A Cross-Sectional Study on the Association Between Risk Factors of Toxoplasmosis and One Health Knowledge in Pakistan

Toxoplasmosis is a zoonotic disease caused by Toxoplasma gondii, a protozoan that infects warm-blooded animals and humans. Approximately one third of the global population is infected by T. gondii. We conducted a cross-sectional study to assess the risk factors and One Health knowledge of toxoplasmosis in Rawalpindi and Islamabad, Pakistan. From July through December 2020, we collected data using questionnaires. The results showed that 60% of participants had heard or read about the disease, 23.3% of participants had no knowledge about the disease, and 16.8% participants were not sure about the disease. More than half of the participants (53.3%) reported that toxoplasmosis was caused by toxins, 5.3% reported that toxoplasmosis was an animal disease, 13.8% reported that toxoplasmosis was a human disease, 65.8% reported that it was both an animal and human disease, and 15.3% reported that it was neither an animal nor a human disease. Approximately 80.5% of participants reported that individuals acquired toxoplasmosis by changing cat litter. Our study findings revealed a low level of knowledge and awareness about toxoplasmosis among males. Therefore, there should be awareness programs to educate individuals about the risks of this deadly disease and to provide information on the major routes of transmission.


INTRODUCTION
Toxoplasmosis is a zoonotic disease caused by the intracellular protozoan Toxoplasma gondii (1). T. gondii is an obligate intracellular parasite that naturally exists in one of three forms: (1) oocysts, which release sporozoites, are only produced in the small intestines of cats and are released into the environment through their feces; (2) tissue cysts, which release bradyzoites; and (3) tachyzoites, which are the proliferative form (2). Type I, II, and III strains of T. gondii have been identified in Europe, parts of Asia, and US where type II strain is mostly involved in human toxoplasmosis (3). Type I and type III strains are prevalent in Central and South America (4). Approximately 33% of the total human population has been affected by T. gondii (1). Countries in North America, Southeast Asia, Northern Europe, and Saharan African have low prevalence rates (10% to 30%), Central and Southern Europe have moderate prevalence rates (30% to 50%), and tropical African countries and Latin America have high prevalence rates of toxoplasmosis (5). The seroprevalence of toxoplasmosis was 29.45% from Southern Punjab, Pakistan (6). In Pakistan, Khyber Pakhtunkhwa has 40.6% of the seroprevalence of toxoplasmosis in women with poor obstetric history (7).
In humans, toxoplasmosis is transmitted by consuming raw or inadequately cooked meat (8), by inadvertently ingesting oocysts passed into feces by cats, either in a cat litter box or outdoors in the soil (9), and from mother to her unborn fetus (10). T. gondii infection, which is a life-threatening disease, results in retinal infection in both healthy and immunocompromised individuals (11). In immunocompromised individuals, toxoplasmosis is mostly asymptomatic (12); however, 10% of those infected may develop lymphadenitis, ocular toxoplasmosis (chorioretinitis), and mild flu-like and/or mononucleosis-like symptoms (13).
Due to their non-specificity, the clinical symptoms of toxoplasmosis are not reliable for diagnosis. While traditional diagnostic methods are based on serological tests and bioassays, a variety of molecular methods have been recently used for diagnosis of toxoplasmosis (14). Some of the diagnostic tests for toxoplasmosis include microscopy (15), bioassays (16), dye test (17), modified agglutination test (18), latex agglutination test (19), indirect hemagglutination test (20), indirect fluorescent antibody test (21), enzyme-linked immunosorbent assay (22), immunosorbent agglutination assay (23), immunochromatographic test (24), piezoelectric immunoagglutination assay (25), Western blot (26), and avidity test (27). Pharmaceutical interventions against toxoplasmosis include either a combination of pyrimethamine and sulfadiazine with folic acid or a combination of pyrimethamine and macrolide antibiotics or lincosamide. For congenital toxoplasmosis, pregnant women are treated with spiramycin (12).
Toxoplasmosis, which affects both animals and humans, causes major economic losses (28). In the livestock sector of Pakistan, different diseases cause annual economic loss of 79 billion Pakistani rupees (PKR) (29). Despite having such significant impact, very few studies have explored the prevalence of toxoplasmosis in Pakistan. Therefore, we conducted a study to determine the knowledge, attitudes, and practices of toxoplasmosis among university students of twin cities, Rawalpindi and Islamabad, Pakistan. Table 1 presents the sociodemographic characteristics of the participants (n = 400). Most of the participants (86%) were females. The majority of the participants (65.5%) were Abbreviations: WHO, World Health Organization; NZDs, Neglected Zoonotic Diseases; T. gondii, Toxolasma gondii; DALYs, Disability Adjusted Life Years.

Association Among Different Variables Based on ANOVA
We used one-way ANOVA to determine whether there were any statistically significant differences among the means of three or more independent groups. We used six specific independent variables, i.e., age, sex, ethnicity, education, religion, and marital status, and five dependent variables, i.e., knowledge, attitudes, practices, risk factors, and One Health. Our ANOVA results revealed that age was associated (p < 0.05) with attitudes and One Health; however, there were no significant associations with sex. Ethnicity was associated (p < 0.05) with knowledge and One Health; religion was associated (p < 0.05) with One health; and marital status was associated (p < 0.05) with knowledge, attitudes, risk factors, and One health. Likewise, the education of the participants was associated (p < 0.05) with knowledge, risk factors, and One Health ( Table 7).

DISCUSSION
Toxoplasmosis is a major global zoonotic disease that has a deleterious effect on human health, with severe consequences in immunocompromised, pregnant women (10). Consumption of contaminated raw meat, water, fruits, and vegetables; contact with cats; and exposure to soil contaminated with cat feces are the main transmission routes (11). Out of 400 participants, 240 (60%) were aware of toxoplasmosis. Similar findings have been reported in Northeast Ethiopia (1).
Our study findings revealed that 87.3 and 85.5% of participants washed their hands after gardening and changing the cat litter, respectively. Additionally, 89% of participants thoroughly cooked meat prior to consumption, and 86.3% avoided drinking raw milk. A study from Ethiopia reported that among pregnant women, 77.6% washed their hands after gardening, 64.7% washed their hands after changing the cat litter, and 62.2% washed their hands after handling raw meat. Furthermore, 85.9% of the pregnant women reported that they did not avoid drinking untreated water (1). In our study, 80% of participants considered toxoplasmosis to be a dangerous disease, and 33.5% reported that they had not consumed undercooked meat. In contrast, a study reported that 51.4% of participants did not consider toxoplasmosis to be a severe disease. Additionally, 48% individuals were unsure whether toxoplasmosis was spread via consumption of inadequately washed vegetables (30). Our study showed that 81.8% of participants washed their kitchen utensils after contact with raw meat or unwashed fruits and vegetables. Similar findings were obtained in Brazil, where 24.7% of pregnant women reported washing kitchen utensils (31). Approximately 30% of the participants did not allow anyone else to change the cat litter box. Similar findings have been reported in a study conducted   in Northeast Ethiopia where 51.3% women responded that they did not allow someone else to change the cat litter box (1). Most of the participants (76.8%) reported that toxoplasmosis was acquired by consuming raw/undercooked meat. These findings were consistent with those of a study carried out in Mexico, where more than half of the respondents correctly defined the routes of transmission: (1) consumption of raw or undercooked foods, unwashed fruits and vegetables and (2) direct contact with cats (32). In our study, 69.5% of participants considered blood transfusion to be a cause of toxoplasmosis. In one of the surveys, 27.7% of the participants did not assume that blood transfusion could spread toxoplasmosis, and 38.5% believed that it could be transmitted from the mother to her fetus (33). Approximately 68.3% of participants responded that gardening without gloves could be a transmission source of toxoplasmosis. In a study conducted in the US, 29% of the participants thought that toxoplasmosis could be transmitted by gardening without gloves (34). Our study findings showed that immunocompromised pregnant women had a high risk of toxoplasmosis similar to the findings of Desta who reported there is a high risk of toxoplasmosis in immunocompromised, pregnant women (77.9%) (1). The majority (58%) of participants reported that toxoplasmosis is a zoonotic infection. A previous study reported that 33.82% of participants were aware that toxoplasmosis is a zoonotic disease (1).

Strength, Limitations, and Future Recommendations
The limited amount of knowledge about toxoplasmosis emphasized to provide and promote health education regarding toxoplasmosis especially awareness regarding transmission of disease in the pregnant women. It is important to improve primary health care system of the country for the better control, management, and prevention of the disease. Moreover, it is stressed that in the study population to commence health education and awareness campaigns for the community and to design relevant policies for the guidance of the government and stakeholders to reduce the risk of disease. In the study design, the use of close questionnaire is one of the limitation, where free form response was not allowed. In our study included the participants from university which is not representative of the situation of whole country. The strength of the study is maximum number of female participants and preliminary study on the knowledge about toxoplasmosis among university students in Pakistan.

Study Area
We conducted a cross-sectional analysis in Islamabad and Rawalpindi district of Punjab, Pakistan, also known as twin cities. The terrain consists of plains and mountains in the metropolitan area of Islamabad and Rawalpindi. In the mountainous terrain of Margala hills is the northern part of the metropolitan area, while Rawalpindi is situated on the Pothohar plateau (35).

Participants
The study participants included students from universities of the twin cities that were enrolled in different degree programs (Bachelors, Masters, Ph.D., and Post doc). The sample size was calculated using Raosoft software (http://www.raosoft.com/ samplesize.html; 5% margin of error, 95% confidence level, and 50% response distribution). Four hundred questionnaires were randomly distributed and filled by the participants. We collected data from July through December 2020.

Sample Size
A questionnaire was designed to access the knowledge, attitude, practices, risk factor and one health regarding toxoplasmosis. A total of 400 questionnaires were administrated. The questionnaire was categories into the following sections as demography (n = 17), knowledge (n = 34), attitude (n = 19), practices (n = 11), risk factors (n = 8), and one health (n = 7).

Data Collection
We developed a structured questionnaire to collect the data. After obtaining verbal informed consent from the participants, we conducted interviews. A team was trained for interviews, data collection, and record keeping. A supervisor routinely coordinated the interview process to ensure adequate data collection and record maintenance. The purpose of study was explained to the participants. The questionnaire consisted of six sections. The first section was on the socio-demographics of the participants. The second section was on the knowledge on toxoplasmosis, including common signs, symptoms, and diagnostic tests used for toxoplasmosis. The third section was on the attitudes and perceptions toward toxoplasmosis. The fourth section was on practices performed when toxoplasmosis was either suspected or diagnosed. The fifth section was on major risk factors of the disease, and the sixth section was on One Health questions regarding toxoplasmosis.

Statistical Analysis
We generated a database using Excel (Microsoft, Redmond, WA, USA) and calculated basic frequencies. We used descriptive statistics to initially analyze the data and classified the variables   into independent and dependent variables. We performed statistical analysis using Jamovi software (version 1.6.7; https:// www.jamovi.org) to observe the factors involved in the occurrence of toxoplasmosis. The relationship between various factors influencing knowledge, attitudes, and practices was analyzed. For data analysis, we used Chi square test, one-way analysis of variance (ANOVA), and log-linear regression.

CONCLUSIONS
There is a low level of knowledge and awareness regarding toxoplasmosis among males. Therefore, there should be awareness programs to educate individuals about the risks of this deadly disease and to provide information on the major routes of transmission. Our study highlights the need of toxoplasmosis awareness to reduce the burden and economic impact of the disease.

DATA AVAILABILITY STATEMENT
The original contributions presented in the study are included in the article/supplementary material, further inquiries can be directed to the corresponding author/s.

ETHICS STATEMENT
The studies involving human participants were reviewed and approved by the Ethics Committee of COMSATS University. The patients/participants provided their written informed consent to participate in this study.

AUTHOR CONTRIBUTIONS
HA and KS designed and supervised the study. TM and KS performed the data collection. KS, SS, SA, MA, HA, and JC conducted statistical and data analysis. SN drafted the manuscript. SS and JC performed critical revisions. All authors read and approved the final manuscript. The funders had no role in the study design, the data collection and analysis, the decision to publish, or the preparation of the manuscript.