Chromatographic immunoassay based detection of human toxoplasmosis in District Mardan , Khyber Pakhtunkhawa , Pakistan

This study was carried out to determine the sero-prevalence of human Toxoplasmosis in district Mardan, Pakistan. A total of 600 human blood samples were collected by random sampling method. Blood samples were analyzed for Toxoplasma gondii infection and anti-T. gondii IgM and IgG antibodies by using Latex agglutination test (LAT) and Chromatographic technique, respectively. Out of 600 individuals, 105 (17.5%) were sero-positive for T. gondii. Males showed slightly higher (18.4%) prevalence as compared to female (16%), but statistically the difference was non-significant (X= 0.394, P = 0.5302). Sero-prevalence was high (29.41%) in age group 3145 years and lowest (10%) in age group ≤1-15years. A non-significant difference (P > 0.05) of sero-positivity was observed between rural (18.06%) and urban (15.56%) areas. Seropositivity for IgG was high (24%) in age group 31-45. IgM anti-body showed highest seropositivity (14%) in age group 61-75. Of various risk factors, a significant association (P = 0.0001) was found between sero-positivity and no hand washing after contact with animal, soil or other contaminated objects. However, there was no significant relation between sero-positivity of T. gondii and educational status of subjects (p>0.05).


Introduction
Toxoplasmosis is a widely prevalent, zoonotic infection, caused by an obligate, intracellular protozoan parasite, commonly cosmopolitan in its distribution across the globe [1-3].The disease condition is of considerable importance, particularly in hot and humid climates [4,5] infecting about one third of the human population at global level [6,7].The sero-prevalence of toxoplasmosis in human populations is quite high in developing world, ranging up to 80% as compared to developed world [8].Felines or cats are the definitive hosts as it has been shedding the oocysts of T. gondii in its feces in a routine excretion.Other domestic animals like cattle, goats, sheep, pigs, mice, rats and rodents are also known for transmitting toxoplasmosis to human [3, 9, 10].Placental transmission is also evident from mother to fetus whereas blood transfusion is the other possible cause [3,11,12].However, the major risk factor for human toxoplasmosis is the consumption of undercooked meat or food stuffs contacted by any intermediate host (

Materials and methods
The present study investigated the seroprevalence of T. gondii antibodies in human population of District Mardan, Khyber Pakhtunkhwa Pakistan.A total of 600 blood samples were collected by random sampling method.The study area was enough vast, that is why random sampling method was the best choice for conducting such a human oriented study.Blood samples were collected from male and female hosts by visiting different urban and rural areas of the district.Data was collected on properly designed questionnaire.Positive results were noticeable in one minute.The manifestation of any reddishpurple color in the T bands, apart from intensity, was considered as presence of the band.

Statistical analysis
To consolidate the results and check its statistical significance, chi square test was applied to calculate the P-value.The level of significance was selected <0.05.Data was analyzed through computer software SPSS (version 10.0-registered).

Risk factors for transmission
In relation to mode of transmission, there were 411 individuals noted for contact with animals.Out of 41, 19% were sero-positive for the parasite antibodies.Subjects having no association with the animals showed only 14% sero-positivity.It seems that animals' contact is positively associated factor with the infection.But difference observed in both groups was statistically non-significant (X 2 =1.46,P= 0.2269).People used to wash their hands after animal and soils contact showed 10% sero-positivity and those not washing their hands were with 28% seropositive.An extremely significant difference (X 2 =22.36,P = 0.0001) was observed between the groups.Most of the infected individuals (18%) were unaware about the infection and mode of toxoplasmosis transmission.While those having knowledge about the transmission mode were with 14% sero-positivity.But the difference was again statistically non-significant (X 2 = 0.491, P = 0.4835).There was also a slight difference (X 2 =0.016,P = 0.8993) in the sero-positivity of individuals using unwashed raw vegetables and fruits.And those using washed vegetables and fruits were having 18% and 17% ratios respectively for toxoplasmosis.People with soil contact were with higher (08%) sero-positivity than those having no contact with (4.75%) soil.However the difference is statistically insignificant (Table 5).The result shows, the prevalence of toxoplasmosis was higher (20%) in individuals, having secondary education than those having higher studies (18%) and none or primary education (having 14% infection).However, there was no statistically significant relation between sero-positivity to T. gondii and educational standard of the study subjects (P > 0.05; P = 0.3848).Regarding sero-prevalence in different sexes, our data recoded a slightly higher prevalence in male sex as compared to female sex.However, the difference was statistically non-significant (p> 0.05).The possible explanation for the slight change may be the difference in human activities and immune system [28], Klein had reported similar findings of higher occurrence of infections in males as compared to females due to immune system differences [31].The intrinsic physiological changes between females and males might be another reason in difference of frequency in different sexes [32].High rate of T. gondii infection in males was also reported from Punjab, Pakistan [16] which is in agreement with this findings.Our findings ( The effects of various factors such as the animal contact, hand washing after contact, knowledge about transmission modes, unwashed raw vegetables or fruits consumption, gardening or contact with soil were studied (Table 5).A significant association (P= 0.0001) was found between hand washing after contact with soil, animal or infection.Previous studies support the association between these factors and toxoplasmosis infection [36].In the current study, no significant connection (P>0.05) was observed between the T. gondii seropositivity and such factors due to small sample size.

Conclusions
It is concluded that the risk of infection increases with the increase in age, nonwashing of hands after animal and soil contact.Men remain slightly at higher risk as compared to females.Infection occurs in urban areas as well as in rural.IgG seropositivity is high in 31-45 age group, while IgM sero-positivity recorded high in old age group (61-75).Public awareness is sought through health programmes, as the infection is prevalent equally in both educated and non-educated subjects.Preventive measures should be taken to ensure public health and avoid its detrimental health effects.Hands should properly be washed, undercooked meal should be avoided, proper hand washing is required and animals contact should be avoided.

Authors' contributions
Conceived and designed the experiments: M Zahid, Performed the experiments: M Shah & S Attaullah, Analyzed the data: M Haroon & B Ali, Contributed reagents/ materials/ analysis tools: B Bibi & A Hussain, Wrote the paper: M Zahid, & M Shah.