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Clinical Biochemistry 41 (2008) 65 – 68
Immunological features of visceral leishmaniasis may mimic systemic lupus erythematosus Lazaros I. Sakkas a,⁎, Maria Boulbou a , Despina Kyriakou a , Irene Makri a , Chrysanthi Sinani a , Anastasios Germenis b , Nikolaos Stathakis a a
Department of Internal Medicine and Rheumatology, Thessaly University School of Medicine and University Hospital of Larisa, Larisa 41110, Greece Laboratory of Immunology and Histocompatibility, Thessaly University School of Medicine and University Hospital of Larisa, Larisa 41110, Greece
Received 2 August 2007; received in revised form 14 September 2007; accepted 11 October 2007 Available online 25 October 2007
Abstract Objective: Visceral leishmaniasis (VL), caused by the intracellular parasite Leishmania, can present with fever, splenomegaly, pancytopenias, hypergammaglobulinemia, and autoantibody production. These features may mimic systemic lupus erythematosus (SLE). The objective was to study features of VL that shared with and differed from SLE. Design and methods: A small retrospective study of six patients with VL diagnosed in a University Hospital between 2001 and 2007. Results: All patients had cytopenias, firm splenomegaly, high acute phase reactants, and activation of the coagulation cascade. Hypergammaglobulinemia was detected in five patients. Direct Coombs test was positive in all patients, anti-nuclear antibodies were detected in five patients, anti-smooth muscle antibodies (ASMA) in four patients, and IgM rheumatoid factor (RF) in four patients. Anti-dsDNA antibodies were detected in one patient and IgM anti-cardiolipin antibodies were detected in one patient. Conclusion: Autoantibodies are frequently detected in VL and may mimic SLE, but massive firm splenomegaly, very high acute phase reactants, and activation of coagulation system with high D-dimers point toward infection. © 2007 The Canadian Society of Clinical Chemists. Published by Elsevier Inc. All rights reserved. Keywords: Autoantibody; Coombs test; Cytopenia; Splenomegaly; Visceral leishmaniasis
Introduction Visceral leishmaniasis (VL) is an acute or chronic systemic disease caused by the protozoan Leishmania species, mainly Leishmania donovani. Leishmania species are obligate intracellular parasites that are transmitted to humans by sandflies as promastigotes. Promastigotes are phagocytozed by macrophages and are transformed into amastigotes that multiply and, after rupture of macrophages, infect other macrophages in the reticulo-endothelial system of liver, spleen, and bone marrow . The immune response to Leishmania includes T-cell and Bcell responses. T-cell immune responses can be divided into two polar types, TH1 and TH2, depending on the cytokines produced. TH1 cells produce the pro-inflammatory cytokines
⁎ Corresponding author. Thessaly University School of Medicine, 22 Papakyriazi street, Larisa 412 22, Greece. Fax: +30 2410 670243. E-mail address: [email protected]
interleukin (IL)-2, and interferon-γ (IFNγ), whereas TH2 cells produce the anti-inflammatory cytokines IL-4, IL-13, and IL-5. IL-10, produced by TH2 cells in mice, that can be produced by both TH1 and TH2 in humans . In experimental Leishmania infection, a TH1–TH2 polarization has been established as an important element for the eradication or persistence of parasites. Genetically resistant mice develop a TH1 immune response with IFNγ production, whereas susceptible mice develop a TH2 response with IL-4, IL-13, and IL-10 production . In human VL, IL-10 has been associated with active disease  or disease relapse . VL is a major public health problem in tropical and subtropical countries but, with human travel and climatic changes, it occurs worldwide. It often remains asymptomatic but may have a life-threatening course with fever, hepatosplenomegaly and wasting. Some laboratory findings of VL, including cytopenias and antinuclear antibodies, resemble those of rheumatic autoimmune diseases, particularly systemic lupus erythematosus (SLE) [6,7].
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L.I. Sakkas et al. / Clinical Biochemistry 41 (2008) 65–68
We retrospectively analyzed clinical and laboratory, including immunological, features of patients with VL and focused on findings that help clinicians to differentiate VL from SLE. Patients and methods Six patients with VL were diagnosed at the Department of Internal Medicine and Rheumatology, University Hospital of Larisa between 2001 and 2007. Demographic and laboratory findings are shown in Table 1. Disease duration was 20 days in three patients (#1, 3, 5), 30 days in two patients (#2, 4), and 45 days in one patient (#6). In all patients, massive and firm splenomegaly was palpated. All patients underwent routine hematological and biochemistry tests, serological tests for viruses and bacteria, PPD skin test, as well as blood and urine cultures, blood coagulation tests, and bone marrow aspiration. Autoantibodies were tested in all patients. Anti-nuclear antibodies (ANA) were tested by indirect immunofluorescence on Hep2 cells. Anti-dsDNA antibodies were tested by immunofluorescence on crithidia and by enzymelinked immunosorbent assay (ELISA). IgM rheumatoid factor (RF) was tested by immunonephelometry (Dade Behring). Antineutrophil cytoplasmic antibodies (ANCA), which are necrotizing vasculitis-associated antibodies, were tested by indirect immunofluorescence staining. In addition, the specificities of ANCA against proteinase-3 (PR3) and myeloperoxidase (MPO) were tested by ELISA.
Table 1 Demographic and laboratory parameters in 6 patients with visceral leishmaniasis Feature
Age/sex Hct (%) WBC PLT ANA (pattern)
28/F 29.6 2400 105 1:160 Diffuse
17/F 22 1500 97 1:1280 Nucleolar
56/M 36 1900 68 1:640 Nucleolar
71/M 33 4000 109 1:320 Speckled
79/F 23 900 65 1:80
a-dsDNA a-CL (IgM) dCoombs Other Abs
– – + (IgG) – 12 ND 1400 14.0 389 430.3 7227 1.46 28.1
– – ++ (IgG) ASMA (1:80) 60 166/21 4200 8.9 1804 388 906 1.18 41.0
1:40 35 ND ASMA
IgM RF C3/C4 IgG CRP Ferritin Fibrinogen D-dimers INR APTT (s)
– – ++ (IgG) ASMA (1:320) 153 110/13 3780 1.6 96 302 885 1.26 41.9
54/M 34 2500 122 1:640 Nucleolar dif, cyto – – +++ (IgG) ASMA (1:320) 107 ND 5660 1.6 1558 208 ND 1.29 50.8
– – ++ (IgG) aANCA (1:40) 131 49/11 2530 9.5 631 176 5800 1.14 52.7
b9.50 ND 2770 11.3 1260 557.6 3850 1.33 34.3
WBC count (× 103/mm3); PLT count (× 103/mm3). ANA(cut off, 1:80); RF = rheumatoid factor (cut off, 15 IU/mL); CRP = C-reactive protein (cut off, 0.5 mg/dL); C3 (normal, 90–180 mg/dL); C4 (normal, 10–40 mg/dL); Ferritin (cut off, 150 ng/mL); Fibrinogen (normal, 212–488 mg/dL); D-dimers (cut off, 0.3 mg/L); aCL = anti-cardiolipin antibodies (normal, b20 MPLU/mL); aANCA = atypical ANCA (normal, b1:20); ASMA = anti-smooth muscle antibodies (normal b1:20), dif = diffuse, cyto = cytoplasmic.
Results Diagnosis of VL was accomplished by demonstration of Leishmania species in Giemsa-stained bone marrow aspirates (patients #1, 2, 3, 5, 6). In one patient (#4) without Leishmania parasites on bone marrow aspirate, diagnosis was established by high serum anti-leishmanial antibodies. Autoantibodies were detected in all patients (Table 1). Most frequently, these autoantibodies were SLE-associated antibodies: ANA in five patients, positive direct Coombs test in all patients, anti-dsDNA antibodies in one patient, and IgM anti-cardiolipin antibody in one patient. Anti-smooth muscle antibodies (ASMA), associated with autoimmune hepatitis, were detected in four patients. IgM rheumatoid factor (RF) was present in four patients. Atypical ANCA, that have a cytoplasmic-like pattern on immunofluorescence staining but did not recognize PR3 on ELISA, were detected in one patient. The acute phase reactants C-reactive protein (CRP) and ferritin were high. There was evidence of activation of the coagulation pathways with increase of D-dimers and prolongation of prothrombin time (PT, international normalization ratio, INR) and activated partial thromboplastin time (APTT). One patient had concomitant low serum fibrinogen levels, indicative of disseminated intravascular coagulation. All patients were treated successfully with liposomal amphotericin B, and had an uneventful recovery. Discussion B cell responses in VL are exemplified by anti-Leishmania antibodies and hypergammaglobulinemia. Autoantibody production has also been described [6,7]. Some of these autoantibodies, such as ANA, anti-dsDNA antibodies, and positive direct Coombs test, are SLE-associated autoantibodies. In particular, anti-dsDNA antibodies, as detected in one of our patients, have a high specificity for SLE. ASMA, detected in four patients, are frequented detected in autoimmune hepatitis, which is also associated with SLE. Rheumatoid arthritis-related antibodies, such as IgM rheumatoid factor (RF-IgM) and anticyclic citrullinated peptide (CCP) antibodies, have also been reported in VL. In a small study of 10 patients with VL, RF-IgM and anti-CCP antibodies were detected in nine and three patients, respectively . It should be noted that anti-CCP antibodies have 95% specificity for rheumatoid arthritis . In our study, IgM-RF was positive in 4 out of 6 patients. Atypical ANCA, detected in one patient, have a cytoplasmic-like staining on immunofluorescence that can be misinterpreted as cANCA, which are associated with systemic necrotizing vasculitis (i.e. Wegener's granulomatosis). However, cANCA recognize PR3 on ELISA in Wegener's granulomatosis, whereas atypical ANCA do not recognize PR3 . This re-emphasizes the importance of ELISA for PR3, when ordering ANCA. Other immunological abnormalities published as case reports in VL include type-II cryoglobulinemia  and monoclonal gammopathy . Polyclonal B cell activation with circulating immune complexes and autoimmunity was described in VL more than 20 years ago . Polyclonal activation of B cells can occur
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through extensive cross-linking of membrane immunoglobulin (Ig) by microbial antigens or through stimulation by cytokines produced by other cells . For instance, Trypanosoma cruzi glutamate dehydrogenase stimulates a T cell-independent polyclonal activation of B cells through macrophage production of IL-10 and the B cell activating factor, BAFF . Also, repeated administration of staphylococcal enterotoxin B superantigen to mice results in the production of TH2 cytokines (IL-4 and IL-10), polyclonal B cell activation, and anti-mouse autoantibodies . IL-10 can be derived from CD4+CD25− TH1 cells and CD4+CD25+ T regulatory cells, as has been shown in mice, and from macrophages [3,17]. In human VL, IL-10 production by macrophages can be induced by IgG-containing immune complexes . These IgG-containing immune complexes also switch off macrophage production of IL-12 , and are associated with peak parasitemia, and absent delayed-type hypersensitivity (DTH) responses to Leishmania antigens in patients with VL . It should be noted that IL-12 induces naive T cells to differentiate into TH1 cells and produce IFNγ. With resolution of human disease, IgG decreases and DTH reappears. In SLE there is also polyclonal activation of B cells, associated with a deviation of TH1 to TH2 cells , and increased production of IL-10 and IL-6 by B cells and monocytes . Cross-reactivity between Leishmania and human antigens can be another mechanism for autoantibody production in VL. Immunization of A/J mice with cardiac myosin or Trypanosoma cruzi antigen induces bi-directionally cross-reactive immune responses . This autoimmune response apparently requires a proper MHC background since it does not occur in C57BL/6 mice . Anti-ribosomal antibodies, present in patients with SLE, recognize the epitope motif GFGLFD in the C-termini of ribosomal phosphoproteins. This motif is also present in proteins of Leishmania species . Hypergammaglobulinemia, low serum C3 and C4 complement levels, and serum autoantibodies, such as ANA, antidsDNA antibodies, and positive direct Coombs reaction are SLE-related immunological findings and may lead to misdiagnosis of VL for SLE. Furthermore, there may be additional features that are shared between VL and SLE, such as fever, splenomegaly, pericardial effusion , and pancytopenias. VL can occur in patients with SLE and mimic exacerbation of SLE  or in patients with RA on anti-TNFα therapy  and may mimic drug-induced lupus. Distinguishing VL from SLE is of paramount importance because an immunosuppressive treatment for SLE is detrimental to a patient with VL. The following features help in differential diagnosis: (a) The spleen can be massively enlarged (more than 8 cm below the left costal margin) and firm in texture in VL, whereas it is hardly palpable and soft in SLE; (b) Peripheral blood cytopenias require a bone marrow examination that includes a careful search for parasites; (c) SLE may have additional features, such as butterfly rash, symmetrical polyarthritis, alopecia, poly-serositis, and glomerulonephritis, not present in VL; (d) Serum levels of acute phase reactants are very high in VL and usually normal in SLE. In SLE, small increases of CRP may be found when arthritis, serositis, or concomitant infection is present. In our small series, serum
levels of CRP were increased in all VL patients. Serum ferritin, an acute phase reactant, was also increased in five patients. In two of our patients, serum ferritin levels were very high (N1500 ng/mL). Among rheumatic diseases, very high serum ferritin levels are frequently found in adult onset Still's disease . (e) Activation of coagulation pathways with PT and APTT prolongation is suggestive of VL. In infections, IL-6 and other mediators induce monocyte tissue factor and activate the coagulation pathways. D-dimers were increased in all 4 patients tested, whereas concomitant decreased serum fibrinogen, as detected in one patient, was indicative of disseminated intravascular coagulation. In patients with SLE, APTT prolongation occurs when anti-phospholipid syndrome is present, with anticardiolipin antibodies and/or lupus anticoagulant positive. In the latter case, D-dimers and fibrinogen are normal. (f). Hypergammaglobulinemia occurs in both VL and SLE, but it is usually massive only in VL. Massive hypergammaglobulinemia can also be found in autoimmune hepatitis. In conclusion, although there are features of VL, such as fever, splenomegaly, cytopenias, and SLE-related autoantibodies, that resemble SLE, there are some clues, including massive firm splenomegaly, very high acute phase reactants, activation of the coagulation cascade, and massive hypergammaglobulinemia, that point toward infection. References  Murray HW, Berman JD, Davies CR, Saravia NG. Advances in leishmaniasis. Lancet 2005;366:1561–77.  Windhagen A, Anderson DE, Carrizosa A, Williams RE, Hafler DA. IL-12 induces human T cells secreting IL-10 with IFN-γ. J Immunol 1996;157: 1127–31.  Roberts MTM. Current understandings on the immunology of leishmaniasis and recent developments in prevention and treatment. Br Med Bull 2006;75–76:115–30.  Ghalib HW, Piuvezam MR, Skeiky YA, Siddig M, Hashim FA, el Hassan AM, et al. Interleukin 10 production correlates with pathology in human Leishmanis donovani infections. J Clin Invest 1993;92:324–9.  Babaloo Z, Kaye PM, Eslami MB. Interleukin 13 in Iranian patients with visceral leishmaniasis: relationship to other TH2 and TH1 cytokines. Trans R Soc Med Hyg 2001;95:85–8.  Voulgari PV, Pappas G, Liberopoulos EN, Elisaf M, Skopouli FN, Drosos AA. Visceral leishmaniasis resembling systemic lupus erythematosus. Ann Rheum Dis 2004;63:1348–9.  Ossadron A, Bompane D, Alessandri C, Marocchi E, Conti F, Vaesini G. Leishmania in systemic lupus erythemasosus mimicking an exacerbation. Clin Exp Rheumatol 2006;24:186–90.  Atta AM, Carvalho EM, Jeronimo SM, Sousa Atta ML. Serum markers of rheumatoid arthritis in visceral leishmaniasis: rheumatoid factor and anticyclic citrullinated peptide antibody. J Autoimmun 2007;28:55–8.  Alexiou I, Germenis A, Ziogas A, Theodoridou K, Sakkas LI. Diagnostic value of anti-cyclic citrullinated peptide antibodies in Greek patients with rheumatoid arthritis. MBC Musculoskelet Dis 2007;8:37–43.  Bosch X, Guilabert A, Font J. Anti-neutrophil cytoplasmic antibodies. Lancet 2006;368:404–18.  Rizos E, Dimos G, Liberopoulos EN, Elisaf M, Drosos AA. Cryoglobulinemic purpura in visceral leishmaniasis. Rheumatol Int 2005;25:469–71.  Randi ML, Ruzzon E, Tezza F, Pacquola E, Fabris F. Monoclonal gammopathy in human leishmaniasis. Neth J Med 2006;64:50–1.  Galvao-Castro B, Sa Ferreira JA, Marzochi KF, Marzochi MC, Coutinho SG, Labert PH. Polyclonal B cell activation, circulating immune complexes and autoimmunity in human American visceral leishmaniasis. Clin Exp Immunol 1984;56:58–66.
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