Culture negative endocarditis- an approach to laboratory diagnosis. Dr Shradha Subedi Microbiology Registrar Westmead Hospital, Sydney, NSW

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1 Culture negative endocarditis- an approach to laboratory diagnosis Dr Shradha Subedi Microbiology Registrar Westmead Hospital, Sydney, NSW

2 Definition and epidemiology Infective endocarditis (IE), whereby 3 or more sets of blood cultures (both aerobic and anaerobic) are negative despite prolonged incubation is defined as culture negative IE. Accounts for 3-31% of all cases of IE. More recent studies show lower prevalence. High morbidity and mortality Delayed diagnosis and difficulty with determining adequacy of treatment. Liaison between the clinicians, microbiology and tissue pathology laboratory essential 1. Fournier PE et al. Clin Infect Dis 2010, 51(2): Brouqui P et al. Clin Microbiol Rev 2001, 14(1):

3 Aetiology Prior antibiotic use and sub-optimal specimen collection accounts for 35-40% Causative organism is usually identified in 2/3rds of the cases and include: Intracellular pathogens, eg Coxiella burnetii, Legionella spp, Bartonella spp Fastidious bacteria, eg. HACEK group, nutritionally variant streptococci Difficult to culture and non-culturable microorganisms eg. Fungi, mycobacteria, Tropheryma whipplei. Non-infective endocarditis Marantic and those in the setting of auto-immune diseases 1. Fournier PE et al. Clin Infect Dis 2010, 51(2): Brouqui P et al. Clin Microbiol Rev 2001, 14(1): Badour LM et al. Circulation 2015, 132(15):

4 Diagnostic approaches Clinical history, microbiology, other laboratory investigations, eg. rheumatoid factor, imaging (echocardiogram, cardiac MRI), tissue pathology. Microbiology Culture based methods Extended incubation, specialised media Molecular methods Serology

5 Culture based method Conventional blood culture systems At least 3 sets of aerobic and anaerobic blood culture Each set should contain ~20 mls of blood Age/weight based for children Incubation for 5 days adequate for most organisms including HACEK. Prolonged incubation (21 days) and blind subculture into specialised media may yield growth of suspected organisms in some cases. Culture of resected valves or diseased tissue specimen and prosthetic devices solid and broth media. 1. Fournier PE et al. Clin Infect Dis 2010, 51(2):

6 Culture based- specialised media Mycobacteria and fungi Specialised media for mycobacteria and some fungi (moulds and dimorphic) For example: BD Bactec myco/f lytic blood culture bottles Middlebrook 7H9 and brain heart infusion broth formulation Incubation for 6 weeks Recovery of mycobacteria, yeast and fungi from blood stream and sterile sites. 1. Brouqui P et al. Clin Microbiol Rev 2001, 14(1): Badour LM et al. Circulation 2015, 132(15): Jorgensen JH et al. Manual of Clinical Microbiology vol. 1. Washington DC: ASM Press; 2015.

7 Culture based-specialised media Media supplementation Nutritionally variant Streptococci- pyridoxal or cysteine Co-cultivation with Staphylococci or use of pyridoxal impregnated disks or specially supplemented or enriched media. Legionella species Subculture blood culture or excised heart valves into buffered charcoal yeast extract (BCYE) Mycoplasma species Commercial blood culture broth contains sodium polyanetholsulfonate as an anticoagulant agent, which is inhibitory to the growth of Mycoplasma species Culture of excised valve into specialised A7 medium 1. Brouqui P et al. Clin Microbiol Rev 2001, 14(1): Jorgensen JH et al. Manual of Clinical Microbiology vol. 1. Washington DC: ASM Press; 2015.

8 Culture based- other techniques Isolator blood culture system Blood is collected in a tube containing lysing solution. Tube centrifuged and pellet is inoculated into specialised media (eg. SAB, LJ media) Higher pick up rates of dimorphic fungi, Bartonella spp. Lower pick up rates for anaerobes, Haemophilus spp and pneumococci. More labor intensive, greater risk of laboratory acquired infections. Not routinely done in diagnostic laboratories in Australia. 1. Jorgensen JH et al. Manual of Clinical Microbiology vol. 1. Washington DC: ASM Press; 2015.

9 Extended and blind subcultures yay or nay?? Forward KR, 2006 Single center Canadian study 507 blood cultures for patients with suspected IE handled using extended sub-culture (24 days) and blind subcultures at day 10 into a chocolate slope and further incubation for 14 days. Approach only yielded growth of contaminant organisms and no difference in patient outcomes were achieved by extending incubation time. Fastidious organisms recovered within 5 days incubation. 1. Forward KR. Can J Infect Dis Med Microbiol May-Jun; 17(3):

10 1. Baron JE et al. Clin Infect Dis (2005) 41 (11): Extended/blind subcultures yay or nay?? Baron JE et al, patients with suspected IE over 3 years. Extend incubation period over 21 days. Sub-culture at 3 and 10 days BCYE agar (14 days) Rabbit blood agar (21 days total) 2 chocolate agar plates (21 days total) 1 SAB agar plate (4 weeks) 1 lowenstein jensen media (6 weeks total) Anaerobic bottle- brucella blood agar with hemin and vitamin K (incubated anaerobically for 6 days) Acridine orange stain at days 3 and 10 Lysis centrifugation samples obtained on all and inoculated into the same media as above.

11 1. Baron JE et al. Clin Infect Dis (2005) 41 (11): Extended/blind subcultures yay or nay?? Baron JE et al, 2005 results: Most fastidious organisms including HACEK grew within 5 days with the standard blood culture protocol. 15 isolates recovered from 215 patients using special protocol. 12/15 were contaminant type organisms. 2 isolates of M. avium complex from Lowenstein Jensen media- HIV patient with profound immunosuppression. Legionella species from both isolator tube and blind sub cultures into BCYE. Each request cost US$ in materials and technologists time.

12 Extended/blind subcultures yay or nay?? Extended incubation of blood cultures beyond 5 days is rarely useful. Specialised methods/ media, however may have a higher yield of picking up organisms. They are very resource and time consuming to routinely adapt in all patients with suspected endocarditis. Should be utilised on a case by case basis after consultation with an infectious diseases specialist and in conjunction with other laboratory methods.

13 1. Brouqui P et al. Clin Microbiol Rev 2001, 14(1): Serology Recommended for IE due to organisms that are difficult to grow- eg. Coxiella burnetii, Legionella species, Mycoplasma species and Bartonella species. A single high titre or a four-fold rise in titre from blood samples taken 2-4 weeks apart in time. Many assays are in use: Immunofluorescent antibody, compliment fixation test, EIA

14 Serology Immunofluorescent assay (IFA) Used commonly to aid diagnosis in patients with IE due to C. burnetii, Bartonella spp, Legionella spp. Q fever: IFA can distinguish between antibody responses to phase 1 and phase 2 antigens. Very high phase 1 IgG titres of >/= 800 is highly suggestive of Q fever endocarditis. Cutoff used for phase I IgA titre varies from studies to studies Can monitor response to therapy

15 Brouqui P et al. Clin Microbiol Rev 2001, 14(1): Serology- Q fever Complement fixation test Measures mostly phase I and phase II IgG; less effective for detecting IgM. Does not permit immunoglobulin subclass analysis of Q fever antibody responses. Less sensitive compared to IFA. Enzyme immunoassay Can differentiate immunoglobulin subclasses. Unable to measure changing antibody patterns

16 Edouard S et al. J Clin Microbiol 2015, 53(3): Bartonella endocarditis Serology Immunofluorescent antibody assay commonly used. High titres of >800 associated with endocarditis with 95% positive predictive value 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 1. Brouqui P et al. Clin Microbiol Rev 2001, 14(1): Serology Legionella endocarditis Indirect immunofluorescent antibody test Titre >256 is considered significant. Positive predictive value of serology in the diagnosis of endocarditis due to legionella species is not known. Serologic testing is unable to detect antibody to all legionella species. The role of antibody detection to Mycoplasma and Chlamydia spp is also unclear, with the relatively few case reports of mycoplasma endocarditis usually relying on molecular diagnoses.

18 1. Fournier PE et al. Clin Infect Dis (2010) 51 (2): Molecular methods Various nucleic acid amplification tests can be used on blood specimen and explanted valvular tissue. Genus and/or species specific PCR and a pan-bacterial or pan fungal PCR assay may be used. Study by Fournier et al 740 cases of culture neg IE Multi-modal diagnostic strategies PCR identified a causative pathogen in 109 seronegative cases, including 106 by PCR of valvular tissues and 3 by PCR of blood only.

19 Fournier et al 1. Fournier PE et al. Clin Infect Dis (2010) 51 (2):

20 Molecular methods Particularly useful in patients who have received prior antimicrobials or have negative or equivocal serologies. Sensitivity of PCR is variable; species-specific PCR assays have higher sensitivity than broad-range PCR. Sensitivity on valvular tissue is higher than blood. False positive results may result from contamination or detection of non-viable organisms.

21 Brouqui P et al. Clin Microbiol Rev 2001, 14(1): Histopathology Examination of clinical specimen, especially explanted heart valve tissue Conventional haematoxylin and eosin (H&E) stain Inflammation, tissue invasion and necrosis Presence of microorganisms e.g fungal elements or suggestion of a particular diagnosis eg. granulomas in mycobacterial or Bartonella infections Non-infectious diagnoses- eg. Libman sacks

22 Brouqui P et al. Clin Microbiol Rev 2001, 14(1): Histopathology Special stains Gram stain- differentiate gram positive from gram negative organisms. Periodic acid-schiff (PAS) stain for detection of T. whipplei granules within characteristic foamy macrophages. Acid fast stains for Mycobacteria. Gimenez stain for C. burnetii and Legionella spp. Warthin-Starry silver stains demonstrate characteristic granular organisms of Bartonella spp. Methenamine silver stains for fungi. Immunohistochemical stains for various organisms

23 Q fever- histology section from liver Maurin M et al. CMR 1999 vol. 12 no

24 Warthin-Starry stain of a lymph node section- Bartonella species

25 Whipples- PAS positive granules

26 Inacio RC et al: Int J Infect Dis 2015, 38: Newer methods Sonication of explanted cardiac implant devices. Study by Inacio et al 80 explanted devices from 40 patients (20 with infection, 20 without any infection), 7 negative control. Positive culture defined as 2 or more specimen type positive from the same patient. Sonication fluid culture more sensitive compared to conventional culture in (65% vs 50%; p=0.04). Bacterial cell count in sonicate fluid much higher than conventional culture.

27 Newer methods Metagenomics approaches Imai A et al (Japan) 2 cases of culture positive IE (1 blood culture pos, 2 nd blood culture and valve culture pos) Metagenomic analysis using NGS technology in resected heart valves. Both yielded positive result concordant with traditional culture. Proof of concept that it works and can be employed in the future. Unlikely to surpass pan bacterial PCR and sequencing in the near future. Imao A et al. Int Journal of Cardiology (172), e288 e289

28 Conclusion Laboratory approach to culture neg IE requires liaison between the clinician, microbiology laboratory and tissue pathology. Systematic approach including conventional culture with the use of specialised media based on history and clinical suspicion is recommended. Serology and molecular diagnostic tests provide additional value. Newer methods including metagenomics approaches are in progress.