Toxocara infection: seroprevalence and associated risk factors among primary school children in central China

Toxocariasis is a zoonotic disease that poses a threat to public health worldwide. In the present study, we investigated the seroprevalence of Toxocara infection among primary school children in Henan province, central China, which was previously unknown. Sera from 2451 primary school children were collected from September 2015 to October 2018, and evaluated for anti-Toxocara antibodies by enzyme-linked immunosorbent assay (ELISA). The overall seroprevalence of Toxocara infection was 5.14% (126/2451). The main risk factors related to Toxocara infection identified in this study were the age of children, residence area of children, contact with cats or dogs, and exposure to soil. Hand washing before eating was considered to be a protective factor. These findings demonstrate that Toxocara infection is relatively common among primary school children in Henan province.


Introduction
Toxocariasis is a worldwide zoonotic infection caused by the ascarid larvae of the Toxocara genus, including Toxocara canis (T. canis) and Toxocara cati (T. cati). Toxocara canis acts as the most frequent cause of toxocariasis, whereas T. cati is less common [16]. Their definitive hosts are domestic dogs and cats, respectively. Humans are accidental hosts who become infected by ingesting infective eggs or undercooked meat/viscera of infected paratenic hosts. After ingestion, the eggs hatch and larvae migrate through the intestine and can be carried to multiple organs (heart, liver, lungs, muscle, brain, and eyes) via the bloodstream, causing local reactions and mechanical damage that causes clinical toxocariasis [28]. Infection in humans leads to various disorders accompanied by relevant manifestations. There are four commonly described disorders: convert toxocariasis (CT), neurotoxocariasis (NT) (e.g., eosinophilic meningoencephalitis), ocular larva migrans (OLM), and visceral larva migrans (VLM) [21,28].
Children usually become infected by accidentally ingesting embryonated eggs of T. canis or T. cati from the contaminated environment (e.g., soil and water) [24,27] and dirty hands or, occasionally, by eating invertebrates, such as earthworms [4]. Consumption of undercooked meat from paratenic hosts that contain encapsulated larvae can also result in toxocariasis [14,34].
In China, T. canis and T. cati have been widely detected in dogs and cats, respectively [9,32,36]. An increasing number of clinical cases of toxocariasis have been reported in children (Table 1; [18,20,33,[37][38][39]). However, little is known about the seroprevalence of Toxocara infection among children in China (Table 2; [5,6,19,20]). Most surveys were published in local journals in Chinese, and are not readily accessible to international readers. Furthermore, reports on Toxocara seroprevalence among students in primary school in Henan province, central China were still lacking. Consequently, the aim of the current study was to investigate Toxocara seroprevalence and relevant risk factors among students in primary school in Henan province.

Ethics statements
In the current study, all protocols were reviewed and approved by the Ethics Committee of Xinxiang Medical University (Ref. no. 2015018).

Study site and sample collection
Blood samples were collected from 2451 primary school children in four cities of Henan province, as previously described [31]. The same sera were used in both studies.

Antibodies to Toxocara
A commercial Toxocara IgG ELISA kit (Diagnostic Automation Inc., Woodland Hills, CA, USA) was used to detect anti-Toxocara IgG antibodies. Both negative and positive controls were provided in the kit and used in each test. Samples were considered positive on the basis of absorption that was no less than 0.3 OD units. Samples with inconclusive results were tested again. All the operations were performed according to the manufacturer's instructions [5,35]. The sensitivity and specificity of the ELISA were 87.5% and 93.3%, respectively.

Statistical analysis
Statistical analysis was performed using SPSS 20 software for Windows (SPSS Inc., Chicago, IL, USA). Statistical analyses of Toxocara prevalence in different variables were performed using a v 2 -test. A p-value of less than 0.05 was deemed statistically significant. Table 3, the overall seroprevalence of Toxocara infection among primary school children in Henan province was 5.14%. Toxocara seroprevalence among the children living in Xinxiang, Zhengzhou, Zhumadian and Nanyang were 3.08%, 5.90%, 4.52%, and 7.08%, respectively.

As shown in
The prevalence of Toxocara antibodies varied significantly with the place of residence (p = 0.014).
The seroprevalence of Toxocara infection in children living in rural areas was significantly higher than those living in urban areas (v 2 = 4.799, p = 0.028), and the rate was significantly higher in children in contact with cats (v 2 = 4.328, p = 0.037) and dogs (v 2 = 10.051, p = 0.002). Moreover, the probability of infection with Toxocara was significantly increased in children in contact with soil compared to those with no contact (5.69% vs. 3.64%, v 2 = 4.153, p = 0.042). Children with the behavior of washing hands before eating exhibited lower seropositive rates for Toxocara than those without (4.26% vs. 7.02%, v 2 = 8.306, p = 0.004).

Discussion
The overall seroprevalence for Toxocara infection was 5.14% in primary school children in Henan province. The seroprevalence obtained in this study was lower than that of other provinces in China such as Sichuan (10.96% and 11.49%) [19,20], and Shandong and Jilin provinces (19.27%) [6]. Compared to other countries and regions, the total Toxocara seropositive rate in Henan province was also lower than 86.1% reported in children aged 7-17 years from Makoko, an urban slum community in Nigeria [12], 86.75% among students in primary schools from the capital area of the Republic of the Marshall Islands [10], 12.02% in the Amecameca and Chalco regions of México [7], 29.0% in Aragua state, Venezuela [22], and 10.0% in Serbia [11]. However, the seropositive rate was higher than that observed in Iran (1.39-3.8%) [13,15,16]. Several factors such as age of children, sample sizes, the specificity and sensitivity of the detection methods used, various climatic and geographical conditions, hygiene habits, and lifestyle of the population assessed could have contributed to the differences observed in seroprevalence rates.
The present study showed that geographical origin is a risk factor. The differences in seropositive rates among the different regions of Henan may be attributed to local food habits, climate condition, densities of population, and the number of stray dogs and cats. In this study, the seroprevalence value Zhang et al. [37] for boys was nearly equal to that of girls, indicating that the exposure levels of both sexes were very similar, and thus male or female sex was not a critical risk factor associated with Toxocara infection. This finding was consistent with other similar reports [7,13]. It has been found in numerous studies that Toxocara seroprevalence is positively correlated with the age of children [12,30]. In the current study, similarly, Toxocara seroprevalence also exhibited a positive correlation with the age of children, with a progressive and significant pattern. It has been hypothesized that this phenomenon results from increasing years of exposure as the child grows, and a generally highly contaminated environment.
The present study found that children with a history of contact with dogs or cats showed a higher tendency to acquire Toxocara infection than those who do not. A number of studies showed the presence of embryonated T. canis and T. cati eggs on the hair of dogs and cats, respectively, indicating that direct contact with dogs or cats may be a potential route of infection [1,8,23]. If children touch dogs and cats frequently, the possibility of accidentally ingesting embryonated T. canis or T. cati eggs from the hair increases, which results in a higher risk of infection. The seropositive rate of Toxocara in children living in rural areas was significantly higher than that in urban areas, which was consistent with other reports [16,29]. The explanation for this observation is that the higher number of stray and domesticated dogs and cats in rural areas may have increased the degree of environmental contamination with Toxocara eggs. Poor hygiene habits in rural areas can also lead to a higher incidence of toxocariasis.
Soil contamination with Toxocara eggs is considered to be the main source of human infections [2,25,26]. The present study also revealed that exposure to soil was related to Toxocara infection in primary school children, indicating that soil contamination by dog and cat feces is widespread in these areas. In the future, more research is needed to evaluate the  prevalence of Toxocara eggs in the soil of local parks and primary schools. Additionally, hand washing before eating has been verified as a protective factor related to Toxocara seroprevalence in this study. This finding is in line with other similar surveys [3,17].

Conclusion
In conclusion, the present study revealed for the first time that Toxocara infection in primary school children is relatively common in Henan province, China. Prevention approaches including cleaning hands after contact with soil, cats or dogs and before eating, reducing soil contamination by dog or cat feces in public areas, and treating dogs and cats with anthelmintics to reduce Toxocara burdens, can be beneficial to minimize exposure to Toxocara spp.

Conflict of interest statement
We declare that we have no conflict of interest.