Prevalence of Giardia duodenalis among Asian children: a systematic review and meta-analysis

Abstract Giardia duodenalis is one of the major causes of diarrhea among children. We performed a systematic review and meta-analysis to assess the prevalence of G. duodenalis and associated risk factors among Asian children. We searched online databases (PubMed, Scopus and Web of Science) and Google Scholar search engine for studies published from 1 January 2000 to 15 March 2022 that measured the prevalence of G. duodenalis among Asian children. Accordingly, the pooled prevalence and 95% CIs were estimated using a random-effects meta-analysis model for the included studies. A total of 182 articles from 22 Asian countries met the inclusion criteria. The pooled prevalence of G. duodenalis infection among Asian children was estimated as 15.1% (95% CI 14.1 to 16%). The highest and lowest pooled prevalence values of G. duodenalis infection were estimated for Tajikistan and China as 26.4% (95% CI 22.9 to 30%) and 0.6% (95% CI 0.001 to 1.02%), respectively. The infection had a higher prevalence in males than in females (OR=1.24; 95% CI 1.16 to 1.31; p<0.001), which was statistically significant. Giardiasis is common among Asian children, hence, a prevention and control scheme of this protozoan in children should be considered by health officials and health policymakers, especially in Asian countries where the prevalence is highest.


Introduction
Diarrhea is one of the major causes of morbidity and mortality among children, particularly those living in underdeveloped or developing nations. 1 -3 Accordingly, unsafe water, sanitation and childhood malnutrition are the leading risk factors for diarrhea, especially in south Asia and sub-Saharan Africa. 1 , 4 Overall, diarrhea is the cause of mortality of about 480 000 young children worldwide, and in 2019 was responsible for 9% of all deaths among children aged < 5 y ( https://data.unicef.org/topic/childhealth/diarrhoeal-disease/).According to WHO reports in 2010, giardiasis is estimated to cause approximately 28.2 million cases of diarrhea. 5 , 6Among infectious agents, giardiasis is one of the major causes of diarrhea in developing countries. 7Giardia duodenalis is an intestinal protozoan parasite with a worldwide distribution in human and animals. 8 -10The fecal-oral route is the main transmission mode of G. duodenalis . 8 , 10 , 11Drinking water, food and vegetables contaminated by G. duodenalis cysts are the main sources of transmission. 10 , 12 , 13The distribution of G. duodenalis in different communities is related to sanitation status, health measures and the quality of the drinking water. 14In this regard, the prevalence of G. duodenalis is much lower in developed countries than in less-developed geographical areas. 7 , 15Symptomatic giardiasis is significantly associated with worsening of nutritional status among children aged < 5 y in Asian and African countries. 16he common clinical manifestations of giardiasis include greasy diarrhea (steatorrhea), nausea, vomiting, abdominal bloating, cramps, malabsorption and weight loss. 17 , 18Moreover, chronic giardiasis is related to food allergies, 19 irritable bowel syndrome, 20 chronic fatigue syndrome 21 and arthritis, 22 as well as growth deficiency in children. 23Previous studies have shown that individuals with a weak immune system, such as pregnant women and immunocompromised patients (e.g. with cancer, HIV/AIDS, etc.), are at a higher risk of G. duodenalis infection. 10 , 24 -26Also, children are at a higher risk of G. duodenalis infection. 10 , 24 -26Children have more contact with different environmental sources and also S. Kalavani et al.   have immature immune systems to fight infections. 27 , 28Therefore, this group has a higher probability of contracting infectious agents.According to a recent meta-analysis, 29 the risk factors for giardiasis were determined as exposure to sewage/wastewater, untreated drinking water and recreational waters.Interestingly, contact with pets was found to be a significant risk factor for giardiasis in children.Travel abroad was a risk factor for giardiasis in industrialized countries. 29A previous meta-analysis by Muhsen and Levine 7 revealed the association between endemic pediatric diarrhea and giardiasis in developing countries.Asia is the world's largest continent with the highest human population.A large number of underdeveloped or developing countries are located in Asia.Therefore, determination of the epidemiological patterns of G. duodenalis infection in children is necessary to design future control programs and preventive measures to reduce the incidence of the infection in Asian countries.To address this gap, we designed a systematic review and meta-analysis to assess the prevalence of G. duodenalis and associated risk factors in children in Asia.

Information sources and systematic search
This systematic review and meta-analysis was followed based on the Preferred Reporting Items for Systematic Reviews and Metaanalyses (PRISMA) protocol. 30Published literature on the prevalence of G. duodenalis in children in Asia was retrieved through three major databases (i.e.PubMed, Scopus and Web of Science) and Google Scholar search engine from 1 January 2000 to 15 March 2022.The search process was accomplished using Medical Subject Headings (MeSH) terms alone or in combination: ('Intestinal protozoa' OR ' Giardia ' OR 'Giardiasis') AND ('Prevalence' OR 'Epidemiology') AND ('Children').Moreover, the reference lists of all the selected articles were hand-searched to find other relevant articles or their citations by searching in Google Scholar.

Inclusion criteria, study selection and data extraction
To assess article eligibility based on the determined inclusion criteria, all the papers were reviewed by two independent reviewers and possible contradictions among studies were removed by discussion and consensus.The inclusion criteria for this systematic review were: (1) full texts or abstracts published in English from Asia; (2) peer-reviewed original research papers or short reports; (3) cross-sectional studies that estimated the prevalence of Giardia in children (aged ≤18 y); (4) utilizing fecal microscopy, coproantigen or molecular diagnostic methods; (5) reports with information on the total sample size and positive samples; and (6) published online from 1 January 2000 to 15 March 2022.Those papers without full-text accessibility or papers that did not meet the above criteria were excluded.Next, the desired data were gathered precisely using a data extraction form including each first author's surname, the year in which the study was conducted and the publication year, countries, provinces or cities, the types of method used, total sample sizes, the number of positive samples, types of children, gender and age of the children, as well as the presence or absence of diarrhea.

Study quality assessment
The Joanna Briggs Institute (JBI) checklist was applied for the risk of bias (internal validity) assessment of the included articles. 31his checklist comprises 10 questions, with four options consisting of Yes, No, Unclear and Not applicable.In summary, a study can be awarded a maximum of one star for each numbered item.Those papers with a total score of either 4-6 or 7-10 points were specified as being of moderate or high quality, respectively.Based on the score each paper obtained, the authors decided to either include (4-10 points) or exclude ( ≤3 points) it.

Meta-analysis
For each of the included studies, the point estimates and their respective 95% CIs were calculated using a random effect model (REM).The REM allows for a distribution of true effect sizes between articles.To visualize possible heterogeneity among the included studies, forest plot analysis was used.The heterogeneity index among the included studies was defined using the I 2 index and Tau squared to reveal the variation in study outcomes between individual studies. 32 , 33Univariate and multivariable metaregression analysis was used to estimate the effects of probable factors in heterogeneity. 33To investigate the effect of each study on the pooled estimation of prevalence, the sensitivity analysis method was used by removing studies one by one.The robustness of each model was evaluated and, finally, the most favorable model was chosen.
Using subgroup analyses, the pooled prevalence of Giardia infection was estimated according to countries, types of diagnostic methods, types of children and the periods of studies.An OR (and the corresponding 95% CI) was calculated for each study to assess the association between Giardia spp.prevalence and risk factors such as sex (male and female) and place of living (rural and urban).
We used funnel plots and Egger's test for examining funnel plot asymmetry to assess the risk of bias due to missing results in a synthesis.Furthermore, the trim-and-fill method was used to estimate the number of censored studies and correct the overall estimate. 33oreover, because of different sensitivities and specificities of diagnostic methods, we assumed that our results would be 'apparent' prevalence rates, which did not represent true prevalence rates.The prevalence of G. duodenalis in children in different countries of Asia was demonstrated as a world map using ArcGIS 10.3 software ( https://www.arcgis.com).This meta-analysis was conducted with Stata version 16 software and the trial version of comprehensive meta-analysis software (version 3, BIOSTAT, Englewood, NJ, USA) version 3. p < 0.05 was considered significant.

Characteristics of the eligible studies
A flowchart depicting the identification process of the qualifying studies is presented in Figure 1 .In brief, the systematic search identified 5162 potentially relevant articles.After removing duplicates and/or non-eligible papers, 182 articles  1.The results of quality assessment according to JBI with references for eligible studies are depicted in Supplementary Table 1.The articles included in the current meta-analysis displayed acceptable quality.

The pooled prevalence of G. duodenalis in children
We estimated that the pooled prevalence of G. duodenalis for children in Asia, using REM, was 15.1% (95% CI 14.1 to 16%) (Q statistic = 37713.61,d.f.= 182, p < 0.0001, I 2 = 99.5%,Tau squared = 0.0033); after sensitivity analysis using one-by-one removal of studies, the best robustness model was selected.
According to I 2 = 99.5% and the significance of the Q statistic, it was detected that heterogeneity between studies was high, so univariate and multivariable meta-regression models were used to discover the causes of heterogeneity (Table 1 ).Univariate meta-regression analysis showed that year of study (p < 0.001) could be the source of heterogeneity.In the next step, the multivariable meta-regression showed that year of study (Bcoefficient = −0.00494,p < 0.001) might have been the source of heterogeneity.This means that a change in the publication year can decrease the prevalence by −0.00494.Furthermore, the multivariable meta-regression analysis did show a significant differ-ence in the location of studies, sample size, type of study population and year of study (p < 0.05) (Table 1 ).

Publication bias
The funnel plots and Egger's test show that there is publication bias for studies reporting the prevalence of G. duodenalis in children (bias = 9.22034, 95% CI 7.377599 to 11.0631; p < 0.001) (Figure 5 ).According to the trim-and-fill method, 90 hypothetical censored studies were estimated using non-parametric methods and regarded in the corrected meta-analysis.Therefore, the overall prevalence in children corrected by the REM based on the trimand-fill model was estimated to be 3.04% (95% CI 2.05 to 4.03%) (Table 3 ).

Discussion
This is the first study to estimate the prevalence of G. duodenalis among children in Asian countries.The prevalence values obtained for G. duodenalis in the meta-analysis were relatively high, especially in studies that used the PCR method (Table 2 ).Traditionally, the microscopy detection method using staining procedures is considered the gold standard for the detection of cysts and/or trophozoites of G. duodenalis . 34However, molecular methods are preferred for conducting research activities because they have higher sensitivity and specificity and interpretation of the results is easier. 35 , 36Accordingly, the pooled prevalence provided by molecular methods could be closer to the true preva-lence.For a deeper understanding of this issue, it is necessary for researchers to use molecular methods in addition to microscopy methods.
When considering the prevalence of G. duodenalis in different Asian countries, the highest infection rates were found in Tajikistan (one study with a pooled prevalence of 26.4%) and Malaysia (four studies with a pooled prevalence of 26%).By contrast, infection rates in China (four studies with a pooled prevalence of 0.6%) and Saudi Arabia (seven studies with a pooled prevalence of 6.04%) were relatively low.Several environmental and sociodemographic parameters are complicated in the different prevalence rates obtained, including climatic condition, parasite control measures, the Human Development Index and the use of diverse diagnostic methods in different regions.In addition to all these factors, it is necessary to conduct more studies in Asian countries, and those countries that have not conducted any research on this issue should consider doing so; thus, a deeper understanding of the prevalence of G. duodenalis in children in different parts of Asia will be obtained.
Considering the year of publication (Table 2 ), during 2021 and 2022, the pooled prevalence of G. duodenalis infection decreased.This can be attributed to the increased health knowledge awareness of people. 37 , 38Furthermore, during the COVID-19 pandemic, because of enhanced personal and social hygiene, most foodand waterborne infectious diseases were relatively reduced. 39 , 40he results of this meta-analysis study, especially the pooled prevalence rates based on the year of publication, should be interpreted with caution, as a source of heterogeneity has been suggested by meta-regression analysis (Table 1 ).Factors such as the number of published articles and the sample size of studies each year may play a role in causing heterogeneity.The current risk factor analyses showed that males and people living in rural areas are at a greater risk of exposure to G. duodenalis , which might be explained by lower personal hygiene scores and more contact with G. duodenalis cyst-contaminated water and vegetables.
The current study has a number of limitations.First, despite our comprehensive search, there was a paucity or absence of data for a number of countries, and many of the available studies had limited sample sizes and a lack of data on sociodemographic and/or risk factors.Moreover, in some countries, only one or two eligible studies were identified, which could compromise somewhat the interpretation of current estimates.Second, the studies included were undertaken during different time periods, with an absence of recent data for some countries, limiting the accuracy of inter-regional comparisons.Third, there was high heterogeneity in this meta-analysis, although we investigated its possible source by performing meta-regression analysis.
In summary, a prevention and control scheme of G. duodenalis in children should receive greater attention from health officials and health policymakers, especially in Asian countries where the prevalence is highest.

Figure 2 .
Figure 2. Prevalence of G. duodenalis in children based in countries in Asia.

Figure 4 .
Figure 4. Association between living are and prevalence of G. duodenalis in children.

Figure 5 .
Figure 5. Funnel plot with pseudo 95% confidence limits for the detection of publication bias in this meta-analysis.

Table 2 .
Pooled prevalence of G. duodenalis in children in Asia

Table 3 .
Comparison of common and corrected meta-analysis results for publication bias