Donna M. Wolk

Pathogen Detection in the Genomic Era

In the 21st century, one of the greatest challenges to public health and clinical microbiologists is the rapid detection and identification of emerging and reemerging pathogens. Complex factors such as genetic variation in the host and pathogen, environmental changes, population pressures, and global travel can all influence the emergence of infectious diseases. The SARS epidemic of 2003 highlighted the potential of an emerging pathogen to spread globally in a very short time frame (Peruski and Peruski, 2003). The diagnostics of such infectious diseases has been greatly affected in the past 20 years. No longer is cultivation and microscopy the only means of detecting infectious agents. With the introduction of molecular diagnostics, the ability to detect minute amounts of microbial nucleic acids in clinical specimens has revolutionized clinical microbiology. In particular, the utility of PCR allows the detection and quantitation of specific agents in a matter of hours. PCR sequencing of specific segments of nucleic acid allows for the determination of specific drug resistance that now aids in guiding viral therapies.

5 Identification of Pathogens by Nonculturing Molecular Techniques

This chapter will focus on the use of non-culture-based methods to identify prokaryotic human pathogens. The methods discussed rely primarily on nucleic acid detection and analysis for pathogens commonly encountered in diagnostic and clinical researchsettings.Diagnosticcapabilitiesinclinicalmicrobiology have exponentially grown due to the impact of molecular methods and tools, like (but not limited to) polymerase chain reaction (PCR) and DNA sequencing, for the detection of human pathogens. Increasingly, technologies such as mass spectrometry and next-generation sequencing that have been incorporated into routine research laboratory use for sometime will be implemented for routine use in a clinical setting along with PCR-based assays. In addition, the pace and continued development of molecular methods for clinical applications of pathogen detection may ultimately transform diagnostic microbiology into a ‘‘culture less’’ diagnostic science, in the future.

16S rRNA gene sequencing on a benchtop sequencer: accuracy for identification of clinically important bacteria

Test the choice of 16S rRNA gene amplicon and data analysis method on the accuracy of identification of clinically important bacteria utilizing a benchtop sequencer.