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Sensitive PCR-restriction fragment length polymorphism assay for detection and genotyping of Giardia duodenalis in human feces.

ABSTRACT: 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.

SUBMITTER: Amar CF

PROVIDER: S-EPMC153413 | biostudies-literature | 2002 Feb

REPOSITORIES: biostudies-literature

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Sensitive PCR-restriction fragment length polymorphism assay for detection and genotyping of Giardia duodenalis in human feces.

Amar C F L CF Dear P H PH Pedraza-Díaz S S Looker N N Linnane E E McLauchlin J J

Journal of clinical microbiology 20020201 2

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 speci ...[more]

PMID: 11825955

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Project description:Giardia duodenalis is one of the main enteric pathogens associated with diarrheal disease. In developing countries, giardiasis is a major public health concern, particularly in children under five years of age. This study aimed to evaluate the occurrence and genetic diversity of G. duodenalis causing human infections in Shushtar County, Southwestern Iran. Individual faecal specimens were collected from 1,163 individuals (male/female ratio: 0.9; age range 2-75 years) with (n = 258) and without (n = 905) gastrointestinal symptoms living in rural and urban settings during the period 2017-2018. Conventional (sucrose flotation and microscopy) methods were used for the initial detection of G. duodenalis cysts in faecal specimens. Microscopy-positive samples were confirmed by PCR amplification and sequencing of the small subunit rRNA (ssu rRNA) gene of the parasite. A multilocus genotyping (MLG) scheme targeting the triose phosphate isomerase (tpi), the glutamate dehydrogenase (gdh), and the beta-giardin (bg) genes was used for genotyping purposes. Giardia duodenalis cysts were detected in 7.7% (90/1,163) of samples by microscopy, of which 82 were confirmed by ssu-PCR. Successful amplification and sequencing results were obtained for 23.2% (19/82), 9.8% (8/82), and 8.5% (7/82) of the confirmed samples at the tpi, gdh, and bg loci, respectively. MLG data for the three loci were available for two samples only. Out of the 24 samples genotyped at any loci, 50% (12/24) were identified as assemblage A and the remaining half as assemblage B. Overall, AII was the most prevalent sub-assemblage detected (41.7%, 10/24), followed by BIII (25.0%, 6/24), discordant BIII/BIV (5/24) or AII/AIII (2/24) sequences, and BIV (1/24). No significant correlation was demonstrated between a given assemblage/sub-assemblage and the occurrence of clinical symptoms. No genotypes adapted to animal hosts other than humans (e.g. assemblages C-F) were found circulating in the investigated human population, suggesting that transmission of human giardiasis in this Iranian region is primarily of anthroponotic nature. Further molecular-based studies are needed to confirm and expand these results, and to ascertain the presence and public health relevance of the parasite in environmental (e.g. drinking water) samples.

Dataset Information

Sensitive PCR-restriction fragment length polymorphism assay for detection and genotyping of Giardia duodenalis in human feces.

Publications

Sensitive PCR-restriction fragment length polymorphism assay for detection and genotyping of Giardia duodenalis in human feces.

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