Parasitologists United Journal

ORIGINAL ARTICLE
Year
: 2014  |  Volume : 7  |  Issue : 2  |  Page : 104--109

The epidemiology of Giardia intestinalis assemblages A and B among Egyptian children with diarrhea: A PCR-RFLP-based approach


Nora L El-Tantawy, Amira I Taman 
 Department of Parasitology, Faculty of Medicine, Mansoura University, Mansoura, Egypt

Correspondence Address:
Nora L El-Tantawy
Department of Medical Parasitology, Faculty of Medicine, Mansoura University, 35516, Mansoura
Egypt

Abstract

Background The protozoan parasite Giardia intestinalis is a common childhood infection in developing countries that causes diarrheal illness. The majority of G. intestinalis isolates from humans are grouped into two distinct genetic assemblages A and B. The molecular epidemiological studies on G. intestinalis assemblages in humans are limited in Egypt. Objective This study was conducted to estimate the detection rate of G. intestinalis infection among a cohort of children suffering from diarrhea in the Dakahalia governorate, Egypt, and to correlate between clinical giardiasis and Giardia spp. assemblages in positive stool samples by PCR-restriction fragment length polymorphism (PCR-RFLP). Participants and methods A total of 311 diarrheal stool samples were examined microscopically for Giardia spp. infection. DNA samples were isolated from the stools of 103 (33.12%) positive samples with G. intestinalis, amplified with PCR, and digested with the XhoI enzyme for RFLP. Results Of the 103 samples, 64 (62.14%) were found to be assemblage B, whereas 32 samples (31.07%) belonged to assemblage A. Mixed genotype A and B was present in three samples (2.91%), and four samples (3.88%) were of undetermined Giardia spp. assemblage. The detection rate of assemblage B was higher in samples from children with persistent diarrhea, whereas assemblage A detection rate was higher in samples from acute diarrhea. Conclusion G. intestinalis causing diarrhea in children in the Dakahalia governorate, Egypt, predominantly belongs to assemblage B, indicating that human-to-human method of infection is more common than zoonotic method.



How to cite this article:
El-Tantawy NL, Taman AI. The epidemiology of Giardia intestinalis assemblages A and B among Egyptian children with diarrhea: A PCR-RFLP-based approach.Parasitol United J 2014;7:104-109


How to cite this URL:
El-Tantawy NL, Taman AI. The epidemiology of Giardia intestinalis assemblages A and B among Egyptian children with diarrhea: A PCR-RFLP-based approach. Parasitol United J [serial online] 2014 [cited 2020 Apr 1 ];7:104-109
Available from: http://www.new.puj.eg.net/text.asp?2014/7/2/104/149557


Full Text

 Introduction



The flagellated protozoan Giardia spp. is one of the most common intestinal parasites affecting humans worldwide. It is estimated that 200 million people in the developing countries of Africa, Asia, and Latin America have symptomatic giardiasis [1],[2] . Giardiasis affects all ages; however, the infection rate is significantly higher in children [3] , with a prevalence rate of up to 30% in children below the age of 10 years [4] . Giardiasis is transmitted by the fecal-oral route following direct or indirect exposure to Giardia spp. cyst, through the ingestion of contaminated food, drinking contaminated water, and direct contact with infected individuals or contaminated fomites [5],[6],[7] .

Giardia intestinalis has been divided into a number of genotypes (A-H) [8],[9] . Further work using small-subunit rRNA (SSU rRNA), triosephosphate isomerase (tpi), and glutamate dehydrogenase (GDH) gene confirmed the presence of two major genotypes among the human isolates named as A and B [1],[10] , which were later divided into several subassemblages [11],[12] . These two major human assemblages showed variable distribution among studies even within the same country [13] .

Giardiasis causes a wide number of gastrointestinal symptoms ranging from a mild self-limiting illness to a persistent illness lasting for several months [14] , and is considered the most important nonviral infectious agent that causes worldwide diarrhea in humans [15],[16] . It is responsible for 2.5 million cases of diarrhea and nutritional deficiencies in children living in developing countries every year [17] . The symptomatology of giardiasis has been attributed to many factors such as host response, dose of infection, and Giardia spp. genotype [18] . Attempts to associate symptoms with genotype in human infections have not provided a definitive answer to date [19],[20] . Researchers from Australia, Bangladesh, Peru, Spain, and Great Britain found that assemblage A is associated with severe clinical symptoms than assemblage B [19],[21],[22],[23] , whereas this distribution was reversed in the Netherlands and Ethiopia [24],[25] , and no association was found in Brazil [26] .

Although G. intestinalis assemblages' genotypic separation is relatively established [27] , the clinical or epidemiologic significance of infection with these assemblages is poorly understood [28] . It is questionable whether G. intestinalis' infectivity and pathogenicity vary in hosts of different ages, or whether there is an association of clinical symptoms, pathogenicity, or zoonotic potential with assemblage or genotype [29],[30] .

The aim of our study was to estimate the detection rate of giardiasis among a cohort of children suffering from diarrhea in the Dakahalia governorate, Egypt, and to study the associated risk factors. In addition, an attempt to determine the Giardia spp. genotypes present in this group was made to find a possible correlation between diarrhea and G. intestinalis assemblages.

 Participants and methods



Type of the study

Descriptive study.

Study population and sample collection

From July 2012 to August 2013, the study was conducted on 311 children (158 boys and 153 girls) complaining of diarrhea, with ages ranging from 3 to 12 years. They were from attendants of Mansoura Children Hospital (MCH), Dakahalia governorate, Egypt. Demographic and lifestyle information was obtained through a survey questionnaire. The questionnaire included age, sex, address, source of water for drinking and washing, contact with animals, and washing of vegetables and fruits before eating. Details of the number of stools passed per day and other associated symptoms of fever, abdominal pain, flatulence, or vomiting were also recorded. From each child, fresh stool samples were collected in 50 ml screw-capped containers and divided into two parts: the first part was examined for parasitic infections, whereas the second part was stored without preservatives at −20°C for DNA extraction. Diarrhea was defined as a passage of three loose stools in a 24-h period followed by at least 2 days without diarrhea [31] . According to WHO criteria, diarrheal episodes were classified as acute diarrhea that lasts for less than 2 weeks, and persistent diarrhea that lasts for 14 days or more [32] .

Smear samples from the first part were examined by wet preparation and Lugol's iodine, followed by formalin ethyl acetate concentration technique [33] . Smears were also stained using the modified Ziehl Nielsen procedure [34] , and examined by light microscopy under the magnification of ×1000. Smears were reported as positive if cysts or trophozoites were detected. In addition, parasitological examinations for Entamoeba histolytica, Cryptosporidium spp., Cyclospora cayetanensis, Microsporidium spp., and helminth infections were conducted. Only Giardia spp. positive samples, which were negative for other intestinal parasites, were used for DNA extraction.

DNA extraction

This was performed according to Volotão et al. [35] . In brief, 5 g of preserved stool was suspended in 15 ml distilled water, and then filtered through four layers of gauze to remove coarse materials. The filtrate was centrifuged for 5 min at 800 g, the supernatant was decanted, and the washing step was repeated many times to remove debris. Finally, the pellet was used for DNA extraction using QIAamp DNA mini kit (Qiagen, Hilden, Germany), according to the manufacturer's instructions. DNA was eluted in 100 μl of elution buffer and DNA samples were stored at −20°C till use in PCR analysis.

PCR amplification

A segment of the 683 bp of triosephosphate isomerase (tpi) gene from G. intestinalis isolates was amplified by PCR reaction according to Baruch et al. [36] . Two oligonucleotide primers were used: a forward primer (5′-ATGCCTGCTCGTCGCCCCTTC-3′) and a reverse primer (5′-CACTGGCCAAGCTTCTCGCAG-3′). The reaction mixture was as follows: 10 μl of 10′ PCR buffers, 200 nmol/l of each of 6 mmol/l MgCl 2 and deoxynucleoside triphosphate, 100 nmol/l of each primer, 2.5 U of Platinum Pfx DNA polymerase (Invitrogen, Carlsbad, California, USA), and 2 μl of DNA template. The reaction mixture was completed to 50 μl using DNase-RNase-free water. The PCR was carried out using the following conditions: initial denaturation for 5 min at 95°C followed by 30 cycles each consisting of 95°C for 1 min, 45°C for 1 min, and 72°C for 1 min. The final extension was for 10 min at 72°C.

Restriction digestion and gel electrophoresis

For the restriction reaction, 5 μl of the PCR product was digested using 20 U of XhoI enzyme (Invitrogen) and 2 μl of 10 reaction buffer in a reaction mixture of 20 μl. The reaction was incubated at 37°C for 1 h. The digestion product was run on 4% agarose gel and visualized using ethidium bromide. A molecular size marker ΦX 174 RF DNA/HaeIII fragments (Invitrogen) was used to assess the size of the PCR product.

Statistical analysis

Data were analyzed using the SSPS version 11.5 (SPSS Inc., Chicago, Illinois, USA). The risk factors were estimated with odds ratio, and the significance was tested using 95% confidence interval and P-value. The significance was defined as P-value less than 0.05.

Ethical consideration

An informed consent was obtained from children's guardians and human experimentation guidelines were followed for conducting this study. Cases that proved positive for any parasitic infection were treated according to their clinical conditions in the Pediatric Department.

 Results



Of the 311 examined diarrheal stool specimens, 103 (33.12%) were found to be infected with Giardia spp. by microscopic examination. The organism was identified more frequently in stools from children with persistent diarrhea (56/103; 54.37%) than in stools from children with acute diarrhea (47/103; 45.63%). In addition, 96.12% of the positive cases complained of abdominal pain, 64.08% of flatulence, 39.81% of vomiting, and 32.04% of fever ([Table 1]).{Table 1}

Of these 103 positive samples, 58 (56.31%) were male and 45 (43.69%) were female. The highest rate of infection in our study was in the 3-7 years old age group (P < 0.05). Sex, residence, drinking piped water, and contact with household pets showed insignificant association with giardial diarrhea (P > 0.05), whereas eating raw vegetables and fruits showed significant association (P < 0.05) ([Table 2]).{Table 2}

PCR products with expected size (683 bp) of Giardia spp. isolates, digested with XhoI to differentiate assemblage A and B by restriction fraction length polymorphism (RFLP), proved that those of assemblage A were resistant to cleavage ([Figure 1]), and those of assemblage B cleaved into two fragments (442 and 241 bp). All 103 positive samples for G. intestinalis identified by microscopy were also positive by PCR technique. The results of the RFLP analysis demonstrated that 32/103 samples (31.07%) belonged to assemblage A, whereas 64/103 (62.14%) belonged to assemblage B. Samples with mixed genotype A and B (3/103; 2.91%), and those with undetermined Giardia spp. assemblage (4/103; 3.88%) had persistent diarrhea. In children suffering from acute diarrhea assemblages A and B recorded 48.94 and 51.06%, respectively, and in those with persistent diarrhea assemblage B predominated (71.43%) as compared with 16.07% assemblage A ([Table 3]).{Figure 1}{Table 3}

 Discussion



G. intestinalis is a worldwide intestinal parasite with high prevalence rate in developing countries, where standard sanitation conditions and proper hygiene practices are lacking [37] . G. intestinalis pathogen is capable of causing diarrheal illness among young children [38] , and it has a complex of several assemblages, of which assemblages A and B are predominantly associated with human infections [39],[40] .

In our study, G. intestinalis infection gave a detection rate of 33.12% among a cohort of children suffering from acute and persistent diarrhea in the Dakahalia governorate. This result is in harmony with 33% reported by Shukry et al. [41] among a sample of Cairo residents, and 34.6% reported for another sample from Egypt using PCR [42] . Nazeer et al. [43] also reported 30.5 and 37.1% by microscopy versus multiplex real-time PCR among a sample of diarrhea patients in Cairo and the Egyptian governorates of Fayoum and Benha. However, it is somewhat more than the 24.7% recorded in the Behera governorate [44] .

Herein, we evaluated the distribution of assemblages A and B in positive giardial diarrhea in the Dakahalia governorate. Our observations revealed that the majority of Giardia spp. isolates were assemblage B genotype (62.14%); this comes in accordance with another study carried out in Kafr El Sheik governorate, in Egypt, that reported (66.7%) predominance of assemblage B [45] . On the contrary, Helmy et al. [46] , also using the tpi gene amplification, reported 75.6% of their study population in Cairo belonging to assemblage A and only 19.5% belonging to assemblage B. The reasons behind the geographic variation in the predominance of the Giardia spp. assemblages are still unclear. It may be explained by the difference in the methods of transmission. It has been known that assemblage A is mostly responsible for zoonotic transmission, with a wide range of animals acting as reservoir hosts, whereas assemblage B is most likely transmitted from human to human. However, assemblage B has been reported in some animals and may represent a zoonotic potential as well [47],[48] .

In our study, persistent diarrhea proved more common than the acute form in children suffering from giardiasis with apparent predominance of assemblage B, whereas assemblage A was mostly associated with acute diarrhea. This finding is consistent with previous studies carried out in the Netherlands [24] and Ethiopia [25] .

In our study, eating fresh fruits and vegetables was a significant risk factor for Giardia spp. infection. It is believed that this association is owing to eating insufficiently cooked vegetables or eating fresh fruits with contaminated hands. Contaminated hands are implicated to play a major role in the transmission of communicable fecal-oral-transmitted diseases in developing countries [49],[50],[51],[52] .

 Conclusion



Giardia spp. is a prevalent protozoan among children complaining of diarrhea in the Dakahalia governorate, Egypt. This result documents that assemblage B is more predominant in cases with persistent diarrhea, whereas assemblage A is more common in acute diarrhea. In addition, our results indicate that assemblage B is the most common genotype in all detected Giardia spp. cases reflecting the human-to-human method of infection in children in our locality rather than zoonotic method. Thus, infection control measures and the development of awareness strategies to improve sanitation and health education to children and their mothers should be considered.

 Acknowledgements



Conflicts of interest

There are no conflicts of interest.

References

1Thompson RCA, Hopkins RM, Homan WL. Nomenclature and genetic groupings of Giardia infecting mammals. Parasitol Today; 2000; 16:210-213.
2 Yason JA, Rivera WL. Genotyping of Giardia duodenalis isolates among residents of slum area in Manila, Philippines. Parasitol Res; 2007; 101:681-687.
3 Keystone JS, Yang J, Grisdale D, Harrington M, Pillon L, Andreychuk R. Intestinal parasites in metropolitan Toronto day-care centres. Can Med Assoc J; 1984; 131:733-735.
4 Havsa MC, Watson JC, Beach MJ. Giardiasis surveillance-USA, 1998-2002. MMWR Surveill Summ; 2005; 54:9-16.
5 Gardner TB, Hill DR. Treatment of giardiasis. Clin Microbiol Rev; 2001; 14:114-128.
6 Monis PT, Thompson RCA. Cryptosporidium and Giardia-zoonosis: fact or fiction? Infect Genet Evol; 2003; 3:233-244.
7 Xiao L, Fayer R, Ryan U, Upton SJ. Cryptosporidium taxonomy: recent advances and implications for public health. J Clin Microbiol; 2004; 17:72-97.
8 Monis PT, Caccio SM, Thompson RC. Variation in Giardia: towards a taxonomic revision of the genus. Trends Parasitol; 2009; 25:93-100.
9 Feng, Y, Xiao L. Zoonotic potential and molecular epidemiology of Giardia species and giardiasis. Clin Microbiol Rev; 2011; 24:110-140.
10Lalle M, Pozio E, Capelli G, Bruschi F, Crotti D, Caccio SM. Genetic heterogeneity at the b-giardin locus among human and animal isolates of Giardia duodenalis and identification of potentially zoonotic sub genotypes. Int J Parasitol; 2005; 35:207-213.
11Thompson JD, Higgins DG, Gibson TJ. Improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice. Nucleic Acids Res; 1994; 22: 4673-4680.
12Monis PT, Andrews RH, Mayrhofer G, Ey PL. Genetic diversity within the morphological species Giardia intestinalis and its relationship to host origin. Infect Genet Evol; 2003; 3:29-38.
13Caccio SM, Thompson RC, McLauchlin J, Smith HV. Unravelling Cryptosporidium and Giardia epidemiology. Trends Parasitol; 2005; 21: 430-437.
14Farthing MJG. Giardiasis as a disease. In: RCA Thompson, JA Reynoldson, AJ Lymbery (editors.). Giardia: from molecules to disease. CAB, Wallingford; 1994, 15-37.
15Meyer EA. Taxonomy and nomenclature. In: EA Meyer (editor)., Giardiasis. Elsevier, Amsterdam. 1990; 51-60.
16Thompson RCA, Reynoldson JA, Lymbery AJ. Giardia from molecules to disease and beyond. Parasitol Today; 1993; 9:313-315.
17Younas M, Shah S, Talaat A. Frequency of Giardia intestinalis infection in children with recurrent abdominal pain. J Pak Med Assoc; 2008; 58:171-174.
18Pierce KK, Kirkpatrick BD. Update on human infections caused by intestinal protozoa. Curr Opin Gastroenterol; 2009; 25:12-17.
19Sahagun J, Clavel A, Goni P, Seral C, Llorente MT. Correlation between the presence of symptoms and the Giardia duodenalis genotype. Eur J Clin Microbiol Infect Dis; 2008; 27:81-83.
20Robertson LJ. Giardia and Cryptosporidium infections in sheep and goats: a review of the potential for transmission to humans via environmental contamination. Epidemiol Infect; 2009; 137:913-921.
21Haque R, Roy S, Kabir M, Stroup SE, Mondal D. Giardia assemblage A infection and diarrhea in Bangladesh. J Infect Dis; 2005; 192:2171-2173.
22Haque R, Mondal D, Karim A, Molla IH, Rahim A. Prospective case-control study of the association between common enteric protozoal parasites and diarrhea in Bangladesh. Clin Infect Dis; 2009; 48:1191-1197.
23Breathnach AS, McHugh TD, Butcher PD. Prevalence and clinical correlations of genetic subtypes of Giardia intestinalis in an urban setting. Epidemiol Infect; 2010; 138:1459-1467.
24Homan WL, Mank TG. Human giardiasis: genotype linked differences in clinical symptomatology. Int J Parasitol; 2001; 31:822-826.
25Gelanew T, Lalle M, Hailu A, Pozio E, Caccio SM. Molecular characterization of human isolates of Giardia duodenalis from Ethiopia. Acta Trop; 2007; 102:92-99.
26Kohli A, Bushen OY, Pinkerton RC, Houpt E, Newman RD. Giardia duodenalis assemblage, clinical presentation and markers of intestinal inflammation in Brazilian children. Trans R Soc Trop Med Hyg; 2008; 102: 718-725.
27Guy RA, Xiao C, Horgen PA Real-time PCR. assay for detection and genotype differentiation of Giardia lamblia in stool specimens. J Clin Microbiol; 2004; 42:3317-3320.
28Cedillo-Rivera R, Darby JM, Enciso-Moreno JA, Ortega-Pierres G, Ey PL. Genetic homogeneity of axenic isolates of Giardia intestinalis derived from acute and chronically infected individuals in Mexico. Parasitol Res; 2003; 90:119-123.
29Sackey ME, Weigel MM, Armijos RX. Predictors and nutritional consequences of intestinal parasitic infections in rural Ecuadorian children. J Trop Pediatr; 2003; 49:17-23
30Stuart JM, Orr HJ, Warburton FG, et al. Risk factors for sporadic giardiasis: a case-control study in south western England. Emerg Infect Dis; 2003; 9:229-233.
31Newman RD, Moore SR, Lima AA, Nataro JP, Guerrant RL, Sears CL. A longitudinal study of Giardia lamblia infection in north-east Brazilian children. Trop Med Int Health; 2001; 6:624-634.
32World Health Organization (WHO). Persistent diarrhoea in children in developing countries: memorandum from a WHO meeting. Bull World Health Organ; 1988; 66:709-717.
33Garcia LS, Shimizu R. Comparison of clinical results for the use of ethyl acetate and diethyl ether in the formalin-ether sedimentation technique performed on polyvinyl alcohol-preserved specimens. J Clin Microbiol; 1981; 13:709-713.
34Ridley DS, Hawgood BC. The value of formol-ether concentration of fecal cysts and ova. J Clin Pathol; 1956; 9:74-76.
35Volotão AC, Costa-Macedo LM, Haddad FS, Brandao A, Peralta JM, Fernandes O. Genotyping of Giardia duodenalis from human and animal samples from Brazil using beta-giardin gene: a phylogenetic analysis. Acta Trop; 2007; 102:10-19.
36Baruch AC, Isaac-Renton J, Adam RD. The molecular epidemiology of Giardia lamblia: a sequence-based approach. J Infect Dis; 1996; 174 :233-236.
37Marshall MM, Naumovitz D, Ortega Y, Sterling CR. Waterborne protozoan pathogens. Clin Microbiol Rev; 1997; 10:67-85.
38Haque R. Human intestinal parasites. J Health Popul Nutr; 2007; 25: 387-391.
39Homan WL, van Enckevort FH, Limper L. Comparison of Giardia isolates from different laboratories by isoenzyme analysis and recombinant DNA probes. Parasitol Res; 1992; 78:316-323.
40Mayrhofer G, Andrews RH, Ey PL, Chilton NB. Division of Giardia isolates from humans into two genetically distinct assemblages by electrophoretic analysis of enzymes encoded at 27 loci and comparison with Giardia muris. Parasitology; 1995; 111:11-17.
41Shukry S, Zaki AM, DuPont HL, Shoukry I, el Tagi M, Hamed Z. Detection of enteropathogens in fatal and potentially fatal diarrhea in Cairo, Egypt. J Clin Microbiol; 1986; 24:959-962.
42Foronda P, Bargues MD, Abreu-Acosta N, et al. Identification of genotypes of Giardia intestinalis of human isolates in Egypt. Parasitol Res; 2008; 103:1177-1181.
43Thien H, El-Sibaei MM, Abdel-Hamid MY, Tawfik RA, Tannich E. Use of multiplex real-time PCR for detection of common diarrhea causing protozoan parasites in Egypt. Parasitol Res; 2013; 112:595-601.
44Curtale F, Nabil M, el Wakeel A, Shamy MY. Anaemia and intestinal parasitic infections among school age children in Behera Governorate, Egypt. J Trop Pediatr; 1998; 44: 323-328.
45Amer SE. Genotypic and phylogenetic characterization of Giardia intestinalis from human and dairy cattle in Kafr El Sheikh Governorate, Egypt. J Egypt Soc Parasitol; 2013; 43:133-146.
46Helmy MM, Abdel-Fattah HS, Rashed L. Real-time PCR/RFLP assay to detect Giardia intestinalis genotypes in human isolates with diarrhea in Egypt. J Parasitol; 2009; 95:1000-1004.
47Van Keulen H, Macechko PT, Wade S, Schaaf S, Wallis PM, Erlandsen SL. Presence of human Giardia in domestic, farm and wild animals, and environmental samples suggests a zoonotic potential for giardiasis. Vet Parasitol; 2002; 108:97-97.
48Trout JM, Santín M, Greiner E, Fayer R. Prevalence and genotypes of Giardia duodenalis in post-weaned dairy calves. Vet Parasitol; 2005; 130:177-183.
49Knight SM, Toodayan W, Caique WC, Kyi W, Barnes A, Desmarchelier P. Risk factors for the transmission of diarrhoea in children: a case-control study in rural Malaysia. Int J Epidemiol; 1992; 21:812-818.
50Velema J, van-Wijnen G, Bult P, van-Naerssen T, Jota S. Typhoid fever in Ujung Pandang, Indonesia: high-risk groups and high-risk behaviours. Trop Med Int Health; 1997; 2:1088-1094.
51Hoque BA, Chakraborty J, Chowdhury JT, et al. Effects of environmental factors on child survival in Bangladesh: a case control study. Public Health; 1999; 113: 57-64.
52Hussein-Gasem M, Dolmans W, Keuter M, Djokomoeljanto R. Poor food hygiene and housing risk factors for typhoid fever in Semarang, Indonesia. Trop Med Int Health; 2001; 6: 484-490.