Identification of genotypes of Giardia duodenalis human isolates in Isfahan, Iran, using polymerase chain reaction - Restriction Fragment Length polymorphism

Abstract

Background:Giardia duodenalis is one of the most prevalent intestinal parasites of human. It also infects a wide range of mammals. Two genotype of G.duodenalis (A and B) were commonly reported among humans with different frequency of distribution in different geographical locations. This work was conducted to discriminate genotypes of Giardia duodenalis human isolates in Isfahan city using PCR- RFLP. This is the first molecular study on human isolates of G.duodenalis in the area.Methods:Samples were collected from different health centers of Isfahan city during June 2011 and February 2012. From 175 Giardia positive stool samples 67 specimens were selected randomly. Cysts of Giardia positive samples were concentrated by flotation sucrose. Extraction of genomic DNA from trophozoite and cysts was performed using QIAamp Stool Mini kit with a modified protocol. PCR- RFLP method was used to amplify a fragment of 458bp at the glutamate dehydrogenase locus, and restriction enzymes BspLI and RsaI differentiated human genotypes A and B and their subgroups.Results:PCR – RFLP assay of 67 isolates showed 40(59.7%) isolates as Genotype A group II, 23(34.32%) samples as Genotype B Group III and two (2.98%) sample as Genotype B group IV. Mixed genotype of (AII and B) was detected only in two isolates (2.98%).Conclusions:PCR – RFLP assay targeting gdh locus is a sensitive tool and discriminates genotypes, sub genotypes and mixed type of G.duodenalis. Results of our study suggest both anthroponotic and zoonotic origins for the infections respectively.

Molecular identification of Giardia duodenalis in Ecuador by polymerase chain reaction-restriction fragment length polymorphism

“…A variety of molecular tools including polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP), isoenzymes analysis and sequence analysis of several genes have provided genotypic information about Giardia duodenalis (Read et al 2004, Cordón et al 2008, Tungtrongchitr et al 2010, Feng & Xiao 2011, Pestehchian et al 2012). Based on these analyses, isolates of G. duodenalis have been classified into seven genetic assemblages, A-G (Thompson et al 2000, Read et al 2004).…”

“…PCR-RFLP has been used previously to target the glutamate dehydrogenase ( gdh ) locus . This gene has been shown to be a reliable, easy and cost-effective method to identify G. duodenalis isolates directly from faeces (Thompson et al 2000, Cordón et al 2008, Fallah et al 2008, Tungtrongchitr et al 2010, Pestehchian et al 2012). Stud-ies on the relationship between parasite genotype and symptoms have been mixed, though a genotypic analysis using PCR-RFLP amplification of the gdh gene did find a strong genetic link with clinical symptomatology in 18 Dutch patients (Homan & Mank 2001).…”

Molecular epidemiology and multilocus genotyping of Giardia duodenalis in individuals attending major public hospitals in Shiraz, southwestern Iran: A public health concern

“…Nevertheless, there is a dearth of molecular epidemiological studies utilizing the MLG system in Iran. The available molecular data on giardiasis in Iran are limited to a few studies that utilized PCR-RFLP and nested-PCR techniques to examine specific loci such as gdh , tpi , and/or bg ( Pestehchian et al, 2012 ; Tappeh et al, 2014 ). Previous investigations of human giardiasis in Iran, specifically in Shiraz, primarily relied on microscopy, with molecular methods being used only for single-locus analyses ( Bahrami et al, 2017 ; Kasaei et al, 2018 ; Mahmoudi et al, 2020 ; Rayani et al, 2014 ).…”

Comparison and evaluation of four methods for extracting DNA from Giardia duodenalis cysts for PCR targeting the tpi gene

“…Pestechian et al (2014), reported the successful identification of genotypes of G. duodenalis in Isfahan, Iran by using the glass beads and freeze-thaw cycles with the QIA gene kit (QIA gene, Germany). Furthermore, Babaei et al (2011) and Pestehchian et al (2012), used the glass beads, freeze-thaw cycles and QIA gene kit (QIA gene, Germany) to remove the PCR inhibitor. In developing countries, usually for DNA extraction, Phenol-Chloroform Isoamyl Alchol (PCI) is used that this methods is not suitable for DNA extraction from blood smear or small helminthes, and also PCI is toxic as well (Blagg et al 1955;Planelles et al 1996).…”

“…Additionally, almost 280 million new cases of the disease were reported without any clinical sign of giardiasis (Lane and Lloyd 2002;WHO 1996). High prevalence of giardiasis can be seen in tropical and developing countries such as Isfahan, Iran (Pestehchian et al 2012), where infections are associated with poor personal hygiene, sanitary conditions, poor water quality control and overcrowding. Clinical manifestations of giardiasis vary from asymptomatic infection (60-70 % of the cases) to acute or chronic diarrhea with malabsorption (WHO 1996).…”