dRapid and accurate detection of Shiga toxin-producing Escherichia coli (STEC) of all serotypes from patients with diarrhea is critical for medical management and for the prevention of ongoing transmission. In this prospective study, we assessed the performance of a multiplex, real-time PCR assay targeting stx 1 and stx 2 for the detection of O157 and non-O157 STEC in diarrheal stool samples enriched in Gram-negative broth. We show that the assay is 100% sensitive (95% confidence interval [CI], 89.1% to 100%) and 98.5% specific (95% CI, 90.6% to 99.9%) based on a panel of 40 known STEC-positive specimens and 65 known negative specimens. During a 2-year postvalidation period, the assay detected more positive samples from patients in northern California than did culture and PCR testing performed at a public health reference laboratory, with a positive predictive value of 95.6% (95% CI, 87.6% to 99.1%). Serotyping data showed an incidence rate of 51.2% for non-O157 STEC strains, with 5.8% of patients (1/17) with non-O157 strains and 42.9% (6/14) with O157 strains (P ‫؍‬ 0.03) developing hemolytic-uremic syndrome. The findings from this study underscore the recommendations of the CDC for laboratories to test all diarrheal stool samples from patients with acute community-acquired diarrhea for non-O157 STEC in addition to the O157 serotype by using a sensitive assay. Additionally, a survey of 17 clinical laboratories in northern California demonstrated that nearly 50% did not screen all stool specimens for the presence of Shiga toxins, indicating that many clinical microbiology laboratories still do not routinely screen all stool specimens for the presence of Shiga toxins as recommended in the 2009 CDC guidelines. Shiga toxin-producing Escherichia coli (STEC) infections are a major cause of bacterial gastroenteritis throughout the world and can be complicated by hemorrhagic colitis and hemolyticuremic syndrome (HUS) (1, 2). Although previously the majority of reported cases were attributed to E. coli O157:H7 and many recent studies indicate higher incidence rates of HUS associated with E. coli O157:H7 (3, 4), non-O157 STEC serotypes are emerging as important etiological agents of both sporadic cases and community outbreaks of diarrhea (3-8). Most notably, in May and June 2011, a large outbreak of E. coli O104:H4-associated diarrheal illness in Germany led to HUS in over 800 patients, many of whom were adults, and ultimately resulted in 54 deaths (5, 9). This outbreak and others have underscored the fact that severe disease and HUS can occur in cases related to non-O157 or O157 E. coli strains and that adults as well as children are susceptible to these complications (8-10). Timely laboratory identification of STEC has important implications for outbreak containment and patient management, including prompt parenteral hydration, monitoring for development of HUS, and avoidance of antibiotics and antidiarrheal agents, which can exacerbate disease (1). Importantly, a number of studies have shown that STECrelated illnesses...