INTRODUCTION — Infective endocarditis (IE) is a diagnostic challenge in some patients. Identification of the etiologic agent is critical to selecting an appropriate treatment, as the fatality rate remains high [1]. The proportion of IE that is without an etiologic diagnosis varies from country to country and among different centers in the same country. These variations reflect the local epidemiology of IE, diagnostic criteria used, initiation of antibiotics in patients prior to obtaining blood cultures, and the diagnostic protocol used to establish an etiology [2].

The epidemiology and microbiology of culture-negative endocarditis will be reviewed here. Criteria for the diagnosis of IE and treatment are discussed separately. (See "Clinical manifestations and evaluation of adults with suspected left-sided native valve endocarditis" and "Antimicrobial therapy of left-sided native valve endocarditis" and "Antimicrobial therapy of prosthetic valve endocarditis".)

EPIDEMIOLOGY — The epidemiology of blood culture-negative infective endocarditis (IE) varies by country and host, as exposure to infection with highly fastidious bacteria (many zoonotic) or fungi depends on whether the organism is endemic to the area and whether the host is particularly susceptible to infection with the organism.

Definition — Blood culture-negative IE is defined as endocarditis without etiology following inoculation of at least three independent blood samples in a standard blood-culture system with negative cultures after seven days of incubation and subculturing [2].

Incidence — Cultures remain negative in 2 to 7 percent of patients with IE even when the utmost care is taken in obtaining the proper number and volume of blood cultures and patients with prior antibiotic treatment are excluded; the frequency is higher in patients who have already been treated with antibiotics [3-8].

Cultures are negative in IE for three major reasons:

●Previous administration of antimicrobial agents

●Inadequate microbiological techniques

●Infection with highly fastidious bacteria or nonbacterial pathogens (eg, fungi)

The incidence of culture-negative IE varies by country, with a higher proportion of culture-negative IE in resource-limited settings [5,6,9-14]. The variability in incidence is illustrated by the following studies:

●A case series from Algeria found 62 of 110 (56 percent) cases of IE were culture negative [9].

●A similar rate was found in South Africa, 26 of 47 (55 percent), and, in Pakistan, 32 of 66 (48 percent) patients with definite IE were culture negative [10,11].

●A 10-year national prospective survey in the Slovak Republic found that 133 of 339 (39 percent) cases of IE were culture negative [12].

●A two-year multicenter prospective study in Italy found 37 of 147 (25 percent) patients with a definite diagnosis of IE by Duke criteria were culture negative [13].

●A case series from Sweden identified 116 of 487 (24 percent) episodes of IE were culture negative; however, if patients who had received antibiotic treatment were excluded, the proportion of culture-negative IE was 64 of 487 (13 percent) [5].

●A second study from Spain addressed the differences in rates of culture-negative endocarditis in patients with or without previous antibiotics; rates in patients who did or did not receive antibiotic treatment were 20 of 107 (19 percent) compared with 6 of 107 (6 percent) [6].

●A case series from Japan noted 170 of 848 (20 percent) patients with IE were culture negative [15].

●A case series from France reported 79 of 427 (18.5 percent) patients with IE were culture negative [2].

●A case series from the United Kingdom found that 63 of 516 (12 percent) cases with IE were culture negative [14].

The differences in frequency of culture-negative IE in countries reflect the increased incidence of fastidious zoonotic agents (eg, Bartonella spp, Coxiella burnetii, or Brucella spp) causing human infection in resource-limited settings [9], the differences in microbiologic techniques, and, perhaps, the availability of antibiotics without prescription in some countries.

Risk factors — Risk factors for culture-negative IE include the risk of exposure to fastidious organisms including zoonotic agents, underlying valvular heart disease, and the presence of a pacemaker [14,16-18]. Culture-negative IE due to Aspergillus has also been reported in a patient with a left ventricular assist device [19]. There is no convincing evidence that endocarditis involving the tricuspid valve is more likely to be culture negative [20].

Clinical clues to the diagnosis — Inquiry about the following exposures or conditions may be useful during the assessment of individual patients:

●Contact with or occupational exposure to farm animals such as sheep, cattle, and goats, particularly to their products of conception (Brucella spp, Coxiella); such contact may be indirect through aerosolization of placental or parturient fluids or contaminated soil or straw (Coxiella).

●Contact with the human body louse or homeless shelters (Bartonella quintana).

●Cat contact or ownership (Bartonella henselae).

●Ingestion of unpasteurized milk (Brucella and Coxiella) or cheese or insufficiently cooked meat (Brucella spp).

●Immunosuppression, including HIV infection (fungi, Coxiella).

●Chronic alcoholism (B. quintana).

●Recent or prolonged antibiotic use.

●Travel to the Middle East or other areas where brucellosis is common.

●Origin from North Africa where B. quintana is common.

●Occupational exposure to soil or farm animals (Tropheryma whipplei).

●Occupational exposure to abattoirs (Coxiella).

●Laboratory exposure to pathogens (Histoplasma, Coxiella).

MICROBIOLOGY — The HACEK (Haemophilus aphrophilus [subsequently called Aggregatibacter aphrophilus and Aggregatibacter paraphrophilus]; Actinobacillus actinomycetemcomitans [subsequently called Aggregatibacter actinomycetemcomitans]; Cardiobacterium hominis; Eikenella corrodens; and Kingella kingae) organisms were traditionally thought to be the most common agents of culture-negative endocarditis. However, studies have found that the HACEK organisms can be easily isolated with current blood culture systems when incubated for at least five days [21,22]; therefore, they are no longer important causes of blood culture-negative infective endocarditis (IE).

In retrospective study at three university medical centers between 2003 and 2004, findings included [22]:

●The mean and median times to detection of HACEK isolates from blood cultures were 3.4 and 3.0 days, respectively.

●None of 407 blood cultures in patients with suspected culture-negative endocarditis grew HACEK or other bacteria with extended incubation (10 to 14 days).

The most common causative agents of blood culture-negative IE are fastidious organisms (eg, zoonotic agents and fungi) and Streptococcus spp in patients who have received previous antibiotic treatment.

Large series of culture-negative endocarditis include:

●A study including 759 individuals with endocarditis; of these, a cause was identified in 63 percent of cases [23]. These included 19 patients with noninfective endocarditis (7 marantic, 9 systematic lupus erythematosus, 2 rheumatoid arthritis, 1 Behçet diseases), 229 with Q fever, 86 with Bartonella, 12 with T. whipplei, 8 with fungal infection, and 2 with Finegoldia magna [23]. Rare organisms from this series included Legionella pneumophila, Mycobacterium tuberculosis, and Abiotrophia spp.

●A study including 819 cases of suspected blood-culture negative IE: comprehensive diagnostic testing revealed the following: (1) endocarditis was excluded by pathologic findings in 60 patients; (2) 19 patients did not have IE; (3) an etiologic agent was identified in 476 patients; and (4) no etiologic agent was identified in the remaining 264 patients (of which 73 [28 percent] had definite endocarditis). Of the 476 patients who had an etiologic agent identified, 229 had Q fever, 86 were infected with a Bartonella spp, 70 had a common bacterial agent, 12 had T. whipplei infection, and 8 had fungal endocarditis [23].

●A study including 177 patients with culture-negative endocarditis; an etiologic agent was identified in 70 percent of cases using multimodal diagnostic strategy [8].

The utility of the above observations may not be applicable to other geographic areas, as the prevalence of infection due to pathogens such as C. burnetii and Bartonella spp varies widely between geographic locations and epidemiologic settings.

C. burnetii may cause 3 to 10 percent of cases of IE in European countries other than France [24]. The annual incidence of Q fever has been estimated to be one per one million inhabitants per year in France, Israel, and Switzerland [25]. Q fever was less common in northern Europe and the United Kingdom but is emerging in Netherlands following a large outbreak [26]. (See "Q fever endocarditis".)

The prevalence of Bartonella IE varies in Europe, increasing from north to south. The prevalence of Bartonella IE is reported as less than 1 percent in northern Europe, 1 percent in England, 3 percent in France and Germany, and ≥10 percent in Tunisia and Algeria [9,27]. Most of the cases found in Northern Africa are caused by B. quintana. (See "Endocarditis caused by Bartonella".)

T. whipplei may be a more important cause of culture-negative IE than previously recognized. In an observational cohort study including 255 patients with evidence of bacterial colonization on explanted valve tissue, T. whipplei was isolated in 6 percent of cases in Germany [28]. It was the most commonly isolated pathogen in individuals with culture-negative IE and the fourth most commonly isolated pathogen overall. In a subsequent study, T. whipplei was linked clinically to previous chronic arthralgias [29]. Fungi are a common cause of postoperative nosocomial blood culture-negative endocarditis [30].

Mycobacteria (including M. tuberculosis and Bacille-Calmette Guérin) are rare causes of IE [7,31]. In addition, IE due to Mycobacterium chimaera and Mycobacterium abscessus has been described in association with airborne transmission from contaminated heater-cooler unit water tanks during open cardiac surgery [32-36]. (See "Overview of nontuberculous mycobacterial infections", section on 'M. chimaera associated with cardiac surgery'.)

Cutibacterium (formerly Propionibacterium) acnes is a rare cause of IE that may be missed if routine blood cultures are discarded after five days of incubation. In one series including 1325 episodes of definite IE by Duke Criteria between 2007 and 2015 reported by Cleveland Clinic, 24 cases due to C. acnes were observed [37]. The median time to detection of growth in blood cultures was seven days (range three to nine days). The median time to positivity of valve cultures was 5.5 days (range 3 to 28 days). Six patients had negative blood and valve cultures but positive bacterial 16s rDNA sequencing results. [38,39]

DIAGNOSIS — Diagnostic tests for culture-negative endocarditis include special culturing techniques (eg, shell vial and lysis centrifugation), molecular techniques (eg, polymerase chain reaction [PCR] and serologic assays), and histopathology evaluation of valvular tissue when surgical excision is performed.

Histopathology — Explanted valve tissue should be evaluated with histopathologic examination, as well as PCR testing (see 'Polymerase chain reaction' below). Histopathology can be useful for pathogen identification of infective endocarditis (IE) as well as noninfectious mimics including marantic endocarditis, rheumatic endocarditis, and myxoma.

Special tissue staining (Gram, Giemsa, acid-fast, Warthin-Starry, and Periodic acid-Schiff stain) may allow a presumptive identification of the etiologic agent [24]. In addition, immunohistochemistry with specific polyclonal or monoclonal antibodies can identify specific causative agents including Bartonella spp [40], C. burnetii [41], and T. whipplei [42,43].

Autoimmunohistochemistry is a novel method to detect microorganisms that uses the patient's own serum [44]. The rate of detection is higher than culture and similar to PCR.

Fluorescence in situ hybridization has been used to identify the cause of culture-negative IE [45].

Molecular techniques

Polymerase chain reaction — Most data on PCR for the diagnosis of culture-negative endocarditis come from its use in testing explanted valve tissue [16,46-52], although PCR testing of serum samples may also be useful [53].

Broad-range PCR is a highly sensitive technique that amplifies small quantities of bacterial or fungal DNA; if combined with sequencing, it can identify a specific organism [48,54,55]. Molecular testing of excised valve material using PCR is most useful in cases in which definitive microbiologic diagnosis cannot be established based on culture or serology alone. PCR is particularly useful in blood culture-negative patients with previous antibiotic exposure, since bacterial DNA frequently persists even when organisms are present in quantities too low to be detected via culture. However, because bacterial DNA may persist after eradication of viable organisms, and because sample contamination may be associated with false-positive results, PCR findings should be interpreted in the context of other clinical information and should not be used to guide duration of therapy.

There are broad-range PCR techniques for amplifying 16SrDNA (for bacteria) or 18SrDNA (for fungi), which can then be sequenced for pathogen identification. The most frequently identified bacteria have included streptococci, enterococci, staphylococci, Bartonella spp, and T. whipplei [8,16,46-51,53,56]. (See "Endocarditis caused by Bartonella" and "Q fever endocarditis" and "Whipple's disease".)

The sensitivity of PCR for pathogen identification in culture-negative endocarditis ranges from 40 to 60 percent, the specificity is laboratory dependent but reaches nearly 100 percent in some settings [46,47]. These studies used a gold standard of Duke criteria together with histopathologic examination. In comparison, valve culture rates have a sensitivity of up to 13 percent, and histopathology sensitivity can vary from 12 to 63 percent [46,47]. Specific-pathogen PCR has proven most sensitive than broad spectrum PCR [8,57].

Metagenomic sequencing of valve tissue to establish etiologic diagnosis of infective endocarditis has been described [58].

Serologic assays — The etiologic agents in culture-negative IE best identified by serology include C. burnetii, Bartonella spp, Legionella spp, and Brucella spp [2]. (See appropriate topic reviews.)

C. burnetii phase I antibody titers >800 are diagnostic for Q fever endocarditis. The presence of immunoglobulin (Ig) G antiphospholipid during acute Q fever is highly predictive of evolution to endocarditis [59]. Bartonella IgG levels ≥800 are diagnostic for Bartonella spp [2]. However, patients with Q fever may have low-level cross-reacting antibodies to Bartonella spp and/or Chlamydia spp [24]. Furthermore, the role of Chlamydia spp in the etiology of IE is controversial and difficult to ascertain because of the cross-reacting antibodies to Bartonella spp [24].

Western blot serology may be helpful for diagnosis of enterococcus and Streptococcus gallolyticus endocarditis, which may be blood culture negative [60].

Imaging techniques — Fluorodeoxyglucose positron emission tomography with computed tomography (FDG-PET-CT) has been used to identify infected cardiac devices, periprosthetic valve abscesses, mycotic aneurysms, and prosthetic heart valve infections and may contribute to diagnosis, specifically for fastidious organisms, such as T. whipplei and C. burnetii [61-63]. FDG-PET-CT has favorable positive and negative predictive values in patients with prosthetic valve IE (>90 percent) but is less useful in patients with native valve IE [64].

DIFFERENTIAL DIAGNOSIS — The differential diagnosis of culture-negative infective endocarditis (IE) includes conditions that produce noninfectious valvular or intracardiac vegetations. These include [23]:

●Antiphospholipid syndrome – Antiphospholipid syndrome is characterized by thromboses, thrombocytopenia, livedo reticularis, and/or stroke. Ischemic stroke may occur due to in situ thrombosis or embolism arising from valvular heart disease. The diagnosis of antiphospholipid syndrome is established by the presence of antiphospholipid antibodies. Antiphospholipid syndrome may occasionally be associated with Q fever [65]. (See "Clinical manifestations of antiphospholipid syndrome".)

●Acute rheumatic fever – Clinical manifestations of acute rheumatic fever include arthritis, carditis and valvulitis, central nervous system involvement, erythema marginatum, and subcutaneous nodules. Cardiac involvement may include verrucous vegetations, which can be visualized echocardiographically. The diagnosis evaluation includes establishing the diagnosis of group A streptococcal infection (via throat culture, streptococcal antigen test, or antistreptolysin O titer), measurement of acute phase reactants, and assessment of cardiac function. (See "Acute rheumatic fever: Clinical manifestations and diagnosis".)

●Atrial myxoma – Clinical manifestations of atrial myxoma resemble symptoms of mitral valve obstruction; these include dyspnea, hemoptysis, atrial fibrillation, and thromboembolism. In addition, constitutional symptoms and laboratory abnormalities suggestive of connective disease may be observed, and electrocardiography may demonstrate left atrial hypertrophy. Atrial myxoma may be visualized echocardiographically. (See "Cardiac tumors", section on 'Myxomas'.)

●Nonbacterial thrombotic endocarditis (NBTE; also known as marantic endocarditis or Libman-Sachs endocarditis) – NBTE refers to a spectrum of noninfectious lesions of the heart valves seen in the setting of advanced malignancy, systemic lupus erythematosus, or hypercoagulable state. The major clinical manifestations result from systemic emboli. Echocardiography may be performed to detect valvular vegetations. (See "Nonbacterial thrombotic endocarditis" and "Non-coronary cardiac manifestations of systemic lupus erythematosus in adults", section on 'Valvular disease'.)

●Vasculitis – Various disorders associated with vasculitis such as polyarteritis nodosa, Behçet disease, or granulomatosis with polyangiitis may present with symptoms and signs that mimic culture-negative IE. The clinical manifestations and diagnosis of these disorders are discussed separately. (See "Overview of and approach to the vasculitides in adults".)

●Temporal arteritis – Clinical manifestations of temporal arteritis include headache, abrupt onset of visual disturbances, symptoms of polymyalgia rheumatica, fever, anemia, and elevated inflammatory markers. The diagnosis is established via temporal artery biopsy. (See "Diagnosis of giant cell arteritis".)

●Connective tissue disease – Symptoms of connective tissue disease include fatigue, myalgia, arthralgia, and fever. The diagnosis is established based on clinical manifestations and antibody titers. (See "Definition and diagnosis of mixed connective tissue disease".)

●Cholesterol emboli syndrome – Cholesterol embolism occurs when atherosclerotic plaque is disrupted and cholesterol crystals embolize distally. Clinical manifestations depend on the location of the embolic source, extent of embolization, and degree of preexisting disease in the affected vascular bed; they may include systemic or cerebral embolism. (See "Embolism from atherosclerotic plaque: Atheroembolism (cholesterol crystal embolism)".)

●Mural thrombi associated with cardiomyopathy or heart failure – There is an increased propensity for left ventricular thrombus formation in patients with left ventricular systolic dysfunction. This may be associated with systemic or cerebral embolism. The diagnosis is established by echocardiography. (See "Left ventricular thrombus after acute myocardial infarction".)

●Lambl's excrescences – Tiny mobile strands on cardiac valves may be visualized via transesophageal echocardiography and mistaken for vegetations; they probably represent a normal degenerative process. (See "Role of echocardiography in infective endocarditis", section on 'Differential diagnosis of cardiac masses identified by echocardiogram'.)

●Paradoxical emboli due to right-to-left cardiac shunts – Patients with an atrial septal defect or patent foramen ovale with a right-to-left shunt are at risk for stroke due to paradoxical embolization. These lesions may be diagnosed via echocardiography. (See "Clinical manifestations and diagnosis of atrial septal defects in adults".)

●Atrial fibrillation – Patients with atrial fibrillation may present with palpitations, tachycardia, fatigue, lightheadedness, dyspnea, and/or an embolic event. The diagnosis is established via electrocardiogram; echocardiography is useful to identify the presence of left atrial thrombi. (See "Overview of atrial fibrillation".)

In addition to the above conditions, allergic endocarditis to pork in patients with prosthetic valve endocarditis has been described [66].

SOCIETY GUIDELINE LINKS — Links to society and government-sponsored guidelines from selected countries and regions around the world are provided separately. (See "Society guideline links: Treatment and prevention of infective endocarditis".)

SUMMARY

●Blood culture-negative infective endocarditis (IE) is defined as endocarditis without etiology following inoculation of three independent blood samples in a standard blood culture system with negative cultures after seven days of incubation and subculturing. However, HACEK (Haemophilus aphrophilus [subsequently called Aggregatibacter aphrophilus and Aggregatibacter paraphrophilus]; Actinobacillus actinomycetemcomitans [subsequently called Aggregatibacter actinomycetemcomitans]; Cardiobacterium hominis; Eikenella corrodens; and Kingella kingae) organisms often grow within five days using the BACTEC system. (See 'Definition' above.)

●Cultures remain negative in 2 to 7 percent of patients with IE even when the utmost care is taken in obtaining the proper number and volume of blood cultures and patients with prior antibiotic treatment are excluded; the frequency is higher in patients who have already been treated with antibiotics. (See 'Incidence' above.)

●Cultures are negative in patients with IE for three major reasons:

•Previous administration of antimicrobial agents

•Inadequate microbiological techniques

•Infection with highly fastidious bacteria (eg, streptococci or Cutibacterium) or nonbacterial pathogens (eg, fungi)

A noninfectious cause of endocarditis must be investigated when blood culture are negative. (See 'Incidence' above.)

●The incidence of culture-negative IE is variable, with a higher proportion of IE being culture-negative in resource-limited settings. The differences in frequency reflect the increased incidence of fastidious zoonotic agents causing human infection in resource-limited settings, the differences in microbiologic techniques, and, perhaps, the availability of antibiotics without prescription in some countries. (See 'Incidence' above.)

●The most common causative agents of blood culture-negative IE are fastidious organisms (eg, zoonotic agents and fungi) and Streptococcus spp in patients who have received previous antibiotic treatment. (See 'Microbiology' above.)

●The local prevalence of infection with pathogens such as Coxiella burnetii and Bartonella spp, the most common agents of culture-negative endocarditis, varies widely in different geographic locations and epidemiologic settings. (See 'Microbiology' above.)

●Serology and polymerase chain reaction on blood samples or removed valves help to identify fastidious pathogens. (See 'Molecular techniques' above.)