Sympatric occurrence of Taenia solium, T. saginata, and T. asiatica, Thailand.

We confirmed sympatric occurrence of Taenia solium, T. saginata, and T. asiatica in western Thailand. DNA analysis of morphologically identified T. saginata, in a dual infection with T. solium, indicated it was T. asiatica. To our knowledge, this report is the first of T. asiatica and a dual Taenia infection from Thailand.

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Taeniid tapeworm infections in the human intestine are caused by Taenia solium, T. saginata, and T. asiatica in Asia and the Pacific (1-3). Taeniasis caused by T. solium is a serious public health problem worldwide because eggs and proglottids expelled in the stool can infect humans through contamination of the environment and cause fatal neurocysticercosis. Neurocysticercosis cases caused by T. solium have increased in non-taeniasis-endemic areas (3-5).

A related taeniid tapeworm, Asian Taenia (= T. asiatica), has been described in Taiwan and the Republic of Korea (1-3,6-8). Although T. asiatica is phylogenetically closely related and is considered to be a sister species of T. saginata (1-3,6,7), the important intermediate host for T. asiatica is the domestic pig and the metacestodes mainly develop in the pigs' liver (6). The morphologic characteristics of adult T. asiatica are very similar to those of T. saginata. Morphologic differentiation by either scolex or gravid proglottid of these 2 species is practically impossible (1,3). On the basis of molecular analysis of taeniid isolates from Asia and the Pacific, T. asiatica is distributed in Taiwan, the Republic of Korea, Malaysia, People's Republic of China, Philippines, Indonesia, and Vietnam (1-3,6-11). However, there has been no evidence of the distribution of T. asiatica in Thailand (8,12).

The Study

The field investigation was conducted during 2002-2005 in communities in Thong Pha Phum District, 150 km northwest of Kanchanaburi Province, Thailand, close to the Myanmar border (Figure 1). The region is mountainous terrain that acts as a natural border between Thailand and Myanmar, and it mostly comprises natural parks and a water reservoir. The major studied population is Karen; Mon and Thai are minorities. In our study, molecular identification of these taeniid samples was conducted in Asahikawa, Japan in 2006. The study team, the Faculty of Tropical Medicine at Mahidol University, obtained ethical approval for a human stool survey for control of helminthic infections.

[FIGURE 1 OMITTED]

All taeniasis patients were informed of the study objectives and worm collection procedure through discussions, including about expulsion of proglottids. Tapeworms, either with scolices or without scolices, were expelled from 24 persons with taeniasis after the persons received 2 g of niclosamide or 40 mg/kg of praziquantel and a purgative. A total of 29 scolices were confirmed. Scolices with hooks expelled from 5 patients (nos. 2, 4, 7, 9, 11) were identified as T. solium. Scolices without hooks from 12 patients were identified as T. saginata because no molecular evidence on the distribution of T. asiatica in Thailand exists and all Taenia scolices without hooks were confirmed to be T. saginata in other areas in Thailand (8,10,12). Morphologic identification of the species was based on scolex only. Specimens without scolices were not identified morphologically. Most patients (12/17) harbored a single scolex. However, several patients harbored 2-6 scolices, including 1 dual infection with 2 T. solium and 1 T. saginata (patient 7) (Table). Patients were 7-60 years of age; 16 were male, and 8 were female.

Nineteen Taenia samples were fixed in 80% ethanol and kept at -20[degrees]C until use. Four samples were fixed in 10% formalin. All scolices were fixed with alcohol-formalin-acetic acid and stained with acetocarmine for morphologic comparative examination. One scolex with or without hooklets each from a patient 7 was further processed for molecular studies.

DNA samples were extracted from taeniid proglottids except for patient 7, for whom DNA was extracted from 2 scolices. DNeasy Blood and Tissue Kit (QIAGEN, Hilden, Germany) was used for the samples kept in ethanol. A DNA Isolator PS kit (Wako Pure Chemicals, Osaka, Japan) and DEXPAT (TaKaRa Shuzo, Shiga, Japan) were used for the formalin-fixed proglottids. DNA samples from 2 scolices expelled from patient 7 stained with acetocarmine were prepared by using DEXPAT and 0.05 N NaOH/1% sodium dodecyl sulfate containing proteinase K. Mitochondrial DNA diagnosis of ethanol-fixed samples was performed by multiplex PCR by using cytochrome c oxidase subunit 1 gene (cox1), except for the use of a forward primer (5'-TTATTTATTTACGTCAATCTTATTG-3', positions 561-585) for T. asiatica (10). The formalin-fixed and acetocarmine-stained specimens were identified by base excision sequence scanning thymine-base (BESS T-base) analysis that used either cox1 or cytochrome b gene (cob) (13). For BESS T-base analysis, the following primers were used: F3 (5'-TATTTGATCGTAAATTTAGTTCT3', corresponding to nucleotide (nt) positions 629-651) and R7 (5'-ATTAACACATAAACCTCGGGA-3', nt positions 740-720) for cox1 of T. solium, F1 (5'-GTCAAAAGATTCTTTTTTTACTTGGT-3', nt positions 180-205) and R2 (5'-CCCTTCTTTCTATAACTTGAATAAT-3', nt positions 305-281) for cob for T. solium. DNA sequencing of the products amplified by multiplex PCR was performed for confirmation. DNA samples for sequencing were prepared with a BigDye Terminator v3.1 Cycle Sequencing Kit (Applied Biosystems, Foster City, CA, USA). DNA sequencing was performed on an ABI PRISM 310 Genetic Analyzer (Applied Biosystems), and the nucleotide sequence data were analyzed by using DNASTAR version 3.75 (DNASTAR Inc., Madison, WI, USA).

Multiplex PCR applied on 19 proglottids from 17 patients with cox1 (10) showed that 5, 7, and 7 proglottids were T. solium (Asian genotype) (10,13), T. saginata, and T. asiatica, respectively (data not shown). These results were supported by DNA sequencing of the amplicons (data not shown). By contrast, small sizes of 112-bp cox1 products were successfully amplified from samples taken from patients 3-6. These samples had been preserved in 10% formalin for years and BESS T-base analysis indicated that they were T. solium (Asian genotype) (Figure 2A) (14). BESS T-base analysis showed that scolices with and without hooklets from a dual infection (patient 7) were T. solium (Asian genotype) and T. asiatica, respectively (Figure 2B and C). To our knowledge, this is the first report demonstrating a dual infection with T. solium and T. asiatica in which 3 human taeniid cestodes are sympatrically distributed (1).

[FIGURE 2 OMITTED]

Conclusions

We documented sympatric distribution of T. solium, T. saginata, and T. asiatica in western Thailand on the basis of mitochondrial DNA analysis. Our study indicated that 53.3% (8/15) of taeniid specimens expected to be T. saginata were T. asiatica and that both T. asiatica and T. saginata are codistributed in Kanchanaburi Province. Although T. solium taeniasis has seldom been reported in the literature in Thailand (15), our study has shown infection with T. solium (Asian genotype) in 11 (45.8%) of 24 Taenia-infected patients. The number of T. solium organisms expelled from taeniasis patients varied from 1 to 6, and [greater than or equal to] 2 tapeworms were found in 36.4% (4/11) of T. solium taeniasis patients. In addition, we confirmed a dual infection with T. solium and T. asiatica (in patient 7). This experience indicates that molecular analysis is preferable and necessary for precise re-identification of so-called T. saginata in Asia and the Pacific (1).

Although T. solium cysticercosis in humans has not been reported in this study area, these populations appear to pose a risk for environmental contamination and person-to-person spread of T. solium leading to cysticercosis in humans and swine. Raw or inadequately cooked beef, pork, or pig viscera, and fresh blood are commonly consumed by local people in the study areas, and consequently they are at high risk of acquiring taeniasis. Therefore, to improve sanitation and quality of life, sustainable health education should be introduced and stressed to the population in the community.

Acknowledgments

We thank Vajiralongkom Dam for accommodations during our field work and Peter M. Schantz for his comments and suggestions for improving this article.

The field survey in Thailand from 2002 until 2005 was funded by Mahidol University research grant 02011285-0002 to J.W. The molecular work was supported by a grant-in-aid for Scientific Research from the Japan Society for Promotion of Science (JSPS) to A.I. (14256001, 17256002) and to M.O. (18406008) and by a JSPS-Asia/Africa Sciences Platform Fund (2006-2008) to A.I.

Dr Anantaphruti is an associate professor in the Department of Helminthology, Faculty of Tropical Medicine, Mahidol University, Bangkok, Thailand. Her research interests include epidemiology, drug trials, and immunodiagnosis of helminthic infections, particularly cestode zoonoses.

References

(1.) Ito A, Nakao M, Wandra T. Human taeniasis and cysticercosis in Asia. Lancet. 2003;362:1918-20.

(2.) Ito A, Wandra T, Yamasaki H, Nakao M, Sako Y, Nakaya K, et al. Review: cysticercosis/taeniasis in Asia and the Pacific. Vector Borne Zoonotic Dis. 2004;4:95-107.

(3.) Ito A, Craig PS, Schantz PM. Taeniasis/cysticercosis and echinococcosis with focus on Asia and the Pacific. Parasitol Int. 2006;55: S1-308.

(4.) Schantz PM, Moore AC, Munoz JL, Hartman B J, Schaefer JA, Aron AM, et al. Neurocysticercosis in an orthodox Jewish community in New York City. N Engl J Med. 1992;327:692-5.

(5.) Hira PR, Francis I, Abdella NA, Gupta R, Ai-Ali FM, Grover S, et al. Cysticercosis: imported and autochthonous infections in Kuwait. Trans R Soc Trop Med Hyg. 2004;98:233-9.

(6.) Fan PC. Taiwan Taenia and taeniasis. Parasitol Today. 1988;4:86-8.

(7.) Eom KS, Rim HJ. Morphological descriptions of Taenia asiatica sp. n. Korean J Parasitol. 1993;31:1-6.

(8.) Bowles J, McManus DE Genetic characterization of the Asian Taenia, a newly described taeniid cestode of humans. Am J Trop Med Hyg. 1994;50:33-44.

(9.) De NV, Hoa LT, Doanh NQ, Ngoc NB, Cong LD. Report on a new species of Taenia (Taenia asiatica) in Hanoi, Vietnam. J Malaria Parasit Dis Control. 2001;3:80-5.

(10.) Yamasaki H, Allan JC, Sato MO, Nakao M, Sako Y, Nakaya K, et al. DNA differential diagnosis of taeniasis and cysticercosis by multiplex PCR. J Clin Microbiol. 2004;42:548-53.

(11.) Wandra T, Depary AA, Sutisna P, Margono SS, Suroso T, Okamoto M, et al. Taeniasis and cysticercosis in Bali and North Sumatra, Indonesia. Parasitol Int. 2006;55:S155-60.

(12.) Morakote N, Wijit A, Uparanukraw P. Further search for Taenia saginata asiatica in Chiang Mai, Thailand. Ann Trop Med Parasitol. 2000;94:521-4.

(13.) Nakao M, Okamoto M, Sako Y, Yamasaki H, Nakaya K, Ito A. A phylogenetic hypothesis for the distribution of two genotypes of the pig tapeworm Taenia solium worldwide. Parasitology. 2002;124:657-62.

(14.) Yamasaki H, Nakao M, Sako Y, Nakaya K, Sato MO, Mamuti W, et al. DNA differential diagnosis of human taeniid cestodes by base excision sequence scanning thymine-base reader analysis with mitochondrial genes. J Clin Microbiol. 2002;40:3818-21.

(15.) Anantaphruti MT. Human taeniasis in Thailand. In: Ito A, Wen H, Yamasaki H, editors. Asian Parasitology, vol. 2. Taeniasis/cysticercosis and echinococcosis in Asia, Chiba (Japan): FAP Journal Ltd; 2005. p. 89-98.

Address for correspondence: Akira Ito, Department of Parasitology, Asahikawa Medical College, Midorigaoka-Higashi 2-1-1-1, Asahikawa 078-8510, Japan; email: [e-mail]

Malinee T. Anantaphruti, * ([dagger]) (1) Hiroshi Yamasaki, ([dagger]) (1) Minoru Nakao, ([dagger]) Jitra Waikagul, * Dorn Watthanakulpanich, * ([dagger]) Supaporn Nuamtanong, * Wanna Maipanich, * Somchit Pubampen, * Surapol Sanguankiat, * Chatree Muennoo, * Kazuhiro Nakaya, ([dagger]) Marcello O. Sato, ([dagger]) ([double dagger]) Yasuhito Sako, ([dagger]) Munehiro Okamoto, [section] and Akira Ito ([dagger]) (1)

* Mahidol University, Bangkok, Thailand; ([dagger]) Asahikawa Medical College, Asahikawa, Japan; ([double dagger]) Universidade Federal do Tocantins, Araguaina, Brazil; and ([section]) Tottori University, Tottori, Japan

(1) These authors contributed equally to this article.

Table. Characteristics of 24 taeniasis cases, Thailand, 2002-2005 *

        Patient                    No. scolex/
                                  proglottids
No.   Sex  Age, y   Year            expelled

1      M     60     2003    1 scolex without hook
2      F     34     2005    3 scolices with hooks
3      M     55     2002   Segment without scolex
                             ([dagger])
4      M     29     2002   6 scolices with hooks
5      F     38     2003   Segment without scolex
                             ([dagger])
6      F     46     2002   Segment without scolex
                             ([dagger])
7      F     28     2002    2 scolices with hooks,
                            1 scolex without hook
8      M     NK     2002   Segment without scolex
                             ([dagger])
9      M     47     2004     1 scolex with hooks
10     M     7      2004   Segment without scolex
                             ([dagger])
11     M     43     2004    3 scolices with hooks
12     M     10     2004   Segment without scolex
                             ([dagger])
13     M     40     2003   Segment without scolex
                             ([dagger])
14     F     NK     2002   Segment without scolex
                             ([dagger])
15     M     40     2003    1 scolex without hook
16     M     55     2004    1 scolex without hook
17     F     30     2004    1 scolex without hook
18     M     13     2004    1 scolex without hook
19     M     45     2005    1 scolex without hook
20     F     42     2003    3 scolices without hook
21     F     28     2005    1 scolex without hook
22     M     60     2005    1 scolex without hook
23     M     32     2005    1 scolex without hook
24     M     37     2005    1 scolex without hook

Patient     Morphologic
          identification    Preservative       Molecular
No.          of scolex          used        identification

1         Taenia saginata        NA               NT
2            T. solium           NA               NT
3               --            Formalin         T. solium
4            T. solium        Formalin         T. solium
5               --            Formalin         T. solium
6               --            Formalin         T. solium
7           T. solium,          AFA            T. solium
            T. saginata         AFA           T, asiatica
                                           ([double dagger])
8               --            Ethanol          T. solium
9            T. solium        Ethanol          T. solium
10              --            Ethanol          T. solium
11           T. solium        Ethanol          T. solium
12              --            Ethanol          T. solium
13              --            Ethanol         T. saginata
14              --            Ethanol         T. saginata
15          T. saginata       Ethanol         T. saginata
16          T. saginata       Ethanol         T. saginata
17          T. saginata       Ethanol         T. saginata
18          T. saginata       Ethanol         T. saginata
19          T. saginata       Ethanol         T. saginata
20          T. saginata       Ethanol         T. asiatica
21          T. saginata       Ethanol         T, asiatica
                                           ([double dagger])
22          T. saginata       Ethanol         T, asiatica
                                              T, asiatica
23          T. saginata       Ethanol      ([double dagger])
24          T. saginata       Ethanol         T, asiatica
                                           ([double dagger])

* Year refers to year when specimen was collected;
NA, not available; NT, not tested; Formalin, 10% formalin;
AFA, alcohol-formalin-acetic acid; NK, not known; ethanol,
80% ethanol.

([dagger]) These segments without scolices were not examined for
morphologic identification.

([double dagger]) These cases (boldface) were identified as
T. saginata morphologically but were confirmed to be T. asiatica
by mitochondrial DNA analysis.

([section]) Three worms fixed separately.