Eun Hee Shin

High Prevalence of Haplorchis taichui, Phaneropsolus molenkampi, and Other Helminth Infections among People in Khammouane Province, Lao PDR

The prevalence of liver and intestinal helminth infections, including Opisthorchis, Haplorchis, Phaneropsolus, hookworms, Enterobius, and Taenia, was surveyed in Khammouane province, Lao PDR. Fecal specimens were collected from 1,242 people (590 men and 652 women) in 3 Mekong riverside villages and were examined by the Kato-Katz thick smear technique. The overall helminth egg positive rate was 81.1%. The positive rate for small trematode eggs, including Opisthorchis viverrini, heterophyids, and lecithodendriids, was 81.1% and the positive rate for hookworms was 6.7%. To obtain adult worms, 35 people who were positive for small trematode eggs were treated with 20-30 mg/kg praziquantel and 10-15 mg/kg pyrantel pamoate, and then purged. Diarrheic stools were collected from 33 of these people and searched for helminth parasites using a stereomicroscope. Mixed infections with various helminths (Haplorchis taichui, Haplorchis yokogawai, Prosthodendrium molenkampi, Phaneropsolus bonnei, echinostomes, hookworms, Trichostrongylus spp., Trichuris trichiura, Enterobius vermicularis, and/or Taenia saginata) were found. The total number of helminth specimens collected was 20,907 (approximately 634 per person). The most common species was H. taichui, followed by P. molenkampi, O. viverrini, P. bonnei, E. vermicularis, hookworms, and Trichostrongylus spp. These results show that diverse species of intestinal nematodes, trematodes, and cestodes are infecting humans in Khammouane province, Lao PDR.

Serologic Survey of Toxoplasmosis in Seoul and Jeju-do, and a Brief Review of Its Seroprevalence in Korea

Knowledge of the prevalence of human Toxoplasma gondii infection is required in the Republic of Korea. In this study, we surveyed the seroprevalence of T. gondii infection and analyzed the risk factors associated with seropositivity among residents in 2 administrative districts; Seoul and the island of Jeju-do, which have contrasting epidemiologic characteristics. Sera and blood collected from 2,150 residents (1,114 in Seoul and 1,036 in Jeju-do) were checked for IgG antibody titers using ELISA and for the T. gondii B1 gene using PCR. In addition, participants completed a questionnaire that solicited information on gender, age, occupation, eating habits, history of contact with animals, and travel abroad. The T. gondii B1 gene was not detected in all residents examined. However, ELISA showed 8.0% (89 of 1,114 sera) positive for IgG antibodies against T. gondii in Seoul and 11.3% (117 of 1,036 sera) in Jeju-do. In both districts, the positive rates were higher in males than in females, and those 40-79 years of age showed higher rates than other ages. In Seoul, residents older than 70 years of age showed the highest positive rate, 14.9%, whereas in Jeju-do the highest prevalence, 15.6%, was in those in their sixties. The higher seropositive rate in Jeju-do than in Seoul may be related to eating habits and occupations. The present results and a review of related literature are indicative of an increased seroprevalence of T. gondii in Korea in recent years.

Adult Opisthorchis viverrini Flukes in Humans, Takeo, Cambodia

To the Editor: Opisthorchis viverrini and Clonorchis sinensis, the 2 major species of small liver flukes (family Opisthorchiidae), cause chronic inflammation in the bile duct, which leads to cholangitis and cirrhosis of the liver, and are a predisposing factor for cholangiocarcinoma (1). Human infections with O. viverrini flukes are found along riverside areas of Indochina (Thailand, Lao People’s Democratic Republic [PDR], and Vietnam) (2). Small trematode eggs (length 20–32 μm) have been found in human fecal samples in Cambodia (1,3,4). During 1981–1982, two of 102 Cambodian refugees in the United States were found to be positive for C. sinensis (likely O. viverrini) eggs (3). Egg-positive cases were later detected in several provinces of Cambodia (4,5). Presence of O. viverrini flukes in Cambodia was verified by detection of metacercariae in freshwater fish in a lake on the border between Takeo and Kandal Provinces and by isolation of adult flukes in experimentally infected hamsters (6). In May 2010, we analyzed fecal samples from 1,993 persons in 3 villages (Ang Svay Chek, Kaw Poang, and Trartang Ang) in the Prey Kabas District, Takeo Province, Cambodia, ≈45 km south of Phnom Penh, to confirm the presence of O. viverrini flukes among humans. We found an egg-positive rate of 32.4% for small trematode eggs. Because these eggs may be those of Haplorchis spp. flukes (H. taichui, H. pumilio, and H. yokogawai) and lecithodendriid flukes (Prosthodendrium molenkampi and Phaneropsolus bonnei) (1), we attempted to detect adult flukes that are responsible for these eggs. Six of the small trematode egg–positive villagers, 1 man and 5 women (age range 16–72 years), who had occasional epigastric discomfort were selected for anthelmintic treatment, purgation, and recovery of adult worms. Fecal examination and anthelmintic treatment of villagers were approved by the Ministry of Health, Cambodia, under the agreement with the Korea–Cambodia International Collaboration on Intestinal Parasite Control in Cambodia (2006–2011). After obtaining informed consent, the villagers were treated with a single oral dose of praziquantel, 40 mg/kg (Shinpoong Pharmaceutical Co., Seoul, South Korea), and given a purgament (solution containing 30–40 g MgSO4). Feces was obtained 3 or 4 times in a 2–3-hour period after purgation, pooled individually, and processed as described (7). Worms obtained were fixed with 10% formalin, stained with acetocarmine, and identified by morphologic features. A total of 34 O. viverrini adult worms were obtained from the 6 villagers (14, 9, 5, 3, 2, and 1 from each villager, respectively). No other species of trematodes were obtained. Five worms were lanceolate and had a mean length of 9.5 mm (range 6.5–12.0 mm), a mean width of 1.5 mm (range 1.2–1.7 mm), and 2 characteristic 4–5-lobulated testes (Figure, panel A). Ten eggs in uteri were 27 μm long (range 25–29 μm) and 15 μm wide (range 13–16 μm). Figure A) Adult Opisthorchis viverrini liver fluke (length 12.0 mm) isolated from a human after chemotherapy and purgation in Takeo Province, Cambodia, showing the characteristic morphology of the two 4–5-lobulated testes. B) Metacercaria of O. viverrini ... To detect the source of infection, 2 freshwater fish species, Puntioplites proctozysron (n = 5) and Cyclocheilichthys apagon (n = 10), were caught in nearby Ang Svay Chek village and examined for O. viverrini metacercariae by using a digestion technique (8). A total of 50 metacercariae (Figure, panel B) were obtained from 5 P. proctozysron fish and fed to a hamster. Six weeks later, 42 young O. viverrini flukes (Figure, panel C) were isolated from the biliary tract of the hamster. Our study identified only O. viverrini infections in humans in Cambodia. However, eggs of other hepatic and intestinal flukes also can be found in humans (1). In Thailand, Vietnam, and Lao PDR, opisthorchiids (O. viverrini and C. sinensis), heterophyids (Haplorchis spp., Centrocestus formosanus, and Stellantchasmus falcatus), and lecithodendriids have been found in humans (1,7,9). In several provinces in Lao PDR, mixed infections with O. viverrini and heterophyids or lecithodendriids were common (7,9), and the relative prevalence of each fluke species varied by locality. In Vientiane, Lao PDR, O. viverrini was the predominant species, whereas in Saravane Province, H. taichui predominated (7). In a mountainous area of Phongsaly Province, H. taichui and H. yokogawai worms were obtained from 10 villagers; however, no O. viverrini worms were detected (10). Thus, in Cambodia, the presence of human infections with intestinal flukes, including Haplorchis spp. and lecithodendriids, cannot be ruled out.

Prevalence of the intestinal flukes Haplorchis taichui and H. yokogawai in a mountainous area of Phongsaly Province, Lao PDR.

Phongsaly Province, located in the northernmost area of Lao PDR, was previously suggested to be endemic for the liver fluke Opisthorchis viverrini infection. To confirm, or rule out, this suggestion, the Phonxay village in the Khoua District, Phongsaly Province, was selected for a survey. Ten volunteers (8 men and 2 women aged 31-57 years) who consumed raw freshwater fish and had gastrointestinal troubles were treated with a single dose of praziquantel (40 mg/kg) and pyrantel pamoate (10 mg/kg) and purged with magnesium sulfate to recover any worm parasites. Eight of the 10 volunteers expelled 1 or more species of trematodes, nematodes, or cestodes (worm positive rate; 80%). The worms were morphologically identified as H. taichui (861 worms from 8 people), H. yokogawai (59 from 6 people), Phaneropsolus bonnei (1 from 1 person), Trichostrongylus sp. (2 from 2 people), Ascaris lumbricoides (2 from 1 person), Enterobius vermicularis (11 from 3 people), and Taenia saginata (1 strobila with scolex from 1 person). The results indicate that the mountainous area of Phongsaly Province, Lao PDR, is not endemic for the liver fluke but endemic for intestinal flukes, in particular, Haplorchis taichui and H. yokogawai.

Echinostome Infections in the Striped-Field Mouse, Apodemus agrarius, and the Ussuri White-Toothed Shrew, Crocidura lasiura, Caught Near the Demilitarized Zone, Gyeonggi-do (Province), Republic of Korea

A total of 1,498 small mammals (rodents and insectivores), including Apodemus agrarius (n = 1,366), Crocidura lasiura (54), Mus musculus (32), Micronytus fortis (28), Eothenomys regulus (9), Micronys minutes (6), and Cricetulus triton (3), were live-trapped in Gyeonggi-do (Province) (Paju-si, Pocheon-gun, and Yeoncheon-gun) near the demilitarized zone (DMZ) from December 2004 to September 2005. A. agrarius was found to be infected with 3 species of echinostomes (Echinostoma hortense, Echinostoma cinetorchis, and Euparyphium murinum), while C. lasiura was infected with 1 species (Echinochasmus japonicas) of echinostome. Other mammals were free from echinostome infections. Total 16 E. hortense were detected in 7 (0.5%) mice, 9 E. cinetorchis from 5 (0.4%), and 3 E. murinum from 2 (0.1%) out of 1.366 A. agrarius examined. E. japonicus was found only in 1 (1.9%; total 3 specimens) C. lasiura. These results demonstrate that A. agrarius and C. lasiura, inhabiting near the DMZ of Gyeonggi-do serve as the natural definitive hosts for several species of echinostomes, although their infection rates are low. This is the first record of natural infections of A. agrarius with E. cinetorchis and C. lasiura with E. japonicus in the Republic of Korea.

Intestinal nematodes from small mammals captured near the demilitarized zone, Gyeonggi province, Republic of Korea.

A total of 1,708 small mammals (1,617 rodents and 91 soricomorphs), including Apodemus agrarius (n = 1,400), Microtus fortis (167), Crocidura lasiura (91), Mus musculus (32), Myodes (= Eothenomys) regulus (9), Micromys minutus (6), and Tscherskia (= Cricetulus) triton (3), were live-trapped at US/Republic of Korea (ROK) military training sites near the demilitarized zone (DMZ) of Paju, Pocheon, and Yeoncheon, Gyeonggi Province from December 2004 to December 2009. Small mammals were examined for their intestinal nematodes by necropsy. A total of 1,617 rodents (100%) and 91 (100%) soricomorphs were infected with at least 1 nematode species, including Nippostrongylus brasiliensis, Heligmosomoides polygyrus, Syphacia obvelata, Heterakis spumosa, Protospirura muris, Capillaria spp., Trichuris muris, Rictularia affinis, and an unidentified species. N. brasiliensis was the most common species infecting small mammals (1,060; 62.1%) followed by H. polygyrus (617; 36.1%), S. obvelata (370; 21.7%), H. spumosa (314; 18.4%), P. muris (123; 7.2%), and Capillaria spp. (59; 3.5%). Low infection rates (0.1-0.8%) were observed for T. muris, R. affinis, and an unidentified species. The number of recovered worms was highest for N. brasiliensis (21,623 worms; mean 20.4 worms/infected specimen) followed by S. obvelata (9,235; 25.0 worms), H. polygyrus (4,122; 6.7 worms), and H. spumosa (1,160; 3.7 worms). A. agrarius demonstrated the highest prevalence for N. brasiliensis (70.9%), followed by M. minutus (50.0%), T. triton (33.3%), M. fortis (28.1%), M. musculus (15.6%), C. lasiura (13.2%), and M. regulus (0%). This is the first report of nematode infections in small mammals captured near the DMZ in ROK.

Detection of Gnathostoma spinigerum Third-Stage Larvae in Snakeheads Purchased from a Central Part of Myanmar

To examine the infection status of freshwater fish with Gnathostoma spp. larvae in Myanmar, we purchased 15 snakeheads, Channa striatus, from a local market in a suburban area of Naypyidaw, the new capital city. Two larval gnathostomes were collected using an artificial digestion technique, and observed by a light microscope and a scanning electron microscope. The size of an intact larva was 2.65 mm long and 0.32 mm wide. The characteristic morphology of the larvae included the presence of a long esophagus (0.80 mm long), 2 pairs of cervical sacs (0.43 mm long), and a characteristic head bulb with 4 rows of hooklets. The number of hooklets in the 1st, 2nd, 3rd, and 4th row was 45, 48, 50, and 52, respectively. Based on these morphological characters, the larvae were identified as the advanced 3rd-stage larvae of Gnathostoma spinigerum. This is the first report of detection of G. spinigerum 3rd-stage larvae in the central part of Myanmar. Our study suggests that intake of raw meat of snakehead fish in Myanmar may result in human gnathostomiasis.

In vitro inhibition of Toxoplasma gondii by the anti-malarial candidate, 6-(1,2,6,7-tetraoxaspiro[7.11]nonadec-4-yl)hexan-1-ol.

An anti-malarial candidate, 6-(1,2,6,7-tetraoxaspiro[7.11]nonadec-4-yl)hexan-1-ol (N-251), was studied to characterize its potential as a novel anti-Toxoplasma gondii drug. In the present study, IC50 and LC50 of N-251 on host cells and T. gondii were compared to those of artemisinin and sulfadiazine. The IC50 on Huh-7 cells was 10.19μg/ml, 67.69μg/ml and 310.17μg/ml for N-251, artemisinin, and sulfadiazine, respectively. The LC50 for anti-T. gondii effect was shown to be 1.11μg/ml, 5.79μg/ml, and 5.45μg/ml for N-251, artemisinin and sulfadiazine, respectively. N-251 concentration causing complete parasiticidal effect with minimal cytotoxicity on host cells was determined to be 5μg/ml. Additionally, the anti-T. gondii effect of N-251 was confirmed by ultrastructural changes, loss of organelles, degenerated morphology and the increase of amylopectin as detected by transmission electron microscope (TEM). Accordingly, the present study suggests that the anti-malarial synthetic endoperoxide, N-251, is an emerging drug candidate more effective than artemisinin and sulfadiazine.

CD8+ T-cell Activation in Mice Injected with a Plasmid DNA Vaccine Encoding AMA-1 of the Reemerging Korean Plasmodium vivax

Relatively little has been studied on the AMA-1 vaccine against Plasmodium vivax and on the plasmid DNA vaccine encoding P. vivax AMA-1 (PvAMA-1). In the present study, a plasmid DNA vaccine encoding AMA-1 of the reemerging Korean P. vivax has been constructed and a preliminary study was done on its cellular immunogenicity to recipient BALB/c mice. The PvAMA-1 gene was cloned and expressed in the plasmid vector UBpcAMA-1, and a protein band of approximately 56.8 kDa was obtained from the transfected COS7 cells. BALB/c mice were immunized intramuscularly or using a gene gun 4 times with the vaccine, and the proportions of splenic T-cell subsets were examined by fluorocytometry at week 2 after the last injection. The spleen cells from intramuscularly injected mice revealed no significant changes in the proportions of CD8+ T-cells and CD4+ T-cells. However, in mice immunized using a gene gun, significantly higher (P<0.05) proportions of CD8+ cells were observed compared to UB vector-injected control mice. The results indicated that cellular immunogenicity of the plasmid DNA vaccine encoding AMA-1 of the reemerging Korean P. vivax was weak when it was injected intramuscularly; however, a promising effect was observed using the gene gun injection technique.