Limited diagnostic possibilities for bloodstream infections with broad‐range methods: A promising PCR/electrospray ionization‐mass spectrometry platform is no longer available

Abstract Fast and accurate detection of causative agents of bloodstream infections remains a challenge of today's microbiology. We compared the performance of cutting‐edge technology based on polymerase chain reaction coupled with electrospray ionization‐mass spectrometry (PCR/ESI‐MS) with that of conventional broad‐range 16S rRNA PCR and blood culture to address the current diagnostic possibilities for bloodstream infections. Of 160 blood samples tested, PCR/ESI‐MS revealed clinically meaningful microbiological agents in 47 samples that were missed by conventional diagnostic approaches (29.4% of all analyzed samples). Notably, PCR/ESI‐MS shortened the time to positivity of the blood culture‐positive samples by an average of 34 hr. PCR/ESI‐MS technology substantially improved current diagnostic tools and represented an opportunity to make bloodstream infections diagnostics sensitive, accurate, and timely with a broad spectrum of microorganisms covered.

a very favorable outcome of a multicentre clinical trial (Vincent et al., 2015). The method is capable of detecting over 800 bacterial and Candida species associated with BSI and shortening the TAT by up to 6 hr (Bacconi et al., 2014). The width of detection, unprecedented for a commercial test, matches the conventional broad-range PCR strategy (Tkadlec et al., 2019) and goes far beyond the possibilities of any pathogen-specific PCR.
We aimed to evaluate the utility of the PCR/ESI-MS system in a clinical setting by comparing its real-life performance with conventional culture-dependent (BC) and culture-independent (16S ribosomal RNA PCR) broad-range tests.
Within 12 months, we examined 166 blood samples from 137 patients (median age: 64 years; range 22-94 years; 72% males) who had been hospitalized with suspected BSI at intensive care units of Motol University Hospital in Prague.
A BC was performed by using BACTEC™ FX (Becton Dickinson) with one pair of aerobic and anaerobic BC bottles collected. These BC bottles were incubated for 5 days before being concluded as negative. Additional (mycotic) BC bottles were collected if patients were suspected of a mycotic infection, and these bottles were incubated for up to 14 days before being concluded as negative. Positive BC bottles were streaked out on solid media, and upon overnight culture, the microorganisms were identified by using a MALDI-TOF mass spectrometer Biotyper v 3.1 (Bruker Daltonics). Two additional EDTA tubes were collected at the time of the BC blood draw. One tube was subjected to panbacterial/panfungal 16S/18S PCR assay (hereafter 16S-PCR) using UMD-SelectNA™ kit with Add-On10 extension (Molzym) to process up to 10 ml of whole blood as previously described (Tkadlec et al., 2019). The other tube with 5 ml whole blood was stored frozen at −20°C for up to 3 months until being processed with the PCR/ESI-MS system. The samples were run with the BAC BSI assay according to the manufacturer's instructions (Abbott Molecular).
We assessed every PCR/ESI-MS result against the BC and 16S-PCR results, which are collectively referred to as standard-of-care (SoC) tests. When a discrepancy between PCR/ESI-MS and SoC occurred, the patient's medical records were carefully reviewed to determine whether the unique positivity of PCR/ESI-MS meant a false negativity of the SoC tests and vice versa. Detection of typical contaminating microbiota by any of the tested methods was not regarded as a relevant finding unless it was supported by microbiological investigation of other materials from the same patient. The unique positivity obtained by PCR/ESI-MS was con- Reliable and timely diagnostics of BSI-causing agents are critical for correct patient management. BC is the gold standard for BSI diagnostics, but due to its limitations, alternative detection systems are being sought. We tested the performance of PCR/ESI-MS as a novel BSI diagnostic method and found that: (a) The method was able to detect clinically relevant causes of BSI in 30% of samples F I G U R E 1 PCR/ESI-MS results categorized by the level of agreement with the standard-of-care methods (i.e., blood culture and 16S-PCR) and by its utility as defined by added value and/ or failure (please note that two samples fulfilled criteria for both categories and are labeled as IDs 51 and 52 in Appendix in Table A1) (that would be determined as negative by the SoC methods); and (b) PCR/ESI-MS exhibited a considerably shorter time to positivity when compared to that of BC.
However, it is important to note that the majority of our patients were on antibiotic therapy at the time of sample collection and that the higher PCR/ESI-MS positivity rate could be also attributed to the detection of free circulating DNA, not necessarily of viable microbial cells. However, positivity of PCR/ESI-MS was already associated with increased mortality in patients with suspected sepsis, indicating the ability of the technology to correctly identify critically ill patients

CO N FLI C T O F I NTE R E S T
The authors declare no conflict of interest.

E TH I C S S TATEM ENT
Study protocol had been reviewed and approved by the institutional Ethics Committee of the Motol University Hospital, Prague, Czech Republic (date of the application for approval: 17 July 2014).
All subjects or their legal representatives provided written informed consent.

DATA AVA I L A B I L I T Y S TAT E M E N T
The datasets generated and/or analyzed during the current study are not publicly available due to confidentiality reasons but are available from the corresponding author upon reasonable request.