Differential diagnosis of Taenia asiatica using multiplex PCR

Taenia asiatica and T. saginata are frequently confused tapeworms due to their morphological similarities and sympatric distribution in Asian regions. To resolve this problem, a high-resolution multiplex PCR assay was developed to distinguish T. asiatica infections from infection with other human Taenia tapeworms. For molecular characterization, the species specificity of all materials used was confirmed by sequencing of the cox1 gene. Fifty-two samples were analyzed in this study, comprising 20 samples of T. asiatica genomic DNA from China, Korea, and the Philippines; 24 samples of T. saginata from Belgium, Chile, China, Ethiopia, France, Indonesia, Korea, Laos, the Philippines, Poland, Taiwan, Thailand, and Switzerland; and 10 samples of T. solium from Cape Verde, China, Honduras, and Korea. The diagnostic quality of the results obtained using PCR and species-specific primers designed from valine tRNA and NADH genes was equal to that based on the nucleotide sequencing of the cox1 gene. Using oligonucleotide primers Ta4978F, Ts5058F, Tso7421F, and Rev7915, the multiplex PCR assay was useful for the differentially diagnosing T. asiatica, T. saginata, and T. solium based on 706-, 629-, and 474-bp bands.     

Sympatric Distribution of Three Human Taenia Tapeworms Collected between 1935 and 2005 in Korea

Taeniasis has been known as one of the prevalent parasitic infections in Korea. Until recently, Taenia saginata had long been considered a dominant, and widely distributed species but epidemiological profiles of human Taenia species in Korea still remain unclear. In order to better understand distribution patterns of human Taenia tapeworms in Korea, partial nucleotide sequences of mitochondrial cox1 and ITS2 (internal transcribed spacer 2) were determined, along with morphological examinations, on 68 Taenia specimens obtained from university museum collections deposited since 1935. Genomic DNA was extracted from formalin-preserved specimens. Phylogenetic relationships among the genotypes (cox1 haplotype) detected in this study were inferred using the neighbor-joining method as a tree building method. Morphological and genetic analyses identified 3 specimens as T. solium, 51 specimens as T. asiatica, and 14 specimens as T. saginata. Our results indicate that all 3 Taenia tapeworms are sympatrically distributed in Korea with T. asiatica dominating over T. saginata and T. solium.     

Taenia asiatica: the Most Neglected Human Taenia and the Possibility of Cysticercosis

Not only Taenia solium and Taenia saginata, but also Taenia asiatica infects humans. The last species is not included in the evaluation of the specificity of the immunodiagnostic techniques for taeniasis/cysticercosis. There is currently no specific immunodiagnostic method for T. asiatica available. Therefore, due to the fact that molecular techniques (the only tool to distinguish the 3 Taenia species) are normally not employed in routine diagnostic methods, the 2 questions concerning T. asiatica (its definite geographic distribution and its ability to cause human cysticercosis), remain open, turning T. asiatica into the most neglected agent of human taeniasis-cysticercosis.     

Human Taeniasis in the Republic of Korea: Hidden or Gone?

History and current status of human taeniasis in the Republic of Korea, due to Taenia solium, Taenia asiatica, and Taenia saginata, are briefly reviewed. Until the 1980s, human taeniasis had been quite common in various localities of Korea. A study from 1924 reported 12.0% egg prevalence in fecal examinations. Thereafter, the prevalence of Taenia spp. ranged from 3% to 14% depending on the time and locality. Jeju-do, where pigs were reared in a conventional way, was the highest endemic area of taeniasis. An analysis of internal transcribed spacer 2 and mitochondrial cytochrome c oxidase 1 genes of 68 taeniasis cases reported from 1935 to 2005 in Korea by a research group revealed the relative occurrence of the 3 Taenia spp. as follows: T. solium (4.4%), T. asiatica (75.0%), and T. saginata (20.6%). However, national surveys on intestinal helminths conducted every 5 years on randomly selected people revealed that the Taenia egg prevalence dropped from 1.9% in 1971 to 0.02% in 1997 and finally to 0.0% in 2004. With the exception of 3 egg-positive cases reported in 2008 and 2 worm-proven cases in 2011, no more cases have been officially recorded. Based on these surveys and also on other literature, it can be concluded that taeniasis has virtually disappeared from Korea, although a few sporadic cases may remain hidden. Human cysticercosis is also expected to disappear within a couple of decades in Korea.     

Current Status of Taeniasis in Thailand

Taeniasis is prevalent in all regions of Thailand, except the South. Infections were more frequently found in males than females of any age from 7-83 years. Taenia saginata is the most common species throughout the country. Taenia asiatica was reported only in the province of Kanchanaburi in the Central region. Co-infections, with Taenia solium and T. asiatica or T. solium and T. saginata, were found. Hybridization between T. asiatica and T. saginata is evidence that co-infection is never found between these 2 species. Finding more than 1 worm in a single patient was not entirely rare. Genetic variation was found without correlation to its geographic distribution in T. saginata, whereas no variation was found in T. asiatica.     

Historical Overview of Taenia asiatica in Taiwan

An overview of the epidemiological, biological, and clinical studies of Taenia and taeniasis in Taiwan for the past century is presented. The phenomenal observations that led to the discovery of Taenia asiatica as a new species, which differ from Taenia solium and Taenia saginata, are described. Parasitological surveys of the aborigines in Taiwan revealed a high prevalence of taeniasis, which might be due to the culture of eating raw liver of hunted wild boars. Chemotherapeutic deworming trials involving many patients with taeniasis were discussed. Praziquantel was found to be very effective, but sometimes complete worms could not be recovered from the feces after treatment, probably due to the dissolution of the proglottids. Atabrine, despite some side effects, can still be used, in properly controlled dosages, as the drug of choice for human T. asiatica infection if we need to recover the expelled worms for morphological examinations. Research results on the infection of T. asiatica eggs from Taiwan aborigines in experimental animals were also noted. Since the pig serve as the natural intermediate host of T. asiatica and the predilection site is the liver, a differential comparison of other parasitic pathogens that might cause apparently similar lesions is also presented.     

Four Cases of Taenia saginata Infection with an Analysis of COX1 Gene

Human taeniases had been not uncommon in the Republic of Korea (=Korea) until the 1980s. The prevalence decreased and a national survey in 2004 revealed no Taenia egg positive cases. However, a subsequent national survey in 2012 showed 0.04% (10 cases) prevalence of Taenia spp. eggs suggesting its resurgence in Korea. We recently encountered 4 cases of Taenia saginata infection who had symptoms of taeniasis that included discharge of proglottids. We obtained several proglottids from each case. Because the morphological features of T. saginata are almost indistinguishable from those of Taenia asiatica, molecular analyses using the PCR-RFLP and DNA sequencing of the cytochrome c oxidase subunit 1 (cox1) were performed to identify the species. The PCR-RFLP patterns of all of the 4 specimens were consistent with T. saginata, and the cox1 gene sequence showed 99.8-100% identity with that of T. saginata reported previously from Korea, Japan, China, and Cambodia. All of the 4 patients had the history of travel abroad but its relation with contracting taeniasis was unclear. Our findings may suggest resurgence of T. saginata infection among people in Korea.     

Epidemiological understanding of Taenia tapeworm infections with special reference to Taenia asiatica in Korea.

In endemic areas of Taenia tapeworms in Korea, most of the reports showed that T. saginata was dominant over T. solium, but eating pigs is the dominant habit over eating cattle. Why do they have more T. saginata despite lower consumption of beef? This problem actually has long been recognized but until recently there has been no intensive trial to give a scientific explanation on this epidemiological enigma. By summing up the data published between the years 1963 and 1999, the ratio of armed versus unarmed tapeworms in humans was estimated at approximately 1:5. The ratio of pig-eaters versus cattle-eaters, however, was approximately 5:1. This inconsistency could be explained with the recently described T. asiatica, which infects humans through the eating of pig''s viscera. We re-evaluate the importance of the consumption of visceral organ of pigs, leading us to an improved epidemiological understanding of the T. asiatica infection together with co-existing T. saginata and T. solium in Korea.     

Molecular Approaches to Taenia asiatica

Taenia solium, T. saginata, and T. asiatica are taeniid tapeworms that cause taeniasis in humans and cysticercosis in intermediate host animals. Taeniases remain an important public health concerns in the world. Molecular diagnostic methods using PCR assays have been developed for rapid and accurate detection of human infecting taeniid tapeworms, including the use of sequence-specific DNA probes, PCR-RFLP, and multiplex PCR. More recently, DNA diagnosis using PCR based on histopathological specimens such as 10% formalin-fixed paraffin-embedded and stained sections mounted on slides has been applied to cestode infections. The mitochondrial gene sequence is believed to be a very useful molecular marker for not only studying evolutionary relationships among distantly related taxa, but also for investigating the phylo-biogeography of closely related species. The complete sequence of the human Taenia tapeworms mitochondrial genomes were determined, and its organization and structure were compared to other human-tropic Taenia tapeworms for which complete mitochondrial sequence data were available. The multiplex PCR assay with the Ta4978F, Ts5058F, Tso7421F, and Rev7915 primers will be useful for differential diagnosis, molecular characterization, and epidemiological surveys of human Taenia tapeworms.     

Genetic Diversity of Taenia asiatica from Thailand and Other Geographical Locations as Revealed by Cytochrome c Oxidase Subunit 1 Sequences

Twelve 924 bp cytochrome c oxidase subunit 1 (cox1) mitochondrial DNA sequences from Taenia asiatica isolates from Thailand were aligned and compared with multiple sequence isolates from Thailand and 6 other countries from the GenBank database. The genetic divergence of T. asiatica was also compared with Taenia saginata database sequences from 6 different countries in Asia, including Thailand, and 3 countries from other continents. The results showed that there were minor genetic variations within T. asiatica species, while high intraspecies variation was found in T. saginata. There were only 2 haplotypes and 1 polymorphic site found in T. asiatica, but 8 haplotypes and 9 polymorphic sites in T. saginata. Haplotype diversity was very low, 0.067, in T. asiatica and high, 0.700, in T. saginata. The very low genetic diversity suggested that T. asiatica may be at a risk due to the loss of potential adaptive alleles, resulting in reduced viability and decreased responses to environmental changes, which may endanger the species.     

State of the Art of Taenia solium as Compared to Taenia asiatica

Three species of tapeworms infect humans in their adult stage (Taenia solium, Taenia saginata and Taenia asiatica). The 3 are flat, opaque white or yellowish, and exceptional long segmented parasites, measuring 1 to 12 m in their adult stage. In this review, the development of the knowledge regarding the first species, mainly focused on understanding how the larval stage or cysticercus is transmitted to humans, is described. The second species is a cosmopolitan parasite that only causes taeniosis and not cysticercosis; therefore, it will not be included. Information on the third species, which is presently being produced, since this species was recognized as such only at the end of the 20th century, will be discussed at the end of this review.     

Nanobodies, a promising tool for species-specific diagnosis of Taenia solium cysticercosis

Taenia solium cysticercosis is a major helminth zoonosis in developing countries. Pigs are the intermediate hosts mediating transmission of infection. Specific assays to diagnose living cysts in pigs are lacking. The monoclonal-based antigen detection ELISA is genus-specific and cross-reactions with Taenia hydatigena hamper the use of this test to screen pigs. We, therefore, aimed to introduce nanobodies, camelid-derived single-domain antibodies specific for T. solium cysticercosis, to develop unambiguous tests. Nanobodies were cloned following immunization of two dromedaries with T. solium antigen and eight T. solium-specific nanobodies were selected after phage display. Their binding characteristics and potential for the diagnosis of porcine cysticercosis were investigated. The nanobodies do not cross-react with T. hydatigena, Taenia saginata, Taenia crassiceps or Trichinella spiralis and were categorized into four epitope-binding groups. The target protein was identified as 14 kDa diagnostic glycoprotein (Ts14), but the nanobodies also reacted with other proteins of the same family. Nanobodies were tested in a sandwich ELISA with cyst fluid, and one particular nanobody detected its cognate serum antigens in a species-specific inhibition ELISA. Considering their beneficial production and stability properties, these highly specific nanobodies constitute a promising tool to diagnose cysticercosis after further improvement of the sensitivity and future assay validation.     

NADH dehydrogenase subunit 1 and cytochrome c oxidase subunit I sequences compared for members of the genus Taenia (Cestoda)

Nine members of the genus Taenia (Taenia taeniaeformis, Taenia hydatigena, Taenia pisiformis, Taenia ovis, Taenia multiceps, Taenia serialis, Taenia saginata, Taenia solium and the Asian Taenia) were characterised by their mitochondrial NADH dehydrogenase subunit 1 gene sequences and their genetic relationships were compared with those derived from the cytochrome c oxidase subunit I sequence data. The extent of inter-taxon sequence difference in NADH dehydrogenase subunit 1 (5.9–30.8%) was usually greater than in cytochrome c oxidase subunit I (2.5–18%). Although topology of the phenograms derived from NADH dehydrogenase subunit 1 and cytochrome c oxidase subunit I sequence data differed, there was concordance in that T. multiceps, T. serialis (of canids), T. saginata and the Asian Taenia (of humans) were genetically most similar, and those four members were genetically more similar to T. ovis and T. solium than they were to T. hydatigena and T. pisiformis (of canids) or T. taeniaeformis (of cats). The NADH dehydrogenase subunit 1 sequence data may prove useful in studies of the systematics and population genetic structure of the Taeniidae.     

Infection of Taenia asiatica in a Bai Person in Dali,China

We report here a human case of Taenia asiatica infection which was confirmed by genetic analyses in Dali, China. A patient was found to have symptoms of taeniasis with discharge of tapeworm proglottids. By sequencing of the mitochondrial cytochrome c oxidase subunit 1 (cox1) gene, we observed nucleotide sequence identity of 99% with T. asiatica and 96% with T. saginata. Using the cytochrome b (cytb) gene, 99% identity with T. asiatica and 96% identity with T. saginata were found. Our findings suggest that taeniasis of people in Dali, China may be mainly caused by T. asiatica.     

Current Status of Human Taeniasis in Lao People's Democratic Republic

Human taeniasis was investigated in Lao People''s Democratic Republic (Lao PDR) between 2000 and 2011 as part of the nation''s helminthiasis survey. A total of 55,038 inhabitants, including 29,846 school children, were examined using the Kato-Katz and scotch-tape anal swab method, and morphological observation of adult worms. Molecular identification of Taenia tapeworms was performed by multiplex PCR or DNA sequence analysis of the mitochondrial cox1 gene. Taenia eggs were present at a rate of 1.5% (845/55,038) in the subject population. Adult tapeworms were identified as T. solium or T. saginata by analyzing the collectable stool specimens (n=126). Three specimens identified as T. solium were found in Luang Prabang, while the remaining 123 specimens, which were T. saginata, were found in Bokeo, Bolikhamxay, Champasak, Houaphan, Khammouane, Luang Namta, Luang Prabang, Oudomxay, Phongsaly, Saysomboune, Saravane, Savannakhet, Xayaboury, Xekong, Xieng Khouang Province, and Vientiane Municipality.     

Current Status of Taeniasis and Cysticercosis in Vietnam

Several reports on taeniasis and cysticercosis in Vietnam show that they are distributed in over 50 of 63 provinces. In some endemic areas, the prevalence of taeniasis was 0.2-12.0% and that of cysticercosis was 1.0-7.2%. The major symptoms of taeniasis included fidgeted anus, proglottids moving out of the anus, and proglottids in the feces. Clinical manifestations of cysticercosis in humans included subcutaneous nodules, epileptic seizures, severe headach, impaired vision, and memory loss. The species identification of Taenia in Vietnam included Taenia asiatica, Taenia saginata, and Taenia solium based on combined morphology and molecular methods. Only T. solium caused cysticercosis in humans. Praziquantel was chosen for treatment of taeniasis and albendazole for treatment of cysticercosis. The infection rate of cysticercus cellulosae in pigs was 0.04% at Hanoi slaughterhouses, 0.03-0.31% at provincial slaughterhouses in the north, and 0.9% in provincial slaughterhouses in the southern region of Vietnam. The infection rate of cysticercus bovis in cattle was 0.03-2.17% at Hanoi slaughterhouses. Risk factors investigated with regard to transmission of Taenia suggested that consumption of raw meat (eating raw meat 4.5-74.3%), inadequate or absent meat inspection and control, poor sanitation in some endemic areas, and use of untreated human waste as a fertilizer for crops may play important roles in Vietnam, although this remains to be validated.     

Interaction and ovicidal activity of nematophagous fungus Pochonia chlamydosporia on Taenia saginata eggs

The ovicidal activity of the nematophagous fungi Pochonia chlamydosporia (isolates VC1 and VC4), Duddingtonia flagrans (isolate AC001) and Monacrosporium thaumasium (isolate NF34) on Taenia saginata eggs was evaluated under laboratory conditions. T. saginata eggs were plated on 2% water-agar with fungal isolates and controls without fungus and examined after 5, 10 and 15 days. At the end of the experiment P. chlamydosporia showed ovicidal activity against T. saginata eggs (p < 0.05), mainly for internal egg colonization with results of 12.8% (VC1) and 2.2% (VC4); 18.1% (VC1) and 7.0% (VC4); 9.76% (VC1) and 8.0% (VC4) at 5, 10 and 15 days, respectively. The other fungi showed only lytic effect without morphological damage to the eggshell. Results demonstrated that P. chlamydosporia was effective in vitro against T. saginata eggs unlike the other fungi.     

Molecular and serological survey on taeniasis and cysticercosis in Kanchanaburi Province,Thailand

A community-based field survey on taeniasis and cysticercosis was performed in two villages in Thong Pha Phum District, Kanchanaburi Province, central Thailand, where 3 Taenia species, T. solium, T. saginata and T. asiatica, are sympatrically occurring. Four (0.6%) out of 667 stool samples were egg-positive for Taenia sp. by Kato–Katz technique. Three out of those four persons and other three persons who were Taenia egg-negative but having a recent (< 1 year) history of discharging worms in stool were treated with niclosamide. One Taenia egg-positive woman was not treated because of severe ascites. After treatment, three persons expelled long strobilae with scolices and two persons expelled strobilae without scolex. One Taenia egg-positive person did not expel any worms post-treatment. Among 5 persons, four expelled a single worm, whereas one expelled multiple worms, may be 6 worms but not confirmed by detection of scolices. One scolex was armed with hooklets, whereas 2 others did not. Multiplex PCR of 10 expelled proglottids (including 6 estimated worms from one patient) revealed that one sample was T. solium, one T. saginata, and 8 T. asiatica. A total of 159 residents agreed to receive a serological test for cysticercosis. By ELISA using partially purified glycoprotein antigen, 9 cases, 5 and 4 from villages A and B respectively, were found to be sero-positive. The five and an additional sample on the border line from village A were evaluated using confirmative immunoblot using recombinant chimeric antigen. Among the six samples, four including the border line sample were confirmed to be cysticercosis by immunoblotting. One of the 4 persons had neurological symptoms with nodular lesions in the brain by computed tomography. These 4 confirmed or suspected cysticercosis cases were free of T. solium worms, but two of them including confirmed NCC case had a past (> 1 year) history of expelling proglottids in the stool.     

Recent Situation of Taeniasis in Mongolia (2002-2012)

Epidemiological situation of taeniasis in Mongolia was assessed based on mitochondrial DNA identification of the parasite species. Multiplex PCR was used on a total of 194 proglottid specimens of Taenia species and copro-PCR and loop-mediated isothermal amplification (LAMP) assays were utilized for detection of copro-DNA of 37 fecal samples from taeniasis patients submitted to the Mongolian National Center for Communicable Diseases (NCCD) from 2002 to 2012. In addition, 4 out of 44 calcified cysts in beef kept in formalin since 2003 were evaluated for histopathological confirmation of cattle cysticercosis. All proglottid specimens and stool samples were confirmed to be Taenia saginata by multiplex PCR and by copro-PCR and LAMP, respectively. Cysts collected from cattle were morphologically confirmed to be metacestodes of Taenia species. T. saginata taeniasis was identified from almost all ages from a 2-year-old boy up to a 88-year-old woman and most prominently in 15-29 age group (37%, 74/198) followed by 30-44 age group (34.8%, 69/198 ) from 15 of Mongolia''s 21 provinces, while cattle cysticerci were found from 12 provinces. The highest proportion of taeniasis patients was in Ulaanbaatar, the capital of Mongolia.