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The article explores infective endocarditis (IE) caused by rare and fastidious bacteria, which frequently lead to culture-negative cases and complicate the diagnostic process. While IE is typically diagnosed through persistent bacteremia with gram-positive cocci and the detection of vegetations via echocardiography, advanced echocardiographic techniques—particularly transesophageal echocardiography—significantly enhance the sensitivity of the Duke criteria. However, echocardiography is more effective in confirming the presence of IE rather than identifying the specific microbial cause, with approximately 24% of proven IE cases being misclassified as “possible” when blood cultures are negative.

**Uncommon Pathogens and Specialized Diagnostics:**

Uncommon pathogens such as **Bartonella spp.**, **Coxiella burnetii**, **Lactobacillus**, **Legionella spp.**, **Chlamydia psittaci**, and members of the **HACEK** group necessitate specialized diagnostic approaches. For instance, *Bartonella spp.*, including *B. henselae* and *B. quintana*, are small facultative intracellular gram-negative bacteria that cause various clinical syndromes. *B. henselae* is associated with cat scratch disease and endocarditis in patients with prior valve injury and cat exposure, whereas *B. quintana* is linked to trench fever and endocarditis in homeless or alcoholic individuals exposed to body lice. Specialized diagnostics for these pathogens involve serological tests (e.g., immunofluorescence assays), polymerase chain reaction (PCR) amplification of specific genes (such as the 16S rRNA gene), special staining techniques like Warthin-Starry, and optimized culture conditions.

Additionally, **Whipple's Disease** caused by *Tropheryma whippelii* presents unique diagnostic challenges. Diagnosed through the presence of PAS-positive macrophages in duodenal or valvular biopsy specimens and confirmed by PCR and sequencing, Whipple's disease endocarditis is likely underreported due to its subtle presentation and reliance on invasive diagnostic methods.

**Legionella spp.** endocarditis, though exceedingly rare, is associated primarily with nosocomial infections and prosthetic valves, requiring specialized culture techniques such as buffered charcoal yeast extract (BCYE) media and PCR for accurate identification. **Chlamydia psittaci** endocarditis is often misdiagnosed due to cross-reactivity with Bartonella spp. in serological tests, necessitating advanced molecular techniques for definitive identification. **Coxiella burnetii** (Q fever) endocarditis is another significant etiology, particularly prevalent in individuals with occupational exposure to farm animals, diagnosed through high-phase I IgG and IgA antibodies, PCR, and histologic observation of infected tissues.

**HACEK Group Bacteria:**

The HACEK group comprises fastidious, small gram-negative bacilli primarily from the oropharyngeal flora, responsible for approximately 3% of all endocarditis cases. This group includes *Haemophilus parainfluenzae*, *H. influenzae*, *H. aphrophilus*, *H. paraphrophilus*, *Actinobacillus actinomycetemcomitans*, *Cardiobacterium hominis*, *Eikenella corrodens*, and *Kingella spp.* These organisms are slow-growing, with growth enhanced by carbon dioxide, and require specific culture conditions. Each HACEK organism demonstrates unique epidemiological and clinical characteristics:

- **Haemophilus paraphrophilus**: Rare, often associated with mitral valve prolapse and previous valve abnormalities. Clinical features include fever, cardiac murmurs, and embolic events. Valve replacement is required in 24% of cases.
  
- **Haemophilus aphrophilus**: Commonly linked to dental procedures and upper respiratory infections. Echocardiography detects vegetations in 70% of patients. Improved diagnostic delay has led to better prognoses in recent reports.

- **Haemophilus parainfluenzae**: Presents insidiously, often following dental procedures. Embolic complications are frequent, with a case fatality rate between 10-35%.

- **Actinobacillus actinomycetemcomitans**: Associated with periodontal disease and prior dental infections. Affects young to middle-aged patients, with a case fatality rate of 9-15% and valve replacement required in 28% of cases.

- **Cardiobacterium hominis**: Linked to dental infections and underlying heart disease, predominantly affecting the aortic valve. Mortality is around 13%, with 30% requiring valve replacement.

- **Eikenella corrodens**: Often found in intravenous drug users due to syringe contamination. Generally causes a mild course with a 15% fatality rate and rare need for valve replacement.

- **Kingella spp.**: *Kingella kingae* primarily affects young children, while *Kingella denitrificans* is less common. In older patients, prosthetic valves are a significant risk factor.

**Effective identification strategies** include obtaining multiple blood cultures under stringent conditions—typically three sets within 24 to 48 hours with adequate blood volume—to establish culture-positive endocarditis. Prior antibiotic therapy, common in many patients, can significantly reduce blood culture yields. To mitigate this, methods such as diluting blood samples in broth, using antimicrobial agent removal devices, and employing specialized culture media are essential for isolating fastidious organisms.

**Clinical Presentations and Diagnostic Challenges:**

IE caused by these atypical organisms may present with subacute and insidious symptoms, often lacking conventional signs like fever and cardiac murmurs, especially in elderly patients or those who have received prior antibiotics. Additional clinical manifestations include:

- **Digital Clubbing**: Observed in one-third of patients, a higher rate than typically seen in other forms of endocarditis.
  
- **Purpuric Rash**: Found in one-fifth of patients, usually occurring on the extremities and mucosa. Skin biopsies reveal immune complex vasculitis.
  
- **Renal Involvement**: Proliferative glomerulonephritis has been reported, increasing the risk of renal insufficiency. Laboratory findings may include microscopic hematuria, elevated serum hepatic transaminases, lactate dehydrogenase, creatinine phosphokinase, cryoglobulins, and hyperglobulinemia.
  
- **Embolic Manifestations**: Occur in about 20-41% of patients, involving cerebral and extremity vessels. Arterial embolism may present as stroke, potentially requiring embolectomy or amputation.
  
- **Pulmonary and Pleural Manifestations**: Can be complications of endocarditis.

**Specific Presentations in Rare IE Types:**

- **Whipple's Disease Endocarditis**: May include systemic symptoms like diarrhea, weight loss, neurological manifestations, and acute congestive heart failure.
  
- **Legionella Endocarditis**: Associated with chronic symptoms such as low-grade fever, night sweats, weight loss, and signs of congestive heart failure without embolic events.
  
- **Coxiella burnetii Endocarditis (Q Fever)**: Typically affects individuals with preexisting valvular injuries, presenting with prolonged fever, cardiac dysfunction, splenomegaly, and hepatomegaly. Laboratory findings often include high-phase IgG and IgA antibodies, circulating immune complexes, rheumatoid factor, anemia, thrombocytopenia, hyperglobulinemia, and various autoantibodies.

Advanced molecular and histological techniques have substantially improved the identification and management of these challenging IE cases. Techniques such as PCR amplification of the 16S rRNA gene, immunohistologic testing, and specialized histologic stains (e.g., Warthin-Starry, Gimenez) are invaluable. Additionally, thorough patient histories regarding animal contact, occupational exposures, and potential sources of infection are crucial for accurate diagnosis.

**Treatment and Prognosis:**

Treatment of IE caused by rare pathogens like *Bartonella spp.* typically involves a combination of antibiotics tailored to the specific organism's susceptibility profile. For instance:

- **Bartonella Endocarditis**: Often treated with aminoglycosides in combination with doxycycline or ceftriaxone for a prolonged course.
  
- **Whipple's Disease Endocarditis**: Requires prolonged antibiotic therapy targeting *T. whippelii*, often necessitating surgical intervention such as valve replacement due to extensive valvular damage.
  
- **Legionella Endocarditis**: Treated with macrolides or fluoroquinolones.
  
- **Coxiella burnetii Endocarditis (Q Fever)**: Typically requires doxycycline combined with hydroxychloroquine for an extended duration (at least 18 months). This combination has proven more effective than doxycycline with quinolones, reducing relapse rates and shortening therapy duration. Serum antibody surveillance and plasma level monitoring of hydroxychloroquine are essential. Surgery, such as valve replacement, is frequently required across these atypical infections due to extensive damage.

The prognosis can be poor, especially in cases with delayed diagnosis and treatment, as evidenced by higher mortality rates among certain populations like homeless individuals with *B. quintana* endocarditis. However, advancements in diagnostic and therapeutic strategies have improved outcomes for many patients.

**Other Rare Etiologies:**

Beyond Bartonella and the newly detailed pathogens, other rare causative agents of IE include:

- **Mycobacterium spp.**: Acid-alcohol-resistant bacteria causing exceptionally rare forms of endocarditis, often associated with prosthetic valves or medical procedures. Diagnosis typically relies on histologic examination and specialized culture techniques, with combination antibiotic therapy recommended.
  
- **Mycoplasma spp.**: Cell wall-deficient bacteria implicated in very few reported cases of IE. Detection is challenging due to their inability to be visualized by Gram stain and growth in routine cultures. Strategies for isolation include specific agar media and PCR techniques.
  
- **Whipple's Disease Bacterium (*Tropheryma whippelii*)**: A rare systemic disorder that can present with endocarditis among other symptoms. Diagnosis may involve PCR and specialized culture methods, with limited treatment data available.

**Conclusion:**

The integration of enhanced echocardiographic methods with the Duke criteria, combined with advanced microbiological and molecular diagnostics, significantly improves the detection and management of infective endocarditis caused by less common and fastidious bacterial agents. Understanding the epidemiology, clinical presentations, and appropriate diagnostic and therapeutic strategies for these rare pathogens is essential for improving patient outcomes. Active investigation for atypical organisms, especially in culture-negative cases, and a high index of suspicion based on patient history and clinical features are crucial for timely and accurate diagnosis and effective treatment.
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In older patients, the most prevalent underlying heart disease factor was prosthetic valve replacement, which was observed in one-third of patients.

Poor dental hygiene or oral surgery have been reported to be associated with infection.

However, it is interesting that Kingella spp.

predominantly colonize the upper respiratory tract, in particular the tonsils.

Rather than being a primary pathogen, this organism probably exploits mucosal impairment that results from viral infection.

Thus, concomitant stomatitis, pharyngitis, or other varicella virus-induced buccal ulcers have been reported in children with Kingella bacteremia303.

A fatality rate of 16% has been reported in K. kingae endocarditis, and central nervous system emboli were detected in 25% of cases.

(ii) Diagnosis and echocardiography.

Because of slow growth, HACEK group bacteria should be considered in the differential diagnosis of culture-negative endocarditis.

Although the mean duration for incubation of blood cultures until detection of growth is 3 to 5 days, up to 30 days may be required, as reported with A. actinomycetemcomitans endocarditis.

Nevertheless, blood cultures are often positive; for example, blood cultures are positive in 90% of cases of A. actinomycetemcomitans endocarditis when incubated for longer than 8 days 107.

In fact, no significant difference in bacterial growth was found between the Septicheck bottle (Becton Dickinson, Cookeysville, Md.)

containing 70 ml of tryptic soy broth and the Isolator tube (Wampole Laboratories, Cranbury, N.J.) 54, 190.

Culture and identification of bacteria from the HACEK group has been reviewed elsewhere 35, 194, 242.

Briefly, bacteria can be identified after subculture on 5 to 8% sheep blood and chocolate agar at 35 to 37°C for 48 to 72 h in an aerobic atmosphere containing 5 to 10% CO2.

Most species of Haemophilus grow well on conventional chocolate agar but do not grow at all on standard 5% sheep blood agar.

All species of Haemophilus require either exogenous hemin (X factor) or NAD (V factor).

Haemophilusspp.

can be grown on a nutrient-rich, nonselective medium lacking X and V factors (e.g., Mueller-Hinton agar) by applying a commercially available filter paper disk saturated with X, V, or X and V factors to the surface medium.

This technique is useful for identification ofHaemophilus at the level species.

Several commercially available kits identify and biotype Haemophilus spp., such as the API 10E and API 20E systems (Biomérieux Vitek, St. Louis, Mo.)

and the HNID system (Dade Behring, West Sacramento, Calif.35.

A. actinomycetemcomitans is positive for both the oxidase and alkaline phosphatase reactions.

Nitrates are reduced to nitrite but not gas, no growth is observed on Simmons citrate medium, the arginine dihydrolase reaction is negative, and no acid is produced from adonitol and sorbose 195.

Biochemical properties used to differentiate members of the HACEK group are listed in Table 5.

However, under some circumstances, identification is difficult and molecular techniques using 16S rRNA gene analysis may be useful (Fig.

7) 53, 113.

Valve vegetations are detected by echocardiography in 60 to 85% of patients and are characteristically thought to be large15.

No correlation between the size of the vegetations as determined by transthoracic echocardiography and the subsequent rate of emboli was found 54.

Care must be taken when diagnosing polymicrobial endocarditis due to bacteria from the oropharynx because prolonged incubation may allow the recovery of an associated HACEK microorganism.

This is important because the HACEK group organisms could be resistant to ampicillin and/or clindamycin, which may explain therapeutic failures.

Table 5.

(iii) Treatment and outcome.

Medical treatment alone or associated with surgical treatment can cure 82 to 87% of patients with endocarditis caused by HACEK group organisms 15.

In the past, ampicillin plus gentamicin was the treatment of choice.

However, β-lactamase-producing organisms in this group have prompted a change in treatment strategy, which should now include a β-lactamase-stable cephalosporin in place of ampicillin109.

The American Heart Association recommends that native-valve and prosthetic-valve endocarditis be treated for 4 and 6 weeks, respectively.

The recommended prophylaxis for dental procedures is currently amoxicillin.

For allergic patients, clindamycin or pristinamycin are alternatives, but it is important to note that 40% of A. actinomycetemcomitans strains are resistant to both antibiotics and E. corrodens is resistant to clindamycin, suggesting the use of tetracycline as yet another alternative107.

Campylobacter fetus.

Campylobacter fetus is a small gram-negative bacterium.

C. fetusendocarditis has been reported in 21 patients 77.

Among these, males were more often involved, previous valve disease was noted in 30%, and prosthetic-valve endocarditis was reported only twice.

One-third of the patients suffered from concurrent illness (hepatic cirrhosis, alcoholism, connective tissue disease, or tuberculosis)77.

Death occurred in 25% of patients.

The current recommended treatment is imipenem, cilastatin, and gentamicin77.

Pasteurella spp.

Pasteurellaspp.

are small, gram-negative, nonmotile coccobacilli that are distributed worldwide.

These organisms are part of the normal flora of the nasopharynx or the gastrointestinal tract in wild animals and in 50% of cats and dogs; they are rarely present in the respiratory tracts of healthy humans.

Endocarditis due to Pasteurellaspp.

has been reported in 21 cases in the literature 200, 255, 287.

Common characteristics include close contact with a cat or a dog in 44% of reported cases and hepatic cirrhosis in 6%.

Only one case involved a prosthetic valve.

Other species ofPasteurella such as Pasteurella dagmatis, Pasteurella haemolytica, and Pasteurella gallinarum may also cause endocarditis 255.

Death occurred in 31% of patients and was more likely to be related to diagnostic delay than to antibiotic susceptibility of the bacteria.

Brucella spp.

Brucella spp.

are small, facultative, gram-negative, intracellular bacteria that cause abortion in domesticated animals: B. abortus in cattle,B.

suis in swine, and B. melitensis in goats and sheep.

Most cases of human brucellosis are caused by B. melitensis, a pathogen of sheep, which is the most virulentBrucella sp.

(i) Epidemiology.

Brucellosis, primarily a worldwide zoonosis caused by Brucella spp., is transmitted to humans mainly through ingestion of contaminated milk or milk products or by close contact with infected livestock or their tissues or secretions.

It is important to note that a similar epidemiologic situation is observed with Coxiella burnetii, the agent of Q fever.

However, the degree of disease control in animals varies among countries, accounting for discrepancies in the number of reported cases, with most being reported from Spain and Italy.

The disease, which is rarely fatal, may take a febrile acute, subacute, or chronic course.

Endocarditis is a rare complication of brucellosis and occurs in 0.3 to 0.6% of patients but is the most frequent cause of death.

Endocarditis due to Brucella spp.

varies from less than 1 to 4% of all cases of bacterial endocarditis depending on the geographic area 82.

We recorded 120 published cases ofBrucella endocarditis 4, 58, 85, 129, 252; A. Micozzi, M. Venditti, G. Gentile, N. Alessandri, M. Santero, and P. Martino, Letter, Eur.

J. Clin.

Microbiol.

Infect.

Dis.9:440–442, 1990; C. Sungur, A. Sungur, G. Gedikoglu, A. Usubutun, U. Yasavul, C. Turgan and S. Caglar, Letter, Nephron67:234–235, 1994).

(ii) Signs and symptoms.

Most patients with B. melitensis endocarditis follow a subacute course over a period of 2 to 10 weeks 83.

Fever, generalized aches, cardiac murmur, and enlargement of the liver and spleen are the most prominent clinical findings.

On rare occasions, Brucella endocarditis follows an afebrile course, complicated by disseminated intravascular coagulation 5.

In patients with prosthetic valves, relapsing bacteremia after appropriate treatment for acute brucellosis is an important clue to the diagnosis of endocarditis.

In a review on zoonotic endocarditis, Fernandez Guerrero reported that 70% of the 20 patients they monitored in Madrid developed cardiac failure82.

Thus, one should consider the diagnosis ofBrucella endocarditis in an epidemiologically exposed patient who presents with febrile cardiac failure.

(iii) Pathology and echocardiography.

Brucella endocarditis is a destructive process predominantly involving the aortic valve and perivascular tissues129, 211.

Evidence of underlying valvular disease, including prosthetic valves, is noted in two-thirds of patients129.

The valvular lesions have been described as bulky and ulcerative with gross abscesses of the myocardium, microabscesses within the cusps, destruction of the commissures, and calcification211.

Myocardial abscesses have been found in 43% of patients dying of Brucella endocarditis 211; aortic root abscess seems to be a common complication when the aortic valve is involved 4.

Ring abscesses leading to valve detachment occur in most instances of prosthetic valve endocarditis caused by B. melitensis83.

These observations help to explain the high fatality rate for Brucellaendocarditis.

(iv) Diagnosis.

Diagnosis depends on the isolation of Brucella spp.

from blood cultures or cardiac tissue.

Isolation of Brucella spp.

is hazardous, and the physician should inform the microbiologist of such a suspected diagnosis.

Blood cultures are positive for Brucella in more than 80% of cases if the incubation time is prolonged to 4–6 weeks.

Culture of vegetations commonly yields Brucella spp.

even after prolonged antimicrobial chemotherapy 83.

Historically, culture of blood for Brucella has been performed by broth methods that required prolonged incubation and the use of blind subculture.

Brucella spp.

grow on most standard laboratory media including blood, chocolate, and tryptic soy agar when incubated at 35°C in 5 to 10% CO2.

Culture has also been performed with the biphasic Ruiz-Castaneda bottle.

Currently available methods that have been advocated include both lysis-centrifugation (Isolator) blood cultures and the use of automated, continuous-monitoring blood culture instrumentation such as EPS, Bactec NR, Bactec NR660, or BacT/Alert.

A shorter isolation time for Brucella spp.

was obtained by the lysis-concentration technique than by using Bactec NR198.

These systems may yield positive cultures in 4 to 10 days, but prolonged incubation (4 to 6 weeks) may be needed, particularly in patients treated with antimicrobial agents145.

In laboratory practice, it is still recommended to perform both early and terminal subculture from the bottle in the ESP, Bactec NR, Bactec NR660, or BacT/Alert system when brucellosis is suspected and to hold these bottles for a minimum of 21 days.

In the absence of bacteriologic confirmation, serologic tests are used to make a presumptive diagnosis 304.

Although several methods have been developed to detect IgA, IgG, and IgM, such as IFA, ELISA, and Western blots analysis, the standard tube agglutination test is still the reference test.

Most cases of active infection will be associated with titers of 1:160 or greater.

The physician should be aware of the serologic cross-reactions that exist between Brucella,Yersinia, and Francisella spp.

that may lead to confusion in etiologic diagnosis 64.

(v) Treatment.

Although antimicrobial agent treatment can result in sterilization of valve vegetations83, only a few patients with definite endocarditis caused by Brucella spp.

have been cured by antibiotics alone (16, 44; Micozzi et al., Letter), and before the introduction of open-heart surgery, the mortality ofBrucella endocarditis was greater than 80%.

The current recommendation is combined medical and surgical treatment, especially with infected prosthetic valves 129, 252.

Quinolones are very efficient in vitro but have been very disappointing in clinical trials, with failure rates of 16 to 66% in acute brucellosis; therefore, they should not be used 150.

Most authors recommended treatment of Brucella endocarditis with a combination of doxycycline and either rifampin or streptomycin for several months.

It is advisable to extend therapy for a minimum of 6 to 8 weeks postoperatively 82.

A progressive drop in antibody titer is evidence that the patient is cured.

Patients with a relapse or a failure have high levels of IgG remaining 31.

Bordetella spp.

Only one case ofBordetella holmesii endocarditis has been reported in the literature 272.

It occurred in a patient with a bicuspid aortic valve who received radiation therapy for stage IIA Hodgkin's lymphoma.

Diagnosis was confirmed by the visualization of a vegetation on the aortic valve at transeosophagal echocardiography.

The organism was identified by molecular biology techniques after isolation from blood culture 272.

Francisella tularensis.

Only a single case of infective endocarditis due to F. tularensis has been reported.

The organisms were detected on day 9 after sampling.

The diagnosis would not be possible without extended incubation of blood in a radiometric blood culture system (Bactec) 271.

Aeromonas hydrophila.

Aeromonas hydrophila is a waterborne gram-negative bacillus associated with gastroenteritis and soft tissue infections.

Only two cases of A. hydrophila endocarditis have been reported in the literature, both in cirrhotic patients.

One patient had chronic renal failure treated by hemodialysis, and one patient had multiple malignancies including colon carcinoma 205.

Sources of infection for A. hydrophila may include alligators, piranha, and the extensive use of medicinal leeches for the treatment of edema in burn patients78.

Yersinia enterocolitica.

Yersinia enterocolitica, a member of the Enterobacteriaceae, causes bacteremia and septicemia that are usually associated with a predisposing underlying disease such as an iron-overloading state (hemoglobin disease, chronic hemolytic anemia, hemochromatosis, acute iron poisoning, deferoxamine therapy, alcoholism, and cirrhosis), immunosuppressive therapy, and immunocompromise such as that due to HIV infection.

Twelve cases of Y. enterocolitica endocarditis have been reported in the literature, including two prosthetic-valve infections 101.

The involved serotypes were O:3, O:8, and O:9, which are termed virulent serotypes and represent the most frequent causes of sporadic human disease 101.

Rheumatic heart disease was reported in three patients.

Etiologic diagnosis was achieved by blood culture in all cases.

Of the 12 patients, 3 died, none of whom was treated appropriately.

The patients who recovered were treated with a broad-spectrum cephalosporin and gentamicin101.

Salmonella spp.

Salmonella spp.

are causative agents of intravascular infections such as endarteritis or aneurysms, but endocarditis has been recognized since 1967, when the first cases were reported 40.

Since that time, 33 cases have been reported in the literature, but a number of these reports do not contain enough information for review purposes 42, 66, 80, 81, 102, 236, 250.

The most frequently observed species isSalmonella enterica serovar Enteritidis, with S. enterica serovars Choleraesuis Typhi being reported only rarely102.

Endocarditis occurs mostly during or after a concomitant Salmonella infection such as gastroenteritis in predisposed patients 42.

Because patients with AIDS are more likely to have non-Typhi Salmonella bacteremia, they are at particular risk for endocarditis 80, 81.

This is especially true if they have used intravenous drugs and have a previously damaged tricuspid valve 66.

In patients without HIV infection, non-Typhi Salmonella endocarditis occurs in prosthetic valves 42 in 15% of patients, in patients with congenital heart defects in 5%, and less frequently in patients with other valve abnormalities including rheumatic disease and mitral valve prolapse 236.

Echocardiography usually demonstrates large vegetations, and blood cultures are positive.
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