oxygen species (ROS)- and endoplasmic reticulum (ER) stress-mediated apoptosis signaling pathways were found to be part of the in vitro cytotoxic effect of PQ. Moreover, PQ treatment had a clinically positive effect in a nonobese diabetic (NOD)/SCID xenograft PEL mouse model, showing a reduction in tumor growth and an improvement in survival. Finally, an exploratory proof-of-concept clinical trial in four patients harboring severe KS was conducted, with the main objectives to assess the efficacy, the safety, and the tolerability of PQ, and which demonstrated a positive efficacy on Kaposi’s sarcoma-related lesions and lymphedema

Nosocomial transmission clusters and lineage diversity characterized by SARS-CoV-2 genomes from two large hospitals in Paris, France, in 2020

International audienceFrance went through three deadly epidemic waves due to the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), causing major public health and socioeconomic issues. We proposed to study the course of the pandemic along 2020 from the outlook of two major Parisian hospitals earliest involved in the fight against COVID-19. Genome sequencing and phylogenetic analysis were performed on samples from patients and health care workers (HCWs) from Bichat (BCB) and Pitié-Salpêtrière (PSL) hospitals. A tree-based phylogenetic clustering method and epidemiological data were used to investigate suspected nosocomial transmission clusters. Clades 20A, 20B and 20C were prevalent during the spring wave and, following summer, clades 20A.EU2 and 20E.EU1 emerged and took over. Phylogenetic clustering identified 57 potential transmission clusters. Epidemiological connections between participants were found for 17 of these, with a higher proportion of HCWs. The joint presence of HCWs and patients suggest viral contaminations between these two groups. We provide an enhanced overview of SARS-CoV-2 phylogenetic changes over 2020 in the Paris area, one of the regions with highest incidence in France. Despite the low genetic diversity displayed by the SARS-CoV-2, we showed that phylogenetic analysis, along with comprehensive epidemiological data, helps to identify and investigate healthcare associated clusters