Blood Culture-Negative Endocarditis: Historical and Future Perspectives

1. Blood Culture-Negative Endocarditis: Historical and Future Perspectives Tracy Lemonovich, MD Clinical Instructor, Department of Medicine UH Case Medical Center Jan 18, 2011

2. Learning Objectives Discuss the historical perspectives of endocarditis Describe the current epidemiology of infective endocarditis Discuss advances in diagnostic strategy for blood culture-negative endocarditis

3. Gustav Mahler, 1907, Vienna Opera HouseGustav Mahler, 1907, Vienna Opera House

4. ��a loud systolic murmur over the precordium, a history of prolonged low grade fever, a palpable spleen, characteristic petechiae on the conjunctiva and skin, and slight clubbing of the fingers. Libman telephoned me to bring the paraphernalia and culture media required for a blood culture� George Baehr George Baehr, assistant to Emmanuel Libman, recounting his experience in Feb 1911 when Libman was called in to consult on Gustav Mahler�s case. Mahler had been ill for at least several weeks, and his condition appeared far advanced at the time he was evaluated by Dr. Libman. George Baehr, assistant to Emmanuel Libman, recounting his experience in Feb 1911 when Libman was called in to consult on Gustav Mahler�s case. Mahler had been ill for at least several weeks, and his condition appeared far advanced at the time he was evaluated by Dr. Libman.

5. �Attenuated streptococcus in blood of subacute bacterial endocarditis�, considerable terminology confusion over streptococci at the time, ultimately viridans streptococci Mahler returned to Europe, traveled to Vienna via the Orient Express, ultimately died in May 1911 at age 51 of complications related to endocarditis: pulmonary edema and uremia despite use of antistreptococcal vaccine treatment�Attenuated streptococcus in blood of subacute bacterial endocarditis�, considerable terminology confusion over streptococci at the time, ultimately viridans streptococci Mahler returned to Europe, traveled to Vienna via the Orient Express, ultimately died in May 1911 at age 51 of complications related to endocarditis: pulmonary edema and uremia despite use of antistreptococcal vaccine treatment

6. History of Infective Endocarditis (IE) 1542: Jean Fernel, first published report of endocarditis 1542-1800s: Anatomical observations- abnormalities of endocardium/valves at autopsy Late 1800s/early 1900s: William Osler and Thomas Horder elucidate pathophysiology; clinical diagnostic criteria Jean Fernel: Renaissance physician to Kings Francis I and Henry II of France William Osler: wrote Gulstonian lectures in 1885, attempted to incorporate known information regarding endocarditis Thomas HorderJean Fernel: Renaissance physician to Kings Francis I and Henry II of France William Osler: wrote Gulstonian lectures in 1885, attempted to incorporate known information regarding endocarditis Thomas Horder

7. History of IE Diagnostics 1800s: Auscultation for detection of cardiac murmurs 1830-40s: Elevated body temperature important 1870s: Microscopic visualization of bacteria in vegetations 1880s: Birth of bacteriology; routine use of blood cultures 1800s: Invention of cylindrical stethoscope by Theophile Laennec in France 1816 1800s: Invention of cylindrical stethoscope by Theophile Laennec in France 1816

8. History of IE Diagnostics 1976: Use of transthoracic echocardiogram (TTE) in diagnosis 1988: Superior sensitivity of transesophageal echocardiogram (TEE) over TTE Late 1990s-present: Use of molecular diagnostics Incorporation of echocardiographic findings into new diagnostic criteria for IE (Duke criteria, 1994) Use of molecular diagnostics for diagnosis of blood culture-negative endocarditisIncorporation of echocardiographic findings into new diagnostic criteria for IE (Duke criteria, 1994) Use of molecular diagnostics for diagnosis of blood culture-negative endocarditis

9. Current Diagnostic Criteria: modified Duke Criteria

10. Modified Duke Criteria Major clinical criteria Blood culture findings positive for IE Typical organism from 2 separate blood cultures or persistently positive cultures Single blood culture for Coxiella burnetii or phase I IgG >1:800 Evidence of endocardial involvement Oscillating intracardiac mass on valve or supporting structures, in path of regurgitant jets, or on implanted material Abscess New dehiscence of prosthetic valve Typical organisms: viridans strep, Strep bovis, HACEK, Staph aureus, community-acquired EnterococciTypical organisms: viridans strep, Strep bovis, HACEK, Staph aureus, community-acquired Enterococci

11. Modified Duke Criteria Minor clinical criteria Predisposition: predisposing heart condition or intravenous drug use Fever, temp >38�C Vascular phenomena, emboli, mycotic aneurysm, intracranial/conjunctival hemorrhage, Janeway lesions Immunologic phenomena: glomerulonephritis, Osler nodes, Roth spots, RF Microbiologic evidence not meeting major criteria

12. Left: Janeway lesions in patient with S. aureus endocarditis, septic emboli phenomena, histology shows subcutaneous abscess, non-painful Top right: Roth spot in patient with viridans strep endocarditis, lymphocytes with edema and hemorrhage in the retina, immunologic Bottom right: Osler nodes, classically immunologic phenomena, likely have immune-complex mediated component initiated by microemboli, small painful nodules on palmar surfaces of fingers and toes, wax and waneLeft: Janeway lesions in patient with S. aureus endocarditis, septic emboli phenomena, histology shows subcutaneous abscess, non-painful Top right: Roth spot in patient with viridans strep endocarditis, lymphocytes with edema and hemorrhage in the retina, immunologic Bottom right: Osler nodes, classically immunologic phenomena, likely have immune-complex mediated component initiated by microemboli, small painful nodules on palmar surfaces of fingers and toes, wax and wane

13. Clinical and Laboratory Findings of 2781 Patients with Definite Endocarditis

14. What is the Burden of Disease? Usual incidence 2-7 cases/100,000 person-years Diagnostic criteria and reporting variable Only 20% of clinically diagnosed cases definite IE 10-20,000 new cases/year in US No significant change in overall incidence last 30 years AHA estimates 10-20,000 new cases annuallyAHA estimates 10-20,000 new cases annually

15. What is the Burden of Disease? In-hospital mortality of IE 15-20% One year mortality approaching 40% Despite advances in diagnostics and therapy, no change in mortality last 25 years

16. Changing Epidemiology of Endocarditis

17. Patient Risk Factors Aging population Underlying valvular disease Shift from rheumatic heart disease to degenerative heart disease Congenital heart disease Mitral valve prolapse Prior IE IV drug use (IVDU) Aging population: mean age of pts has gradually increased, now >50% in pts older than 50, uncommon in children (except with congenital structural defects ie. septal defects and associated repair) Rheumatic heart disease remains most common underlying valvular condition in developing countries, but <5% of IE in US currently Congenital heart disease- patent ductus arteriosus, VSD, coarctation of the aorta, bicuspid aortic valve, tetrology of FallotAging population: mean age of pts has gradually increased, now >50% in pts older than 50, uncommon in children (except with congenital structural defects ie. septal defects and associated repair) Rheumatic heart disease remains most common underlying valvular condition in developing countries, but <5% of IE in US currently Congenital heart disease- patent ductus arteriosus, VSD, coarctation of the aorta, bicuspid aortic valve, tetrology of Fallot

18. Medical Advancements and Risk of IE�Health-Care Associated IE� IE attributed to health-care related exposure in 25% of patients Medical advancements Prosthetic valves Implantable intracardiac devices Indwelling vascular catheters Increasing use of invasive procedures ?Advances in immune suppressive therapies Increasing age of pts with valvular heart disease lead to increased use of prosthetic valves, prosthetic IE accounts of 20% of IE in recent large study Indwelling vascular catheters includes hemodialysis catheters, graftsIncreasing age of pts with valvular heart disease lead to increased use of prosthetic valves, prosthetic IE accounts of 20% of IE in recent large study Indwelling vascular catheters includes hemodialysis catheters, grafts

19. Health-Care Associated IE In N. America, >35% of IE health-care associatedIn N. America, >35% of IE health-care associated

20. Changing Microbiology of IE Staphylococcus aureus now the most common cause worldwide, 31% of patients Other gram positives important Viridans strep, coagulase-negative staph, Enterococcus Fastidious organisms HACEK 2% (0.3% in N. America) Haemophilus, Aggregatibacter, Cardiobacterium, Eikenella, Kingella Fungi/yeast 2% Culture negative 10% Textbooks often still site viridans strep as most common etiology based on data that is now decades oldTextbooks often still site viridans strep as most common etiology based on data that is now decades old

21. Microbiologic etiology of IE

22. Blood Culture-Negative Endocarditis (BCNE) Endocarditis in which no causative organism can be grown in blood sample using usual lab methods Accounts for 2.5%-31% of all IE, depending on case series Strict diagnostic criteria for endocarditis usually results in lower estimated rates of BCNE, ie. 5-10% if definite modified Duke criteria are usedStrict diagnostic criteria for endocarditis usually results in lower estimated rates of BCNE, ie. 5-10% if definite modified Duke criteria are used

23. Factors Contributing to Sterility of Blood Cultures Antibiotic administration preceding blood cultures Right-sided endocarditis Fastidious slow-growing bacteria Non-bacterial organisms i.e. fungi Non-infective endocarditis or incorrect diagnosis

24. Most Common Identified Microbiologic causes of BCNE Coxiella burnetii 3-48% Bartonella species 10-28% Staphylococcus species 2-11% Streptococcus species 1-6% HACEK 0.5-3% Fungi 1-6% Candida, Aspergillus, Cryptococcus, endemic fungi, others Tropheryma whipplei 0.3-3% Others: Legionella, Chlamydia, Brucella Percent ranges based on different series of BCNEPercent ranges based on different series of BCNE

25. Microbiologic Causes of BCNE Zoonotic agents important (Coxiella, Bartonella, Brucella) Limited data from US, North America Geographic epidemiology of zoonoses, fungi Antibiotics prior to blood cultures often a contributor (~50% in most recent series)

26. Coxiella burnetii (Q fever) Febrile illness that occurs worldwide Animal reservoir cattle, sheep, goats Humans infected by inhalation contaminated aerosols Average 50 cases/year Q fever in US Endocarditis main manifestation of chronic Q fever Humans usually have direct contact with infected animals, but indirect exposure can occur as well (outbreaks along roadways where animal skin, contaminated straw are transported), can also be acquired via drinking contaminated milk, skinning of contaminated hides, contact with parturient animals (organism can be re-activated in pregnancy), blood transfusionHumans usually have direct contact with infected animals, but indirect exposure can occur as well (outbreaks along roadways where animal skin, contaminated straw are transported), can also be acquired via drinking contaminated milk, skinning of contaminated hides, contact with parturient animals (organism can be re-activated in pregnancy), blood transfusion

27. Coxiella burnetii Endocarditis Rarely reported in US, but likely under diagnosed Usually diagnosed by serologic assay, phase I IgG titer >1:800 Major Duke diagnostic criteria PCR testing and immunohistochemistry of valve also have been used Q fever IgG criteria added as a modification to the Duke diagnostic criteria PCR testing of blood, valve tissueQ fever IgG criteria added as a modification to the Duke diagnostic criteria PCR testing of blood, valve tissue

28. Coxiella burnetii Endocarditis Immunohistochemical stain from resected cardiac valve from patient with Q fever endocarditis, using mouse monoclonal antibody and hematoxylin counterstainImmunohistochemical stain from resected cardiac valve from patient with Q fever endocarditis, using mouse monoclonal antibody and hematoxylin counterstain

29. Bartonella species Endocarditis linked to B. henselae and B. quintana Both species globally endemic B. henselae transmission to humans via cats Etiology of cat scratch disease B. quintana cause of trench fever Vector human body louse

30. Bartonella Endocarditis B. quintana associated with alcoholism, homelessness Significant proportion afebrile, advanced valvular disease, embolic phenomenon Diagnosed with culture, serologic assay IgG >1:800, PCR testing, or histology/immunohistochemistry of valve Rare to grow with routine bacterial protocols since difficult to culture in <7 days; visible by Warthin-Starry staining PCR testing of blood, valve tissueRare to grow with routine bacterial protocols since difficult to culture in <7 days; visible by Warthin-Starry staining PCR testing of blood, valve tissue

31. Bartonella Endocarditis Immunohistochemical stain for Bartonella henselae from resected cardiac valve using rabbit polyclonocal antibody and hematoxylin counterstainImmunohistochemical stain for Bartonella henselae from resected cardiac valve using rabbit polyclonocal antibody and hematoxylin counterstain

32. Fungal Endocarditis and BCNE Candida most common cause of fungal IE ~70% Most have positive blood cultures Often related to central venous catheters, cardiac surgery, chemotherapy, IVDU Non-candidal fungal IE unlikely to be blood culture positive Fungal IE common cause of prosthetic valve BCNE- 16% in recent series Likely >75% of patients with candida IE have positive blood cultures using standard methodsLikely >75% of patients with candida IE have positive blood cultures using standard methods

33. Fungal Endocarditis and BCNE Aspergillus 2nd most common cause of fungal IE after cardiac surgery Immune suppression important risk factor Mortality high (80% in one series) Diagnosis by valve tissue staining/culture, PCR, serology, ?galactomannan antigen PCR of blood, valve tissuePCR of blood, valve tissue

34. Fungal Endocarditis and BCNE Other important fungi Cryptococcus Rare cause of IE, blood cultures often positive, serum antigen, valve tissue staining/culture Endemic fungi: Histoplasma, Coccidioides Rare, but likely underestimated Diagnosis by valve tissue staining/culture, urine/serum antigen, PCR testing, serology supportive Others: Saccharomyces, Cladosporium, others Endemic fungi may be underdiagnosed, especially in endemic areasEndemic fungi may be underdiagnosed, especially in endemic areas

35. Histoplasma capsulatum Endocarditis Hematoxylin and eosin stain of the aortic prosthetic valve vegetation; original magnification X100. Insets show appearance of yeast forms in the vegetation by tissue Gram stain (upper inset) and Gomori methenamine silver stain (lower inset); original magnification X1,000 Hematoxylin and eosin stain of the aortic prosthetic valve vegetation; original magnification X100. Insets show appearance of yeast forms in the vegetation by tissue Gram stain (upper inset) and Gomori methenamine silver stain (lower inset); original magnification X1,000

36. Tropheryma whipplei May be more frequent cause of BCNE than previously thought- 2.6% in recent series May be only manifestation of Whipple�s disease Improved diagnostics PAS staining of valve tissue, PCR testing, immunohistochemistry PCR testing of blood, valve tissuePCR testing of blood, valve tissue

37. T. whipplei Endocarditis Left: Immunohistochemical stain of Tropheryma whipplei from cardiac valve using rabbit polyclonal antibody and hematoxylin counterstain Right: Section of aortic valve with vegetation, inflammatory infiltrate of lymphocytes, few granulocytes, and numerous macrophages with cytoplasm positive for Periodic Acid Schiff (PAS) staining, original magnification X350, inset high-power magnification of macrophages containing characteristic rod-shaped inclusion bodies, original magnification x731Left: Immunohistochemical stain of Tropheryma whipplei from cardiac valve using rabbit polyclonal antibody and hematoxylin counterstain Right: Section of aortic valve with vegetation, inflammatory infiltrate of lymphocytes, few granulocytes, and numerous macrophages with cytoplasm positive for Periodic Acid Schiff (PAS) staining, original magnification X350, inset high-power magnification of macrophages containing characteristic rod-shaped inclusion bodies, original magnification x731

38. Other Microbiologic Causes of BCNE Legionella species Rare cause of IE, described as cause of prosthetic valve IE Diagnosis by culture (difficult), serology, urinary antigen, PCR testing Brucella melitensis Rare (~1% of BCNE); endemic to Mediterranean, Middle East, Asia, Africa Acquired via animal exposure, unpasteurized milk Diagnosis by blood culture (variable), serology, PCR testing

39. Other Microbiologic Causes of BCNE Chlamydophila (formerly Chlamydia) species Rare but reported in literature Serology may cross-react with Bartonella antibodies Mycoplasma species Also rare but well-described Diagnosis by serology, PCR testing Viruses Cause of myocarditis (enteroviruses) but not BCNE

40. Non-Infectious Causes of BCNE Likely an important cause of BCNE, prevalence not well known 2.5% of BCNE in recent series Marantic, Libman-Sacks/autoimmume (SLE, rheumatoid arthritis, Behcet�s, anti-phospholipid antibody-related) Diagnosis: clinical signs/symptoms, detection of autoantibodies Picture: Libman-Sacks endocarditis of mitral valve in SLE patientPicture: Libman-Sacks endocarditis of mitral valve in SLE patient

41. Diagnostic Studies for BCNE Blood cultures Routine extended incubation does not improve yield HACEK organisms easily isolated with 5 day incubation in current blood culture systems Terminal subculture, lysis centrifugation culture may improve yield of certain organisms Brucella, fungi Study of 407 blood cultures in 2003-2004 in patients with suspected BCNE, none grew HACEK or other fastidious bacteria with extended incubation of 10-14 days. HACEK organisms isolated in mean of 3.4 days.Study of 407 blood cultures in 2003-2004 in patients with suspected BCNE, none grew HACEK or other fastidious bacteria with extended incubation of 10-14 days. HACEK organisms isolated in mean of 3.4 days.

42. Diagnostic Studies for BCNE Serologic testing Molecular testing Blood, valve tissue, embolic vegetations Valve tissue PCR sensitivity 40-60%, specificity near 100% False negative= pre-operative antibiotics False positive= non-viable bacteria after treatment, contaminated tissue PCR sensitivity/specificity compared to gold standard of Duke criteria combined with histopathologic examinationPCR sensitivity/specificity compared to gold standard of Duke criteria combined with histopathologic examination

43. Diagnostic Strategy for BCNE Largest diagnostic case series of BCNE 819 cases evaluated 2001-2009 Most cases from France Definite and possible IE by modified Duke criteria Largest series in which PCR detection from valvular biopsies performed Use of several new diagnostic techniques

44. Serological Testing Coxiella burnetii Bartonella quintana and henselae Legionella pneumophila Brucella melitensis Mycoplasma pneumoniae 47.8% of pts with microbiological diagnosis made by serology C. burnetii serology part of modified Duke criteria Coxiella burnetii (phase I IgG titer >1:800) Bartonella quintana and henselae (IgG >1:800) Legionella pneumophila (total Ab titer >1:256) Brucella melitensis (titer >1:200) Mycoplasma pneumoniae IgG, IgM C. burnetii serology part of modified Duke criteria Coxiella burnetii (phase I IgG titer >1:800) Bartonella quintana and henselae (IgG >1:800) Legionella pneumophila (total Ab titer >1:256) Brucella melitensis (titer >1:200) Mycoplasma pneumoniae IgG, IgM

45. Molecular Testing Blood Broad-range PCR for bacteria (16s rRNA) and fungi (18s rRNA), some viruses Included specific primers for Coxiella, Bartonella sp., T. whipplei, Chlamydia sp., CMV, Enterovirus If valve tissue available: Broad-range PCR for bacteria and fungi If other testing negative, primer extension enrichment reaction (PEER) PCR PEER= used to detect and isolate DNA or RNA from complex nucleic acid mixturesPEER= used to detect and isolate DNA or RNA from complex nucleic acid mixtures

46. Other Valvular Testing All suitable specimens cell cultured Bacteria detected identified by PCR testing Histopathological analysis Autoimmunohistochemistry Done for specimens in which all other techniques negative

47. Additional Testing All patients tested for rheumatoid factor, antinuclear antibodies, anti-DNA antibodies If all testing negative, physicians contacted regarding diagnosis of neoplastic or autoimmune disease made elsewhere

48. Distribution of 819 Suspected BCNE Cases

49. Yield of Additional Diagnostic Testing Most diagnoses made by serological testing (47.8% of cases with microbiologic diagnosis) Chronic Q fever 77%, Bartonella 22.5% PCR second best diagnostic technique Blood poor sensitivity: 36 of 257 (13.5%) specimens tested positive Valve PCR high yield: 157 of 227 (69%) specimens positive Strep, fastidious bacteria; no viruses identified No additional cases of Coxiella identified not found by serology Broad-range PCR for fungi of blood did identify etiology in some patients PEER and autoimmunohistochemistry provided diagnoses for only 4 and 1 patients, respectively, not made by other techniquesNo additional cases of Coxiella identified not found by serology Broad-range PCR for fungi of blood did identify etiology in some patients PEER and autoimmunohistochemistry provided diagnoses for only 4 and 1 patients, respectively, not made by other techniques

50. Proposed Diagnostic Strategy for BCNE

51. Limitations of New Diagnostics In most recent study, 36.5% of cases still undiagnosed after extensive testing Range 22-83% in various studies Overall sensitivity of diagnostic strategy varies by population Lower sensitivity if definite AND possible endocarditis included Poor sensitivity of commercially available PCR detection kits of blood Poor sensitivity blood PCR may be factor in cases in which valve tissue unable to be tested (227 of 745 cases in Fournier series)Poor sensitivity blood PCR may be factor in cases in which valve tissue unable to be tested (227 of 745 cases in Fournier series)

52. Is this strategy applicable to our US population? Lower incidence of zoonoses in US vs. Europe (Coxiella, Bartonella) Likely under reported/diagnosed: Coxiella highly prevalent in US cattle, 22% veterinarians seropositive ?Similar rates of fungal etiologies Higher in N. America in studies of culture positive IE Availability of PCR testing

53. Future Challenges in BCNE Additional studies of epidemiology of BCNE in US/North America Use of serologic testing for zoonoses based on exposure history vs. universal testing Role of non-infectious causes of BCNE Specificity of auto-antibody testing Non-infectious causes of BCNE may be higher prevalence than previously consideredNon-infectious causes of BCNE may be higher prevalence than previously considered

54. Future Challenges in BCNE Improvements in PCR testing techniques, availability Development of highly sensitive PCR assays of blood for staph, strep Role of fungal serological testing, antigen assays Urine/serum antigens, galactomannan, (1,3)�-D-glucan levels Proposed modification of Duke criteria to include molecular testing Highly sensitive blood PCR for staph, strep helpful for patients treated with antibiotics prior to blood culturesHighly sensitive blood PCR for staph, strep helpful for patients treated with antibiotics prior to blood cultures

55. Questions?