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Clinical Microbiology Reviews, January 2001, p. 177-207, Vol. 14, No. 1
0893-8512/01/$04.00+0   DOI: 10.1128/CMR.14.1.177-207.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.

Endocarditis Due to Rare and Fastidious Bacteria

P. Brouqui* and D. Raoult

Unité des Rickettsies, CNRS UPRESA 6020, Faculté de Médecine, 13385 Marseille Cedex 5, France

SUMMARY
INTRODUCTION
APPROACH TO ETIOLOGIC DIAGNOSIS OF INFECTIVE ENDOCARDITIS
    Medical History
    Clinical Approach
    Echocardiography
STRATEGIES FOR IDENTIFICATION OF THE ETIOLOGIC AGENT OF INFECTIVE ENDOCARDITIS
    Culture Methods
        Blood cultures.
        Culture of resected valves or biopsy specimens.
        Tissue cell culture.
    Histologic Testing
    Immunohistologic Testing
    Electron Microscopy
    PCR Amplification
    Serologic Testing
ETIOLOGY OF ENDOCARDITIS DUE TO LESS COMMON FASTIDIOUS BACTERIAL AGENTS
    Agents of Blood Culture-Negative Endocarditis
        Bartonella spp.
        Mycobacterium spp.
        Mycoplasma spp.
        Whipple's disease bacterium.
        Legionella spp.
        Chlamydia psittaci.
        Coxiella burnetii (Q fever agent).
    Gram-Negative Bacilli
        HACEK group bacteria.
        Campylobacter fetus.
        Pasteurella spp.
        Brucella spp.
        Bordetella spp.
        Francisella tularensis.
        Aeromonas hydrophila.
        Yersinia enterocolitica.
        Salmonella spp.
        Klebsiella spp. and other rarely isolated enterics.
        Streptobacillus moniliformis.
    Gram-Negative Cocci
        Neisseria gonorrhoeae.
        Other Neisseria spp.
    Gram-Positive Bacilli
        Listeria monocytogenes.
        Lactobacillus spp.
        Nocardia spp.
        Erysipelothrix rhusiopathiae.
        Clostridium spp.
        Nontoxigenic Corynebacterium diphtheriae.
    Gram-Positive Cocci
        Abiotrophia spp.
        Gemella spp.
        Stomatococcus mucilaginosus.
CONCLUSION AND PERSPECTIVES
ACKNOWLEDGMENTS
REFERENCES


SUMMARY
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The etiologic diagnosis of infective endocarditis is easily made in the presence of continuous bacteremia with gram-positive cocci. However, the blood culture may contain a bacterium rarely associated with endocarditis, such as Lactobacillus spp., Klebsiella spp., or nontoxigenic Corynebacterium, Salmonella, Gemella, Campylobacter, Aeromonas, Yersinia, Nocardia, Pasteurella, Listeria, or Erysipelothrix spp., that requires further investigation to establish the relationship with endocarditis, or the blood culture may be uninformative despite a supportive clinical evaluation. In the latter case, the etiologic agents are either fastidious extracellular or intracellular bacteria. Fastidious extracellular bacteria such as Abiotrophia, HACEK group bacteria, Clostridium, Brucella, Legionella, Mycobacterium, and Bartonella spp. need supplemented media, prolonged incubation time, and special culture conditions. Intracellular bacteria such as Coxiella burnetii cannot be isolated routinely. The two most prevalent etiologic agents of culture-negative endocarditis are C. burnetti and Bartonella spp. Their diagnosis is usually carried out serologically. A systemic pathologic examination of excised heart valves including periodic acid-Schiff (PAS) staining and molecular methods has allowed the identification of Whipple's bacillus endocarditis. Pathologic examination of the valve using special staining, such as Warthin-Starry, Gimenez, and PAS, and broad-spectrum PCR should be performed systematically when no etiologic diagnosis is evident through routine laboratory evaluation.


INTRODUCTION
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Infective endocarditis (IE) is usually suspected in a patient with fever and a new or changing cardiac murmur and is diagnosed based on the presence of a vegetation on echocardiography and positive blood cultures. Diagnosis of endocarditis is usually easy in febrile patients with a continuous bacteremia and the presence of vegetation on echocardiography or on gross examination or histologic testing of the removed valve. However, although numerous clinical situations lead to a high degree of suspicion of endocarditis, culture or histologic examination does not confirm the diagnosis.

Although fever is the single most common finding in endocarditis, it may be absent in the elderly or in patients given previous antibiotic therapy before presentation or it may be intermittent or low-grade as for Q fever endocarditis 29. Cardiac murmur is the second most frequent finding in endocarditis. However, it is not usually present at the initial stage of right-sided endocarditis, and new or changing murmurs are detected in only 40% of patients with endocarditis 261; this rate is even lower in the elderly. Despite the fact that transesophageal echocardiography is more sensitive than transthoracic echocardiography, a vegetation is rarely detected in Q fever or Whipple's disease 29, 221. Sterile blood cultures have been noted for 2.5 to 31% of patients with endocarditis 286. Blood cultures are frequently sterile when antibiotic therapy was administered before sampling and in patients with subacute right-sided endocarditis, mural endocarditis, and endocarditis caused by slow-growing or fastidious organisms such as anaerobes, the HACEK group, Abiotrophia spp., Brucella spp., Bartonella spp., Legionella spp., and Mycoplasma spp. or when obligate intracellular organisms such as Coxiella burnetii are involved 3, 124. These bacteria require specific media and conditions such as L-cysteine-enriched medium for Abiotrophia spp., buffered charcoal yeast extract (BCYE) agar for Legionella spp., or special culture conditions favorable for anaerobes or intracellular bacteria. Moreover, in some cases, slow-growing bacteria require incubation times as long as 6 weeks 179. In such situations, infective endocarditis remains a diagnostic challenge.

To both assist physicians in establishing the final diagnosis of endocarditis and allow comparisons of published cases, diagnostic criteria have been defined 70, 122, 123, 290. For many years, the Beth Israel criteria 290 were the only recognized diagnostic criteria. In 1994, Durack et al. from the Duke Endocarditis Service 70 added echocardiographic findings and other clinical and laboratory data to the well-established clinical and microbiological criteria. More recently, additional criteria have been proposed to improve the list 147, such as including C. burnetii serology or culture as additional major criteria 86.

According to the Duke Endocarditis Service, the diagnosis of IE is definite (i) when a microorganism is demonstrated by culture or histologic testing in a vegetation, an embolism, or an intracardiac abscess; (ii) when active endocarditis is confirmed by histologic examination of the vegetation or intracardiac abscess; or (iii) in the presence of two major clinical criteria, one major and three minor criteria, or five minor criteria (the major and minor Duke criteria are listed in Table 1). The diagnosis of IE is rejected when a firm alternate diagnosis explains the manifestations of endocarditis, when the fever resolves with antibiotic therapy for 4 days or less, or when no pathologic evidence of infective endocarditis is found at surgery or autopsy after antibiotic therapy for 4 days or less 70.

                              
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TABLE 1.   Terminology used for diagnostic criteria for infective endocarditisa (Modified Duke's Endocarditis Service) 70, 86

Consequently, suspected cases of endocarditis should be investigated until all clinical, epidemiologic, echocardiographic, and laboratory data are compiled in order to be able to establish the diagnostic score.

New etiologic agents of endocarditis such as Bartonella spp. and the Whipple's disease bacillus 233 have been identified by PCR amplification and sequence analysis of the amplified DNA. These molecular tools have considerably improved the detection and the identification of noncultivable causative agents of endocarditis 88. This technique is particularly useful for microbial identification in excised infected valves, even after antibiotic therapy. PCR amplification and subsequent sequence analysis of amplified genes has also been used to better identify fastidious organisms such as Gemella spp. 152. New cultivation techniques, especially tissue cell culture, have also been successfully used for isolation of fastidious organism such as Bartonella 65 or strict intracellular pathogens such as C. burnetii and Chlamydia spp. 229, 253. Tissue cell culture has been greatly improved by using the shell vial technique 229. Culture of the removed valve has been shown to be much more efficient than blood culture in recovering the organisms in patients with culture-negative endocarditis 192, 193. The use of enriched media for specific microorganisms such as nutritionally deficient Streptococcus spp., Legionella spp., or Mycobacteria spp. has also been helpful in the etiologic diagnosis of endocarditis. The availability of these improved and new diagnostic tools has considerably enlarged the spectrum of bacteria involved in the etiologic diagnosis of endocarditis. Herein, we review the epidemiology, clinical presentation, and diagnosis of endocarditis caused by rare and fastidious organisms and then focus on the strategies used for the recovery and identification of the etiologic agents of IE. This review has been based on a MEDLINE search from 1966 to 1999 using Internet Grateful Med search V2.6.2 from the National Library of Medicine with the following key words: negative blood culture or culture-negative plus endocarditis or endocarditis plus (name of the bacteria and/or the disease). When a bacterium had different names, such as Abiotrophia spp., all known synonyms were used. The research was carried out without restriction, except that endocarditis or the name of the bacteria should be included in the title. Only papers written in English or French were selected. This research yielded 1,056 references, from which 304 have been selected.


APPROACH TO ETIOLOGIC DIAGNOSIS OF INFECTIVE ENDOCARDITIS
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Once the diagnosis of IE is suggested or confirmed, the causative agent can be specifically investigated by evaluation of the medical history, clinical examination, and echographic findings (Fig. 1).


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FIG. 1.   Probable bacterial etiology of endocarditis depending on the patient history and epidemiologic situation. IVDU/IVDA, intravenous drug user/abusers.

Medical History

The patient's medical history is an important clue for the etiological diagnosis of IE. Contact with animals is an important feature; for example, contact with a cat (including scratches or bites) suggests Bartonella henselae or Pasteurella spp., while contact with cattle should suggest a role for C. burnetii or Brucella spp. Parrots, pheasants, and pigeons have been reported to play a role in Chlamydia psittaci IE 253. Contact with domestic animals such as swine, fish, and poultry is frequently reported in the history of patients with Erysipelothrix endocarditis. Consequently, occupational exposure is an important finding. For example, farmers and veterinarians are exposed to both C. burnettii and Erysipelothrix. The latter is a causative agent of endocarditis in butchers, fishermen, and even in homemakers by contact with organic matter in which the organism is commonly found 230. Contact with arthropods such as lice and fleas should also be considered. The human body louse is involved in the transmission of Bartonella quintana 27, whereas cat fleas are involved in the epidemiology of B. henselae infections 143. Some pathogens are mostly nosocomial in origin. For example, IE following open heart surgery should suggest Legionella spp., Mycobacterium spp., Nocardia spp., or fastidious fungi, especially if routine blood cultures are negative 160, 281. Most reported cases of nosocomial endocarditis occur as small outbreaks, and all available epidemiologic information should be obtained for a diagnosis of postoperative IE. Since a large number of pathogens involved in IE are of oral origin, a history of a dental procedure is an important feature. HACEK group bacteria are often found to be pathogens in young adults after dental procedures, but Lactobacillus spp. and Gemella spp. have also been reported in such situations 152, 270. All information on underlying conditions should be obtained from the patient or from the patient's medical history. Patients with prosthetic valves are at particular risk for IE. Of note, patients with prosthetic valves for whom blood cultures are negative are likely to be infected by C. burnetii or B. henselae as etiologic agents, since more than 55% of C. burnetii endocarditis occurs in patients with prosthetic valves 266. Underlying immunocompromising conditions, including AIDS, cancer, and lymphoma, have been reported in 9% of C. burnetii endocarditis patients, especially in the absence of underlying valvular disease. A medical history of cancer, especially adenocarcinoma associated with disseminated intravascular coagulation, should also suggest the possibility of marantic or nonthrombotic endocarditis, a diagnosis of exclusion when all other investigations are nonrevealing 161. In a similar manner, IE can be suspected in patients with collagen vascular diseases such as systemic lupus erythematosus, particularly if associated with the presence of antiphopholipid antibodies such as in Libman-Sack endocarditis 238. Even if noninfective endocarditis is suspected, it is important to remember that the valvular lesions induced by inflammatory disease may facilitate the infection of the inflamed valve or graft and cause secondary IE. Patients should be questioned about their life habits, especially their sexual practices. A few decades ago, Neisseria gonorrhoeae IE was reported very frequently in sexually active patients; active surveillance and prompt treatment has now significantly reduced this rate. In a similar way, behaviors such as intravenous drug use and alcohol abuse should be evaluated. Intravenous drug users are at risk for Neisseria sicca, Clostridium spp., and Eikenella corrodens IE related to injection 8, 56, 118. E. corrodens has been reported in patients who clean needles with their saliva. For chronic alcoholics, the involved pathogens are more likely to be related to the underlying immunocompromising condition rather than to specific practices. B. quintana, Erysipelothrix rhusiopathiae, Campylobacter fetus, Aeromonas hydrophila, Pasteurella spp., and Listeria monocytogenes are most commonly found 38, 65, 77, 104. Portal hypertension and splenic shunt, which occurs with cirrhosis in these patients, may allow prolonged bacteremia and subsequent valvular infection. Social conditions may impart specific risks. For example, homeless persons are often chronic alcoholics and may harbor lice, allowing the transmission of B. quintana infection, the major fastidious etiologic agent of IE in the homeless 27. Finally, the relative frequency of the microorganism as a cause of IE should be considered in the laboratory investigation. For example, it is noteworthy that C. burnetii is as frequently recognized as an etiologic agent of IE as are all the HACEK group bacteria together (Table 2).

                              
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TABLE 2.   Relative frequency of bacteria as etiologic agents of IEa

Clinical Approach

Some clinical presentations are characteristic and should indicate possible IE. Febrile congestive heart failure is a frequent mode of presentation for patients with endocarditis caused by fastidious organisms, especially C. burnetii and Bartonella spp., but may also be present with Whipple's disease or HACEK group endocarditis. In fact, it is likely that this mode of presentation is related to a diagnostic delay. Emboli are reported in more than 50% of patients infected with microorganisms such as with Corynebacterium diphtheriae, N. sicca, and Haemophilus paraphrophilus 46, 278; R. Lopez-Velez, J. Fortun, C. de Pablo, and J. Martinez Beltran, Letter, Clin. Infect. Dis. 18:660-661, 1994). When these emboli involve the brain, they may mimic an acute cerebrovascular accident or stroke syndrome, but fever should suggest the diagnosis of endocarditis. Pulmonary manifestations such as repetitive bronchopneumonia are common in right-sided endocarditis. Such findings in intravenous drug users or in patients with intravenous catheters should suggest a diagnosis of right-sided endocarditis. Staphyloccocus spp., Candida spp., Eikenella corrodens, and Salmonella enterica serovar enteritidis in patients with AIDS and N. sicca and Clostridium spp. in other patients are potential etiologic agents. It is important to note that the absence of cardiac murmur does not rule out the diagnosis of IE and can suggest ride-side endocarditis. Genital organs should be examined, since most cases of gonoccocal endocarditis are associated with active sexually transmitted diseases. Concomitant gastroenteritis, sinusitis, or pneumonitis should suggest Salmonella spp., H. influenzae, or Chlamydia psittaci as etiologic agents, respectively, while upper respiratory tract infection and pharyngitis is frequently observed in patients with H. aphrophilus endocarditis and in intravenous drug users with C. diphtheriae endocarditis. In patients with Klebsiella pneumoniae endocarditis, urinary tract infection is common 9, and during pregnancy, Listeria monocytogenes is a possible etiologic agent. Finally, culture-negative endocarditis associated with chronic diarrhea should suggest Whipple's disease.

Echocardiography

Over the past 15 years, a number of investigations have confirmed the important role of transthoracic echocardiography in the diagnosis and management of IE. Echocardiography must be performed in all patients suspected of having IE. If transthoracic echocardiography is noncontributory but there is still a high suspicion of IE, transesophageal echocardiography should be performed. Compared to transthoracic echocardiography, transesophageal echocardiography has improved the sensitivity in defining both vegetative lesions and perivalvular infections, particularly in mural abscesses in patients with IE (sensitivity, 80 to 90%) 237, 247. Except in a few cases where vegetations are typically large, such as in Brucella 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 criteria 111. 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
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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 disease 298. 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 series 298, 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 Abiotrophia spp. 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/ml 235. Therefore, the major problems with recognition of Abiotrophia 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 most Mycobacterium spp. have been isolated in conventional blood culture systems including ISOSAT 92 and Bactec 254, 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 culture 277. 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 weeks 225.


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FIG. 2.   Abiotrophia spp. (formerly known as nutritionally deficient streptococci) showing satellite growth with Staphylococcus aureus. Photo courtesy of B. La Scola (Unité des Rickettsies, Marseilles, France).

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 manner 159, 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 of C. burnetii from heparinized blood or resected heart valves 29, and it has also been successfully used for isolation of Bartonella spp. and Chlamydia psittaci isolation 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 for Chlamydia psittaci, and ECV cells for Bartonella spp., 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 Bartonella endocarditis 225. The Ziehl-Nielsen stain is used for detection of acid-fast bacteria, especially Mycobacterium spp. 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 many C. 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.


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FIG. 3.   Whipple's disease endocarditis showing foamy PAS-positive macrophages (arrow). Magnification, ×1,000. Photo courtesy of H. Lepidi (Unité des Rickettsies, Marseilles, France).


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FIG. 4.   Warthin-Starry silver stain of a Bartonella quintana-infected valve. Note the cluster of bacteria in black within the vegetation. Magnification, ×100. Photo courtesy of H. Lepidi.

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 Chlamydia endocarditis 301.

Immunohistologic Testing

Immunohistologic methods have been used for the detection of C. burnetii, Bartonella spp., and Chlamydia 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.


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FIG. 5.   Immunohistochemical demonstration of Coxiella burnetii in a heart valve of a patient with Q fever endocarditis. Magnification, ×600. Immumoalkaline phophatase staining was used.

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 of Bartonella 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 IE 103. 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, and Mycoplasma 87.

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 against C. 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 Duke 70 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 for Bartonella-induced endocarditis may not reliably distinguish between antibody responses to B. henselae and B. quintana or between those to Bartonella spp. and Chlamydia spp. 176, 225. The most common serologic methods used are the tube agglutination test for Brucella melitensis infections, indirect immunofluorescence for Legionella pneumophila, ELISA for Mycoplasma pneumoniae, and complement fixation, ELISA, or indirect immunofluorescence for Chlamydia spp.


ETIOLOGY OF ENDOCARDITIS DUE TO LESS COMMON FASTIDIOUS BACTERIAL AGENTS
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References

Agents of Blood Culture-Negative Endocarditis

The agents of blood culture-negative endocarditis are listed in Table 3 and discussed below.

                              
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TABLE 3.   Agents of blood culture-negative endocarditis

Bartonella spp. Bartonella spp. are small facultative intracellular bacteria that stain gram negative and belong to the alpha 2 division of the proteobacteria (Fig. 6). These organisms cause various clinical syndromes in immunocompetent and immunocompromised hosts. B. henselae causes cat scratch disease, meningoencephalitis, and prolonged fever in immunocompetent patients and bacillary angiomatosis and hepatic peliosis in human immunodeficiency virus HIV-infected patients 179. B. quintana causes trench fever, lymphadenopathy with fever, and bacillary angiomatosis. Endocarditis is caused by both B. henselae and B. quintana, and it has been found to be caused by B. elizabethae 51 and B. vinsonii 239 in only one reported case each.


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FIG. 6.   Phylogeny of the representative members of the alpha 2 group of proteobacteria and location of Bartonella spp. based on analysis of the 16S rRNA sequence by the neighbor-joining method.

(i) Epidemiology. The epidemiology and clinical manifestations of Bartonella infections have been recently reviewed 175, 179. Bartonella infections were first reported from the United States and France, but many recent reports showed that Bartonella spp. are distributed worldwide. B. henselae is transmitted to humans by a cat scratch or bite or by cat fleas. B. quintana is transmitted by the human body louse. The reservoir of B. henselae is the cat, which is chronically bacteremic, and humans are likely to be the reservoir of B. quintana. Review of the literature and personal unpublished observations found a total of 54 cases of Bartonella endocarditis, 8 of which were caused by B. henselae 10, 63, 112, 126, 225, 20 were caused by B. quintana 30, 65, 131, 168, 225, 257, 258, 1 was caused by B. elizabethae 51, one was caused by B. vinsonii 239, and 20 were not characterized at the species level since they were diagnosed by serologic testing alone 75, 172, 225; unpublished data). Bartonella infection was diagnosed in 3 of 86 patients with endocarditis in Marseilles (3.5%), 3 of 123 patients with endocarditis in Halifax (2.4%), and 4 of 90 patients in Lyons (4.4%) 225. Bartonella endocarditis accounted for 10 of 299 cases of endocarditis (3%), being as frequent as C. burnetii (Q fever) endocarditis (unpublished data). Meta-analyses of these reported cases showed that B. henselae endocarditis occurs in 87% of patients with previous valve injury whereas only 30% of patients with B. quintana endocarditis have this underlying condition. Predisposing factors for B. quintana endocarditis are homelessness and alcoholism, a condition found in 70% of patients and associated with exposure to body lice 26. Consequently, epidemiology helps to distinguish between the two infections, one caused by B. quintana mainly in alcoholic homeless persons exposed to body lice and without previous valve injury and the other caused by B. henselae in patients with underlying valve injury who have had contact with a cat. B. quintana endocarditis has been reported in only one HIV-infected patient 257. The mean age of patients with Bartonella endocarditis is 48 years. These patients are significantly younger than control patients with IE caused by other microorganims (P. E. Fournier and D. Raoult, submitted for publication). The gender proportion is 85% male.

(ii) Signs and symptoms. Because Bartonella spp. cause a subacute insidious endocarditis, the diagnosis is usually considerably delayed, which may explain why most patients present with acute cardiac failure 75, 225. Fever higher than 38°C is found in most patients. Patients usually present with cardiac murmur, dyspnea on exertion, and bibasilar lung rales, suggesting global cardiac failure. Aortic valves are more often involved, and prosthetic valve endocarditis has been reported in only 3 of 32 patients. Embolic phenomena have been reported in 41% of patients and may initiate the clinical manifestations. Nonblanching purpura and petechiae are frequently observed on the lower extremities, palms, and conjunctiva. Laboratory data are not specific and a include normal white blood cell WBC count, thrombocytopenia (platelet count, <150 × 109/liter), and elevated blood creatinine levels in some patients with severe cardiac insufficiency.

(iii) Diagnosis. Pathologic diagnosis after valve surgery was performed in 86% of patients, and the remaining patients all had one major criterion (echocardiographic vegetations) and at least three minor criteria. In fact, the diagnosis of endocarditis is quite obvious in such patients, but blood cultures are usually negative. In such situations, especially in alcoholic homeless persons with body lice or in patients with previous valve injury who own cats, a diagnosis of Bartonella endocarditis is likely. Etiologic diagnosis is usually established by serologic testing, inoculation of the blood or resected valve into tissue culture and blood agar, PCR detection of the citrate synthase gene with identification of DNA by sequence analysis, or Warthin-Starry silver staining with immunohistochemical demonstration of the bacteria in valves. Serologic testing is carried out by an immunofluorescence assay (IFA). Low-level cross-reactions with C. burnetii may occur 151, and significant cross-reactions may be seen with Chlamydia pneumoniae 176. In one case, B. quintana endocarditis was diagnosed by detecting bacteria by PCR in the removed valve despite the fact that the patient did not demonstrate any antibodies to Bartonella spp. 225. For this reason, attempts to isolate the bacteria are necessary. The most reliable diagnostic tool, aside from serologic testing, is PCR of freshly removed valves. In our experience, a PCR amplicon was obtained in 72% of specimens even though 42% of valves were sampled during antibiotic therapy (P. E. Fournier, unpublished data). In fact, the lack of sensitivity of blood culture is probably related to the previous antibiotic therapy 153.

(iv) Echocardiography and pathology. The frequency of emboli is related to the large size of the vegetations in Bartonella endocarditis. this explains the efficiency of echocardiography, which identifies vegetations in 100% of B. henselae and 96% of B. quintana endocarditis patients 225; P. E. Fournier et al., unpublished data). Examination of removed valves showed that Bartonella spp. cause significant valvular tissue destruction. No well-formed or suppurative granulomas are observed. Typical lobular vascular proliferations, as found in cutaneous bacillary angiomatosis, are not observed. Gram staining, PAS staining, and Grocott-Gomori staining of the valve are not useful in detecting the bacteria, whereas Giemsa and, especially, Wharthin-Starry stains reveal many characteristic granular organisms in the valve vegetation or the valve (Fig. 4). Masses of bacteria occupy an extracellular location, mainly in the fibrin deposits. No organisms are found within areas of inflammation. Immunohistologic staining has been successfully used to demonstrate B. quintana or B. henselae in human cardiac valve tissues (H. Lepidi et al., unpublished data).

(v) Treatment and prognosis. In vitro antibiotic susceptibility of nine isolates of B. quintana, three isolates of B. henselae, one isolate of B. elizabethae, and one isolate of B. bacilliformis showed that all were highly susceptible to beta -lactam agents, aminoglycosides, macrolides, tetracyclines, and rifampin. They were less susceptible to penicillin, narrow-spectrum cephalosporins, and clindamycin 177. Considerable variation was noted in their susceptibility to fluoroquinolones compounds. Cocultivation of Bartonella spp. with eukaryotic cells did not change the bacteriostatic activity of the antibiotics 194. However, it is interesting that only aminoglycosides (gentamicin, amikacin, and tobramycin) were bactericidal on either axenic medium or in cell cultures. Clinical data on effective treatment of Bartonella-induced endocarditis are scarce. Of 42 patients reviewed recently, 76% were treated with aminoglycosides, 74% were treated with a beta -lactam compound, 35% were treated with a tetracycline, 20% were treated with vancomycin, 16% were treated with a fluoroquinolone, and 14% were treated with rifampin (Fournier and Raoult, submitted). Although a standard regimen for the antibiotic treatment of Bartonella endocarditis has not been established, based on the relevant clinical data and the in vitro activity of antimicrobial compounds, we suggest an aminoglycoside in combination with either doxycycline or ceftriaxone for a prolonged course 175. In addition to antibiotic treatment, surgery is often required owing to the extensive valve damage. In our series, valve replacement was performed in 80% of cases. It is likely that severe damage and delay in diagnosis contributed to the poor prognosis of Bartonella endocarditis. Of the 53 patients for whom the outcome was known, 42 (80%) survived and 11 died. The mortality rate observed among patients with B. quintana endocarditis was almost three times higher than that observed among the controls (Fournier and Raoult, submitted). Half of the patients who died and only one-third of survivors were homeless, suggesting that the lack of medical care for homeless people leads to delay in diagnosis and treatment and subsequently a poor outcome for Bartonella endocarditis.

Mycobacterium spp. Mycobacteria are acid-alcohol-resistant bacteria that are visualized by the Ziehl-Nelsen stain. Tuberculous valvular endocarditis is exceptionally rare; only 16 cases have been reported in the literature 47. It usually presents in the context of miliary tuberculosis, and in all but one case the diagnoses were made at autopsy. The rare cases reported involved congenital defects of the aortic valves, prosthetic valves, and ventriculoatrial shunts. However, because of its rarity, there is still uncertainty whether true tuberculous endocarditis exists as a clinical entity 47.

Nonturberculous mycobacterial endocarditis involves mostly porcine or prosthetic valves. To our knowledge, only two cases involving damaged native valves have been reported 96, 254. In the literature, we found seven cases of prosthetic-valve endocarditis due to Mycobacterium chelonei 92, 160, 241, 288, six cases of prosthetic-valve endocarditis due to Mycobacterium fortuitum 6, 43, 146, 254, one case of prosthetic-valve endocarditis due to Mycobacterium gordonae 159, and one case of Mycobacterium avium-intracellulare-induced native-valve endocarditis (149; J. Butany, Letter, Con. J. Cardiol. 9:214, 252, 1993). Reported infections primarily involve atypical, rapidly growing strains that probably were inoculated at valve replacement 293. The ability of mycobacterial organisms to cause protracted disease is well known. Prosthetic-valve endocarditis due to mycobacteria is a rare, highly fatal infection that occur as small nosocomial outbreaks in which seasonal environmental conditions, contaminated bone wax, water baths, or surgical personnel are clearly epidemiologic factors. The disease occurs within 6 months of valve replacement and can be traced to valve contamination at surgery. In some cases, Mycobacterium spp. were found to contaminate porcine valves after inadequate sterilization during manufacturing and storage 241. Other cases have been associated with sternal wound infections following surgery 146. These patients presented with symptoms typical of endocarditis, such as fever, chills, night sweats, anorexia, and headaches 43, 146. The role of M. avium complex in causing endocarditis is still debated (117, 149; Butany, Letter), and misdiagnoses could result from laboratory contamination by water 117.

(i) Diagnosis. Mycobacterial endocarditis may be diagnosed by blood culture 92, 254, 288, 293; Middlebrook solid culture medium and the nonradiometric Bactec 9000 MB system have been shown to be equally effective in isolation of nontuberculous Mycobacterium spp. from blood 130. However, the diagnosis is frequently achieved by histologic examination of the resected valve or at autopsy by Ziehl-Neelsen staining of acid-fast bacilli. Mycobacterium spp. have been isolated from valve vegetations, bone marrow, urine, sputum 92, 159, and lung biopsy specimens 92.

(ii) Treatment. Combination therapy for infections due to Mycobacterium spp. seems advisable, since single-agent therapy has been shown to select for the emergence of resistant mutants 274. However, there are no controlled studies regarding therapy. Courses longer than 6 months may be needed for endocarditis. Consideration should be given to resection of the focus of infection and to removing foreign material in patients who are surgical candidates 288, 294.

Mycoplasma spp. Despite the fact that endocarditis due to cell wall-deficient bacteria has been suggested since 1978 215, endocarditis due to Mycoplasma spp. has been reported in the literature only twice 45, 217. The first report described a patient with postrheumatic heart disease; the diagnosis was based on serologic test results alone, and no culture was reported. He was treated with 12 × 106 U of benzylpenicillin and 280 mg of gentamicin daily for 6 weeks and then given a 4-week course of oxytetracycline 217. The other case was reported in 1989 45 and involved a 25-year-old woman with a history of SLE, treated with corticosteroids, in whom Streptoccocus sanguis aortic and mitral valve endocarditis developed. Aortic and mitral prosthetic valve replacements were performed for acute cardiac failure. During penicillin treatment, she developed a sternal wound infection and fever. Swabs of the sternal wound and blood cultures were sterile. The prosthetic valves were replaced owing to significant valve dysfunction and renewed cardiac failure. At surgery there was no evidence of sternal or mediastinal infection, but extensive dehiscence of the aortic valve suture line was noted with multiple small abscesses and fistulous tracts extending from the aortic annulus. The mitral valves appeared grossly infected. Mycoplasma hominis was isolated from a portion of the mitral annulus. The presence of sodium polyanetholsulfonate in the blood culture media might be responsible for the lack of M. hominis growth 55. This patient was cured with clindamycin and rifampin administered for 6 weeks followed by a 4-week course of oral doxycycline 45. The role of Mycoplasma spp. in culture-negative endocarditis is unknown and very probably underestimated. This small organism cannot be detected by Gram stain, and growth is not detected in routine blood culture systems. Likewise, Mycoplasma spp. can be difficult to detect in paraffin-embedded valve tissues when using nonspecific staining techniques. Strategies for isolation of this pathogen in patients with culture-negative endocarditis should include systematic subculture of blood and excised valves. Inoculation should be made onto specific Mycoplasma agar media such as SP 4 glucose (pH 7.5), which can be used for both M. pneumoniae and M. hominis, provided that arginine is added for the latter organism. Mycoplasma serologic testing should be evaluated. PCR techniques will be probably very helpful in detecting Mycoplasma spp. in blood or in infected valves.

Whipple's disease bacterium. Whipple's disease is a rare systemic disorder presenting with arthralgia, diarrhea, abdominal pain, generalized lymphadenopathy, progressive weight loss, and central nervous system involvement. This disease is caused by a gram-positive bacterium that has been identified by sequence analysis of the 16S rRNA gene 234. The name Tropheryma whippelii has been suggested for cases when the bacteria have not successfully been established in culture 234. Recently, the Whipple's disease bacterium was propagated in an interleukin-4-treated macrophage cell culture but showed only limited propagation 251. In 2000, we reported the successful isolation and establishment of a strain of Whipple's disease bacterium obtained from the valve of a patient with blood culture-negative endocarditis 222. Up to one-third of patients with Whipple's disease present with concomitant cardiac symptoms, mostly attributed to endocarditis, myocarditis, pericarditis, or coronary lesions (125, 181, 188; M. Jeserich, C. Ihling, and C. Holubarsch, Letter, Ann. Intern. Med. 126:920, 1997). More recently, the relationship between endocarditis and the Whipple's disease bacillus was established in a patient who presented with diarrhea, fever, grand mal seizure, and a history of a 20-kg weight loss over 4 years 297. The diagnosis was based upon the presence of numerous PAS-positive macrophages in the duodenal biopsy specimen. After successful antibiotic therapy, he developed acute congestive heart failure due to aortic valve endocarditis, requiring urgent prosthetic valve replacement. Histologic examination revealed PAS-positive macrophages scattered throughout the entire valve (Fig. 3). DNA prepared from paraffin-embedded valve tissues identified T. whippelii by PCR and sequencing of the PCR product. Interestingly, PAS-positive granules have been demonstrated in the brains of patients with and without cerebral manifestation of the disease 84, and rheumatoid factor is found in 8% of these patients. It is likely that Whipple's disease endocarditis is much more frequent than currently reported. Recently, four cases were reported in patients without fever or evidence of systemic Whipple's disease 110. Before pathologic examination of the resected valves, which established the diagnosis, one patient fulfilled none of the Duke criteria, two patients fulfilled one major Duke criteria, and one patient fulfilled two Duke criteria (one major and one minor). Without examination of the excised valves, the diagnosis of IE could not have been made, indicating that Duke criteria for endocarditis are not pertinent for Whipple's disease endocarditis 110, 221. This underlined the fact that occurrence of the disease is probably underestimated and must depend on an active search for the cause by physician. In a recent, not yet published review on Whipple's disease endocarditis, we found that data for 34 patients had already been published with enough details to include the valvular lesion as a part of the disease and that those patients presented with aortic damage (14 of 34), mitral damage (5 of 34), tricuspid valve damage (2 of 34), mitral and aortic valve damage (5 of 34), mitral and tricuspid valve damage (3 of 34), and damage to three valves (5 of 34). The diagnosis is currently based on the histologic observation of PAS-positive macrophages in the surgically resected valve tissues and by PCR identification from either the valve or a duodenal biopsy specimen. Cultures on axenic media, using chocolate or Columbia sheep blood agar incubated at 32 or 37°C under 5% CO2, or in a microaerophilic or anaerobic atmosphere were unsuccessful. Similarly, cultures on cell culture medium and cell culture medium with lysates of HEL cells incubated at 32 or 37°C under 5% CO2 were not able to recover the bacilli. However, Whipple's bacilli can be cultured using human fibroblast cells lines (HEL and MRC5) which are grown in minimal essential medium with 10% fetal calf serum and 2 mM L-glutamine without antibiotics; a control strain is available for the scientific community (222). By observing the flask monolayer under an inverted microscope, a cytopathic effect could be observed after several weeks; small coarse dark inclusions and large coarse round structures were detected within cells. The cultured bacterium can been identified by PCR and sequencing of the 16S rRNA.

Legionella spp. Legionella spp. are small, facultative, gram-negative intracellular bacteria that cause nosocomial pneumonia and, rarely, disease of extrapulmonary sites 165. Endocarditis due to Legionella spp. has been cited in only three publications. In 1984, McCabe reported the first case of Legionella pneumophila endocarditis in a patient with porcine aortic and mitral valve prostheses 182. In 1988, Tompkins et al. reported seven cases of nosocomial prosthetic-valve endocarditis at Stanford University Hospital Center. L. pneumophila was isolated in two cases, L. dumofii was isolated in three cases, both species were identified in one case, and an undefined Legionella sp. was serologically diagnosed in one case 281. In 1994, Park et al. reported one case of serologically diagnosed Legionella micdadei prosthetic-valve endocarditis 208.

(i) Epidemiology. The natural habitat for L. pneumophila appears to be water, and numerous outbreaks of nosocomial legionellosis due to contaminated water distribution systems have been reported. The most frequently implicated reservoirs for Legionella spp. are cooling towers and hot-water tanks 268. In the report of Tompkins et al., L. pneumophila was repeatedly isolated from drinking-water sites in the Medical Center, but these investigators were unable to clarify whether these bacteria were inoculated during or after surgery 281. All documented cases reported in the literature were nosocomial in origin.

(ii) Signs and symptoms. Patients with Legionella endocarditis often have chronic symptoms, including low-grade fever, night sweats, weight loss, malaise, and symptoms of congestive heart failure. Anemia was reported as a frequent feature, and its severity appeared to be correlated with the duration of the infection. Leukocyte counts were normal, but thrombocytopenia was observed. Unlike prosthetic-valve endocarditis caused by fungi, Legionella endocarditis has not been associated with embolic phenomena.

(iii) Echocardiography and pathology. Similar to the situation reported for Q fever, Legionella endocarditis vegetations are rarely reported on echocardiography, including transesophageal echocardiography. Only three of the nine patients reported in the literature were diagnosed by echocardiography 281. Small vegetations were observed on excised valves in five of six surgically treated patients 281. The small size of these vegetations may explain the lack of embolic manifestations and the low echocardiographic sensitivity.

(iv) Diagnosis. Legionella endocarditis should be suspected in febrile patients with prosthetic valves and negative blood cultures when serologic testing does not support a diagnosis of Q fever. Since most of reported cases are nosocomial in origin, Legionella endocarditis is likely to occur in small outbreaks. Diagnostic delays vary from 3 to 19 months after surgery. Definitive diagnosis may be made by cultivation of the excised valve, a method that was successful in seven of eight cases, and by blood culture, which was successful in three of eight cases 182, 281. In fact, when endocarditis caused by Legionella spp. is suspected, the clinical microbiology laboratory should be contacted. Legionella spp. will grow in most commercial automated blood culture systems, but the amount of growth may be inadequate to attain preestablished thresholds for detection. Consequently, periodic subcultures of incubating blood cultures from those systems to the preferred medium for Legionella spp.---buffered charcoal yeast extract (BCYE)---is recommended 15. Serum antibody titers are usually high in cases of Legionella endocarditis, but the role of serologic testing has not yet been evaluated and the positive predictive value is not known. PCR assays have been used to detect Legionella spp. in clinical samples 268. Despite the high specificity of PCR, it is no more sensitive than culture 268.

(v) Prognosis, prevention, and treatment. None of the nine reported patients with Legionella endocarditis died, but six patients required prosthetic valve replacement. Erythromycin at 2 g/day intravenously with rifampin at 600 to 1,200 mg/day or ciprofloxacin at 600 mg/day intravenously was used in all patients except for one, who was treated with doxycycline at 200 mg twice a day 208. The duration of therapy was at least 5 months, and no relapses were observed 281.

Chlamydia psittaci. Chlamydia spp. are obligate intracellular organisms that can be grown only in tissue culture. The intracellular location explains the difficulty in isolation and identification and the difficulty in investigating its role as a causative agent of endocarditis. Chlamydia spp. have often been suggested to be causative agents of endocarditis (18, 24, 60, 61, 89, 132, 136, 156, 201, 231, 253, 285, 291, 295; R. Norton, S. Schepetiok, and T. W. Kok, Letter, Lancet 345:1376-1377, 1995. D. Dumont, D. Mathieu, M. Alemanni, F. Eb, and G. Manigand, Letter, Press Med. 19:1054, 1990). However, in a recent review of 10 patients reported to have chlamydial endocarditis, 8 were finally diagnosed with Bartonella endocarditis after their sera were tested by antibody cross-absorption and Western immunobloting 176, 225. Interestingly, epidemiologic data for three of these eight patients were comparable to those reported for Bartonella endocarditis patients, including homelessness and alcoholism 172, 176. Therefore, by using serologic testing only, it is difficult to know how many patients really have chlamydial endocarditis.

Culture-confirmed chlamydial endocarditis has been identified in only one patient 253, in whom Chlamydia psittaci was cultured from throat and blood. Even in this case, the diagnosis could not be totally accepted since the cultured microorganism was identified by immunologic techniques only, and still might have represented a Bartonella sp. or atherosclerosis-related Chlamydia pneumoniae 219.

The recent report of a serologic cross-reaction between Chlamydia and Bartonella suggest that serologic results should be carefully interpreted 176. In patients with suspected Chlamydia endocarditis, Bartonella spp. should be excluded by careful serologic analysis including cross-absorption assays and attempts at isolation of Bartonella spp. Direct identification of microorganisms by monoclonal antibody immunohistochemistry in resected valves may help, but the sensitivity and specificity of such techniques have not yet been evaluated 253. In the only reported case involving isolation of the organism, whole blood was collected in the absence of anticoagulant, the serum was removed, and the clot was liquefied in a vortex in the presence of glass beads before the suspension was centrifuged to remove debris 253. The suspension was inoculated onto L929 cells in a shell vial, and the organisms were detected with a rabbit polyclonal antibody directed against C. psittaci. However, it is again important to note that cross-reactions of Chlamydia spp. with Bartonella spp. using rabbit polyclonal antibody may preclude a definitive identification. Monoclonal antibodies, cross-absorption of sera with Western immunoblots, and/or molecular techniques such as PCR with hybridization, which is now commercially available, should be used to clearly identify Chlamydia spp. in patients with endocarditis.

Coxiella burnetii (Q fever agent). Q fever endocarditis is caused by the obligate intracellular pathogen C. burnetii. This bacterium lives and multiplies in the phagolysosomes of infected cells at pH 4.8. In culture, it demonstrates phase variation (phase 1 to phase 2) that is equivalent to the lipopolysaccharide smooth-rough phase variation of members of the family Enterobactericeaceae, an extremely valuable property for diagnosis. Only C. burnetii cells expressing phase 1 lipopolysaccharide are infectious. Only patients with chronic Q fever present with high-level phase I immunoglobulin G (IgG) and IgA antibodies.

(i) Epidemiology. Q fever is prevalent in all countries where it has been studied
227. At the time of this writing, 359 cases of Q fever endocarditis have recorded throughout the world, with 249 being diagnosed in our laboratory 228. Q fever is a zoonosis that is widespread throughout the world and can present as an acute or chronic disease. Culture-negative endocarditis is the most prevalent chronic form and represents 78% of cases of chronic Q fever. Q fever endocarditis represents 8.7 to 11% of all reported Q fever cases 170, 207, causing 3 to 5% of all cases of endocarditis in France, Israel, and Great Britain 207. The incidence of the disease has been determined to be 1 case per million inhabitants per year in France. The reservoir of C. burnetii is only partially known. Although farm animals such as cattle, goats, and sheep are considered primary reservoirs, pets including cats and dogs have also been reported to be infected, explaining urban outbreaks 171. When infected, all of these mammals shed the desiccation-resistant organisms in urine, feces, milk, and birth products 12. The extreme resistance to physical agents allows C. burnetii to survive for a long period in the environment. Subsequently, efficient transmission and infection may be wind-borne over long distances owing to the ability of this organism to persist in an environment that lacks mammalian hosts 266, 276. In humans, infection usually results from inhalation of dust contaminated with parturient fluids of infected livestock. In France, 80.2% of patients with endocarditis had an identified risk exposure; 59.6% of patients reported animal exposures, and half of these reported exposures to sheep 29. Infection by consuming raw milk occurs in only 9.6% of patients 29. Interestingly, only 30% of endocarditis patients live in rural areas, indicating that contamination occurs mainly during short exposures such as farm visits or other recreational activities. Occupational exposure is found in 12.5% of endocarditis patients, of whom most are male farmers 29. Together, occupational risk, rural life, and raw milk are significantly associated with C. burnetii endocarditis compared with endocarditis of other etiologies (D. Raoult, unpublished data). Two-thirds of patients are male (gender ratio, 2.49). The relative risk of contracting Q fever endocarditis is five times higher in the 60- to 69-year age range, whereas patients younger than 40 years have only a minor risk. Underlying disease is found in 90% of patients 266. Most patients (88.5%) have a preexisting valvular injury.

Underlying heart diseases may be congenital, rheumatic, degenerative, or syphilitic. Aortic and mitral valves are equally involved, but tricuspid valve endocarditis has been reported only once, in a child with a congenital fistula 166. Valvular prostheses are present in 55.7% of patients. Immunocompromising conditions such as cancer, leukemia, and chronic renal failure with dialysis are found in 9% of Q fever endocarditis patients. Immunocompromise is especially frequent in endocarditis patients without preexisting valvular injury, being present in five of seven patients in one study 29, indicating that immunity plays a critical role in the clinical expression of Q fever 223.

(ii) Signs and symptoms. Q fever is a severe and often fatal disease that is often associated with a long diagnostic delay. Complaints are related either to the heart (cardiac failure, valve dysfunction) or to a general illness such as low-grade and intermittent fever, fatigue, and weight loss. Because these symptoms are not specific and vegetations are rarely detected by echocardiography, diagnosis is often made late, with a mean delay of 12 months. Fever and acute cardiac failure are the most frequent signs of Q fever endocarditis and are observed in 68 and 67% of patients, respectively. Cardiac failure is usually diagnosed with general manifestations of dyspnea, acute pulmonary edema, angina, and palpitation. Fever is often low-grade (38 to 38.5°C), remittent, and well tolerated, explaining why few patients directly seek medical intervention for this alone. The presence of splenomegaly can be very prominent and is correlated with diagnostic delays 259, sometimes leading to a misdiagnosis of hematologic malignancy 28. Hepatomegaly is also correlated with diagnostic delay, and when it is present, the liver is generally hard and considerably enlarged. Histologic test results for the liver in endocarditis are not specific; granulomas may be observed, but the fibrin ring surrounding the granulomas, with a lipid vacuole in the center giving the typical "doughnut" aspect frequently observed in acute Q fever, have never been found in the livers of patients with endocarditis 57, 283. Digital clubbing is seen in one-third of patients, a higher rate than is generally observed for other types of endocarditis 266. A purpuric rash is found in one-fifth of patients and generally occurs on the extremities and the mucosa 266. When a skin biopsy is performed, histologic examination of the specimen shows immune complex vasculitis. Renal involvement can occur, and proliferative glomerulonephritis has been reported 212, 280, 284, increasing the risk of renal insufficiency. Embolic manifestations occur in about 20% of patients and can involve cerebral and upper- or lower-extremity vessels. Embolectomy or amputation may be required. One typical manifestation of arterial embolism is stroke. Pulmonary and pleural manifestations can be observed as a complication of endocarditis 169.

Clues to the diagnosis of Q fever endocarditis include valvular heart disease, such as valvular dysfunction, in association with an unexplained infectious or inflammatory syndrome 227. This could be a purpuric rash, renal failure, stroke, or, more frequently, progressive heart failure. Some patients require several valve replacement surgeries before the diagnosis is established. Laboratory findings during Q fever endocarditis 29, 266 include circulating immune complexes (in 89% of patients), rheumatoid factor (in 83%), anaemia (<10 mg/100 ml) (in 60%), thrombocytopenia (<150,000/mm3) (in 50%), and microscopic hematuria (in 38%). Serum hepatic transaminase concentrations, particularly aspartate aminotransferase and alkaline phosphatase, are elevated in most patients 29. Lactate dehydrogenase and creatinine phosphokinase levels are also frequently increased, as are cryoglobulin levels 266. Hyperglobulinemia is observed in 94% of patients. This is reported to be helpful when the globulin fraction represents more than 50% of the total protein concentration. In this case, a polyclonal increase in the IgG and IgA levels is noted. The longer the diagnostic delay, the higher the globulin concentration becomes. Anti-smooth muscle antibodies, circulating anticoagulant antibodies, antimitochondrial antibodies, a positive Coombs test, and low titers of antinuclear antibodies may be observed (158; L. Elovaer-Blanc, C. Andre, E. S. Zafrani, M. F. Saint-Marc Girardin, M. Govault-Helmann, and D. Dhumeaux, Letter, Gastroenterol. Clin. Biol. 8:980, 1984).

(iii) Pathophysiology and echocardiography. Patients suffering from Q fever endocarditis have profound lymphocyte unresponsiveness to C. burnetii that results in a lack of macrophage activation 244. Peripheral blood mononuclear cells from patient with Q fever endocarditis produce large amounts of interleukin-10 and transforming growth factor beta  that may play a role in the survival of C. burnetii in macrophages 37. A very high level of specific antibodies is found in the humoral immune response in Q fever endocarditis. These antibodies are not protective and, in association with C. burnetii antigen, result in immune complexes that are responsible for many aspects of the disease 49. The general pathologic changes of valves in patients with Q fever endocarditis differs from those seen in patients with other causes of endocarditis in that Q fever vegetations often have a nodular appearance and a smooth surface. In some cases, the valves even appear normal on gross examination. By histologic testing, the valves demonstrate a mixture of acute and chronic inflammation with fibrin deposits, necrosis, and fibrosis; no well-formed granulomas are observed. C. burnetii can be detected in valves by immunohistochemistry, which shows that bacteria occur nearly exclusively in macrophages at sites of inflammation and valvular injury and only in the vegetations (Fig. 5) 25. Transthoracic echocardiography detects vegetations in only 12.5% of patients 29. In fact, Turck et al. reported that of 10 valves from patients with Q fever endocarditis examined either at autopsy or after surgery, small vegetations with insignificant valve damage were observed in 5 283. The use of transesophageal echocardiography appears to be promising 137.

(iv) Diagnosis. Q fever should be considered in all patients with culture-negative endocarditis. A specific diagnosis is easily made by serologic testing. Antibody to phase 1 and phase 2 antigens may be determined using a variety of tests. The reference technique is IFA, which should distinguish between titers of IgM, IgG, and IgA antibodies. Q fever endocarditis is characterized by a very high titer of antibody to both phase 1 and phase 2 antigens of C. burnetii. An anti-phase 1 IgG antibody titer of >= 800 plus IgA antibody >= 100 is highly predictive and sensitive 279. IgM titers may vary. In these situations, a single serum sample is sufficient for the diagnosis of Q fever endocarditis. C. burnetii may be isolated from the blood in 53% of untreated patient 193 and from the valves even in treated patients 192. Isolation of C. burnetii in cell culture should be restricted to laboratories equipped for isolation of dangerous pathogens. In our laboratory, culture of C. burnetii is obtained by inoculation of human fibroblasts grown in shell vials 229. After 6 days, infected tissue culture cells are revealed by IFA with rabbit polyclonal antibodies or mouse monoclonal antibodies. The diagnosis of Q fever endocarditis may also be established by demonstrating C. burnetii in heart </