Molecular Diagnosis of Taenia saginata Tapeworms from Two Residents of Northern Cambodia.

Taenia saginata infection has seldom been reported in Cambodia. In this study, we performed a survey of intestinal parasites in 1,156 residents of Preah Vihear and Stung Treng Provinces in 2018. The results revealed that 26 (2.4%) cases were positive for Taenia spp. eggs. In order to obtain the strobilae of the tapeworms, 2 patients in Preah Vihear were treated with praziquantel and purged with magnesium salts. The proglottids expelled after the medication were morphologically and molecularly analyzed to determine the species. The main uterine lateral braches in gravid proglottids were >15 in number suggesting that they are either T. saginata or Taenia asiatica. The sequences of the mitochondrial cytochrome c oxidase subunit 1 (cox1) gene and 2 nuclear loci, elongation factor-1 alpha (ef1) and ezrin-radixin-moesin-like protein (elp), were identical to the sequences of T. saginata available in GenBank but distant from Taenia solium, T. asiatica, and T. saginata-T. asiatica hybrid. This is the first report of the presence of T. saginata in the northern part of Cambodia bordering Lao PDR based on a molecular confirmation.

Taenia saginata, Taenia asiatica, and Taenia solium are 3 tapeworm species that can cause zoonotic infections in humans. They have unique life cycles, with humans as the only definitive host [1]. Beef is the infection source for T. saginata, whereas T. asiatica and T. solium infections are contracted by pork. The distribution of human taeniases is worldwide, and T. saginata is the most prevalent species [2].

Little has been known about human Taenia tapeworm infections in Cambodia with the exception of 2 studies reported in 2011 and 2014 [3,4]. According to the reports, fecal examinations of residents in Koh Kong Province [3] and 18 nationwide provinces [4] revealed the egg positive rate of Taenia spp. to be 1.5% (43/2,824) and 0.4% (115/32,201), respectively. In Koh Kong Province, eggs of Taenia spp. from 21 individuals were molecularly analyzed, and T. saginata (n=19) and T. solium (n=2) were identified by cox1 sequencing and multiplex PCR [3]. However, no other information is available regarding the species of Taenia in other localities of Cambodia. In this study, we molecularly confirmed T. saginata proglottids expelled from 2 residents after praziquantel treatment and purging in a northern part of Cambodia.

In May 2018, the Institute of Parasitic Diseases, Korea Association of Health Promotion (KAHP), in cooperation with the National Center for Parasitology, Entomology and Malaria Control, Cambodia, conducted a survey on intestinal parasitic infections in 2 northern provinces (5 villages each), Preah Vihear and Stung Treng (IRB no. 099NECHR, approved by National Ethics Committee for Health Research, Cambodia). A total of 1,156 fecal samples were collected from the residents and examined by the Kato-Katz thick smear method. The results revealed that the overall egg positive rate of Taenia spp. was 2.4% (26/1,156); 1.7% (6/359) in Preah Vihear and 2.5% (20/797) in Stung Treng (Table 1). A small village named Kampong Sangkae located in the northeastern part of Preah Vihear and bordered with Lao PDR showed a fairly high prevalence (3.4%, 3/89) of Taenia spp. eggs. This village was selected for further analysis of Taenia tapeworms in this study.

Table 1

Infection status of tapeworms in 2 northern provinces of Cambodia

Province	Village	No. of people examined	No. of Taenia spp. egg positive cases (%)
Preah Vihear	Kampong Pou	52	0 (0.0)
	Kampong Chey	42	2 (4.8)
	Kampong Sangkaea	89	3 (3.4)
	Kampong Sralau	74	0 (0.0)
	Kampong Sami	50	1 (2.0)
	Subtotal	359	6 (2.0)
Stung Treng	O’ Chay	125	6 (4.8)
	Kanhchanh Tuek	114	4 (3.5)
	Ti Team	93	0 (0.0)
	Srae Russei	204	4 (2.0)
	Peam Khes	261	6 (2.3)
	Subtotal	797	20 (2.5)
Total		1,156	26 (2.4)

The village where residents were recruited for praziquantel treatment and purging to recover adult tapeworms.

Two egg positive cases from this village were treated with 10–15 mg/kg praziquantel (Shinpoong Pharm. Co., Seoul, Korea) followed by purging with 30–40 g MgSO4. The 2 tapeworm strobilae (1 was with scolex) (Fig. 1A, B) discharged from the 2 cases were stored in 70% ethanol until morphological and molecular analyses. The scolex revealed 4 suckers but no recognizable rostellum nor hooklets (Fig. 1B). Several proglottids from each of the 2 cases were fixed in 10% formalin and stained with Semichon’s acetocarmine, and morphologically observed using a light microscope. They were identified as either T. saginata or T. asiatica since they had >15 main uterine lateral branches filled with eggs (Fig. 1C) [5].

Fig. 1

(A) A complete tapeworm strobila, including the scolex (inset), of Taenia saginata discharged from a resident in Kampong Sangkae village, Preah Vihear Province, Cambodia. Scale bar=2 cm. (B, C) Enlarged views of the scolex and a gravid proglottid taken with a stereomicroscope (Leica, Wetzlar, Germany). Morphological characteristics of the unarmed scolex with no distinct rostellum (B) and the high number (>15) of main uterine lateral branches (C) designated the tapeworm as either T. saginata or T. asiatica and far from T. solium. Scale bar=1 mm in (B) and 3 mm in (C).

The genomic DNA of each tapeworm segment was extracted using the DNeasy Blood & Tissue kit (QIAGEN, Hilden, Germany) following the manufacturer’s instructions. PCR amplification and sequencing of mitochondrial cytochrome c oxidase (cox1, 1,620 bp), nuclear elongation factor-1α (ef1, 1,090 bp), and nuclear ezrin-radixin-moesin (ERM)-like protein (elp, 1,160 bp) genes were performed on each tapeworm sample with primers and conditions reported in a previous study [6]. A phylogenetic tree of cox1 gene constructed from the representative selection of sequences available in GenBank, using the maximum-likelihood method available in MEGA and employing Tamura-nei model of nucleotide substitution with 1,000 bootstrap replications, showed that our samples were both identical with T. saginata (Fig. 2). The sequence homology (cox1) was 99.7% with T. saginata and 94.8–95.2% with T. asiatica. In addition, our samples contained the nuclear loci alleles ef1C/ef1C and elpC/elpC, which are homozygous for T. saginata based on the interpretation using Sanger chromatogram analysis (Table 2) [6]. This demonstrates that our samples are not a hybrid between T. saginata and T. asiatica but ‘pure T. saginata’.

Fig. 2

A phylogenetic tree of 2 tapeworm samples obtained in this study in relation to tapeworm species drawn with cox1 DNA sequences in GenBank. Black dots indicate 2 samples identified in this study. Clonorchis sinensis was used as an outgroup. Scale bar indicates nucleotide substitutions per site.

Table 2

Genotype of tapeworm samples from 2 patients

Patient no.	mtDNA type (cox1)	Genotypea at ef1 locus	Genotypea at elp locus
1	T. saginata	ef1C/ef1C (T. saginata)	elpC/elpC (T. saginata)
2	T. saginata	ef1C/ef1C (T. saginata)	elpC/elpC (T. saginata)

The allele types of these samples were analyzed following the previous study [6].

Human taeniases are known to be endemic in Southeast Asia, including Myanmar [7] and Lao PDR [8]. In Cambodia, a few previous surveys reported low-grade prevalence of Taenia spp. among the residents in several provinces [3,4]. Molecular studies on the species were reported in a paper in which the genomic DNA was extracted from eggs collected from the feces [3]. In our study, we obtained gravid proglottids from 2 patients and observed that they had more than 15 main uterine lateral branches being consistent with T. saginata or T. asiatica but not with T. solium. To determine whether they are T. saginata or T. asiatica, it was necessary to perform molecular analyses [2].

Interestingly, hybrid descendants of T. saginata and T. asiatica were reported from humans in Thailand [6] and Lao PDR [8]. Therefore, we conducted molecular analyses of cox1, ef1, and elp genes to rule out this hybrid issue. The cox1 sequences revealed high homologies with T. saginata but low homologies with T. asiatica. The nuclear gene ef1 is known to have 3 alleles (ef1A, ef1B, and ef1C), and another nuclear gene elp has 4 alleles (elpA, elpB, elpC, and elpD); T. saginata has only 1 ef1 allele (ef1C) and 3 elp alleles (elpA, elpC, and elpD), whereas T. asiatica has 2 ef1 alleles (ef1A and ef1B) and 2 elp alleles (elpA and elpB) [6]. Our samples had ef1C and elpC alleles, and thus we could confirm them not a hybrid of the 2 species but pure T. saginata.

The geographical distribution of 3 human Taenia spp. is closely related to cultural characteristics of the people, which include the traditional food habit of consuming undercooked meat, including beef and pork, infected with viable metacestodes [8]. Our study suggests the necessity of continued surveillance of human Taenia tapeworm infections in Cambodia.