Bacterial cultures are negative in over 13% of cases of Cardiovascular Implantable Electronic Device (CIED) Infections. Broad-range PCR and Sanger sequencing could potentially establish a microbiologic diagnosis in these patients. We aim to evaluate and compare detection and sequencing performance of primers targeting two different hypervariable regions of the 16S ribosomal RNA (rRNA) gene on sonicate fluid from extracted CIEDs.
Methods: Samples of sonicate fluid from extracted cardiac devices of patients with suspected CIED infection have been collected in our laboratory from 2012 through 2017. We selected 39 of these samples and classified them as culture-positive or culture-negative based on the results of conventional culture. ZymoBIOMICS DNA miniprep Kit was utilized for DNA extraction with modifications. Quantitative Real-Time PCR was performed on a Roche LightCycler 1.0 instrument. Annealing temperature of 65°C was used for the primers targeting the V3-V4 16S rRNA hypervariable region and 62°C for the V1-V3 primers according to previously published protocols. Samples with crossing point (Cp) of <32 or <3 Cps below the negative control were sent for bidirectional Sanger sequencing. Sequences were aligned and edited using Sequencher 5.0 software. Contiguous assemblies were queried using the NCBI BLAST database and results interpreted using CLSI guidelines. The organism identified by sequencing was compared to the results of conventional culture.
Results: Of the 39 samples, 23 were culture-positive and 16 were culture-negative. Of those 23 culture-positive, 19 were PCR-positive using both sets of primers and sequencing of these samples identified the same organism reported on conventional culture. Two out of the 23 samples were only PCR-positive using the V1-V3 primers. In the culture negative group, 2 specimens were PCR-positive using both sets of primers, identifying Staphylococcus aureus in both. The remaining samples were PCR-negative.
Conclusion: Our results suggest that primer set amplifying the V1-V3 region of the 16S rRNA gene leads to slightly better detection results compared to the V3-V4 primer set used. Molecular testing performed on sonicate fluid from extracted devices may identify pathogens in cases of culture-negative CIED infection.
Z. Esquer Garrigos,
K. Greenwood-Quaintance, None
S. Cunningham, None
P. Vijayvargiya, None
M. Thoendel, None
P. A. Friedman, Medtronic, Guidant, Astra Zeneca: Consultant , Consulting fee . Medtronic, Astra Zeneca via Beth Israel, Guidant, St Jude, Bard: Investigator , Research grant .
R. Patel, CD Diagnostics, BioFire, Curetis, Merck, Hutchison Biofilm Medical Solutions, Accelerate Diagnostics, Allergan, and The Medicines Company: Grant Investigator , Research grant - monies paid to Mayo Clinic . Curetis, Specific Technologies, Selux Dx, GenMark Diagnostics, PathoQuest and Genentech: Consultant and Scientific Advisor , Consulting fee - monies paid to Mayo Clinic . ASM and IDSA: Travel reimbursement and editor's stipends , Travel reimbursement and editor's stipends . NBME, Up-to-Date and the Infectious Diseases Board Review Course: Varies , Honoraria . Mayo Clinic: Employee , Salary .