Elsevier

Experimental Parasitology

Volume 121, Issue 2, February 2009, Pages 151-156
Experimental Parasitology

Differential diagnosis of Taenia asiatica using multiplex PCR

https://doi.org/10.1016/j.exppara.2008.10.014Get rights and content

Abstract

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.

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 (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 above-mentioned 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, Yamasaki et al., 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 assay that could be used for the differential diagnosis of human-infecting Taenia tapeworms.

Section snippets

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),

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. saginata-specific primers (Ts5058F and Rev7915) and T. solium-specific primers (Tso7421F and Rev7915). The specificity

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,

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 Galán-Puchades (Spain) for helping collection of samples used

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    T. asiatica can be differentiated from T. solium and T. saginata by examination of morphological characteristics such as the scolex, mature and gravid proglottids in the adult stage, and the scolex and bladder surface in the larval stage (Eom et al., 1992; Eom and Rim, 1993). To date, T. asiatica has been identified in Korea (Eom and Rim, 1993; Jeon et al., 2008), Taiwan (Fan et al., 1995; Ooi et al., 2013), the Philippines (Fan et al., 1992, 2006; Gonzalez et al., 2010; Hinz, 1985; Jeon et al., 2009; Leon, 2005; Okamoto et al., 2010), China (Chen et al., 2003; Eom et al., 2002; Jeon et al., 2009; Li et al., 2006; Liu et al., 2011; Mou et al., 2007; Nkouawa et al., 2012; Wang and Bao, 2003; Yamane et al., 2012; Yan et al., 2013; Yang et al., 2012; Zhang et al., 1999, 2006), Thailand (Anantaphruti et al., 2007, 2010), Indonesia (Jeon et al., 2009; Simanjuntak et al., 1997), Vietnam (Somers et al., 2007; Willingham et al., 2003; Xuan et al., 2005), Japan (Jeon et al., 2011; Taniyama et al., 2019; Tsuboi et al., 2018; Yamasaki, 2013), Lao PDR (Conlan et al., 2011; Sato et al., 2018), Nepal (Devleesschauwer et al., 2012), and India (Singh et al., 2016). In Korea, all 3 Taenia tapeworms are sympatrically distributed with T. asiatica, with domination over T. saginata and T. solium (Jeon et al., 2008).

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