|Year : 2014 | Volume
| Issue : 4 | Page : 383-386
Research on Toxocara Canis antibodies obtained from patients with eosinophilia
E Artinyan, H Kirkoyun Uysal, O Akgul, S Altiparmak, YA Oner
Department of Parasitology, Medical Microbiology, Istanbul Faculty of Medicine, Istanbul University Istanbul, Turkey
|Date of Submission||21-Mar-2013|
|Date of Acceptance||09-Aug-2013|
|Date of Web Publication||4-Oct-2014|
H Kirkoyun Uysal
Department of Parasitology, Medical Microbiology, Istanbul Faculty of Medicine, Istanbul University Istanbul
Source of Support: None, Conflict of Interest: None
Background: Eosinophils may suggest the presence of a great variety of anomalies whereupon allergies, malignancies, certain tissue disorders, idiopathic hypereosynophilic syndrome and parasitic infections (with the exception of protozoons) can be cited as a few. Although the clinical manifestations may differ, the eosinophils level is quite an important data in cases related to the helminth infections. Similarly, in parasitic infections related to larva migrans (visceral, cutaneous, ocular), the eosinophils level is again a primary indicator and its evident cause is the roundworm Toxocara spp. Aim: The aim of this study was to evaluate the significance characteristics of Toxocara spp. in patients with eosonophilia. Materials and Methods: In our study, serums were collected from 93 patients of various age groups with eosonophilia (10% and above) while visiting Istanbul University Medical Faculty due to various complaints. Results: Serum samples were treated with Toxocara IgG ready ELISA kit and Toxocara western blot IgG ready kit. Based on the ELISA method; out of 93 patients, 30 patients (32.3%) were positive. Western blot technique; 45 (48.4%) were positive with anti-toxocara IgG antibodies. Conclusion: Results point out to western blot technique being more sensitive and superior on a scale of (P < 0.0001) when compared with the ELISA method.
Keywords: ELISA, Toxocara, western blot
|How to cite this article:|
Artinyan E, Uysal H K, Akgul O, Altiparmak S, Oner Y A. Research on Toxocara Canis antibodies obtained from patients with eosinophilia. Indian J Med Microbiol 2014;32:383-6
|How to cite this URL:|
Artinyan E, Uysal H K, Akgul O, Altiparmak S, Oner Y A. Research on Toxocara Canis antibodies obtained from patients with eosinophilia. Indian J Med Microbiol [serial online] 2014 [cited 2020 May 28];32:383-6. Available from: http://www.ijmm.org/text.asp?2014/32/4/383/142239
| ~ Introduction|| |
Eosinophils comprise 2-5% of leucocyte cells and their levels in blood are strictly controlled.  This percentage increase in peripheral blood cells is known as eosynophillia. Eosinophils may suggest the presence of a great variety of anomalies whereupon allergies, malignancies, certain tissue disorders, idiopathic hypereosynophilic syndrome and parasitic infections (with the exception of protozoons) can be cited as a few. , Although the clinical manifestations may differ, the eosinophils level is quite an important data in cases related to the helminth infections.  Eosinophils may either have a harmful effect due to their pro-inflamatory tendency or a beneficial one due to their anti-parasitic nature. Similarly, in parasitic infections related to larva migrans (visceral, cutaneous, ocular), the eosinophils level is again a primary indicator and its evident cause is the roundworm Toxocara spp.
Larva migrans can be defined as Toxocara spp. moving inwards to the internal organs, eyes or the epiderm as a result of which certain pathological disorders start to demonstrate. The laboratory data are indicated by eosinophils, leucocytosis, hypergammaglobinemia (IgG, IgM), elevated serum IgE levels (average 850IU/ml) and positive Toxocara antibody titrates. ,,
| ~ Materials and Methods|| |
In our study, sera were collected from 93 patients of various age groups with eosinophilia (10% and above) while visiting Istanbul University Medical Faculty due to various complaints (criteria of patients such as tiredness, abdominal pain, high levels of total IgE and variety of allergic symptoms). Serum samples were treated according to the test procedure set by r-Biopharm RIDASCREEN (Germany) Toxocara IgG ready ELISA kit. Purified antigens are coated to a microwell plate. Antibodies in the patient samples bind to the antigens and are determined during the second step by using enzyme-labelled protein A. The enzyme converts the colourless substrate to a blue end product. The enzyme reaction is stopped by adding sulphuric acid and the colour of the mixture switches from blue to yellow at the same time. The final measurement is carried out at 450 nm on a photometer using a reference wavelength ≥620 nm. The cut-off value measured as median of optic density of two negative controls by adding 0.150 optic density (OD) value that was proposed in test procedure. Optic densities of serum samples, which were recorded before, sample ratio obtained by dividing to cut-off value. According to procedure, ratios are accepted as <0.9 negative, ratios >1.1 as positive and ratios 0.9-1.1 as equivocal. Sample ratio higher than 1.1 were found in 45 of 93 serum samples in the study. Specificity of test is 98.4% and sensitivity of test is 100%.
Moreover, the same 93 patient's sera were treated with Toxocara western blot (WB) IgG (TXA-WB24G) ready kit by LDBIO DIAGNOSTIC (France). The remarkable sensitivity of the immunoblot technique and its usefulness for demonstrating differences in antigen recognition by antibodies led to the use of it for Toxocara serology testing. In brief, the strips were incubated for 2h at room temperature, with sera at a dilution of 1/100. Before being washed at least three times in phosphate buffered saline (PBS) containing 0.1% Tween 20, the strips were further incubated for 2h at room temperature with the second antibody, anti-human immunoglobulin G peroxidase conjugate (dilution 1/1000). After washing as mentioned previously, the substrate diaminobenzidine was added and the reaction stopped with several washes in distilled water. A positive conclusion was reached when anti-Toxocara IgG antibodies in serum were present in minimum two bands out of four based on the 24, 28, 30 and 35 kDa molecular weight over the strips.
Purified antigens remains complex including specific and non-specific reactions that may give rise to cross reactions (especially with other helminthiasis). However, WB IgG assay allows to the differentiation between anti-Toxocara specific and non-specific anti-ES antigen immunological response. Very sensitive and by definition specific, it is proposed as a confirmatory test for Toxocara serology.
| ~ Results|| |
Out of 93 patients, 45 (48.4%) were positive with anti-Toxocara IgG antibodies, 48 (51.6%) were negative. Based on the ELISA method; 30 patients (32.3%) were positive and 63 patients negative (67.7%). As per the WB technique; 45 patients (48.4%) were assessed positive as minimum two of the bands indicated a weight of 24, 28, 30, 35 kDA and 48 patients (51.6%) were negative as no molecular bands of Toxocara specific antibodies were detected [Figure 1].
|Figure 1: (a) Westernblot bands - patient sample (b) Westernblot bands - control|
Click here to view
| ~ Discussion|| |
Toxocariasis is a zoonosis, which gets contaminated primarily via the soil. Children of certain geographical areas and with a history of pica - also known as ingesting soil - are especially prone to higher risks. Of the nematodes belonging to the Toxocara species, the two - namely the Toxocara canis and the T. cati - are agents of larva migrans.
In a study conducted in 2000 in Poland by Dr. Agata, serum samples from 62 children were collected in an area known to have a history of dog excrete contamination and analysed as per the Toxocara IgG antibodies.  Antibody titrates were found to be high in five children. Pica and dog ownership are determined to be major risk factors. None of the five seropositive children were found to have previous or current symptoms. The study indicated that even in areas known to have a wider contamination probability, asymptomatic cases could be encountered.
In 1989 an epidemiological study by Garcia et al., in western Spain, dog excretes, sand particles and human waste were analysed as per the T. canis eggs and antibodies. Results varied 29.4-33% among dogs, 3.7-9% in sand and 4.6-8.5% in sera analysed with the ELISA method. 
According to an eco-epidemiological study of contamination of soil with parasites in Argentina (2004) 226 topsoil samples were studied, 100 positive infective forms of intestinal parasites and 39 contained more than one species of pathogen.  This study was proved 29 Toxocara sp. In a study conducted in Taiwan, 329 serum samples were studied; 252 (76.6%) were positive for Toxocara IgG antibody for mountain aboriginal schoolchildren with the TcES-ELISA method.  In a study conducted in Brazil with children from varying socio-economic backgrounds, research was conducted on anti-Toxocara with the ELISA method and it was determined that children of lower social status had 21% higher seropositive results as opposed to the children of higher social status having only 3%. Indisputably, it was concluded that being seropositive, especially among the poorer families, had a higher risk factor. 
In a study by Radman et al., in 2000 in Argentina's La plata region, 156 patient's serum of various age groups were analysed for their anti-Toxocara antibodies based on the ELISA method, irrespective of patient's sex or manifestation of the symptoms. A total of 39% of the population were found to be seropositive with 84% of them having clinical symptoms and 41% being asymptomatic. 
In a 2009 study, seropravalence of T. canis infection among asymptomatic children with eosinophilia in Croatia by Sviben et al., serum specimens were tested for the presence of T. canis IgG antibodies using ELISA and WB method. Their results showed a high seroprevalence rate of T. canis infections among children with eosinophilia. 
According to an epidemiological study carried out in 1980 in the suburbs of Pennsylvania USA, although the patients did not have any clinical indications of ocular or visceral larva migrans, the seropositive presence of anti-Toxocara IgG at a substantial percentage of 54% proved to be quite high even though results were not reinforced by the symptoms from people living especially in suburbia. 
In an epidemiological study conducted in 2010 by Yazar et al., in Turkey, a total of 112 patient's serum were investigated by ELISA and anti-Toxocara IgG antibodies were found positive in 24 (21.4%) patients.  But we found that Toxocara IgG antibody was 32.3% with eosinophylia.
In a study conducted in 1999 by Gόngφr et al., in Turkey, 37 infants suffering from stomachalgia for undetermined reasons have taken the ELISA test and shown an anti T. canis IgG seroprevalence of 51.35%. This result led to the fact that children suffering from unresolved pain in the stomach for a long time should undergo tests for Toxocara antibodies. 
As per a study by Bacchli et al., it is certainly advisable to consider toxocariasis in patients with epileptic seizures and eosinophilia. In 2003, a 2-year-old healthy infant was diagnosed with eosonophilic meningoencephilitis caused by T. canis. Even though the child lacked peripheral eosinophilia, the number of eosinophils in Cerebrospinal fluid (CSF) was determined to be 28%. The ELISA method showed anti-Toxocara antibodies both in blood and in CSF. The study strongly underlined the necessity to consider T. canis for a definite diagnosis of eosonophilic meningoencephilitis or menengitis. 
In a study conducted in 2009 on Sri Lankan children with asthma, the study results showed Toxocara seropositivity in children with asthma was 29% and was significantly more than Toxocara seropositivity among non-asthmatic children. 
Recently, a correlation was determined between Toxocara prevalence and asthma, elevation in serum IgE levels, allergen-specific IgE and eosonophilia. Also noticed was the fact that allergic problems surfaced more frequently in children right after a Toxocara infection. According to a study done in 1997 in the Netherlands, a high correlation existed between allergic reactions and T. canis being seropositive. Hypereosinophilia must definitely be considered and requires long and complicated procedures. Both the clinician and the laboratory supervisor should follow results in close contact. Thorough information is also required especially on the symptoms and the epidemiological history of the patients. In identifying toxocariasis, the most frequently used serologic test is the ELISA method as well as the examination of antibodies against the T. canis excretory-secretory antigens. WB technique is advised for final diagnosis in patients with positive anti-Toxocara results from the ELISA test. 
In a study carried out by Magnaval et al., in 1991, the credibility of the WB technique is emphasised for the prevention of cross reaction of antibodies, which can possibly be formed especially in other helminth infections. In the immunodiagnosis of human toxocariasis, the WB technique (IgG specific) should be preferred as it is more sensitive, specific and unaffected by cross reactions. 
In a study conducted in Hungary on 6985 asymptomatic patients, patient sera were examined for anti-Toxocara IgG through the ELISA method and 28.3% of patients were found positive. The positive serum samples with borderline results were furthermore confirmed with the WB technique. The importance of the WB technique is especially emphasised in the study. 
Two independent studies performed by Lynch et al., in two different time periods indicated that in cases where the anti-Toxocara IgG seroprevalence is higher, the ELISA method can give contradictory results in terms of cross reactions and that it is more advisable to use the WB technique with its more reliable results. 
| ~ Conclusion|| |
Toxocariasis carries a major health risk in developing countries and is mainly encountered among infants. The seroprevalence of Toxocara spp. is known to change according to national identities, study groups and socio-cultural background. Lack of hygiene, lower socio-economic status, geographic location, co-living with pets, consumption of raw vegetables grown in contaminated fields, etc., are all underlying factors behind this illness. In most cases, seropositive results are associated with asymptomatic manifestations. Leucocytes, eosinophilia, stomachalgia, ocular distress, lung and liver dysfunctions, neurological disorders are many of the most encountered symptoms in patients.
Among patients included in our study, just because of mere hypereosinophilia, only 48.4% were found to have positive T. canis antibodies. Results point out to WB technique being more sensitive and superior on a scale of (P < 0.0001) when compared with the ELISA method.
Conclusively, toxocariasis is highly likely to be encountered when analysing the eosinophilia present in blood counts of asymptomatic patients. Among the serological diagnostic methods, as opposed to the ELISA method, the WB technique is more sensitive and specific and in the meantime more reassuring due to the lack of cross reactions.
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