Analysis on the detection rate of Giardia lamblia in children of Sichuan province of China

Objective To investigate the detection rates of Giardia lamblia in children of Sichuan province and analyze the susceptible population and the cause of infection. Methods The routine fecal examination of a total of 11575 cases were done by normal wet smear microscopy, and the suspected cases were stained with iodine in outpatient children of West China Second University Hospital, Sichuan University from September 1st, 2020 to August 31th, 2021. Results In the stools of 11,515 outpatient children, 11 cases of cysts or trophozoites of G. lamblia were detected, among which 9 cases were from ethnic minority patients and 2 case were from Han patients. The G. lamblia detection rate of Han patients was 0.019%, while that of ethnic minority patients was 0.968%, between which there was a statistically signicant difference (P<0.05).


Introduction
Giardia lamblia, also known as Giardia duodenalis, is one of the most susceptible intestinal parasitic protozoa in children [1]. G. lamblia parasitizes the human small intestine, gallbladder, mainly in the duodenum, causing abdominal pain, diarrhea, vomiting and indigestion-based symptoms, called giardiasis [2][3]. G. lamblia is a agellate that does not invade epithelial cells and reproduces asexual only by binary division. They produce resistance stages (Giardia cysts) and are released into the environment as a mode of infection for these intestinal parasitic protozoa [3].G. lamblia exhibit a biphasic lifestyle, i.e. dormant cysts or reproductive trophozoites, which propagate by the dichotomy of the trophozoites. Cysts can be found in stools with normal hardness, and trophozoites may be found in cases of diarrhea. At the same time, G. lamblia is a zoonotic intestinal parasitic protozoa that may be transmitted from humans to animals [4][5].
G. lamblia is one of the most prevalent parasite infections in the world. It is also a common intestinal protozoa that infects both humans and animals [6]. About 200 million people worldwide are infected with G. lamblia every year, with a high incidence among tourists. So, diarrhea caused by G. lamblia is also called "tourist diarrhea" [7]. The survey of G. lamblia infection status in 11 provinces (or municipalities and autonomous regions) of China shows that the infection rate is 0-16.2%, of which the infection rate of HIV patients and immunode ciency populations is higher [8][9]. However, the ethnic distribution of giardiasis in Sichuan province of China and the difference in detection rates of different ethnic groups have not been reported. This study was to carry out routine fecal examinations for outpatient children in our hospital and investigate the detection rates of giardiasis in Sichuan province, and to analyze the susceptible people and susceptible causation of this disease.

Patients
A total of 11575 cases of outpatient children aged 0-to 12-year-old (mean ±SD 6.85±4.41 years) in West China Second University Hospital, Sichuan University from September 1st, 2020 to August 31th, 2021 were enrolled in this study. Inclusion criteria were as follow: 1) Informed consents were acquired from patients and their guardians, and patients were from 0-to 12-year-old; 2) Patients who had clinical symptoms of abdominal pain, diarrhea, vomiting and indigestion-based symptoms, etc.; 3) Outpatient children with recent diagnosis in our hospital and who were not previously treated with anti-infective drugs for intestinal tract. Exclusion criteria were as follow: 1) Patients who were treated with anti-infective agents for intestinal tract, or with other agents that would cause interference for diagnosis; 2) Patients who were over 12-year-old.

Methods
The routine fecal examination of a total of 11575 cases were done by normal wet smear microscopy, and the suspected cases were stained with iodine. Among which, there were 10,645 Han patients (mean ±SD 6.91±4.68 years) and 930 ethnic minority patients(mean ±SD 5.96±4.05 years) .The found of Giardium cysts or trophozoites were diagnosed as positive giardiasis cases( Figure 1). And then, the nationality, month of birth, living environment, hygiene habits, current medical history, and previous history of all patients were investigated. In our study, the written informed consent was obtained from all participants and the privacy rights of them were also reserved. And all procedures and protocols are in accordance with the Helsinki Declaration as revised in 2013.The study protocol(Medical Research 2020, No.40)was approved by the Institutional Review Board(IRB) of the West China Second University Hospital, Sichuan University on 26th July 2020 before study initiation.

Statistical analysis
The database was established using Excel sheet to calculate the positive rate of giardiasis. The positive proportion of Han and minority outpatient children was calculated using the χ 2 test correction formula.

Results
In the stools of 11,515 outpatient children, 11 cases of cysts or trophozoites of G. lamblia were detected, among which 9 cases were of ethnic minority patients and 2 case was of Han patients. Among the 9 ethnic minority giardiasis patients, 6 were Tibetans and 3 were Yi nationalities. The total G. lamblia detection rate was 0.095%. The detection rate of G. lamblia of Han patients was 0.019%, meanwhile that of the ethnic minority patients was 0.968% (Table 1), between which there was a statistically signi cant difference (χ 2 = 81.12, P<0.05). The detection rates of G. lamblia in children of different nationalities were shown in Table 2.

Discussion
G. lamblia is a common intestinal parasitic protozoa associated with diarrhoea disease, and also a zoonotic parasite divided into ve genotype assemblages of A-E. Studies have shown that the infection rate of G. lamblia of patients with HIV and the immunode ciency population is higher [7][8].At the same time, the research results showed that G. lamblia infection existed in HIV/AIDS patients in Guangxi, and the genotype was mainly assemblage B, and host-speci c assemblage C was found [5]. Genotype of G. lamblia isolated from different hosts suggests that the host species with the largest infection range of assemblages A and B, appears to be the primary (or possibly only)assemblage of G. lamblia. At least in some cases of wild mammalian assemblage A or B infection, there is evidence that the infection was caused by environmental contamination from the G. lamblia cysts of human origin [11].
The analytic method of G. lamblia directly related to its sensitivity, speci city and detection rate. Currently, G. lamblia assays include direct microscopic, staining, immunological methods and molecular diagnosis. The traditional microscopy method is the "gold standard" for G. lamblia detection. However, it is timeconsuming and low detection rate [12]. Immunological methods, such as immuno-uorescence, ELISA, have improved sensitivity and speci city compared with traditional microscopy. But the former cannot distinguish insect species, the latter is prone to cross-reactions, all of them cannot accurately quantify the number of G. lamblia, and were used poorly in mildly infected fecal samples [13]. With high sensitivity and good speci city, the uorescent quantitation PCR (FQ-PCR) method, which is one of the preferred methods for the quantitative detection of G. lamblia, can detect a large number of samples simultaneously [14]. In recent years, isothermal nucleic acid ampli cation technology has been widely used in the eld of prevention and control of infectious disease, mainly including chain replacement ampli cation, recombinase polymerase ampli cation technology and loop-mediated isothermal ampli cation (LAMP).The LAMP technology has a high degree of speci city and sensitivity, good stability and repetitive in the gene sequence analysis of triose phosphate isomerase (TPI) of G. lamblia [15].
In our study, 9 of the 11 giardiasis patients detected in our hospital were ethnic minority children (6 Tibetans and 3 Yi nationalities), and 2 was Han patients. The detection rate of G. lamblia in minority patients is higher than that in Han patients. Meanwhile, one of the Han patients had a life history in Yi nationality area and G. lamblia have not been detected in other minority children. Considering the intermittent nature of the discharge of the stools and the inexperience of the inspectors, the detection rate of G. lamblia in Sichuan Province may be higher [16]. Through analyzing their life history and susceptible reasons, the pathogeny of giardiasis was related to health conditions, economic status, cultural level, unclean eating habits and living habits in ethnic minority areas, as well as related to the animal husbandry that the local residents mainly engaged in [17]. G. lamblia has a simple life history and spreads extremely fast. People are infected mainly by ingestion of water and food contaminated with cysts. Improper management of human and animal stools, without washing hands before and after meals, and lack of awareness of epidemic prevention constitute important risk factors for G. lamblia infection.
The parents of children work outside, leading to the patients stay in the rural areas under the care of other generations of elders. Due to poor care, the patients may have no personal health position. The children of Kindergarten, nursery, and primary school live together, and play toys with each other can also cause potential risk factors spreading from person to person [18]. 9 outpatient children with giardiasis were less than 5 years old in our hospital, 2 patients was aged over 5 years (12- [20].All these suggest that children are more susceptible to giardiasis. The outpatient children in our hospital are mainly derived from all over Sichuan Province, and many kinds of intestinal protozoa and sporozoa have been detected in their stools. Children with giardiasis often intermittently discharge cysts or trophozoites, which are prone to repeated infection. It is suggested that the primary hospitals should conduct regular census and repeated screening of the population. Insect repellent measures should be taken immediately if the con rmed cases are found. Health and epidemic prevention departments should strengthen the fecal management of human and animal hosts, do a good job in environmental sanitation, and prevent water being polluted. Children should be cultivated to develop good personal hygiene and living habits, and reduce the risk of fecal-hand-mouth-oral transmission and human and animal transmission. At the same time, it is recommended that the local Center for Disease Control and Prevention(CDC) publicize and popularize the relevant knowledge of parasitic diseases to children and their parents in remote areas. For inspectors, we should improve our professional ability and detection experience to reduce the missed detection rate. Immunology and molecular diagnostic techniques based on traditional microscopy should be increased to improve the sensitivity and speci city of G. lamblia detection. It is suggested to strengthen the professional training and assessment of the relevant inspectors, and to further improve the detection rate and diagnosis rate of giardiasis.

Conclusions
There was high detection rate of G. lamblia in ethnic minority patients, which was mainly related to its personal hygiene, living habits and eating habits. It is suggested to strengthen the epidemic prevention and management in ethnic minority areas. And children's good hygiene habits should be cultivated, so as to reduce the spread and prevalence of giardiasis.

Declarations
Ethics approval and consent to participate All procedures performed in studies involving human participants were in accordance with the ethical standards of the institutional and/or national research committee and with the 1964 Helsinki declaration and its later amendments or comparable ethical standards.

Consent for publication
Informed consent was obtained from all individual participants included in the study.

Competing interests
All authors declares that he/she has no competing of interest.

Funding
This research received no speci c grant from any funding agency in the public, commercial, or not-forpro t sectors.

Authors' contributions
Zhengqiang Hu drafted the manuscript. Zhengqiang Hu and Yonglin Zhong participated to acquisition of data. Jie Li generated the experimental results. Yunxia Li designed the study and reviewed the manuscript for intellectual content. All authors approved the nal version of the manuscript.
Project administration: Jie Li.