Approach to the Laboratory Diagnosis of Infective Endocarditis SWACM 2018

Transcription

1 Approach to the Laboratory Diagnosis of Infective Endocarditis SWACM 2018 Rachael Liesman, PhD, D(ABMM) Director, Clinical Microbiology Department of Pathology and Laboratory Medicine University of Kansas Medical Center

2 Financial Disclosures Nothing to disclose

3 Learning Objectives 1. Recognize the most common causes of infective endocarditis 2. Discuss the role of blood cultures in the diagnosis of infective endocarditis 3. Describe the advantages and disadvantages of serological and molecular testing for the diagnosis of culture-negative endocarditis

4 Endocarditis Pathogenesis of infective endocarditis Infection of the endocardial surface of the heart Mitral valve most often affected (33-45%) Aortic valve (25-38%) Tricuspid valve (0-10%) Incidence 6-15 per 100,000 population

5 Werdan K, et al. Nature Reviews Cariology 2014; 11:35 Endocarditis Pathogenesis Endothelial injury Mechanical Obstructive lesions Aberrant blood flow High velocity blood flow Congenital defects Inflammatory Pathogen damage Focal deposition of platelets and fibrin

6 Werdan K, et al. Nature Reviews Cariology 2014; 11:35 Endocarditis Pathogenesis Endothelial injury Focal deposition of platelets and fibrin Exposure Transient bacteremia (skin / mucosal membrane trauma) Distant focal infection

7 Werdan K, et al. Nature Reviews Cariology 2014; 11:35 Endocarditis Pathogenesis Endothelial injury Focal deposition of platelets and fibrin Exposure Transient bacteremia Vegetation Formation Adherence to platelet-fibrin aggregate Proliferation Dependent on pathogen virulence factors (e.g., adhesions) Formation of vegetation Immunologically protected space High organism burden Embolization

8 Endocarditis Pathogenesis Endothelial injury Focal deposition of platelets and fibrin Exposure Transient bacteremia Vegetation Formation Adherence to platelet-fibrin aggregate Proliferation Dependent on pathogen virulence factors (e.g., adhesions) Formation of vegetation Immunologically protected space High organism burden Mitral valve vegetation Embolization

9 Murdoch DR et al, Arch Intern Med 2009; 169(5) Endocarditis Clinical Presentation Fever 96% New / worsening murmur 65-90% Inflammatory markers 60-70%

10 Endocarditis Traditional classification Acute Fulminant course: high fever, systemic disease, leukocytosis Subacute / Chronic Indolent course: low grade fever, weight loss, vague systemic complaints S. aureus, S. pyogenes, S. pneumoniae, N. gonorrhoeae viridans group Streptococci

11 Murdoch DR et al, Arch Intern Med 2009; 169(5) Endocarditis Changing epidemiology Shifts in etiologies Factors affecting changing epidemiology aging population ~50% patient >50yo changes in nature of underlying heart disease incidence of acute rheuma c fever incidence of degenera ve heart disease prosthetic valve surgery IV drug use increased health care contact & invasive therapeutic modalities

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13 Endocarditis Epidemiology: native valve endocarditis Native valve endocarditis ~70% of infectious endocarditis ~50% require surgical intervention 17% in-hospital mortality Causes Gram positive cocci (83%) staphylococci (37%) S. aureus (28%) streptococci (35%) viridans group (21%) enterococci (11%) Murdoch DR et al, Arch Intern Med 2009; 169(5)

14 Endocarditis LATE EARLY Epidemiology: prosthetic valve endocarditis within 60d post surgery intraoperative contamination nosocomial S. epidermidis (24%) S.Cite: aureus (24%)Med 2009 Arch Intern streptococci (12%) community acquired S. aureus (23%) streptococci (22%) Cite: Arch Intern Med 2009 enterococcus (12%) * Pettersson GB et al. J Thoracic and Cardio Surg April 2014 Murdoch DR et al, Arch Intern Med 2009; 169(5) > 60d post surgery *

15 Endocarditis Epidemiology: IV drug use 7% (2000) -> 12% (2013) increases among younger population, whites, females increased incidence of right sided (tricuspid valve) IE (30-70%) Predisposing factors Direct seeding of bloodstream Normal skin and oral flora Contaminated syringes: up to 108 organisms/ml Skin and soft tissue abscesses at injection sites High rates of S. aureus nasal and skin colonization Organisms contaminating drugs or paraphernalia Valvular damage due to injection of particulate matter (e.g., talc) Microbiology S. aureus (CA-MRSA) Streptococci, enterococci Rare organisms: Eikenella corrodens, P. aeruginosa, Serratia spp., Candida, GNBs Wurcel AG et al. Open Forum Inf Dis 2016

16 Endocarditis Modified Duke Criteria

17 Endocarditis Modified Duke Criteria minor MAJOR Microbiological criteria Typical microorganisms consistent with IE from 2 separate blood cultures: viridans streptococci, S. bovis, HACEK group, S. aureus, enterococci Microorganisms consistent with IE from persistently positive blood cultures Coxiella burnetii anti-phase I IgG antibody titer >1:800 Positive blood cultures not meeting major criteria Serological evidence of active infection with organism consistent with IE Li JS et al. Clin Infect Dis 2000

18 Identification of a causative pathogen is integral to appropriate choice and duration of antimicrobial therapy

19 Endocarditis Blood cultures Key Concepts Draw before antibiotics 5 day incubation Support growth of Candida spp. Support growth of HACEK organisms (Haemophilus, Aggregatibacter, Cardiobacterium, Eikenella, Kingella) Support growth of nutritionally variant streptococci (Abiotrophia, Granulicatella species) Extend if DDx include C. acnes (rare; positivity range 4-14d) Notify lab if DDx includes select agent (e.g., Brucella spp.) Petti et al. J Clin Microbiol 2006 Bourbeau et al J Clin Microbiol Sohail et al. Clin Microbiol Infect 2009

20 Endocarditis Blood cultures Key Concepts Draw before antibiotics 5 day incubation 2 bottle sets, 2 peripheral draws 1 aerobic bottle, 1 anaerobic bottle Increases sensitivity (40mL total) Allows for differentiation between true pathogen and skin flora contaminant Volume is key!! 10ml per bottle for adults Lose sensitivity with underfilled blood bottles Patel R, et al. J Clin Microbiol Dec;49(12): Bourbeau et al J Clin Microbiol. 2005

21 Endocarditis Blood cultures Key Concepts Draw before antibiotics 5 day incubation 2 bottle sets, 2 peripheral draws Volume is key!! Fungal endocarditis Rare (evaluate patient specific risk factors) Serology Specialized blood cultures (e.g., lysis-centrifugation)

22 Endocarditis Blood cultures Blood culture spacing American Heart Association & European Society of Cardiology 3 sets of blood cultures, with the first and last at least 1h apart British Society for Antimicrobial Chemotherapy subacute: 3 sets of blood cultures, with 6h between sets severe: 2 sets of blood cultures, with 1h apart CLSI M47: Principles and Procedures for Blood Cultures if negative at 5d, blind subculture to chocolate agar Questionable value Bacteremia is (almost) constant in IE Not part of routine practice Baddour L et al. Circulation 2015 Eur Heart J 2015;36 Gould et al. J Antimicrob Chemother 2012 Weinstein MP et al. Rev Infect Dis 1983 Jan CLSI M

23 Culture Negative Endocarditis Blood cultures negative in 2-40% of cases Depends heavily on adherence to appropriate blood culture recommendations 1. Previous or concurrent administration of antimicrobials 2. Infection with an organism that does not grow in blood cultures

24 Culture Negative Endocarditis Causative agents Most common Coxiella burnetii Bartonella species Tropheryma whipplei Rare Mycoplasma hominis Legionella pneumophila Brucella species Chlamydia/Chlamydophila species Mycoplasma pneumoniae Questionable due to use of questionable diagnostics (cross reactive serology)

25 Culture Negative Endocarditis Coxiella burnetti Obligate intracellular pathogen parasitophorous vacuole in host cell cytoplasm Disease Q fever Query: etiology unknown Queensland: first identified in Queensland Australia (1935) Acute Disease: (40-50%) influenza-like illness, varies Chronic Disease: infection >6mo (2-5%), endocarditis, vascular infection Select Agent/Occupational risk: highly infectious, easily aerosolized Exposures zoonotic; farm animals, pets Life cycle stages Large cell variant: metabolically active, vegetative form Small cell variant: spore-like, extracellular survival / transmissible form Image: Beth Fischer, Rocky Mountain Laboratories

26 Culture Negative Endocarditis Coxiella burnetti Laboratory Diagnosis Serology Indirect immunofluorescence Phase I and phase II IgM and IgG Phase variation of cell wall lipopolysaccharide (LPS) Phase I: virulent II IgG Phase II: avirulent / antigenically active II IgM Temporal immune response Phase II IgM -> Phase II IgG -> Phase I IgM -> Phase I IgG Major Duke criteria anti-phase I IgG 1:800 van Wijk et al, Transfus Med Hemother 2011 I IgM I IgG

27 Culture Negative Endocarditis Bartonella species 2nd most frequent after Coxiella (European) possibly most common in US B. henselae Transmission: cat flea (Ctenocephalides felis), cat scratches / bites Exposures: cats Acute Disease: Cat Scratch Disease B. quintana Transmission: body louse (Pediculus humanus humanus) Exposures: homeless, substance abusers, poor sanitation / hygiene Acute Disease: Trench fever

28 Culture Negative Endocarditis Bartonella species Endocarditis Presentation Subacute +/- fever high incidence of emboli (~40-50%) previous valvular damage (50-60%) Laboratory Diagnosis Serology Immunofluorescence assay Proposed Duke criteria: 1:800 IgG titer Limitations seroreactivity within population (Bh IgG 1:128) cross reactivity Chlamydia / Chlamydophila within Bartonella species Coxiella burnetii (lower)

29 Culture Negative Endocarditis Tropheryma whipplei Slender rods Trilaminar envelope reacts well to periodic acid-schiff (PAS) stain Reservoir Environment related to soil-dwelling actinobacteria Asymptomatic human carrier state 4-12% (France) Disease Classic Whipple s Disease Weight loss, diarrhea, arthropathies, varied systemic symptoms Diagnosis by biopsy of the duodenum/jejunum Rare Localized Whipple s Disease Endocarditis (also, CNS, lymph nodes, bone, eye) +/- CWD symptoms Dutly F and Altwegg M. Clin Microbiol Rev Jul Fenollar F et al. J Infect Dis Mar

30 Culture Negative Endocarditis Tropheryma whipplei Laboratory Diagnosis Serology unavailable rarity of disease difficulty of interpretation asymptomatic carrier state CWD associated with decreased total IgG reaction Histopathological evaluation Cell envelope material in macrophages forms PAS-positive inclusions immunohistochemistry, if available T. whipplei specific PCR Fenollar F, et al. Clin Infect Dis. 2009, Sept Weisman A, et al. Can J Infect Dis Med Microbiol. 2013

31 Culture Negative Endocarditis Serologic Testing Serology Summary Serologic testing as part of routine IE workup identification of etiologic agent in an additional 8% of cases Blood culture negative endocarditis identification of etiologic agent in 50-76% of cases C. burnetii Duke criteria: 1:800 anti-phase I IgG antibodies Bartonella species Serologic criteria proposed, not yet incorporated into Duke criteria Raoult D, et al. J Clin Microbiol. 2005, Oct Houpikian P and Raoult D. Medicine. 2005, 84.

32 Culture Negative Endocarditis Evaluation of excised valvular tissue Surgical intervention performed in 24-53% of cases of infective endocarditis Evaluation of excised valvular tissue Histopathology Culture Molecular testing

33 Culture Negative Endocarditis Histologic Evaluation Vegetation fibrin, platelets, clusters / colonies of bacteria Common histology stains Hematoxylin and eosin (H&E) organisms usually purple/blue Tissue Gram stain Gram variability common after antibiotic therapy Grocott-Gomori methenamine silver (GMS) stains both fungi and bacteria periodic acid-schiff (PAS) stains fungi, T. whipplei

34 Culture Negative Endocarditis Histologic Evaluation Acute endocarditis (e.g., S. aureus) large, friable lesions acute inflammation (neutrophils) large colonies of microorganisms Subacute endocarditis (e.g. viridians streptococci) lesions may be smaller or not grossly visible acute inflammation colonies of organisms evidence of healing (fibrin deposition, monocytes)

35 Culture Negative Endocarditis Culture of excised valvular tissue Culture Pathological criterion by Duke s criteria important for antimicrobial susceptibility Gram stain in Micro > sens than tissue Gram stain (81 vs 67%) Low sensitivity positive cultures in 8-33% of cases High rate of false positivity 39% valve culture grew an organism 10/28 (36%) considered falsely pos 28% expected negative cultures were positive Munoz P, et al. J Clin Micro, Sept 2008

36 Culture Negative Endocarditis Molecular testing of excised valvular tissue Single target PCR Developed for C. burnetii, Bartonella, T. whipplei, C. acnes, M. hominis Laboratory developed tests only Not widely accessible Sensitivity tissue > blood Bartonella 92% tissue vs 33% blood Positive PCR in blood may be helpful in the absence of excised tissue

37 Culture Negative Endocarditis Molecular testing of excised valvular tissue broad range bacterial amplification and sequencing aka, 16S sequencing, universal PCR targets 16S rrna gene, detects any bacterial pathogen (including mycobacteria) 16S rrna encodes a subunit of the bacterial ribosome unique to prokaryotes (no cross reactivity with human genes) multiple copy numbers per bacterial cell (helps with sensitivity) Protocol Extract DNA Amplify 16S rrna gene Sequencing amplicon (Sanger or next generation sequencing) Compare sequence to database of known organism 16S rrna gene sequences

38 Culture Negative Endocarditis Molecular testing of excised valvular tissue broad range bacterial amplification and sequencing Variable sensitivity: 33-90% culture of valves: sensitivity 8-33% tissue > blood (66% vs 14%) High specificity Contamination rare Limitations False positives: Long term persistence of bacterial DNA False negatives: Sampling errors large portion / entire valve sent to micro vegetation not always readily visible, variable experience of techs microorganisms not homogenously distributed

39 Culture Negative Endocarditis Molecular testing of excised valvular tissue Broad range, but for fungi Same concept as bacterial 16S amp/seq, different target (ITS1/2, D1/D2 regions) Not recommended routinely due to rarity of fungal endocarditis Specificity may be an issue 53% (8/15) positive sequencing results were considered contaminants Shrestha NK, et al. Ann Thorac Surg 2015(99)

40 Culture Negative Endocarditis Molecular testing of excised valvular tissue Why not test all specimens with broad range sequencing? Sensitivity Single target > Multiplex >> broad range Bartonella endocarditis, molecular testing of valve tissue 92% by Bartonella specific PCR 60% by broad range bacterial amplification/sequencing Broad range bacterial amplification/sequencing Edouard S, et al. J Clin Microbiol. 2008(53) Targeted PCR

41 Endocarditis Testing algorithm for the ID of the microbiological etiology Liesman RM. JCM 2017

42 Endocarditis Proposed algorithm

43 Endocarditis Proposed algorithm

44 Endocarditis Proposed algorithm

45 Endocarditis Proposed algorithm

46 Endocarditis Unmet needs Improved strategies to promote appropriate blood culture collection Before antimicrobials Volume (do we need 3 sets?) Contamination rate Options for patients with culture negative IE who do not undergo surgery ~50% of patients Culture negative, serology negative, then what? Molecular diagnostics (+ resistance markers) from blood Additional studies on usefulness of valve cultures

47 Questions