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Chapter 49 : Molecular Approaches to the Diagnosis of Sepsis
Category: Clinical Microbiology; Bacterial Pathogenesis
This chapter summarizes the pros and cons of such alternative methods for detection of microorganisms in blood and discusses the perspectives of rapid molecular diagnosis of sepsis. Various methods have been assessed to reduce the time required for identification of microorganisms in blood cultures, including hybridization techniques, PCR-based applications, and spectrometric analysis. Among the hybridization techniques suited to identification of microorganisms in blood cultures are fluorescence in situ hybridization (FISH) and chemiluminescent probe matrices. To further reduce time to diagnosis of bloodstream infection (BSI), molecular methods may be applied directly to blood samples, without prior cultivation of microorganisms. Determination of susceptibility to antimicrobial treatment in sepsis is currently performed on cultured strains by determination of the MICs of a broad spectrum of different antibiotics. Current methods of molecular detection of sepsis concentrate on detection of DNA of the microorganism in the blood by both qualitative and quantitative approaches. In addition, alternative DNA extraction methods and multiplex PCR approaches are a first step towards point-of-care testing. For rapid detection of sepsis this is a promising approach, but a real implementation remains to be seen. Rapid molecular detection of the causative pathogen of sepsis is within reach, but many studies are required before clinical implementation. Several techniques are being tested for this application, but the best technique has not been determined. In general, all these promising tests will add to but not replace conventional blood culture as long as phenotypic susceptibility testing is needed.
Phases in the routine diagnostic work-up of bloodstream infection at which molecular methods can be used as alternatives to conventional culture. Heavy arrows indicate currently available applications; the dashed arrow indicates potential application. CPA, chemiluminescent probe assay; HRM, high-resolution melting; SNP, single nucleotide polymorphisms.
Principle of FISH. Microbial cells are heat fixed on a glass slide, followed by chemical permeabilization. After application of probes, the slide is incubated in a water bath or microwave oven. The temperature increase results in binding of probe to complementary rRNA. Unbound probe is washed off, and the fluorescence of the bound probe is visualized with the microscope. Modified with permission from R. P. Peters et al., Neth. J. Crit. Care (2009).
Example of three T-RFLP patterns discriminating MRSA, methicillin-sensitive S. aureus, and Staphylococcus epidermidis. A molecular size marker is present in each pattern, showing the length of the product in base pairs. Adapted with permission from Christensen et al. ( 10 ).
Melting-curve analysis of Enterococcus/Streptococcus spp. PCR showed different melting points for streptococci and enterococci as well as differentiation of E. faecalis and Enterococcus faecium. BC, blood culture. Adapted with permission from Wellinghausen et al. ( 113 ).
Courses of temperature and S. aureus BDL in a patient with S. aureus bacteremia. S. aureus was isolated from all corresponding blood cultures except those obtained on day 2. The mean BDL was calculated when the BDL was available for two simultaneously obtained blood samples. Antimicrobial treatment was administered as indicated. Days are on the x axis. Adapted with permission from Peters et al. ( 82 ).
Use of FISH or chemiluminescent probe matrix for identification of microorganisms in blood cultures
Characteristics of studies on direct detection of bacteremia in blood with eubacterial PCR assay