Trematode egg internal transcribed space-2 sequence based analysis for diagnosis of Opisthorchis viverrin and Opisthorchis sinensis infections

doi:10.12140/j.issn.1000-7423.2023.04.005

Abstract

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

CLC Number:

R383.22，R383.25

CAI Zhanghuang, ZHANG Zhiping, ZHUO Mingying, GUO Liping, FU Zhihui, LIU Yiruo. Trematode egg internal transcribed space-2 sequence based analysis for diagnosis of Opisthorchis viverrin and Opisthorchis sinensis infections[J]. CHINESE JOURNAL OF PARASITOLOGY AND PARASITIC DISEASES, 2023, 41(4): 427-433.

Figures/Tables 5

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References 22

[1]	Qiu ZZ, Li QK, Liu W. Fauna sinica Inverterata Vol.52. Platyhelminthes Trematoda Dgenea (Ⅲ)[M]. Beijing: Science Press, 2019: 553-576. (in Chinese)
	(邱兆祉, 李庆奎, 刘巍. 中国动物志无脊椎动物第五十二卷扁形动物门吸虫纲复殖目 (三)[M]. 北京: 科学出版社, 2019: 553-576.)
[2]	Saijuntha W, Sithithaworn P, Kiatsopit N, et al. Liver flukes: Clonorchis and Opisthorchis[J]. Adv Exp Med Biol, 2019, 1154: 139-180.
[3]	Suwannatrai A, Saichua P, Haswell M. Epidemiology of Opisthorchis viverrini infection[J]. Adv Parasitol, 2018, 101: 41-67.
[4]	Traub RJ, Macaranas J, Mungthin M, et al. A new PCR-based approach indicates the range of Clonorchis sinensis now extends to Central Thailand[J]. PLoS Negl Trop Dis, 2009, 3(1): e367. doi: 10.1371/journal.pntd.0000367
[5]	Wang XX, He X, Pan WQ. Parasite infections with cancer[J]. Chin Trop Med, 2019, 19(4): 392-395, 400. (in Chinese)
	(王小溪, 何兴, 潘卫庆. 寄生虫感染与癌症[J]. 中国热带医学, 2019, 19(4): 392-395, 400.) doi: 10.13604/j.cnki.46-1064/r.2019.04.22
[6]	Traub RJ, Macaranas J, Mungthin M, et al. A new PCR-based approach indicates the range of Clonorchis sinensis now extends to Central Thailand[J]. PLoS Negl Trop Dis, 2009, 3(1): e367. doi: 10.1371/journal.pntd.0000367
[7]	Sato M, Thaenkham U, Dekumyoy P, et al. Discrimination of O. viverrini, C. sinensis, H. pumilio and H.taichui using nuclear DNA-based PCR targeting ribosomal DNA ITS regions[J]. Acta Trop, 2009, 109(1): 81-83. doi: 10.1016/j.actatropica.2008.09.015
[8]	Lv GL, Yang YC, Jiang ZH, et al. Study on identification of Clonorchis sinensis and Haplorchis taichui by analysis of egg ITS-2 sequence[J]. Int Med Chin, 2014(2): 128-131, 123. (in Chinese)
	(吕国丽, 杨益超, 蒋智华, 等. 虫卵ITS-2序列分析鉴别华支睾吸虫和扇棘单睾吸虫的研究[J]. 内科, 2014(2): 128-131, 123.)
[9]	Lee JJ, Jung BK, Lim H, et al. Comparative morphology of minute intestinal fluke eggs that can occur in human stools in the Republic of Korea[J]. Korean J Parasitol, 2012, 50(3): 207-213. doi: 10.3347/kjp.2012.50.3.207
[10]	Xie JL, Yu DJ, LiuGA, et al. Co-infection of Strongyloides stercoralis and Clonorchis sinensis: one case report and literature review[J]. Chin J Infect Control, 2022, 21(1): 92-96. (in Chinese)
	(谢金玲, 余德基, 刘国安, 等. 粪类圆线虫和华支睾吸虫合并感染1例并文献复习[J]. 中国感染控制杂志, 2022, 21(1): 92-96.)
[11]	Chen X, Feng Z, Qiu LZ. Progress in diagnosis of posterior orchis in musk cat[J]. Fore Med (Parasit Dis Book), 2004, 31(1): 1-5. (in Chinese)
	(陈叙, 冯正, 裘丽姝. 麝猫后睾吸虫病的诊断研究进展[J]. 国外医学 (寄生虫病分册), 2004, 31(1): 1-5.)
[12]	Kim EM, Verweij JJ, Jalili A, et al. Detection of Clonorchis sinensis in stool samples using real-time PCR[J]. Ann Trop Med Parasitol, 2009, 103(6): 513-518. doi: 10.1179/136485909X451834
[13]	Belizario VY Jr, de Leon WU, Bersabe MJ, et al. A focus of human infection by Haplorchis taichui (Trematoda : Heterophyidae) in the southern Philippines[J]. J Parasitol, 2004, 90(5): 1165-1169. doi: 10.1645/GE-3304RN pmid: 15562620
[14]	Lin JX, Li YS, Cheng YZ, et al. Report on human body experimental infection with Metorchis orientalis[J]. Straits J Pre Med, 2001, 7(2): 9-11. (in Chinese)
	(林金祥, 李友松, 程由注, 等. 东方次睾吸虫(Metorchis orientalis)人体实验感染报告[J]. 海峡预防医学杂志, 2001, 7(2): 9-11.)
[15]	Lin JX, Li LS, Chen BJ, et al. Morphological observations of five small fluke pathogens in humans[J]. J Trop Med, 2006, 6(2): 194-196. (in Chinese)
	(林金祥, 李莉莎, 陈宝建, 等. 人体5种小型吸虫病原形态观察[J]. 热带医学杂志, 2006, 6(2): 194-196.)
[16]	Lin RQ, Ai L, Zou FC, et al. A multiplex PCR tool for the specific identification of Oesophagostomum spp. from pigs[J]. Parasitol Res, 2008, 103(4): 993-997. doi: 10.1007/s00436-008-1070-9 pmid: 18581142
[17]	Zhu XQ, D'Amelio S, Gasser RB, et al. Practical PCR tools for the delineation of Contracaecum rudolphii A and Contracaecum rudolphii B (Ascaridoidea : Anisakidae) using genetic markers in nuclear ribosomal DNA[J]. Mol Cell Probes, 2007, 21(2): 97-102. doi: 10.1016/j.mcp.2006.08.004
[18]	Ai L, Dong SJ, Zhang WY, et al. Specific PCR-based assays for the identification of Fasciola species: their development, evaluation and potential usefulness in prevalence surveys[J]. Ann Trop Med Parasitol, 2010, 104(1): 65-72. doi: 10.1179/136485910X12607012373713
[19]	Huang SY, Tang JD, Song HQ, et al. A specific PCR assay for the diagnosis of Clonorchis sinensis infection in humans, cats and fishes[J]. Parasitol Int, 2012, 61(1): 187-190. doi: 10.1016/j.parint.2011.07.010
[20]	Tang YZ, Hao YH, Sun QS, et al. Identification of Clonorchis sinensis eggs in Jilin and establishment of PCR detection[J]. Hubei Agric Sci, 2013, 52(7): 1601-1603, 1606. (in Chinese)
	(唐艺芝, 郝玉花, 孙青松, 等. 吉林地区华支睾吸虫虫卵的鉴定及PCR检测方法的建立[J]. 湖北农业科学, 2013, 52(7): 1601-1603, 1606 )
[21]	Yang QL, Shen JQ, Jiang ZH, et al. Identification of metacercariae based on ribosomal DNA ITS region and COX1 gene[J]. Chin J Parasitol Parasit Dis, 2014, 32 (3): 217-230. (in Chinese)
	(杨庆利, 申继清, 蒋智华, 等. 基于核糖体DNA ITS区和COX1基因鉴别华支睾吸虫囊蚴[J]. 中国寄生虫学与寄生虫病杂志, 2014, 32(3): 217-230.)
[22]	Chen BJ, Xie HG, Xie XL, et al. Investigation of Clonorchis sinensis infection in the national surveillance site of Pucheng County, Fujian Province during 2016—2020[J]. Chin J Parasitol Parasit Dis, 2021, 39(5): 716-718, inside back cover. (in Chinese)
	(陈宝建, 谢汉国, 谢贤良, 等. 2016—2020年福建省浦城县华支睾吸虫病国家监测点人群感染情况分析[J]. 中国寄生虫学与寄生虫病杂志, 2021, 39(5): 716-718, 封三.)

病例 Case	虫卵数量 No. eggs	长/μm Length/μm	宽/μm Width/μm	长宽比 Length-width ratio	浮德-梅勒尼指数 FMI
病例1 Case 1	12	27.19 ± 1.68	13.65 ± 0.61	1.99 ± 0.15	5 075.90 ± 599.70
病例2 Case 2	4	26.58 ± 1.68	14.93 ± 0.67	1.78 ± 0.14	5 931.78 ± 622.32
病例3 Case 3	21	26.86 ± 2.70	14.78 ± 1.36	1.82 ± 1.71	5 971.41 ± 1 596.86
平均 Average	37	26.94 ± 2.28	14.43 ± 1.22	1.88 ± 0.18	5 676.69 ± 1 317.97
病例4 Case 4	6	29.61 ± 1.77	13.67 ± 0.34	2.17 ± 0.17	5 528.59 ± 244.47
病例5 Case 5	9	29.96 ± 0.81	14.71 ± 0.81	2.04 ± 0.55	6 487.60 ± 495.87
病例6 Case 6	2	28.86 ± 0.49	14.83 ± 0.76	1.95 ± 0.07	6 361.17 ± 762.70
平均 Average	17	29.70 ± 1.21^a	14.36 ± 0.70	2.07 ± 0.14^a	6 134.25 ± 626.27

病例 Case	年龄/岁 Age/year	性别 Gender	职业 Occupation	现居住地 Current residence	原始居住地Original habitation	入境年份Year of entry	初次粪检（改良加藤厚涂片法）Initial fecal examination (Kato-Katz method)		复检（粪检 + PCR）Retrial（fecal examination + PCR）
病例 Case	年龄/岁 Age/year	性别 Gender	职业 Occupation	现居住地 Current residence	原始居住地Original habitation	入境年份Year of entry	时间 Time	结果 Result	时间 Time	结果 Result
病例1 Case 1	37	女 Female	农民 Farmer	松溪县郑墩镇 Songxi County Zhengdun Town	柬埔寨 Cambodia	2013	2020.11	华支睾吸虫C. sinensis	2020.12	麝猫后睾吸虫O. viverrin
病例2 Case 2	27	女 Female	农民 Farmer	浦城县水北镇 Pucheng County Shuibei Town	柬埔寨 Cambodia	2014	2017.11	华支睾吸虫C. sinensis	2020.12	麝猫后睾吸虫O. viverrin
病例3 Case 3	42	女 Female	农民 Farmer	松溪县茶坪乡 Songxi County Chaping Town	柬埔寨 Cambodia	2014	2020.11	华支睾吸虫C. sinensis	2020.12	麝猫后睾吸虫O. viverrin
病例4 Case 4	53	男 Male	农民 Farmer	浦城县仙阳镇 PuchengCounty Xianyang Town	浦城县 Pucheng County	无境外史 No foreign history	2017.11	华支睾吸虫C. sinensis	2020.12	华支睾吸虫C. sinensis
病例5 Case 5	49	女 Female	农民 Farmer	松溪县松源乡 Songxi County Songyuan Town	松溪县 Songxi County	无境外史 No foreign history	2020.11	华支睾吸虫C. sinensis	2020.12	华支睾吸虫C. sinensis
病例6 Case 6	52	男 Male	农民 Farmer	浦城县仙阳镇 Pucheng County Xianyang Town	浦城县Pucheng County	无境外史 No foreign history	2018.1	华支睾吸虫C. sinensis	2020.12	华支睾吸虫C. sinensis