Human Toxocarosis Research Articles

Migratory pattern of zoonotic Toxocara cati and T. canis in experimentally infected pigs

BackgroundOver a billion people are infected with Toxocara canis or T. cati, the roundworms of dogs and cats. Historically, T. canis has been considered the main species responsible for human toxocarosis, but as serodiagnosis cannot discriminate between the two species, this remains unresolved. We used pigs as a relevant large animal model for human infection to assess the migratory pattern of T. cati and T. canis.MethodsPigs were inoculated with T. cati or T. canis eggs or PBS (negative controls) and necropsied 14 or 31 days later. Different organs and tissues were examined for parasites and pathological changes.ResultsOverall, the two parasite species had a similar migration pattern reaching multiple organs and tissues, including the mesenteric lymph nodes, liver, lungs, and diaphragm. We recovered larvae of both species in the brain, suggesting that T. cati also can cause neurological toxocarosis in humans. Both species induced systemic eosinophilia and histopathological changes in the lungs, livers, and mesenteric lymph nodes.ConclusionThis study emphasises the importance of T. cati as a zoonotic agent and the need to develop diagnostic methods that can differentiate between sources of infection in humans.

Gastrointestinal parasites of cats in Egypt: high prevalence high zoonotic risk

BackgroundSeveral gastrointestinal parasites that infect cats pose potential health threats for humans and animals. The present study is the first to report gastrointestinal (GIT) parasites in feces of stray cats from Gharbia governorate, Egypt. Findings were combined with those published in the earlier surveys from various Egyptian governorates, and various meta-analyses were conducted to underline the parasitic zoonoses from cats in Egypt.ResultsOut of 143 samples tested in Gharbia, 75 (52.4%) were found infected with 13 different parasites. Co-infections were observed in 49.3% of positives. Several parasites were detected, e.g., Toxocara cati (30.0%), Toxascaris leonina (22.4%), hookworms (8.4%), taeniids (4.2%), Strongyloides spp. (2.1%), Physaloptera spp. (2.1%), Alaria spp. (1.4%) and Dipylidium caninum (0.7%). Opisthorchis-like eggs were found in a single sample being the first report from cats in Africa. Oocysts of 4 coccidian parasites were identified, and a few Toxoplasma gondii-like oocysts were detected in 2 samples (1.4%). Results of the meta-analysis illustrated that occurrence of T. gondii oocysts in feces of cats from Egypt may have been overestimated in earlier studies; 1432 cats have been tested and displayed a 5 times higher pooled prevalence (11.9%) than the published global pooled prevalence for T. gondii oocysts in cats. This overestimation might have occurred because some small-sized oocysts that belong to other coccidian parasites were mis-identified as T. gondii. Toxocara cati had a high pooled prevalence (22.5%) in cats from Egypt, which is even greater than the published pooled prevalence in cats globally; however, several reports from Egypt have neglected the role of T. cati in human toxocarosis. Dipylidium caninum displayed also a high prevalence (26.7%).ConclusionSeveral zoonotic parasite species have been found in stray cats from Egypt, raising concerns about the risks to the Egyptian human population as well as environmental contamination. Prompt surveillance supervised by the government and accompanied by data dissemination will be helpful for developing effective control strategies.

Obtaining immunodiagnostic preparations from <i>Toxocara canis</i> antigens for serodiagnostics of toxocariasis in human

Toxocariasis is a human helminthic invasion that has a wide range of hosts with epizootic distribution. The populational seropositivity in countries with a temperate climate comprises about 37%, whereas in regions with tropical climate up to 92%. Almost all age groups of the population are at risk of invasion by toxocars. The immunological method currently retains diagnostic significance for toxocariasis, because the reaction of immune system against helminths is accompanied by developing sensitization, and techniques as well as methods of collecting clinical material to identify the migratory form of toxocarosis in humans are time-consuming and invasive. The purpose of this study is to develop an immunobiological preparation based on excretory-secretory antigens from Toxocara canis larvae for serodiagnostics of larva migrans syndrome in human by using enzyme-linked immunoassay (ELISA). Antigens were obtained from Toxocara canis larvae by culturing its eggs isolated from the uterus of female nematodes in the glutamine-supplemented medium. The preparation was purified from ballast substances by centrifugation at 8000 g for 20 minutes followed by filtration through microfiltration membrane with a pore diameter of 0,050,15 microns, type MFAS-P-1 (manufactured by CJSC STC Vladipor). Blood serum of student volunteers from countries with high incidence rate of toxocariasis were tested to detect T. canis-specific antibodies. ELISA with commercial antigens Toxocara-IgG-ELISA-BEST kit was used for selection of seropositive and seronegative sera also used as a control. Diagnostic antigenic preparations with different protein concentrations were prepared to examine seropositive sera for determining optimal dose of the antigenic drugs. Results. ELISA with standard test kit allowed to detect Toxocara-IgG antibodies in 20 sera (8%) from 250 samples, what indicates about antigenic stimulation of the immune system by helminths and suspected former disease. Analyzing experimental samples allowed to find the number of seropositive data correlated with antigenic protein concentration in preparations: at a concentration of 1,96 g/ml 5,2% of positive results were detected, 1,71 g/ml 3.2%, 0.33 g/ml 0.8% (Pearsons correlation coefficient 0,94). The number of positive sera detected with commercial antigen in ELISA kit was identical to that of positive sera detected by an experimental antigen sample with a protein concentration of 2,49 g/ml. Thus, the immunodiagnostic preparations obtained by the experimental method based on the excretory-secretory antigens from Toxocara canis are characterized by high sensitivity and can be used to detect Toxocara-IgG antibodies in ELISA. The antigen preparation protein concentration of at least 2.49 g/ml is optimal and correlates whit sensitivity of the commercial antigen of the standard ELISA.

Study of IgG avidity and the level of specific IgA antibodies and their significance in the diagnosis of human toxocarosis

Toxocarosis is a zoonotic disease caused by migration and subsequent localization of nematode larvae of Toxocara spp. in human organs and tissues, which is manifested with development of various non-specific clinical symptoms. Main diagnostic methods are serological and consists in proving the presence of anti-Toxocara IgG antibodies in patient's sera. In humans, anti-Toxocara IgG has been shown to persist in the serum for a long time and cannot be used to distinguish between past and recent infection.Aim of the present work is to investigate the diagnostic significance of the specific IgG avidity level, determined by an immuno enzyme test developed by us, and the presence of anti-Toxocara IgA for distinguishing between acute and chronic toxocarosis.The study included 130 patients with positive results in routine serological ELISA and Western blot tests and with clinical symptoms of visceral and ocular toxocarosis. The results revealed low IgG avidity (≤40%) in nine (7.3%) and presence of anti-Toxocara IgA antibodies in 36 (26.2%) of the subjects. Low avidity of IgG antibodies was found only in the first tests, and a presence of specific IgA for up to 9 months. The results of our study give us reason to believe that determination of the IgG avidity in toxocarosis is of greater diagnostic value than the presence of IgA to establish the stage of the disease.

Role of cats in human toxocarosis.

Toxocara cati, the feline ascarid, is ubiquitous in domestic cats globally and is increasingly recognised as an important zoonotic species. In the definitive host, infections with the adult ascarid usually do not present any clinical signs; if clinical signs do appear, it is usually in kittens infected with T. cati, especially by the transmammary route. Diseases may include cachexia, a pot-bellied appearance, respiratory disorders, diarrhoea, vomiting, among other signs, and these may present as early as 3 weeks of age. However, infections with Toxocara spp. larvae in paratenic hosts (including humans and many other animals), can result in serious complications from the migration of larvae. Historically, there has been an assumption that Toxocara canis was the most likely cause of Toxocara spp.-related disease; while it is probably true that T. canis is responsible for the majority of infections, it is important that those caused by T. cati are accurately identified so that the contribution of this parasite to human disease can be established and then handled appropriately. Overall, the detection of infections in cats and the control of parasite stages in the environment are essential to minimise the infection risk to other animals or humans.

Токсокароз на юге России: эпидемиологические и экологические аспекты

Toxocarosis is a widespread parasitic infection and a significant healthcare challenge. Humans can be infected through contacts with animals and soil contaminated by eggs of Toxocara сanis. Children aged 3–5 years are particularly vulnerable population because of their active contact with soil. Veterinary specialists, people working in dog shelters, zoos, groceries, and people who have gardens are also at risk of infection. Objective. To analyze the epidemiological situation of toxocarosis in the South of Russia. Materials and methods. The Laboratory of Sanitary and Parasitological Monitoring, Medical Parasitology, and Immunology of Rostov Research Institute of Microbiology and Parasitology, Federal Service for Surveillance on Consumer Rights Protection and Human Wellbeing, has been continuously monitoring the situation with parasitoses in the Southern Federal District for many years. A total of 1,026 samples of soil were collected in 2012–2020. We performed serological and epidemiological examination for toxocarosis among residents of this Federal District. Results. We found that the number of seropositive individuals and soil contamination with the eggs of Toxocara spp. in the Southern Federal District were high and were not consistent with the official figures of toxocarosis incidence. This suggests that the true level of toxocarosis incidence is significantly higher than that officially reported for this territory. Conclusion. Our study confirmed that environment contamination with the eggs of Toxocara spp. is the main risk factor for toxocarosis in humans. Key words: toxocarosis, Toxocara spp., epidemiological situation, enzyme-linked immunosorbent assay, sanitary and parasitological examination, soil

Role of cats in human toxocarosis

Toxocara cati, the feline ascarid, is ubiquitous in domestic cats globally and is increasingly recognised as an important zoonotic species. In the definitive host, infections with the adult ascarid usually do not present any clinical signs; if clinical signs do appear, it is usually in kittens infected with T. cati, especially by the trans-mammary route. Diseases may include cachexia, a pot-bellied appearance, respiratory disorders, diarrhoea, vomiting, among other signs, and these may present as early as 3 weeks of age. However, infections with Toxocara spp. larvae in paratenic hosts (including humans and many other animals), can result in serious complications from the migration of larvae. Historically, there has been an assumption that Toxocara canis was the most likely cause of Toxocara spp.-related disease; while it is probably true that T. canis is responsible for the majority of infections, it is important that those caused by T. cati are accurately identified so that the contribution of this parasite to human disease can be established and then handled appropriately. Overall, the detection of infections in cats and the control of parasite stages in the environment are essential to minimise the infection risk to other animals or humans.

Coprological survey of endoparasite infections in owned dogs and owners' perceptions of endoparasite control in Belgium and the Netherlands.

Infections with endoparasites are common in dogs. Some of these parasites are potentially zoonotic and therefore a public health concern. A survey was conducted in twenty-six small animal practices to evaluate the prevalence of endoparasites in Belgian and Dutch owned dogs older than 6months as well as risk factors associated with infection. Out of 239 faecal samples screened (168 in Belgium and 71 in the Netherlands), 18 dogs were tested positive for at least one type of endoparasite with three dogs co-infected with two parasitic species. Toxocara sp. was the most frequently found endoparasite (4.6%). Three other dogs were positive for Angiostrongylus vasorum (1.4%) using the Baermann method and confirmed in one dog by the Angiodetect® test. Age and predation behaviour were identified as two risk factors associated with endoparasite infection. Although the majority (77%) of the owners in this study reported to administer at least one anthelmintic treatment per year, only a minority of them (24.3%) were aware of the risk to human health, indicating that owner awareness is sub-optimal. For dog owners, human toxocarosis and other potential zoonoses remain an underestimated health concern. The implementation of sustainable parasite control strategies should be promoted taking also into account the public health risk.

Toxocariasis and the clinical spectrum.

Toxocara canis and T. cati are among the most widely distributed helminthic species in the world with a high zoonotic impact. Millions of people are infecteda and hundreds of thousands are suffering from toxocarosis, a disease encompassing four different entities: larva migrans visceralis (VLM) syndrome, ocular larva migrans (OLM) syndrome, covert toxocarosis (covT), common toxocarosis (comT) and neurotoxocarosis (NT). Toxocara infections in humans may remain clinically inapparent but may also induce severe diseases. This contribution gives a synoptic overview of the most important historical, clinical, diagnostic and therapeutical aspects of toxocarosis in humans.

Seroprevalence of human toxocarosis in Europe: A review and meta-analysis.

Toxocarosis is distributed worldwide and represents the most prevalent zoonotic helminth infection in industrialized countries, thereby posing a substantial risk for public health. Thus, toxocarosis is one of CDC's Neglected Parasitic Infections that has been targeted for public health action. This systematic review and meta-analysis summarizes Toxocara seroprevalence in general populations from Europe, populations suspected of Toxocara infection and defined risk groups. Random-effects meta-analysis of proportions based on 41 publications resulted in an overall seroprevalence of 6.2% (95% CI: 4.7-8.3%) in the general population of Europe. Subgroup analysis according to decades (1970-2010s) revealed a significant increase in seroprevalence, with the highest value (12.4%; 95% CI: 6.5-22.3%) in the 2010s (χ2=17.87, df=4, P=0.001). There were no significant differences between pooled prevalence rates of European sub-regions (χ2=3.01, df=3, P=0.389). Furthermore, meta-analysis of seroprevalence according to age groups, based on data from 22 publications, indicated a significantly higher pooled seroprevalence of 14.9% (95% CI: 8.5-24.8%) in people more than 50 years of age than in younger age cohorts (χ2=8.33, df=2, P=0.016). Occupational groups exposed to contaminated soil and infected animals bear substantial risk for acquiring Toxocara infection. Due to the close link between animal infection and human Toxocara exposure, a "One Health" approach for the prevention of Toxocara infection in both humans and animals is required.

Seroprevalence and incidence of human toxocarosis in Russia.

Overall incidence of toxocariasis in Russia is low and varies between 1.6 and 2.7 per 100,000, while in several hyper-endemic regions, such as Altay, Kurgan, Perm and Udmurtia, it reaches 43 per 100,000. The seroprevalence of toxocariasis in published references was on average 16% and varied across the regions of Russia from negligible in North Siberia to 40% in southern regions of West Siberia. Seroprevalence in adults in five regions of Russia identified in this study was on average 20%, and varied from 3% in Yakutia (north of East Siberia) to 36% in Rostov-on-Don, South Russia. There was no correlation between seroprevalence and reported incidence of toxocariasis; however, the pattern of seroprevalence variation could be linked to Toxocara prevalence in dogs. Toxocariasis seroprevalence has more than doubled over the last 20 years. Diagnostic antibody titres (1:800 or more) were found in 3.6% of sera, suggesting about five million of acute Toxocara invasions per year.

Clinical and epidemiological aspects of human toxocarosis in the Astrakhan region

Analyzed the clinical and epidemiological aspects of human toxocariasis in the Astrakhan region. It was established that from 2013 to 2017 in its territory 34 cases of human toxocariasis were recorded. In the structure of the total number of helminthoses for the analyzed period, the share of toxocariasis accounted for 0,2% of cases and 0,2% of the total number of all parasitosis recorded in the Astrakhan region. Both adults and children were affected by toxocariasis. Thus, the share of the adult population was 82,4%, incl. persons employed in production (working population) - 67,6%, pensioners- 11,8% and unemployed - 2,9%. The proportion of children in the overall incidence of toxocarcosis was 17,6%, of which11,8% attended kindergartens and secondary school 2,9%. And in one case the child was unorganized - he was at home withhis parents - 2.9%. The main complaints of people seeking medical care were: cough - 23,5%, general weakness - 17,6%,pain in the epigastric region and temperature rise to subfebrile numbers - 11,8%, allergic rhinitis and muscle pain and joints- by 5,9%, as well as decreased appetite, allergic rashes on the skin of varying degrees of intensity, feeling of suffocation andweight loss - by 2,9%.When ascertaining the epidemiological history, it was found that the probable cause of the development of the diseasecould be: not following personal hygiene rules - 94,1%, eating unwashed fruits and vegetables - 88,2%, close contact withdomestic dogs - 52,9%, onigophagy - 20,6%, contact with domestic cats - 14,7%, geophagy and the habit of licking hands- 8,8% each, and contact with stray dogs - 5,9%. Personal hygiene rules were observed in 5,9% of patients. Generally, thecurrent situation on human toxocariasis in the Astrakhan region remains highly relevant. The main causes of the diseasewere close contact with both domestic and stray animals, as well as non-compliance with the rules of personal hygiene. Thepresence of diseased antibody titers in the formulation of enzyme immunoassay 1:800 and above, as well as the presence ofa clinical picture of the disease, indicates the direct presence of parasitic invasion in the examined.

Prevalence of Toxocara eggs in the soil of public parks of Khorramshahr city, southwest Iran

Human toxocarosis is a chronic zoonotic disease, which creates various syndromes in humans such as visceral larva migrans, ocular larva migrans, and neurological toxocarosis. This disease is commonly contracted through ingestion of eggs from the Toxocara species. The eggs are transmitted to humans through contact with soil that contaminated with feces of infected animals by Toxocara species worm. This study, investigated the soil from various parks and public areas of Khorramshahr city southwest of Iran for Toxocara egg contamination. One-hundred and fifty soil samples were collected from 21 parks and green public areas from April to July 2018 and examined by the modified floatation method using saturated sucrose solution. The Toxocara eggs were isolated in 27 (18%) out of 150 samples collected. Based on Chi-square test, none of the factors: location of sampling (in city and the suburb), the presence of dogs and cats and the size of the sampling location; was not significant difference by soil contamination by Toxocara eggs. The presence of Toxocara eggs in Khorramshahr parks soil and green public areas is one of the main risk factors for catch the toxocarosis. This is important to develop preventive measures in the city and suburb areas to reduce the Environmental contaminations and thus reduce the incidence of disease in humans.

Assessment of the diagnostic value of specific anti-Toxocara IgA in Slovakian patients suspected to have toxocarosis

Human toxocarosis is one of the most widespread and prevalent helminthic zoonosis in many countries, including Slovakia. The aim was to evaluate the usefulness of IgA anti-Toxocara antibody detection in the serodiagnosis of toxocarosis. The levels of specific IgA antibodies were determined by excretory-secretory (ES)-enzyme-linked immunosorbent assay (ELISA). The IgA seropositivity in IgG anti-Toxocara seropositive patients (n = 52) was 32.7% and found to be highest in the oldest age groups (P = 0.026). The presence of IgA in suspected patients for toxocarosis were evaluated in respect to some characteristics of examined persons. Substantially higher IgA seropositivity was detected in patients with increased total IgE (44.8%) than in subjects with a normal level of IgE (17.4%; P = 0.036). No associations (P > 0.05) were found between IgA seropositivity and sex, level of specific IgG antibodies, avidity of IgG, eosinophilia, domicile, geophagia, traveling abroad, dog/cat ownership, or clinical symptoms. The IgA-ELISA showed sensitivity of 57.1% and specificity of 100%. Mild correlations (r = 0.302, r = 0.305, r = - 0.409) were observed between the levels of anti-Toxocara IgA antibodies and age, the amounts of eosinophils and IgA antibody levels, the amounts of eosinophils, and the values of IgG avidity, respectively. The presence of anti-Toxocara IgA may facilitate the diagnosis of toxocarosis and may well be useful for the determination of acute Toxocara infection. Moreover, this test should be accompanied by other immunological markers of examined patients (e.g., increased total IgE, eosinophilia, and low-avidity IgG antibodies).

Sero-detection of Toxocara canis infection in human with T.canis recombinant arginine kinase, cathepsin L-1 and TES-26 antigens.

Three recombinant antigens viz. arginine kinase, cathepsin L-1 and TES-26 of Toxocara canis were expressed in Escherichia coli and evaluated for their potential in the detection of T. canis larval infection in human in immunoglobulin G-enzyme linked immunosorbent assay (IgG-ELISA). Results of the IgG-ELISA with the above recombinant antigens were confirmed with commercially available IgG detection kit for T. canis infection used as a standard test. All three recombinant antigens were 100% sensitive in the detection of positive cases (n = 6) of T. canis infection in human and were screened for their cross-reactivity in human patients with history of Toxoplasma gondii, Plasmodium vivax, Entamoeba histolytica, hydatid and hookworm infections. The recombinant TES-26 antigen showed higher specificity and cross-reacted with T. gondii infection sera only. However, arginine kinase and cathepsin L-1 recombinant antigens showed cross-reactions with sera of patients infected with T. gondii, P. vivax and E. histolytica but not with the patient sera infected with hydatid and hookworm. These results show that recombinant TES-26 is a potential diagnostic candidate antigen for human toxocarosis caused by migrating T. canis larvae.

Limits of laboratory diagnosis in human Toxocarosis

Limits of laboratory diagnosis in human Toxocarosis

Human case of visceral larva migrans syndrome: pulmonary and hepatic involvement

SummaryVisceral Larva Migrans (VLM) syndrome is commonly caused by larvae of roundwormsToxocara canisorToxocara cati. Human toxocarosis is a soil-transmitted zoonosis, which may result in partial or general pathological changes in host tissues. We reported a case of 14-year-old boy presented with severe dry cough without dyspnea, mild chest and abdominal pain with general fatigue. Examination of peripheral blood showed marked increase in eosinophils. The chest radiography showed an infiltrative shadow in the lung fields. Chest CT demonstrated multiple opacities in both lungs. Abdominal CT showed multiple low attenuation areas in the liver. Ultrasound guided liver biopsy revealed granulomas with severe eosinophilic infiltration. The boy was treated with albendazole and responded radically. It is worth mentioning that this is the first case of hepato-pulmonary VLM syndrome in Egypt.

Toxocara and toxocarosis a roundtable discussion

Foreword Toxocara spp. infection in dogs and cats is often taken for granted, although it can cause illness in puppies/kittens, and insufficient research has been carried out in recent decades. Human infection with these parasites, toxocarosis, has been recognised as a neglected disease. The recent publication of a draft genome for T. canis may lead to new targets for development of vaccines and treatments, and new insights into epidemiology. Greater awareness of the level and consequences of human toxocarosis are needed. A One Health approach is essential and veterinary professionals can play an important role in public health education.

Toxocara canis: anthelmintic activity of quinone derivatives in murine toxocarosis.

Human toxocarosis is a chronic tissue parasitosis most often caused by Toxocara canis. The seroprevalence can reach up to 50%, especially among children and adolescents. The anthelmintics used in the treatment have moderate efficacy. The aim of this study was to evaluate the in vitro and in vivo anthelmintic activity of quinones and their derivatives against T. canis larvae and the cytotoxicity of the larvicidal compounds. The compounds were evaluated at 1 mg mL(-1) concentration in microculture plates containing third stage larvae in an Roswell Park Memorial Institute (RPMI) 1640 environment, incubated at 37 °C in 5% CO2 tension for 48 h. Five naphthoxiranes were selected for the cytotoxicity analysis. The cell viability evaluated by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide and lactate dehydrogenase assays using murine peritoneal macrophages isolated from C57BL/6 mice revealed that the naphthoxiranes (1 and 3) were less cytotoxic at a concentration of 0.05 mg mL(-1). The efficacy of naphthoxiranes (1 and 3) was examined in murine toxocarosis also. The anthelmintic activity was examined by evaluating the number of larvae in the brain, carcass, liver, lungs, heart, kidneys and eyes. Compound (3) demonstrated anthelmintic activity similar to that of albendazole by decreasing the number of larvae in the organs of mice and thus could form the basis of the development of a new anthelmintic drug.

Immunodiagnostic approaches for the detection of human toxocarosis

Human toxocarosis is an important zoonosis caused by larvae of Toxocara canis/cati. The objective was to evaluate the role of IgG anti-Toxocara antibody detection and the specific IgG avidity in diagnostics of human toxocarosis. Anti-Toxocara IgG antibodies and IgG avidity were evaluated by excretory-secretory (ES)-enzyme-linked immunosorbent assay (ELISA). The IgG anti-Toxocara seroprevalence in people (n = 7678) from western Slovakia was 15.3% and found to be highest in the oldest age groups. The presence of low- IgG avidity in 179 suspected patients for toxocarosis was evaluated in relation to sex, age, IgG antibody levels, eosinophilia, increased total IgE, domicile, geophagia, dog/cat ownership, anamnesis. Low- IgG avidity index was found in 30.7% of the patients. The low- IgG avidity in eosinophilic group (42.1%) was significantly higher than in non-eosinophilic group (22.0%; P = 0.043). Substantially higher eosinophilia was detected in children (under 10 years old; 55.6%) than in adults (aged ≥ 41 years; 17.6%; P = 0.009). Significant difference between seroprevalence of total IgE in patients coming from towns (48.8%) and patients from villages (21.3%) was established (P = 0.007). Mild negative correlation (r = −0.477, P = 0.043) was observed between the amounts of eosinophils and the values of IgG avidity. The sensitivity and specificity of IgG avidity assay were 43.8% and 83.3%, respectively. Our results suggest that besides anti-Toxocara IgG, measurement of IgG avidity may be useful for the determination of acute toxocarosis. Moreover, these tests should be accompanied by other immunological markers and determinants of examined patients such as eosinophilia, increased total IgE and age.