High resolution melting real-time PCR for genotyping of Giardia lamblia assemblages A and B regardless of parasite load.

Giardia lamblia is a globally distributed fecal-oral transmitted enteroparasite. G. lamblia is phylogenetically divided into assemblages A to H. Assemblages A and B have high zoonotic potential, as reported in humans and other animals, including dogs and cats. Currently, G. lamblia genotyping is performed using gene sequencing, which is an expensive technique, especially in developing countries where higher frequencies of the protozoan have been reported. Real-time PCR with High Resolution Melting (qPCR-HRM) is a sensitive method for detecting polymorphisms and is used for differentiate species, subspecies, and assemblages. This study aimed to standardize and validate the qPCR-HRM technique for genotyping G. lamblia in human fecal samples, regardless of the parasite load. qPCR-HRM was standardized using the β-giardin target, and validation was performed using DNA extracted from fecal samples (n = 76). Sanger sequencing of the same genetic target was used the gold standard. Genotyping of the samples (assemblages A and B) was performed using both techniques, and qPCR-HRM demonstrated 98.08% concordance with the sequencing results. Only one sample showed a discrepancy in the assemblage determination between the techniques. DNA sequencing failed to characterize 24 samples from patients with low or moderate parasite loads. Thus, qPCR-HRM successfully differentiate 23 samples into assemblage A and one as assemblage B. In this context, when compared to sequencing, the qPCR-HRM technique proved to be effective for genotyping G. lamblia, even in samples with a low parasite load, with the potential to significantly reduce execution time and financial investment required to identify G. lamblia assemblages. This tool can be strategic for strengthening public health policies for the surveillance and control of giardiasis.