Entamoeba histolytica is an invasive, water- and food-borne protozoan parasite and a significant contributor to parasitic disease mortality worldwide. In India, where transmission occurs primarily through the fecal-oral route, prevalence estimates range from 3 to 23 percent, with particularly high rates among children aged five to twelve. Clinical manifestations span from asymptomatic carriage to severe complications including extra-intestinal abscesses, perforating bowel disease, and toxic megacolon. The stakes of accurate diagnosis are heightened by the existence of morphologically identical but clinically distinct relatives: Entamoeba dispar is generally non-pathogenic, and Entamoeba moshkovskii, long considered a free-living species, has increasingly been implicated in gastrointestinal symptoms. Because these organisms are indistinguishable under the microscope, conventional microscopy cannot tell a patient who needs anti-amoebic therapy from one who does not.

The diagnostic challenge is especially acute for patients with ulcerative colitis. Symptoms of amoebic colitis—bloody diarrhea, abdominal pain, mucosal inflammation—overlap substantially with those of active UC, and the two conditions can coexist. Escalating immunosuppressive therapy in a patient harboring an untreated invasive parasite can be counterproductive or even catastrophic; fulminant amoebic colitis following corticosteroid therapy has been documented in the literature. Yet the role of parasitic infections during IBD flares has been comparatively neglected, with most research focusing on bacterial pathogens. The study team, led by Devyani Sharma of the Post Graduate Institute of Medical Education and Research (PGIMER) in Chandigarh, set out to determine whether real-time LAMP could close this gap in an endemic setting.

Between January 2021 and January 2023, the researchers collected 494 stool samples across four groups: 117 patients with ulcerative colitis diagnosed according to European Crohn’s and Colitis Organisation criteria, 177 patients with persistent or chronic diarrhea but no IBD diagnosis, 100 asymptomatic community participants recruited from rural villages, and 100 diseased controls with gastrointestinal symptoms other than diarrhea. Every sample was subjected to four diagnostic approaches in parallel: light microscopy after concentration, conventional PCR targeting the small subunit ribosomal RNA gene, real-time PCR with SYBR Green detection and melt curve analysis, and real-time LAMP run on a portable fluorometer. A subset of positive samples was then sequenced and subjected to phylogenetic analysis to confirm species identification.

The real-time LAMP assay was designed around two genetic targets. The first, the serine-rich Entamoeba histolytica protein gene (SREHP), is specific to E. histolytica. The second, the large subunit ribosomal RNA gene (LSU rRNA), is shared across the three Entamoeba species of interest. Because the annealing temperatures of the two amplicons were too close to allow reliable differentiation in a single duplex reaction, the team ran the two primer sets as parallel monoplex reactions in the same run—two tubes per sample. The logic of the readout is elegant: samples positive for E. histolytica amplify both targets and produce two fluorescent peaks, while E. dispar and E. moshkovskii amplify only the LSU rRNA target and yield a single peak. The reaction runs isothermally at 63 degrees Celsius for 60 minutes, eliminating the need for the repeated thermal cycling that PCR demands.

The performance figures are striking. Using Bayesian latent class analysis—a statistical framework that accounts for the fact that no single test is a perfect gold standard—the researchers calculated sensitivity of 39.4 percent for microscopy, 84.3 percent for conventional PCR, and 99.3 percent for both real-time PCR and real-time LAMP. Specificity for real-time LAMP ranged from 99.4 to 100 percent. Five samples that conventional PCR missed were successfully detected by both real-time methods, likely because PCR inhibitors present in some fecal samples suppressed the conventional reaction. The limit of detection told a similar story: conventional PCR could detect 1 nanogram of parasite DNA, real-time PCR reached 1 picogram, and real-time LAMP pushed down to 10 femtograms—the most sensitive of the three molecular techniques.

Turnaround time is where LAMP pulls decisively ahead. Real-time LAMP delivered results in about 60 minutes, compared with roughly 120 minutes for real-time PCR and 200 minutes for conventional PCR. The amplification is carried out by Bst DNA polymerase using outer and inner primers in a strand-displacement reaction that proceeds at a single constant temperature, meaning the assay can run on simple equipment—a water bath, a heat block, or the battery-powered portable fluorometer used in this study. The closed-tube format minimizes the risk of aerosol contamination from amplified DNA, and minimal post-amplification processing makes the workflow suitable for busy, resource-limited laboratories. These are precisely the conditions found in tertiary care hospitals across low- and middle-income countries, where patient load is high and molecular infrastructure may be scarce.

The clinical findings carry their own weight. Overall, 33 of 494 samples—6.7 percent—tested positive for Entamoeba by the real-time methods: 23 with E. histolytica, 9 with E. dispar, and 1 with E. moshkovskii. Among the 117 UC patients, 20 were positive, 17 of them with E. histolytica. Notably, patients with active UC had a higher incidence of E. histolytica infection—13 of 88—than those in remission, at 4 of 29. E. histolytica rates in the UC group were significantly higher than in the control groups (p<0.001), though the difference between active disease and remission did not reach statistical significance (p=0.898). Microscopy, meanwhile, identified cysts or trophozoites in only 15 samples and missed 20 of the 33 molecularly confirmed cases, underscoring how much the field loses when it relies on the microscope alone.

The authors are careful about the boundaries of their conclusions. With only 33 positive cases, the similar diagnostic sensitivity of real-time LAMP and real-time PCR may reflect limited statistical power, and larger multicentric studies are needed to confirm the equivalence. The parallel monoplex format, while necessary for accurate species differentiation with the available master mix chemistry, doubles reagent consumption per sample; the researchers suggest that the instrument’s dual-channel fluorescence system with sequence-specific probes could enable single-tube multiplexing in future iterations. The study was cross-sectional and diagnostic in design, so it could not establish whether E. histolytica infection drives UC disease activity or merely coexists with it, nor could it assess the effect of anti-amoebic treatment on UC outcomes. Cost-effectiveness analysis was not performed, and the authors flag it as a priority for further work.

Even with these caveats, the implications for clinical practice are concrete. The findings support considering molecular testing in microscopy-negative cases, particularly in high-incidence areas and among high-risk patients such as those with ulcerative colitis experiencing diarrhea exacerbations. Before escalating immunosuppressive therapy, the data suggest, clinicians in endemic regions should have access to a rapid, sensitive test that can distinguish a pathogenic invader from a harmless commensal. Real-time LAMP, with its hour-long turnaround, portable hardware, and PCR-equivalent accuracy, offers a plausible route to that standard—one that could reshape how amoebic infection is ruled out in the populations least equipped to wait for an answer.

Subject of Research: Evaluation of real-time LAMP for detecting and differentiating Entamoeba species in stool samples from ulcerative colitis patients and controls in North India

Article Title: Evaluation of real-time LAMP assay for detection of Entamoeba species in clinical stool samples from a tertiary care center in North India

Article References: Sharma, D., Dutta, U., Rattan, D., Kaur, U., Inoue, S., Hamano, S., Jain, R., Sandha, K. S., Sehgal, R., & Lakshmi, P. V. M. (2026). Evaluation of real-time LAMP assay for detection of Entamoeba species in clinical stool samples from a tertiary care center in North India. Frontiers in Parasitology, 5, Article 1937802. https://doi.org/10.3389/fpara.2026.1937802

Image Credits: AI Generated

DOI: 10.3389/fpara.2026.1937802

Keywords: Entamoeba histolytica, real-time LAMP, molecular diagnostics, ulcerative colitis, amoebiasis, stool testing, parasitology, India, diagnostic sensitivity, inflammatory bowel disease, isothermal amplification, Entamoeba dispar