99. Clinical Experience using Broad-Range PCR-based Sequencing for Pathogen Identification
Session: Oral Abstract Session: Diagnostics: Typing/Sequencing
Thursday, October 8, 2015: 10:30 AM
Room: 7--AB
Background: Broad-range polymerase chain reaction (PCR)-based DNA sequencing (“universal PCR sequencing”) has been adopted as a powerful tool for identifying bacterial and fungal organisms from clinical samples, particularly in settings with high suspicion for infection but negative cultures. Several prospective studies have examined the sensitivity and specificity of this approach in defined clinical settings, however little is known about its real-world performance.

Methods: We conducted a retrospective chart review over 3 years to evaluate the utility of universal PCR-sequencing when ordered at the discretion of clinicians at our institution. A total of 163 fluid or tissue samples from various anatomic sites (most common: CNS, lung, lymph nodes) were collected for identification of bacterial, mycobacterial, and/or fungal organisms, by universal PCR sequencing of conserved regions of the 16S, hsp65/rpoB, and/or 23S/ITS genes, respectively.

Results: In this study, 26 /163 samples (16%) were true positives, and 8/163 (5%) were false positives. The rate of positive results varied by type of tissue collected (range 6-33%, lowest for CNS tissue, highest for cardiac and orthopedic tissue). Positive rates were significantly increased when organisms were visible on microbiology smears (50% versus 12%, p value < 0.01) and when high levels of inflammation were seen on histology (25% versus 4%, p value 0.02). There was poor concordance between culture and PCR results: 3 of 13 (23%) culture-positive samples were also PCR-positive, while only 3 of 21 (15%) PCR-positive results were also culture-positive. Notably, the proportion of true positive results was not significantly different depending on whether the tissue sent for sequencing was fresh versus formalin-fixed paraffin-embedded (FFPE), or from a fine-needle aspirate versus a tissue biopsy. However, 7 of the 8 samples with a false positive result were FFPE tissue.

Conclusion: Taken together, our data offer a window into understanding the clinical utility of universal PCR sequencing in a real-world setting, and may help to guide recommendations and set expectations regarding the value of this emerging technology in clinical practice.

Rachel Rutishauser, MD, PhD1, Steve Miller, MD, PhD2 and Jennifer Babik, MD, PhD1, (1)Department of Medicine, Division of Infectious Diseases, UCSF, San Francisco, CA, (2)Department of Laboratory Medicine, UCSF, San Francisco, CA


R. Rutishauser, None

S. Miller, None

J. Babik, None

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