A Multiplex PCR for the Simultaneous Detection and Genotyping of the Echinococcus Granulosus Complex

Overview

Journal PLoS Negl Trop Dis

Specialties Infectious Diseases
Tropical Medicine

Date 2013 Jan 26

PMID 23350011

Citations 27

Authors

Ghalia Boubaker

Natalia Macchiaroli

Laura Prada

Marcela A Cucher

Mara C Rosenzvit

Iskender Ziadinov

Peter Deplazes

Urmas Saarma

Hamouda Babba

Bruno Gottstein

Markus Spiliotis

Affiliations

Soon will be listed here.

Abstract

Echinococcus granulosus is characterized by high intra-specific variability (genotypes G1-G10) and according to the new molecular phylogeny of the genus Echinococcus, the E. granulosus complex has been divided into E. granulosus sensu stricto (G1-G3), E. equinus (G4), E. ortleppi (G5), and E. canadensis (G6-G10). The molecular characterization of E. granulosus isolates is fundamental to understand the spatio-temporal epidemiology of this complex in many endemic areas with the simultaneous occurrence of different Echinococcus species and genotypes. To simplify the genotyping of the E. granulosus complex we developed a single-tube multiplex PCR (mPCR) allowing three levels of discrimination: (i) Echinococcus genus, (ii) E. granulosus complex in common, and (iii) the specific genotype within the E. granulosus complex. The methodology was established with known DNA samples of the different strains/genotypes, confirmed on 42 already genotyped samples (Spain: 22 and Bulgaria: 20) and then successfully applied on 153 unknown samples (Tunisia: 114, Algeria: 26 and Argentina: 13). The sensitivity threshold of the mPCR was found to be 5 ng Echinoccoccus DNA in a mixture of up to 1 µg of foreign DNA and the specificity was 100% when template DNA from closely related members of the genus Taenia was used. Additionally to DNA samples, the mPCR can be carried out directly on boiled hydatid fluid or on alkaline-lysed frozen or fixed protoscoleces, thus avoiding classical DNA extractions. However, when using Echinococcus eggs obtained from fecal samples of infected dogs, the sensitivity of the mPCR was low (<40%). Thus, except for copro analysis, the mPCR described here has a high potential for a worldwide application in large-scale molecular epidemiological studies on the Echinococcus genus.

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References

Guarnera E, Parra A, Kamenetzky L, Garcia G, Gutierrez A . Cystic echinococcosis in Argentina: evolution of metacestode and clinical expression in various Echinococcus granulosus strains. Acta Trop. 2004; 92(2):153-9. DOI: 10.1016/j.actatropica.2004.06.008. View

Aaty H, Abdel-Hameed D, Alam-Eldin Y, El-Shennawy S, Aminou H, Makled S . Molecular genotyping of Echinococcus granulosus in animal and human isolates from Egypt. Acta Trop. 2011; 121(2):125-8. DOI: 10.1016/j.actatropica.2011.10.014. View

Stefanic S, Shaikenov B, Deplazes P, Dinkel A, Torgerson P, Mathis A . Polymerase chain reaction for detection of patent infections of Echinococcus granulosus ("sheep strain") in naturally infected dogs. Parasitol Res. 2004; 92(4):347-51. DOI: 10.1007/s00436-003-1043-y. View

Breyer I, Georgieva D, Kurdova R, Gottstein B . Echinococcus granulosus strain typing in Bulgaria: the G1 genotype is predominant in intermediate and definitive wild hosts. Parasitol Res. 2004; 93(2):127-30. DOI: 10.1007/s00436-004-1116-6. View

Logotheti M, Kotsovili-Tseleni A, Arsenis G, Legakis N . Multiplex PCR for the discrimination of A. fumigatus, A. flavus, A. niger and A. terreus. J Microbiol Methods. 2008; 76(2):209-11. DOI: 10.1016/j.mimet.2008.10.006. View

Huttner M, Siefert L, Mackenstedt U, Romig T . A survey of Echinococcus species in wild carnivores and livestock in East Africa. Int J Parasitol. 2009; 39(11):1269-76. DOI: 10.1016/j.ijpara.2009.02.015. View

Soriano S, Pierangeli N, Pianciola L, Mazzeo M, Lazzarini L, Saiz M . Molecular characterization of Echinococcus isolates indicates goats as reservoir for Echinococcus canadensis G6 genotype in Neuquén, Patagonia Argentina. Parasitol Int. 2010; 59(4):626-8. DOI: 10.1016/j.parint.2010.07.003. View

Mrad S, Filisetti D, Oudni M, Mekki M, Belguith M, Nouri A . Molecular evidence of ovine (G1) and camel (G6) strains of Echinococcus granulosus in Tunisia and putative role of cattle in human contamination. Vet Parasitol. 2005; 129(3-4):267-72. DOI: 10.1016/j.vetpar.2005.02.006. View

Sadjjadi S . Present situation of echinococcosis in the Middle East and Arabic North Africa. Parasitol Int. 2005; 55 Suppl:S197-202. DOI: 10.1016/j.parint.2005.11.030. View

10.

Maillard S, Benchikh-Elfegoun M, Knapp J, Bart J, Koskei P, Gottstein B . Taxonomic position and geographical distribution of the common sheep G1 and camel G6 strains of Echinococcus granulosus in three African countries. Parasitol Res. 2006; 100(3):495-503. DOI: 10.1007/s00436-006-0286-9. View

11.

Kamenetzky L, Gutierrez A, Canova S, Haag K, Guarnera E, Parra A . Several strains of Echinococcus granulosus infect livestock and humans in Argentina. Infect Genet Evol. 2003; 2(2):129-36. DOI: 10.1016/s1567-1348(02)00131-4. View

12.

Eckert J, Thompson R . Intraspecific variation of Echinococcus granulosus and related species with emphasis on their infectivity to humans. Acta Trop. 1997; 64(1-2):19-34. DOI: 10.1016/s0001-706x(96)00635-3. View

13.

Battelli G . Echinococcosis: costs, losses and social consequences of a neglected zoonosis. Vet Res Commun. 2009; 33 Suppl 1:47-52. DOI: 10.1007/s11259-009-9247-y. View

14.

Nakao M, Yanagida T, Okamoto M, Knapp J, Nkouawa A, Sako Y . State-of-the-art Echinococcus and Taenia: phylogenetic taxonomy of human-pathogenic tapeworms and its application to molecular diagnosis. Infect Genet Evol. 2010; 10(4):444-52. DOI: 10.1016/j.meegid.2010.01.011. View

15.

Gasser R, Zhu X, McManus D . Dideoxy fingerprinting: application to the genotyping of Echinococcus. Int J Parasitol. 1998; 28(11):1775-9. DOI: 10.1016/s0020-7519(98)00126-x. View

16.

Schneider R, Gollackner B, Edel B, Schmid K, Wrba F, Tucek G . Development of a new PCR protocol for the detection of species and genotypes (strains) of Echinococcus in formalin-fixed, paraffin-embedded tissues. Int J Parasitol. 2008; 38(8-9):1065-71. DOI: 10.1016/j.ijpara.2007.11.008. View

17.

Kitamura S, Jung S, Oh M . Differentiation of lymphocystis disease virus genotype by multiplex PCR. J Microbiol. 2006; 44(2):248-53. View

18.

Siles-Lucas M, Benito M, Cuesta-Bandera C . Echinococcus granulosus: genomic and isoenzymatic study of Spanish strains isolated from different intermediate hosts. Vet Parasitol. 1996; 63(3-4):273-82. DOI: 10.1016/0304-4017(95)00903-5. View

19.

Zhang G, Guan Y, Sheng H, Zheng B, Wu S, Xiao H . Multiplex PCR and oligonucleotide microarray for detection of single-nucleotide polymorphisms associated with Plasmodium falciparum drug resistance. J Clin Microbiol. 2008; 46(7):2167-74. PMC: 2446905. DOI: 10.1128/JCM.00081-08. View

20.

Bardonnet K, Benchikh-Elfegoun M, Bart J, Harraga S, Hannache N, Haddad S . Cystic echinococcosis in Algeria: cattle act as reservoirs of a sheep strain and may contribute to human contamination. Vet Parasitol. 2003; 116(1):35-44. DOI: 10.1016/s0304-4017(03)00255-3. View