new insight into the diagnosis of fastidious bacterial endocarditis

M I N I R E V I E W

New insight into the diagnosis offastidiousbacterial endocarditisPhilippe Brouqui & Didier Raoult

Service des Maladies Infectieuses et Tropicales, CHU Nord AP-HM & Unite des rickettsies, Faculte de Medecine,

Universite de la Mediterranee, Marseilles, France

Correspondence: Philippe Brouqui, Service

des Maladies Infectieuses et Tropicales, CHU

Nord AP-HM & Unite des rickettsies, CNRS

UMR 6020, IFR 48, Faculte de Medecine,

Universite de la Mediterranee, Marseilles,

France. Tel.: 133 491 324375;

fax: 133 491 830390;

e-mail: [email protected]

Received 4 November 2005; accepted 14

November 2005.

First published online 8 February 2006.

doi:10.1111/j.1574-695X.2006.00054.x

Editor: Willem van Leeuwen

Keywords

culture-negative endocarditis; Bartonella sp;

Coxiella burnetii; PCR; whipple’s disease;

HACEK.

Abstract

Sterile blood cultures are noted in one third of patients with infectious endocardi-

tis. Although in half of cases this is due to previous antibiotic therapy, in the other

half, the aetiology of culture-negative endocarditis is intracellular bacteria such as

Coxiella burnetii or fastidious growing bacteria. Although it was previously

considered that the prevalence of such organisms was identical throughout the

world, recent investigations on Bartonella endocarditis clearly showed that the

aetiology of culture-negative endocarditis is likely to be strongly related to

epidemiology of the agent in each country. During the past decade the use of

molecular techniques such as PCR with subsequent sequencing to detect or to

identify bacteria in valves from patients with infectious endocarditis have

considerably improved the aetiological diagnosis. This is especially true in the case

of culture-negative endocarditis following earlier antibiotic therapy. However, the

fact that DNA remnants of past endocarditis can be detected some time after the

acute episode, when the patient has been cured, suggests that the predictive value

of these techniques along with the traditional histology and culture need to be

evaluated closely.

Introduction

Infective endocarditis can be evoked in a patient by fever

and a new or changing cardiac murmur. The diagnosis is

most often based upon the detection of vegetation on the

cardiac valves using echocardiography and positive blood

culture. However, numerous situations in which blood

culture or echocardiography are not able to confirm the

diagnosis, lead to a high degree of suspicion of endocarditis.

Fever, the most common finding in endocarditis, may not

be present in the elderly or in patients given antibiotic

therapy before presentation, or in Whipple’s disease (Ri-

chardson et al., 2003) and it may be low-grade or inter-

mittent in Q fever endocarditis (Brouqui et al., 1993).

Cardiac murmur is the second most frequent finding in

endocarditis but may be absent in the initial stage of right

side endocarditis. A new or changing murmur is detected in

40% of patients with endocarditis only (Stamboulian &

Carbone, 1997). Echocardiography has assumed a central

role in the diagnosis of suspected endocarditis. Transeoso-

phagal echocardiography is more invasive and expensive

than transthoracic echocardiography but reportedly is more

efficient in detecting smaller lesions. Its role in routine

screening for endocarditis is controversial. The sensitivity

of echocardiography depends upon the causative organism,

the operator and the quality of the echocardiograph. In a

recent study, vegetation was detected in 87% and 75% of

cases of Bartonella and Whipple’s disease endocarditis,

respectively, and in only 13% of Q fever cases (Fenollar

et al., 2001), while it was detected in 39% of positive blood

culture endocarditis (Watanakunakorn & Burkert, 1993).

However, with the use of modern diagnosis techniques

that will be reviewed below, especially PCR and culture of

infected valves, the number of cases without a detected

aetiology dropped from 27% to 9% and 1.4% in the last

published series (Hoen & Alla, 2002; Lamas & Eykyn, 2003;

Werner et al., 2003a; Houpikian & Raoult, 2004). Antibiotic

treatment preceded blood culture in 45–60% of cases of

culture-negative endocarditis (CNE) (Lamas & Eykyn, 2003;

Werner et al., 2003a). Another cause of CNE is subacute

right side endocarditis and mural endocarditis (Brouqui &

Raoult, 2001). Slow-growing and fastidious organisms re-

present approximately 5–15% of infective endocarditis and

50% of CNE. The most frequently reported of these are

FEMS Immunol Med Microbiol 47 (2006) 1–13 c� 2006 Federation of European Microbiological SocietiesPublished by Blackwell Publishing Ltd. All rights reserved

Coxiella burnetii, Brucella spp., Abiotrophia spp., HACEK

group endocarditis and Listeria monocytogenes (Brouqui &

Raoult, 2001). More recently discovered, an increase in

endocarditis due to the fastidious Bartonella spp. has been

reported (Fournier et al., 2001). Some of these slow-growing

bacteria require specific media such as L-cysteine-enriched

medium for Abiotrophia spp., special culture conditions for

anaerobes or intracellular bacteria and incubation times as

long as 6 weeks (Maurin & Raoult, 1996). The lack of

systematic serological testing for Bartonella spp. and C.

burnetii in CNE, as well as the length of incubation of blood

cultures, are important limiting factors in the aetiological

diagnosis of CNE and may explain the variation in the

proportion of CNE in patients with infectious endocarditis

reported in the literature (Werner et al., 2003a). In such

situations, infective endocarditis remains a diagnostic chal-

lenge.

This review is based on a previous review (Brouqui &

Raoult, 2001) completed using a Medlines search with the

following keywords from 1998–2004: culture-negative en-

docarditis (n = 161), diagnosis (n = 130), echocardiography

(n = 41), PCR (n = 40). Articles have been selected on the

basis of the scope of the study, the impact factor and the half

life citation of the journal, as well as the accessibility of the

journal.

Epidemiology

Endocarditis with a negative blood culture account for

2.5–48% of cases (Tariq et al., 2004). Developing countries

have the highest rate of CNE, but it is usually admitted that

one major explanation for this is the lack of efficient

microbiology. However, in a comparative study made by

the same research group on the aetiology of endocarditis in

Slovakia, CNE accounted for 26.7% in the 1991–1997 period

and for 53.3% of cases in the 1998–2001 period, despite use

of better bacteriological techniques (Krcmery et al., 2003).

Interestingly, no serological testing was performed for

Coxiella burnetii and Bartonella in that study and the

respective part these two major infectious agents play in

CNE is unknown. This suggests that epidemiological

factors interfere with the aetiology of CNE. In two recent

studies of our laboratory, we demonstrated that the pre-

valence of Bartonella spp. as an aetiological agent of CNE

varies greatly depending on the country. In Tunisia and

Algeria, Bartonella endocarditis accounts for 9.8% and 15%

of all endocarditis, respectively (Znazen et al., 2005; Bensli-

mani et al., 2005). The poor living conditions in some

countries such as Algeria, where louse-borne typhus has re-

emerged, may explain the high prevalence of Bartonella

quintana in CNE.

Several studies on Bartonella endocarditis have previously

been published, reporting prevalences of 0–4.5% of all

infective endocarditis. In France, Bartonella species account

for 3% of infective endocarditis in Marseilles and for 4.5% in

Lyon (Raoult et al., 1996). In Germany, 3% of all endocardi-

tis cases investigated retrospectively by PCR on valve tissues

were attributed to Bartonella (with 2.6% attributed to

Bartonella henselae and 0.4% attributed to Bartonella quin-

tana). In Sweden, Bartonella endocarditis was reported in

only one case in 1997 and a study on 334 patients with

infective endocarditis did not find any cases of Bartonella

(Werner et al., 2003b). In the UK, a recent study showed that

Bartonella species accounts for 1.1% of infective endocardi-

tis (Lamas & Eykyn, 2003). These data suggest a north to

south gradient in the distribution of Bartonella infections

(Fig. 1). One may also suggest that the prevalence of C.

burnetii as an aetiological agent of CNE may vary depending

on the level of exposure to cattle. As a consequence, the

prevalence of the aetiological agent in CNE is likely to be

dependent upon environmental exposure and may vary

greatly between countries.

Diagnosis of infectious endocarditis

Role of echocardiography in the diagnosis ofinfective endocarditis

Transeosophagal echocardiography (TEE) has been reported

to have a better sensitivity than transthoracic echocardio-

graphy (TTE) (Jacob & Tong, 2002), most studies reporting

sensitivities of 30–50% and 85–100% for TTE and transeo-

sophagal echocardiography, respectively. Transeosophagal

echocardiography appears to be especially useful for the

diagnostic evaluation of patient with suspected prosthetic

valve endocarditis (Roe et al., 2000). Transeosophagal echo-

cardiography is also superior to TTE in detecting mechan-

ical complications such as valve perforation and chordal

rupture (Jacob & Tong, 2002). As a general rule, TTE should

be performed in anyone with suspected endocarditis (Ro-

bles, 2003). Transeosophagal echocardiography should be

performed first in patients with possible endocarditis with

clinical criteria, or in cases of prosthetic valve endocarditis

or in cases of complicated infective endocarditis (Li et al.,

2000).

New approaches of diagnostic scores

To both assist physicians in establishing the final diagnosis

of endocarditis and to allow comparison of published cases,

diagnostic scores have been defined. For many years, the

Beth Israel criteria were the only recognized diagnostic

criteria (Von Reyn et al., 1981). The use of echocardiography

has led to the inclusion of echocardiographic findings in the

criteria of the Duke endocarditis service (Durack et al.,

FEMS Immunol Med Microbiol 47 (2006) 1–13c� 2006 Federation of European Microbiological SocietiesPublished by Blackwell Publishing Ltd. All rights reserved

2 P. Brouqui & D. Raoult

1994). According to the Duke endocarditis service, the

diagnosis of infective endocarditis is definite: (1) when a

microorganism is demonstrated by culture or histological

testing of a vegetation, an embolism, or an intracardiac

abscess; (2) when active endocarditis is confirmed by

histological examination of vegetation or intracardiac ab-

scess; or (3) when two major criteria and three minor

criteria or five minor criteria are met (Durack et al., 1994).

Evaluation of these criteria in patients with pathologically

proven endocarditis showed that 24% of patients were

misclassified as ‘possible’ despite the use of Duke criteria

especially in case of CNE or Q fever infective endocarditis

(Habib et al., 1999). The overall sensitivity of the Beth Israel

and Duke criteria was evaluated to be 60% and 80%,

respectively (Lamas & Eykyn, 1997; Habib et al., 1999).

Consequently, several researchers have tried to improve the

value of these criteria and have suggested modifications that

take into account several clinical or microbiological criteria.

Among these proposed modifications are Coxiella burnetii

serology or the molecular detection of the aetiological agent

in the removed valve as major criteria (Fournier et al., 1996;

Li et al., 2000). In a study of 20 patients with infective

endocarditis confirmed at pathological examination, the

Duke endocarditis criteria misclassified four of them, all Q

fever endocarditis with C. burnetii IgG Phase I 4 800. The

authors conclude that including C. burnetii IgG Phase I

4800 or a single positive blood culture for C. burnetii as a

major criterion in the Duke criteria would improved their

sensitivity (Fournier et al., 1996). In another study of 100

cases of proven native valve endocarditis, the addition of

some minor criteria increased the sensitivity from 83% to

94% (Lamas & Eykyn, 1997). An often-heard criticism of the

Duke criteria is the over broad categorization of the group

‘possible infective endocarditis’. In 2000, a redefinition of

this group was proposed, based upon the review of the Duke

University data on more than 800 patients. This category has

been proposed to include patients with one major criterion

and one minor criterion or three minor criteria (Li et al.,

2000). Several issues remained including the relative risk of

infective endocarditis in the cases of Staphylococcus aureus

bacteraemia and the relative role of transeosophagal echo-

cardiography. Previous studies have shown that S. aureus

infective endocarditis is unlikely when the bacteraemia

is nosocomially acquired and a primary focus, such as

<1%

1-2

3 - 4

5 -10

> 10 %

Fig. 1. Prevalence (%) of Bartonella quintana endocarditis in Europe and North Africa.


3Diagnosis of fastidious bacterial endocarditis

intravascular device, is present at the time of bacteraemia (Li

et al., 2000). As a result, the original Duke criteria consid-

ered blood cultures that were positive for S. aureus to be a

major criterion only if community acquired in the absence

of primary focus (Durack et al., 1994). In a review of the

Duke endocarditis service database, 13% of patients with

nosocomially acquired S. aureus bacteraemia developed

infective endocarditis whether this bacteraemia was cathe-

ter-related or not. The authors suggest that S. aureus

bacteraemia should be considered a major criterion regard-

less of whether the infection is nosocomially acquired or a

removable source of infection is present (Li et al., 2000). The

Duke University Medical Center maintains prospective

databases on all echocardiograms performed since 1994.

Using these databases they suggest that transthoracic echo-

cardiography should be used at first in all patients except

those for whom transeosophagal echocardiography is re-

commended (patients with prosthetic valves, those rated as

at least ‘possible infective endocarditis’ by clinical criteria, or

those with complicated infective endocarditis such as para-

valvular abscess). In some cases of afebrile chronic endocar-

ditis caused by slow growing fastidious organisms such as

Whipple’s disease bacilli, the diagnosis remains a challenge

and is made only at surgery or autopsy (Gubler et al., 1999).

The revised Duke criteria are summarized in Tables 1 and 2

(Li et al., 2000).

Table 1. Definition of infective endocarditis according to the proposed

modified Duke criteria (Li et al., 2000)

Definite infective endocarditis

Pathological criteria

Microorganisms demonstrated by culture or histological examination

of a vegetation, a vegetation that has embolized, or an intracardiac

abscess specimen; or

Pathological lesions; vegetation or intracardiac abscess confirmed by

histological examination showing active endocarditis

Clinical criteria�

Two major criteria; or

One major and three minor criteria; or

Five minor criteria

Possible infective endocarditis

One major and one minor criterion; or

Three minor criteria

Rejected

Firm alternate diagnosis explaining evidence of infective endocarditis; or

Resolution of infective endocarditis syndrome with antibiotic therapy for

�4 days; or

No pathological evidence of infective endocarditis at surgery or autopsy,

with antibiotic therapy for � 4 days; or

Does not meet criteria for possible infective endocarditis, as above

�See Table 3 for definitions of major and minor criteria.

Table 2. Definition of terms used in the proposed modified Duke criteria for the diagnosis of infective endocarditis (infective endocarditis)

Major criteria

� Blood culture positive for infective endocarditis:

� Typical microorganisms consistent with infective endocarditis from two separate blood cultures: Viridans streptococci, Streptococcus bovis, HACEK

group, Staphylococcus aureus; or community-acquired enterococci, in the absence of a primary focus; or

� Microorganisms consistent with infective endocarditis from persistently positive blood cultures, defined as follows:

At least two positive cultures of blood samples drawn 412 h apart; or

All of three or a majority of Z4 separate cultures of blood (with first and last samples drawn at least 1 h apart)

� Single positive blood culture for Coxiella burnetii or antiphase I IgG antibody titre 41 : 800

� Evidence of endocardial involvement

� Echocardiogram positive for infective endocarditis [TEE recommended in patients with prosthetic valves, rated at least ‘possible infective

endocarditis’ by clinical criteria, or complicated IE (paravalvular abscess); TTE as first test in other patients], defined as follows:

Oscillating intracardiac mass on valve or supporting structures, in the path of regurgitant jets, or on implanted material in the absence of an

alternative anatomic explanation; or Abscess; or new partial dehiscence of prosthetic valve

� New valvular regurgitation (worsening or changing of pre-existing murmur not sufficient)

Minor criteria

� Predisposition, predisposing heart condition or injection drug use

� Fever, temperature 438 1C

� Vascular phenomena, major arterial emboli, septic pulmonary infarcts, mycotic aneurysm, intracranial haemorrhage, conjunctival haemorrhages, and

Janeway’s lesions

� Immunological phenomena: glomerulonephritis, Osler’s nodes, Roth’s spots and rheumatoid factor

� Microbiological evidence: positive blood culture but dues not meet a major criterion as noted above� or serological evidence of active infection with

organism consistent with infective endocarditis

� Echocardiographic minor criteria eliminated

TEE, transesophageal echocardiography; TTE, transthoracic echocardiography; IE, infective endocarditis.�Excludes single positive cultures for coagulase negative staphylococci and organisms that do not cause endocarditis.



Conditions leading to negative bloodculture

Fastidious agents

Intracellular agents

Several intracellular agents have been reported to be in-

volved in infective endocarditis (Table 3). The most fre-

quently detected is Coxiella burnetii, the agent of Q fever, but

others have been reported, such as Tropheryma whippelii and

Chlamydia pneumoniae (Fenollar et al., 2001; Gdoura et al.,

2002).

Q fever has been found in all the countries where it has

been investigated. More than 400 cases of Q fever endocar-

ditis have been found, 359 in the literature and more than

60 new cases diagnosed in our laboratory (Houpikian &

Raoult, 2004). Q fever endocarditis is often a severe disease

associated with a long diagnostic delay. Q fever represents

5% of endocarditis cases in France. It occurs almost exclu-

sively in patients with previous a cardiac defect or in

immunocompromised patients, and Q fever endocarditis is

being recognized increasingly all over the world. The clinical

presentation has changed over the last 30 years. With faster

diagnoses, the prevalence of heart failure, hepatomegaly,

inflammatory syndrome, anaemia and leucopaenia and

abnormal liver function tests have decreased significantly

(Houpikian et al., 2002). Diagnosis based on serum anti-

body response to C. burnetii phase I and II should be carried

out systematically in confronting CNE as it represents the

main aetiological diagnosis.

Whipple’s disease endocarditis is a specific entity. A

previous valvular disease is found in only 13% of patients

compared to 52% and 88% in Bartonella and Q fever

endocarditis, respectively (Fenollar et al., 2001). The absence

of fever is a characteristic of Whipple’s endocarditis and is

reported in only 26% of patients. In the majority of cases the

endocarditis is associated with other signs of Whipple’s

diseases such as diarrhoea, arthralgia, abdominal pain and

lymphadenopathy (48%). Death occurs in 57% of cases.

Vegetation is detected by echocardiography in 75% of cases.

Anaemia (80%) and hypereosinophilia (40%) are specifi-

cally associated with Whipple’s disease (Fenollar et al.,

2001).

Chlamydia spp. have often been suggested to cause CNE

but this has rarely been proven. Most reported cases were

due to serological cross-reaction with Bartonella spp. (Brou-

qui & Raoult, 2001). To our knowledge there are two

documented cases of chlamydial endocarditis reported, one

due to Chlamydia psittaci in which the organism has been

cultured from the blood and another recent case of Ch.

pneumoniae in which it as been molecularly characterized

(Gdoura et al., 2002).

Isolation of these agents requires tissue cell culture

facilities and a laboratory staff experienced in the growth of

intracellular organisms. The availability of sequenced gen-

omes now provides the opportunity to define culture media

for the growth of fastidious pathogens. This has been

applied successfully to the growth of T. whippelii in cell-free

culture medium, creating the opportunity to transfer the

Table 3. Prevalence of bacterial agents involved in culture-negative

endocarditis until 2004 as detected by a search in Medlines with the

following keyword (‘‘bacterial name’’, endocarditis) and no restrictions

Bacteria Agent characteristics

No. of

published

cases

(Medlines

2004)

Coxiella burnetii SIC Gram negative 419

Bartonella spp. FIC Gram negative 120

Brucella spp. FIC Gram negative

bacilli

120

Abiotrophia spp. EX Gram positive

cocci

110

Actinobacillus

actinomycetemcomitans

EX Gram negative

bacilli

102

Haemophilus aphrophilus EX Gram negative

bacilli

78

Cardiobacterium

hominis

EX Gram negative

bacilli

78

Corynebacterium

diphtheriae

EX Gram negative

bacilli

67

Haemophilus

parainfluenzae

EX Gram negative

bacilli

68

Listeria monocytogenes EX Gram positive

bacilli

68

Erysipelothrix

rhusiopathiae

EX Gram positive

bacilli

52

Neisseria spp. EX Gram negative

cocci

o 50

Gemella spp. EX Gram negative

cocci

o 50

Mycoplasma spp. Epicellular no Gram

stain

o 50

Campylobacter spp. EX Gram negative

bacilli

o 50

Pasteurella EX Gram negative

bacilli

o 50

Mycobacterium spp. FIC Ziehl positive

bacilli

o 50

Legionella spp. FIC Gram negative

bacilli

o 50

Whipple’s disease

bacillus

FIC Gram negative

bacilli

o 50

Francisella tularensis FIC Gram negative

bacilli

o 50

Yersinia spp. FIC Gram negative

bacilli

o 50

SIC, strict intracellular; FIC, facultative intracellular; EX, extracellular.



technology to routine laboratories (Renesto et al., 2003). In

the meantime, infective endocarditis caused by these organ-

isms remains the true CNE. Etiological diagnosis of these

intracellular agents relies mostly on serology and molecular

techniques, which will be described below.

Slow-growing bacteria

Several other agents are classified as causing CNE because

the blood cultures are still negative after 48 h of incubation.

This is due to the slow growth of these agents. The most

prevalent organisms in this category are Bartonella spp. and

Brucella spp.

Bartonella endocarditis has been reported in 120 cases in

the literature. Most cases are caused by B. quintana or B.

henselae but other Bartonella spp. have occasionally been

reported. Bartonella quintana has been associated with

body-louse infested alcoholic homeless persons and B.

henselae with patients with a previous valvulopathy and

contact with cats or their fleas (Fournier et al., 2001).

Valvular surgery is needed in 96% of cases. Bartonella spp.

are isolated from blood by prolonged incubation in the

Bactecs system (Becton Dickinson), inoculation on rabbit

blood agar or on tissue cell culture (Brouqui & Raoult, 2001).

Brucella endocarditis represent 1.1% of brucellosis and

3.5% of endocarditis cases in Spain (Reguera et al., 2003).

An occupational exposure is found in almost all patients and

an underlying heart disease is reported in 45% of patients.

The mean duration of symptoms before diagnosis is 3

months. The aortic valve is involved in 80% of cases.

Although some systems such as the lysis concentration

technique lead to shorter isolation time, Brucella spp.

growth takes at least 4–10 days. However, despite the fact

that Brucella endocarditis is generally associated with CNE,

in a recent study, 64% of Brucella endocarditis cases were

culture positive in patients without previous antibiotic

therapy if processed correctly (Reguera et al., 2003). Brucella

endocarditis is severe and has cited as responsible for death

in 80% of patients who died of brucellosis.

Other agents are Mycobacterium spp., Francisella tular-

ensis, HACEK group and Legionella spp. Francisella tular-

ensis was detected in blood after 9 days of incubation,

whereas the mean duration time for HACEK group is 3–5

days, and Actinobacillus actinomycetemcomitans may require

up to 30 days for growth (Houpikian & Raoult, 2003a). In

some situations, Brucella spp. as well as Bartonella spp. were

recovered from tissue cell culture only (Rovery et al., 2003).

Anaerobic bacteria

Endocarditis caused by anaerobes is uncommon and usually

associated with CNE. Propionibacterium spp. and the

Bacteroides fragilis group are the cause of most cases of

anaerobic endocarditis (Bisharat et al., 2001). Although

anaerobes are usually detected in anaerobic blood culture

media, subculture and isolation in nonselective culture

media under anaerobic conditions is slow and requires an

incubation time of at least 5 days.

Nutritionally deficient bacteria

Abiotrophia species formerly known as nutritionally defi-

cient streptococci can be detected in routine blood culture in

2 or 3 days. However, subculture usually requires supple-

mentation of blood agar or broth with pyridoxal hydro-

chloride or L-cysteine at 1–1000 mg mL�1. Legionella species

required buffered charcoal yeast extract (BCYE) agar. Myco-

plasma spp. grow better in media such as SP4 glucose at pH

4.5. Mycobacterium spp. have been isolated in conventional

blood culture systems with prolonged incubation time, but

special media such as Middlebrook 7H13 broth should be

considered especially for Mycobacterium tuberculosis. Most

Haemophilus spp. grow well on conventional chocolate agar,

but require either exogenous haemin (X factor) or NAD

(V factor).

Previous antibiotic therapy

Previous antibiotic therapy is noted in two thirds of patients

with CNE (Brouqui & Raoult, 2001; Lamas & Eykyn, 2003).

The duration of previous antimicrobial therapy is an

important factor. If antibiotics are given for only 2–3 days,

blood cultures that were initially negative rapidly become

positive. However, after longer noncurative courses of

therapy, some blood cultures remained negative for a

number of weeks (Tunkel & Kaye, 1992).

Other conditions (right heart)

Blood cultures are frequently negative in patients with right-

side endocarditis and mural endocarditis (Brouqui &

Raoult, 2001). Of 11 nondrug addict patients with tricuspid

valve endocarditis diagnosed with two-dimensional echo-

cardiography, seven had S. aureus positive blood culture and

four were culture negative (Naidoo, 1993).

Strategies developed for the diagnosis ofCNE

From blood specimens

Blood culture

Quantitative culture techniques show that blood from

patients with infective endocarditis contains 1–10 bacteria

per mL, and this quantity remains constant during the

course of the disease. Because of the approximate



correlation between the yield of the bacteria from blood and

the volume of blood drawn, it has been recommended that

at least 10 mL of blood be obtained for each culture. The use

of more than three blood cultures will not improve diag-

nosis, and therefore culture of three sets (anaerobic and

aerobic) of blood drawn with an interval of at least 1 h

within a 24–48 h period are normally sufficient to establish

the diagnosis of culture positive endocarditis and, conver-

sely, to indicate a possible diagnosis of culture negative

endocarditis (Brouqui & Raoult, 2001; Houpikian & Raoult,

2003a). Incubation time is one of the major limiting factors

for recovery of fastidious organisms. Several of them may

require a number of weeks to grow and in patients with

suspected endocarditis with negative blood culture the

incubation time can be as long as 30–42 days. If antibiotics

have not been previously administered and the blood

cultures are negative, a fastidious growing organism should

be suspected.

Tissue cell culture

Tissue cell culture was developed initially for isolation of

strict intracellular bacteria. Use of the ‘shell vial’ technique,

adapted from a technique used for isolation of cytomegalo-

virus, has resulted in the isolation of C. burnetii, recognized

today as one of the most common aetiological agents of CNE

(Marrero & Raoult, 1989; Brouqui & Raoult, 2001). Coxiella

burnetii can be isolated from blood in 53% of untreated

patients (Musso & Raoult, 1995) but this should be restricted

to biosafety level 3 equipped laboratories. A large number of

cell lines can be used. L929 cells, Vero, and human embryonic

lung fibroblast (HEL cells) are used for C. burnetii, L929 for

Chlamydia spp., and ECV cells for Bartonella spp. (Brouqui &

Raoult, 2001). This technique has been applied successfully

for the isolation of other agents such as Bartonella spp. and

Whipple’s disease bacilli in CNE (Drancourt et al., 1995;

Raoult et al., 2000; Fenollar et al., 2001). In patients with

Bartonella spp. endocarditis the sensitivity of the shell vial

assay when inoculated with blood was 28%, compared with

only 5% when cultured onto agar plates. The most efficient

method for recovering Bartonella spp. from blood in the case

of endocarditis was the subculture in shell vial of the aerobic

Bactec Pluss (Becton Dickinson) blood culture broth on day

7 (LaScola & Raoult, 1999).

Serology

Of the fastidious organisms causing CNE, serum antibody

testing is available for Chlamydia spp., Legionella, Brucella,

Bartonella and C. burnetii. The serological tests are included

as a part of the Duke criteria for the diagnosis of infective

endocarditis but their predictive value differs. The good

predictive value of positive serology to C. burnetii has led

authors to propose that a single titre of C. burnetii antibody

IgG phase 1–800 be a major criterion for infective endocar-

ditis (Fournier et al., 1996; Li et al., 2000). For C. burnetii,

the most reliable and commonly used methods are indirect

immunofluorescence and the complement fixation test

(Houpikian & Raoult, 2003a). At present, the reference

technique is indirect immunofluorescence assay. Both C.

burnetii Nine-Mile strain Phase I and Phase II are used as

antigens. Antibodies to Phase I and II can be determined in

the IgG, IgM and IgA classes. Q fever endocarditis is

characterized by a very high titre of anti-Phase I antibodies;

an IgG anti-Phase I antibody titre of: 1600 is considered to

be highly predictive and sensitive, with a 98% positive

predictive value, whereas anti-Phase I IgA and IgM titres do

not contribute usefully to diagnosis (Dupont et al., 1994). A

single serum sample is sufficient for the diagnosis of Q fever

endocarditis. Cross-reactions, however, may be a source of

confusion when interpreting serological results. These vary

according the serological technique employed. They have

been described between C. burnetii and either Legionella or

Bartonella species, but a differential diagnosis is easily

established when quantitative antibody titres against both

Phase I and II of C. burnetii antigens are determined.

Currently, both indirect immunofluorescent assay and

enzyme-linked immunosorbent assay (ELISA) are used in

the diagnosis of Bartonella infection to detect specific

antibodies. Current serological tests may not distinguish

reliably between antibody responses to B. quintana and B.

henselae, although antibody cross-absorption and Western

immunoblotting allow differentiation of the serological

responses to these two species (Fig. 2) (Houpikian & Raoult,

2003b). Furthermore, cross-reactions may occur at a low

level with C. burnetii (La Scola & Raoult, 1996) and

significantly with Chlamydia spp. (Maurin et al., 1997).

Patients with proven B. quintana endocarditis have been

reported with IgG titres of 41 : 256 against Ch. pneumoniae

and titres of 1 : 64 against Chlamydia trachomatis and Ch.

psittaci (Drancourt et al., 1995). Absorption of the sera with

Ch. pneumoniae did not reduce the high antibody titres

against B. quintana, but absorption with B. quintana elimi-

nated reactivity with the Ch. pneumoniae antigen. This

cross-reactivity was confirmed using immunoblotting

(Drancourt et al., 1995). Taken altogether, an indirect

immunofluorescence assay antibody titre toward Bartonella

spp. 41 : 800 has a predictive value of 95% to detect

Bartonella infection in patients with endocarditis, leading

to the suggestion that Bartonella serology should be in-

cluded as a major criterion in the Duke criteria (Fournier

et al., 2002).

In a series of 10 patients reported to have Chlamydia

endocarditis, eight were finally diagnosed with Bartonella

endocarditis after testing their sera by cross-absorption

procedures and western immunoblotting. Because of the



serological cross-reaction describe below, an elevated titre of

antibody to Chlamydia spp. in a patient with CNE should

prompt Bartonella antibody testing.

Although several methods such as indirect immunofluor-

escent assay, ELISA and Western blot analysis have been

developed to detect specific antibodies to Brucella spp., the

tube agglutination test is still the reference test. It can be used

to make a presumptive diagnosis in the absence of bacter-

iological confirmation because most cases of active infection

will be associated with titres 41 : 160 (Young, 1991). The

physician should be aware of serological cross-reactions that

exist between Brucella, Yersinia and Francisella species that

can lead to confusion in the aetiological diagnosis.

Molecular detection

Considerable efforts have been recently made to establish

the aetiological diagnosis of CNE. Molecular techniques by

using PCR with subsequent sequencing of the amplicon and

gene analysis and comparison in the data bank have allow to

a considerable number of new aetiological diagnoses of CNE

(Mueller et al., 1999; Millar et al., 2001; Casalta et al., 2002;

Gauduchon et al., 2003; Lang et al., 2004). In our series, PCR

amplification using universal primers in valves removed at

surgery allowed 100% aetiological diagnosis in patients

classified with possible or definite endocarditis, including

those who had blood or valve material that cultured negative

(Millar et al., 2001). Although the sensitivity and specificity

of PCR is well established in resected valves (see below), the

usefulness of this technique in blood is still debated (Bos-

shard et al., 2003). The low specificity of PCR in blood is

likely due to pre-PCR contamination. When universal 16S

rRNA gene PCR is coupled with a simple pre-PCR deconta-

mination step by enzymatic digestion, the specificity of the

PCR was 100% in patients with culture positive endocardi-

tis, providing a promising diagnostic in CNE (Rothman

et al., 2002). Molecular methods seem particularly indicated

in CNE, both due to previous antibiotic therapy and due to

fastidious organisms (Podglajen et al., 2003). In their study,

Millar et al. (2001) recovered DNA of possible causal agents

in 10 of the 25 CNE blood cultures they tested. Three were

Streptococcus spp. in three patients who had received pre-

vious antibiotic therapy (Millar et al., 2001). Blood samples

from two patients who had been treated previously with

antibiotics were negative with PCR, but Streptococcus spp.

was detected in the valve (Khulordava et al., 2003). While

waiting for more data, broad range PCR of blood in cases of

CNE should be restricted to CNE patients who have

previously received antibiotics and who are Bartonella and

C. burnetii seronegative. Recently, Light Cycler Nested PCR

(LCN-PCR) in sera from patients with a proven diagnosis of

either Bartonella or C. burnetii endocarditis has been

reported to be more sensitive and specific than other

traditional methods such as culturing or PCR of EDTA-

treated blood (Zeaiter et al., 2003; Fenollar et al., 2004).

From cardiac valves

Histology

Because histopathology can confirm the diagnosis by reveal-

ing valvular inflammation, the vegetation, the organisms or

MWM 1 2 3 4 5 MWM MWM

Not adsorbed Adsorbed with 1 Adsorbed with 2

1 : Bartonella quintana 2 : Bartonella henselae (houston)3 : Bartonella elizabethae 4 : Bartonella vinsonii subps berkofii 5 : Bartonella vinsonii subps arupensis

1 2 3 4 5 1 2 3 4 5

Fig. 2. Western immunoblot with cross ad-

sorption for the species-specific diagnosis of

Bartonella endocarditis. Antigens used are Bar-

tonella quintana (L1), Bartonella henselae

Houston (L2), Bartonella elizabethae (L3) Bar-

tonella vinsonii spp. berkofii (L4) and Bartonella

vinsonii spp. arupensi (L5). The patient’s serum

was not adsorbed, adsorbed with B. quintana

or adsorbed with B. henselae. Note that all

reactions disappeared when adsorbed with B.

quintana, whereas when adsorbed with B.

henselae a reaction appears with B. quintana,

indicating that the specific antibodies con-

tained in the patient’s sera are those toward B.

quintana.



other changes consistent with infective endocarditis, the

histology of the resected valve remains the gold standard

for the diagnosis of infective endocarditis and is a major

criterion of the Duke classification.

Nonspecific staining

Endocarditis can be identified histologically with haematox-

ylin–eosin (H&E) staining by demonstration of an inflam-

matory reaction in valvular tissue and vegetation. The

presence of vegetation and a significant inflammation with

up to 2% polymorphonuclear neutrophil leucocytes is a key

in the diagnosis of endocarditis and should be considered a

major criterion (Lepidi et al., 2005). Eight nonspecific stains

can be used to detect bacteria and fungi in paraffin sections

of CNE valve specimens: Giemsa, Brown–Brenn and

Brown–Hopps tissue Gram stains, periodic acid-Schiff,

Grocott–Gomori methenamine silver, Warthin–Starry,

Gimenez, and Ziehl–Neelsen stains. Although the Giemsa

stain appears to be the most sensitive, the most popular and

widely used histological method for detection of bacteria is

the tissue Gram stain. In CNE due to previous antibiotic

therapy, the Gram stain appears to be helpful for further

identification. The diagnosis of T. whippelii endocarditis can

usually be made with periodic acid-Schiff staining, which

reveals numerous characteristic periodic acid-Schiff positive

granules. Silver impregnation using the Warthin–Starry

stain is among the most sensitive methods for detection

of bacteria, including those that stain weakly with a

tissue Gram stain, such as Bartonella sp. Silver impregnation

is not specific, staining virtually all bacteria, spirochetes,

and fungi. The Gimenez stain is a good method for the

Legionella species. The Ziehl–Neelsen stain is used for

detection of acid-fast bacteria, especially mycobacteria.

Sometimes, antibiotics induce changes in bacterial mor-

phology and staining properties which lead to an erroneous

diagnosis of a yeast or fungal infection. Histological evalua-

tion in infective endocarditis has recently been reviewed

(Lepidi et al., 2002).

Immunohistochemistry

The successful isolation and cultivation of C. burnetii, B.

quintana, B. henselae and T. whippelii has made it possible

to generate polyclonal rabbit or monoclonal mouse anti-

bodies to these bacteria. Specific detection of these micro-

organisms in tissues may now be achieved by using these

antibodies for immunohistology. Intracellular bacteria such

as C. burnetii or T whippelii are demonstrated in large

numbers within swollen histiocytes, whereas Bartonella spp.

are found in extracellular locations without inflammatory

infiltrates (Brouqui et al., 1994; Lepidi et al., 2000, 2003)

(Fig. 3).

Culture

Pathogens can also be isolated from resected valves or biopsy

specimens by inoculation onto agar or tissue culture. C.

burnetii, Mycobacterium spp., Brucella, Legionella, Bartonella

and Whipple’s disease bacteria have been isolated in this

manner. In Bartonella endocarditis, the sensitivity of the

shell vial culture of valve biopsy is greater than that of blood

culture (44% vs. 28%) (LaScola & Raoult, 1999). No patient

with prior antibiotic therapy yielded a positive blood

culture; this does not affect isolation of Bartonella from the

resected valve, although one must not allow the strain to

become established in culture (LaScola & Raoult, 1999).

Among 35 valves resected in patients with Whipple’s disease

endocarditis, 34 were positive at histological examination,

eight using PCR and two in culture (Fenollar et al., 2001).

Thus surgically removed material should be systematically

cultured in appropriate medium when possible. Serological

testing may help to indicate the appropriate media (Brouqui

& Raoult, 2001).

Molecular detection

Molecular detection in heart valves is certainly the most

sensitive tool today for the diagnosis of CNE. PCR is

especially helpful in the aetiological diagnosis of CNE with

prior antibiotic therapy. In a study of 49 patients with CNE

for whom the infected valve was available for diagnostic

tests, the sensitivity, specificity and predictive positive and

negative values were 17.6%, 88.9%, 75% and 36%, respec-

tively, with valve culture compared with 82.6%, 100%, 100%

and 76.5% with PCR (Bosshard et al., 2003). The most

common situation is that of an organism identified in the

valve by histology or culture and characterized by PCR.

Recently, the detection of S. pneumoniae rpoB DNA in a

histologically normal heart valve of a man, who had

Fig. 3. Immunohistological demonstration of Whipple’s disease bacillus

in cardiac valve using species-specific monoclonal antibody. Magnifica-

tion � 400. Courtesy of Dr Hubert Lepidi, CNRS UMR 6020.



presented with pneumococcal endocarditis 7 years earlier,

raised the question of the persistence of DNA in the absence

of any evidence of infection (Branger et al., 2003). In a recent

series, we report that bacterial DNA is detected in 60% of

patients with infective endocarditis while on antibiotic

therapy, and that this DNA was still detected in 37% of

patients who had completed their treatment (Rovery et al.,

2005).

Perspective in the diagnosis of CNE

The most important new trend in the diagnosis of CNE in

the last decade is the use of molecular techniques. Molecular

detection of microorganisms either by broad range PCR or

specific PCR has led to detection or characterization of a

considerable number of new organisms responsible for

fastidious endocarditis. This technique is by especially by in

CNE with previous antibiotic treatment. PCR has been

helpful for the diagnosis of streptococcal endocarditis

in patients receiving antibiotic therapy, for fastidious

streptococci, Tropheryma whippelii endocarditis, Myco-

plasma spp. and Mycobacterium spp. endocarditis and

Bartonella spp. and C. burnetii when serology was not tested

(Table 4).

The sensitivity of this technique is much greater in the

resected valves than in blood. A number of authors recently

proposed modifying the Duke criteria to include molecular

results as a major criterion (Millar et al., 2001; Millar &

Moore, 2004). However, the recently published detection of

remnant DNA in patients with previously cured infectious

endocarditis (Branger et al., 2003; Rovery et al., 2004) raised

the question of the specificity of such technique. This

specificity may be raised to 100% if interpreted following a

predefined procedure (Fig. 4) (Greub et al., 2005). Histolo-

gical examination of the valve remains the gold standard for

the diagnosis of infective endocarditis, and the molecular

Negative

CERTAIN

Yes

PositiveCongruent sequences

PositiveCongruent sequences

Negative

PCR targeting a third gene

Negative

PCR targeting a 2nd gene

Probable

No

Confirmed ?Serology

Valve or blood culture

Yes NO

PositiveReliable ?

PCR targeting 16 s r RNA gene

Fig. 4. Proposed protocol for polymerase chain reaction testing of cardiac valves in culture-negative endocarditis.



diagnosis of CNE should interpreted carefully as molecular

detection cannot be considered a major criterion.

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Tropheryma whippelii 11

Mycoplasma 1

Mycobacterium spp. 1

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performed

Coxiella burnetii 11 When serology is not

performed



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