260 Chapter 11 Rapid microbiologic diagnostic approaches Blood cultures (BCs), aiming to detect viable microorganisms in blood, are still considered as the “gold standard” for the microbiological diagnosis of bloodstream infections (BSIs) during sepsis [7]. However, this culture-based method suffers from important limitations, such as false-negative results due to ongoing antimicrobial therapy, and long time to positivity (usually from 12h to 72h). These drawbacks reflect the low numbers of circulating microorganisms (from 1 to 10 CFU/ml), the possible presence of fastidious or uncultivable pathogens, and inadequate blood sample volumes (especially in children) [8-10]. Therefore, the overall sensitivity of BCs is sub-optimal; BC positivity can be as low as 30-40% in septic patients [11]. However, BCs are relatively inexpensive and use widely available/accepted technologies. Furthermore, BCs facilitate the evaluation of pathogen antimicrobial susceptibility. Much can still be gained by optimizing BC processes, such as adequate blood sample volume collection before the start of antibiotics, more rapid work-up and faster result reporting to clinicians [12]. To achieve a faster and more accurate diagnosis of sepsis, several rapid, usually molecular diagnostic tests (RDTs) have been developed for BSI diagnosis [8, 13-15]. Pathogen-specific assays have a limited role due to the high variety of pathogens potentially responsible for sepsis, but depending on the clinical and epidemiological context, rapid tests to confirm or rule out malaria, Dengue fever or other non-bacterial infections that can lead to sepsis may have focussed roles. For bacterial sepsis, many broad-range and multiplex polymerase chain reaction (PCR) assays are commercially available. From a practical point of view, RDTs for bacteraemia and fungaemia can be divided into two main categories: assays performed on positive BC bottles, and those performed directly on blood samples [13-15]. RDTs are also used on samples from other sites, such as respiratory secretions or urine. These RDTs are outside the scope of this paper. For in vitro diagnostic use on positive BCs, there are different types of approaches: non-molecular, PCR-based and non-PCR-based methods (Table 1) [8, 13-20]. The time to initiating appropriate treatment when the results of RDTs performed on positive BCs are included (time to positivity 12-72 hours) adds ~0.2-3 hours (Table 1). The use of matrixassisted laser desorption/ionization–time of flight (MALDI-TOF) mass spectrometry directly from positive BCs has been extensively investigated, since it is a fast, simple and cost-effective approach; for example, using either a commercially-available (Sepsityper® kit, Bruker Daltonics) or laboratory-developed technique [21]. There are varied PCRbased assays for the identification of bacteria and fungi from positive BCs, but with similar analytical performances (sensitivity and specificity usually >90%). Quantitative PCR assays can also be used to measure bacterial load, which correlates with severity of sepsis; for example, such biomarkers can estimate the risk of mechanical ventilation, septic shock and mortality in patients with pneumococcal pneumonia in the Emergency Department [22]. Finally, fluorescent in situ hybridization (FISH) using peptide nucleic acid (PNA) probes (AdvanDx) can also be applied to positive BCs. The fastest strategy to identify microorganisms is by direct detection of DNA from blood, since this avoids the enrichment step in BCs. The turn-around time of RDTs performed directly on blood samples ranges from 3-12 hours (Table 2). However, this approach has different drawbacks, such as false-positive results due to contaminating bacterial or fungal DNA (from the environment, from PCR reagents or from dead
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