Background: Cholera surveillance relies on clinical diagnosis of acute watery diarrhea. Suspected cholera case definitions have high sensitivity but low specificity, challenging our ability to characterize cholera burden and epidemiology. Our objective was to estimate the proportion of clinically suspected cholera that are true Vibrio cholerae infections and identify factors that explain variation in positivity. Methods and findings: We conducted a systematic review of studies that tested ≥10 suspected cholera cases for V. cholerae O1/O139 using culture, PCR, and/or a rapid diagnostic test. We searched PubMed, Embase, Scopus, and Google Scholar for studies that sampled at least one suspected case between January 1, 2000 and April 19, 2023, to reflect contemporary patterns in V. cholerae positivity. We estimated diagnostic test sensitivity and specificity using a latent class meta-analysis. We estimated V. cholerae positivity using a random-effects meta-analysis, adjusting for test performance. We included 119 studies from 30 countries. V. cholerae positivity was lower in studies with representative sampling and in studies that set minimum ages in suspected case definitions. After adjusting for test performance, on average, 52% (95% credible interval (CrI): 24%, 80%) of suspected cases represented true V. cholerae infections. After adjusting for test performance and study methodology, the odds of a suspected case having a true infection were 5.71 (odds ratio 95% CrI: 1.53, 15.43) times higher when surveillance was initiated in response to an outbreak than in non-outbreak settings. Variation across studies was high, and a limitation of our approach was that we were unable to explain all the heterogeneity with study-level attributes, including diagnostic test used, setting, and case definitions. Conclusions: In this study, we found that burden estimates based on suspected cases alone may overestimate the incidence of medically attended cholera by 2-fold. However, accounting for cases missed by traditional clinical surveillance is key to unbiased cholera burden estimates. Given the substantial variability in positivity between settings, extrapolations from suspected to confirmed cases, which is necessary to estimate cholera incidence rates without exhaustive testing, should be based on local data. Using combined data extracted from 119 studies from 30 countries, Kirsten E. Wiens and colleagues estimate the true burden Vibrio Cholerae positivity. Author summary: Why was this study done?: Cholera surveillance typically relies on the clinical diagnosis of acute watery diarrhea (i.e., "suspected cholera"), but this definition has a low specificity for cholera. Our goal was to estimate the proportion of suspected cholera cases that are true Vibrio cholerae infections and identify factors that contribute to variation in observed positivity. What did the researchers do and find?: We conducted a systematic review of studies from 2000 to 2023 that tested suspected cholera cases for V. cholerae infection using one of 3 different laboratory tests. We included 119 studies from 30 countries and found that, on average, half of suspected cholera cases represented true V. cholerae infections, after accounting for laboratory test accuracy. We also found high variability between studies and that the odds of a suspected case being a true infection were higher during outbreaks compared to non-outbreak settings. What do these findings mean?: Our findings suggest that burden estimates based solely on suspected cases may overestimate the incidence of medically attended cholera by 2-fold. The high variability across studies suggests also that local testing data should be used to inform assumptions about positivity when exhaustive testing is not feasible. A limitation of our approach was that we could not account for cases missed by clinical surveillance, which is crucial for unbiased overall cholera burden estimates and an important area for future work. [ABSTRACT FROM AUTHOR]