Molecular and Microbiologic Diagnosis of Bacterial Endocarditis
Sequencing of 16S rDNA, and of sodA_int and rpoB_int in some cases, was applied to DNA from heart valves of 46 patients (36 with definite and 10 with possible endocarditis). Sequence-based identifications were compared with those obtained with conventional methods. Among the 36 definite cases, 30 had positive blood cultures and 6 had negative cultures. Among the 30 positive cases, sequencing of 16S rDNA permitted identification of species, genus, or neither; sodA_int and rpoB_int sequencing was necessary for species identification in 8 cases. Species identifications were identical in only 61.5%, when conventional techniques and DNA sequencing were used. In five of the six blood culture-negative endocarditis cases, sequencing identified Bartonella quintana,B. henselae, and Streptococcus gallolyticus. Our results demonstrate a clear benefit of molecular identification, particularly in cases of blood culture-negative endocarditis and of possible endocarditis, to confirm or invalidate the diagnosis. Moreover, in 19.4% of the definite cases, the improvement in species identification by sequencing led to improved patient management.

According to the earliest published report on the subject, the prevalence of blood culture-negative endocarditis once ranged from 2.5% to 31%. In more recent studies, approximately 9% is the reported rate. One explanation for the improvement in the bacteriologic diagnosis of endocarditis is better knowledge of its clinical symptoms and risk factors, which has encouraged earlier blood culture. Another reason is the improvement in bacterial culture techniques, with prolonged incubation times, presence of carbon dioxide, enriched culture media, and timed subcultures. Thus, the isolation of fastidious microorganisms including Abiotrophia (new genus Granulicatella) and the HACEK group, has improved dramatically, and organisms frequently missed with the use of earlier blood culture techniques are now recognized. Another improvement is the use of specific serologic tests for certain microorganisms. Such tests, associated with cell cultures, are now recommended for patients with blood culture-negative endocarditis for which Coxiella burnetii and Bartonella spp. are the suspected causative organisms. Despite these improvements, the diagnosis of blood culture-negative endocarditis remains a challenge. The absence of positive culture is most frequently due to previous antimicrobial drug treatment or to bacterial species that are difficult to grow or that remain nonculturable in the laboratory.

To overcome these problems, molecular techniques using broad-range DNA primers for amplification of bacterial 16S rDNA directly from clinical samples and subsequent nucleotide sequencing have been proposed to establish the infectious etiology. This approach, combined with sodA_int, encoding superoxide dismutase and rpoB_int, encoding the ß sub-unit of RNA polymerase sequencing when 16S rDNA sequences were not sufficiently discriminating, was used here 1) to evaluate the bacterial content of 46 resected heart valves from patients operated on for endocarditis, 2) to compare the results with bacteriologic and histologic findings from heart valves and from preceding blood cultures, and 3) to analyze the data with respect to the clinical background of the patients, including the modified Duke criteria.