Differential Diagnosis of Human Sparganosis Using Multiplex PCR

Human sparganosis was diagnosed by morphological and genetic analyses in Korea. The complete mitochondrial genomes of Spirometra erinaceieuropaei and S. decipiens isolated in Korea have been recorded. Present study was performed to provide information to diagnose the etiologic agent of sparganosis by multiplex PCR using mitochondrial genome sequences of S. erinaceieuropaei and S. decipiens. In an effort to examine the differential diagnosis of spirometrid tapeworms, multiplex PCR assays were performed on plerocercoid larvae of S. erinaceieuropaei and S. decipiens. The PCR products obtained using species-specific primers were positively detected in all PCR assays on mixture of S. erinaceieuropaei and S. decipiens DNA. S. erinaceieuropaei-specific bands (239 bp and 401 bp) were obtained from all PCR assays using a mixture of S. erinaceieuropaei-specific primers (Se/Sd-1800F and Se-2018R; Se/Sd-7955F and Se-8356R) and S. erinaceieuropaei template DNA. S. decipiens-specific bands (540 bp and 644 bp) were also detected in all PCR assays containing mixtures of S. decipiens-specific primers (Se/Sd-1800F and Sd-2317R; Se/Sd-7955F and Sd-8567R) and S. decipiens template DNA. Sequence analyses on these species-specific bands revealed 100% sequence identity with homologous regions of the mtDNA sequences of S. erinaceieuropaei and S. decipiens. The multiplex PCR assay was useful for differential diagnosis of human sparganosis by detecting different sizes in species-specific bands.

Genetic Identification of Spirometra decipiens Plerocercoids in Terrestrial Snakes from Korea and China

Human sparganosis is a zoonotic disease caused by infection with larval forms (procercoid/plerocercoid) of Spirometra spp. The purpose of this study was to identify Spirometra spp. of infected snakes using a multiplex PCR assay and phylogenetic analysis of mitochondrial DNA sequence data from the spargana of terrestrial snakes obtained from Korea and China. A total of 283 snakes were obtained that included 4 species of Colubridae comprising Rhabdophis tigrinus tigrinus (n=150), Dinodon rufozonatum rufozonatum (n=64), Elaphe davidi (n=2), and Elaphe schrenkii (n=7), and 1 species of Viperidae, Agkistrodon saxatilis (n=60). The snakes were collected from the provinces of Chungbuk, Chungnam, and Gyeongbuk in Korea (n=161), and from China (n=122). The overall infection rate with spargana was 83% (235/283). The highest was recorded for D. rufozonatum rufozonatum (100%), followed by A. saxatilis (85%) and R. tigrinus tigrinus (80%), with a negative result for E. davidi (0%) and E. schrenkii (0%). The sequence identities between the spargana from snakes (n=50) and Spirometra erinaceieuropaei (KJ599680) or S. decipiens (KJ599679) control specimens were 90.8% and 99.2%, respectively. Pairwise genetic distances between spargana (n=50) and S. decipiens ranged from 0.0080 to 0.0107, while those between spargana and S. erinaceieuropaei ranged from 0.1070 to 0.1096. In this study, all of the 904 spargana analyzed were identified as S. decipiens either by a multiplex PCR assay (n=854) or mitochondrial cox1 sequence analysis (n=50).

Mitochondrial Genome Sequences of Spirometra erinaceieuropaei and S. decipiens (Cestoidea: Diphyllobothriidae)

The present study was performed to compare the mitochondrial genomes between 2 Spirometra tapeworms, Spirometra erinaceieuropaei and Spirometra decipiens (Cestoidea: Diphyllobothriidae), which larval stages are important etiological agents of sparganosis in humans. For each species, the full mitochondrial genome was amplified in 8 overlapping fragments using total genomic DNA purified from a single worm as the template. The mitochondrial genomes were 13,643 bp (S. erinaceieuropaei) and 13,641 bp (S. decipiens) in length and contained 36 genes; 12 protein-coding genes, 2 ribosomal RNA (rRNA, small and large subunits), and 22 transfer RNAs (tRNAs). The 12 protein-coding genes constituted 10,083 bp (S. erinaceieuropaei) and 10,086 bp (S. decipiens) of their respective mitochondrial genomes. The tRNA genes, ranging in length from 56 to 70 bp, were identified based on putative secondary structures such as the typical cloverleaf shape. A total of 23 intergenic sequences, varying from 1 to 204 bp in size, were interspersed in S. erinaceieuropaei (total, 504 bp) and S. decipiens (total, 496 bp) mtDNA. The 12 protein-coding genes of S. erinaceieuropaei and S. decipiens differed by 12.4%, whereas the overall difference in mtDNA sequence between S. erinaceieuropaei and S. decipiens was 12.9%. Thus, from the standpoint of the mitochondrial genome, S. decipiens represents a valid species that can be distinguished from S. erinaceieuropaei.

Human Infections with Spirometra decipiens Plerocercoids Identified by Morphologic and Genetic Analyses in Korea

Tapeworms of the genus Spirometra are pseudophyllidean cestodes endemic in Korea. At present, it is unclear which Spirometra species are responsible for causing human infections, and little information is available on the epidemiological profiles of Spirometra species infecting humans in Korea. Between 1979 and 2009, a total of 50 spargana from human patients and 2 adult specimens obtained from experimentally infected carnivorous animals were analyzed according to genetic and taxonomic criteria and classified as Spirometra erinaceieuropaei or Spirometra decipiens depending on the morphology. Morphologically, S. erinaceieuropaei and S. decipiens are different in that the spirally coiled uterus in S. erinaceieuropaei has 5-7 complete coils, while in S. decipiens it has only 4.5 coils. In addition, there is a 9.3% (146/1,566) sequence different between S. erinaceieuropaei and S. decipiens in the cox1 gene. Partial cox1 sequences (390 bp) from 35 Korean isolates showed 99.4% (388/390) similarity with the reference sequence of S. erinaceieuropaei from Korea (G1724; GenBank KJ599680) and an additional 15 Korean isolates revealed 99.2% (387/390) similarity with the reference sequences of S. decipiens from Korea (G1657; GenBank KJ599679). Based on morphologic and molecular databases, the estimated population ratio of S. erinaceieuropaei to S. decipiens was 35: 15. Our results indicate that both S. erinaceieuropaei and S. decipiens found in Korea infect humans, with S. erinaceieuropaei being 2 times more prevalent than S. decipiens. This study is the first to report human sparganosis caused by S. decipiens in humans in Korea.

Morphologic and Genetic Identification of Diphyllobothrium nihonkaiense in Korea

Diphyllobothrium nihonkaiense was first described by Yamane in 1986 but the taxonomical features have been obscure due to lack of critical morphologic criteria in its larval and adult stages. In Korea, this tapeworm had long been known as Diphyllobothrium latum. In this study, we observed 62 specimens collected from Korean residents and analyzed them by morphological features and nucleotide sequences of mitochondrial cox1 gene as well as the ITS1 region. Adult tapeworms were examined after carmine or trichrome stain. Longitudinal sections of the gravid proglottids showed an obtuse angle of about 150 degree between the cirrus sac and seminal vesicle. This angle is known as a major differential point compared with that of D. latum. Nucleotide sequence differences between D. latum and the specimens from Koreans represented 17.3% in mitochondrial DNA cox1 gene. Sequence divergence of ITS1 among 4 Korean isolates was 0.3% and similarity was 99.7% with D. nihonkaiense and D. klebanovskii. All of the Korean specimens analyzed in this study were identified as being D. nihonkaiense (n = 62). We propose its Korean name as "Dong-hae-gin-chon-chung" which means 'long tapeworm of the East Sea' for this newly analyzed diphyllobothriid tapeworm in Korea.

Sympatric Distribution of Three Human Taenia Tapeworms Collected between 1935 and 2005 in Korea

Taeniasis has been known as one of the prevalent parasitic infections in Korea. Until recently, Taenia saginata had long been considered a dominant, and widely distributed species but epidemiological profiles of human Taenia species in Korea still remain unclear. In order to better understand distribution patterns of human Taenia tapeworms in Korea, partial nucleotide sequences of mitochondrial cox1 and ITS2 (internal transcribed spacer 2) were determined, along with morphological examinations, on 68 Taenia specimens obtained from university museum collections deposited since 1935. Genomic DNA was extracted from formalin-preserved specimens. Phylogenetic relationships among the genotypes (cox1 haplotype) detected in this study were inferred using the neighbor-joining method as a tree building method. Morphological and genetic analyses identified 3 specimens as T. solium, 51 specimens as T. asiatica, and 14 specimens as T. saginata. Our results indicate that all 3 Taenia tapeworms are sympatrically distributed in Korea with T. asiatica dominating over T. saginata and T. solium.