Skip to main content Accessibility help
×
Home

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

  • Fan Chen (a1), Lei Liu (a2), Qili He (a1), Yan Huang (a1), Wentao Wang (a3), Guo Zhou (a4), Wenjie Yu (a1), Wei He (a1), Qi Wang (a1), Guangjia Zhang (a1), Sha Liao (a1), Ruirui Li (a1), Liu Yang (a1), Renxin Yao (a1), Qian Wang (a1) and Bo Zhong (a1)...

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.

Copyright

Corresponding author

Author for correspondence: Fan Chen, E-mail: 1149837551@qq.com; Qian Wang, E-mail: 2781958232@qq.com and Bo Zhong, E-mail: 402417235@qq.com

Footnotes

Hide All
*

These authors contributed equally to this work.

Footnotes

References

Hide All
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.
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, 016.
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.
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.
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, 795802.
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.
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, 38173824.
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, 15. doi: 10.1017/S0022149X18000883.
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, 386390.
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, 493498.
Elnifro, EM, Cooper, BD, Ashshi, AM and Klapper, P (2000) Multiplex PCR: optimization and application in diagnostic virology. Clinical Microbiology Reviews 13, 559570.
Henegariu, O and Al, E (1997) Multiplex PCR: critical parameters and step-by-step protocol. Biotechniques 23, 504511.
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, 693698.
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, 191200.
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, 585590.
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, 4448.
McManus, DP, Zhang, W, Li, J and Bartley, PB (2003) Echinococcosis. Lancet 362, 12951304.
McManus, DP, Gray, DJ, Zhang, W and Yang, Y (2012) Diagnosis, treatment, and management of echinococcosis. British Medical Journal 344, e3866e3866.
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, 247256.
Nakao, M, Sako, Y, Yokoyama, N, Fukunaga, M and Ito, A (2000) Mitochondrial genetic code in cestodes. Molecular and Biochemical Parasitology 111, 415424.
Nakao, M, Lavikainen, A, Yanagida, T and Ito, A (2013) Phylogenetic systematics of the genus Echinococcus (Cestoda: Taeniidae). International Journal for Parasitology 43, 10171029.
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, 584586.
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, 578582.
Polz, MF and Cavanaugh, CM (1998) Bias in template-to-product ratios in multitemplate PCR. Applied and Environmental Microbiology 64, 37243730.
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, 139152.
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, 14. doi: 10.1017/S0022149X18000536.
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, 711.
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, S233S236.
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, 292294.
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, 881892.
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, 330333.
Zhang, W and McManus, DP (2006) Recent advances in the immunology and diagnosis of echinococcosis. FEMS Immunology and Medical Microbiology 47, 2441.
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.
Zhenghuan, W, Xiaoming, W and Xiaoqing, L (2008) Echinococcosis in China, a review of the epidemiology of Echinococcus spp. EcoHealth 5, 115126.

Keywords

Metrics

Full text views

Total number of HTML views: 0
Total number of PDF views: 0 *
Loading metrics...

Abstract views

Total abstract views: 0 *
Loading metrics...

* Views captured on Cambridge Core between <date>. This data will be updated every 24 hours.

Usage data cannot currently be displayed