Sensitive universal detection of blood parasites by selective pathogen-DNA enrichment and deep amplicon sequencing

Abstract

Background Targeted amplicon deep sequencing (TADS) has enabled characterization of diverse bacterial communities, yet the application of TADS to communities of parasites has been relatively slow to advance. The greatest obstacle to this has been the genetic diversity of parasitic agents, which include helminths, protozoa, arthropods, and some acanthocephalans. Meanwhile, universal amplification of conserved loci from all parasites without amplifying host DNA has proven challenging. Pan-eukaryotic PCRs preferentially amplify the more abundant host DNA, obscuring parasite-derived reads following TADS. Flaherty et al. (2018) described a pan-parasitic TADS method involving amplification of eukaryotic 18S rDNA regions possessing restriction sites only in vertebrates. Using this method, host DNA in total DNA extracts could be selectively digested prior to PCR using restriction enzymes, thereby increasing the number of parasite-derived reads obtained following NGS. This approach showed promise though was only as sensitive as conventional PCR. Results Here, we expand on this work by designing a second set of pan-eukaryotic primers flanking the priming sites already described, enabling nested PCR amplification of the established 18S rDNA target. This nested approach facilitated introduction of a second restriction digestion between the first and second PCR, reducing the proportional mass of amplifiable host-derived DNA while increasing the number of PCR amplification cycles. We applied this method to blood specimens containing Babesia, Plasmodium, various kinetoplastids, and filarial nematodes and confirmed its limit of detection (LOD) to be approximately 10-fold lower than previously described, falling within the range of most qPCR methods. Conclusions The assay detects and differentiates the major malaria parasites of humans, along with several other clinically important blood parasites. This represents an important step towards a TADS-based universal parasite diagnostic (UPDx) test with a sufficient LOD for routine applications.

Simulation study and comparative evaluation of viral contiguous sequence identification tools

“…The tools used in the study shown in Table 1 were tested on their performance to identify viral elements from assembled contigs in the simulations. The tools used in this study to identify viral contigs were Vibrant (Version 1.2.0), VirSorter, VirSorter2, VirFinder, DeepVirFinder, MetaPhinder, Kraken 2, Phybrid, and a BLAST search using identified proteins from the Earth Virome Pipeline [ 15 , 16 , 19 , 28 ].…”

“…MetaPhinder, Phybrid, Phybrid Proteins, and Vibrant were run with default parameters [ 13 , 15 ]. Double-stranded DNA phages and single stranded DNA viruses were selected with the groups parameter of VirSorter2 as described by the authors [ 16 ]. Kraken 2 was run with default parameters using the minikraken database from March 2020 [ 21 ].…”

“…Unlike prophage identification methods that use viral domain enrichment or presence/absence to calculate a score, a new method, called Vibrant, uses domain abundances in a neural network framework to classify contigs having more than 4 proteins [ 15 ]. VirSorter2 follows a similar methodology, using domain percentages, gene content features, and key homology genes in a tree-based machine learning framework to classify viral reads [ 16 ].…”

Priming for Life: Early Life Nutrition and the Microbiota-Gut-Brain Axis

“…Recently, sparse but viable microbiota have been identified in the fetal gut at mid-gestation in humans [ 75 ]. However, this clinical study has been recently heavily criticised by other researcher of the field [ 103 , 104 ] who claim that contamination of the samples during analysis is the most probable explanation for the bacteria found in the fetal gut. Researchers who analysed meconium (the first faecal sample of the new-born) found low amounts of viable bacteria present but confirmed that the main bacterial colonization of the gut happens at the time of birth [ 105 ].…”

Molecular diagnosis of infectious parasites in the post-COVID-19 era

“…NGS has been used for the detection of Leishmania in the bone marrow of visceral leishmaniasis,[ 77 ] accurate diagnosis of neurocysticercosis infection in patients with atypical clinical manifestations,[ 78 ] diarrheal infections,[ 74 ] and detection of blood-borne helminths and protozoa. [ 79 80 ]…”