In vitro Culture and Multilocus Genotyping of Giardia duodenalis Trophozoites obtained from Human Fecal Samples in Southwest of Iran

Abstract

Introduction: The enteric protozoa, Giardia duodenalis (G. duodenalis), consists of eight distinct assemblages (A-H) with identical morphological characteristics and a direct life cycle. Successful axenic cultivation of this parasite is an important preliminary step for biological, drug resistance and phylogenetic studies. Moreover, G. duodenalis exhibits great genetic and biotypic diversity.Aim The current study aimed to evaluate in vitro culture and multilocus genotyping of G. duodenalis trophozoites obtained from human fecal samples in southwest Iran.Methods: Thirty human fecal specimens containing G. duodenalis cysts were collected from Ahvaz city (southwest of Iran). The purification of cysts was carried out by the sucrose flotation technique. The cysts were inoculated in a modified TYI-S-33 medium and was daily monitored for the development and viability of trophozoites. After extracting DNA, gdh, bg and tpi genes were evaluated using molecular techniques (the semi-nested PCR for gdh gene and the nested PCR for tpi and bg genes). Eventually, the amplified fragments were sequenced and then, the phylogenetic tree was drawn.Results: Of 30, the trophozoites were encysted from five samples. All three genes were detected in two cases of five samples using molecular techniques. The multilocus phylogenetic analysis demonstrated that all the two samples belonged to assemblage A and sub-assemblage ARConclusion: Our findings indicated the presence of different numbers of trophozoites with variable development and survival rates in modified TYI-S-33 medium. Furthermore, the multilocus genotyping showed that these trophozoites belonged to assemblage A and sub-assemblage AR