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Introduction: There is currently no protocol for the initiation of extra corporeal cardiopulmonary resuscitation (ECPR) in out of hospital cardiac arrest (OHCA) in Atlantic Canada. Advanced care paramedics (ACPs) perform advanced cardiac life support in the prehospital setting often completing the entire resuscitation on-scene. Implementation of ECPR will present a novel intervention that is only available at the receiving hospital, altering how ACPs manage selected patients. Our objective is to determine if an educational program can improve paramedic identification of ECPR candidates. Methods: An educational program was delivered to paramedics including a short seminar and pocket card coupled with simulations of OHCA cases. A before and after study design using a case-based survey was employed. Paramedics were scored on their ability to correctly identify OHCA patients who met the inclusion criteria for our ECPR protocol. Scores before and after the education delivery were compared using a two tailed t-test. A 6-month follow-up is planned to assess knowledge retention. Qualitative data was also collected from paramedics during simulation to help identify potential barriers to implementation of our protocol in the prehospital setting. Results: Nine advanced care paramedics participated in our educational program. Mean score pre-education was 9.7/16 (61.1%) compared to 14/16 (87.5%) after education delivery. The mean difference between groups was 4.22 (CI = 2.65-5.80, p = 0.0003). There was a significant improvement in the paramedics’ ability to correctly identify ECPR candidates after completing our educational program. Conclusion: Paramedic training through a didactic session coupled with a pocket card and simulation appears to be a feasible method of knowledge translation. 6-month retention data will help ensure knowledge retention is achieved. If successful, this pilot will be expanded to train all paramedics in our prehospital system as we seek to implement an ECPR protocol at our centre.
Innovation Concept: Free open access medical education (FOAM) is a quickly growing field. While there is an abundance of resources online, and on social media, the quality of those resources should always be questioned and reviewed. Furthermore, as medical learners progress in their training, they become lead consumers and producers of FOAM. Our educational innovation concept was the introduction of two FOAM streams into our residency program to assist learners to produce their own content with mentorship from our emergency medicine faculty. Methods: Medical students and residents training in the emergency department were encouraged to submit content to either our department website in the form of a clinical PEARL, or a research paper to the departmental Cureus online journal. All website content was reviewed by an attending physician and all Cureus content was submitted for further peer review and publication if approved. All published content was shared on social media through our department's Twitter account. A select number of residents were also mentored in reviewing and editing FOAM content and publishing it to our departmental website. Curriculum, Tool or Material: sjrhem.ca is the Saint John Regional Hospital Department of Emergency Medicine's website. A portion of the website is dedicated to posts arising from departmental rounds, case reviews as well as posts from learners in the form of clinical PEARLS. They are designed as succinct and informative clinical summaries and allow learners to share their content to a wider audience online. Cureus.com is an online journal of medical science, with a dedicated Dalhousie Emergency Medicine Channel. The editors are local emergency medicine faculty and senior residents, while reviewers are independent. In the last year, the clinical pearls received 5672 views, and the Cureus channel received 1143 content views. Conclusion: Feedback from learners regarding publication of their own FOAM has been positive and has allowed them to share their content to a much wider audience through our Departmental Website, Cureus Channel and Twitter stream. Furthermore, we are helping to prepare residents to produce their own high quality content, allowing our FOAM program to grow.
Introduction: Although use of point of care ultrasound (PoCUS) protocols for patients with undifferentiated hypotension in the Emergency Department (ED) is widespread, our previously reported SHoC-ED study showed no clear survival or length of stay benefit for patients assessed with PoCUS. In this analysis, we examine if the use of PoCUS changed fluid administration and rates of other emergency interventions between patients with different shock types. The primary comparison was between cardiogenic and non-cardiogenic shock types. Methods: A post-hoc analysis was completed on the database from an RCT of 273 patients who presented to the ED with undifferentiated hypotension (SBP <100 or shock index > 1) and who had been randomized to receive standard care with or without PoCUS in 6 centres in Canada and South Africa. PoCUS-trained physicians performed scans after initial assessment. Shock categories and diagnoses recorded at 60 minutes after ED presentation, were used to allocate patients into subcategories of shock for analysis of treatment. We analyzed actual care delivered including initial IV fluid bolus volumes (mL), rates of inotrope use and major procedures. Standard statistical tests were employed. Sample size was powered at 0.80 (α:0.05) for a moderate difference. Results: Although there were expected differences in the mean fluid bolus volume between patients with non-cardiogenic and cardiogenic shock, there was no difference in fluid bolus volume between the control and PoCUS groups (non-cardiogenic control 1878 mL (95% CI 1550 – 2206 mL) vs. non-cardiogenic PoCUS 1687 mL (1458 – 1916 mL); and cardiogenic control 768 mL (194 – 1341 mL) vs. cardiogenic PoCUS 981 mL (341 – 1620 mL). Likewise there were no differences in rates of inotrope administration, or major procedures for any of the subcategories of shock between the control group and PoCUS group patients. The most common subcategory of shock was distributive. Conclusion: Despite differences in care delivered by subcategory of shock, we did not find any significant difference in actual care delivered between patients who were examined using PoCUS and those who were not. This may help to explain the previously reported lack of outcome difference between groups.
Introduction: Point of care ultrasound has been reported to improve diagnosis in non-traumatic hypotensive ED patients. We compared diagnostic performance of physicians with and without PoCUS in undifferentiated hypotensive patients as part of an international prospective randomized controlled study. The primary outcome was diagnostic performance of PoCUS for cardiogenic vs. non-cardiogenic shock. Methods: SHoC-ED recruited hypotensive patients (SBP < 100 mmHg or shock index > 1) in 6 centres in Canada and South Africa. We describe previously unreported secondary outcomes relating to diagnostic accuracy. Patients were randomized to standard clinical assessment (No PoCUS) or PoCUS groups. PoCUS-trained physicians performed scans after initial assessment. Demographics, clinical details and findings were collected prospectively. Initial and secondary diagnoses including shock category were recorded at 0 and 60 minutes. Final diagnosis was determined by independent blinded chart review. Standard statistical tests were employed. Sample size was powered at 0.80 (α:0.05) for a moderate difference. Results: 273 patients were enrolled with follow-up for primary outcome completed for 270. Baseline demographics and perceived category of shock were similar between groups. 11% of patients were determined to have cardiogenic shock. PoCUS had a sensitivity of 80.0% (95% CI 54.8 to 93.0%), specificity 95.5% (90.0 to 98.1%), LR+ve 17.9 (7.34 to 43.8), LR-ve 0.21 (0.08 to 0.58), Diagnostic OR 85.6 (18.2 to 403.6) and accuracy 93.7% (88.0 to 97.2%) for cardiogenic shock. Standard assessment without PoCUS had a sensitivity of 91.7% (64.6 to 98.5%), specificity 93.8% (87.8 to 97.0%), LR+ve 14.8 (7.1 to 30.9), LR- of 0.09 (0.01 to 0.58), Diagnostic OR 166.6 (18.7 to 1481) and accuracy of 93.6% (87.8 to 97.2%). There was no significant difference in sensitivity (-11.7% (-37.8 to 18.3%)) or specificity (1.73% (-4.67 to 8.29%)). Diagnostic performance was also similar between other shock subcategories. Conclusion: As reported in other studies, PoCUS based assessment performed well diagnostically in undifferentiated hypotensive patients, especially as a rule-in test. However performance was similar to standard (non-PoCUS) assessment, which was excellent in this study.
Introduction: Patients with chronic diseases are known to benefit from exercise. Such patients often visit the emergency department (ED). There are few studies examining prescribing exercise in the ED. We wished to study if exercise prescription in the ED is feasible and effective. Methods: In this pilot prospective block randomized trial, patients in the control group received routine care, whereas the intervention group received a combined written and verbal prescription for moderate exercise (150 minutes/week). Both groups were followed up by phone at 2 months. The primary outcome was achieving 150 min of exercise per week. Secondary outcomes included change in exercise, and differences in reported median weekly exercise. Comparisons were made by Mann-Whitney and Fishers tests (GraphPad). Results: Follow-up was completed for 22 patients (11 Control; 11 Intervention). Baseline reported median (with IQR) weekly exercise was similar between groups; Control 0(0-0)min; Intervention 0(0-45)min. There was no difference between groups for the primary outcome of 150 min/week at 2 months (Control 3/11; Intervention 4/11, RR 1.33 (95%CI 0.38-4.6;p=1.0). There was a significant increase in median exercise from baseline in both groups, but no difference between the groups (Control 75(10-225)min; Intervention 120(52.5-150)min;NS). 3 control patients actually received exercise prescription as part of routine care. A post-hoc comparison of patients receiving intervention vs. no intervention, revealed an increase in patients meeting the primary target of 150min/week (No intervention 0/8; Intervention 7/14, RR 2.0 (95%CI 1.2-3.4);p=0.023). Conclusion: Recruitment was feasible, however our study was underpowered to quantify an estimated effect size. As a significant proportion of the control group received the intervention (as part of standard care), any potential measurable effect was diluted. The improvement seen in patients receiving intervention and the increase in reported exercise in both groups (possible Hawthorne effect) suggests that exercise prescription for ED patients may be beneficial.
Introduction: Electrocardiographic (ECG) rhythms are used during resuscitation (ACLS) to guide resuscitation, and often to determine futility. Survival rates to hospital discharge have been reported to be higher for patients with PEA than asystole in out-of-hospital cardiac arrest. This study examines how well the initial ECG cardiac rhythm represents actual cardiac activity as determined by point of care ultrasound (PoCUS). Methods: A database review was completed for patients arriving to a tertiary ED in asystole or PEA arrest, from 2010 to 2014. Patients under 19y or with a previous DNR were excluded. Patients were grouped into those with cardiac activity (PEA) and asystole on ECG; as well as whether cardiac activity was seen on PoCUS during the arrest. Data was analyzed for visualized cardiac activity on PoCUS. Results: 186 patients met the study criteria. Those with asystole on ECG were more likely to have no cardiac activity than those with PEA (Odds 7.21 for initial PoCUS; 5.45 for any PoCUS). The sensitivity of ECG rhythm was 80.49% and 82.12%, specificity was 77.91% and 54.28%, positive predictive value was 94.28% and 88.57%, and negative predictive value was 30.43% and 41.30% for cardiac activity on initial PoCUS and on any PoCUS respectively. The positive and negative likelihood ratios for ECG were 3.47 and 0.25 for activity on initial PoCUS. The positive and negative likelihood ratios for activity on any PoCUS were 1.78 and 0.33. Conclusion: Our results suggest that although most patients with asystole on ECG demonstrate no cardiac activity, a small number actually had activity on PoCUS. This supports the use of PoCUS during cardiac arrest, in addition to ECG, to identify patients with ongoing mechanical cardiac activity.
Introduction: Situational Awareness is the ability to identify, process, and comprehend the critical elements of information about the patient condition, stability, the operational environment and an appropriate clinical course. The Situational Awareness Global Assessment Tool (SAGAT) is a validated tool for measuring situational awareness. The SAGAT tool was measured during a series of standardized high fidelity advanced airway management simulations in multidisciplinary teams in New Brunswick Emergency Departments delivered by two simulation programs Methods: Thirty eight simulated emergency airway cases were performed in situ in Emergency Departments and in learning centers in Southern New Brunswick from September 2015 to October 2017. Eight standardized cases were used whose educational objectives were to develop the optimization of critically ill patients prior to induction, to deliver patient-centered anesthesia and to choose an appropriate airway strategy. Learner profiles collected. Cases were divided into two groups; those that contained critical errors and those that did not based on video assessment. Critical errors were defined as failure of 1) Oxygenation 2) Shock correction 3) Induction dose estimation 4) Choice of airway management paradigm. The SAGAT has a maximum score of 13 and was assessed by research nurses after each case for all participants. SAGAT scores were non-normally distributed, so results were expressed as medians with interquartile ranges. Mann Whitney U tests were used to calculate statistical significance. Results: Results. Of the 38 cases, 14 contained one more critical errors. The median SAGAT score in the group that contained critical errors was 8 +/− 2 (IQR). The median SAGAT Score in the group that contained no critical errors was 11 +/− 2 (IQR). The median scores we significantly different with a p-value of 0.02. Conclusion: In this study in simulated emergency cases, higher SAGAT scores were associated with teams leaders that did not commit safety critical errors. This work is the initial analysis to develop standards for Simulated team performance in Emergency Department teams.
Introduction: Ultrasonography (US), performed in the Emergency Department (ED) by Emergency Physicians, is well established. Educational studies have shown some promise in training paramedics in US use. We have developed and piloted a novel curriculum for paramedic US education. Methods: Based on an informal needs assessment, an US curriculum for paramedics was developed to include: Basic principles, Focused assessment with sonography for trauma (FAST), cardiac, and vascular access. Participants included ED-based and pre-hospital paramedics including all paramedics with critical care training who routinely perform vascular access and procedural sedation within our ED. Comparisons were made using paired non-parametric tests (GraphPad). Results: Participants (N=9) were provided pre- reading materials prior to completing a 6-hour course, consisting of a mix of didactic and practical sessions with live models and vascular access phantoms. Each module was introduced with a 30 minute didactic session, led by an Emergency Physician trained in US, followed immediately by a 1 hour hands-on session lead by either an Emergency Physician or an Emergency Medicine Resident at a learner to instructor ratio of 3:1. At the end of the course, participants were asked to complete a short 10 minute survey that included (1) an assessment of the course quality with regard to preparatory material and course content/delivery (4 point Likert scale; excellent, good, fair, poor); (2) self reported US knowledge pre and post course on a scale of 1-10 (10 high, 1 low); (3) general yes/no questions related to the future of ECCU paramedical and (4) a subjective written section for additional comments. All participants rated the content favourably: 97% scoring it as excellent, and 3% as good. The participants median self-reported US knowledge score increased from 2/10 (IQR 2-3) to 8/10 (IQR 7.25-8; p=0.009) post- course. All comments from the text field were positive in nature. Conclusion: We report a paramedic US course curriculum, which when piloted resulted in high learner satisfaction and a high rate of self reported improvement in US knowledge. Further study will include an assessment of knowledge acquisition and practical performance. Future modifications in our curriculum will be based on needs assessment and may include additional modules.
Introduction: The decision as to whether to end resuscitation for pre-hospital cardiac arrest (CA) patients in the field or in the emergency department (ED) is commonly made based upon standard criteria. We studied the reliability of several easily determined criteria as predictors of resuscitation outcomes in a population of adults in CA transported to the ED. Methods: A retrospective database and chart analysis was completed for patients arriving to a tertiary ED in cardiac arrest, between 2010 and 2014. Patients were excluded if aged under 19. Multiple data were abstracted from charts using a standardized form. Regression analysis was used to compare criteria that predicted return of spontaneous circulation (ROSC) and survival to hospital admission (SHA). Results: 264 patients met the study inclusion criteria. Logistic regression was used to identify predictors of ROSC and SHA. The criteria that emerged as significant predictors for ROSC included; longer ED resuscitation time (Odds ratio 1.11 (1.06- 1.18)), witnessed arrest (Odds ratio 9.43 (2.58- 53.0)) and having an initial cardiac rhythm of Pulseless Electrical Activity (Odds Ratio 3.23 (1.07-9.811)) over Asystole. Receiving point of care ultrasound (PoCUS; Odds ratio 0.22 (0.07-0.69)); and having an initial cardiac rhythm of Pulseless Electrical Activity (Odds Ratio 4.10 (1.43-11.88)) were the significant predictors for SHA. Longer times for ED resuscitation was close to reaching significance for predicting SHA Conclusion: Our results suggest that both fixed and adaptable factors, including increasing resuscitation time, and PoCUS use in the ED were important independent predictors of successful resuscitation. Several commonly used criteria were unreliable predictors.
Introduction: Traditionally, out of hospital cardiac arrests (CA) have poor outcomes. Incorporation of extracorporeal cardiopulmonary resuscitation (ECPR) is being used increasingly to supplement ACLS to provide better outcomes for patients. Current literature suggests potentially improved outcomes, including neurological function. We assessed the feasibility of introduction of ECPR to a regional hospital using a 4-phase study. We report phase-1, an estimation of the number of potential candidates for ECPR in our setting. Methods: Following development and agreement on local criteria for selection of patients for ECPR using a modified Delphi Technique, inclusion and exclusion criteria were applied retrospectively, to a database comprising 4 years of emergency department (ED) cardiac arrests (n=395). This provided estimates of the number of patients who would have qualified for EMS transport for ECPR and initiation of ECPR in the ED. Results: Application of criteria would result in 20.0% (95% CI 16.2-24.3%) of CA being transported to the ED for ECPR (mean 18.5 patients per year). In the ED 4.6% (95% CI 2.83-7.26%) would be eligible to receive ECPR (4.3 patients per year). Incorporating downtime criteria, 3.0% (95% CI 1.6-5.3%) qualify. After considering local in-house cardiac catheterization hours 9.4% (95% CI 6.8-12.9%) and 5.4% (95% CI 3.5-8.2%), without and with EMS rhythm assumptions respectively, would be eligible for transport. For placement on pump, 3.0% (95% CI 1.6-5.3%) and 2.4% (95% CI 1.2-4.6%), without and with use of total downtime respectively, were eligible. Conclusion: If historical patterns of CA were to continue, we believe that an ECPR program may be feasible in our regional hospital setting, with a small number of selected cardiac arrest patients meeting eligibility for transportation and initiation of ECPR. These numbers suggest that an ECPR program would not be resource intensive, yet would be sufficiently busy to maintain adequate team competency.
Introduction: Extracorporeal cardiopulmonary resuscitation (ECPR), a method of cardiopulmonary bypass, is increasingly being used to supplement traditional CPR to improve outcomes for cardiac arrest (CA). CA and particularly out of hospital CA (OHCA) have poor outcomes. Prior to development of a 3 phase ECPR program in a Canadian regional hospital, we wished to identify and optimize a practical selection process (inclusion and exclusion criteria) for patients who may benefit from ECPR. Methods: Using a locally modified Delphi technique, we followed a literature review to construct a proposed set of evidence based criteria with a questionnaire, where inclusion and exclusion criteria were scored by a selected group of 13 experts. Following 3 rounds, and additional review by an international expert in the field of ECPR, consensus was achieved for patient selection criterion. Results: First round responses achieved 87.5% agreement for selection of exclusion criteria. Inclusion criteria had agreement 62.5%. Responses to the second round for selection of inclusion criteria were unanimous at 100% with the exception of age parameters (<65 years vs. <70 years). The third and final set of criteria achieved 100% consensus though subsequent expert review refined a single exclusion criteria (asystole). Agreed inclusion criteria were: witnessed CA, age <70, refractory arrest, no flow time <10min, total downtime <60min, and a cardiac or select non-cardiac etiology (PE, drug OD, poisoning, hypothermia). Exclusion criteria were : unwitnessed arrest, asystole, certain etiologies (uncontrolled bleeding, irreversible brain damage, trauma), and comorbidities (severe disability limiting ADLs, standing DNR, palliation). Simplified criteria for EMS transport included witnessed OHCA, age, and no flow time. Conclusion: Selection criteria of candidates for ECPR are important components for any program. Expert consensus review of current evidence is an effective method for development of ECPR selection criteria.
Introduction: Situational awareness (SA) is the team understanding patient stability, presenting illness and future clinical course. Losing SA has been shown to increase safety-critical events in multiple industries. SA can be measured by the previously validated Situational Awareness Global Assessment Tool (SAGAT). Checklists are used in many safety-critical industries to reduce errors of omission and commission. An RSI checklist was developed from case review and published evidence.The New Brunswick Trauma Program supports an inter-professional simulation-based medical education program Methods: Simulations were facilitated in three hospitals in New Brunswick from April 2017 to October 2017. Learner profiles were collected. The SAGAT tool was completed by a research nurse at the end of each scenario. SAGAT scores were non-normally distributed, so results were expressed as medians and interquartile ranges. Mann Whitney U tests were used to calculate statistical significance. To understand the effect of the of an RSI checklist a comparison was made between SAGAT scores at baseline in scenario 1, and the same first scenario completed after a washout period. A Poisson regression analysis will be used to account for the effect of confounding variables in further analyses. Results: The group was composed of Registered Nurses (8), Physicians (7), and Respiratory Therapists (2). Situational awareness increased significantly with the use of an RSI checklist after 1 day of 4 simulations. The washout period ranged between 5 weeks and 8 weeks. The baseline situational awareness of the whole group during scenario 1 was 9 +/− 0.5 (median, IQR), and with the RSI checklist was 12 +/−1 (median, IQR). The difference was highly statistically significant, p=< 0.001. This level of situational awareness using checklist is comparable to the SAGAT scores after 10 scenarios. Conclusion: In this provisional analysis, the use of an RSI checklist was associated with an increase in measured situational awareness. Higher levels of situational awareness are associated with greater patient safety. A Poisson regression model will be used to understand the confounding effects of user expertise and the likely interaction with simulation exposure.
Introduction: Situational awareness (SA) is essential for maintenance of scene safety and effective resource allocation in mass casualty incidents (MCI). Unmanned aerial vehicles (UAV) can potentially enhance SA with real-time visual feedback during chaotic and evolving or inaccessible events. The purpose of this study was to test the ability of paramedics to use UAV video from a simulated MCI to identify scene hazards, initiate patient triage, and designate key operational locations. Methods: A simulated MCI, including fifteen patients of varying acuity (blast type injuries), plus four hazards, was created on a college campus. The scene was surveyed by UAV capturing video of all patients, hazards, surrounding buildings and streets. Attendees of a provincial paramedic meeting were invited to participate. Participants received a lecture on SALT Triage and the principles of MCI scene management. Next, they watched the UAV video footage. Participants were directed to sort patients according to SALT Triage step one, identify injuries, and localize the patients within the campus. Additionally, they were asked to select a start point for SALT Triage step two, identify and locate hazards, and designate locations for an Incident Command Post, Treatment Area, Transport Area and Access/Egress routes. Summary statistics were performed and a linear regression model was used to assess relationships between demographic variables and both patient triage and localization. Results: Ninety-six individuals participated. Mean age was 35 years (SD 11), 46% (44) were female, and 49% (47) were Primary Care Paramedics. Most participants (80 (84%)) correctly sorted at least 12 of 15 patients. Increased age was associated with decreased triage accuracy [-0.04(-0.07,-0.01);p=0.031]. Fifty-two (54%) were able to localize 12 or more of the 15 patients to a 27x 20m grid area. Advanced paramedic certification, and local residency were associated with improved patient localization [2.47(0.23,4.72);p=0.031], [-3.36(-5.61,-1.1);p=0.004]. The majority of participants (78 (81%)) chose an acceptable location to start SALT triage step two and 84% (80) identified at least three of four hazards. Approximately half (53 (55%)) of participants designated four or more of five key operational areas in appropriate locations. Conclusion: This study demonstrates the potential of UAV technology to remotely provide emergency responders with SA in a MCI. Additional research is required to further investigate optimal strategies to deploy UAVs in this context.
Introduction: With hospital occupancy rates frequently approaching 100%, even small variations in daily admission numbers can have a large impact. The ability to predict variance in emergency admission rates would provide administrators with a significant advantage in managing hospital daily bed requirements. There is a growing interest in patterns of hospital admissions, and many EDs utilize historical admission patterns to attempt to predict daily bed requirements. Previous studies have utilized patient demographics and past medical history to develop an admission likelihood model. We wished to examine the predictive strength of individual CEDIS presenting complaints (PC) on admission likelihood Methods: Using a database analysis of over 285,000 ED presentations (2013-2017), we calculated visit frequencies and admission rates by PC. Using a logistic regression analysis PCs were ordered from high to medium predictive strength. Results: Of 285,155 presentations, there were 38,090 hospital admissions, a rate of 13.36%. Based on the number of visit frequencies and admission rates, the PCs demonstrating high predictive strength were Direct Referral (effect=0.36, binomial CI: 0.28 to 0.44); Shortness of Breath (0.32: 0.26 to 0.41); General Weakness; Weakness/Query CVA; & Chest Pain Cardiac Features (each 0.30: 0.25 to 0.42); Altered level of consciousness (0.24: 0.16 to 0.31); and Confusion (0.18: 0.08 to 0.26). With our sample size, all remaining CEDIS PCs had low predictive value (the effect is <0.1), or were not predictive at all. Conclusion: We have demonstrated that, for our population, certain PCs are associated with an increased likelihood of admission and have quantified this effect using logistic regression analysis. Variance from the average daily admission rate may be predicted, in our population, by identifying these PCs at registration.We plan to develop a tool, based on this data and implemented at registration, to predict cumulative likely daily admission requirements as patients present over a 24hr period.
Genotypes of the myxosporean parasite Ceratonova shasta are defined by the number of ATC repeats in the parasite's ribosomal DNA internal transcribed spacer region 1. These genotypes correlate with specific salmonid fish hosts. We observed coho salmon (Oncorhynchus kisutch) and rainbow trout (Oncorhynchus mykiss) with mixtures of genotypes II and III, and assumed that this was a consequence of fish having an aggregate infection from multiple individual parasites. We hypothesized that although multiple ITS copies are present within a parasite spore, the DNA sequences of these copies are identical, and thus individual C. shasta spores are a single genotype. We tested this by extracting and sequencing DNA from individual myxospores. We trialed three approaches for in-tube DNA extraction; digestion with proteinase K was superior to simply rehydrating spores, or incubation in the buffer. Sequences from 14 myxospores were each a mixture of genotypes II and III. Therefore, intra-genomic ribosomal DNA variants exist within individual parasite spores, and II and III should no longer be regarded as discrete C. shasta genotypes. This single-spore genotyping approach will be a useful tool for testing validity of other C. shasta genotypes, and for correctly matching genotype with phenotype for mixed infections of other myxozoan species.
We describe a new freshwater myxosporean species Ceratomyxa gracillima n. sp. from the gall bladder of the Amazonian catfish Brachyplatystoma rousseauxii; the first myxozoan recorded in this host. The new Ceratomyxa was described on the basis of its host, myxospore morphometry, ssrDNA and internal transcribed spacer region (ITS-1) sequences. Infected fish were sampled from geographically distant localities: the Tapajós River, Pará State, the Amazon River, Amapá State and the Solimões River, Amazonas State. Immature and mature plasmodia were slender, tapered at both ends, and exhibited vermiform motility. The ribosomal sequences from parasite isolates from the three localities were identical, and distinct from all other Ceratomyxa sequences. No population-level genetic variation was observed, even in the typically more variable ITS-1 region. This absence of genetic variation in widely separated parasite samples suggests high gene flow as a result of panmixia in the parasite populations. Maximum likelihood and maximum parsimony analyses placed C. gracillima n. sp. sister to Ceratomyxa vermiformis in a subclade together with Ceratomyxa brasiliensis and Ceratomyxa amazonensis, all of which have Amazonian hosts. This subclade, together with other Ceratomyxa from freshwater hosts, formed an apparently early diverging lineage. The Amazonian freshwater Ceratomyxa species may represent a radiation that originated during marine incursions into the Amazon basin that introduced an ancestral lineage in the late Oligocene or early Miocene.