DEMOGRAPHIC DRIVERS OF DIAGNOSTIC POSITIVITY: A MULTI-MODAL EVALUATION OF ENTAMOEBA HISTOLYTICA DETECTION BY MICROSCOPY, ELISA, AND PCR IN SYMPTOMATIC CHILDREN

Abstract

Entamoeba histolytica (E. histolytica) is a leading cause of intestinal parasitic infection in children living in low resource settings with inadequate sanitation. Accurate and timely diagnosis is critical for effective case management, yet the comparative diagnostic performance of routinely available methods relative to molecular reference standards remains insufficiently characterized in South Punjab, Pakistan. In order to determine the accuracy, positive predictive value, negative predictive value and degree of correlation between light microscopy, ELISA and PCR as the gold standard for diagnosis of E. histolytica in children presenting symptoms, as well as epidemiologic factors associated with these positive cases, A hospital-based cross-sectional study was conducted at Nishtar Hospital, Multan, enrolling 120 symptomatic children aged 1-18 years by consecutive sampling. Fresh stool specimens were simultaneously examined by light microscopy (wet mount and formalin-ethyl acetate concentration technique), species-specific ELISA antigen detection, and PCR targeting the 18S ribosomal RNA gene. Diagnostic performance metrics and epidemiological associations were computed against PCR as the gold standard.PCR confirmed E. histolytica in 38.3% (n=46) of participants. ELISA yielded 100% positivity across all samples (sensitivity 100%, specificity 0%, PPV 38.3%), indicating a complete absence of discriminatory capacity. Microscopy achieved a sensitivity 76.1%, specificity 29.7%, PPV 40.2%, and NPV 66.7%. ROC analysis demonstrated poor discriminative ability: microscopy AUC 0.53 (95% CI 0.42-0.64, p=0.214) and ELISA AUC 0.50 (95% CI 0.50-0.50, p=1.000). Rural residence (88.2%), large household size (>10 members, 82.1%), and the 6-12-year age group (85.2%) were significantly associated with higher infection rates. Neither microscopy nor ELISA achieved acceptable diagnostic accuracy relative to PCR. PCR should be adopted as the confirmatory standard in clinical and epidemiological settings. Integration of molecular diagnostics with community-level WASH interventions is urgently needed to reduce the burden of pediatric amebiasis in South Punjab.