Multiplex PCR for gastrointestinal parasites in stool: Benchmarking against direct microscopy and simplex PCR

Abstract

Purpose: To develop and validate a multiplex conventional PCR assay to simultaneously detect Cryptosporidium spp., Entamoeba histolytica, and Giardia lamblia in diarrheal samples as a rapid, cost-effective, and sensitive diagnostic tool for prevalent co-infections for improved diagnostic accuracy and efficiency in resource-limited settings.

Methods: Stool samples collected from patients with gastrointestinal symptoms after taking written consent, processed via wet mount, iodine mount, and PCR assays. Cohen's kappa statistical analysis was done to test agreement.

Result: Among 240 patients, 28.75% showed intestinal protozoa via Microscopy; Single-plex and multiplex PCR demonstrated 100% concordance, detecting 27.9%; confirmed by sequencing. Highest parasite positivity was observed in transplant and immunocompromised patients, with moderate to almost perfect agreement between microscopy and molecular methods.

Conclusion: Multiplex-conventional PCR offers superior sensitivity and specificity over microscopy and 100% concordance with single-plex PCR, enabling rapid, cost-effective diagnosis of multiple parasites from single stool sample. Its adoption could revolutionize parasitic infection management in routine diagnostics.

Keywords: Diagnosis; Intestinal protozoans; Microscopy; Multiplex-conventional PCR; Resource-limited settings.