Taenia saginata: differential diagnosis of human taeniasis by polymerase chain reaction-restriction fragment length polymorphism assay

Speciation of Taenia in human stool is important because of their different clinical and epidemiological features. DNA analysis has recently become possible which overcomes the problems of differentiating human taeniid cestodes morphologically. In the present study, we evaluated PCR coupled to restriction fragment length polymorphism to differentiate Taenia solium from Taenia saginata eggs present in fecal samples from naturally infected patients. A different DraI-RFLP pattern: a two-band pattern (421 and 100 bp) for T. saginata and a three-band pattern (234, 188, and 99 bp) for T. solium was observed allowing the two species to be separated. The lower detection limit of the PCR-RFLP using a non-infected fecal sample prepared with a given number of T. saginata eggs was 34 eggs in 2 g stool sediment. The 521 bp mtDNA fragment was detected in 8 out of 12 Taenia sp. carriers (66.6%). Of these, three showed a T. solium pattern and five a T. saginata pattern.     

Molecular identification of Taenia tapeworms by Cox1 gene in Koh Kong, Cambodia

We collected fecal samples from 21 individuals infected with Taenia tapeworms in Koh Kong Province, Cambodia, and performed nucleotide sequencing of the cox1 gene and multiplex PCR on the eggs for DNA differential diagnosis of human Taenia tapeworms. Genomic DNA was extracted from the eggs of a minimum number of 10 isolated from fecal samples. Using oligonucleotide primers Ta7126F, Ts7313F, Tso7466F, and Rev7915, the multiplex PCR assay proved useful for differentially diagnosing Taenia solium, Taenia saginata, and Taenia asiatica based on 706, 629, and 474 bp bands, respectively. All of the Taenia specimens from Kho Kong, Cambodia, were identified as either T. saginata (n=19) or T. solium (n=2) by cox1 sequencing and multiplex PCR.     

Morphologic and genetic identification of Taenia tapeworms in Tanzania and DNA genotyping of Taenia solium

Species identification of Taenia tapeworms was performed using morphologic observations and multiplex PCR and DNA sequencing of the mitochondrial cox1 gene. In 2008 and 2009, a total of 1,057 fecal samples were collected from residents of Kongwa district of Dodoma region, Tanzania, and examined microscopically for helminth eggs and proglottids. Of these, 4 Taenia egg positive cases were identified, and the eggs were subjected to DNA analysis. Several proglottids of Taenia solium were recovered from 1 of the 4 cases. This established that the species were T. solium (n = 1) and T. saginata (n = 3). One further T. solium specimen was found among 128 fecal samples collected from Mbulu district in Arusha, and this had an intact strobila with the scolex. Phylegenetic analysis of the mtDNA cox1 gene sequences of these 5 isolates showed that T. saginata was basal to the T. solium clade. The mitochondrial cox1 gene sequences of 3 of these Tanzanian isolates showed 99% similarity to T. saginata, and the other 2 isolates showed 100% similarity to T. solium. The present study has shown that Taenia tapeworms are endemic in Kongwa district of Tanzania, as well as in a previously identified Mbulu district. Both T. solium isolates were found to have an "African/Latin American" genotype (cox1).     

Taenia solium and Taenia saginata: identification of sequence characterized amplified region (SCAR) markers

Cysticercosis is one of the most important zoonosis, not only because of the effects on animal health and its economic consequences, but also due to the serious danger it poses to humans. The two main parasites involved in the taeniasis-cysticercosis complex in Brazil are Taenia saginata and Taenia solium. Differentiating between these two parasites is important both for disease control and for epidemiological studies. The purpose of this work was to identify genetic markers that could be used to differentiate these parasites. Out of 120 oligonucleotide decamers tested in random amplified polymorphic DNA (RAPD) assays, 107 were shown to discriminate between the two species of Taenia. Twenty-one DNA fragments that were specific for each species of Taenia were chosen for DNA cloning and sequencing. Seven RAPD markers were converted into sequence characterized amplified region (SCAR) markers with two specific for T. saginata and five specific for T. solium as shown by agarose gel electrophoresis. These markers were developed as potential tools to differentiate T. solium from T. saginata in epidemiological studies.     

Characterization and detection of a newly described Asian taeniid using cloned ribosomal DNA fragments and sequence amplification by the polymerase chain reaction.

DNA isolated from a newly described taeniid from Taiwan which shows adult characters indistinguishable from those of Taenia saginata was compared to DNA from T. saginata and nine other cestodes by restriction endonuclease digestion of genomic DNA and Southern blot analysis using 32P-labeled total cestode RNA and cloned ribosomal RNA gene fragments as probes. Hybridization patterns of Taiwan Taenia DNA revealed distinct variations from that of T. saginata and Taenia solium as well as all other cestode DNAs examined; however, similarities in restriction maps and sequence data between cloned ribosomal gene fragments from Taiwan Taenia (pTTr 3.1) and T. saginata (pTSgr 3.1 and PTSgr 2.4, respectively) suggest close evolutionary relatedness between these two taeniids. DNA sequence amplification from genomic DNA using oligonucleotide primers homologous to regions on both the 2.4- and 3.1-kb fragments was able to delineate between Taiwan Taenia and T. saginata by generating 1.0- and 0.29-kb fragments, respectively. Results demonstrated that Taiwan Taenia is not exclusive to Taiwan but exists in other parts of Eastern Asia and that adult morphology is insufficient for its detection in other locations. Results further support biological data indicating that Taiwan Taenia and T. saginata, although similar morphologically, are distinct genotypes.     

Experimental human infection with Asian Taenia saginata metacestodes obtained from naturally infected Korean domestic pigs.

The infectivity of metacestodes of Asian Taenia saginata, now tentatively called Taenia saginata taiwanensis, in human host was confirmed. The metacestodes used in experimental infection were collected from the livers of naturally infected domestic pigs at an abattoir in Cheongju City, Korea. The first gravid proglottid was spontaneously discharged 76 days after infection. Two worms were recovered two years later by chemotherapy. The scolex was unarmed. The number of main uterine branches, varying from 16 to 21, was similar to that of classical Taenia saginata. The liver of pigs was confirmed to be an infection source of Asian T. saginata in Korea.     

The Asian Taenia and the possibility of cysticercosis

In certain Asian countries, a third form of human Taenia, also known as the Asian Taenia, has been discovered. This Asian Taenia seems to be an intermediate between Taenia solium and T. saginata since in morphological terms it is similar to T. saginata, yet biologically, as it uses the same intermediate host (pigs), it is more akin to T. solium. Taenia solium causes human cysticercosis, while T. saginata does not. It is not known whether the Asian taeniid is able to develop to the larval stage in humans or not. The arguments proposed by those authors who consider it unlikely that the Asian Taenia causes human cysticercosis are: (a) its molecular similarities with T. saginata; (b) the absence of cases of human cysticercosis in populations where the Asian adult is highly prevalent; and (c) the unsupporting results derived from an experimental infestation study. These three arguments are debated, although bearing in mind that at present there is still no clear scientific data to support that human cysticercosis can be caused by the Asian Taenia.     

Oral elimination of strobila segments of Taenia saginata]

Within a 14-24 hours lapse after gynecological operative surgery, a 27-year-old woman eliminated with vomit two large taperworm pieces, measuring 3 and 2 meters respectively. At the laboratory they were identified as Taenia saginata strobila with immature, mature and gravid proglottids. The patient informed that she liked to eat raw beef. Treatment consisted in a single dose of praziquantel: 15 mg/kg body weight. Parasitological controls (two series of three fecal samples each), performed three months later, resulted negative for Taenia eggs.     

Detection of Paragonimus heterotremus eggs in experimentally infected cats by a polymerase chain reaction-based method

A polymerase chain reaction (PCR) procedure for the detection of Paragonimus heterotremus eggs in stool samples was developed and compared with Stoll's egg count method. The primers were designed on the basis of a previously constructed pPH-13-specific DNA probe, which produced an approximate 0.5-kb amplified product. This PCR method could detect as few as 5 eggs in 0.6 g of artificially inoculated feces of a healthy control cat or as little as 1 x 10(-4) ng of P. heterotremus genomic DNA. The assay had 100% sensitivity in all infected cats. The method did not yield an approximate 0.5-kb product with DNA from other parasites such as Gnathostoma spinigerum, Trichinella spiralis, Fasciola gigantica, Echinostoma malayanum, Opisthorchis viverrini, Dirofilaria immitis, and Taenia saginata; exceptions were Paragonimus siamensis and Paragonimus westermani. In addition, no genomic DNA from Escherichia coli, Burkholderia pseudomallei, Acinetobacter anitratus, Mycobacterium tuberculosis, Staphylococcus aureus, beta-Streptococcus grA, and Proteus mirabilis or from the vertebrate and invertebrate hosts of P. heterotremus was amplified in the PCR assay. This assay has great potential for application in clinical epidemiological studies.     

Taenia asiatica and Taenia saginata: genetic divergence estimated from their mitochondrial genomes

We conducted a differential identification of Taenia asiatica and Taenia saginata, through the mapping of mitochondrial genomes and the sequencing of the cox1 and cob genes. The entire mitochondrial genomes of T. asiatica and T. saginata were amplified by long-extension PCR and cloned; each was approximately 14 kb in size. Restriction maps of T. asiatica and T. saginata mitochondrial genomes were then constructed using 13 restriction enzymes. The resulting restriction patterns enable us to estimate their genetic divergence at 4.8%. The actual sequence divergence was computed 4.5% from the cox1 gene, and 4.1% from the cob gene. These results support the designation of T. asiatica as a separate species from T. saginata.     

Portrait of human tapeworms

The tapeworms of the genus Taenia that infect human beings are T. solium, T. saginata and T. saginata asiatica. Taenia solium and T. saginata exhibit unequivocal features that characterize them; in contrast, only recent DNA studies, morphological characteristics, and epidemiological and sanitary aspects indicate that T. saginata asiatica is a subspecies of T. saginata. These 3 tapeworms occur in humans in their adult stage, and the intermediate hosts are pigs for T. solium and T. saginata asiatica and cows for T. saginata. Their identification is crucial considering the migratory increase from Asia to the Western Hemisphere and the fact that these tapeworms coexist in the same environment in Asia; furthermore, it is estimated that movement in both directions across the United States-Mexico border exceeds 200 million persons per yr, and thus, opportunities for acquiring and transporting T. solium infections are multiplied. It is not easy to distinguish among these tapeworms; therefore, a comparative diagram of the 3 parasites is shown in this article, which will facilitate their identification. All morphological features, some of which allow for identification, are clear and can be easily distinguished among the 3 tapeworms.     

Complete sequence of the mitochondrial genome of Taenia saginata: comparison with T. solium and T. asiatica

The complete sequence of the Taenia saginata mitochondrial genome was determined, and its organization and structure were compared to other human-tropic Taenia tapeworms for which complete mitochondrial sequence data were available. The mitochondrial genome was 13,670 bp long, contained 12 protein-coding genes, two ribosomal RNAs (rRNAs, a small and a large subunit), and 22 transfer RNAs (tRNAs). It did not encode the atp8 gene. Overlapping regions were found between nad4L and nad4, nad1 and trnN, and cox1 and trnT. The ATG initiation codon was used for 10 protein-coding genes, and the GTG initiation codon was used for the remaining 2 genes (nad4 and atp6). The size of the protein-coding genes of the three human Taenia tapeworms did not vary, except for Taenia solium nad1 (891 aa) and nad4 (1212 aa) and Taenia asiatica cox2 (576 aa). The tRNA genes were 57-75 bp long, and the predicted secondary structures of 18 of these genes had typical clover-leaf shapes with paired dihydrouridine (DHU) arms. The genes in all human Taenia tapeworms for the two mitochondrial rRNA subunits rrnL and rrnS are separated by trnC. The putative T. saginata rrnL and rrnS are 972 and 732 bp long, respectively. The non-coding regions of the mt genome of T. saginata consisted of 2 regions: a short non-coding region (SNR, 66 nucleotides) and a long non-coding region (LNR, 159 nucleotides). The overall sequence difference in the full mitochondrial genome between T. saginata and T. asiatica was 4.6%, while T. solium differed by 11%. In conclusion, the complete sequence of the T. saginata mitochondrial genome will serve as a resource for comparative mitochondrial genomics and systematic studies of the parasitic cestodes.     

A loop-mediated isothermal amplification method for a differential identification of Taenia tapeworms from human: Application to a field survey

In this study, we applied a loop-mediated isothermal amplification method for identification of human Taenia tapeworms in Tibetan communities in Sichuan, China. Out of 51 proglottids recovered from 35 carriers, 9, 1, and 41 samples were identified as Taenia solium, Taenia asiatica and Taenia saginata, respectively. Same results were obtained afterwards in the laboratory, except one sample. These results demonstrated that the LAMP method enabled rapid identification of parasites in the field surveys, which suggested that this method would contribute to the control of Taenia infections in endemic areas.     

Asian (Taiwan) Taenia: species or strain?

Recent studies on the epidemoiological pattern of taenisis in Southeast Asia have indicated the existence of a third form of human Taenia which is distinguishable from Taenia saginata and T. solium. Don McManus and Josephine Bowles here review how new genetic evidence supports earlier conclusions that the Asian Taenia is a distinct entity but is closely related T. saginata, and suggests its taxonomic classification as a subspecies or strain of T. saginata is more appropriate than formal designation as a new species.     

Recent hybridization between Taenia asiatica and Taenia saginata

Five Taenia tapeworms collected from humans in Tibetan Plateau, Sichuan, China, where three species of human Taenia are sympatrically endemic, were examined for the mitochondrial cox1 gene and two nuclear genes, ef1 and elp. Phylogenetic analyses of these genes revealed that two adult worms showed nuclear-mitochondrial discordance, suggesting that they originated from hybridization between Taenia saginata and Taenia asiatica. One of two worms had T. asiatica-type mtDNA, whereas another worm had T. saginata-type mtDNA, indicating that reciprocal hybridization between T. saginata and T. asiatica could occur. The worm having T. asiatica-type mtDNA was heterozygous at both nuclear loci with T. saginata-type alleles and T. asiatica-type alleles. In another worm, the ef1 locus was heterozygous with a T. saginata-type alleles and T. asiatica-type alleles, while the elp locus was homozygous with T. saginata-type alleles. Self-fertilization is the main reproductive method of the genus Taenia. Since self-fertilization represents a type of inbreeding, each locus in the offspring would become homozygous over generations with genetic drift. The fact that some nuclear loci are still heterozygous means that hybridization might have occurred recently. Hybridization between T. asiatica and T. saginata is probably an ongoing event in many areas in which they are sympatrically endemic.     

Experimental infection of pigs and cattle with eggs of Asian Taenia saginata with special reference to its extrahepatic viscerotropism.

Asian Taenia saginata, tentatively called Taenia saginata taiwanensis, has been described to be infected in its metacestode stage only in the liver of intermediate host animals. Experimentally, however, we found that the metacestodes of the Asian Taenia saginata are also infected in other viscera than the liver of pigs (Landrace-Duroc-Hampshire) 4 days to 4 months postinoculation (PI). Viscerotropism of cysticercosis was apparent because a majority (70.7%) of the non-calcified cysticerci were found in the livers while a minority were found in extrahepatic organs such as the omentum (19.2%), lungs (8.1%) and serosa of colon (2.0%). When experimentally infected to cattle, Asian T. saginata cysticerci were also observed calcified in the livers. On the other hand, classical Taenia saginata metacestodes infected the muscles and viscera of the Holstein-Friesian cattle whereas no infection was observed in experimental pigs. Extrahepatic metacestodes of Asian T. saginata, which were obtained from an experimental pig were confirmed to be infective to a male volunteer. This extrahepatic viscerotropism of Asian T. saginata metacestodes in experimental pigs explains well the transmission modes of Asian T. saginata among people considering the eating habits.     

Comparison of conventional techniques to differentiate between Taenia solium and Taenia saginata and an improved polymerase chain reaction-restriction fragment length polymorphism assay using a mitochondrial 12S rDNA fragment

Given the constraints of classical diagnostic methods, i.e., morphological and isoenzymatic studies of proglottids, a polymerase chain reaction test complemented with restriction enzyme analysis has been modified by redesigning one of the primers to reduce nonspecific amplifications experienced when using field samples. The use of these new, highly cestode-specific primers and the restriction enzyme Ddel led to the development of a diagnostic assay that clearly distinguishes between Taenia saginata and T. solium proglottids in field samples. This assay confirms the presence of T. saginata in Ecuador. DNA amplification of some of these taeniids showed different patterns, suggesting the possibility that strain differences exist. These results demonstrate the need for development of useful molecular assays as reliable tools for epidemiological studies on cestodes.     

Detection of methylated tissue factor pathway inhibitor 2 and human long DNA in fecal samples of patients with colorectal cancer in China

Background: To investigate the feasibility of detecting methylated tissue factor pathway inhibitor (TFPI2) and quantifying human long DNA with fluorescent quantitative Alu PCR in fecal DNA as a non-invasive screening tool for colorectal cancer (CRC). Materials and Methods: Methylation-specific PCR (MSP) was performed to analyze TFPI2 gene promoter methylation status in a blinded fashion in stool samples taken from 30 endoscopically diagnosed healthy controls, 20 patients with adenomas, and 60 patients with colorectal cancer. Real-time Alu PCR was used to quantify human long DNA. Results: The specificity of fecal TFPI2 MSP assay and long DNA assay was 100% and 83.3%, respectively. The sensitivity of fecal TFPI2 MSP assay and long DNA assay was 68.3% and 53.3%, respectively. The sensitivity of fecal DNA assay (either marker being positive) was 86.7%, which was high for CRC. Conclusions: Our results have demonstrated the feasibility of using TFPI2 methylation and quantify human long DNA with fluorescent quantitative Alu PCR in fecal samples as a new noninvasive test for CRC.     

Further molecular discrimination of Spanish strains of Echinococcus granulosus

We have designed two polymerase chain reaction (PCR) primer sets (PEg9F1-PEg9R1 and PEg16F1-PEg16R1) and two PCR protocols (Eg9-PCR and Eg16-PCR) for discrimination of Echinococcus granulosus genotypes. The oligonucleotide sequences originate from two E. granulosus DNA multiplex-PCR amplification fragments, previously reported, that allows species-specific discrimination between Taenia saginata, Taenia solium, and E. granulosus. The Eg9-PCR, Eg16-PCR, and Eg9-PCR linked restriction fragment length polymorphism (RFLP) analysis was used to characterize 53 E. granulosus isolates from the central region of Spain, highly endemic for echinococcosis. The analysis resulted in: (i) the discrimination of E. granulosus from Echinococcus multilocularis; (ii) the characterisation and discrimination of discrete E. granulosus strains from Spain; and (iii) the identification of two distinct genotypes within E. granulosus Spanish pig isolates. To further characterize the genetic variants in pigs, fragments of the NADH dehydrogenase I (ND1) and the cytochrome c oxidase subunit I (CO1) genes were amplified from parasite DNA and sequenced. The results again revealed the presence of two distinct genotypes: the G1 (sheep-dog strain) and G7 (pig-dog strain) genotypes. This observation could have important consequences for human health in Spain. Furthermore, the Eg9-PCR, Eg16-PCR, and Eg9-PCR-RFLP protocols can be used as additional methods to discriminate various E. granulosus genotypes.