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.

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.

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.

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.

Systems biology studies of adult paragonimus lung flukes facilitate the identification of immunodominant parasite antigens.

BACKGROUND: Paragonimiasis is a food-borne trematode infection acquired by eating raw or undercooked crustaceans. It is a major public health problem in the far East, but it also occurs in South Asia, Africa, and in the Americas. Paragonimus worms cause chronic lung disease with cough, fever and hemoptysis that can be confused with tuberculosis or other non-parasitic diseases. Treatment is straightforward, but diagnosis is often delayed due to a lack of reliable parasitological or serodiagnostic tests. Hence, the purpose of this study was to use a systems biology approach to identify key parasite proteins that may be useful for development of improved diagnostic tests. METHODOLOGY/PRINCIPAL FINDINGS: The transcriptome of adult Paragonimus kellicotti was sequenced with Illumina technology. Raw reads were pre-processed and assembled into 78,674 unique transcripts derived from 54,622 genetic loci, and 77,123 unique protein translations were predicted. A total of 2,555 predicted proteins (from 1,863 genetic loci) were verified by mass spectrometric analysis of total worm homogenate, including 63 proteins lacking homology to previously characterized sequences. Parasite proteins encoded by 321 transcripts (227 genetic loci) were reactive with antibodies from infected patients, as demonstrated by immunoaffinity purification and high-resolution liquid chromatography-mass spectrometry. Serodiagnostic candidates were prioritized based on several criteria, especially low conservation with proteins in other trematodes. Cysteine proteases, MFP6 proteins and myoglobins were abundant among the immunoreactive proteins, and these warrant further study as diagnostic candidates. CONCLUSIONS: The transcriptome, proteome and immunolome of adult P. kellicotti represent a major advance in the study of Paragonimus species. These data provide a powerful foundation for translational research to develop improved diagnostic tests. Similar integrated approaches may be useful for identifying novel targets for drugs and vaccines in the future.

Preparation of colloidal gold immunochromatographic strip for detection of Paragonimiasis skrjabini.

BACKGROUND: Paragonimiasis is a food-borne trematodiasis, a serious public health issue and a neglected tropical disease. Paragonimus skrjabini is a unique species found in China. Unlike paragonimiasis westermani, it is nearly impossible to make a definitive diagnosis for paragonimiasis skrjabini by finding eggs in sputum or feces. Immunodiagnosis is the best choice to detect paragonimiasis skrjabini. There is an urgent need to develop a novel, rapid and simple immunoassay for large-scale screening patients in endemic areas. METHODOLOGY/PRINCIPAL FINDINGS: To develop a rapid, simple immunodiagnostic assay for paragonimiasis, rabbit anti-human IgG was conjugated to colloidal gold particles and used to detect antibodies in the sera of paragonimiasis patients. The synthesis and identification of colloidal gold particles and antibody-colloidal gold conjugates were performed. The size of colloidal gold particles was examined using a transmission electron microscope (TEM). The average diameter of colloidal gold particles was 17.46 nm with a range of 14.32-21.80 nm according to the TEM images. The formation of antibody-colloidal gold conjugates was monitored by UV/Vis spectroscopy. Excretory-secretory (ES) antigen of Paragonimus skrjabini was coated on nitrocellulose membrane as the capture line. Recombinant Staphylococcus protein A was used to prepare the control line. This rapid gold immunochromatographic strip was assembled in regular sequence through different accessories sticked on PVC board. The relative sensitivity and specificity of the strip was 94.4% (51/54) and 94.1% (32/34) respectively using ELISA as the standard method. Its stability and reproducibility were quite excellent after storage of the strip at 4°C for 6 months. CONCLUSIONS/SIGNIFICANCE: Immunochromatographic strip prepared in this study can be used in a rapid one-step immunochromatographic assay, which is instantaneous and convenient.

Angiographic findings in 2 children with cerebral paragonimiasis with hemorrhage.

Hemorrhagic events associated with cerebral paragonimiasis are not rare, especially in children and adolescents; however, angiographic evidence of cerebrovascular involvement has not been reported. The authors describe angiographic abnormalities of the cerebral arteries seen in 2 children in whom cerebral paragonimiasis was associated with hemorrhagic stroke. The patients presented with acute intracerebral and subarachnoid hemorrhage. Angiography revealed a beaded appearance and long segmental narrowing of arteries, consistent with arteritis. In both patients, involved vessels were seen in the area of the hemorrhage. The vascular changes and the hemorrhage, together with new lesions that developed close to the hemorrhage and improved after praziquantel treatment, were attributed to paragonimiasis. Further study of the frequency and mechanism of hemorrhagic cerebrovascular complications associated with cerebral paragonimiasis is needed.

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.

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.

Human paragonimiasis in Viet Nam: epidemiological survey and identification of the responsible species by DNA sequencing of eggs in patients' sputum.

Parasitological and sero-epidemiological surveys for human paragonimiasis were conducted in three provinces of Viet Nam. A total of 590 participants from two known endemic areas of human paragonimiasis (Sinho district of Laichau province and Lucyen district of Yenbai province) and from Dakrong district of Quangtri province where we recently found crab hosts heavily infected with Paragonimus westermani metacercariae. By multiple dot-ELISA screening, 28 (12.7%) out of 220 participants in Sinho district of Laichau province and 4 (3.3%) out of 120 participants in Lucyen district of Yenbai province were proven to be antibody-positive against the Paragonimus antigen. None of the 250 sera of the residents in Dakrong, Quangtri province, gave sero-positivity. Among a total of 32 sero-positive patients Paragonimus eggs were found in 6 cases. ITS2 sequences were successfully determined from a single Paragonimus egg from each patient. The results of homology search by BLAST and alignment clearly confirmed that Paragonimus eggs collected from 6 patients were all of Paragonimus heterotremus. The pathogenicity of P. westermani for human paragonimiasis in Viet Nam is still questionable and needs to be explored in the future.

Diversidad enzimática de la fracción soluble de un aislado venezolano de adultos Paragonimus sp / Enzymatic diversity of enzymes in the soluble fraction of adult worms from a Venezuelan isolate of Paragonimus sp

Paragonimus sp. es un trematodo que causa inflamación crónica del pulmón en mamíferos carnívoros y en el hombre, constituyendo un problema de salud pública en países asiáticos y latinoamericanos. Los trematodos poseen enzimas que facilitan la penetración y migración en diferentes hospederos a fin de garantizar su ciclo evolutivo. Con el objetivo de evaluar la diversidad enzimática de la fracción soluble (FSPA, 100.000 g) de un aislado venezolano de adultos de Paragonimus sp. se realizaron determinaciones enzimáticas a diferentes pH, usando curvas de calibración (A 405 nm vs. nmol) para interpolar la absorbancia de grupos p-nitrofenol o p-nitroanilina liberados por la hidrólisis de sustratos sintéticos; se utilizaron también sustratos 2-naftilamídicos y 2-naftólicos para determinar esterasas, peptidasas, fosfomonoesterasas y glicosidasas. Se demostró que fosfohidrolasas, glicosidasas y peptidasas están presentes en la FSPA, destacándose la β-NAG (0,55 μmol/h/mg, pH 5,5) y la cistein proteasa (0,4 μmol/h/mg, pH 5,5) como las actividades más elevadas, señalando la importancia funcional de la actividad glicosídica y peptídica en este parásito, las cuales están probablemente relacionadas con su hábitat y su necesidad de degradación de secreciones pulmonares. Estos resultados representan los primeros estudios enzimáticos registrados en vermes adultos de un aislado venezolano de Paragonimus sp. obtenidos del reservorio Didelphis marsupialis

Paragonimus sp. is a trematode that causes chronic inflammation of the lung in carnivorous mammals and humans, which constitute a public health problem in Asian and Latin American countries. Trematodes have enzymes that facilitate their penetration and migration through different host organs to ensure their life cycle. To evaluate the enzymatic diversity of the soluble fraction (FSPA, 100,000 g) of a Venezuelan isolate of Paragonimus sp. adult worms, several enzyme determinations were conducted at different pH. The activities of enzymes releasing p-nitrophenol or p-nitroanilina from the corresponding dye-related synthetic peptides were assessed by interpolating absorbance (A 405 nm) values in the corresponding calibration curve (A 405 nm vs. nmol); on the other hand, absorbances82 Bol. Mal. Salud Amb.Diversidad enzimatica de Paragonimus sp. en Venezuelaof 2-naphtylamine and 2-naphtols released from another series of synthetic substrates were read at different wavelengths between 450 nm and 620 nm to assess for the activity of the corresponding hydrolases. Phosphohydrolase, glycosidase and peptidase activities were detected in FSPA, β-N-acetyl-β-D-glucosaminidase (0.55 μmol/h/mg, pH 5.5) and cystein protease (0.4 μmol/h/mg, pH 5.5) being higher than all the other detected activities. These activities are probably related to the adult worm habitat and its need for glycan and peptide degradation of lung secretions. These results represent the first enzymatic study done with a Venezuelan isolate of adult Paragonimus sp. worms collected from the common reservoir Didelphis marsupialis

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.

Paragonimiasis in Cameroon: molecular identification, serodiagnosis and clinical manifestations.

Paragonimiasis is a common parasitic zoonosis in Cameroon and neighbouring countries in Western Africa. Serum, sputum and faecal samples were collected in an endemic area of South West Province, Cameroon, after administration of a questionnaire to identify individuals with appropriate symptoms and histories. Microscopic examination revealed eggs in sputum from 16 people, but none in any faecal sample. These 16 were among the 25 and 26 people, respectively, positive by ELISA and by immunoblot using Paragonimus africanus crude antigens. Copro-DNA detection was attempted using 23 faecal samples (18 from sputum egg-negative and five from sputum egg-positive individuals). Copro-DNA was detected in four of the five sputum egg-positive individuals. These results strongly suggest that: (1) serology is much more sensitive than sputum examination for diagnosis of paragonimiasis; and (2) a copro-DNA test may be more sensitive than a microscopic search for eggs in faeces. Molecular sequence data from ITS2 and cox1 genes confirmed that adult worms experimentally raised in cats were P. africanus and that eggs from sputum or other worm products from human faeces also belonged to this species. Based on these results, 26 of 168 persons (15.5%) were diagnosed as suffering from paragonimiasis.

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.

Pulmonary paragonimiasis with coincidental malignant mesothelioma.

A 72-year-old man patient was referred to our institution for evaluation and treatment of right pleural effusion. Eosinophilic pleural effusion and peripheral eosinophilia were identified during the course of hospitalization. Pulmonary paragonimiasis was confirmed by the presence of paragonimus-specific IgG antibodies for Paragonimus (P.) westermani and P. miyazakii in his serum. Although Praziquantel, a highly effective agent for the treatment of lung flukes was repeatedly administered, the pleural effusion did not subside and the patient's condition gradually deteriorated until his death due to circulatory insufficiency. Postmortem examination revealed malignant mesothelioma of the sarcomatous type encasing the right lung and heart. Cardiac involvement accompanied with old and recent-onset myocardial ischemic changes resulted in death of this patient. Here, we report a very rare case of malignant mesothelioma with a concomitant infection of parasitic lung fluke.