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Journal of Clinical Microbiology, February 2002, p. 446-452, Vol. 40, No. 2
0095-1137/01/$04.00+0     DOI: 10.1128/JCM.40.2.446-452.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.

Sensitive PCR-Restriction Fragment Length Polymorphism Assay for Detection and Genotyping of Giardia duodenalis in Human Feces

C. F. L. Amar,1,2 P. H. Dear,3 S. Pedraza-Díaz,1 N. Looker,4 E. Linnane,5 and J. McLauchlin1*

Food Safety Microbiology Laboratory, Division of Gastrointestinal Infections, Public Health Laboratory Service, Central Public Health Laboratory, London NW9 5HT,1 School of Chemical and Life Sciences, University of Greenwich, Woolwich, London SE18 8PF,2 Medical Research Council Laboratory of Molecular Biology, Cambridge CB2 2QH,3 Public Health Laboratory, Glan Clwyd District Hospital, Rhyl, Denbighshire LL18 5UJ,4 Bro Taf Health Authority, Temple of Peace and Health, Cathays Park, Cardiff CF1 3NW, United Kingdom5

Received 13 September 2001/ Returned for modification 28 October 2001/ Accepted 18 November 2001

An assay that uses heminested PCR-restriction fragment length polymorphism analysis for the detection and genotyping of Giardia duodenalis on the basis of polymorphism in the triose phosphate isomerase (tpi) gene was developed. This assay was evaluated with DNA extracted from purified parasite material, bacterial cultures, whole human feces containing G. duodenalis and other parasites, and their corresponding immunofluorescence-stained fecal smears on glass microscope slides. The assay was specific and discriminated between G. duodenalis assemblages A and B. RFLP analysis further distinguished two groups (designated groups I and II) within assemblage A. Among 35 DNA samples extracted from whole feces from patients with confirmed sporadic giardiasis, the tpi gene was amplified from 33 (94%). Of these, nine (27%) samples contained assemblage A group II, 21 (64%) contained assemblage B, and 3 (9%) contained a mixture of assemblage A group II and assemblage B. The tpi gene of G. duodenalis assemblage B was amplified from 21 of 24 (88%) DNA samples extracted from whole feces from patients with confirmed cases of infection in a nursery outbreak. No amplification was detected from the remaining three DNA samples. Overall, analysis of DNA extracted from material recovered from stained microscope slides identified identical G. duodenalis genotypes in 35 (65%) of the 54 samples for which a genotype was established with DNA from whole feces. The heminested PCR method developed is sensitive, simple, and rapid to perform and is applicable for the analysis of other intestinal pathogens.

* Corresponding author. Mailing address: Food Safety Microbiology Laboratory, Division of Gastrointestinal Infections, PHLS Central Public Health Laboratory, 61 Colindale Ave., London NW9 5HT, United Kingdom. Phone: 44 20 8200 4400, ext. 3505. Fax: 44 20 8358 3112. E-mail: jmclauchlin{at}phls.nhs.uk.

Journal of Clinical Microbiology, February 2002, p. 446-452, Vol. 40, No. 2
0095-1137/01/$04.00+0     DOI: 10.1128/JCM.40.2.446-452.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.



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