Seroprevalence of Toxocara antibodies among patients suspected of ocular toxocariasis in Slovenia

Ocular toxocariasis named also ocular larva migrans is caused by larvae of the roundworm Toxocara spp. The purpose of this study was to find out the seroprevalence of Toxocara antibodies in patients suspected of ocular toxocariasis. Between January 2001 and December 2003, sera from 239 ocular patients, aged 3 to 80 years, were examined by ELISA and confirmed by Western blot test. Out of the 239 patients, 172 (72%) were seronegative and 67 (28%) were Toxocara seropositive; 95% CI (22-34%). The median age of Toxocara seropositive patients was 37.6 years. There was no significant difference in the number of Toxocara positive sera between the younger age group (< or = 14 years) and the older age group (> 14 years), p > 0.05. A high rate of Toxocara seropositivity in ocular patients should alert the ophthalmologists in Slovenia to include toxocariasis in the differential diagnosis of eye diseases more frequently.     

Toxocara canis: proteolytic enzymes secreted by the infective larvae in vitro

Second-stage larvae of the dog nematode Toxocara canis are infective to man and cause the syndromes of visceral larva migrans and ocular toxocariasis. Larvae cultured in vitro secrete proteases which degrade components of a model of extracellular matrix and basement membranes. These enzymes have been characterized using a variety of techniques. Multiple enzyme activities were demonstrated by substrate gel electrophoresis, associated with proteins of molecular weights of 120 and 32 kDa. The enzyme activity was inhibited both in substrate gels and in a radiogelatin microplate assay by phenylmethylsulfonyl fluoride. Optimal activity occurred at pH 9, with minor activities apparent at pH 5 and 7; the relationship between these proteolytic activities is currently under investigation.     

The epidemiology and public health importance of toxocariasis: A zoonosis of global importance

Toxocariasis, caused by infection with larvae of Toxocara canis, and to a lesser extent by Toxocara cati and other ascaridoid species, manifests in humans in a range of clinical syndromes. These include visceral and ocular larva migrans, neurotoxocariasis and covert or common toxocariasis. Toxocara canis is one of the most widespread public health and economically important zoonotic parasitic infections humans share with dogs, cats and wild canids, particularly foxes. This neglected disease has been shown through seroprevalence studies to be especially prevalent among children from socio-economically disadvantaged populations both in the tropics and sub-tropics and in industrialised nations. Human infection occurs by the accidental ingestion of embryonated eggs or larvae from a range of wild and domestic paratenic hosts. Most infections remain asymptomatic. Clinically overt infections may go undiagnosed, as diagnostic tests are expensive and can require serological, molecular and/or imaging tests, which may not be affordable or available. Treatment in humans varies according to symptoms and location of the larvae. Anthelmintics, including albendazole, thiabendazole and mebendazole may be given together with anti-inflammatory corticosteroids. The development of molecular tools should lead to new and improved strategies for the treatment, diagnosis and control of toxocariasis and the role of other ascaridoid species in the epidemiology of Toxocara spp. Molecular technologies may also help to reveal the public health importance of T. canis, providing new evidence to support the implementation of national control initiatives which have yet to be developed for Toxocara spp. A number of countries have implemented reproductive control programs in owned and stray dogs to reduce the number of young dogs in the population. These programs would positively impact upon T. canis transmission since the parasite is most fecund and prevalent in puppies. Other control measures for T. canis include the regular and frequent anthelmintic treatment of dogs and cats, starting at an early age, education and enforcement of laws for the disposal of canine faeces, dog legislation and personal hygiene. The existence of wild definitive and paratenic hosts complicates the control of T. canis. Increasing human and dog populations, population movements and climate change will all serve to increase the importance of this zoonosis. This review examines the transmission, diagnosis and clinical syndromes of toxocariasis, its public health importance, epidemiology, control and current research needs.     

Toxocariasis in North America: A Systematic Review

Toxocariasis is an important neglected tropical disease that can manifest as visceral or ocular larva migrans, or covert toxocariasis. All three forms pose a public health problem and cause significant morbidity in areas of high prevalence. To determine the burden of toxocariasis in North America, we conducted a systematic review of the literature following PRISMA guidelines. We found 18 articles with original prevalence, incidence, or case data for toxocariasis. Prevalence estimates ranged from 0.6% in a Canadian Inuit community to 30.8% in Mexican children with asthma. Commonly cited risk factors included: African-American race, poverty, male sex, and pet ownership or environmental contamination by animal feces. Increased prevalence of Toxocara spp. infection was linked in a group of case control studies conducted in Mexico to several high risk groups including waste pickers, asthmatic children, and inpatient psychiatry patients. Further research is needed to determine the true current burden of toxocariasis in North America; however the prevalence estimates gathered in this review suggest that the burden of disease is significant.     

Development of a Luminex Bead Based Assay for Diagnosis of Toxocariasis Using Recombinant Antigens Tc-CTL-1 and Tc-TES-26

The clinical spectrum of human disease caused by the roundworms Toxocara canis and Toxocara cati ranges from visceral and ocular larva migrans to covert toxocariasis. The parasite is not typically recovered in affected tissues, so detection of parasite-specific antibodies is usually necessary for establishing a diagnosis. The most reliable immunodiagnostic methods use the Toxocara excretory-secretory antigens (TES-Ag) in ELISA formats to detect Toxocara-specific antibodies. To eliminate the need for native parasite materials, we identified and purified immunodiagnostic antigens using 2D gel electrophoresis followed by electrospray ionization mass spectrometry. Three predominant immunoreactive proteins were found in the TES; all three had been previously described in the literature: Tc-CTL-1, Tc-TES-26, and Tc-MUC-3. We generated Escherichia coli expressed recombinant proteins for evaluation in Luminex based immunoassays. We were unable to produce a functional assay with the Tc-MUC-3 recombinant protein. Tc-CTL-1 and Tc-TES-26 were successfully coupled and tested using defined serum batteries. The use of both proteins together generated better results than if the proteins were used individually. The sensitivity and specificity of the assay for detecting visceral larval migrans using Tc-CTL-1 plus Tc-TES-26 was 99% and 94%, respectively; the sensitivity for detecting ocular larval migrans was 64%. The combined performance of the new assay was superior to the currently available EIA and could potentially be employed to replace current assays that rely on native TES-Ag.     

Visceral larva migrans (toxocariasis) in Toronto.

A 7-year-old child was admitted to Toronto''s Hospital for Sick Children in 1976 with symptoms and laboratory findings compatible with visceral larva migrans, a disease usually caused by Toxocara canis. This prompted a search for other cases seen at the hospital during the period 1952 through 1978. Only 18 cases were discovered that met at least three of six criteria and thus were considered possible or probably cases of the disease. Three possible cases of ocular toxocariasis during the same period were also uncovered. Fever was the commonest presenting symptom. Eosinophilia, leukocytosis and hyperglobulinemia were the most frequent laboratory findings. In view of the small number of cases found in 27 years at this large pediatric hospital with a broad referral base, it is concluded that visceral larva migrans poses little risk to the health of children in the Toronto area.     

Demonstration of antibodies during experimental toxocarosis using antigenic extract prepared from the adult parasit]

The detection of antibodies in subjects with visceral larva migrans syndrome caused by Toxocara canis is usually carried out with antigenic larvae, whose preparation presents some difficulties. Results obtained using extracts prepared from adult parasites show that such antigens are quite suitable for the detection of antibodies present in visceral larva migrans syndrome. This phenomenon is easily understood when one considers that there are numerous antigenic points in common between the different stages of the evolution of this parasite.     

An improved method to obtain antigen-excreting Toxocara canis larvae

Toxocara canis is a dog helminth which causes visceral larva migrans (VLM) when infecting humans as a larva. The infection is demonstrated by detecting IgG antibodies against excretory-secretory larval antigens (ESLA) in serum by ELISA. The production of ESLA involves the collection of adult worms from dog puppy stools, the separation of eggs from dissected uteri, and the in vitro growing of egg-derived larvae, following the time-consuming and laborious protocol described by De Savigny [De Savigny, D.H., 1975. In vitro maintenance of T. canis larvae and a simple method for the production of Toxocara ES antigen for the uses in serodiagnostic tests for visceral larva migrans. Journal of Parasitology 61, 781-782]. In this work, an improved protocol for obtaining T. canis larvae is described. The modifications proposed improved the efficiency of the original De Savigny method in three ways: (i) increasing the parasite yield up to five fold, (ii) improving the larval purity, and (iii) markedly reducing the execution time of the protocol.     

Analysis of Toxocara canis larval excretory-secretory antigens: physicochemical characterization and antibody recognition

Toxocara canis larval excretory-secretory antigens (TEX) were resolved by gradient pore polyacrylamide gel electrophoresis and analyzed using silver, periodic acid-Schiff, and immunoperoxidase stains. At least 15 bands between 29 and 94 kilodaltons (kDa) were detected by silver stain, all of which were recognized by antibodies in serum of a patient with visceral larva migrans. Immunoperoxidase stain detected an additional band at 92 kDa and 4-6 others above 200 kDa. Periodic acid-Schiff stain also detected the high molecular weight components, but did not detect constituents of approximately 53 and 57 kDa. Immunoperoxidase stain using antibody from the vitreous fluid of an ocular larva migrans patient detected 2 TEX components, approximately 76 and 80 kDa. Antigens were compared with respect to batch of larvae and age of larvae in culture. Qualitative differences that correlated with batch were found in the number of constituents above 200 kDa, and in 1 component of 78 kDa. Qualitative differences were noted in many minor components, some of which appeared to correlate with age of larvae in culture. Major TEX constituents were recognized consistently by antibody, regardless of batch or age of larvae. Total protein production per larva was approximately 8 ng/day, and was consistent over time. There was no evidence of neutral proteases in TEX.     

Visceral larva migrans: examinations by means of enzyme-linked immunosorbent assay of human sera for antibodies to excretory-secretory antigens of the second-stage larvae of Toxocara canis

An enzyme-linked immunosorbent assay is described for the detection of serum antibodies to visceral larva migrans (Toxocariasis). Excretory-secretory antigens of the second-stage larvae of Toxocara canis were used as antigen to coat the polystyrene plates. With sera from patients high antibody titers were observed in both ocular and visceral disorders. Cross-reactions due to other parasitic infections could be excluded, including other migrating larval infections such as ascariasis, trichinellosis, strongyloidiasis, filariasis, and anisakiasis. In a small seroepidemiologic survey of healthy primary schoolchildren, a remarkably high percentage (7.1) reacted positively to this method. These children showed eosinophilia as compared to the seronegative group. The data were compared with those observed in other countries and the results prompt reconsideration of the significance of T. canis for public health.     

Five cases of ocular toxocariasis confirmed by serology

We report 5 cases of ocular toxocariasis in Korean adults complaining of visual impairment along with floating or bubbling sensation. Fundoscopic examination revealed a retinal detachment along with exudate in 4 cases. They all showed typical reaction by ELISA and immunoblot against Toxocra excretory-secretory antigen. One case showed high level of anti-Toxocara IgE antibodies (34,000 Toxocara units/L) as well as increased level of serum total IgE antibodies and the specific IgE antibodies for 3 inhalant antigens, suggesting that high level of anti-Toxocara IgE antibodies was associated with an atopic status. Clinical manifestations were improved after the sequential use of steroids then mebendazole. We also suggest that ocular toxocariasis should be thoroughly investigated even when an evocative uniocular inflammatory lesion is encountered in peripheral retina without a systematic disease.     

Nematodes transmitted to man by fish and aquatic mammals

Zoonotic nematodes may cause disease in man through migrating larva (larva migrans), through direct infection or possibly through allergic responses. The parasitic genera Ancylostoma, Uncinaria, Bunostomum and Toxocara can cause larva migrans. The cod worm (Phocanema decipiens) a parasite found in fish and seals, can infect man, as can Anisakis, Dioctophyme renale and Gnathostoma hispidium larvae obtained from eating raw fish.     

A familial case of visceral toxocariasis due to consumption of raw bovine liver

We present 3 adult cases of visceral toxocariasis from the same family, who each consumed thin slices of raw bovine liver weekly, and developed eosinophilia and multiple small lesions in their livers and lungs. Serological examinations using the larval excretory–secretory product of Toxocara canis strongly indicated infection with Toxocara species larvae. The patients responded well to treatment with albendazole. Ingestion of raw liver from paratenic animals is considered to be a common transmission route of human toxocariasis, especially in adults.     

Toxocara canis: search for a potential drug amongst beta-carboline alkaloids--in vitro and mouse studies

The goal of this study was to search for new treatments for Toxocara canis using both in vitro and in vivo experiments. We specifically looked for a treatment for T. canis larva migrans, and examined beta-carboline alkaloids (17 compounds) with various structural modifications, both in in vitro and in vivo experiments. In the in vitro experiments, screening for nematocidal activity on the T. canis second stage larvae, cytotoxic activity, and immune activity in the host were undertaken. Compound 17 was selected, as it exhibited nematocidal activity for T. canis larvae and did not have any cytotoxic or immunosuppressive activity in the host. The effectiveness of compound 17 was then examined using T. canis larvae infected mice in in vivo experiments. To evaluate the anthelmintic effect, the relative mobility value for the larvae was examined in addition to the number of larvae in the brain, skeletal muscle, and liver. Compound 17 was also examined in both free and liposome-entrapped (LE) forms. Polyethylene glycol (PEG)-LE compound 17 showed an anthelmintic effect in which the number of larvae in the brain was decreased compared free albendazole. PEG-LE compound 17 also effectively suppressed the mobility of the larva in brain and skeletal muscle. The experimental procedure employed assisted in the discovery of this potential candidate and is a promising approach for finding alternative therapeutic regimens for T. canis larva migrans.     

Toxocariasis in humans: clinical expression and treatment dilemma

A new scheme of clarifying clinical forms of toxocariasis is proposed to include: (i) systemic forms: classical VLM and incomplete VLM; (ii) compartmentalized forms: ocular and neurological toxocariasis; (iii) covert toxocariasis; and (iv) asymptomatic toxocariasis. The following markers are helpful in defining clinical forms namely, patient characteristics and history, clinical symptoms and signs, positive serology, eosinophilia and increased levels of IgE. Amongst the available drugs albendazole is the most commonly used, although other benzimidazole compounds have a similar efficacy. The recommended dose of albendazole is 15 mg kg(-1) body weight daily for 5 days and in some cases with VLM syndrome the treatment needs to be repeated. An evaluation of treatment efficacy can be made by observing a rise in eosinophilia within a week followed by any improvement in clinical symptoms and signs, lower eosinophilia and serological tests taken over a period of at least 4 weeks. In addition to clinical rationales for the specific treatment of VLM and OLM, preventive treatment needs to be considered bearing in mind the increasing risk of larvae localizing in the brain during the course of an infection. To reduce migration of Toxocara larvae a single course of albendazole is suggested in cases where eosinophilia and serology are at least moderately positive.     

A Seroepidemiological Survey of Toxocariasis among Eosinophilia Patients in Chungcheongnam-do

There have been several reports on the relationship between toxocariasis and eosinophilia, but all have been limited to the areas of Seoul or Gangwon-do. In the present study, we investigated the seroprevalence of toxocariasis among eosinophilia patients in Chungcheongnam-do, the central district of Korea. Among the 101 patients tested, 51 (50.5%) were identified as positive by Toxocara ELISA, and 46 (45.5%) were confidently diagnosed with toxocariasis because of absence of any other cause of eosinophilia. Whereas 22 of 42 seropositive patients (52.3%) had a recent history of consuming raw livers, especially the cow liver, only 1 of 25 seronegative patients (4%) had done so (P<0.01). From these results, we could confirm that toxocariasis is related to eosinophilia, and infer that ingestion of raw cow liver plays a vital role in the transmission of toxocariasis in Chungcheongnam-do.     

Toxocara canis: monoclonal antibodies to larval excretory-secretory antigens that bind with genus and species specificity to the cuticular surface of infective larvae

When maintained in culture, the infective-stage larvae of Toxocara canis produce a group of excretory-secretory antigens. Monoclonal antibodies to these antigens have been produced and partially characterized. Hybridomas were made using spleens from mice that had been given 250 embryonated eggs of T. canis followed by immunization with excretory-secretory antigens. Monoclonal antibodies were first screened against excretory-secretory antigens using an indirect enzyme-linked immunosorbent assay. Those antibodies positive in this assay were then screened against the surfaces of formalin-fixed, infective-stage larvae using an indirect fluorescent antibody assay. The two monoclonal antibodies showing fluorescence were also tested against the surfaces of infective-stage larvae of Toxocara cati, Baylisascaris procyonis, Toxascaris leonina, Ascaris suum, a Porrocaecum sp., and Dirofilaria immitis. One of these two antibodies bound to the surface of T. canis and T. cati while the other bound only to the surface of T. canis; neither were reactive with the other ascaridoid larvae or the larvae of D. immitis. Enzyme-linked immunoelectrotransfer blotting techniques were used to demonstrate that the cross-reactive antibody recognized antigens with molecular weights of about 200 kDa while the more specific monoclonal antibody recognized antigens with approximate molecular weights of 80 kDa. The specificity of these two antibodies for T. canis and T. cati should prove helpful in the development of more specific assays for the diagnosis of visceral and ocular larva migrans.     

Baylisascaris procyonis (Nematoda: Ascarididae) larva migrans in free-ranging wildlife in Orange County,California

Baylisascaris procyonis is well recognized as a cause of visceral (VLM), ocular, and neural (NLM) larva migrans in birds and mammals, including man. A study of the prevalence of larva migrans in free-ranging wildlife associated with raccoon latrines as well as a retrospective study of wildlife mortalities with neurological disease was conducted in 2000 in Orange County, California. Eighty-seven birds of 18 species and 64 mammals of 8 species were found to have NLM or VLM or both. NLM clinical signs included convulsions, torticollis, opisthotonus, head-tilts, circling, ataxia, paralysis, and visual defects. NLM lesions were characterized by focally disseminated, frequently linear, "tracklike" areas of parenchymal degeneration with varying degrees of astrocytosis in the white matter of the cerebrum, cerebellum, brain stem, and spinal cord. Larvae were rarely found in these lesions but were rather isolated in the brain-spinal cord parenchyma. At least 1 larva was isolated by digestion from each case of NLM and identified as Baylisascaris sp., most likely B. procyonis. VLM lesions consisted of granulomatous reactions surrounding intact or degenerative larvae in the parenchyma of the liver, kidney, diaphragm, and, occasionally, the lymph nodes. This report broadens the range of species of wild birds and mammals that have been found to be susceptible to larva migrans caused by B. procyonis and reaffirms the importance of raccoon latrine sites as contaminative foci for wildlife.     

Serodiagnosis of Toxocariasis by ELISA Using Crude Antigen of Toxocara canis Larvae

Toxocariasis is a worldwide zoonosis caused by larvae of ascarid nematodes of dogs or cats, Toxocara canis or T. cati. Diagnosis of human toxocariasis currently relies on serology that uses T. canis excretory-secretory antigen to detect specific IgG antibodies by ELISA. We investigated the serodiagnostic efficacy of ELISA using crude antigen of T. canis larvae (TCLA). Serum specimens of 64 clinically confirmed toxocariasis, 115 healthy controls, and 119 other tissue-invading helminthiases were screened by ELISA using TCLA. The ELISA using TCLA showed 92.2% (59/64 patient samples) sensitivity and 86.6% (103/119) specificity. Its positive diagnostic predictivity was 78.7% and negative predictivity was 97.8%. No serum of healthy controls reacted but that of anisakiasis (45.5%), gnathostomiasis (19.2%), clonorchiasis (15.8%), sparganosis (11.1%), and cysticercosis (6.3%) cross-reacted. Immunoblot analysis on TCLA recognized antigenic proteins of 28- and 30-kDa bands in their dominant protein quantity and strong blotting reactivity. The present results indicate that the ELISA using our TCLA antigen is acceptable by the sensitivity and specificity for serodiagnosis of human toxocariasis. ELISA with TCLA is recommended to make differential diagnosis for patients with any sign of organ infiltration and eosinophilia.     

Toxocara canis: Larval migration dynamics, detection of antibody reactivity to larval excretory-secretory antigens and clinical findings during experimental infection of gerbils (Meriones unguiculatus)

In this study, Mongolian gerbils were used to analyse features of Toxocara infection that included larval migration, humoral immune responses to Toxocara canis excretory-secretory antigens (TES) and aspects of host physiology. At day 10 post-infection (p.i.) most larvae were in the intestine and the lungs while later the total number of larvae was higher in the carcass tissue; the number of larvae per gram of tissue was lower elsewhere other than in the brain. Infected animals showed several neurological abnormalities, an early increase in leukocyte and neutrophil levels, two peaks of peripheral eosinophilia (5 and 40 d.p.i.) and high antibody levels against TES in the circulation and in the vitreous humor. A sequential recognition of eight T.canis larval antigens with MW from 24 to 200 kDa was detected by Western blot. The results obtained in this study further support the use of gerbils as an experimental model for systemic, ocular and cerebral toxocariasis.