Detection of Human Paragonimiasis by ELISA Using Recombinant Paragonimus westermani Cysteine Protease 7.

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.

The first case report of Paragonimus westermani infection diagnosed by transbronchial lung cryobiopsy.

We report a case of pulmonary paragonimiasis diagnosed by transbronchial lung cryobiopsy (TBLC). TBLC is likely to be a superior method to transbronchial forceps biopsy because TBLC can get larger specimens, resulting in a higher chance of containing the eggs. A male patient aged 57 years presented with hemoptysis and dyspnea on exertion. His initial chest computed tomography scans showed a cavitary nodule with a peripheral ground-glass appearance, leading to a prescription of an oral antibiotic, with an initial assumption of pneumonia. A follow-up chest computed tomography, however, revealed an appearance of a new nodule adjacent to the original nodule. TBLC and transbronchial forceps biopsy were done to rule out lung cancer and eventually, the eggs of Paragonimus westermani were found using TBLC. Praziquantel was prescribed, showing improvements in symptoms and chest X-ray findings. TBLC has more potential to be utilized as a diagnostic method than transbronchial forceps biopsy because it has a better chance to confirm pulmonary paragonimiasis, which can be initially suspected as pulmonary tuberculosis or lung cancer.

Distribution of Pseudorabies Virus Antigen in Hunting Dogs with Concurrent Paragonimus westermani Infection.

Following isolation of pseudorabies virus (PRV) from two hunting dogs in Oita prefecture, Japan, we investigated the PRV antigen distribution in the tissues of the infected animals. At necropsy, PRV-associated lesions included facial oedema, tonsillar and meningeal congestion, blotchy haemorrhages on the pericardium and mitral valves, and incomplete splenic contraction in one dog, with less prominent findings in the other dog. Multiple pulmonary nodules were seen in both cases, caused by the diploid form of Paragonimus westermani lung flukes, as confirmed by PCR-restriction fragment length polymorphism and gene sequencing analyses. Histological examination revealed that the PRV infection was associated with lesions of non-suppurative encephalitis in the brainstem. PRV antigen was detected in the cerebrum, cerebellum, brainstem, submucosal and myenteric plexuses, and mononuclear cells, mainly in the bone marrow, lymph nodes, tonsils and spleen tissues. There was evidence of PRV dissemination to the brain via the trigeminal or olfactory routes, in addition to possible spread to lymphoid organs via infected mononuclear cells.

Paragonimus westermani metacercariae in two freshwater crab species in Kagoshima Prefecture, Japan, as a possible source of infection in wild boars and sika deer.

Paragonimiasis is a particular foodborne parasitic disease that is endemic to southern Kyushu, including Kagoshima Prefecture, Japan. We previously detected Paragonimus westermani triploid larvae in meat samples obtained from wild boars and sika deer hunted in Akune City, Kagoshima Prefecture. These mammals act as paratenic hosts and their meat is a source of human paragonimiasis. Paratenic host mammals and humans become infected with the lung fluke, P. westermani, following consumption of second intermediate hosts, freshwater crab species, namely, Geothelphusa dehaani or Sawagani in Japanese, and Eriocheir japonica or Mokuzugani in Japanese. Therefore, this study aimed to investigate the current infection status of P. westermani in freshwater crabs in Akune City. We collected freshwater crabs from 15 locations and found that the prevalence of P. westermani metacercariae was 1.6% for Sawagani (15 of 941 examined) and 22.1% for Mokuzugani (21 of 95 examined). Based on the morphological characterization of metacercariae and molecular analyses of the internal transcribed spacer 2 region and mitochondrial 16S rRNA gene region using PCR-restriction fragment length polymorphism and sequencing, all metacercariae were identified as the triploid form of P. westermani. These results indicate that Sawagani and Mokuzugani serve as second intermediate hosts to maintain the life cycle of triploid P. westermani. Further, infection in crabs potentially leads to subsequent P. westermani infections in wild mammals, including wild boars and sika deer, both of which are considered important types of game meat in Japan.

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.

An outbreak of Paragonimus westermani infection among Cambodian technical intern trainees in Japan, exhibiting various extrapulmonary lesions.

We encountered an outbreak of paragonimiasis among Cambodian technical intern trainees (TITs) at a food-processing factory in Fukuoka, Japan. The patients were 20-28 years old, seven females and two males, who had been in Japan for one to four years. All of them had consumed raw or undercooked Japanese mitten crab they purchased at a local grocery store near their training place. CT images showed multiple lesions not only in the lungs but in the extrapulmonary organs as well, such as subcutaneous tissues, abdominal muscles, and mesentery, in most of the patients. Their medical records indicated that all of them acquired infection in Japan, not in Cambodia. Diagnosis was made serologically and the patients were treated with praziquantel successfully. Foreign workers and TITs are increasing in Japan so rapidly, that food borne-infections, including paragonimiasis, should be considered in people from developing countries who have exotic dietary habits.

Pleuropulmonary Paragonimiasis with Multiple Nodules in the Pleura.

An asymptomatic 47-year-old woman was admitted with pleural effusion and pulmonary infiltrates 1 month after ingesting raw wild boar and deer meat. Both her blood and pleural fluid were eosinophilic. Thoracoscopy revealed multiple nodules of the pleura, and biopsy samples of the nodules showed necrosis with epithelioid cell granulomas. An enzyme-linked immunosorbent assay was positive for antibodies against Paragonimus westermani, and the patient was successfully treated with praziquantel. This is the first reported case of pulmonary or pleuropulmonary paragonimiasis where several pleural nodules were observed. The detection of pleural nodules on thoracoscopy can contribute to the prompt and accurate diagnosis of paragonimiasis.

[Complete mitochondrial genomes of Paragonimus westermani in China and phylogenetic analysis of various geographical isolates].

OBJECTIVE: To compare the complete mitochondrial genome sequences of two phenotypes of Paragonimus westermani isolated from Fujian Province with different sizes of metacercariae, and perform a phylogenetic analysis of various geographical isolates of P. westermani from Asia, so as to identify the possible genetic characteristics associated with the P. westermani phenotypes. METHODS: P. westermani metacercariae with different sizes (large metacercariae, 380-420 µm in diameter; small metacercariae, 320-340 µm) isolated from freshwater crabs were used to infect dogs, and the eggs and adult worms of P. westermani were collected from the dog stool samples and lung tissues. Then, the egg size and morphology were compared. In addition, genomic DNA was extracted from the adult worms of the two phenotypes of P. westermani and used for the PCR amplification to yield the complete mitochondrial genome sequence. Sequence structure and phylogenetic analyses were performed based on the complete mitochondrial genome of P. westermani. RESULTS: Following infection with large and small P. westermani metacercariae, the adult worms recovered from the dog lung had a thick body, and had oral and ventral suckers. The ventral sucker was located slightly in front of the midline of the body, and testes, ovary and vitelline gland were seen in the adult worms. Following fixation, the adults appeared oval, with an approximately 1.7∶1 of the length-width ratio. The length and width of the eggs isolated from the fecal samples of dogs infected with large and small P. westermani metacercariae varied significantly, and the large metacercariae produced bigger eggs than the smaller metacercariae. Based on the morphological features of adults and eggs and the ITS2 sequences, both phenotypes were identified as P. westermani. The complete mitochondrial genome sequence analysis of adults showed almost consistent sequences in the protein-coding region of the mitochondrial genome of adult worms derived from large and small metacercariae, with a major variation seen in the former non-coding region. Sliding window analysis revealed the most polymorphic region within the ND4 gene across the mitochondrial genome from various geographical isolates of P. westermani, and phylogenetic analysis showed that both phenotypes were clustered into the Chinese branch of P. westermani, which was close to the Japanese branch and distinct from the South/Southeast Asian branch. CONCLUSIONS: The genetic distance between the phenotypes of P. westermani isolated from Fujian Province is near at a mitochondrial genome level, with no remarkable genetic differentiation seen; however, the mutation and structural changes in the non-coding region may result in the phenotypic variations. In addition, there is a distinct variation of the evolutionary rate in the mitochondrial coding genes, suggesting the selection of appropriate molecular markers during the phylogenic researches.

DNA detection of Paragonimus westermani: Diagnostic validity of a new assay based on loop-mediated isothermal amplification (LAMP) combined with a lateral flow dipstick.

Paragonimus westermani (P. westermani) is widely spread in Asian countries and is one of the most important causative agents for lung fluke diseases. The prevention and control of Paragonimiaisis mainly depends on the accurate diagnosis and effective treatment. In this study, we developed a loop-mediated isothermal amplification (LAMP) assay targeted to a portion of the Ty3/gypsy-like LTR retrotransposon (Rn1) sequence coupled with a lateral flow dipstick (LFD) for the rapid detection of P. westermani-specific amplicons. The positive LAMP products were biotin-labeled and hybridized with a fluorescein isothiocyanate-labeled probe which could be visually detected by LFD. No cross-reaction were observed with other parasitic pathogens including Trichinella spiralis, Anisakis simplex, Schistosoma japonicum and Gnathostoma spinigerum, but this LAMP assay could not distinguish P. westermani with Paragonimus skrjabini and Paragonimus heterotremus. The detection limit of the LAMP assay for P. westermani was 2.7 fg/µL, while that of PCR method was 27 fg/µL. LAMP method was applied to detect P. westermani genomic DNA in blood samples form experimental infected dogs, and results showed the parasite was detectable as early as week 2. LAMP-LFD assay applicability was successfully tested in dog blood samples collected from five cities (Wenzhou, Hangzhou, Huzhou, Jiaxing and Shaoxing) in Zhejiang province. In summary, the established LAMP-LFD assay targeted to the Rn1 sequence is a rapid and convenient method for specific detection of P. westermani.

[Clinical and imaging features of thirty cases of paragonimiasis westermani].

OBJECTIVE: To analyze the clinical manifestations and imaging characteristics of pulmonary and extra pulmonary paragonimiasis westermani. METHODS: A retrospective analysis was performed of 30 patients diagnosed by clinical features, laboratory serological tests and surgical pathology. RESULTS: The symptoms of the lung included mainly chest distress, fever, chest pain, cough and expectoration, and dyspnea. The extra pulmonary symptoms included abdominal pain, vomiting, diarrhea, poor appetite, emaciation, both lower extremities asthenia, headache, dizziness, epileptic seizures, and subcutaneous migratory masses. The laboratory examination showed that the eosinophil numbers of serum and pleural effusion of all the thirty patients were increased, and the eggs of Paragonimus westermani were found by the stool tests in four cases. The chest CT tests found abnormal nodules, ground glass changes, insect damages, pleural effusion, "tunnel" signs, and "halo" signs. Cranial CT and MRI showed intracranial hemorrhage foci, and extensive "finger-like" edema. Abdominal CT showed serpentine deformation and "tunnel" signs in the hepatic and spleen capsules. CONCLUSIONS: Paragonimiasis westermani is a multiple organ system involved infection, and it has complex and varied clinical manifestations. The "tunnel" sign and serpentine deformations in the intracranial part, lung, liver and spleen are important imaging manifestations of the disease.

Analysis of COI and ITS2 regions of DNA obtained from Paragonimus westermani eggs in ancient coprolites on Joseon dynasty mummies.

The genetic information of ancient Paragonimus westermani, the oriental lung fluke infecting over 20 million people worldwide, has not been thoroughly investigated thus far. We analysed genetic markers (COI and ITS2) of P. westermani from coprolite specimens (n = 6) obtained from 15th to 18th century Korean mummies. Our results indicated that all P. westermani sequences were generally distinct from the other species of the genus Paragonimus. The sequences were clustered into three groups: Group I for East Asia; Group II for South and Southeast Asia; and Group III for India and Sri Lanka. In this study, we found that ancient P. westermani sequences in Korea belong to Group I, adding invaluable information to the existing knowledge of Paragonimus paleogenetics.

Whole-genome sequence of the oriental lung fluke Paragonimus westermani.

Background: Foodborne infections caused by lung flukes of the genus Paragonimus are a significant and widespread public health problem in tropical areas. Approximately 50 Paragonimus species have been reported to infect animals and humans, but Paragonimus westermani is responsible for the bulk of human disease. Despite their medical and economic importance, no genome sequence for any Paragonimus species is available. Results: We sequenced and assembled the genome of P. westermani, which is among the largest of the known pathogen genomes with an estimated size of 1.1 Gb. A 922.8 Mb genome assembly was generated from Illumina and Pacific Biosciences (PacBio) sequence data, covering 84% of the estimated genome size. The genome has a high proportion (45%) of repeat-derived DNA, particularly of the long interspersed element and long terminal repeat subtypes, and the expansion of these elements may explain some of the large size. We predicted 12,852 protein coding genes, showing a high level of conservation with related trematode species. The majority of proteins (80%) had homologs in the human liver fluke Opisthorchis viverrini, with an average sequence identity of 64.1%. Assembly of the P. westermani mitochondrial genome from long PacBio reads resulted in a single high-quality circularized 20.6 kb contig. The contig harbored a 6.9 kb region of non-coding repetitive DNA comprised of three distinct repeat units. Our results suggest that the region is highly polymorphic in P. westermani, possibly even within single worm isolates. Conclusions: The generated assembly represents the first Paragonimus genome sequence and will facilitate future molecular studies of this important, but neglected, parasite group.

Paragonimus westermani infection manifesting as a pulmonary cavity and adrenal gland mass: A case report.

We report a case of Paragonimus westermani infection simultaneously affecting two separate organs that presented as both a pulmonary cavity and adrenal mass in an immunocompromised host. A 65-year-old male with a previous kidney transplant visited our clinic because of hemoptysis. Chest computed tomography (CT) showed a pulmonary cavity and right adrenal gland mass. The Aspergillus antigen titer in bronchial lavage fluid was elevated and showed positive conversion. It was necessary to differentiate lung cancer with adrenal gland metastasis from a fungal infection with an adrenal gland adenoma. Positron emission tomography CT suggested benign disease, and it was misdiagnosed as pulmonary aspergillosis based on the elevated Aspergillus antigen titer in the bronchial lavage fluid. Owing to the adverse effects of anti-fungal treatment, the patient underwent wedge resection of the lung and P. westermani was confirmed. A careful history revealed that the patient had eaten raw freshwater crabs 3 years earlier, and a test for serum antibodies to P. westermani was positive. Despite treatment with praziquantel, the adrenal mass persisted on 3-month follow-up CT. A right adrenalectomy was performed and a P. westermani infection was confirmed.

Cerebral Paragonimiasis With Hemorrhagic Stroke in a Developed Country.

Paragonimiasis is a food-borne parasitic disease caused by Paragonimus lung flukes, which are epidemic in Asia. Cerebral paragonimiasis accounts for <1% of symptomatic paragonimiasis but is the most common extrapulmonary infection. Cerebral paragonimiasis often mimics stroke and sometimes causes severe neurological sequelae. A 61-year-old woman was admitted to the hospital for severe headache. A head computed tomography scan revealed intracerebral hemorrhage with subarachnoid hemorrhage. The patient also had lesions in the lungs. She frequently ate Japanese mitten crab. Peripheral blood examination results of increased eosinophilia and immunological testing results confirmed the diagnosis of Paragonimus westermani infection. The patient was successfully treated with praziquantel as the first-line agent. Cerebral paragonimiasis is currently rare in developed countries; however, it is an important disease to consider.

Takotsubo cardiomyopathy associated with Paragonimiasis westermani.

An 11-year-old boy collapsed during morning assembly at his junior high school. The automated external defibrillator detected ventricular fibrillation and provided shock delivery. He was successfully resuscitated and reverted to sinus rhythm. Electrocardiography showed ST-T elevation in the precordial leads. Echocardiography and angiography demonstrated akinesia of the apex and mid-wall of the left ventricle with preserved contraction of the basal segments, which suggested Takotsubo cardiomyopathy. The patient and his family had often eaten uncooked crab, and his father had a past history of infection with Paragonimiasis westermani. The patient had had a persistent cough and chest pain for several weeks. Chest radiograph showed cystic cavities in the left upper lung. Microbiological examination of the sputum demonstrated an egg of P. westermani and immunological assay showed a raised antibody titre to P. westermani. On the12th day of admission, he developed seizures, and magnetic resonance imaging demonstrated cerebral involvement. After the administration of praziquantel for 3 days, the clinical manifestations improved immediately, and echocardiography normalised within 3 weeks. The patient was discharged on the 32nd day + and follow-up was normal. Takotsubo cardiomyopathy following a potentially fatal arrhythmia is a rare cardiac complication associated with pulmonary and central nervous system infection by P. westermani.