|Year : 2018 | Volume
| Issue : 2 | Page : 193-196
Detection and genetic characterization of Giardia intestinalis in children with gastrointestinal symptoms by PCR RFLP in Sikkim, India
Nongmaithem Onila Chanu1, T Shantikumar Singh1, Sudip Dutta2
1 Department of Microbiology, Sikkim Manipal Institute of Medical Sciences, Gangtok, Sikkim, India
2 Department of Paediatrics, Sikkim Manipal Institute of Medical Sciences, Gangtok, Sikkim, India
|Date of Web Publication||20-Jun-2018|
Nongmaithem Onila Chanu
Department of Microbiology, Sikkim Manipal Institute of Medical Sciences, Tadong, Gangtok, Sikkim
Source of Support: None, Conflict of Interest: None
| Abstract|| |
Background: This study is aimed to detect Giardia and genetically characterize Giardia intestinalis among children in Sikkim, India.
Materials and Methods: A total of 400 stool samples were collected from the children (<15 years) with gastrointestinal symptoms, who attended Central Referral Hospital and Sir Thodup Namgyal Memorial Hospital. Giardia cysts were detected by microscopy from the stool samples and were genetically characterized by polymerase chain reaction–restriction fragment length polymorphism (PCR-RFLP) targeting glutamate dehydrogenase gene (gdh). Results: Giardia cysts were detected in 20/400 (5% incidence rate). Out of 20 samples, 13 (65%) were successfully assayed by PCR-RFLP. The PCR product of gdh gene was digested by BsPL1 and RsaI and isolated 53.7% of BIV, 38.5% of AII and B mixed, and 7.8% of BIII assemblages. There was no AI assemblage found in this study. Diarrhea and abdominal pain were the common complaints associated with giardiasis. Conclusions: PCR-RFLP targeting gdh gene locus is a reliable, easy, and cost-effective method to identify G. intestinalis and its assemblages. This is the first report on the prevalence and genetic variability of human giardiasis in symptomatic children in Sikkim, India.
Keywords: Children, gastrointestinal, genotyping, Giardia intestinalis, Sikkim
|How to cite this article:|
Chanu NO, Singh T S, Dutta S. Detection and genetic characterization of Giardia intestinalis in children with gastrointestinal symptoms by PCR RFLP in Sikkim, India. J Nat Sc Biol Med 2018;9:193-6
|How to cite this URL:|
Chanu NO, Singh T S, Dutta S. Detection and genetic characterization of Giardia intestinalis in children with gastrointestinal symptoms by PCR RFLP in Sikkim, India. J Nat Sc Biol Med [serial online] 2018 [cited 2019 Nov 12];9:193-6. Available from: http://www.jnsbm.org/text.asp?2018/9/2/193/234712
| Introduction|| |
Giardia intestinalis (synonyms Giardia lamblia and Giardia duodenalis) is one of the most common intestinal parasites which affects children worldwide., It causes giardiasis which results in malnutrition, stunted growth, and cognitive impairment. Giardiasis in human is mostly transmitted through ingestion of cysts contaminated with food and water. The genus Giardia belongs to phylum Sarcomastigophora, class Zoomastigophora, and a member of the order Diplomonadida. Eight assemblages (A-H) have been described, of which all human isolates are characterized into either assemblage A or B. Assemblage AI is also detected in livestock, cats, and dogs, whereas assemblage AII is exclusively in humans. Approximately 200 million people have symptomatic giardiasis in Asia, Africa, and Latin America and about 500,000 new cases are reported each year among children. Till date, the genotyping studies of G. intestinalis are relied on using β-giardin, gdh, Tpi, SSU, RNA, eflα, and variant surface protein (vsp) genes from different hosts., Distribution of the human-associated assemblage varies geographically, and the use of primers based on Tpi marker detected more mixed infection with assemblage A and B than other primers., The study is aimed to identify the G. intestinalis assemblage and subassemblage in children with gastrointestinal symptoms in Sikkim, India.
| Materials and Methods|| |
This prospective study was conducted using stool samples from 400 children attending Sir Thodup Namgyal Memorial Hospital and Central Referral Hospital located in East Sikkim, during April 2015–April 2017. Children in the age group of 0–15 years, who presented with gastrointestinal symptoms (diarrhea, vomiting, nausea, weight loss, fatigue, and bloating), were investigated for intestinal parasitic infection before administration of medication. This study was approved by the University Ethical Committee. Written informed consent was obtained from parents/guardian.
Stool sample collection and processing
The children/guardians were given a labeled, leak-proof container with a plastic scoop (HiMedia) to collect sample as per the standard procedure of the WHO and processed within 4 h of collection. The samples were examined microscopically after formal ether concentration technique using saline and iodine mount coverslip preparations.
DNA was extracted directly from the specimen using commercial kit Helini pure fast stool DNA Minispin Prep Kit (Catalog no. 2006; Helini biomolecules, Chennai, India) using the manufacturer's instruction. To enhance the recovery of DNA, before adding the lysis buffer and proteinase K, the suspension of the stool-processing buffer and the stool were subjected to freezing and thawing process (−80/+80) with added glass beads (HiMedia) for 10 min alternatively for 1 h and the final elution was made at 50 μl and stored at −20°C.
Polymerase chain reaction amplification
The amplification of gdh was performed by a semi-nested polymerase chain reaction (PCR) to get the fragment of approximately 432 bp. The primers used in the first PCR reaction were GDHeF, GDHiR, and GDHiF, and GDHiR was used in second PCR reaction as previously described. PCR reaction mixtures consisted of 10 μl of Red Dye PCR Master Mix (Helini biomolecules), 2.5 μl of each primer pairs, and 10 μl of extracted DNA. The reactions were carried out in 25 μl volume. The amplification conditions were as follows: the first initial denaturation at 95°C for 5 min followed by 35 cycles (denaturation at 95°C for 30 s, annealing at 58°C for 30 s, extension for 72°C for 30 s, and final extension at 72°C for 5 min). All PCR reactions were carried out using Eppendorf Master Cycler. 10 μl of sterile water as a negative control and 10 μl of positive template as positive control was used (Helini biomolecules). Electrophoresis of PCR products wase performed on 2% agarose gel containing ethidium bromide and visualized under UV transilluminator.
Restriction fragment length polymorphism analysis
Amplification products of 20 Giardia-positive samples were performed. Genotyping by restriction fragment length polymorphism (RFLP) digestion method to distinguish between Giardia assemblages was achieved using Bspl I (10 U/μl, Thermo Fisher Scientific, USA) restriction enzyme to differentiate between Group I and II of assemblages A and B and by Rsal restriction enzymes (10 U/μl, Thermo Fisher Scientific) to distinguish between subassemblages BIII and BIV. Restriction condition was performed according to the manufacturer's instruction. Restriction fragment was separated by 15% polyacrylamide gel electrophoresis using 100 bp DNA ladder (Helini's biomolecules, Chennai, India) as size standard and visualized.
The statistical analysis was performed using GraphPad Software Inc., San Diego, USA. The association between the two categorical variables was analyzed by Chi-squared test or Fisher's exact test as appropriate. Different variables were summarized using frequency tables. P ≤ 0.05 is considered statistically significant.
| Results|| |
Out of the 400 children, Giardia cysts were detected in 5% of the participants (20/400) by microscopy in which 65% (13/20) were successfully assayed by PCR-RFLP-targeting gdh genes. The PCR product of gdh gene was digested by BsPL1 and Rsal to differentiate between the isolates of A and B subassemblages. Out of 65% Giardia positive, 53.7% of BIV, 38.5% of AII and B mixed, and 7.8% of BIII assemblages were isolated [Table 1]. AI assemblage was not observed in this study. The predicted restriction fragment sizes are listed in [Table 2]. Higher prevalence of giardiasis was from the age group of 6–10 years and the infection rate was similar in male and female participants [Table 3]. BIV genotype was observed in the age group of 1–13 years, BIII genotype was observed in one 4-year-old patient, and mixed assemblage of BIV and B genotype was observed in 9–12-year-old children. Diarrhea and abdominal pain was the common complaint associated with giardiasis in this study.
|Table 1: Genotypes of Giardia lamblia by polymerase chain reaction-restriction fragment length polymorphism targeting glutamate dehydrogenase genes|
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|Table 2: Giardia intestinalis predicted fragment size of sub assemblages after digesting with restriction enzymes|
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| Discussion|| |
G. intestinalis causes human giardiasis and is the most common intestinal parasite of human especially in children. It is more common in young children which is due to the consumption of food/water contaminated with Giardia cysts.
In our study, assemblage B (61.5%) was the predominant genotype observed followed by a mixed assemblage of BIV and BIII (53.7% and 7.8%). Similar observations were previously reported in which assemblage B (61%) was observed to be predominant with 38% BIII assemblages., Although one study has reported a 93.02% prevalence of assemblages B, all these studies were targeting gdh genes. However, several other studies have reported the assemblage A as the predominant genotype.,,, Such differences in the genotypes observed may reflect the geographical variation in the prevalence in the intestinal parasites and are hence valuable in designing effective therapeutic approaches.
The molecular genotyping in our study reveals that the human isolate of Giardia is associated with the assemblage A and B only which are related to human infections. However, false-negative result was also reported when the gdh gene was amplified by nested PCR. Some studies also reported that tpi PCR was better able to resolve mix infections with assemblage A and B since both the gdh and β-giardia PCR-RFLP typed these samples as either assemblage A or B infection alone. There was also a mixed infection found in this study between AII and B. This could be due to amplification of single genotype over other at a specific locus. The data from this study are in agreement with several previous studies from other geographical locations.,,,,,,, In this study, we were unable to amplify 7/20 of the Giardia positive by microscopy. This could be due to low DNA yield from fecal samples, either due to degradation of fecal sample and/or presence of some PCR inhibitors (lipids, hemoglobin, bile salts, polysaccharides from mucus, bacteria, and food degradation product).,
In this study, the highest prevalence of giardiasis was observed in the age group of 6–10 years. Other studies have been reported that the higher prevalence seen in 2–8 years, 1–13 years, or 0–4 years.,,
| Conclusions|| |
The predominant G. intestinalis genotype found in the Sikkim, Indian region, was BIV assemblages and not anthroponotic assemblages. However, the reasons for the exact transmission of mixed assemblages are unclear. Nevertheless, studies based on different assemblage-specific genotyping based on β-giardin, tpi, and small subunit ribosomal RNA genes will be necessary in future.
Financial support and sponsorship
Conflicts of interest
There are no conflicts of interest.
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[Table 1], [Table 2], [Table 3]