Molecular Methods to Detect Vibrio cholerae and Associated Bacteriophages among Diarrheal Patients in Bangladesh

Molecular diagnostics to detect Vibrio cholerae (Vc) may be negatively impacted by pathogen-specific lytic bacteriophage (phage) predation. To address this problem, phage detection as a proxy for pathogen detection has been proposed. However, efforts to modernize cholera diagnostics with molecular tools require addressing knowledge gaps on best practices to detect Vc and associated bacteriophages. We conducted polymerase chain reaction (PCR), quantitative PCR (qPCR), and nano-liter (nl) qPCR targeting Vc and known phages (ICP1/2/3) on stool samples collected from patients admitted at hospitals across Bangladesh. Of 4,975 patients enrolled, 2,574 diarrheal samples were collected and over 65,000 reactions were conducted, including replicates. We analyzed the results for target-specific assay alignment and then used machine learning to determine the effect of phage predation on Vc-assay alignment. Standard curve analyses were used to set qPCR-positivity thresholds at 7.3x105 CFU/mL for Vc and 1.7x103, 9.3x103, and 3.0x105 PFU/mL for ICP1, ICP2, and ICP3, respectively. Among 2,462 samples assayed by qPCR, target detection was 25.3% (623), 7.8% (193), 0.5% (13), and 5.8% (144) for Vc, ICP1, ICP2, and ICP3, respectively. There was strong alignment between assays for Vc detection ({kappa}=0.785) and moderate alignment for phage detection ({kappa}=0.609, 0.593, and 0.533 for ICP1/2/3, respectively). Phages were ranked as the first (ICP1) and third (ICP3) effectors of Vc diagnostic alignment. These findings provide insights on how to prioritize molecular methods in the cholera field as well as related less tractable diseases facing similar diagnostic challenges. IMPORTANCEThis paper presents a comprehensive comparison of molecular methods to detect Vibrio cholerae (Vc) and associated bacteriophage (phage) which can be used as a proxy for pathogen detection. This initiative is an important step towards modernizing cholera diagnostics with molecular tools. In this study, we found that quantitative polymerase chain reaction (qPCR) represents a reasonable approach to detect Vc and associated phages balancing assay performance, cost, and accessibility. A key additional finding was that phage predation was found to be a leading factor that impacts the alignment of molecular methods to detect Vc. While we recommend qPCR be added to the cholera diagnostic toolkit, the effects of phage predation need to be accounted for in the development and evaluation of cholera diagnostics. These findings have applicability to less tractable disease where diagnostics share similar vulnerabilities.