Genotyping of Giardia duodenalis cysts by new real-time PCR assays for detection of mixed infections in human samples

Abstract

Of the seven genetic groups, or assemblages, currently recognized in the Giardia duodenalis species complex, only assemblages A and B are associated with human infection, but they also infect other mammals. Recent investigations have suggested the occurrence of genetic exchanges among isolates of G. duodenalis, and the application of assemblage-specific PCR has shown both assemblages A and B in a significant number of human infections. In this work, three real-time quantitative (qPCR) assays were developed to target the G. duodenalis triose phosphate isomerase, glutamate dehydrogenase, and open reading frame C4 sequences. Primers were designed to allow the specific amplification of the DNA of assemblage A or B and to generate products distinguishable by their melting curves or, after qPCR, by their sequences, sizes, or restriction patterns. The assays showed full specificity and detected DNA from a single trophozoite (4 to 8 target copies). We applied these assays, as well as a TaqMan assay that targets the beta-giardin gene, to genomic DNA extracted from 30 human stools and to Giardia cysts purified by immunomagnetic capture from the same samples. Simultaneous detection of both assemblages was observed in a large number of DNAs extracted from stools, and experiments on the cysts purified from the same samples showed that this was essentially attributable to mixed infections, as only one assemblage was detected when dilutions of cysts were tested. In a few cases, detection of both assemblages was observed even when single cysts were tested. This result, which suggests the presence of recombinants, needs to be confirmed using more accurate methods for cyst separation and enumeration. The assays described in this study can be used to detect Giardia cysts infectious to humans in samples from animals and in water and food.

FIG. 1.

Schematic representation of the amplification curves (left), melting curves (middle), and electrophoretic separation of products (right) obtained using the gdh, orfC4, and tpi qPCR assays. In gel electrophoresis of gdh products, lane M is the 50-bp size ladder; lanes 1 and 6 show the assemblage A product (180 bp); lanes 2, 3, and 7 show the assemblage B product (133 bp); lanes 4, 5, and 8 show the presence of both assemblages A and B; and lane 9 shows the negative control. In gel electrophoresis of orfC4 products, lane M is the 50-bp size ladder; lanes 1 and 7 show the assemblage A product (103 bp); lanes 3, 4, 5, and 8 show the assemblage B product (171 bp); lanes 2, 6, and 9 show the presence of both assemblages A and B; and lane 10 shows the negative control. In gel electrophoresis of tpi amplicons, the product from assemblage A is not cut by AluI (lane 1) but is cut by HincII into two fragments of 47 bp and 31 bp (lane 2), whereas the B product is cut by AluI into two fragments of 45 bp and 32 bp (lane 3) but is not cut by HincII (lane 4).

FIG. 2.

Standard curves of the different qPCR assays obtained using serial dilutions of freshly collected trophozoites of the G. duodenalis WB strain using assemblage A-specific primer pairs. The number of trophozoites (in 10 base log units) is indicated on the x axis, while the number of qPCR cycles is indicated on the y axis. The slopes, intercepts, and correlation coefficients are indicated for each target gene.