Unknown Circovirus in Immunosuppressed Patient with Hepatitis, France, 2022.

Hepatitis of undetermined origin can be caused by a wide variety of pathogens, sometimes emerging pathogens. We report the discovery, by means of routine shotgun metagenomics, of a new virus belonging to the family Circoviridae, genus Circovirus, in a patient in France who had acute hepatitis of unknown origin.

Viral genomic, metagenomic and human transcriptomic characterization and prediction of the clinical forms of COVID-19.

COVID-19 is characterized by respiratory symptoms of various severities, ranging from mild upper respiratory signs to acute respiratory failure/acute respiratory distress syndrome associated with a high mortality rate. However, the pathophysiology of the disease is largely unknown. Shotgun metagenomics from nasopharyngeal swabs were used to characterize the genomic, metagenomic and transcriptomic features of patients from the first pandemic wave with various forms of COVID-19, including outpatients, patients hospitalized not requiring intensive care, and patients in the intensive care unit, to identify viral and/or host factors associated with the most severe forms of the disease. Neither the genetic characteristics of SARS-CoV-2, nor the detection of bacteria, viruses, fungi or parasites were associated with the severity of pulmonary disease. Severe pneumonia was associated with overexpression of cytokine transcripts activating the CXCR2 pathway, whereas patients with benign disease presented with a T helper "Th1-Th17" profile. The latter profile was associated with female gender and a lower mortality rate. Our findings indicate that the most severe cases of COVID-19 are characterized by the presence of overactive immune cells resulting in neutrophil pulmonary infiltration which, in turn, could enhance the inflammatory response and prolong tissue damage. These findings make CXCR2 antagonists, in particular IL-8 antagonists, promising candidates for the treatment of patients with severe COVID-19.

Omicron induced distinct immune respiratory transcriptomics signatures compared to pre-existing variants in critically ill COVID-19 patients.

Severe coronavirus disease 2019 (COVID-19) is related to dysregulated immune responses. We aimed to explore the effect of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants on the immune response by nasopharyngeal transcriptomic in critically-ill patients. This prospective monocentric study included COVID-19 patients requiring intensive care unit (ICU) admission between March 2020 and 2022. Patients were classified according to VOC (ancestral, Alpha, Delta, and Omicron). Eighty-eight patients with severe COVID-19 were included after matching (on prespecified clinical criteria). Profiling of gene expression markers of innate and adaptive immune responses were investigated by respiratory transcriptomics at ICU admission. Eighty-eight patients were included in the study after matching (ancestral [n = 24], Alpha [n = 24], Delta [n = 22], and Omicron [n = 18] variants). Respiratory transcriptomic analysis revealed distinct innate and adaptive immune profiling between variants. In comparison with the ancestral variant, there was a reduced expression of neutrophil degranulation, T cell activation, cytokines signalling pathways in patients infected with Alpha and Delta variants. In contrast, there was a higher expression of neutrophil degranulation, T and B cells activation, and inflammatory interleukins pathways in patients infected with Omicron. To conclude, Omicron induced distinct immune respiratory transcriptomics signatures compared to pre-existing variants in patients with severe COVID-19, pointing to an evolving pathophysiology of severe COVID-19 in the Omicron era.

BNT162b2 Messenger RNA Vaccination Did Not Prevent an Outbreak of Severe Acute Respiratory Syndrome Coronavirus 2 Variant 501Y.V2 in an Elderly Nursing Home but Reduced Transmission and Disease Severity.

We report an outbreak of severe acute respiratory syndrome coronavirus 2 501Y.V2 in a nursing home. All nonvaccinated residents (5/5) versus half of those vaccinated with BNT162b2 (13/26) were infected. Two of 13 vaccinated versus 4 of 5 nonvaccinated residents presented severe disease. BNT162b2 did not prevent the outbreak, but reduced transmission and disease severity.

Persistent SARS-CoV-2 Alpha Variant Infection in Immunosuppressed Patient, France, February 2022.

We describe persistent circulation of SARS-CoV-2 Alpha variant in an immunosuppressed patient in France during February 2022. The virus had a new pattern of mutation accumulation. The ongoing circulation of previous variants of concern could lead to reemergence of variants with the potential to propagate future waves of infection.

Genetic and Phenotypic Study of the Pectobacterium versatile Beta-Lactamase, the Enzyme Most Similar to the Plasmid-Encoded TEM-1.

Genus Pectobacterium bacteria include important agricultural pathogens. Pectobacterium versatile isolates contain a chromosome-borne beta-lactamase, PEC-1. This enzyme is the closest relative of TEM-1, a plasmid-borne beta-lactamase widespread in the Enterobacterales. We performed bioinformatics and phenotypic analyses to investigate the genetic and phenotypic features of PEC-1 and its frequency and ability to spread within genus Pectobacterium. We also compared the characteristics of PEC-1 and TEM-1 and evaluated the likelihood of transfer. We found that blaPEC-1 was present principally in a small number of genetic environments in P. versatile. Identical blaPEC-1 genetic environments were present in closely related species, consistent with the high frequency of genetic exchange within the genus Pectobacterium. Despite the similarities between PEC-1 and TEM-1, their genetic environments displayed no significant identity, suggesting an absence of recent transfer. Phenotypic analyses on clonal constructs revealed similar hydrolysis spectra. Our results suggest that P. versatile is the main reservoir of PEC-1, which seems to transfer to closely related species. The genetic distance between PEC-1 and TEM-1, and the lack of conserved elements in their genetic environments, suggest that any transfer that may have occurred must have taken place well before the antibiotic era. IMPORTANCE This study aimed to compare the chromosomal beta-lactamase from Pectobacterium versatile, PEC-1, with the well-known and globally distributed TEM-1 in terms of genetic and functional properties. Despite the similarities between the enzymes, we obtained no definitive proof of gene transfer for the emergence of blaPEC-1 from blaTEM-1. Indeed, given the limited degree of sequence identity and the absence of a common genetic environment, it seems unlikely that any transfer of this gene has occurred recently. However, although blaPEC-1 was found mostly in one specific clade of the P. versatile species, certain isolates from other closely related species, such as Pectobacterium brasiliense and Pectobacterium polaris, may also carry this gene inserted into common genetic environments. This observation suggests that genetic exchanges are frequent, accounting for the diffusion of blaPEC-1 between isolates from different Pectobacterium species and, potentially, to exogenous mobile genetic elements.

CHM mutation spectrum and disease: An update at the time of human therapeutic trials.

Choroideremia is an X-linked inherited retinal disorder (IRD) characterized by the degeneration of retinal pigment epithelium, photoreceptors, choriocapillaris and choroid affecting males with variable phenotypes in female carriers. Unlike other IRD, characterized by a large clinical and genetic heterogeneity, choroideremia shows a specific phenotype with causative mutations in only one gene, CHM. Ongoing gene replacement trials raise further interests in this disorder. We describe here the clinical and genetic data from a French cohort of 45 families, 25 of which carry novel variants, in the context of 822 previously reported choroideremia families. Most of the variants represent loss-of-function mutations with eleven families having large (i.e. ≥6 kb) genomic deletions, 18 small insertions, deletions or insertion deletions, six showing nonsense variants, eight splice site variants and two missense variants likely to affect splicing. Similarly, 822 previously published families carry mostly loss-of-function variants. Recurrent variants are observed worldwide, some of which linked to a common ancestor, others arisen independently in specific CHM regions prone to mutations. Since all exons of CHM may harbor variants, Sanger sequencing combined with quantitative polymerase chain reaction or multiplex ligation-dependent probe amplification experiments are efficient to achieve the molecular diagnosis in patients with typical choroideremia features.

Novel SARS-CoV-2 Variant Derived from Clade 19B, France.

We report a novel severe acute respiratory syndrome coronavirus 2 variant derived from clade 19B (HMN.19B variant or Henri Mondor variant). This variant is characterized by the presence of 18 amino acid substitutions, including 7-8 substitutions in the spike protein and 2 deletions. These variants actively circulate in different regions of France.

WDR34, a candidate gene for non-syndromic rod-cone dystrophy.

Rod-cone dystrophy (RCD), also called retinitis pigmentosa, is characterized by rod followed by cone photoreceptor degeneration, leading to gradual visual loss. Mutations in over 65 genes have been associated with non-syndromic RCD explaining 60% to 70% of cases, with novel gene defects possibly accounting for the unsolved cases. Homozygosity mapping and whole-exome sequencing applied to a case of autosomal recessive non-syndromic RCD from a consanguineous union identified a homozygous variant in WDR34. Mutations in WDR34 have been previously associated with severe ciliopathy syndromes possibly associated with a retinal dystrophy. This is the first report of a homozygous mutation in WDR34 associated with non-syndromic RCD.

Assessment of Bacterial Colonization of Intracranial Pressure Transducers: A Prospective Study.

OBJECTIVES: Cerebral infections related to the presence of an intraparenchymal intracranial pressure transducer (ICPT) are rare. We assessed the incidence of ICPT-related infections and colonization using culture, molecular biology, and electron microscopy. METHODS: All consecutive patients in a neurosurgical intensive care unit who had an ICPT inserted between March 2017 and February 2018 were prospectively included. Presence of colonization on the ICPTs was assessed after removal using culture, scanning electron microscopy (SEM), and next-generation sequencing (NGS). RESULTS: Fifty-three ICPTs (53 patients), indwelling for a median of 4 (range 3-7) days, were studied. Median patient follow-up was 3 months. SEM, microbial culture, and NGS were performed for 91%, 79%, and 72% of ICPTs, respectively; 28 ICPTs (53%) were assessed using all three techniques. No patient developed ICPT-related infection. Microbial cultures were positive for two of the ICPTs (5%); colonization was identified on all ICPTs using NGS and SEM. Mature biofilm was observed on 35/48 (73%) of ICPTs. A median of 10 (8-12) operational taxonomic units were identified for each ICPT, most being of environmental origin. There was no association between biofilm maturity and antimicrobial treatment or duration of ICPT insertion. Antimicrobial treatment was associated with decreased alpha and beta-diversity (p = 0.01). CONCLUSIONS: We observed no ICPT-related cerebral infections although colonization was identified on all ICPTs using NGS and SEM. Mature biofilm was the main bacterial lifestyle on the ICPTs.

Mutated CCDC51 Coding for a Mitochondrial Protein, MITOK Is a Candidate Gene Defect for Autosomal Recessive Rod-Cone Dystrophy.

The purpose of this work was to identify the gene defect underlying a relatively mild rod-cone dystrophy (RCD), lacking disease-causing variants in known genes implicated in inherited retinal disorders (IRD), and provide transcriptomic and immunolocalization data to highlight the best candidate. The DNA of the female patient originating from a consanguineous family revealed no large duplication or deletion, but several large homozygous regions. In one of these, a homozygous frameshift variant, c.244_246delins17 p.(Trp82Valfs*4); predicted to lead to a nonfunctional protein, was identified in CCDC51. CCDC51 encodes the mitochondrial coiled-coil domain containing 51 protein, also called MITOK. MITOK ablation causes mitochondrial dysfunction. Here we show for the first time that CCDC51/MITOK localizes in the retina and more specifically in the inner segments of the photoreceptors, well known to contain mitochondria. Mitochondrial proteins have previously been implicated in IRD, although usually in association with syndromic disease, unlike our present case. Together, our findings add another ultra-rare mutation implicated in non-syndromic IRD, whose pathogenic mechanism in the retina needs to be further elucidated.

Fatal Encephalitis Caused by Cristoli Virus, an Emerging Orthobunyavirus, France.

We report the discovery of a new orthobunyavirus, Cristoli virus, by means of shotgun metagenomics. The virus was identified in an immunodepressed patient with fatal encephalitis. Full-length genome sequencing revealed high-level expression of a virulence factor, possibly explaining the severity of the infection. The patient's recent history suggests circulation in France.

Fatal Measles Inclusion-Body Encephalitis in Adult with Untreated AIDS, France.

We report a fatal case of measles inclusion-body encephalitis occurring in a woman from Romania with AIDS. After an extensive but unsuccessful diagnostic evaluation, a pan-pathogen shotgun metagenomic approach revealed a measles virus infection. We identified no mutations previously associated with neurovirulence.

Phenotype Analysis of Retinal Dystrophies in Light of the Underlying Genetic Defects: Application to Cone and Cone-Rod Dystrophies.

Phenotypes observed in a large cohort of patients with cone and cone-rod dystrophies (COD/CORDs) are described based on multimodal retinal imaging features in order to help in analyzing massive next-generation sequencing data. Structural abnormalities of 58 subjects with molecular diagnosis of COD/CORDs were analyzed through specific retinal imaging including spectral-domain optical coherence tomography (SD-OCT) and fundus autofluorescence (BAF/IRAF). Findings were analyzed with the underlying genetic defects. A ring of increased autofluorescence was mainly observed in patients with CRX and GUCY2D mutations (33% and 22% of cases respectively). "Speckled" autofluorescence was observed with mutations in three different genes (ABCA4 64%; C2Orf71 and PRPH2, 18% each). Peripapillary sparing was only found in association with mutations in ABCA4, although only present in 40% of such genotypes. Regarding SD-OCT, specific outer retinal abnormalities were more commonly observed in particular genotypes: focal retrofoveal interruption and GUCY2D mutations (50%), foveal sparing and CRX mutations (50%), and outer retinal atrophy associated with hyperreflective dots and ABCA4 mutations (69%). This study outlines the phenotypic heterogeneity of COD/CORDs hampering statistical correlations. A larger study correlating retinal imaging with genetic results is necessary to identify specific clinical features that may help in selecting pathogenic variants generated by high-throughput sequencing.

MERTK mutation update in inherited retinal diseases.

MER tyrosine kinase (MERTK) encodes a surface receptor localized at the apical membrane of the retinal pigment epithelium. It plays a critical role in photoreceptor outer segment internalization prior to phagocytosis. Mutations in MERTK have been associated with severe autosomal recessive retinal dystrophies in the RCS rat and in humans. We present here a comprehensive review of all reported MERTK disease causing variants with the associated phenotype. In addition, we provide further data and insights of a large cohort of 1,195 inherited retinal dystrophies (IRD) index cases applying state-of-the-art genotyping techniques and summarize current knowledge. A total of 79 variants have now been identified underlying rod-cone dystrophy and cone-rod dystrophy including 11 novel variants reported here. The mutation spectrum in MERTK includes 33 missense, 12 nonsense, 12 splice defects, 12 small deletions, two small insertion-deletions, three small duplications, and two exonic and three gross deletions. Altogether, mutations in MERTK account for ∼2% of IRD cases with a severe retinal phenotype. These data are important for current and future therapeutic trials including gene replacement therapy or cell-based therapy.

Ultra-deep sequencing improves the detection of drug resistance in cellular DNA from HIV-infected patients on ART with suppressed viraemia.

Background: Standard genotypic tests performed on HIV DNA from patients on suppressive ART, with previous resistance-associated mutations (RAMs) detected in their plasma, underestimate resistance. We thus compared ultra-deep sequencing (UDS) with bulk sequencing of DNA to detect RAMs previously identified in plasma. Methods: We sequenced the DNA of 169 highly treatment experienced patients with suppressed viraemia (ANRS 138-EASIER trial). Protease (PR) and reverse transcriptase (RT) genes from HIV DNA were sequenced by bulk sequencing and UDS, comparing 1% and 20% as thresholds of detection for UDS. Results: Patients were highly treatment experienced (13.6 years). UDS of DNA was successful for the RT and PR genes in 133 (79%) and 137 (81%) patients, respectively. The detection of RAMs was similar by bulk sequencing and UDS with a 20% cut-off. However, the detection of RAMs by UDS with a 1% cut-off was significantly higher than that of bulk sequencing for RT codons D67N (65.4% versus 52.3%), M184V (66.2% versus 52.3%), L210W (48.9% versus 36.4%) and T215Y (57.9% versus 42.1%) and PR codons M46I (46% versus 26%), I54L (12.4% versus 3.9%), V82A (44.5% versus 29.9%) and L90M (57.7% versus 42.5%). Conclusions: Genotypic resistance testing of cellular HIV DNA of well-controlled patients should use UDS technology with a sensitivity threshold of 1% to improve the detection of the resistant reservoir.

Expanding the Mutation Spectrum in ABCA4: Sixty Novel Disease Causing Variants and Their Associated Phenotype in a Large French Stargardt Cohort.

Here we report novel mutations in ABCA4 with the underlying phenotype in a large French cohort with autosomal recessive Stargardt disease. The DNA samples of 397 index subjects were analyzed in exons and flanking intronic regions of ABCA4 (NM_000350.2) by microarray analysis and direct Sanger sequencing. At the end of the screening, at least two likely pathogenic mutations were found in 302 patients (76.1%) while 95 remained unsolved: 40 (10.1%) with no variants identified, 52 (13.1%) with one heterozygous mutation, and 3 (0.7%) with at least one variant of uncertain significance (VUS). Sixty-three novel variants were identified in the cohort. Three of them were variants of uncertain significance. The other 60 mutations were classified as likely pathogenic or pathogenic, and were identified in 61 patients (15.4%). The majority of those were missense (55%) followed by frameshift and nonsense (30%), intronic (11.7%) variants, and in-frame deletions (3.3%). Only patients with variants never reported in literature were further analyzed herein. Recruited subjects underwent complete ophthalmic examination including best corrected visual acuity, kinetic and static perimetry, color vision test, full-field and multifocal electroretinography, color fundus photography, short-wavelength and near-infrared fundus autofluorescence imaging, and spectral domain optical coherence tomography. Clinical evaluation of each subject confirms the tendency that truncating mutations lead to a more severe phenotype with electroretinogram (ERG) impairment (p = 0.002) and an earlier age of onset (p = 0.037). Our study further expands the mutation spectrum in the exonic and flanking regions of ABCA4 underlying Stargardt disease.

Whole-exome sequencing identifies KIZ as a ciliary gene associated with autosomal-recessive rod-cone dystrophy.

Rod-cone dystrophy (RCD), also known as retinitis pigmentosa, is a progressive inherited retinal disorder characterized by photoreceptor cell death and genetic heterogeneity. Mutations in many genes have been implicated in the pathophysiology of RCD, but several others remain to be identified. Herein, we applied whole-exome sequencing to a consanguineous family with one subject affected with RCD and identified a homozygous nonsense mutation, c.226C>T (p.Arg76(∗)), in KIZ, which encodes centrosomal protein kizuna. Subsequent Sanger sequencing of 340 unrelated individuals with sporadic and autosomal-recessive RCD identified two other subjects carrying pathogenic variants in KIZ: one with the same homozygous nonsense mutation (c.226C>T [p.Arg76(∗)]) and another with compound-heterozygous mutations c.119_122delAACT (p.Lys40Ilefs(∗)14) and c.52G>T (p.Glu18(∗)). Transcriptomic analysis in mice detected mRNA levels of the mouse ortholog (Plk1s1) in rod photoreceptors, as well as its decreased expression when photoreceptors degenerated in rd1 mice. The presence of the human KIZ transcript was confirmed by quantitative RT-PCR in the retina, the retinal pigment epithelium, fibroblasts, and whole-blood cells (highest expression was in the retina). RNA in situ hybridization demonstrated the presence of Plk1s1 mRNA in the outer nuclear layer of the mouse retina. Immunohistology revealed KIZ localization at the basal body of the cilia in human fibroblasts, thus shedding light on another ciliary protein implicated in autosomal-recessive RCD.