BACKGROUND:Ocular emergencies comprise 2-3% of emergency department (ED) visits, with retinal detachment requiring emergency surgery. Two-dimensional ultrasound is a rapid bedside tool but is highly operator dependent. OBJECTIVE:We determined three-dimensional ultrasound (3DUS) feasibility, acceptability, and usability in eye pathology detection using the ophthalmologist examination as reference standard. METHODS:We performed a prospective, blinded cohort study of a 3DUS-enabling device in 30 eye clinic and ED patients with visual symptoms and calculated 3DUS performance characteristics. Two expert readers interpreted the 3DUS images for pathology. All participants completed surveys. RESULTS:3DUS sensitivity was 0.81, specificity 0.73, positive predictive value 0.54, negative predictive value 0.91, and likelihood ratio (LR)+/LR- 3.03 and 0.26, respectively. Novice and expert sonographers had "substantial" agreement in correct diagnosis of abnormal vs. normal (κ = 0.68, 95% confidence interval 0.48-0.88). Most patients indicated that 3DUS is fast, comfortable, helps them understand their problem, and improves provider interaction/care, and all sonographers agreed; 4/5 sonographers felt confident performing ultrasound. Expert readers correctly identified an abnormal eye in 83/120 scans (76%) and correct diagnosis in 72/120 scans (65%), with no statistical difference between novice (79%; 69%) and expert (72%; 61%) sonographers (p = 0.39, p = 0.55), suggesting reduced operator dependence. Reader diagnosis confidence and image quality varied widely. Image acquisition times were fast for novice (mean 225 ± 83 s) and expert (201 ± 51) sonographers, with fast expert reader interpretation times (225 ± 136). CONCLUSIONS:A 3DUS-enabling device demonstrates a sensitivity of 0.81 and specificity of 0.73 for disease detection, fast image acquisition, and may reduce operator dependence for detecting emergent retinal pathologies. Further technological development is needed to improve diagnostic accuracy in identifying and characterizing retinal pathology.

Operator dependence limits the reliability and accuracy of two-dimensional (2D) ultrasound (US) for nonexpert users, including emergency physicians. Methods to reduce operator dependence could improve the utility of point-of-care (POC) US for many emergency applications. Three-dimensional (3D) US techniques might achieve this goal, as they do not require operator skill and knowledge to identify and record specific planes of interest during image acquisition, which is the case for 2DUS. Instead, like computed tomography (CT), 3DUS allows reconstruction of any desired plane within the field of view (FOV) after standardized acquisition of a region of interest, and without the operator recognizing specific anatomy and selecting desired planes at the time of acquisition. Integrated 3DUS systems with specialized 3D transducers are costly and lack a portable form factor, but low-cost 3DUS by augmentation of 2DUS is an alternative. In these augmentation systems, an inexpensive orientation device is mounted externally on any 2DUS transducer, allowing use of existing, portable point-of-care equipment. 2D source images plus orientation data allow reconstruction of 3DUS volumes.1, 2 Reliability of novice-acquired 3DUS using low-cost 2D augmentation in obstetric evaluation is high, suggesting reduced operator dependence.3 We chose to explore novice 3DUS by low-cost 2D augmentation in carotid artery stenosis (CAS) for several reasons: data suggesting enhanced reliability with 3DUS,4 our experience with cervical vascular 3DUS using an augmentation system,5 and the clinical importance of carotid disease and stroke. Improving the diagnosis of carotid stenosis could have wide benefits, as atherosclerotic carotid stenosis is responsible for 15% to 20% of ischemic strokes, accounting for 80% of all strokes.6, 7 Stroke was the second leading cause of death worldwide in 2019, and survivors face significant morbidity.8 Early detection and management of CAS are important, as about 16% of patients with asymptomatic stenosis of greater than 60% will experience a stroke within 5 years.7 In the emergency department (ED), detection of CAS could impact management of patients who present with acute stroke-like symptoms. The purpose of our study was to evaluate the diagnostic accuracy and interreader reliability of novice-acquired POC 3DUS for evaluation of extracranial CAS. We hypothesized that accuracy measures would not differ between novice-acquired carotid 3DUS and cervical CT angiography (CTA), a common clinical reference standard in our ED. Institutional review board approval was obtained for this HIPAA-compliant study. Each subject or their legal guardian provided written informed consent prior to enrollment. We adhered to the Standards for Reporting of Diagnostic Accuracy Studies (STARD). Between December 2019 and March 2020, a prospective convenience sample of patients with concurrent cervical CTA was enrolled in the ED or following admission to the stroke service. A novice (third-year medical student, <10 hours of US training) acquired carotid 3DUS using a 2DUS linear probe and external orientation sensor, mounted in a 3D-printed guide (Data Supplement S1, Figures S1–S3, available as supporting information in the online version of this paper, which is available at http://onlinelibrary.wiley.com/doi/10.1111/acem.14320/full), as previously described.1, 2, 5 After brief training on the research system and location of the extracranial carotid artery and bifurcation, the user captured the region of interest by fanning the sensor-equipped transducer across the lateral neck over a period of approximately 15 seconds, with no effort made to record a specific image plane or structure. Both carotid arteries were scanned in all participants, with most arteries scanned two or more times in the same session to ensure capture of an adequate FOV. Total acquisition time (3DUS) and interpretation times (3DUS, CTA) were recorded. 3DUS were displayed in cardinal plane stacks for blinded interpretation by board-certified radiologists (5 and 7 years of US experience) and US fellowship–trained emergency medicine physicians (4, 5, and 8 years of US experience). CTAs were displayed for blinded interpretation by board-certified neuroradiologists (5 and 13 years of CTA experience). Readers of both modalities evaluated for any extracranial CAS. 3DUS sensitivity, specificity, positive likelihood ratio (+LR), and negative likelihood ratio (–LR) were calculated on a per-vessel basis with the most experienced CTA reader interpretation as the reference standard. The disease-positive state was defined as the presence of any stenosis. For an individual 3DUS reader, when more than one image volume representing a vessel was interpreted, the vessel was considered positive for stenosis if any volume was read as positive. Recognizing that CTA is an imperfect clinical reference standard,9 we also compared interreader agreement for 3DUS versus 3DUS, 3DUS versus CTA, and CTA versus CTA. To evaluate interreader agreement outside of chance, we used Cohen's kappa (κ). Interpretation was based on an established agreement scale (≤0.00 no agreement, 0.01–0.20 slight, 0.21–0.40 fair, 0.41–0.60 moderate, 0.61–0.80 substantial, 0.81–1.00 almost perfect). We prospectively determined a sample size of 100 vessels for kappa of 0.8, alpha of 0.05, and power of 0.8. Due to COVID-19, enrollment was halted at 60 vessels (144 3DUS, 32 CTAs). Median time between CTA and 3DUS was 1 day (IQR = 0–5). Subject demographics are presented in the Table S1. A total of 116 CTA interpretations, 288 3DUS interpretations by radiologists, and 160 3DUS interpretations by US-trained emergency physicians were analyzed. Radiologist 3DUS interpretations had per-vessel sensitivity of 0.84 (95% CI = 0.73 to 0.93), specificity of 0.59 (95% CI = 0.45 to 0.72), +LR of 2.07 (95% CI = 1.48 to 2.91), and –LR 0.26 (95% CI = 0.14 to 0.50). US-trained emergency physician 3DUS readers had sensitivity of 0.67 (95% CI = 0.50 to 0.80), specificity of 0.85 (95% CI = 0.71 to 0.94), +LR 4.56 (95% CI = 2.11 to 9.84), and –LR 0.39 (95% CI = 0.25 to 0.61). Interreader agreement values were as follows: 3DUSradiologists κ = 0.39 (95% CI = 0.19 to 0.60 [fair]), 3DUSemergency physicians κ = 0.42 (95% CI = 0.09 to 0.75 [moderate]), CTA-3DUSradiologists κ = 0.41 (95% CI = 0.29 to 0.54 [moderate]), CTA-3DUSemergency physicians κ = 0.49 (95% CI = 0.35 to 0.62 [moderate]), and CTA κ = 0.81 (95% CI = 0.67 to 0.96 [almost perfect]). Median total 3DUS acquisition time per subject was 8.8 minutes (IQR = 7.2–10.5 minutes). Median 3DUS interpretation time (one vessel) was 2.9 minutes (IQR = 2.0–4.6 minutes) versus median CTA interpretation time (two vessels) 4.6 minutes (IQR = 2.4–7.0 minutes). Readers rated best image quality as “excellent/5” or “good/4” on a 5-point scale, citing inadequate FOV and poor image quality as the most common limitations. Selected image examples are shown in Figure 1 (additional content is available online in Data Supplement S1). In this convenience sample, novice-acquired POC 3DUS showed fair to moderate diagnostic accuracy and reliability when interpreted by radiologists and US-trained emergency physicians. Additional improvements would be required for application of this technique to patient care. For the purposes of this study, we treated CTA as the reference standard, with an assumed sensitivity and specificity of 100%. However, a meta-analysis found that on a per-patient basis, CTA is only 77% sensitive for diagnosing 70% to 99% carotid stenosis, compared to 89% sensitivity for 2D-Doppler US (DUS). When assessing for moderate stenosis (50%–69%), sensitivities of both CTA (36%) and 2D-DUS (67%) are even lower.9 A recent study of 2D POCUS by emergency physicians found similar sensitivity (70.0%, 95% CI = 34.8% to 93.3%), specificity (86.7%, 95% CI = 75.4% to 94.1%), LRs (+LR = 5.3, 95% CI = 1.2–9.3; –LR = 0.4, 95% CI = 0.0 to 0.7), and agreement (κ = 0.68, 95% CI = 0.46 to 0.90). That study used a higher threshold for disease (≥50% stenosis based on CTA vs. any stenosis in ours).10 Direct comparison using a threshold of ≥50% was not possible in our study due to the low number of patients (10%) and vessels (5%) with high-grade extracranial CAS. Whereas our study examined presence or absence of CAS, future studies should evaluate diagnosis of >50% or >70%, common thresholds in prior research.9, 10 Our study evaluated the performance of novice-acquired 3DUS. Previous 2DUS studies used more highly trained operators (e.g., sonographers, emergency medicine residents, and emergency physicians with fellowship training in POCUS).9, 10 Given these differences, the performance of the novice sonographer in our study is promising. Future studies should include a larger number of operators of varying experience to allow interoperator comparison of the acquisition phase of imaging. The low cost and portability of 3DUS by 2D augmentation make it suitable for use in low-resource environments. Compatibility with existing 2D systems could reduce cost and training compared to dedicated 3DUS systems used in previous studies. In an ED setting, 3D POCUS could allow for improved speed in diagnosis of CAS or other conditions by eliminating the need for patient transport to and from the radiology suite. The brief acquisition and interpretation times suggest feasibility in the ED. While we did not achieve the hypothesized diagnostic performance, studying a higher-risk population might reveal benefits. Future studies should train operators to systematically acquire volumes spanning from skull base to base of the neck to improve FOV. Additional training for readers may also improve performance. 3DUS volumes allow depiction of any desired plane, enabling orientation of images to match the path of a blood vessel (Videos S1 and S2). Focusing on abbreviated training for readers, we did not apply this feature, which might aid more accurate interpretation of vessel cross-sections and stenosis in subsequent research. Improvements in acquisition and reconstruction are also possible, including roll-and-yaw correction using existing sensor data. Our study may have failed to detect benefits of 3DUS because the diagnostic challenge did not leverage the potential of the technology. 3D images provide orientation and context and allow accurate and precise measurement of volume.2 Future studies could focus on clinical targets requiring these capabilities, such as procedure guidance or monitoring volume of traumatic parenchymal hematoma. Elena Drews participated in design of the study and data collection and analysis and drafted the manuscript; Benjamin Wildman-Tobriner participated in study design and ultrasound interpretations; Amanda Mathews was involved with data collection and ultrasound interpretations; Joao Ricardo N. Vissoci was involved with study design and statistical analysis; Kevin Kalisz was involved with ultrasound interpretations; Timothy J. Amrhein and Walter Wiggins participated in computed tomography interpretations; Nada El Husseini contributed to study design and participant recruitment; Jacob Nast, Brandon T. Ruderman, Rebecca Theophanous, and Erica Peethumnongsin participated in ultrasound interpretations; Nayara Fernandes participated in statistical analysis; Joshua S. Broder was involved in study design and oversight and drafted the manuscript; all authors read and approved the final manuscript. Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.

The pigmentation mutation speck is a commonly used recombination marker characterized by a darkly pigmented region at the wing hinge. Identified in 1910 by Thomas Hunt Morgan, speck was characterized by Sturtevant as the most 'workable' mutant in the rightmost region of the second chromosome and eventually localized to 2-107.0 and 60C1-2. Though the first speck mutation was isolated over 115 years ago, speck is still not associated with any gene. Here, as part of an undergraduate-led research effort, we show that speck is encoded by the Arylalkylamine N-acetyltransferase 1 (AANAT1) gene. Both alleles from the Morgan lab contain a retrotransposon in exon 1 of the RB transcript of the AANAT1 gene. We have also identified a new insertion allele and generated multiple deletion alleles in AANAT1 that all give a strong speck phenotype. In addition, expression of AANAT1 RNAi constructs either ubiquitously or in the dorsal portion of the developing wing generates a similar speck phenotype. We find that speck alleles have additional phenotypes, including ectopic pigmentation in the posterior pupal case, leg joints, cuticular sutures and overall body color. We propose that the acetylated dopamine generated by AANAT1 decreases the dopamine pool available for melanin production. When AANAT1 function is decreased, the excess dopamine enters the melanin pathway to generate the speck phenotype.

History of present illness: A 64-year-old woman with a remote history of breast cancer presented to the emergency department with one day of worsening right-sided weakness and headaches. She had been seen two weeks prior for similar symptoms and underwent unenhanced brain computed tomography (CT), which revealed a left parietal mass suspected to represent new metastatic disease. The patient presented on this visit not only with weakness of her right upper and lower extremity, but was found to be febrile, tachycardic and exhibiting lethargy. Peripheral white blood cell count (WBC) was 27.1 (x109/L). Given her rapidly declining clinical course and expected time to complete magnetic resonance imaging (MRI) of the brain, the decision was made to first perform an emergent contrast-enhanced brain CT to further evaluate the mass. The contrast-enhanced brain CT revealed a large rim-enhancing left parietal lesion (Figures 1 and 2). Significant findings: A non-contrast CT (Figure 1) revealed a large hypoattenuating left parietal lesion. When the CT was enhanced with intravenous contrast (Figure 2), the same lesion showed peripheral rim enhancement, suggestive of a brain abscess. Discussion: Intracranial abscess is a rare yet potentially devastating disease process that can often be difficult to diagnosis, with an incidence of about 0.4-0.9 cases per 100,000 people.1 Mortality rates are about 15% within the past decade, but can increase up to 85% if the abscess ruptures. The most common mechanisms for formation of intracranial abscesses include direct spread from local infections (odontogenic infections or sinusitis), hematogenous spread (congenital heart disease, endocarditis, dental procedures, or intrapulmonary shunting), or penetrating trauma.2 Intracranial abscesses are more likely to occur in severely immunosuppressed patients, after neurosurgical procedures, or in head trauma. About 32%-60% of brain abscesses are polymicrobial, but Viridans streptococci, a group of aerobic gram-positive cocci, is one of the most commonly isolated microorganisms, especially from intraoral sources.3,4 The most common presenting symptom in patients is a headache, and the classic triad of headache, fever, and focal neurologic deficits is present in only about 20% of cases.5 Neurologic findings often take days to weeks to manifest and will vary based on the location of the abscess. Up to 25% of patients will also present with seizures.1 Classic CT features favoring an abscess include a continuous thin rim of peripheral enhancement and thinning along the medial wall.6 However, these are nonspecific and frequently difficult to differentiate from high-grade neoplasms, metastases, infarcts, or hematomas. MRI with gadolinium is therefore significantly more sensitive and specific for diagnosing and differentiating a brain abscess from primary or metastatic cancer. Prospective studies report a sensitivity and specificity of about 96% for MRI when combined with diffusion-weighted imaging.1,5 In this case, the patient underwent operative stereotactic drainage of the lesion. A biopsy was negative for malignancy, but bacterial cultures grew Viridans streptococci. Additional imaging of the chest revealed a large pulmonary arteriovenous malformation as the suspected etiology for the abscess. The patient was treated with intravenous vancomycin and ceftriaxone and ultimately discharged to rehabilitation for physical therapy.

Many essential aspects of genome function, including gene expression and chromosome segregation, are mediated throughout development and differentiation by changes in the chromatin state. Along with genomic signals encoded in the DNA, epigenetic processes regulate heritable gene expression patterns. Genomic signals such as enhancers, silencers, and repetitive DNA, while required for the establishment of alternative chromatin states, have an unclear role in epigenetic processes that underlie the persistence of chromatin states throughout development. Here, we demonstrate in fission yeast that the maintenance and inheritance of ectopic heterochromatin domains are independent of the genomic sequences necessary for their de novo establishment. We find that both structural heterochromatin and gene silencing can be stably maintained over an ~10-kb domain for up to hundreds of cell divisions in the absence of genomic sequences required for heterochromatin establishment, demonstrating the long-term persistence and stability of this chromatin state. The de novo heterochromatin, despite the absence of nucleation sequences, is also stably inherited through meiosis. Together, these studies provide evidence for chromatin-dependent, epigenetic control of gene silencing that is heritable, stable, and self-sustaining, even in the absence of the originating genomic signals.