[Clinical analysis of 17 cases of paragonimiasis].

OBJECTIVE: To investigate the clinical features, diagnosis and treatment of paragonimiasis, so as to improve the prevention and treatment of it. METHODS: The clinical data of paragonimiasis patients were collected and retrospectively analyzed. RESULTS: Totally 17 patients were diagnosed as paragonimiasis and the main clinical features of 11 patients were cough, chest pain and fever, and the pleural effusion was found in 13 cases. Peripheral blood eosinophil percentages of all patients were significantly increased, and the detections of antibody IgG againstParagonimus parasite of ELISA method were positive in all patients. All the patients were cured after praziquantel treatment and no recurrence found in the follow-up visit. CONCLUSIONS: The clinical features of paragonimiasis patients are diverse, and pleural effusion is quite common in imaging examinations. The eosinophil percentages and antibody detections have important values for the diagnosis of paragonimiasis. Praziquantel is an effective medicine in the treatment.

[Dot immuno-gold filtration assay in the diagnosis of suspected paragonimiasis and evaluation of chemotherapeutic effect].

OBJECTIVE: To evaluate the usefulness of dot immuno-gold filtration assay(DIGFA) for the diagnosis of Paragonimus infection. METHODS: During 2003 to 2006, 72 cases suspected of paragonimiasis in Zhejiang Province were examined with DIGFA for rapid detection of specific antibodies against Paragonimus (Pw-DIGFA). The diagnosis was primarily established with the presence of antibodies, experience of ingesting raw freshwater crabs or crayfishes and clinical presentations. The cases were treated with praziquantel and followed-up at 3 and/or 6 months post-treatment. Antibody level in patients (pre- and post-treatment) were detected in parallel and analyzed comparatively by Pw-DIGFA and ELISA. RESULTS: The result of detection by Pw-DIGFA was in agreement with that of ELISA. 28 of 72 cases were antibody positive and 44 cases were negative. Among the 28 positives, 26 cases had a history of eating raw freshwater crab or crayfishes and the other 2 cases drank freshwater from brook before. 21 cases showed paragonimiasis-related clinical symptoms such as low-grade fever, cough, or changes in image examination, while the other 7 cases showed only eosinophilia in peripheral blood (15%-70%). The mean absorbance values (A450) of positive sera, negative sera and normal sera tested by ELISA were 1.7361, 0.2973 and 0.2657 respectively. There was significant difference between the positive cases and the negative cases (t=12.047, P<0.01) and no significant difference between the negative cases and normal controls (t=1.919, P>0.05). At 3 month post-treatment, serum antibody in 5 cases whose clinical symptoms and physical signs relieved or disappeared decreased 2-5 titers and that of one case who relapsed with new signs increased by one titer. In Pw-DIGFA, the dot color of 5 cured cases showed a little weaker than that of pre-treatment and the relapsed case displayed similar response. At 6 month post-treatment, 7 sera of clinically cured cases showed significantly weaker response than that of pre-treatment. The antibodies of those sera dropped 3-6 titers. CONCLUSION: Pw-DIGFA is of supplementary value for clinical diagnosis of paragonimiasis. Antibody detection by pre- and post-treatment using Pw-DIGFA shows potential for the evaluation of therapeutic effect.

Effects of L-type Ca2+ channel antagonists on in vitro excystment of Paragonimus ohirai metacercariae induced by sodium cholate.

The inhibitory effects of L-type Ca2+ channel antagonists on Na cholate-induced in vitro excystment (CIIE) of Paragonimus ohirai metacercariae were studied. At concentrations of 10 microM, nicardipine and nimodipine inhibited CIIE completely and by approximately 92%, respectively. Nitrendipine and (+/-)-verapamil inhibited CIIE by about one half and one third, respectively. Nifedipine and diltiazem did not inhibit CIIE significantly. At higher concentrations, nitrendipine at 20 microM completely inhibited CIIE, and (+/-)-verapamil at 40 microM inhibited CIIE by 93%. Nifedipine and diltiazem inhibited CIIE only slightly and little, respectively, even at 40 microM. Complete inhibition by nicardipine at 10 microM required preincubation of metacercariae with the antagonist for 15 min. The inhibitory effects of nicardipine and nimodipine were reversible, and most of the nimodipine-treated metacercariae could excyst within 1 h after being washed, but the nicardipine-treated ones started to excyst 1 h after washing. Nicardipine suppressed the active movement of encysted juveniles evoked by Na cholate, whereas nimodipine did not suppress this significantly. These results suggested that L-type Ca2+ channels appeared to be involved in CIIE of P. ohirai metacercariae and that the inhibitory effect of the channels was due primarily to factors other than the inhibition of muscular activity, probably involving the secretion and release of enzymes lytic against the metacercarial cyst wall.

Effects of blockers of Ca2+ channels and other ion channels on in vitro excystment of Paragonimus ohirai metacercariae induced by sodium cholate.

The inhibitory effects of various ion channel blockers were examined on in vitro excystment of Paragonimus ohirai metacercariae induced by a bile salt, sodium cholate. At a concentration of 10 microM, bepridil, a non-selective Ca(2+) channel blocker, completely inhibited in vitro excystment, whereas TEA, lidocaine, and R(+)-IAA-94, channel blockers against K(+), Na(+) and Cl(-) ions, respectively, benzamil, an Na(+)/H(+) and Na(+)/Ca(2+) ion exchanger blocker, and R(+)-DIOA, a [K(+), Cl(-)] cotransporter inhibitor, did not. Considering the previous result that Ca(2+) ionophores are also efficient inducing factors for in vitro excystment of P. ohirai metacercariae and the present result, bile salts appear to induce the excystment of P. ohirai metacercariae through evoking the Ca(2+) channels of target cells within the metacercarial juveniles.

Paragonimiasis cases recently found among immigrants in Japan.

OBJECTIVE: Paragonimiasis is a typical food-borne parasitic disease mainly endemic in Southeast Asia. In Japan, the disease has been re-emerging since the 1980s. In addition, recently we encountered an increasing number of immigrants with paragonimiasis in Japan. In this study we summarized the clinical features of immigrants. PATIENTS AND METHODS: Among a total of 152 paragonimiasis cases referred to and diagnosed in our laboratory during 1998 to 2002, 18 were immigrants. Their clinical features including laboratory data such as eosinophilia and total IgE level were gathered from the consultation sheets from attending physicians. RESULTS: Among a total of 18 immigrant cases, 16 were from China and 2 from Thailand. A majority of immigrants had eaten freshwater crabs. Most of the Chinese patients were infected as small groups of family and/or compatriots. Chest radiographic findings were variable and multiple lung lesions were seen in about one-half of the patients. About 80% of patients had peripheral blood eosinophilia and 65% had elevated serum IgE level. CONCLUSION: The clinical features of paragonimiasis in immigrants in Japan were much more severe compared to those of Japanese patients.

In vitro excystation of Paragonimus heterotremus metacercariae.

In vitro excystation studies were carried out on the metacercariae cysts of Paragonimus heterotremus obtained from naturally infected crabs Potamon spp. The effects of elastase, trypsin, trypsin-dog bile, trypsin-bile salt, and dithiothreitol (DTT) were examined. The trypsin-dog bile medium stimulated maximum excystation. Of the media that contained 1 mM DTT, the optimum conditions for the excystation were shown to be pH 9, temperature of 39-40 C, and osmolarity of 250-350 mOsm. The DTT acceleration was antagonized by all of the following 6 protease inhibitors: leupeptin (0.5-4 microg/ml), L-trans-epoxysuccinyl leucylamido (4-guanidine) butane (1-8 microM), N-tosyl-L-phenylalanine chloromethyl ketone (0.1-0.4 mM), N alpha-p-tosyl-L-lysine chloromethyl ketone (25-200 microg/ml), iodoacetic acid (0.5-4 mM), and phenylmethylsulfonyl fluoride (1-4 mM). These results suggest that a number of extrinsic and intrinsic factors may modulate excystation.

Effect of ionophores on in vitro excystment of Paragonimus ohirai metacercariae.

Ca2+ ionophores (A23187 and ionomycin) induced in vitro excystment of Paragonimus ohirai metacercariae, whereas Na+ ionophore (monensin) and K+ ionophores (nigericin and valinomycin) did not. The effect of A23187 on in vitro excystment was dependent on the concentration of the ionophore. A high concentration of 1 microM or more induced rapid excystment, but resulted in the death of metacercarial juveniles. The appropriate concentration of 0.2 microM induced in vitro excystment by 5 h after incubation with little or no damage to the juveniles.

[Effect of triclabendazole on the ultrastructure of body wall and vitelline cells of Paragonimus westermani].

AIM: To observe the ultrastructural changes in the body wall and the vitelline cells of Paragonimus westermani in vitro and in vivo before and after triclabendazole treatment. METHODS: The worms were obtained from in vitro and in vivo tests. All of the samples were processed by conventional techniques, and observed by scanning electron microscopy (SEM) and transmission electron microscopy (TEM). RESULTS: The external plasma membrane and matrix were cracked or disappeared after the treatment. The necrosis of the muscular layer differed. The cell membranes of cortex and vitelline cells were damaged. Nuclear membrane was damaged partially, heterochromatin solidified and condensed to brim and dissolved. The Golgi complex disappeared, endoplasmic reticulum expanded, mitochodria denatured and dissolved. The damage was more serious in vivo than in vitro. CONCLUSION: Triclabendazole is remarkablely effective against Paragonimus westermani by damaging the body wall and vitelline cells, mainly affecting the nuclei, membrane structures and microtubular system.

Excystment of Paragonimus westermani metacercariae by endogenous cysteine protease.

To infect definitive or paratenic hosts, metacercariae of Paragonimus westermani should excyst in the host intestine. Optimum conditions for the excystment have been known to be pH 8-9 and a temperature of 40 C. Under these conditions, excystment of P. westermani metacercariae was accelerated in the presence of 1 mM dithiothreitol (DTT). The DTT acceleration was antagonized dose-dependently by cysteine protease inhibitors of L-trans-epoxysuccinylleucylamido(4-guanidino)butane (E-64, 2-20 microM) or leupeptin (0.1-1 mM), suggesting that certain cysteine proteases of the metacercaria are involved in excystment. Protease activities were detected in excretory-secretory products (ESP) of newly excysted metacercariae. Two distinct proteases were purified by DEAE anion-exchange chromatography of the ESP. While a 27-kDa protease exhibited endodipeptidolytic activity at pH 5-8.5 and remained stable at neutral pH for 3 days, the 28-kDa enzyme was stable at pH 5-7.5, with lower activity at pH 8.5. Both proteases hydrolyzed collagen, fibronectin, and myosin within 1 hr at pH 8. These results suggest that cysteine proteases secreted by P. westermani metacercariae modulate excystment.

Effective pretreatment by cysteine proteinase inhibitor for improved analysis of protein components of trematodes on SDS-PAGE.

Since Fasciola sp. contained proteolytic enzyme(s), it was confirmed that degradation took place in protein components in extracts of the liver flukes, which resulted in lack of clarity of sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE). Degradation was shown to occur mostly during a heating process of the extract samples. The proteolytic activity in the extracts was completely blocked and electrophoretic patterns were improved only by the use of cysteine proteinase inhibitor N-[N-(L-3-trans-carboxyoxiran-2-carbonyl)-L-leucyl]-agmatine (E-64). Great improvement was also noted in electrophoretic patterns of the extracts of other trematodes, such as Paragonimus westermani, P. miyazakii and Clonorchis sinesis, when their extracts were treated with E-64.

Further studies on the effect of cyclosporin A on the course of Paragonimus infection in rats.

Paragonimus ohirai-infected rats were treated with cyclosporin A (CyA) at different times during the course of infection. CyA (5 x 80 mg/kg) affected the worm recovery, growth and maturation rates of P. ohirai with respect to control values. This tendency was most remarkable in animals treated 15 days and more after infection with CyA (groups B, +15 to +19 days; C, +25 to +29; D, +35 to +39 and E, +45 to +49). In group A (0 to +4), however, the drug did not affect markedly the growth and maturation of worms, although it significantly lowered worm recovery rates. CyA administration also affected normal migration of P. ohirai in the highly susceptible host (rat), when the drug was administered during the peritoneal and/or liver phase of infection. Thus, in this P. ohirai/rat model, CyA significantly reduced worm recovery rates, and affected the growth, maturation and migration of the worms depending on the time of administration.

The effects of triclabendazole on the lung fluke, Paragonimus uterobilateralis in the experimental host Sigmodon hispidus.

The new veterinary drug triclabendazole (Fasinex) was tested for its anthelmintic activity against adult Paragonimus uterobilateralis in experimentally infected cotton rats, Sigmodon hispidus. After a single dose of 50, 100 or 150 mg/kg b.w. triclabendazole or after two doses of 5 or 10 mg/kg b.w. given at an interval of 24 hours, 56-61% of the flukes were dead at three to five weeks after treatment. Two doses of 50 or 75 mg/kg b.w. triclabendazole killed all flukes. The flukes, which were still alive three to five weeks after treatment, were significantly smaller than the un-treated controls. Histological sections from these flukes showed severely damaged tissues. It is assumed that these flukes would have died later.

Effects of the two new anthelmintic agents CGP 6140 and CGP 20376 on adult Paragonimus uterobilateralis in the experimental host Sigmodon hispidus.

The new compounds CGP 6140 and CGP 20376 were tested for their anthelmintic activity against adult Paragonimus uterobilateralis in experimentally infected Sigmodon hispidus. Both drugs were effective at a dosage of 2 x 100 mg/kg b.w. given orally with an interval of six hours between the doses. This regimen killed 80% of the flukes collected on Days 7 and 11 after treatment with CGP 6140 and 100% of the flukes collected on Days 5, 7, and 11 after therapy with CGP 20376. Both compounds showed no effect at single doses of 25, 50 and 100 mg/kg or with 12.5 and 25 mg/kg/day given on four consecutive days.

Paragonimiasis: experimental and clinical experience with praziquantel in Korea.

The effect of praziquantel (2-cyclohexylcarbonyl-1,2,3,6,7,11b-hexahydro-4H-pyrazino[2,1-a]++ +isoquinolin- 4-one, EMBAY 8440, Biltricide) on the ultrastructure of Paragonimus westermani was investigated in vitro and in vivo. In in vitro experiments, P. westermani was less sensitive to praziquantel. Only very few vacuoles were formed in the tegumental syncytium after incubation in 100 micrograms/ml for 60 min. However, in vivo the parasite was successfully destroyed by praziquantel. In fine structural changes of P. westermani obtained from experimentally infected dogs after treatment, bleb-like structures were formed at the papillae near the two suckers and bubble-like vacuoles and vesicles in the tegumental syncytium were observed along the basement membrane being detached from the circular muscles of the worm. It was also observed that many host cells invaded the exposed muscular layer of the worm after exfoliation of the tegument and the interior of the parasite was heavily damaged by those host cells. In our clinical trials, complete cure was obtained in 10 patients given praziquantel at a dosage of 3 X 25 mg/kg body weight for 3 consecutive days and with a dosage of 3 X 25 mg/kg for 2 consecutive days, 18 (85.7%) of 21 patients were cured. On the other hand, the dosage of 3 X 25 mg/kg on one day cured 15 (71.4%) out of 21 patients. Nine uncured cases were treated again with dosages of 3 X 25 mg/kg for 2 or 3 consecutive days. Only two out of nine failed to be completely cured and a very small number of eggs were detected again in their sputum and faeces.(ABSTRACT TRUNCATED AT 250 WORDS)