Haff Disease in Salvador, Brazil, 2016-2021: Attack rate and detection of toxin in fish samples collected during outbreaks and disease surveillance.

Background: From late 2016 to early 2021, cases of Haff disease, a rare cause of rhabdomyolysis, possibly due to poisoning by palytoxin-like compounds in seafood, were detected in Salvador, Brazil. Surveillance was established to detect additional cases aiming at describing the clinical characteristics of the cases, identifying associated factors, estimating disease attack rate, and investigating the presence of biotoxins and trace metals in selected fish specimens obtained from cases. Method: Between December/2016-January/2021, surveillance investigated Haff disease suspected cases, and obtained clinical and fish samples to test. Findings: Of 65 cases investigated during the 2016-2017 outbreak, 43 (66%) had high creatine phosphokinase (CPK) levels. Among those with laboratory-confirmed rhabdomyolysis, 38 (88%) were hospitalized, 11 (26%) required intensive care, and three (7%) dialysis. Ingestion of marine fish 24h before disease onset was reported by 74% of the cases with elevated CPK and by 41% of those without CPK measurement (P=0·02). Attack rate for individuals who ate fish related to the outbreak was 55%. Following this outbreak, surveillance identified 12 suspected cases between 2017-2019, and a second outbreak in 2020-2021, with 16 laboratory-confirmed rhabdomyolysis patients (five required intensive care; one died). No traces of ciguatoxins and metals were detected in fish specimens obtained in 2016, found to be Seriola rivoliana. Some fish samples from 2020 were screened for palytoxin (PlTX)-like compounds and contained detectable levels of molecule fragments characteristics of isobaric PlTX, ovatoxin-a (OVTX-a), OVTX-b and OVTX-d. Interpretation: These findings support the hypothesis that compounds related to PlTX accumulated in marine fish may be the toxic agent causing the disease. Haff disease is a life-threatening condition, requiring clinical suspicion for patients with sudden-onset myalgia following fish ingestion. Suspected cases should be reported to health authorities for investigation.

Antioxidant responses and okadaic acid accumulation in Laeonereis acuta (Annelida) exposed to the harmful dinoflagellate Prorocentrum cf. lima.

We evaluated the accumulation of okadaic acid (OA), a diarrhetic toxin, and the antioxidant responses in the marine annelid Laeonereis acuta exposed to the benthic toxigenic dinoflagellate Prorocentrum cf. lima. Nontoxic Tetraselmis sp. was used as a control diet. Living cells of the two algae were supplied as food to animals kept in agar medium for 72 h. To assess the significance of the observed effects, our experimental design treated the algal species (diet), algal cell densities, and exposure time as fixed factors. Responses of the organisms were assessed through oxidative stress biomarkers (glutathione-S-transferase [GST], catalase [CAT], reduced glutathione [GSH] and lipid peroxidation [LPO]). Toxin accumulation was measured by LC-MS/MS in whole-body homogenates after 12, 24 and 72 h of exposure. Worms exposed to the toxigenic dinoflagellate gradually accumulated OA, with toxin levels directly related to the cell density of Prorocentrum cf. lima. Worms fed with Prorocentrum cf. lima exhibited decreased CAT activity, increased LPO levels - both interactively affected by algal species and time - and decreased GSH levels, which were interactively affected by algal species and cell density. Higher LPO levels, along with the inhibition of CAT and GSH, clearly indicated an oxidative stress situation in worms exposed to the toxigenic dinoflagellate. Laeonereis acuta accumulated moderate OA levels and may act as a vector of OA to food webs in estuarine areas under high Prorocentrum cf. lima abundance.

Toxin accumulation, detoxification and oxidative stress in bivalve (Anomalocardia flexuosa) exposed to the dinoflagellate Prorocentrum lima.

Prorocentrum lima is a cosmopolitan benthic dinoflagellate capable of producing the diarrhetic shellfish toxins (DSTs) okadaic acid (OA) and dinophysistoxin (DTX). These compounds may cause oxidative stress and accumulate in bivalve tissues, which become vectors of intoxication to human consumers. We investigated DST accumulation, detoxification and oxidative stress biomarkers in clams (Anomalocardia flexuosa) experimentally exposed to P. lima cells or their compounds. Experimental diets consisted of 6000 cells mL-1 of the non-toxic chlorophyte Tetraselmis sp. (C; control condition), and combinations of C with 10 P. lima cells mL-1 (T10), 100 P. lima cells mL-1 (T100), or to a toxin concentration of ∼4 µg OA L-1 and ∼0.65 µg DTX-1 L-1 (T100d). Clams were exposed to these diets for 7 days (uptake phase), followed by a 7-day depuration period. No DSTs were detected in clams exposed to treatments C (control) nor to T100d (dissolved compounds) during either uptake or detoxification phase. Conversely, clams exposed to T10 or T100 accumulated, on average, up to 2.5 and 35 µg DST kg-1 in their whole bodies at the end of the uptake phase. These concentrations are ∼64 and ∼4.5 times lower than the regulatory level of 160 µg OA kg-1, respectively. Accumulated OA quotas were 12-22 times higher in the digestive gland (DG) than in remaining tissues over the uptake phase. Quick toxin transformation was indicated by the early detection of conjugated compounds - DTX-1 and OA esters - in the DG after 6 h of exposure, with OA-ester representing the main compound (30 - 100 %) in that tissue over the experiment. During the depuration period, detoxification rates represented 0.024 h-1, 0.04 h-1 and 0.052 h-1 for OA, DTX-1 and OA-ester, respectively. The activities of catalase, glutathione S-transferase, glutathione peroxidase and the levels of oxidative stress by lipoperoxidation varied similarly in the DG of A. flexuosa individuals subjected to T100, T100d and the control condition. However, contrasting antioxidant responses were measured in those exposed to T10. These findings indicate that no oxidative stress was primarily induced by DST-producing dinoflagellates in this clam species under laboratory conditions representative of toxic bloom situations. Even though, possible interactions should be considered under multistressor scenarios.

Hydrocarbon and sewage contamination near fringing reefs along the west coast of Havana, Cuba: A multiple sedimentary molecular marker approach.

Organic contamination is a major environmental concern in coastal regions, and it can be evaluated by the determination of aliphatic hydrocarbons (AHs), polycyclic aromatic hydrocarbons (PAHs), faecal sterols and linear alkylbenzenes (LABs). The concentrations of these organic markers were obtained from nine surface sediment samples to evaluate a possible contamination near a fringing reef on the west coast of Havana, Cuba. The AH levels ranged from 1.24 to 135.6 µg g-1, the PAH levels were up to 2133 ng g-1, the faecal sterol levels ranged from 0.03 to 1.54 µg g-1, and the total LAB levels were up to 22.7 ng g-1. The highest concentrations were obtained at sites close to Havana Bay and at the sources of untreated sewage input. A decreasing concentration gradient was observed from Havana Bay to the outer sites. Although only two sites presented high levels of contamination, untreated sewage discharged close to the fringing reef may affect its environment.

Diel Variations in Cell Abundance and Trophic Transfer of Diarrheic Toxins during a Massive Dinophysis Bloom in Southern Brazil.

Dinophysis spp. are a major source of diarrheic toxins to marine food webs, especially during blooms. This study documented the occurrence, in late May 2016, of a massive toxic bloom of the Dinophysis acuminata complex along the southern coast of Brazil, associated with an episode of marked salinity stratification. The study tracked the daily vertical distribution of Dinophysis spp. cells and their ciliate prey, Mesodinium cf. rubrum, and quantified the amount of lipophilic toxins present in seston and accumulated by various marine organisms in the food web. The abundance of the D. acuminata complex reached 43 × 104 cells·L−1 at 1.0 m depth at the peak of the bloom. Maximum cell densities of cryptophyceans and M. cf. rubrum (>500 × 104 and 18 × 104 cell·L−1, respectively) were recorded on the first day of sampling, one week before the peak in abundance of the D. acuminata complex. The diarrheic toxin okadaic acid (OA) was the only toxin detected during the bloom, attaining unprecedented, high concentrations of up to 829 µg·L−1 in seston, and 143 ± 93 pg·cell−1 in individually picked cells of the D. acuminata complex. Suspension-feeders such as the mussel, Perna perna, and barnacle, Megabalanus tintinnabulum, accumulated maximum OA levels (up to 578.4 and 21.9 µg total OA·Kg−1, respectively) during early bloom stages, whereas predators and detritivores such as Caprellidae amphipods (154.6 µg·Kg−1), Stramonita haemastoma gastropods (111.6 µg·Kg−1), Pilumnus spinosissimus crabs (33.4 µg·Kg−1) and a commercially important species of shrimp, Xiphopenaeus kroyeri (7.2 µg·Kg−1), only incorporated OA from mid- to late bloom stages. Conjugated forms of OA were dominant (>70%) in most organisms, except in blenny fish, Hypleurochilus fissicornis, and polychaetes, Pseudonereis palpata (up to 59.3 and 164.6 µg total OA·Kg−1, respectively), which contained mostly free-OA throughout the bloom. Although algal toxins are only regulated in bivalves during toxic blooms in most countries, including Brazil, this study indicates that human seafood consumers might be exposed to moderate toxin levels from a variety of other vectors during intense toxic outbreaks.