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Chapter 26 : Molecular and Mass Spectrometry Detection and Identification of Causative Agents of Bloodstream Infections

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Abstract:

Bloodstream infections (BSIs) are severe diseases associated with a high morbidity and mortality, which increases with the delay until administration of the first appropriate antibiotic (1–8). For this reason, empiric treatments made of broad-range anti-infectious compounds or made of a combination of antimicrobials are started immediately after the sampling of blood bottles. BSIs can be caused by various microorganisms. In the absence of microbiological documentation, physicians suspect a BSI on the basis of clinical symptoms, which trigger the start of empirical treatments. The clinical presentations are multiple and include fever or hypothermia, increases in heart rate, change in inflammatory variable (C-reactive protein, procalcitonin, and white blood cell count increase), and organ failure (2, 9). These symptoms are generally nonspecific and only suggest bloodstream dissemination. Empirical treatments are made of broad-spectrum antibiotics on the basis of the clinical and epidemiological data, but this does not exclude any risk of inappropriate initial treatment.

Citation: Opota O, Jaton K, Prod'hom G, Greub G. 2016. Molecular and Mass Spectrometry Detection and Identification of Causative Agents of Bloodstream Infections, p 336-361. In Persing D, Tenover F, Hayden R, Ieven M, Miller M, Nolte F, Tang Y, van Belkum A (ed), Molecular Microbiology. ASM Press, Washington, DC. doi: 10.1128/9781555819071.ch26
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FIGURE 1

Different molecular technologies used for the detection and identification of microbes during bloodstream infections. Molecular methods for the diagnosis of BSI include nucleic acid-based methods and non-nucleic acid-based methods. Nucleic acid amplification-based techniques can be applied on positive blood cultures or used directly on blood, whereas non-nucleic acid amplification-based techniques such as FISH and microarray or non-nucleic acid-based methods such as MALDI-TOF MS can be used only on positive blood culture. Adapted from reference .

Citation: Opota O, Jaton K, Prod'hom G, Greub G. 2016. Molecular and Mass Spectrometry Detection and Identification of Causative Agents of Bloodstream Infections, p 336-361. In Persing D, Tenover F, Hayden R, Ieven M, Miller M, Nolte F, Tang Y, van Belkum A (ed), Molecular Microbiology. ASM Press, Washington, DC. doi: 10.1128/9781555819071.ch26
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Image of FIGURE 2
FIGURE 2

Conventional and molecular methods for the identification of microorganisms from positive blood culture. When a blood culture is detected as positive, several strategies are available to identify the incriminated microorganism after the initial Gram staining, as follows. ( ) Identification directly from the positive blood culture using nucleic acid-based methods. ( ) Identification after the microbe's enrichment, namely a purified bacterial pellet suitable for MALDI-TOF MS analysis and some automated identification/antibiotic susceptibility testing approaches such as Vitek2 and Phoenix and rapid biochemical tests HMRZ, based on the chromogenic cephalosporin HMRZ-86 {(7R)-7-[2-(aminothiazol-4-yl)-(z)-2-(1-carboxy-1-ethylethoxyimino) acetamido]-3-(2,4-dinitrostyryl)-3-cephem-4-carboxylic acid trifluoroacetate, E-isomer}, and ESBLNDP (ESBL Nordmann-Poirel-Dortet). ( ) Identification after a subculture. To date, the automated system Vitek2 has been validated on short subcultures as well as the rapid biochemical tests HMRZ and CARNP (carbapenemase test Nordmann-Poirel); long subcultures are suitable for any type of analysis including phenotypic characterization; automated systems such as Vitek2, Phoenix, and MicroScan WalkAway; as well as PCR followed by sequencing. Adapted from reference .

Citation: Opota O, Jaton K, Prod'hom G, Greub G. 2016. Molecular and Mass Spectrometry Detection and Identification of Causative Agents of Bloodstream Infections, p 336-361. In Persing D, Tenover F, Hayden R, Ieven M, Miller M, Nolte F, Tang Y, van Belkum A (ed), Molecular Microbiology. ASM Press, Washington, DC. doi: 10.1128/9781555819071.ch26
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Image of FIGURE 3
FIGURE 3

Microbial identification from positive blood culture using MALDI-TOF MS. The sample that is deposited on the MALDI-TOF multiwell plate can be a bacterial pellet obtained by centrifugation and erythrocyte lysis. Alternatively, the sample can be bacteria obtained after a subculture. Proteins are then extracted and embedded in a matrix directly on the multiwell conductive metal plate and submitted to the MALDI-TOF MS, which separates the proteins according to their molecular weights and their charges. This generates a mass spectrum (MS), which in a second stage is compared to a database of spectra. This analysis provides the identification of the microorganism with a confidence score, which allows acceptance at the species or at the genus level. The identification is interpreted according to the Gram staining and to the clinical presentation. Adapted with permission from reference .

Citation: Opota O, Jaton K, Prod'hom G, Greub G. 2016. Molecular and Mass Spectrometry Detection and Identification of Causative Agents of Bloodstream Infections, p 336-361. In Persing D, Tenover F, Hayden R, Ieven M, Miller M, Nolte F, Tang Y, van Belkum A (ed), Molecular Microbiology. ASM Press, Washington, DC. doi: 10.1128/9781555819071.ch26
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Image of FIGURE 4
FIGURE 4

Schematic workflow of PCR/ESI-MS. (a) Nucleic acids are extracted from the sample, directly from whole blood, and (b) amplified by multiple PCR using multiple pairs of primers; each color represents a different primer. After amplification, the molecular mass of the amplicon(s) is precisely determined using ESI-MS (c, d, and e), from which (f) the base composition of the amplicon(s) is deduced. Finally, (g) information—base composition—obtained from one or more amplicon(s) is compared with a database, which provides the identification with a confidence score. Adapted from reference .

Citation: Opota O, Jaton K, Prod'hom G, Greub G. 2016. Molecular and Mass Spectrometry Detection and Identification of Causative Agents of Bloodstream Infections, p 336-361. In Persing D, Tenover F, Hayden R, Ieven M, Miller M, Nolte F, Tang Y, van Belkum A (ed), Molecular Microbiology. ASM Press, Washington, DC. doi: 10.1128/9781555819071.ch26
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