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Differential diagnosis of Taenia asiatica using multiplex PCR
Experimental Parasitology 121 (2009) 151–156
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Differential diagnosis of Taenia asiatica using multiplex PCR Hyeong-Kyu Jeon a, Jong-Yil Chai b, Yoon Kong c, Jitra Waikagul d, Bounnaloth Insisiengmay e, Han-Jong Rim f, Keeseon S. Eom a,* a
Department of Parasitology and Medical Research Institute, Chungbuk National University College of Medicine, Gaeshin-Dong 12, Chongju, Chungbuk 361-763, Republic of Korea Department of Parasitology and Tropical Medicine, Seoul National University College of Medicine, and Institute of Endemic Diseases, Seoul National University Medical Research Center, Seoul, Republic of Korea c Department of Molecular Parasitology and Center for Molecular Medicine, Sungkyunkwan University School of Medicine and Samsung Biomedical Research Institute, Suweon, Republic of Korea d Department of Helminthology, Faculty of Tropical Medicine, Mahidol University, Bangkok 10400, Thailand e Department of Hygiene and Prevention, Ministry of Public Health, Vientiane, Lao People’s Democratic Republic f Department of Parasitology, Korea University College of Medicine, Seoul, Republic of Korea b
a r t i c l e
i n f o
Article history: Received 17 April 2008 Received in revised form 24 October 2008 Accepted 28 October 2008 Available online 5 November 2008 Keywords: Taenia asiatica T. saginata T. solium Multiplex PCR Differential diagnosis
a b s t r a c t 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. Ó 2008 Published by Elsevier Inc.
1. Introduction Taenia solium, T. saginata, and T. asiatica are cestodes that cause taeniasis in humans and cause cysticercosis in intermediate host animals (cows and pigs). Among them, T. asiatica is the most recently described human Taenia tapeworm, with its morphological characteristics and life cycle having been reported. The morphological characteristics of this adult tapeworm include an unarmed rostellum, a large of number of uterine twigs, and a posterior protuberance on the gravid proglottids (Eom and Rim, 1993). The larval tapeworm of T. asiatica (Cysticercus viscerotropica) also differs microscopically from that of T. saginata, in that it has wart-like formations on the external surface of the bladder wall. The phylogenetic relationships between taeniid species have also been studied base on various morphological and molecular characteristics, which has indicated that T. asiatica is a distinct sister species of T. saginata (De Queiroz and Alkire, 1998; Eom et al., 2002; Hoberg et al., 2000; Jeon et al., 2005, Jeon and Eom, 2006). Epidemiologically, T. asiatica has been found in China, Indonesia, Korea, the Philippines, Taiwan, Thailand, and Vietnam * Corresponding author. Fax: +82 43 272 1603. E-mail address: [email protected] (K.S. Eom). 0014-4894/$ - see front matter Ó 2008 Published by Elsevier Inc. doi:10.1016/j.exppara.2008.10.014
(Fan et al., 1995; Simanjuntak et al., 1997; Eom and Rim, 2001; Eom et al., 2002; Willingham et al., 2003; Li et al., 2006; Anantaphruti et al., 2007). T. asiatica and T. saginata exhibit morphological similarities, particularly in the eggs and proglottids of adult worms, which cause frequent confusion in the abovementioned Asian countries where the distributions of the two tapeworms overlap. Consequently, it is necessary to be able to accurately distinguish between these tapeworms. Molecular diagnostic methods have been developed for the rapid and accurate detection of species. Molecular approaches to the differential diagnosis of T. saginata and T. asiatica have been developed, including the use of sequence-specific DNA probes, PCR coupled to restriction fragment length polymorphisms (Bowles and MacManus, 1994; Yamasaki et al., 2002; McManus, 2006), and multiplex PCR (Gonzales et al., 2004; Yamasaki et al., 2004). A mitochondrial-based multiplex PCR has been applied to diagnose of three human Taenia tapeworms and two genotypes of T. solium (Yamasaki et al., 2004, 2007). In the present study, we used species-specific primers based on the mitochondrial sequences for T. asiatica, T. saginata and T. solium, and amplified fragments of different lengths from other species. Our method employs a high-resolution multiplex PCR
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assay that could be used for the differential diagnosis of humaninfecting Taenia tapeworms.
Table 1 Taenia tapeworm samples in the present study. No
Status of samples
Locality
cox1 sequence and multiplex PCR finding
1 2
Korea (Chuncheon) Korea (Chongju)
T. asiatica T. asiatica*
Korea Korea Korea Korea Korea Korea Koera Korea Koera Koera Korea Koera Koera Koera Koera China
T. T. T. T. T. T. T. T. T. T. T. T. T. T. T. T.
asiatica asiatica asiatica asiatica asiatica asiatica asiatica asiatica asiatica asiatica asiatica asiatica asiatica asiatica asiatica asiatica*
China (Binyang) Philippines Belgium Belgium
T. T. T. T.
asiatica asiatica saginata saginata*
23 24 25
Proglottid Proglottid, eggsb Proglottid Proglottid Proglottid Proglottid Proglottid Proglottid Proglottid Proglottid Proglottid Proglottid Proglottid Proglottid Proglottid Proglottid Proglottid Proglottid, cysticercusb Proglottid Proglottid Proglottid Proglottid, cysticercusb Proglottid Proglottid Proglottid
T. saginata T. saginata T. saginata
26
Proglottid
27
Proglottid
28
Proglottid
29 30 31 32 33 34 35 36 37 38 39 40
Proglottid Proglottid Proglottid Proglottid Proglottid Peoglottid Proglottid Proglottid Proglottid Proglottid Proglottid Proglottid
41 42 43 44 45 46 47
Proglottid Proglottid Proglottid Proglottid Proglottid Proglottid Proglottid, cysticercusb Proglottid Proglottid Proglottid Proglottid Proglottid, cysticercusb Proglottid Proglottid
Belgium China (Beijing) China (Hongshui, Gauangxi) China (Hongshui, Gauangxi) China (Hongshui, Gauangxi) China (Hongshui, Gauangxi) Chile Ethiopia Indonesia France Korea (Busan) Korea (Jeju) Laos (Savanakhet) Laos (Savanakhet) Laos (Savanakhet) Philippines Poland Swiss (Natural History Museum) Taiwan Thailand Thailand Thailand Cape Verde China (Henan) China (Luzhai)
2. Materials and methods 2.1. Parasites The parasites were collected from 54 individuals having proven parasitosis, and selected on the basis of a parasitological examination and identification of eggs, larvae, or adult worms. Fifty-two samples were analyzed in this study, comprising 20 samples of T. asiatica genomic DNA from China (n = 2), Korea (n = 17) and the Philippines (n = 1); 24 samples of T. saginata from Belgium (n = 3), Chile (n = 1), China (n = 5), Ethiopia (n = 1), France (n = 1), Indonesia (n = 1), Korea (n = 2), Laos (n = 3), Poland (n = 1), the Philippines (n = 1), Taiwan (n = 1), Thailand (n = 3), and Switzerland (n = 1); and 10 samples of T. solium from Cape Verde (n = 1), China (n = 7), Honduras (n = 1) and Korea (n = 1). For molecular characterization, all of the materials used (i.e., proglottids, cysticerci, and eggs) were previously confirmed from taeniid tapeworms by sequencing of the cox1 gene (Table 1). The negative control samples used were T. crassiceps (donated by Dr. Y. Kong, Korea), T. taeniaeformis (donated by Dr. W.M. Sohn, Korea), and Echinococcus multilocularis (donated by Dr. M. Kamiya, Japan). 2.2. Fecal samples Fecal samples were obtained from 30 individuals infected with helminthes from Khammouane province, LaoPDR, in 2003. The stool samples from eight Taenia infections were collected from carriers who discharged gravid proglottids. Taenia-positive fecal samples were identified using eggs and adult worms by morphological examination and multiplex PCR. These fecal samples were stored at 20 °C until use. 2.3. DNA extraction Total genomic DNA was extracted from adult worms, eggs, and metacestodes using the DNeasy Tissue Kit (Qiagen, Valencia, CA, USA) according to the manufacturer’s instructions and used as template DNA for the PCR. A single frozen specimen was chopped into small pieces and then digested in tissue lysis buffer of the DNA extraction kit. A single worm was enzymatically digested in 180 ll of a lysis solution (ATL buffer, Qiagen), then 20 ll of proteinase K (50 lg/ml) was added, and the sample was incubated at 56 °C for 2–3 h with vortexing every 30 min. Genomic DNA of eggs was extracted using the QIAamp Stool Kit (Qiagen). A mixture comprising 200 mg of stool sample in 200 ll of buffer (ASL, Qiagen) was incubated for 5 min at 95 °C. One inhibition tablet (inhibitEX, Qiagen) was then added, and the supernatant collected by centrifugation was put in a lysis buffer after adding 200 ll of buffer (AL, Qiagen) containing guanidine hydrochloride and 15 ll of RNase A (100 mg/ ml) and was mixed for 15 s, and then incubated at 70 °C for 10 min. Ethanol (200 ll) was added, and the mixture was vortexed for 15 s and then added to a DNA-binding column and spun down for 1 min. The column was then washed several times using AW1 and AW2 buffers (Qiagen). The genomic DNA extract was diluted to a working concentration of 50 ng/ll, and 1 ll of it was used as a template in a PCR. 2.4. Primer design and multiplex PCR Three species-specific forward primers were prepared based on the nucleotide sequences of valine transfer RNA (tRNA) and NADH dehydrogenase subunit 2 from the human taeniid cestodes. The three forward primers were designed to amplify products of differ-
3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22
48 49 50 51 52 53 54
(Chuncheon) (Wanju) (Hwasun) (Hwasun) (Hwasun) (Youngju) (Wando) (Chuncheon) (Ansan) (Cheonan) (Cheju) (Cheju) (Cheju) (Cheju) (Cheju) (Luzhai)
China (Guangxi) China (Tiandong) China (Tiandong) China (Sanjiang) China (Tongliao, Nei Mongu) Korea (Cheju) Honduras
GenBanka
AF445798
AY684274 AY195858
T. saginata T. saginata T. saginata T. T. T. T. T. T. T. T. T. T. T. T.
saginata saginata saginata saginata saginata saginata saginata saginata saginata saginata saginata saginata
T. T. T. T. T. T. T.
saginata saginata saginata saginata solium solium solium*
T. T. T. T. T.
solium solium solium solium solium*
DQ08966
T. solium T. solium
* The life cycle was confirmed in experimental animals.:7-day-old domestic male pig (Duroc–Landrace–Yorkshire) was infected with 500,000 of T. asiatica eggs and the liver was observed after 45 days for T. asiatica (No. 18), and 9-day-old male calf (Holstein-Friesian) was infected with 500,000 of T. saginata eggs and the muscle was observed after 100 days for T. saginata (No. 22). a These numbers correspond to the lane numbers in Figures. b GenBank database Accession Number for the mitochondrial cox1 gene.
ent size as fellow: (1) Ta4978F, specific for T. asiatica (50 -GGG TTT AAG TTA TAA ATG TGA TGT-30 ; nucleotides 4978 to 5001 from
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GenBank Accession No. AF445798); (2) Ts5058F, specific for T. saginata (50 -ACT ACA TTT GGT TTG TTT TTG TAG-30 ; nucleotides 5058 to 5081 from AY684274); and (3) Tso7421F, specific for T. solium (50 -CTA GGC CAC TTA GTA GTT TAG TTA-30 ; nucleotides 7421 to 7444 from AB086256). The reverse primer, Rev7915 (50 -CAT AAA ACA CTC AAA CCT TAT AGA-30 ; nucleotides 5659 to 5685 from AF445798, nucleotides 5657 to 5683 from AY684274, and nucleotides 7870 to 7895 from AB086256), was from a highly conserved sequences common to all of these cestodes (Fig. 1). The PCR was performed in a 50 ll reaction volume consisting of 10 units of TaKaRa LA Taq polymerase (TAKARA SHUZO, Otsu, Japan), 25 mM MgCl2, 2.5 mM dNTP, 10 pmol of each primer and 100 ng of genomic DNA. The optimal ratio of forward-to-reverse primers was 1:3 (0.5 pmol of forward primer, 1.5 pmol of reverse primer). The multiplex PCR protocol consisted of 35 cycles of predenaturation (3 min at 94 °C) denaturation (30 s at 94 °C), annealing (40 s at 45 °C), and extension (60 s at 72 °C), followed by 1 cycle of 5 min at 72 °C.
153
3. Results 3.1. Specificity of multiplex PCR The results of PCR amplification of human-infecting Taenia tapeworm DNA using species-specific primers and multiplex PCR are presented in Fig. 2. A band specific to T. asiatica was obtained in all PCR reactions using T. asiatica-specific primers (Ta4978F and Rev7915). Bands specific to T. saginata and T. solium were also detected in all PCR reactions containing mixtures of T. saginataspecific primers (Ts5058F and Rev7915) and T. solium-specific primers (Tso7421F and Rev7915). The specificity of PCR products was confirmed by cloning and sequencing. Species-specific products with molecular sizes of 706-, 629-, and 474-bp were amplified from T. asiatica (Fig. 2A, lane 1), T. saginata (Fig. 2A, lane 2), and the Asian genotype of T. solium (Fig. 2A, lane 3) in regular PCR reactions, respectively. The diagnostic, species-specific PCR fragments derived using species-specific primers were similar to those
Fig. 1. Relative positions of the species-specific primers used for multiplex PCR in mitochondrial genomes of Taenia asiatica, T. saginata, and T. solium. Forward primers specific to T. asiatica (Ta4978F), T. saginata (Ts5058F), and T. solium (Tso7421F) were designed using the positions between valine transfer RNA and NADH dehydrogenase subunit 2 regions, and reverse primer (Rev7915) common to these species was designed from NADH dehydrogenase subunit 1 region.
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Taenia tapeworms (Fig. 2B, lane 1). Performing multiplex PCR with a mixture of T. asiatica and T. saginata template DNA using the species-specific primer sets generated PCR products of different sizes in species-specific bands (Fig. 2B, lane 2). On the other hand, multiplex PCR with a mixture either T. asiatica and T. solium template DNAs (Fig. 2B, lane 3) or T. saginata and T. solium template DNAs (Fig. 2B, lane 4) was generated clear PCR bands depending on the template present. 3.2. Differential diagnosis of human taeniid tapeworms by multiplex PCR
Fig. 2. Amplification of diagnostic fragments of human-infecting Taenia tapeworms using species-specific primers and multiplex PCR. (A) Lane M, DNA size marker (100-bp ladder); lanes 1–3, PCR products using species-specific primers; lanes 4–6, multiplex PCR; lanes 1 and 4, T. asiatica; lanes 2 and 5, T. saginata; 3 and 6, T. solium. (B) Multiplex PCR with a mixture of all primers and genomic DNA from Taenia tapeworms used in the present study. Lane M, DNA size marker (100-bp ladder); lane 1, T. asiatica, T. saginata, and T. solium; lane 2, T. asiatica and T. saginata; lane 3, T. asiatica and T. solium; lane 4, T. saginata and T. solium.
obtained using multiplex PCR (Fig. 2A, lanes 4–6). In order to differentially diagnose of Taenia tapeworm infections in a single test, multiplex PCR was performed using a mixture of all primers and genomic DNA of Taenia tapeworms (Fig. 2B). Three set of primers (Ta4978F, Ts5058F, and Tso7421F, each with Rev7915) were combined in a single reaction using a mixture of template DNA of three
The 706-bp diagnostic band was generated by multiplex PCR of samples from Asian carriers of T. asiatica from China, Korea and the Philippines. Furthermore, T. asiatica was detected in samples from different areas of China and the Philippines. A 629-bp diagnostic band was detected in the T. saginata samples from Belgium, China, Chile, France, Indonesia, Laos, the Philippines, Poland, Switzerland, Taiwan and Thailand; whereas in the T. solium samples, a 474-bp diagnostic band was detected in the American, African and Asian genotypes. The China isolates were collected from Beijing, Henan, Hongshui, Luzhai, Sanjiang, and the Tiandong province. The diagnostic bands of different species of human taeniid cestodes were observed in all of the samples in this study, including those with distinct genotypes or from diverse geographical regions. No products from negative control samples were amplified by multiplex PCR using any of the primer sets (data not shown). 3.3. Differential diagnosis of tapeworm carriers by multiplex PCR with copro-DNA In order to detect Taenia infections, multiplex PCR was applied to DNA samples prepared from 30 cases with a minimum of 24 helminth eggs per gram of feces. Fig. 3 shows the results obtained by multiplex PCR with copro-DNA extracted from fecal samples of T. saginata tapeworm infections in Khammouane, Lao PDR. The 629-bp diagnostic band unique to T. saginata was clearly demon-
Fig. 3. Differential diagnosis of tapeworms by multiplex PCR on stool specimens. Helminth infections in Lao PDR: lane M, DNA size marker (100-bp ladder); lanes 2, 4, 6, 8, 9, 13, 16, and 28, T. saginata (A and B).
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strated in 8 of 30 cases subjected to multiplex PCR for copro-DNA from egg-positive cases (Fig. 3; lanes 2, 4, 6, 8, 9, 13, 16, and 28). In other words, all eight fecal samples confirmed microscopically as Taenia sp. could be detected and confirmed as T. saginata by this new PCR. The minimum number of Taenia eggs for positive reactions was as low as 10, and all other groups were also positive, containing 50, 100, and 1000 eggs per test. 4. Discussion Identification of species based on the band size in PCR analysis is correlated with species identification based on cox1 sequence analysis. We examined species-specific sequence variations among taeniid cestodes by sequencing 337-bp of the human taeniid mitochondrial cox1 gene. Intraspecies variation in cox1 was tested in 20 isolates of T. asiatica, 22 isolates of T. saginata and 10 isolates of T. solium (Table 1). No nucleotide difference was detected among the 20 isolates of T. asiatica, majority of them was from Korea though, while 1–3 nucleotides (0.2–0.8%) differed in 11 isolates of T. saginata, and 3–6 (0.9–1.7%) differed in 10 isolates of T. solium. The analysis of partial cox1 gene sequences in our study indicated that T. solium isolates from various geographical regions belonged to two genotypes-Cape Verde and Honduras in one type and Korea and China type in the other type-as Nakao et al. (2002) described, while the T. asiatica and T. saginata isolates were almost identical. The DNA sequences of T. saginata cox1 differed slightly variation in 11 isolates from different geographical regions, whereas T. asiatica samples from three different localities were almost identical. The overall sequence difference in mitochondrial cox1 sequences between T. saginata and T. asiatica was 4.6%. The nucleotide sequence of cox1 is thought to be generally conserved. In the present study, the species-specific forward primers (Ta4978F, Ts5058F, and Tso7421F) used in this multiplex PCR assay were designed by using sequences from the mitochondrial valinetRNA, and the reverse primer (Rev7915) was derived from a highly conserved sequences in the atp6 gene. Our differential diagnosis by multiplex PCR provided good resolution of T. saginata and T. asiatica tapeworms possibly due to forward primers that differed by 8-bp out of 24-bp primers including 5-bp deletions especially in T. asiatica between valine tRNA and NADH2. PCR products derived from DNA isolated from fecal samples were detectable where T. saginata infections were present at a minimum concentration of 10 eggs per gram and target gene fragments could be reliably detected at this concentration. Although our methods are effective for differential diagnosis, the assay might not always be suitable for the detection of eggs, since eggs or proglottids are only intermittently present in stools. In this study, the eggs were detected by the Kato–Katz stool examination method, which multiplies the number of eggs counted in the entire field of 41.7 mg of pressed stool smear by 24 in order to obtain the number of eggs per gram of feces. Taeniasis and cysticercosis remain a significant public health concern in regions of Asia, Africa, Eastern Europe, and Central and South America. Ingestion of the eggs of T. solium, T. saginata, and T. asiatica results in bovine or porcine cysticercosis as is known. In pigs metacestodes of T. asiatica are found in visceral organs such as liver, omentum, serosa and lung. If we assume that the metacestode of T. asiatica (C. viscerotropica) follows the same developmental course as observed in intermediate host animals, this could also be the hepatic cysticercosis in human hosts also, although human infectivity by the eggs of T. asiatica still remains controversial (Ito, 1992). The clinical importance of infections by T. asiatica and the potential for cysticercosis attributable to T. asiatica in humans needs further study.
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In the present study, the quality of the differential diagnosis obtained by PCR using species-specific primers was equal in quality to that which is based on nucleotide sequence analysis of the mitochondrial cox1 gene. The multiplex PCR assay with the Ta4978F, Ts5058F, Tso7421F, and Rev7915 primers will be useful for the differential diagnosis, molecular characterization, and epidemiological surveys of T. asiatica, T. saginata, and T. solium. Acknowledgments This work was supported by a research grant from Chungbuk National University in 2006. Parasite materials used in this study were provided by the Parasite Resource Bank of Korea National Research Resource Center (R21-2005-000-10007-0), Republic of Korea. We also thank Drs. Sun Huh, Young-Bae Chung, Hyeon-Jong Yang (Korea); Yichao Yang (China), Stanny Geerts (Belgium), René Houin (France), Alain de Chambrier (Switzerland), M. Teresa GalánPuchades (Spain) for helping collection of samples used in this study. References Anantaphruti, M.T., Yamasaki, H., Nakao, M., Waikagul, J., Watthanakulpanich, D., Nuamtanong, S., Maipanich, W., Pubampen, S., Sanguankiat, S., Muennoo, C., Nakaya, K., Sato, M.O., Sako, Y., Okamoto, M., Ito, A., 2007. Sympatric occurrence of Taenia solium, T. Saginata, and T. asiatica, Thailand. Emerging Infectious Disease 13, 1413–1416. Bowles, J., MacManus, D.P., 1994. Genetic characterization of the Asian Taenia, a newly described taeniid cestodes of human. American Journal of Tropical Medicine and Hygiene 50, 33–44. De Queiroz, A., Alkire, N.L., 1998. The phylogenetic placement of Taenia cestodes that parasitize humans. Journal of Parasitology 84, 379–383. Eom, K.S., Rim, H.J., 1993. Morphologic descriptions of Taenia asiatica sp.. N. Korean Journal of Parasitology 31, 1–6. Eom, K.S., Rim, H.J., 2001. Epidemiological understanding of Taenia tapeworms infections with special reference to Taenia asiatica in Korea. Korean Journal of Parasitology 39, 267–284. Eom, K.S., Jeon, H.K., Kong, Y., Hwang, U.K., Yang, Y., Li, X., Xu, L., Feng, Z., Rim, H.J., 2002. Identification of Taenia asiatica in China: molecular, morphological and epidemiological analysis of a Luzhai isolate. Journal of Parasitology 88, 758– 764. Fan, P.C., Lin, C.Y., Chen, C.C., Chung, W.C., 1995. Morphological description of Taenia saginata asiatica (Cyclophyllidea: Taeniidae) from man in Asia. Journal of Helminthology 69, 299–303. Gonzales, L.M., Montero, E., Morakote, N., Pueute, S., De Serra, T., Lopez-Velez, R., McManus, D.P., Harrison, L.J.S., Parkhouse, R.M.E., Garate, T., Tuesta, J.L.D., 2004. Differential diagnosis of Taenia saginata and Taenia saginata asiatica taeniasis through PCR. Diagnostic Microbiology and Infectious Disease 49, 183–188. Hoberg, E.P., Jones, A., Rausch, R.L., Eom, K.S., Gardner, S.L., 2000. A phylogenetic hyphothesis for species of the genus Taenia (Eucestoda: Taeniidae). Journal of Parasitology 86, 89–98. Ito, A., 1992. Cysticercosis in Asian-Pacific regions. Parasitology Today 8, 181– 183. Jeon, H.K., Lee, K.H., Kim, K.H., Hwang, W.U., Eom, K.S., 2005. Complete sequence and structure of the mitochondrial genome of the human tapeworm, Taenia asiatica (Platyhelminthes; Cestoda). Parasitology 130, 717–726. Jeon, H.K., Eom, K.S., 2006. Taenia asiatica and Taenia saginata: Genetic divergence estimated from their mitochondrial genomes. Experimental Parasitology 113, 58–61. Li, T., Craig, P.S., Ito, A., Chen, X., Qiu, D., Qiu, J., Sato, M.O., Wandra, T., Bradshaw, H., Li, L., Yang, Y., Wang, Q., 2006. Taeniasis/cysticercosis in a Tibetan population in Sichuan province, China. Acta Tropica 100, 223–231. McManus, D.P., 2006. Molecular discrimination of taeniid cestodes. Parasitology International 55, 31–37. Suppl.. Nakao, M., Okamoto, M., Sako, Y., Yamasaki, H., Nakaya, K., Ito, A., 2002. A phylogenetic hypothesis for the distribution of two genotypes of the pig tapeworm Taenia solium worldwide. Parasitology 124, 657–662. Simanjuntak, G.M., Margono, S.S., Okamoto, M., Ito, A., 1997. Taeniasis/cysticercosis in Indonesia as an emerging disease. Parasitology Today 13, 321–323. Willingham, A.L., De, N.V., Doanh, N.Q., Cong le, D., Dung, T.V., Dorny, P., Cam, P.D., Dalsgaard, A., 2003. Current status of cysticercosis in Vietnam. Southeast Asian Journal of Tropical Medicine and Public Health 3, 159–163. Yamasaki, H., Nakao, M., Sako, Y., Nakaya, K., Sato, M.O., Mamuti, W., Okamoto, M., Ito, A., 2002. DNA differential diagnosis of human taeniid cestodes by base excision sequence scanning thymine-base reader analysis with mitochondrial genes. Journal of Clinical Microbiology 40, 3818–3821.
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Differential diagnosis of Taenia asiatica using multiplex PCR Hyeong-Kyu Jeon a, Jong-Yil Chai b, Yoon Kong c, Jitra Waikagul d, Bounnaloth Insisiengmay e, Han-Jong Rim f, Keeseon S. Eom a,* a
Department of Parasitology and Medical Research Institute, Chungbuk National University College of Medicine, Gaeshin-Dong 12, Chongju, Chungbuk 361-763, Republic of Korea Department of Parasitology and Tropical Medicine, Seoul National University College of Medicine, and Institute of Endemic Diseases, Seoul National University Medical Research Center, Seoul, Republic of Korea c Department of Molecular Parasitology and Center for Molecular Medicine, Sungkyunkwan University School of Medicine and Samsung Biomedical Research Institute, Suweon, Republic of Korea d Department of Helminthology, Faculty of Tropical Medicine, Mahidol University, Bangkok 10400, Thailand e Department of Hygiene and Prevention, Ministry of Public Health, Vientiane, Lao People’s Democratic Republic f Department of Parasitology, Korea University College of Medicine, Seoul, Republic of Korea b
a r t i c l e
i n f o
Article history: Received 17 April 2008 Received in revised form 24 October 2008 Accepted 28 October 2008 Available online 5 November 2008 Keywords: Taenia asiatica T. saginata T. solium Multiplex PCR Differential diagnosis
a b s t r a c t 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. Ó 2008 Published by Elsevier Inc.
1. Introduction Taenia solium, T. saginata, and T. asiatica are cestodes that cause taeniasis in humans and cause cysticercosis in intermediate host animals (cows and pigs). Among them, T. asiatica is the most recently described human Taenia tapeworm, with its morphological characteristics and life cycle having been reported. The morphological characteristics of this adult tapeworm include an unarmed rostellum, a large of number of uterine twigs, and a posterior protuberance on the gravid proglottids (Eom and Rim, 1993). The larval tapeworm of T. asiatica (Cysticercus viscerotropica) also differs microscopically from that of T. saginata, in that it has wart-like formations on the external surface of the bladder wall. The phylogenetic relationships between taeniid species have also been studied base on various morphological and molecular characteristics, which has indicated that T. asiatica is a distinct sister species of T. saginata (De Queiroz and Alkire, 1998; Eom et al., 2002; Hoberg et al., 2000; Jeon et al., 2005, Jeon and Eom, 2006). Epidemiologically, T. asiatica has been found in China, Indonesia, Korea, the Philippines, Taiwan, Thailand, and Vietnam * Corresponding author. Fax: +82 43 272 1603. E-mail address: [email protected] (K.S. Eom). 0014-4894/$ - see front matter Ó 2008 Published by Elsevier Inc. doi:10.1016/j.exppara.2008.10.014
(Fan et al., 1995; Simanjuntak et al., 1997; Eom and Rim, 2001; Eom et al., 2002; Willingham et al., 2003; Li et al., 2006; Anantaphruti et al., 2007). T. asiatica and T. saginata exhibit morphological similarities, particularly in the eggs and proglottids of adult worms, which cause frequent confusion in the abovementioned Asian countries where the distributions of the two tapeworms overlap. Consequently, it is necessary to be able to accurately distinguish between these tapeworms. Molecular diagnostic methods have been developed for the rapid and accurate detection of species. Molecular approaches to the differential diagnosis of T. saginata and T. asiatica have been developed, including the use of sequence-specific DNA probes, PCR coupled to restriction fragment length polymorphisms (Bowles and MacManus, 1994; Yamasaki et al., 2002; McManus, 2006), and multiplex PCR (Gonzales et al., 2004; Yamasaki et al., 2004). A mitochondrial-based multiplex PCR has been applied to diagnose of three human Taenia tapeworms and two genotypes of T. solium (Yamasaki et al., 2004, 2007). In the present study, we used species-specific primers based on the mitochondrial sequences for T. asiatica, T. saginata and T. solium, and amplified fragments of different lengths from other species. Our method employs a high-resolution multiplex PCR
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assay that could be used for the differential diagnosis of humaninfecting Taenia tapeworms.
Table 1 Taenia tapeworm samples in the present study. No
Status of samples
Locality
cox1 sequence and multiplex PCR finding
1 2
Korea (Chuncheon) Korea (Chongju)
T. asiatica T. asiatica*
Korea Korea Korea Korea Korea Korea Koera Korea Koera Koera Korea Koera Koera Koera Koera China
T. T. T. T. T. T. T. T. T. T. T. T. T. T. T. T.
asiatica asiatica asiatica asiatica asiatica asiatica asiatica asiatica asiatica asiatica asiatica asiatica asiatica asiatica asiatica asiatica*
China (Binyang) Philippines Belgium Belgium
T. T. T. T.
asiatica asiatica saginata saginata*
23 24 25
Proglottid Proglottid, eggsb Proglottid Proglottid Proglottid Proglottid Proglottid Proglottid Proglottid Proglottid Proglottid Proglottid Proglottid Proglottid Proglottid Proglottid Proglottid Proglottid, cysticercusb Proglottid Proglottid Proglottid Proglottid, cysticercusb Proglottid Proglottid Proglottid
T. saginata T. saginata T. saginata
26
Proglottid
27
Proglottid
28
Proglottid
29 30 31 32 33 34 35 36 37 38 39 40
Proglottid Proglottid Proglottid Proglottid Proglottid Peoglottid Proglottid Proglottid Proglottid Proglottid Proglottid Proglottid
41 42 43 44 45 46 47
Proglottid Proglottid Proglottid Proglottid Proglottid Proglottid Proglottid, cysticercusb Proglottid Proglottid Proglottid Proglottid Proglottid, cysticercusb Proglottid Proglottid
Belgium China (Beijing) China (Hongshui, Gauangxi) China (Hongshui, Gauangxi) China (Hongshui, Gauangxi) China (Hongshui, Gauangxi) Chile Ethiopia Indonesia France Korea (Busan) Korea (Jeju) Laos (Savanakhet) Laos (Savanakhet) Laos (Savanakhet) Philippines Poland Swiss (Natural History Museum) Taiwan Thailand Thailand Thailand Cape Verde China (Henan) China (Luzhai)
2. Materials and methods 2.1. Parasites The parasites were collected from 54 individuals having proven parasitosis, and selected on the basis of a parasitological examination and identification of eggs, larvae, or adult worms. Fifty-two samples were analyzed in this study, comprising 20 samples of T. asiatica genomic DNA from China (n = 2), Korea (n = 17) and the Philippines (n = 1); 24 samples of T. saginata from Belgium (n = 3), Chile (n = 1), China (n = 5), Ethiopia (n = 1), France (n = 1), Indonesia (n = 1), Korea (n = 2), Laos (n = 3), Poland (n = 1), the Philippines (n = 1), Taiwan (n = 1), Thailand (n = 3), and Switzerland (n = 1); and 10 samples of T. solium from Cape Verde (n = 1), China (n = 7), Honduras (n = 1) and Korea (n = 1). For molecular characterization, all of the materials used (i.e., proglottids, cysticerci, and eggs) were previously confirmed from taeniid tapeworms by sequencing of the cox1 gene (Table 1). The negative control samples used were T. crassiceps (donated by Dr. Y. Kong, Korea), T. taeniaeformis (donated by Dr. W.M. Sohn, Korea), and Echinococcus multilocularis (donated by Dr. M. Kamiya, Japan). 2.2. Fecal samples Fecal samples were obtained from 30 individuals infected with helminthes from Khammouane province, LaoPDR, in 2003. The stool samples from eight Taenia infections were collected from carriers who discharged gravid proglottids. Taenia-positive fecal samples were identified using eggs and adult worms by morphological examination and multiplex PCR. These fecal samples were stored at 20 °C until use. 2.3. DNA extraction Total genomic DNA was extracted from adult worms, eggs, and metacestodes using the DNeasy Tissue Kit (Qiagen, Valencia, CA, USA) according to the manufacturer’s instructions and used as template DNA for the PCR. A single frozen specimen was chopped into small pieces and then digested in tissue lysis buffer of the DNA extraction kit. A single worm was enzymatically digested in 180 ll of a lysis solution (ATL buffer, Qiagen), then 20 ll of proteinase K (50 lg/ml) was added, and the sample was incubated at 56 °C for 2–3 h with vortexing every 30 min. Genomic DNA of eggs was extracted using the QIAamp Stool Kit (Qiagen). A mixture comprising 200 mg of stool sample in 200 ll of buffer (ASL, Qiagen) was incubated for 5 min at 95 °C. One inhibition tablet (inhibitEX, Qiagen) was then added, and the supernatant collected by centrifugation was put in a lysis buffer after adding 200 ll of buffer (AL, Qiagen) containing guanidine hydrochloride and 15 ll of RNase A (100 mg/ ml) and was mixed for 15 s, and then incubated at 70 °C for 10 min. Ethanol (200 ll) was added, and the mixture was vortexed for 15 s and then added to a DNA-binding column and spun down for 1 min. The column was then washed several times using AW1 and AW2 buffers (Qiagen). The genomic DNA extract was diluted to a working concentration of 50 ng/ll, and 1 ll of it was used as a template in a PCR. 2.4. Primer design and multiplex PCR Three species-specific forward primers were prepared based on the nucleotide sequences of valine transfer RNA (tRNA) and NADH dehydrogenase subunit 2 from the human taeniid cestodes. The three forward primers were designed to amplify products of differ-
3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22
48 49 50 51 52 53 54
(Chuncheon) (Wanju) (Hwasun) (Hwasun) (Hwasun) (Youngju) (Wando) (Chuncheon) (Ansan) (Cheonan) (Cheju) (Cheju) (Cheju) (Cheju) (Cheju) (Luzhai)
China (Guangxi) China (Tiandong) China (Tiandong) China (Sanjiang) China (Tongliao, Nei Mongu) Korea (Cheju) Honduras
GenBanka
AF445798
AY684274 AY195858
T. saginata T. saginata T. saginata T. T. T. T. T. T. T. T. T. T. T. T.
saginata saginata saginata saginata saginata saginata saginata saginata saginata saginata saginata saginata
T. T. T. T. T. T. T.
saginata saginata saginata saginata solium solium solium*
T. T. T. T. T.
solium solium solium solium solium*
DQ08966
T. solium T. solium
* The life cycle was confirmed in experimental animals.:7-day-old domestic male pig (Duroc–Landrace–Yorkshire) was infected with 500,000 of T. asiatica eggs and the liver was observed after 45 days for T. asiatica (No. 18), and 9-day-old male calf (Holstein-Friesian) was infected with 500,000 of T. saginata eggs and the muscle was observed after 100 days for T. saginata (No. 22). a These numbers correspond to the lane numbers in Figures. b GenBank database Accession Number for the mitochondrial cox1 gene.
ent size as fellow: (1) Ta4978F, specific for T. asiatica (50 -GGG TTT AAG TTA TAA ATG TGA TGT-30 ; nucleotides 4978 to 5001 from
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GenBank Accession No. AF445798); (2) Ts5058F, specific for T. saginata (50 -ACT ACA TTT GGT TTG TTT TTG TAG-30 ; nucleotides 5058 to 5081 from AY684274); and (3) Tso7421F, specific for T. solium (50 -CTA GGC CAC TTA GTA GTT TAG TTA-30 ; nucleotides 7421 to 7444 from AB086256). The reverse primer, Rev7915 (50 -CAT AAA ACA CTC AAA CCT TAT AGA-30 ; nucleotides 5659 to 5685 from AF445798, nucleotides 5657 to 5683 from AY684274, and nucleotides 7870 to 7895 from AB086256), was from a highly conserved sequences common to all of these cestodes (Fig. 1). The PCR was performed in a 50 ll reaction volume consisting of 10 units of TaKaRa LA Taq polymerase (TAKARA SHUZO, Otsu, Japan), 25 mM MgCl2, 2.5 mM dNTP, 10 pmol of each primer and 100 ng of genomic DNA. The optimal ratio of forward-to-reverse primers was 1:3 (0.5 pmol of forward primer, 1.5 pmol of reverse primer). The multiplex PCR protocol consisted of 35 cycles of predenaturation (3 min at 94 °C) denaturation (30 s at 94 °C), annealing (40 s at 45 °C), and extension (60 s at 72 °C), followed by 1 cycle of 5 min at 72 °C.
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3. Results 3.1. Specificity of multiplex PCR The results of PCR amplification of human-infecting Taenia tapeworm DNA using species-specific primers and multiplex PCR are presented in Fig. 2. A band specific to T. asiatica was obtained in all PCR reactions using T. asiatica-specific primers (Ta4978F and Rev7915). Bands specific to T. saginata and T. solium were also detected in all PCR reactions containing mixtures of T. saginataspecific primers (Ts5058F and Rev7915) and T. solium-specific primers (Tso7421F and Rev7915). The specificity of PCR products was confirmed by cloning and sequencing. Species-specific products with molecular sizes of 706-, 629-, and 474-bp were amplified from T. asiatica (Fig. 2A, lane 1), T. saginata (Fig. 2A, lane 2), and the Asian genotype of T. solium (Fig. 2A, lane 3) in regular PCR reactions, respectively. The diagnostic, species-specific PCR fragments derived using species-specific primers were similar to those
Fig. 1. Relative positions of the species-specific primers used for multiplex PCR in mitochondrial genomes of Taenia asiatica, T. saginata, and T. solium. Forward primers specific to T. asiatica (Ta4978F), T. saginata (Ts5058F), and T. solium (Tso7421F) were designed using the positions between valine transfer RNA and NADH dehydrogenase subunit 2 regions, and reverse primer (Rev7915) common to these species was designed from NADH dehydrogenase subunit 1 region.
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Taenia tapeworms (Fig. 2B, lane 1). Performing multiplex PCR with a mixture of T. asiatica and T. saginata template DNA using the species-specific primer sets generated PCR products of different sizes in species-specific bands (Fig. 2B, lane 2). On the other hand, multiplex PCR with a mixture either T. asiatica and T. solium template DNAs (Fig. 2B, lane 3) or T. saginata and T. solium template DNAs (Fig. 2B, lane 4) was generated clear PCR bands depending on the template present. 3.2. Differential diagnosis of human taeniid tapeworms by multiplex PCR
Fig. 2. Amplification of diagnostic fragments of human-infecting Taenia tapeworms using species-specific primers and multiplex PCR. (A) Lane M, DNA size marker (100-bp ladder); lanes 1–3, PCR products using species-specific primers; lanes 4–6, multiplex PCR; lanes 1 and 4, T. asiatica; lanes 2 and 5, T. saginata; 3 and 6, T. solium. (B) Multiplex PCR with a mixture of all primers and genomic DNA from Taenia tapeworms used in the present study. Lane M, DNA size marker (100-bp ladder); lane 1, T. asiatica, T. saginata, and T. solium; lane 2, T. asiatica and T. saginata; lane 3, T. asiatica and T. solium; lane 4, T. saginata and T. solium.
obtained using multiplex PCR (Fig. 2A, lanes 4–6). In order to differentially diagnose of Taenia tapeworm infections in a single test, multiplex PCR was performed using a mixture of all primers and genomic DNA of Taenia tapeworms (Fig. 2B). Three set of primers (Ta4978F, Ts5058F, and Tso7421F, each with Rev7915) were combined in a single reaction using a mixture of template DNA of three
The 706-bp diagnostic band was generated by multiplex PCR of samples from Asian carriers of T. asiatica from China, Korea and the Philippines. Furthermore, T. asiatica was detected in samples from different areas of China and the Philippines. A 629-bp diagnostic band was detected in the T. saginata samples from Belgium, China, Chile, France, Indonesia, Laos, the Philippines, Poland, Switzerland, Taiwan and Thailand; whereas in the T. solium samples, a 474-bp diagnostic band was detected in the American, African and Asian genotypes. The China isolates were collected from Beijing, Henan, Hongshui, Luzhai, Sanjiang, and the Tiandong province. The diagnostic bands of different species of human taeniid cestodes were observed in all of the samples in this study, including those with distinct genotypes or from diverse geographical regions. No products from negative control samples were amplified by multiplex PCR using any of the primer sets (data not shown). 3.3. Differential diagnosis of tapeworm carriers by multiplex PCR with copro-DNA In order to detect Taenia infections, multiplex PCR was applied to DNA samples prepared from 30 cases with a minimum of 24 helminth eggs per gram of feces. Fig. 3 shows the results obtained by multiplex PCR with copro-DNA extracted from fecal samples of T. saginata tapeworm infections in Khammouane, Lao PDR. The 629-bp diagnostic band unique to T. saginata was clearly demon-
Fig. 3. Differential diagnosis of tapeworms by multiplex PCR on stool specimens. Helminth infections in Lao PDR: lane M, DNA size marker (100-bp ladder); lanes 2, 4, 6, 8, 9, 13, 16, and 28, T. saginata (A and B).
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strated in 8 of 30 cases subjected to multiplex PCR for copro-DNA from egg-positive cases (Fig. 3; lanes 2, 4, 6, 8, 9, 13, 16, and 28). In other words, all eight fecal samples confirmed microscopically as Taenia sp. could be detected and confirmed as T. saginata by this new PCR. The minimum number of Taenia eggs for positive reactions was as low as 10, and all other groups were also positive, containing 50, 100, and 1000 eggs per test. 4. Discussion Identification of species based on the band size in PCR analysis is correlated with species identification based on cox1 sequence analysis. We examined species-specific sequence variations among taeniid cestodes by sequencing 337-bp of the human taeniid mitochondrial cox1 gene. Intraspecies variation in cox1 was tested in 20 isolates of T. asiatica, 22 isolates of T. saginata and 10 isolates of T. solium (Table 1). No nucleotide difference was detected among the 20 isolates of T. asiatica, majority of them was from Korea though, while 1–3 nucleotides (0.2–0.8%) differed in 11 isolates of T. saginata, and 3–6 (0.9–1.7%) differed in 10 isolates of T. solium. The analysis of partial cox1 gene sequences in our study indicated that T. solium isolates from various geographical regions belonged to two genotypes-Cape Verde and Honduras in one type and Korea and China type in the other type-as Nakao et al. (2002) described, while the T. asiatica and T. saginata isolates were almost identical. The DNA sequences of T. saginata cox1 differed slightly variation in 11 isolates from different geographical regions, whereas T. asiatica samples from three different localities were almost identical. The overall sequence difference in mitochondrial cox1 sequences between T. saginata and T. asiatica was 4.6%. The nucleotide sequence of cox1 is thought to be generally conserved. In the present study, the species-specific forward primers (Ta4978F, Ts5058F, and Tso7421F) used in this multiplex PCR assay were designed by using sequences from the mitochondrial valinetRNA, and the reverse primer (Rev7915) was derived from a highly conserved sequences in the atp6 gene. Our differential diagnosis by multiplex PCR provided good resolution of T. saginata and T. asiatica tapeworms possibly due to forward primers that differed by 8-bp out of 24-bp primers including 5-bp deletions especially in T. asiatica between valine tRNA and NADH2. PCR products derived from DNA isolated from fecal samples were detectable where T. saginata infections were present at a minimum concentration of 10 eggs per gram and target gene fragments could be reliably detected at this concentration. Although our methods are effective for differential diagnosis, the assay might not always be suitable for the detection of eggs, since eggs or proglottids are only intermittently present in stools. In this study, the eggs were detected by the Kato–Katz stool examination method, which multiplies the number of eggs counted in the entire field of 41.7 mg of pressed stool smear by 24 in order to obtain the number of eggs per gram of feces. Taeniasis and cysticercosis remain a significant public health concern in regions of Asia, Africa, Eastern Europe, and Central and South America. Ingestion of the eggs of T. solium, T. saginata, and T. asiatica results in bovine or porcine cysticercosis as is known. In pigs metacestodes of T. asiatica are found in visceral organs such as liver, omentum, serosa and lung. If we assume that the metacestode of T. asiatica (C. viscerotropica) follows the same developmental course as observed in intermediate host animals, this could also be the hepatic cysticercosis in human hosts also, although human infectivity by the eggs of T. asiatica still remains controversial (Ito, 1992). The clinical importance of infections by T. asiatica and the potential for cysticercosis attributable to T. asiatica in humans needs further study.
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In the present study, the quality of the differential diagnosis obtained by PCR using species-specific primers was equal in quality to that which is based on nucleotide sequence analysis of the mitochondrial cox1 gene. The multiplex PCR assay with the Ta4978F, Ts5058F, Tso7421F, and Rev7915 primers will be useful for the differential diagnosis, molecular characterization, and epidemiological surveys of T. asiatica, T. saginata, and T. solium. Acknowledgments This work was supported by a research grant from Chungbuk National University in 2006. Parasite materials used in this study were provided by the Parasite Resource Bank of Korea National Research Resource Center (R21-2005-000-10007-0), Republic of Korea. We also thank Drs. Sun Huh, Young-Bae Chung, Hyeon-Jong Yang (Korea); Yichao Yang (China), Stanny Geerts (Belgium), René Houin (France), Alain de Chambrier (Switzerland), M. Teresa GalánPuchades (Spain) for helping collection of samples used in this study. References Anantaphruti, M.T., Yamasaki, H., Nakao, M., Waikagul, J., Watthanakulpanich, D., Nuamtanong, S., Maipanich, W., Pubampen, S., Sanguankiat, S., Muennoo, C., Nakaya, K., Sato, M.O., Sako, Y., Okamoto, M., Ito, A., 2007. Sympatric occurrence of Taenia solium, T. Saginata, and T. asiatica, Thailand. Emerging Infectious Disease 13, 1413–1416. Bowles, J., MacManus, D.P., 1994. Genetic characterization of the Asian Taenia, a newly described taeniid cestodes of human. American Journal of Tropical Medicine and Hygiene 50, 33–44. De Queiroz, A., Alkire, N.L., 1998. The phylogenetic placement of Taenia cestodes that parasitize humans. Journal of Parasitology 84, 379–383. Eom, K.S., Rim, H.J., 1993. Morphologic descriptions of Taenia asiatica sp.. N. Korean Journal of Parasitology 31, 1–6. Eom, K.S., Rim, H.J., 2001. Epidemiological understanding of Taenia tapeworms infections with special reference to Taenia asiatica in Korea. Korean Journal of Parasitology 39, 267–284. Eom, K.S., Jeon, H.K., Kong, Y., Hwang, U.K., Yang, Y., Li, X., Xu, L., Feng, Z., Rim, H.J., 2002. Identification of Taenia asiatica in China: molecular, morphological and epidemiological analysis of a Luzhai isolate. Journal of Parasitology 88, 758– 764. Fan, P.C., Lin, C.Y., Chen, C.C., Chung, W.C., 1995. Morphological description of Taenia saginata asiatica (Cyclophyllidea: Taeniidae) from man in Asia. Journal of Helminthology 69, 299–303. Gonzales, L.M., Montero, E., Morakote, N., Pueute, S., De Serra, T., Lopez-Velez, R., McManus, D.P., Harrison, L.J.S., Parkhouse, R.M.E., Garate, T., Tuesta, J.L.D., 2004. Differential diagnosis of Taenia saginata and Taenia saginata asiatica taeniasis through PCR. Diagnostic Microbiology and Infectious Disease 49, 183–188. Hoberg, E.P., Jones, A., Rausch, R.L., Eom, K.S., Gardner, S.L., 2000. A phylogenetic hyphothesis for species of the genus Taenia (Eucestoda: Taeniidae). Journal of Parasitology 86, 89–98. Ito, A., 1992. Cysticercosis in Asian-Pacific regions. Parasitology Today 8, 181– 183. Jeon, H.K., Lee, K.H., Kim, K.H., Hwang, W.U., Eom, K.S., 2005. Complete sequence and structure of the mitochondrial genome of the human tapeworm, Taenia asiatica (Platyhelminthes; Cestoda). Parasitology 130, 717–726. Jeon, H.K., Eom, K.S., 2006. Taenia asiatica and Taenia saginata: Genetic divergence estimated from their mitochondrial genomes. Experimental Parasitology 113, 58–61. Li, T., Craig, P.S., Ito, A., Chen, X., Qiu, D., Qiu, J., Sato, M.O., Wandra, T., Bradshaw, H., Li, L., Yang, Y., Wang, Q., 2006. Taeniasis/cysticercosis in a Tibetan population in Sichuan province, China. Acta Tropica 100, 223–231. McManus, D.P., 2006. Molecular discrimination of taeniid cestodes. Parasitology International 55, 31–37. Suppl.. Nakao, M., Okamoto, M., Sako, Y., Yamasaki, H., Nakaya, K., Ito, A., 2002. A phylogenetic hypothesis for the distribution of two genotypes of the pig tapeworm Taenia solium worldwide. Parasitology 124, 657–662. Simanjuntak, G.M., Margono, S.S., Okamoto, M., Ito, A., 1997. Taeniasis/cysticercosis in Indonesia as an emerging disease. Parasitology Today 13, 321–323. Willingham, A.L., De, N.V., Doanh, N.Q., Cong le, D., Dung, T.V., Dorny, P., Cam, P.D., Dalsgaard, A., 2003. Current status of cysticercosis in Vietnam. Southeast Asian Journal of Tropical Medicine and Public Health 3, 159–163. Yamasaki, H., Nakao, M., Sako, Y., Nakaya, K., Sato, M.O., Mamuti, W., Okamoto, M., Ito, A., 2002. DNA differential diagnosis of human taeniid cestodes by base excision sequence scanning thymine-base reader analysis with mitochondrial genes. Journal of Clinical Microbiology 40, 3818–3821.
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