Bacteriology
Pathogen enrichment from human whole blood for the diagnosis of bloodstream infection: Prospects and limitations

https://doi.org/10.1016/j.diagmicrobio.2018.11.015Get rights and content

Abstract

Blood culture represents the current reference method for the detection of bacteria or fungi in the circulation. To accelerate pathogen identification, molecular diagnostic methods, mainly based on polymerase chain reaction (PCR), have been introduced to ensure early and targeted antibiotic treatment of patients suffering from bloodstream infection. Still, these approaches suffer from a lack of sensitivity and from inhibition of PCR in a number of clinical samples, leading to false negative results. To overcome these limitations, various approaches aiming at the enrichment of pathogens from larger blood volumes prior to the extraction of pathogen DNA, thereby also depleting factors interfering with PCR, have been developed. Here, we provide an overview of current systems for diagnosing bloodstream infection, with a focus on approaches for pre-analytical pathogen enrichment, and highlight emerging applications of pathogen depletion for therapeutic purposes as a potential adjunctive treatment of sepsis patients.

Introduction

Sepsis, a life-threatening organ dysfunction caused by a dysregulated host response to infection, afflicts 18 million people worldwide each year, with mortality rates of 30 to 50% even in state-of-the-art intensive care, and its incidence continues to increase (Fleischmann et al., 2016, Martin, 2012, Mayr et al., 2014, Mellhammar et al., 2016, Singer et al., 2016).

Over 600 pathogen species have been associated with sepsis (Vincent et al., 2009). Gram-positive bacteria account for more than half of all sepsis cases, followed by gram-negative bacteria and fungi (Martin, 2012, Vincent et al., 2006). The spread of antibiotic-resistant pathogens, evidenced by the global emergence of methicillin-resistant Staphylococcus aureus or by increasing resistances among gram-negative bacilli and common respiratory pathogens, poses particular challenges to the treatment of bloodstream infections.

Current Guidelines for the Management of Severe Sepsis and Septic Shock (Rhodes et al., 2017) recommend immediate antibiotic therapy, which can significantly improve the survival of septic patients (Ferrer et al., 2014, Gaieski et al., 2010, Garnacho-Montero et al., 2006, Zhang et al., 2015). To ensure rapid onset of therapy, antibiotic treatment is initiated based on clinical and epidemiological information, and adjusted or de-escalated following identification of the etiologic agents. Culture-based diagnosis (blood culture) remains the reference standard to identify the causative pathogens in bloodstream infection (Lamy et al., 2016, Opota et al., 2015). Blood culture methods have been optimized to increase their sensitivity and specificity, but still, up to 50% of suspected bloodstream infections occur with negative blood culture, which can delay adequate antibiotic therapy. This can be due to the presence of slow-growing or intracellular pathogens, such as Rickettsia spp., Bartonella spp., Coxiella spp., Mycoplasma spp., or Chlamydia spp. (Fenollar and Raoult, 2007, Lamas and Eykyn, 2003, Lamy et al., 2016), or to antibiotic treatment initiated before blood sampling.

Molecular diagnostic methods, in particular polymerase chain reaction (PCR), are less sensitive to previously initiated antibiotic treatment than blood culture, and circumvent time-consuming and potentially pre-selecting culture steps (Dark et al., 2015, Stevenson et al., 2016), but PCR-based pathogen detection may be inhibited (Mancini et al., 2010) by sample matrix components, such as non-target DNA (Doring et al., 2008), heparin (Djordjevic et al., 2006, Garcia et al., 2002), immunoglobulins (Al-Soud and Radstrom, 2001), or iron associated with hemoglobin and lactoferrin. Efficient protocols for pre-analytical sample processing and pathogen enrichment are therefore required, as summarized in Fig. 1.

In this review, we provide an overview of current systems for the diagnosis of bloodstream infection. The most common approaches relying on pathogen identification after blood culture are summarized in Table 1 and have been reviewed extensively elsewhere (Dubourg and Raoult, 2016). Here, we focus on pathogen identification without previous blood culture, as compiled in Table 2. We particularly address methods relying on pre-analytical pathogen enrichment and compare their workflow to blood culture based approaches (Fig. 2).

Section snippets

Pathogen enrichment for diagnostic purposes

Pathogen enrichment prior to DNA extraction aims to increase the ratio of pathogens to non-target cells and to deplete factors potentially interfering with PCR (Beutler et al., 1990, Doring et al., 2008, Garcia et al., 2002, Mancini et al., 2010). Pathogen enrichment may further be required in sepsis patients, where pathogen loads of less than one CFU/mL whole blood have been reported (Arpi et al., 1989, Mermel and Maki, 1993), which is below the detection limit of current PCR methods (Ginn et

Pathogen depletion from whole blood for therapeutic purposes

The increasing emergence of multi-drug resistant microbial pathogens and the threat of pan-resistant bacteria require a consideration of non-antibiotic approaches as adjuvant treatments or as alternative therapies. Potential non-antibiotic options to treat serious bacterial infection include inhibitors of bacterial quorum sensing, lytic bacteriophages, liposome-based cytotoxin inhibitors, advanced immunotherapies, as well as extracorporeal therapies, such as hemoadsorption, which aims at

Summary

A number of molecular diagnostic systems for bloodstream infection have been developed that exploit selective lysis of blood cells and centrifugation to enrich pathogens and deplete factors inhibiting or interfering with PCR prior to analysis. The clinical performance of these systems is currently evaluated in comparison to blood culture or to first generation molecular diagnostics, such as the SeptiFast system (Roche, Basel, Switzerland). Beyond diagnostics, the capture and depletion of

Declaration

Availability of data and materials

All data included in this article are publicly available, as this review summarizes published data.

Authors' contributions

MP compiled the review and wrote the first draft. AS supported with writing and with the preparation of the figures. DOH reviewed the draft and provided input from a medical perspective. VW extensively reviewed and critically revised the manuscript. All authors read and approved the final manuscript.

Competing interests

This work was supported

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