Development and evaluation of a novel fast broad-range 16S ribosomal DNA PCR and sequencing assay for diagnosis of bacterial infective endocarditis: multi-year experience in a large Canadian healthcare zone and a literature review

Abstract

BackgroundThe study aimed to explore the sensitivity and specificity of a novel fast 16S rDNA PCR and sequencing assay for the improved diagnosis of infective endocarditis (IE) in patients with suspected native or prosthetic heart valve (HV) infection over a multi-year period at our cardiovascular center.MethodsSixty-eight patients were prospectively enrolled who underwent HV replacement for suspected or confirmed IE between February 1, 2009 and September 1, 2014. Patient demographics, medical co-morbidities, Duke’s criteria, culture results, and antibiotic therapy were collected by detailed chart reviews. Dual-priming oligonucleotide primers targeted to 500 bps of the V1-V3 region of the 16S rRNA gene were used to perform fast broad-range 16S rDNA PCR and Sanger sequencing on ribosomal DNA extracted from HV tissues. The performance/diagnostic efficiency of the molecular test was evaluated against blood cultures and Gram stain and culture of HV tissue in patients’ with definite IE according to Duke’s criteria.ResultsFifty patients (73.5 %) had definite IE and another 8 (11.8 %) had possible IE according to Duke’s criteria. Cardiac surgery was delayed an average of 15.4 days from the time of the patient’s last positive blood culture, and appropriate antibiotic therapy was given in the pre-operative period. While 44/50 (88 %) patients had a positive blood culture, HV tissue culture was only positive in 23 (46 %) of them. Molecular testing of all HV tissues had sensitivity, specificity, NPV and PPV of 92, 77.8, 77.8 and 92 % compared to 44, 100, 39.1 and 100 % respectively for culture for diagnosis of definite IE. For prosthetic HV tissue, 16S rDNA PCR had sensitivity of 93 % and specificity of 83 % compared to 35 and 100 % respectively for culture. A literature review showed that the diagnostic accuracy of our novel fast broad-range 16S rDNA PCR assay was similar or better than that of previously published studies.ConclusionsThis novel fast broad-range 16S rDNA PCR/sequencing test had superior sensitivity compared to tissue Gram stain and culture for identifying underlying bacterial pathogen in both native and prosthetic valve endocarditis.

Added diagnostic value and impact on antimicrobial therapy of 16S rRNA PCR and amplicon sequencing on resected heart valves in infective endocarditis: a prospective cohort study

“…Analytical performance of 16S rRNA PCR on excised heart valves from patients with IE has been evaluated in several studies [5,6]. Overall, VC seems less sensitive (range 8%e62%) than 16S rRNA PCR (range 41%e100%).…”

Molecular identification of pathogens from excised heart valves of infective endocarditis cases

“…Heart valve culture had a less optimal range of pathogen recovery (11.2-32.3%) compared to either blood culture or broad-range 16S rRNA gene PCR/sequencing on heart valve tissue (Miller et al, 2016). Overall, these reports (Li et al, 2000).…”

“…Overall, these reports (Li et al, 2000). However, heart valve culture has a much lower pathogen recovery rate (11.2-32.3%) compared to broad-range 16S rRNA gene PCR on heart valve tissue (41.2-100%) (Miller et al, 2016 LightCycler® SeptiFast, has been tested for diagnosis of IE (Cambau et al, 2017. Leli et al, 2014, Menacacci et al, 2012 …”

The diagnosis of microorganism involved in infective endocarditis (IE) by polymerase chain reaction (PCR) and real‐time PCR: A systematic review

“…In 11 of these studies, valve sample culture and blood culture were used to perform the molecular tests. The blood sample culture is important since it can determine the causative organism that induces the disease and this facilitates the appropriate prescription and administration of the required antibiotic resulting in more effective treatment [5,6,27,40,41]. However, in 2.5%e31% of cases of infective endocarditis, blood cultures are negative [3,5,27,42].…”

“…The analysis of the results of 12 studies indicated that the sensitivity of molecular methods is higher than that of the conventional blood culturing methods so that the highest sensitivity belonged to Marín's study with 96% sensitivity [36] while the PCR results in some cases have been even negative. Marín and Miller attribute the negative results of PCR test to the presence of low amounts of microorganisms in the sample or to the high probability of sampling error [36,41]. Kotilainen et al stated in their study that negative PCR result does not mean that the patients have responded appropriately to antibiotics [50].…”