Comparative evaluation of four genetic markers for molecular detection of Giardia duodenalis in human fecal samples

Giardia duodenalis is globally distributed intestinal protozoan parasite responsible for giardiasis causing various clinical manifestations ranging from asymptomatic to chronic diarrhea and malabsorption. Accurate diagnosis is crucial for effective clinical management and prevention of transmission especially in cases with low-parasite burden where false-negative results remain a challenge.

In this study, the diagnostic performance of four commonly used molecular markers: triosephosphate isomerase (tpi), small subunit ribosomal RNA (ssu-rRNA), glutamate dehydrogenase (gdh), and β-giardin (bg) was systematically evaluated using nested PCR in human fecal samples (n = 60), comprising microscopy confirmed Giardia-positive (n=40) and microscopy-negative samples (n=20). Sensitivity, specificity, PPV, NPV and limit of detection (LOD) were determined for each genetic marker.

Among the four molecular markers assessed, tpi gene showed the highest sensitivity (100%) and lowest LOD (10⁻⁸ ng/µl). The ssu-rRNA gene demonstrated sensitivity of 92.5% with LOD of 10⁻⁵ ng/µl, followed by gdh, which showed 85% sensitivity and LOD of 10⁻⁴ ng/µl. The bg gene exhibited the lowest sensitivity (72.5%) with LOD of 10⁻³ ng/µl. Although all targets showed 100% analytical specificity, the clinical diagnostic specificity of tpi was 80% which was lower compared to ssu-rRNA (90%), gdh (95%) and bg (95%).

The tpi gene showed the highest sensitivity and lowest LOD. Although its diagnostic accuracy (80%) was lower than other genes, this likely reflects its ability to detect low-parasite burden infections missed by microscopy. Therefore, tpi is the most suitable marker for sensitive detection, while ssu-rRNA, gdh, and bg may be used as complementary confirmatory markers.