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Candida spp. are increasingly important pathogens in neonatal intensive care units (NICU). Prior colonization is a major risk factor for candidemia, but few studies have focused on risk factors for colonization, particularly in NICU patients.A prospective, multicenter cohort study was performed in six NICUs to determine risk factors for Candida colonization. Infant gastrointestinal tracts were cultured on admission and weekly until NICU discharge and health care worker hands were cultured monthly for Candida spp.The prevalence of Candida spp. colonization was 23% (486 of 2157 infants); 299 (14%), 151 (7%) and 74 (3%) were colonized with Candida albicans, Candida parapsilosis and other Candida spp., respectively. Multiple logistic regression analysis adjusting for length of stay, birth weight ≤1000 g and gestational age <32 weeks revealed that use of third generation cephalosporins was associated with either C. albicans (155 incident cases) or C. parapsilosis (104 incident cases) colonization. Use of central venous catheters or intravenous lipids were risk factors for C. albicans, whereas delivery by cesarean section was protective. Use of H2 blockers was an independent risk factor for C. parapsilosis. Of 2989 cultures from health care workers’ hands, 150 (5%) were positive for C. albicans and 575 (19%) for C. parapsilosis, but carriage rates did not correlate with NICU site-specific rates for infant colonization.We speculate that NICU patients acquire Candida spp. , particularly C. parapsilosis, from the hands of health care workers. H2 blockers, third generation cephalosporins and delayed enteral feedings alter gastrointestinal tract ecology, thereby facilitating colonization.