Seroprevalence and Hematological Investigation of Toxoplasmosis in Women of Lahore, Pakistan

Background. Toxoplasma gondii is responsible for toxoplasmosis infection. Human beings and most warm-blooded animals are infected by this parasite, though the primary host of this parasite is the felid family. The current study was designed to assess the seroprevalence of toxoplasmosis and to investigate the hematological changes in the female human population of Lahore, Pakistan. Methodology. For this research, 150 blood samples were collected from women being treated at Sir Ganga Ram Hospital Lahore, along with other details. Of these, 90 samples were selected for the analysis of hematological changes by using a hematology analyzer. The serum of these samples was analyzed to estimate the seroprevalence of toxoplasmosis by using the ELISA technique. All the information was collected with the help of a questionnaire and analyzed to find out the risk factors. Results . The overall prevalence of toxoplasmosis in the female human population in Lahore was found to be 27%. The prevalence rate was 31% and 24% among pregnant and non-pregnant women, respectively. Similarly, women who underwent abortion had a high prevalence rate (66.6%) as compared to normal pregnant women (25.6%). In pregnant women, infection was more prevalent in the third trimester of pregnancy (43.7%), as compared to the first (28.5%) and second (20%) trimesters. The prevalence rate was higher in those women who had contact with cats or any other pet animal. The hematological parameters of the samples were also examined. In seropositive women, the levels of Hb and PCV declined, while the counts of lymphocytes and neutrophils considerably increased. Abnormal concentration levels of ALT and AST enzymes were also observed in seropositive women. Conclusion . This study revealed a higher prevalence of toxoplasmosis in pregnant women. Therefore, clinical screening should be encouraged for this infection


Highlights
• All serum samples were analyzed by using ELISA technique • Infection is more prevalent during the third trimester of pregnancy • Infected female showed a decreased Hb and PCV level • Infection alters the concentration level of ALT and AST enzyme

INTRODUCTION
Toxoplasma gondii is the pathogen that causes toxoplasmosis.This parasite infects human beings and other warm-blooded animals, although the felid family is the parasite's primary host [1].Still, the infection caused by this parasite is prevalent among human beings.Clear clinical distinctions are seen between human beings, species of birds, and sea and land mammals.This infection occurs worldwide, especially in regions with a moist and warm climate [2].
When the infection occurs with some other infection, such as HIV, then it causes serious complications including abortion and birth defects [3], transmission of HBV, and reproductive disorders [4].If a parasite infects immune-impaired persons, then it would be released uncontrollably because of the rupturing of tissue cysts in the brain [5].It results in an infected central nervous system, hemiparesis, headache, ataxia, seizures, facial weakness, and an altered mental status.If it is not treated, then the infection may cause fatal toxoplasmic encephalitis [6].
According to one systematic review with meta-and modelling-analysis, the global IgM seroprevalence in 2020 was 1.9%.The Eastern Mediterranean had the highest regional IgM seroprevalence (4.1%), whereas the Americas had the lowest (1.1%).The global seroprevalence of IgG was 32.9%.The Americas had the greatest prevalence (45.2%), while the Western Pacific had the lowest (11.2%) [7].This infection is also responsible for the deaths of animals, including livestock.Dairy goats are infected with T. gondii which results in reproductive loss [8] and also causes serious concern for public health [9].Tissue cysts of T. gondii are present in food animals and vary with species, such as sheep, pigs, and goats have a high rate of presence, while rabbits, horses, poultry, and dogs have a low rate of presence.Buffalo and cattle have a high rate of seroprevalence but rarely develop tissue cysts.The meat of sheep, pigs, and goats may contain tissue cysts if they are seropositive because these animals have a high rate of tissue cysts [10].
Maternal contamination by T. gondii amid pregnancy may have genuine ramifications for the baby, extending from premature delivery, focal sensory system association, retino choroiditis, and subclinical contamination during childbirth with visual ailments [11].Amid the disease, the parasite needs to transpose the intestinal boundary to spread all through the body, which might be a trigger for a provocative response [12].Through PCR, the rate of toxoplasma disease was discovered to be 20% in butchers and 22% in wild oxen [13].One investigation showed that TgPF could be a promising immunization competitor against interminable toxoplasmosis, which can be additionally used to create multiepitope antibody definitions in nourishment-delivering creatures against T. gondii disease [14].
The current study was undertaken to assess the prevalence and risk factors related to toxoplasma infection in individuals from the female human population of Lahore, Pakistan.It also aimed to investigate the hematological changes in toxoplasmosis-infected women and analyzed the said changes in their biochemical parameters.

MATERIALS AND METHODS
Blood samples of 150 women were collected at 11:00 am from Sir Ganga Ram Hospital Lahore, Pakistan.Preliminary information about hygienic conditions, contact with cats and other animals, use of drinking water from different sources, and the consumption of meat and vegetables was gathered through a designed questionnaire.Fresh blood was used for the analysis of hematological parameters in the selected women.A focused selection technique was employed to choose 90 samples, based on a questionnaire demonstrating their engagement with risk factors, for Toxoplasma-specific IgG antibody detection.All serum samples were analyzed for the detection of specific anti-Toxoplasma gondii IgG antibodies by using enzyme linked immuno-sorbant assay (ELISA).It took two months to complete the research.
The site for venipuncture was chosen at the counter cubital fossa region of the arm, where the middle cubital, cephalic, and basilic veins lie genuinely near the surface.The analysis of hematological parameters was done by using fresh anticoagulant blood in a hematology analyzer (Sysmex KX-21).Four (04) indicators were chosen for analysis, namely the number of neutrophils, the number of lymphocytes, the concentration of hemoglobin (Hb), and the packed cell volume (PCV) to assess changes in individuals infected with toxoplasma IgG antibodies.
An ELISA Immuno Explorer Kit, a commercial ELISA kit manufactured by Bio-Rad, was used to measure toxoplasmaspecific IgG antibodies.Toxo G Index of 1.00 or greater or a WHO IU/ml value greater than 32 IU/ml was considered seropositive.It indicated prior exposure to the toxoplasma infection.Toxo G index less than 0.90 indicated the absence of prior exposure to toxoplasma (<32 IU/ml).
Liver enzymes known as alanin aminotranferase (ALT) and aspartate aminotranferase (AST) have an average concentration level of 4 IU/L to 41 IU/L and 8 IU/L to 31 IU/L in women, respectively.Toxoplasmosis can cause changes in these levels, affecting ALT and AST.Biochemical analysis was performed on infected serum samples using the AST Assay Kit model number MET-5127 and the ALT Activity Assay Kit model number MET-5123, both manufactured by CELL BIOLABS, INC.For analysis, the colorimetric method was used.With the help of Chemistry Analyzer, the sample and the proper reagent were mixed to produce a reaction that yielded a color.The intensity of the color obtained was determined by the analyte's concentration.

RESULTS
Among all the selected women, 27% (n=25) presented specific antibodies to T. gondii.In the remaining 72% (n=65), these antibodies were not found.Among the total infected samples, 31% (n=14) were of pregnant women and 24% (n=11) were of non-pregnant women presenting specific antibodies to T. gondii.So, the prevalence rate was higher in pregnant women as compared to non-pregnant women (Figure 1).The study found older women to be more susceptible to toxoplasma due to a longer exposure time, with a higher rate of prevalence of the parasite, as shown in Table 1, Figure 2, and Figure 3. Out of a total of 45 pregnant women, 6 (66.6%) cases were reported in those women who had an abortion history and underwent premature miscarriage of their last baby, with some of them experiencing miscarriage more than once.The study found that toxoplasmosis prevalence varied across all trimesters of pregnancy, with a significant increase in seropositivity in the third trimester.Pregnant women in the third trimester had 43.7% prevalence, while those in the second and first trimesters had 20% and 28.5% prevalence of T. gondii, respectively.
Due to their frequent contact with vegetables during food preparation and poor cleanliness, low-income and unskilled housewives had a higher incidence rate.If hand hygiene is lacking, veggies might spread infection.Women who drink tap water instead of boiling or filtered water are more likely to have T. gondii than those who have pets (Table 3).Anti-coagulated blood samples were used to determine neutrophil count, erythrocyte count, lymphocyte count, the concentration of blood hemoglobin (Hb), and packed cell volume (PCV) by using the hematological analyzer.Hematological changes were observed in infected women as some parameters decreased and others increased in all samples.Their values were noted and the results were evaluated.The concentration of Hb decreased in 96% (n=24) seropositive cases.PCV level also decreased in mostly infected (n=20) samples as in 80% of cases.Overall, its level in all the samples was low.Out of the total infected samples, lymphocyte count was normal in some samples.Although, it increased to a high value from the normal value in 52% of cases.Overall, neutrophil count was normal in all samples, although in 80% of seropositive cases its value increased (Figure 4).Out of the total infected samples (n=14), 6% were infected with ALT and 8% were infected with AST, as shown in Table 4. Out of the total infected samples (n=11), 5% were infected with ALT and 6% were infected with AST, as shown in Table 4.

Statistical
analysis showed a significant difference among pregnant and non-pregnant women, risk factors, age groups, and in between trimesters.The prevalence rate was higher in pregnant women, housewives, those women who had contact with cats, and those who used tap water.Among age groups, it was found that older age groups were more susceptible to T. gondii.

DISCUSSION
The current study was conducted to find out the prevalence of T. gondii in female human population of Lahore and to evaluate the resultant hematological and biochemical changes in them.The results showed that approximately 31% of pregnant women and 24% of non-pregnant women had IgG antibodies to T. gondii.The overall prevalence in the all-female human population was 27%.
A study examined T. gondii infection in pregnant women and infants in Lorestan province, Western Iran using ELISA and PCR assays.The results showed infection in the umbilical cord and mother at 7.2% and 5.2%, respectively.Anti-toxoplasma IgG was found in 34.7% of samples, while IgM was found in 5.2% [15].
The transmission pathways, trimesters, and contamination of T. gondii were studied in North-West Pakistan.Maternity centers collected 733 blood samples of which 18.41% were infected, 7% were infected during the first trimester and 31% were infected during the third.A study in Baghdad, Iraq found 26% prevalence of toxoplasmosis in the female human population, which is similar to the current study's findings [18].A researcher discovered T. gondii in non-pregnant women in southern Punjab, testing them for IgG toxoplasma antibodies, confirming the results of the current study [19].Another found the same rate of prevalence of toxoplasmosis (24%) in non-pregnant women [20].Still another research found the same rate of prevalence for pregnant women [21].In one study, pregnant women were tested for the detection of toxoplasma IgG and the same results were found [22].Another study found a similar prevalence of toxoplasmosis in pregnant women in Tanzania.According to this study, the prevalence rate of T. gondii in pregnant women was 31% [23].
Polymerase chain response-based strategies for toxoplasmosis discovery, including creative, subjective, and quantitative RT-PCR approaches, have proven to be valuable in pathogen location and quality articulation examinations [24].PCR is the best examination to identify fetal contamination due to its high affectability and specificity, demonstrating parasitemia half a month before clinical signs [25].
A team of researchers worked on toxoplasmosis to check its seroprevalence in Muzaffargarh Kallarwali village.They reported 33% prevalence in non-pregnant women [26].However, acute T. gondii was found in early pregnancy in Kuwait.This investigation tested pregnant women for IgG toxoplasma antibodies and demonstrated that 53% of women were seropositive for these antibodies [27].According to a similar research, 74% of pregnant women were seropositive for T. gondii [28].An examination showed 54% of pregnant women as positive for specific T.
gondii antibodies [29].Furthermore, a study discovered 71% seroprevalence of toxoplasma among French women and 51% seroprevalence among immigrant women in the Paris area [30].
In the current study, it was observed that the prevalence of T. gondii increases with age.A study conducted almost two decades ago also demonstrated the infection of T. gondii as more prevalent in persons among older age groups as compared to persons among younger age groups [31].Other studies on pregnant women found that infection increases significantly with age [32].
In the current study, hematological analysis was performed in women infected with T. gondii specific IgG antibody.It was found that hemoglobin (Hb) concentration and packed cell volume (PCV) significantly decreased in these women, while lymphocyte and neutrophil count increased.Another study detected significant decreases in Hb and PCV in patients who were infected with T. gondii [33].Another examination also reported the same results [34].Biochemical changes were also analyzed in seropositive women, although AST and ALT levels did not significantly vary in infected women.This A study of 242 participants found 82.7% toxoplasmosis prevalence, with 62.8% toxoplasma IgG, 11.6% IgM, and 8.3% IgG/IgM, along with significant risk factors including cat presence, meat consumption, and blood transfusion history [36].The study found that secondary education and cat contact are major risk factors for T. gondii IgG and IgM seroprevalence in 400 first-trimester pregnant women [37].Another study involving 244 women aged 16 to 43 years found that 22.1% had IgG anti-T.gondii antibodies, while none had IgM anti-T.gondii antibodies [38].
A case-control study involving 50 healthy women and 135 women with abortion history found that anti-toxoplasma IgM and anti-toxoplasma IgG antibodies were positive in 51% and 8% of cases respectively, along with a higher seroprevalence in women with abortion history [39].Most studies show that higher seropositivity of T. gondii is associated with low education, age, cat contact, and consumption of semi-cooked or raw meat [40].

Conclusion
This study concludes that pregnant women (with the prevalence rate of 31%) are at a higher risk of toxoplasmosis than non-pregnant women (with the prevalence rate of 24%).The prevalence of T. gondii antibodies was found to be higher in older participants in this study.Hematological parameters of the samples were analyzed and it was observed that Hb and PCV levels decreased significantly, while lymphocyte and neutrophil counts increased or remained normal in infected women.Altered concentration levels of ALT and AST enzymes were seen in seropositive women.In pregnant women, infection was found to be more prevalent in the third trimester of pregnancy as compared to the first and second trimesters.Women who underwent abortion had a high prevalence rate as compared to women who did not.The prevalence rate was higher in housewives because of direct contact with vegetables during food preparation and contact with cats or any other pet animal.It is suggested that further studies should be carried out regarding the prevalence of toxoplasmosis among higher-risk groups including pregnant, aborted, and diseased females.

Muhammad Yaser Khan
Occupational transmission of toxoplasmosis is essential.For the identification of toxoplasmosis, latex agglutination test (LAT) and polymerase chain response (PCR) (in light of the enhancement of dull B1 quality of T. gondii) were utilized in this study.The study depended on the aggregate of 100 examples from 50 butchers and 50 wild oxen.

Figure 1 .
Figure 1.Number of Infected Female by Toxoplasmosis Population

Figure 4 .
Figure 4. Changes in Hematological Parameters in Seropositive Females study's findings are similar to the results of Kadir et al. (2013) [35].

Table 1 .
Age Wise Seroprevalence of Toxoplasmosis in Females Figure 2. Comparison of Toxoplasmosis among Three Major Age Groups in Pregnant Women in Terms of Percentage Figure 3. Percentage of the Examined and Infected-Non-Pregnant Females in Relation to Age Table 2. Trimester Wise and According to Abortion History Seroprevalence of Toxoplasmosis in Females

Table 3 .
Seroprevalence of Toxoplasmosis in Females with Respect to Occupation, Contact with Cats and Source of Drinking Water