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The article examines infective endocarditis (IE) caused by rare and fastidious bacteria, which often lead to culture-negative cases and complicate diagnosis. While IE is usually identified through continuous bacteremia with gram-positive cocci and echocardiographic detection of vegetations, uncommon pathogens such as Bartonella, Coxiella burnetii, Lactobacillus, and members of the HACEK group require specialized diagnostic approaches. These include serological tests, polymerase chain reaction (PCR), special staining techniques, and enhanced culture conditions. Clinical presentation of IE caused by these organisms may lack typical symptoms like fever and cardiac murmurs, particularly in the elderly or those with prior antibiotic use. Comprehensive diagnostic criteria, including the Duke criteria, alongside advanced molecular and histological methods, have improved the identification and management of atypical IE cases. Additionally, thorough patient history regarding animal contact, occupational exposures, and potential sources of infection is crucial for accurate diagnosis.
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Except in a few cases where vegetations are typically large, such as inBrucella endocarditis, echocardiographic findings are more effective in establishing a diagnosis of IE than in determining the microbial etiology.

Recently, the diagnostic value of echocardiography was confirmed to improve the sensitivity of the Duke's criteria111.

Transesophageal echocardiography is particularly important in patients with negative blood cultures, but even under these conditions, 24% of patients with proven IE were misclassified as having “possible” IE 111.

STRATEGIES FOR IDENTIFICATION OF THE ETIOLOGIC AGENT OF INFECTIVE ENDOCARDITIS
Culture Methods
Blood cultures.

Quantitative culture techniques show that blood from patients with IE contain 1 to 10 bacteria per ml and that this quantity remains constant during the course of the disease298.

In 1947, Salazar Mallen et al.

reported that 19 of 24 patients with a positive culture from at least one site had positive venous blood cultures, none with negative blood cultures had a positive arterial culture, and 5 with both negative venous and arterial cultures had positive bone marrow cultures 245.

From these studies it appears that arterial cultures add nothing to the diagnosis of IE and that in rare cases with persistently negative venous blood cultures, bone marrow cultures may yield diagnostic information.

Moreover, in a series of 82 patients with IE, 52 (63%) were diagnosed from the first culture, 78% from two blood cultures, 82% from three, and 91% from four 36.

Most patients with culture-positive endocarditis are persistently culture positive even if only one, two, or three cultures are obtained 296.

Culture-negative endocarditis is best defined when negative blood cultures (more that three venous blood culture of at least 5 ml each) are obtained repetitively.

Therefore, the data support the concept that cultures (anaerobic and aerobic) of three sets of blood drawn within a 24- to 48-h period are sufficient to establish a diagnosis of culture-positive endocarditis 36.

Because of the approximately linear relationship between the yield of bacteria from blood and the volume of blood, some authors recommend that at least 10 ml of blood be obtained for each culture and that as much as 30 ml of blood be obtained for each culture in an adult 184, 296.

When clinical and laboratory findings suggest endocarditis but blood cultures remain negative, attempts to elicit a history of prior antibiotic therapy should be made.

Hampton and Harrison in 1967 studied 107 patients with IE 114 and reported that 59% had positive blood cultures and 41% had negative blood cultures.

The 96 available records showed that 71 patients had been given antibiotics before blood culture, including 50% of the culture-positive group and 67% of the culture-negative group.

In a New York series298, prior administration of antibiotics reduced the incidence of positive blood cultures in patients with documented streptoccocal endocarditis from 97 to 91%.

Another study showed that 62% of 52 patients with culture-negative endocarditis had previously received antibiotics compared with only 31% of 84 patients who had positive blood cultures 213.

In 1982, Pazin et al.

reported that 64% of 88 cultures from 17 patients who received antibiotics before hospitalization were positive compared with 100% from 15 patients who had not received antibiotics 210.

The duration of prior antimicrobial therapy also appears to be an important factor.

If antibiotics are given for only 2 to 3 days, blood cultures that were initially negative rapidly become positive.

However, after longer noncurative courses of therapy, some blood cultures remained negative for weeks (A. R. Tunkel and D. Kaye, Editorial, N. Engl.

J. Med.

326:1215–1217, 1992).

If prior antibiotic therapy has been documented, neutralization or diminution of the presence of antibiotics in blood may be accomplished by diluting the blood in broth in a ratio of 1:10 and by incorporating sodium polyanetholsulfonate, which inactivates aminoglycosides in the broth.

Another approach to secure cultivation from the blood of previously treated patients is to process the blood in an antimicrobial agent removal device with cationic and polymeric adsorbent resins in saline with sodium polyanetholsulfonate 296.

The resins have been pretreated to prevent bacterial retention; they remove up to 100 μg of antibiotics per ml as well as some bacterial inhibitors present in the blood 256, 296.

If previous antibiotic therapy has not been administered and the blood cultures are negative, fastidious organisms should be suspected, and special cultivation methods are needed.

Although Abiotrophiaspp.

grow well in the presence of fresh human blood, they usually fail to grow in subcultures on conventional agar media.

They do grow as satelliting colonies around a Staphylococcus aureus streak on blood agar (Fig.

2) and in medium supplemented with pyridoxal hydrochloride at 1 to 1,000 μg/ml235.

Therefore, the major problems with recognition ofAbiotrophia spp.

are the failure to grow when subcultured and the possibility that the sample may be regarded as containing only nonviable contaminants that are seen microscopically.

The simplest approach to isolation is to inoculate the blood culture broth onto blood agar that is cross-streaked with S. aureus.

Although uncommon, Legionella spp.

should be considered in patients with prosthetic valve endocarditis and negative blood cultures; isolation requires special techniques such as BCYE agar and an incubation period of 15 days 281.

Although mostMycobacterium spp.

have been isolated in conventional blood culture systems including ISOSAT 92 and Bactec254, the use of Bactec 13A bottles containing Middlebrook 7H13 broth should be considered, especially for Mycobacterium tuberculosis.

Brucella spp.

may be easily isolated using the Bactec NR blood culture system, but the use of lysis concentration (Isolator) system significantly shortens the time to isolation (3.5 versus 14 days) 145, 198.

Bartonella spp.

have been isolated from blood by prolonged incubation in the Bactec system, inoculation on rabbit blood agar, or tissue cell culture277.

In fact, they grow well in automated blood culture systems, but the small amount of CO2 produced fails to trigger the alert system 277.

Consequently, both acridine orange and Gimenez staining of the blood with subculture on blood agar should be performed if cultures remain negative after 3 weeks225.

Fig.

2.

Culture of resected valves or biopsy specimens.

Pathogens can also be isolated from resected valves or biopsy specimens by inoculation onto agar or into tissue culture.

This is particularly helpful and efficient, probably because of the large number of organisms in the tissue.

C. burnetii, Mycobacterium spp., Brucella spp.,Legionella spp., and numerous other fastidious bacteria have been identified as causative agents of endocarditis in this manner159, 192, 225, 281.

Thus, surgically resected materials should be cultured in appropriate medium when possible.

In some instances, positive serologic test results for agents such as C. burnetii, Legionella spp., or Brucella spp.

may be helpful in delineating the appropriate media to be employed.

Tissue cell culture.

Tissue cell culture is the only method for isolation of obligate intracellular pathogens.

This procedure may be hazardous and should be attempted only in laboratories equipped for biosafety level 3 (BSL3) pathogens.

The shell vial technique has been reported as the best method for isolation ofC.

burnetii from heparinized blood or resected heart valves29, and it has also been successfully used for isolation of Bartonella spp.

and Chlamydia psittaciisolation 63, 65, 253.

Basically, different cell lines, chosen depending on the potential bacterial etiology, e.g., L929, Vero, and HEL cells for C. burnetii, L929 cells forChlamydia psittaci, and ECV cells for Bartonellaspp., are grown on coverslips in shell vials until a confluent monolayer is obtained.

An inoculum of 200 μl of leukocyte-rich plasma from heparinized blood or a portion of the removed, infected valve is inoculated onto the tissue culture cells and centrifuged at 700 ×g for 1 h, after which the remaining plasma is removed and replaced with fresh culture medium and the culture is incubated at 37°C in a CO2 incubator.

On days 3, 6, and 15, coverslips are removed and growth is detected by indirect immunofluorescence either with antibodies that react with the bacteria suspected or with a 1:200 dilution of the patient's serum.

When growth is observed, bacteria are subcultured and identified.

Histologic Testing
Histologic examination of the removed valve is critical.

Standard stains such as hematoxylin and eosin can reveal whether the histologic findings from the valve are compatible with the diagnosis of IE.

If no inflammation is observed in the valve, the diagnosis of IE should be reevaluated or excluded.

Histologic parameters are included among the Duke diagnostic criteria for IE.

Hematoxylin and eosin staining, the first step in the histologic examination, allows recognition of a consistent pattern of inflammation.

Numerous special histologic stains are available, several of which are particularly appropriate for IE.

Tissue Gram stains allow the differentiation of gram-positive and gram-negative microorganisms and, in the hands of a skilled observer, allow a rapid preliminary identification of the organisms based on their morphology 301.

However, this technique has limitations since the causative bacteria may lack a cell wall (Mycoplasma spp.).

The periodic acid-Schiff (PAS) stain is especially valuable for Whipple's disease and demonstrates PAS-positive foamy histiocytes variably surrounded by infiltrates of mixed neutrophils, lymphocytes, and mononuclear cells (Fig.3) 301.

This stain may also be used to detect fungi.

The Giemsa stain not only allows the detection of bacteria, including Bartonella spp., but also stains white blood cells and therefore highlights the inflammatory infiltrate in vegetations 301.

The Warthin-Starry technique, a silver impregnation method, is among the most sensitive methods for detection of bacteria, even those which stain weakly with a tissue Gram stain, such as Bartonella spp.

(Fig.

4).

Using this method, we have demonstrated massive vegetations on the valve surface with extensive destruction of the underlying valve tissue and the presence of numerous bacteria in Bartonellaendocarditis 225.

The Ziehl-Nielsen stain is used for detection of acid-fast bacteria, especially Mycobacteriumspp.

The Gimenez stain is a good method for the detection of C. burnetii and Legionella spp.

In Q fever endocarditis, the vegetations, which are often smooth and nodular, are frequently infiltrated with mononuclear inflammatory cells containing manyC.

burnetii cells 25.

Another stain of interest is the Grocott-Gomori methenamine silver stain, which provides the best contrast of the special fungal stains 301.

Fig.

3.

Fig.

4.

Other stains are occasionally used.

The acridine orange stain, a stain used in the nonspecific-fluorescence diagnostic technique, may also be used to detect any living microorganism in biopsy specimens.

The Kinyoun stain or the Machiavello stain may be used to demonstrate large macrophages containing dark red granules in Chlamydiaendocarditis 301.

Immunohistologic Testing
Immunohistologic methods have been used for the detection ofC.

burnetii, Bartonella spp., andChlamydia spp.

in valvular tissues 25, 75.

Several techniques are available, including a capture enzyme-linked immunosorbent/immunofluorescent assay (ELISA/ELIFA) system, direct immunofluorescence using fluorescein-conjugated monoclonal antibodies, and immunoperoxidase staining (Fig.

5)25.

Fig.

5.

Electron Microscopy
Although tedious and time-consuming, electron microscopy should be able to reveal microorganisms undetectable by molecular or immunological methods.

IE use should be reserved for cases when all these other techniques fail.

PCR Amplification
The advent of DNA amplification methods for detecting microorganisms has raised great interest among infectious-disease physicians and clinical microbiologists.

Such techniques, particularly PCR, have now been adapted for use with most types of clinical materials, including valvular specimens.

For example, detection of a portion of the 16S rRNA gene has been used successful in the identification of the Whipple's disease bacterium as an etiologic agent of endocarditis (297; K. H. Wilson, R. B. Blitchington, R. Frothingham, and J.

A. Wilson, Letter, N. Engl.

J. Med.328:62, 1993).

This is also true for some cases ofBartonella endocarditis 225.

The main advantages of these methods are that they are culture independent and that almost all bacteria can be detected in a single reaction through the incorporation of broad-spectrum primers.

The organisms from which amplified DNA is derived can easily be identified by sequence analysis followed by comparison of the nucleotide sequence with a large database that contains sequences of the targeted gene from many other bacteria.

Goldenberger et al.

amplified bacterial DNA from 15 valvular samples and concluded that this technique was both easy and reliable when applied to surgically removed heart valves from patients with IE103.

However, a limitation of this system is the number and quality of DNA sequences available in databases.

For instance, some of the reference sequences in the GenBank and the EMBL databases are too short or contain too many undetermined nucleotides for confident assignment of clinically derived sequences.

Moreover, another likely limitation of molecular detection is the quality and nature of the infectious material collected (vegetation or piece of valve).

Some clinical materials, especially blood, may contain substances which limit the sensitivity of PCR.

Contamination may also occur, and therefore caution must be exercised in the interpretation of PCR-based sequence analyses when the organism has not been observed in stained valve tissues.

Currently, specific primers are available for most bacterial genera, including Chlamydia, Brucella,Legionella, Mycobacterium, andMycoplasma87.

In contrast to its use directly on clinical samples, PCR with subsequent sequencing of the amplified gene can be used for identification of bacteria isolated by culture methods.

This is particularly useful when the bacteria demonstrate unusual biochemical or staining characteristics.

For example, molecular identification has successfully identified Gemella spp.

in three cases of endocarditis that were reported to be due to small “gram-negative coccobacilli” 152.

Such techniques for detection and molecular characterization should be used each time biochemical identification test fail.

Serologic Testing
In our laboratory, testing for culture-negative endocarditis includes the systematic determination of antibody titers againstC.

burnetii, Bartonella spp., Mycoplasma pneumoniae, Legionella pneumophila,Chlamydia spp., and Brucella melitensis, which are among the most common pathogens in culture-negative IE.

These serologic tests are included as diagnostic criteria for IE in the Duke70 and the modified Duke 86 criteria.

However, in other instances, one must be cautious when relying solely on serologic methods to diagnose endocarditis because of cross-reactions.

For example, currently available serologic tests forBartonella-induced endocarditis may not reliably distinguish between antibody responses to B. henselae and B. quintana or between those to Bartonella spp.

andChlamydia spp.

176, 225.

The most common serologic methods used are the tube agglutination test forBrucella melitensis infections, indirect immunofluorescence for Legionella pneumophila, ELISA forMycoplasma pneumoniae, and complement fixation, ELISA, or indirect immunofluorescence for Chlamydia spp.

ETIOLOGY OF ENDOCARDITIS DUE TO LESS COMMON FASTIDIOUS BACTERIAL AGENTS
Agents of Blood Culture-Negative Endocarditis
The agents of blood culture-negative endocarditis are listed in Table 3 and discussed below.

Table 3.

Bartonella spp.

Bartonella spp.
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