Development and evaluation of a unique molecular assay for both infective endocarditis diagnosis and antimicrobial susceptibility testing

Abstract

Infective Endocarditis (IE) is one of the main causes of mitral and aortic insufficiency. IE is a disease with a high mortality rate which causes damages to both endocardium and devices attached to the heart, such as prosthetics valves. The increased incidence of IE is associated with health elevated costs and prolonged hospitalization. Despite the advances in recent years, there are still some limitations that prevent a rapid, sensitive and specific diagnosis of IE diagnosis. Therefore, IE requires still the development of a tool of diagnosis with accuracy and speed, since the correct choice of antibiotic therapy and surgical indication depend on the etiologic agent. The aim of the present study is to develop a unique molecular method that allows a rapid identification of both the etiologic agent and the antimicrobial resistance genes of IE. The patients admitted to Dante Pazzanese Institute of Cardiology with clinical suspicion for IE will be included in the study. They will be divided into two groups (IE and controls) according to both blood culture and echocardiography results. Peripheral blood and tissue samples (from patients undergoing surgical heart valve replacement) will be used for classical and molecular diagnosis of IE. The tissues samples will be sent to the laboratory of molecular biology in vials containing the RNAlater® for immediate DNA stabilization. DNA will be extracted from peripheral blood and valve tissue samples using QIAamp DNA Mini Kit ®. The molecular identification of etiologic agent and the antimicrobial susceptibility test will be performed by amplification of DNA sequences coding for both 16S ribosomal RNA (16S rRNA) and molecules that confer antimicrobial drugs resistance, respectively. The amplification will be performed by Real-Time PCR using the Custom Microbial DNA qPCR Arrays and RotorGene Q® platform. With the results of this study, we hope to develop a fast and accurate diagnostic tool which will be useful for early identification of IE etiologic agents, including those that are responsible for high rates of negative blood culture endocarditis; In addition, the detection of antimicrobial resistant genes will contribute to the best drug terapy choice and thus to avoid the prolongation of both drug therapy and hospitalization as well as the unnecessary exposure of patients to toxic drugs, and also will contribute to the reduction of number of relapse IE cases. Thus, it is expected that this study results will improve the life quality of IE patient. (AU)