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Traditionally, infective endocarditis (IE) has been microbiologically diagnosed by blood cultures or serology. However, conventional microbiologic methods do not always provide an etiologic diagnosis. We conducted the current study to evaluate the usefulness of a universal real-time polymerase chain reaction (PCR) of the 16S rRNA gene followed by sequencing for the diagnosis of IE in explanted heart valve tissue (HV) as part of the routine of a clinical microbiology laboratory, and to compare it with conventional culture of blood or HV.We prospectively analyzed 177 HV samples by universal PCR and sequencing: 48 were from 35 patients with definite IE and 129 were from 120 patients without IE. Specific PCR tests were used when necessary to confirm broad-range PCR results.For the 35 patients with IE, all of the HV samples except for 2 from the same patient gave positive PCR results. The microorganisms identified matched those isolated by blood culture in 31 cases. The other 3 patients had negative blood culture IE, but PCR made possible the detection of Tropheryma whipplei, Bartonella quintana, and Streptococcus gallolyticus. For the negative control group, universal PCR was completely negative in 123 of the 129 samples. Sensitivity, specificity, and negative and positive predictive values of this real-time PCR method were 96%, 95.3%, 98.4%, and 88.5%, respectively, for the diagnosis of IE, using the Duke criteria to define IE and using blood culture results to identify etiologic microorganisms. Conventional HV culture correlated poorly with blood cultures and molecular techniques, and frequently represented tissue contamination resulting from valve handling.Our universal PCR method has proved to be more sensitive, specific, and rapid than conventional culture methods, and should therefore be included as a new major Duke criterion for the diagnosis of IE. According to the results of the current study, this technique should be used to supplement blood and HV culture. Conventional HV cultures are frequently responsible for false-positive and false-negative results, and are not always useful to establish the etiology of IE.