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Updated: Jun 4, 2025
Laboratory Techniques Used to Maintain and Differentiate Biotypes of Vibrio cholerae Clinical and Environmental Isolates
Published on: May 30, 2017
Assembly and performance of a cholera RDT prototype that detects both Vibrio cholerae and associated bacteriophage as
Md Abu Sayeed1,2, Imrul Kayes Nabil3, Piyash Bhattacharjee3
1Department of Pediatrics, University of Florida, Gainesville, Florida, USA.
Abstract:
Cholera rapid diagnostic tests (RDTs) are vulnerable to virulent bacteriophage predation. We hypothesized that an enhanced cholera RDT that detects the common virulent bacteriophage ICP1 might serve as a proxy for pathogen detection. We previously developed a monoclonal antibody (mAb) to the ICP1 major capsid protein. Our objective was to design and assemble a first-of-its-kind RDT that detects both a bacterial pathogen (Vibrio cholerae) and associated virulent bacteriophage (ICP1). Candidate mAbs were expanded to increase design options and evaluated by immunological assays (ELISA; western blot). A subset of mAbs were selected for gold conjugation and printing on the RDT. The detection limit of the prototype RDTs was determined in diarrheal stools with the addition of ICP1. Three mAb candidates were developed and evaluated for the capsid decoration protein (ORF123) and tail fiber protein (ORF93), and the prior mAb for the major capsid protein (ORF122). A single mAb sandwich RDT prototype for ORF122 was able to detect ICP1; RDTs with mAbs to ORF123 and ORF93 failed to detect ICP1 in single- or dual-sandwich configurations. Biologically relevant concentrations for ICP1 were detected only after boiling the stool with ICP1; analysis by electron microscopy (EM) suggested increased epitope availability after boiling. In this study, we demonstrate a proof of concept for a functional RDT that can detect both the primary pathogen and a common virulent bacteriophage as a proxy for pathogen detection. Further optimization is required before scaled production and implementation.IMPORTANCEThis paper represents an important step forward to address the vulnerability of cholera RDTs to the effects of phage predation on the target Vibrio cholerae. The assembly and evaluation of an RDT that detects both the primary pathogen and a phage as a proxy for the primary pathogen is an innovative solution. When optimized and evaluated in clinical studies, this tool may become critical in the cholera response tool kit as well as represent a diagnostic proof-of-concept for other infectious agents.
Insights
This study developed an enhanced cholera rapid diagnostic test (RDT) that detects both Vibrio cholerae bacteria and the ICP1 bacteriophage. This innovative RDT aims to overcome RDT vulnerabilities to phage predation for improved cholera detection.
Area of Science:
- Infectious disease diagnostics
- Microbiology
- Immunological assays
Background:
- Cholera rapid diagnostic tests (RDTs) are susceptible to bacterial predation by virulent bacteriophages.
- Virulent bacteriophages can compromise the accuracy of standard cholera diagnostics.
- Detecting bacteriophages may serve as an indirect indicator of bacterial presence.
Purpose of the Study:
- To design and assemble a novel RDT capable of detecting both *Vibrio cholerae* and the ICP1 bacteriophage.
- To evaluate the efficacy of monoclonal antibodies (mAbs) targeting different bacteriophage proteins for RDT development.
- To establish a proof-of-concept for a dual-detection RDT addressing phage predation vulnerability.
Main Methods:
- Development and expansion of candidate monoclonal antibodies (mAbs) against ICP1 bacteriophage proteins (major capsid protein ORF122, ORF123, ORF93).
- Evaluation of mAbs using immunological assays including ELISA and western blotting.
- Assembly of prototype RDTs using gold-conjugated mAbs and determination of detection limits in spiked diarrheal stool samples, including heat treatment.
Main Results:
- A single mAb sandwich RDT prototype targeting the ICP1 major capsid protein (ORF122) successfully detected ICP1.
- RDTs utilizing mAbs against other ICP1 proteins (ORF123, ORF93) failed to detect the bacteriophage.
- Optimal detection of ICP1 in stool samples required boiling, which likely enhanced epitope accessibility as suggested by electron microscopy.
Conclusions:
- A proof-of-concept RDT has been successfully developed to detect both the cholera pathogen and a common virulent bacteriophage.
- This dual-detection RDT offers a potential solution to overcome the limitations of current cholera RDTs vulnerable to phage predation.
- Further optimization and clinical validation are necessary for the scaled production and implementation of this innovative diagnostic tool.
