PREreview del Development of Loop Mediated Isothermal Amplification for Rapid and Sensitive Detection of Enteroaggregative Escherichia coli (EAEC)

Development of Loop Mediated Isothermal Amplification for Rapid and Sensitive Detection of Enteroaggregative Escherichia coli (EAEC)

Summary

The preprint "Development of Loop Mediated Isothermal Amplification for Rapid and Sensitive Detection of Enteroaggregative Escherichia coli (EAEC)" presents a practical and cost-effective molecular diagnostic method for detecting EAEC, an important cause of diarrheal disease worldwide. In this study, the authors developed and optimized a LAMP assay targeting the aaic gene, which is a conserved genetic marker associated with EAEC. The researchers evaluated multiple reaction components, including MgSO₄, dNTP concentrations, Bst polymerase, and incubation temperature and duration. They then compared the performance of the assay to conventional PCR using 60 bacterial isolates that included EAEC strains, other diarrheagenic E. coli pathotypes, and several non–E. coli species. The results demonstrate exceptionally high diagnostic accuracy. The LAMP assay achieved 100% sensitivity and 100% specificity, and it displayed a substantially lower detection limit than PCR. The assay could detect genomic DNA at 0.098 pg per reaction and as few as 81 CFU/mL in bacterial cultures. These findings suggest that LAMP provides not only faster results but also superior analytical sensitivity compared to PCR. The manuscript is clearly written, well-organized, and provides extensive experimental detail. The authors effectively describe the public health need for rapid, low-cost diagnostic tools, particularly in settings where PCR is not feasible. Overall, the study positions this LAMP assay as a promising diagnostic option for both clinical laboratories and field-based detection.

Suggested Revisions

Although the manuscript is strong and well executed, several areas would benefit from additional clarification and expansion to improve clarity and reproducibility.

• Comment: The rationale for selecting the aaic gene as the assay target would be clearer if the authors briefly compared it to other commonly used EAEC markers, such as aggR, aaiA, or aap.

• Recommendation: The authors should clearly state why AAIC was chosen over these alternatives to help readers understand the comparative advantages of this gene for diagnostic purposes.

• Comment: The manuscript would benefit from a more detailed description of the primer design process. Although the authors state that they used online tools to generate and screen primers, they do not describe whether they assessed the potential for secondary structures, primer-dimer formation, cross-reactivity, or melting temperature mismatches.

• Recommendation: The authors should include information about in-silico verification, such as BLAST analysis, to confirm specificity against other bacterial genomes, to strengthen confidence in the assay's precision and reduce uncertainty about potential off-target amplification.

• Comment: Greater detail regarding sample selection would improve transparency. The study includes 60 isolates, but the distribution across pathogen types is uneven, with relatively few representatives of some E. coli pathotypes.

• Recommendation: The authors should explain how isolates were selected and acknowledge how this sample composition may influence estimates of sensitivity and specificity to provide readers with a more complete understanding of the study's limitations.

• Comment: The authors could enhance the discussion by describing the assay's robustness and real-world applicability. It would be helpful to know how sensitive the reaction is to temperature fluctuations, whether rapid or crude extraction methods could be used in place of full purification, and to what extent the assay is compatible with minimal equipment in field or point-of-care settings.

• Recommendation: The authors should expand the discussion to include these practical considerations to help readers envision the assay's real-world use and improve understanding of its field deployment potential.

• Comment: The section on statistical analysis would benefit from a brief interpretation of the reported metrics. While the authors present Cohen's kappa and confidence intervals, they do not explain why these metrics were selected or how to interpret a kappa value of 1.

• Recommendation: The authors should clarify why Cohen's kappa was selected as the primary statistical metric and explain how the small number of EAEC isolates affects the confidence intervals to help readers better assess the strength of the diagnostic claims.

• Comment: The discussion of LAMP performance in stool samples would be stronger if the authors provided more detailed explanations of how stool inhibitors affect amplification.

• Recommendation: The authors should describe the types of inhibitors commonly present in stool and strategies to mitigate their effects to improve readers' understanding of the assay's current readiness for clinical implementation. The authors should also discuss how these challenges compare to other stool-based LAMP assays to further contextualize the findings.

Overall Recommendation

Overall, this preprint offers a valuable and technically sound proof-of-concept for a LAMP-based diagnostic test capable of detecting EAEC rapidly and with exceptional sensitivity. The study demonstrates that LAMP can outperform PCR in both speed and analytical sensitivity, making it a promising tool for clinical diagnostics and for use in low-resource environments where conventional PCR is not feasible. The manuscript is clearly written, methodologically detailed, and addresses an important global health need. The suggested revisions relate primarily to expanding methodological transparency and strengthening the discussion around assay robustness and clinical applicability. These additions would not alter the central conclusions of the study but would enhance its clarity, reproducibility, and real-world relevance. With these minor revisions, the manuscript is well-positioned for publication and represents an important step toward accessible, field-deployable molecular diagnostics for enteric pathogens.

Competing interests

The author declares that they have no competing interests.

Use of Artificial Intelligence (AI)

The author declares that they used generative AI to come up with new ideas for their review.