Molecular identification of Taenia tapeworms by Cox1 gene in Koh Kong, Cambodia.

We collected fecal samples from 21 individuals infected with Taenia tapeworms in Koh Kong Province, Cambodia, and performed nucleotide sequencing of the cox1 gene and multiplex PCR on the eggs for DNA differential diagnosis of human Taenia tapeworms. Genomic DNA was extracted from the eggs of a minimum number of 10 isolated from fecal samples. Using oligonucleotide primers Ta7126F, Ts7313F, Tso7466F, and Rev7915, the multiplex PCR assay proved useful for differentially diagnosing Taenia solium, Taenia saginata, and Taenia asiatica based on 706, 629, and 474 bp bands, respectively. All of the Taenia specimens from Kho Kong, Cambodia, were identified as either T. saginata (n=19) or T. solium (n=2) by cox1 sequencing and multiplex PCR.

Since 2006, intestinal parasite control has been performed among primary school children in Cambodia by the Korea Association of Health Promotion (KAHP) and the National Center for Parasitology, Entomology, and Malaria Control, Cambodia (CNM). These organizations carried out a parasitological survey of inhabitants and primary school children in Koh Kong, Kam Pot, and Kampong Som Provinces, and performed stool examinations using the Kato-Katz smear and scotch-tape anal swab methods. Since 1988 in Cambodia, parasitic helminth infections have revealed malaria, schistosomiasis, and other intestinal helminthic infections [1-4]. Previous studies reported that the intestinal helminth eggs detected were Ascaris lumbricoides, Echinostoma sp., hookworms, Hymenolepis nana, Opisthorchis sp., Rhabditis sp., and Trichuris trichiura, and protozoan cysts were Entamoeba histolytica, Entamoeba coli, Giardia lamblia, and Iodamoeba buetschlii. The overall infection rate of intestinal parasites was 54.2% in 2002 [5], 2-40% in 2003 [6], and 25.7% in 2004 [7]. However, there are few reports on taeniasis in Cambodia. Epidemiologically, Taenia solium and Taenia saginata are found worldwide and Taenia asiatica is found mostly in Asian countries, including Korea, China, Taiwan, Thailand, Indonesia, Vietnam, Japan, and the Philippines [8]. We have been monitoring the epidemiological status of T. solium, T. saginata, and T. asiatica infections in Cambodia, and now it is considered necessary to clarify the distribution of these tapeworms in Cambodia. To identify Taenia species, we analyzed nucleotides for sequence variations, and performed multiplex PCR using the copro-DNA from the eggs.

We collected a total of 2,824 fecal specimens from Koh Kong (n=904), Kam Pot (n=1,002), and Kampong Som (n=918). Of them, 2,052 specimens were collected from primary school children, and the remaining 772 specimens were from inhabitants. We collected Taenia sp. eggs from 21 patients in Koh Kong (Table 1). The eggs were detected by the Kato-Katz smear technique, in which the number of eggs counted in the entire field of 41.7 mg of stools is multiplied by 24 in order to obtain the number of eggs per gram of feces. The Taenia eggs were isolated from the stools and subjected to DNA sequencing and multiplex PCR for differential diagnosis according to the protocols already established [9].

Table 1

Helminth egg positive rates in the feces of schoolchildren and residents in Koh Kong, Kam Pot, and Kampong Som, Cambodia in 2007

Al, Ascaris lumbricoides; Hw, hookworms; Tt, Trichuris trichiura; Ov, Opisthorchis viverrini; Hn, Hymenolepis nana; Ev, Enterobius vermicularis; Ech, Echinostoma sp.; Others, unidentified parasite eggs.

The overall helminth egg positive rate was 33.1% (934/2,824) and 1.5% (43/2,824) for the Taenia sp. eggs. Other helminth eggs detected were Ascaris lumbricoides 15.8% (445/2,824), hookworms 9.6% (270/2,824), Trichuris trichiura 1.0% (27/2,824), Enterobius vermicularis 2.3% (64/2,824), and echinostomes 0.4% (11/2,824). Multiple parasite infections were found in 5.9% (168/2,824) of the samples. Taenia eggs had a positive rate of 4.6% (42/904) inhabitants in Koh Kong Province (Table 1).

The PCR amplification and direct sequencing of the cox1 target fragment (456 bp in length corresponding to the positions 67-522 bp of the cox1 gene) were performed using the total genomic DNA extracted from the eggs of parasites found in patients. The cox1 sequences (456 bp) of the code number 97 and 166 showed 99% similarity with the reference sequences of T. solium (GenBank AB086256), and the other code numbers 70, 72, 76, 77, 83, 86, 89, 128, 129, 135, 139, 167, 180, 186, 190, 194, 196, 197, and 198 showed 99% similarity with T. saginata (GenBank AY684274). The diagnostic quality of the results obtained using multiplex PCR and species-specific primers were equal to that based on the nucleotide sequencing of the cox1 gene. All of the 21 Taenia specimens from Koh Kong were identified by cox1 sequencing and multiplex PCR as either T. saginata (n=19) or T. solium (n=2) (Table 2).

Table 2

Sample specimens of Koh Kong inhabitants analyzed in this study

aKato-Katz method; Al, Ascaris lumbricoides.

The most prevalent parasitic helminths in Cambodia were soil-transmitted nematodes, such as Ascaris, hookworms, and Trichuris. In addition, Opisthorchis viverrini, intestinal trematodes, and schistosomes are also found along the Mekong River [4]. Cestodes dominantly known were Hymenolepis nana; however, Taenia tapeworm species were not well recognized until 2006. In our study, Taenia eggs were detected in schoolchildren and other inhabitants in Koh Kong and Kampong Som provinces and were genetically analyzed, but adult tapeworms were not collected. In Cambodia, according to a KAHP report (unpublished), the overall prevalence rate of Taenia tapeworms ranged from 0.3% to 1.5% between 2006 and 2009. In the present study, a higher prevalence of Taenia sp. was found in Koh Kong inhabitants who habitually eat raw beef and pork.

The diagnosis of Taenia tapeworm infections is usually based on microscopic detection of eggs or discharged proglottids in the stool. Coprological examination based on the morphology of tapeworm proglottids has a low sensitivity, and the eggs are morphologically indistinguishable. Molecular diagnostic methods, including the use of sequence specific DNA probes, PCR coupled to restriction fragment length polymorphism, and multiplex PCR, are highly useful for differential diagnosis of Taenia tapeworm eggs in stools. However, these methods may not be always suitable unless eggs and particles of proglottids are released into the stool. In our study, we collected 42 stool samples but 21 cases lacked the fecal volume for DNA extraction. The DNA recovery yield increased using ethanol precipitation of eggs in stools, and PCR was usually performed secondarily or thirdly for DNA sequencing. PCR products derived from fecal samples were detectable at a minimum number of 10 eggs.

There are countries showing sympatric distributions of 3 human Taenia tapeworms in South and East Asian regions. However, in this study, only T. saginata (n=19) and T. solium (n=2) were identified by cox1 sequencing and multiplex PCR. The distribution of T. asiatica in Cambodia is yet to be cleared.