Morphological and Molecular Characterization of Paragonimus skrjabini Complex from Yunnan, China: A Brief Report.

PURPOSE: To perform environmental sampling and molecular identification of Paragonimus in endemic regions, which may help in minimizing transmission among humans. METHODS: Mountain crabs from the genus Potamiscus were collected and the encysted metacercariae were extracted and subjected to morphological identification, followed by animal inoculation in Sprague-Dawley (SD) rats. After 112 days of infection, animals were killed and adult worms were extracted from lungs and muscles. The morphology of adult worms was characterized by microscopy and molecular identification was done by polymerase chain reaction, followed by sequencing of cox1 and ITS2 genes. Phylogenetic analysis was done by maximum parsimony method. RESULTS: A total of 447 crabs were captured from the streams of Tongchang Town, Jinping County, Yunnan Province, China. The infection rate was found to be 41% (186 out of 447 crabs). The metacercariae of Paragonimus skrjabini was identified by the characteristics round or spherical encysted form measuring 410 to 460 × 400 to 460 µm. After animal infection in SD rats, adults were presumptively confirmed to be P. skrjabini, which was also confirmed by gene amplification and sequence analysis of cox1 and ITS2 regions. Paragonimus skrjabini clustered with previously reported P. skrjabini from Yunnan and Vietnam. The confidence values of their branches were > 95%. Phylogenetic analysis of the ITS2 region revealed two distinct clusters with distinct geographical grouping. Phylogenetic analysis with the combined data sets reiterated the geographical grouping with P. skrjabini from Yunnan clustering with strains from Vietnam. CONCLUSION: Metacercariae of P. skrjabini was discovered in freshwater crabs in Yunnan province, China, and the strains were phylogenetically related to P. skrjabini from Vietnam.

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.

Morphological and Molecular Characterization of Paragonimus Species Isolated from Freshwater Crabs in Southern Yunnan, China.

Paragonimus species are highly prevalent in various regions of China. The study's objective is to isolate and identify Paragonimus from natural habitats and compare the phylogenetic diversity of Paragonimus in southern Yunnan province, China. Metacercariae of Paragonimus was isolated from crabs, and morphologic identification was performed by microscopy. Metacercariae were injected into experimental Paragonimus free Sprague Dawley rats. After 114 days, adult worms and eggs were isolated from multiple organs. Morphologic identification confirmed the initial identification. DNA was extracted from 5 adult worms, and molecular characterization was performed by amplification and sequencing of CO1 and ITS2 regions, followed by phylogenetic analysis. Out of 447 crabs captured, 186 crabs were found to be infected. A total of 4 species of Paragonimus was observed from naturally infected crabs. Paragonimus microrchis (2), Paragonimus heterotremus (1), Paragonimus proliferus (1), and Paragonimus skrjabini (1) were isolated and identified. A total of 32 sequences were downloaded from the National Center for Biotechnology Information, and 5 sequences generated in the study were used for phylogenetic analysis. In the phylogenetic tree of the CO1 gene, Paragonimus proliferus, Paragonimus heterotremus, and Paragonimus skrjabini were clustered with the same species, and the confidence values of their branches were >95%. A congruent phylogenetic relationship was observed with the ITS2 phylogenetic tree. In the phylogenetic tree constructed with the combined dataset of CO1 and ITS2 datasets, Paragonimus proliferus, Paragonimus heterotremus, and Paragonimus skrjabini clustered with the same species, and their branch confidence values were >94%. Paragonimus microrchis clustered with Paragonimus bangkokensis in both datasets. Phylogenetic analysis revealed robustness of the double loci method as against the single-locus method with either CO1 or ITS2 alone. Paragonimus species isolated from the southern Yunnan province, China, was phylogenetically diverse, and the analysis revealed the clustering of multiple species of Paragonimus isolated from different geographic locations.