Utilization of Molecular and Serological Methods to Investigation Toxoplasma gondii in Healthy Apparently Students in Babylon Province

Hayam Khalis Al-Masoudi
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Keywords : Toxoplasma gondii, nested PCR, ELISA, latex test.
Medical Journal of Babylon  12:4 , 2016 doi:1812-156X-12-4
Published :17 January 2016

Abstract

Toxoplasmosis, an infection caused by Toxoplasma gondii, is generally asymptomatic or is associated with mild, non-specific clinical manifestations in immunocompetent patients. The present study consider the first trial in Babylon province to detection of toxoplasmosis among healthy university and secondary students, from February to May 2015, collect 112 blood sample from healthy apparently students. The results of serological test revealed that 57/112 (50.8%) was positive for T. gondii in LAT test, (25%) for university students and (25.8%) for secondary students, there isa significant difference (p? 0.05) between male (27.6%) and female (23.2%) in toxoplasmosis infection, where as 21.4% was positive for ELISA in which 13.8% positive for IgG antibodies and 8.0% positive for IgM, while nPCR results showed 19 out of 112(16.9%) was positive to toxoplasmosis. The presence of T. gondii antibodies in the serum regarded as the important criterion for the diagnosis of toxoplasmosis but these methods insufficient to diagnosis, it must be combine with molecular methods such as polymerase chain reaction which detect T. gondii DNA in five or ten parasites are present in blood.

Introduction

Toxoplasmosis is a disease that results from infection with the Toxoplasma gondii parasite, one of the most common parasite in word. It may cause flu-like symptoms in some people, but most people affected never develop sign and symptoms [1]. Toxoplasmsis in generally transmitted through oral route (ingestion of raw or undercooked meat, or meat-derived product, ingestion of unwashed fruits or vegetable or ingestion of contaminated ca feces) or by accidentally ingesting Toxoplasma gondii cysts. Also Toxoplasmosis can be transmitted congenitally from mother to fetus through the placenta[2]. T. gondii has a wide variety of hosts, as almost all warm blooded animals can be infected. Sexual replication of the parasite occurs only in domestic cats and wild felidae (definite hosts), while asexual replication occurs in both intermediate and final hosts . Oocysts are passed in the feces of cats and become infectious within 21 days of being shed.Tachyzoites survive and multiply only in an intra -cellular location while tissue cysts containing few or many bradyzoites occur in the tissues of infected animals within a week of infection [1]. Diagnosis of T. gondii can be made by direct observation of the parasite in stained tissue sections, cerebrospinal fluid or other biopsy material, however these techniques are used less frequently because of the difficulty of obtaining these specimens, also parasite can be isolated from blood or other body fluids but this process can be difficult and require considerable time[3]. Use of serelogical test for demonstration of specific antibody to T. gondii is the initial and primary method of diagnosis. Different serologic test often measure different antibodies that possess unique patterns of rise and fall with time after infection [4]. Serodiagnosis of acute toxoplasmosis is based on the demonstration of significant increase in specific IgG or IgM antibodies level, however the prevalence of high T. gondii IgG antibody titer among individuals in most population and the sustained persistence of specific IgM antibodies in some peoples have complicated the interpretation of serological tests when acute toxoplasmosis is suspected. Many studies have shown high prevalence of T.gondii antibodies in healthy voluntary blood donors in urban Karnataka [5]. [6] used the titer of IgG and IgM antibodies as a test for diagnosis of T. gondii among couples, while [7]showed higher prevalence of Toxoplasmosis among health voluntary blood donors when used Elisa test for IgG and IgM antibodies. Also serelogical test such as latex agglutination test and Elisa were used for detection of T. gondii antibodies in meats [8]. In recent studies used molecular methods based on polymerase chian reaction (PCR)for detection of Toxoplasmosis, these methods are simple, sensitive, reproducible and can be applied to all clinical sample[9]. These methods based on detection of a specific DNA sequence of B1 gene using different assay and protocol such as conventional PCR, nested PCR and real-time PCR. Many studies used B1gene as a target gene, [10] concluded that nested PCR assay in blood has advantage in detection of recent and active toxoplasmosis. While [11] confirm that real-time PCR was a rapid, sensitive and quantitative methods for detection of T. gondii DNA in blood. This study aimed to investigation the possible presence of Toxoplasma gondi in healthy apparently students by using serological and molecular methods.

Materials and methods

A total of 112 blood sample were randomly collected from apparently healthy voluntaries (male and female) from February - May 2015, for detection of Toxoplasmosis in healthy student in Babylon province. Voluntaries were divided into two groups, university student and secondary student. Five ml of venous blood was collected for using in serological and genetic detection of Toxoplasma gondiias following:
1-Serological diagnosis
A-Latex agglutination test (LAT):
   The latex agglutination test (HumatexToxo)  was used as screening test for serological demonstration of T. gondii  according to manufactures  instruction.
B-Enzyme linked immunosorbent assay (ELISA):
   This test was done by using standard commercial kit (Biokit diagnostic company, Spain) specific to T. gondii IgG and IgM antibodies according to manufactures instruction.
2-Genetic diagnosis
A-DNA extraction:
     DNA was extracted from whole blood by using DNA extraction kit (Bioneer, korea) according to manufactures instruction.
B- detection of T. gondii DNA by nested PCR assay :
nested PCR assay was used for amplification targeting B1 gene for T. gondii by using set of primers (580bp and 531bp) depended on [12].




Results

The results of our experience by using serological ( LAT and ELISA) and nested PCR test for the identification of T. gondii in blood of healthy apparently students showed that 57/112 (50.8%) were positive to T. gondii by using LAT test, this results agree with [8], over all the (112) healthy students voluntaries the percentage of infection in male was (10.7%) while in female (14.3%) in university students. Were as secondary students revealed (16.9%) in male and (8.9%) in female (table 3). The infection of male was significantly higher (p? 0.05) than female (31/54 for male and 26/58 for female) but there are no significant different (p? 0.05) between university and secondary students . This result was in agree with [14] which showed that male in puberty age are more susceptible than female to get Toxoplasmosis infection or may be some of these infection occur in earlier age. study of [15] confirm that male is more susceptible than female to many types of parasites, sex differences in exposure as well as susceptibility to parasites probably contribute to sex-based differences in the intensity and prevalence of parasite, males are more likely to engage in behaviors such as aggression, dispersal and grouping that increase the likelihood of contact with parasites. The prevalence of toxo-plasmosis in healthy apparently students may be associated with exposure to risk factors, such as contact with contaminated soil, eat contaminated meat or vegetable, or unhygienic food preparation. Early study reported that T. gondiitachyzoites may be isolated from raw chicken egg laid by hens with experimentally induced infection [16]. Also animal meat may sever as potential source of infection for students, this results confirm by study of [12] who proved the presence of Toxoplasma B1gene in blood, liver, heart, pectoral muscles and small intestine of avian (chicken, turkey, geese and ducks) in middle Euphrates region. We screened 112 blood sample by using serological and genetic tests, table (4) showed that 57/112 sample were positive for LAT, 24/112 were positive for ELISA while nPCR results showed 19/112 were positive.in our study we used different methods to increase the accuracy of Toxoplasomsis diagnosis in healthy students.

Discussions

The results of our experience by using serological ( LAT and ELISA) and nested PCR test for the identification of T. gondii in blood of healthy apparently students showed that 57/112 (50.8%) were positive to T. gondii by using LAT test, this results agree with [8], over all the (112) healthy students voluntaries the percentage of infection in male was (10.7%) while in female (14.3%) in university students. Were as secondary students revealed (16.9%) in male and (8.9%) in female (table 3). The infection of male was significantly higher (p? 0.05) than female (31/54 for male and 26/58 for female) but there are no significant different (p? 0.05) between university and secondary students . This result was in agree with [14] which showed that male in puberty age are more susceptible than female to get Toxoplasmosis infection or may be some of these infection occur in earlier age. study of [15] confirm that male is more susceptible than female to many types of parasites, sex differences in exposure as well as susceptibility to parasites probably contribute to sex-based differences in the intensity and prevalence of parasite, males are more likely to engage in behaviors such as aggression, dispersal and grouping that increase the likelihood of contact with parasites. The prevalence of toxo-plasmosis in healthy apparently students may be associated with exposure to risk factors, such as contact with contaminated soil, eat contaminated meat or vegetable, or unhygienic food preparation. Early study reported that T. gondiitachyzoites may be isolated from raw chicken egg laid by hens with experimentally induced infection [16]. Also animal meat may sever as potential source of infection for students, this results confirm by study of [12] who proved the presence of Toxoplasma B1gene in blood, liver, heart, pectoral muscles and small intestine of avian (chicken, turkey, geese and ducks) in middle Euphrates region. We screened 112 blood sample by using serological and genetic tests, table (4) showed that 57/112 sample were positive for LAT, 24/112 were positive for ELISA while nPCR results showed 19/112 were positive.in our study we used different methods to increase the accuracy of Toxoplasomsis diagnosis in healthy students.

Conclusions

N/A

References

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