Alessandra L. Morassutti

Eosinophilic meningitis caused by Angiostrongylus cantonensis: an emergent disease in Brazil.

Eosinophilic meningitis (EoM) is an acute disease that affects the central nervous system. It is primarily caused by infection with the nematode Angiostrongylus cantonensis. This infection was previously restricted to certain Asian countries and the Pacific Islands, but it was first reported in Brazil in 2007. Since then, intermediate and definitive hosts infected with A. cantonensis have been identified within the urban areas of many states in Brazil, including those in the northern, northeastern, southeastern and southern regions. The goals of this review are to draw the attention of the medical community and health centres to the emergence of EoM in Brazil, to compile information about several aspects of the human infection and mode of transmission and to provide a short protocol of procedures for the diagnosis of this disease.

Characterization of Angiostrongylus cantonensis excretory-secretory proteins as potential diagnostic targets

Angiostrongyliasis results from infections with intra-arterial nematodes that accidentally infect humans. Specifically, infections with Angiostrongylus cantonensis cause eosinophilic meningitis and Angiostrongylus costaricensis infections result in eosinophilic enteritis. Immunological tests are the primary means of diagnosing infections with either pathogen since these parasites are usually not recoverable in fecal or cerebrospinal fluid. However, well-defined, purified antigens are not currently available in sufficient quantities from either pathogen for use in routine immunodiagnostic assays. Since A. costaricensis and A. cantonensis share common antigens, sera from infected persons will recognize antigens from either species. In addition to their potential use in angiostrongyliasis diagnosis, characterization of these proteins that establish the host-parasite interphase would improve our understanding of the biology of these parasites. The main objective of the present work was to characterize A. cantonensis excretory-secretory (ES) products by analyzing ES preparations by two-dimensional gel electrophoresis coupled with immunoblotting using pools of positive sera (PS) and sera from healthy individuals (SC). Protein spots recognized by PS were excised and analyzed by electrospray ionization (ESI) mass spectrometry. MASCOT analysis of mass spectrometry data identified 17 proteins: aldolase; CBR-PYP-1 protein; beta-amylase; heat shock protein 70; proteosome subunit beta type-1; actin A3; peroxiredoxin; serine carboxypeptidase; protein disulfide isomerase 1; fructose-bisphosphate aldolase 2; aspartyl protease inhibitor; lectin-5; hypothetical protein F01F1.12; cathepsin B-like cysteine proteinase 1; hemoglobinase-type cysteine proteinase; putative ferritin protein 2; and a hypothetical protein. Molecular cloning of these respective targets will next be carried out to develop a panel of Angiostrongylus antigens that can be used for diagnostic purposes and to further study host-Angiostrongylus interactions.

The 31-kDa Antigen of Angiostrongylus cantonensis Comprises Distinct Antigenic Glycoproteins

Human angiostrongyliasis results from accidental infection with Angiostrongylus, an intra-arterial nematode. Angiostrongylus cantonensis infections result in eosinophilic meningitis, and A. costaricensis infections cause eosinophilic enteritis. Immunological methodologies are critical to the diagnosis of both infections, since these parasites cannot be isolated from fecal matter and are rarely found in cerebrospinal fluid samples. A. costaricensis and A. cantonensis share common antigenic epitopes which elicit antibodies that recognize proteins present in either species. Detection of antibodies to a 31-kDa A. cantonensis protein present in crude adult worm extracts is a sensitive and specific method for immunodiagnosis of cerebral angiostrongyliasis. The objective of the present work was to isolate and characterize the 31-kDa proteins using soluble protein extracts derived from adult female worms using both one- (1DE) and two-dimensional (2DE) gel electrophoresis. Separated proteins were blotted onto nitrocellulose and probed using sera from infected and non-infected controls. The 31-kDa band present in 1DE gels and the 4 spots identified in 2DE gels were excised and analyzed by electrospray ionization mass spectrometry. Using the highest scores obtained following Mascot analysis, amino acid sequences were obtained that matched four unique proteins: tropomyosin, the 14-3-3 phosphoserine-binding protein, a protein containing a nascent polypeptide-associated complex domain, and the putative epsilon subunit of coatomer protein complex isoform 2. Oxidative cleavage of diols using sodium m-periodate demonstrated that carbohydrate moieties are essential for the antigenicity of all four spots of the 31-kDa antigen. In this article we describe the identification of the 31-kDa antigen, and provide DNA sequencing of the targets. In conclusion, these data suggest that reactivity to the 31-kDa proteins may represent antibody recognition of more than one protein, and recombinant protein-based assays for cerebral angiostrongyliasis diagnosis may require eukaryotic expression systems to maintain antigenicity.

Longitudinal clinical and serological survey of abdominal angiostrongyliasis in Guaporé, southern Brazil, from 1995 to 1999

Abdominal angiostrongyliasis is a zoonotic infection caused by Angiostrongylus costaricensis, a nematode with an intra-vascular location in the mesentery. Our objective was to address several aspects of the natural history of this parasitosis, in a longitudinal clinical and seroepidemiological study. A total of 179 individuals living in a rural area with active transmission in southern Brazil were followed for five years (1995-1999) resulting in yearly prevalence of 28.2%, 4.2%, 10%, 20.2% and 2.8% and incidences of 0%, 5.9%, 8% and 1.5%, respectively. Both men and woman were affected with higher frequencies at age 30-49 years. In 32 individuals serum samples were collected at all time points and IgG antibody reactivity detected by ELISA was variable and usually persisting not longer than one year. Some individual antibody patterns were suggestive of re-infection. There was no association with occurrence of abdominal pain or of other enteroparasites and there was no individual with a confirmed (histopathologic) diagnosis. Mollusks were found with infective third-stage larvae in some houses with an overall prevalence of 16% and a low parasitic burden. In conclusion, abdominal angiostrongyliasis in southern Brazil may be a frequent infection with low morbidity and a gradually decreasing serological reactivity.

Interface Molecules of Angiostrongylus cantonensis: Their Role in Parasite Survival and Modulation of Host Defenses.

Angiostrongylus cantonensis is a nematode parasite that causes eosinophilic meningoencephalitis in humans. Disease presents following the ingestion of third-stage larvae residing in the intermediate mollusk host and disease manifests as an acute inflammation of the meninges characterized by eosinophil infiltrates which release a battery of proinflammatory and cytotoxic agents in response to the pathogen. As a mechanism of neutralizing these host defenses, A. cantonensis expresses different molecules with immunomodulatory properties that are excreted or secreted (ES). In this paper we discuss the role of ES proteins on disease exacerbation and their potential use as therapeutic targets.

Workshop on research priorities for management and treatment of angiostrongyliasis(1).

In a concluding session of the workshop, the participants developed a list of 115 research and outreach needs, outlining the top 5-7 needs in each of 8 areas (Table). For complete information, including presenter details and abstracts, visit the workshop website at www.hawaii.edu/cowielab/Angio%20website%20home.htm.

Update on Baylisascariasis, a Highly Pathogenic Zoonotic Infection

SUMMARY Baylisascaris procyonis, the raccoon roundworm, infects a wide range of vertebrate animals, including humans, in which it causes a particularly severe type of larva migrans. It is an important cause of severe neurologic disease (neural larva migrans [NLM]) but also causes ocular disease (OLM; diffuse unilateral subacute neuroretinitis [DUSN]), visceral larva migrans (VLM), and covert/asymptomatic infections. B. procyonis is common and widespread in raccoons, and there is increasing recognition of human disease, making a clinical consideration of baylisascariasis important. This review provides an update for this disease, especially its clinical relevance and diagnosis, and summarizes the clinical cases of human NLM and VLM known to date. Most diagnosed patients have been young children less than 2 years of age, although the number of older patients diagnosed in recent years has been increasing. The recent development of recombinant antigen-based serodiagnostic assays has aided greatly in the early diagnosis of this infection. Patients recovering with fewer severe sequelae have been reported in recent years, reinforcing the current recommendation that early treatment with albendazole and corticosteroids should be initiated at the earliest suspicion of baylisascariasis. Considering the seriousness of this zoonotic infection, greater public and medical awareness is critical for the prevention and early treatment of human cases.

Detection of anti-oxidant enzymatic activities and purification of glutathione transferases from Angiostrongylus cantonensis.

There are several anti-oxidant enzyme families that play pivotal roles in facilitating the survival of parasites. Glutathione transferases (GSTs) are members of the anti-oxidant family that can detoxify a broad range of exogenous or endogenous compounds including reactive oxidative species. GSTs have been studied as vaccine candidates, immunodiagnostic markers and as treatment targets. Helminths of the genus Angiostrongylus live inside arteries of vertebrates and two main species are associated with accidental human infections: Angiostrongylus costaricensis adult worms live inside the mesenteric arteries and larvae of Angiostrongylus cantonensis become trapped in the central nervous system vasculature. Since the interactions between angiostrongylid nematodes and their vertebrate hosts are poorly understood, this study characterized the anti-oxidant enzymatic activities of A. cantonensis from female worms by collecting excreted and secreted (ES) and total extract (TE) molecules. Catalase (CAT) and superoxide dismutase (SOD) activities were found both in the ES and TE while glutathione peroxidase (GPX) and GST were found only in the TE. GSTs were purified by glutathione agarose affinity column (AcGST) and the pool of eluted GSTs was analyzed by mass spectrometry (LC-MS/MS) and de novo sequencing (Masslynx software). Sequences from two peptides (AcGSTpep1 and AcGSTpep2) present high identity to the N-terminal and C-terminal from sigma class GSTs of nematodes. It is known that these GST enzymes are associated with host immune regulation. Furthermore, understanding the role of parasite-derived anti-oxidant molecules is important in understanding host-parasite interactions.

High throughput sequencing of the Angiostrongylus cantonensis genome: a parasite spreading worldwide

Angiostrongylus cantonensis is a parasitic nematode of rodents and a leading aetiological agent of eosinophilic meningitis in humans. Definitive diagnosis is difficult, often relying on immunodiagnostic methods which utilize crude antigens. New immunodiagnostic methods based on recombinant proteins are being developed, and ideally these methods would be made available worldwide. Identification of diagnostic targets, as well as studies on the biology of the parasite, are limited by a lack of molecular information on Angiostrongylus spp. available in databases. In this study we present data collected from DNA random high-throughput sequencing together with proteomic analyses and a cDNA walking methodology to identify and obtain the nucleotide or amino acid sequences of unknown immunoreactive proteins. 28 080 putative ORFs were obtained, of which 3371 had homology to other deposited protein sequences. Using the A. cantonensis genomic sequences, 156 putative ORFs, matching peptide sequences obtained from previous proteomic studies, were considered novel, with no homology to existing sequences. Full-length coding sequences of eight antigenic target proteins were obtained. In this study we generated not only the complete nucleotide sequences of the antigenic protein targets but also a large amount of genomic data which may help facilitate future genomic, proteomic, transcriptomic or metabolomic studies on Angiostrongylus.

Acute and chronic exposure to Tyrophagus putrescentiae induces allergic pulmonary response in a murine model.

Background Tyrophagus putrescentiae (Tp) is a source of aeroallergen that causes allergic diseases. Objective To describe an acute and chronic murine model of allergic asthma with Tp extract with no systemic sensitization and no use of adjuvant. Methods Mites from dust sample were cultured and a raw extract was produced. Female BALB/c mice (6-8 weeks) were challenged intranasally with Tp extract or Dulbeccos phosphate-buffered saline, for 10 consecutive days (acute protocol) or for 6 weeks (chronic protocol). Twenty-four hours after the last intranasal challenge, bronchoalveolar lavage fluid (BALF) was performed for total and differential cells count, cytokine analysis, and eosinophil peroxidase activity. Lung tissue was also removed for histopathologic analysis. Results Tp extract has shown a significant increase in total cells count from BALF as well as an increase in absolute eosinophils count, eosinophil peroxidase activity, interleukin (IL)-5 and IL-13 levels, in both acute and chronic protocols. Peribronchovascular infiltrate, goblet cells hyperplasia and collagen deposition were shown in the airways of acute and chronic Tp-exposed mice. Conclusion Our data suggest that the intranasal exposure to Tp extract, with no systemic sensitization and no use of adjuvants, induces a robust allergic inflammation in the lungs of mice, in both acute and chronic models. Our Tp extract seems to be a potent allergen extract which may be used in asthma model studies.