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

An indirect in-house immunofluorescent assay was developed in order to assess the serological status of COVID-19 patients in Marseille, France. Performance of IFA was compared to a commercial ELISA IgG kit. We tested 888 RT-qPCR-confirmed COVID-19 patients (1302 serum samples) and 350 controls including 200 sera collected before the pandemic, 64 sera known to be associated with nonspecific serological interference, 36 sera from non-coronavirus pneumonia and 50 sera from patient with other common coronavirus to elicit false-positive serology. 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. IFA presented substantial agreement (86%) with ELISA EUROIMMUN SARS-CoV-2 IgG kit (Cohen's Kappa = 0.61). The presence of antibodies was then measured at 3% before a 5-day evolution up to 47% after more than 15 days of evolution. We observed that the rates of seropositivity 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 detection anti-SARS-CoV-2 antibodies is useful as a marker associated with COVID-19 severity. The IFA assay reported here is useful for monitoring SARS-CoV-2 exposure at the individual and population levels.

Hepatitis A outbreak in HIV-infected patients in Southeastern France: questions and responses?

During the 2017 European hepatitis A (HA) outbreak we assessed HA incidence in our cohort of 2300 HIV-infected patients, implemented preventive measures and evaluated practices and knowledge on sexually transmitted diseases (STD). HA incidence was assessed between 1 January 2017 and 31 December 2017 and included all symptomatic patients with virologically confirmed HA. Preventive measures consisted in identifying at risk and not immunised patients to propose them a free HAV vaccination, and an anonymous survey related to transmission routes of STD and to sexual behaviours. Twenty HA were diagnosed. All were homosexual men recently diagnosed with HIV and another STD. None were vaccinated against hepatitis A virus (HAV). Hospitalisation was required for 52%. We identified 250 patients at risk to acquire HAV and invited them to a free immunisation program. A total of 110 (44%) were vaccinated, of whom 74 responded to our survey. A majority of them (84%) reported recent active anal and oral sexuality with multiple (52%) male partners (81%), and ChemSex consumption (14%). Internet was the meeting link for 58%. Another STD history was found in 69%. One third of these individuals had no idea about STD transmission modes. This HA outbreak pointed the insufficient vaccine coverage against HAV and knowledge on STD, which may be improved by Internet.

Salt in stools is associated with obesity, gut halophilic microbiota and Akkermansia muciniphila depletion in humans.

BACKGROUND/OBJECTIVES: High salt intake has been linked to several diseases including obesity and an increased risk of death; however, fecal salinity and the ability of salt to alter the gut microbiota, which was recently identified as an instrumental factor for health and disease, remains poorly explored. METHODS/SUBJECTS: We analyzed the fecal samples of 1326 human individuals for salinity by refractometry, 572 for gut microbiota by culturomics, and 164 by 16S rRNA-targeted metagenomics. Geographical origin, age, gender, and obesity were tested as predictors of fecal salinity and halophilic diversity. All halophilic isolates were characterized by taxonogenomics and their genome sequenced. RESULTS: Fecal salinity was associated with obesity independently of geographical origin, gender, and age. The first 2 human-associated halophilic archaeal members were isolated along with 64 distinct halophilic species, including 21 new species and 41 known in the environment but not in humans. No halophiles grow in less than 1.5% salinity. Above this threshold, the richness of the halophilic microbiota was correlated with fecal salinity (r = 0.58, p < 0.0001). 16S metagenomics linked high fecal salinity to decreased diversity (linear regression, p < .035) and a depletion in anti-obesity Akkermansia muciniphila and Bifidobacterium, specifically B. longum and B. adolescentis. Genomics analysis suggested that halophilic microbes are not only transient passengers but may be residents of the human gut. CONCLUSIONS: High salt levels are associated with alteration of the gut microbial ecosystem and halophilic microbiota, as discovered during this study. Further studies should clarify if the gut microbiota alterations associated with high salt levels and the human halophilic microbiota could be causally related to human disease, such as obesity.

Emergence of Clostridium difficile tcdC variant 078 in Marseille, France.

The purpose of this investigation was to evaluate the epidemiology of hypervirulent Clostridium difficile ribotypes from January 2013 to February 2017 in the Marseille area of southern France. By using the Xpert Clostridium difficile Epi polymerase chain reaction (PCR) assay and sequencing the tcdC gene, we characterised C. difficile isolates from symptomatic patients diagnosed with C. difficile infection (CDI) in Marseille university hospitals. We first tested retrospectively 278 C. difficile samples isolated from January 2013 to December 2014 and observed a high prevalence of isolates with tcdC mutations and deletions previously described in both hypervirulent ribotypes RT027 and RT078 (16.4% and 10.7%, respectively). We highlighted the co-circulation of these two hypervirulent C. difficile tcdC variants (tV) with distinct epidemiological characteristics. While an RT027 outbreak occurred mainly as healthcare-associated infection in the elderly, CDI caused by tV078 occurred mainly in a younger population as community-associated infection. From January 2016, a systematic survey of these two hypervirulent C. difficile ribotypes revealed the emergence of CDI caused by tV078, currently being more prevalent than RT027 in the Marseille area. The present study is the first report of the emergence of CDI caused by tV078 in southern France. We showed the simultaneous circulation and sequential spread of hypervirulent ribotypes, such as RT027 and tV078. This emphasises the need for an efficient surveillance system for CDI with ribotyping and an optimised management of CDI caused by hypervirulent strains.

Aerobic culture of methanogenic archaea without an external source of hydrogen.

Culturing methanogenic archaea is fastidious, expensive, and requires an external source of hydrogen and carbon dioxide. Until now, these microorganisms have only been cultivated under strictly anaerobic conditions. We previously developed a single versatile culture medium containing sugars and anti-oxydants for cultivating all human known methanogens. Performing aerobic cultures in the presence of Bacteroides thetaiotaomicron, which produces hydrogen, allows for cultivation of Methanobrevibacter smithii which itself produces methane. To obtain colonies, we cultivated M. smithii in an agar plate in the upper part of a double chamber flask with a liquid culture of B. thetaiotaomicron in the lower compartment. We subsequently cultured four other methanogenic species for the first time and successfully isolated 13 strains of M. smithii and nine strains of Methanobrevibacter oralis from 100 stools and 45 oral samples. This procedure allows aerobic isolation and antibiotic susceptibility testing. This changes the ability to routinely study methanogens, which have been neglected in clinical microbiology laboratories and may be useful for biogas production.

Dramatic reduction in Clostridium difficile ribotype 027-associated mortality with early fecal transplantation by the nasogastric route: a preliminary report.

Clostridium difficile ribotype 027 (CD027)-associated diarrhea preferentially affects elderly patients and causes a high mortality rate. Fecal microbiota transplantation has become an alternative treatment for recurrent C. difficile infections. An outbreak of CD027 infections has occurred in Marseille since March 2013. From March to November 2013, we treated patients using only antibiotics or fecal microbiota transplantation after at least three relapses. Beginning in November 2013, we performed early transplantation using a nasogastric tube during the first week of infection, in combination with antibiotic treatment. Sixty-one patients with a mean age of 84 years were hospitalized, including 42 patients treated only with antibiotics, three with tardive transplantation, and 16 with early transplantation. The patients were comparable in clinical involvement. The global mortality rate was 3/16 (18.75 %) among the patients treated by early transplantation and 29/45 (64.4 %) among the patients only treated by antibiotics or by tardive transplantation (p < 0.01). Among these 45 patients, 23 (51 %) died at day 31, including 17 who died at day 7. Early fecal transplantation was associated with a significantly reduced mortality rate, with only one patient dead at day 31 (6.25 %). In a Cox model, early transplantation was the only independent predictor of survival (hazard ratio 0.18, 95 % confidence interval 0.05-0.61, p = 0.006). Six of the 16 patients (37.5 %) needed a second transplantation before symptom resolution. Early fecal microbiota transplantation in combination with antibiotics should be the first-line treatment for CD027 infections.

Japanese encephalitis in a French traveler to Nepal.

Japanese encephalitis is frequent in Asia, with a severe prognosis, but rare in travelers. Culex mosquitoes transmit Japanese encephalitis virus. Risk factors are destination, duration of stay, summer and fall seasons, outdoor activities, and type of accommodation. We report the case of a French traveler to Nepal with neutralization-based serological confirmed Japanese encephalitis. He presented classical clinical (viral syndrome before an encephalitis status with behavioral disorder, global hypotonia, mutism, movement disorders, seizure, and coma), radiological (lesions of thalami, cortico-spinal tracts, and brainstem) and biological features (lymphocytic meningitis). Nowadays, the presence of Japanese encephalitis virus in Nepal, including mountain areas, is established but Japanese encephalitis remains rare in travelers returning from this area and neurologist physicians need to become familiar with this. We recommend vaccination for travelers spending a long period of time in Nepal and having at-risk outdoor activities.

Aerobic culture of anaerobic bacteria using antioxidants: a preliminary report.

Antioxidants have been shown to help the growth of anaerobic bacteria. We were able to grow six anaerobe species (including Fusobacterium necrophorum and Ruminococcus gravus) and seven aerobic species all aerobically in Schaedler agar tubes and agar plates with high doses of ascorbic acid and/or glutathione. This may deeply change strategies for culturing bacteria.

The gut microbiota of a patient with resistant tuberculosis is more comprehensively studied by culturomics than by metagenomics.

Gut microbiota consists of 10(10) bacteria per gram of stool. Many antibiotic regimens induce a reduction in both the diversity and the abundance of the gut flora. We analyzed one stool sample collected from a patient treated for drug-resistant Mycobacterium tuberculosis and who ultimately died from pneumonia due to a Streptococcus pneumoniae 10 months later. We performed microscopic observation, used 70 culture conditions (microbial culturomics) with identification by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF) and 16S rRNA amplification and sequencing, pyrosequencing, and 18S rRNA amplification and clone sequencing. Electron and optical microscopic observations revealed the presence of yeast, but no bacterial species were observed. By culture, only 39 bacterial species were identified, including one new species, as well as three species that have not been previously observed in the human gut. The pyrosequencing showed only 18 phylotypes, detecting a lower number of bacterial species than the culture techniques. Only two phylotypes overlapped with culturomics. In contrast, an amount of chloroplasts was found. Additionally, specific molecular eukaryote detection found three fungal species. We recovered, for the first time, more cultivable than non-cultivable bacterial species in a patient with a low bacterial load in the gut, demonstrating the depth bias of pyrosequencing. We propose that the desertification of gut microbiota in this patient is a reflection of the total body microbiota and may have contributed to the invasive infection of S. pneumoniae. This finding suggests that caution should be applied when treating patients with broad-spectrum antibiotics, and preventive measures should be taken in order to avoid invasive infection.

Culturomics identified 11 new bacterial species from a single anorexia nervosa stool sample.

The rebirth of bacterial culture has been highlighted successively by environmental microbiologists, the design of axenic culture for intracellular bacteria in clinical microbiology, and, more recently, by human gut microbiota studies. Indeed, microbial culturomics (large scale of culture conditions with the identification of colonies by MALDI-TOF or 16S rRNA) allowed to culture 32 new bacterial species from only four stool samples studied. We performed culturomics in comparison with pyrosequencing 16S rRNA targeting the V6 region on an anorexia nervosa stool sample because this clinical condition has never been explored before by culture, while its composition has been observed to be atypical by metagenomics. We tested 88 culture conditions generating 12,700 different colonies identifying 133 bacterial species, with 19 bacterial species never isolated from the human gut before, including 11 new bacterial species for which the genome has been sequenced. These 11 new bacterial species isolated from a single stool sample allow to extend more significantly the repertoire in comparison to the bacterial species validated by the rest of the world during the last 2 years. Pyrosequencing indicated a dramatic discrepancy with the culturomics results, with only 23 OTUs assigned to the species level overlapping (17 % of the culturomics results). Most of the sequences assigned to bacteria detected only by pyrosequencing belonged to Ruminococcaceae, Lachnospiraceae, and Erysipelotrichaceae constituted by strictly anaerobic species, indicating the future route for culturomics. This study revealed new bacterial species participating significantly to the extension of the gut microbiota repertoire, which is the first step before being able to connect the bacterial composition with the geographic or clinical status.

Genome analysis of Lachnoclostridium phocaeense isolated from a patient after kidney transplantation in Marseille.

Lachnoclostridium phocaeense is a new species in the genus Lachnoclostridium. Lachnoclostridium phocaeense is a Gram-positive anaerobic rod. This strain, Marseille-P3177T (CSUR = P3177) with the below described genome was isolated from the urine sample of a women after kidney transplantation. The strain genome is 3 500 754 bp long with 50.62% G + C content and consists of a single contig (GenBank accession number NZ_LT635479.1).

Luxibacter massiliensis gen. nov., sp. nov., a new bacterium isolated from the human gut microbiota.

An anaerobic facultative Gram-stain positive bacterium was isolated from human gut microbiota. Strain Marseille-P5551T was considered to be a new genus within the phylum Firmicutes, as it exhibits a 91.87% similarity level with Faecalicatena orotica (NR_117129.1), the phylogenetically closest related species. The draft genome size of strain Marseille-P5551T is 4 142 938 bp with 44.4% of G + C content. We hereby suggest the creation of Luxibacter massiliensis gen. nov., sp. nov., as a new bacterial genus.

Bacterial culture through selective and non-selective conditions: the evolution of culture media in clinical microbiology.

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.

Taxono-genomics and description of Haloimpatiens massiliensis sp. nov., a new bacterium isolated from the gut of a healthy infant.

A polyphasic taxono-genomic strategy was used to describe a new bacterium, strain Marseille-P1935; isolated from the gut of a healthy infant. 16S rRNA sequencing showed that the isolate belongs to the genus Haloimpatiens in the family Clostridiaceae. Phenotypic analysis and whole-genome sequence analyses confirm the status of the new species. We propose the creation of the new species Haloimpatiens massiliensis strain Marseille-P1935T (= CSURP1935T; = DSM100591T).

Description of Gracilibacillus phocaeensis sp. nov., a new halophilic bacterium isolated from Senegalian human stool.

Using the taxonogenomics method, we describe Gracilibacillus phocaeensis strain Marseille-P3801, a new species previously isolated from a salty stool of a 20-year-old man from N'Diop, Senegal. It is a Gram-positive, aerobic and motile bacillus. The major fatty acids are C15:0-anteiso (59%), C16:0 (16%) and C17:0-anteiso (11%). Strain Marseille-P3801 exhibits a 98.45% sequence similarity with Gracilibacillus thailandensis strain TP2-8, the phylogenetically closest species. Its genome is 4.66 Mb with 39.6 mol% G + C content.

Clinical efficacy of chloroquine derivatives in COVID-19 infection: comparative meta-analysis between the big data and the real world.

In the context of the current coronavirus disease 2019 (COVID-19) pandemic, we conducted a meta-analysis on the effects of chloroquine derivatives in patients, based on unpublished and published reports available publicly on the internet as of 27 May 2020. The keywords 'hydroxychloroquine', 'chloroquine', 'coronavirus', 'COVID-19' and 'SARS-Cov-2' were used in the PubMed, Google Scholar and Google search engines without any restrictions as to date or language. Twenty studies were identified involving 105 040 patients (19 270 treated patients) from nine countries (Brazil, China, France, Iran, Saudi Arabia, South Korea, Spain and the USA). Big data observational studies were associated with conflict of interest, lack of treatment dosage and duration, and absence of favourable outcome. Clinical studies were associated with favourable outcomes and details on therapy. Among clinical studies, three of four randomized controlled trials reported a significant favourable effect. Among clinical studies, a significant favourable summary effect was observed for duration of cough (OR 0.19, p 0.00003), duration of fever (OR 0.11, p 0.039), clinical cure (OR 0.21, p 0.0495), death (OR 0.32, p 4.1 × 10-6) and viral shedding (OR 0.43, p 0.031). A trend for a favourable effect was noted for the outcome 'death and/or intensive care unit transfer' (OR 0.29, p 0.069) with a point estimate remarkably similar to that observed for death (∼0.3). In conclusion, a meta-analysis of publicly available clinical reports demonstrates that chloroquine derivatives are effective to improve clinical and virological outcomes, but, more importantly, they reduce mortality by a factor of 3 in patients with COVID-19. Big data are lacking basic treatment definitions and are linked to conflict of interest. The retraction of the only big data study associated with a significantly deleterious effect the day after (June 5, 2020) the acceptance of the present work (June 4, 2020) confirms the relevance of this work.

Neoactinobaculum massilliense gen. nov., a new genesis and Pseudopropionibacterium massiliense sp. nov., a new bacterium isolated from the human oral microbiota.

Neoactinobaculum massilliense gen. nov., strain Marseille-P6182T (= CSUR P6182) and Pseudopropionibacterium massiliense sp. nov., strain Marseille-P6184T (= CSUR P6184) are a new bacterial genus and new bacterial species belonging to the Actinobacteria phylum that have been isolated from the human oral microbiota.

Lysinibacillus timonensis sp. nov., Microbacterium timonense sp. nov., and Erwinia mediterraneensis sp. nov., three new species isolated from the human skin.

Lysinibacillus timonensis strain Marseille-P5727T (=CSURP5727), Microbacterium timonense strain Marseille-P5731T (=CSURP5731) and Erwinia mediterraneensis strain Marseille-P5165T (=CSURP5165) are three new species isolated from the human skin.

Isolation and culture of Methanobrevibacter smithii by co-culture with hydrogen-producing bacteria on agar plates.

OBJECTIVES: Methanogenic Archaea are considered as extremely oxygen-sensitive organisms, and their culture is fastidious, requiring specific equipment. We report here conditions allowing the cultivation of Methanobrevibacter smithii in an anaerobic chamber without the addition of hydrogen. METHODS: We first enriched the stool sample in an anaerobic liquid medium. To cultivate M. smithii with Bacteroides thetaiotaomicron and other hydrogen-producing bacteria on solid medium in an anaerobic chamber, we divided the agar plates into two compartments and seeded each strain on each compartment. Methane production was assessed by gas chromatography, and the growing colonies were authenticated by MALDI-TOF MS. RESULTS: We successfully cultured M. smithii from a liquid culture medium inoculated with stool collected from a healthy donor in an anaerobic chamber. The isolation in pure culture permitted successful culture on agar medium by our performing a co-culture with B. thetaiotaomicron. We also successfully tested the co-cultivation of M. smithii with other known hydrogen-producing bacteria. Gas chromatographic tests showed that these strains produced hydrogen in different amounts. Agar colonies of methanogens were obtained by co-culture with these bacteria, and methane production was detected. CONCLUSIONS: We propose a new approach to isolate and cultivate new strains of M. smithii by using a co-culture-based technique that can facilitate and make available the isolation of new methanogenic Archaea strains in clinical microbiology laboratories.