Over the last 30 years, molecular diagnostics have played an increasingly important role in the diagnosis of IE (29
). The data indicate that PCR/ESI-MS has potential for use as a diagnostic tool for IE. PCR/ESI-MS has the potential to decrease turnaround time to 12 to 16 h compared to 24 to 48 h for standard tissue processing and culture (providing it is performed on fresh tissue). Beyond organism detection and classification, select antimicrobial resistance is also defined.
A major benefit of this method over other molecular methods is the ability to detect more than one organism simultaneously in a sample. ESI-MS may be more sensitive than other molecular methods. Imrit et al. identified causative organisms of IE using FFPE heart valves coupled with 16S rRNA gene sequencing. They found that the length of time the heart valves were stored in the paraffin block affected the sensitivity of the assay, with 70% positivity for valves stored for less than 5 years versus 18% positivity for those stored longer (12
). This is in contrast to the data presented herein, which showed no difference in detection between FFPE heart valves stored for more than and less than a decade (P
This is the first large-scale PCR/ESI-MS study to use FFPE samples of infected tissue. A case report has described the use of PCR/ESI-MS to detect Bartonella
species in an abdominal aortic mycotic aneurysm (30
), and a recent publication has described the use of this technology for the analysis of synovial fluid from subjects with prosthetic joint infections (25
). Organisms detected in this study included common IE pathogens (S. aureus
, S. epidermidis
, and E. faecalis
) and not-so-common pathogens (T. whipplei
, Granulicatella adiacens
, G. morbillorum
, C. hominis
, and M. fortuitum
), which speaks to the potential applications of the assay studied.
In eight cases with concordant microbiology, an additional bacterium was detected (G. adiacens [n = 1], S. epidermidis [n = 1], and Candida tropicalis [n = 6]) using PCR/ESI-MS; these apparently false-positive results may relate to contamination of the tissue block during storage, sectioning, or processing for testing or PCR contamination. The specimens studied were inherently nonsterile and subject to contamination during cutting of the blocks (i.e., from technologists or razor blades). Further, PCR/ESI-MS is not a closed system, so it allows for possible amplified product contamination. All C. tropicalis detection occurred with a single assay plate, a finding that strongly supports contamination.
Not all IE cases were detected, possibly because the valves studied came from FFPE blocks. Fixation with formalin degrades DNA, lowering the yield of amplifiable DNA compared to that of fresh tissue (33
). Although not evaluated herein, testing of fresh tissue is a possible clinical application of this assay. Finally, it is possible that the infectious agent was not equally distributed throughout the valves, in which case, due to sampling error, a 40-μm cut may have missed the microbial DNA.
Misidentification of VGS as S. pneumoniae, noted in four instances, was likely due to an error in the identification algorithms used. This can possibly be corrected by targeting a more specific gene for S. pneumoniae during the upfront PCR step or by reporting all S. pneumoniae detected as “VGS/S. pneumoniae.” Identification of T. whipplei IE from a case originally misdiagnosed through blood culture as S. lugdunensis IE illustrates that this system can identify and diagnose cases that might otherwise be missed using conventional methods.
There are several limitations of the technology evaluated and of the study design. DNA extracted from FFPE tissue was diluted to have enough volume to distribute to 16 PCR wells, potentially impacting assay sensitivity. As a consequence, some positive specimens may not have been identified due to low concentrations of target DNA. Formalin fixation may lead to DNA cross-linking, preventing PCR amplification. The PCR products generated with the assay studied herein are very short (~80 to 120 bp), maximizing the likelihood of detection since cross-linking occurs at approximately every 100 bp. A major limitation in study design was that only culture-positive valves were studied; future studies should assess culture-negative valves. Finally, limited antimicrobial resistance markers were studied, although mecA, vanAB, and blaKPC do represent key early decision markers for the inclusion or exclusion of related antimicrobials in the treatment regimen.
In summary, PCR/ESI-MS might be a useful tool in the diagnosis of IE cases where a microbiologic etiology is not defined by current laboratory methods. If these findings are supported by those demonstrated in future investigations, this tool might have a meaningful impact on both early and subsequent antimicrobial selection, which might positively influence patient outcomes.