Cristina Carranza

Ciguatera Fish Poisoning, Canary Islands

To the Editor: Ciguatera outbreaks usually occur in the area between 35° north and 35° south latitude, mainly in the Caribbean, Indo-Pacific islands, and the Indian Ocean (1–5) (Figure). Occasionally, ciguatera poisoning has been reported outside disease endemic areas, such as the Bahamas, Canada, or Chile, but no case had been described in the West African region until now. European and Spanish cases have been rarely described and are mainly associated with seafood imported from disease-endemic regions (6). n n n nFigure n nWorldwide distribution of ciguatera. Gray indicates coral reef regions located between 35° north and 35° south latitudes; brown indicates disease-endemic areas of ciguatera; red circle indicates Canary Islands (latitude 28°06´ ... n n n nCiguatera fish poisoning is a clinical syndrome caused by eating contaminated fish (1). The causative toxins of its clinical manifestations are ciguatoxins (7). These toxins are transmitted by dinoflagellates of the species Gambierdiscus toxicus, which lives adhered to damaged coral reefs in tropical seas (2). Herbivorous fish species accumulate toxins in their musculature, liver, and viscera after ingesting dinoflagellates. Larger marine carnivores eat contaminated fish and concentrate ciguatoxins (1,2). n nMore than 425 species of fish are associated with ciguatera poisoning in humans. The most commonly implicated fish are barracuda, red snapper, grouper, amberjack, sea bass, surgeonfish, and moray (eel) (2,3). In January 2004, 2 fishermen captured a 26-kg amberjack (local name: Medregal Negro; scientific name: Seriola Rivoliana) while scuba diving along the coast of the Canary Islands, Spain. The fishermen filleted the fish and stored fillets in a household freezer. Within a few days, one of the fishermen and 4 family members consumed some fish, and neurologic and gastrointestinal symptoms developed within 30 minutes to 28 hours. The 5 family members sought treatment at the emergency room of Hospital de Fuerteventura and the Outpatient Clinic of Infectious Diseases and Tropical Medicine Service of Hospital Insular de Las Palmas. n nThe 5 family members exhibited a combination of gastrointestinal (diarrhea [4 persons], nausea/vomiting [3 persons], metallic taste [1 person]), cardiologic (heart rhythm disturbances [2 persons]), systemic (fatigue [5 persons], itching [3 persons], dizziness (1 person]), and neurologic manifestations (myalgia [3 persons], peripheral paresthesia [3 persons], perioral numbness [2 persons], and reversal of hot and cold sensations [3 persons], which is pathognomonic of ciguatera poisoning). These clinical observations and laboratory data were collected from a prospective questionnaire filled in by physicians at the patients first visits. No hematologic or biochemical abnormalities were detected in any patient. Based upon the symptomatic profiles, relationships of the patients, and their common dietary histories, ciguatera intoxication was diagnosed in all. None of the patients required hospitalization. The neurologic and gastrointestinal symptoms resolved over several weeks, but intermittent recurrence of some symptoms, at lower intensities, was noted for several months. n nA portion of the implicated fish was recovered from freezer storage at the fishermans home. A solid-phase membrane immunobead assay with a monoclonal antibody directed against Pacific ciguatoxins and related polyether toxins was used to detect ciguatoxins or other antigenically related substances in fish tissues. Results were positive. n nA 150-g sample of the fish was delivered to the US Food and Drug Organizations Gulf Coast Seafood Laboratory, Dauphin Island, Alabama, USA, for sodium channel–specific in vitro assay (8) and liquid chromatography–mass spectrometry (LC/MS/MS) analysis. Assay results were positive and the ciguatoxin content of the fish sample was estimated to be 1.0 ppb (ng/g). Caribbean ciguatoxin (CCTX-1: MH+ m/z 1141.6) was confirmed by LC/MS/MS by using multiple reaction monitoring (9). The amount of ciguatoxin in the fish tissue estimated by in vitro assay was low, and close to the limit the LC/MS/MS method can detect. At least 2 additional toxins were detected in the fish sample by in vitro assay of liquid chromatography fractions. We cannot rule out the possibility that these toxins represent new ciguatoxinlike structures unique to the eastern Atlantic. Further studies are necessary to elucidate all toxins implicated in this outbreak. n nClassic symptoms of ciguatera developed in our patients after eating a fish they captured in the Canary Islands, which are not in the ciguatera-endemic zone (Figure). The preliminary results of this outbreak investigation suggest the presence of ciguatoxins or ciguatoxinlike structures in fish from temperate waters of the eastern Atlantic. Ciguatera poisoning is a matter of public health concern and residents of coastal West Africa and the regional island archipelagos could be a new community at risk for this seafood intoxication syndrome. We emphasize that ciguatera poisoning is a debilitating disease, and therapeutic intervention strategies are very limited (10).

Helminth-related Eosinophilia in African immigrants, Gran Canaria.

Of 788 recent African adult immigrants to Las Palmas de Gran Canaria, 213 (27.0%) had eosinophilia. The most frequent causes were filariasis (29.4%), schistosomiasis (17.2%), and hookworm infection (16.8%). Stool microscopy and filarial and schistosomal serologic tests gave the highest diagnostic yield. Country of origin and eosinophil count were associated with specific diagnoses.

Manejo práctico de una eosinofilia

In this first part of this paper we review the definition of eosinophilia and their classification according to the degree of elevation of eosinophils/microL. Aetiological factors related with eosinophilia were described in three groups of patients: a). autochthonous non-infected by HIV; b). HIV-infected; and c). arrived from tropical countries (imported eosinophilia). We included an algorithmic approach to the diagnosis, including the diagnostic studies that should be performed in patients with or without organ involvement. Pathological consequences of eosinophilia are indicated in the next part of the paper. Finally, therapeutical options used in patients with eosinophilia are reviewed, with an special emphasis on antihelminthic therapies and the management of the hypereosinophilic syndromes.

Detection and discrimination of Loa loa, Mansonella perstans and Wuchereria bancrofti by PCR-RFLP and nested-PCR of ribosomal DNA ITS1 region.

The ribosomal deoxyribonucleic acid (DNA) internal transcribed spacer region (ITS1) of two filarial nematodes, Loa loa and Mansonella perstans, was amplified and further sequenced to develop an species-specific polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) protocol for the differentiation of both species from Wuchereria bancrofti, three filarial nematodes with blood circulating microfilariae. The ITS1-PCR product digested with the restriction endonuclease Ase I generated an specific diagnostic pattern for each of the three species. Moreover, three new specific nested-PCRs, targeting the ITS1 region, for differential detection of L. loa, M. perstans and W. bancrofti were developed and used when the ITS1-PCR products were insufficient for the Ase I enzymatic digestion. These filarial species-specific molecular protocols were evaluated in forty blood samples from African adult immigrants attending in the Hospital Insular of Gran Canaria, Canarias, Spain.

Rickettsia massiliae in the Canary Islands.

To the Editor: Rickettsia massiliae was recently recognized as a human tick-borne spotted fever group rickettsia (1). We report the finding of R. massiliae in Rhipicephalus pusillus ticks from Gran Canaria, Canary Islands, Spain. Introduction of this pathogen into the Canary Islands is thought to have resulted from translocation of the European wild rabbit Oryctolagus cuniculus (Linnaeus), a preferred host of R. pusillus ticks (www.kolonin.org/16_4.html), from the Iberian Peninsula 600 years ago (2). n nWe collected questing adult ticks in 2008 in Gran Canaria and identified 2 tick species, Hyalomma lusitanicum (n = 82 [46 females]) and R. pusillus (n = 8 [5 females]). Whole ticks were preserved in 70% ethanol and used for DNA extraction by using TriReagent (Sigma, St. Louis, MO, USA) according to the manufacturer’s instructions. We identified rickettsial sequences by using PCR primers that amplify fragments of 16S rRNA, ompB, atpA, dnaA, dnaK, and recA genes (Table). Amplicons were cloned into pGEM-T (Promega, Madison, WI, USA), and 3 independent clones were sequenced from both ends for each gene marker. Sequence similarity search was performed by using BLAST (www.ncbi.nlm.nih.gov). Rickettsial DNA was detected in 2 R. pusillus males only; sequences were identical in both ticks. Fragments of 16S rRNA were 99% identical to the R. massiliae strain Mtu5 ({type:entrez-nucleotide,attrs:{text:CP000683,term_id:157843848}}CP000683) isolated from R. sanguineus ticks in southern France (3), and fragments of ompB, atpA, dnaA, dnaK, and recA genes were 100% identical to the R. massiliae strain Bar29 ({type:entrez-nucleotide,attrs:{text:AF123710,term_id:6969935}}AF123710, {type:entrez-nucleotide,attrs:{text:AY124739,term_id:32345011}}AY124739, {type:entrez-nucleotide,attrs:{text:DQ821798,term_id:110433410}}DQ821798, {type:entrez-nucleotide,attrs:{text:DQ821828,term_id:110433470}}DQ821828, and {type:entrez-nucleotide,attrs:{text:AY124750,term_id:32345033}}AY124750, respectively), previously isolated from R. sanguineus ticks in Catalonia, Spain (4) (Table). n n n nTable n nRickettsia massiliae PCR conditions and amplicon size, Canary Islands, 2008* n n n nR. massiliae was first isolated in 1992 from R. sanguineus ticks collected near Marseille, France (5). Since then, the pathogen has been identified in different Rhipicephalus species in France, Greece, Portugal, Switzerland, Spain, North and Central Africa, Argentina, and the United States (6,7). R. massiliae has been identified in southern Spain (8) but not in the Canary Islands. R. pusillus ticks are commonly found in southern Europe (Portugal, Spain, and France) and northern Africa (Tunisia and Morocco). All stages of these ticks inhabit burrows of wild rabbits and feed on them (www.kolonin.org/16_4.html). n nWild rabbits were introduced into the Canary Islands at the end of 14th century during colonization by the kingdom of Castilla. Colonists were asked to bring rabbit couples with them to provide food in the islands (2), a practice continued by new colonists because of their interest in hunting this rabbit species. Introduction of wild rabbits by colonists led to establishment of parasites, such as helminths, coccidia, and viruses in the Canary Islands (9). R. pusillus, a common ectoparasite (tick) that feeds on wild rabbits on the Iberian Peninsula, was also introduced this way. R. massiliae could have been introduced in the islands by infected R. pusillus ticks or by infected wild rabbits if this species serves as a natural reservoir host for the pathogen. n nTo find evidence for this hypothesis, we tested blood and liver samples of 150 wild rabbits from both Canary Islands and Andalucia (southern Spain) by using Rickettsia-specific PCR primers (Table). No R. massiliae DNA was detected in the rabbit samples tested, suggesting that the pathogen probably was introduced in the Canary Islands with infected R. pusillus ticks feeding on rabbits. Alternatively, R. massiliae infection levels in wild rabbits may be below the PCR detection limit and were not detected. n nThe Canary Islands are a popular tourist destination. The presence of R. massiliae in the islands constitutes a risk for human infection and should be considered in hospital diagnostic and wildlife management strategies. As with other Rhipicephalus spp., R. pusillus ticks could feed on humans under certain circumstances (10). Our results emphasize the risks associated with unsupervised animal translocations, a factor that probably plays a role in the introduction of ticks and tick-borne pathogens in different parts of the world.

In vivo inhibition of inducible nitric oxide synthase decreases lung injury induced by Toxocara canis in experimentally infected rats.

The direct effect of nitric oxide (NO) on the viability of Toxocara canis larvae was studied. We observed that the nitric oxide donors, SIN‐1 and SNOG, exert no cytotoxic effect on the in vitro viability of T. canis larvae. In addition, we developed a model in rats to elucidate the role of NO during T. canis infection. We evaluated different indicators in four experimental groups: morphological parameters, the total number cells and cell types recovered, nitrite and protein concentration, lactate dehydrogenase and alkaline phosphatase enzymatic activity in the bronchoalveolar lavage fluid, lung index and detection of anti‐T. canis specific antibodies. We observed significant differences between non‐infected and infected groups. The infected animals treated with the inducible nitric oxide synthase (iNOS) inhibitor aminoguanidine were less damaged than infected, non‐treated animals. Our results suggest that the in vivo inhibition of the synthesis of NO triggered by iNOS diminishes the deleterious effects of the parasite upon the host, especially the vascular alterations in the lungs. We could show that in vivo production of NO induced by infection with T. canis results in direct host damage. Thus, this induction may constitute an evasion/adaptation mechanism of the parasite.

Antigens from Ascaris suum trigger in vitro macrophage NO production

We investigated the in vitro effect of total excretory/secretory and somatic antigens from Ascaris suum adults (ESA and SA) and larvae 3 (ESL3 and SL3), and of 10 purified protein fractions from ESA components on rat alveolar macrophage nitric oxide (NO) production. Our results showed that in vitro incubation of macrophages with SA and SL3 antigens of A. suum did not result in NO release from cells, whereas incubation with ESA or ESL3 antigens resulted in the stimulation of NO production by these cells, both in a specific (inhibited by l‐NAME and l‐canavanine) and dose‐dependent manner. In addition, we could demonstrate that a purified ESA fraction consisting of three Coomassie‐stained bands of approximately 37, 44 and 46 kDa is involved in the in vitro triggering of NO production by host cells. These three bands were subjected to MALDI‐peptide mass fingerprint, showing similarities with phosphoglycerate kinase, elongation factor Tu and enolase molecules, respectively. Future studies will focus on the characterization of these parasite‐derived molecules.

Evaluation of the role of angiogenic factors in the pathogenesis of schistosomiasis

Schistosomiasis is one disease produced by helminths, which affect many people in tropical areas. Granuloma formation is the main mechanism involved in the pathogenesis of this disease. Experimental studies have demonstrated angiogenesis (blood vessels formation from pre-existing vessels) in the initial phase of granuloma formation. In the present work, VEGF (vascular endothelial growth factor) levels were analyzed in sera from people diagnosed with different helminthic infections. Patients with schistosomiasis and filariasis had significantly high VEGF levels in compared with healthy people and patients diagnosed with hookworms. In addition, the effects of angiogenesis inhibition using anti-angiogenic factors (endostatin) were evaluated in a schistosomiasis murine model. A lesion decrease was observed in mice infected with Schistosoma mansoni and treated with endostatin. Finally, mechanisms of angiogenesis induction were studied and observed that cercariae antigens stimulated the angiogenic factors by host alveolar macrophages.

Application of an ELISA test using schistosoma bovis adult worm antigens in travellers and immigrants from a schistosomiasis endemic area and its correlation with clinical findings

We have recently evaluated an ELISA for the diagnosis of human schistosomiasis using S. bovis adult worm antigens (AWA Sb), showing a sensitivity of 94% and a specificity of 97% for patients diagnosed by egg detection. Nevertheless, the comparison of this AWA Sb ELISA with direct parasitological findings as the gold standard could introduce a selection bias, due to the well-known lack of sensitivity of direct methods in the detection of acute schistosomiasis and of low burden infections. The objective of the present work is to compare it with parasitological methods and commercial indirect haemagglutination test using S. mansoni antigens (WA Sm IHA) in 254 immigrants and travellers with different clinical settings; in addition, to find specific bands in the EITB of different phases of schistosomiasis. The AWA Sb ELISA showed 72% of seropositivity in patients with Katayama fever, while patients with eosinophilia and genito-urinary complaints showed 27% and 93%, respectively. The diagnosis yield was globally higher than direct egg detection or WA Sm IHA test with regard to the clinical setting. Finally, the utilization of EITB with S. bovis AWA permits the confirmation of diagnosis in chronic and acute phases of the disease.