Characterization and localization of antigens for serodiagnosis of human paragonimiasis.

Paragonimiasis is a foodborne trematode infection that affects 23 million people, mainly in Asia. Lung fluke infections lead frequently to chronic cough with fever and hemoptysis, and are often confused with lung cancer or tuberculosis. Paragonimiasis can be efficiently treated with praziquantel, but diagnosis is often delayed, and patients are frequently treated for other conditions. To improve diagnosis, we selected five Paragonimus kellicotti proteins based on transcriptional abundance, recognition by patient sera, and conservation among trematodes and expressed them as His-fusion proteins in Escherichia coli. Sequences for these proteins have 76-99% identity with amino acid sequences for orthologs in the genomes of Paragonimus westermani, Paragonimus heterotremus, and Paragonimus miyazakii. Immunohistology studies showed that antibodies raised to four recombinant proteins bound to the tegument of adult P. kellicotti worms, at the parasite host interface. Only a known egg antigen was absent from the tegument but present in developing and mature eggs. We evaluated the diagnostic potential of these antigens by Western blot with sera from patients with paragonimiasis (from MO and the Philippines), fascioliasis, and schistosomiasis, and with sera from healthy North American controls. Two recombinant proteins (a cysteine protease and a myoglobin) showed the highest sensitivity and specificity as diagnostic antigens, and they detected antibodies in sera from paragonimiasis patients with early or mature infections. In contrast, antibodies to egg yolk ferritin appeared to be specific marker for patients with adult fluke infections that produce eggs. Our study has identified and localized antigens that are promising for serodiagnosis of human paragonimiasis.

Evaluation of IgG4 subclass antibody detection by peptide-based ELISA for the diagnosis of human paragonimiasis heterotrema.

A synthetic peptide was prepared based on the antigenic region of Paragonimus westermani pre-procathepsin L, and its applicability for immunodiagnosis for human paragonimiasis (due to Paragonimus heterotremus) was tested using an ELISA to detect IgG4 antibodies in the sera of patients. Sera from other helminthiases, tuberculosis, and healthy volunteers were used as the references. This peptide-based assay system gave sensitivity, specificity, accuracy, and positive and negative predictive values of 100%, 94.6%, 96.2%, 100%, and 88.9%, respectively. Cross reactivity was frequently seen against the sera of fascioliasis (75%) and hookworm infections (50%). Since differential diagnosis between paragonimiasis and fascioliasis can be easily done by clinical presentation and fascioliasis serology, this cross reaction is not a serious problem. Sera from patients with other parasitoses (0-25%) rarely responded to this synthetic antigen. This synthetic peptide antigen seems to be useful for development of a standardized diagnostic system for paragonimiasis.

Analysis of the misdiagnosis of 8 adult cases of paragonimiasis with lung masses as the main manifestation in Xishuangbanna, Yunnan.

OBJECTIVE: To summarize the clinical characteristics of adult cases of paragonimiasis with lung masses as the main manifestation in Xishuangbanna, Yunnan Province, analyze the causes of misdiagnosis, and improve the levels of clinical diagnosis and treatment. METHOD: We conducted a retrospective analysis of the clinical data and diagnosis and treatment of 8 adult cases of paragonimiasis with lung masses as the main manifestation that were diagnosed in the Oncology Department of People's hospital of Xishuangbanna Dai Autonomous Prefecture from July 2014 to July 2019. RESULT: All 8 patients were from epidemic paragonimiasis areas and had a confirmed history of consuming uncooked freshwater crabs. The clinical manifestations were mainly fever, dry cough, and chest pain. The disease durations were long, and peripheral blood eosinophil counts were elevated. The cases had been misdiagnosed as pneumonia or pulmonary tuberculosis. After years of anti-inflammatory or anti-tuberculosis treatment, the symptoms had not improved significantly. Patients eventually sought treatment from the oncology department for hemoptysis. Chest computed tomography showed patchy consolidation in the lungs, with nodules, lung masses, and enlarged mediastinal lymph nodes. CONCLUSION: Paragonimiasis is a food-borne parasitic disease. Early clinical manifestations and auxiliary examination results are nonspecific. The parasite most often invades the lungs, and the resulting disease is often misdiagnosed as pneumonia, pulmonary tuberculosis, or lung cancer (Acta Trop 199: 05074, 2019). To avoid misdiagnosis, clinicians should inquire, in detail, about residence history and history of unclean food and exposure to infected water and make an early diagnosis based on the inquired information and imaging examination results. For patients who have been diagnosed with pneumonia or pulmonary tuberculosis and whose symptoms do not improve significantly after anti-inflammatory or anti-tuberculosis treatments, their epidemiological history should be traced to further conduct differential diagnosis and avoid misdiagnosis.

Paragonimiasis in a person whose symptoms were shown 22 years after emigrating to Japan from Laos.

We report a patient, a 52-year-old man from Laos, who had come to Japan at 30 years of age, but had maintained a habit of eating raw freshwater crabs. The patient visited a physician for left chest pain in January 2007. Infiltration and mass-like shadows were noted in the left superior and inferior lobes on chest X-ray. Diagnosis could not be made by bronchial brushing, but eggs were present in sputum cytology 3 days after bronchoscopy. Therefore, paragonimiasis was diagnosed. The peripheral eosinophil count had increased to 2550/µl and the serum IgE level was elevated, at 71000 IU/ml. Multiple-dot enzyme-linked immunosorbent assay (ELISA) for specific IgG antibodies in serum was positive for Paragonimus westermani and P. miyazakii. Paragonimiasis may have been caused by the style of Laotian cooking without heating. Because the habit of eating raw freshwater crabs is common in Laos, Laos is one of the countries where paragonimiasis is prevalent. For patients from Laos with lung diseases, differentiation including paragonimiasis is required.

First findings on the seroepidemiology of human paragonimosis at the anti-tuberculosis centre of Divo, Repubuc of Ivory Coast (West Africa).

An epidemiological study was carried out in 2004-2005 at the anti-tuberculosis centre of Divo (Ivory Coast) to collect sera from patients who consulted for tuberculosis suspicion and to estimate the seroprevalence of human paragonimosis in the context of a systematic screening. No Paragonimus egg was found in the stools and/or sputa of the 167 persons investigated. In contrast, 41 sera were ascertained with antibodies against Paragonimus africanus using ELISA testing. As the optical density (OD) values related to seropositive findings were found under 0.6 (the minimal OD to detect an active paragonimosis), the above antibody titres might originate from patients in chronic or in convalescent stages, or might result of cross reactions with trematodes. Concomitantly, dissection of local crabs (Callinectes marginatus) demonstrated the presence of Paragonimus metacercariae in six out of 34 examined. The parasite burdens in crabs ranged from two to 35 cysts with a mean diameter of 302 microm. In Ivory Coast, the locality of Divo must be considered an at-risk zone in reason of the presence of anti-Paragonimus antibodies in several human sera and the presence of infected crabs at the local market.

ELISA based on a recombinant Paragonimus heterotremus protein for serodiagnosis of human paragonimiasis in Thailand.

BACKGROUND: Paragonimus heterotremus is the main causative agent of paragonimiasis in Thailand. In Western blot diagnostic assays for paragonimiasis, the 35 kDa band present in crude P. heterotremus somatic extracts represents one of the known diagnostic bands. This study aimed to use a P. heterotremus cDNA library to create a recombinant version of this antigen for use in immunodiagnosis of paragonimiasis. METHODS: To accomplish this aim a cDNA expression library was constructed from adult worm mRNA and immuno-screened using antibodies from mice that had been immunized with the 35 kDa antigen. Screening resulted in the identification of an immunoreactive protein encoded by clone CE3, which contained an inserted sequence composed of 1292 base pairs. This clone was selected for use in the construction of a recombinant P. heterotremus protein because of its similarity to proactivator polypeptide. For recombinant protein expression, the CE3 gene sequence was inserted into the plasmid vector pRset and the resulting product had the expected molecular weight of 35 kDa. An IgG-ELISA based on the CE3 recombinant protein was evaluated by using sera from healthy individuals, from patients with paragonimiasis and other parasitic infections. This ELISA was performed by using human sera diluted at 1:2000, an optimized antigen concentration of 1 µg/ml, and anti-human IgG diluted at 1:4000. RESULTS: The cut-off optical density value was set as the mean + 2 standard deviations (0.54), which resulted in the test having a sensitivity of 88.89% and a specificity of 95.51%. The recombinant antigen could react with antibodies from P. heterotremus, P. pseudoheterotremus and P. westermani infections. Cross-reactivity occurred with a few cases of Blastocystis hominis infection (2/3), Bancroftian filariasis (1/10), opisthorchiasis (3/10), strongyloidiasis (4/10) and neurocysticercosis (1/11). CONCLUSIONS: Given the high test sensitivity and specificity, reflected in the low level of heterologous infection cross-reactivity (11/215 serum samples), observed in the IgG-ELISA, this 35 kDa antigen may be useful for the detection of paragonimiasis.

[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.

Evaluation of human IgG subclass antibodies in the serodiagnosis of Paragonimiasis heterotremus.

Immunoglobulin G subclass antibodies (IgG1, IgG2, IgG3, and IgG4) responses to the excretory-secretory antigens of the lung fluke, Paragonimus heterotremus, were analyzed using the immunoblotting technique in an attempt to further improve the sensitivity and specificity for serodiagnosis of human paragonimiasis. Serum samples from patients with proven paragonimiasis, from patients with other parasitic infections, pulmonary tuberculosis and from healthy counterparts were analyzed. The results indicate that immunoblotting for the detection of IgG4 antibodies to an excretory-secretory product of P. heterotremus of an approximate molecular mass of 31.5 kDa, is the most reliable test. It gives accuracy, sensitivity, specificity, and positive and negative predictive values of 97.6%, 100%, 96.9%, 90% and 100%, respectively.

A rare case of paragonimiasis miyazakii with lung involvement diagnosed 7 years after infection: A case report and literature review.

We report a rare case of pulmonary paragonimiasis caused by Paragonimus miyazakii that showed pulmonary manifestations and a long-term clinical course after infection. A 45-year-old Japanese male developed cough and dyspnea in 2004 and was diagnosed with eosinophilic pneumonia. He had been treated with low-dose oral corticosteroid for 7 years. He recalled that he had consumed a large amount of raw freshwater crab (Geothelphusa dehaani) several weeks before he had been admitted for the first time, and that had been the only occasion when he had eaten this meat. The patient was referred to our hospital due to persistent hemoptysis, and his chest computed tomography scan showed pulmonary nodules and cavities, and his serum total IgE level was elevated. Bronchoscopy was performed, and ova were detected in the bronchoalveolar lavage fluid. The morphological examination of the ova and immunoserological examination yielded typical findings of P. miyazakii. Treatment with praziquantel improved his chest radiographic findings and a decrease of serum total IgE, as well as the values of immunoserological examination for P. miyazakii. The clinical course of this patient indicated that he had been infected with P. miyazakii for 7 years at least, which is unusual for paragonimiasis miyazakii.

Induction of resistance in Oncomelania hupensis nosophora against Schistosoma japonicum, but not against Paragonimus ohirai, using irradiated miracidia.

Oncomelania hupensis nosophora snails sensitized with X-irradiated Schistosoma japonicum miracidia demonstrated resistance against a following challenge infection with non-irradiated homologous miracidia. The resistance in O. h. nosophora against S. japonicum was acquired within 1 day of sensitization, and it was strongest in a group challenged at an interval of 3 days. The resistance persisted for at least 4 weeks. Histological examinations revealed amoebocyte accumulation around the challenged S. japonicum sporocysts. On the other hand, when O. h. nosophora sensitized by exposure to X-irradiated P. ohirai or S. japonicum miracidia were subsequently challenged with normal P. ohirai miracidia, no resistance was observed, although they expressed the resistance against heterologous S. japonicum infection.

Studies on immunodiagnosis of human paragonimiasis and specific antigen of Paragonimus heterotremus.

Adult Paragonimus heterotremus were recovered from the lungs and pleural cavity of cats orally infected with metacercariae. The worms were ground and extracted with distilled water. The soluble crude antigen (CA) contained about 40% proteins which could be fractionated by gel filtration on Sephadex G-200 into three profiles namely the F1, F2 and F3. The CA and its Sephadex profiles were used in an indirect enzyme-linked immunosorbent assay (ELISA) for detecting antibodies to P. heterotremus in three groups of patients, i.e. patients whose sputum and/or faeces revealed P. heterotremus eggs (group 1), patients with other parasitic infections (group 2), bacterial proven tuberculosis patients (group 3) and healthy, parasite-free controls (group 4). The sensitivity and specificity of the assay when the F1 was used as the antigen were 100%. Western blot analysis revealed that specific antigen of P. heterotremus was a non-protein component of Mr35 kDa.

Excretory/secretory antigens of adult Paragonimus ohirai recognized by a monoclonal antibody.

An IgM monoclonal antibody (mAb) that reacted with the gut epithelium and luminal contents of adult Paragonimus ohirai was prepared from a mouse immunized with the excretory/secretory (ES) products of the parasite. The mAb reacted with the same sites not only of immature worms and newly excysted juveniles of P. ohirai, but also of adult worms of other Paragonimus species, such as P. westermani and P. miyazakii. The mAb did not react with adult antigens prepared from any other helminth parasites. Thus, the mAb is a genus-specific antibody against the gut-associated antigen. Immunoblotting revealed that two antigens with molecular masses (M(r)s) of 27 and 29 kDa were present in the ES products of adult P. ohirai, whereas four antigens with M(r) 27, 29, 36, and 38 kDa were detected i adult worm extract. Partially purified fractions containing acidic and neutral cysteine proteinases have 27 and 29 kDa protein molecules reactive with the mAb. The possibilities that the 36 and 38 kDa antigens may be precursors of these proteinases are discussed.

Enzyme-linked immunosorbent assay using cysteine proteinase antigens for immunodiagnosis of human paragonimiasis.

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.

Diagnosis of paragonimiasis by immunoblot.

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%).

Characterization of stage-specific antigens of Paragonimus westermani.

In order to develop a serodiagnostic assay to detect antigens of Paragonimus westermani in biological specimens, we generated monoclonal antibodies to antigens of metacercariae and adult worms, and partially characterized the epitopes recognized by representative Mabs. Metacercarial stage-specific determinants were periodate-sensitive and protease-resistant, indicating that they are carbohydrate epitopes. In contrast, adult worm-specific determinants resisted periodate treatment but were sensitive to proteases, indicating that they are polypeptides. The majority of cross-reactive Mabs studied were directed against phosphorylcholine determinants. When used in a dot-ELISA, Paragonimus-specific Mabs could detect 0.3-7 ng/ml parasite antigen in human sera.

The antigens of Paragonimus westermani, Schistosoma mansoni, and Fasciola hepatica adult worms. Evidence for the presence of cross-reactive antigens and for cross-protection to Schistosoma mansoni infection using antigens of Paragonimus westermani.

The presence of cross-reacting antigens between Paragonimus westermani, Schistosoma mansoni, and Fasciola hepatica adult worms was demonstrated by Ouchterlony immunodiffusion and enzyme-linked immunosorbent assay (ELISA). A serum bank was developed against the three trematode genera to serve as probes to determine the presence of cross-reacting antibodies to P. westermani worm extracts. In this manner, it was possible to demonstrate that antigens common to F. hepatica and S. mansoni tegument were also present in P. westermani worm extracts. Likewise, it was possible to demonstrate that the F. hepatica antigens which bind to Concanavalin A, as well as the subfraction which in isoelectric focusing has a pI of 4.2, were also found in P. westermani worms. Also, a monospecific polyclonal serum to a Fasciola/Schistosoma cross-reacting antigen and the anti-P. westermani serum both reacted in Ouchterlony immunodiffusion with the P. westermani antigenic extract, each producing a line which linked with each other indicating common antigenic determinants and suggesting a common antigen among the digenetic trematodes. Finally, the P. westermani antigenic extracts induced in mice the production of antibodies which reacted with S. mansoni adult worm antigens by ELISA. As all of the Fasciola and Schistosoma sera were prepared against antigenic preparations which induced in mice protection to challenge infection with S. mansoni, this suggested that the P. westermani worms also contain protective antigens against S. mansoni. Immunity to Schistosoma mansoni infection was induced in mice by vaccination with Paragonimus westermani whole worm extracts (PwWWE). Immunized mice showed as high as a 67% worm burden reduction over controls. High doses of PwWWE did not confer protection to S. mansoni infection. Thus, in this study, immunity in heterologous systems was demonstrated and the existence of a common protective antigen shared by the digenetic trematodes was suggested.

Parasite-specific IgE and IgG levels in the serum and pleural effusion of paragonimiasis westermani patients.

In seven patients with paragonimiasis westermani, parasite-specific IgE and IgG levels in sera and pleural effusion were determined by an enzyme-linked immunosorbent assay (ELISA). The sensitivity to adult excretory-secretory (E-S) antigen was compared with the sensitivity to whole worm extract antigen, and the former was more sensitive in both an IgE-ELISA and IgG-ELISA. Both parasite-specific IgE and IgG could be detected by ELISA at levels much higher than those in control subjects using E-S antigen. When specific IgE and IgG levels in sera and pleural effusion of individual patients were compared, the latter had higher values. The difference between levels of specific IgE in pleural effusion and serum did not correlate with that of specific IgG. These results indicate that specific IgE and IgG antibodies form locally, i.e., in the lung, and that pleural effusions from patients with paragonimiasis are more suitable than serum for immunodiagnosis.

Diagnosis of active Paragonimus westermani infections with a monoclonal antibody-based antigen detection assay.

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.