A real-time assemblage-specific PCR assay for the detection of Giardia duodenalis assemblages A, B and E in fecal samples.

Giardiosis is a common gastrointestinal infection caused by the flagellate Giardia duodenalis, and affects both humans and animals, worldwide. Animals are infected with both zoonotic and host-specific G. duodenalis assemblages, and their role in the transmission of the infection to humans has been a subject of intense research and debate. Conventional PCR assays are appropriate to determine G. duodenalis assemblages, but lack sensitivity for the detection of mixed infections. Previous surveys demonstrated the occurrence of mixed infections with G. duodenalis assemblage A and B in humans, and with assemblages A and E in cattle, but are likely to be underestimated. In this study, we designed a set of assemblage-specific primers by exploiting sequence variability in homologous genes from assemblages A, B and E. Primers were designed to amplify fragments of different size that generated different melting curves from each assemblage in real-time PCR (rt-PCR) experiments. The assay has been tested on a large panel of human and farm animal isolates, and shown to possess high specificity (no cross reactions observed) and sensitivity (detection limit close to 20 copies). Therefore, this assay can be useful to detect zoonotic and host-specific G. duodenalis assemblages in fecal samples from farm animals, particularly when a large number of samples is to be tested.

Detection of Giardia duodenalis assemblages A and B in human feces by simple, assemblage-specific PCR assays.

The flagellated protozoan Giardia duodenalis is a common gastrointestinal parasite of mammals, including humans. Molecular characterizations have shown the existence of eight genetic groups (or assemblages) in the G. duodenalis species complex. Human infections are caused by assemblages A and B, which infect other mammals as well. Whether transmission routes, animal reservoirs and associations with specific symptoms differ for assemblage A and assemblage B is not clear. Furthermore, the occurrence and clinical significance of mixed (A+B) infections is also poorly understood. To date, the majority of PCR assays has been developed to identify all G. duodenalis assemblages based on the use of primers that bind to conserved regions, yet a reliable identification of specific assemblages is better achieved by ad hoc methods. The aim of this work was to design simple PCR assays that, based on the use of assemblage-specific primers, produce diagnostic bands of different lengths for assemblage A and B. We first generated novel sequence information from assemblage B, identified homologous sequences in the assemblage A genome, and designed primers at six independent loci. Experiments performed on DNA extracted from axenic cultures showed that two of the six assays can detect the equivalent of a single cyst and are not negatively influenced by disproportions between DNA of each assemblage, at least up to a 9:1 ratio. Further experiments on DNAs extracted from feces showed that the two assays can detect both assemblages in single tube reactions with excellent reliability. Finally, the robustness of these assays was demonstrated by testing a large collection of human isolates previously typed by multi-locus genotyping.