关键词: 麝猫后睾吸虫, 华支睾吸虫, PCR扩增, 形态学, 流行病学调查, 诊断

Abstract:

Objective To explore the diagnosis and differentiation between Opisthorchis viverrin and Opisthorchis sinensis infection through morphological examination of small trematode eggs, egg internal transcribed space 2 (ITS-2) sequence amplification and epidemiological investigation. Methods The epidemiological data of 6 trematode egg positive cases diagnosed by the modified Kato-Katz thick smear method were collected, and positive fecal samples were collected for 3 consecutive days for collection of eggs by washing with water and sedimentation prior to microscopy. The eggs DNA was extracted, amplified and sequenced using universal primers for the rDNA ITS-2 region. The product sequences were aligned in the NCBI database to identify the species, and a phylogenetic tree based on ITS-2 sequences was constructed using the neighbor-joining method. The diagnosis was reached based on data analysis of the egg morphology, gene sequence alignment and epidemiological findings. Results Microscopically, the eggs in the fecal samples of cases 1 to 3 were similar to those of cases 4 to 6 in the shell, morphology, color, egg cover, acromion, hair and small wart at the bottom of the eggs. The egg length, width and aspect ratio in the fecal samples from cases 1 to 3 were (26.94 ± 2.28) μm, (14.43 ± 1.22) μm, 1.88 ± 0.18. Compared with (29.70 ± 1.21) μm, (14.36 ± 0.70) μm and 2.07 ± 0.14 in cases 4 to 6. The difference between length and aspect ratio was significant (t = 4.318, 4.816; P < 0.01). The former is shorter and wider than the latter, The Forde-Meleni index (FMI) of the two eggs was 5 676.69 ± 1 317.97 and 6 134.25 ± 626.27 (t = 0.428, P > 0.05). In cases 1 to 3, PCR and sequencing results showed that the ITS-2 fragments were 355, 362 and 359 bp, respectively, with the sequence identity of 99.14%, 98.86% and 99.39% to C. sinensis (GenBank No. MK886663). In cases 4 to 6, ITS-2 gene fragments were 394, 395 and 396 bp, respectively, with the sequence identity of 100%, 99.49% and 100% to O. viverrin (GenBank No. OK103575.1). The phylogenetic tree showed that the ITS-2 sequence of the eggs in cases 1 to 3 clustered on a branch with C. muscidiode and the ITS-2 sequence of the eggs in cases 4 to 6 clustered on a branch with C. sinensis. The epidemiological investigation results showed that cases 1 to 3 were all foreign migrant women from Cambodia who had the habit of eating pickled freshwater fish since childhood; cases 4 to 6 were all local farmers. Combining the morphological, molecular biology and epidemiological findings, cases 1 to 3 were diagnosed as imported testicular fluke infection, and cases 4 to 6 were C. sinensis infection. Conclusion Combining morphological detection as well as epidemiological investigation and analysis, trematode egg ITS-2 amplification and sequencing could effectively differentiate C. sinensis and O. viverrin in infection.

Key words: Opisthorchis viverrin, Opisthorchis sinensis, PCR amplification, Morphology, Epidemiological investigation, Diagnosis