Epidemiology of Toxoplasmosis among the Pakistani Population: A Systematic Review and Meta-Analysis

Toxoplasma gondii is an intracellular obligate parasite that causes toxoplasmosis, a zoonotic infection that affects warm-blooded animals and humans worldwide. To comprehensively characterize the disease condition in Pakistan for future reference, we ascertained the prevalence of Toxoplasma infection and predisposing factors in the Pakistani population over a 20-year period. We systematically reviewed research articles published in English (2000–2020) from PubMed and Google Scholar. The search results 26 publications involving 10,924 people and 2611 seropositive cases. The toxoplasmosis seropositivity rate was higher in women (25.44%) as compared to men (21.48%) and were statistically significant (p < 0.001). Furthermore, seropositivity was high among people with direct contact with cats, who consumed uncooked meat and raw vegetables, had poor education, and lived in rural areas. The 35–65-year age group had the highest prevalence rate of T. gondii infection. Toxoplasma infection was significantly more prevalent in Khyber Pakhtunkhwa province (25.87%) than in Punjab (20.42%) (p < 0.001). This is the first comprehensive analysis of T. gondii infection epidemiology in Pakistan. It reveals a high frequency of infection among women. We strongly encourage further research to aid patient care and the development of more efficient diagnostic tests and preventative techniques.


Discussion
It is critical to know how common Toxoplasma infection is among the general population of Pakistan. Using data from the literature obtained from different provinces, the present systematic review and meta-analysis assessed the epidemiology and prevalence of T. gondii infection in the general population of Pakistan. In a recent review, Rostami and colleagues summarized that the incidence rates of T. gondii infection among pregnant women were highest in the South American (56.2%) and African (48.7%) regions, while the lowest frequency was reported in the Western Pacific region (11.8%) [47]. Similarly, T. gondii seroprevalence was highest (75-85%) in Latin America, Central and Eastern Europe, and Southeast Asia [48]. Our meta-analysis findings reveal that approximately 24% of the general population of Pakistan was seropositive for the toxoplasmosis infection. Our findings support the widely held belief that approximately one-third of humanity is infected by T. gondii [49]. When it comes to the possibility of contracting toxoplasmosis, the most dangerous risk factors are eating raw or undercooked meat, having close contact with cats and soil, and engaging in inadequate hygiene standards [50][51][52]. Pakistan is classified as a country with a low-middle income; nevertheless, despite this, it is one of the top 10 nations in the world that does not have enough access to clean water. In addition, the lack of knowledge and the budgetary constraints in the health system in this nation all contribute to the spread of this infection [37,53], making the situation even more direr.
Epidemiological studies have been used for ascertaining T. gondii endemicity in humans and animals. There is a dearth of information on its seroprevalence [54,55]. Appropriate data on parasite prevalence and transmission routes would enable accurate risk assessment in pregnant women and aid the creation and implementation of parasitic infection control and preventive strategies [40,55]. Here, we determined that the total incidence and epidemiology of toxoplasmosis in Pakistan is higher in the female

Discussion
It is critical to know how common Toxoplasma infection is among the general population of Pakistan. Using data from the literature obtained from different provinces, the present systematic review and meta-analysis assessed the epidemiology and prevalence of T. gondii infection in the general population of Pakistan. In a recent review, Rostami and colleagues summarized that the incidence rates of T. gondii infection among pregnant women were highest in the South American (56.2%) and African (48.7%) regions, while the lowest frequency was reported in the Western Pacific region (11.8%) [47]. Similarly, T. gondii seroprevalence was highest (75-85%) in Latin America, Central and Eastern Europe, and Southeast Asia [48]. Our meta-analysis findings reveal that approximately 24% of the general population of Pakistan was seropositive for the toxoplasmosis infection. Our findings support the widely held belief that approximately one-third of humanity is infected by T. gondii [49]. When it comes to the possibility of contracting toxoplasmosis, the most dangerous risk factors are eating raw or undercooked meat, having close contact with cats and soil, and engaging in inadequate hygiene standards [50][51][52]. Pakistan is classified as a country with a low-middle income; nevertheless, despite this, it is one of the top 10 nations in the world that does not have enough access to clean water. In addition, the lack of knowledge and the budgetary constraints in the health system in this nation all contribute to the spread of this infection [37,53], making the situation even more direr.
Epidemiological studies have been used for ascertaining T. gondii endemicity in humans and animals. There is a dearth of information on its seroprevalence [54,55]. Appropriate data on parasite prevalence and transmission routes would enable accurate risk assessment in pregnant women and aid the creation and implementation of parasitic infection control and preventive strategies [40,55]. Here, we determined that the total incidence and epidemiology of toxoplasmosis in Pakistan is higher in the female population (25.44%) than in the male population (21.48%), while combining male and female participants resulted in a low prevalence rate (15.67%) that was significantly different (p < 0.0001). Prevalence was predicted to be higher in women (25.44%), while the frequency was comparatively low in men (21.48%). In 2011, a study was performed in Kohat, KPK, to determine the seroprevalence and predisposing factors for T. gondii in pregnant women. Overall seropositivity was 14.4% (26 of 180) [15]. In 2016, a comparable study performed in the same zone obtained 733 random selections from prenatal facilities. Overall, seroprevalence was observed in 81.41% of pregnant women examined [31]. In a recent investigation, toxoplasmosis was prevalent in 24.36% of the male population [56]. Another study reported that the incidence rate of toxoplasmosis between 2000 and 2004 in the male population of the Czech Republic was 23% [57]. Our findings demonstrate that toxoplasmosis seroprevalence in the male population in Pakistan was 21.48%, which is in accordance with earlier research.
People with direct contact with cats, who ate undercooked meat and raw vegetables, and those with low levels of education (illiterate) had much higher T. gondii infection rates, with a statistically significant difference (p < 0.0001) between them ( Table 2). A variety of risk factors have led to a high rate of toxoplasmosis. Reduced host immune responses, drinking polluted water and eating contaminated fruit and vegetables, owning pet cats, consuming undercooked meat, and coming into contact with the flesh or viscera of infected animals are all risk factors that increase toxoplasmosis occurrence [58,59]. Rostami et al. [47] have indicated a high incidence of congenital toxoplasmosis and acute Toxoplasma infection in people in low-income countries, and consuming raw or undercooked meat in the daily diet could be a significant predictor of toxoplasmosis. This is in line with our findings that people who directly touched cats, ate raw or undercooked meat and vegetables, or were illiterate had significantly higher T. gondii infection rates. Toxoplasma oocysts excreted in cat feces are more likely to survive and spread if the habitat contains atmospheric agents that aid their sporulation, such as sufficient humidity, which increases the risk of human and animal infection. As mentioned earlier, behavior is influenced indirectly by risk factors such as occupation, gender, geography, and educational attainment, influencing infection prevalence [59,60].
The subgroup analysis revealed higher toxoplasmosis prevalence in the rural population (16.65%) than in the urban population (8.54%), with no significant differences (p > 0.005). Cats and infected hunting animals are common in rural regions, as are higher oocyst survival rates in waste soil, a significant number of rural residents, inadequate health literacy, and restricted accessibility to health care facilities [60,61]. The subgroup analysis also revealed that prevalence estimates increased with age, consistent with previously reported data [47,58]. The data support our findings of inequalities among countries with different economic statuses. Overall, the total prevalence of toxoplasmosis in Pakistan is 23.9%. In contrast, the prevalence is lower in the US (10.8%), Mexico (20.26%), and China (6.31%) due to better hygiene practices, effective public awareness campaigns, accurate and consistent screening procedures for reproductive-age women and pregnant women, modernized livestock farming, well-established surveillance systems, and enhanced meat evaluation rules [4,62,63]. In addition, the likelihood of toxoplasmosis transmission by blood donation from healthy and asymptomatic carriers of infection adds to the worldwide prevalence, particularly in locations where toxoplasmosis is considerably more widespread [64]. People who test positive for Toxoplasma during the acute stage of the disease may have contributed to the expansion of toxoplasmosis through blood donation. Africa and Asia reported the highest and lowest global burdens of toxoplasmosis in blood donors at 46% (95% CI = 14-78) and 29% (95% CI = 23-35), respectively [65,66]. The highest toxoplasmosis seroprevalence was detected in Brazil (75%) and Ethiopia (73%) [65]. Pakistan is one of the top poultry producers in the world and chicken is the simplest source of meat, accounting for approximately 28% of high meat output. Increased chicken-eating appears to enhance the likelihood of T. gondii transmission to humans [67].
Here, toxoplasmosis seropositivity was 6%, 19.07%, 25.82%, and 19.93%, respectively, in the 25-40-, 35-, 35-65-, and >66-year age groups, respectively, indicating that T. gondii infection frequency is highest in the 35-65-year age group. According to research in Pakistan, infection was highest in the 26-40-year age cohort, accounting for 54.55% of the examined population, whereas the 41-55-year age group had low infection seropositivity, accounting for approximately 21.87%. The 15-25-year age group, on the other hand, had a moderate infection incidence of approximately 23.63%. Up to 76.1%, 45.1%, and 72.1% of infants in Tehsil Charsadda, Tangi, and Tehsil Shabqadar, respectively, were born with birth anomalies [40]. Similarly, another study from Pakistan reported that the 31-40-year age group had the highest toxoplasmosis incidence (33.33%, 30/110), followed by the 21-30-year age group (30%, 33/110) [46]. A similar pattern was reported in the US; while the frequency of toxoplasmosis may be declining, approximately 14% of people are seropositive by the age of 40 years [4].
The clinical manifestations of a T. gondii infection are not helpful in the diagnosis because they are not distinct [68]. The specific diagnosis of a T. gondii infection often relies on bioassays and serological testing, even though both methods have their limits in identifying or discriminating parasite subtypes [69,70]. The conventional toxoplasmosis diagnostic methods encompass etiological, immunologic, and imaging techniques. The advances in molecular technologies that can amplify parasite nucleic acids have led to significant advancements in the diagnostic process for toxoplasmosis. Among all these diagnostic methods, polymerase chain reaction (PCR) has the higher sensitivity and specificity in diagnosing the Toxoplasmosis [71,72]. In this meta-analysis, anti-T. gondii IgG serum antibody level was measured in different studies using a number of serological diagnostic tests. Enzyme-linked immunosorbent assay (ELISA, 24.92%, n = 5240) was the most used detection method, followed by the latex agglutination test (LAT, 28.35%, n = 4585), lateral flow chromatographic immunoassay (LFCI, 20.31%, n = 1216), immunochromatographic assay (ICA, 43.47%, n = 920), rapid diagnostic test (RDT, 2.35%, n = 170), and the indirect fluorescent antibody test (IFA, 18.41%, n = 733).
Our subgroup analysis revealed that toxoplasmosis seropositivity frequency increased with advancing age, which is consistent with previously reported data [73]. Assessing the incidence of latent toxoplasmosis in different parts of the country can aid risk factor identification and the implementation of better policies and programs for reducing the disease burden. Therefore, we believe that our findings will aid future risk mapping and modeling. We also discovered regional differences and other parameters linked to the incidence of latent toxoplasmosis. Examining the underlying causes of this heterogeneity, particularly across locations or countries with similar features, would aid the development of better public health strategies. For example, if two countries had identical characteristics but vastly differing prevalence rates, lessons could be learned and implemented to reduce the burden of toxoplasmosis. Accordingly, a global approach to examining these variances and their consequences is required to inform action. Toxoplasmosis epidemiology varies geographically, requiring further epidemiological studies on socio-economic factors. These are most likely due to disparities in public health and hygiene facilities, socio-economic background, social and cultural conditions, and climate. Epidemiological research in Latin/Caribbean countries has revealed a high prevalence of T. gondii in feral cats, implying that the environment may be severely contaminated with oocysts, contaminating plants and people who touch the soil [74]. In contrast, this systematic review and meta-analysis have a number of limitations. For instance, although a complete search was done utilizing two databases, it is possible that some data were overlooked since certain findings were printed in local journals in languages rather than English; hence, local language articles were omitted.
To summarize, this systematic review and meta-analysis focused on the epidemiology and geographical location of T. gondii in Pakistan. Public understanding, training courses, and sanitary practice are all beneficial for guiding people toward cooked foods and cleaned vegetables, handling feral cats, and treating sick pet cats in a timely and proper manner. Intensive monitoring and routine inspections of food manufacturing businesses will enhance hygiene standards in abattoirs and the sanitary disposal of viscera, as would sophisticated screening programs utilizing modulus diagnostic tests, as shown in Table 1. Despite extensive research on T. gondii infection in humans, blood donors, pregnant women, immunocompromised patients, and animals (sheep, goats, cattle, and cats), there are no systematic reviews or meta-analyses on toxoplasmosis in ingested meats, or birds or other zoonotic hosts in Pakistan. In the future, we believe it would be beneficial for researchers and policymakers to concentrate their focus on the neglected populations.

Material and Methods
This was a systematic review for determining the prevalence of diseases caused by T. gondii in the Pakistani population (men and women). To reduce the potential for bias, data collection and eligibility criteria were designed for evaluating the crude prevalence (percentage of positive cases) and weighted population prevalence (proportion of positive cases) for every study. The frequency of infection over 20 years was depicted using forest plots.

Search Strategy
The search was limited to articles written in the English language. PubMed and Google Scholar were searched for papers published in 2000-2020 on toxoplasmosis infection prevalence, diagnostic tests, and risk factors for our systematic review and meta-analysis to determine T. gondii infection frequency in people in Pakistan. For data gathering, the English databases were searched using the following terms: "toxoplasmosis", "T. gondii", "humans", "prevalence", "epidemiology", and "Pakistan" alone or in combination.

Data Collection
All 26 studies obtained were meticulously examined. The following information was extracted: year of publication, study demographic characteristics, author(s) name, study location, sample size, case numbers, diagnostic tests, and risk variables.

Inclusion and Exclusion Criteria
The inclusion criteria were established by abstract review, publication reading, and title screening. Duplicate papers, irrelevant data, inadequate data, and human-based research that did not focus on T. gondii-related disorders were excluded; studies conducted in Pakistan were retrieved.

Meta-Analysis
The meta-analysis was performed using R. First, T. gondii infection prevalence was determined using binomial distribution,p1 = x n (1: Where x is the number of positive cases and n is the total number of examined cases) and the standard error se = pq n for each study was computed. Next, the weighted prevalence of T. gondii was calculated for gender (female, male, and both), contact with animals, undercooked food, provinces (KPK and Punjab), and sample size (<200, 200-400, 400-600, and >600 people). For the above variables, we used a single-proportion meta-analysis. The Freeman-Tukey Double arcsine transformation was considered for the proportions and the inverse variances were estimated with the DerSimonian-Laird method. For the meta-analyses of location (rural and urban) and education (literate and illiterate), the odds ratios were calculated for the pooled effect size. Variance was estimated using the Sidik-Jonkman method and 0.1 increments were added for continuity correction of zero cells. The Cochran Q test and I 2 statistic were used to determine between-study heterogeneity and forest plots (described as ES (effect size) with a 95% confidence interval (95% CI)) were constructed as a graphical summary of the results.

Conclusions
This review examines the current state of T. gondii infection in the Pakistani population. Toxoplasmosis was more common in women than in men. The high prevalence of T. gondii infection in Pakistan is a serious public health concern. Therefore, health measures should be taken to prevent and control the disease. Our findings provide important statistics and information on the prevalence of T. gondii infection, which could be helpful in infection management and control. In addition to serological techniques, more research is required to employ PCR tests to assess infection accurately. We also recommend multiple precautionary measures should be followed to control the risk factors of toxoplasmosis, including properly cooking meat and vegetables, using gloves while gardening, proper handwashing, and maintaining hygienic conditions.