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international publications on paragonimiasis in Ecuador, epidemiological records from the Ministry of Public Health and unpublished research data was conducted to summarise the current status of the parasite/disease.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.

Massive pleural effusion due to paragonimiasis: Biochemical, cytological, and parasitological findings.

Paragonimiasis is an important food-borne parasitic zoonosis caused by trematode species of the genus, Paragonimus occurring in many parts of the world except in Australia and Antarctica. In India, it is an emerging parasitic disease, which is endemic in the northeast states where people have a common practice of eating raw or inadequately cooked freshwater crabs. In these states, Paragonimus heterotremus has been identified as the major causative agent of the human paragonimiasis. The most common clinical form of the disease is pulmonary paragonimiasis; however, extra-pulmonary manifestations are not uncommon. Here, we report a case of primary massive unilateral pleural effusion due to paragonimiasis. The diagnosis was confirmed by finding Paragonimus ova in the pleural fluid. The patient was successfully treated with repeated thoracocentesis and a course of praziquantel.

Paragonimus & paragonimiasis in India.

Ever since the discovery of the first indigenous case in 1981, paragonimiasis has gained recognition as a significant food borne parasitic zoonosis in India. The data available on the occurrence of paragonimiasis, until today, may be just the tip of an iceberg as the study areas covered were restricted to Northeast Indian States. Nevertheless, the results of research on paragonimiasis in India have revealed valuable information in epidemiology, life cycle, pathobiology and speciation of Indian Paragonimus. Potamiscus manipurensis, Alcomon superciliosum and Maydelliathelphusa lugubris were identified as the crab hosts of Paragonimus. Paragonimus miyazakii manipurinus n. sub sp., P. hueit’ungensis, P. skrjabini, P. heterotremus, P. compactus, and P. westermani have been described from India. P. heterotremus was found as the causative agent of human paragonimiasis. Ingestion of undercooked crabs and raw crab extract was the major mode of infection. Pulmonary paragonimiasis was the commonest clinical manifestation while pleural effusion and subcutaneous nodules were the common extra-pulmonary forms. Clinico-radiological features of pulmonary paragonimiasis simulated pulmonary tuberculosis. Intradermal test, ELISA and Dot-immunogold filtration assay (DIGFA) were used for diagnosis and epidemiological survey of paragonimiasis. Phylogenitically, Indian Paragonimus species, although nested within the respective clade were distantly related to others within the clade.

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.

Molecular discrimination between individual metacercariae of Paragonimus heterotremus and P. westermani occurring in Thailand.

To accurately discriminate between individual metacercariae of Paragonimus heterotremus and P. westermani occurring in Thailand, polymerase chain reaction (PCR)-based molecular methods were established and subjected to an evaluation. We first amplified and sequenced the second internal transcribed spacer (ITS2) region of the nuclear ribosomal DNA of the two species. Based on their nucleotide differences, P. heterotremus and P. westermani were unequivocally discriminated from each other. These nucleotide differences were further utilized to select the ApaL1 endonuclease site for PCR-restriction fragment length polymorphism (PCR-RFLP) analyses and to design species-specific primers for multiplex PCR reactions. Both PCR-RFLP and multiplex PCR methods allowed a more rapid and labor-effective species discrimination. Furthermore, the multiplex PCR method enabled the most efficient discrimination because species identification involved a single round of PCR in a single tube. In Thailand, P. heterotremus is the only species affecting humans. Thus, the methods established in the present study can be used as reliable tools to identify the lung fluke metacercariae that cause human disease.