Sn-HDP2 cPCR and Sn-HDP2 qPCR amplified T. saginata and T. solium DNA with an analytical sensitivity of 40 fg and 400 fg, respectively, which was maintained in both protocols. In the case of T. saginata DNA in particular, sensitivity was comparable to that reported for other molecular markers [19,20,21].
Sn-HDP2 cPCR had previously been successfully used for the diagnosis of neurocysticercosis based on cerebrospinal fluid samples from infected Mexican patients [11]. At that time, a species-specific amplification protocol was not needed, as T. solium larva is the only taeniid that can invade the human central nervous system. However, in the case of taeniasis, which is produced by both taeniids, Sn-HDP2 cPCR yielded an amplicon with an identical size for both T. saginata and T. solium DNA [9, 10]; sequencing of the amplification product was essential for the specific identification of the taeniid [11, 12]. A new diagnostic amplification protocol was set up to circumvent the sequencing step by taking advantage of the differences in HDP2 in nucleotides between the two species. The novel Sn-HDP2 qPCR showed a distinct melting temperature (Tm) according to the Taenia species analyzed (Tm of 83.30 °C for T. saginata and 86.00 °C for T. solium), and the difference in Tm had already been detected in the first amplification (Fig. 1b). Furthermore, for proglottids, the first reaction usually yielded conclusive diagnostic results in both amplification protocols, as was the case in fecal samples when the parasite material was sufficient. In previous years, other HDP2 cPCRs protocols were applied for identification of T. saginata in fecal samples artificially spiked with known numbers of T. saginata eggs [22]. Moreover, as indicated above, amplifications of several molecular targets such as mitochondrial genes (cox1, nad5, nad1, 12S), and ribosomal ITS1 region and 28S and 5.8S genes [19, 21, 23] were described in various amplification protocols (cPCR, qPCR, LAMP) using mono- or multiplex systems, with very good results for the identification of Taenia spp. [19,20,21, 23, 24]. In comparison with Triplex Taq-Man (T3qPCR) [21], Sn-HDP2 qPCR did not need a labeled probe, although it did not include an internal PCR control and was not checked with T. asiatica DNA.
In order to confirm the applicability of Sn-HDP2 cPCR and Sn-HDP2 qPCR in the diagnosis of infected human samples, proglottids and fecal samples from taeniasis patients were examined using both PCR assays. The results obtained are summarized in Table 1. With proglottids, the two PCRs were able to diagnose taeniasis in the first amplification as follows: T. saginata in patients from Europe (n = 9), South America (n = 3), Africa (n = 1) and from undetermined regions (n = 4), with ages ranging from 15 to 84 years; T. solium in a 43 year-old Latin American individual. In the case of fecal samples, the PCRs yielded positive results, and T. saginata was identified, even in stools negative for taeniid eggs (Case 3 and Case 10, Table 1). The identification was confirmed by agarose gel electrophoresis of the cPCR amplification product (Fig. 1a) followed by amplicon sequencing. Molecular approaches have already been used in human samples for the diagnosis of taeniasis in endemic regions, with excellent specificity and sensitivity [20, 21, 23]. The molecular identification of a T. solium tapeworm carrier in the present study is noteworthy, given the epidemiological consequences of the transmission of cysticercosis [25] and the possible need for surveillance measures in countries where the parasite has been eliminated. Today, migration from taeniid-endemic regions could extend transmission [26]. In addition, the frequent diagnosis of T. saginata in the taeniasis patients studied, in accordance with recent data from Europe [27], suggests the need for better reporting of taeniasis/bovine cysticercosis if the disease is to be better controlled [25].
In-depth analysis and characterization of the properties of the HDP2 homolog of T. solium were performed through cloning of the DNA sequence by screening a T. solium gDNA library with the T. saginata HDP2 homolog [14] and through direct cPCR-cloning using T. solium gDNA and specific ribosomal DNA primers [28]. A positive recombinant phage (phage clone #1, c.14–15 kb) and a specific genomic amplicon (c.3.0 kb) were obtained (Fig. 2). To determine which region of the phage fragment corresponded to the HDP2 sequence, phage clone #1 DNA was digested by the SalI restriction enzyme. The digestion yielded four fragments (Fig. 2a: a.1), two of which were hybridized with the T. saginata HDP2 sequence probe, namely, subclones #1.1 and #1.2 (Fig. 2a: a.2). T. solium phage subclones #1.1 and #1.2 were 2081 bp and 2799 bp long, respectively (Fig. 2b), and showed high similarities in their DNA sequences (GenBank: subclone #1.1: KY750552; subclone #1.2: KY750553). They also exhibited a high similarity with the DNA sequence of the T. solium ribosomal amplicon (3122 bp) cloned by cPCR (Fig. 2c), with few nucleotide substitutions and small deletions (GenBank: amplicon: KY750551). The DNA similarities discovered and the BLAST analysis data confirmed that T. solium HDP2 (3.12 kb) was a ribosomal gene, as was previously suggested for other taeniid homologous HDP2 molecules [13], when significant similarities between T. saginata HDP2 and the repetitive ribosomal sequences Taiwan Taenia pTTr 3.1, T. saginata pTSgr 3.1 and pTSgr 2.4, were demonstrated [29]. The T. solium fragment (3.12 kb) and subclones #1.1 and #1.2, in comparison with the T. saginata HDP2, lacked the first 900 nt at the 5′ end, as described elsewhere [9], and showed nucleotide substitutions and small deletions. Specifically, the largest deletion was located in T. solium HDP2 clones (from 590 to 620 nt).
The T. solium HDP2 structure within the ribosomal tandem repeats was further studied by restriction enzyme mapping based on the characteristics of both phage subclones and amplicon sequences. Thus, the rough size of cloned phage 1 was 14–15 kb. Together with the characteristics of standard rRNA genes determined by BLAST analysis, this phage included two partial copies of the ribosomal amplicon HDP2 (3.12 kb) at its 5′ and 3′ ends and the phage subclones #1.2 (2.79 kb) and #1.1 (2.08 kb), which were located in two different and contiguous non-transcribed sequence/external transcribed repeat (NTS/ETS) regions (Fig. 2b). Since information on the structural organization of rDNA sequences in Taenia species is poor, these data make it possible to conclude that the phage 1 sequence corresponded to a T. solium ribosomal DNA repeat plus an NTS/ETS region (Fig. 2b), with the HDP2 fragment being an NTS/ETS unit, according to the description made for the sequence and structure of the Caenorhabditis elegans rDNA repeat [30]. Therefore, the genomic variations detected between the characteristics of HDP2 in both taeniids could explain the following: (i) the different hybridization profiles found when the T. saginata and T. solium gDNAs were hybridized with the labeled T. saginata HDP2 DNA sequence [8, 9]; (ii) the non-amplification of T. solium DNA by the R1F1-HDP2-PCR [9]; and (iii) the differential sensitivity of taeniids determined by the amplification protocols described in the present paper.
Finally, the repetitive nature of HDP2 sequences (ribosomal NTS/ETS units) and the differences in nucleotide composition between Taenia species would explain the diagnostic properties of the molecule as a target in high-sensitivity, species-specific amplification protocols.
