Search

Paragonimiasis is an important but neglected foodborne trematodiasis caused by Paragonimus mexicanus in Costa Rica. Immunological techniques for diagnosing this parasitosis in humans do not exist in Central America. The objective of the present study was to use recombinant Paragonimus westermani cysteine protease 7 to standardize an ELISA for the detection of antibodies against Paragonimus spp. Human sera positive for P. westermani, P. mexicanus, or Paragonimus spp., human sera infected with other helminths, as well as sera of healthy humans without parasitic infections, were analyzed. The sensitivity of the ELISA was 92.9%, and the specificity was 91.9%. This report is the first to describe the development of an ELISA for the diagnosis of Paragonimus spp. in Costa Rica and Central America. Using this ELISA in the health system of Costa Rica is recommended to detect infections.

We report an unusual case of paragonimiasis in a Nepali patient presenting with massive pericardial effusion and pericardial tamponade. The patient reported neither the consumption of crabs or crayfish nor the consumption of wild animal meat, which are the usual sources of infection. It is suspected that the source of infection was instead the ingestion of raw live slugs as part of a traditional medicine treatment.

Abstract. We studied the value of an IgG Western blot (WB) with Paragonimus kellicotti (Pk) antigen for diagnosis of North American paragonimiasis. The test was evaluated with sera from patients with Pk and Paragonimus westermani infections, with control sera from patients with other helminth infections, and sera from healthy Americans. All 11 proven Pk infection sera and two samples from suspected cases that were negative by P. westermani WB at the Centers for Disease Control and Prevention (CDC) contained antibodies to antigens at 34 kDa and at 21/23 kDa. Seven of 7 P. westermani sera contained antibodies to the 34 kDa antigen, but only 2 recognized the 21/23 kDa doublet. No control samples were reactive with these antigens. Antibody reactivity declined after praziquantel treatment. Thus, the P. kellicotti WB appears to be superior to P. westermani WB for diagnosing Pk infections, and it may be useful for assessing responses to treatment.

Paragonimiasis is an important food-borne parasitic zoonosis caused by infection with lung flukes of the genus Paragonimus. In Southeast Asia, Paragonimus heterotremus is the only proven causative pathogen. Recently, a new Paragonimus species, P. pseudoheterotremus, was found in Thailand. This species is genetically similar to P. heterotremus and is considered as a sister species. However, infectivity or pathogenicity of P. pseudoheterotremus to humans remains unclear. We report the first confirmed human pulmonary paragonimiasis case caused by P. pseudoheterotremus infection. After polymerase chain reaction/sequencing of the DNA extracted from Paragonimus eggs in the sputum of the patient, partial internal transcribed spacer 2 and cytochrome c oxidase subunit 1 sequences were approximately identical (98-100%) with those of P. pseudoheterotremus. For P. heterotremus, the partial internal transcribed spacer 2 sequence was approximately identical (99-100%), but the partial mitochondrial cytochrome c oxidase subunit 1 sequence showed a similarity of 90-95%.

The cysteine proteinases of Paragonimus westermani are known to play important roles in invasion and pathogenesis to hosts and in immune modulation and nutrient uptake. In this study, we have cloned a new cysteine proteinase of P. westermani, PwCP2, from adult worms and tested its diagnostic usefulness. The PwCP2 gene had an open reading frame of 816 base pairs and a conserved catalytic triad of cysteine, histidine, and asparagine residues. The mature form of recombinant PwCP2 (rPwCP2) lacking a proregion was overexpressed in Escherichia coli and used to produce antiserum. Western blot and immunohistochemical analyses using this antiserum showed that PwCP2 was expressed as a mature form, 24-kD product in a crude extract and in the excretory-secretory product of P. westermani, and was localized mainly in the intestinal epithelium of the adult worm. Western blot analysis using the rPwCP2 showed not only high sensitivity (90%) and specificity (100%) to sera from patients with paragonimiasis westermani, but also no cross-reactivity with sera from patients with clonorchiasis, sparganosis, or cysticercosis. Furthermore, an enzyme-linked immunosorbent assay using rPwCP2 exhibited a sensitivity of 93% and a specificity of 93% with sera of rats infected with P. westermani metacercariae. These results suggest that the excretory-secretory PwCP2 can be used for the diagnosis of paragonimiasis.

A 67-year-old Korean woman attended our hospital complaining of a severe headache. A brain computed tomography scan showed conglomerated, high-density, calcified nodules in the left temporo-occipito-parietal area and high-density subarachnoid hemorrhage in the basal cisterns. Magnetic resonance imaging of the brain shows multiple conglomerated iso- or low-signal intensity round nodules with peripheral rim enhancement. She underwent craniotomies to clip the aneurysm and remove the calcified masses. Paragonimus westermani eggs were identified in the calcified necrotic lesions. Results of parasitic examinations on the sputum and an enzyme-linked immunosorbent assay for P. westermani were all negative. The patient presented with headache and dizziness that had occurred for more than 30 years. She had not eaten freshwater crayfish or crabs. However, she had sometimes prepared raw crabs for several decades. Overall, this case was diagnosed as chronic cerebral paragonimiasis, in which she may have been infected through the contamination of utensils during the preparation of the second intermediate hosts, combined with a cerebral hemorrhage.

A 66-year-old man visited our hospital with primary complaint of cough. Chest roentgenogram showed slight pleural effusion and pneumothorax in the left lung. Eosinophilia (22.8%) was also found in his peripheral blood. Multiple-dot enzyme-linked immunosorbent assay (dot-ELISA) for the detection of parasite-specific immunoglobulin (Ig) G antibody was used to screen his serum against various parasitic diseases, but no significant binding was observed with any of the 12 parasite antigens examined, including those of Paragonimus westermani and P. miyazakii. Although he seemed to have been spontaneously cured without treatment, a nodular shadow appeared in the right upper medial lung field on the chest roentgenogram 6 months later. This time, his serum was positive for anti-P. westermani IgG antibody by the same method. A reexamination of the first and second admission serum samples for parasite-specific IgM and IgG antibodies revealed significant level of IgM antibody in the serum of the first admission, which had decreased at the time of the second admission. Conversely, the level of IgG antibody, which was low at the first admission, became dominant in the second admission serum 6 months later. These results clearly show that although the dot-ELISA to detect IgG antibody is generally useful for screening and detecting paragonimiasis, detection of IgM antibody seems to be a useful aid and should also be included in immunoserological diagnosis, especially if the patient is considered to be in the early stage of infection.

An ELISA was developed using chicken cystatin as a capture agent for the immunodiagnosis of paragonimiasis and fascioliasis. The assay detected specific antibodies to fluke cysteine proteinases without the need for purified proteinases. An ELISA plate was sensitized with chicken egg white cystatin, incubated with excretory-secretory (ES) products of adult flukes, and standard ELISA procedures were then followed. The ELISA plates incubated with the ES products of Paragonimus westermani and Fasciola sp. showed high reactivity to sera from patients with paragonimiasis westermani and fascioliasis, respectively. The capture ELISA showed little cross-reactivity with sera from patients with paragonimiasis and fascioliasis, which showed immunodiagnostic cross-reactivity in a conventional ELISA using crude fluke antigens. Moreover, the capture ELISA showed little reactivity with sera from patients with five other helminth diseases and from healthy volunteers. Omitting either sensitization with cystatin or incubation with fluke ES products abolished high ELISA reactivity, as did a prior exposure of the ES products to cystatin. The addition of papain to an incubation solution of the ES products greatly reduced ELISA reactivity. Incubating the cystatin-sensitized plates with partially purified cysteine proteinases from flukes instead of the ES products also maintained a similar high ELISA reactivity. These results indicate that the cystatin capture ELISA elicits a cystatin and fluke cysteine proteinase antigen-mediated reaction and measures fluke cysteine proteinase-specific antibodies. Prior exposure to low molecular weight inhibitors of cysteine proteinases and other proteinases, such as E-64, leupeptin, aprotinin, and pepstatin, had no effect, suggesting that these inhibitors can be added to cysteine proteinase preparations to prevent autoproteolysis. This assay has good sensitivity and high specificity and is useful for the immunodiagnosis of paragonimiasis and fascioliasis.

Monoclonal antibodies (MAbs) specific to the lung fluke (Paragonimus heterotremus) were produced against the soluble metabolic products (excretory-secretory antigen). Three hybrids secreting MAbs specific for P. heterotremus antigens were identified by an indirect enzyme-linked immunosorbent assay (ELISA) against a panel of homologous and 24 heterologous parasite antigens and Mycobacterium tuberculosis. Of the three specific clones, clone 10F2, which was IgG1 producing and which gave immune complex bands with 31.5-kD and 22-kD polypeptides by gel electrophoresis and immunoblotting, was selected for further characterization and evaluation of its possible diagnostic potential. The result obtained from an indirect immunofluorescent antibody test suggested that MAb 10F2 reacted with mucosa and contents of the worm's intestine. The antibody could be readily used to prepare an affinity-purified antigen for use in an indirect ELISA that was highly sensitive and specific for the detection of circulating antibody in sera of paragonimiasis patients.

An enzyme-linked immunsorbent assay (ELISA) using worm extract antigens from lung flukes of Paragonimus westermani provided good sensitivity to sera from patients with paragonimiasis westermani but high cross-reactivity with sera from most fascioliasis patients and some patients with onchocerciasis or clonorchiasis. To improve the specificity, we tested an ELISA using fluke cysteine proteinases as antigens. Cysteine proteinases were partially purified from the excretory/secretory products of P. westermani by 40-75% ammonium sulfate fractionation, hydrophobic chromatography, and arginine affinity chromatography. An ELISA using the enzyme preparation not only had increased sensitivity to paragonimiasis westermani sera but also reduced cross-reactivity with the fascioliasis, onchocerciasis, and clonorchiasis sera to negligible levels. The reactivity of the ELISA to paragonimiasis miyazakii sera was similar to that of paragonimiasis westermani sera. A proteinase preparation from P. ohirai, which can be obtained easily from infected rats, provided similar results. Therefore, the ELISA using cysteine proteinases of Paragonimus could not distinguish the parasite species with which patients were infected, but it is a valuable assay with which to immunodiagnose paragonimiasis even when the proteinases are prepared from nonhuman species.

We evaluated the performance of Dot-enzyme-linked immunosorbent assays designed to detect species- and stage-specific antigens of Paragonimus westermani in sera with monoclonal antibodies as serodiagnostic tests for active paragonimiasis. Sera from all donors with parasitologically confirmed infections with P. westermani contained adult worm antigens, as did a high proportion of sera from persons suspected to be infected with this parasite. A smaller proportion of these sera also contained metacercarial stage-specific antigens. Sera from donors with other helminth infections, with confirmed pulmonary tuberculosis, or from healthy Chinese donors were nonreactive in the assay. Treatment of experimentally infected animals with praziquantel triggered a marked but transient increase in serum levels of adult P. westermani antigens, which then gradually disappeared within the next two months. The results of our studies indicate that the antigen-detection assay we have developed is a highly specific and sensitive diagnostic test for active infections with P. westermani.

A sensitive and specific immunoblot assay was used to rapidly and accurately diagnose paragonimiasis. The immunoreactivity of a complex Paragonimus westermani Chaffee antigen was evaluated by SDS-PAGE and Western blot analysis. Initial probing with pooled human serum from proven Paragonimus infections revealed many bands, including a significant antibody response to an approximately 8,000 molecular weight (8 kDa) protein. Forty-three of 45 proven paragonimiasis serum specimens had antibodies to this diagnostic band. Of 29 normal serum specimens and 210 serum specimens from patients with other parasitic and nonparasitic infections, only 1 serum, from a schistosomiasis haematobium patient, reacted positively. These results indicate that our immunoblot for paragonimiasis, which uses a comparatively crude antigen, is highly sensitive (96%) and specific (99%).