Albanian j. agric. sci. 2014;(Special edition)

Agricultural University of Tirana

(Open Access)

RESEARCH ARTICLE

Immunological reactions caused by helminthes and diagnosis of parasites using different methods LEDIA VASJARI1*, VALBONA GJONI2, MIRELA LIKA (ÇEKANI)1, ARTAN TREBICKA1 1

University of Tirana, Faculty of Natural Sciences, Department of Biology, Bulevardi “Zog I”, Tirana, Albania.

2

Public Health Institution, Department of Infecticous Diseases and Epidemiology, Laboratory of Parasitology, Rruga

“Aleksandër Moisiu”, No.12, Tirana, Albania. * Corresponding author e-mail: [email protected]

Abstract Helminthes are one of the causer agents of the type I reactions of hypersensitivity. In this article will review the findings of recent human studies of the association between helminth parasite infections and allergy and discuss their potential relevance to public health. The parasitic worms are an important risk factor for anaphylaxy reaction, but this could be explained by an enhanced ability of atopics to produce IgE. The human immune response to helminth infections is associated with elevated levels of IgE, tissue eosinophilia and mastocytosis. The involvement of immunologic mechanisms in the pathogenesis which are caused by worms in the gastrointestinal tract are associated with anaphylaxy response. For this study, faeces were collected primary in children aged 1-15 years old, because the worms are most frequent in these ages. Through the coproscopic method were analyzed as biological materials, the feces of 300 children for the presence of protozoa’s eggs, helminthes larva, trophosoids, cists, etc. We have taken the photo of the positive cases. The analysis have been done in the Parasitological Laboratory of the Institute of Public Health, Tirana through the method of concentration with floatation in sulphat zinc; the permanent color as Ziehln-Neelsen stain, Giemsa stain, Blumetilen, etc. We have used the color methods and blood striche to diagnose the eosiniphilia presence. To determine the IgE are used the EIA kits. The level of the eosinophyle and IgE in the blood is performed in 152 individes who had been positive cases by helminths. Keywords: Immunoglobulin E, helminthes, eosinophils, gastro-intestinal tract.

1. Introduction Gastro-intestinal parasites are the causative agent of infections in more than 3 billion people worldwide. The majority of cases occur in developing countries, mostly composed of migrant population. According to recent studies, only 10% of these populations are carriers of 70% of intestinal helminthes [1, 2]. One of the characteristics associated with allergic diseases caused by parasites, is an increase in the concentration of IgE in the serum of patients. Since many immunoglobulins are closely related to specific diseases, their use as diagnostic markers has been successful [6]. In different allergic diseases the possibility of experiencing an elevated serum immunoglobulin E is high [8]. In populations of industrialized countries where parasitic infections frequencies are low, type I hypersensitivity reactions show a high frequency of immunoglobulin E action [3]. While in less developed countries, especially agrarian countries, parasitic infections are a major cause of the increased concentration of serum immunoglobulin E. 289

Immunoglobulin E is one of the five classes of human antibodies. As well as other immunoglobulins, IgE is produced by B cells, also known as plasma cells. Unlike other immunoglobulins, the concentration of circulating IgE is very low, less than 1 U/ml (1U = 2.4ng). Usually, IgE reaches high values between the ages of 5-7 years old. In children between 10 and 14 years old, IgE levels in the blood are higher than in adults. Over 70 years of age, IgE levels might somehow decrease and can possibly be lower than its concentration in adults younger than age 40 years. The synthesizing rate of IgE by the body is low. The concentration of circulating IgE is very low because mast cells are characterizes by a high IgEaffinity through the ε heavy chains of their FcεRI receptor. The IgE antibody binds to both mast cells and basophils and activating in the same time eosinophils. Once associated to mast cells, eosinophils have a half-life longer than 10 days. This binding stimulate mast cells to degranulate. Degranulation is a process associated by the release of histamine and other biological substances which induce anaphylactic

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response. Increase of sensitivity, due to histamine release, is necessary for recruiting serum components in the same place where the parasite is. While IgE is bound to the parasite and also to eozinophils, the latter release their granule products against helminthes. These products are toxic and may kill, damage or mislead parasites acting in this way as a defense mechanism of the host. Thus the elevated IgE concentration and eosinophilia phenomenon become evident. Eosinophilia is defined as an increase in the number of eosinophils, more than 600 cells per microliter in individuals infected with helminthes. The study and diagnosis of infections with modern microbiological methods is very important, especially in this period of development for our country. Parasites have been and still are one of the main factors causing infections in the population. Presenting a great interest due to the high risk that these infections show, they became the subject of many analyzes, studies, scientific papers etc. This paper expands even more the knowledge of their biological terms . 2. Materials and methods For the realization of this topic were obtained biological samples, faeces and blood (serum), which are collected by the respective procedures and stored under proper conditions. This work was conducted in the period March 2011 - June 2012 and was conducted at the Laboratory of Parasitology, Department of Infectious Diseases and Epidemiology, Institute of Public Health , Tirana, and near a Clinical, Microbiological-, Biochemical- and ImmunologicalLaboratory, in Tirana . For this study the required biological materials, faeces and blood, were collected according to the respective procedures and stored under proper conditions. This work was conducted in the period between March 2011 and June 2012 in part in the Parasitological Laboratory, Department of Infective Diseases and Epidemiology, Institute of Publlic Health, Tirana; and in part in a ClinicMicrobiological-Biochemical- and Immunological Laboratory, Tirana. In the period between July 2011 and June 2012 , a total of 1133 faeces samples suspected for parasite presence were analyzed using the formalin-ether sedimentation methodsh total suspected presence of parasites [7].

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Between December 2011- April 2012 75 individuals who were positive for the presence of parasites were analyzed and subjected to further analysis for the determination of the total serum IgE. In parallel they were also examined for the level of eosinophils in the blood [4, 5]. To achieve the objectives of our study we used three techniques : 1. Formalin-ether sedimentation method for diagnosis of parasites.This type of procedure can define all kinds of worm eggs, larvae and protozoan cysts . 2. ELISA to determine the total IgE concentration (kit DiaMetra) . 3. The method to determine the level of eosinophils in the blood of infected individuals. To analyze the number of eosinophils in the blood of patients who have tested positive for parasites, first must be perform the procedure of drawing-blood. 3. Results and discussions Our study was concepted, developed and realized in the main categories: • The study and diagnostication of parasites, which are often the cause of gastro-intestinal diseases. • The study of first type hypersensitivity and the determination of imunoglobulin IgE as a result of the parasites in the human organism. • Determination of eosinophils, along with IgE, in the diagnosed individuals with parasites as a result of the reactions caused by them. The first part of the study describes the results of the prevalence of parasitic infections of the gastrointenstinal tract as well as their transmitting dynamics in the district of Tirana. The results of these examinations are being submitted here, as well as the occurrence frequencies of the parasitic infections (Figure 1). These results are being analyzed according to the months, gender (Figure 2), age etc. .Figure 1 shows the incidence and percentage of the parasitic infections in the individuals which are being analyzed. These infections were identified in 335 individuals (31.33%) out of 1133 analyzed samples. These data include both women and men which have different ages and residence. These high percentages show that parasitosis are a potential cause of infections within a population. The data from graphic 2 show that 55.5% of the women are positive, or a frequency of 197 women out of the 355 persons who are infected with parasitosis.

Immunological reactions caused by helminthes and diagnosis of parasites using different methods

In men parasitosis were found in 44.5% of the positive samples or in 158 individuals. Until now there hasn't been found a connection between sex the frequency of parasitosis, since they are nearly equal in percentage. This is clear taking in consideration their way of infecting people. The infection of the host is caused by the larvae and never from the eggs. The larvae is able to penetrate the skin of the feet and then to go through the body, and with the help of the vascular system they spread until finally settling in the gut where the larvae matures and becomes a worm. Figure number 3 shows the absolute frequencies of the infected according to the parasitic species and the months of the year. From the graphic we deduce that the largest number of the infected is seen during spring, between February and May 2012. This season has optimal environmental conditions (optimal temperature, soil with humidity, heat and shade) which allows the fertile eggs of the parasites to hatch and to become infectious within a certain period of time varying from a couple of days to a couple of weeks. The worst parasite is Giardia lamblia which constitutes 61.69% percent of the cases, or 219 individuals with this infection. This parasite is often found in humans in April, with 37 identified cases. Another parasite often found is Blastocysis homminis with 13.52% and it's peak is in May and June, each with 6 infected individuals.

31.33%

68.66% Positive Negative Figure 1. Percentages of parasitic infection

55.5%

60.0%

55.5%

44.5%

50.0%

44.5%

40.0% 30.0% 20.0% 10.0% 0.0% Females

Males

Positive

Figure 2. Parasitic infections for males and females. 40 35 30 25 20 15 10 5 0

Giardia lamblia

Entamoeba

Blastocystis

Trichiuris trichiura

Ascaris lumbricoides

Figure 3. Distribution of parasitic infections according to various times of the year and parasites species

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Infections with helminthes which are transmitted by natural pollution (such as Ascaris lumbricoides, Trichiuris trichiura, Hymenolepis nana) were found in 28 individuals, or 7,88% percent of the examined cases. The prevalence of infections by more than one species was 1,69% or 6 individuals out the 355 examined sampled. Out of these 6 individuals were carriers of two parasitic species: 1. Ascaris lumbricoides and Giardia lamblia 2. Enterobius vermicularis and Giardia lamblia 3. Trichiuris trichiura and Ascaris lumbricoides 4. Blastocystis sp and Enterobius sp (2 individuals)

Only one individual (Figure. 4) was a carrier of three parasitic species Blastocystis hamminis, Enterobius vermicularis and Ascaris lumbricoides.

Figure 4. Images of 3 parasitic species in a single individual (Photo V. Gjoni)

150 100 50 0

M

F

Figure 5. Absolute frequencies of accurence of eight different species of parasites devided by gender

From the results of figure 5, it is clear that despite the species of the parasite, the infections are found in men and women in nearly equal percentages. The above table shows a bigger number of infected men than women. These results are not definitive that men are more prone than women to these infections, given the fact that there were more men samples than women's. Type I hypersensitivity reactions can be seen when the antigen responsible for the anaphylactic response has leaked from the parasite to the guts. The immune response for these worms induces the release of IgE and as result of the binding of IgE with the worms, histamines and other intermediates are released triggering an anaphylactic response. By using the ELISA method we determined the totals IgE of 75 individuals who were earlier diagnosed with parasites. From the results shown in figure 6 we can get an overview of how immunoglobulin E values vary in individuals belonging to four different groupages.

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15.00% 10.00% 5.00% 0.00% Gr. I

Interval I of IgE

Gr. II

Interval III of IgE

Gr. III

Gr. IV

Interval II of IgE

Interval IV of IgE

Figure 6. Absolute frequencies of IgE levels according to groupages Gr. I (0-3 years old), Gr. II (4-7 years old), Gr. III (8-11 years old), Gr. IV (12-15 years old), Interval I IgE (165-175 UI/ml), Interval II IgE (176-186 UI/ml), Interval III IgE (187197 UI/ml), Interval IV IgE (197-207 UI/ml).

It can be noted that higher concentrations of IgE are associated with the second groupage (4-7 years old) and the third one (8-11 years old), which are also

Immunological reactions caused by helminthes and diagnosis of parasites using different methods

the groups more affected from parasitic infections. A considerable number of individuals that belong to the second and third group respectively 15 and 12 individuals (respectively 20% and 16% of the 75 analyzed individuals), present a high concentration of immunoglobulin E, from 187 to 197 UI/ml in their serum. This result is consistent with the fact that higher values of total serum IgE is observed at age 5-7 years old, for helminthes infection. The lowest total serum immunoglobulin E concentration were found in the first and fourth groupages (0-3 years old; 12-15 years old). This might be related to the slow production of IgE in children younger than 3 years old, as their immune response is poor.

cases, values in the serum ranged from 0.7 to 0.8 x103 /mm3 cells. Only in 21.3% (16 individuals) belonging to the four groupages, the concentration of eosinophils was 0.9x103 /mm3 cells. The increase in the number of eosinophils more than 0.6x103 /mm3 cells leads to the hypothesis that eosinophilia is concomitant to the immune response induced by parasitic worms. 15.00% 10.00% 5.00% 0.00% Gr. I

6 5 4 3 2 1 0

Gr. II

Interval I of IgE

Interval III of IgE Figure

8.

Absolute

Gr.III

Gr. IV

Interval II of IgE

frequencies

of

eosinophils level according to groupages Gr. I (0-3 years old), Gr. II (4-7 years old), Gr. III (8-11 years old), Gr. IV (12-15 years old), Interval I (0.5-0.6 x 10³/mm3), Interval II (0.7-0.8 x 10³/mm3), Interval III ( ≥0.9 x 10³/mm3) Interval I IgE Interval III IgE

Interval II IgE Interval IV IgE

10 8 6 4 2 0

Figure 7. Absolute frequencies of IgE in different months Interval I IgE (165-175 UI/ml), Interval II IgE (176-186 UI/ml), Interval III IgE (187197 UI/ml), Interval IV IgE (197-207 UI/ml).

For nine months, August 2009-April 2010, the serum of 75 patients experiencing parasitic infections were analyzed. The highest concentration of IgE were found in the samples collected in April, August, September and October, that is also the peak of protozoa and helminthes blooming (Figure 7). There is an increase in the permeability due to histamine release, that is neccessary to recruit serum components, including IgE, where parasitic worms are present. Antibodies bind to the surface of worms and through chemotactic effects they recruit eosinophils. Eosinophil determination was performed in 75 individuals, previously diagnosed with parasitic infection and high levels of serum IgE. Figure 8 and 9 show the variability of eosinophils concentration in the blood or serum of infected patients. It can be noted that 49.3% (37 individuals) of 293

Interval I of eosinophils Interval III of eosinophils

Figure

9.

Absolute

Interval II of eosinophils

frequencies

of

eosinophils level in different months

In the following studies regarding infectious diseases, it would be interesting to search for a connection between different groupages and occurrence of parasitosis. Of the same importance is the correlation between the concentration of immunoglobulin E, and therefore the number of eosinophils in the blood of infected patients, and cases of parasitic infection. In this first study, we were not able to accomplish this feat due to the relatively small number of samples for a reliable and significant statistical elaboration and also due to the lack of

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control serum samples (individuals who show no signs of parasitic infections).

References: 1. Abbas A, Leitchman H: The parasites immunology. In: Cellular and Molecular Immunology. 5th Edition; 2007: 438-447

4. Conclusions Intestinal parasite cause Type I Hypersensitivity reactions, and patients experiencing parasitosis show a rise in the number of immunoglobulin E and eosinophils. Intestinal parasitism has a high prevalence in the first groupage (1-5 years old) showing a 42% of infected cases. For the second groupage (6-11 year old), the frequency of this type of infection start to decrease, but is still too high, around 31%. This is due to a more active lifestyle of children included in the second group and the high possibility to be exposed to different sources of infection. Parasitosis among children represent a potential danger to their health. The highest concentration of immunoglobulin E antibodies were encountered among children aged 411 years old. A rise in the number of eosinophils were found especially in individuals aged 4-7 years old, 36 out of 75 cases, and also from 8 to 11 years old, 21 out of 75 cases in total. Increased number of eosinophils to more than 0.6x103 cells/mm3, in individuals diagnosed positive for parasitosis, is considered as concomitant to the immune response caused by intestinal parasitic worms. Gardia lambia, Blastocytic hominis and Entamoeba spp. were the parasites showing the greater frequency respectively 62%, 13.5% and 7.9%. These parasites were found mostly during spring, from March to July.

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2. Dushniku N: Të dhëna praktike mbi shpeshtësinë e takimit të protozoarëve intestinal tek të sëmurët e shtruar me çrregullime gastro-intestinale. In: Buletini i Universitetit Shtetëror të Tiranës. XVII; 1997: 157 3. Homburger HA, Henry JB: Allergic diseases. In: Clinical Diagnosis and Management by Laboratory Methods. 19th Edition; 1996: 10511063 4. Lika (Çekani) M: Gjaku dhe hematopojeza, leukocitet. In: Histologjia; 2007: 110-111 5. Lika (Çekani) M, Bërxholi K: Reaksionet anafilaktike. In: Imunologjia; 2004: 134-136 6. Marchand F, Mecheri S, Guilloux L, Lannascoli B, Weyer A, Blank U: Human serum IgEmediated mast cell degraluation shows poor correlation to allergen-specific IgE content. Allergy 2003, 58(10): 1037-1043 7. Papajorgji M, Kero A: Metodat e përcaktimit të parazitëve. In: Manual bazë mbi metodat doagnostike dhe mikrobiologjike; 2001: 163-186 8. Winter E, Hardt N, Fahrman S: Immunoglobulin E: Importance in Parasitic Infections and Hypersensitivity Responses. Archives of Pathology & Laboratory Medicine 2000, 124:1382-1385