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There is significant heterogeneity in testing strategies used for Clostridium difficile infection (CDI) in Quebec, Canada.Molecular assays are increasingly used as single tests and in multistep algorithms.There are significant associations between CDI incidence rates and diagnostic tests.Fragmentation and confounders prevent retrospective adjustment of surveillance data.This study highlights potential threats to the validity of CDI surveillance programs.Several Clostridium difficile infection (CDI) surveillance programs do not specify laboratory strategies to use. We investigated the evolution in testing strategies used across Quebec, Canada, and its association with incidence rates.Cross-sectional study of 95 hospitals by surveys conducted in 2010 and in 2013-2014. The association between testing strategies and institutional CDI incidence rates was analyzed via multivariate Poisson regressions.The most common assays in 2014 were toxin A/B enzyme immunoassays (EIAs) (61 institutions, 64%), glutamate dehydrogenase (GDH) EIAs (51 institutions, 53.7%), and nucleic acid amplification tests (NAATs) (34 institutions, 35.8%). The most frequent algorithm was a single-step NAAT (20 institutions, 21%). Between 2010 and 2014, 35 institutions (37%) modified their algorithm. Institutions detecting toxigenic C difficile instead of C difficile toxin increased from 14 to 37 (P < .001). Institutions detecting toxigenic C difficile had higher CDI rates (7.9 vs 6.6 per 10,000 patient days; P = .01). Institutions using single-step NAATs, GDH plus toxigenic cultures, and GDH plus cytotoxicity assays had higher CDI rates than those using an EIA-based algorithm (P < .05).Laboratory detection of CDI has changed since 2010. There is an association between diagnostic algorithms and CDI incidence. Mitigation strategies are warranted.