Comparison of Two Target Genes for Detection and Genotyping of Giardia lamblia in Human Feces by PCR and PCR-Restriction Fragment Length Polymorphism - Archive ouverte HAL Accéder directement au contenu
Article Dans Une Revue Journal of Clinical Microbiology Année : 2005

Comparison of Two Target Genes for Detection and Genotyping of Giardia lamblia in Human Feces by PCR and PCR-Restriction Fragment Length Polymorphism

Résumé

A PCR assay targeting the tpi gene was developed to detect and to genotype Giardia lamblia in human feces. Our assay was specific and discriminated between G. lamblia assemblages A and B. G. lamblia cysts isolated from human feces were also analyzed with two previously described PCR-restriction fragment length poly-morphism (RFLP) assays, which are based on the detection of tpi or gdh genes. These RFLP analyses distinguished groups I and II within assemblage A or groups III and IV within assemblage B. Among 26 fecal samples from patients with sporadic giardiasis diagnosed by hospital laboratories, the tpi gene was amplified from 25 (96%) with our PCR assay, whereas only 21 (81%) samples were positive when the gdh gene was targeted. Of the 25 positive samples, nine (36%) contained assemblage A and 16 (64%) contained assemblage B. Thus, RFLP analysis classified eight samples (32%) in assemblage A group II, eight (32%) in assemblage B group III, and five (20%) in assemblage B group IV. The group could not be specified for four samples. The tpi and gdh genes of G. lamblia assemblage B were amplified from 14 (93%) of 15 samples collected only from French soldiers coming back from the Ivory Coast. All of these contained assemblage B group III. The PCR method developed is sensitive, simple, and specific and shows that the tpi gene is well adapted for G. lamblia genotyping. The intestinal protozoan Giardia lamblia (synonyms, G. in-testinalis and G. duodenalis [1]) is a cosmopolitan parasite frequently involved in human parasitic gastroenteritis throughout the world. Transmission of the G. lamblia cyst to humans occurs mainly following ingestion of contaminated water. Clinical manifestations of symptomatic giardiasis include greasy stools, flatulence, diarrhea, and abdominal cramps (9). However , the majority of cases are asymptomatic or minimally symptomatic in immunocompetent individuals. Among the six species identified in the Giardia genus, only G. lamblia infects humans and numerous other mammals as well (1, 25). Moreover, isolates of G. lamblia are classified into seven assemblages, based on the characterization of the gluta-mate dehydrogenase (gdh), small-subunit (SSU) rRNA, and triosephosphate isomerase (tpi) genes (12, 18, 20, 21). Assemblages A and B infect humans and a broad range of other hosts, including livestock, cats, dogs, and wild mammals. The assemblage A isolates have been further grouped into subgroups I and II. The assemblage B isolates have been separated into subgroups III and IV (17, 24). Genetic assemblages C, D, E, F, and G appear to be host restricted to domestic animals, livestock , and wild animals (19, 21). At present, antigen detection immunoassays for Giardia are used as the routine diagnostic procedure of choice in many hospitals and public health laboratories (8, 13, 27). However, these methods are unable to differentiate between the genetic asemblages of Giardia lamblia. Molecular detection methods based on PCR have been developed to detect G. lamblia cysts in feces. These techniques have numerous advantages in terms of sensitivity, speed, and specificity in comparison to conventional methodologies (3, 16). Moreover, these molecular techniques may allow the genotyping of Giardia lamblia cysts (3, 4, 20). We previously described a method for the successful extraction and detection of giardial DNA from naturally contaminated wastewater (5). In the present study, we evaluated primers for rapid and sensitive classification of G. lamblia cysts from human feces into assemblages A and B. These primers were previously designed for the detection and quantification of G. lamblia assemblages A and B in environmental samples by real-time PCR (data not shown). In this study, we evaluated the distribution of these major assemblages in sporadic human giardiasis in France and also in samples from 15 French soldiers coming back from the Ivory Coast. Moreover, the simultaneous use of our assay and two previously described PCR-restriction fragment length polymorphism (RFLP) assays allowed comparison of the tpi and gdh genes for detection and genotyping of G. lamblia. MATERIALS AND METHODS Purified suspensions. Giardia lamblia assemblage B cysts, produced by passage of human strain H3 (10) of G. lamblia through Mongolian gerbils, were obtained from Waterborne Inc. (New Orleans, La.). Prior to delivery, cysts were 95 to 99% purified by sucrose and Percoll density gradient centrifugation and washing with water, and then stored in phosphate-buffered saline (PBS, pH 7.4) with antibiotics. Giardia lamblia (assemblage A) cysts were purified from one human feces sample by the ethyl acetate procedure (see below) followed by * Corresponding author. Mailing address: LCPME-UMR 7564 CNRS-UHP, Equipe Microbiologie et Physique, Faculté de Pharmacie,

Domaines

Parasitologie

Dates et versions

hal-01792661 , version 1 (15-05-2018)

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Citer

I. Bertrand, Laetitia Albertini, Janine Schwartzbrod. Comparison of Two Target Genes for Detection and Genotyping of Giardia lamblia in Human Feces by PCR and PCR-Restriction Fragment Length Polymorphism. Journal of Clinical Microbiology, 2005, 43 (12), pp.5940 - 5944. ⟨10.1128/JCM.43.12.5940-5944.2005⟩. ⟨hal-01792661⟩
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