Introduction

Waterborne infections are enteric diseases that are called gastroenteritis in advanced countries. These are very frequent diseases and various reports suggest that their outbreaks in adults occur at least two times a year [1–6]. One of the most noteworthy characteristics of Giardia, a waterborne parasite that causes giardiasis, is that its eradication rate by sterilization is very low compared with other pathogens. A typical example of its outbreak occurred in Bergen (Norway) in 2004, when 2400 patients were infected and most outbreaks were caused by inflow of ranch wastewater, defective drinking water pipes, and inadequate water purification treatment [7,8].

In May 2010, cases of acute diarrhea were reported in Korea. Consequently, environmental research on the outbreak and epidemiological research on the patients with diarrhea were conducted. This report presents the outbreak of the first waterborne infection caused by the parasite Giardia lamblia.

Materials and Methods

2.1. DNA extraction

DNA was extracted from the 120 L of collected water which was centrifuged at 10,000 x g for 30 min. The supernatant was completely removed, and DNA was extracted from the precipitate using a QIAamp DNA stool mini kit (QIAGEN, Hilden, Germany). After centrifugation, the pellet precipitate was transferred to a 2 ml Eppendorf tube and completely dissolved in 1.4 ml of the ASL buffer from the kit. The solution was subjected to five freeze/thaw iterations and then incubated at 95 °C for 10 min. The remaining DNA extraction steps followed the manufacturer’s instructions. The extracted DNAs were used as templates in the primary PCR reactions.

2.2. PCR reaction

PCR reactions were conducted in a total of 25 ul, which included PCR premix (GenDEPOT, Barker, TX, USA), 2 ul (∼100 ng) of extracted DNA, and 10 pmol of each primer (Table 1). The PCR reactions were conducted with a G-STORM apparatus (Gene Technologies, England). The DNAs were denatured for 7 min at 94 °C, followed by 35 cycles of 60 sec at 94 °C, 60 sec at 60 °C or 52 °C, and 90 sec at 72 °C. Finally, reactions were incubated for 10 min at 72 °C. Nested PCR was conducted with 1 ul of the primary PCR product as template. After the nested PCR, the final product was electrophoresed in a 2% agarose gel and stained with ethidium bromide. Bands were visualized under ultraviolet light, and photos were taken using a gel documentation system (Syngene, Cambridge, UK).

Results

People in the villages of Baekun-dong, Baekunmeon, Jinan-gun, and Jeollabuk-do use ground water and provisionally supplied water. Seven of the eight households (25 people) that used provisionally supplied water complained of abdominal pain accompanied by diarrhea from around 8:00 AM on the morning of April 12, 2010. The village chief reported their case to the local health district board, which sent a local epidemiological investigator for investigation. The investigator asked the 25 people to fill out a questionnaire. It was verified that they had not eaten meals together. Approximately 1500 L of the water from the common provisional water supply system was then collected and analyzed by polymerase chain reaction (PCR). Fecal samples of the 25 people in eight households (patient group: nine people; asymptomatic control group: 16 people), who were using the provisionally supplied water, were collected. Those who had at least one of the following symptoms were considered patients: vomiting, abdominal pain, febricity, chillness, or a sense of residual stool. Jeollabuk-do Institute of Health and Environment Research conducted the PCR test to detect the presence of G. lamblia, while the Korea Centers for Disease Control and Prevention conducted a confirmation test.

Out of the 25 who used provisionally supplied water, three among the nine in the patient group and four among the 16 in the control group tested positive for G. lamblia infection, according to the results of PCR analysis targeting the β-giardin gene. A sequence analysis of the β-giardin gene was also positive for G. lamblia infection. The incidence rate was 36% with nine symptomatic patients out of the 25 who used the provisional water supply system being exposed to risk factors. They mostly had diarrhea and abdominal pain, followed by a sense of residual stool and vomiting, and they had diarrhea a maximum of six times (Figure 1).

Discussion

The causative pathogen of this mass diarrhea was confirmed to be G. lamblia and this was the first epidemiological investigation of mass diarrhea caused by this parasite. The Centers for Disease Control and Prevention investigated the rates of infections caused by waterborne protozoans (G. lamblia, Cryptosporidium parvum, and Entamoeba histolytica) in 96 hospitals and clinics for 3 years from 2004 to 2006. The overall positive rate stood at approximately 0.17%; detection of G. lamblia among these parasites accounted for 55% and its outbreak was most frequent during the period from April to May [9]. It is to be noted that the outbreak presented in this report also occurred in April. Cheun et al (2010) reported G. lamblia infection with viral infection in patients, and the mixed infection rate with norovirus was 4.7%. Tests to identify 10 causative bacteria of food poisoning and norovirus were performed on fecal samples of patients with diarrhea. While three patients tested positive for norovirus, the other patients were negative for all the other bacteria. Giardiasis infection is more frequent in developing countries than in developed countries and in Europe approximately 3–14% of patients with human immunodeficiency virus (HIV) infection/acquired immunodeficiency syndrome were reported to be positive for the parasite [10]. In Korea, 1.5% of patients with HIV have been reported to be infected with Giardia[11] and genetic classification to Nash’s group was conducted by separating the parasite from outpatients in 1999. However, genetic classification of G. lamblia was not carried out due to the shortage of its DNA in this study.

Because of this outbreak, provisional water supply was stopped, and public health education for residents on the safety of heating drinking water was conducted. Water to the households was supplied through Ginan-gun water tank trucks, while kitchens and rest rooms were chlorinated, quarantined, and disinfected. Eventually, G. lamblia was not detected. Sodium hypochlorite is only used to disinfect small-scale water supply systems such as provisionally supplied water or well water in Korea [12, 13]. This is because the pathogen is highly resistant to temperature, solar irradiation, sodium hypochlorite, and chlorine dioxide treatments on a large scale [14–17]. However, this outbreak was through the provisional water supply system using unsterilized valley water.

In Korea, the testing and treatment of diarrhea has been focused mainly on viruses and bacteria, and currently there is a lack of tests to diagnose diarrhea caused by protozoans. According to the 2009 water and foodborne epidemiology annual report [18], diseases from unknown pathogens accounted for 40% of infections and in most cases, diarrhea resulting from causative waterborne parasitic infections was suspected. Finally, this outbreak by G. lamblia suggests the importance of continuous monitoring of waterborne and foodborne parasites in order to prevent and control the spread of parasites.

Acknowledgements

This work was supported by a grant from the Korea National Institute of Health (Grant No. NIH-091-4800-4847-302), and Jeollabuk-do Institute of Health and Environment Research (Grant No. JBIHER-200-207-03).

References

• 1. Feachem RG, Hogan RC, Merson MH. Diarrhoeal disease control: reviews of potential interventions. Bull World Health Organ 1983;61:637−40. PMID: 6354505.PubMedPMC
• 2. Yason JA, Rivera WL. Genotyping of Giardia duodenalis isolates among residents of slum area in Manila, Philippines. Parasitol Res 2007;8;101:681−7. PMID: 17401723.ArticlePubMed
• 3. Thompson RC. Giardiasis as a re-emerging infectious disease and its zoonotic potential. Int J Parasitol 2000;11;30:1259−67. PMID: 11113253.ArticlePubMed
• 4. Hellard ME, Sinclair MI, Hogg GG, Fairley CK. Prevalence of enteric pathogens among community based asymptomatic individuals. J Gastroenterol Hepatol 2000;3;15:290−3. PMID: 10764030.ArticlePubMed
• 5. Olsen SJ, MacKinnon LC, Goulding JS, et al. Surveillance for foodborne-disease outbreaks—United States, 1993–1997. MMWR CDC Surveill Summ 2000;3;49:1−62. PMID: 10789699.
• 6. Lee SH, Levy DA, Craun GF, et al. Surveillance for waterborne-disease outbreaks—United States, 1999–2000. MMWR Surveill Summ 2002;11;51:1−47. PMID: 12489843.
• 7. Nygard K, Schimmer B, Sobstad O, et al. A large community outbreak of waterborne giardiasis-delayed detection in a non-endemic urban area. BMC Public Health 2006;5;6:141−50. PMID: 16725025.ArticlePubMedPMC
• 8. Karanis P, Kourenti C, Smith H. Waterborne transmission of protozoan parasites: a worldwide review of outbreaks and lessons learnt. J Water Health 2007;3;5:1−38. PMID: 17402277.Article
• 9. Cheun HI, Cho SH, Lee JH, et al. Infection status of hospitalized diarrheal patients with gastrointestinal protozoa, bacteria, and viruses in the Republic of Korea. Korean J Parasitol 2010;6;48:113−20. PMID: 20585526.ArticlePubMedPMC
• 10. Feng Y, Xiao L. Zoonotic potential and molecular epidemiology of Giardia species and giardiasis. Clin Microbiol Rev 2011;1;24:110−40. PMID: 21233509.ArticlePubMedPMC
• 11. Guk SM, Seo M, Park YK, et al. Parasitic infections in HIV-infected patients who visited Seoul National University hospital during the period 1995–2003. Korean J Parasitol 2005;3;43:1−5. PMID: 15793352.ArticlePubMedPMC
• 12. Park SJ, Yong TS, Yang HW, et al. Axenic cultivation and characterization of Giardia lamblia isolated from humans in Korea. Korean J Parasitol 2005;3;43(1). 1−5. PMID: 15793352.ArticlePubMedPMC
• 13. Korea Ministry of Environment. The regulation of water quality standard and test in drinking water. 4th ed. Report No. 439. Sejong: Ministry of Environment; 2011. (in Korean).
• 14. Winiecka-Krusnell J, Linder E. Cysticidal effect of chlorine dioxide on Giardia intestinalis cysts. Acta Trop 1998;7 30 70(3). 369−72. PMID: 9777721.ArticlePubMed
• 15. Kim KJ, Hong WS, Lee KS. Disinfection characteristics of waterborne pathogenic protozoa Giardia lamblia. Biotechnol Bioprocess Eng 2001;4;6(2). 95−9.Article
• 16. Ongerth JE, Johnson RL, Macdonald SC, et al. Back-country water treatment to prevent giardiasis. Am J Public Health 1989;12;79:1633−7. PMID: 2817191.ArticlePubMedPMC
• 17. McGuigan KG, Méndez-Hermida F, Castro-Hermida JA, et al. Batch solar disinfection inactivates oocysts of Cryptosporidium parvum and cysts of Giardia muris in drinking water. J Appl Microbiol 2006;8;101(2). 453−63. PMID: 16882154.ArticlePubMed
• 18. Korea Centers for Disease Control and Prevention. Water and food-borne epidemiology annual report. Osong: Korea Centers for Disease Control and Prevention; 2011. p 4in Korean.

Figure 1

(A) Main symptoms in infected patients and their frequencies; (B) frequency of diarrhea in infected patients/day.

Table 1

Primer sets used for detection of Giardia lamblia

Target gene	Primer name	Sequence	PCR product size (bp)	Tm (°C)
β-giardin Accession no. M36728	G376A F	5′-CCA TCC ATA ACG ACG CCA TCG CGG CTC TC-3′	415	60
	GGR789-809B R	5′-GGC GCT TAG TGC TTT GTG ACC-3′
	G376B F	5′-CGA CGC CAT CGC GGC TCT CAG GAA GGA GG-3′	374	60
	G759A R	5′-CGC CCT GGA TCT TCG AGA CGA CGT CCT-3′

Figure & Data

References

Citations

Citations to this article as recorded by  

• Waterborne Cryptosporidium and Giardia in resources of MENA: a systematic review and meta-analysis
  Layla Ben Ayed, Shahira Abdelaziz Ali Ahmed, Sonia Boughattas, Panagiotis Karanis
  Journal of Water and Health.2024;[Epub]     CrossRef
• Molecular detection and assemblage analysis of the intestinal protozoan Giardia duodenalis in wild boars in Korea
  Haeseung Lee, Dongmi Kwak
  Frontiers in Veterinary Science.2023;[Epub]     CrossRef
• A scoping review of risk factors and transmission routes associated with human giardiasis outbreaks in high-income settings
  Sarah Krumrie, Paul Capewell, Alison Smith-Palmer, Dominic Mellor, Willie Weir, Claire L. Alexander
  Current Research in Parasitology & Vector-Borne Di.2022; 2: 100084.     CrossRef
• Prevalence and molecular characterization of Giardia duodenalis in small ruminants of Shiraz, southwestern Iran: A zoonotic concern
  Ali Asghari, Farzad Mahdavi, Laya Shamsi, Mohammad Hossein Motazedian, Qasem Asgari, Saeed Shahabi, Behnam Mohammadi-Ghalehbin, Alireza Sadrebazzaz
  Comparative Immunology, Microbiology and Infectiou.2022; 86: 101819.     CrossRef
• Genotypic and Epidemiologic Profiles of Giardia duodenalis in Four Brazilian Biogeographic Regions
  Deiviane A. Calegar, Beatriz C. Nunes, Kerla J. L. Monteiro, Polyanna A. A. Bacelar, Brenda B. C. Evangelista, Mayron M. Almeida, Jurecir Silva, Jéssica P. Santos, Márcio N. Boia, Lauren H. Jaeger, Filipe A. Carvalho-Costa
  Microorganisms.2022; 10(5): 940.     CrossRef
• Molecular Identification of Parasitic Protozoa Sarcocystis in Water Samples
  Živilė Strazdaitė-Žielienė, Agnė Baranauskaitė, Dalius Butkauskas, Elena Servienė, Petras Prakas
  Veterinary Sciences.2022; 9(8): 412.     CrossRef
• Assessment microbial risks for the presence of Cryptosporidium spp. and Giardia spp. based on the surveillance of the water supply systems in Colombia, 2014–2018
  Blanca Lisseth Guzmán Barragán, Marcelo de Souza Lauretto, Maria Tereza Pepe Razzolini, Adelaide Cássia Nardocci, Karen Vanessa Marimón Sibaja
  Water Environment Research.2022;[Epub]     CrossRef
• A Giardiasis Case Diagnosed by Wright’s Stain and BioFire FilmArray Gastrointestinal Panel Assay Using Stool Sample
  Kui Hyun Yoon
  Laboratory Medicine Online.2022; 12(4): 315.     CrossRef
• Diarrhoea among Children Aged 5 Years and Microbial Drinking Water Quality Compliance: Trends Analysis Study in South Africa (2008–2018)
  Mohora Feida Malebatja, Mpata Mathildah Mokgatle
  International Journal of Environmental Research an.2022; 20(1): 598.     CrossRef
• DNA-based detection of Leptospira wolffii, Giardia intestinalis and Toxoplasma gondii in environmental feces of wild animals in Korea
  Priyanka KUMARI, Kyung Yeon EO, Woo-Shin LEE, Junpei KIMURA, Naomichi YAMAMOTO
  Journal of Veterinary Medical Science.2021; 83(5): 850.     CrossRef
• Multilocus Genotyping of Giardia duodenalis Occurring in Korean Native Calves
  Sang-Ik Oh, Suk-Han Jung, Han-Kyoung Lee, Changyong Choe, Tai-Young Hur, Kyoung-Min So
  Veterinary Sciences.2021; 8(7): 118.     CrossRef
• Development of a simple, rapid multiplex PCR tool kit by using the 16S rRNA gene for the identification of faecal and non-faecal coliforms in drinking water
  A. Shiva Shanker, N. Rajesh, Pavan Kumar Pindi
  Water Supply.2021; 21(7): 3319.     CrossRef
• Use of the bivalve Dreissena polymorpha as a biomonitoring tool to reflect the protozoan load in freshwater bodies
  Elodie Géba, Dominique Aubert, Loïc Durand, Sandy Escotte, Stéphanie La Carbona, Catherine Cazeaux, Isabelle Bonnard, Fanny Bastien, Mélissa Palos Ladeiro, Jitender P. Dubey, Isabelle Villena, Alain Geffard, Aurélie Bigot-Clivot
  Water Research.2020; 170: 115297.     CrossRef
• Multilocus genotyping of Giardia duodenalis from pigs in Korea
  Haeseung Lee, ByeongYeal Jung, Jun-Sik Lim, Min-Goo Seo, Seung-Hun Lee, Kwang-Ho Choi, Mi-Hye Hwang, Tae-Hwan Kim, Oh-Deog Kwon, Dongmi Kwak
  Parasitology International.2020; 78: 102154.     CrossRef
• Multilocus genotyping and risk factor analysis of Giardia duodenalis in dogs in Korea
  Ha-Young Kim, Haeseung Lee, Seung-Hun Lee, Min-Goo Seo, Seonju Yi, Jong Wan Kim, Chung Hyun Kim, Yu-Ran Lee, ByungJae So, Oh-Deog Kwon, Dongmi Kwak
  Acta Tropica.2019; 199: 105113.     CrossRef
• Molecular Prevalence and Genotypes of Cryptosporidium parvum and Giardia duodenalis in Patients with Acute Diarrhea in Korea, 2013-2016
  Da-Won Ma, Myoung-Ro Lee, Sung-Hee Hong, Shin-Hyeong Cho, Sang-Eun Lee
  The Korean Journal of Parasitology.2019; 57(5): 531.     CrossRef
• The effect of water source and soil supplementation on parasite contamination in organic vegetable gardens
  Fernanda Pinto Ferreira, Eloiza Teles Caldart, Roberta Lemos Freire, Regina Mitsuka-Breganó, Felipe Machado de Freitas, Ana Carolina Miura, Marcelle Mareze, Felippe Danyel Cardoso Martins, Mariana Ragassi Urbano, Adilson Luiz Seifert, Italmar Teodorico Na
  Revista Brasileira de Parasitologia Veterinária.2018;[Epub]     CrossRef
• Identification of zoonotic Giardia duodenalis in Korean native calves with normal feces
  Yoon-Joo Lee, Du-Gyeong Han, Ji-Hyoung Ryu, Jeong-Byoung Chae, Joon-Seok Chae, Do-Hyeon Yu, Jinho Park, Bae-Keun Park, Hyeon-Cheol Kim, Kyoung-Seong Choi
  Parasitology Research.2018; 117(6): 1969.     CrossRef
• Waterborne transmission of protozoan parasites: Review of worldwide outbreaks - An update 2011–2016
  Artemis Efstratiou, Jerry E. Ongerth, Panagiotis Karanis
  Water Research.2017; 114: 14.     CrossRef
• Prevalence and molecular characterisation of Giardia duodenalis in calves with diarrhoea
  S. H. Lee, D. VanBik, H. Y. Kim, A. Cho, J. W. Kim, J. W. Byun, J. K. Oem, S. I. Oh, D. Kwak
  Veterinary Record.2016; 178(25): 633.     CrossRef
• Occurrence of Cryptosporidium spp. and Giardia spp. in a public water-treatment system, Paraná, Southern Brazil
  Jonatas Campos Almeida, Felippe Danyel Cardoso Martins, José Maurício Ferreira Neto, Maíra Moreira dos Santos, João Luis Garcia, Italmar Teodorico Navarro, Emília Kiyomi Kuroda, Roberta Lemos Freire
  Revista Brasileira de Parasitologia Veterinária.2015; 24(3): 303.     CrossRef
• A Case of Colitis by <i>Giardia lamblia</i> Infection with Atypical Presentation
  Soo Kyoung Choi, Youn Hee Lee, Jinhee Lee, Seon Ho Park, Mi Hee Kim, Ok-Ran Shin, Young-Seok Cho
  The Korean Journal of Medicine.2015; 89(3): 312.     CrossRef
• On the track for an efficient detection of Escherichia coli in water: A review on PCR-based methods
  Diana Mendes Silva, Lucília Domingues
  Ecotoxicology and Environmental Safety.2015; 113: 400.     CrossRef
• Genotyping and subtyping Cryptosporidium parvum and Giardia duodenalis carried by flies on dairy farms in Henan, China
  Zifang Zhao, Haiju Dong, Rongjun Wang, Wei Zhao, Gongyi Chen, Shouyi Li, Meng Qi, Sumei Zhang, Fuchun Jian, Jinfeng Zhao, Longxian Zhang, Haiyan Wang, Aiqin Liu
  Parasites & Vectors.2014;[Epub]     CrossRef
• Consistency of direct microscopic examination and ELISA in detection of Giardia in stool specimen among children
  Zohreh Torabi, Ali Niksirat, Saeideh Mazloomzadeh, Akefeh Ahmadiafshar
  Asian Pacific Journal of Tropical Disease.2014; 4: S725.     CrossRef