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A multiplex PCR for the identification of Echinococcus multilocularis, E. granulosus sensu stricto and E. canadensis that infect human

Published online by Cambridge University Press:  31 July 2019

Fan Chen Open the ORCID record for Fan Chen [Opens in a new window] ,
Lei Liu ,
Qili He ,
Yan Huang ,
Wentao Wang ,
Guo Zhou ,
Wenjie Yu ,
Wei He ,
Qi Wang  and
Guangjia Zhang
...Show all authors
Fan Chen*
Affiliation:
Sichuan Center for Disease Control and Prevention, Sichuan, China
Lei Liu
Affiliation:
Sichuan Provincial Orthopedic Hospital, Sichuan, China
Qili He
Affiliation:
Sichuan Center for Disease Control and Prevention, Sichuan, China
Yan Huang
Affiliation:
Sichuan Center for Disease Control and Prevention, Sichuan, China
Wentao Wang
Affiliation:
West China Hospital, Sichuan, China
Guo Zhou
Affiliation:
Sichuan Provincial People's Hospital, Sichuan, China
Wenjie Yu
Affiliation:
Sichuan Center for Disease Control and Prevention, Sichuan, China
Wei He
Affiliation:
Sichuan Center for Disease Control and Prevention, Sichuan, China
Qi Wang
Affiliation:
Sichuan Center for Disease Control and Prevention, Sichuan, China
Guangjia Zhang
Affiliation:
Sichuan Center for Disease Control and Prevention, Sichuan, China
Sha Liao
Affiliation:
Sichuan Center for Disease Control and Prevention, Sichuan, China
Ruirui Li
Affiliation:
Sichuan Center for Disease Control and Prevention, Sichuan, China
Liu Yang
Affiliation:
Sichuan Center for Disease Control and Prevention, Sichuan, China
Renxin Yao
Affiliation:
Sichuan Center for Disease Control and Prevention, Sichuan, China
Qian Wang*
Affiliation:
Sichuan Center for Disease Control and Prevention, Sichuan, China
Bo Zhong*
Affiliation:
Sichuan Center for Disease Control and Prevention, Sichuan, China
*
Author for correspondence: Fan Chen, E-mail: [email protected]; Qian Wang, E-mail: [email protected] and Bo Zhong, E-mail: [email protected]
Author for correspondence: Fan Chen, E-mail: [email protected]; Qian Wang, E-mail: [email protected] and Bo Zhong, E-mail: [email protected]
Author for correspondence: Fan Chen, E-mail: [email protected]; Qian Wang, E-mail: [email protected] and Bo Zhong, E-mail: [email protected]
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Abstract

Echinococcus granulosus sensu stricto (s.s.), Echinococcus multilocularis and Echinococcus canadensis are the common causes of human echinococcosis in China. An accurate species identification tool for human echinococcosis is needed as the treatments and prognosis are different among species. The present work demonstrates a method for the simultaneous detection of these three Echinococcus species based on multiplex polymerase chain reaction (mPCR). Specific primers of this mPCR were designed based on the mitochondrial genes and determined by extensive tests. The method can successfully detect either separated or mixed target species, and generate expected amplicons of distinct size for each species. Sensitivity of the method was tested by serially diluted DNA, showing a detection threshold as less as 0.32 pg for both E. granulosus s.s. and E. canadensis, and 1.6 pg for E. multilocularis. Specificity assessed against 18 other parasites was found to be 100% except weakly cross-react with E. shiquicus. The assay was additionally applied to 69 echinococcosis patients and 38 healthy persons, confirming the high reliability of the method. Thus, the mPCR described here has high application potential for clinical identification purposes, and can further provide a useful tool for evaluation of serology and imaging method.

Keywords

E. canadensisE. granulosus sensu strictoE. multilocularishuman echinococcosismultiplex PCR
Type
Research Article
Information
Parasitology , Volume 146 , Issue 12 , October 2019 , pp. 1595 - 1601
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Copyright
Copyright © Cambridge University Press 2019 

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Footnotes

*

These authors contributed equally to this work.

References

Bart, JM, Abdukader, M, Zhang, YL, Lin, RY, Wang, YH, Nakao, M, Ito, A, Craig, PS, Piarroux, R, Vuitton, DA and Wen, H (2006) Genotyping of human cystic echinococcosis in Xinjiang, PR China. Parasitology 133, 571.Google Scholar
Brunetti, E, Kern, P and Vuitton, DA (2010) Expert consensus for the diagnosis and treatment of cystic and alveolar echinococcosis in humans. Acta Tropica 114, 0–16.Google Scholar
Bharathi, MJ, Rameshkumar, G, Ramakrishnan, R, Reddy, YCV, Shivkumar, C and Ramesh, S (2013) Comparative evaluation of uniplex, nested, semi-nested, multiplex and nested multiplex PCR methods in the identification of microbial etiology of clinically suspected infectious endophthalmitis. Current Eye Research 38, 13.Google Scholar
Boubaker, G, Macchiaroli, N, Prada, L, Cucher, MA and Spiliotis, M (2013) A multiplex PCR for the simultaneous detection and genotyping of the Echinococcus granulosus complex. PLoS Neglected Tropical Diseases 7, e2017.Google Scholar
Boufana, B, Umhang, G, Qiu, J, Chen, X, Lahmar, S, Boué, F, Jenkins, D and Craig, P (2013) Development of three PCR assays for the differentiation between Echinococcus shiquicus, E. granulosus (g1 genotype), and E. multilocularis DNA in the co-endemic region of Qinghai-Tibet plateau, China. American Journal of Tropical Medicine and Hygiene 88, 795–802.Google Scholar
Cong-Nuan, L, Zhong-Zi, L, Li, L, Hong-Bin, Y, David, B, Meng-Tong, L, Jin-Zhong, C, Yan-Lei, F, Jian-Qiu, L, Bao-Quan, F, Yu-Rong, Y, Donald, PM and Wan-Zhong, J (2015) Discrimination between E. granulosus sensu stricto, E. multilocularis and E. shiquicus using a multiplex PCR assay. PLoS Neglected Tropical Diseases 9, e0004084.Google Scholar
Chaabane-Banaoues, R, Oudni-M'rad, M, M'rad, S, Amani, H, Mezhoud, H and Babba, H (2016) A novel PCR-RFLP assay for molecular characterization of Echinococcus granulosus sensu lato and closely related species in developing countries. Parasitology Research 115, 3817–3824.Google Scholar
Cao, M, Chen, K, Li, W, Ma, J, Xiao, Z, Wang, H and Gao, J (2018) Genetic characterization of human-derived hydatid fluid based on mitochondrial gene sequencing in individuals from northern and western China. Journal of Helminthology, 1–5. doi: 10.1017/S0022149X18000883.Google Scholar
Dong, X, Cheng, G and Jian, W (2000) Simultaneous identification of five bifidobacterium species isolated from human beings using multiple PCR primers. Systematic and Applied Microbiology 23, 386–390.Google Scholar
Dinkel, A, Kern, S, Brinker, A, Oehme, R, Amélie, V, Giraudoux, P, Mackenstedt, U and Romig, T (2011) A real-time multiplex-nested PCR system for coprological diagnosis of Echinococcus multilocularis and host species. Parasitology Research 109, 493–498.Google Scholar
Elnifro, EM, Cooper, BD, Ashshi, AM and Klapper, P (2000) Multiplex PCR: optimization and application in diagnostic virology. Clinical Microbiology Reviews 13, 559–570.Google Scholar
Henegariu, O and Al, E (1997) Multiplex PCR: critical parameters and step-by-step protocol. Biotechniques 23, 504–511.Google Scholar
HołodyZaręba, J, Zaręba, KP and Kędra, B (2013) Assessment of the accuracy of preoperative imaging methods in the diagnosis of hepatic single-chamber echinococcosis. Polski Przeglad Chirurgiczny 85, 693–698.Google Scholar
Jacobs, DE, Zhu, X, Gasser, RB and Chilton, NB (1997) PCR-based methods for identification of potentially zoonotic ascaridoid parasites of the dog, fox and cat. Acta Tropica 68, 191–200.Google Scholar
Li, T, Ito, A, Nakaya, K, Qiu, J, Nakao, M, Zhen, R, Xiao, N, Chen, XW, Giraudoux, P and Craig, PS (2008) Species identification of human echinococcosis using histopathology and genotyping in northwestern China. Transactions of the Royal Society of Tropical Medicine & Hygiene 102, 585–590.Google Scholar
Ma, J, Wang, H, Lin, G, Zhao, F, Li, C, Zhang, T, Ma, X, Zhang, Y, Hou, Z, Cai, H, Liu, P and Wang, Y (2015) Surveillance of Echinococcus isolates from Qinghai, China. Veterinary Parasitology 207, 44–48.Google Scholar
McManus, DP, Zhang, W, Li, J and Bartley, PB (2003) Echinococcosis. Lancet 362, 1295–1304.Google Scholar
McManus, DP, Gray, DJ, Zhang, W and Yang, Y (2012) Diagnosis, treatment, and management of echinococcosis. British Medical Journal 344, e3866–e3866.Google Scholar
Mesut, B, Gulbiz, KM, Sabri, Y, Erdem, Y, Ravza, Y, Dilek, S, Murat, A and Oguz, BE (2016) Multimodality imaging in diagnosis and management of alveolar echinococcosis: an update. Diagnostic and Interventional Radiology 22, 247–256.Google Scholar
Nakao, M, Sako, Y, Yokoyama, N, Fukunaga, M and Ito, A (2000) Mitochondrial genetic code in cestodes. Molecular and Biochemical Parasitology 111, 415–424.Google Scholar
Nakao, M, Lavikainen, A, Yanagida, T and Ito, A (2013) Phylogenetic systematics of the genus Echinococcus (Cestoda: Taeniidae). International Journal for Parasitology 43, 1017–1029.Google Scholar
Orkhontuul, S, Batbold, B, Avaajigmed, L, Tugbayar, T, Tsetsegdelger, M, Tsogtsaikhan, S, Enkhsaikhan, L, Yanagida, T, Nishikawa, Y and Ito, A (2018) Cerebral cystic echinococcosis in Mongolian children caused by, Echinococcus canadensis. Parasitology International 67, 584–586.Google Scholar
Piccoli, L, Bazzocchi, C, Brunetti, E, Mihailescu, P, Bandi, C, Mastalier, B, Cordos, I, Beuran, M, Popa, LG, Meroni, V, Genco, F and Cretu, C (2013) Molecular characterization of Echinococcus granulosus in south-eastern Romania: evidence of G1–G3 and G6–G10 complexes in humans. Clinical Microbiology and Infection 19, 578–582.Google Scholar
Polz, MF and Cavanaugh, CM (1998) Bias in template-to-product ratios in multitemplate PCR. Applied and Environmental Microbiology 64, 3724–3730.Google Scholar
Schweiger, A, Grimm, F, Tanner, I, Müllhaupt, B, Bertogg, K, Müller, N and Deplazes, P (2012) Serological diagnosis of echinococcosis: the diagnostic potential of native antigens. Infection 40, 139–152.Google Scholar
Sarink, M, Koelewijn, R, Slingerland, B, Tielens, A, Van Genderen, P and Van Hellemond, J (2018) Performance of the commercially available SERION ELISA classic Echinococcus igg test for the detection of cystic echinococcosis in clinical practice. Journal of Helminthology 93, 1–4. doi: 10.1017/S0022149X18000536.Google Scholar
Wang, N, Wang, Y, Ye, Q, Yang, Y, Wan, J, Guo, C, Zhan, J, Gu, X, Lai, W, Xie, Y, Peng, X and Yang, G (2018) Development of a direct PCR assay to detect Taenia multiceps, eggs isolated from dog feces. Veterinary Parasitology 251, 7–11.Google Scholar
Xiao, N, Qiu, J, Nakao, M, Li, T, Yang, W, Chen, X, Schantz, PM, Craig, PS and Ito, A (2006a) Echinococcus shiquicus, a new species from the Qinghai–Tibet plateau region of China: discovery and epidemiological implications. Parasitology International 55, S233–S236.Google Scholar
Xiao, N, Nakao, M, Qiu, J, Budke, CM and Giraudoux, P (2006b) Dual infection of animal hosts with different Echinococcus species in the eastern Qinghai-Tibet plateau region of China. American Journal of Tropical Medicine and Hygiene 75, 292–294.Google Scholar
Yang, GJ, Liu, L, Zhu, HR, Griffiths, SM, Tanner, M, Bergquist, R, Utzinger, J and Zhou, XN (2014) China's sustained drive to eliminate neglected tropical diseases. The Lancet Infectious Diseases 14, 881–892.Google Scholar
Yang, D, Zhang, T, Zeng, Z, Zhao, W, Zhang, W and Liu, A (2015) The first report of human-derived G10 genotype of Echinococcus canadensis in China and possible sources and routes of transmission. Parasitology International 64, 330–333.Google Scholar
Zhang, W and McManus, DP (2006) Recent advances in the immunology and diagnosis of echinococcosis. FEMS Immunology and Medical Microbiology 47, 24–41.Google Scholar
Zhang, T, Yang, D, Zeng, Z, Zhao, W, Liu, A, Piao, D, Jiang, T, Cai, J, Shen, Y, Liu, H and Zhang, W (2014) Genetic characterization of human-derived hydatid cysts of Echinococcus granulosus sensu lato in Heilongjiang Province and the first report of G7 genotype of E. canadensis in humans in China. PLoS One 9, e109059.Google Scholar
Zhenghuan, W, Xiaoming, W and Xiaoqing, L (2008) Echinococcosis in China, a review of the epidemiology of Echinococcus spp. EcoHealth 5, 115–126.Google Scholar