extraction, and setup techniques are critical to performance of these broad-range techniques ■ Single gene targets that can identify genus or species level are more sensitive than broad-range 16S rDNA PCR They are more likely to be successful in acute sepsis and endocarditis, where circulating bacterial numbers may be much higher than in subacute IE However, multiple primers sets are required to amplify the range of pathogens that could be present, increasing complexity and expense ■ In addition to species identification, genetic determinants of antimicrobial resistance, such as mecA (conferring resistance to majority of β-lactam antibiotics) in S aureus, can also be rapidly detected ■ In the future, high-throughput sequencing (or next-generation sequencing) will be routinely available, which can theoretically provide whole-genome analysis of organisms in situ Proteomic technology ■ The majority of currently available techniques utilize MALDI-TOF ■ Can identify causative agent both rapidly and accurately once a BC is positive.97 ■ Direct identification of bacteria in blood (without a culture step) by MALDI-TOF is possible, although not yet developed enough to be useful in clinical practice ■ Accurate determination of minimum inhibitory concentration to key antibiotics used in definitive therapy often still requires isolation and culture of the organism ■ Future developments include use of sophisticated mass spectrometry and proteomic techniques that not only detect the pathogen but also resistance and virulence determinants Investigation of resected material, by both broad-range and pathogenspecific PCRs and mass spectrometry techniques, can be particularly informative in identifying the etiologic agent.98,99 This can be useful to confirm a causative agent where blood (or tissue) cultures may have not been conclusive or may have been mixed,100 or in blood culture negative–endocarditis (BCNE) Blood Culture–Negative Endocarditis BCNE can occur due to: Prior antibiotic administration Suboptimal culture techniques Fastidious, intracellular or factitively intracellular, or require specialized culture techniques or rarely cultivable from BCs, including: ■ Coxiella burnetii ■ Bartonella spp ■ Brucella spp ■ Nutritional variant gram-positive (formerly Streptococci) organisms Abiotrophia sp., Granulicatella spp ■ Mycobacteria spp ■ Mycoplasma spp ■ Legionella spp ■ Tropheryma whipplei ■ Filamentous fungi In such cases, a combined approach, using serologic assays, direct PCR on blood, and molecular testing on resected material can establish the causative agent, which allows for reasonable accuracy of detection rates.101,102 The proportion of BCNE varies quite widely between studies: 26.6% in a mixed adult and pediatric cohort,103 30.2% in a pediatric cohort.16 These might become identified on molecular studies on resected material It is clear that further development of sensitive and accurate diagnostic tests, including molecular and proteomic techniques, are warranted Antimicrobial Therapy Principles Effective management of IE requires at least one bactericidal antimicrobial to be used in high doses that is corrected for age, weight, and adjusted for renal function Treatment of IE is necessarily prolonged because infection is established in a biofilm matrix Organisms contained therein are either tolerant to bactericidal killing or can exist as “persisters.” This is especially relevant for prosthetic material IE; therefore duration of treatment is long (6 weeks or longer) In addition, biofilm and vegetation protect microorganisms from host-mediated clearance mechanisms Current American Heart Association (AHA) and ESC guidelines provide comprehensive protocols for pathogen-specific therapy, but there are important differences, which are detailed in Table 56.6 The suggested antibiotic treatment for streptococcal IE is shown in Table 56.7 and for staphylococcal IE is shown in Table 56.8 The therapy should always be advised by a clinical microbiologist/infectious diseases specialist Table 56.6 Differences and Similarities Between 2015 ESC and Pediatric AHA Guidelines in Antimicrobial Therapy for Infective Endocarditis AHA2 For treatment of streptococcal IE: ■ Short-course (2 weeks) standard regimen for treatment of uncomplicated IE due to oral streptococci in children not recommended due to lack of effectiveness data ■ MICs for highly penicillin susceptibility ≤0.1 mg/L ■ MICs for relatively resistant strains ≥0.2 mg/L Treatment of Staphylococcus spp.: ■ Treat native valve IE with oxacillin (methicillin)-sensitive strains; gentamicin can be used for first 3–5 days of initial treatment, but this may increase likelihood of ototoxicity or renal toxicity ■ No mention of delay of rifampicin to either flucloxacillin or vancomycin in treatment of prosthetic valve endocarditis ■ Daptomycin is now recommended as an alternative agent for treatment of ESC1 For treatment of streptococcal IE: ■ Short-course (2 weeks) standard regimen for treatment of uncomplicated IE not explicitly ruled out ■ MICs for highly penicillin susceptibility ≤0.0125 mg/L ■ MICs for relatively resistant strains 0.250–2.0 mg/L Treatment of Staphylococcus spp.: ■ Treat native valve IE with oxacillin (methicillin)-sensitive strains; gentamicin not recommended due to lack of evidence of efficacy and toxicity concerns ■ For prosthetic valve endocarditis, addition of rifampicin to either flucloxacillin or vancomycin can be delayed until 3– 5 days of effective therapy with either of these agents The rationale supporting this recommendation is based on the antagonistic effect of the antibiotic combinations with rifampin against planktonic/replicating bacteria and the synergy seen against dormant bacteria within the biofilm ■ Daptomycin is now recommended as an alternative agent for treatment of staphylococcal IE for penicillin-allergic (anaphylaxis) patients and in treatment of methicillinresistant staphylococci when vancomycin cannot be used Daptomycin may be superior to vancomycin for treatment of either MSSA (if penicillin allergic) or MRSA IE when ... The proportion of BCNE varies quite widely between studies: 26.6% in a mixed adult and pediatric cohort,103 30.2% in a pediatric cohort.16 These might become identified on molecular studies on resected material... advised by a clinical microbiologist/infectious diseases specialist Table 56.6 Differences and Similarities Between 2015 ESC and Pediatric AHA Guidelines in Antimicrobial Therapy for Infective Endocarditis AHA2 For treatment of