Low seroprevalence and poor specificity of antineutrophil cytoplasmic antibodies in tuberculosis

L. Teixeira, A. Mahr, F. Jaureguy1, L.-H. Noël4, H. Nunes2, A. Lefort3, S. Barry, P. Deny1 and L. Guillevin

Department of Internal Medicine, Unité de Recherche Clinique et Thérapeutique (EA 3409), 1 Department of Bacteriology–Virology, 2 Department of Pneumology, 3 Department of Infectious and Tropical Diseases, Hôpital Avicenne, AP–HP, Université Paris-Nord, Bobigny and 4 Department of Immunology, Hôpital Necker, AP-HP, Paris, France.

Correspondence to: A. Mahr, Department of Internal Medicine, Hôpital Cochin, 27, rue du Faubourg Saint-Jacques, 75679 Paris Cedex 14, France. E-mail: alfred.mahr{at}cch.ap-hop-paris.fr


    Abstract
 Top
 Abstract
 Introduction
 Patients and methods
 Results
 Discussion
 References
 
Objectives. Recently published findings suggested that antineutrophil cytoplasmic antibodies (ANCA), particularly those with a cytoplasmic (C-ANCA) labelling pattern and targeting proteinase 3 (anti-PR3), might be markers of tuberculosis (TB). This is a critical issue, because C-ANCA/anti-PR3 were considered to be a highly specific hallmark of Wegener's granulomatosis or microscopic polyangiitis and because TB may clinically mimic Wegener's granulomatosis. We therefore undertook a study with the aim of investigating further the prevalence and specificity of ANCA in TB.

Methods. We evaluated serum samples from 67 patients diagnosed with culture-proven TB and 10 previously untested control samples from patients known to be ANCA positive (four Wegener's granulomatosis and two microscopic polyangiitides) or negative. All 77 sera were screened for ANCA using commercially available indirect immunofluorescence (IIF) and enzyme-linked immunosorbent assay (ELISA) for anti-PR3 and antimyeloperoxidase (MPO). IIF-positive and anti-PR3- and anti-MPO-negative sera were also tested for bactericidal/permeability-increasing protein, lactoferrin, elastase and cathepsin G specificities with commercially available ELISA.

Results. IIF detected ANCA in seven (10%) of the TB sera, including three C-ANCA and four atypical perinuclear-labelling ANCA. Only one IIF-negative specimen was anti-PR3 positive in ELISA. ANCA testing of the control sera yielded IIF and ELISA results concordant with previous findings, except for one borderline ELISA.

Conclusion. Our results indicate that TB is associated with low ANCA seroprevalence and poor specificity, with no test serum showing combined C-ANCA/anti-PR3 activity. In a clinical setting of Wegener's granulomatosis/TB mimicry, such combined reactivity would seem to be more suggestive of Wegener's granulomatosis.

KEY WORDS: Antineutrophil cytoplasmic antibodies, Tuberculosis, Prevalence, Specificity


    Introduction
 Top
 Abstract
 Introduction
 Patients and methods
 Results
 Discussion
 References
 
Since they were first described [1], antineutrophil cytoplasmic antibodies (ANCA) have been closely linked to a subset of primary systemic vasculitides, namely Wegener's granulomatosis (WG), microscopic polyangiitis (MPA) and Churg–Strauss syndrome [2, 3]. For these so-called ANCA-associated vasculitides, ANCA detection has become an important diagnostic hallmark, with, in particular, those labelling the cytoplasm of neutrophils in indirect immunofluorescence (IIF) assays (C-ANCA) and targeting proteinase-3 (anti-PR3) in enzyme-linked immunosorbent assay (ELISA) being considered highly specific for WG or MPA [4, 5].

However, the expanded search for ANCA provided evidence that various subtypes of these autoantibodies were also being produced in patients with non-vasculitic diseases [2, 3]. Pertinently, a study recently published in this journal [6] reported an ANCA seroprevalence of 40% in tuberculosis (TB), with 33% of the patients displaying a C-ANCA/anti-PR3 pattern. This observation highlights a crucial issue in view of the potential phenotypic similarities of TB and WG, while emphasizing the poor ability of ANCA to distinguish between these two diseases. Therefore, this study was undertaken to investigate further the prevalence and specificities of ANCA in TB.


    Patients and methods
 Top
 Abstract
 Introduction
 Patients and methods
 Results
 Discussion
 References
 
Patients
This study was conducted in a university hospital, located in a northeastern suburb of Paris, which serves an urban and multiethnic population characterized by a high TB incidence [7].

From the computerized database of the Department of Bacteriology–Virology, we identified all the individuals registered with a Mycobacterium tuberculosis-positive culture during the calendar year 2002. For the 93 subjects responding to that criterion, we searched this department's serum bank for available samples that had been drawn within ±21 days of the time the body fluid or biopsy specimens had been taken that subsequently cultured M. tuberculosis. This procedure was possible because we routinely store (at –20°C) in this serum bank any surplus serum corresponding to all requests for hepatitis B (HBV), C (HCV) and human immunodeficiency virus (HIV) serological testing. We retained 67 serum samples fulfilling these criteria. The medical files of the selected patients were reviewed with respect to demographics, ethnic background, major TB-associated conditions and clinical presentation, therapy and outcome of TB.

To assess the reliability of this study's ANCA testing, we also included 10 control sera. These samples were retrieved from the serum bank of the Department of Internal Medicine that routinely stores sera (at –80°C) from patients with definite or suspected vasculitides. To be selected, these sera had to belong to patients who had had a previous ANCA serology tested under routine clinical conditions by the same laboratory performing the ANCA testing for the present study (see below). Only untested serum aliquots that had been obtained within ±3 days of the date of another sample that had been tested for ANCA were considered. Eventually, we chose four samples from previously C-ANCA/anti-PR3-positive WG patients, two sera from previously perinuclear-labelling (P)-ANCA/anti-MPO–positive MPA patients and four IIF-negative controls from patients diagnosed with ANCA-unrelated non-vasculitic chronic inflammatory diseases.

ANCA-determination methods
The 67 TB and 10 control sera were forwarded to an ANCA reference laboratory (Department of Immunology, Necker Hospital, Paris) subjected to bimonthly quality control, as defined by the United Kingdom National External Quality Assessment Schemes. The laboratory personnel were informed that these sera included a number of controls, but they were unaware of the respective numbers of TB samples and ANCA-positive and -negative controls.

For all sera, ANCA were sought using a commercially available IIF on a substrate of ethanol-fixed human neutrophils. A single observer (L.-H.N.) read all the slides with IIF-positive sera being categorized as: (1) P- (with irregular perinuclear-label clumping and some overflow into the nucleus), (2) atypical P- (with a fine continuous perinuclear ring and no overflow into the nucleus, which, like the cytoplasm, remained totally unlabelled) and (3) C-ANCA. Furthermore, anti-PR3 and anti-MPO antibody specificities of all samples were determined using monospecific commercial ELISA kits and judged positive above a cut-off value of ≥20 arbitrary units (AU)/ml. For patients with a positive IIF but negative PR3/MPO ELISA, potential target antigens—elastase, lactoferrin, bactericidal permeability-increasing (BPI) protein and cathepsin G—were further investigated by ELISA, with an optical density positivity threshold of ≥1.0. All assay kits were purchased from the same manufacturer (Euroimmun, Bioadvance, Emerainville, France) and were run according to the manufacturer's instructions.

Ethical aspects
In accordance with current French law, because no intervention was necessitated by this study, specific ethics committee approval and informed patient consent was not required.


    Results
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 Abstract
 Introduction
 Patients and methods
 Results
 Discussion
 References
 
Characteristics and ANCA determination for TB patients
The principal characteristics of the selected TB patients are summarized in Table 1. For two of the 67 patients, medical charts were inaccessible and clinical data retrieval was incomplete. Based on the remaining 65 individuals, six had extrapulmonary TB, including four lymphadenitides, one peritonitis and one pleuropericarditis. Six other patients had TB involving more than two of the following tissues/organs: the lungs, pleura, peritoneum, pericardium, lymph nodes, brain, spine, hip, liver and/or spleen. A prior episode of TB, which had preceded the current infection by 2–31 yr, was noted for six patients. None of the 65 patients presented clinical signs suggestive of an associated vasculitis. Except for one patient lost-to-follow-up, all had received antituberculous agents and were considered to have responded favourably. For 13 patients, the examined sera had been obtained after the initiation of TB therapy: nine patients had been treated for 1–7 days and four patients for 13–16 days (data missing for two patients).


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TABLE 1. Main characteristics of 67 patients with culture-proven TB

 
Results of ANCA serology for all 67 TB serum samples are reported in Table 2. The mean (±S.D.) time interval between the sampling of these sera and the material that cultured M. tuberculosis was 1.7 ± 5.4 days. IIF detected seven (10%) positive sera: three (4%) C-ANCA and four (6%) atypical P-ANCA. For two of the three C-ANCA-positive samples, immunofluorescence was described as being weak. Anti-PR3/anti-MPO ELISA were positive for a single (1%) patient (anti-PR3 >200 AU/ml), whose serum had been negative in the IIF assay. Screening by ELISA of the seven samples that were IIF positive and anti-PR3/anti-MPO negative for other potential specificities was negative. Among the seven IIF-positive samples, three were from patients with severe co-morbidities, i.e. two HIV infections (one concurrent with chronic HBV infection) and one ulcerative colitis. Moreover, two of the seven had started anti-TB therapy 2 or 13 days prior to serum sampling. For the single anti-PR3-positive sample, no severe associated disease or concurrent medication could be identified.


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TABLE 2. Breakdown of IIF–ELISA ANCA-testing results for 67 patients with culture-positive TB

 
ANCA determination in control samples
Concerning the six controls known to be ANCA positive, IIF was positive for all, with concordant labelling patterns (four C-ANCA, two P-ANCA). ELISA also yielded highly concordant results for five sera, with ANCA titres similar to those obtained previously (with paired values of 131/84, 121/130, 138/106, >200/>200 and 132/134 AU/ml). Conversely, for the remaining patient, whose previous sample had been considered to have borderline MPO specificity (25 AU/ml), the new ELISA was negative with an anti-MPO titre of 10 AU/ml. For the four formerly ANCA-negative controls, no ANCA were detected by IIF or anti-PR3/anti-MPO ELISA.


    Discussion
 Top
 Abstract
 Introduction
 Patients and methods
 Results
 Discussion
 References
 
Compared with the observations reported by Flores-Suarez et al. [6], our findings, based on 67 patients with culture-proven M. tuberculosis infections, seem to indicate that TB is associated with a low ANCA seroprevalence. Indeed, although 10% of our TB serum samples were IIF positive, ELISA specificity testing yielded only one PR3-positive serum and no combined IIF–ELISA positivity was detected.

The reasons for the opposing findings between our study and that from Mexico [6] are unclear. It is unlikely that geographical factors could have accounted for these discrepancies, because we also included patients with diverse geographical backgrounds and a scenario of an ANCA autoimmune response varying widely among ethnic groups or within different strains of a potentially antibody-triggering micro-organism remains unsubstantiated. The hypothesis of TB-associated diseases acting as confounders also seems improbable, given that our study contained an even higher proportion of subjects with potentially ANCA-inducing underlying conditions, e.g. HIV or HCV infections. As pointed out by Flores-Suarez et al. [6], an alternative explanation raises the possible effect of exposure to antituberculous agents, since the majority of sera they analysed were obtained under therapy. Indeed, ANCA have been associated with various drugs, e.g. hydralazine and propylthiouracil [8] but, to the best of our knowledge, not with anti-TB agents.

Although so far unconfirmed, the report by Flores-Suarez et al. [6] revives the question of the capacity of ANCA to specifically diagnose a vasculitis. It is widely accepted that the P-ANCA/anti-MPO pattern, which is most typically encountered in MPA and Churg–Strauss syndrome [2, 3], carries limited specificity because this ANCA type may also be detected in non-vasculitic autoimmune diseases [2, 3]. Conversely, based on a meta-analysis of earlier investigations, C-ANCA were estimated to be 98% specific for WG [4] and another study found combined C-ANCA/anti-PR3 positivity to be 99% specific for a group of systemic vasculitides mainly consisting of WG and MPA [5]. These figures may nonetheless be criticized as being overestimates, since the former was derived from many controls, including either healthy individuals or subjects with diseases that would not clinically be confounded with WG [4], and because the latter investigation used a high ELISA cut-off threshold that had been defined a posteriori to yield high specificity [5].

The vasculitis specificity of ANCA was further undermined by the multitude of observations of these autoantibodies being detectable during the course of bacterial [9–12], viral [13–18], fungal [19] or parasitic [20, 21] infections. Analysis of those publications showed that their infection-associated ANCA mainly had typical or atypical P-ANCA labelling [10, 12, 14–16, 18, 21] and reactivities against MPO [10, 15], BPI protein [16, 18], cathepsin G [16, 21], lactoferrin [14] or unidentified antigens [12, 14, 19]. But the supposedly more vasculitis-specific C-ANCA/anti-PR3 pattern has also been described in this setting of infection [9, 11, 13, 15, 17, 20], in particular in as many as 75% of patients with invasive hepatic amoebiasis [20], 56% of chronic hepatitis C infections [17] and up to 4% of HIV infections [14]. However, the clinical relevance of detecting ANCA during those infections might be tempered by the fact that many of the infections occur predominantly within restricted geographical areas, can be diagnosed by appropriate microbiological and serological techniques and have an absence of symptoms suggestive of systemic vasculitis.

In contrast, the issue of ANCA also being a marker of TB has major clinical implications. TB is a ubiquitous granulomatous disease commonly manifesting cavitated lung nodules. Consequently, distinguishing between TB and WG can be difficult at disease onset [22, 23], thereby conferring a potentially elementary differential diagnostic role onto ANCA testing. Supported by our results, those of smaller studies published as abstracts [24, 25] and the few available case reports describing ANCA in TB [26, 27], we postulate that the presence of C-ANCA/anti-PR3 or P-ANCA/anti-MPO would make a diagnosis of TB rather unlikely. As previously emphasized [5], our findings that three of the investigated TB sera containing exclusively C-ANCA reactivity and another one with high anti-PR3-ANCA positivity alone reinforces that the highest vasculitis specificity can be obtained only with combined IIF–ELISA positivity.

More than 20 yr after the initial description of ANCA, their diagnostic value is still uncertain. Many of the data accumulated to date would suggest that neither a P-ANCA/anti-MPO nor a C-ANCA/anti-PR3 pattern is able to ensure per se a diagnosis of ANCA-associated vasculitis and—except when confronted with a very high diagnostic pre-test probability—should not be used as a substitute for a histological confirmation [4, 28]. While accepting that ANCA cannot replace biopsy, further assessment of the ANCA specificity should focus on the careful choice of controls that may clinically and histologically mimic any ANCA-associated vasculitis and for which the initiation of immunosuppressive therapy would be particularly detrimental. Therefore, in addition to TB, bacterial endocarditis, leprosy and histoplasmosis might represent such differential diagnostic challenges, all the more since they have been reported to be potentially also associated with ANCA [9, 10, 12, 29, 30]. However, particularly for bacterial endocarditis, the ANCA seroprevalence has not yet been formally estimated. In light of the dramatic therapeutic consequences of misdiagnosing TB as an ANCA-associated vasculitis, further studies are warranted to confirm our findings of ANCA being uncommon in and poorly specific to TB.


    Acknowledgments
 
The authors are indebted to Dr M. Bentata (Bobigny) for help in retrieving clinical data, M. C. Tamby (Paris) who manages the serum bank of the Department of Internal Medicine, and Ms J. Jacobson for editorial assistance.

The authors have declared no conflicts of interest.


    References
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 Abstract
 Introduction
 Patients and methods
 Results
 Discussion
 References
 

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Submitted 6 June 2004; revised version accepted 5 September 2004.



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