S01.KL2 NEXT GENERATION MOLECULAR DIAGNOSTICS FOR ORTHOPEDIC INFECTIONS

Abstract

First generation molecular diagnostics based on PCR suggested that the routine culture of bacteria was inadequate for the detection of many pathogens, particularly after antibiotic treatment or when associated with chronic infection and biofilm growth. These techniques, however, suffered from their own problems. False negative results were caused by inhibitors of the PCR process and by the overly specific nature of most simplex assays which require an a priori assumption on the part of the investigator as to which species to test for. False positives resulted from contamination, or carryover, of amplified DNA. Recently several new technologies have been developed and have resulted in “next generation” tests that overcome the problems associated with the earlier methods. We will provide an overview of two of these technologies and present our experience in their application to the diagnosis of orthopedic infections associated with arthroplasties and external fixations. 454-based deep 16S rDNA sequencing provides for a comprehensive and quantitative analysis of all bacterial species present in clinical specimens regardless of whether the species present have been previously identified. The results of this test can be used to improve the specificity of other tests such as the Ibis Universal Biosensor. The Ibis Universal Biosensor T-5000 system uses a highly multiplex PCR front end which is coupled to a highly sensitive electron spray ionization (ESI) time-of-flight (TOF) mass spectrometer (MS) which provides for the exact base composition of the amplified DNA permitting species and even strain-specific identification of bacterial and fungal pathogens through an interface with a massive DNA sequence database. This system therefore provides both great breadth of coverage, with exquisite specificity. Moreover, this system can identify multiple species within a specimen providing a rapid analysis of polymicrobial infections.