A global genotyping survey of Strongyloides stercoralis and Strongyloides fuelleborni using deep amplicon sequencing

Abstract

Strongyloidiasis is a neglected tropical disease caused by the human infective nematodes Strongyloides stercoralis, Strongyloides fuelleborni fuelleborni and Strongyloides fuelleborni kellyi. Previous large-scale studies exploring the genetic diversity of this important genus have focused on Southeast Asia, with a small number of isolates from the USA, Switzerland, Australia and several African countries having been genotyped. Consequently, little is known about the global distribution of geographic sub-variants of these nematodes and the genetic diversity that exists within the genus Strongyloides generally. We extracted DNA from human, dog and primate feces containing Strongyloides, collected from several countries representing all inhabited continents. Using a genotyping assay adapted for deep amplicon sequencing on the Illumina MiSeq platform, we sequenced the hyper-variable I and hyper-variable IV regions of the Strongyloides 18S rRNA gene and a fragment of the mitochondrial cytochrome c oxidase subunit 1 (cox1) gene from these specimens. We report several novel findings including unique S. stercoralis and S. fuelleborni genotypes, and the first identifications of a previously unknown S. fuelleborni infecting humans within Australia. We expand on an existing Strongyloides genotyping scheme to accommodate S. fuelleborni and these novel genotypes. In doing so, we compare our data to all 18S and cox1 sequences of S. fuelleborni and S. stercoralis available in GenBank (to our knowledge), that overlap with the sequences generated using our approach. As this analysis represents more than 1,000 sequences collected from diverse hosts and locations, representing all inhabited continents, it allows a truly global understanding of the population genetic structure of the Strongyloides species infecting humans, non-human primates, and domestic dogs.

Machine learning-based analyses support the existence of species complexes for Strongyloides fuelleborni and Strongyloides stercoralis

“…In cases where the HVR-I, HVR-IV and/or cox1 sequences were available from the same individual worm, the complete or partial genotype of these worms was recorded (Supplementary File S1, Tabs B and E, column F) after the genotyping approach by Barratt et al (2019b) and Jaleta et al (2017). In instances where specimens were genotyped using polymerase chain reaction (PCR) products amplified directly from stool (Barratt et al, 2019b;Beknazarova et al, 2019), only specimens obtaining a single haplotype for each locus were included in this analysis. The rationale for this criterion was that for amplicons sequenced directly from stool where multiple haplotypes were detected, the underlying genotype of individual worms cannot be elucidated.…”

“…Haplotypes were identified in each specimen using a recently described genotyping system (Jaleta et al, 2017;Barratt et al, 2019b) with some modifications (Fig. 1).…”

“…This requirement considers the pertinent observations of Hasegawa et al (2009), regarding HVR-IV, indicating that 'its nucleotide arrangements are mostly species specific'. It also considers that Part B of HVR-I can differentiate lineages A and B of S. stercoralis (Nagayasu et al, 2017;Barratt et al, 2019b). Therefore, part B of HVR-IV was set as a supplementary data requirement to compensate for information lost in the event of a truncated cox1 sequence.…”

“…The classification was performed for S. stercoralis and S. fuelleborni separately using an epsilon value of 0.05 for Plucinski's naïve Bayes classifier (refer to: https://github.com/Joel-Barratt/ Eukaryotyping), and the resultant pairwise distance matrices (a pairwise matrix is the standard output of our ML method -Supplementary File S1, Tabs C and F) were clustered. Clustering was performed using the agglomerative nested approach in the 'agnes' R package, utilizing Ward's clustering method as described here (Barratt et al, 2019b). The 'ggtree' R package was then used to generate cluster dendrograms.…”

“…Unfortunately, this practice would result in a loss of meaningful information when analyzing Strongyloides 18S rDNA HVRs. Furthermore, Strongyloides genotyping surveys Barratt et al (2019b) and Jaleta et al (2017). This scheme was expanded here to include haplotype XV of 18S HVR-I (GenBank Accession: MT436714), identified in genomes sequenced from Japanese humans by Kikuchi et al (2016).…”