Validation of Nanopore long-read sequencing to resolve RPGR ORF15 genotypes in individuals with X-linked retinitis pigmentosa.

X-linked retinitis pigmentosa (XLRP) is characterized by progressive vision loss leading to legal blindness in males and a broad severity spectrum in carrier females. Pathogenic alterations of the retinitis pigmentosa GTPase regulator gene (RPGR) are responsible for over 70% of XLRP cases. In the retina, the RPGRORF15 transcript includes a terminal exon, called ORF15, that is altered in the large majority of RPGR-XLRP cases. Unfortunately, due to its highly repetitive sequence, ORF15 represents a considerable challenge in terms of sequencing for molecular diagnostic laboratories. However, in a recent preliminary work Yahya et al. reported a long-read sequencing approach seeming promising. Here, the aim of the study was to validate and integrate this new sequencing strategy in a routine screening workflow. For that purpose, we performed a masked test on 52 genomic DNA samples from male and female individuals carrying 32 different pathogenic ORF15 variations including 20 located in the highly repetitive region of the exon. For the latter, we have obtained a detection rate of 80-85% in males and 60-80% in females after bioinformatic analyses. These numbers raised to 100% for both status after adding a complementary visual inspection of ORF15 long-reads. In accordance with these results, and considering the frequency of ORF15 pathogenic variations in XLRP, we suggest that a long-read screening of ORF15 should be systematically considered before any other sequencing approach in subjects with a diagnosis compatible with XLRP.

Unraveling ice multiplication in winter orographic clouds via in-situ observations, remote sensing and modeling.

Recent years have shown that secondary ice production (SIP) is ubiquitous, affecting all clouds from polar to tropical regions. SIP is not described well in models and may explain biases in warm mixed-phase cloud ice content and structure. Through modeling constrained by in-situ observations and its synergy with radar we show that SIP in orographic clouds exert a profound impact on the vertical distribution of hydrometeors and precipitation, especially in seeder-feeder cloud configurations. The mesoscale model simulations coupled with a radar simulator strongly support that enhanced aggregation and SIP through ice-ice collisions contribute to observed spectral bimodalities, skewing the Doppler spectra toward the slower-falling side at temperatures within the dendritic growth layer, ranging from -20 °C to -10 °C. This unique signature provides an opportunity to infer long-term SIP occurrences from the global cloud radar data archive, particularly for this underexplored temperature regime.

Clinical features and treatment outcomes of bone and joint nontuberculous mycobacterial infections according to immune status: a 9-year retrospective observational cohort.

OBJECTIVES: Nontuberculous mycobacteria (NTM) bone and joint infections (BJIs) are uncommon. We evaluated the characteristics of BJIs and identified differences according to immune status. METHODS: We performed a multicenter retrospective study in France involving patients with documented NTM BJI over a 9-year period. We collected the clinical and microbiological characteristics, management, and clinical outcomes of the patients. RESULTS: Overall, 95 patients were included, of whom 50.5% (48/95) were immunosuppressed. Tenosynovitis was more frequent in the immunocompetent group, and native arthritis more common in the immunosuppressed group. Mycobacerium marinum and M. abscessus complex were significantly more frequent in the immunocompetent group, and M. avium and M. xenopi were significantly more frequent in the immunosuppressed group. The combination of antibiotherapy with surgery tended to be more frequent in the immunocompetent than the immunosuppressed group (63.8% (30/47) vs 47.8% (22/46), respectively); of the latter, 45.7% (21/46) received antimicrobial therapy alone, a higher frequency than in the immunocompetent group (23.4%, 11/47). The median duration of antimicrobial treatment was similar in the two groups (11 months). Mortality was significantly higher in the immunosuppressed group. CONCLUSIONS: Although the clinical presentations and the NTM species involved in BJI differed according to immune status, most recovered completely after treatment.

Single-stage revision for total shoulder arthroplasty infection. Results at a minimum 2 years follow-up.

INTRODUCTION: Similar to the management of periprosthetic joint infections of the lower limb, one-stage revision in total shoulder arthroplasty (TSA) infections is an option that has been highlighted in scientific publications since the early 2010s. However, there are only a few studies which validate this treatment and determine its scope of application in relation to two-stage treatment. HYPOTHESIS: Single-stage revision for infected TSA is a reliable treatment allowing good infection control and satisfactory functional results. METHODS: This single-center retrospective series of 34 consecutive patients operated on between 2014 and 2020 for a one-stage prosthetic revision was evaluated at a minimum of 2 years of follow-up. All of the patients included underwent revision shoulder arthroplasty during this period with the diagnosis of infection confirmed by microbiological analysis of surgical samples. Patients who did not benefit from a bipolar revision were excluded. All patients were followed at least 2 years after the intervention. Clinically suspected recurrence of infection was confirmed by a periprosthetic sample under radiographic guidance. Functional clinical outcomes as well as mechanical complications were also reported. RESULTS: The average follow-up was 40.4 months (24-102±21.6). A septic recurrence was observed in three patients (8.8%). A mechanical complication was present in four patients (14.7%), and three (11.8%) required at least one surgical revision. The mean Constant-Murley score at the last follow-up was 49 (42-57±21.83). DISCUSSION: Single-stage revision for shoulder periprosthetic joint infection results in a success rate of 91.2% with satisfactory functional results after more than 2 years of follow-up. LEVEL OF EVIDENCE: IV; retrospective study.

A prospective multicentre evaluation of BioFire® Joint Infection Panel for the rapid microbiological documentation of acute arthritis.

OBJECTIVES: To assess the performance of the rapid syndromic BioFire® Joint Infection Panel (BF-JIP) to detect bacterial and fungal pathogens, as well as antibiotic resistance genes, directly in synovial fluid specimens collected from patients with acute arthritis. METHODS: The study was conducted in six French bacteriological laboratories. To assess the performances of BF-JIP, results were compared with those of synovial fluid 14-day culture and, in case of discrepancy, with those of complementary molecular methods and intraoperative samples. A total of 308 synovial fluid specimens were tested after collection from 308 adults and children presenting with clinical and biological suspicion of acute arthritis; patients presenting with acute periprosthetic joint infection were included according to the European Bone and Joint Infection Society 2021 criteria. RESULTS: Only one specimen failed (no result). On the basis of the consolidated data, the BF-JIP was concordant with the 14-day culture in 280 (91.2%) of the 307 specimens finally included in the study. The positive percentage agreement was 84.9% (95% CI, 78.8-89.8%) and the negative percentage agreement was 100% (95% CI, 97.2-100%). The positive predictive value was extremely high (100%; 95% CI, 97.6-100%), whereas the negative predictive value was lower (82.6%; 95% CI, 75.7-88.2%), partially explained by the missing target species in the panel. DISCUSSION: The BF-JIP showed high performances to detect pathogens involved in acute arthritis.

Features of cryptococcosis among 652 HIV-seronegative individuals in France: a cross-sectional observational study (2005-2020).

OBJECTIVES: We aimed to describe features and outcomes of cryptococcosis among HIV-seronegative individuals in a large surveillance network for cryptococcosis in France. METHODS: We included incident cases of cryptococcosis in HIV-seronegative individuals from 2005 to 2020. We compared patient characteristics, disease presentations, cryptococcal antigen results, and induction antifungal treatments according to underlying disease. We examined factors associated with 90-day mortality. Among patients with disseminated infections, we investigated whether receipt of flucytosine and polyene combination was associated with lower mortality. RESULTS: Among 652 individuals, 209 (32.1%) had malignancy, 130 (19.9%) were solid-organ transplant recipients, 204 (31.3%) had other immunocompromising conditions, and 109 (16.7%) had no reported underlying factor. The commonest presentations were disseminated infections (63.3%, 413/652) and isolated pulmonary infections (25.3%, 165/652). Solid-organ transplant patients were most likely to have disseminated infections and a positive serum cryptococcal antigen result. Patients with malignancy were older and less likely to receive a flucytosine-containing regimen for disseminated infections than others (58.7%, 78/133 vs. 73.2%, 194/265; p 0.029). The crude 90-day case-fatality ratio was 27.2% (95% CI, 23.5%-31.1%). Age ≥60 years (aOR: 2.75 [1.78-4.26]; p < 0.001), meningitis/fungaemia (aOR: 4.79 [1.80-12.7]; p 0.002), and malignancy (aOR: 2.4 [1.14-5.07]; p 0.02) were associated with higher 90-day mortality. Receipt of flucytosine and polyene combination was associated with lower 90-day mortality (aOR: 0.40 [0.23-0.71]; p 0.002) in multivariable analysis and inverse probability of treatment weighted analysis (aOR: 0.45 [0.25-0.80]; p 0.006). DISCUSSION: HIV-seronegative individuals with cryptococcosis comprise a wide range of underlying conditions with different presentations and outcomes, requiring a tailored approach to diagnosis and management.

Overview on biotics development.

Although probiotics have been used in food products and supplements for decades, there has been a considerable increase in their use more recently. Recent technological advances have thus led to major advances in knowledge of the gut microbiota, enabling a significant development of biotics. In this review, we discuss the uses of traditional probiotics but also the discovery of next-generation probiotics that could be used as live biotherapeutics. These novel preventive and therapeutic strategies hold promise for the treatment of numerous diseases such as inflammatory bowel diseases such as Crohn's disease and ulcerative colitis. Probiotic bacteria can be consumed alone, or in combination with prebiotics as synbiotics, or mixed with other probiotic strains to form a consortium for enhanced effects. We also discuss the benefits of using postbiotics.

The Economic Impacts of COVID-19 on Autistic Children and Their Families.

PURPOSE: We used data from the National Survey of Children's Health to (1) examine differences in economic hardship and safety net program use after the implementation of federal relief efforts, and (2) assess whether the COVID-19 pandemic exacerbated autism-based disparities in hardship and program use. METHODS: We examined five dimensions of economic hardship (poverty, food insecurity, medical hardship, medical costs, and foregone work) and four safety net programs (cash assistance, Supplemental Nutrition Assistance Program (SNAP), Special Supplemental Nutrition Program for Women, Infants and Children (WIC), and free or reduced-cost meals). First, we calculated adjusted prevalence and odds ratios to compare pre-COVID (2018-2019) and during COVID (2021) outcomes by autism status. Next, we calculated the adjusted odds of each outcome among autistic children compared to those of children with and without other special healthcare needs at both time points. RESULTS: COVID-19 exacerbated autism-based disparities in food insecurity, SNAP, and public health insurance, but alleviated inequities in medical hardship, foregone work, and cash assistance. Autistic children did not experience declines in food insecurity or increases in SNAP like other children; medical hardship and foregone work decreased more for autistic children; and the magnitude of autism-based differences in public coverage significantly increased during the pandemic. CONCLUSION: Federal relief efforts likely improved economic outcomes of children; however, these effects varied according to type of hardship and by disability group. Efforts to promote economic well-being among autistic populations should be tailored to the financial challenges most salient to low-income autistic children, like food insecurity.

Cryptococcus neoformans Infections Differ Among Human Immunodeficiency Virus (HIV)-Seropositive and HIV-Seronegative Individuals: Results From a Nationwide Surveillance Program in France.

Among 1107 cryptococcosis cases from the French surveillance network (2005-2020), the proportion of HIV-seronegative individuals has recently surpassed that of HIV-seropositive individuals. We observed marked differences in patient characteristics, disease presentations, cryptococcal antigen results, infecting species, and mortality according to HIV serostatus.

Low-Income Households of Children With Autism and the Economic Safety Net.

OBJECTIVE: This paper examines the distribution, parameters, and determinants of safety net program use among a nationally representative sample of low-income children with autism spectrum disorder (ASD). METHODS: We used data from the 2021 National Survey of Children's Health to produce population estimates of material hardship and safety net program use among 554 low-income households of children with ASD, ages 3 to 17 years, relative to 2831 children with other special health care needs (SHCN) and 8758 children with no SHCN of the same age. Design-adjusted multivariate logistic regression models identified predictors of cash assistance, Supplemental Nutrition Assistance Program, and disconnection from both. RESULTS: There were few significant differences in material hardship between children with ASD and those with other SHCN, although children with ASD experienced significantly higher levels of hardships compared to children with no SHCN. Having a child with ASD did not significantly increase the odds of safety net use. Health insurance and household income were stronger predictors of use than disability. Nine percent of disconnected children lived in households under 100% federal poverty level and experienced some type of material hardship. CONCLUSIONS: Future research about the economic security of children with ASD and their families could focus on the following 3 areas of inquiry: assess how race, ethnicity, or socioeconomic position interact with disability to influence safety net program use; examine the intersection between Medicaid and safety net programs at the state and national levels; and identify specific subgroups of children at risk for disconnection and understand why they are not accessing benefits.

Functional analysis of germline VANGL2 variants using rescue assays of vangl2 knockout zebrafish.

Developmental studies have shown that the evolutionarily conserved Wnt Planar Cell Polarity (PCP) pathway is essential for the development of a diverse range of tissues and organs including the brain, spinal cord, heart and sensory organs, as well as establishment of the left-right body axis. Germline mutations in the highly conserved PCP gene VANGL2 in humans have only been associated with central nervous system malformations, and functional testing to understand variant impact has not been performed. Here we report three new families with missense variants in VANGL2 associated with heterotaxy and congenital heart disease p.(Arg169His), non-syndromic hearing loss p.(Glu465Ala) and congenital heart disease with brain defects p.(Arg135Trp). To test the in vivo impact of these and previously described variants, we have established clinically-relevant assays using mRNA rescue of the vangl2 mutant zebrafish. We show that all variants disrupt Vangl2 function, although to different extents and depending on the developmental process. We also begin to identify that different VANGL2 missense variants may be haploinsufficient and discuss evidence in support of pathogenicity. Together, this study demonstrates that zebrafish present a suitable pipeline to investigate variants of unknown significance and suggests new avenues for investigation of the different developmental contexts of VANGL2 function that are clinically meaningful.

USH2A variants causing retinitis pigmentosa or Usher syndrome provoke differential retinal phenotypes in disease-specific organoids.

There is an emblematic clinical and genetic heterogeneity associated with inherited retinal diseases (IRDs). The most common form is retinitis pigmentosa (RP), a rod-cone dystrophy caused by pathogenic variants in over 80 different genes. Further complexifying diagnosis, different variants in individual RP genes can also alter the clinical phenotype. USH2A is the most prevalent gene for autosomal-recessive RP and one of the most challenging because of its large size and, hence, large number of variants. Moreover, USH2A variants give rise to non-syndromic and syndromic RP, known as Usher syndrome (USH) type 2, which is associated with vision and hearing loss. The lack of a clear genotype-phenotype correlation or prognostic models renders diagnosis highly challenging. We report here a long-awaited differential non-syndromic RP and USH phenotype in three human disease-specific models: fibroblasts, induced pluripotent stem cells (iPSCs), and mature iPSC-derived retinal organoids. Moreover, we identified distinct retinal phenotypes in organoids from multiple RP and USH individuals, which were validated by isogenic-corrected controls. Non-syndromic RP organoids showed compromised photoreceptor differentiation, whereas USH organoids showed a striking and unexpected cone phenotype. Furthermore, complementary clinical investigations identified macular atrophy in a high proportion of USH compared with RP individuals, further validating our observations that USH2A variants differentially affect cones. Overall, identification of distinct non-syndromic RP and USH phenotypes in multiple models provides valuable and robust readouts for testing the pathogenicity of USH2A variants as well as the efficacy of therapeutic approaches in complementary cell types.

Unrealized Cross-System Opportunities to Improve Employment and Employment-Related Services Among Autistic Individuals.

Policy Points Employment is a key social determinant of health and well-being for the estimated 5.4 million autistic adults in the United States-just as it is for citizens without disabilities. Evaluation and monitoring of publicly funded employment services is paramount given the dramatic increases in adults with autism who need job supports. Vocational Rehabilitation agencies appeared to be absorbing short-term employment needs of autistic people, but Medicaid was severely lacking-and losing ground-in serving those who need longer-term employment services. Across both Vocational Rehabilitation and Medicaid, we estimated that only 1.1% of working-age autistic adults who potentially need employment services are actually receiving them-leaving an estimated 1.98 million autistic individuals without the employment services that are associated with achievement of well-being. CONTEXT: Employment is a key social determinant of health. As such, high rates of unemployment, underemployment, and poverty across the rapidly growing autistic population are concerning. A web of publicly funded services exists to support the employment, and associated health and well-being, of United States citizens with autism and other intellectual and developmental disabilities, namely through Vocational Rehabilitation (VR) and Medicaid home- and community-based services (HCBS) waivers. Given an absence of overarching surveillance of employment services, this study aimed to characterize the distribution of autistic service users across Medicaid versus VR, understand the types of employment services utilized within these programs and expenditures, and assess overall capacity to provide employment services as needs continue to increase. METHODS: This study examined the distribution of employment services among autistic people compared with those with intellectual disability using 2008-2016 data from the Centers for Medicare & Medicaid Services and the Rehabilitation Services Administration. Estimated need for employment services among autistic individuals was compared with capacity derived from VR service counts and a review of HCBS waivers. FINDINGS: The number of autistic people served through VR tripled during the study years, whereas those served through Medicaid only increased slightly. VR spending increased by 384% over the study years, whereas Medicaid costs decreased by 29%. Across VR and Medicaid, we estimated that only 1.1% of working-age autistic adults who needed employment services received them. CONCLUSIONS: Although VR appeared to be absorbing short-term employment needs of autistic individuals, Medicaid was severely lacking-and losing ground-in serving those who needed longer-term employment services. VR far outpaced Medicaid in both the number of autistic people served and total expenditures across the study years. However, an estimated 1.98 million autistic adults did not receive employment services that could be critical to improving their health and well-being.

In Vivo Emergence of Dual Resistance to Rifampin and Levofloxacin in Osteoarticular Cutibacterium avidum.

Cutibacterium avidum is an emerging causative agent of orthopedic device-related infections (ODRIs). There are no guidelines for the antimicrobial treatment of C. avidum ODRI, but oral rifampin is frequently used in combination with a fluoroquinolone following intravenous antibiotics. We describe the in vivo emergence of combined resistance to rifampin and levofloxacin in a C. avidum strain isolated from a patient with early-onset ODRI treated with debridement, antibiotic treatment, and implant retention (DAIR) using rifampin combined with levofloxacin as the oral treatment. Whole-genome sequencing of C. avidum isolates before and after antibiotic exposure confirmed strain identity and identified new mutations in rpoB and gyrA, leading to amino acid substitutions previously reported to be associated with resistance to rifampin (S446P) and fluoroquinolones (S101L), respectively, in other microbial agents, in the posttherapy isolate. Aside from the molecular insights reported here, this study highlights potential limitations of the combination of oral rifampin and levofloxacin in patients undergoing a DAIR procedure for C. avidum ODRI and the potential need to evaluate specific optimal therapy for emerging ODRI pathogens. IMPORTANCE In this study, we report for the first time the in vivo emergence of dual resistance to levofloxacin and rifampin in C. avidum isolated from a patient who received both antibiotics orally in the setting of a salvage debridement and implant retention of an ODRI. Aside from the molecular insights reported here, this study highlights potential limitations of the combination of oral rifampin and levofloxacin in patients undergoing these surgical procedures and the potential need to evaluate specific optimal therapy for emerging ODRI pathogens.

Identification and in vivo functional investigation of a HOMER2 nonstop variant causing hearing loss.

DFNA68 is a rare subtype of autosomal dominant nonsyndromic hearing impairment caused by heterozygous alterations in the HOMER2 gene. To date, only 5 pathogenic or likely pathogenic coding variants, including two missense substitutions (c.188 C > T and c.587 G > C), a single base pair duplication (c.840dupC) and two short deletions (c.592_597delACCACA and c.832_836delCCTCA) have been described in 5 families. In this study, we report a novel HOMER2 variation, identified by massively parallel sequencing, in a Sicilian family suffering from progressive dominant hearing loss over 3 generations. This novel alteration is a nonstop substitution (c.1064 A > G) that converts the translational termination codon (TAG) of the gene into a tryptophan codon (TGG) and is predicted to extend the HOMER2 protein by 10 amino acids. RNA analyses from the proband suggested that HOMER2 transcripts carrying the nonstop variant escaped the non-stop decay pathway. Finally, in vivo studies using a zebrafish animal model and behavioral tests clearly established the deleterious impact of this novel HOMER2 alteration on hearing function. This study identifies the fourth causal variation responsible for DFNA68 and describes a simple in vivo approach to assess the pathogenicity of candidate HOMER2 variants.

Genetic Evaluation of Prelingual Hearing Impairment: Recommendations of an European Network for Genetic Hearing Impairment.

The cause of childhood hearing impairment (excluding infectious pathology of the middle ear) can be extrinsic (embryofoetopathy, meningitis, trauma, drug ototoxicity, noise trauma, etc [...].

Evaluating the Transition from Targeted to Exome Sequencing: A Guide for Clinical Laboratories.

The transition from targeted to exome or genome sequencing in clinical contexts requires quality standards, such as targeted sequencing, in order to be fully adopted. However, no clear recommendations or methodology have emerged for evaluating this technological evolution. We developed a structured method based on four run-specific sequencing metrics and seven sample-specific sequencing metrics for evaluating the performance of exome sequencing strategies to replace targeted strategies. The indicators include quality metrics and coverage performance on gene panels and OMIM morbid genes. We applied this general strategy to three different exome kits and compared them with a myopathy-targeted sequencing method. After having achieved 80 million reads, all-tested exome kits generated data suitable for clinical diagnosis. However, significant differences in the coverage and PCR duplicates were observed between the kits. These are two main criteria to consider for the initial implementation with high-quality assurance. This study aims to assist molecular diagnostic laboratories in adopting and evaluating exome sequencing kits in a diagnostic context compared to the strategy used previously. A similar strategy could be used to implement whole-genome sequencing for diagnostic purposes.

CEP162 deficiency causes human retinal degeneration and reveals a dual role in ciliogenesis and neurogenesis.

Defects in primary or motile cilia result in a variety of human pathologies, and retinal degeneration is frequently associated with these so-called ciliopathies. We found that homozygosity for a truncating variant in CEP162, a centrosome and microtubule-associated protein required for transition zone assembly during ciliogenesis and neuronal differentiation in the retina, caused late-onset retinitis pigmentosa in 2 unrelated families. The mutant CEP162-E646R*5 protein was expressed and properly localized to the mitotic spindle, but it was missing from the basal body in primary and photoreceptor cilia. This impaired recruitment of transition zone components to the basal body and corresponded to complete loss of CEP162 function at the ciliary compartment, reflected by delayed formation of dysmorphic cilia. In contrast, shRNA knockdown of Cep162 in the developing mouse retina increased cell death, which was rescued by expression of CEP162-E646R*5, indicating that the mutant retains its role for retinal neurogenesis. Human retinal degeneration thus resulted from specific loss of the ciliary function of CEP162.

The Medical Home and Mental Health Services in Children and Youth with Special Health Care Needs.

BACKGROUND AND OBJECTIVES: Children and youth with special health care needs (CYSHCN) commonly experience mental health concerns, but conditions are often not identified or treated within primary care. Mental health care is often not a primary focus of pediatric primary care, but the medical home model has potential to address these concerns more adequately. The purpose of this study is to examine the relationship between the medical home and use of mental health services in CYSHCN. METHODS: Data came from the Medical Expenditure Panel Survey years 2015-2017, a nationally representative survey of health and healthcare in US families. The study included CYSHCN ages 6-17. We compared the use of mental health services, expenditures, and psychotropic medications across CYSHCN with and without a medical home using multivariable regression. RESULTS: 45% of CYSHCN received care within a medical home. CYSHCN with and without a medical home reported similar frequency of office-based mental health visits (21.2% versus 25.2%), average expenditures for visits ($147 versus $128), and psychotropic medications (11.9% versus 15.1%). Medical home status was not associated with office-based mental health visits, use of psychotropic medications, or cost for either. CONCLUSIONS: CYSHCN with mental health care needs face barriers to satisfactory care. Creating better connections between primary and mental health care could help to ameliorate this problem. Findings suggest the medical home, a more comprehensive primary care model, may not address mental health care needs of CYSHCN.