Newly isolated Drexlerviridae phage LAPAZ is physically robust and fosters eradication of Klebsiella pneumoniae in combination with meropenem.

Due to the spread of multidrug resistance there is a renewed interest in using bacteriophages (briefly: phages) for controlling bacterial pathogens. The objective of this study was the characterization of a newly isolated phage (i.e. phage LAPAZ, vB_KpnD-LAPAZ), its antimicrobial activity against multidrug resistant Klebsiella pneumoniae and potential synergistic interactions with antibiotics. LAPAZ belongs to the family Drexlerviridae (genus: Webervirus) and lysed 30 % of tested strains, whereby four distinct capsular types can be infected. The genome consists of 51,689 bp and encodes 84 ORFs. The latent period is 30 min with an average burst size of 27 PFU/cell. Long-term storage experiments show that LAPAZ is significantly more stable in wastewater compared to laboratory media. A phage titre of 90 % persists up to 30 min at 50 ˚C and entire phage loss was seen only at temperatures > 66 ˚C. Besides stability against UV-C, antibacterial activity in liquid culture medium was consistent at pH values ranging from 4 to 10. Unlike exposure to phage or antibiotic alone, synergistic interactions and a complete bacterial eradication was achieved when combining LAPAZ with meropenem. In addition, synergism with the co-presence of ciprofloxacin was observed and phage resistance emergence could be delayed. Without co-addition of the antibiotic, phage resistant mutants readily emerged and showed a mixed pattern of drug sensitivity alterations. Around 88 % became less sensitive towards ceftazidime, meropenem and gentamicin. Conversely, around 44 % showed decreased resistance levels against ciprofloxacin. Whole genome analysis of a phage-resistant mutant with a 16-fold increased sensitivity towards ciprofloxacin revealed one de novo frameshift mutation leading to a gene fusion affecting two transport proteins belonging to the major facilitator-superfamily (MFS). Apparently, this mutation compromises ciprofloxacin efflux efficiency and further studies are warranted to understand how the non-mutated protein might be involved in phage-host adsorption.

Thrombotic microangiopathy in patients with sickle cell disease.

OBJECTIVE: To describe two cases of patients who had thrombotic microangiopathy (TMA) associated with sickle cell disease (SCD). CASE DESCRIPTION: Both patients started with a painful crisis and had acute chest syndrome during hospitalization. They showed significant worsening of hemolytic anemia, with very high levels of lactate dehydrogenase, thrombocytopenia, lowered level of consciousness, organ damage and the presence of schistocytes in peripheral blood. Due to the possibility of TMA, despite the very rare association with SCD, they were treated with fresh frozen plasma replacement and plasmapheresis, with good response. COMMENTS: TMA is a serious, life-threatening disease, characterized by microangiopathic hemolytic anemia, thrombocytopenia, and organ damage. The association of SCD and TMA is difficult to diagnose, since they can share a similar clinical presentation. Recognizing this association and promptly instituting treatment may impact the survival of these patients.

Thrombotic microangiopathy in patients with sickle cell disease / Microangiopatia trombótica em pacientes com doença falciforme

ABSTRACT Objective: To describe two cases of patients who had thrombotic microangiopathy (TMA) associated with sickle cell disease (SCD). Case description: Both patients started with a painful crisis and had acute chest syndrome during hospitalization. They showed significant worsening of hemolytic anemia, with very high levels of lactate dehydrogenase, thrombocytopenia, lowered level of consciousness, organ damage and the presence of schistocytes in peripheral blood. Due to the possibility of TMA, despite the very rare association with SCD, they were treated with fresh frozen plasma replacement and plasmapheresis, with good response. Comments: TMA is a serious, life-threatening disease, characterized by microangiopathic hemolytic anemia, thrombocytopenia, and organ damage. The association of SCD and TMA is difficult to diagnose, since they can share a similar clinical presentation. Recognizing this association and promptly instituting treatment may impact the survival of these patients.

RESUMO Objetivo: Descrever dois casos de pacientes que apresentaram microangiopatia trombótica (MAT) associada à doença falciforme (DF). Descrição do caso: Ambos os pacientes iniciaram com crise dolorosa e apresentaram síndrome torácica aguda durante a internação. Eles apresentaram piora significativa da anemia hemolítica, com níveis muito elevados de lactato desidrogenase, trombocitopenia, rebaixamento do nível de consciência, lesão de órgãos e presença de esquistócitos no sangue periférico. Diante da possibilidade de MAT, apesar da associação muito rara com DF, eles foram tratados com reposição de plasma fresco congelado e plasmaférese, com boa resposta. Comentários: A MAT é uma doença grave e com risco de vida, caracterizada por anemia hemolítica microangiopática, trombocitopenia e danos a órgãos. A associação de DF e MAT é de difícil diagnóstico, pois as duas podem ter apresentação clínica semelhante, portanto reconhecer essa associação e instituir o tratamento prontamente pode ter grande impacto na sobrevida desses pacientes.

Evaluation of Capacity for Implementation of Pediatric-to-Adult Care Transition for Sickle Cell Disease in Brazil

Implementation of HCT programs is a high priority in Brazil, given the growing survival rates for children with SCD. Our data highlights a well-connected community of healthcare workers, but also reveals that the current level of HCT activities for SCD in Brazil is low. This underscores the urgent need for transition intervention. The key facilitators for successful HCT implementation in Brazil are strong leadership and a motivated workforce. However, community participation and finances are weak points that require implementation strategies during program implementation. Compared to the US, both Brazilian institutions scored significantly lower on all components needed for transition, emphasizing the need for intervention. This project is the first to evaluate the readiness for HCT implementation in a low- and middle-income country. Designing a context-specific HCT program that suits low-resource settings will improve outcomes for individuals with SCD in the country. (AU)

Differences between real-world and score-based decision-making in the microsurgical management of patients with unruptured intracranial aneurysms.

BACKGROUND: Management of unruptured intracranial aneurysms (UIAs) is complex, balancing the risk of rupture and risk of treatment. Therefore, prediction scores have been developed to support clinicians in the management of UIAs. We analyzed the discrepancies between interdisciplinary cerebrovascular board decision-making factors and the results of the prediction scores in our cohort of patients who received microsurgical treatment of UIAs. METHODS: Clinical, radiological, and demographical data of 221 patients presenting with 276 microsurgically treated aneurysms were collected, from January 2013 to June 2020. UIATS, PHASES, and ELAPSS were calculated for each treated aneurysm, resulting in subgroups favoring treatment or conservative management for each score. Cerebrovascular board decision-factors were collected and analyzed. RESULTS: UIATS, PHASES, and ELAPSS recommended conservative management in 87 (31.5%) respectively in 110 (39.9%) and in 81 (29.3%) aneurysms. The cerebrovascular board decision-factors leading to treatment in these aneurysms (recommended to manage conservatively in the three scores) were: high life expectancy/young age (50.0%), angioanatomical factors (25.0%), multiplicity of aneurysms (16.7%). Analysis of cerebrovascular board decision-making factors in the "conservative management" subgroup of the UIATS showed that angioanatomical factors (P=0.001) led more frequently to surgery. PHASES and ELAPSS subgroups "conservative management" were more frequently treated due to clinical risk factors (P=0.002). CONCLUSIONS: Our analysis showed more aneurysms were treated based on "real-world" decision-making than recommended by the scores. This is because these scores are models trying to reproduce reality, which is yet not fully understood. Aneurysms, which were recommended to manage conservatively, were treated mainly because of angioanatomy, high life expectancy, clinical risk factors, and patient's treatment wish. The UIATS is suboptimal regarding assessment of angioanatomy, the PHASES regarding clinical risk factors, complexity, and high life expectancy, and the ELAPSS regarding clinical risk factors and multiplicity of aneurysms. These findings support the need to optimize prediction models of UIAs.

Functional capacity and quality of life in children and adolescents with sickle cell anemia.

OBJECTIVE: To evaluate the functional and exercise capacity, lung function, quality of life of children and adolescents with sickle cell anemia (SCA HbSS) and to test the reproducibility of functional capacity tests in this population. METHOD: Cross-sectional study with volunteers with SCA Hb-SS (SCAG), aged 6-18 years matched in age and gender to the control group (CG) with healthy individuals. Spirometry, 5-repetition sit-to-stand test (5STS-test), modified shuttle test (MST), and Pediatric Quality of Life Questionnaire (PedsQL) were performed. The reproducibility of 5STS-test and MST was evaluated: RESULTS: Forty eight volunteers of SCAG and 48 of CG were evaluated. Lung function of SCAG (FVC: 92 ± 15% pred.; FEV1 /FVC: 84 ± 8% pred.) was worse than the CG (104 ± 15% pred.; FEV1 /FVC: 90 ± 6% pred.) p < 0.05. SCAG had worse functional capacity registered by distance walked: 576 m (515-672 m) and 5STS-test: 8 s (7.4-8.9 s) compared with the CG who showed distance walked: 1010 m (887-1219 m) and 5STS-test: 7 s (7.0-8.1 s), p < 0.001. SCAG had worse quality of life compared to CG, p < 0.05. The reproducibility of MST (ICC 0.99 (0.98-0.99 IC-95%)) and 5STS-test (ICC 0.80 (0.69-0.88) was considered good, p < 0.001. CONCLUSION: Children and adolescents with sickle cell anemia presented worse capacity to walk or run, and to perform sit-to-stand test when compared with their control peers. Additionally, they have poorer quality of life. The MST and 5STS-test showed good reproducibility to be applied in pediatric individuals with SCA.

Characterization of Atmospheric Processes of Brevetoxins in Sea Spray Aerosols from Red Tide Events.

Atmospheric processes can affect the longevity of harmful toxins in sea spray aerosols (SSA). This study characterized the degradation of brevetoxin (BTx) in SSA under different environmental conditions. The samples of seawater collected during a Karenia brevis bloom in Manasota, Florida, were nebulized into a large outdoor photochemical chamber to mimic the atmospheric oxidation of aerosolized toxins and then aged in the presence or absence of sunlight and/or O3. Aerosol samples were collected during the aging process using a Particle-Into-Liquid Sampler. Their BTx concentrations were measured using an enzyme-linked immuno-sorbent assay (ELISA) and high-performance liquid chromatography/tandem mass spectroscopy. The BTx ozonolysis rate constant measured by ELISA was 5.74 ± 0.21 × 103 M-1 s-1. The corresponding lifetime for decay of 87.5% BTx in the presence of 20 ppb of O3 was 7.08 ± 0.26 h, suggesting that aerosolized BTx can still travel long distances at night before SSA deposition. BTx concentrations in SSA decreased more rapidly in the presence of sunlight than in its absence due to oxidation with photochemically produced OH radicals.

Validation of the Clavien-Dindo grading system of complications for microsurgical treatment of unruptured intracranial aneurysms.

OBJECTIVE: Microsurgery plays an essential role in managing unruptured intracranial aneurysms (UIAs). The Clavien-Dindo classification is a therapy-oriented grading system that rates any deviation from the normal postoperative course in five grades. In this study, the authors aimed to test the applicability of the Clavien-Dindo grade (CDG) in patients who underwent microsurgical treatment of UIAs. METHODS: The records of patients who underwent microsurgery for UIAs (January 2013-November 2018) were retrieved from a prospective database. Complications at discharge and at short-term follow-up (3 months) were rated according to the Clavien-Dindo system. Patient outcomes were graded using the modified Rankin Scale (mRS) and the National Institutes of Health Stroke Scale (NIHSS). A descriptive statistic was used for data analysis. RESULTS: Overall, 156 patients underwent 157 surgeries for 201 UIAs (size range 4-42 mm). Thirty-nine patients (25%) had complex UIAs. An adverse event (CDG ≥ I) occurred in 21 patients (13.5%) by the time of discharge. Among these, 10 patients (6.4%) presented with a new neurological deficit. Significant correlations existed between a CDG ≥ I and an increase in mRS and NIHSS scores (p < 0.001). Patients treated for complex aneurysms had a significantly higher risk of developing new neurological deficits (20.5% vs 1.7%, p = 0.007). At the 3-month follow-up, a CDG ≥ I was registered in 16 patients (10.3%); none presented with a new neurological deficit. A CDG ≥ I was associated with a longer hospital length of stay (LOS) (no complication vs CDG ≥ I, 6.2 ± 3.5 days vs 9.3 ± 7.7 days, p = 0.02). CONCLUSIONS: The CDG was applicable to patients who received microsurgery of UIAs. A significant correlation existed between CDG and outcome scales, as well as LOS. The aneurysm complexity was significantly associated with a higher risk for new neurological deficit.

COVID-19 Transmission in a Psychiatric Long-Term Care Rehabilitation Facility: An Observational Study.

OBJECTIVE: To report the clinical characteristics and transmission rate of coronavirus disease 2019 (COVID-19) in a community inpatient long-term care psychiatric rehabilitation facility designed for persons with serious mental illness to provide insight into transmission and symptom patterns and emerging testing protocols, as well as medical complications and prognosis. METHODS: This study examined a cohort of 54 residents of a long-term care psychiatric rehabilitation program from March to April 2020. Baseline demographics, clinical diagnoses, and vital signs were examined to look for statistical differences between positive versus negative severe acute respiratory syndrome coronavirus 2 (SARS-COV-2) groups. During the early phase of the pandemic, the facility closely followed the local shelter-in-place order (starting March 19, 2020) and symptom-based testing. RESULTS: Of the residents, the primary psychiatric diagnoses were schizoaffective disorder: 28 (51.9%), schizophrenia: 21 (38.9%), bipolar I disorder: 3 (5.5%), and unspecified psychotic disorder: 2 (3.7%). Forty (74%) of 54 residents tested positive for SARS-COV-2, with a doubling time of 3.9 days. There were no statistical differences between the positive SARS-COV-2 versus negative groups for age or race/ethnicity. Psychiatric and medical conditions were not significantly associated with contracting SARS-COV-2, with the exception of obesity (n = 17 [43%] positive vs n = 12 [86%] negative, P = .01). Medical monitoring of vital signs and symptoms did not lead to earlier detection. All of the residents completely recovered, with the last resident no longer showing any symptoms 24 days from the index case. CONCLUSION: Research is needed to determine optimal strategies for long-term care mental health settings that incorporate frequent testing and personal protective equipment use to prevent rapid transmission of SARS-COV-2.

The effects of prolonged exposure to hypoxia and Florida red tide (Karenia brevis) on the survival and activity of stone crabs.

Florida red tides are harmful algae blooms caused by the dinoflagellate Karenia brevis, which occur along Florida's gulf coast almost annually. In recent years Florida red tide blooms have become more common, frequent, and intense. Florida's southwest coast, from Manatee to Collier County, has experienced repeated and prolonged K. brevis blooms since 2011 with the most recent bloom in 2017 lasting 17 months and resulting in both hypoxic and anoxic events. We therefore determined the survival and level of lethargy (e.g., lack of responsiveness or reduction in behavioral reactions) of sublegal stone crabs to K. brevis and hypoxia as both singular and simultaneous stressors. Crabs were randomly assigned to one of six treatments that included: 1) high concentration of toxic K. brevis (> 1 million cells L-1) maintained at normoxic levels (7.2 mg L-1 ± S.D. 0.47 dissolved oxygen), 2) moderate hypoxia (1.6 mg L-1 ± S.D. 0.42 dissolved oxygen) with no K. brevis, 3) moderate hypoxia (1.5 mg L-1 ± S.D. 0.43 dissolved oxygen) with a high concentration of K. brevis, 4) severe hypoxia with no K. brevis (0.69 mg L-1 ± S.D. 0.36 dissolved oxygen), 5) severe hypoxia (0.63 mg L-1 ± S.D. 0.40 dissolved oxygen) with a high concentration of K. brevis, and 6) a normoxic control (7.3 mg L-1 ± S.D. 0.61 dissolved oxygen) with no K. brevis. Survival and stone crab lethargy or responsiveness was monitored every 10-12 h for six days. Crabs simultaneously exposed to K. brevis and severe hypoxia exhibited a 43% decrease in survival and experienced increased lethargy within 24 h relative to the control (7% decrease in survival, no increase in lethargy). The increase in stress level and sluggish behavior during exposure to hypoxia was evident by a general lack of responsiveness or movement which indicates that nearshore populations of stone crabs are unlikely to emigrate away from such conditions suggesting that future harvests may be reduced following prolonged K. brevis blooms and hypoxic events.

Development of machine learning-based preoperative predictive analytics for unruptured intracranial aneurysm surgery: a pilot study.

BACKGROUND: The decision to treat unruptured intracranial aneurysms (UIAs) or not is complex and requires balancing of risk factors and scores. Machine learning (ML) algorithms have previously been effective at generating highly accurate and comprehensive individualized preoperative predictive analytics in transsphenoidal pituitary and open tumor surgery. In this pilot study, we evaluate whether ML-based prediction of clinical endpoints is feasible for microsurgical management of UIAs. METHODS: Based on data from a prospective registry, we developed and internally validated ML models to predict neurological outcome at discharge, as well as presence of new neurological deficits and any complication at discharge. Favorable neurological outcome was defined as modified Rankin scale (mRS) 0 to 2. According to the Clavien-Dindo grading (CDG), every adverse event during the post-operative course (surgery and not surgery related) is recorded as a complication. Input variables included age; gender; aneurysm complexity, diameter, location, number, and prior treatment; prior subarachnoid hemorrhage (SAH); presence of anticoagulation, antiplatelet therapy, and hypertension; microsurgical technique and approach; and various unruptured aneurysm scoring systems (PHASES, ELAPSS, UIATS). RESULTS: We included 156 patients (26.3% male; mean [SD] age, 51.7 [11.0] years) with UIAs: 37 (24%) of them were treated for multiple aneurysm and 39 (25%) were treated for a complex aneurysm. Poor neurological outcome (mRS ≥ 3) was seen in 12 patients (7.7%) at discharge. New neurological deficits were seen in 10 (6.4%), and any kind of complication occurred in 20 (12.8%) patients. In the internal validation cohort, area under the curve (AUC) and accuracy values of 0.63-0.77 and 0.78-0.91 were observed, respectively. CONCLUSIONS: Application of ML enables prediction of early clinical endpoints after microsurgery for UIAs. Our pilot study lays the groundwork for development of an externally validated multicenter clinical prediction model.

Karenia brevis causes high mortality and impaired swimming behavior of Florida stone crab larvae.

The dinoflagellate Karenia brevis causes harmful algal blooms commonly referred to as red tides that are prevalent along Florida's gulf coast. Severe blooms often cause fish kills, turbid water, and hypoxic events all of which can negatively impact local fisheries. The stone crab, Menippe mercenaria, is a ˜$25 million per year fishery that occurs primarily along Florida's gulf coast. On the west Florida shelf, red tides occur from fall through spring, although severe blooms can occur during the summer. During the summer, stone crabs are reproductive and release larvae that are transported offshore where K. brevis blooms originate. This study determined the effects of K. brevis exposure on the survivorship, vertical swimming behavior, and oxygen consumption of stage-1 larval stone crabs. Survivorship was determined by exposing larvae to high (> 1 × 106 cells L-1) and medium (˜1 × 105 cells L-1) K. brevis concentrations for 96-hrs and were compared to controls that had no algae present. Larval swimming behavior (i.e., geotaxis) and oxygen consumption were monitored after 6-hr exposure to K. brevis. After 96-hrs of exposure, mortality was 100% and 30% for larvae in the high and medium concentrations of K. brevis, respectively, relative to the control. Larval swimming behavior was reversed in the K. brevis treatment; however oxygen consumption rates did not differ among treatments. These results suggest that severe blooms during the summer may reduce larval supply and serve as a potential bottleneck for new individuals recruiting into the fishery in years following a K. brevis bloom.

Cardiac Arrest During Spine Surgery in the Prone Position: Case Report and Review of the Literature.

OBJECTIVE: Intraoperative cardiac arrest (CA) is usually attributable to pre-existing disease or intraoperative complications. In rare cases, intraoperative stress can demask certain genetic diseases, such as catecholaminergic polymorphic ventricular tachycardia (CPVT). It is essential that neurosurgeons be aware of the etiologies, risk factors, and initial management of CA during surgery with the patient in the prone position. METHODS: We present a case of CA directly after spinal fusion for lumbar spondylolisthesis and review the literature on cardiac arrests during spinal neurosurgery in the prone position. We focus on etiologies of CA in patients with structurally normal hearts. RESULTS: After resuscitation, a 53-years-old female patient achieved return of spontaneous circulation after 17 minutes, without any neurologic deficits and with substantial improvement of functional disability and pain scores. Extensive imaging, stress testing, and genetic screening ruled out common etiologies of CA. In this patient with a structurally normal heart, CPVT was established as the most likely cause. We identified 18 additional cases of CA associated with spinal neurosurgery in the prone position. Most cases occurred during deformity or fusion procedures. Commonly reported etiologies of CA were air embolism, hypovolemia, and dural traction leading to vasovagal response. In patients with structurally normal hearts, inherited arrhythmia syndromes including CPVT, Brugada syndrome, and long QT syndrome should be included in the differential diagnosis and specifically included in testing. CONCLUSIONS: Although intraoperative CA is rare during spine surgery, neurosurgeons should be aware of the etiologies and the specific difficulties in the management associated with the prone position.

The effects of red tide (Karenia brevis) on reflex impairment and mortality of sublegal Florida stone crabs, Menippe mercenaria.

The Florida stone crab, Menippe mercenaria, is a major commercial fishery that occurs primarily along Florida's west coast, where harmful algal blooms of Karenia brevis frequently develop. To determine sublethal and lethal effects of K. brevis on M. mercenaria, we exposed sublegal stone crabs to three seawater treatments in laboratory conditions: no K. brevis (control), a low-toxin K. brevis strain (Wilson LT), and a toxic K. brevis (New Pass strain). Total food consumed, reflex impairment and survivorship of each crab was monitored throughout the nine-day experiment. Crabs in the toxic treatment consumed 67% less food. The probability of an individual losing a reflex significantly increased with time (days), and there was a 42% decrease in survivorship in the toxic treatment. This is the first study to demonstrate negative effects of K. brevis on the stone crab, presenting the critical need of further investigation to fully understand how red tide may impact sustainability of the fishery.

Maternal transfer and sublethal immune system effects of brevetoxin exposure in nesting loggerhead sea turtles (Caretta caretta) from western Florida.

Blooms of Karenia brevis (also called red tides) occur almost annually in the Gulf of Mexico. The health effects of the neurotoxins (i.e., brevetoxins) produced by this toxic dinoflagellate on marine turtles are poorly understood. Florida's Gulf Coast represents an important foraging and nesting area for a number of marine turtle species. Most studies investigating brevetoxin exposure in marine turtles thus far focus on dead and/or stranded individuals and rarely examine the effects in apparently "healthy" free-ranging individuals. From May-July 2014, one year after the last red tide bloom, we collected blood from nesting loggerhead sea turtles (Caretta caretta) on Casey Key, Florida USA. These organisms show both strong nesting and foraging site fidelity. The plasma was analyzed for brevetoxin concentrations in addition to a number of health and immune-related parameters in an effort to establish sublethal effects of this toxin. Lastly, from July-September 2014, we collected unhatched eggs and liver and yolk sacs from dead-in-nest hatchlings from nests laid by the sampled females and tested these samples for brevetoxin concentrations to determine maternal transfer and effects on reproductive success. Using a competitive enzyme-linked immunosorbent assay (ELISA), all plasma samples from nesting females tested positive for brevetoxin (reported as ng brevetoxin-3[PbTx-3] equivalents [eq]/mL) exposure (2.1-26.7ng PbTx-3eq/mL). Additionally, 100% of livers (1.4-13.3ng PbTx-3eq/mL) and yolk sacs (1.7-6.6ng PbTx-3eq/mL) from dead-in-nest hatchlings and 70% of eggs (<1.0-24.4ng PbTx-3eq/mL) tested positive for brevetoxin exposure with the ELISA. We found that plasma brevetoxin concentrations determined by an ELISA in nesting females positively correlated with gamma-globulins, indicating a potential for immunomodulation as a result of brevetoxin exposure. While the sample sizes were small, we also found that plasma brevetoxin concentrations determined by an ELISA in nesting females significantly correlated with liver brevetoxin concentrations of dead-in-nest hatchlings and that brevetoxins could be related to a decreased reproductive success in this species. This study suggests that brevetoxins can still elicit negative effects on marine life long after a bloom has dissipated. These results improve our understanding of maternal transfer and sublethal effects of brevetoxin exposure in marine turtles.

Compositional changes in neurotoxins and their oxidative derivatives from the dinoflagellate, Karenia brevis, in seawater and marine aerosol.

The harmful alga, Karenia brevis, produces a suite of polyether neurotoxins, brevetoxins or PbTx, that cause marine animal mortality and neurotoxic shellfish poisoning (NSP). A characteristic of K. brevis blooms is associated airborne toxins that result in severe respiratory problems. This study was undertaken to determine the composition of aerosolized brevetoxins and oxidative derivatives to which beachgoers are exposed during a K. brevis bloom. The suite of brevetoxins and derivatives in seawater is comprised of intra-cellular (IC) and extra-cellular (EC) compounds. We hypothesized that aerosolized compounds are generated primarily from EC, hydrophobic compounds in seawater by bubble-mediated transport. Thus the composition of aerosolized brevetoxins and derivatives, to which beachgoers are exposed, would reflect the EC composition of the source matrix (the local surf zone). Brevetoxins were extracted from water collected along the shore and from marine aerosols along Siesta Beach and Lido Beach in Sarasota, FL, USA, during K. brevis blooms. Water samples were further processed into IC and EC components. The primary brevetoxins observed in water and air included PbTx-1, -2, -3, -PbTx-2-carboxylic acid, and brevenal. Oxidation and/or hydrolysis products of PbTx-1, -2, -3 and -7 were also found in EC water and in aerosol, but not IC.

Inland Transport of Aerosolized Florida Red Tide Toxins.

Florida red tides, an annual event off the west coast of Florida, are caused by the toxic dinoflagellate, Karenia brevis. K. brevis produces a suite of potent neurotoxins, brevetoxins, which kill fish, sea birds, and marine mammals, as well as sickening humans who consume contaminated shellfish. These toxins become part of the marine aerosol, and can also be inhaled by humans and other animals. Recent studies have demonstrated a significant increase in symptoms and decrease lung function in asthmatics after only one hour of beach exposure during an onshore Florida red tide bloom.This study constructed a transect line placing high volume air samplers to measure brevetoxins at sites beginning at the beach, moving approximately 6.4 km inland. One non-exposure and 2 exposure studies, each of 5 days duration, were conducted. No toxins were measured in the air during the non-exposure period. During the 2 exposure periods, the amount of brevetoxins varied considerably by site and by date. Nevertheless, brevetoxins were measured at least 4.2 kilometers from the beach and/or 1.6 km from the coastal shoreline. Therefore, populations sensitive to brevetoxins (such as asthmatics) need to know that leaving the beach may not discontinue their environmental exposure to brevetoxin aerosols.

Cellular metabolism of brevetoxin (PbTx-2) by a monocyte cell line (U-937).

Blooms of Karenia brevis produce brevetoxins which cause neurotoxic shellfish poisoning and respiratory symptoms in humans as well as harmful effects on sea life. To investigate potential effects of brevetoxins on immune system components, a monocyte cell line (U-937) was exposed in vitro to PbTx-2. U-937 cells metabolized PbTx-2 through cellular detoxification mechanisms, as evidenced by depletion of intracellular glutathione and formation of glutathione and cysteine conjugates. Total intracellular glutathione was significantly decreased in toxin-treated cells compared to control cells, as measured using an enzymatic recycling method. LC/MS was used to detect the following brevetoxin metabolites: a cysteine-PbTx-2 conjugate (m/z 1018) and two putative glutathione-PbTx-2 conjugates (m/z 1204 and 1222). During 3h incubation, glutathione conjugates were detectable as early as 1h and increased in concentration after 2 and 3h. A cysteine-PbTx-2 conjugate appeared after 2h and increased in concentration after 3h. Detectable levels of brevetoxin conjugates were present in response to toxin concentrations of 1muM. Depletion of intracellular glutathione and formation of brevetoxin metabolites, with changes in concentrations over time, suggest immune cells (U-937) have important cellular detoxification pathways for PbTx-2.

Monitoring of brevetoxins in the Karenia brevis bloom-exposed Eastern oyster (Crassostrea virginica).

Brevetoxin uptake and elimination were examined in Eastern oyster (Crassostrea virginica) exposed to recurring blooms of the marine alga Karenia brevis in Sarasota Bay, FL, over a three-year period. Brevetoxins were monitored by in vitro assays (ELISA, cytotoxicity assay, and receptor binding assay) and LC-MS, with in vivo toxicity of shellfish extracts assessed by the traditional mouse bioassay. Measurements by all methods reflected well the progression and magnitude of the blooms. Highest levels recorded by mouse bioassay at bloom peak were 157 MU/100g. Oysters were toxic by mouse bioassay at levels >or=20 MU/100g for up to two weeks after bloom dissipation, whereas brevetoxins were measurable by in vitro assays and LC-MS for several months afterwards. For the structure-based methods, summed values for the principal brevetoxin metabolites of PbTx-2 (cysteine and cysteine sulfoxide conjugates), as determined by LC-MS, were highly correlated (r(2)=0.90) with composite toxin measurements by ELISA. ELISA and LC-MS values also correlated well (r(2)=0.74 and 0.73, respectively) with those of mouse bioassay. Pharmacology-based cytotoxicity and receptor binding assays did not correlate as well (r(2)=0.65), and were weakly correlated with mouse bioassay (r(2)=0.48 and 0.50, respectively). ELISA and LC-MS methods offer rapid screening and confirmation, respectively, of brevetoxin contamination in the oyster, and are excellent alternatives to mouse bioassay for assessing oyster toxicity following K. brevis blooms.