Incidence of Giardia lamblia subspecies by PCR-RFLP in stool specimens of hospitalized children at Urmia Mutahhari Hospital, West Azerbaijan Province, Iran

Hazrati Tappeh, KH and Manafi, G and Asgharzadeh, M and Manafi, F (2014) Incidence of Giardia lamblia subspecies by PCR-RFLP in stool specimens of hospitalized children at Urmia Mutahhari Hospital, West Azerbaijan Province, Iran. Iranian Journal of Parasitology, 9 (4). pp. 541-547.

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Abstract

Giardia lamblia is one of the most prevalent intestinal flagellate protozoa that infects a wide range of vertebrate hosts causing severe intestinal disorder in children. This study was performed to determine subspecies of G.lamblia by the PCRRFLP method, targeting the glutamate dehydrogenase(gdh)locus, in hospitalized children at Urmia Mutahhari Hospital, West Azerbaijan Province,Iran and determining the infection transformational storages in this area. Methods: Overall,720 stool specimens were collected from the hospitalized children, 34 samples were positive and Giardia cysts were detected under the microscope. Cysts were partially purified by the sucrose density gradient method and then washed with sterile distilled water to remove effectively the PCR inhibitors. Genomic DNA of G. lamblia isolates was extracted by freeze-thaw cycles followed by phenol/ chloroform/ isoamyl alcohol method. The single step PCR-RFLP assay was used to differentiate the assemblages between A and B, which were found in humans. In this method, 432 bp expected size was amplified, and then for detection of subspecies, specific restriction RsaI and BspLI enzymes were used. Results: Totally 34 samples were positive in terms of Giardia cyst out of 720 examined samples microscopically, so the parasite spread rate is reported 4.72%. Analysis PCR-RFLP on these samples revealed that 28 samples (93.3%) have the genotype BIII and 2 samples (6.7%) belong to the subgroup BIV. Conclusion: PCR-RFLP is a proper analytical method for determining the genotype among parasite types, using the glutamate dehydrogenizes zone's genes. Based on the results, an animal origin of infection cycle is suggested.

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