Molecular determination of the origin of acephalic cysticercus.

Acephalic cysticercus (Ac), a rarely developed multilobulated and nonencysted form of larval Taenia, causes hydrocephalus or adhesive arachnoiditis in the ventricles and subarachnoidal space that often lead to fatal outcome in affected patients. Ac has been proposed to originate from T. solium on the basis of morphological features, while no molecular data supporting the presumption have been available. In the present study, we investigated the immunological properties as well as molecular characteristics of Ac that was obtained surgically from 6 patients. Immunoblotting of the cyst fluid from Ac samples demonstrated the constitutive expression of a T. solium metacestode (TsM) 10 kDa protein. Specific antibodies against the truncated 10 kDa protein, which appears to be species specific for TsM cysticercosis, were detected in both serum and cerebrospinal fluid samples of Ac patients. Nucleotide sequences of mitochondrial cytochrome c oxidase subunit I (COI) and NADH dehydrogenase subunit 1 (ND1) genes of Ac were almost identical to those of T. solium but differed substantially from those of the other Taenia species. In phylogenetic analysis, Ac clustered with T. solium in a well-supported clade. Our results strongly suggest that Ac may have originated from T. solium.

Analysis of polymorphic regions of Plasmodium vivax Duffy binding protein of Korean isolates.

The present study was designed to investigate polymorphism in Duffy binding protein (DBP) gene of Plasmodium vivax isolates of Korea. Thirty samples were obtained from P. vivax patients in Yonchon-gun, Kyonggi-do in 1998. The PCR products of the samples were subjected to sequencing and hybridization analyses of the regions II and IV of P. vivax DBP gene. Two genotypes, SK-1 and SK-2, were identified on the basis of amino acid substitution and deletion. The genotype of 10 isolates was SK-1 and that of 20 isolates was SK-2. Most of the predicted amino acids in the region II of DBP gene were conserved between the Korean isolates and Belem strain except for 4-5 amino acid substitutions. In the region IV of DBP, a 6-bp insert that was shown in the Sal-1 allele type was found in SK-1, and a 27-bp insert that was shown in the Papua New Guinea allele type was found in SK-2. In conclusion, the present findings suggest that two genotypes of P. vivax coexist in the endemic area of Korea.

Analysis of polymorphic region of GAM-1 gene in Plasmodium vivax Korean isolates.

The identification, characterization and quantification of Plasmodium sp. genetic polymorphism are becoming increasingly important in the vaccine development. We investigated polymorphism of Plasmodium vivax GAM-1 (PvGAM-1) gene in 30 Korean isolates. The polymorphic region of the PvGAM-1 gene, corresponding to nt 3792-4029, was amplified using polymerase chain reaction (PCR) followed by sequencing. All of the P. vivax Korean isolates were one type of GAM-1 gene, which were identical to that of the Belem strain. It is suggested that PvGAM-1 could not be used as a genetic marker for identifying or classifying P. vivax Korean isolates. It revealed that the polymorphic pattern was acquired basically by duplication and modification or deletion event of a 33 bp-motif fragment ended by poly guanine (G) and that there were at least three complete and one partial 33 bp-motif sequences within the polymorphic region in the longest cases such as those of South Korean and Belem isolates. In addition, we clustered P. vivax isolates with parsimonious criteria on the basis of PvGAM-1 polymorphic patterns (insertion/deletion patterns).

Border malaria characters of reemerging vivax malaria in the Republic of Korea.

Since 1993, the number of vivax malaria cases has increased every year in the northern part of the Republic of Korea (ROK). This study was designed to characterize factors related to the reemergence of malaria in the ROK. A total of 21 cases diagnosed in 1993 and 1994 distributed sporadically in the narrow zone along the demilitarized zone (DMZ). Of total 317 civilian inhabitant cases reported in 1994-1997, 287 cases were studied and 80.8% of them resided within 10 km from the southern border of the DMZ. The frequency distribution of anti-Plasmodium vivax antibody titers using indirect fluorescent antibody test was compared in three villages in relation with distance from the DMZ. The number of inhabitants with high antibody titers was larger in the village nearest to the border than that in more distant villages. The present results highly suggested that the reemerging vivax malaria start in the border area, most possibly caused by infected mosquitoes which flew across the border. This pattern of transmission repeated year after year.

Two new genotypes of Plasmodium vivax circumsporozoite protein found in the Republic of Korea.

The gene encoding Plasmodium vivax circumsporozoite protein (PvCSP) exhibits polymorphism in many geographical isolates. The present study was designed to investigate polymorphism in PvCSP gene of P. vivax isolates in Korea. Thirty isolates, obtained from indigenous cases in Yonchon-gun, Kyonggi-do in 1997, were subjected for sequencing and RFLP analysis of the repeat and post-repeat regions of PvCSP gene and two genotypes (SK-A and SK-B) were identified. The genotype of 19 isolates was SK-A and that of 11 isolates was SK-B. Although the number of 12-base repeats present in SK-A was three while two were found in a Chinese strain CH-5, the repeat sequence of SK-A was identical to that of CH-5 except for one base substitution. Compared with known data there was no identical isolates with SK-B, but the sequence of SK-B was similar to that of a North Korean (NK) isolate. These results indicate that two genotypes of PvCSP coexist in the present epidemic area of Korea and the present parasite may originate from East Asia. RFLP would be useful to classify genotypes of P. vivax population instead of gene sequencing.

Current status of vivax malaria among civilians in Korea.

A result of national malaria surveillance in Korean civilians was described. Since a case of indigenous vivax malaria was detected in 1993, a total of 2,198 cases was confirmed by blood smear up to 1997. Of them, 1,548 cases were soldiers serving in the demilitarized zone (DMZ), while 650 cases were civilians. Number of civilian cases was 3 in 1994, 19 in 1995, 71 in 1996, and 557 in 1997. Of them, 239 were ex-soldiers who discharged after military service in the prevalent areas such as Paju, Yonchon, Kimpo, Kangwha, Tongduchon in Kyonggi-do and Chorwon in Kangwon-do while 308 patients were civilian residents in the prevalent areas. Seventy-two patients, living nationwide, had a history of visiting the prevalent areas during transmission season. Only 32 civilian patients denied any relation with the prevalent areas. As a whole, a half of the civilian cases was diagnosed when living in non-prevalent areas. Male patients in their twenties was the highest in number. Annual parasite index is steadily elevated in residents living in the prevalent areas. Monthly incidence showed an unimodal distribution, forming a peak in August. Ex-soldiers exhibited a delayed incubation ranging from 153 to 452 days (279 +/- 41 days). The time required for diagnosis was shortened from 23.6 days in 1995 to 13.7 days in 1997. Although the current epidemic of vivax malaria started as a border malaria, it seems highly probable that vivax malaria is established in the local areas and responsible for at least a part of transmission.

Immunoblot patterns of clonorchiasis.

Clonorchis sinensis is a liver fluke which is the most prevalent helminth of humans in Korea. The better diagnostic measure of clonorchiasis is required for its nationwide control program. The present study observed antigenic bands of C. sinensis and reacting immunoglobulins in serum of infected residents. Adult C. sinensis were recovered from experimentally infected rabbits and soluble crude extract of the worms was used as the antigen after supplementation of E-64, a cysteine proteinase inhibitor. SDS-PAGE of the crude antigen resolved more than 20 protein bands between 200 and 14 kDa. The sera of infected humans collected at an endemic village showed specific IgG and IgE antibodies but little IgM and IgA antibodies. The protein bands of 94, 80, 72, 68, 52, 47, 43, 37, 34, and 28-25 kDa strongly reacted with serum Ig(GMA) or IgG antibody and 64, 62, 52, 47, 44, 34, 28, and 26 kDa bands reacted with serum IgE antibody. However, the 94, 80, 72, 68, 64, 62, 52, 47, and 40 kDa bands of C. sinensis antigen were found non-specific. The protein bands of 43, 34, and 28-25 kDa of C. sinesis are primary target molecules of further analysis.

Two vivax malaria cases detected in Korea.

On June and July 1994, two cases of vivax malaria were consecutively diagnosed at the Yongsan Hospital, Chung-Ang University in Seoul. The first patient was a soldier serving in western parts of the demilitarized zone (DMZ) while the second case was a resident of a village near DMZ. Neither patients had history of being abroad. Republic of Korea (ROK) has been free of malaria since the mid-1970s except for imported cases. The two vivax malaria cases, together with an additional patient detected in 1993, occurred in relatively small areas near DMZ. This necessitated an epidemiologic surveillance. When medical records and blood smears in the areas were examined, no other cases were found. Of 7,723 mosquitoes collected by a black light trap for two nights in June, 7,066 (91.5%) were Anopheles sinensis. In order to evaluate a significance of the recent malaria occurrence, a surveillance system should be operated in the areas.

High prevalence of Gymnophalloides seoi infection in a village on a southwestern island of the Republic of Korea.

Gymnophalloides seoi (Digenea: Gymnophallidae), a new human intestinal trematode reported from a Korean woman who complained of epigastric discomfort, has been shown to be highly prevalent among the villagers of a southwestern island of the Republic of Korea. For the detection of human infections, fecal examinations were conducted on the inhabitants of a seashore village, where the first patient with a G. seoi infection had resided. Of 98 inhabitants examined, 70 (71.4%) were infected with various intestinal parasites; among them, G. seoi showed the highest rate of egg positivity 48 of 98 (49.0%). Individual worm burdens of G. seoi, as measured by collection of adult flukes after anthelmintic treatment and purgation, ranged from 106 to 26,373 specimens (average per infected case = 3,326). There was no sex-related difference in the prevalence of G. seoi, and the age distribution of the infected cases showed a relatively even pattern. This study confirms that human infection by G. seoi is not an incidental one and provides the first evidence of its high prevalence on this Korean island.

[Infection status with the metacercariae of heterophyid flukes in the brackish water fish from Haenam-gun, Chollanam-do, Korea].

An epidemiological survey was performed to know the infection status of heterophyid fluke metacercariae in the brackish water fishes purchased from a local market in Haenam-gun, Chollanam-do, Korea from November to December, 1991. A total 20 out of 30 shads (Konosirus punctatus) examined were infected with the metacercariae of H. continua (average number of larvae per an infected fish: 2.7). Of 20 perches (Lateolabrax japonicus), 19 were infected with the metacercariae of H. continua (average number per an infected fish: 17.2). Of 30 mullets (Mugil cephalus) examined, 11 were infected with the metacercariae of H. nocens (average number per an infected fish: 51.5), 10 mullets harboured 31 and 349 metacercariae of P. summa and S. fuscatum respectively. Of 30 gobies (Acanthogobius flavimanus) examined, 3 were infected with the metacercariae of H. nocens (average number per an infected fish: 32.0), 8 with those of H. continua (average number per an infected fish: 12.6) and 3 with those of S. fuscatum. From the above results, it has been confirmed that the brackish water fish from Haenam-gun, Chollanam-do are infected with numerous metacercariae of H. nocens, H. continua, P. summa and S. fuscatum.

Larval Gnathostoma nipponicum found in the imported Chinese loaches.

Six early third-stage larvae of Gnathostoma nipponicum were recovered from the muscle of 376 loaches, Misgurnus anguillicaudatus, imported from China. They were 614 x 114 microns in average size, almost colorless except brownish intestine, and encircled by about 229 transverse rows of minute cuticular spines. Their head bulbs provided with 3 rows of hooklets, of which average number were 34.5 on the first, 36.7 on the second and 39.7 on the third. Accordingly, it is revealed that the Chinese loach is a natural second intermediate host of G. nipponicum and G. nipponicum has been distributed somewhere in China. This parasite may infect human when the imported loaches are consumed raw.

Mucosal mast cell responses to experimental Metagonimus yokogawai infection in rats.

Intestinal mucosal mast cell (MMC) responses were studied in rats experimentally infected with Metagonimus yokogawai (Digenea: Heterophyidae). Twenty Sprague-Dawley rats were fed each 2,500 metacercariae isolated from the sweetfish and sacrificed on the week 1, 2, 3 and 4 post-infection (PI). Recovery of worms was performed from the small intestine of each rat. To visualize the MMCs, duodenal and jejunal (upper, middle and lower) tissue sections were made and stained with alcian blue/safranine-0. The average worm recovery rates were 16.2% and 13.8% on the week 1 and week 2, respectively, but they decreased rapidly to 4.1% and 4.2% on the week 3 and week 4 PI, respectively, which indicate spontaneous worm expulsion after the week 2. The MMC number in the infected rats was, compared with uninfected controls, significantly increased in the whole small intestine, through the whole period of observation. The peak level of mastocytosis was observed on the week 3 PI. It is strongly suggested that MMCs might be involved in the expulsion process of flukes from the rat intestine.

Turnover of biliary epithelial cells in Clonorchis sinensis infected rats.

We performed bromodeoxyuridine (BrdU) staining to observe the proliferation pattern of epithelial cells on the biliary mucosa in Clonorchis sinensis infection. Albino rats were infected with 100 metacercariae each and their livers were processed for histopathological observation after BrdU injection. Five to six sites in the liver of a rat were selected for paraffin section, and stained immunohistochemically to visualize BrdU incorporating cells. The flukes were mainly in the common bile duct and right or left hepatic bile ducts. The proportion of stained epithelial cells in the infected bile ducts where the worms were found on the section was 2.9-10.2% at 1 week after infection, 7.3-12.8% at 2 weeks, 7.3-13.4% at 5 weeks, and 8.4-14.8% at 15 weeks while in the non-infected ducts 0 to 2.7% cells were stained. The stained cells were mainly at the base of the mucosal layer. It is suggested that mucosal epithelial cells of the bile ducts infected with C. sinensis become hyperplastic mainly by direct and local stimulation of the worms.

Karyotypes of Pneumocystis carinii from Korean rats.

Molecular karyotyping was applied to Pneumocystis carinii(Pc) from two strains of experimental rats, Sprague Dawley(SD) and Fisher(F), in Korea. Field inversion gel electrophoresis and contour clamped homogeneous electric field electrophoresis resolved 15 chromosomal bands from the Pc. The size of the bands was estimated 270kb to 684kb from SD rats, and 273kb to 713 kb from F rats. The bands of 283 kb from SD rats and of 273 kb from F rats stained more brightly suggesting duplicated bands. Total number of chromosomes was at least 16, and total genomic size was estimated 7 x 10(6) bp. All of the bands from F rats hybridized to the probe of repeated DNA sequences of Pc and the band of 448 kb size was proved to contain rDNA sequences, but Pc. chromosome bands from SD rats showed no reactions to the probes. The 2 different karyotypes of P. carinii from 2 strains of rats were maintained consistently for 2 years.

[Experimental life history of Echinostoma hortense].

The complete life cycle of Echinostoma hortense has been maintained in the laboratory, using Lymnaea pervia snails and Rana nigromaculata tadpoles as the first and second intermediate hosts. ICR mice was used as the definitive host. Within the egg of E. hortense, the miracidium was fully matured in 13 days of incubation at 29-30 degrees C. The miracidium was 93.8 x 53.6 microns in average size, covered with numerous cilia of 7-11 microns length. The epidermal plates were arranged in 6-8-4-2 formula. The first generation rediae (1.19 x 0.27 mm in average size) were observed in 14 days after miracidial challenge to the snails, and the second generation rediae (1.40 x 0.26 mm in average size) in 30 days. The average size of the cercaria was 295.5 x 145.0 microns. Their head crown was poorly developed, and collar spines were not yet observed. After a cercarial challenge to the tadpoles, all of the tadpoles became infected and the average worm recovery rate was 88.5%. The majority of the metacercariae (75.5%) were recovered from the muscle of the tadpole's posterior body and the rest (24.3%) from their gills. The metacercariae from the tadpoles were elliptical, and 167.7 x 129.9 microns in average size. The recovery rate of adults from the mice was different by the age of the metacercariae grown in the tadpoles. The metacercariae younger than 5 hrs could not infect mice whereas those older than 6 hrs could infect mice. The recovery rate became higher as the metacercaria matured, with the peak recovery rate of 90.0% at the metacercarial age of 9 days. Thereafter the recovery rate decreased to 55.0% at the age of 50 days. As shown by the above results, the whole life cycle of E. hortense has been completed in the laboratory. At least 55-58 days were required to maintain one egg-to-egg cycle of E. hortense.

Immunohistochemical study on the antigenicity of each organ structure of Clonorchis sinensis.

An immunohistochemical study was performed to demonstrate comparative antigenicity of each body structure of the liver fluke, Clonorchis sinensis, such as the digestive tract, reproductive organs, excretory system, tegument and suckers. Indirect immunoperoxidase technique was applied, using formalin-fixed and paraffin-embedded sections of C. sinensis as the antigen. Pooled cat sera obtained 10 weeks after an experimental infection with C. sinensis and peroxidase-conjugated goat anti-cat IgG were used as the primary and secondary antibodies, respectively. The intensity of immunohistochemical stain was very sensitive upon the titers of the primary and secondary antibodies, and their optimum dilutions were found to be 1:1,000-1:2,000 and 1:1,000, respectively. The intestinal epithelial cells, intestinal content and excretory bladder showed strong positive coloring reactions even at lower titer (1:2,000) of the primary antibody, whereas the uterine wall and eggs, vitelline glands, and male reproductive organs showed only weak positive reactions despite an increase in the antibody titer (1:1,000). On the other hand, the suckers, tegument, subtegumental cells and other parenchyma portions did not reveal any positive immunoperoxidase reaction at the same antibody titers. From the above results, it is highly suggested that the most potent antigenicity of C. sinensis occur from their excretory-secretory substances originated from the digestive and excretory organs.

[Chronological observation of intestinal lesions of rats experimentally infected with Echinostoma hortense].

Intestinal histopathological changes due to infection with Echinostoma hortense (Trematoda) were studied in rats after experimental infection with the metacercariae. The metacercariae were obtained from the tadpoles of Rana nigromaculata, a second intermediate host infected in the laboratory. Total 18 albino rats (Sprague-Dawley) were given 200 matacercariae each and sacrificed on the day 1, 3, 7, 11, 22 or 44 post-infection (PI). Segments of the small intestine at 1, 3, 5, 8 and 30 cm posterior to the pylorus (PTP) were resected and studied histopathologically. 1. The flukes were seen to have intruded into the intervillous space in the upper small intestine at early stages (1-3 days PI), however, they were located mainly in the intestinal lumen at later stages (7-44 days PI). The flukes were sucking and destroying the epithelial layers of villi with their oral and ventral suckers. 2. Histopathological changes of the intestine were recognizable in as early as 1-3 days after infection, and the changes became severer as the infection progressed. 3. The intestinal mucosa was histopathologically characterized by villous atrophy and crypt hyperplasia throughout the infection period. Major villous changes were blunting, fusion, severe destruction and loss of epithelial layers of villi. Villous/crypt (V/C) height ratio was remarkably reduced from 3:1 in controls to 1:1 in severely infected animals. In the stroma of villi, inflammatory cell infiltrations, vascular congestion, edema, and/or fibrosis were recognized. The goblet cells were increased in number after 11 days PI.(ABSTRACT TRUNCATED AT 250 WORDS)

[A human case of tick bite by Ixodes nipponensis on the scalp].

A human case of tick bite on the scalp was found at a local hospital on June, 1984. The patient, 63-year old female, was attacked by a tick while working in a farm forest which located in the suburbs of Seoul. The clinical complaint was a (worm) mass on the scalp which she thought as a tumor. On admission the patient complained of facial edema and general malaise. After removal of the mass (tick), small bleeding and discoloration were observed around the biting site. The tick was morphologically examined and identified as Ixodes nipponensis. This is the 4th human case of tick bite reported in the literature of Korea.