Diagnostic Value of 16S Ribosomal RNA Gene Polymerase Chain Reaction/Sanger Sequencing in Clinical Practice

Abstract

Background: Accurate microbiologic diagnosis is important for appropriate management of infectious diseases. Sequencing-based molecular diagnostics are increasingly used for precision diagnosis of infections. However, their clinical utility is unclear.

Methods: We conducted a retrospective analysis of specimens that underwent 16S ribosomal RNA (rRNA) gene polymerase chain reaction (PCR) followed by Sanger sequencing at our institution from April 2017 through March 2019.

Results: A total of 566 specimens obtained from 460 patients were studied. Patients were considered clinically infected or noninfected based on final diagnosis and management. In 17% of patients, 16S rRNA PCR/sequencing was positive and in 5% of patients, this test led to an impact on clinical care. In comparison, bacterial cultures were positive in 21% of patients. Specimens with a positive Gram stain had 12 times greater odds of having a positive molecular result than those with a negative Gram stain (95% confidence interval for odds ratio, 5.2-31.4). Overall, PCR positivity was higher in cardiovascular specimens (37%) obtained from clinically infected patients, with bacterial cultures being more likely to be positive for musculoskeletal specimens (P < .001). 16S rRNA PCR/sequencing identified a probable pathogen in 10% culture-negative specimens.

Conclusion: 16S rRNA PCR/sequencing can play a role in the diagnostic evaluation of patients with culture-negative infections, especially those with cardiovascular infections.

Keywords: 16S rRNA gene PCR; Sanger sequencing; bacterial infections; broad range bacterial PCR; molecular diagnostics.

Figure 1.

Culture and polymerase chain reaction (PCR) results by clinical diagnosis. Specimens with PCR inhibitors (n = 10) and specimens without bacterial cultures (n = 15) are not included. Abbreviations: CNS, central nervous system; CV, cardiovascular; MSK, musculoskeletal.