Distribution of Toxoplasma gondii IgM and IgG antibody seropositivity among age groups and gestational periods in pregnant women

Background: Toxoplasmosis is a globally distributed parasitic disease. The present study aimed to estimate the prevalence and geographic distribution of toxoplasmosis as well as determine the percentage of toxoplasmosis-associated IgM and IgG seropositivity among different age groups. In addition, it aimed to estimate the proportion of toxoplasma IgM seropositivity among pregnancy trimesters. Methods: A total of 500 pregnant women were included in this study. From each participant, a 5-ml venous blood sample was collected and centrifuged to obtain serum that was tested for Toxoplasma gondii IgM and IgG antibodies using immunochromatographic testing and ELISA. Results: The overall seroprevalence of toxoplasmosis was 24.8%. Out of the total of 500 participants, only 8% had a serological marker of acute toxoplasmosis). There is a statistically significant difference in the seroprevalence of disease among the study areas. Amongst positive cases of every trimester, 54.34% of first trimester positive cases had a serologic marker for acute toxoplasmosis. Conclusions: In this study, there is a high prevalence of toxoplasmosis. Therefore, it is necessary to test every pregnant woman for toxoplasmosis and distinguish the type of infection, as well as the conduction of public health education programs to generate the awareness.


Introduction
Toxoplasmosis is a widely distributed zoonotic illness causes by Toxoplasma gondii, an obligate intracellular parasite 1,2 . Globally, the distribution of this disease is extremely variable even inside the countries 3,4 . In all host species, including humans, Toxoplasmosis is generally acquiring either vertically from mother to fetus (congenital infection), or through ingestion of oocysts in contaminated food or water 5 . Rarely, T. gondii can transmit through organ transplantation and the transfusion of infected blood 6,7 . Following ingestion, the intestinal epithelium is the primary portal of entrance for T. gondii; next, it spreads to other tissues, where it can cause more severe pathogenesis 8,9 . If toxoplasmosis is acquired during pregnancy, severe infection may develop, especially in immunocompromised individuals, such as those with defects in T-cell-mediated immunity 10 . In patients with AIDS, toxoplasmosis may lead to life-threatening disease 11 . For example, cerebral focal lesions are caused by cerebral toxoplasmosis (CT) in HIV-infected patients 12 .
The signs and symptoms of this illness are markedly divergent and range from asymptomatic to serious infection 13 . This variation depends on several factors includes inoculums size, virulence of the strain of toxoplasma, the individual's genetic background and the status of the immune system of the infected individual 14 . In addition, since the organism has an affinity for muscular and neural tissues as well as the other visceral organs, many hosts harboring latent tissue cysts following toxoplasmosis 15 .
Fetuses may acquire toxopasmosis through the placenta during pregnancy 16  Our study was undertaken to determine the prevalence and geographic distribution of toxoplasmosis as well as to estimate the seropositivity of toxoplasma antibodies among different age groups. It also attempted to identify the percentage of toxoplasma IgM seropositivity (indicative of acute infection) among different pregnancy trimesters.

Methods
This a descriptive cross-sectional hospital-based study carried out in the District Head quarter Hospital (Mansehra, Hazara, Pakistan) and Ayub Medical Complex Hospital (Abbottabad, Khyber Pakhtunkhwa, Pakistan) over a period of 4 months (April to July 2015).

Study population and sample size
Our study included pregnant women of different trimesters, ages and ethnic groups who visited our study areas hospitals; the only eligibility criteria were pregnancy and visiting the hospitals in our study area. Patients were recruited by the researchers face-to-face. During this study duration, a total of 500 pregnant women (convenience sample) fulfilled the inclusion criteria. Out of the total of participants, 204 were recruited from Abbottabad and 296 from Mansehra district.

Laboratory analysis
A total of 5 ml venous blood was collected from each participant using a sterile syringe and transferred to a blood container without anticoagulant, allowed to clot at room temperature for 15 minutes, then centrifuged at 3000 rpm for 10 minutes to obtain serum, which was transferred into a 1.5ml microcentrifuge tube and stored at −80°C for further analysis. In this study, every sample was screened and confirmed for toxoplasmosis through the serological tests.

Screening
All sera samples were screened for T. gondii IgG and IgM antibodies using Rapid Diagnostic immunochromatographic test (Tox IgG/IgM Rapid Test Dip strip, CTK BIOTECH, San Diego, USA) according to manufacturer instructions. In order to avoid false-positive results due to the incomplete specificity of the screening test, every positive sample was further subject to confirmation step by ELISA. Each positive individual also answered a questionnaire concerning their age, trimester and whether they had been in recent contact with animals (Supplementary File 1).

Confirmation
Following the screening, all the positive samples (n=150) were further confirmed to toxoplasmosis using IgM and IgG ELISA kit (Monobind, San Diego, USA) according to the manufacturer protocol. The positive ELISA test for T. gondii IgG titers indicates the chronic infection, whereas with high IgM titers indicate the recent or acute infection. All ELISA tests were performed in triplicate.

Amendments from Version 2
In response to reviewer, we have removed the animal contact part from the abstract, results, discussion, conclusion and dataset. Consequently, authors made minor changes in some words and sentences of the abstract, discussion, and conclusion. Additionally, we changed the title for the dataset.

Ethical statement
Our study was approved by the Ethics Review Committee of Hazara University. Further approval was provided by the administration of Ayub Medical Complex Hospital. From every participant, written informed consent was obtained for conduction of the study. In addition, all the performed steps in this study were completely in accordance with the Helsinki Declaration and the rules defined by the World Medical Association, including samples collection and processing.

Statistical analysis
The obtained results were analyzed by Graph Pad Prism 5 (Graph Pad Software, La Jolla, CA, USA). A χ 2 test was involved to check the statistical differences in seropositivity and negativity of anti-toxoplasma antibodies among the participants of different study areas and gestational periods, at 95% level of significance. Moreover, ANOVA has tested the statistical difference of these antibodies among the participants of every age group. The difference was considered statistically significant when P <0.05.

Seroprevalence of toxoplasmosis
Out of 500 women, using ELISA the overall seroprevalence of toxoplasmosis was 24.8% (124/500). Statistically significant differences were observed between the seroprevalence of disease in Abbottabad and Mansehra district (Figure 1). In addition, the prevalence of toxoplasma antibodies among pregnant women revealed out of the total of 500 participants, only 8% had a serological marker of acute toxoplasmosis (Figure 2).
Toxoplasma antibodies seropositivity among age groups and gestational periods in overall positive cases Among the positive cases (n=124), the seropositivity of toxoplasma antibodies was shown to be statistically significant different among different age groups (Table 1). There was also a statistically significant difference in the seropositivity of toxoplasma IgM (indicating acute infection) between different gestational trimesters, the highest level of IgM seropositivity was observed in first trimester (54.34%) (Figure 3).    Moreover, our study revealed that the geographic distribution of toxoplasmosis is significantly different among the study areas. Out of the 296 participants analyzed from Mansehra and 204 from Abbottabad, the overall prevalence of toxoplasmosis was 15% and 38.7%, respectively. The higher prevalence in Abbottabad when compared with Mansehra may because Abbottabad is an area where agricultural practices are common, and domestic animals like cats and goats were generally kept in or near the homes. Thus, contact with these animals may be the main risk factor of the disease. In addition, low educational and socioeconomic level may have contributed.
In our study, a high percentage of IgM seropositivity was reported in the 1st trimester, which indicated a high prevalence of acute toxoplasmosis or recent infection in this trimester compared with the others. Furthermore, as reported in this study, there is a mild difference in the seropositivity of toxoplasma antibodies among age groups, which requires further study to assess whether, is there any significant association exists between toxoplasmosis and age.
Usually T. gondii does not cause clinical illness in the majority of animal species 35 . Human often acquires this infection from animals by ingestion of improperly cooked or raw animal meat, or via consumption of contaminated food and water with animal's waste 14 . However, there is a need for detailed knowledge about the risk factors of toxoplasmosis. Previously, it was reported that some risk factors are associated with toxoplasmosis, such as owning cats 36 . Additionally, the previous study revealed that that contact with domestic animals may associate with this disease 37,38 . Therefore, the next study studies should evaluate the role of cats contact in disease development.
In this study, a high prevalence of toxoplasmosis was revealed. Moreover, in the first and second trimester of pregnancy, the prevalence of acute toxoplasmosis seems to be higher compare with a third. Thus it is necessary to test every pregnant woman for toxoplasmosis and distinguish the type of infection. In addition, urgent treatment and medicine are essential to decrease the risk of intra-uterine infection and congenital toxoplasmosis. Additionally, there is a need to conduct public health education to create greater awareness about the disease, its transmission, symptoms, and prevention. In addition, screening of T. gondii infection and maternal care should be considered as the main stratagem to reduce the risks of congenital toxoplasmosis.

Grant information
The author(s) declared that no grants were involved in supporting of this work. The manuscript is written in a good structure and presents useful data including the rate of antiantibodies (IgG and IgM) in sera of pregnant women referred to hospitals of two districts in Toxoplasma Pakistan. The manuscript however requires some corrections and modifications as follows: The authors should consider that "toxoplasmosis" is not necessarily "illness or disease" as such they have mentioned in many parts of the manuscript. Toxoplasmosis is an infection which is mostly latent and does not lead to disease or illness in most of immunocompetent individuals. Also, some other issues in the Introduction are scientifically susceptible, e.g. toxoplasma acquisition in fetuses during delivery (mentioned in the 3 paragraph of the Introduction); to my knowledge, this issue is not documented in the literature.
The basis of data arrangement, analysis and discussion about percentage of Toxoplasma gondii antibodies in different age groups (Table 1) is not valid. For this issue, all 500 study cases must be primarily divided into different age groups, then the percentage of seropositivity (IgG, IgM, IgG+IgM) in all initial samples of each group should be calculated and consequently compared and analyzed.
There is no scientific reason to indicate possible relation between infection and Toxoplasma contact with animals like caw, buffalo, goats, and dogs, except for cats which are a definitive host of and shed oocysts. The only way of passing infection from these Toxoplasma Toxoplasma animals to humans, is ingestion of undercooked meats or rarely through contact of open wounds with meats contaminated with bradyziotes, but not other types of contact with these animals or their wastes. So, this variable (animal contact) is not wise to be included in the manuscript and is recommended to be removed from all parts of the manuscript.
Miss-citation is the case for some facts in the manuscript. For example, cerebral Toxoplasma toxoplasmosis is referred to Kristiah (2009), which is not the right citation; while it was originally reported and discussed by other researchers, i.e. Luft and Remington (1988 ) and Luft and Remington (1992 ).
English writing correction is required in some parts of the manuscript text.