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. Video Abstract.

Keywords: Amplicon sequencing; Blood microbiota; Molecular diagnosis; Molecular parasitology; Parasite biodiversity; Parasite detection.

Fig. 1

Graphical representation of our modified UPDx assay involving nested PCR amplification and two restriction digestion steps and its comparison to earlier UPDx assays. a Conventional PCR with universal primers amplifies primarily host DNA and yields sequencing reads almost entirely derived from the host. b Selective amplification of non-host eukaryote DNA via host-specific restriction enzyme digestion prior to PCR alters the ratio of amplifiable host to parasite DNA, increasing the relative mass of parasite-derived amplicon post-PCR and improving the sensitivity of parasite detection via NGS. c Modification of the assay described in b to a nested approach, facilitating an additional restriction enzyme digestion and additional amplification cycles, reduces the number of host-derived reads and enhances detection of parasite reads via NGS

Fig. 2

UPDx with double digestion significantly reduces the number of human-derived reads recovered following TADS. Restriction enzyme digestion yields a marked reduction in the percentage of human-derived 18S rDNA reads in parasite-infected blood samples following PCR and TADS. Human-derived read reduction is most pronounced in samples undergoing both restriction Digest 1 and Digest 2, as evidenced by the percentage of human-derived reads in most blood samples containing apixomplexan (a) and kinetoplastid (b) parasites. However, no visible reduction in human reads is observed in human blood displaying low parasitemia microfilarial infections (c, 2-way ANOVA with Tukey’s multiple comparisons posttest, **** p < 0.0001, *** p < 0.001, ** p < 0.01, n = 3, mean ± SD)

Fig. 3

UPDx with double digestion increases the number of parasite-derived reads recovered following TADS. Restriction enzyme digestion yields a substantial increase in the percentage of parasite-derived 18S rDNA reads from parasite-infected whole blood samples following PCR and TADS. The increase in parasite reads is most pronounced in samples undergoing both restriction Digest 1 and Digest 2, as evidenced by the percentage of parasite-derived reads recovered from blood samples containing apixomplexans (a), kinetoplastids (b), and microfilariae (c), (statistical analysis involved a 2-way ANOVA with Tukey’s multiple comparisons posttest, **** p < 0.0001, *** p < 0.001, ** p < 0.01, n = 3, mean ± SD)

Fig. 4

UPDx has a limit of detection similar to standard qPCR. a Serial dilutions of P. falciparum 3D7 parasites in whole human blood were processed using the original universal parasite detection method (grey bars), the nested method with DNA Digest 1 only (light-colored bars), the nested method with DNA Digest 2 only (medium-colored bars), or the nested method with both DNA Digest 1 and DNA Digest 2 (dark bars). Statistical significance of differences between conditions was assessed using a 2-way ANOVA with Tukey’s multiple comparisons posttest, p < 0.0001, n = 3, mean ± SD. b Nested PCR with both restriction Digest 1 and Digest 2 detected P. falciparum at a limit of 0.58 parasites/μL in 2/3 samples (each point is the average of 2 or 3 replicates, error bars are ± 1 SD, circles = parasites were detected in 3/3 replicates, squares = parasites were detected in 2/3 replicates, and triangles = parasites were detected in fewer than 2 replicates). c Analysis of the same samples by qPCR demonstrated that positive amplification curves were observed between 0.58 and 5.8 parasites/μL (each point represents the average of three replicates, error bars are ± SD, diamonds are positive controls; Pos. 1 and Pos. 2, circles = detection of parasites in 3/3 replicates, squares = detection of parasites in 2/3 replicates, and triangles = detection of parasites in 0/3 replicates). Percentage total reads refers to the percentage of all reads generated that were used to construct haplotypes identical to P. falciparum 18S sequences with the GenBank Accessions XR_002966654.1 and XR_002273081.2

Fig. 5

The UPDx amplicon differentiates several taxa of clinically important parasites. Clustering of sequences generated for a range of parasites (Table 2) demonstrates that the UPDx amplicon can differentiate some taxa to the species level, but not all. This segment of the 18S rDNA gene differentiates the most important Plasmodium species that infect humans but does not differentiate most Leishmania species that infect humans. It does not differentiate subspecies of T. brucei from some trypanosomes of veterinary importance, such as T. evansi and T. equiperdum, but it clearly differentiates T. cruzi. Filarial nematodes of the family Onchocercidae are differentiated beyond the family level, but not to the genus level. Babesia species commonly infecting humans are divided into three sequence types based on the haplotypes detected: one for Babesia microti, another for Babesia duncani, and a third type that includes Babesia divergens and the B. divergens-like MO1 type. Sequences generated in this study are shaded blue and include the haplotypes detected in clinical specimens 1 to 18 listed in Table 2