The lung fluke, Paragonimus, and lung fluke disease, were reviewed, especially on the works performed in Korea. Among 43 species of Paragonimus recorded in the world literature, P. westermani, P. pulmonalis, and P. iloktsuenensis are known to distribute in Korea. Biological studies on P. westermani have revealed that its snail intermediate host is Semisulcospira spp. and major second intermediate hosts are crabs such as Eriocheir japonicus and E. sinensis or crayfish such as Cambaroides similis and C. dauricus. The final hosts other than man are dogs, cats, pigs, and wild animals. Paragonimiasis has been known to distribute widely over the south Korea, however, the prevalence among people and intermediate hosts is gradually decreasing in recent years. In Korean people, the lung fluke have caused pulmonary infections in a great many of cases but as well extrapulmonary ones including cerebral, spinal, subcutaneous, hepatic, splenic, abdominal, urinary, gynecologic, and other types. The definite diagnosis of paragonimiasis can be executed by the recovery of eggs from sputum or feces of patients, however, immunological methods such as intradermal test, complement-fixation test, precipitin reactions, immunofluorescent techniques, enzyme-linked immunosorbent assay (ELISA) are greatly helpful for egg-negative or extrapulmonary cases. Various drugs have been introduced for the treatment of paragonimiasis, and among them, bithionol, niclofolan and praziquantel have shown high therapeutic efficacy. The most recommendable one is considered praziquantel. For control of this disease, mass chemotherapy of patients seems to be the most efficient and feasible measure.

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Paragonimus and Paragonimiasis in Korea

Human paragonimiasis is caused by nine species of Paragonimus, namely, P westermani, P africanus, P heterotremus, P kellicotti, P mexicanus, P siamensis, P skrjabini, P skrjabini miyazakii, and P uterobilateralis Cerebral or spinal involvements are most common in P westermani, and can also occur in P skrjabini, P skrjabini miyazakii, and P mexicanus In P westermani, cerebral paragonimiasis comprises about 45% of all extrapulmonary paragonimiasis cases, and accounts for about 1% of all paragonimiasis patients In cerebral paragonimiasis, seizure, headache, visual disturbance, and motor and sensory disturbances are the five major clinical symptoms The most commonly performed diagnostic procedures for cerebral infections are serological tests to detect circulating antibodies or antigens using ELISA or immunoblotting, and radiological examinations, including plain skull x-rays, brain CT, and MR scans The drug of choice is praziquantel at the dose of 25 mg/kg three times daily for 2–3 days In severe infections, a second set of treatment may be needed Triclabendazole can be used in P uterobilateralis, P mexicanus, and P skrjabini infections with the dose of 10 mg/kg twice a day; however, its efficacy in P westermani infection, in particular cerebral infections, remains to be elucidated

Chapter 23 – Paragonimiasis

ELISA-inhibition test using Paragonimus westermani specific monoclonal antibody (Mab) was investigated to improve the diagnostic specificity of paragonimiasis. By cell fusion, one hybridoma clone secreting anti-P. westermani specific Mab was selected (Pwa-14), which reacted on bands of 28 kDa, 42.5 kDa, 89 kDa and 120.5 kDa. IFA showed Pwa-14 was located at the vitelline follicles. By micro-ELISA, 100% of 22 paragonimiasis cases were found positive, but 5 of 40 clonorchiasis cases (12.5%), 3 of 26 cysticercosis cases (7.7%) showed false positive. None of 10 sparganosis patients or 28 normal controls reacted positively. On the other hand, by ELISA-inhibition test using a P. westermani specific Mab, 100% of paragonimiasis cases were found positive, and there were no positive in cysticercosis, sparganosis cases or normal controls, except 2 (5.0%) false-positive sera of 40 clonorchiasis cases. The ELISA-inhibition test using a Mab showed higher specificity in comparison with micro-ELISA for serodiagnosis of human paragonimiasis.

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Serodiagnosis of human paragonimiasis by ELISA-inhibition test using monoclonal antibodies

To observe antibody changes after praziquantel treatment in paragonimiasis, a total of 46 serum samples from 13 serologically diagnosed patients was collected for 4-28 months. The specific antibody (IgG) levels were measured by enzyme-linked immunosorbent assay (ELISA). All but one patient who needed retreatment became symptom-free within a week. Antibody levels were dropped near to or below a cut-off absorbance (abs.) of 0.25 in varying intervals from 4 to 18 months. Of 9 patients who were retested within 3 months, 5 revealed temporary elevation of antibody level. After the elevation, the levels began to decline slowly to negative ranges. If treated earlier after symptoms developed, the temporary elevation did not occur and intervals to negative conversion were shorter. By sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE)/immunoblot, antigen-antibody reactions in individual patient faded gradually without significant changes in reacting antigen bands.

Antibody changes in paragonimiasis patients after praziquantel treatment as observed by ELISA and immunoblot.

Diagnosis of early paragonimiasis is difficult because parasitological evidence is not easily obtained Antibody tests have been proposed as a good substitute for classical diagnostic techniques Using the crude extracts of Paragonimus westermani eggs, metacercariae, 4- and 7-week juveniles, and 16-week adults as antigens, we observed the early antibody responses Sera were obtained from 4 experimental cats, fed 50 metacercariae each, at intervals until 13 weeks post-infection Antibody (IgG) responses were identified by ELISA using extracts of 4-week juveniles, followed by those of 7- and 16-week worms Antibody responses were minimal against the metacercarial extracts Antibodies to P westermani egg extracts were elevated after 10 weeks post-infection In immunoblot analysis, more than nine protein bands in 4-week juveniles reacted with the early infection sera Antigenic proteins in adult worms were different from those of juveniles After four weeks of infection, 32 and 35 kDa bands in the adult extracts were increasingly reactive Egg specific proteins at 28, 46 and 94 kDa were reactive only after 10 weeks Antigenic components reacting to the early infection sera changed during the maturation stages of P westermani; almost all juvenile antigens were replaced by adult antigen components

Variation of antigenic proteins of eggs and developmental stages of Paragonimus westermani

In order to observe the antigenic fractions in saline extract of adult Paragonimus westermani, proteins in the crude extract were separated by sodium dodecyl sulfate-polyacylamide gel electrophoresis(SDS.PAGE) in reducing conditions. The separated protein fractions were transferred to nitrocellulose paper on which 20 sera from human paragonimiasis were reacted and immunoblottrd. Out of 15 stained protein bands in SDS-PAGE, 7 reacted with infected sera while 8 did not. Additionally, 7 unstained protein bands in SDS-PAGE reacted with the sera. Of 14 reacted bands, 30 kilodalton(kDa) band was the most frequently reacted (95%) and was a strong antigen. Protein bands of 23 and 46 kDa were also strong antigens. Bands of over 150 kDa, 120 kDa, 92 kDa, 86 kDa, 74 kDa, 62 kDa, 51 kDa, 32 kDa, 28 kDa, 16.5 kDa and 15.5 kDa were also reactive but their frequencies of the reaction were variable. Key words: Paragonimus westermani, human paragonimiasis, antigenic proteins, SDS-PAGE/ immunoblot

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Sung Hwan Kim

Papers

Immunoblot observation of antigenic protein fractions in Paragonimus westermani reacting with human patients sera.