Molecular techniques such as broad-range amplification of 16S rRNA gene sequences allow a culture independent approach for detection of infectious agents. Over the past decade this strategy has resulted in the recognition of a number of uncultured or difficult to culture pathogens. A variety of pitfalls need to be addressed before postulating a causal relationship between the nucleic acid sequence of an uncultured agent and a clinical disease. In principle, cultural procedures for recovery of bacterial pathogens are well developed and established. Several conditions may result in non recognition of an infectious pathogen in the diagnostic laboratory, such as tedious isolation procedures or previous antibiotic treatment. Certain microbes may require special culture conditions and failure to consider these pathogens may result in negative culture. Broad-range 16S rRNA gene amplification is particularly useful for detecting infectious agents of unknown identity under disease conditions in which there is circumstantial evidence for a bacterial etiology, e.g. infectious endocarditis. Molecular diagnostic procedures have left the stage of a scientific challenge and have become a technology issue. Problems to be addressed in this regard include contamination of gene amplification reagents with exogenous nucleic acids and the quality of public data bases. Besides mere laboratory investigations studying the performance of molecular diagnostic techniques, the clinical utility of molecular diagnostic procedures has to be addressed.
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