GIARDIA DUODENALIS GLUTAMATE DEHYDROGENASE AND TRIOSE-PHOSPHATE ISOMERASE PCR-RFLP GENOTYPING EFFICIENCY AND PARASITE DENSITY

Abstract

Background. Giardia duodenalis is an intestinal protozoan parasite, with human infections predominantly caused by assemblages A and B. Genotyping of G. duodenalis infections commonly relies on GDH and TPI gene targets using PCR-RFLP and sequencing approaches, which are widely applied across diverse epidemiological settings. However, the performance and discriminatory power of these molecular tools can vary depending on parasite density, DNA quality, and the infecting assemblage, with assemblage B often demonstrating higher genetic heterogeneity. As such this study evaluates the effectiveness of GDH- and TPI-PCR-RFLP in characterizing G. duodenalis in relation to parasite density in patients with giardiasis at Busia county referral hospital, Kenya.
Methodology This hospital-based cross-sectional study was done at Busia County Referral Hospital from 2017 to 2020. A total of 147 patients referred to the clinical laboratory for stool analysis were recruited into the study. Genomic DNA was isolated from stool samples of 88 patients who tested positive for G. duodenalis by microscopic examination. The isolates were genotyped at the GDH and TPI gene loci using a semi-nested PCR-RFLP technique. Genotyping agreement between the GDH and TPI gene loci was analysed using Cohen's kappa statistics. Whereas parasite density were compared across the assemblages and sub-assemblages by Kruskal wallis and post hot dunnes analysis.

Results. This study showed that GDH and TPI single locus genotyping demonstrated no agreement in overall DNA amplification (Cohen’s kappa, 0.125; P = 0.228) and assemblage (Cohen’s kappa, 0.024; P = 0.234) genotyping success. Nevertheless, moderate or lack of agreement between GDH and TPI was detected for assemblage (Cohen’s kappa, 0.435; P < 0.001) and sub-assemblage (Cohen’s kappa, 0.027; P = 0.117) genotypes, respectively. The parasite density was marginally significantly higher in GDH (P = 0.067) but significantly lower in TPI (P = 0.032) in amplified cases. Furthermore, the parasite densities were significantly different amongst the assemblages (P = 0.014) and sub-assemblages (P = 0.003). Post-hoc analyses indicated a significantly higher parasite density in assemblage B relative to assemblage A (P < 0.001); BIII sub-assemblage compared to AI (P < 0.001), AIII (P < 0.001), and mixed AI/AIII (P < 0.001) sub-assemblages; as well as BIV versus AI (P < 0.001) sub-assemblages.
Conclusions. The findings show the influence of parasite density and genetic heterogeneity on genotyping efficiency and support the use of multi-locus approaches for more reliable characterisation of G. duodenalis infections.