INTRODUCTION
Giardiasis, a common intestinal protozoal infection caused by Giardia duodenalis, has gained attention as a neglected disease in both developed and developing countries.1 Clinical presentations of giardiasis vary from asymptomatic carriage to acute and chronic diarrheal illness. Young children are at high risk of chronic infection resulting in nutritional malabsorption and failure to thrive.2 Infection is acquired by the ingestion of viable cysts, which are transmitted through fecal–oral contamination. Waterborne outbreaks of giardiasis have been reported in developed countries including the U.S. and also countries in Europe, while the major risk factors in low-income countries were sanitation and personal hygiene.3
G. duodenalis isolated from humans and other mammals is morphologically identical. Molecular studies revealed a substantial level of genetic diversity between G. duodenalis isolates. G. duodenalis recovered from humans fall into the two major genetic assemblages, i.e., A and B. Other distinct groups, assemblages C, D, E, F, and G, are animal-specific.3 However, assemblage A and B have also both been isolated from animals, indicating that these 2 assemblages have zoonotic potential. The existence of subgenotypes within each assemblage was also identified. Assemblage A comprises two distinct clusters, AI and AII, while assemblage B consists of BIII and BIV. To date, only one study showed strong evidence supporting zoonotic transmission of giaridiasis.4
Most studies in Thailand examined the prevalence of giardiasis especially in schoolchildren and in day-care centers.5–7 However, at the present time, no information on risk factors of giardiasis in Thailand is available. In the present study, we conducted a cross-sectional study of giardiasis in rural primary schoolchildren to determine the prevalence and the associated risk factors. In addition, genotypic characterization of G. duodenalis isolates from these children was also performed. The epidemiology of the infection with different Giardia genotypes would significantly contribute to the understanding of its sources and modes of transmission.
MATERIALS AND METHODS
Study population.
This cross-sectional study was undertaken in a rural community, Chacheongsao province, Central Thailand, in February 2005. The research protocol was approved by the Ethical Committee of the Medical Department, Royal Thai Army. This community comprised approximately 5,000 people. Most adults were farmers and laborers whose parents had migrated from northeastern Thailand. They have maintained their northeastern tradition and culture, such as dialect and dietary habits. We aimed at studying the prevalence and the risk factors for giardiasis in primary schoolchildren because of our survey in 2002 showing highest prevalence of giardiasis in this group. A large primary school located in the center of the community consisted of 793 students. A total of 531 stool specimens were collected from these children who voluntarily enrolled into the study with the informed consent of their parents.
Stool collection and examination.
Stool specimens were examined for intestinal parasites immediately after the collection by wet-smear preparation in normal saline and Lugol’s iodine solution. All specimens were then processed with the formalin/ethyl acetate sedimentation concentration and the sodium nitrate flotation techniques to detect G. duodenalis and other intestinal parasites. Blastocystis hominis was identified after a short-term in vitro cultivation using Jones’ medium supplemented with 10% horse serum because this method is more sensitive than wet-smear preparation and concentration techniques.8,9 The cultures were incubated at 37°C for 48–72 hours and then examined at 10× and 40× magnification under a light microscope. Every stool specimen was examined for Cryptosporidium spp. and microsporidia using modified acid-fast and Gram-chromotrope staining, respectively.
Genotypic characterization.
The purified Giardia cysts were washed thrice with phosphate-buffered saline (PBS). DNA extraction was performed using FTA filter paper as previously described.10 Genotypic characterization of G. duodenalis was determined by polymorphic sites using semi-nested PCR of a 432-bp region of the glutamate dehydrogenase (gdh) gene and PCR-RFLP method described by Read et al. (2004).11 Briefly, amplification of the gdh gene was performed using primer pairs of GDHeF/GDHiR and GDHiF/GDHiR. A total mixture of 50 μL contained DNA template using a piece of FTA filter paper, 1× PCR buffer, 1.0 U of Taq polymerase, 1.5 mM MgCl2, 0.2 mM dNTP, and 25 pmol of each primer. The PCR condition was as follows: 1 cycle of 94°C for 2 min, 56°C for 1 min and 72°C for 2 min, followed by 55 cycles of 94°C for 30 s, 56°C for 20 s and 72°C for 45 s and a final extension of 72°C for 7 min. RFLP analysis was performed by digesting 10 μL of the PCR product with 5 U of NlaIV in 1× enzyme buffer (New England Biolabs, Ipswich, MA) in a final volume of 20 μL for 3 h at 37°C. PCR products and restriction fragments were separated by electrophoresis in 2% agarose gel. Gels were stained with ethidium bromide and visualized under UV light and documented on high-density printing paper by using a UVIsave gel-documentation system (UVItech, Cambridge, England). DNA sequencing of PCR products was also performed to compare results with the sequences of the following GenBank entries: L40509 (G. duodenalis assemblage AI), L40510 (G. duodenalis assemblage AII), AF069059 (G. duodenalis assemblage BIII), and L40508 (G. duodenalis assemblage BIV). Multiple alignment and restriction map analysis were performed using BioEdit (version 7) software.
Questionnaires.
To determine the risk factors and outcomes of giardiasis, standardized questionnaires for collection of demographic data, sanitary behaviors including cooking and eating habits, source and treatment method of drinking water, pets or animal contact, and also history of present gastrointestinal symptoms were used in this study. Diarrhea was defined as a change in their normal pattern of bowel movements and at least 3 loose stools during a 24-h period, and dysentery was defined as at least one passage of mucous bloody stool in 1 day. The weight and the height of each student were recorded at school to determine their nutritional status using Thailand’s standard growth curve, Ministry of Public Health, Thailand, 1999. Parents of the enrolled students were asked to complete the questionnaires.
Statistical analysis.
The association between potential risk factors and G. duodenalis carriage was assessed by the χ2 test with a 95% confidence interval. Univariate analysis was performed using EpiInfo, version 6.04b. Odds ratios with 95% confidence intervals and P values were calculated to compare outcomes among study groups. Logistic regression using SPSS for Windows, version 9.6, was performed for multivariate analysis to assess the independent association of risk factors and G. duodenalis.
RESULTS
Of 793 students, 531 (67%) students were voluntarily enrolled into the study. The prevalences of parasitic infections in the study population are shown in Table 1. Protozoal infections were predominant in this group. B. hominis was the most common intestinal parasite found in this study (8.1%). G. duodenalis was the second most common, with a prevalence of 6.2%. Other common intestinal parasites reported in children, such as Ascaris lumbricoides, Cryptosporidium spp., and microsporidia, were not found. Those who were infected with pathogenic parasites were treated with appropriate antiparasitic drugs.
Genotypic characterization.
Of 33 samples, PCR amplification of the gdh gene was successful from 12 samples (42.4%). PCR-RFLP using NlaIV endonuclease and sequence analysis of the PCR products led to the identification of the G. duodenalis genotypes found in schoolchildren as assemblage A, sub-genotype II in 41.7% (5/12) of the group and assemblage B, subgenotype IV in the other 58.3% (7/12) of the group.
Characteristics of the enrolled students.
Table 2 shows the characteristics of the enrolled students. These schoolchildren ranged in age from 5 to 14 years old. Giardiasis was more prevalent in younger students. Prevalence in the students of age 5–9 years was 9.0%, which was significantly higher than those over 9 years old (2.9%) (P = 0.004, χ2 test). The prevalences of giardiasis were not significantly different between groups consisting of differences in sex, nutritional status, and clinical symptoms (Table 2).
Risk factors of giardiasis.
Table 3 shows univariate analysis of the risk factors for giardiasis in this population; it pointed at children ranging in age from 5 to 9 years old, households with ≥ 3 children under age 12 years, a low parental educational level (less than primary school), and people living in proximity to dogs for more than once a week. These groups had a greater risk of acquiring giardiasis. Multivariate analysis also confirmed that these risk factors were independently associated with giardiasis in this population (Table 4). Children whose parents had a lower educational level than primary school were at 2.4 times a greater risk of getting infected with G. duodenalis. Children of age 5–9 years old had a 1.3 times greater risk of getting infected. Having more than 3 children per household of an age under 12 years old increased by 2.5-fold the risk of contracting Giardia. Those who had history of contacting dogs more than once a week had a 2.3-fold greater risk for getting the infection. In addition, drinking bottled water was identified as the risk factor for giardiasis. Washing hands before every meal had protective effect against contracting Giardia.
Because only 12 samples were genotypically identified, we were unable to statistically determine the association between genotypes and risk factors. Considering these 12 characterized samples, those who consumed boiled or filtered water (4 of 12) had assemblage B, subgenotype IV of G. duodenalis. For those who did not consume boiled or filtered water, 5 and 3 children had assemblage A, subgenotype II and assemblage B, subgenotype IV of G. duodenalis, respectively. In addition, 2 of them consumed bottled water, which was identified as a risk for acquiring the infection, harbored assemblage A, sub-genotype II of G. duodenalis.
DISCUSSION
In this population, intestinal protozoal infections, i.e., blastocystosis and giardiasis, were predominant. These findings contrasted with most surveys done in Thailand, which showed a higher prevalence of helminthiasis among Thai schoolchildren.5,12 The biannual mass chemotherapy for helminthic infection using albendazole in this population, as directed by the parasitic control program of the Ministry of Public Health, might account for our findings. This policy might also have affected the level of G. duodenalis infection in this population as well because albendazole can be used for the treatment of giardiasis.13 The prevalence of giardiasis in Thai schoolchildren was previously reported in a few studies. In 1989, a survey in primary schoolchildren in Chiangmai, Northern Thailand, showed a prevalence of 7.7%,14 while in 2002, a survey in Nan, another province in Northern Thailand, showed a prevalence of 5.5%.5 However, the data in the present study cannot be compared with those previous studies because G. duodenalis was detected with different methods. Most studies only used wet preparations that might underdetect the prevalence of giardiasis. We used the flotation technique, a more sensitive method, for detection of G. duodenalis.
Most cases in this population were asymptomatic, so we made the choice of conducting a cross-sectional rather than a case-control study. Because giardiasis is a chronic disease, some of the cases might not be recent infections. This would mean that persons who answered questions related to specific risks might have suffered from recall bias. However, most of the questions used in our study appeared to be general rather than specific in nature. Hence, the results should not be affected by this possibility. In several studies, outbreak of giardiasis especially in developed countries usually indicated waterborne transmission. Significant risks identified in these studies included drinking untreated water, swallowing water while swimming, and even drinking tap water.15–20 In the present study, multivariate analysis showed that drinking bottled water presented a 2.5 times greater risk of acquiring giardiasis. However, most of the bottled water consumed by this population was distributed by regional water companies, and the quality of the bottled water has not been investigated in this study. The safety of the drinking water is ensured by filtration processes because cysts are relatively resistant to chlorine and ozone.21
In this study, we used PCR-RFLP of the ghd gene to characterize G. duodenalis. Although amplification of the gdh gene was less sensitive than other gene loci, such as SSU-rRNA gene, subgenotypes of Giardia could be differentiated using a single gene locus.10,11 Only 42.2% of positive specimens (12 of 33) were successfully characterized for their genotype. From our findings, it might be postulated that, in this population, only assemblage A, subgenotype II of G. duodenalis was transmitted via water. However, there was no statistical measurement to support this finding because the sample size was too small. In addition, examination of water for G. duodenalis with genotypic characterization should be done.
Both assemblage A, subgenotype II and assemblage B, sub-genotype IV of G. duodenalis can be identified both in humans and animals; hence, it is possible that these genotypes are zoonoses. However, epidemiological evidence supporting zoonotic transmission is rather limited. Until recently, molecular epidemiological evidence strongly supported giardiasis as a zoonosis. The study by Traub et al. (2004) showed that humans owning dogs infected with Giardia had a greater risk of getting parasitized than those who had not. In addition, some humans and dogs living in the same household harbored the same genotypes of G. duodenalis.4 A recent study in Thailand showed that some dogs living in temple communities might serve as a source of G. duodenalis for human infection because infected human cases in this community contained assemblage A, which is similar to that found in dogs.22 In the present study, we found that schoolchildren who had close contact with dogs had an approximately 2 times greater risk of acquiring giardiasis. In addition, assemblage A, subgenotype II and assemblage B, subgenotype IV were identified in these schoolchildren. Thus, both epidemiological data and G. duodenalis assemblages identified in these children might support the zoonotic transmission from dogs to human.
A study of clinical presentations of giardiasis in Dutch patients showed that assemblage A isolates were detected in patients with intermittent diarrhea, while assemblage B isolates were present in patients with persistent diarrhea.24 Another case-control study showed the significant association between assemblage A, subgenotype AII infections and diarrhea.25 In contrast, Cedillo-Rivera and colleagues (2003) reported that assemblages A and B exhibit no apparent differences in virulence, suggesting that host factors play a dominant role in determining the clinical course of the infection.26 In this study, no specific symptoms were significantly observed in those infected children. In addition, all 12 children whose Giardia was characterized had no GI symptoms. Thus, further studies need to be carried out to confirm the correlation of the assemblages and their clinical symptoms.
Person-to-person transmission is well recognized in crowded populations with exposure to infected persons. In developed countries, several reports put the emphasis on this mode of transmission in day-care centers.27 In developing countries, person-to-person transmission could occur in the community, especially in households with numerous children.28 The prevalence of giardiasis in this population was significantly greater in younger children. Thus, it is not surprising that having ≥ 3 children under age 12 years and sharing the same house presented a 2.5 times greater risk of getting the infection. This indicates that person-to-person transmission is also an important transmission pathway of giardiasis in this community.
Personal hygiene is a critical factor for children with giardiasis worldwide. In this study, children who washed their hands before meals were at a lower risk for Giardia infection. Educational achievement, especially for those affected children, can only occur effectively through the support of a school health program.22 To ensure that children are being adequately cared for at home, provision of health education to parents or caretakers needs to be addressed, particularly for parents who have had no formal education, as children under their care have a higher risk of getting infected with Giardia.
Most epidemiological studies of giardiasis indicated waterborne, foodborne, or person-to-person transmission, depending on the study population.23 Multiple modes of transmission of giardiasis were previously reported in only a few studies, most of which were conducted in developed countries.18,19,29,30 Two case-control studies were conducted in Auckland, New Zealand, in which infected cases included those of different regions, so it should not be surprising to identify multiple modes of transmission of giardiasis.18,19 White et al. (1989) and Katz et al. (2006) also reported multiple modes of transmission of giardiasis in rather specific populations, i.e., residents, employees and children of nursing home and country-club residents, respectively.29,30 Our study was conducted in a different setting, i.e., a rural community in Thailand, where the evidence of multiple modes of transmission of giardiasis, including person-to-person, zoonotic, and waterborne transmission, could be observed. This indicates that the nature of transmission of giardiasis varied depending on each population. Hence, control strategies of giardiasis in each population should be based on the epidemiological information for each population.
Intestinal parasitic infections in 531 students
| Intestinal parasitic infection | Number | Percent |
|---|---|---|
| Blastocystis hominis | 43 | 8.1 |
| Giardia duodenalis | 33 | 6.2 |
| Opisthorchis viverrini | 21 | 3.9 |
| Trichuris trichiura | 8 | 1.5 |
| Hookworm | 7 | 1.3 |
| Enterobius vermicularis | 3 | 0.6 |
| Strongyloides stercoralis | 2 | 0.4 |
| Other | 17 | 3.2 |
Characteristics of enrolled students and prevalence of Giardia duodenalis infection
| Characteristics | Total (%) | No. of G. duodenalis positive (%) | P value |
|---|---|---|---|
| Age group (years) | |||
| ≤ 7 | 103 (19.4) | 9 (8.7) | |
| 8 | 97 (18.3) | 10 (10.3) | |
| 9 | 90 (16.9) | 7 (7.8) | |
| 10 | 114 (21.5) | 2 (1.7) | |
| 11 | 64 (12.0) | 3 (4.7) | |
| ≥ 12 | 63 (11.9) | 2 (3.2) | 0.093 |
| Sex | |||
| Male | 280 (52.7) | 19 (6.8) | |
| Female | 251 (47.3) | 14 (5.6) | 0.594 |
| Parent occupation(s) | |||
| Agricultural | 486 (91.5) | 32 (6.6) | |
| Other | 45 (8.5) | 1 (2.2) | 0.345 |
| Diarrhea | |||
| No | 449 (84.6) | 29 (6.5) | |
| Yes | 82 (15.4) | 4 (4.9) | 0.804 |
| Nutritional status | |||
| Normal | 484 (91.1) | 29 (6.0) | |
| Low normal and underweight | 47 (8.9) | 4 (8.5) | 0.521 |
| Total | 531 (100) | 33 (6.2) | |
Univariate analysis of risk factors of Giardia duodenalis infection
| Giardia duodenalis | ||||
|---|---|---|---|---|
| Characteristics | Negative | Positive | Crude odds ratio (95% CI) | P value |
| Age group (years) | ||||
| > 9 | 234 (97.1) | 7 (2.9) | 1 | 0.004 |
| 5–9 | 264 (91) | 26 (9.0) | 3.3 (1.4–7.7) | |
| Sex | ||||
| Female | 234 (94.4) | 14 (5.6) | 1 | 0.565 |
| Male | 261 (93.2) | 19 (6.8) | 1.2 (0.6–2.5) | |
| No. of children of age < 12 years in home | ||||
| < 3 | 365 (95.3) | 18 (4.7) | 1 | 0.02 |
| ≥ 3 | 133 (89.9) | 15 (10.1) | 2.3 (1.1–4.7) | |
| Parent education | ||||
| At least primary school | 435 (94.8) | 24 (5.2) | 1 | |
| No education | 63 (87.5) | 9 (12.5) | 2.6 (1.2–5.8) | 0.017 |
| Keeping dog(s) at home | ||||
| No | 160 (96.4) | 6 (3.6) | 1 | |
| Yes | 338 (92.6) | 27 (7.4) | 2.1 (0.9–5.3) | 0.094 |
| Close contact to dog | ||||
| < 1/week | 303 (95.6) | 14 (4.4) | 1 | |
| ≥ 1/week | 195 (91.1) | 19 (8.9) | 2.1 (1.0–4.3) | 0.037 |
| Washing hands before meal | ||||
| Occasionally | 348 (92.3) | 29 (7.7) | 1 | |
| Every time | 150 (97.4) | 4 (2.6) | 0.3 (0.1–0.9) | 0.029 |
| Drinking bottled water | ||||
| No | 362 (95.0) | 19 (5.0) | 1 | |
| Yes | 136 (90.7) | 14 (9.3) | 1.9 (0.9–4.0) | 0.062 |
Multivariate analysis of risk factors of Giardia duodenalis infection*
| Characteristics | Adjusted odds ratio | 95% CI | P value |
|---|---|---|---|
| * Adjusted for sex, parent income, and parent occupation(s). | |||
| Age group (years) | |||
| > 9 | 1 | ||
| 5–9 | 1.3 | 1.0–1.6 | 0.045 |
| Close contact to dog(s) | |||
| < 1/week | 1 | ||
| ≥ 1/week | 2.3 | 1.1–4.9 | 0.025 |
| No. of children of age < 12 years in home | |||
| < 3 | 1 | ||
| ≥ 3 | 2.5 | 1.2–5.2 | 0.017 |
| Washing hands before meal | |||
| Occasionally | 1 | ||
| Every time | 0.3 | 0.09–0.8 | 0.022 |
| Parent education | |||
| At least primary school | 1 | ||
| No education | 2.4 | 1.0–5.6 | 0.041 |
| Drinking bottled water | |||
| No | 1 | ||
| Yes | 2.5 | 1.2–5.5 | 0.016 |
Address correspondence to Saovanee Leelayoova, Department of Parasitology, Phramongkutklao College of Medicine, 315 Ratchawithi Road, Ratchathewi, Bangkok 10400, Thailand. E-mail: s_leelayoova@scientist.com
Authors’ addresses: Supawat Ratanapo, Suthipong Soontrapa, Chakri Faithed, and Ram Rangsin, Department of Military and Community Medicine, Phramongkutklao College of Medicine, Bangkok 10400, Thailand. Mathirut Mungthin, Tawee Naaglor, Phunlerd Piyaraj, Paanjit Taamasri, and Saovanee Leelayoova, Department of Parasitology, Phramongkutklao College of Medicine, 315 Ratchawithi Rd., Ratchathewi, Bangkok 10400, Thailand, Telephone/ Fax: +662 354 7761, E-mail: s_leelayoova@scientist.com.
Acknowledgments: We acknowledge all participants and staff of this primary school. The authors thank Francois Le Berre for his assistance in the preparation of the manuscript.
Financial support: This study was financially supported by the Thailand Research Fund (BRG 4880003).
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