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.

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.