Blood Culture-Negative Endocarditis

Blood culture-negative endocarditis is often severe and difficult to diagnose. Infective blood culture-negative endocarditis is classified into three main categories: (1) bacterial endocarditis with blood cultures sterilized by previous antibacterial treatment; (2) endocarditis related to fastidious microorganisms in which prolonged incubation is necessary; (3) true blood culture-negative endocarditis, due to intra-cellular bacteria that cannot be routinely cultured in blood with currently available. There are two major etiologies for noninfective endocarditis: (1) nonbacterial thrombotic endocarditis and (2) endocarditis related to systemic diseases (SLE and Behcet disease). Team approach including cardiologists, infection disease (ID) specialists, microbiologists, pathologist and immunologist is crucial for diagnosis and management of blood culture-negative endocarditis as it needs elegant and high-quality modern technics of histology, molecular analysis and essential epidemiological information.


Introduction
Blood culture-negative IE (BCNIE) refers to infective endocarditis (IE) in which no causative microorganism can be grown using the usual blood culture methods. BCNIE accounts for 5-10% of all cases of endocarditis [1]. This variation is caused by differences in the diagnostic criteria and sampling strategies used. A European study included 820 cases indicated 20% of

Diagnostic approach
Definitions of the terms used in the European Society of Cardiology 2015 [4] modified criteria adapted from modified Duke Criteria [3] were shown in Table 1. Diagnosis of IE is drawn as follows:

Definition
Pathological criteria: Microorganisms demonstrated by culture or on histological examination of a vegetation, a vegetation that has embolized, or an intracardiac abscess specimen; or pathological lesions; vegetation or intracardiac abscess by histological examination showing active endocarditis.
Clinical criteria: two major criteria; or one major criterion and three minor criteria or five minor criteria.
Possible IE: One major criterion and one minor criterion or three minor criteria.
Rejected IE: Firm alternate diagnosis; or Resolution of symptoms suggesting IE with antibiotic therapy for ≤4 days; or No pathological evidence of IE at surgery or autopsy, with antibiotic therapy for ≤4 days; or Does not meet criteria for possible IE, as above.
When blood culture is negative, systematic diagnostic approach should be performed for rapid and correct management of BCNIE. Diagnostic work-up in blood culture-negative endocarditis is shown in Figure 1 [1,4].

Past history and clinical examination
A precise interview about epidemiological factors, history of prior infections, exposure to antimicrobials, should be made in all patients with suspected BCNIE [1,4].
Previous exposure to antibiotics is the most common cause of BCNE, and even a short course of antibiotics can cause long-lasting suppression of bacterial activity. A history of animal exposures may predispose to certain microbiologic etiologies. Immunosuppression or prolonged antibiotic therapy suggests endocarditis due to fungi. The epidemiological clues for defining the etiology of BCNIE are shown in

Blood culture
BCNIE occurs frequently (45-60%) by common and easily grown staphylococci or streptococci in patients with preceding administration of antibiotics as it reduces the recovery rate of bacteria by 35-40% [5,6]. In these cases, withdrawing antibiotics and repeating blood cultures are preferable methods to diagnose if the patient status allowed. The use of specific blood culture bottles for fastidious microorganisms is not recommended recently [1,4,5]. The extended incubation is applied only when cultures remain sterile after 48-72 h. Sophisticated automated systems allow isolating most pathogens that can grow slowly including Candida sp., deficient streptococci and HACEK group bacteria(Haemophilus, Aggregatibacter (previously Actinobacillus), Cardiobacterium, Eikenella, Kingella). Extending culture beyond 5 days is not contributive [1,[4][5][6][7][8]. The popular pathogens such as staphylococci, streptococci and enterococci are usually identified within 48 h. The European guidelines recommend that clinicians require prolonged incubation of vials only in the rare cases of cultures remaining negative at 48-72 h and if the diagnosis of IE remains plausible [4,8].

Serology
The list of serological tests to be performed in case of blood culture-negative endocarditis used to include: Legionella pneumophila, Mycoplasma hominis, Chlamydophila pneumoniae, Brucella sp., Coxiella burnetii (C. burnetii), and Bartonella sp. Two major series showed that only Bartonella sp. and C. burnetii serological tests are contributive: 348 cases of suspected BCNIE were investigated between 1983 and 2001, the diagnosis was documented by serological tests in 268 cases (77%),

Evaluation of valve tissue
The more frequent use of valve replacement in the acute phase of infective endocarditis and the advent of molecular biology techniques have revolutionized the diagnosis of blood culture-negative endocarditis: PCR systems based on universal bacterial 16S ribosomal RNA have demonstrated excellent sensitivity and specificity [8,9], as well as PCR targeting bacteria specifically responsible for endocarditis with negative blood culture: Bartonella sp., C. burnetii [10] and Tropheryma whipplei (T. whipplei) [11].
Moreover, the microscopic examination of valves after Gram staining, and cultures on appropriate media provide important information not only for the identification of the pathogen involved when the data were not available preoperatively [12], but also information on its viability at the time of valve replacement, which will impact the duration of post-replacement treatment [11,13]. The histological analysis of valves is not contributive to diagnose except some rare diagnoses such as porcine bioprosthesis endocarditis mediated by allergy to porcine proteins [22,23]. Summary of diagnostic procedure of rare pathogens of BCNIE is shown in Table 3.

Empirical therapy
Selection of medical therapy for patients with BCNIE is difficult. Some of the laboratorybased diagnostic techniques to define fastidious or rare pathogens are not available in most clinical laboratories. It consumed considerable time for completion of testing if specimens are sent to a referral laboratory. Patients with BCNIE are often treated empirically for the more common bacterial causes of IE during the waiting time. There is a need to provide empirical antimicrobials for all likely pathogens, though certain therapeutic agents, including aminoglycosides, have potentially toxic effects. Consultation with an ID specialist to define the most appropriate choice of therapy is recommended. Once additional clinical and laboratory data were brought, initial empirical therapy should be changed to more specific treatment.
For patients with acute (days) clinical presentations of native valve infection, coverage for S aureus, β-hemolytic streptococci, and aerobic Gram-negative bacilli is reasonable. Empirical coverage could include vancomycin and cefepime as an initial regimen [1,4,14]. For patients with a subacute (weeks) presentation of native valve IE, empirical coverage of S. aureus, Viridance group streptococci (VGS), HACEK, and enterococci is reasonable. One treatment option could include vancomycin and ampicillin-sulbactam to provide some coverage for these organisms [1,4,14]. For patients with culture-negative prosthetic valve IE, coverage for staphylococci, enterococci, and aerobic Gram-negative bacilli is reasonable if the onset of symptoms is within 1 year of prosthetic valve placement. A regimen could include vancomycin, rifampin, gentamicin [1,4,14]. If symptom onset is >1 year after valve placement, then IE is more likely to be caused by staphylococci, VGS, and enterococci, and antibiotic therapy for these potential pathogens is reasonable [1,4,14]. One initial treatment option could include vancomycin and ceftriaxone. If subsequent blood culture results or other laboratory methodologies define a pathogen, then empirical therapy should be changed to focused therapy that is recommended for the specific pathogen identified.

Antibiotic treatment for fastidious microorganisms
HACEK Gram-negative bacilli are fastidious organisms, and the laboratory should be made aware that infection with these agents needs consultation to specialist. Because of slow growth, standard MIC tests may be difficult to interpret. Some HACEK-group bacilli produce betalactamases, and ampicillin is therefore no longer the first-line option. They are susceptible to ceftriaxone, other third-generation cephalosporins and quinolones; the standard treatment is ceftriaxone 2 g/day for 4 weeks in native valve endocarditis and for 6 weeks in prosthetic valve endocarditis. If they do not produce beta-lactamase, ampicillin (12 g/day i.v. in four or six doses) plus gentamicin (3 mg/kg/day) divided into two or three doses for 4-6 weeks is an option [1,4,13]. Ciprofloxacin (400 mg/8-12 h i.v. or 750 mg/12 h orally) is a less wellvalidated alternative. Clinical outcome of HACEK endocarditis is favorable.
In cases with fungi, mortality is very high, and treatment necessitates combined antifungal administration and surgical valve replacement. Antifungal therapy for Candida sp. includes liposomal amphotericin B with or without flucytosine or an echinocandin at high doses; and for Aspergillus spp., voriconazole is the drug of choice and some experts recommend the addition of an echinocandin or amphotericin B. Suppressive long-term treatment with oral azoles (fluconazole for Candida and voriconazole for Aspergillus) is recommended [1,4,14]. Consultation with an infectious doctor specialist in the Endocarditis Team is recommended.

Specific therapy for true culture-negative microorganisms
The recommended therapy for true culture-negative microorganisms in the European guidelines 2015 is shown in Table 4 [4,12]. Consultation with ID specialist is highly recommended for the treatment of these special organisms. This is an area with a very limited level of evidence. The treatment of T. whipplei endocarditis has not been standardized. Doxycycline + hydroxychloroquine for 12-18 months, with monitoring of plasma levels of these two agents (objective: achieving plasma concentrations of 0.8-1.2 mg/L for hydroxychloroquine, and < 5 mg/L for doxycycline), and of negativation of samples initially positive for T. whipplei was proposed. The treatment of Bartonella sp. endocarditis is a beta-lactam antibiotic (amoxicillin or ceftriaxone) or doxycycline for 4 weeks in combination with gentamicin for the first 2 weeks [1,4,14] the treatment of C. burnetii endocarditis, is doxycycline + hydroxychloroquine until a phase1 antibody rate <800 is reached for IgG, and <50 for IgM and IgA [1,4,14].

Surgical treatment of blood culture-negative IE
There is no specific recommendation for surgical treatment of BCNIE: cardiac surgery indications rely on the same criteria that apply for any type of endocarditis (heart failure, uncontrolled infection, risk of embolism [1,4,15]). However, an additional argument for the surgical treatment of BCNIE is the ability to harvest valve tissue, which often finally allows microbiological documentation.

Noninfective endocarditis
When all microbiological assays are negative, the diagnosis of noninfectious endocarditis should systematically be considered (Figure 1).

Nonbacterial thrombotic endocarditis
Nonbacterial thrombotic endocarditis (marantic endocarditis, Trousseau syndrome) is observed in 1.2% of patients with active cancer at autopsy [16]. Usually, the single or multiple small vegetation-like lesions are observed predominantly on the mitral and aortic valves with no underlying valve diseases. These are associated with an underlying hypercoagulable state that justifies routine anticoagulation. Control of pathologically altered coagulation mechanism is essential for the treatment and the prognosis is poor without resolving the problem. The differential diagnosis with an infectious cause of BCNIE is often difficult, and the prognosis is poor [17]. The initial lesion is usually breast, lung, prostate, ovarian or colon cancer. However, it should not be forgotten that undiagnosed infective endocarditis is also common in cancer patients with sterile blood cultures and/or fastidious organisms that are difficult to identify by conventional methods.

Systemic diseases
Inflammatory diseases can cause endocarditis and produce a syndrome similar to culturenegative IE. Perhaps the one most often encounter is antiphospholipid antibody (APA) syndrome [18], which has been described as both a primary and a secondary syndrome of systemic lupus erythematosus(SLE) and malignancies. Sterile valvular vegetations form and often embolize, clinically mimicking in many respects with IE. The mitral valve is most often affected, and valvular regurgitation is the frequent functional abnormality. To complicate matters, the APA syndrome may also develop secondary to IE [19].
In patients with SLE, valve abnormalities are common (15-75% of autopsy series, depending on the severity of the disease), but rarely progress to a clinical stage of Libman-Sacks endocarditis [20]. The patients are usually young individuals with a very severe lupus poorly controlled by treatments. Immunological manifestations (Osler nodes) and embolism (stroke, often in combination with an antiphospholipid syndrome) may be observed. Valve lesions are mainly found in the left heart. Endocardium involvement may occur in Behҫet's disease [21]. It is a disease of young ± male patients with a predominantly aortic involvement. Endocardium involvement in Behҫet's disease is a poor prognostic factor. The treatment is of course should be targeted on the systemic disease (immune-suppressants, immune-modulators) with lifelong curative anticoagulation. Checkup for antinuclear antibodies as well as antiphospholipid antibody {anticardiolipin antibodies [immunoglobulin (Ig) G and anti-b2-glycoprotein 1 antibodies [IgG and IgM]} should be performed for the patients who are suspected to have noninfective endocarditis.

Allergy for porcine valve
When the patient has a porcine bioprosthesis implanted during last 6 months, anti-pork antibodies should be sought [22,23] to consider allergy for the valve.

Conclusion
Blood culture-negative endocarditis is still a clinical challenge with heterogeneous pathology. Remarkable progress has been made in methodologies to evaluate the main etiologies in past two decades. Team approach including cardiologists, infectious disease specialists, microbiologists and immunologist is crucial for the correct diagnosis that is able to reach rapidly the new diagnostic microbiological techniques, and high-quality epidemiological information.