Supplementary file 1_Evaluation of real-time LAMP assay for detection of Entamoeba species in clinical stool samples from a tertiary care center in North India.docx

All samples were analyzed using microscopy, conventional PCR, real-time PCR, and real-time LAMP, followed by phylogenetic analysis for species confirmation. For real-time LAMP, the SREHP gene and LSU rRNA gene reactions were performed separately in single run to ensure accurate identification. Statistical evaluation was performed using Bayesian latent class analysis.</p>Results<p>The sensitivity of microscopy, conventional PCR, real-time PCR, and real-time LAMP was calculated to be 39.4% 84.3%, 99.3%, and 99.3%, respectively, with 99.4–100% specificity for real-time LAMP.

Five samples missed by conventional PCR were successfully detected by both real-time PCR and real-time LAMP. The overall infection rate across was 6.7% (33/494), with differential detection of Entamoeba species. Samples positive for E. histolytica showed amplification of both SREHP and LSU rRNA genes, whereas E. dispar and E.moshkovskii showed amplification of the LSU rRNA gene only.

Among patients with UC, those with active disease had a higher incidence of E. histolytica infection (13/88) than those in remission (4/29).</p>Interpretation<p>Real-time LAMP demonstrated specificity and sensitivity of 99.3% (92.5–100) equivalent to that of real-time PCR with an added advantage of shorter turnaround time than conventional PCR and real-time PCR (60 minutes vs. 200 minutes vs 120 minutes), facilitating prompt differential diagnosis of Entamoeba species in patients with UC.

These finding support considering molecular tests in microscopy-negative cases to improve detection and differentiation of Entamoeba species in high-incidence areas and among high-risk patients. However, cost effectiveness and downstream impact on clinical outcomes warrant further study.</p>