Soil-transmitted Helminthiasis, Intestinal Protozoa and Clonorchiosis Infections in Southeast China: A Cross-sectional Study

Background: The large scale epidemiology of parasitic diseases in Zhejiang Province has not been investigated since the second national survey. Methods: We investigated the distribution of three types of parasitic diseases (i.g. soil-transmitted helminthiasis (STH), intestinal protozoa, and clonorchiosis) in Zhejiang province from late 2014 to 2015. Kato-Katz technique was used for STH and Clonorchis. Sinensis detection, transparent adhesive paper anal swab was used for pinworm detection, and iodine smear was for protozoa detection. Hookworm positive samples need to be cultured for identication of Ancylostmoma duodenale or Necator americanus. Results: 23,552 participants were included in this study (19,935 from rural area and 3,617 from urban area). The overall prevalence of any intestinal helminth infection was 1.800%. Seven species of helminth were found: A.duodenale, N.americanus, Trichuris trichiura, Ascaris lumbricoides, C. sinensis, Fasciolopsis buski and pinworm. The prevalence of STH infection was 1.711% (1.941% in rural area, 0.442% in urban area). Hookworm was the most prevalent infection (1.584%). Only 2.79% children were found for pinworm infection in rural area. 0.396% of the rural participants were infected protozoa, and Endolimax nana was the most prevalent (0.231%). C. Sinensis infected only one man. 89.135% of participants had good personal hygiene habits. The awareness of C. Sinensis was 24.467% and 45.963% in rural and in urban area, respectively. Conclusions: Comparing to the second national survey, the prevalence of STH and protozoa infections declined greatly and C. Sinensis infection was still rare in Zhejiang Province. However, hookworm, especially N.americanus was still a parasitic threat to population health. More health education about fertilization and farming habits should be provided in rural areas. The awareness of hookworm and C. Sinensis should be reinforced in both rural and urban areas. protozoa pinworm infection. Permanent residents in for both and C. sample from


Background
Parasite disease is a major component of Neglected Tropical Diseases (NTDs) that affect the health for more than one million population globally [1]. It especially affects low-income populations in developing regions of Africa, Asia, and the Americas [1]. Helminthiasis, protozoa, and clonorchiosis are three types of parasite diseases continuously found in China but their prevalence were likely to be underestimated because their symptoms are not obvious at early stage of infection.
Soil-transmitted helminth (STH) is widely distributed in tropical and subtropical areas. Moreover, the incidence of STH infection is highly overlapped with areas being lack of sanitation facilities. Thereby STH is prevalent among population in poverty [2]. In the year of 2010, approximately 5.3 billion people in total and 1.0 billion school-aged children lived in areas considered as habitats of at least one STH species, and the 69% of them were living in Asia [3]. In the year of 2003, the prevalence of all species of STH (there are four main species of STH: roundworm (Ascaris lumbricoides), whipworm (Trichuris trichiura) and hookworms (Necator americanus and Ancylostoma duodenale) [4]) was highest in China, followed by Southeast Asia and India [5]. The data of STH species are limited. The data of South Asia and South East Asia show that in the year of 2018, roundworm was the most common cause of STH (18% of all cases), followed by whipworm (14%) and hookworm (12%) [6]. China conducted two national surveys in 1992 and 2001-2004 respectively. The overall prevalence of roundworm, whipworm and hookworm was 47.0%, 18.8%, and 17.2% in 1992 [7], 12.72%, 4.63% and 6.12% in 2003 [8]. However, the prevalence of STH is lack of investigation in recent years. One possibility is that because the economy and sanitation improved unequally in different areas of China, the distribution of STH should shift from it in decades ago.
Cryptosporidium, the cause of cryptosporidiosis, is one of the most common causes of diarrhea and gastroenteritis in humans and brings a great health burden to children and those who have impaired or weakened immune system [11][12][13]. The incidence rates of cryptosporidiosis range from 1.4% to 10.4% in different countries, and the rate was higher in low-income countries (5-10%) than in developed area (1%) [14]. G.intestinalis can cause giardiasis, which is another common cause of chronic diarrhea. The cases of giardiasis are mainly in developing countries and children. In developing countries, the childhood giardiasis prevalence is 20-30%, which is only 2-3% in high-income countries [13]. The amebiasis caused by E. histolytica affects 50 million people and causes 100,000 deaths people annually in worldwide [15]. In China, the infection rates of G.intestinalis was 2.52% and of E. histolytica was 0.95% according to the national survey conducted in 1992 [7], but the prevalence declined signi cantly in the national survey conducted during 2001-2004. For example, in Zhejiang province, the prevalence of G.intestinalis decreased from 3.85-1.30% and the prevalence of E. histolytica decreased from 1.5-0.14% between the two surveys [16].
Human liver uke is another NTD threating people's health in East Asia. Clonorchis sinensis (C. sinensis) is the most common type of human liver uke. The infection of Clonorchis sinensis (C. sinensis) often develop server consequence. The infection may result in the severe complication of cholangiocarcinoma which was classi ed as a group 1 biological carcinogen by the International Agency of Cancer Research in 2009 [17]. In addition, due to its high prevalence, C. sinensis caused a great health burden in East Asian countries such as China. Approximately 15 million people were infected with C. sinensis globally in 2004 [18], with 85% of infections were occurred in China [19]. In China, the infection rate of C. sinensis was 0.365% [20] in the year of 1992 and the rate was increased to 0.58% [8] in early 2000s. There were two epidemic zones of C. sinensis in China: the rst zone was the Heilongjiang and Liaoning provinces in northern China, and the second zone was the Guangdong and Guangxi Provinces in southern China. Zhejiang province is located in the southeast area of China. The prevalence of C. sinensis is generally low in Zhejiang province. In the second national survey, the prevalence of C. sinensis infection for Zhejiang prevalence was only 0.01% [16], which was much lower than the national average 0.58% [8]. However, the transmission of parasites and the composition of population changed rapidly in the recent decades. It is necessary to conduct another survey to know if the distribution of C. sinensis in Zhejiang province has been changed consequentially [21].
To our knowledge, there is no large-scale study investigated the epidemiology of the three species of parasites introduced above (STH, intestinal protozoa, and clonorchiosis) since the two national surveys completed in 1992 and 2003 respectively. Moreover, the overall incidence of parasitic diseases decreased greatly due to the improvement of public health condition and monitoring system in China. However, speci c areas might have increased risk for particular types of parasitic diseases because of the change of climate and demographics. The aim of this study was to describe the distribution of the three types of parasites in Zhejiang province. This study can provide reference for the health burden and prevention of intestinal parasitic infections in Zhejiang province.

Study Sites
An extensive eld sample collection and survey were conducted in 34 counties of all 89 counties in Zhejiang province from late 2014 to 2015 ( Figure. 1).
Zhejiang province located in the southeast area of China, and it features a monsoon season, temperate climate, and mountainous landscape. Each study county had one study site, which was assigned to rural site or (and) urban site. All of study sites were investigated for STH, and C. Sinensis and rural sites were investigated for intestinal protozoa as well. The survey conducted in rural counties was primarily focusing on STH and intestinal protozoa, while the survey conducted in urban counties was primarily focusing on C. Sinensis. The urban study sites were selected by the national Center for Disease Control and Prevention based on previous study results of 27 provinces in 2004, and the smallest selected unit was the neighborhood. The rural study sites were selected based on a multi-stage sampling method: counties in Zhejiang province were divided into three or four levels based on the county-level per capita net income (GDP) (data from 2011 Zhejiang provincial statistic yearbook). In each GDP level, one town was randomly selected, and then, one village was randomly selected from the town. In each neighborhood of urban and each village of rural, two-hundred and fty subjects were sampled by randomly selecting families.
Once the family was selected, all of people in this family will be asked to participant. Additional le 1 and 1aditional le 2 Table showed the study sites in rural area and urban area, respectively.

Study Population and Sample Collection
Permanent residents in selected rural sites were screened for STH, C. Sinensis and intestinal protozoa infection. Children of 3-6 years old were additionally examined for pinworm infection. Permanent residents in urban sites were screened for both STH and C. Sinensis infection. About 30g stool sample were taken from each participant and an additional anal swab was taken from each child participant.

Ethics Approval and Consent to Participate
This study was part of the Epidemiology Survey of Important Parasitic Diseases in China, which has been approached by the Ethical Review Committee of National Institute of Parasitic Disease, Chinese Center of Disease Control and Prevention. The form of inform consent was not emphasized, written or verbal were both acceptable. Because some old participates, especially in rural areas were illiterate, in order to unify the inform consent form in Zhejiang Province, verbal informed consent was obtained from all participants before the sample collection and the face-to-face survey. Study staff, for example, the head of village, explained the content in presence of villagers, if they agreed to participate the study, they would sent the sample by themselves or on behalf of their children.

Sample Examination
For each stool sample, two thick-smear slides were processed using the modi ed Kato-Katz technique under light microscope at 10×and 40× objectives for eggs detection. Meanwhile, one slide was processed using iodine solution under light microscope at 40× objectives for cysts of protozoa. The mean number of eggs on two Kato-Katz slides was calculated if any helminth egg was detected. The result was reported as positive or negative for cysts of protozoa.
Pinworm eggs were examined under light microscope at 10× using the transparent adhesive paper anal swab method and reported as positive or negative in detection.

Sample Examination
After collection, stool samples were cultured to detect hookworms using the following steps. About 0.5g of each stool sample was spread on a piece of spindly lter paper. This disposal was placed in a cone-shaped tube with cold boiled water at the bottom of the tube. Then, the sample was incubated for 4 days at 30°C. During the incubation period, water was added if necessary to prevent the tube drying up. Finally, after spontaneous sedimentation, the water at the bottom was collected and examined under an anatomical lens for A. duodenale or N. americanus larvae. The species name and the number of larvae were recorded. At most 50 stool samples conducted culture in a study county. The intensity of infection was measured by eggs per gram (EPG), EPG=mean eggs of two slides of one sample*24. Intensity of hookworm: 0<EPG<400, intensity=light; 400≤EPG≤3000, intensity= moderate; EPG>3000, intensity= heavy; intensity of A.lumbricoides: 0<EPG<5000, intensity=light; 5000≤EPG≤50000, intensity= moderate; EPG>50000, intensity= heavy; intensity of T. trichiura: 0<EPG<1000, intensity=light; 1000≤EPG≤10000, intensity= moderate; EPG>10000, intensity= heavy.

Quality control
Two trained laboratory staff read two Kato-Katz slides of each stool sample, and an parasitologist would check the slides when the results reported by the two trained staff were inconsistent. An experienced parasitologist would re-observed iodine slide if the cysts of protozoa was detected.

Questionnaire survey
The questionnaires (additional le 3 and additional le 4) were developed for this study and the surveys were conducted in 17 counties that randomly selected from the 34 study counties. 60 subjects among those under stool examination in each study unit were surveyed about the alimentary habits, but the sanitary behaviors were only surveyed in rural area.

Statistical analysis
Filled questionnaire and laboratory examination results were entered parallelly by two research staffs using Epi-Info 3.5.4 (CDC, GA, USA), and the data entered by them were compared to avoid discrepancies, logical errors and missing values. Statistical analysis was conducted using SPSS 24 (SPSS Inc., IL, USA) software. The study county distribution and hookworm infection distribution were mapped using open-source QGIS 2.18.27 software. Pearson's chi-squared test (c2) was used to compare the crude associations between binary and independent variable. For all the tests, the signi cance level was set to 5% (p<0.05).

Characteristics of study sites
Seventy-eight villages in rural area and 14 urban neighborhoods were selected in this study. The landscapes of selected villages (rural study sites) were mountainous (35.9897%), hilly (32.051%), and plain (25.641%); and the main landscape of urban study sites was plain (42.86%). More than a half of (58.33%) study villages do not have modern toilet. Most villages (90.476%) can access to tap water. No villages have the habit of eating raw meat, but there was one village in Wenzhou city have the habit of eating raw freshwater sh and shrimp. Local drinking and eating habits are displayed in Table 1. Farming was the predominant type of occupation in rural areas. In urban, farming and being retired were the two most common occupations. The proportions of participants with illiteracy, completed primary school and middle school were similar in rural and urban areas, but the proportion of participants with degree of high school, college and above were signi cantly higher in urban than rural area.  Table 3). Hookworm was the most prevalent parasite (1.584%), followed by T. trichiura (0.085%) and A.lumbricoides (0.055%). The prevalence of hookworm was much higher in rural area (1.791%) than it in urban area (0.442%). T. trichiura and A.lumbricoides were only found in rural area. Among participants with parasite infection, most of them has only one type of infection. Three participants (0.909%) had mix infection of T. trichiura infection and A. lumbricoides. No one had more than two types of infection. Figure 2 displayed the distribution of hookworm infection among all study counties. Hookworm infection had higher prevalence in counties located in the central and western Zhejiang. For example, Yongkang was a county in central of Zhejiang with the highest hookworm prevalence (10.246%), followed by Kaihua (6.796%) and Jiande County (6.570%). Hookworm infection was not detected in ten counties (29.412%). Most infections of hookworm (70.241%), T.trichiura (100%), and A.lumbricoides (92.308%) were of light intensity (Table 4). Male (60.590%), old (> 60) participants and farmers had higher prevalence of hookworm infection (Table 5). Three hundred and thirty of 357 hookworm positive stool samples in rural areas were cultured and larvae were found in 79.9% samples. N. americanus was the predominant (75.934%) species in Zhejiang Province ( Table 6). The average number of hookworm eggs on the slide of larvae negative samples was 6.186. Although the maximal number of hookworm eggs was high (60), most of them (62.791%.) had less than 3 eggs on a slide.

Prevalence of protozoa infection
Nine species of protozoa were detected in rural area. There were 0.396% of rural participants were infected with protozoa, and Endolimax nana was the most prevalent (0.231%) species, followed by Entamoeba histolytica (0.045%) and Entamoeba hartmani (0.040%) ( Table 7). Entamoeba coli, Entamoeba polecki, Giardia intestinalis, Trichomonas hominis, lodamoeba butschli, Blastocystis hominis were also detected but the prevalence were low (Table 7). Three participants from different villages have the multiple infection of hookworm and protozoa.

Sanitary behaviors and alimentary habits
There were 2,485 participants completed the questionnaire survey of the behaviors related to STH infection in rural study areas. The majority of them (89.135%) had good personal hygiene such as wash hands before eating or after using the toilet. However, there were still 12.515% people in rural area had the habits of drinking unboiled water. Moreover, 21.127% participants still used human stools to fertilize crops and 22.777% participants did not wear shoes when farming. These two kinds of behaviors could increase the risk of STH infection.
There were 2,485 in rural area and 483 participants in urban area completed the survey of the knowledge, risky behaviors, and alimentary habits related to C. Sinensis infection. The knowledge of C. Sinensis' transmission route and its damage to health showed signi cant difference between rural and urban participants ( Table 8). The proportion of participants using separated cutting board for raw and cooked food was not signi cantly different between rural and urban. Small proportion of participants (14.004% in rural area 9.317% in urban area) have the habits of eating raw freshwater sh and shrimp. The vast majority people would like to take medicine if they were told to be infected with C. Sinensis (Table 8).

Discussion
As a part of the third national parasitic disease survey, more than one third of counties in Zhejiang province were covered in this study. Zhejiang province is one of the smallest provinces in China with 74.63% lands are mountains and hills [22]. Generally, the study counties have access to clean water, fairly hygiene condition and rare special eating habits in Zhejiang province.
According to the sex and age distribution of participants, we found that women were more willing to participate the survey in either rural or urban area. The average age of rural participants was higher than that of urban area. It is due to that most old people in rural area stayed in hometown while most younger people moved to urban for work. The urban area in this study included many previous suburban areas because of the accelerated process of urbanization.
Therefore, the most common job among urban participants was still farming. In addition, to some extent, it could explain the reason that the rate of having low-level education rate was similar among rural and urban participants. However, people in urban area have better access to higher education, which may be the reason that urban participants have higher rates of high school or higher degrees than rural ones.
The overall prevalence of intestinal parasite infections in Zhejiang province decreased dramatically from 22.84% [16] in the second national survey (2003) to 2.123% in this study. Nationally,the prevalence of intestinal parasite infections was 3.93% in rural area in 2015 [23]. Compared to neighboring countries, the overall prevalence of STH infection was much higher: 59.9% in Malaysia [24], 75.6% in India [25], 31% in Philippines [26]. The prevalence of all types of STH infection was signi cantly higher in rural area than that in urban area. The causes of STH infection has changed greatly in Zhejiang since the second national survey [16]. In Zhejiang, there were 17 species of parasites detected in the second national survey [16] and 16 species of parasites (7 species of helminth and 9 protozoa) detected in this study. Although the number of species found in the two surveys were close, the prevalence showed great differences. The prevalence decreased dramatically from 6.84-0.055% for A.lumbricoides, from 3.64-0.085% for T. trichiura, from 8.18-1.584% for hookworm, from 24.42-2.79% for pinworm, from 0.14-0.045% for Entamoeba histolytica, from 1.00-0.015% for Giardia intestinalis [16]. The top cause of STH infection switched from multiple helminth species to hookworm, and the prevalence of hookworm was higher than that of those helminth species. A.lumbricoides and T. trichiura were no longer the leading cause of helminth infection in Zhejiang Province, but the hookworm did. In children, the infection of pinworm was well controlled.
The substantial reduction of prevalence may attribute to the rapid economic development [27], increased access to tap water and sanitary toilet, as well as improvement of personal hygiene habit such as wash hands. According to the provincial statistical yearbook, the per capita income increased 452.4% for urban residents and increased 486.7% for rural residents. The rate of having access to tap water in rural area was 80% in nationwide at the year of 2017 [28].
In Zhejiang rural tap water supply rate reached 99% in 2017 [29]. A meta-analysis study showed that use of piped water was associated with reduced likelihood of A.lumbricoides infection and T.trichiura infection but little evidence for similar association for hookworm infection [30]. Although tap water was supplied to more and more villages, the habits of drinking unboiled water from well or pond may still introduce some protozoa infection. In addition to tap water, the rate of access to sanitary toilet showed a rapid growth from 20.9% in 1996 to 78.43% in 2015 in nationwide [31]. In Zhejiang, coverage rate of sanitary toilet had already reached 94.60% households in 2011 [32]. However, in some rural area, villagers, especially seniors, were not willing to use modern sanitary toilet and used to apply fresh stool as fertilization. Furthermore, the habit of barefooted farming would increase their hookworm infection risk. In rural area, man was the main labor force in the eld. These habits and customs may explain the higher prevalence of hookworm in male, the old and farmers.
Hookworm took the place of other STH and became the main parasitic threaten to Zhejiang people. The prevalence of hookworm increase inversely with latitude. In South Asia and South East Asia, hookworm (12%) is the third most prevalent type of STH; The country-level prevalence of hookworm was highest in Laos, Vietnam and Cambodia [6]. A.duodenale and N.americanus were both endemic in Zhejiang Province, but N.americanus was the predominant species.
In neighboring Jiangxi Province, which located southwest of Zhejiang, N.americanus had a higher proportion of hookworm (80.41%) [33]. In Thailand, N.americanus was the main hookworm identi ed in northeast, while only N.americanus was detected in the south [34]. In the rst national survey, there was already a trend for a higher prevalence of N.americanus in the south, while A.duodenale is predominant in the north [7].
Questionnaire survey about C. Sinensis revealed that although urban participants had more knowledge regarding the Clonorchiosis or C. Sinensis than rural participants, the overall awareness rate in urban participants was still less than a half. More than a half of participants would like to try raw freshwater sh and shrimp without knowing that those were the transmission route of parasites. Interestingly, 9.317% urban participants eaten raw freshwater sh and shrimp, and this rate increased to 12.215% among participants knowing the risk of C. Sinensis infection. However, the situation was opposite in rural participants. Maybe the knowledge and health education were not the main factors in uencing people's behavior, curiousness in novelty, the con dence to cure the disease in urban area might enhance the courage of trying risky food.

Conclusion
In summary, the results of this study indicated that the prevalence of STH and protozoa infections had sharply declined and C. Sinensis infection was still rare in Zhejiang. However, hookworm, especially N.americanus was still a parasitic threat to population health. More health education about fertilization and farming habits should be provided, and the awareness of hookworm and C. Sinensis should be reinforced, and regular medication should be supervised instead of self-medication, in order to decrease hookworm infection further.

Declarations
Ethics approval and consent to participate The collection of samples used in this study and the questionnaire survey were conducted with the permission of participants or their parents. The research protocol was reviewed and approved by the Ethical Review Committee of National Institute of Parasitic Disease, Chinese Center of Disease Control and Prevention. Verbal informed consent was obtained from all participants before the sample collection and the face-to-face survey.
Written informed consent for publication was obtained from all participants.
Availability of data and materials The datasets analyzed during the current study are not publicly available due to data sharing need the approval of a liation but are available from the corresponding author on reasonable request.

Funding
This research received funding from Chinese Central Government's transferring payment.
Authors' contributions YF, KgY, HlC, XZ, QyL, XxW performed the research. YF performed the data analysis and wrote the manuscript. WR and LnY performed the on-site organization and quality control. XyZ modi ed the grammar and words of the manuscript. All authors read and approved the nal manuscript.

Availability of data and materials
The datasets analysed during the current study are not publicly available due to data sharing need the approval of a liation but are available from the corresponding author on reasonable request.