Molecular screening of Entamoeba spp. (E. histolytica, E. dispar, E. coli, and E. hartmanni) and Giardia intestinalis using PCR and sequencing

M Tokoro; T Mizuno; X Bi; S A Lacante; C Jiang; R N Makunja

doi:10.1016/j.mex.2023.102361

Molecular screening of Entamoeba spp. (E. histolytica, E. dispar, E. coli, and E. hartmanni) and Giardia intestinalis using PCR and sequencing

MethodsX. 2023 Sep 9:11:102361. doi: 10.1016/j.mex.2023.102361. eCollection 2023 Dec.

Authors

M Tokoro¹, T Mizuno¹, X Bi¹, S A Lacante¹, C Jiang¹, R N Makunja¹

Affiliation

¹ Department of Global Infectious Diseases, Graduate School of Medical Sciences, Kanazawa University, Japan.

Abstract

A wide range of intestinal protozoan parasites inhabit the human gut. To establish a more comprehensive molecular screening, we designed PCR-sequencing screening methods for Entamoeba spp., including commensal species, and Giardia intestinalis, and performed such methods using 174 stool samples collected from Kenyan children. The prevalences of the target species were as follows: E. histolytica (2/174, 1.1%), E. dispar (20/174, 11.5%), E. coli (107/174, 61.5%), E. hartmanni (77/174, 44.3%), and G. intestinalis (54/174, 31.0%). PCR amplicons specific to G. intestinalis was differentiated to assemblages A (8/174, 4.6%) and B (46/174, 26.4%). PCR specificity for Entamoeba spp. was quite high, except for some cross-reactions between E. hartmanni detection primers and G. intestinalis, although the false-positive amplicons were discernible by the band size. The 18S rRNA PCR primers that was designed by Monis et al. in 1999 for G. intestinalis, have specificity issue, therefore amplicon sequencing was essential not only to determine assemblage classifications but also to confirm the positive results by eliminating potential non-specific reactions. The detection sensitivity of both the Entamoeba universal PCR and the G. intestinalis PCR was more than 100 copies of the target loci, which is sufficient for detecting a single trophozoite or cyst of both species.

Keywords: Entamoeba; Giardia; Gut protozoan flora; Molecular epidemiology; PCR-sequencing; PCR-sequencing screening methods for Entameoba spp. and Giardia intestinalis.