Current status of Paragonimus and paragonimiasis in Ecuador

A review of national and international publications on paragonimiasis in Ecuador, epidemiological records from the Ministry of Public Health and unpublished research data was conducted to summarise the current status of the parasite/disease. The purpose of the review is to educate physicians, policy-makers and health providers on the status of the disease and to stimulate scientific investigators to conduct further research. Paragonimiasis was first diagnosed in Ecuador 94 years ago and it is endemic to both tropical and subtropical regions in 19 of 24 provinces in the Pacific Coast and Amazon regions. Paragonimus mexicanus is the only known species in the country, with the mollusc Aroapyrgus colombiensis and the crabs Moreirocarcinus emarginatus, Hypolobocera chilensis and Hypolobocera aequatorialis being the primary and secondary intermediate hosts, respectively. Recent studies found P. mexicanus metacercariae in Trichodactylus faxoni crabs of the northern Amazon. Chronic pulmonary paragonimiasis is commonly misdiagnosed and treated as tuberculosis and although studies have demonstrated the efficacy of praziquantel and triclabendazole for the treatment of human infections, neither drug is available in Ecuador. Official data recorded from 1978-2007 indicate an annual incidence of 85.5 cases throughout the 19 provinces, with an estimated 17.2% of the population at risk of infection. There are no current data on the incidence/prevalence of infection, nor is there a national control programme.

Paragonimus paishuihoensis Metacercariae in Freshwater Crabs, Potamon lipkei, in Vientiane Province, Lao PDR

Among Paragonimus species, P. paishuihoensis is one of the most mysterious and poorly understood species. Metacercariae are characterized by having a unique dendritically branched excretory bladder. However, the morphology of the adult worm remains unknown. To date, metacercariae of this species have been reported only in China and Thailand. In this study, we first found P. paishuihoensis metacercariae in freshwater crabs, Potamon lipkei, in Hinheub District, Vientiane, Lao PDR, with a prevalence of 77.7% and the average intensity of 10.3 (range 1-28) metacercariae per crab. The molecular data based on ITS2 and CO1 markers indicated that P. paishuihoensis from Laos and Thailand were almost completely identical and were close to members of the Paragonimus bangkokensis/Paragonimus harinasutai complex. Attempts to infect experimental animals (cats, dogs, and rats) with P. paishuihoensis were unsuccessful, suggesting that these animals might be unsuitable definitive hosts for the species. Further studies are necessary to elucidate the taxonomic status and life cycle of P. paishuihoensis.

Avaliação microbiológica e parasitológica de carne de caranguejo comercializada em São Luís, MA / Microbiological and parasitological evaluation of crab meat commercialized in São Luís, MA

O caranguejo é um produto que participa da culinária maranhense, sendo de fácil aquisição em feiras e mercados. Por ser um alimento normalmente consumido cru ou após leve fervura, torna-se um veículo de disseminação de microorganismos enteropatogênicos. O estudo objetivou avaliar a qualidade microbiológica da carne de caranguejo comercializada em supermercados e feiras da cidade de São Luís, MA.

A Case of Pulmonary Paragonimiasis with Involvement of the Abdominal Muscle in a 9-Year-Old Girl

In Korea, many people enjoy eating raw or underkooked freshwater crayfish and crabs which unfortunately may cause paragonimiasis. Here, we describe a case of pulmonary and abdominal paragonimiasis in a 9-year-old girl, who presented with a 1-month history of abdominal pain, especially in the right flank and the right inguinal area, with anorexia. A chest radiograph revealed pleural effusion in both lungs, and her abdominal sonography indicated an inflammatory lesion in the right psoas muscle. Peripheral blood analysis of the patient showed hypereosinophilia (66.0%) and an elevated total serum IgE level (>2,500 IU/ml). The pleural effusion tested by ELISA were also positive for antibodies against paragonimiasis. Her dietary history stated that she had ingested raw freshwater crab, 4 months previously. The diagnosis was pulmonary paragonimiasis accompanied by abdominal muscle involvement. She was improved after 5 cycles of praziquantel treatment and 2 times of pleural effusion drainage. In conclusion, herein, we report a case of pulmonary and abdominal paragonimiasis in a girl who presented with abdominal pain and tenderness in the inguinal area.

Discovery of Maritrema jebuensis n. sp. (Digenea: Microphallidae) from the Asian Shore Crab, Hemigrapsus sanguineus, in Korea

Maritrema spp. (Digenea: Microphallidae) are parasites of birds, but have not been found in the Republic of Korea. In this study, metacercariae of Maritrema sp. were discovered in the Asian shore crab, Hemigrapsus sanguineus, caught in the mud-flats of Jebu-do, Hwasung-gun, Gyeonggi-do, and the adult flukes were confirmed by experimental infection into mice. Based on the symmetric ribbon-like vitellarium, adult flukes of Maritrema sp. were identified, but did not belong to previously described species in terms of the following morphologic characteristics: ceca reaching to the lateral wall at the anterior border of the ovary; ventral sucker larger than oral sucker; a prominent metraterm; and vitellarium forming a complete ring. Hence, we named this microphallid M. jebuensis n. sp. after the island where the second intermediate hosts were collected. From this study, it has been shown that Maritrema sp. is distributed in Korea and transmitted by the Asian shore crab, H. sanguineus.

Surveys of Gynaecotyla squatarolae and Microphallus koreana (Digenea: Microphallidae) Metacercariae in Two Species of Estuarine Crabs in Western Coastal Areas, Korea

To figure out the geographical distribution of Gynaecotyla squatarolae and Microphallus koreana metacercariae in Korea, shore crabs of southwestern coastal areas were examined. Eight coastal areas in Inchon-si (A), Gyeonggi-do (B), Chungcheongnam-do (C, D, and E), Jeollabuk-do (F), and Jeollanam-do (G and H) were selected, and 2 kinds of crabs, Macrophthalmus dilatatus and/or Macrophthalmus japonicus, were caught. After transportation to the laboratory, 15 crabs per each group were grouped and ground in a mortar and pestle, and examined for microphallid metacercariae. In M. dilatatus, G. squatarolae metacercariae were recovered from 3 (C, E, and H) out of 6 regions, but M. koreana metacercariae were not recovered. In the case of M. japonicus, G. squatarolae metacercariae were recovered from 6 (B, D, E, F, G, and H) of 7 areas surveyed, and M. koreana matacercariae were detected from 5 regions (A, B, D, F, and H). These results indicate that the life cycle of G. squatarolae is maintained in the western coastal areas using M. dilatatus and M. japonicus as intermediate hosts, while that of M. koreana is maintained only using M. japonicus.

Infection Status of Freshwater Crabs and Crayfish with Metacercariae of Paragonimus westermani in Korea

The present study investigated the infection status of Paragonimus westermani metacercariae in freshwater crabs (n = 363) and crayfish (n = 31) from October 2007 to October 2008 using the crush method. All of the freshwater crabs, Eriocheir japonicus, were negative for P. westermani metacercariae while 10 (32.3%) of the 31 examined crayfish were positive. The 10 positive crayfish were caught in Haenam, Jeollanam-do, and there were 8-59 (mean 28.4) metacrcariae per infected crayfish. These results suggest that P. westermani metacerariae are still transmitted by crayfish enzootically in southern Korea, and that freshwater crabs may transmit metacercariae only on rare occasions.

Paragonimiasis prevalences in Saraburi Province, Thailand, measured 20 years apart.

Saraburi Province, Central Thailand has been a paragonimiasis-endemic area since 1956. This study compared the prevalences of human paragonimiasis in two villages near Chet Khot Waterfall, Kaeng Khoi District, investigated in 1984-1985 and 2005. The results from the 1980s showed 6.3% and 1% of villagers were positive for Paragonimus eggs in sputum and stool, respectively. In 2005, Paragonimus eggs were not found in feces or sputum. An IgG-ELISA for paragonimiasis was conducted on 33 serum samples collected in the 1980s, 23 collected in 2005 and 25 diagnosed with other parasitic infections. Ninety percent of the samples from the eighties were positive for paragoimiasis, and 43% from 2005 were positive, equivalent to 10.9% and 4.9% of the total population examined in the 1980s and 2005, respectively. Serodiagnosis is currently the best method for detecting paragonimiasis. The positive cases in the 1980s were age 10-60 years and in 2005 were age 34- 67-years-old. The prevalence and intensity of Paragonimus metacercariae in fresh Waterfall crabs collected from Chet Khot Waterfall were significantly lower in the 1980s than in 2005. The prevalence of paragonimiasis in this endemic area has decreased to the level that no egg-producing cases were detected. No infections were found in villagers age < 30 years, despite the high density of metacercariae in the crabs, indicating a change in the habit of eating raw food among the younger people.

Microphallus koreana n. sp. (Trematoda: Microphallidae) Transmitted by a Marine Crab, Macrophthalmus dilatatus

Microphallus species occur primarily as intestinal parasites of birds and mammals, and metacercariae of a new species belonging to this genus have been discovered from the crab, Macrophthalmus dilatatus, in the Republic of Korea. The metacercaria of this fluke was round with 2 thick walls, and the excysted one had mature genital organs. The adult flukes recovered from experimentally infected chicks had numerous intrauterine eggs, well-developed pars prostatica, widely bifurcating ceca, and prominent uterine bulge. After observing internal structures, it was concluded that this species is different from any other known Microphallus spp. Based on the morphology of metacercariae and adult flukes, we describe this specimen as a new species, Microphallus koreana n. sp.

Skin Test for Paragonimiasis among Schoolchildren and Villagers in Namback District, Luangprabang Province, Lao PDR

As a part of a broader effort to determine the status of Paragonimus species infection in Lao PDR, an epidemiological survey was conducted on villagers and schoolchildren in Namback District between 2003 and 2005. Among 308 villagers and 633 primary and secondary schoolchildren, 156 villagers and 92 children evidenced a positive reaction on a Paragonimus skin test. Only 4 schoolchildren out of 128 skin test-positive cases had Paragonimus sp. eggs in their sputum, all of which was collected on 1 day. Several types of crabs, which were identified as the second intermediate host of the Paragonimus species, were collected from markets and streams in a paragonimiasis endemic area for the inspection of metacercariae. Among the examined crabs, only "rock crabs" (Indochinamon ou) harbored Paragonimus sp. metacercariae, and it is speculated that the life cycle of Paragonimus sp. was maintained via rock crabs in Namback District, Lao PDR.

Tegumental Ultrastructure of Adult Gynaecotyla squatarolae (Digenea: Microphallidae)

Gynaecotyla squatarolae (Digenea: Microphallidae) adult flukes were recovered from experimental chicks at day 4-6 post-infection and their tegumental ultrastructure was observed with a scanning electron microscopy. They were pyriform in shape, and their anterior halves were concaved ventrally. The whole body surface was covered with tegumental spines, which were wide and 16-17 digitated between oral and ventral suckers. The density of spines and number of digits decreased posteriorly. The oral sucker was subterminal and the excretory pore was at the posterior end of the worm. wo ventral suckers were similar in appearance and protruded near midline of the worm. The genital atrium was dextral to the small ventral sucker. The dorsal surface was covered with tegumental spines, but the spines were sparser than on the ventral surface. On the middle portion of the dorsal surface, a small opening presumed to be the Laurer's canal was een. From these findings, it has been confirmed that the adult G. squatarolae has unique characteristics in the surface ltrastructure.

Gynaecotyla squatarolae (Digenea: Microphallidae) from rats experimentally infected with metacercariae from the shore crab, Macrophthalmus dilatatus

Metacercariae of Gynaecotyla squatarolae (Digenea: Microphallidae) were discovered from the shore crab, Macrophthalmus dilatatus, purchased at a market in a coastal town of Taean-Eup, Chungcheongnam-do, Republic of Korea. Their adult flukes were confirmed by experimental infection of rats. The metacercariae of G. squatarolae were elliptical (391.1 x 362.5 micrometer), and the excysted metacercariae had progenetic genital organs, including the ovary and testes. To obtain adult flukes, 6 Sprague-Dawley rats were fed 500 metacercariae each, and killed at days 2, 4, and 6 post-infection. The adult flukes were identified as G. squatarolae (Yamaguti, 1934) Yamaguti, 1939, based on morphological characters, including 2 ventral suckers (1 large and 1 small), a large genital atrium equipped with the cirrus and the metraterm, separated male and female genital pores, a transversely long cirrus pouch, and extensive vitelline follicles. In the present study, it has been first proven that the shore crab M. dilatatus is a second intermediate host for G. squatarolae in the Republic of Korea.

Application of multiplex pcr for species discrimination using individual metacercariae of Paragonimus occurring in Thailand.

A total of 6 lung fluke species have been documented in Thailand, of which P. heterotremus is the most important, since it affects humans. Although P. westermani is found as metacercariae in the same crab species as P. heterotremus in Thailand, human infections with P. westermani have not been confirmed. To accurately discriminate between the individual metacercariae of these two species, we established a multiplex PCR method. Using this method, two products each were amplified from the metacercarial DNA samples of P. heterotremus (ca. 310 and 520 bp) and P. westermani (ca. 140 and 520 bp). In contrast, 520-bp products alone were found to be generated from the DNA samples of P. siamensis, P. bangkokensis and P. harinasutai, 3 other species of lung flukes known to occur in Thailand. Digestion of these 520-bp products with the restriction enzyme ScrFI could unequivocally discriminate species by the number and size of the produced band(s): 3 bands (ca. 60, 210 and 250 bp) for P. harinasutai, 2 bands (ca. 250 and 270 bp) for P. bangkokensis, and an uncut band (520 bp) for P. siamensis. The established multiplex PCR used in combination with restriction enzyme digestion (PCR-RFLP with ScrFI) is effective for discriminating the 5 different species of lung flukes occurring in Thailand, even at the metacercarial stage.

A new record of Paragonimus other than P. westermani in southern Thailand.

Field surveys of Paragonimus in Surat Thani Province, southern Thailand, revealed a new record of a lung fluke species other than P. westermani. The metacercariae were obtained from the crab, Ranguna smalleyi. The cysts of the metacercariae were spherical in shape and the larval body in the cysts contained pinkish granules. Fully mature adult worms were obtained from experimental infections with a rat and a ferret. The adult worms from the two host animals resembled each other, except for size, and had the anatomical characteristics of P. bangkokensis, ie the cuticular spines were arranged mainly in groups, the ovaries were highly branched, while the testes were more simply divided. Chromosomal preparations of the testes showed a haploid number of 11. As no sequence data of P. bangkokensis has been deposited in the GenBank/EMBL/DDBJ nucleotide database, the ITS2 region was sequenced using the metacercariae as starting material. A similarity search of P. bangkokensis ITS2 sequence using the BLAST program revealed that there was only one base difference between this population and P. harinasutai occurring in central Thailand. The result may suggest a close relationship between P. bangkokensis and P. harinasutai. This is the first description of Paragonimus species other than P. westermani occurring in southern Thailand.

Possible discovery of Chinese lung fluke, Paragonimus skrjabini in Manipur, India.

To obtain more information about Paragonimus species prevalent in Manipur, India, hundreds of freshwater crabs, Potamiscus manipurensis, were captured from mountain streams in the Motbung Mountains in Senapati District, from December 1997 to January 1998. Crab extracts were prepared by digestion, differential filtration, and sedimentation. The filtered sediments were critically examined under a stereomicroscope. Isolated Paragonimus metacercariae were used for morphological study and animal experimentation. Forty-seven metacercariae were fed orally to a 3-month-old male puppy of local breed; at autopsy 155 days after inoculation, 12 adult worms were recovered; 2 were free in the thoracic cavity and 5 pairs were in lung cysts. Two adult worms were flattened and fixed in 70% ethanol and the remaining worms were put directly into 70% ethanol and preserved until July 2005. The former 2 worms were stained with borax carmine for morphological study at Sikkim Manipal Institute of Medical Sciences, Sikkim, India. Two adult worms in the latter group were sent to the National Institute of Infectious Diseases, Tokyo, Japan, for morphological identification and DNA sequencing. The morphological features of the borax carmine-stained worms were characteristic of P. skrjabini, although no genetic material for PCR amplification and sequencing could be extracted from the worm.

A new species of Microphallus (Trematoda: Microphallidae) from Venezuela

During 1997-1999, a total of 94 crabs, Uca rapax were collected from La Sabana, La Ceiba and El Paujil, Sucre State, Venezuela. Of these 36 were infected with metacercariae. Two parasites were located in the abdominal muscles and one under the tissue of carapace and gonad. These metacercariae grew to adults in the following genera: Levinseniella, Microphallus and Maritrema, in the period of 2-5 days after feeding experimentally to the rat Rattus norvegicus, mice Mus musculus and duck Cairinia moschata. Specimens of the genus Microphallus were described herein as a new species M. sabanensis. The life cycle of M. sabanensis sp.nov. were studied experimentally using rat, mice and duck. All developmental stages and the adult are described. In addition, M. sabanensis was collected from wild birds Anas discors, Pluvialis squatarola, Butorides striatus, Egretta caerulea and Nycticorax violaceus from the same localities.

Helminths of freshwater animals from five provinces in northern Thailand.

Seven species of fishes, 6 species of mollusks, 1 species of crab, and 1 species of shrimp were collected from 12 natural freshwater sites from 5 provinces: Lumpang, Phrae, Phayao, Chiang Rai and Chiang Mai during the Rainy season of 2000. Fishes, Channa striatus, Dermogenus pusillus, Oxyeleotris marmoratus, Trichogaster trichopterus and Trichopsis vittatus were examined for helminths and 34.69% (17/49) were infected. There were 2 genera of monogenean identified as follows: Dactylogyrus and Trianchoratus; 3 genera of metacercariae were as follows: Acanthostomum, Posthodiplostomum and Stellantchasmus. Furthermore, 1 genus of Acanthocephala was found from fishes to be Pallisentis sp. The prevalence of infection in mollusks was 6.20% (17/274); Pleurolophocercous and Furcocercous cercariae were observed only in one species of mollusks, Melanoides sp. A crab and a shrimp were negative.

A survey of helminths in freshwater animals from some areas in Chiang Mai.

Thirteen freshwater animals consisting of 8 species of fish, 1 species of shrimps and crabs and 3 species of mollusks collected during September 2000 from 8 sites in 4 districts of Chiang Mai Province: Hang Dong, San Pa Tong, Chom Tong and Hot were investigated for helminthes. Two metacercariae were recovered from 4 species of fish with the prevalence of 80.32%; Metacercariae of Stellantchasmus falcatus were found in the body cavity and under the scales of Dermogenus pusillus (39.34%) and the metacercariae of Haplorchoides sp were found only under the scales of Mystacoleucus marginatus, Systomus orphoides and Rasbora paviei, (40.98%). Adult of Acanthocephala, Pallisentis sp also recovered from the intestine of Trichopsis vittatus (3.29%). Moreover, Pleurolophocercous cercaria and redia were found in mollusk (Lymnaea sp) with the prevalence of 45.65%.

Paragonimosis in the Cajabamba and Condebamba districts, Cajamarca, Peru

Stool samples from 409 pre-school and school students, living in six villages of the Cajabamba and Condebamba districts, Cajamarca, Perú, were examined using wet preparations and Lumbreras' method, looking for Paragonimus eggs. Fecal and sputum samples from two children (0.5 percent) of 6 and 8 year-old showed eggs of Paragonimus. One hundred and twenty freshwater crabs, Hypolobocera chilensis eigenmanni, were collected from the Condebamba valley and 21 (17.5 percent) of them were infected with P. mexicanus (syn. P. peruvianus) metacercariae. Our results show the persistence of Paragonimus in human beings and in the main source of infection, the crabs

Epidemiology, symptoms and treatment of paragonimiasis in Sin Ho district, Lai Chau province, Vietnam.

Sin Ho is a district of the northern mountainous province of Lai Chau, Vietnam, where the people have the habit of eating undercooked crabs. A study on paragoniamiasis carried out from 1994 to 1995 with 1,642 persons in this endemic area, showed that the rate of eating raw-crab was 72.5%. Crab examination (Ranguna kimboiensis) showed an infection rate of Paragonimus metacercaria of 98.1%. With 624 stool samples examined by Kato technique, the infection rate of Paragonimus in humans was 6.4%. With 338 sputum samples examined by direct and centrifuge methods, the infection rate of Paragonimus was 7.4%. Most of the patients were children (63.2%). The infection rate of Paragonimus in dogs was 18.2 - 33.3%. Adult worms, collected from the dogs in the field and from the cats in laboratory, were identified as Paragonimus heterotremus. The main symptoms of Paragonimus patients were cough and hemoptysis (92%), discontinuously developed (96%), without fever (94%), chest pain (70%), pleural effusion (26%), neurogical symptoms (8%), eosinophilia (88.9%), nodular ring shadows in the lungs, as shown by chest X-ray examination and more in lower lobe, (76.2%). Paragonimiasis patients were treated by (a) Praziquantel 25 mg/kg/day x 3 days; the cure rate was 68.8%. (b) Praziquantel 50 mg/kg/day x 3 days; the cure rate was 75%.