Species Detection within the Echinococcus granulosus sensu lato Complex by Novel Probe-Based Real-Time PCRs

Infections with eggs of Echinococcus granulosus sensu lato (s.l.) can cause cystic echinococcosis in intermediate host animals and humans. Upon ingestion of viable eggs, oncospheres hatch from the eggs and subsequently develop into fluid-filled larval cysts, most frequently in the liver or the lungs. The slowly growing cysts progressively interfere with organ function. The risk of infection is determined by the host range of the parasite, its pathogenicity and other epidemiologically relevant parameters, which differ significantly among the five species within the E. granulosus s.l. complex. It is therefore essential to diagnose the correct species within E. granulosus s.l. to help understand specific disease epidemiology and to facilitate effective implementation of control measures. For this purpose, simple, fast and cost-effective typing techniques are needed. We developed quantitative real-time polymerase chain reactions (qPCRs) to target polymorphic regions in the mitochondrial genome of E. granulosus s.l. In a single-step typing approach, we distinguished E. granulosus s.l. members in four epidemiologically relevant subgroups. These were E. granulosus sensu stricto, E. equinus, E. ortleppi and the E. canadensis cluster. The technique also allowed identification and differentiation of these species from other Echinococcus or Taenia taxa for samples isolated from cysts or faeces.

Previously, E. granulosus was considered to be a single species, subdivided into "strains" and genotypes (G1 to G8, G10 and the "lion strain") [4], most of them associated with specific  The mitochondrial genomes of species and intraspecific genotypes from the E. granulosus s. l. complex were screened for polymorphic regions that could be used as targets for specific PCR primer pairs and corresponding probes. Identified regions and verified primers and probes for all four species, E. granulosus s.s. (G1-G3), E. equinus (G4), E. ortleppi (G5), and E. canadensis (G6-10), are shown in Table 1 and Figure 1. We used reference samples of known tapeworm species and genotypes to test the newly identified primer pairs and probes. To ensure the identity of the reference material, tapeworm-specific DNA was amplified by conventional PCR and the resulting amplicons were then analysed by Sanger sequencing. The conventional PCR and sequencing results confirmed the identity of the DNA reference samples in all cases (Figure 2, sequencing results not shown).
Pathogens 2020, 9, x FOR PEER REVIEW 5 of 17 We used reference samples of known tapeworm species and genotypes to test the newly identified primer pairs and probes. To ensure the identity of the reference material, tapewormspecific DNA was amplified by conventional PCR and the resulting amplicons were then analysed by Sanger sequencing. The conventional PCR and sequencing results confirmed the identity of the DNA reference samples in all cases (Figure 2, sequencing results not shown).

Figure 2.
Reference DNA amplification of specific polymerase chain reaction (PCR) products of Echinococcus granulosus sensu lato. PCR products from reference DNAs obtained from Echinococcus granulosus sensu lato species amplified by specific PCR (Cest4, Cest5 (Trachsel et al., 2007) The bioinformatically selected primer pairs were then examined by SYBR ® green qPCR to test if they specifically amplified sequences from the genome of the targeted species. We found that the selected primer pairs for E. granulosus s.s. (G1-G3) and E. equinus (G4) specifically amplified their respective reference DNA and generated amplicons that were distinguishable by their melting peak temperatures from amplicons generated using samples with DNA from closely related Echinococcus spp. and Taenia spp. (Figure 3A,B). Furthermore, with the E. granulosus s.s. (G1-G3) primers, the melting temperature of 81 °C was distinguishable from the melting temperatures observed in amplicons generated with the same primer pair in reference samples of E. equinus (G4), E. ortleppi (G5) and E. canadensis (G6-10) at 79-80 °C ( Figure 3A). The selected primer pair designed for E. equinus (G4) generated a specific PCR product and did not cross-react with samples of the other E. granulosus s.l. species or E. multilocularis ( Figure 3B).
By contrast, primers designed to amplify sequences of E. ortleppi (G5), targeting a polymorphic sequence in Nad5, also yielded non-specific PCR products in the E. canadensis (G6-10) reference samples and vice versa, i.e., primers designed to generate E. canadensis (G6-10) amplicons from Cox3 also amplified E. ortleppi (G5) reference DNA. Whilst the selected primer pairs amplified PCR products in E. granulosus DNA samples, the resulting melting curves of all of the products were hardly distinguishable at the observed temperatures of 78 to 78.5 °C for E. canadensis (G6-10) and 79 °C for E. ortleppi (G5) ( Figure 3C,D). Nevertheless, the primers were selected for further testing in the TaqMan qPCR with their corresponding probes.
Additional primer and probe combinations that were initially selected in silico, but did not satisfactorily amplify sequences from the targeted species during SYBR ® green, or in further TaqMan ® qPCR experiments, are shown in the supplementary Table S1.
In summary, amplicon generating primer pairs were identified for all targeted species (Table 1). Conventional SYBR ® green qPCR was sufficient to differentiate E. granulosus s. s. (G1-G3) and E. equinus (G4) using primers targeting polymorphic regions in Cox1 (Table 1) based on the generated amplicon alone. Using the same method, primers targeting regions in Nad5 in E. ortleppi (G5) and in Cox3 in E. canadensis (G6-8, G10) amplified successfully, but cross-reacted and required additional testing to separate the respective genotypes. The bioinformatically selected primer pairs were then examined by SYBR ® green qPCR to test if they specifically amplified sequences from the genome of the targeted species. We found that the selected primer pairs for E. granulosus s.s. (G1-G3) and E. equinus (G4) specifically amplified their respective reference DNA and generated amplicons that were distinguishable by their melting peak temperatures from amplicons generated using samples with DNA from closely related Echinococcus spp. and Taenia spp. (Figure 3A,B). Furthermore, with the E. granulosus s.s. (G1-G3) primers, the melting temperature of 81 • C was distinguishable from the melting temperatures observed in amplicons generated with the same primer pair in reference samples of E. equinus (G4), E. ortleppi (G5) and E. canadensis (G6-10) at 79-80 • C ( Figure 3A). The selected primer pair designed for E. equinus (G4) generated a specific PCR product and did not cross-react with samples of the other E. granulosus s.l. species or E. multilocularis ( Figure 3B).
By contrast, primers designed to amplify sequences of E. ortleppi (G5), targeting a polymorphic sequence in Nad5, also yielded non-specific PCR products in the E. canadensis (G6-10) reference samples and vice versa, i.e., primers designed to generate E. canadensis (G6-10) amplicons from Cox3 also amplified E. ortleppi (G5) reference DNA. Whilst the selected primer pairs amplified PCR products in E. granulosus DNA samples, the resulting melting curves of all of the products were hardly distinguishable at the observed temperatures of 78 to 78.5 • C for E. canadensis (G6-10) and 79 • C for E. ortleppi (G5) ( Figure 3C,D). Nevertheless, the primers were selected for further testing in the TaqMan qPCR with their corresponding probes.
Additional primer and probe combinations that were initially selected in silico, but did not satisfactorily amplify sequences from the targeted species during SYBR ® green, or in further TaqMan ® qPCR experiments, are shown in the supplementary Table S1.
In summary, amplicon generating primer pairs were identified for all targeted species (Table 1). Conventional SYBR ® green qPCR was sufficient to differentiate E. granulosus s. s. (G1-G3) and E. equinus (G4) using primers targeting polymorphic regions in Cox1 (Table 1) based on the generated amplicon alone. Using the same method, primers targeting regions in Nad5 in E. ortleppi (G5) and in Cox3 in E. canadensis (G6-8, G10) amplified successfully, but cross-reacted and required additional testing to separate the respective genotypes.

Detection of Echinococcus Species by TaqMan ® Quantitative PCRs
To increase the specificity of Echinococcus spp. target DNA detection, the four selected primer pairs were combined with amplicon-specific TaqMan ® DNA probes (Table 1), and used to analyse serially diluted DNA reference samples ( Figure 4). TaqMan ® qPCR assays for E. granulosus s. s. (G1-G3) and E. equinus (G4) specifically identified target DNA in the corresponding reference samples. The selected probes for both subgroups did not bind DNA from the other E. granulosus s.l. species, from E. multilocularis spp. or from Taenia spp. (Table 2). Primers and probes for detecting E. granulosus s.s. (G1-G3) also amplified DNA from the Gx, strain sample, indicating that the assay is also applicable to other genotypes of E. granulosus s.s.

Detection of Echinococcus Species by TaqMan ® Quantitative PCRs
To increase the specificity of Echinococcus spp. target DNA detection, the four selected primer pairs were combined with amplicon-specific TaqMan ® DNA probes (Table 1), and used to analyse serially diluted DNA reference samples ( Figure 4). TaqMan ® qPCR assays for E. granulosus s.s. (G1-G3) and E. equinus (G4) specifically identified target DNA in the corresponding reference samples. The selected probes for both subgroups did not bind DNA from the other E. granulosus s.l. species, from E. multilocularis spp. or from Taenia spp. (Table 2). Primers and probes for detecting E. granulosus s.s. (G1-G3) also amplified DNA from the G x, strain sample, indicating that the assay is also applicable to other genotypes of E. granulosus s.s. The addition of TaqMan ® probes to qPCR reactions with selected G5_qPCR primer pairs for E. ortleppi (G5) also showed specific amplification of the DNA target region. No cross-reactions were recorded with DNA samples from the remaining Echinococcus spp. and Taenia spp. (Table 2). Thus, it appears that the addition of sequence-specific DNA probes ameliorated the cross-reactivity with the E. canadensis cluster observed in the qPCR system. The addition of TaqMan ® probes to qPCR reactions with selected G5_qPCR primer pairs for E. ortleppi (G5) also showed specific amplification of the DNA target region. No cross-reactions were recorded with DNA samples from the remaining Echinococcus spp. and Taenia spp. (Table 2). Thus, it appears that the addition of sequence-specific DNA probes ameliorated the cross-reactivity with the E. canadensis cluster observed in the qPCR system.
TaqMan ® qPCR with the G6_G10_qPCR primer set targeting the E. canadensis (G6-8, G10) cluster resulted in amplification of a probe-binding PCR product ( Figure 4C,D). However, this assay also cross-reacted with the E. ortleppi (G5) DNA sample and thus did not allow differentiation of these two species (Table 2). To distinguish E canadensis (G6-8, G10) from G5 samples, we combined the G5_qPCR with the G6_G10_qPCR primer pairs and probes in a TaqMan ® qPCR (G5_G10_qPCR) Pathogens 2020, 9, 791 8 of 17 duplex format. With this format, DNA obtained from E. canadensis (G6-8, G10) is amplified only by a single fluorescence curve, which is specific for the E. canadensis (G6-8, G10). However, when the duplex qPCR amplifies an E. ortleppi (G5) DNA sample, both, the E. ortleppi (G5)-specific probe and the probe specific for E. canadensis (G6-8, G10) bind to the target region in the E. ortleppi (G5) DNA amplifying two fluorescence curves, as the primer and probe for E. canadensis (G6-8, G10) also amplified and detected E. ortleppi (G5) ( Table 2). Therefore, the duplex TaqMan ® qPCR format of G5_G10_qPCR primer and probes allowed diagnostic differentiation of E. ortleppi (G5) from the E. canadensis (G6-8, G10) cluster (Table 2). To characterise the diagnostic TaqMan ® qPCR assays further, the analytical efficiency and the limit of detection (LoD) of the qPCR reactions for each subgroup were determined by testing DNA extracted from clinical samples as cloned PCR products (plasmid DNA) ( Table 3, Figures 4 and 5).    The analytical efficiency of TaqMan ® qPCR assays varied between 94% and 107%. The LoD of the assays varied between 0.6 and 1.4 copies/µL ( Table 3). The specificity in G1_3_qPCR, G4_qPCR and G5_qPCR was 100%. G6_10_qPCR cross-reacted with E. ortleppi (G5) samples (Table 2) and required a duplex assay design with G5_qPCR for diagnostic purposes.
In summary, sequence-specific DNA probe-based TaqMan ® qPCR assays were established that identified four species within E. granulosus s.l. in reference samples. These samples were further differentiated from other Echinococcus and Taenia species. (Table 2).

Detection of E. granulosus s.l. Species in Faecal Samples
The reference DNA samples used in this study for the development of qPCR assays that differentiate four Echinococcus species was derived from cyst wall material of metacestodes isolated from infected hosts. While cysts may serve as a sample matrix for the detection of E. granulosus s.l. species, it is diagnostically relevant that the assays also amplify target DNA extracted from other relevant matrices such as faecal matter. To test the performance of the TaqMan ® qPCR assays when used with faecal samples, fox faeces were spiked with serially diluted reference DNA to simulate the testing of faeces from definitive hosts infected with members of E. granulosus s.l. A known quantity of standardised, heterologous plasmid DNA with matching primers and probes was included as an internal control (IC) in the qPCR mixture [24] to control for PCR inhibition by faecal factors. Inclusion of the IC also allowed testing the assays in duplex (G1_3_qPCR or G4_qPCR in combination with IC-qPCR) and triplex (G5_G10_qPCR and IC-qPCR) TaqMan ® qPCR formats (see Table 1). All four assays targeting E. granulosus s.s. (G1-G3), E. equinus (G4), E. ortleppi (G5) and E. canadensis (G6-8, G10) respectively, amplified the specific tapeworm DNA region from each analysed faecal sample type (Table 4). Combining E. ortleppi, E. canadensis as well as IC primers and respective probes in a triplex qPCR differentiated E. ortleppi (G5) and E. canadensis (G6-8, G10) DNA in faecal samples (Table 4). Probe recognition of IC DNA and quantification showed that no inhibition of the amplification process had occurred in these faecal samples and that the selected primer probe mixtures performed reliably with samples prepared in a matrix of faecal matter under duplex or triplex qPCR conditions. The Cq values generated from the re-extracted tapeworm DNA from faeces were much higher than those from the non-faecal control samples i.e., DNA extracted from hydatid cyst material. This suggests that the quantities of re-extracted tapeworm DNA from faeces were much lower than from non-faecal control samples, possibly due to loss of DNA during the faecal DNA extraction process (Table 4).

Discussion
Cystic echinococcosis is a globally important parasitic zoonosis that requires well-informed prevention and control measures based on reliable and efficient diagnosis of the various causative agents [25,26].
By designing TaqMan ® -qPCR probes that directly identify polymorphic genome regions among the four most important species of the E. granulosus s.l. complex, we simplify and enhance current diagnostic procedures in multiple ways. The advantages include increased sensitivity, increased specificity, the ability to quantify sample DNA, reduced time-cost to achieve a diagnostic result and potentially reduced processing and equipment costs due to focusing on a single technology. Taken together, these methodological properties should significantly facilitate the process of establishing diagnostic capacities for the detection of E. granulosus s.l. in a field laboratory settings and also facilitate higher sample throughput for epidemiological studies [22,23].
We discriminate the individual CE agents diagnostically by targeting genetic variability of the mitochondrial genome. Using a bioinformatics approach, we first identified polymorphic regions in the genes Cox1, Cox3 and Nad5 (Figure 1), which have previously emerged as suitable targets for E. granulosus s.l. genotyping [8,27]. Selected primer-probe combinations for all but one species were specific, as the single-plex qPCR system for E. canadensis (G6-8, G10) cross-reacted with E. ortleppi (G5) samples ( Table 2). One obvious possible explanation is the close relationship among these species, resulting in a low degree of polymorphism between E. ortleppi and E. canadensis in the Cox3 region targeted by G6-10 primers and probes. However, the non-specific G6-10-TaqMan ® probe directed at Cox3 differed from its corresponding G5 sequence by four nucleotides (G > A, G > A, C > T, C > T), whereas the specific G5-Nad5-probe differed only by three nucleotides when compared to its corresponding G6-10 sequence (C > T, C > T, G > A) (Figure 1). Therefore, it appears that nucleotide polymorphism alone is not sufficient to determine probe specificity in this context and that other single stranded DNA-affinity mechanisms, such as steric nucleotide relations [28], interact to overcome the mismatching probe-sequence in the G6-10 qPCR system. Furthermore, to avoid cross reactivity between G5 and E. canadensis complex (G6-10), we tested eight additional primers or primer/probe combinations derived from polymorphic regions of four mitochondrial genes, which unfortunately failed to eliminate this cross-reaction (Table S1). Our observations on this cross-reactivity are in accord with phylogenetic studies on the genome level that demonstrated a close relationship between E. ortleppi and E. canadensis [4,10]. Born from necessity, we therefore developed a duplex qPCR format, which now enabled successful G5 and G6-10 diagnosis for both samples in a single, parallel step.
Whilst phylogenetic relationships may contribute to diagnostic cross-reactivity, testing of E. granulosus s.s. haplotypes, which do not precisely fit with sequences of G1 and G3, but clearly belong to that species (here named Gx), showed that our assay correctly classified a "non-conventional" echinococcal isolate (Figure 2). Phylogenetic studies of E. granulosus s.s. isolates found that a large proportion of the identified haplotypes were not homologous with G1 and G3 in the classical sense of the G-nomenclature, yet they clearly belonged to the E. granulosus s.s. cluster [4,10]. Here, we classify the G x -isolate that could not be assigned to the conventional G1-3 genotypes previously [4,10] as E. granulosus s.s., which indicates a broad applicability of the G1_3_qPCR assay in the context of the natural genetic diversity in this subgroup (for an alternative interpretation of genotypes G1 and G3 see [7]). The described qPCR system includes all species and genotypes of E. granulosus s.l. except E. felidis. However, this species, which is not known to be zoonotic, has a very fragmented distribution range in sub-Saharan Africa as it apparently depends on the presence of its principle definitive host, the lion. In any case, the genetic structure of African echinococcosis seems to be more complex than that found elsewhere and is in need of further research. This is illustrated by the presence of a highly divergent zoonotic genotype, G-Omo, in north-eastern Africa [27]. This genotype was only tentatively retained in E. granulosus s.s. pending further epidemiological information [27]. Interestingly, DNA of G-Omo did not react in the E. granulosus s.s. qPCR (as E. felidis, which belongs to the same species cluster), which supports a separate identity of this taxon. Likewise, E. canadensis may in future have to be split in two species, namely the wildlife-transmitted G8 and G10, and the largely domestically transmitted G6/7 genotype group [29]. This will not diminish the value of the qPCR, as both genotype clusters are allopatrically distributed and occur in very different epidemiological settings, with only a limited area of possible overlap in the Asian part of the Russian Federation [4].
The TaqMan ® -probe-based qPCR assays developed in this study show good efficiency, analytical specificity as well as diagnostic sensitivity when used with DNA obtained from cysts. Therefore, they are suitable for diagnosis of Echinococcus species in intermediate hosts, including humans and may be further validated in the field in different parts of the world under various conditions. Our findings in spiked faecal samples suggest that the duplex qPCR should also be applicable for the detection of E. granulosus s.l.-egg DNA in definitive hosts. Detection and genotyping of Echinococcus spp. DNA in faecal samples would be particularly useful when examining larger populations of living animals in the field for epidemiological studies. Whilst we characterised the LoD of the assays by using cloned amplicons (Table 3, Figures 4 and 5), their methodological and diagnostic sensitivity with faecal samples containing parasite eggs remains to be determined. A similar standardised, cloned-amplicon-based approach could be used to quantify target DNA in sample matrix of interest when using our assays. For instance, we observed an approx. 100-fold loss of specific DNA qPCR detection in spiked faecal samples (approx. 7 Cq-values) when compared to similar DNA quantities analysed in cyst material ( Table 4). Inclusion of the IC-plasmid internal control suggested that the DNA loss occurred at the extraction step, as the amplification in faecal and non-faecal samples was comparable (Table 3). This observation further showed that faecal inhibitors known to potentially interfere with PCR amplification [30], had no noticeable effect in our qPCR system. However, faecal samples from other animals could harbour another combination of the inhibitory components, which may negatively influence the amplification process of the assays [30].
Due to the different designs of the experiments conducted to establish the already published PCRs, the assays are difficult to compare with TaqMan qPCRs described here concerning their diagnostic performance. Other sample matrices with different concentrations of target DNA, different materials, e.g., master mixes and PCR cyclers were applied.
We anticipate that single-step genotyping techniques for Echinococcus granulosus s.l. complex diagnosis by DNA-probe-based qPCR will complement the available methods, improve case reporting on the genotype level, advance our knowledge on the epidemiology of these parasites and ultimately support effective control of CE.

Primer and DNA Probe Design for qPCRs
For a bioinformatic prediction of suitable regions in the mitochondrial genome of Echinococcus spp., relevant sequences were downloaded from the public NCBI database. Accession numbers are listed in Table 1 and sequence analysis is summarised in Figure 1. Mitochondrial genomes were aligned using the "MAFFT" algorithm embedded in the Geneious Prime ® (Version 2019.2.3) software suite. The program was used with default settings, whereby selection of the appropriate algorithm for the alignment was set to automatic. The Primer 3 algorithm, embedded in Geneious Prime ® , was then applied to all aligned sequences to predict potential targets for qPCR-based diagnosis. Thereafter, the in silico predicted qPCR targets were further analysed to find suitable polymorphic regions that might allow differentiating E. granulosus s.l. species of interest from others within the E. granulosus s.l. complex. To validate selected primer sequences in vitro, their performance was assessed in a SYBR ® Green qPCR assay as described below.

Reference DNA Samples and Faecal Spiking
To develop and validate the qPCR, we utilised a panel of reference DNA samples from members of the E. granulosus s.l. complex and related cestode species as outgroup controls. We included E. granulosus s.s. (G1-3), E. equinus (G4), E. ortleppi (G5) and genotypes of the E. canadensis cluster (G6, G7, G8 and G10) ( Table 5). To verify the identity of the taxa in the reference material, we genotyped all DNA samples by conventional PCR (Figure 2) as described in this manuscript and shipped the obtained amplicons to Eurofins Genomics (Ebersberg, Germany) for Sanger sequencing. To minimise the cross-contamination risk among reference samples, the DNA quantity was reduced by diluting samples 1:100. These dilutions were used as working samples in all experiments. To determine the limit of detection (LoD) and the analytical efficiency of the TaqMan ® qPCR assays, DNA samples were further diluted in ten-fold steps up to 1:10 −7 in Tris-EDTA buffer with 2 µg/µL Bovine Serum Albumin (BSA) (Carl Roth, Karlsruhe, Germany) [31]. As faecal matter is known to contain PCR inhibitory components [30], spiked faecal samples from a confirmed E. multilocularis-, and E. granulosus s.l.-free red fox (Vulpes vulpes) were prepared. To test whether E. granulosus s.l. species could be differentiated in DNA samples extracted from faecal samples, 200 mg of faeces was spiked with reference DNA that had been serially diluted in ten-fold steps up to 1:10 −4 . Each sample was prepared in duplicate. Spiked faecal samples were then processed using the ZR Faecal DNA MiniPrep TM kit (Zymo Research, Freiburg, Germany) to carry out DNA extraction as recommended in the kit manual.

Conventional PCR
To confirm the identity of the respective Echinococcus spp. in reference DNA samples, conventional PCR was performed as described elsewhere [20,30]. In brief, the following primers were used to amplify E. multilocularis DNA sequences (H15 F [5-CCATATTACAACAATATTCCTATC-3],