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Development of a rapid and sensitive method for the isolation and diagnosis of microorganisms in human blood

Abstract

Blood stream infections (BSI) are severe diseases causing high morbidity and mortality and are a major public health problem worldwide. The effective diagnosis of the microorganisms causing BSI is the most critical and most difficult problem in the treatment of blood infections. Currently, the gold standard for diagnosis is blood culture, however there are a number of disadvantages associated with this method including the fact that it is time consuming (up to 5 days for results) and has low sensitivity. These issues are associated with a delay in administration of the first adequate anti-infectious agent, resulting in a mortality rate as high as 60%.The use of molecular detection techniques for BSI diagnosis offers a number of advantages over the traditional blood culture method. Molecular methods are much more rapid, require a smaller volume for blood collection, and allow for increased sensitivity. Some BSI diagnostic methods based on molecular detection techniques such as PCR have been developed, however most of these assays isolate the DNA template from a positive blood culture, thus still requiring time for pathogen growth. Therefore, molecular techniques applied directly to whole blood samples offer the best option for rapid diagnosis. Recently some methods have been developed that rely on direct lysis of the pathogen in the blood sample, extraction of the DNA template, PCR amplification and pathogen identification. While there has been some limited success with these methods, there are still issues associated with sensitivity. Therefore, a method for the rapid and sensitive diagnosis of the microorganisms causing BSI is still needed. The most critical step is the purification of microbial nucleic acids from blood. The quantity of microbes present in blood during BSIs ranges from 1 CFU/mL to 1 x 104 CFU/mL; therefore the DNA isolation method used for molecular diagnosis must be sensitive enough to isolate from low amounts. Rapid and sensitive diagnosis of the pathogens causing blood stream infections (BSI) is critical. In this project, a novel method to detect microorganisms in blood for BSI diagnosis will be developed. First, a sensitive kit will be developed to isolate DNA from all the microorganisms present in blood. Panels will then be made to allow for the detection of the most common microorganisms causing BSI found in Brazil using real-time PCR technology, as well as to screen for antibiotic resistance. (AU)

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