Hidden Diversity within Common Protozoan Parasites as Revealed by a Novel Genomotyping Scheme

Abstract

Giardia duodenalis (syn. G. lamblia, G. intestinalis) is the causative agent of giardiasis, one of the most common diarrheal infections in humans. Evolutionary relationships among G. duodenalis genotypes (or subtypes) of assemblage B, one of two genetic assemblages causing the majority of human infections, remain unclear due to poor phylogenetic resolution of current typing methods. Here, we devised a methodology to identify new markers for a streamlined multi-locus sequence typing (MLST) scheme based on comparisons of all core genes against the phylogeny of whole-genome sequences (WGS). Our analysis identified three markers with comparable resolution to WGS data. Using newly designed PCR primers for our novel MLST loci, we typed an additional 68 strains of assemblage B. Analyses of these strains and previously determined genome sequences showed that genomes of this assemblage can be assigned to 16 clonal complexes, each with unique gene content that is apparently tuned to differential virulence and ecology. Obtaining new genomes of Giardia spp. and other eukaryotic microbial pathogens remains challenging due to difficulties in culturing the parasites in the laboratory. Hence, the methods described here are expected to be widely applicable to other pathogens of interest and advance understanding of their ecology and evolution. IMPORTANCE Giardia duodenalis assemblage B is a major waterborne pathogen and the most commonly identified genotype causing human giardiasis worldwide. The lack of morphological characters for classification requires the use of molecular techniques for strain differentiation, however, the absence of scalable and affordable NGS-based typing methods has prevented meaningful advancements in high resolution molecular typing for further understanding of the evolution and epidemiology of Assemblage B. Prior studies have reported high sequence diversity but low phylogenetic resolution at standard loci in Assemblage B, highlighting the necessity of identifying new markers for accurate and robust molecular typing. Data from comparative analyses of available genomes in this study identified three loci that together form a novel high-resolution typing scheme with high concordance to whole-genome-based phylogenomics and which should aid in future public health endeavors related to this parasite. In addition, data from newly characterized strains suggest evidence of biogeographic and ecologic endemism.

Multilocus Genotyping of Giardia duodenalis in Mostly Asymptomatic Indigenous People from the Tapirapé Tribe, Brazilian Amazon

“…Finally, the typing scheme used in the present study may lack enough phylogenetic resolution to correctly differentiate between sub-assemblage BIII and BIV sequences. This issue has been highlighted in recent molecular studies for assemblage B and assemblage A sequences [46,47]. This important point emphasises the need of identifying new markers and of developing novel methods for MLST purposes.…”

Cryptosporidium and Giardia in Livestock in Tigray, Northern Ethiopia and Associated Risk Factors for Infection: A Cross-Sectional Study

“…Of the 81 Giardia-positive calf samples from the four districts, 31 were selected for molecular characterisation, of which 18 provided good sequence results; 14 were Assemblage E, three were Assemblage A, and one was Assemblage B. Subtyping the Assemblage A isolates revealed that two were AI and one could not be subtyped. Subtyping of the Assemblage B using multilocus sequence typing showed a close relationship with Algerian samples (37). Out of 11 Giardia-positive calf samples from urban farms, molecular characterisation was successful for six and all were Assemblage E.…”

Uncovering the genetic diversity of Giardia intestinalis in isolates from outbreaks in New Zealand

“…The subtyping in this study was carried out at only the gdh locus. This presents a potential limitation since other studies have shown that sequencing typing at different loci can result in the assignation of multiple subtypes [ 2 , 35 ] and is why more studies are utilising multilocus sequence typing (MLST) [ 36 ]. However, this study sought to compared data from NGS to samples that had previously been characterised by Sanger sequencing at the gdh locus.…”