Laboratory Approach to the Diagnosis of Culture-Negative Infective Endocarditis

HLC 2322 1–9

REVIEW

Heart, Lung and Circulation (2017) xx, 1–9 1443-9506/04/$36.00 http://dx.doi.org/10.1016/j.hlc.2017.02.009

3

Laboratory Approach to the Diagnosis of Culture-Negative Infective Endocarditis

4

S. Subedi a, Z. Jennings a, S.C.-A. Chen a[14_TD$IF],b,c*

1 2

5 6 7 8 9 10 11

Q2 Q1

a

Centre for Infectious Diseases and Microbiology Laboratory Services, Institute of Clinical Pathology Medical Research, Westmead Hospital, Sydney, NSW, Australia b Q3 Sydney Medical School, University of Sydney, Sydney, NSW, Australia c Centre for Infectious Diseases and Microbiology, Westmead Hospital and the Marie Bashir Institute for Infectious Diseases and Biosecurity, University of Sydney, Sydney, NSW, Australia Received 27 January 2017; accepted 1 February 2017; online published-ahead-of-print xxx

Blood-culture negative endocarditis (BCNE) accounts for up to 35% of all cases of infective endocarditis (IE), a serious life-threatening condition with considerable morbidity and mortality. Rapid detection and identification of the causative pathogen is essential for timely, directed therapy. BCNE presents a diagnostic and therapeutic challenge. Causes of BCNE are varied including: prior treatment with antibiotic agents prior to blood culture collection; sub-optimal specimen collection; and/or infection due to fastidious (eg. nutritionally variant streptococci), intracellular (eg. Coxiella burnetii, Bartonella species) or non-culturable or difficult to culture organisms (eg. Mycobacteria, Tropheryma whippelii and fungi); as well as non-infective aetiologies. Here, we review aetiological and diagnostic approaches to BCNE including newer molecular based techniques, with a brief summary of imaging investigation and treatment principles.

Q4

Q5

Keywords

12 13 14 15

[2_TD$IF]Introduction Q6 Q7

16 17 18 19 20 21 22 23 24 25 26 27 28 29

infective endocarditis  blood culture negative endocarditis  nuclear amplification tests  device associated infections  Q fever  endocarditis

Q8

Infective endocarditis (IE) is a serious, life-threatening condition associated with significant morbidity and mortality [1–3]. Rapid detection and identification of the causative pathogen is essential in ensuring timely and directed therapy. Diagnosis of IE is usually made by a combination of clinical, echocardiographic, histological and microbiological criteria as set out in the modified Duke’s criteria [2,4]. However, IE, whereby no causative microorganism is grown from blood culture or from diseased cardiac tissue by standard laboratory methods, may occur. ‘‘Blood culture negative endocarditis” (BCNE) [3,5] which accounts for 2.1–35% of all IE cases remains a diagnostic and therapeutic challenge [5–7]. The causes of BCNE are varied. Although receipt of antibiotic agents prior to blood culture collection is the most common cause (35–40%), sub-optimal specimen

collection and/or infection due to fastidious, intracellular or non-culturable organisms are other causes of BCNE [5,6]. In addition, non-infective aetiologies such as marantic endocarditis and those in the setting of autoimmune diseases such as lupus are also included in the umbrella of BCNE [3,5,6]. Diagnostic approaches to BCNE include both culture-, and non-culture based methods including the use of serological tests and of molecular techniques such as those that employ broad range polymerase chain reaction (PCR) assays on affected heart valves and other cardiac tissue [2,3,6,8]. To this end, liaison between the physician and the microbiology and tissue pathology laboratory is critical to ensure appropriate diagnostic investigations are performed. Here we review the aetiology, and microbiological diagnostic approaches including newer molecular based techniques of BCNE, with a brief summary of imaging investigation and treatment principles.

*Corresponding author [1_TD$IF]at: Centre for Infectious Diseases and Microbiology Laboratory Services, Institute of Clinical Pathology Medical Research, Westmead Hospital, Westmead, NSW, Australia. Tel: +61 2 984 56255. Email: [email protected] © 2017 Published by Elsevier B.V. on behalf of Australian and New Zealand Society of Cardiac and Thoracic Surgeons (ANZSCTS) and the Cardiac Society of Australia and New Zealand (CSANZ).

Please cite this article in press as: Subedi S, et al. Laboratory Approach to the Diagnosis of Culture-Negative Infective Endocarditis. Heart, Lung and Circulation (2017), http://dx.doi.org/10.1016/j.hlc.2017.02.009

30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47

HLC 2322 1–9

2

S. Subedi et al.

[3_TD$IF]Aetiology

48 49

69 70

There are a numerous causes of BCNE resulting from infection with organisms that are either difficult to grow or which are non-culturable. Typically, in patients presenting with suspected IE, at least three sets of blood cultures (including an aerobic and anaerobic bottle in each set) from separate venipuncture sites should be obtained, with the first and last samples drawn at least one hour apart [2,9]. The more common microbial aetiologies of BCNE (see Table 1) [2,3,5] include intracellular pathogens such as Coxiella burnetti, Bartonella, Legionella, Mycoplasma and Chlamydia species [6]. Non-culturable organisms, or organisms that are difficult to culture are best exemplified by Tropheryma whippleii, fungi and mycobacterial species, especially non-tuberculous mycobacteria and less commonly Mycobacterium tuberculosis [5,6]. Fastidious organisms may also pose a problem in yielding a positive blood culture and these include members of the HACEK group (Haemophilus species, Aggregatibacter species, Cardiobacterium hominis, Eikenella corrodens and Kingella species) and nutritionally variant streptococci. In one study, where comprehensive serologic, molecular and histopathological methods were used to investigate the cause of BCNE, the causative agent was found only in 62.7% of patients [5].

71

Intracellular Pathogens

72 73

[15_TD$IF]Coxiella burnetii Coxiella burnetii is an obligate intracellular pathogen and causes Q fever. It is found worldwide except in New Zealand [10,11]. In acute infection, Q fever has protean manifestations and may include febrile illness with myalgia, headaches and hepatitis, often mistaken for a viral infection. If untreated, Q fever can have fatal consequences [10,12] and lead to the chronic phase, most often manifesting as BCNE, however, osteomyelitis and granulomatous hepatitis can also occur. Patients with Q fever endocarditis frequently have minimal valvular changes seen on echocardiography [12]. Serology is the mainstay of diagnosis of Q fever endocarditis (see, Laboratory Diagnosis: General). Culture of C. burnetii from blood cultures or affected heart valves may be attempted in specialised laboratories, and is not routinely performed in the diagnostic laboratory [13]. Immunohistochemistry on resected heart valves and special Gimenez stain is often used to aid histological diagnosis of C. burnetiiinfected tissues [6]. The presence of doughnut granulomas in histological sections are highly suggestive of Q fever endocarditis. Molecular methods for diagnosis include broad range PCR or C. burnetii-specific PCR on freshly excised heart valve tissue [6,14,15]. Treatment of Q fever endocarditis is listed in Table 1.

50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 66 67 68

Q9

74 75 76 77 78 79 80 81 82 83 84 85 86 87 88 89 90 91 92 93 94 95 96 97 98 99 100 101

Q10

Bartonella Species Bartonella spp. are small intracellular Gram-negative bacteria that cause a range of infections in immunocompetent and immunocompromised hosts. Bartonella henselae is transmitted to humans by a cat scratch or bite or by cat fleas whilst B. quintana is transmitted by the human body louse [13,16].

Bartonella henselae typically causes cat scratch disease, bacillary angiomatosis and hepatic peliosis in human immunodeficiency virus (HIV) infected patients whilst B. quintana causes trench fever, lymphadenopathy with fever, and bacillary angiomatosis. Both species, and rarely other species B. elizabethae and B. vinsonii, can cause endocarditis [6,8,17]. Patients with Bartonella endocarditis usually have negative blood cultures and diagnosis is made using a combination of serology, PCR/DNA sequencing and histological examination of resected heart valve tissue [6,15] (see also Laboratory Diagnosis below). Cell culture-based methods are required to isolate the organism from heart valves or blood cultures and hence are not readily adaptable to the diagnostic laboratory [13]. Table 1 summarises the treatment of Bartonella endocarditis.

102

Legionella Species Legionella species cause pneumonia and Legionnaires’ disease but rarely extra-pulmonary infections including endocarditis, myocarditis, peritonitis or pyelonephritis [13]. To date, only 19 cases of endocarditis due to Legionella species have been reported [6,18–20]. Patients often present with chronic symptoms of low-grade fever, weight loss, malaise, night sweats and symptoms of congestive cardiac failure. Echocardiography may not reveal distinct vegetation, similar to patients with Q fever endocarditis [6,19,21]. Endocarditis due to Legionella species should be suspected in cases of BCNE where serological tests for Q fever and Bartonella return negative. Endocarditis due to Legionella species can be nosocomial in origin following cardiothoracic surgery and diagnosis may take months or years [18,21]. Community-acquired cases have had a preceding history of pneumonia and the diagnosis is often made during that admission. As before, diagnosis can be made by one or more of cultures of the resected valve or periodic subculture of the incubating blood culture broth onto specialised media supportive for Legionella [6,13], molecular methods and serology [6,19].

117 118

Chlamydia Species Chlamydia species are a rare cause of BCNE. To date, approximately 15 cases have been reported [22–26]. Because Chlamydia species are obligate intracellular organisms, they can only be grown on tissue culture. As a result, serological diagnostic methods are the mainstay of diagnosis (see Laboratory Diagnosis: Serology). This approach, however, is problematic as Chlamydia antigens often cross-react with those of other microorganisms including Bartonella spp [6]. Because most early case reports of Chlamydia endocarditis were diagnosed by serological tests, the certainty of diagnosis can be questioned [23,25] Other diagnostic methods include identification of the organism by using immunohistochemistry in resected valves or by molecular methods [6,22].

139 140

Mycoplasma Species Mycoplasma species are also rare causes of BCNE with nine cases reported to date; 8/9 were due to Mycoplasma hominis and 1/9 due to Mycoplasma pneumonia [27–29]. All but one

153 154

Please cite this article in press as: Subedi S, et al. Laboratory Approach to the Diagnosis of Culture-Negative Infective Endocarditis. Heart, Lung and Circulation (2017), http://dx.doi.org/10.1016/j.hlc.2017.02.009

103 104 105 106 107 108 109 110 111 112 113 114 115 116

119 120 121 122 123 124 125 126 127 128 129 130 131 132 133 134 135 136 137 138

141 142 143 144 145 146 147 148 149 150 151 152

155 156

Causative agents of culture negative endocarditis: frequency, diagnostic methods, treatment.

Causative agent

Approximate

Microbiological/histopathological investigations

Treatment

Serology

Doxycycline + Hydroxycholoroquine

frequency (%) of total BCNE Coxiella burnetii

37

(Q fever) Bartonella species

Histopathology Immunohistochemistry, PCR of resected surgical material 12

Blood cultures

Ceftriaoxne IV or Doxycycline orally + Gentamicin IV

Culture of resected tissue Serology PCR of resected material Legionella species

<1%

Blind sub cultures of blood culture into specialised media

Erythromycin or azithromycin + Rifampicin or ciprofloxacin

Serology Mycoplasma species

<1

Immunohistochemistry and PCR of resected surgical material Serology, culture, immunohistochemistry and PCR of resected

Newer fluoroquinolones (eg. Moxifloxacin, laevofloxacin)

surgical material Chlamydia species

<1

Serology, tissue culture, immunohistochemistry and PCR of

Mycobacteria

<1

Mycobacterial blood cultures, mycobacterial culture and PCR

resected surgical material According to species identified and susceptibility pattern

of resected surgical tissue, histopathology Tropheryma whippleii

Fungi

2-3

1-2

Histopathology and immunohistochemistry of resected

Penicillin G and streptomycin IV for 2 weeks followed by Co-

surgical material, PCR of resected surgical material.

trimoxazole long term or Doxycycline and hydroxycholoroquine long term

Blood cultures (including fungal blood cultures), culture of

Prolonged anti-fungal therapy based on identification of the

resected surgical material. Pan-fungal PCR of resected tissue.

organism and susceptibility pattern

Serum galactomanan and aspergillus specific PCR on resected tissue (for Aspergillus species) Other fastidious bacteria No cause found

2-3 37

Blood cultures, culture of resected tissue, PCR of resected

Antibiotic therapy based on organism identification and

surgical material

susceptibility

Blood and tissue cultures, serology, broad range (bacterial, fungal, mycobacterial) and specific PCR on tissues targeting

Empiric and ongoing therapy based on clinical presentation and suspected culprit pathogen.

culture negative pathogens, immunohistochemistry and histopathology on valvular tissue. Non infectious

2-3

Blood and tissue cultures, serology and PCR to exclude

As per diagnosis

infectious aetiology Autoimmune screen, histopathology Abbreviations: Polymerase chain reaction, PCR.

HLC 2322 1–9

Table 1

Laboratory Approach to the Diagnosis of Culture-Negative Infective Endocarditis

Please cite this article in press as: Subedi S, et al. Laboratory Approach to the Diagnosis of Culture-Negative Infective Endocarditis. Heart, Lung and Circulation (2017), http://dx.doi.org/10.1016/j.hlc.2017.02.009

Q1

3

HLC 2322 1–9

4

157 158 159 160 161 162 163 164 165 166 167 168 169 170 171 172 173 174 175 176 177 178 179 180 181 182 183 184 185 186 187 188 189 190 191 192 193 194 195 196 197 198 199 200 201 202 203 204 205 206 207 208 209

S. Subedi et al.

case occurred following cardiac surgery, suggesting a nosocomial aetiology. Most cases reported have relied either on serology techniques or the use of broad range bacterial PCR to detect/identify Mycoplasma as the causative pathogen [28,29].

Non-Culturable or Difficult to Culture Organisms Mycobacteria Infective endocarditis due to Mycobacterial species is mostly caused by non-tuberculous mycobacteria with rapidly growing mycobacteria more commonly implicated than slow growing non-tuberculous mycobacteria [30–36]. Endocarditis due to M. tuberculosis has been reported in only a handful of cases, mostly in the setting of tuberculosis in military personnel [35,37,38]. Symptom onset and progression in mycobacterial endocarditis is insidious with delayed diagnosis. Risk factors include preceding central venous access devices including haemodialysis catheters, surgical procedures such as mammoplasty, arthroplasty and cardiothoracic operations, and underlying immunocompromise including HIV, haematological malignancies, solid organ transplant or patients on tumour necrosis factor blockers [6,39]. Blood culture positivity rates for mycobacterial endocarditis vary with native valve endocarditis more likely to yield positive blood cultures [39]. Diagnosis is made typically by a combination of culture-based, histological and molecular methods (see Laboratory Diagnosis). Since 2011, several multi-country outbreaks of M. chimaera invasive cardiovascular infections including IE have been reported [40]. The point source has been linked with the heater cooler units used during cardiopulmonary bypass operation. The time from the operation till disease onset has ranged from one to four years. Clinical manifestations include prosthetic valve endocarditis, vascular graft infection and/or manifestations of disseminated mycobacterial infection, including embolic and immunologic manifestations [32,41] In general, non-tuberculous mycobacteria are refractory to multiple antimicrobial agents, and endocarditis caused by these micro-organisms is associated with high morbidity and mortality[39]. See Table 1 for summary. [16_TD$IF]Tropheryma whippelii Whipples disease is a chronic multi-systemic disease caused by the bacterium Tropheryma whippeii. and manifests with a myriad of symptoms including weight loss, diarrhea, arthralgia and abdominal pain [42,43]. Atypical presentations may include involvement of the heart, lungs or central nervous system. Endocarditis is rare, and most commonly affects older Caucasian men [42,44,45]. Diagnosis requires a high index of suspicion, using a combination of histological and non-culture-based approaches to confirm the cause of IE. Treatment is summarised in Table 1. Fungi Fungi account for approximately 1–2% of all cases of IE [1,46,47] with Candida albicans the most common causative

agent (approximately 25% of cases of fungal endocarditis). However, other Candida species can also cause endocarditis (25%) as can Aspergillus species, especially Aspergillus fumigatus. The remaining 25% of cases are caused by a wide variety of other fungi [2,46]. Risk factors for fungal endocarditis include presence of prosthetic heart valves, intravenous drug use, prior antibiotic therapy, parenteral nutrition, prolonged indwelling central venous catheter, diabetes mellitus and immunocompromising conditions. Nosocomial infection, especially following cardiac surgery has been reported [46,47]. Diagnosis is often delayed, as blood cultures are only positive in 50% of patients with the majority of fungi grown in blood culture being Candida species. Fungal blood cultures may be superior to standard blood cultures for detecting fungaemia [48], but in general, routine blood culture media sufficiently supports the growth of yeasts. Prolonged antifungal therapy is required for treatment (Table 1).

210

Other Fastidious Bacteria These bacteria include microorganisms in the HACEK group, Brucella species and nutritionally variant streptococci. Whilst they can usually be cultured on the modern blood culture medium, they will require supplementation of the culture medium with growth nutrients as described below.

228 229

[4_TD$IF]Clinical Diagnosis The most widely accepted clinical definition of IE is based on the modified Duke criteria which stratifies patients as having definitive or possible IE or rejecting the diagnosis of IE [49]. These criteria are summarised in Table 2 [50].

[5_TD$IF]Laboratory Diagnosis

211 212 213 214 215 216 217 218 219 220 221 222 223 224 225 226 227

230 231 232 233

234 235 236 237 238

239

The diagnostic approach to the cause of BCNE will be dependent on the clinical setting and most likely diagnosis. However, overall, a combination of both culture, and non culturebased approaches are typically employed. The relative importance of various approaches varies with the causative pathogen. In essence, all of histopathological, culture, serological and molecular methods are employed.

240

Histopathology

247

Examination of clinical specimens in particular, heart valve tissue, for the presence of microorganisms and their identification provides definitive evidence of IE, and is considered the gold standard approach [3]. Conventional haematoxylin and eosin (H&E) stains are important in confirming the presence of inflammation, tissue invasion and necrosis and may sometimes provide clues to the presence of particular microorganisms e.g fungal elements or a particular diagnosis eg. granulomas in mycobacterial or Bartonella disease [6,13]. Importantly, it also assists in diagnosing non-infectious causes of valvular lesions including nonbacterial thrombotic endocarditis, rheumatic endocarditis and myxoma. The use

248

Please cite this article in press as: Subedi S, et al. Laboratory Approach to the Diagnosis of Culture-Negative Infective Endocarditis. Heart, Lung and Circulation (2017), http://dx.doi.org/10.1016/j.hlc.2017.02.009

241 242 243 244 245 246

249 250 251 252 253 254 255 256 257 258 259

HLC 2322 1–9 Laboratory Approach to the Diagnosis of Culture-Negative Infective Endocarditis

5

Table 2 Modified Duke Criteria. Classification

Definite IE

 Pathologic criteria  Two major criteria  One major and three minor clinical criteria Possible IE

 One major and one minor clinical criteria  Three minor clinical criteria Rejected IE

   

Alternate diagnosis made Resolution of clinical manifestations after
Pathologic criteria

Histopathology demonstrating vegetation of abscess with active endocarditis

Major criteria

A positive blood culture for infective endocarditis as defined by the recovery of:

Micro-organisms demonstrated by culture or histopathology of a vegetation or intracardiac abscess

 A typical microorganism from two separate blood cultures in the absence of a primary focus

(viridans streptococci b Streptococcus bovis, HACEK group, or community-acquired Staphylococcus aureus or enterococci) or  A persistently positive blood culture defined as the recovery of a microorganism consistent with endocarditis from either blood cultures drawn more than 12 h apart or all three or a majority of four or more separate blood cultures with the first and last drawn at least 1 h apart. Evidence of endocardial involvement:

 Positive echocardiogram for infective endocarditis (i) 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 (ii) Abscess, or (iii) New partial dehiscence of prosthetic valve or  New valvular regurgitation (increase or charge in pre-existing murmur not sufficient) Serology for Q fever phase 1 IgG antibodies at  800 Minor criteria

Predisposition: predisposing heart condition or intravenous drug use Fever: 38  C (100.4  F) Vascular phenomena: major arterial emboli, septic pulmonary infarcts, mycotic aneurysm, intracranial haemorrhage, conjunctival haemorrhages, Janeway lesions, Immunologic phenomena: glomerulonephritis, Osler’s nodes, Roth spots, rheumatoid factor Microbiologic evidence: positive blood culture but not meeting major criterion as noted above* or serologic evidence of an active infection with organism consistent with infective IE with infective endocarditis but not meeting major criterion as noted above

*

260 261 262 263 264

Excluding single positive blood cultures for coagulase-negative staphylococci and organisms that do not cause endocarditis.

of organism-specific stains is essential to detect presence of bacteria (Gram stain) and to allow differentiation of Gram positive from Gram negative organisms. Specialised stains may allow presumptive identification of organisms, ie Periodic acid-Schiff (PAS) stain for detection of T. whippeli

granules within characteristic foamy macrophages [6,43], acid fast stains for Mycobacteria, the Gimenez stain for C. burnetii and Legionella, and methenamine silver stains for fungi. Pathologic examination resected heart valve tissue Warthin-Starry silver staining often demonstrate

Please cite this article in press as: Subedi S, et al. Laboratory Approach to the Diagnosis of Culture-Negative Infective Endocarditis. Heart, Lung and Circulation (2017), http://dx.doi.org/10.1016/j.hlc.2017.02.009

265 266 267 268 269

HLC 2322 1–9

6

270

S. Subedi et al.

276

characteristic granular organisms of Bartonella spp. in the valve where Bartonella BCNE is suspected [17_TD$IF][6]. Immunohistochemical staining is sometimes performed in specialised laboratories with specific monoclonal or polyclonal antibodies which may allow identification to a species level. Such stains have been developed for Bartonella spp., C. burnettii, and T. whipplei [6,42].

277

Culture

278 279

Conventional laboratory blood culture systems can isolate the common causes of infective endocarditis including HACEK organisms within five days of incubation. Blood culture media include antibiotic binding resins to diminish inhibitory antibiotic effect [13]. To maximise rates of recovery, three sets of both aerobic and anaerobic blood culture bottles should be collected. Prolonged incubation should be requested in cases of suspected IE, to allow time for fastidious organisms to grow. Blind subculture (after three weeks’ incubation) from blood culture media to specialised enrichment media may increase rates of bacterial isolation, particularly with consideration to Bartonella spp. Culture of resected valve and other diseased tissue specimens and prosthetic devices should also be undertaken if indicated. Of note, specialised media may be required to recover more fastidious organisms and based on detailed patient history, clinician liaison with the microbiology service is essential. Knowledge of patient travel, animal contacts, occupational and recreational activities is critical. Fastidious organisms such as nutritionally variable streptococci may fail to grow on subculture without additional Vitamin B6, or cysteine [6] and Abiotrophia spp. requires blood agar plates streaked with another organism, S. aureus to support its growth [6,13]. Brucella spp. are also relatively fastidious but may be isolated from blood culture in over 80% of cases [6] if incubation is prolonged to four to six weeks. If Legionella IE is suspected, the optimum culture medium includes charcoalcontaining agar such as buffered charcoal yeast extract agar with prolonged (15-day) incubation [6,13] Mycolytic blood culture bottles containing Middelbrook 7H13 media should be collected in cases of suspected mycobacterial infection, and in the case of excised valves, cultured onto Middlebrook agar or Lowenstein Jensen medium. In particular, mycobacterial culture media should be included in routine set up for cultures from porcine and prosthetic valves, highlighted by the recent Mycobacterium chimera infections associated with cardiac bypass circuits [32,40]. Because Mycoplasma species cannot be detected by gram stain, they are difficult to observe and are easily missed on routine bacteriological media. Further, commercial blood culture broth contains sodium polyanetholsulfonate as an anticoagulant agent, which is inhibitory to the growth of Mycoplasma species [13]. Culture of excised heart valves into specialised A7 medium has been used in some case reports to successfully grow these microorganisms. Finally, cell culture media to grow organisms such as Bartonella spp., Chlamydia, C. burnettii and T. whippeli are not available in clinical

271 272 273 274 275

280 281 282 283 284 285 286 287 288 289 290 291 292 293 294 295 296 297 298 299 300 301 302 303 304 305 306 307 308 309 310 311 312 313 314 315 316 317 318 319 320 321 322 323 324

laboratories and culture for these agents of IE are not routinely undertaken.

325

Serology

327

Serological tests are particularly useful in the context of BCNE where detection of organism-specific antibody, often requiring either a single high titre or demonstration of an interval four-fold rise in titre from blood samples taken two to four weeks apart in time is typically diagnostic of recent infection. Serological tests are useful for epidemiological purposes but only allow for retrospective diagnosis in the majority of cases of IE. There are multiple laboratory methods available for serologic testing which are not necessarily directly comparable, and a rise in titre requires testing of both the ‘acute’ and ‘convalescent’ blood sample using the same assay. In cases of BCNE, serological test methods are available and are generally recommended for suspected Q fever endocarditis, and for IE due to Bartonella, Brucella, Chlamydia, Legionella and Mycoplasma species [6,12,17,24]. Coxiella burnettii antibodies are detected by immunofluorescence assay, which is able to distinguish between antibody responses to the two main C. burnetii antigens the phase 1 and phase 2 antigen [6]. These antigens represent lipopolysaccharides on the cell wall of the metabolically inactive (or small call variant form of the bacteria) [51]. The phase 1 antigen is associated with virulence and macrophage immunomodulation whilst the phase 2 antigen is avirulent in immunocompetent hosts. Very high titres of antibody to phase 1 and phase 2 antigens of C. burnetii characterise Q fever endocarditis with phase 1 IgG titre >/ = 800 and IgA titre >/ = 100 being highly sensitive for Q fever endocarditis [6,12,14]. Conversely, IgM levels are generally low. Response to therapy can also be monitored with Q fever serology [13]. Bartonella endocarditis is also frequently diagnosed by serological methods. Serologic testing is typically by immunofluorescent assays (IFA) [17,52] with high titres of >800 associated with endocarditis with 95% positive predictive value [17]. However, titres <800 do not exclude endocarditis; in a study by Edouard et al. approximately 40% of patients with Bartonella endocarditis did not meet this cut-off [17]. Active endocarditis due to Brucella spp. also usually results in antibody titres in excess of 180 [6]. Tube agglutination methods are the gold standard, with IFA and ELISA in common use [13]. Legionella serology is generally conducted via indirect fluorescent antibody test, with titre >/ = 256 considered significant. Serology is however, used in adjunct to support the diagnosis rather for diagnosis of acute infection, the positive predictive value of serology in the diagnosis of endocarditis due to Legionella species is not known [6]. The role of antibody detection to Mycoplasma and Chlamydia is also unclear, with the relatively few case reports of mycoplasma endocarditis usually relying on molecular diagnoses [53]. Limitations of serologic investigations are well known for their slow turn-around times but they may also be problematic with regards to cross reactivity (especially seen with low

328

Please cite this article in press as: Subedi S, et al. Laboratory Approach to the Diagnosis of Culture-Negative Infective Endocarditis. Heart, Lung and Circulation (2017), http://dx.doi.org/10.1016/j.hlc.2017.02.009

326

329 330 331 332 333 334 335 336 337 338 339 340 341 342 343 344 345 346 347 348 349 350 351 352 353 354 355 356 357 358 359 360 361 362 363 364 Q11

365 366 367 368 369 370 371 372 373 374 375 376 377 378 379

HLC 2322 1–9 Laboratory Approach to the Diagnosis of Culture-Negative Infective Endocarditis

380

383

titres), particularly between Bartonella and Chlamydia spp. [22]. Cross reactions have been reported between these organisms and various others including Pseudomonas, Haemophilus, and Bordetella spp [13].

384

Molecular Methods

385

Various nucleic amplification tests (NAT) can be used to detect and identify microorganisms in explanted valve tissue and less commonly, from blood samples and blood cultures. Genus-, and species-specific PCR assay as well as pan-bacterial (and pan-mycobacterial) or panfungal approaches may be used. Many laboratories in Australia employ a pan-bacterial PCR assay where the 16S ribosomal RNA (rRNA) region of bacteria is amplified and then sequenced to obtain an organism identity. The 16S rRNA gene region conserved within all bacteria but with sufficient sequence variation to allow differentiation to a genus, if not, species level. The sequence of the amplified gene product is compared to archived sequences of known bacterial isolates in gene repositories such as the Genbank database (NCBI; https://www. ncbi.nlm.nih.gov/pmc/articles/PMC1829013/) to obtain an identification number. Similarly the internal transcriber spacer (ITS) or other rRNA gene regions are amplified and then sequenced in cases of suspected fungal IE [54]. Molecular methods are most useful in the context where there is a high suspicion for a particular cause of BCNE and/ or in patients with positive or equivocal serologic test results. Polymerase chain reaction-based assays may then be employed to confirm infection due to, for example, Bartonella or Brucella spp. Molecular detection methods are also useful in patients who have been receiving antimicrobials as bacterial DNA persists below levels detectable by culture [55]. Specificity approaches 100% for molecular assays, however sensitivity is highly variable, with species-specific assay being more sensitive than those detecting organisms only to ‘‘genus” level or those detecting ‘‘all” bacteria/fungi. Bartonella specific PCR has sensitivity for valvular biopsy specimens of up to 92%, significantly higher than for serum or whole blood samples (36% and 33%, respectively) [17]. Polymerase chain reaction can be conducted from valvular tissue specimens for Brucella spp., Legionella sp., Mycoplasma spp., and T. whippelli, however sensitivity from valve specimens is unclear. Molecular methods are not without limitations. False positive results may occur through detection of non-viable organism DNA or contamination during the collection process. Any molecular result therefore requires careful clinical correlation. This is particularly challenging when low virulence organisms such as coagulase negative Staphylococci are detected from prosthetic valve tissue. Fournier et al. assessed the clinical utility of 16S rRNA in 819 prospectively diagnosed cases of culture negative endocarditis in conjunction with traditional histopathology and serology investigations to identify micro-organisms [5]. A responsible organism was established in 62.7% of cases using conventional methods compared to 66% via 16S PCR (150 of 227 tissue valve

381 382

386 387 388 389 390 391 392 393 394 395 396 397 398 399 400 401 402 403 404 405 406 407 408 409 410 411 412 413 414 415 416 417 418 419 420 421 422 423 424 425 426 427 428 429 430 431 432 433 434

7

samples). Only three samples from peripheral blood were PCR positive indicating this approach rarely contributes to diagnosis. The Kemp study analysed 13 culture negative cardiac tissue samples from cases with proven endocarditis using 16S PCR [56]. Streptococcal species DNA was detected in five samples, demonstrating the utility of 16S PCR to detect bacterial DNA after commencement of antibiotics. Bacterial detection by 16S may significantly alter antimicrobial treatment; seven of 46 patients in a five-year prospective study conducted by Marsch et al. underwent treatment changes due to positive 16S results. Detection of DNA, however, may not represent treatment failure as bacterial DNA has been identified from valvular tissue up to seven years after successful treatment of endocarditis [56]. Kemp also highlighted the risk of false positive results after conducting 16S rRNA testing on 36 valve tissue samples from patients without IE which identified Proprionibacterium acnes from seven samples.

435

Imaging

454

Echocardiograpy remains the most common imaging method for diagnosis of IE and fulfills a major Duke’s criterion. Transthoracic echocardiography (TTE) for detection of vegetations, peri-valvular and mural abscesses has sensitivity of approximately 80% and is recommended for all patients with clinically suspected infective endocarditis [57–62]. If the TTE is negative and clinical suspicion remains high, diagnostic sensitivity is improved to 90–100% by transoesophageal echocardiography (TOE) [57,62]. Importantly a negative TOE provides a negative predictive value of 86– 97%, however, in a study assessing 65 patients with negative initial TOE, five (7.6%) developed visible lesions on repeat imaging one to two weeks later [63]. When assessing for pacemaker lead vegetations, TOE is markedly more sensitive than TTE, >90% versus 30% respectively [64]. Cardiac MRI using three dimensional phase contrast is an emerging technique which can demonstrate valvular abnormalities. Other imaging modalities including ultrasound, computed tomography (CT), or magnetic resonance imaging (MRI) may demonstrate evidence of end organ infarction due to systemic embolisation fulfilling minor Dukes criterion [65].

455

Other Diagnostic Criteria

476

Rheumatoid factor was elevated in 38% vs 68% of culture negative versus culture positive IE (p value <0.001) in a study of 221 Danish cases of IE [66]. The Spanish collaborative endocarditis study which included 2000 prospectively recruited patients also found rheumatoid factor elevated in 6.9% of BCNE compared with 8.4% in those with an organism identified on culture [8].

477

Treatment

484

Treatment of patients with BCNE depends on the likely (if not cultured or detected) or causative agent, if known. After adequate blood culture collection, empiric therapy with

485 486

Please cite this article in press as: Subedi S, et al. Laboratory Approach to the Diagnosis of Culture-Negative Infective Endocarditis. Heart, Lung and Circulation (2017), http://dx.doi.org/10.1016/j.hlc.2017.02.009

436 437 438 439 440 441 442 443 444 445 446 447 448 449 450 451 452 453

456 457 458 459 460 461 462 463 464 465 466 467 468 469 470 471 472 473 474 475

478 479 480 481 482 483

487

HLC 2322 1–9

8

488

S. Subedi et al.

494

intravenous benzylpenicillin, flucloxacillin and gentamicin is recommended in patients with native valve endocarditis. In patients with prosthetic heart valves or intra cardiac devices, vancomycin and gentamicin is generally the recommended empiric choice [2,3]. Directed therapy and duration of therapy depends on the causative agent of BCNE [2,3] and is summarised in Table 1.

495

Device Associated Infections

496 497

519 520

Infections of the cardiovascular implantable electronic devices (CIED), which include permanent pacemakers, ventricular assist devices and implantable cardiovascular devices range from exit and pocket site infections to blood stream infection with IE [67]. The rate of CIED infections has increased significantly in the last two decades [68,69]. Staphylococcus species, including coagulase negative Staphylococcus account for 60–80% of cases of CIED infections. Other aetiological agents include enterococci, Propionobacterium species, Corynebacterium species, Gram negative bacteria and Candida species [69]. As for BCNE, diagnosis of CIED infections requires a combination of culture and non-culture based methods. In addition to taking blood cultures, ultrasound scan is recommended for suspected pocket infections [2]. Transoesophageal echocardiogram has a higher sensitivity for detecting CIED infections than transthoracic echocardiogram. Cultures of explanted tissues from infected sites and lead tip should be set up for bacterial culture, as well as mycobacterial and fungal cultures. Molecular diagnostic methods as described above may be useful in identifying the causative pathogen if blood and tissue cultures are negative [2]. Although not routinely available in many diagnostic laboratories, sonication fluid culture from removed cardiac implants can also improve the microbiological diagnosis of CIED [70].

521

[6_TD$IF]Conclusions

489 490 491 492 493

498 499 500 501 502 503 504 505 506 507 508 509 510 511 512 513 514 515 516 517 518

522

529

Culture negative endocarditis remains a challenging clinical condition. Best practice requires a thorough systematic approach guided by patient history including histopathology, culture based, molecular and serologic investigations. Liaison between pathology and cardiology services is critical to ensuring optimal investigation and treatment and that the appropriate specimens are collected, cultured and examined in the optimal manner.

530

References

523 524 525 526 527 528

531 532 533 534 535 536 537 538 539

[1] Murdoch DR, Corey GR, Hoen B, Miro JM, Fowler Jr VG, Bayer AS. Clinical presentation, etiology, and outcome of infective endocarditis in the 21st century: the International Collaboration on Endocarditis-Prospective Cohort Study. Arch Intern Med 2009;169(5):463–73. [2] Baddour LM, Wilson WR, Bayer AS, Fowler Jr VG, Tleyjeh IM, Rybak MJ. Infective Endocarditis in Adults: Diagnosis, Antimicrobial Therapy, and Management of Complications: A Scientific Statement for Healthcare Professionals From the American Heart Association. Circulation 2015;132(15):1435–86.

[3] Habib G, Hoen B, Tornos P, Thuny F, Prendergast B, Vilacosta I, et al. Guidelines on the prevention, diagnosis, and treatment of infective endocarditis (new version 2009): the Task Force on the Prevention, Diagnosis, and Treatment of Infective Endocarditis of the European Society of Cardiology (ESC). Endorsed by the European Society of Clinical Microbiology and Infectious Diseases (ESCMID) and the International Society of Chemotherapy (ISC) for Infection and Cancer. Eur Heart J 2009;30(19):2369–413. [4] Topan A, Carstina D, Slavcovici A, Rancea R, Capalneanu R, Lupse M. Assesment of the Duke criteria for the diagnosis of infective endocarditis after twenty-years. An analysis of 241 cases. Clujul Med 2015;88(3):321–6. [5] Fournier PE, Thuny F, Richet H, Lepidi H, Casalta JP, Arzouni JP. Comprehensive diagnostic strategy for blood culture-negative endocarditis: a prospective study of 819 new cases. Clin Infect Dis 2010;51(2):131– 40. [6] Brouqui P, Raoult D. Endocarditis due to rare and fastidious bacteria. Clin Microbiol Rev 2001;14(1):177–207. [7] Lamas CC, Fournier PE, Zappa M, Brandao TJ, Januario-da-Silva CA, Correia MG. Diagnosis of blood culture-negative endocarditis and clinical comparison between blood culture-negative and blood culture-positive cases. Infection 2016;44(4):459–66. [8] Diez-Villanueva P, Munoz P, Marin M, Bermejo J, de Alarcon Gonzalez A, Farinas MC. Infective endocarditis: Absence of microbiological diagnosis is an independent predictor of inhospital mortality. Int J Cardiol 2016;220:162–5. [9] Lamas CC, Eykyn SJ. Blood culture negative endocarditis: analysis of 63 cases presenting over 25 years. Heart 2003;89(3):258–62. [10] Raoult D, Marrie T. Q fever. Clin Infect Dis 1995;20(3):489–95. quiz 496. [11] Raoult D. From Cat scratch disease to Bartonella henselae infection. Clin Infect Dis 2007;45(12):1541–2. [12] Anderson A, Bijlmer H, Fournier PE, Graves S, Hartzell J, Kersh GJ, et al. Diagnosis and management of Q fever–United States, 2013: recommendations from CDC and the Q Fever Working Group. MMWR Recomm Rep 2013;62(RR-03):1–30. [13] Jorgensen JHPM, Carroll KC, Funke G, Landry ML, Richter SS, Warnock DW. Manual of Clinical Microbiology, vol. 1. Washington DC: ASM Press; 2015. [14] Landais C, Fenollar F, Thuny F, Raoult D. From acute Q fever to endocarditis: serological follow-up strategy. Clin Infect Dis 2007;44(10):1337– 40. [15] Siciliano RF, Castelli JB, Mansur AJ, Pereira dos Santos F, Colombo S, do Nascimento EM. Bartonella spp and Coxiella burnetii Associated with Community-Acquired, Culture-Negative Endocarditis, Brazil. Emerg Infect Dis 2015;21(8):1429–32. [16] Chomel BB, Kasten RW, Williams C, Wey AC, Henn JB, Maggi R. Bartonella endocarditis: a pathology shared by animal reservoirsand patients. Ann N Y Acad Sci 2009;1166:120–6. [17] Edouard S, Nabet C, Lepidi H, Fournier PE, Raoult D. Bartonella, a common cause of endocarditis: a report on 106 cases and review. J Clin Microbiol 2015;53(3):824–9. [18] Massey R, Kumar P, Pepper JR. Innocent victim of a localised outbreak: legionella endocarditis. Heart 2003;89(5):e16. [19] Pearce MM, Theodoropoulos N, Noskin GA, Flaherty JP, Stemper ME, Aspeslet T. Native valve endocarditis due to a novel strain of Legionella. J Clin Microbiol 2011;49(9):3340–2. [20] Samuel V, Bajwa AA, Cury JD. First case of Legionella pneumophila native valve endocarditis. Int J Infect Dis 2011;15(8):e576–7. [21] Tompkins LS, Roessler BJ, Redd SC, Markowitz LE, Cohen ML. Legionella prosthetic-valve endocarditis. N Engl J Med 1988;318(9):530–5. [22] Gdoura R, Pereyre S, Frikha I, Hammami N, Clerc M, Sahnoun Y. Culture-negative endocarditis due to Chlamydia pneumoniae. J Clin Microbiol 2002;40(2):718–20. [23] Brearley BF, Hutchinson DN. Endocarditis associated with Chlamydia trachomatis infection. Br Heart J 1981;46(2):220–1. [24] Etienne J, Ory D, Thouvenot D, Eb F, Raoult D, Loire R. Chlamydial endocarditis: a report on ten cases. Eur Heart J 1992;13(10):1422–6. [25] van der Bel-Kahn JM, Watanakunakorn C, Menefee MG, Long HD, Dicter R. Chlamydia trachomatis endocarditis. Am Heart J 1978;95(5):627–36. [26] Shapiro DS, Kenney SC, Johnson M, Davis CH, Knight ST, Wyrick PB. Brief report: Chlamydia psittaci endocarditis diagnosed by blood culture. N Engl J Med 1992;326(18):1192–5. [27] Scapini JP, Flynn LP, Sciacaluga S, Morales L, Cadario ME. Confirmed Mycoplasma pneumoniae endocarditis. Emerg Infect Dis 2008;14 (10):1664–5. [28] Jamil HA, Sandoe JA, Gascoyne-Binzi D, Chalker VJ, Simms AD, Munsch CM, et al. Late-onset prosthetic valve endocarditis caused by

Please cite this article in press as: Subedi S, et al. Laboratory Approach to the Diagnosis of Culture-Negative Infective Endocarditis. Heart, Lung and Circulation (2017), http://dx.doi.org/10.1016/j.hlc.2017.02.009

540 541 542 543 544 545 546 547 548 549 550 551 552 553 554 555 556 557 558 559 560 561 562 563 564 565 566 567 568 569 570 571 572 573 574 575 576 577 578 579 580 581 582 583 584 585 586 587 588 589 590 591 592 593 594 595 596 597 598 599 600 601 602 603 604 605 606 607 608 609 610 611 612 613 614 615

HLC 2322 1–9 Laboratory Approach to the Diagnosis of Culture-Negative Infective Endocarditis

616 617 618 619 620 621 622 623 624 625 626 627 628 629 630 631 632 633 634 635 636 637 638 639 640 641 642 643 644 645 646 647 648 649 650 651 652 653 654 655 656 657 658 659 660 661 662 663 664 665 666 667 668 669 670 671 672 673 674 675 676 677 678 679

[29]

[30]

[31]

[32]

[33]

[34]

[35] [36]

[37]

[38] [39] [40]

[41]

[42] [43] [44]

[45]

[46] [47]

[48]

[49]

Mycoplasma hominis, diagnosed using broad-range bacterial PCR. J Med Microbiol 2012;61(Pt 2):300–1. Gagneux-Brunon A, Grattard F, Morel J, Suy F, Fuzellier JF, Verhoeven P. Mycoplasma hominis, a Rare but True Cause of Infective Endocarditis. J Clin Microbiol 2015;53(9):3068–71. Bouchiat C, Saison J, Boisset S, Flandrois JP, Issartel B, Dauwalder O, et al. Nontuberculous Mycobacteria: An Underestimated Cause of Bioprosthetic Valve Infective Endocarditis. Open Forum Infect Dis 2015;2(2). ofv047. McMullen AR, Mattar C, Kirmani N, Burnham CA. Brown-Pigmented Mycobacterium mageritense as a Cause of Prosthetic Valve Endocarditis and Bloodstream Infection. J Clin Microbiol 2015;53(8):2777–80. Achermann Y, Rossle M, Hoffmann M, Deggim V, Kuster S, Zimmermann DR. Prosthetic valve endocarditis and bloodstream infection due to Mycobacterium chimaera. J Clin Microbiol 2013;51(6):1769–73. Al-Benwan K, Ahmad S, Mokaddas E, Johny M, Kapoor MM. Diagnosis of endocarditis caused by Mycobacterium abscessus. Ann Saudi Med 2010;30(5):408–11. Spell DW, Szurgot JG, Greer RW, Brown 3rd JW. Native valve endocarditis due to Mycobacterium fortuitum biovar fortuitum: case report and review. Clin Infect Dis 2000;30(3):605–6. Shaikh Q, Mahmood F. Triple valve endocarditis by mycobacterium tuberculosis: a case report. BMC Infect Dis 2012;12:231. Kumar A, Pazhayattil GS, Das A, Conte HA. Mycobacterium neoaurum causing prosthetic valve endocarditis: a case report and review of the literature. Braz J Infect Dis 2014;18(2):235–7. Klingler K, Brandli O, Doerfler M, Schluger N, Rom WN. Valvular endocarditis due to Mycobacterium tuberculosis. Int J Tuberc Lung Dis 1998;2(5):435–7. Liu A, Nicol E, Hu Y, Coates A. Tuberculous endocarditis. Int J Cardiol 2013;167(3):640–5. Yuan SM. Mycobacterial endocarditis: a comprehensive review. Rev Bras Cir Cardiovasc 2015;30(1):93–103. Invasive cardiovascular infection by Mycobacterium chimaera potentially associated with heater-cooler units used during cardiac surgery. [http://ecdc.europa.eu/en/publications/publications/ mycobacterium-chimaera-infection-associated-with-heater-coolerunits-rapid-risk-assessment-30-april-2015.pdf]. Sommerstein R, Ruegg C, Kohler P, Bloemberg G, Kuster SP, Sax H. Transmission of Mycobacterium chimaera from Heater-Cooler Units during Cardiac Surgery despite an Ultraclean Air Ventilation System. Emerg Infect Dis 2016;22(6):1008–13. Fenollar F, Puechal X, Raoult D. Whipple’s disease. N Engl J Med 2007;356(1):55–66. Dutly F, Altwegg M. Whipple’s disease and ‘‘Tropheryma whippelii”. Clin Microbiol Rev 2001;14(3):561–83. Fenollar F, Lepidi H, Raoult D. Whipple’s endocarditis: review of the literature and comparisons with Q fever, Bartonella infection, and blood culture-positive endocarditis. Clin Infect Dis 2001;33(8):1309–16. Fenollar F, Celard M, Lagier JC, Lepidi H, Fournier PE, Raoult D. Tropheryma whipplei endocarditis. Emerg Infect Dis 2013;19(11):1721– 30. Pierrotti LC, Baddour LM. Fungal endocarditis, 1995-2000. Chest 2002;122(1):302–10. Ellis ME, Al-Abdely H, Sandridge A, Greer W, Ventura W. Fungal endocarditis: evidence in the world literature, 1965-1995. Clin Infect Dis 2001;32(1):50–62. McDonald LC, Weinstein MP, Fune J, Mirrett S, Reimer LG, Reller LB. Controlled comparison of BacT/ALERT FAN aerobic medium and BATEC fungal blood culture medium for detection of fungemia. J Clin Microbiol 2001;39(2):622–4. Durack DT, Lukes AS, Bright DK. New criteria for diagnosis of infective endocarditis: utilization of specific echocardiographic findings. Duke Endocarditis Service. Am J Med 1994;96(3):200–9.

9

[50] Li JS, Sexton DJ, Mick N, Nettles R, Fowler Jr VG, Ryan T. Proposed modifications to the Duke criteria for the diagnosis of infective endocarditis. Clin Infect Dis 2000;30(4):633–8. [51] A guide to Q fever and Q fever vaccination. [http://www.meatiesohs. org/files/Q_Fever_booklet.pdf]. [52] Shah SH, Grahame-Clarke C, Ross CN. Touch not the cat bot a glove*: ANCA-positive pauci-immune necrotizing glomerulonephritis secondary to Bartonella henselae. Clin Kidney J 2014;7(2):179–81. [53] Fenollar F, Gauduchon V, Casalta JP, Lepidi H, Vandenesch F, Raoult D. Mycoplasma endocarditis: two case reports and a review. Clin Infect Dis 2004;38(3):e21–4. [54] Halliday CL, Kidd SE, Sorrell TC, Chen SC. Molecular diagnostic methods for invasive fungal disease: the horizon draws nearer? Pathology 2015;47(3):257–69. [55] Duffett S, Missaghi B, Daley P. Culture-negative endocarditis diagnosed using 16S DNA polymerase chain reaction. Can J Infect Dis Med Microbiol 2012;23(4):216–8. [56] Kemp M, Bangsborg J, Kjerulf A, Schmidt TA, Christensen J, Irmukhamedov A. Advantages and limitations of ribosomal RNA PCR and DNA sequencing for identification of bacteria in cardiac valves of danish patients. Open Microbiol J 2013;7:146–51. [57] Evangelista A, Gonzalez-Alujas MT. Echocardiography in infective endocarditis. Heart 2004;90(6):614–7. [58] Rohmann S, Erbel R, Mohr-Kahaly S, Meyer J. Use of transoesophageal echocardiography in the diagnosis of abscess in infective endocarditis. Eur Heart J 1995;16(Suppl B):54–62. [59] Rohmann S, Erbel R, Gorge G, Makowski T, Mohr-Kahaly S, Nixdorff U. Clinical relevance of vegetation localization by transoesophageal echocardiography in infective endocarditis. Eur Heart J 1992;13(4):446–52. [60] Rohmann S, Seifert T, Erbel R, Jakob H, Mohr-Kahaly S, Makowski T. Identification of abscess formation in native-valve infective endocarditis using transesophageal echocardiography: implications for surgical treatment. Thorac Cardiovasc Surg 1991;39(5):273–80. [61] Roldan CA. Diagnostic value of transesophageal echocardiography in Libman-Sacks endocarditis. Minerva Cardioangiol 2009;57(4):467–81. [62] Roldan CA, Qualls CR, Sopko KS, Sibbitt Jr WL. Transthoracic versus transesophageal echocardiography for detection of Libman-Sacks endocarditis: a randomized controlled study. J Rheumatol 2008;35(2):224–9. [63] Sochowski RA, Chan KL. Implication of negative results on a monoplane transesophageal echocardiographic study in patients with suspected infective endocarditis. J Am Coll Cardiol 1993;21(1):216–21. [64] Victor F, De Place C, Camus C, Le Breton H, Leclercq C, Pavin D. Pacemaker lead infection: echocardiographic features, management, and outcome. Heart 1999;81(1):82–7. [65] Thadani SR, Dyverfeldt P, Gin A, Chitsaz S, Rao RK, Hope MD. Comprehensive evaluation of culture-negative endocarditis with use of cardiac and 4-dimensional-flow magnetic resonance imaging. Tex Heart Inst J 2014;41(3):351–2. [66] Siciliano RF, Mansur AJ, Castelli JB, Arias V, Grinberg M, Levison ME. Community-acquired culture-negative endocarditis: clinical characteristics and risk factors for mortality. Int J Infect Dis 2014;25:191–5. [67] Baddour LM, Epstein AE, Erickson CC, Knight BP, Levison ME, Lockhart PB. Update on cardiovascular implantable electronic device infections and their management: a scientific statement from the American Heart Association. Circulation 2010;121(3):458–77. [68] Cabell CH, Heidenreich PA, Chu VH, Moore CM, Stryjewski ME, Corey GR. Increasing rates of cardiac device infections among Medicare beneficiaries: 1990-1999. Am Heart J 2004;147(4):582–6. [69] Voigt A, Shalaby A, Saba S. Rising rates of cardiac rhythm management device infections in the United States: 1996 through 2003. J Am Coll Cardiol 2006;48(3):590–1. [70] Inacio RC, Klautau GB, Murca MA, da Silva CB, Nigro S, Rivetti LA. Microbial diagnosis of infection and colonization of cardiac implantable electronic devices by use of sonication. Int J Infect Dis 2015;38:54–9.

Please cite this article in press as: Subedi S, et al. Laboratory Approach to the Diagnosis of Culture-Negative Infective Endocarditis. Heart, Lung and Circulation (2017), http://dx.doi.org/10.1016/j.hlc.2017.02.009

680 681 682 683 684 685 686 687 688 689 690 691 692 693 694 695 696 697 698 699 700 701 702 703 704 705 706 707 708 709 710 711 712 713 714 715 716 717 718 719 720 721 722 723 724 725 726 727 728 729 730 731 732 733 734 735 736 737 738 739 740 741 742 743