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Background: Opioid overdose deaths have disproportionately impacted Black and Hispanic populations, in part due to disparities in treatment access. Emergency departments (EDs) serve as a resource for patients with opioid use disorder (OUD), many of whom have difficulty accessing outpatient addiction programs. However, inequities in ED treatment for OUD remain poorly understood., Methods: This secondary analysis examined racial and ethnic differences in buprenorphine access using data from EMBED, a study of 21 EDs across five health care systems evaluating a clinical decision support system for initiating ED buprenorphine. The primary outcome was receipt of buprenorphine, ED administered or prescribed. Hospital type (academic vs. community) was evaluated as an effect modifier. Hierarchical models with cluster effects for site and clinician were used to assess buprenorphine receipt by race and ethnicity., Results: Black patients were less likely to receive buprenorphine (6.4% [51/801] vs. White patients 8.5% [268/3154], odds ratio [OR] 0.59, 95% confidence interval [CI] 0.45-0.78). This association persisted after adjusting for age, insurance, gender, clinician X-waiver, hospital type, and urbanicity (adjusted OR [aOR] 0.64, 95% CI 0.48-0.84) but not when discharge diagnosis was included (aOR 0.75, 95% CI 0.56-1.02). Hispanic patients were more likely to receive buprenorphine (14.8% [122/822] vs. non-Hispanic patients, 11.6% [475/4098]) in unadjusted (OR 1.57, 95% CI 1.09-1.83) and adjusted models (aOR 1.41, 95% CI 1.08-1.83) but not including discharge diagnosis (aOR 1.32, 95% CI 0.99-1.77). Odds of buprenorphine were similar in academic and community EDs by race (interaction p = 0.97) and ethnicity (interaction p = 0.64)., Conclusions: Black patients with OUD were less likely to receive buprenorphine whereas Hispanic patients were more likely to receive buprenorphine in academic and community EDs. Differences were attenuated with discharge diagnosis, as fewer Black and non-Hispanic patients were diagnosed with opioid withdrawal. Barriers to medication treatment are heterogenous among patients with OUD; research must continue to address the multiple drivers of health inequities at the patient, clinician, and community level., (© 2023 The Authors. Academic Emergency Medicine published by Wiley Periodicals LLC on behalf of Society for Academic Emergency Medicine.)

Background: Untreated opioid use disorder (OUD) is a significant public health problem. Buprenorphine is an evidence-based treatment for OUD that can be initiated in and prescribed from emergency departments (EDs) and office settings. Adoption of buprenorphine initiation among ED clinicians is low. The EMBED pragmatic clinical trial investigated the effectiveness of a clinical decision support (CDS) tool to promote ED clinicians' behavior related to buprenorphine initiation in the ED. While the CDS intervention was not associated with increased rates of buprenorphine treatment for patients with OUD at intervention ED sites, attending physicians at intervention EDs were more likely to initiate buprenorphine at least once over the duration of the study compared to those in the usual care arms (44.4% vs 34.0%, P = 0.01). This suggests the CDS intervention may be associated with increased adoption of buprenorphine initiation. As a secondary aim, we sought to identify the determinants of CDS adoption, implementation, and maintenance in a variety of ED settings and geographic locations., Methods: We purposively sampled and conducted semi-structured, in-depth interviews with clinicians across EMBED trial sites randomized to the intervention arm from five healthcare systems. Interviews elicited clinician experiences regarding buprenorphine initiation and CDS use. Interviews were analyzed using directed content analysis informed by the Practical, Robust Implementation and Sustainability Model (PRISM). We used a hybrid approach (a priori codes informed by PRISM and emergent codes) for codebook development. ATLAS.ti (version 9.0) was used for data management. Coded data were analyzed within individual interview transcripts and across all interviews to identify major themes. This process involved (1) combining, comparing, and making connections between codes; (2) writing analytic memos about observed patterns; and (3) frequent team meetings to discuss emerging patterns., Results: Twenty-eight interviews were conducted. Major themes that influenced the successful adoption, implementation, and maintenance of the EMBED intervention and ED-initiated BUP were organizational culture and commitment, clinician training and support, the ability to connect patients to ongoing treatment, and the ability to tailor implementation to each ED. These findings informed the identification of implementation strategies (framed using PRISM domains) to enhance the ED initiation of buprenorphine., Conclusion: The findings from this qualitative analysis can provide guidance to build better systems to promote the adoption of ED-initiated buprenorphine., (© 2023. The Author(s).)

The harmful algal bloom species, Karenia brevis , forms annual, often intense blooms in the Gulf of Mexico, particularly along the west Florida shelf. Though the ability of K. brevis blooms to cause mass mortalities in juvenile fish are well documented, the direct effect of bloom concentrations on larval fish has not been studied extensively. To better understand the potential effect of K. brevis on larval fish survival, laboratory spawned red porgy ( Pagrus pagrus ) larvae from 4-26 days post-hatch were exposed to concentrations of K. brevis observed in the field for either 24 or 48 h. This species is representative of fish which spawn in regions of the Gulf of Mexico and whose larvae are epipelagic and may encounter K. brevis blooms. In this study, three different K. brevis strains varying in the amount of brevetoxin produced were tested. Larval survivorship was found to be inversely proportional to the amount of brevetoxin produced by each strain. The EC 50 value from the combined 24 h experiments was ~163,000 K. brevis cells L - 1 , which corresponds to cell concentrations found in moderately dense blooms. Larval mortality also increased substantially in the 48 h versus 24 h exposure treatments. These findings indicate K. brevis blooms have the potential to contribute to natural mortality of fish larvae and further reduce inter-annual recruitment of fishery species whose stocks in the Gulf of Mexico may already be depleted.

Gambierdiscus and Fukuyoa dinoflagellates produce a suite of secondary metabolites, including ciguatoxins (CTXs), which bioaccumulate and are further biotransformed in fish and marine invertebrates, causing ciguatera poisoning when consumed by humans. This study is the first to compare the performance of the fluorescent receptor binding assay (fRBA), neuroblastoma cell-based assay (CBA-N2a), and liquid chromatography tandem mass spectrometry (LC-MS/MS) for the quantitative estimation of CTX contents in 30 samples, obtained from four French Polynesian strains of Gambierdiscus polynesiensis . fRBA was applied to Gambierdiscus matrix for the first time, and several parameters of the fRBA protocol were refined. Following liquid/liquid partitioning to separate CTXs from other algal compounds, the variability of CTX contents was estimated using these three methods in three independent experiments. All three assays were significantly correlated with each other, with the highest correlation coefficient ( r 2 = 0.841) found between fRBA and LC-MS/MS. The CBA-N2a was more sensitive than LC-MS/MS and fRBA, with all assays showing good repeatability. The combined use of fRBA and/or CBA-N2a for screening purposes and LC-MS/MS for confirmation purposes allows for efficient CTX evaluation in Gambierdiscus . These findings, which support future collaborative studies for the inter-laboratory validation of CTX detection methods, will help improve ciguatera risk assessment and management.

Dinoflagellate species are traditionally defined using morphological characters, but molecular evidence accumulated over the past several decades indicates many morphologically-based descriptions are inaccurate. This recognition led to an increasing reliance on DNA sequence data, particularly rDNA gene segments, in defining species. The validity of this approach assumes the divergence in rDNA or other selected genes parallels speciation events. Another concern is whether single gene rDNA phylogenies by themselves are adequate for delineating species or if multigene phylogenies are required instead. Currently, few studies have directly assessed the relative utility of multigene versus rDNA-based phylogenies for distinguishing species. To address this, the current study examined D1-D3 and ITS/5.8S rDNA gene regions, a multi-gene phylogeny, and morphological characters in Gambierdiscus and other related dinoflagellate genera to determine if they produce congruent phylogenies and identify the same species. Data for the analyses were obtained from previous sequencing efforts and publicly available dinoflagellate transcriptomic libraries as well from the additional nine well-characterized Gambierdiscus species transcriptomic libraries generated in this study. The D1-D3 and ITS/5.8S phylogenies successfully identified the described Gambierdiscus and Alexandrium species. Additionally, the data showed that the D1-D3 and multigene phylogenies were equally capable of identifying the same species. The multigene phylogenies, however, showed different relationships among species and are likely to prove more accurate at determining phylogenetic relationships above the species level. These data indicated that D1-D3 and ITS/5.8S rDNA region phylogenies are generally successful for identifying species of Gambierdiscus, and likely those of other dinoflagellates. To assess how broadly general this finding is likely to be, rDNA molecular phylogenies from over 473 manuscripts representing 232 genera and 863 described species of dinoflagellates were reviewed. Results showed the D1-D3 rDNA and ITS phylogenies in combination are capable of identifying 97% of dinoflagellate species including all the species belonging to the genera Alexandrium, Ostreopsis and Gambierdiscus, although it should be noted that multi-gene phylogenies are preferred for inferring relationships among these species. A protocol is presented for determining when D1-D3, confirmed by ITS/5.8S rDNA sequence data, would take precedence over morphological features when describing new dinoflagellate species. This protocol addresses situations such as: a) when a new species is both morphologically and molecularly distinct from other known species; b) when a new species and closely related species are morphologically indistinguishable, but genetically distinct; and c) how to handle potentially cryptic species and cases where morphotypes are clearly distinct but have the same rDNA sequence. The protocol also addresses other molecular, morphological, and genetic approaches required to resolve species boundaries in the small minority of species where the D1-D3/ITS region phylogenies fail., Competing Interests: The authors have read the journal’s policy and have the following competing interests: R. Wayne Litaker is a paid contractor for the National Oceanic and Atmospheric Administration (NOAA).

Ciguatera poisoning is caused by the ingestion of fish or shellfish contaminated with ciguatoxins produced by dinoflagellate species belonging to the genera Gambierdiscus and Fukuyoa . Unlike in the Pacific region, the species producing ciguatoxins in the Atlantic Ocean have yet to be definitely identified, though some ciguatoxins responsible for ciguatera have been reported from fish. Previous studies investigating the ciguatoxin-like toxicity of Atlantic Gambierdiscus species using Neuro2a cell-based assay identified G. excentricus as a potential toxin producer. To more rigorously characterize the toxin profile produced by this species, a purified extract from 124 million cells was prepared and partial characterization by high-resolution mass spectrometry was performed. The analysis revealed two new analogs of the polyether gambierone: sulfo-gambierone and dihydro-sulfo-gambierone. Algal ciguatoxins were not identified. The very low ciguatoxin-like toxicity of the two new analogs obtained by the Neuro2a cell-based assay suggests they are not responsible for the relatively high toxicity previously observed when using fractionated G. excentricus extracts, and are unlikely the cause of ciguatera in the region. These compounds, however, can be useful as biomarkers of the presence of G. excentricus due to their sensitive detection by mass spectrometry.

Objectives: Adoption of emergency department (ED) initiation of buprenorphine (BUP) for opioid use disorder (OUD) into routine emergency care has been slow, partly due to clinicians' unfamiliarity with this practice and perceptions that it is complicated and time-consuming. To address these barriers and guide emergency clinicians through the process of BUP initiation, we implemented a user-centered computerized clinical decision support system (CDS). This study was conducted to assess the feasibility of implementation and to evaluate the preliminary efficacy of the intervention to increase the rate of ED-initiated BUP., Methods: An interrupted time series study was conducted in an urban, academic ED from April 2018 to February 2019 (preimplementation phase), March 2019 to August 2019 (implementation phase), and September 2019 to December 2019 (maintenance phase) to study the effect of the intervention on adult ED patients identified by a validated electronic health record (EHR)-based computable phenotype consisting of structured data consistent with potential cases of OUD who would benefit from BUP treatment. The intervention offers flexible CDS for identification of OUD, assessment of opioid withdrawal, and motivation of readiness to start treatment and automates EHR activities related to ED initiation of BUP (including documentation, orders, prescribing, and referral). The primary outcome was the rate of ED-initiated BUP. Secondary outcomes were launch of the intervention, prescription for naloxone at ED discharge, and referral for ongoing addiction treatment., Results: Of the 141,041 unique patients presenting to the ED over the preimplementation and implementation phases (i.e., the phases used in primary analysis), 906 (574 preimplementation and 332 implementation) met OUD phenotype and inclusion criteria. The rate of BUP initiation increased from 3.5% (20/574) in the preimplementation phase to 6.6% (22/332) in the implementation phase (p = 0.03). After the temporal trend of the number of physician's with X-waiver training and other covariates were adjusted for, the relative risk of BUP initiation rate was 2.73 (95% confidence interval [CI] = 0.62 to 12.0, p = 0.18). Similarly, the number of unique attendings who initiated BUP increased modestly 7/53 (13.0%) to 13/57 (22.8%, p = 0.10) after offering just-in-time training during the implementation period. The rate of naloxone prescribed at discharge also increased (6.5% preimplementation and 11.5% implementation; p < 0.01). The intervention received a system usability scale score of 82.0 (95% CI = 76.7 to 87.2)., Conclusion: Implementation of user-centered CDS at a single ED was feasible, acceptable, and associated with increased rates of ED-initiated BUP and naloxone prescribing in patients with OUD and a doubling of the number of unique physicians adopting the practice. We have implemented this intervention across several health systems in an ongoing trial to assess its effectiveness, scalability, and generalizability., (© 2020 The Authors. Academic Emergency Medicine published by Wiley Periodicals LLC on behalf of Society for Academic Emergency Medicine (SAEM).)

Elevated seawater temperatures are linked to the development of harmful algal blooms (HABs), which pose a growing threat to marine birds and other wildlife. During late 2015 and early 2016, a massive die-off of Common Murres (Uria aalge; hereafter, murres) was observed in the Gulf of Alaska coincident with a strong marine heat wave. Previous studies have documented illness and death among seabirds resulting from exposure to the HAB neurotoxins saxitoxin (STX) and domoic acid (DA). Given the unusual mortality event, corresponding warm water anomalies, and recent detection of STX and DA throughout coastal Alaskan waters, HABs were identified as a possible factor of concern. To evaluate whether algal toxins may have contributed to murre deaths, we tested for STX and DA in a suite of tissues obtained from beach-cast murre carcasses associated with the die-off as well as from apparently healthy murres and Black-legged Kittiwakes (Rissa tridactyla; hereafter, kittiwakes) sampled in the preceding and following summers. We also tested forage fish and marine invertebrates collected in the Gulf of Alaska in 2015-2017 to evaluate potential sources of HAB toxin exposure for seabirds. Saxitoxin was present in multiple tissue types of both die-off (36.4 %) and healthy (41.7 %) murres and healthy kittiwakes (54.2 %). Among birds, we detected the highest concentrations of STX in liver tissues (range 1.4-10.8 μg 100 g -1 ) of die-off murres. Saxitoxin was relatively common in forage fish (20.3 %) and invertebrates (53.8 %). No established toxicity limits currently exist for seabirds, but concentrations of STX in birds and forage fish in our study were lower than values reported from most other bird die-offs in which STX intoxication was causally linked. We detected low concentrations of DA in a single bird sample and in 33.3 % of invertebrates and 4.0 % of forage fish samples. Although these results do not support the hypothesis that acute exposure to STX or DA was a primary factor in the 2015-2016 mortality event, additional information about the sensitivity of murres to these toxins is needed before we can discount their potential role in the die-off. The widespread occurrence of STX in seabirds, forage fish, and invertebrates in the Gulf of Alaska indicates that algal toxins should be considered in future assessments of seabird health, especially given the potential for greater occurrence of HABs in the future., Competing Interests: Declaration of Competing Interest None., (Published by Elsevier B.V.)

Buprenorphine (BUP) can safely and effectively reduce craving, overdose, and mortality rates in people with opioid use disorder (OUD). However, adoption of ED-initiation of BUP has been slow partly due to physician perception this practice is too complex and disruptive. We report progress of the ongoing EMBED ( EMergency department-initiated BuprenorphinE for opioid use Disorder) project. This project is a five-year collaboration across five healthcare systems with the goal to develop, integrate, study, and disseminate user-centered Clinical Decision Support (CDS) to promote the adoption of Emergency Department (ED)-initiation of buprenorphine/naloxone (BUP) into routine emergency care. Soon to enter its third year, the project has already completed multiple milestones to achieve its goals including (1) user-centered design of the CDS prototype, (2) integration of the CDS into an automated electronic health record (EHR) workflow, (3) data coordination including derivation and validation of an EHR-based computable phenotype, (4) meeting all ethical and regulatory requirements to achieve a waiver of informed consent, (5) pilot testing of the intervention at a single site, and (6) launching a parallel group-randomized 18-month pragmatic trial in 20 EDs across 5 healthcare systems. Pilot testing of the intervention in a single ED was associated with increased rates of ED-initiated BUP and naloxone prescribing and a doubling of the number of unique physicians adopting the practice. The ongoing multi-center pragmatic trial will assess the intervention's effectiveness, scalability, and generalizability with a goal to shift the emergency care paradigm for OUD towards early identification and treatment., Trial Registration: Clinicaltrials.gov # NCT03658642., Competing Interests: CONFLICTS OF INTEREST The authors have no financial conflicts or competing interests to disclose.

Paralytic shellfish poisoning (PSP) is precipitated by a family of toxins produced by harmful algae, which are consumed by filter-feeding and commercially popular shellfish. The toxins, including saxitoxin, neosaxitoxin, and gonyautoxins, accumulate in shellfish and cause intoxication when consumed by humans and animals. Symptoms can range from minor neurological dysfunction to respiratory distress and death. There are over 40 different chemical congeners of saxitoxin and its analogs, many of which are toxic and many of which have low toxicity or are non-toxic. This makes accurate toxicity assessment difficult and complicates decisions regarding whether or not shellfish are safe to consume. In this study, we describe a new antibody-based bioassay that is able to detect toxic congeners (saxitoxin, neosaxitoxin, and gonyautoxins) with little cross-reactivity with the low or non-toxic congeners (decarbamoylated or di-sulfated forms). The anti-saxitoxin antibody used in this assay detects saxitoxin and neosaxitoxin, the two most toxic congers equally well, but not the relatively highly toxic gonyautoxins. By incorporating an incubation step with L-cysteine, it is possible to convert a majority of the gonyautoxins present to saxitoxin and neosaxitoxin, which are readily detected. The assay is, therefore, capable of detecting the most toxic PSP congeners found in commercially relevant shellfish. The assay was validated against samples whose toxicity was determined using standard HPLC methods and yielded a strong linear agreement between the methods, with R2 values of 0.94-0.96. As ELISAs are rapid, inexpensive, and easy-to-use, this new commercially available PSP ELISA represents an advance in technology allowing better safety management of the seafood supply and the ability to screen large numbers of samples that can occur when monitoring is increased substantially in response to toxic bloom events.

Blooms of the toxic microalga Karenia brevis occur seasonally in Florida, Texas and other portions of the Gulf of Mexico. Brevetoxins produced during Karenia blooms can cause neurotoxic shellfish poisoning in humans, massive fish kills, and the death of marine mammals and birds. Brevetoxin-containing aerosols are an additional problem, having a severe impact on beachgoers, triggering coughing, eye and throat irritation in healthy individuals, and more serious respiratory distress in those with asthma or other breathing disorders. The blooms and associated aerosol impacts are patchy in nature, often affecting one beach but having no impact on an adjacent beach. To provide timely information to visitors about which beaches are low-risk, we developed HABscope; a low cost (~$400) microscope system that can be used in the field by citizen scientists with cell phones to enumerate K. brevis cell concentrations in the water along each beach. The HABscope system operates by capturing short videos of collected water samples and uploading them to a central server for rapid enumeration of K. brevis cells using calibrated recognition software. The HABscope has a detection threshold of about 100,000 cells, which is the point when respiratory risk becomes evident. Higher concentrations are reliably estimated up to 10 million cells L-1. When deployed by volunteer citizen scientists, the HABscope consistently distinguished low, medium, and high concentrations of cells in the water. The volunteers were able to collect data on most days during a severe bloom. This indicates that the HABscope can provide an effective capability to significantly increase the sampling coverage during Karenia brevis blooms., Competing Interests: The authors have declared that no competing interests exist.

Lionfish, native to reef ecosystems of the tropical and sub-tropical Indo-Pacific, were introduced to Florida waters in the 1980s, and have spread rapidly throughout the northwestern Atlantic, Caribbean Sea and the Gulf of Mexico. These invasive, carnivorous fish significantly reduce other fish and benthic invertebrate biomass, fish recruitment, and species richness in reef ecosystems. Fisheries resource managers have proposed the establishment of a commercial fishery to reduce lionfish populations and mitigate adverse effects on reef communities. The potential for a commercial fishery for lionfish is the primary reason to identify locations where lionfish accumulate sufficient amounts of ciguatoxin (CTX) to cause ciguatera fish poisoning (CFP), the leading cause of non-bacterial seafood poisoning associated with fish consumption. To address this issue, an initial geographic assessment of CTX toxicity in lionfish from the Caribbean and Gulf of Mexico was conducted. Lionfish samples (n = 293) were collected by spearfishing from 13 locations (74 sampling sites) around the Caribbean and Gulf of Mexico between 2012 and 2015. The highest frequencies of lionfish containing measurable CTX occurred in areas known to be high-risk regions for CFP in the central to eastern Caribbean (e.g., 53% British Virgin Islands and 5% Florida Keys). Though measurable CTX was found in some locations, the majority of the samples (99.3%) contained CTX concentrations below the United States Food and Drug Administration guidance level of 0.1 ppb Caribbean ciguatoxin-1 (C-CTX-1) equivalents (eq.). Only 0.7% of lionfish tested contained more than 0.1 ppb C-CTX-1 eq. As of 2018, there has been one suspected case of CFP from eating lionfish. Given this finding, current risk reduction techniques used to manage CTX accumulating fish are discussed., Competing Interests: The Ocean Tester commercial affiliation did not alter our adherence to PLOS ONE policies on sharing data and materials as detailed in the online guide for authors http://journals.plos.org/plosone/s/competing-interests). Patricia Tester was a former NOAA employee who participated in the project before leaving NOAA and founding the Ocean Tester. Ocean Tester did not impose any restrictions on sharing of data and/or materials. The views expressed in this article are those of the authors and do not necessarily reflect the official policy or position of the National Oceanic and Atmospheric Administration, Center for Coastal Fisheries and Habitat Research, nor the U.S. Government. D. Ransom Hardison, William C. Holland, Alex K. Bogdanoff, James A. Morris, Jr., Harold A. Flores Quintana, Christopher R. Loeffler, and R. Wayne Litaker are employees of the U.S. Government. This work was prepared as part of their official duties. Title 17, USC, §105 provides that 'Copyright protection under this title is not available for any work of the U.S. Government.' Title 17, USC, §101 defines a U.S. Government work as a work prepared by a military service member or employee of the U.S. Government as part of that person's official duties.

Dinoflagellate species belonging to the genera Gambierdiscus and Fukuyoa produce ciguatoxins (CTXs), potent neurotoxins that concentrate in fish causing ciguatera fish poisoning (CFP) in humans. While the structures and toxicities of ciguatoxins isolated from fish in the Pacific and Caribbean are known, there are few data on the variation in toxicity between and among species of Gambierdiscus and Fukuyoa. Quantifying the differences in species-specific toxicity is especially important to developing an effective cell-based risk assessment strategy for CFP. This study analyzed the ciguatoxicity of 33 strains representing seven Gambierdiscus and one Fukuyoa species using a cell based Neuro-2a cytotoxicity assay. All strains were isolated from either the Caribbean or Gulf of Mexico. The average toxicity of each species was inversely proportional to growth rate, suggesting an evolutionary trade-off between an investment in growth versus the production of defensive compounds. While there is 2- to 27-fold variation in toxicity within species, there was a 1740-fold difference between the least and most toxic species. Consequently, production of CTX or CTX-like compounds is more dependent on the species present than on the random occurrence of high or low toxicity strains. Seven of the eight species tested (G. belizeanus, G. caribaeus, G. carolinianus, G. carpenteri, Gambierdiscus ribotype 2, G. silvae and F. ruetzleri) exhibited low toxicities, ranging from 0 to 24.5 fg CTX3C equivalents cell-1, relative to G. excentricus, which had a toxicity of 469 fg CTX3C eq. cell-1. Isolates of G. excentricus from other regions have shown similarly high toxicities. If the hypothesis that G. excentricus is the primary source of ciguatoxins in the Atlantic is confirmed, it should be possible to identify areas where CFP risk is greatest by monitoring only G. excentricus abundance using species-specific molecular assays.

Maitotoxins (MTXs) are among the most potent toxins known. These toxins are produced by epi-benthic dinoflagellates of the genera Gambierdiscus and Fukuyoa and may play a role in causing the symptoms associated with Ciguatera Fish Poisoning. A recent survey revealed that, of the species tested, the newly described species from the Canary Islands, G. excentricus , is one of the most maitotoxic. The goal of the present study was to characterize MTX-related compounds produced by this species. Initially, lysates of cells from two Canary Island G. excentricus strains VGO791 and VGO792 were partially purified by (i) liquid-liquid partitioning between dichloromethane and aqueous methanol followed by (ii) size-exclusion chromatography. Fractions from chromatographic separation were screened for MTX toxicity using both the neuroblastoma neuro-2a (N2a) cytotoxicity and Ca 2+ flux functional assays. Fractions containing MTX activity were analyzed using liquid chromatography coupled to high-resolution mass spectrometry (LC-HRMS) to pinpoint potential MTX analogs. Subsequent non-targeted HRMS analysis permitted the identification of a novel MTX analog, maitotoxin-4 (MTX4, accurate mono-isotopic mass of 3292.4860 Da, as free acid form) in the most toxic fractions. HRMS/MS spectra of MTX4 as well as of MTX are presented. In addition, crude methanolic extracts of five other strains of G. excentricus and 37 other strains representing one Fukuyoa species and ten species, one ribotype and one undetermined strain/species of Gambierdiscus were screened for the presence of MTXs using low resolution tandem mass spectrometry (LRMS/MS). This targeted analysis indicated the original maitotoxin (MTX) was only present in one strain ( G. australes S080911&#95;1). Putative maitotoxin-2 (p-MTX2) and maitotoxin-3 (p-MTX3) were identified in several other species, but confirmation was not possible because of the lack of reference material. Maitotoxin-4 was detected in all seven strains of G. excentricus examined, independently of their origin (Brazil, Canary Islands and Caribbean), and not detected in any other species. MTX4 may therefore serve as a biomarker for the highly toxic G. excentricus in the Atlantic area., Competing Interests: The authors declare no conflict of interest. The funding sponsors had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, and in the decision to publish the results.

Species in the epi-benthic dinoflagellate genus Gambierdiscus produce ciguatoxins (CTXs) and maitotoxins (MTXs), which are among the most potent marine toxins known. Consumption of fish contaminated with sufficient quantities of CTXs causes Ciguatera Fish Poisoning (CFP), the largest cause of non-bacterial food poisoning worldwide. Maitotoxins, which can be found in the digestive system of fish, could also contribute to CFP if such tissues are consumed. Recently, an increasing number of Gambierdiscus species have been identified; yet, little is known about the variation in toxicity among Gambierdiscus strains or species. This study is the first assessment of relative CTX- and MTX-toxicity of Gambierdiscus species from areas as widespread as the North-Eastern Atlantic Ocean, Pacific Ocean and the Mediterranean Sea. A total of 13 strains were screened: (i) seven Pacific strains of G. australes, G. balechii, G. caribaeus, G. carpenteri, G. pacificus, G. scabrosus and one strain of an undetermined species (Gambierdiscus sp. Viet Nam), (ii) five strains from the North-Eastern Atlantic Ocean (two G. australes, a single G. excentricus and two G. silvae strains), and (iii) one G. carolinianus strain from the Mediterranean Sea. Cell pellets of Gambierdiscus were extracted with methanol and the crude extracts partitioned into a CTX-containing dichloromethane fraction and a MTX-containing aqueous methanol fraction. CTX-toxicity was estimated using the neuro-2a cytoxicity assay, and MTX-toxicity via a human erythrocyte lysis assay. Different species were grouped into different ratios of CTX- and MTX-toxicity, however, the ratio was not related to the geographical origin of species (Atlantic, Mediterranean, Pacific). All strains showed MTX-toxicity, ranging from 1.5 to 86pg MTX equivalents (eq) cell -1 . All but one of the strains showed relatively low CTX-toxicity ranging from 0.6 to 50 fg CTX3C eq cell -1 . The exception was the highly toxic G. excentricus strain from the Canary Islands, which produced 1426 fg CTX3C eq cell -1 . As was true for CTX, the highest MTX-toxicity was also found in G. excentricus. Thus, the present study confirmed that at least one species from the Atlantic Ocean demonstrates similar toxicity as the most toxic strains from the Pacific, even if the metabolites in fish have so far been shown to be more toxic in the Pacific Ocean., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Objectives: To estimate the proportion of older adults in the emergency department (ED) who are willing and able to use a tablet computer to answer questions., Design: Prospective, ED-based cross-sectional study., Setting: Two U.S. academic EDs., Participants: Individuals aged 65 and older., Measurements: As part of screening for another study, potential study participants were asked whether they would be willing to use a tablet computer to answer eight questions instead of answering questions orally. A custom user interface optimized for older adults was used. Trained research assistants observed study participants as they used the tablets. Ability to use the tablet was assessed based on need for assistance and number of questions answered correctly., Results: Of 365 individuals approached, 248 (68%) were willing to answer screening questions, 121 of these (49%) were willing to use a tablet computer; of these, 91 (75%) were able to answer at least six questions correctly, and 35 (29%) did not require assistance. Only 14 (12%) were able to answer all eight questions correctly without assistance. Individuals aged 65 to 74 and those reporting use of a touchscreen device at least weekly were more likely to be willing and able to use the tablet computer. Of individuals with no or mild cognitive impairment, the percentage willing to use the tablet was 45%, and the percentage answering all questions correctly was 32%., Conclusion: Approximately half of this sample of older adults in the ED was willing to provide information using a tablet computer, but only a small minority of these were able to enter all information correctly without assistance. Tablet computers may provide an efficient means of collecting clinical information from some older adults in the ED, but at present, it will be ineffective for a significant portion of this population., (© 2016, Copyright the Authors Journal compilation © 2016, The American Geriatrics Society.)

Background: Ciguatera is a circumtropical disease produced by polyether sodium channel toxins (ciguatoxins) that enter the marine food chain and accumulate in otherwise edible fish. Ciguatoxins, as well as potent water-soluble polyethers known as maitotoxins, are produced by certain dinoflagellate species in the genus Gambierdiscus and Fukuyoa spp. in the Pacific but little is known of the potential of related Caribbean species to produce these toxins., Methods: We established a simplified procedure for extracting polyether toxins from Gambierdiscus and Fukuyoa spp. based on the ciguatoxin rapid extraction method (CREM). Fractionated extracts from identified Pacific and Caribbean isolates were analysed using a functional bioassay that recorded intracellular calcium changes (Ca2+) in response to sample addition in SH-SY5Y cells. Maitotoxin directly elevated Ca2+i, while low levels of ciguatoxin-like toxins were detected using veratridine to enhance responses., Results: We identified significant maitotoxin production in 11 of 12 isolates analysed, with 6 of 12 producing at least two forms of maitotoxin. In contrast, only 2 Caribbean isolates produced detectable levels of ciguatoxin-like activity despite a detection limit of >30 pM. Significant strain-dependent differences in the levels and types of ciguatoxins and maitotoxins produced by the same Gambierdiscus spp. were also identified., Conclusions: The ability to rapidly identify polyether toxins produced by Gambierdiscus spp. in culture has the potential to distinguish ciguatoxin-producing species prior to large-scale culture and in naturally occurring blooms of Gambierdiscus and Fukuyoa spp. Our results have implications for the evaluation of ciguatera risk associated with Gambierdiscus and related species.

In this study, inter- and intraspecific genetic diversity within the marine harmful dinoflagellate genus Coolia Meunier was evaluated using isolates obtained from the tropics to subtropics in both Pacific and Atlantic Ocean basins. The aim was to assess the phylogeographic history of the genus and to clarify the validity of established species including Coolia malayensis. Phylogenetic analysis of the D1-D2 LSU rDNA sequences identified six major lineages (L1-L6) corresponding to the morphospecies Coolia malayensis (L1), C. monotis (L2), C. santacroce (L3), C. palmyrensis (L4), C. tropicalis (L5), and C. canariensis (L6). A median joining network (MJN) of C. malayensis ITS2 rDNA sequences revealed a total of 16 haplotypes; however, no spatial genetic differentiation among populations was observed. These MJN results in conjunction with CBC analysis, rDNA phylogenies and geographical distribution analyses confirm C. malayensis as a distinct species which is globally distributed in the tropical to warm-temperate regions. A molecular clock analysis using ITS2 rDNA revealed the evolutionary history of Coolia dated back to the Mesozoic, and supports the hypothesis that historical vicariant events in the early Cenozoic drove the allopatric differentiation of C. malayensis and C. monotis., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Ciguatera fish poisoning is an illness suffered by > 50,000 people yearly after consumption of fish containing ciguatoxins (CTXs). One of the current methodologies to detect ciguatoxins in fish is a radiolabeled receptor binding assay (RBA(R)). However, the license requirements and regulations pertaining to radioisotope utilization can limit the applicability of the RBA(R) in certain labs. A fluorescence based receptor binding assay (RBA(F)) was developed to provide an alternative method of screening fish samples for CTXs in facilities not certified to use radioisotopes. The new assay is based on competition binding between CTXs and fluorescently labeled brevetoxin-2 (BODIPY®-PbTx-2) for voltage-gated sodium channel receptors at site 5 instead of a radiolabeled brevetoxin. Responses were linear in fish tissues spiked from 0.1 to 1.0 ppb with Pacific ciguatoxin-3C (P-CTX-3C) with a detection limit of 0.075 ppb. Carribean ciguatoxins were confirmed in Caribbean fish by LC-MS/MS analysis of the regional biomarker (C-CTX-1). Fish (N = 61) of six different species were screened using the RBA(F). Results for corresponding samples analyzed using the neuroblastoma cell-based assay (CBA-N2a) correlated well (R2 = 0.71) with those of the RBA(F), given the low levels of CTX present in positive fish. Data analyses also showed the resulting toxicity levels of P-CTX-3C equivalents determined by CBA-N2a were consistently lower than the RBA(F) affinities expressed as % binding equivalents, indicating that a given amount of toxin bound to the site 5 receptors translates into corresponding lower cytotoxicity. Consequently, the RBA(F), which takes approximately two hours to perform, provides a generous estimate relative to the widely used CBA-N2a which requires 2.5 days to complete. Other RBA(F) advantages include the long-term (> 5 years) stability of the BODIPY®-PbTx-2 and having similar results as the commonly used RBA(R). The RBA(F) is cost-effective, allows high sample throughput, and is well-suited for routine CTX monitoring programs.

Objectives: Musculoskeletal pain is a common reason for emergency department (ED) visit by older adults. Outpatient pain management following ED visits in this population is challenging as a result of contraindications to, and side effects from, available therapies. Shared decision-making (SDM) between patients and emergency physicians may improve patient experiences and health outcomes. Among older ED patients with acute musculoskeletal pain, we sought to characterize their desire for involvement in the selection of outpatient analgesics. We also sought to assess the impact of SDM on change in pain at 1 week, patient satisfaction, and side effects., Methods: This was a prospective study of adults aged 60 years and older presenting to the ED with acute musculoskeletal pain. Participants' desire to contribute to outpatient analgesic selection was assessed by phone within 24 hours of ED discharge using the Control Preferences Scale and categorized as active, collaborative, or passive. The extent to which SDM occurred in the ED was also assessed within 24 hours of discharge using the 9-item Shared Decision Making Questionnaire, and scores were subsequently grouped into tertiles of low, middle, and high SDM. The primary outcome was change in pain severity between the ED visit and 1 week. Secondary outcomes included satisfaction regarding the decision about how to treat pain at home, satisfaction with the pain medication itself, and side effects., Results: Desire of participants (N = 94) to contribute to the decision regarding selection of outpatient analgesics varied: 16% active (i.e., make the final decision themselves), 37% collaborative (i.e., share decision with provider), and 47% passive (i.e., let the doctor make the final decision). The percentage of patients who desired an active role in the decision was higher for patients who were college educated versus those who were not college educated (28% vs. 11%; difference 17%, 95% confidence interval [CI] = 0% to 35%), received care from a nurse practitioner versus a resident or an attending physician (32% vs. 9%; difference 23%, 95% CI = 4% to 42%), or received care from a female versus a male provider (24% vs. 5%; difference 19%, 95% = CI 5% to 32%). After potential confounders were adjusted for, the mean decrease in pain severity from the ED visit to 1-week follow-up was not significantly different across tertiles of SDM (p = 0.06). Higher SDM scores were associated with greater satisfaction with the discharge pain medications (p = 0.006). SDM was not associated with the class of analgesic received., Conclusions: In this sample of older adults with acute musculoskeletal pain, the reported desire of patients to contribute to decisions regarding analgesics varied based on both patient and provider characteristics. SDM was not significantly related to pain reduction in the first week or type of pain medication received, but was associated with greater patient satisfaction., (© 2016 by the Society for Academic Emergency Medicine.)

Background: Clinical trial registries can improve the validity of trial results by facilitating comparisons between prospectively planned and reported outcomes. Previous reports on the frequency of planned and reported outcome inconsistencies have reported widely discrepant results. It is unknown whether these discrepancies are due to differences between the included trials, or to methodological differences between studies. We aimed to systematically review the prevalence and nature of discrepancies between registered and published outcomes among clinical trials., Methods: We searched MEDLINE via PubMed, EMBASE, and CINAHL, and checked references of included publications to identify studies that compared trial outcomes as documented in a publicly accessible clinical trials registry with published trial outcomes. Two authors independently selected eligible studies and performed data extraction. We present summary data rather than pooled analyses owing to methodological heterogeneity among the included studies., Results: Twenty-seven studies were eligible for inclusion. The overall risk of bias among included studies was moderate to high. These studies assessed outcome agreement for a median of 65 individual trials (interquartile range [IQR] 25-110). The median proportion of trials with an identified discrepancy between the registered and published primary outcome was 31%; substantial variability in the prevalence of these primary outcome discrepancies was observed among the included studies (range 0% (0/66) to 100% (1/1), IQR 17-45%). We found less variability within the subset of studies that assessed the agreement between prospectively registered outcomes and published outcomes, among which the median observed discrepancy rate was 41% (range 30% (13/43) to 100% (1/1), IQR 33-48%). The nature of observed primary outcome discrepancies also varied substantially between included studies. Among the studies providing detailed descriptions of these outcome discrepancies, a median of 13 % of trials introduced a new, unregistered outcome in the published manuscript (IQR 5-16%)., Conclusions: Discrepancies between registered and published outcomes of clinical trials are common regardless of funding mechanism or the journals in which they are published. Consistent reporting of prospectively defined outcomes and consistent utilization of registry data during the peer review process may improve the validity of clinical trial publications.

Study Objective: We estimate the prevalence of malnutrition among older patients presenting to an emergency department (ED) in the southeastern United States and identify subgroups at increased risk., Methods: We conducted a cross-sectional study with random time block sampling of cognitively intact patients aged 65 years and older. Nutrition was assessed with the Mini Nutritional Assessment Short-Form (0 to 14 scale), with malnutrition defined as a score of 7 or less and at risk for malnutrition defined as a score of 8 to 11. The presence of depressive symptoms was defined as a Center for Epidemiological Studies Depression-10 score of 4 or more (0 to 10 scale)., Results: Among 138 older adults, 16% (95% confidence interval [CI] 11% to 23%) were malnourished and 60% (95% CI 52% to 68%) were either malnourished or at risk for malnutrition. Seventeen of the 22 malnourished patients (77%) denied previously receiving a diagnosis of malnutrition. The prevalence of malnutrition was not appreciably different between men and women, across levels of patient education, or between those living in urban and rural areas. However, the prevalence of malnutrition was higher among patients with depressive symptoms (52%), those residing in assisted living (44%), those with difficulty eating (38%), and those reporting difficulty buying groceries (33%)., Conclusion: Among a random sample of cognitively intact older ED patients, more than half were malnourished or at risk for malnutrition, and the majority of malnourished patients had not previously received a diagnosis. Higher rates of malnutrition among individuals with depression, difficulty eating, and difficulty buying groceries suggest the need to explore multifaceted interventions., (Copyright © 2014 American College of Emergency Physicians. Published by Elsevier Inc. All rights reserved.)

This study examined the toxicity of six Gambierdiscus species (Gambierdiscus belizeanus, Gambierdiscus caribaeus, Gambierdiscus carolinianus, Gambierdiscus carpenteri, Gambierdiscus ribotype 2 and Gambierdiscus ruetzleri) using a human erythrocyte lysis assay. In all, 56 isolates were tested. The results showed certain species were significantly more toxic than others. Depending on the species, hemolytic activity consistently increased by ∼7-40% from log phase growth to late log - early stationary growth phase and then declined in mid-stationary growth phase. Increasing growth temperatures from 20 to 31 °C for clones of G. caribaeus showed only a slight increase in hemolytic activity between 20 and 27 °C. Hemolytic activity in the G. carolinianus isolates from different regions grown over the same 20-31 °C range remained constant. These data suggest that growth temperature is not a significant factor in modulating the inter-isolate and interspecific differences in hemolytic activity. The hemolytic activity of various isolates measured repeatedly over a 2 year period remained constant, consistent with the hemolytic compounds being constitutively produced and under strong genetic control. Depending on species, greater than 60-90% of the total hemolytic activity was initially associated with the cell membranes but diffused into solution over a 24 h assay incubation period at 4 °C. These findings suggest that hemolytic compounds produced by Gambierdiscus isolates were held in membrane bound vesicles as reported for brevetoxins produced by Karenia brevis. Gambierdiscus isolates obtained from other parts of the world exhibited hemolytic activities comparable to those found in the Caribbean and Gulf of Mexico confirming the range of toxicities is similar among Gambierdiscus species worldwide. Experiments using specific inhibitors of the MTX pathway and purified MTX, Gambierdiscus whole cell extracts, and hydrophilic cell extracts containing MTX, were consistent with MTX as the primary hemolytic compound produced by Gambierdiscus species. While the results from inhibition studies require validation by LC-MS analysis, the available data strongly suggest differences in hemolytic activity observed in this study reflect maitotoxicity., (Published by Elsevier Ltd.)

Ciguatera fish poisoning (CFP) is a serious health problem in tropical regions and is caused by the bioaccumulation of lipophilic toxins produced by dinoflagellates in the genus Gambierdiscus. Gambierdiscus species are morphologically similar and are difficult to distinguish from one another even when using scanning electron microscopy. Improved identification and detection methods that are sensitive and rapid are needed to identify toxic species and investigate potential distribution and abundance patterns in relation to incidences of CFP. This study presents the first species-specific, semi-quantitative polymerase chain reaction (qPCR) assays that can be used to address these questions. These assays are specific for five Gambierdiscus species and one undescribed ribotype. The assays utilized a SYBR green format and targeted unique sequences found within the SSU, ITS, and the D1/D3 LSU ribosomal domains. Standard curves were constructed using known concentrations of cultured cells and 10-fold serial dilutions of rDNA PCR amplicons containing the target sequence for each specific assay. Assay sensitivity and accuracy were tested using DNA extracts purified from known concentrations of multiple Gambierdiscus species. The qPCR assays were used to assess Gambierdiscus species diversity and abundance in samples collected from nearshore areas adjacent to Ft. Pierce and Jupiter, Florida USA. The results indicated that the practical limit of detection for each assay was 10 cells per sample. Most interestingly, the qPCR analysis revealed that as many as four species of Gambierdiscus were present in a single macrophyte sample., (© 2012 Phycological Society of America.)

Dinoflagellates in the genus Gambierdiscus produce toxins that bioaccumulate in tropical and sub-tropical fishes causing ciguatera fish poisoning (CFP). Little is known about the diversity and distribution of Gambierdiscus species, the degree to which individual species vary in toxicity, and the role each plays in causing CFP. This paper presents the first global distribution of Gambierdiscus species. Phylogenetic analyses of the existing isolates indicate that five species are endemic to the Atlantic (including the Caribbean/West Indies and Gulf of Mexico), five are endemic to the tropical Pacific, and that two species, Gambierdiscus carpenteri and Gambierdiscus caribaeus are globally distributed. The differences in Gambierdiscus species composition in the Atlantic and Pacific correlated with structural differences in the ciguatoxins reported from Atlantic and Pacific fish. This correlation supports the hypothesis that Gambierdiscus species in each region produce different toxin suites. A literature survey indicated a >100-fold variation in toxicity among species compared with a 2 to 9-fold within species variation due to changing growth conditions. These observations suggest that CFP events are driven more by inherent differences in species toxicity than by environmental modulation. How variations in species toxicity may affect the development of an early warning system for CFP is discussed., (Published by Elsevier Ltd.)

Purpose: The purpose of this study was to determine the prevalence of lattice retinal degeneration (LRD) in a primary eye care population and to compare this prevalence to that of other studies reported from selected populations. In addition, the percentage of unilateral and bilateral cases was to be determined., Methods: A prospective study design was conceived to examine 600 consecutive patients presenting to the UABSO Primary Care Clinics. Each patient had been appointed for general eye examination. The following data were collected contemporaneously over six months from September 1993 to March 1994: demographics, medical and ocular history; refractive correction (calculated and recorded as spherical equivalent, SE); best-corrected visual acuity; and the presence and features of all ocular fundus findings, as evaluated through a dilated pupil., Results: Of the 600 patients examined, 31 (5.2%) had LRD. Subjects ranged in age from 14 to 78 years (mean, 37.4 +/- 17.9 yrs.). Seventeen (55%) of the patients were white and 14 (45%) were black; 21 patients (68%) were female and 14 (32%) were male. The mean refractive correction (SE) was -1.77 D (range, +2.25 to -8.00 D). The lesions were bilateral in 19 (61.3%) of the patients; unilateral in 12 (38.7%). LRD lesions were observed in the vertical meridian (within 30 degrees of 6 or 12 o'clock) in all patients., Conclusions: The prevalence of LRD in a primary eye care population is comparable to that reported from other, selected populations. The proportion of bilateral cases in our material was greater than that from some other studies. Another item of note from the present data was exclusively vertical geographic location. We attribute the greater prevalence of bilateral cases in this material to the nature of the eye care delivery system.

The confusion test examines ankle dorsiflexion in patients with cerebral palsy. Orthopedists have related this test to swing-phase activity of the tibialis anterior, and have used it as a prerequisite for tendon transfer. To determine the validity of this assumption, ankle dorsiflexion was tested in 47 normal children. Forty-seven percent had a positive, unresisted confusion test, and 97% had a positive, resisted confusion test. Twenty-three patients with cerebral palsy who had a positive confusion test underwent gait analysis. Tibialis anterior electromyographs showed wide variability. Sagittal-plane ankle-movement curves revealed five patterns. Thirty-three percent of the patients showed abnormal swing-phase dorsiflexion, and 61% had abnormal swing-phase plantar-flexion. We conclude that the confusion test evaluates a normal, patterned response, and is positive in most children with cerebral palsy. Although a positive confusion test shows that active ankle dorsiflexion is possible, it is not predictive of swing-phase ankle kinematics.