Assembly and performance of a cholera RDT prototype that detects both Vibrio cholerae and associated bacteriophage as a proxy for pathogen detection

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.

Keywords: Bangladesh; ICP1; RDT; Vibrio cholerae; bacteriophage; cholera; diarrhea; phage; rapid diagnostic test; vibriophage.

FIG 1

IgG antibody responses by ELISA in hybridoma clone culture supernatants derived from mice immunized with recombinant proteins: ICP1 tail fiber ORF69 (A), tail fiber ORF93 (B), and head decoration protein ORF123 (C). X-axis represents the antigens assayed; VCWC, formalin-killed V. cholerae whole cell; BSA, bovine serum albumin. Y-axis is the absorbance at 450 nm read in SYNERGY Mx (BioTek) plate reader.

FIG 2

Testing RDT prototype for ICP1 detection using different boiling periods at a 1:10 dilution of high titer ICP1 (1 × 10⁹ PFU/mL) in 0.5× PBS (A), and then with dilutions ranging from 1:20 (5 × 10⁷ PFU/mL) to 1:500 (2 × 10⁶ PFU/mL) after 10 min boil (B). C, control line; T, test line; -ve, negative result; +ve, positive result. Red lines indicate positive control or test line. Positive C line ensures the validity of the RDT prototype result.

FIG 3

Evaluation of the RDT prototype for ICP1 detection in spiked diarrheal stools (EN70, EN105, and EN122). High-titer ICP1 (1 × 10⁹ PFU/mL) was spiked at 1:10 dilution and boiled at 95°C for 10, 20, and 30 min. C, control line; T, test line; -ve, negative result (none); and +ve, positive result (all). Red lines indicate positive control or test line. Positive C line ensures the validity of the RDT prototype result.

FIG 4

Electron micrographs of the ICP1 preparation (1 × 10⁹ PFU/mL) at a 1:10 dilution without boiling (A,B), with 10 min boiling (C,D), and with 20 min boiling (E,F) after immunolabeling with ICP1ORF122 mAb. Scale bars are embedded in images (A,C,E); paired images with increased resolution shown in lower row (B,D,F).