Expires April 2021
Robin Patel, M.D., is the Division Chair of Clinical Microbiology in the Department of Laboratory Medicine and Pathology in Rochester, Minnesota. She holds the academic rank of Professor of Medicine and Microbiology.
Contact us: MMLHotTopics@mayo.edu.
Transcript and References
Hi, I’m Matt Binnicker, the Director of Clinical Virology and Vice Chair of Practice in the Department of Laboratory Medicine and Pathology at Mayo Clinic.
Infective endocarditis, which involves infection of the inner lining of the heart chambers or valves, is associated with a high mortality rate, with more than one-third of patients affected dying within a year following diagnosis. So, it’s important that health care providers have a good understanding of how this infection is diagnosed so that therapy can be optimized. In this month’s “Hot Topic,” my colleague Dr. Robin Patel will review the laboratory methods used to diagnose infective endocarditis. Specifically, she’ll discuss the role of blood cultures, nucleic acid amplification tests, histopathology, and recently, broad-range bacterial sequencing, and how these methods can assist in the diagnosis of this disease.
I hope you enjoy this month’s “Hot Topic,” and I want to personally thank you for allowing Mayo Clinic the opportunity to be a partner in your patients’ health care.
Thank you, Dr. Binnicker.
My disclosures are shown here.
As you view this presentation, consider the following important points regarding the laboratory testing of endocarditis:
- Which tests should you use?
- When should these tests be used?
The mortality of infective endocarditis is high, with more than one-third of patients affected dying within a year following diagnosis.
Identification of the specific microbial etiology is essential for optimal patient management. Delays in diagnosis contribute to late initiation of effective antimicrobial therapy, influencing morbidity and mortality.
Staphylococci and streptococci combined cause about 80% of cases. Staphylococcus aureus remains the prevailing pathogen, associated with 25% to 30% of cases, while coagulase negative staphylococci cause approximately 11% of cases. Streptococci, primarily viridans streptococci, account for about 30% of cases, with Streptococcus gallolyticus being involved in 20% to 50% of streptococcal cases. Enterococci account for approximately 10% of cases. Gram-negative bacilli account for about 5% of cases, and include the HACEK group organisms Haemophilus, Aggregatibacter, Cardiobacterium, Eikenella, and Kingella species and, less commonly, non-HACEK Gram-negative bacilli, such as the Enterobacteriaceae and non-fermenting Gram-negative bacilli.
A number of “uncultivable” or challenging to cultivate organisms may cause endocarditis, the most common of which are Coxiella burnetii, Bartonella species, and Tropheryma whipplei.
The modified Duke criteria provide a basic framework for diagnosis and definition of endocarditis. The finding of two or more blood cultures positive for a typical microorganism consistent with infective endocarditis is a major criterion for diagnosis, as is positive Q fever serology. Echocardiographic findings are also considered by the Duke criteria, although they are beyond the scope of this “Hot Topic.”
Importance of Blood Cultures
Endocarditis is an endovascular infection associated with the persistent presence of infecting microorganisms in blood. Accordingly, blood cultures are the standard test to determine the microbial etiology. Routine blood cultures incubated on modern automated, continuous-monitoring blood culture systems allow recovery of almost all easily cultivable agents of endocarditis without a need for prolonged incubation or terminal subculture. Blood cultures should be drawn prior to administration of antibiotics.
Blood Cultures Infective Endocarditis Diagnosis
Conventionally, three sets of blood cultures, with each set including one aerobic and one anaerobic bottle, are collected. Alternatively, two sets may be collected, with two aerobic and one anaerobic bottle per set. Yield of blood cultures is directly related to the volume of blood cultured, with properly filled blood-culture bottles being essential.
Most, if not all, blood cultures from patients with endocarditis caused by microorganisms able to grow in blood-culture systems should be positive, provided that they are appropriately collected and drawn prior to the administration of antimicrobial therapy. Separation of blood-culture draws over time is unnecessary.
Standard blood-culture incubation times of five days are adequate for recovery of almost all cultivable causes of endocarditis, including Candida species. The HACEK organisms were classically considered challenging to detect in blood cultures; however, with current blood-culture systems, neither extended incubation nor terminal blind subculture is unnecessary for their recovery. Current blood-culture systems also contain sufficient supplements to support growth of Abiotrophia and Granulicatella species, formerly known as the nutritionally variant streptococci. Brucella species are infrequent causes of endocarditis in the United States; however, their detection in routine blood cultures is typically achieved within the standard five-day incubation period. Cutibacterium acnes deserves special consideration, however, as some strains of this species may require prolonged blood-culture incubation.
Fungal endocarditis is rare and is most commonly caused by the Candida species, which should grow in routine blood cultures. Non-candidal fungal endocarditis is very rare, requires specialized testing, and should only be considered in patients with specific risks after more common etiologies have been excluded.
Blood cultures are negative in 2% to 40% of cases of endocarditis, with some studies reporting higher blood culture-negative rates. The causes of so-called “culture-negative endocarditis” fall into two categories: negative blood cultures due to concomitant or antecedent antibacterial therapy, or the presence of an organism that does not grow in routine blood cultures, with the former being more common.
In patients who have not received antibiotics, the most common etiologies of culture-negative endocarditis are Coxiella burnetii and Bartonella species, with the former accounting for 28% to 37% and the latter for 12% to 28% of cases. Tropheryma whipplei causes up to 6% of culture-negative endocarditis cases. Cutibacterium acnes, a rare cause of endocarditis, may cause culture-negative endocarditis due to the requirement for prolonged blood-culture incubation for growth of some strains. In addition, some strains may not grow in blood cultures. Mycoplasmal endocarditis, while rare, is primarily caused by Mycoplasma hominis and is usually diagnosed using a nucleic acid amplification test.
Culture-Negative Endocarditis Blood Tests
For some organisms that do not grow or do not easily grow in routine blood cultures, serologic evaluation can be helpful. Organisms for which serologic tests have been shown to aid in the diagnosis of endocarditis include Coxiella burnetii and Bartonella species and, in areas where Brucella endocarditis occurs, Brucella species. Serology for Coxiella burnetii is the best-established serologic test for the diagnosis of endocarditis and is included as a major criterion in the modified Duke criteria. In chronic Q fever endocarditis, anti-phase I IgG Coxiella burnetii titers greater than or equal to 1:800 are diagnostic. Nucleic acid amplification tests performed on blood can be helpful for diagnosis of Coxiella burnetii, Bartonella species, and Tropheryma whipplei endocarditis, but their sensitivity is imperfect. Although testing of cardiac valve tissue is more sensitive than testing blood, a PCR result on blood is helpful when positive.
Excised Cardiac Valvular Tissue Histopathology
Surgery is performed in some, but not all, cases of endocarditis. If a microbial diagnosis has not been established at the time of surgery, excised valvular tissue provides a valuable diagnostic specimen. It should be submitted for histopathological and microbiological evaluation. If a microbial diagnosis has already been established, additional microbiology testing is typically unnecessary, but histopathologic evaluation is often still performed.
On gross examination, vegetations may be soft, friable, or firm and vary in size based on the infecting organism. Discrete vegetations are not, however, always present. Representative sections of the valvular material should be processed for histopathology.
On histologic examination of excised valve tissue, patterns and degrees of inflammation will vary depending on the infecting organism.
Endocarditis caused by highly virulent organisms, such as Staphylococcus aureus, is often associated with acute inflammation characterized by extensive neutrophilic infiltration as well as large colonies of microorganism-associated areas of tissue destruction. In cases of subacute endocarditis caused by less virulent organisms, such as viridans streptococci, in addition to focal colonies and neutrophilic infiltration, evidence of healing, including fibrin deposition and mononuclear inflammatory cells, may be present. In endocarditis caused by Bartonella species, Coxiella burnetii, or Tropheryma whipplei, valves primarily show chronic inflammation. Mononuclear, rather than neutrophilic, infiltration predominates. Abundant foamy macrophages are the primary finding in Tropheryma whipplei endocarditis. Macrophages may also be found in Coxiella burnetii endocarditis, although the infiltration is typically less pronounced than with Tropheryma whipplei infection, and the inflammatory response may be mistaken for degenerative changes. Histopathologically, Bartonella endocarditis typically shows marked fibrosis and with minimal vegetation formation, in addition to macrophage and lymphocyte infiltration.
Bacteria may be apparent as basophilic or eosinophilic colonies on hematoxylin and eosin (H&E) stained tissue. Not all bacteria are readily detectable in H&E stained tissue, however; and it is common practice to evaluate a panel of stains when endocarditis is suspected, including Gram and Grocott-Gomori methenamine silver stains. A tissue Gram stain is commonly used but may fail to highlight some organisms, particularly in the setting of prior antibiotic administration. The Grocott-Gomori methenamine silver stain, while classically used for the identification of fungi, may offer increased sensitivity for identification of bacteria in valve tissue. Additional stains that are useful in some settings include Warthin-Starry, Ziehl-Nielson, and periodic acid-Schiff stains. Like Grocott-Gomori methenamine silver, periodic acid-Schiff will highlight most bacteria and may offer increased sensitivity over tissue Gram stain.
Periodic acid-Schiff with diastase is also the stain of choice for visualizing Tropheryma whipplei within foamy macrophages in cases of Whipple’s endocarditis. Although Warthin-Starry stains may be used to identify weakly staining bacteria, such as Bartonella species, staining is non-specific, and heavy background precipitate often renders this stain difficult to interpret. A Ziehl-Nielson stain may be used for the detection of acid-fast bacteria, such as mycobacteria, but these are rare causes of endocarditis.
Caution should be exercised when interpreting staining properties of organisms in valves removed from patients on antimicrobial therapy, as bacterial morphologies and stain properties may be altered. Additionally, the presence of organisms in tissue does not necessarily indicate active endocarditis, as clearance of organisms is delayed compared to sterilization of the vegetation.
Consultation with a pathologist with specialized expertise in infectious diseases pathology is recommended.
Excised Cardiac Valvular Tissue Culture
Several studies have shown that culture of valve tissue suffers from low sensitivity and specificity, with positive cultures in only 6% to 26% of endocarditis cases.
A microorganism different from that identified by blood culture or valve PCR is detected in approximately one-third of cases, suggesting a high rate of false positivity. Cultures of valves from patients without evidence of endocarditis are falsely positive in one-quarter to one-third of cases, due to contamination during processing.
Because of the low specificity of valve cultures, routine culture of valvular tissue removed for reasons other than possible endocarditis is not recommended.
In cases of blood culture-positive endocarditis, results of valve cultures may cause unnecessary confusion if valve cultures generate discrepant results. In cases of blood culture-negative endocarditis, valve tissue culture still suffers from low sensitivity and specificity, although growth of an organism does allow for antimicrobial susceptibility testing. When available tissue is insufficient for all tests of interest, culture should not be prioritized over more sensitive assays, such as molecular testing.
Excised Cardiac Valvular Tissue Nucleic Acid Amplification Tests
Molecular methods used in endocarditis diagnosis include organism-specific PCR and broad range bacterial PCR followed by sequencing. Broad range bacterial PCR, with amplification primers targeting the bacterial 16S ribosomal RNA gene, is a molecular method for detecting bacteria in general. Following amplification, bacterial identification is determined by sequencing amplified DNA followed by comparison of the sequence to established databases. Broad range bacterial PCR and sequencing performed on valve tissue has a reported sensitivity of 33% to 90% and has a high specificity. Therefore, for patients with infective endocarditis who undergo surgical excision of affected valves, and for whom no etiologic agent has yet been identified, testing valvular tissue by broad range bacterial PCR and sequencing is recommended when histopathologic examination of excised tissue shows acute inflammation.
Broad range fungal PCR is technically possible but has low yield for endocarditis diagnosis, due to the rarity of fungi as causes of endocarditis. It is not routinely recommended.
Notably, organism-specific PCR assays demonstrate superior sensitivity compared to broad range bacterial PCR and sequencing. Therefore, for diagnosis of culture-negative endocarditis, broad range PCR should not be performed in lieu of organism-specific PCR. Organism-specific PCR assays targeting Bartonella species, Coxiella burnetii, and Tropheryma whipplei, and possibly Mycoplasma hominis should be considered.
Caution should be exercised in the interpretation of nucleic acid amplification tests from removed valves after completion of antibiotic therapy. Long-term persistence of bacterial DNA may occur in patients who have completed a full course of antibiotic therapy, even cases several years after diagnosis of endocarditis. Conversely, results can be falsely negative due to the presence of PCR inhibitors, microbial nucleic acid below the limit of detection of the assay being used, or sampling error, since microorganisms are often not homogenously distributed in resected valves.
A multimodal testing strategy for the diagnosis of endocarditis, employing culture, serology, histopathology, and molecular analyses, is essential for optimal sensitivity and specificity in identifying an infectious etiology. A proposed testing strategy is shown.
Notably, this algorithm should be applied in the context of clinical evaluation of the patient and other findings, such as echocardiography, supportive of a diagnosis of infective endocarditis. Positive blood cultures are the standard means of microbial diagnosis; blood cultures should be collected prior to initiation of antibiotic therapy. In cases of culture-positive endocarditis, with two or more positive blood cultures, no additional microbiology testing is necessarily needed, although histopathologic evaluation of valvular tissue, if excised, is typically performed to confirm the diagnosis. In cases of culture-negative endocarditis, Coxiella burnetii and Bartonella serology should be performed, and consideration should be given to performing Tropheryma whipplei PCR on blood.
If the patient undergoes valvular resection, histopathology and staining of the resected valve is recommended, with evaluation by a pathologist with specific expertise in infectious diseases pathology. If a microbiological diagnosis has not been established, molecular testing of fresh excised valve tissue (or formalin-fixed, paraffin-embedded valve tissue, if fresh tissue is unavailable) should be guided by histopathologic evaluation and includes broad range bacterial PCR and sequencing, especially in the presence of acute inflammation, as well as specific PCR assays for Tropheryma whipplei, Coxiella burnetii, and Bartonella species. If there is chronic inflammation with macrophage predominance, Tropheryma whipplei PCR is especially recommended.
Ideally, a representative sample of valvular tissue should be specifically collected in the operating room for molecular testing.
- Liesman R, Pritt B, Maleszewski J, Patel R: Laboratory Diagnosis of Infective Endocarditis. J Clin Microbiol. Sept. 2017. 55:2599-2608.