Rounding out her junior year at the University of Florida College of Agricultural and Life Sciences, Michelle Mu celebrated an exciting day with her team as the announcers called her name for the undergraduate research poster competition.
Emerging Pathogens Institute Research Day 2025 differed from the institute’s past events by including a poster competition for undergraduate students. Mu and the international work of other lab members set the standard for collaborative research, shining the spotlight on a terribly unpleasant pathogen: Vibrio cholerae.
Cholera is a diarrheal disease caused by the Gram-negative waterborne pathogen Vibrio cholerae. It infects the intestines, causing aggressive symptoms, such as vomiting, severe diarrhea, dehydration and death. You can get cholera from drinking water or eating food containing bacteria. Globally, there are 1.3 to 4 million reported cases annually, resulting in 21,000 to 143,000 deaths.
Current methods of detecting V. cholerae include polymerase chain reaction (PCR) tests, quantitative PCR (qPCR) tests and traditional culture tests in which scientists attempt to grow the bacterium on a Petri dish. PCR-based approaches offer faster, more sensitive and specific results than culturing the bacterium. However, Mu and the team wanted to observe another detection mechanism: bacteriophages. They also sought to compare the associated costs of these various tests.
“There are different things that can limit the effectiveness of PCR when we’re looking for bacteria like Vibrio cholerae, and one of those things … is the presence of bacteriophages,” Mu said when asked about the query behind this study. “What we hypothesized is that we can use phage detection as a proxy for diagnosing cholera.”
If the phages are present, they must be attacking their respective bacteria. Combined with the bacteriophages as a proxy, the pathogen essentially tells on itself without realizing it. Current testing methods before this study are effective for detecting V. cholerae, but this research shines in its potential to detect trickier pathogens that evade PCR and qPCR tests.
The study included over 2,500 stool samples from patients admitted with diarrheal disease in Bangladesh. When asked about the daunting size of the data pool, Mu reminded us of what many forget in the world of infectious disease research: you’re not only working to study the disease, but also to treat those participating in the study.
“Every time we want to increase [the sample size], … we have to think about ‘Okay, but does this increase in sample size justify the burden that we’re putting on the participants and asking them to participate in this study?’”
The study also found that, although qPCR and nano-liter qPCR (nl-qPCR) require more complex machinery, they offer lower costs per sample — a bonus for labs already possessing the machinery required for these kinds of tests. These findings encourage a new player in detecting V. cholerae infections, which can act quickly once in our system.
“I think this research can definitely improve public health efforts, because by improving our ability to diagnose and detect cholera, we’re better able to understand the burdens of diseases that are being posed on a population at a certain time,” said Mu. “It can also help us, in the future, stay ahead if we are able to catch cholera outbreaks early.”
Comparison of molecular approaches to detect virulent bacteriophage as a proxy for V. cholerae
Collaborators
- Michelle Mu – Department of Pediatrics and Department of Environmental and Global Health, Emerging Pathogens Institute, University of Florida
- Ishae Sriguha – Department of Pediatrics and Department of Environmental and Global Health, Emerging Pathogens Institute, University of Florida
- Sharia Ahmed – Department of Epidemiology, Emory University
- Md Abu Sayeed – Department of Pediatrics and Department of Environmental and Global Health, Emerging Pathogens Institute, University of Florida
- Emilee Cato – Department of Pediatrics and Department of Environmental and Global Health, Emerging Pathogens Institute, University of Florida
- Ashton Creasy-Marrazzo – Department of Pediatrics and Department of Environmental and Global Health, Emerging Pathogens Institute, University of Florida
- Firdausi Qadri – Infectious Diseases Division, International Centre for Diarrhoeal Disease Research
- B. Jesse Shapiro – Department of Microbiology & Immunology, McGill University
- Ashraful Islam Khan – Infectious Diseases Division, International Centre for Diarrhoeal Disease Research
- Eric Nelson – Department of Pediatrics and Department of Environmental and Global Health, Emerging Pathogens Institute, University of Florida
Introduction
Cholera is a diarrheal disease caused by the Gram-negative water-borne pathogen Vibrio cholerae. Globally, there are 1.3-4 million reported cases annually resulting in 21,000-143,000 deaths. Methods to detect V. cholerae and related enteric pathogens include culture, polymerase chain reaction (PCR) and quantitative PCR (qPCR). Nano-liter qPCR (nl-qPCR) is an alternative to qPCR that utilizes a high-throughput platform of nanofluidic volumes and cuts costs six-fold. PCR-based molecular approaches offer faster, more sensitive and more specific results than culture. The primary objective of this study was to compare different modes of molecular detection of V. cholerae and its associated virulent bacteriophages (phages). The secondary objective was to compare associated costs.
Methods
We conducted three different molecular detection assays (PCR, qPCR, nl-qPCR) on 2574 stool samples collected from patients admitted with diarrheal disease in Bangladesh. Targets included V. cholerae and its phages (ICP1/2/3). To evaluate the pairwise agreement, we used Cohen’s kappa. We conducted McNemar’s test to compare qualitative results. Associated costs and comparisons were analyzed based on a per reaction cost (cpr); cost of machinery was excluded.
Results
Comparison between qPCR and nl-qPCR showed strong agreement for tcpA (for V. cholerae, κ=0.886) and moderate agreement for ICP1 (κ=0.767), ICP2 (κ=0.768), and ICP3 (κ=0.597). Three-way analysis among PCR, qPCR, and nl-qPCR showed strong agreement for V. cholerae (κ=0.785) and moderate agreement for ICP1 (κ=0.609), ICP2 (κ=0.593), and ICP3 (κ=0.533). McNemar’s test showed evidence of statistically significant differences in the marginal probabilities of a positive detection by qPCR and nl-qPCR for the targets tcpA and ICP3, but no significant evidence for differences in detection of ICP1 or ICP2. With respect to cost, PCR was most expensive ($1.31 cpr) compared to qPCR ($1.05 cpr) and nl-qPCR ($0.004 cpr); cost attribution for PCR was both the gel matrix and reaction mixture.
Conclusions
The different molecular approaches taken herein showed better congruence for V. cholerae compared to detection of associated phages. Although qPCR and nl-qPCR require more complex machinery, they offer lower costs per sample, increasing scalability when the machinery is pre-existing. These results support both qPCR and nl-qPCR in diagnostic approaches for detecting V. cholerae, as well as its phages as a proxy for pathogen detection. These results may have impact on related diseases that are less tractable to study.