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Giardiasis is a prevalent parasitic diarrheal disease caused by Giardia lamblia , affecting people worldwide. Recently, the availability of several drugs for its treatment has highlighted issues such as multidrug resistance, limited effectiveness and undesirable side effects. Therefore, it is necessary to develop alternative new drugs and treatment strategies that can enhance therapeutic outcomes and effectively treat giardiasis. Natural compounds show promise in the search for more potent anti-giardial agents. Our investigation focused on the effect of Andrographolide (ADG), an active compound of the Andrographis paniculata plant, on Giardia lamblia , assessing trophozoite growth, morphological changes, cell cycle arrest, DNA damage and inhibition of gene expression associated with pathogenic factors. ADG demonstrated anti- Giardia activity almost equivalent to the reference drug metronidazole, with an IC 50 value of 4.99 μM after 24 h of incubation. In cytotoxicity assessments and morphological examinations, it showed significant alterations in trophozoite shape and size and effectively hindered the adhesion of trophozoites. It also caused excessive ROS generation, DNA damage, cell cycle arrest and inhibited the gene expression related to pathogenesis. Our findings have revealed the anti-giardial efficacy of ADG, suggesting its potential as an agent against Giardia infections. This could offer a natural and low-risk treatment option for giardiasis, reducing the risk of side effects and drug resistance. [Display omitted] • The in vitro inhibitory effects of ADG on the proliferation and cell death of Giardia trophozoites. • ADG led to trophozoite shrinkage, membrane rupture and remarkable changes in morphology. • The administration of ADG led to a reduction of cells in the S/G2M phase. • ADG led to the production of excessive intracellular ROS in Giardia trophozoites. [ABSTRACT FROM AUTHOR]

• The muliplex PCR-RFLP method could easily detect and distinguish between human infecting genotypes of Giardia duodenalis i. e., AI/AII and BIII/BIV. • The muliplex PCR method can quickly identify mixed infection cases i.e., A+B • The results obtained by the newly developed method was concordant with previously described method i. e., Reference PCR and sequencing. • The PCR method demonstrated high degree of diagnostic sensitivity (94.2 %), accuracy (97.1 %) and specificity (100 %). Giardia duodenalis is a common cause of diarrheal illness in regions with limited resources. The demand for rapid and cost-effective detection and genotyping methods in large-scale epidemiological studies and clinical diagnostics is imperative. Hence, we developed a multiplex PCR-RFLP technique targeting the tpi gene of G. duodenalis. The assay successfully screened G. duodenalis positive clinical samples (6.33 %; 36/565). It was also able to categorize the isolates into assemblages A (41.66 %; 13/36) and B (58.33 %; 23/36), as well as into subassemblages: AI (13.8 %; 5/36), AII (27.77 %; 10/36), BIII (36.11 %; 15/36) and BIV (22.22 %; 8/36). High diagnostic sensitivity (94.2 %), specificity (100 %) and accuracy (97.1 %) of the PCR assay were obtained, indicating its reliability for diagnosing giardiasis. Notably, the assay demonstrated close concordance with microscopy (κ=0.85) and reference PCR (κ=0.98) results. The optimized method offers a cost-effective and rapid approach for G. duodenalis detection and genotyping, convenient for epidemiological studies and clinical diagnostics. [ABSTRACT FROM AUTHOR]