Agha Sadaruddin ( Pakistan Medical Research Council, Central Research Centre, Islamabad. )
Farida Agha ( Pakistan Medical Research Council, Central Research Centre, Islamabad. )
Farzal Anwar ( Pakistan Medical Research Council, National Institute of Health, Islarnabad. )
Abdul Ghafoor ( Pakistan Medical Research Council, National Institute of Health, Islarnabad. )
Sera, from 270 school children (age 13 to 20 years) residing in suburbs of lslamabad, were investigated for the presence of toxoplasma gondii, lgG antibodies using the enzyme linked immunosorbent assay (ELISA). The overall prevalence was 17.4%. There was no significant difference between the two sexes. Since a positive test result for lgG antibodies at any level does not eliminate the possibility of a current infection, the toxoplasma lgG antibody positive children were further tested for the presence of toxoplasma IgM antibodies by the same technique. An acute infection was indicated in 12.7% (6/47) IgO positive children. This study shows that toxoplasmosis is prevalent in adolescence in Islamabad. The presence of cats and the degree of soil contact appeared compatible with hypothesis of transmission by oocysts. Poor sanitary habits and conditions and water shortage in schools may cause parasitic infection through contact between the children. An improvement in general hygienic conditions is important in reducingthe rate of transmission by oocysts. Further studies are needed to assess the possible age of exposure to this parasite in the paediatric group (JPMA 41: 131, 1991).
The intracellular protozoan parasite, toxoplasma gondii, causes infection in men and animals. The primary ‘hosts which harbour the intestinal, sexual stage are cats1,2. Transmission to humans happens mainly by eating raw or undercooked contaminated meat3,4 , raw cow’s milk and birds eggs, swallowing oocysts dis-charged in faeces of infected cats, inoculation of trophozoites through the skin, or by inhalation5-7. Trans-mission from a mother infected during pregnancy, to the fetus causes congenital toxoplasmosis8-10. Human toxoplasmosis is worldwide. In adolescence and adulthood, most infections are subclinical or run a very mild clinical course11. Toxoplasmosis is a systemic infection, always accompanied by the production of serum anti-bodies at high titre. After the acute stage antibodies persist at lower titre, usually throughout life. The number of seropositive persons in a population, therefore, increases with age. Although antibodies for toxoplasma gondii have been found in the sera of humans and animals throughout the world, the proportion of subjects with positive reactions varies consider-ably by geographic area, age and test method used1,12,13. The risk, by age, of acquiring infection is not uniform throughout the world1 - It has been reported that prevalence of seropositivity among Eskimos is zero, among Brazilians, 72%14. Frequency among the population of the U.S.A ranges from 10 to 20% in young adults and from 35 to 70% in older persons11. Little is known about prevalence of toxoplasma gondii infection in Pakistan. Very few studies have been reported concerning prevalence of toxoplasma gondii antibodies in some population groups15-17. However, no data is available on toxoplasmosis in school children in Pakistan. The present study was undertaken to determine the prevalence of toxoplasmosis in a group of school children for toxoplasma IgG antibodies. Since very high IgG antibody levels may correlate with current infection, IgM testing may be used for differential diagnosis. Detection of IgM antibodies establishes the diagnosis of recently acquired or reactivated infection, but these antibodies soon disappear or decrease to very low levels followed by the appearance of IgG which stays longer. It was also planned to determine toxoplasma gondii IgM antibodies in children who were positive for toxoplasma IgG antibodies.
SUBJECTS AND METHODS
A total of 270 children from schools located in the suburbs of Islamabad city, were investigated, the majority residing in different villages near the schools. They were from lower to lower middle socioeconomic strata, and family sizes varied from 4 to 18 with a mean of 8.6. Some families keep cows and goats as a source of income. Blood samples were collected after seeking written parental permission. All bloods were taken by veinepuncture in sterile plain tubes. Serum was separated by centrifugation and kept at -200C until processed for analysis.
The technique used for antibody detection was enzyme linked inununosorbent assay ELISA (Lab System Toxoplasma gondii IgG, EIA). The principle of the EJA kit is based on an indirect solid-phase enzyme finmunoassay with alkaline phosphatase as the marker enzyme. The colour intensity (at 405 nm) is directly related to the concentration of toxoplasma IgG-class antibodies in the patients’ serum. A set of high positive, low positive and negative controls were also included in each run of sample determinations. Each sample and control was tested in duplicate. The results were expressed in Enzyme Immuno Units (EIU).
The results were calculated by the formula:
EIU = AsampldApc x 100
Asample: mean absorbance of the patients sample
Apc: mean absorbance of the high positive control.
Enzyme Immuno Units (EIU) 20 to 130 were taken as positive, > 130 high positive, 10-19 uncertain positive and below 10 (<10) not detectable. The toxoplasma gondii IgM levels were determined (using Labsystems Toxoplasma gondii 1gM EIA test Kit) only in those sera which were positive for toxoplasma IgG antibodies. Enzyme Immunoassay Units (EIU) greater than 40(EIU>40) were taken as positive for toxoplasma IgM antibodies.
Of the total 270 children, 170 (63%) were boys and 100 (37%) girls. Themean (± SE)agewas 15.07 (± 0.11) years with a range of 13 to 20 years. Majority (68%) of the children were between the age of 13 to 15 years. The toxoplasma gondii IgG antibody was found positive in 47(17.4%) children. Out of these 47 children, 28 were boys and 19 were girls. Of the total 170 boys and 100 girls, 16.5% boys and 19% girls showed positive results. There was no significant difference between the two sexes.
Table I shows the age and sex distribution of 270 children in relation to the positive rate of toxoplasma IgG antibodies. The prevalence of toxoplasma IgG antibodies in boys and girls was higher in the age group 13 to 16 years as compared to older ones. However, in boys in age group 19 to 20 years the prevalence of antibodies was very high (40%) but the number of samples in this group was very small (4/10) and no sample of girls could be obtained in this age group. Of the total 270 children, in 16 (13 boys and 3 girls) the presence of anti-toxoplasma gondii IgG antibody could not be definitely established. These were uncertain positive cases (EIU = 10 to 19). These 16 cases were also tested for the presence of Toxoplasma IgM antibody together with 47 children who showed presence of Toxoplasma IgG antibodies. The prevalence of toxoplasma 1gM antibody in Toxoplasma IgG positive children is shown in Table II.
Of 63 children tested for the presence of toxoplasma 1gM antibody, 7(11%) showed positive results. Out of 47 children positive for IgG antibody, 6(12.7%) showed presence of toxoplasma IgM antibody while in 16 uncertain positive children for toxoplasma IgG antibody, only one (6.2%) had toxoplasma 1gM antibody, which indicated possibility of current infection. No symptoms were elicited that could reasonably be linked to primary infection, in these children, at the time of blood collection. However, 2 children gave the history of hepatitis 3 months back and one had malaria and typhoid 3 months back, remaining gave no history of previous diseases.
The prevalence of toxoplasma in Pakistan has been reported in blood donors15, in some high and low risk groups16, and in pregnant women17, but no data is available in children. The present study revealed presence of toxoplasma gondii IgG antibodies in 17.4% apparently healthy school children in the age group 13 to 20 years. The data is based on a single collection of blood from each individual and due to the lack of cooperation, it was not possible to carry out follow-up studies on the individuals found with positive titers and to obtain blood from children below 13 years of age. Studies have shown considerable variation in the prevalence of toxoplasma antibodies in different age groups, being more prevalent in childhood in some countries18-20. , and in elderly groups in other countries20,21. The present study simply shows prevalence of toxoplasma antibodies in adolescent group, the overall prevalence found in this study higher than in the reported series from China2 and Panama22 and lower than those reported from Africa18,19,23. In the present study no obvious difference was found between the proportion of females as compared to males with antibodies toxoplasma gondii. Similar findings were reported by other workers from different countries20-26. Since very high IgG antibody levels in single samples may correlate with the current infection and a positive test result for IgG antibodies at any level does not eliminate the possibility of a current infection, we have also checked the IgG positive sera for presence of IgM antibodies. An acute toxoplasma gondii infection was found in 6 (12%) out of 47 IgG positive children. One child who showed an uncertain positive result for IgG antibodies was positive for IgM antibodies, which inchcated existence of an acute T. gondii infection. Similar findings were reported by other workers, who relate the high titers to relative recency of primary infection and to high reinfection rates21,22,25. Various studies on the epidemiology of toxoplasmosis have shown that it may be acquired congenitally or by consuming raw and undercooked meat containing cysts or from contact with cats and other animals3,8,27-29. In Pakistan people do not eat pork and meat is usually well cooked, transmission by tissue cysts could be excluded, and it seems consequently improbable that infected meat could be an important source of human infection in our population. However, the consumption of goat’s milk is quite common in our villages and often given to children in cities as well. This milk, if not properly boiled, can also transfer the infection30. As most of the children in present study reside in villages and keep cows and goats, occasional transmission by infected milk cannot be entirely excluded. Since the early studies in U.S1. the life cycle of toxoplasma appears to have been clarified and transmission by cat faeces contaminated soil has been postulated2. It has been reported in Panama that age specific incidence rates were 6.3% to 9.8% per year between 1 and 10 and 11% to 15% per year between 11 and 35 years, thereafter they declined per year, probably due to lesser contact with soil contaminated by cats22. In Pakistan many people keep cats as pets, and they can be seen in and around streets, meat markets and at hotels. Majority of children play outdoors in streets and on the grounds where soil is heavily contaminated with cat faeces. The climate favours long survival of oocysts and as the children come in close contact with the soil, we believe that these circumstances greatly increase the risk of human infection by oocysts. Poor sanitary habits and conditions in schools may cause parasitic infection through contact between the children. Art improvement in day to day hygiene can be of great help in decreasing the transmission by oocysts. This study suggests that toxoplasmosis is present in younger age group and further epidemiological studies are needed to assess the possible age of exposure to this parasite in the paediatric group.
We are grateful to Mr. Sheikh Salahuddin for typing the manuscript.
1. Lamb, GA. and Feldman, HA. Risk in acquiring toxoplasms antibodies. JAMA., 1968;206: 1305.
2. Frenkel, J.K. Toxoplasma in and around us. Blo. Science, 1973; 23: 343.
3. Scheuer-Karpin, R. Toxoplasmosis. from cats. Br.Med.J., 1971; 21: 526.
4. Frenkel, 3K. and Ruis, A. Endemicity of toxoplasmosis in Costa Rica. Am.3. Epidemiol., 1981; 113: 254.
5. Wallace, GD. Isolation of toxoplasma gondii from the feces of naturally infected cats.3.Infect Dis., 1971; 124: 227.
6. Wallace, GD. The role of the cat in the natural history of toxoplasma gondii. Am.J.Trop.Med.Hyg., 1973; 22: 313.
7. Krause, A.C. Toxoplasma in tissues of man and pets. 3. Parasitology 1955; 41: 545.
8. Williams, K.A. Scott, 3M., MacFsrlsne, D.E., Williamson, 3M., Bliss-Jones, T.F. and Williams, H. Congenital toxoplasmosis; a prospective survey in the West of Scotland. 3. Infect., 1981; 3:219.
9. Kimball, A.C., Kean, B.H. and Fuchs, F. Congenital toxoplasmosis: a prospective study of 4048 obstetric patiençs. Am. 3. Obstet, Gynecol., 1971; 11:211.
10. Remington, J.S. and Desmonts, 0. Toxoplasmosis, in infectious diseases of the fetus and newborn infants. edited by Remington J.S. and Klein, 3.0. Philadelphia, Saunders, 1983, p. 142.
11. Remington, J.S. Toxoplasmosis in adult Bull. N.Y. Acad. Med., 1974; 50:211.
12. Feldman, H.A. and Miller, LT. Serological study of toxoplasmosis prevalence. Am. 3. Hyg., 1956; 64:320.
13. Jacobs, L Toxoplasmosis. New Zeal Med. J., 1962; 61: 2.
14. Feldman. H.A. Epidemiology of toxoplasma infections. Epidemiol., Rev., 1982; 4:204.
15. Farzal, A., Khalida, K., Mohammad, N.A. and Tauqeer, A. Toxoplasmosis: Detection of antibodies in blood donors ofKarachi and Islamabad (Pakistan). Pakistan 3. Med. Res., 1990; 29: 23.
16. Abmed, M. and Hatiz., A. Surveillance of toxoplasmosis in different groups. JPMA.,1989; 39: 183.
17. Bad, A. and Khan, Q.A.S. Toxoplasmosis among pregnant women in northern parts of Pakistan. JPMA., 1990; 40: 288.
18. Hinda, J.A. Hasan, H.M., Waraam, Y., Said, F.A. and Gunnel, H. Human toxoplasmosis in Somalia. Prevalence of toxoplasma antibodies in a village in the lower Scebell region and Mogadishu. Trans. K. Soc. Trop. Med. Hyg., 1988; 82: 330.
19. Bowzy, T.R., Camargo, M.E. and Kinyanjui, M. Seroepidemiology of toxoplasma gondii infection in young children in Nairobi, Kenya. Trans. K. Soc. Trop. Med. Hyg., 1986; 80:439.
20. Ronald, C.K., Wong. F.W., Todd, D. and Lam, K.C. Prevalence of toxoplasma gondii antibodies in the Chinese population of Hong Kong. Trans. R. Soc. Trop. Med. Hyg., 1980; 74:351.
21. Jackson, M.H., Hutchison, W.M. and Siim., J.C. A seroepidemiological survey of toxoplasmosis in Scotland and England. Ann. Trop. Med. Paraaitol., 1987; 81: 359.
22. Octavio, ES., Rolando, E.S. and Jacob, K.F. Toxoplasmosis in Panama: A 10-year study. Am. Soc. Trop. Med. Hyg.., 1988; 38:315.
23. Griffin, Land Williams, K.A. Serological and parasitological survey of blood donors in Kenya for toxoplasmosis. Trans. K. Soc. Trop. Med. Hyg., 1983; 77: 763.
24. Mahajan, R.C., Chiktkara, N.L and Jolly, J.G. Serological survey of toxoplasma antibodies in Chandigarh area. Indian J. Med. Res., 1975; 62: 1.
25. Zardi, 0., Adorisio, E., Harare, D. and Nuti, M. Serological survey of toxoplasmosis in Somalia. Trans. K. Soc. Trop. Med, Hyg., 1980:74:577.
26. Behbebani, K. and Al-Karmi, T. Epidemiology of toxoplasmosis in Kuwait. I. Detection of antibodies to toxoplasma gondii and percentage distribution among the inhabitants. Trans. R. Soc. Trop. Med. Hyg., 1980; 74:203.
27. Zardi, 0., Adorisio, E., Andreassi, S. and Kutpnar, H. Summazy of ten years studies on the epidemiology of toxoplasmosis in Italy. Biological Latina, 1972; 23: 39.
28. Hall, S.M. Congenital toxoplasmosis in England, Wales, snd Northern Ireland; some epidemiological problems. Br. Med. J., 1983; 287:453.
29. Wallace, 0.0. Serologic and epidemiologic observations on toxoplasmosis on three Pacific atolls. Am. J. Epidemiol., 1969: 90: 103.
30. Sacks, 3.J., Roberto, R.R> and Brooks, N.F. Toxoplasmosis infection associated with raw goat’s milk. JAMA., 1982; 248: 1728.