Evaluation of real-time LAMP assay for detection of Entamoeba species in clinical stool samples from a tertiary care center in North India
8 affiliations
- 1Department of Medical Parasitology, Post Graduate Institute of Medical Education and Research, Chandigarh, India
- 2Department of Gastroenterology, Post Graduate Institute of Medical Education and Research, Chandigarh, India
- 3Amity School of Biological Sciences, Amity University Punjab, SAS Nagar, India
- 4Department of Immunology, Nagasaki University, Nagasaki, Japan
- 5Department of Parasitology, Institute of Tropical Medicine (NEKKEN), Nagasaki University, Nagasaki, Japan & The Joint Usage/Research Center on Tropical Disease, Institute of Tropical Medicine (NEKKEN), Nagasaki University, Nagasaki, Japan
- 6Department of Microbiology, Civil Hospital, Manimajra, India
- 7Department of Microbiology, Aarupadai Veedu Medical College and Hospital, Pondicherry, India
- 8Department of Public Health and Community Medicine, PGIMER, Chandigarh, India
Abstract
Background: Enteric protozoa infections contribute to diarrheal diseases in low and middle income countries, affecting those with limited access to clean water and sanitation. Entamoeba histolytica is an invasive water and food-borne parasite, associated with mucosal inflammation causing symptoms of amoebic colitis that can overlap with ulcerative colitis (UC). As conventional diagnostic methods such as microscopy cannot differentiate morphologically similar species, diagnostic challenges frequently arise. The present prospective study evaluated the clinical diagnostic performance of Real-Time Loop Mediated Isothermal Amplification Assay (LAMP) for detecting and differentiating Entamoeba species in clinical endemic settings, with a focus on patients with UC.
Methods: The study was conducted at a tertiary care hospital in North India from January 2021 to January 2023. Stool samples (n=494) were collected across four groups: patients with UC (n=117), non-inflammatory bowel disease (IBD) diarrhea patients (n=177), community participants (n=100), and diseased controls (n=100). 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.
Results: 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).
Interpretation: 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.
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BibTeX
@article{sharma2026evaluation,
title = {{Evaluation of real-time LAMP assay for detection of Entamoeba species in clinical stool samples from a tertiary care center in North India}},
author = {Devyani Sharma and Usha Dutta and Divya Rattan and Upninder Kaur and Shinichi Inoue and Shinjiro Hamano and Ruby Jain and Kawaljeet Singh Sandha and Rakesh Sehgal and P. V. M. Lakshmi},
journal = {Frontiers in Parasitology},
year = {2026},
month = oct,
doi = {10.3389/fpara.2026.1937802},
url = {https://doi.org/10.3389/fpara.2026.1937802},
note = {CC BY 4.0},
}
RIS
TY - JOUR
TI - Evaluation of real-time LAMP assay for detection of Entamoeba species in clinical stool samples from a tertiary care center in North India
AU - Devyani Sharma
AU - Usha Dutta
AU - Divya Rattan
AU - Upninder Kaur
AU - Shinichi Inoue
AU - Shinjiro Hamano
AU - Ruby Jain
AU - Kawaljeet Singh Sandha
AU - Rakesh Sehgal
AU - P. V. M. Lakshmi
PY - 2026
DA - 2026/10/01
JO - Frontiers in Parasitology
DO - 10.3389/fpara.2026.1937802
UR - https://doi.org/10.3389/fpara.2026.1937802
AB - Background: Enteric protozoa infections contribute to diarrheal diseases in low and middle income countries, affecting those with limited access to clean water and sanitation. Entamoeba histolytica is an invasive water and food-borne parasite, associated with mucosal inflammation causing symptoms of amoebic colitis that can overlap with ulcerative colitis (UC). As conventional diagnostic methods such as microscopy cannot differentiate morphologically similar species, diagnostic challenges frequently arise. The present prospective study evaluated the clinical diagnostic performance of Real-Time Loop Mediated Isothermal Amplification Assay (LAMP) for detecting and differentiating Entamoeba species in clinical endemic settings, with a focus on patients with UC. Methods: The study was conducted at a tertiary care hospital in North India from January 2021 to January 2023. Stool samples (n=494) were collected across four groups: patients with UC (n=117), non-inflammatory bowel disease (IBD) diarrhea patients (n=177), community participants (n=100), and diseased controls (n=100). 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. Results: 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). Interpretation: 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.
SN - 2813-2424
ER -