An All-in-One Highly Multiplexed Diagnostic Assay for Rapid, Sensitive, and Comprehensive Detection of Intraocular Pathogens.

PURPOSE: Intraocular infections are sight-threatening conditions that can lead to vision loss. Rapid identification of the etiologies plays a key role in early initiation of effective therapy to save vision. However, current diagnostic modalities are time consuming and lack sensitivity and inclusiveness. We present here a newly developed comprehensive ocular panel designed to improve diagnostic yields and provide a tool for rapid and sensitive pathogen detection. DESIGN: Experimental laboratory investigation. METHODS: A panel containing 46 pathogens and 2 resistance/virulence markers that are commonly detected in intraocular infections was developed. Genomic targets were scrutinized for stretches predicted to be specific for a particular species while being conserved across different strains. A set of primers for sample enrichment, and two 50mer NanoString compatible probes were then designed for each target. Probe-target hybrids were detected and quantified using the NanoString nCounter SPRINT Profiler. Diagnostic feasibility was assessed in a pilot clinical study testing samples from infectious retinitis (n = 15) and endophthalmitis (n = 12) patients, for which the etiologies were confirmed by polymerase chain reaction (PCR) or culture. RESULTS: Analytical studies demonstrated highly sensitive detection of a broad spectrum of pathogens, including bacteria, viruses, and parasites, with limits of detection being as low as 2.5 femtograms per reaction. We also found excellent target specificity, with minimal cross-reactivity detected. The custom-designed NanoString ocular panel correctly identified the causative agent from all clinical specimens positive for a variety of pathogens. CONCLUSION: This highly multiplexed panel for pathogen detection offers a sensitive, comprehensive, and uniform assay run directly on ocular fluids that could significantly improve diagnostics of sight-threatening intraocular infections.

Improved Detection of Herpesviruses from Diluted Vitreous Specimens Using Hydrogel Particles.

Infectious uveitis is a sight-threatening infection commonly caused by herpesviruses. Vitreous humor is often collected for molecular confirmation of the causative agent during vitrectomy and mixed in large volumes of buffered saline, diluting the pathogen load. Here, we explore affinity-capture hydrogel particles (Nanotrap®) to concentrate low abundant herpesviruses from diluted vitreous. Simulated samples were prepared using porcine vitreous spiked with HSV-1, HSV-2, VZV and CMV at 105 copies/mL. Pure undiluted samples were used to test capturing capability of three custom Nanotrap particles (red, white and blue) in a vitreous matrix. We found that all particles demonstrated affinity to the herpesviruses, with the Red Particles having both good capture capability and ease of handling for all herpesviruses. To mimic diluted vitrectomy specimens, simulated-infected vitreous were then serially diluted in 7 mL TE buffer. Diluted samples were subjected to an enrichment protocol using the Nanotrap Red particles. Sensitivity of pathogen detection by qPCR in diluted vitreous increased anywhere between 2.3 to 26.5 times compared to non-enriched specimens. This resulted in a 10-fold increase in the limit of detection for HSV-1, HSV-2 and VZV. These data demonstrated that Nanotrap particles can capture and concentrate HSV-1, HSV-2, VZV and CMV in a vitreous matrix.

Molecular characterization of fungal endophthalmitis and keratitis caused by yeasts.

Candida species are the most common causes of sight-threatening fungal ocular infections in temperate regions of the world. Despite their relevance, little is known about the emergence of novel species and the molecular epidemiology of these infections. Here, we molecularly characterized 38 yeast isolates collected from patients diagnosed with endophthalmitis or keratitis at Massachusetts Eye and Ear from 2014 to 2021. Sequencing of the ITS1-5.8S-/ITS2 regions demonstrated that this population of yeasts was dominated by Candida spp. (37 out of 38; 97%), with 58% of the cases caused by C. albicans (n = 22) and the remaining by emerging non-albicans species, predominantly by C. parapsilosis (n = 8) and C. dubliniensis (n = 6). One isolate each was identified as C. tropicalis and Clavispora lusitaniae. Interestingly, all C. dubliniensis were isolated from endophthalmitis and most C. parapsilosis from keratitis. Multilocus sequence typing analysis of C. albicans showed a prevalence of CC-1 isolates that has DST69 as the putative founder, with 64% of them belonging to this clonal complex (CC). Isolates grouped within this cluster were more predominant in endophthalmitis (10 out of 14; 71%). One C. albicans CC-1 isolate was multi-azole resistant. In conclusion, we observed that nearly half of the ocular infections caused by yeasts are associated with C. albicans, with evidence for the emergence of non-albicans species that are differentially enriched in distinct ocular niches. Candida albicans isolates clustered within the predominant CC-1 group were particularly more common in endophthalmitis, demonstrating a potential pattern of ocular disease enrichment within this clade.

Candida species are the most common pathogen responsible for ocular infections in the temperature regions of the world. Here, we sequenced and molecularly characterized the Candida species seen in patients who present to our hospital with infection to understand the species' distribution over time.

Experimental methods to study sleep disruption and immune balance in urban children with asthma.

Study Objectives: We describe research methods developed to examine effects of sleep disruption on changes in immune balance, lung function, and cognitive performance in a sample of urban, ethnically diverse children with persistent asthma. Two case examples (8- and 10-year-old males) are presented to highlight methods of the current study and illustrate effects of experimentally disrupted sleep on the immune balance profile (Th1/Th2 cytokines), key sleep variables from polysomnography data, and lung function in our sample. Methods: Children follow an individualized structured sleep schedule consistent with their habitual sleep need (≥9.5 hours' time in bed) for six days before a laboratory-based experimental sleep protocol. Children then spend two successive nights in the sleep lab monitored by polysomnography: a baseline night consisting of uninterrupted sleep, and a disruption night, during which they are awoken for 2 minutes between 20-minute intervals of uninterrupted sleep. Evening and morning blood draws bracket baseline and disruption nights for immune biomarker assessment. Results: A shift towards immune imbalance following the sleep disruption protocol was observed in these illustrative cases. Conclusions: Data from these case examples provide evidence that the experimental protocol caused disruptions in sleep as observed on polysomnography and had the hypothesized downstream effects on immune balance associated with clinical asthma control. Documenting the effects of sleep disruption on immune function in children with persistent asthma is a crucial step towards understanding associations between sleep, immune balance, and asthma outcomes and provides important information for developing novel interventions for youth with asthma and suboptimal sleep. Clinical Trials: Not applicable.

The Examination of Instrumental Activities of Daily Living and Perceived Adulthood in Emerging Adults.

Emerging adults differ in terms of the extent to which they perceive themselves as adults. We examined how the ability to perform activities related to independent living (i.e., instrumental activities of daily living [IADLs]) was associated with perceived adulthood. Data were collected from 236 emerging adults in college. Results suggested that IADL scores were positively related to perceived adulthood and achieved criteria of adulthood even after controlling for race, year in school, age, and sex. Results are discussed in terms of the development and importance of IADLs during emerging adulthood.

The Influence of Source and Type of Support on College Students' Physical Activity Behavior.

BACKGROUND: College is a place where students develop health behaviors that persist throughout adulthood, yet a large percentage of students are not engaging in adequate physical activity. Social support is associated with physical activity; however, the effects of source (ie, family and friend) and type (ie, companionship, esteem, and informational) are not fully understood. The current study examined the effects of different sources and types of social support on college students' physical activity behaviors. METHODS: An online survey tool collected data from 733 college students enrolled at a Mid-Atlantic university. Structural equation models and a repeated-measures analysis of variance were conducted. RESULTS: Support from family and friends was associated with physical activity behaviors; however, support from friends exerted a stronger effect. Students received different types of support from their network, which differentially influenced physical activity behaviors. Students received higher levels of companionship support from their friends and higher levels of esteem support from their family, which was positively associated with physical activity behaviors. Students received higher levels of informational support from their families, which was negatively associated with physical activity behaviors. CONCLUSIONS: This study highlights the importance of examining the independent effects of source and type of support on college students' physical activity.

Random variability in congenital hypothyroidism from thyroid dysgenesis over 16 years in Québec.

CONTEXT: Research on the etiology of congenital hypothyroidism from thyroid dysgenesis (CHTD) (comprising mostly ectopy and agenesis) over the past decade has focused on genetic mechanisms. However, the possibility that environmental factors might be involved has been raised by studies showing a seasonal variability of the incidence of CHTD. OBJECTIVES: The objective of this study was to assess the variability in incidence of CHTD in the province of Québec, Canada. DESIGN, SETTING, PATIENTS, AND MAIN OUTCOME MEASURE: The Québec provincial newborn screening database was analyzed from January 1990 to December 2005. Only cases of permanent congenital hypothyroidism with thyroid ectopy or agenesis on scintigraphy were analyzed. RESULTS: During the study period, 1,303,341 children were screened, and 424 cases of permanent congenital hypothyroidism were diagnosed, giving an overall incidence of 1:3074. Of these, 306 had CHTD (overall incidence 1:4259) from either ectopy (n = 231) or agenesis (n = 75). Over the 16 yr of the study, this incidence remained stable (P = 0.57), and no significant variability in monthly incidence was found (P = 0.87). CONCLUSIONS: The incidence of CHTD did not vary over the observation period, and its monthly variation was random. Therefore, environmental factors do not appear to play a significant role in the etiology of CHTD.

The effectiveness of anticoagulant and antiplatelet agents in preventing venous thromboembolism during stroke rehabilitation: a historical cohort study.

OBJECTIVE: To determine the effectiveness of anticoagulant and antiplatelet agents in preventing venous thromboembolism (VTE) during stroke rehabilitation. DESIGN: Historical cohort study. SETTING: Acute inpatient rehabilitation hospital. PARTICIPANTS: Consecutive patients (N=1506) with ischemic and hemorrhagic stroke admitted for rehabilitation. INTERVENTIONS: Documented use of anticoagulants (warfarin or anticoagulant doses of heparin), heparin in prophylactic doses, and antiplatelet agents. MAIN OUTCOME MEASURE: Occurrence of deep vein thrombosis detected by ultrasound or venography or pulmonary embolism detected by ventilation perfusion scan, spiral computed tomography, or pulmonary angiography. RESULTS: Fifty-eight VTE events occurred (3.9% incidence or 1.36 events per 1000 patient days), with higher risk in patients with severe stroke. Only therapeutic anticoagulation had a statistically significant protective effect for VTE risk in univariate analysis (odds ratio [OR]=.44; 95% confidence interval [CI],.20-.98). After adjusting for multiple medication use and other factors, including age, stroke onset to admission interval, length of rehabilitation stay, cause of stroke, and admission National Institutes of Health Stroke Scale score, therapeutic anticoagulation gave strong protection against VTE (OR=.37; 95% CI,.15-.88), followed by heparin (OR=.48; 95% CI,.23-.98) but not by antiplatelet agents (OR=.79; 95% CI,.40-1.57). No medications were associated with significant bleeding complications. CONCLUSIONS: Use of therapeutic anticoagulants or prophylactic heparin prevented VTE in stroke patients during inpatient rehabilitation.