Development of a multiplex real-time polymerase chain reaction assay for detection of three common enteric protozoa infection

Enteric protozoa infections remain a significant health challenge, particularly in developing countries. Conventional diagnostic methods such as microscopy or antigen test are time-consuming and have limited sensitivity and specificity, often resulting in misdiagnosis. This study aimed to develop an improved molecular method using a multiplex real-time polymerase chain reaction (qPCR) assay for the simultaneous detection of three common enteric protozoa (Entamoeba histolytica, Giardia lamblia, and Cryptosporidium parvum) in a single tube. Specific primers and TaqMan probes targeting each pathogen and the internal control, labelled with different fluorophores (HEX, ROX, FAM, CY5), were designed. Analytical validation showed a limit of detection (LOD) for E. histolytica and G. lamblia of 1 × 10¹ pg DNA at a 10-fold serial dilution, while C. parvum was detected at 3.2 × 10³ pg at a 1:5 serial dilution. The assay achieved 100% sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV), with no cross-reactivity against non-target organisms in spiked stool sample tests. Compared to conventional methods, multiplex qPCR can simultaneously detect multiple targets, especially in mixed infection samples, with significantly higher sensitivity and specificity, reduces reagent consumption, and shortens turnaround time. Multiplex qPCR is a robust, accurate, and efficient method for alternate diagnosis method of enteric protozoa, allowing timely intervention to reduce the burden on healthcare workers.