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International audience; Background: In Marseille, France, following a first severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) outbreak in March-May 2020, a second epidemic phase occurred from June, involving 10 new variants. The Marseille-4 variant caused an epidemic that started in August and is still ongoing. Methods: The 1038 SARS-CoV-2 whole genome sequences obtained in our laboratory by next-generation sequencing with Illumina technology were analysed using Nextclade and nextstrain/ncov pipelines and IQ-TREE. A Marseille-4-specific qPCR assay was implemented. Demographic and clinical features were compared between patients with the Marseille-4 variant and those with earlier strains. Results: Marseille-4 harbours 13 hallmark mutations. One leads to an S477N substitution in the receptor binding domain of the spike protein targeted by current vaccines. Using a specific qPCR, it was observed that Marseille-4 caused 12-100% of SARS-CoV-2 infections in Marseille from September 2020, being involved in 2106 diagnoses. This variant was more frequently associated with hypoxemia than were clade 20A strains before May 2020. It caused a re-infection in 11 patients diagnosed with different SARS-CoV-2 strains before June 2020, suggesting either short-term protective immunity or a lack of cross-immunity. Conclusions: Marseille-4 should be considered as a major SARS-CoV-2 variant. Its sudden appearance points towards an animal reservoir, possibly mink. The protective role of past exposure and current vaccines against this variant should be evaluated.

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In the present study, we aimed to assess recovery rates at more than six months post-onset of anosmia and ageusia due to COVID-19 infection. Patients who reported anosmia and/or ageusia at the acute phase of a COVID-19 PCR-confirmed episode were retrospectively identified from a cohort of 3,737 patients seen at Hospital University institute, Marseille between 3 March and 27 April, 2020. A total of 200 patients were randomly selected. 75 (37.5%) were lost in follow-up and 125 (62.5%) answered the questionnaire. 68.1% patients recovered their sense of smell and 73.0% their sense of taste during the first six weeks following the onset of symptoms. Anosmia tended to take more time to resolve than ageusia. A total of 30 patients (24.0%) reported persistent taste and smell disorders. Female patients were more likely to report persistent symptoms than male patients (73.3% versus 26.7%, respectively, p value = 0.02). Further studies based on validated discrimination tests including psychophysical tests or electrophysiological methods should be conducted to better characterize long-term olfactory and gustative disorder in COVID-19 patients

Background In France, the combination hydroxychloroquine (HCQ) and azithromycin (AZ) is used in the treatment of COVID-19. Methods We retrospectively report on 1061 SARS-CoV-2 positive tested patients treated with HCQ (200 mg three times daily for ten days) + AZ (500 mg on day 1 followed by 250 mg daily for the next four days) for at least three days. Outcomes were death, clinical worsening (transfer to ICU, and >10 day hospitalization) and viral shedding persistence (>10 days). Results A total of 1061 patients were included in this analysis (46.4% male, mean age 43.6 years – range 14–95 years). Good clinical outcome and virological cure were obtained in 973 patients within 10 days (91.7%). Prolonged viral carriage was observed in 47 patients (4.4%) and was associated to a higher viral load at diagnosis (p

Background An ongoing epidemic of respiratory diseases caused by a novel coronavirus (COVID 2019, SARS-CoV2) started in Wuhan, Hubei, in China at the end of December 2019. The French government decided to repatriate the 337 French nationals living in Wuhan and place them in quarantine in their home country. We decided to test them all for SARS-Cov2 twice in order to reduce anxiety among the population and decision-makers. Methods We investigated the presence of SARS-CoV-19 in asymptomatic carriers by testing all repatriated patients within the first 24 h of their arrival in France and at day 5. Viral RNA was extracted from pooled nasal and oropharyngeal swab fluids or sputum in the absence of nasal/oropharyngeal swabs. Detection of SARS-CoV-2 RNA was then carried out using several real-time reverse transcription (RT)-PCR assays. Results We tested 337 passengers at day 0 and day 5. All the tests for SARS-CoV2 were negative. By optimising the sampling process, sending samples sequentially and reducing the time-scale for biological analysis, we were able to test the samples within 5 h (including sampling, shipment and biological tests). Conclusion Optimising our procedures reduces anxiety and reassures the population and decision makers.

As a result of Next Generation Sequencing methods, metagenomic studies have become increasingly widespread. After being first applied to microbiome description, metagenomics is currently proposed as a diagnostic tool in clinical microbiology, although this application remains confined to the field of research. In this review, we will discuss the application of metagenomics to the detection of bacterial pathogens and demonstrate that the interpretation of the metagenomic results may fluctuate depending on the type of sample analyzed. However, we propose a view of metagenomic application to the evaluation of antimicrobial resistance, epidemic investigations and forensic medicine. Secondly, we present the many limits of metagenomic interpretation and application in routine clinical microbiology. From our perspective, metagenomics is not yet reliable enough for general use in clinical microbiology. Keywords: Metagenomic, Clinical microbiology, Next Generation Sequencing, Diagnosis, Limitation

Objectives The aim was to evaluate the feasibility and diagnostic contribution of protein profiling using matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectrometry (MS) applied to sputum to diagnose pulmonary tuberculosis. Methods Sputum samples collected from patients suspected of having pulmonary tuberculosis were analysed using MALDI-TOF MS. Using the differentially expressed protein peaks, we compared three groups of patients, including those with confirmed pulmonary tuberculosis (PTB), those without tuberculosis but with a lower respiratory tract infection (non-TB LRTI) and those without tuberculosis and without an LRTI (non-TB controls). Results A total of 102 patients included 35 PTB, 36 non-TB LRTI and 31 non-TB controls. The model differentiated between the PTB patients and the non-TB controls using the 25 most differentially expressed protein peaks, with a sensitivity of 97%, 95% CI 85–100%, and a specificity of 77%, 95% CI 59–90%. The model distinguished the PTB patients from the non-TB LRTI patients using the ten most differentially expressed protein peaks, with a sensitivity of 80%, 95% CI 63–92%, and a specificity of 89%, 95% CI 74–97%. We observed that the negative predictive value of MALDI-TOF MS sputum analysis was higher (96%, 95% CI 80–100%) than that of direct sputum microscopic examination and sputum culture (78%, 95% CI 62–89%) for non-TB controls. When MALDI-TOF MS sputum analysis and direct microscopic examination were combined, the negative predictive value reached 94%, 95% CI 80–99%, for non-TB LRTI patients. Discussion These results suggest that MALDI-TOF MS sputum analysis coupled with microscopic examination could be used as a screening tool for diagnosing pulmonary TB.

International audience; The strain GD9(T) is the type strain of the newly proposed species Blautia massiliensis sp. nov., belonging to the family Lachnospiraceae. It was isolated from a fresh stool sample collected from a healthy human using the culturomics strategy. Cells are Gram-negative rods, oxygen intolerant, non-motile and non spore forming. The 16S rRNA gene sequencing showed that strain GD9T was closely related to Blautia luti, with a 97.8% sequence similarity. Major fatty acids were C14:0 (19.8%) and C16:0 (53.2%). Strain GD9T exhibits a genome of 3,717,339 bp that contains 3,346 protein-coding genes and 81 RNAs genes including 63 tRNAs. The features of this organism are described here, with its complete genome sequence and annotation. Compared with other Blautia species which are Gram positive, the strain was Gram negative justifying an emended description of the genus Blautia. (C) 2016 Elsevier Ltd. All rights reserved.

Clostridium difficile infections (CDI) are the first cause of healthcare associated diarrhea in both Europe and the USA, causing between 15,000 and 30,000 deaths annually. Over the age of 65, antibiotic treatments are the two main risk factors of developing CDI. Fecal microbiota transplantation has a major role to play in managing these infections. Gut microbiota dysbiosis associated with CDI has been now comprehensively analyzed. Elderly individuals, patients treated with antibiotics or proton pump inhibitors have a dramatically decreased level of gut microbiota diversity as well as undergoing structural changes in taxa composition. In addition to this decreased diversity, patients with CDI present an increase in species belonging to Proteobacteria and a decrease in Clostridiales Incertae Sedis XI, and some commensal bacteria as Ruminococcaceae , Lachnospiraceae or Bifidobacterium longum for patients with CDI, caused by the 027 ribotype. Fecal microbiota transplantation is followed by a reestablishment of diversity, an increase in Firmicutes and Bacteroidetes and a decrease in Proteobacteria, Enterobacteriaceae and Streptoccaceae . Most of the studies are performed using metagenomics and sometimes yield contradictory results. Large studies, including culture dependent techniques and metagenomics using optimized extraction protocols to limit biases should be designed in order to comprehensively highlight the gut microbiota dysbiosis and consider specific microbiome-based therapeutic approaches.

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International audience

Strain Vm-5(T) was isolated from the stool specimen of a 10-year-old Amazonian boy. This bacterium is a Gram-positive, strictly aerobic rod, motile by a polar flagellum. Here we describe its phenotypic characteristics and complete genome sequence. The 4 353 177 bp long genome exhibits a G + C content of 36.87% and contains 4394 protein-coding and 125 predicted RNA genes. Phylogenetically and genetically, strain Vm-c is a member of the genus Virgibacillus but is distinct enough to be classified as a new species. We propose the creation of V. massiliensis sp. nov., whose type strain is strain Vm-5(T) (CSUR P971 = DSM 28587).

By diversifying culture conditions, in a strategy named culturomics, we were able in a short time to grow 124 new bacterial species from human stools, including 39 strict anaerobes. To describe these microorganisms, we use genome sequencing and MALDI-TOF mass spectrometry. Both tools have been major breakthroughs in clinical microbiology over the past decade, have previously been used for taxonomic purposes, and have the advantage over chemotaxonomic methods and DNA-DNA hybridization, to exhibit an excellent intra- and inter-laboratory reproducibility. We developed a polyphasic taxonomic strategy including MALDI-TOF MS and genomic analyses to describe new bacterial species associated with human beings. This strategy, that we have named taxono-genomics, was used to propose the description of 48 new species, the names of 13 of which have officially been validated. In this manuscript, we briefly reviewed the pros and cons of the currently validated taxonomic tools and propose that genomic sequencing and MALDI-TOF mass spectrometry may be incorporated in the taxonomic classification of prokaryotes.

International audience

An increasing number of scientists are turning to the microbiota to understand and/or explain the origin of various human metabolic or inflammatory diseases. Oxygen-intolerant bacteria represent the major population of the human intestinal microbiota. Their isolation is often difficult or even fastidious. The number of studies showing their beneficial role in human health is growing exponentially. Faecalibacterium prausnitzii and Akkermansia muciniphila are abundantly represented in healthy intestinal microbiota and their imbalance is positively correlated with inflammatory diseases and metabolic disorders (obesity, diabetes, cancers). Their use as probiotics presents very promising results in restoring the balance of microbial flora but also in the treatment of certain pathological conditions. The Christensenellaceae family has recently emerged as a hereditary taxon and studies have shown that its abundance is positively correlated with leanness and controls obesity in recipient mice. Here, we report the different culture strategies and techniques used for their isolation; the role of antioxidants in the survival of these oxygen-sensitive species in clinical sample and their maintenance in culture isolates.

Microbiology has been largely developed thanks to the discovery and optimization of culture media. The first liquid artificial culture medium was created by Louis Pasteur in 1860. Previously, bacterial growth on daily materials such as some foods had been observed. These observations highlighted the importance of the bacteria's natural environment and their nutritional needs in the development of culture media for their isolation. A culture medium is essentially composed of basic elements (water, nutrients), to which must be added different growth factors that will be specific to each bacterium and necessary for their growth.The evolution of bacterial culture through the media used for their culture began with the development of the first solid culture medium by Koch, allowing not only the production of bacterial colonies, but also the possibility of purifying a bacterial clone. The main gelling agent used in solid culture media is agar. However, some limits have been observed in the use of agar because of some extremely oxygen-sensitive bacteria that do not grow on agar media, and other alternatives were proposed and tested. Then, the discovery of antimicrobial agents and their specific targets prompted the emergence of selective media. These inhibiting agents make it possible to eliminate undesirable bacteria from the microbiota and select the bacteria desired. Thanks to a better knowledge of the bacterial environment, it will be possible to develop new culture media and new culture conditions, better adapted to certain fastidious bacteria that are difficult to isolate. Keywords: Culture media, Enriched media, Gelling agents, Liquid and solid media, Selective media

Increasing numbers of sporadic cases of melioidosis in returning travelers have been reported from non-endemic regions.We report a new case and undertook a literature review.Eighty-two travelers with melioidosis infection were included. The mean age was 50.95 years, with only one case15 years. A male predominance was noted, with 66 males (80.5%). Type of travel included tourism (51.2%), family visits (15.8%) and business (14.6%). The most common destinations were Asia (80.5%), America (9.7%) and Africa (7.3%). No cases were documented from Oceania. Underlying conditions were documented in 68 patients, showing a strong association with diabetes (37.8%). Exposure risks were documented in 32 patients, including contact with water. Pulmonary involvement was seen in 41 patients, cutaneous in 23, abdominal in 14, and urogenital in 10 cases. Blood cultures posed the diagnosis in 43 cases. Fifty-seven patients fully recovered, 12 died, and three relapsed. The mortality rate (14.6%) was close to that observed in Australia but lower than series in Southern Asia.Melioidosis should not only be considered in travelers returning from classically considered endemic areas (Australia and South-East Asia) but also from America and Africa, especially in diabetic patients or after contact with water.

From culture to metagenomics, within only 130 years, our knowledge of the human microbiome has considerably improved. With >1000 microbial species identified to date, the gastro-intestinal microbiota is the most complex of human biotas. It is composed of a majority of Bacteroidetes and Firmicutes and, although exhibiting great inter-individual variations according to age, geographic origin, disease or antibiotic uptake, it is stable over time. Metagenomic studies have suggested associations between specific gut microbiota compositions and a variety of diseases, including irritable bowel syndrome, Crohn's disease, colon cancer, type 2 diabetes and obesity. However, these data remain method-dependent, as no consensus strategy has been defined to decipher the complexity of the gut microbiota. High-throughput culture-independent techniques have highlighted the limitations of culture by showing the importance of uncultured species, whereas modern culture methods have demonstrated that metagenomics underestimates the microbial diversity by ignoring minor populations. In this review, we highlight the progress and challenges that pave the way to a complete understanding of the human gastrointestinal microbiota and its influence on human health.

The human gut flora is currently widely characterised using molecular techniques. Microbial culturomics (large-scale culture conditions with identification of colonies using MALDI-TOF or 16S rRNA) is part of the rebirth of bacterial culture that was initiated by environmental microbiologists for the design of axenic culture for intracellular bacteria in clinical microbiology. Culturomics was performed on four stool samples from patients treated with large-scale antibiotics to assess the diversity of their gut flora in comparison with other culture-dependent studies. Pyrosequencing of the V6 region was also performed and was compared with a control group. Gut richness was also estimated by bacterial counting after microscopic observation. In total, 77 culture conditions were tested and 32,000 different colonies were generated; 190 bacterial species were identified, with 9 species that had not been isolated from the human gut before this study, 7 newly described in humans and 8 completely new species. A dramatic reduction in diversity was observed for two of the four stool samples for which antibiotic treatment was prolonged and uninterrupted. The total number of bacteria was generally preserved, suggesting that the original population was replaced but was sustained in size. Discordances between culture and pyrosequencing biodiversity biomarkers highlight the depth of bias of molecular studies. Stool samples studied showed a dramatic reduction in bacterial diversity. Considering the variable antibiotic concentration in the gut, this reduction in the number of species is possibly linked to the production of bacteriocin in the upper digestive tract by specific bacteria, such as Lactobacillus spp.

Summary Background Tropheryma whipplei was recently associated with acute infections as gastroenteritis in children. We hypothesize that T. whipplei may be a contributing microbe in traveler's diarrhea. Method The presence of T. whipplei was investigated by using a specific PCR on stool samples of travelers to Senegal before and after traveling, independently of the occurrence of diarrhea. Results A total of 59 travelers returned both stool samples before and after travel and 16 (27%) experienced diarrhea during travel. Fifty-three patients (89.8%) were negative for T. whipplei before and after travel. Two patients (3.4%) were negative before and positive after travel. Two patients (3.4%) were positive before travel and negative after travel. Finally, two patients (3.4%) were positive before and after travel. There were no carriage differences of T. whipplei , visiting Senegal or staying in France. We found no significant association of T. whipplei carriage and travel-associated diarrhea but this may have been impacted by the use of doxycycline. Conclusions This is the first report of T. whipplei carriage acquired during travel to tropical area. Further study addressing this issue in larger cohorts of travelers to Senegal, including individuals visiting friends and relatives in their country of origin and the protective role of doxycycline malaria chemoprophylaxis may help to understand the potential contribution of T. whipplei to travelers' diarrhea.

Venous thromboses have been associated with tuberculosis, but the relationship with circulating anticoagulant has not been studied yet. In a cohort of 48 patients with tuberculosis, 22.9% of them presented with venous thromboses significantly associated with dose dependent level of antiphosphophatidyl-ethanolamine antibodies. Keywords: Tuberculosis, Deep vein thrombosis, Pulmonary embolism, Antiphospholipds antibodies, Mycobacterium tuberculosis, Anti-phosphatidylethanolamine antibodies

A patient presented diffuse pulmonary parenchymal micronodules. Tropheryma whipplei was detected in the saliva, a bronchial biopsy and bronchoalveolar fluid. PAS staining, immunohistochemistry and PCR for T. whipplei were negative in the duodenal biopsies. T. whipplei was isolated from the bronchoalveolar fluid, reinforcing its role as a respiratory pathogen.

International audience; no abstract

Resume Nous rapportons le cas d’un homme âge de 38 ans qui s’est presente avec une diarrhee severe, une perte ponderale de 10 kg, des paresthesies des chevilles et une faiblesse motrice importante dans le territoire du nerf fibulaire apres l’introduction d’un traitement par corticoides et azathioprine pour sa proteinurie. La coloscopie et la gastroscopie avaient revele un aspect typique d’une maladie de Whipple (MW), associe a une reaction PAS positive et a un aspect specifique a l’immuno-histochimie. Les resultats de la PCR ont ete positifs pour le Tropheryma Whipplei sur des echantillons sanguins, fecaux, salivaires et duodenaux. Le diagnostic de MW avec des manifestations systemiques a ete retenu et un traitement par doxycycline associe a l’hydroxychloroquine a ete introduit, ameliorant les douleurs articulaires, les manifestations cutanees et les symptomes intestinaux. Un mois plus tard, notre patient avait presente une fievre associee a un important syndrome inflammatoire biologique (CRP 150 mg/dL et leucocytes a 16,8 10 9 /L), alors qu’aucune infection n’a ete retrouvee malgre un bilan extensif. Nous avons conclu a un syndrome de restauration de l’immunite (SRI). Les manifestations avaient persiste malgre la majoration des corticoides, la thalidomide a ete introduite a 200 mg/j ameliorant les symptomes. La MW pourrait etre revelee par des symptomes non specifiques tels que la perte ponderale et les arthralgies, mais aussi par plusieurs autres signes trompeurs. Notre observation illustre le polymorphisme des presentations cliniques de la MW et les difficultes diagnostiques qui peuvent etre rencontrees. Il s’agit aussi d’une observation supplementaire en faveur de l’efficacite de la thalidomide dans le SRI observe dans la MW.

We report the case of a 38-year-old man who presented with severe diarrhea, weight loss of 10 kg, ankles paresthesia and severe motor weakness in the left fibular nerve territory after introduction of azathioprine and corticosteroid for proteinuria. Coloscopy and gastroscopy revealed a typical aspect of Whipple disease (WD), associated with both positive PAS staining and specific immunohistochemistry. T. whipplei PCR results were positive in blood, faeces, saliva and duodenal biopsy specimens. Diagnosis of WD with systemic manifestations was retained and doxycycline plus hydroxychloroquine therapy were started. This treatment improved joint pain, and skin and intestinal symptoms. One month later, our patient presented with fever and an important inflammatory syndrome (CRP 150 mg/dL and 16.8 10 9 /L leukocytes), while no infection was found despite a thorough review. We concluded it was an immune reconstitution inflammatory syndrome (IRIS). Manifestations persisted despite increasing corticosteroids and thalidomide (200 mg/day) was introduced with good efficacy on these symptoms. WD may be revealed by non-specific symptoms such as weight loss or arthralgia, but also by many other misleading signs. Our observation illustrates the highly polymorphic clinical presentation of WD, and the diagnostic difficulties that may arise. This is also a new report of thalidomide effectiveness in IRIS in WD.

The first successful culture of Tropheryma whipplei made 10 years ago opened the way for identification of the bacterium and the development of many diagnostic tools. Phylogenetic analyses made it possible to classify it among Gram positive bacilli in the family of Actinomycetes, close to other ubiquitous bacteria of the environment. More than one century later, in the first description of Whipple's disease, T. whipplei was found to be responsible for a broad spectrum of clinical presentations. There is a variable prevalence according to areas or populations, and there are asymptomatic carriers of the bacterium. Whipple's disease is responsible mainly for arthralgia and diarrhea but can involve many organs. T. whipplei can also be responsible for neurological infection, blood culture-negative endocarditis, or uveitis. In addition to histological analyses, molecular tools help the clinician to prove these difficult diagnoses. Failure and relapse data and antibiotic susceptibility tests have allowed to determine a rational treatment.

Introduction Face a l’epidemie de VHA chez les patients HSH decrite en France depuis 2016, nous avons organise une campagne de vaccination(VHAv) en urgence des patients a haut risque associee a une evaluation des bonnes pratiques medicales et de counseling sur les IST. Materiels et methodes A partir des donnees du dossier patient informatise NADIS nous avons selectionne dans notre cohorte de 3200 patients VIH, les patients HSH et bisexuels avec une serologie VHA negative ou non renseignee, pour les contacter (30/06–30/09/2017) leur proposant une VHAv gratuite et un auto-questionnaire anonyme sur leur pratiques sexuelles et leur connaissance des differentes IST. Resultats Parmi nos patients VIH+/HSH et/ou bisexuels ( n = 637), ceux ayant une serologie VHA negative en IgG (169 ; 26 %) ou non renseignee (141 ; 22 %) dans NADIS ont ete soumis a l’approbation du medecin referent avant d’etre inclus dans notre etude et contactes ( n = 250). Au total 110 (44 %) hommes ont eu une VHAv. Leur mediane d’âge etait de 45 ans (17–71), de duree d’infection par le VIH etait courte (7 ans), tous etaient sous ARVc avec une CVVIH 500/mm 3 pour 74 %. La plupart avait une IST en antecedent : syphilis pour 38 %, chlamydiae pour 6 %, HPV pour 14 %, VHB/VHC pour 13 %, et gonococcie pour 2 %. Un total de 74 patients a accepte de repondre a l’autoquestionnaire. La majorite (84 %) a declare avoir eu une sexualite active dans les deux mois precedents avec un homme (77 % d’HSH) ou les deux (7 % de bisexuels) mais rarement avec un seul partenaire stable (32 %). La plupart (43 %) rapportait 1–10 partenaires/2 mois et 9 % plus de 30. Le moyen ou lieu de rencontre etait Internet dans 58 % des cas, les lieux festifs dedies dans 31 % de cas, et les parcs dans 9 % des cas. Seulement 3 % des patients consommaient mensuellement du ChemSex (sexualite sous l’effet de produits chimiques),11 % annuellement, et 28 % ignorait cette pratique. Au cours des deux mois precedents un rapport bucco*genital etait rapporte par 77 % d’entre eux, toujours protege par un preservatif dans 3 % des cas. Une penetration anale a ete rapportee par 76 % des patients avec souvent (24 %) ou toujours (34 %) le port du preservatif. Plus du tiers des patients interroges declarait n’avoir aucune idee des modes de transmission des IST (20 % pour le VHA, 31 %/VHBVHC, 20 %/syphilis,42 %/gonocoque, 39 %/chlamydiae) bien que 69 % d’entre eux en ait deja contracte une (11 %/VHBVHC, 21 %/syphilis,18 %/gonocoque, 39 %/chlamydiae). Conclusion Notre etude a permis de vacciner presque la moitie de nos patients exposes au VHA a l’acme de l’epidemie permettant de reduire la chaine de contamination. En revanche, elle souligne que la « mobilisation medicale + la gratuite + disponibilite du VHAv » n’ont pas suffi a motiver tous les patients. Le Network devrait devenir Le support d’informations et de prevention pour les IST.