Arthritis & Rheumatism DOI 10.1002/art.38389
Brief Report
PREVALENCE OF ANTI-NEUTROPHIL CYTOPLASMIC ANTIBODIES IN INFECTIVE ENDOCARDITIS
Alfred Mahr, MD, PhD1, Frédéric Batteux, MD, PhD2, Sarah Tubiana, PharmD3, Claire Goulvestre, PharmD, PhD2, Michel Wolff, MD4, Thomas Papo, MD5, François Vrtovsnik, MD, PhD6, Isabelle Klein, MD, PhD7, Bernard Iung, MD8, Xavier Duval, MD, PhD3,9,10 and the IMAGE Study Group
1
Department of Internal Medicine, Hospital Saint-Louis, Assistance Publique-Hôpitaux de
Paris, Université Paris 7–Diderot; 2Department of Immunology, Hospital Cochin, Assistance Publique-Hôpitaux de Paris, Université Paris 5–René Descartes; 3Center for Clinical Investigation, Hospital Bichat; 4Intensive Care Unit, 5Department of Internal Medicine, 6 9
Department of Nephrology, 7Department of Radiology, 8Department of Cardiology,
Department of Infectious Diseases, Hospital Bichat-Claude Bichat, Assistance Publique-
Hôpitaux de Paris, Université Paris 7–Diderot; 10INSERM U738, CIC 007 Paris, France
Key words: Infective endocarditis, anti-neutrophil cytoplasmic antibodies, rheumatoid factor, autoantibodies, seroprevalence
Running title: ANCA in infective endocarditis.
Word count (introduction through references): 2,230 words
Financial support: The cohort was funded by a grant from the French Ministry of Health (PHRC AOP 04076).
Corresponding author and address for reprints: Prof. Alfred Mahr, Department of Internal Medicine, Hospital Saint-Louis, Assistance Publique-Hôpitaux de Paris, 1, avenue Claude-Vellefaux, 75475 Paris Cedex 10, France, Phone: +33 1 42499780, Fax: +33 1 424997, E-mail: [email protected]
This article has been accepted for publication and undergone full peer review but has not been through the copyediting, typesetting, pagination and proofreading process which may lead to differences between this version and the Version of Record. Please cite this article as an ‘Accepted Article’, doi: 10.1002/art.38389 © 2014 American College of Rheumatology Received: Jul 17, 2013; Revised: Dec 27, 2013; Accepted: Jan 28, 2014
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ABSTRACT Objectives: Infective endocarditis (IE) is a primary systemic vasculitis mimic and was sporadically reported to produce anti-neutrophil cytoplasmic antibodies (ANCA). Because the frequency of ANCAs in IE is unknown, we assessed the seroprevalence of ANCAs in a large number of cases with IE. Methods: The study was conducted in the framework of a single-center prospective cohort study of incident IE cases. Demographic, clinical, laboratory and microbiological data were collected and MRI of the brain was performed at diagnosis. Among the patients for whom sera had been stored at diagnosis, ANCA were searched by indirect immunofluorescence assay using ethanol-, formalin- and methanol-fixed neutrophils, and anti-proteinase 3 (PR3) and anti-myeloperoxidase (MPO) ELISA. Rheumatoid factor (RF), antinuclear antibodies (ANA), anticardiolipin antibodies (aCL) and serum immunoglobulin (Ig) levels were also measured. Comparisons used Wilcoxon rank-sum and chi-square or Fisher’s exact tests. Results: Among 109 IE cases, 18% had cytoplasmic and/or perinuclear ANCA (C-/P-ANCA) and 8% had PR3-ANCA or MPO-ANCA, some with very high titers. Positivity for both C-/PANCA and PR3-ANCA or MPO-ANCA were found in 6% and RF, ANA and aCL were detected in 35%, 16% and 23% of samples. No consistent clinical IE pattern was observed for the anti-PR3/anti-MPO–positive IE cases whereas C-/P-ANCA were associated with younger age (P = 0.022), more common vegetations (P = 0.043) and above-normal serum IgG levels (P = 0.017). Conclusions: ANCA, including PR3- and MPO-ANCA, occur in a substantial proportion of IE cases. The link between C-/P-ANCA and IE features requires further study. (247 words)
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INTRODUCTION Anti-neutrophil cytoplasmic antibodies (ANCA) target various constituents of human neutrophils. Indirect immunofluorescence assays can distinguish ANCA with cytoplasmic (CANCA) or perinuclear (P-ANCA) staining patterns; the specific self-reactivities can be further characterized by ELISA. ANCA are a major biomarker of ANCA-associated vasculitides (1), up to 95% showing positive serology results depending on the vasculitis type (2). Specifically, ANCA directed against proteinase 3 (PR3-ANCA) and myeloperoxidase (MPO-ANCA) are highly predictive of ANCA-associated vasculitis, although MPO-ANCA are also found in other autoimmune diseases and with infection or drug exposure (2-4). Sporadic case reports have described PR3- or MPO-ANCA in infective endocarditis (IE) (2, 3, 5-7). The production of non–organ-specific autoantibodies in IE is well established for rheumatoid factor (RF) and was suggested by single case series for antinuclear (ANA), anticardiolipin (aCL) and other antiphosphospholipid antibodies (see online supplementary Table 1). The detection of ANCA in IE poses a particular clinical dilemma because the protean presentations of IE with possible joint symptoms, skin vasculitis and nephritis can mimic an ANCA-associated vasculitis. Therefore, a positive ANCA test may result in the initiation of immunosuppression that would be detrimental to patients with IE (7). This study was undertaken to assess the frequency of ANCA in IE. In addition, we investigated the frequencies of other autoantibodies and the association of ANCA and IE manifestations and serum immunoglobulin (Ig) levels.
PATIENTS AND METHODS We included patients with possible or definite IE based on the modified Duke criteria (8) who were enrolled since 2005 in an ongoing single-center inception cohort (ClinicalTrials.gov number: NCT00144885); the present study is based on patients enrolled in the first phase of
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the cohort study (2005–2008). Comprehensive information for all patients on the principal demographic, clinical, laboratory and microbiological information was collected by use of a standardized case report form. The protocol also included MRI of the brain at study inclusion to identify IE-related cerebrovascular complications. At minimum, diffusion and T2*susceptibility sequences were performed on a 1.5 Tesla scanner to identify acute ischemic lesions or microbleeds, respectively. A radiologist blinded to clinical data reviewed all MRI images; the results of imaging studies were previously reported for part of the cohort (9). After obtaining written informed consent from patients, serum samples were taken on study inclusion and stored at –80°C. Serological tests were performed in a central laboratory for the patients for whom stored serum was available in April 2012. ANCA were detected by immunofluorescence assay involving ethanol-, formalin- and methanol-fixed neutrophils (IFI Granulocyte Mosaic2 kit and methanol-fixed granulocyotes, Euroimmun, Germany) and PR3- and MPO-ANCA were detected by use of a commercially available
ELISA
(anti-PR3-hn-hr
[IgG]
and
anti-MPO
[IgG]
kits,
Euroimmun).
Immunofluorescence test results were classified as positive according to a threshold of 1:20 (seen on the ethanol-fixed slides) and were further categorized into weak (1+), strong (2+) and very strong (3+) based on the intensity of immunofluorescence staining. C-ANCA were defined as those displaying cytoplasmic staining on all 3 substrates. P-ANCA were defined by a perinuclear staining pattern on ethanol, which switched to a cytoplasmic pattern and became negative on formalin and methanol fixation, respectively. In compliance with the different definitions given to them, atypical ANCA were considered samples with a positive perinuclear pattern with ethanol or methanol used as fixative but negative staining on formalin-fixed substrates (10) or sera with dual C- and P-ANCA staining (4). Two observers with experience in the interpretation of ANCA immunofluorescence staining results evaluated
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all immunofluorescence tests independently; a third observer was used to resolve disagreements. We also determined positivity for IgM rheumatoid factor (RF), by use of an in-house ELISA assay (11); ANA (IFI HEp-20-10 kit, Euroimmun); and aCL IgG and IgM isotypes (Cardio Lisa kit, Biomedical Diagnostics, France). Samples positive for ANA, defined by titers ≥ 1:160, were assessed for double-stranded anti-DNA antibodies (ETI-dsDNA kit, DiaSorin, Italy) and for reactivity against the extractable nuclear antigens anti-CENP, antiJo1, anti-U1-RNP, anti-Scl70, anti-Sm, anti-SSA and anti-SSB (Varelisa ANA 8 screen, Phadia, Germany). The serum concentrations of IgG, IgA and IgM were quantified by nephelometry (BN ProSpec, Dade Behring, Germany). Serology results are expressed as number (percentage) of patients with positive tests. Circulating IgG, IgA and IgM levels are described as categorical variables, with 15.0, 3.75 and 2.20 g/l, respectively, as maximum cut-off values. Wilcoxon rank-sum tests and chisquare or Fisher’s exact test were used for comparing continuous and categorical variables, respectively. The main comparative analyses investigated the distribution of pre-selected IE characteristics and percentage of subjects with above-normal IgG levels by ANCA status. Two-sided P < 0.05 was considered statistically significant.
RESULTS Among the 136 patients included in the cohort during 2005–2008, sera were available for 109. The main characteristics of these patients, 89 (82%) showing definite and 20 (18%) possible IE, are in Table 1. One patient had a history of combined autoimmune thyroiditis and hepatitis. The pathogens causing IE were Staphylococcus aureus for 33 patients (30%), Streptococcus viridans or S. bovis for 33 (30%), enterococci for 7 (6%) and coagulasenegative Staphylococci for 4 (4%); other microorganisms and ≥ 2 microorganisms were
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identified in 9 patients (8%) and 2 (2%), respectively, and 21 patients (19%) were culturenegative. Cytoplasmic and/or perinuclear ANCA (C-/P-ANCA) were detected in 20 (18%) of the 109 analyzed patients. C-ANCA were found in 14 patients (13%) and P-ANCA in 6 patients (6%) (Table 1) with weak (1+), strong (2+) and very strong (3+) positivity in 10 (50%), 7 (35%) and 3 (15%) cases, respectively. PR3-ANCA and MPO-ANCA were detected in 4 patients each (4% each), some with very high titers (Tables 1 and 2). Samples for 6 patients (6%) were positive on both immunofluorescence assay and ELISA with 3 C-/PR3-, 1 P/MPO- and 2 C-/MPO-ANCA patterns (Table 2). RF, ANA and aCL were detected in 38 (35%), 17 (16%) and 25 (23%) patients, respectively (Table 1). Overall, 63 (58%) and 30 (28%) patients showed ≥ 1 or ≥ 2 of the 4 tested autoantibodies, respectively. In addition to samples found with true C-/P-ANCA, atypical ANCA were found in 5 (5%) cases; none with positive ELISA results for PR3- or MPO-ANCA. The 8 patients with positive PR3- or MPO-ANCA showed no consistent clinical pattern (Table 2). Patients positive for C-/P-ANCA indicated that ANCA-positive cases were younger at diagnosis (P = 0.022) and more frequently had IE fulfilling the modified Duke criteria (P = 0.021) and echocardiography-documented vegetations (P = 0.043) than ANCAnegative patients. None of the other autoantibodies (RF, ANA, aCL) were associated with any of these 3 variables, except for increased IE fulfilling the Duke criteria in cases positive for RF (92% vs. 76%, P = 0.039). C-/P-ANCA positivity was associated with elevated serum IgG levels (P = 0.017) (Table 1 and online supplementary Figure 1). Elevated IgG levels were also found for cases positive for RF (P = 0.003) and aCL (P = 0.006) but not ANA (P = 0.335) (see online supplementary Figure 1). Elevated IgG levels were more common in patients with than without IE fulfilling
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the modified Duke criteria (42% vs. 15% [P = 0.026]) and with than without echocardiographic vegetations (44% vs. 29% [P = 0.018]).
DISCUSSION The findings of this first assessment of the seroprevalence of ANCA in IE suggest that IE is an important cause of ANCA, especially when considering that the incidence of IE in the general population is several-fold higher than that of ANCA-associated vasculitis (12, 13). Among our 109 subjects with IE, 6% showed combined patterns of positivity, including C/PR3-, P-/MPO- and C-/MPO-ANCA, tightly linked with ANCA-associated vasculitis (2-4). An
additional
2% were
positive
for
PR3-
or
MPO-ANCA
but
negative
on
immunofluorescence staining. This pattern has also been linked with ANCA-associated vasculitis (2, 4), although remaining undetected in many routine settings in which ELISA is performed only after positive immunofluorescence screening. The large proportion of patients positive for C-/P-ANCA but not PR3- or MPO-ANCA underlines the poor specificity of this ANCA pattern. As well, the finding that more than half of patients tested positive for at least 1 among ANCA, RF, ANA and aCL confirms the high frequency of IE-associated autoantibody production. The message conveyed by the observation of ANCA synthesis in IE is not easy to decipher. The lack of any clear association between IE characteristics and PR3- or MPOANCA positivity, that is, ANCA types best predicting vasculitis, argues against a pathogenic contribution of ANCA to particular manifestations in IE. Because C-/P-ANCA–positive patients more commonly had echocardiography-documented vegetations than negative patients, ANCA immunogenesis in IE could reflect antigenic stimulation by neutrophilic enzymes released within vegetations. In fact, immuno-biochemical analyses revealed high levels of MPO and elastase within vegetations (14). In such a scenario of prominent
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vegetations representing the cornerstone of ANCA production, an increase in the occurrence of systemic emboli would be expected in C-/P-ANCA–positive IE because the size of vegetations has been shown to predict the occurrence of systemic emboli (15). In our study, C/P-ANCA positive IE indeed was associated although not significantly with ischemic lesions seen on MRI. Alternatively, our finding of higher serum IgG concentrations in positive compared to negative C-/P-ANCA patients, as well as in those positive for RF or aCL, lend support to a theory of autoantibodies becoming detectable in IE in response to a non-specific hyperimmune humoral response (16). ANCA detection has been reported for other prolonged infections such as HIV infection, chronic hepatitis, tuberculosis, leprosy, malaria and invasive amoebiasis (2). Hence, the relationship between ANCA and IE with echocardiographic vegetations might reflect that these cases had a more pronounced inflammatory response and IE cases with than without echocardiographic vegetations indeed more frequently had increased IgG concentrations. The limitations of this study include the possibility that some ANCA tests were positive before the onset of IE, but only 1 patient had a known autoimmune disease and ANCA are not commonly found in healthy individuals (2, 3). No follow-up sera were tested to verify single observations suggesting the disappearance of ANCA with successful IE therapy (6). The prevalence of autoantibodies might have been inflated by preferential recruitment of IE forms with prominent inflammatory responses in tertiary referral centers. Thus, the results for RF, ANA and aCL did not substantially deviate from figures reported by others (see online supplementary Table 1). Finally, we cannot derive from the available data what subset of the analyzed IE cases presented clinical symptoms that truly simulated an ANCA-associated vasculitis and for which a positive ANCA test might have misled the clinicians’ diagnostic work-up.
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To conclude, our findings urge caution in interpreting ANCA positivity in clinical settings reconcilable with a diagnosis of IE. In those instances, IE should be ruled out by echocardiography and blood cultures. Whether ANCA are a reflection of antigenic stimulation
in
large
vegetations,
mere
bystanders
of
transient
polyclonal
hypergammaglobulinemia or the cause or effect of a phenomenon not identified by this study requires further study.
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Jennette JC, Falk RJ, Bacon PA, Basu N, Cid MC, Ferrario F, et al. 2012 revised international chapel hill consensus conference nomenclature of vasculitides. Arthritis Rheum 2013;65:1-11.
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Bosch X, Guilabert A, Font J. Antineutrophil cytoplasmic antibodies. Lancet 2006;368:404-18.
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Hoffman GS, Specks U. Antineutrophil cytoplasmic antibodies. Arthritis Rheum 1998;41:1521-37.
4.
Savige J, Dimech W, Fritzler M, Goeken J, Hagen EC, Jennette JC, et al. Addendum to the International Consensus Statement on testing and reporting of antineutrophil cytoplasmic antibodies. Quality control guidelines, comments, and recommendations for testing in other autoimmune diseases. Am J Clin Pathol 2003;120:312-8.
5.
Choi HK, Lamprecht P, Niles JL, Gross WL, Merkel PA. Subacute bacterial endocarditis with positive cytoplasmic antineutrophil cytoplasmic antibodies and anti-proteinase 3 antibodies. Arthritis Rheum 2000;43:226-31.
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Chirinos JA, Corrales-Medina VF, Garcia S, Lichtstein DM, Bisno AL, Chakko S. Endocarditis associated with antineutrophil cytoplasmic antibodies: a case report and review of the literature. Clin Rheumatol 2007;26:590-5.
7.
Robert SC, Forbes SH, Soleimanian S, Hadley JS. Complements do not lie. BMJ Case Rep 2011;2011.
8.
Li JS, Sexton DJ, Mick N, Nettles R, Fowler VG, Jr., Ryan T, et al. Proposed modifications to the Duke criteria for the diagnosis of infective endocarditis. Clin Infect Dis 2000;30:633-8.
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Duval X, Iung B, Klein I, Brochet E, Thabut G, Arnoult F, et al. Effect of early cerebral magnetic resonance imaging on clinical decisions in infective endocarditis: a prospective study. Ann Intern Med 2010;152:497-504, W175.
10. Savige J, Gillis D, Benson E, Davies D, Esnault V, Falk RJ, et al. International Consensus Statement on Testing and Reporting of Antineutrophil Cytoplasmic Antibodies (ANCA). Am J Clin Pathol 1999;111:507-13. 11. Faith A, Pontesilli O, Unger A, Panayi GS, Johns P. ELISA assays for IgM and IgG rheumatoid factors. J Immunol Methods 1982;55:169-77. 12. Bor DH, Woolhandler S, Nardin R, Brusch J, Himmelstein DU. Infective endocarditis in the u.s., 1998-2009: a nationwide study. PLoS One 2013;8:e60033. 13. Gibelin A, Maldini C, Mahr A. Epidemiology and etiology of wegener granulomatosis, microscopic
polyangiitis,
churg-strauss
syndrome
and
goodpasture
syndrome:
vasculitides with frequent lung involvement. Semin Respir Crit Care Med 2011;32:26473. 14. Al-Salih G, Al-Attar N, Delbosc S, Louedec L, Corvazier E, Loyau S, et al. Role of vegetation-associated protease activity in valve destruction in human infective endocarditis. PLoS One 2012;7:e45695. 15. Vilacosta I, Graupner C, San Roman JA, Sarria C, Ronderos R, Fernandez C, et al. Risk of embolization after institution of antibiotic therapy for infective endocarditis. J Am Coll Cardiol 2002;39:1489-95. 16. Bacon PA, Davidson C, Smith B. Antibodies to candida and autoantibodies in sub-acute bacterial endocarditis. Q J Med 1974;43:537-50.
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Table 1. Baseline characteristics and results of testing for antineutrophil cytoplasmic antibodies (ANCA) and other autoantibodies and measurement of serum immunoglobulin (Ig) levels in 109 patients with newly diagnosed infective endocarditis (IE) and by presence or not of cytoplasmic and/or perinuclear ANCA (C- and/or P-ANCA). Variables*
All patients (n=109)
Patient characteristics Age, years, mean (SD) Male gender Previous IE [108] Injection drug use [108] Interval between first symptoms and IE diagnosis ≥ 1 month [107] Fever ≥ 38°C C-reactive protein level, mg/l, mean (SD) [99] Antibiotic administration–serum sampling interval, days, mean (SD) [103] IE characteristics Definite IE according to modified Duke criteria§ Right-sided IE Prosthetic valve Microorganisms Staphylococcus aureus Streptococcus viridians or S. bovis Other or multiple microorganisms or culture-negative Echocardiography-documented vegetations Length of vegetations, mm, mean (SD) [106] Intracardiac abscess Valvular regurgitation [107] Vascular and immunological features [107] Immunological phenomena** Vascular phenomena† Serum creatinine level, µmol/l, mean (SD) [107] Cerebral MRI findings [103] Microbleeds
C- and/or P-ANCA Positive Negative (n=20) (n=89)
P value
57.5 (15.4) 82 (75) 14 (13) 13 (12) 40 (37) 84 (77) 146.0 (100.7) 7.2 (7.9)
50.1 (13.9) 17 (85) 2 (10) 4 (21) 5 (26) 14 (70) 138.5 (88.6) 6.6 (6.8)
59.2 (15.3) 65 (73) 12 (14) 9 (10) 35 (40) 70 (79) 147.5 (103.5) 7.4 (8.1)
0.022 0.391 1.0 0.239 0.272 0.393 0.922 0.794
89 (82) 9 (8) 33 (30)
20 (100) 4 (20) 5 (25)
69 (78) 5 (6) 28 (31)
0.021 0.057 0.570
33 (30) 33 (30) 43 (39) 78 (72) 9.2 (8.4) 14 (13) 84 (79)
8 (40) 2 (10) 10 (50) 18 (90) 12.6 (10.0) 0 (0) 15(79)
25 (28) 31 (35) 33 (37) 60 (67) 8.5 (7.8) 14 (16) 69 (78)
0.043 0.250 0.069 1.00
11 (10) 30 (28) 168.3 (347.4)
2 (11) 8 (42) 303.6 (740.7)
9 (10) 22 (25) 137.2 (149.4)
1.00 0.132 0.250
59 (57)
10 (56)
49 (58)
0.871
0.092
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ANCA in infective endocarditis (R1 – Clean copy) All patients (n=109)
C- and/or P-ANCA Positive Negative (n=20) (n=89) 14 (78) 45 (53)
P value
Ischemic lesions 59 (57) 0.053 Outcome Surgical treatment (valve repair or replacement) [103] 48 (47) 10 (56) 38 (45) 0.402 In-hospital death 11 (10) 2 (10) 9 (10.0) 1.00 Post-hospitalization death at month 6 of diagnosis [93] 3 (3) 1 (6) 2 (3) 0.436 Autoantibodies ANCA (by indirect immunofluorescence assays) 20 (18) NR NR – C-ANCA 14 (13) NR NR – P-ANCA 6 (6) NR NR – Atypical ANCA (by indirect immunofluorescence assays) 5 (5) NR NR – PR3-ANCA or MPO-ANCA (by ELISA) 8 (7) ND ND – PR3-ANCA 4 (4) ND ND – MPO-ANCA 4 (4) ND ND – Rheumatoid factor 38 (35) 11 (55) 27 (30) 0.037 Anti-nuclear antibodies 17 (16) 0.733 2 (10) 15 (17) ‡ Double-stranded anti-DNA 0 NR NR – Anti-ENA‡ 2 (2) ND ND – Anticardiolipin antibodies (IgG and/or IgM) 25 (23) 6 (30) 19 (21) 0.393 IgG 20 (18) 5 (25) 15 (17) 0.522 IgM 7 (6) 2 (10) 5 (6) 0.610 Circulating Ig levels IgG level ≥ 15.0 g/l 40 (37) 12 (60) 28(31) 0.017 IgA level ≥ 3.75 g/l 42 (39) 9 (45) 33 (37) 0.511 IgM level ≥ 2.2 g/l 19 (17) 6 (30) 13 (15) 0.112 Data are no. (%) unless indicated. NR: not relevant, ND: not done, PR3: anti-proteinase 3, MPO, anti-myeloperoxydase. *Values in square brackets indicate numbers of values missing in the denominator. **Immunologic phenomena (as defined by the modified Duke criteria): glomerulonephritis (n = 3; 3%), Osler nodes (n = 6; 6%), Roth’s spots (n =1; 1%), rheumatoid factor (n =1; 1%); the number of cases with reported rheumatoid factor does not include the test results specifically done in this study. † Vascular phenomena (as defined by the modified Duke criteria) included major arterial emboli (n = 15; 14%), septic pulmonary infarct (n = 8; 7%), mycotic aneurysm (n = 1; 1%), intracranial hemorrhage (n = 3, 2.8%), conjunctival hemorrhage (n = 2; 2%), Janeway lesions (n = 1; 1%). ‡ Tested only for ANA-positive cases. § Reference (8).
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Table 2. Main characteristics of 8 patients with IE and sera positivity for PR3-ANCA or MPO-ANCA.
Variable Patient characteristics Age (years)/gender Injection drug use Interval between first symptoms and IE diagnosis ≥ 1 month Antibiotic administration-serum sampling interval, days Significant history
IE characteristics Definite IE according to modified Duke criteria* Right-sided IE Prosthetic valve Microorganism Vegetations Length of vegetations, mm Intracardiac abscess Vascular and immunological features Immunological phenomena** Vascular phenomena** Serum creatinine level, µmol/l Cerebral MRI findings Microbleeds Ischemic lesions Outcome Surgical treatment (valve repair or replacement) In-hospital death
Patients 1
2
3
4
5
6
7
8
51/male No No
52/male No Yes
65/female No Yes
64/male No No
64/female No No
36/female NA No
67/female No Yes
44/male No Yes
3
0
1
8
3
NA
4
20
Autoimmune thyroiditis and hepatitis
Previous IE (Streptococcus bovis)
Previous IE (enterococci)
Yes†
Yes
Yes
Yes
Yes
Yes
Yes
Yes
No No None identified
No Yes None identified
No No S. bovis
No No B. quintana
Yes 6
No No
Yes 7 No Yes
No
Yes 45 No No
No No Haemophilus aphrophilus Yes 13 Yes
No Yes Enterococci
No
No No Bartonella quintana Yes 11 No No
Yes 8 No Yes
No No Streptococcus viridans Yes 15 No No
No No 141
No No 75
No No 103
No No 86
No No 123
NA NA 78
No No 130
Yes Yes 97
No No
Yes Yes
Yes Yes
No No
No Yes
NA NA
Yes Yes
Yes Yes
Yes No
No No
Yes No
No No
Yes No
No No
No No
Yes No
–
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Variable
Patients
Death at month 6 after diagnosis Autoantibodies and serum Ig levels ANCA by IIF (normal < 1:20)# ANCA by ELISA (normal < 20 UR/ml) Rheumatoid factor (normal < 10 UA/ml) Other autoantibodies
1
2
3
4
5
6
7
8
No
No
No
No
No
NA
No
No
P-ANCA (3+) MPO-ANCA (200 UR/ml) Absent
C-ANCA (1+) MPO-ANCA (21 UR/ml) Absent
C-ANCA (2+) PR3-ANCA (111 UR/ml) Present (130 UA/ml) ANA (titer 1/160)
Absent MPO-ANCA (24 UR/ml) Absent
C-ANCA (3+) PR3-ANCA (200 UR/ml) Present (92 UA/ml)
C-ANCA (2+) MPO-ANCA (93 UR/ml) Present (10 UA/ml) aCL (IgG 59 UPL, IgM 45 UPL; normal < 22 UPL) 12.1 g/l
C-ANCA (2+) PR3-ANCA (49 UR/ml) Present (37 UA/ml)
Absent PR3-ANCA (20 UR/ml) Present (121 UA/ml)
18.6 g/l
28.5 g/l
Circulating IgG level 9.77 g/l 18.7 g/l 51.7 g/l 9.64 g/l 31.5 g/l (normal < 15.0 g/l) aCL: anticardiolipin antibodies, ANA: antinuclear antibodies, Ig: immunoglobulin, IIF: indirect immunofluorescence, NA: information not available. *Reference (8). **Defined by the modified Duke criteria.
This patient had histologically-proven endocarditis. Positive
results
were
categorized
as
weak
(1+),
strong
(2+)
and
very
strong
(3+)
immunofluorescence
staining
intensity.S
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Brief Report
PREVALENCE OF ANTI-NEUTROPHIL CYTOPLASMIC ANTIBODIES IN INFECTIVE ENDOCARDITIS
Alfred Mahr, MD, PhD1, Frédéric Batteux, MD, PhD2, Sarah Tubiana, PharmD3, Claire Goulvestre, PharmD, PhD2, Michel Wolff, MD4, Thomas Papo, MD5, François Vrtovsnik, MD, PhD6, Isabelle Klein, MD, PhD7, Bernard Iung, MD8, Xavier Duval, MD, PhD3,9,10 and the IMAGE Study Group
1
Department of Internal Medicine, Hospital Saint-Louis, Assistance Publique-Hôpitaux de
Paris, Université Paris 7–Diderot; 2Department of Immunology, Hospital Cochin, Assistance Publique-Hôpitaux de Paris, Université Paris 5–René Descartes; 3Center for Clinical Investigation, Hospital Bichat; 4Intensive Care Unit, 5Department of Internal Medicine, 6 9
Department of Nephrology, 7Department of Radiology, 8Department of Cardiology,
Department of Infectious Diseases, Hospital Bichat-Claude Bichat, Assistance Publique-
Hôpitaux de Paris, Université Paris 7–Diderot; 10INSERM U738, CIC 007 Paris, France
Key words: Infective endocarditis, anti-neutrophil cytoplasmic antibodies, rheumatoid factor, autoantibodies, seroprevalence
Running title: ANCA in infective endocarditis.
Word count (introduction through references): 2,230 words
Financial support: The cohort was funded by a grant from the French Ministry of Health (PHRC AOP 04076).
Corresponding author and address for reprints: Prof. Alfred Mahr, Department of Internal Medicine, Hospital Saint-Louis, Assistance Publique-Hôpitaux de Paris, 1, avenue Claude-Vellefaux, 75475 Paris Cedex 10, France, Phone: +33 1 42499780, Fax: +33 1 424997, E-mail: [email protected]
This article has been accepted for publication and undergone full peer review but has not been through the copyediting, typesetting, pagination and proofreading process which may lead to differences between this version and the Version of Record. Please cite this article as an ‘Accepted Article’, doi: 10.1002/art.38389 © 2014 American College of Rheumatology Received: Jul 17, 2013; Revised: Dec 27, 2013; Accepted: Jan 28, 2014
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ABSTRACT Objectives: Infective endocarditis (IE) is a primary systemic vasculitis mimic and was sporadically reported to produce anti-neutrophil cytoplasmic antibodies (ANCA). Because the frequency of ANCAs in IE is unknown, we assessed the seroprevalence of ANCAs in a large number of cases with IE. Methods: The study was conducted in the framework of a single-center prospective cohort study of incident IE cases. Demographic, clinical, laboratory and microbiological data were collected and MRI of the brain was performed at diagnosis. Among the patients for whom sera had been stored at diagnosis, ANCA were searched by indirect immunofluorescence assay using ethanol-, formalin- and methanol-fixed neutrophils, and anti-proteinase 3 (PR3) and anti-myeloperoxidase (MPO) ELISA. Rheumatoid factor (RF), antinuclear antibodies (ANA), anticardiolipin antibodies (aCL) and serum immunoglobulin (Ig) levels were also measured. Comparisons used Wilcoxon rank-sum and chi-square or Fisher’s exact tests. Results: Among 109 IE cases, 18% had cytoplasmic and/or perinuclear ANCA (C-/P-ANCA) and 8% had PR3-ANCA or MPO-ANCA, some with very high titers. Positivity for both C-/PANCA and PR3-ANCA or MPO-ANCA were found in 6% and RF, ANA and aCL were detected in 35%, 16% and 23% of samples. No consistent clinical IE pattern was observed for the anti-PR3/anti-MPO–positive IE cases whereas C-/P-ANCA were associated with younger age (P = 0.022), more common vegetations (P = 0.043) and above-normal serum IgG levels (P = 0.017). Conclusions: ANCA, including PR3- and MPO-ANCA, occur in a substantial proportion of IE cases. The link between C-/P-ANCA and IE features requires further study. (247 words)
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INTRODUCTION Anti-neutrophil cytoplasmic antibodies (ANCA) target various constituents of human neutrophils. Indirect immunofluorescence assays can distinguish ANCA with cytoplasmic (CANCA) or perinuclear (P-ANCA) staining patterns; the specific self-reactivities can be further characterized by ELISA. ANCA are a major biomarker of ANCA-associated vasculitides (1), up to 95% showing positive serology results depending on the vasculitis type (2). Specifically, ANCA directed against proteinase 3 (PR3-ANCA) and myeloperoxidase (MPO-ANCA) are highly predictive of ANCA-associated vasculitis, although MPO-ANCA are also found in other autoimmune diseases and with infection or drug exposure (2-4). Sporadic case reports have described PR3- or MPO-ANCA in infective endocarditis (IE) (2, 3, 5-7). The production of non–organ-specific autoantibodies in IE is well established for rheumatoid factor (RF) and was suggested by single case series for antinuclear (ANA), anticardiolipin (aCL) and other antiphosphospholipid antibodies (see online supplementary Table 1). The detection of ANCA in IE poses a particular clinical dilemma because the protean presentations of IE with possible joint symptoms, skin vasculitis and nephritis can mimic an ANCA-associated vasculitis. Therefore, a positive ANCA test may result in the initiation of immunosuppression that would be detrimental to patients with IE (7). This study was undertaken to assess the frequency of ANCA in IE. In addition, we investigated the frequencies of other autoantibodies and the association of ANCA and IE manifestations and serum immunoglobulin (Ig) levels.
PATIENTS AND METHODS We included patients with possible or definite IE based on the modified Duke criteria (8) who were enrolled since 2005 in an ongoing single-center inception cohort (ClinicalTrials.gov number: NCT00144885); the present study is based on patients enrolled in the first phase of
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the cohort study (2005–2008). Comprehensive information for all patients on the principal demographic, clinical, laboratory and microbiological information was collected by use of a standardized case report form. The protocol also included MRI of the brain at study inclusion to identify IE-related cerebrovascular complications. At minimum, diffusion and T2*susceptibility sequences were performed on a 1.5 Tesla scanner to identify acute ischemic lesions or microbleeds, respectively. A radiologist blinded to clinical data reviewed all MRI images; the results of imaging studies were previously reported for part of the cohort (9). After obtaining written informed consent from patients, serum samples were taken on study inclusion and stored at –80°C. Serological tests were performed in a central laboratory for the patients for whom stored serum was available in April 2012. ANCA were detected by immunofluorescence assay involving ethanol-, formalin- and methanol-fixed neutrophils (IFI Granulocyte Mosaic2 kit and methanol-fixed granulocyotes, Euroimmun, Germany) and PR3- and MPO-ANCA were detected by use of a commercially available
ELISA
(anti-PR3-hn-hr
[IgG]
and
anti-MPO
[IgG]
kits,
Euroimmun).
Immunofluorescence test results were classified as positive according to a threshold of 1:20 (seen on the ethanol-fixed slides) and were further categorized into weak (1+), strong (2+) and very strong (3+) based on the intensity of immunofluorescence staining. C-ANCA were defined as those displaying cytoplasmic staining on all 3 substrates. P-ANCA were defined by a perinuclear staining pattern on ethanol, which switched to a cytoplasmic pattern and became negative on formalin and methanol fixation, respectively. In compliance with the different definitions given to them, atypical ANCA were considered samples with a positive perinuclear pattern with ethanol or methanol used as fixative but negative staining on formalin-fixed substrates (10) or sera with dual C- and P-ANCA staining (4). Two observers with experience in the interpretation of ANCA immunofluorescence staining results evaluated
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all immunofluorescence tests independently; a third observer was used to resolve disagreements. We also determined positivity for IgM rheumatoid factor (RF), by use of an in-house ELISA assay (11); ANA (IFI HEp-20-10 kit, Euroimmun); and aCL IgG and IgM isotypes (Cardio Lisa kit, Biomedical Diagnostics, France). Samples positive for ANA, defined by titers ≥ 1:160, were assessed for double-stranded anti-DNA antibodies (ETI-dsDNA kit, DiaSorin, Italy) and for reactivity against the extractable nuclear antigens anti-CENP, antiJo1, anti-U1-RNP, anti-Scl70, anti-Sm, anti-SSA and anti-SSB (Varelisa ANA 8 screen, Phadia, Germany). The serum concentrations of IgG, IgA and IgM were quantified by nephelometry (BN ProSpec, Dade Behring, Germany). Serology results are expressed as number (percentage) of patients with positive tests. Circulating IgG, IgA and IgM levels are described as categorical variables, with 15.0, 3.75 and 2.20 g/l, respectively, as maximum cut-off values. Wilcoxon rank-sum tests and chisquare or Fisher’s exact test were used for comparing continuous and categorical variables, respectively. The main comparative analyses investigated the distribution of pre-selected IE characteristics and percentage of subjects with above-normal IgG levels by ANCA status. Two-sided P < 0.05 was considered statistically significant.
RESULTS Among the 136 patients included in the cohort during 2005–2008, sera were available for 109. The main characteristics of these patients, 89 (82%) showing definite and 20 (18%) possible IE, are in Table 1. One patient had a history of combined autoimmune thyroiditis and hepatitis. The pathogens causing IE were Staphylococcus aureus for 33 patients (30%), Streptococcus viridans or S. bovis for 33 (30%), enterococci for 7 (6%) and coagulasenegative Staphylococci for 4 (4%); other microorganisms and ≥ 2 microorganisms were
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identified in 9 patients (8%) and 2 (2%), respectively, and 21 patients (19%) were culturenegative. Cytoplasmic and/or perinuclear ANCA (C-/P-ANCA) were detected in 20 (18%) of the 109 analyzed patients. C-ANCA were found in 14 patients (13%) and P-ANCA in 6 patients (6%) (Table 1) with weak (1+), strong (2+) and very strong (3+) positivity in 10 (50%), 7 (35%) and 3 (15%) cases, respectively. PR3-ANCA and MPO-ANCA were detected in 4 patients each (4% each), some with very high titers (Tables 1 and 2). Samples for 6 patients (6%) were positive on both immunofluorescence assay and ELISA with 3 C-/PR3-, 1 P/MPO- and 2 C-/MPO-ANCA patterns (Table 2). RF, ANA and aCL were detected in 38 (35%), 17 (16%) and 25 (23%) patients, respectively (Table 1). Overall, 63 (58%) and 30 (28%) patients showed ≥ 1 or ≥ 2 of the 4 tested autoantibodies, respectively. In addition to samples found with true C-/P-ANCA, atypical ANCA were found in 5 (5%) cases; none with positive ELISA results for PR3- or MPO-ANCA. The 8 patients with positive PR3- or MPO-ANCA showed no consistent clinical pattern (Table 2). Patients positive for C-/P-ANCA indicated that ANCA-positive cases were younger at diagnosis (P = 0.022) and more frequently had IE fulfilling the modified Duke criteria (P = 0.021) and echocardiography-documented vegetations (P = 0.043) than ANCAnegative patients. None of the other autoantibodies (RF, ANA, aCL) were associated with any of these 3 variables, except for increased IE fulfilling the Duke criteria in cases positive for RF (92% vs. 76%, P = 0.039). C-/P-ANCA positivity was associated with elevated serum IgG levels (P = 0.017) (Table 1 and online supplementary Figure 1). Elevated IgG levels were also found for cases positive for RF (P = 0.003) and aCL (P = 0.006) but not ANA (P = 0.335) (see online supplementary Figure 1). Elevated IgG levels were more common in patients with than without IE fulfilling
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the modified Duke criteria (42% vs. 15% [P = 0.026]) and with than without echocardiographic vegetations (44% vs. 29% [P = 0.018]).
DISCUSSION The findings of this first assessment of the seroprevalence of ANCA in IE suggest that IE is an important cause of ANCA, especially when considering that the incidence of IE in the general population is several-fold higher than that of ANCA-associated vasculitis (12, 13). Among our 109 subjects with IE, 6% showed combined patterns of positivity, including C/PR3-, P-/MPO- and C-/MPO-ANCA, tightly linked with ANCA-associated vasculitis (2-4). An
additional
2% were
positive
for
PR3-
or
MPO-ANCA
but
negative
on
immunofluorescence staining. This pattern has also been linked with ANCA-associated vasculitis (2, 4), although remaining undetected in many routine settings in which ELISA is performed only after positive immunofluorescence screening. The large proportion of patients positive for C-/P-ANCA but not PR3- or MPO-ANCA underlines the poor specificity of this ANCA pattern. As well, the finding that more than half of patients tested positive for at least 1 among ANCA, RF, ANA and aCL confirms the high frequency of IE-associated autoantibody production. The message conveyed by the observation of ANCA synthesis in IE is not easy to decipher. The lack of any clear association between IE characteristics and PR3- or MPOANCA positivity, that is, ANCA types best predicting vasculitis, argues against a pathogenic contribution of ANCA to particular manifestations in IE. Because C-/P-ANCA–positive patients more commonly had echocardiography-documented vegetations than negative patients, ANCA immunogenesis in IE could reflect antigenic stimulation by neutrophilic enzymes released within vegetations. In fact, immuno-biochemical analyses revealed high levels of MPO and elastase within vegetations (14). In such a scenario of prominent
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vegetations representing the cornerstone of ANCA production, an increase in the occurrence of systemic emboli would be expected in C-/P-ANCA–positive IE because the size of vegetations has been shown to predict the occurrence of systemic emboli (15). In our study, C/P-ANCA positive IE indeed was associated although not significantly with ischemic lesions seen on MRI. Alternatively, our finding of higher serum IgG concentrations in positive compared to negative C-/P-ANCA patients, as well as in those positive for RF or aCL, lend support to a theory of autoantibodies becoming detectable in IE in response to a non-specific hyperimmune humoral response (16). ANCA detection has been reported for other prolonged infections such as HIV infection, chronic hepatitis, tuberculosis, leprosy, malaria and invasive amoebiasis (2). Hence, the relationship between ANCA and IE with echocardiographic vegetations might reflect that these cases had a more pronounced inflammatory response and IE cases with than without echocardiographic vegetations indeed more frequently had increased IgG concentrations. The limitations of this study include the possibility that some ANCA tests were positive before the onset of IE, but only 1 patient had a known autoimmune disease and ANCA are not commonly found in healthy individuals (2, 3). No follow-up sera were tested to verify single observations suggesting the disappearance of ANCA with successful IE therapy (6). The prevalence of autoantibodies might have been inflated by preferential recruitment of IE forms with prominent inflammatory responses in tertiary referral centers. Thus, the results for RF, ANA and aCL did not substantially deviate from figures reported by others (see online supplementary Table 1). Finally, we cannot derive from the available data what subset of the analyzed IE cases presented clinical symptoms that truly simulated an ANCA-associated vasculitis and for which a positive ANCA test might have misled the clinicians’ diagnostic work-up.
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To conclude, our findings urge caution in interpreting ANCA positivity in clinical settings reconcilable with a diagnosis of IE. In those instances, IE should be ruled out by echocardiography and blood cultures. Whether ANCA are a reflection of antigenic stimulation
in
large
vegetations,
mere
bystanders
of
transient
polyclonal
hypergammaglobulinemia or the cause or effect of a phenomenon not identified by this study requires further study.
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REFERENCES 1.
Jennette JC, Falk RJ, Bacon PA, Basu N, Cid MC, Ferrario F, et al. 2012 revised international chapel hill consensus conference nomenclature of vasculitides. Arthritis Rheum 2013;65:1-11.
2.
Bosch X, Guilabert A, Font J. Antineutrophil cytoplasmic antibodies. Lancet 2006;368:404-18.
3.
Hoffman GS, Specks U. Antineutrophil cytoplasmic antibodies. Arthritis Rheum 1998;41:1521-37.
4.
Savige J, Dimech W, Fritzler M, Goeken J, Hagen EC, Jennette JC, et al. Addendum to the International Consensus Statement on testing and reporting of antineutrophil cytoplasmic antibodies. Quality control guidelines, comments, and recommendations for testing in other autoimmune diseases. Am J Clin Pathol 2003;120:312-8.
5.
Choi HK, Lamprecht P, Niles JL, Gross WL, Merkel PA. Subacute bacterial endocarditis with positive cytoplasmic antineutrophil cytoplasmic antibodies and anti-proteinase 3 antibodies. Arthritis Rheum 2000;43:226-31.
6.
Chirinos JA, Corrales-Medina VF, Garcia S, Lichtstein DM, Bisno AL, Chakko S. Endocarditis associated with antineutrophil cytoplasmic antibodies: a case report and review of the literature. Clin Rheumatol 2007;26:590-5.
7.
Robert SC, Forbes SH, Soleimanian S, Hadley JS. Complements do not lie. BMJ Case Rep 2011;2011.
8.
Li JS, Sexton DJ, Mick N, Nettles R, Fowler VG, Jr., Ryan T, et al. Proposed modifications to the Duke criteria for the diagnosis of infective endocarditis. Clin Infect Dis 2000;30:633-8.
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Duval X, Iung B, Klein I, Brochet E, Thabut G, Arnoult F, et al. Effect of early cerebral magnetic resonance imaging on clinical decisions in infective endocarditis: a prospective study. Ann Intern Med 2010;152:497-504, W175.
10. Savige J, Gillis D, Benson E, Davies D, Esnault V, Falk RJ, et al. International Consensus Statement on Testing and Reporting of Antineutrophil Cytoplasmic Antibodies (ANCA). Am J Clin Pathol 1999;111:507-13. 11. Faith A, Pontesilli O, Unger A, Panayi GS, Johns P. ELISA assays for IgM and IgG rheumatoid factors. J Immunol Methods 1982;55:169-77. 12. Bor DH, Woolhandler S, Nardin R, Brusch J, Himmelstein DU. Infective endocarditis in the u.s., 1998-2009: a nationwide study. PLoS One 2013;8:e60033. 13. Gibelin A, Maldini C, Mahr A. Epidemiology and etiology of wegener granulomatosis, microscopic
polyangiitis,
churg-strauss
syndrome
and
goodpasture
syndrome:
vasculitides with frequent lung involvement. Semin Respir Crit Care Med 2011;32:26473. 14. Al-Salih G, Al-Attar N, Delbosc S, Louedec L, Corvazier E, Loyau S, et al. Role of vegetation-associated protease activity in valve destruction in human infective endocarditis. PLoS One 2012;7:e45695. 15. Vilacosta I, Graupner C, San Roman JA, Sarria C, Ronderos R, Fernandez C, et al. Risk of embolization after institution of antibiotic therapy for infective endocarditis. J Am Coll Cardiol 2002;39:1489-95. 16. Bacon PA, Davidson C, Smith B. Antibodies to candida and autoantibodies in sub-acute bacterial endocarditis. Q J Med 1974;43:537-50.
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Table 1. Baseline characteristics and results of testing for antineutrophil cytoplasmic antibodies (ANCA) and other autoantibodies and measurement of serum immunoglobulin (Ig) levels in 109 patients with newly diagnosed infective endocarditis (IE) and by presence or not of cytoplasmic and/or perinuclear ANCA (C- and/or P-ANCA). Variables*
All patients (n=109)
Patient characteristics Age, years, mean (SD) Male gender Previous IE [108] Injection drug use [108] Interval between first symptoms and IE diagnosis ≥ 1 month [107] Fever ≥ 38°C C-reactive protein level, mg/l, mean (SD) [99] Antibiotic administration–serum sampling interval, days, mean (SD) [103] IE characteristics Definite IE according to modified Duke criteria§ Right-sided IE Prosthetic valve Microorganisms Staphylococcus aureus Streptococcus viridians or S. bovis Other or multiple microorganisms or culture-negative Echocardiography-documented vegetations Length of vegetations, mm, mean (SD) [106] Intracardiac abscess Valvular regurgitation [107] Vascular and immunological features [107] Immunological phenomena** Vascular phenomena† Serum creatinine level, µmol/l, mean (SD) [107] Cerebral MRI findings [103] Microbleeds
C- and/or P-ANCA Positive Negative (n=20) (n=89)
P value
57.5 (15.4) 82 (75) 14 (13) 13 (12) 40 (37) 84 (77) 146.0 (100.7) 7.2 (7.9)
50.1 (13.9) 17 (85) 2 (10) 4 (21) 5 (26) 14 (70) 138.5 (88.6) 6.6 (6.8)
59.2 (15.3) 65 (73) 12 (14) 9 (10) 35 (40) 70 (79) 147.5 (103.5) 7.4 (8.1)
0.022 0.391 1.0 0.239 0.272 0.393 0.922 0.794
89 (82) 9 (8) 33 (30)
20 (100) 4 (20) 5 (25)
69 (78) 5 (6) 28 (31)
0.021 0.057 0.570
33 (30) 33 (30) 43 (39) 78 (72) 9.2 (8.4) 14 (13) 84 (79)
8 (40) 2 (10) 10 (50) 18 (90) 12.6 (10.0) 0 (0) 15(79)
25 (28) 31 (35) 33 (37) 60 (67) 8.5 (7.8) 14 (16) 69 (78)
0.043 0.250 0.069 1.00
11 (10) 30 (28) 168.3 (347.4)
2 (11) 8 (42) 303.6 (740.7)
9 (10) 22 (25) 137.2 (149.4)
1.00 0.132 0.250
59 (57)
10 (56)
49 (58)
0.871
0.092
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ANCA in infective endocarditis (R1 – Clean copy) All patients (n=109)
C- and/or P-ANCA Positive Negative (n=20) (n=89) 14 (78) 45 (53)
P value
Ischemic lesions 59 (57) 0.053 Outcome Surgical treatment (valve repair or replacement) [103] 48 (47) 10 (56) 38 (45) 0.402 In-hospital death 11 (10) 2 (10) 9 (10.0) 1.00 Post-hospitalization death at month 6 of diagnosis [93] 3 (3) 1 (6) 2 (3) 0.436 Autoantibodies ANCA (by indirect immunofluorescence assays) 20 (18) NR NR – C-ANCA 14 (13) NR NR – P-ANCA 6 (6) NR NR – Atypical ANCA (by indirect immunofluorescence assays) 5 (5) NR NR – PR3-ANCA or MPO-ANCA (by ELISA) 8 (7) ND ND – PR3-ANCA 4 (4) ND ND – MPO-ANCA 4 (4) ND ND – Rheumatoid factor 38 (35) 11 (55) 27 (30) 0.037 Anti-nuclear antibodies 17 (16) 0.733 2 (10) 15 (17) ‡ Double-stranded anti-DNA 0 NR NR – Anti-ENA‡ 2 (2) ND ND – Anticardiolipin antibodies (IgG and/or IgM) 25 (23) 6 (30) 19 (21) 0.393 IgG 20 (18) 5 (25) 15 (17) 0.522 IgM 7 (6) 2 (10) 5 (6) 0.610 Circulating Ig levels IgG level ≥ 15.0 g/l 40 (37) 12 (60) 28(31) 0.017 IgA level ≥ 3.75 g/l 42 (39) 9 (45) 33 (37) 0.511 IgM level ≥ 2.2 g/l 19 (17) 6 (30) 13 (15) 0.112 Data are no. (%) unless indicated. NR: not relevant, ND: not done, PR3: anti-proteinase 3, MPO, anti-myeloperoxydase. *Values in square brackets indicate numbers of values missing in the denominator. **Immunologic phenomena (as defined by the modified Duke criteria): glomerulonephritis (n = 3; 3%), Osler nodes (n = 6; 6%), Roth’s spots (n =1; 1%), rheumatoid factor (n =1; 1%); the number of cases with reported rheumatoid factor does not include the test results specifically done in this study. † Vascular phenomena (as defined by the modified Duke criteria) included major arterial emboli (n = 15; 14%), septic pulmonary infarct (n = 8; 7%), mycotic aneurysm (n = 1; 1%), intracranial hemorrhage (n = 3, 2.8%), conjunctival hemorrhage (n = 2; 2%), Janeway lesions (n = 1; 1%). ‡ Tested only for ANA-positive cases. § Reference (8).
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Table 2. Main characteristics of 8 patients with IE and sera positivity for PR3-ANCA or MPO-ANCA.
Variable Patient characteristics Age (years)/gender Injection drug use Interval between first symptoms and IE diagnosis ≥ 1 month Antibiotic administration-serum sampling interval, days Significant history
IE characteristics Definite IE according to modified Duke criteria* Right-sided IE Prosthetic valve Microorganism Vegetations Length of vegetations, mm Intracardiac abscess Vascular and immunological features Immunological phenomena** Vascular phenomena** Serum creatinine level, µmol/l Cerebral MRI findings Microbleeds Ischemic lesions Outcome Surgical treatment (valve repair or replacement) In-hospital death
Patients 1
2
3
4
5
6
7
8
51/male No No
52/male No Yes
65/female No Yes
64/male No No
64/female No No
36/female NA No
67/female No Yes
44/male No Yes
3
0
1
8
3
NA
4
20
Autoimmune thyroiditis and hepatitis
Previous IE (Streptococcus bovis)
Previous IE (enterococci)
Yes†
Yes
Yes
Yes
Yes
Yes
Yes
Yes
No No None identified
No Yes None identified
No No S. bovis
No No B. quintana
Yes 6
No No
Yes 7 No Yes
No
Yes 45 No No
No No Haemophilus aphrophilus Yes 13 Yes
No Yes Enterococci
No
No No Bartonella quintana Yes 11 No No
Yes 8 No Yes
No No Streptococcus viridans Yes 15 No No
No No 141
No No 75
No No 103
No No 86
No No 123
NA NA 78
No No 130
Yes Yes 97
No No
Yes Yes
Yes Yes
No No
No Yes
NA NA
Yes Yes
Yes Yes
Yes No
No No
Yes No
No No
Yes No
No No
No No
Yes No
–
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Variable
Patients
Death at month 6 after diagnosis Autoantibodies and serum Ig levels ANCA by IIF (normal < 1:20)# ANCA by ELISA (normal < 20 UR/ml) Rheumatoid factor (normal < 10 UA/ml) Other autoantibodies
1
2
3
4
5
6
7
8
No
No
No
No
No
NA
No
No
P-ANCA (3+) MPO-ANCA (200 UR/ml) Absent
C-ANCA (1+) MPO-ANCA (21 UR/ml) Absent
C-ANCA (2+) PR3-ANCA (111 UR/ml) Present (130 UA/ml) ANA (titer 1/160)
Absent MPO-ANCA (24 UR/ml) Absent
C-ANCA (3+) PR3-ANCA (200 UR/ml) Present (92 UA/ml)
C-ANCA (2+) MPO-ANCA (93 UR/ml) Present (10 UA/ml) aCL (IgG 59 UPL, IgM 45 UPL; normal < 22 UPL) 12.1 g/l
C-ANCA (2+) PR3-ANCA (49 UR/ml) Present (37 UA/ml)
Absent PR3-ANCA (20 UR/ml) Present (121 UA/ml)
18.6 g/l
28.5 g/l
Circulating IgG level 9.77 g/l 18.7 g/l 51.7 g/l 9.64 g/l 31.5 g/l (normal < 15.0 g/l) aCL: anticardiolipin antibodies, ANA: antinuclear antibodies, Ig: immunoglobulin, IIF: indirect immunofluorescence, NA: information not available. *Reference (8). **Defined by the modified Duke criteria.
This patient had histologically-proven endocarditis. Positive
results
were
categorized
as
weak
(1+),
strong
(2+)
and
very
strong
(3+)
immunofluorescence
staining
intensity.S
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