A simple immune complex dissociation ELISA for leishmaniasis: Standardization of the assay in experimental models and preliminary results in canine and human samples
Highlights
► Leishmania serology is affected by the presence of immune complexes. ► Immune complexes are present in early stage of Leishmania chagasi experimental infection. ► Dissociation of immune complexes allows 2–5% increase in leishmaniasis IgG ELISA.
Introduction
Visceral leishmaniasis (VL), caused in the Old World by Leishmania (Leishmania) donovani and Leishmania (Leishmania) infantum or caused in the New World by Leishmania (Leishmania) chagasi (or L. (L.) infantum), is endemic in South America, and has an estimated annual incidence of 500,000 new human cases worldwide, of which 90% occur in Brazil, Bangladesh, India, Nepal and the Sudan (WHO, 2012). Most cases occur in the poorest of urban communities and are related to both migration and urbanization (Alves and Bevilacqua, 2004). Symptomatic VL is associated with hepatosplenomegaly and chronic fever, depending on disease progression, but most infected individuals remain oligo- or asymptomatic (Oliveira et al., 2010). Dogs are considered the main reservoir of the parasite, especially in South America, where a high level of canine infection is associated with a high risk of human disease (Werneck et al., 2007). VL patients mount large humoral responses characterized by hypergammaglobulinemia, which appears to be inefficient against parasites; however, the high concentration of antibodies and circulating antigen can result in the formation of circulating immune complexes (CIC) (Lambert et al., 1981). CIC can mask the actual antibody concentration present in a sample when assayed using conventional methods (Gustaw et al., 2008).
The “gold standard” for the diagnosis of VL is based on the detection of parasites in organ aspirates from the spleen, liver, lymph nodes and bone marrow. The technique is specific but invasive, and accidents occasionally occur due to bleeding, as visceral leishmaniasis causes thrombocytopenia (Chappuis et al., 2007). Serological tests for specific IgG are widely used in the epidemiological surveillance of VL, including indirect immunofluorescence, direct agglutination and ELISA, but all of these methods cross-react with other Leishmania parasites that are frequently found in the same endemic areas of South America (Romero et al., 2009). Additionally, serological tests also present difficulties with regard to sensitivity, specificity and efficiency due to the use of several antigen sources, such as promastigotes, amastigotes from culture or recombinant proteins (Porrozzi et al., 2007).
IgG ELISA is considered a valuable tool in the diagnosis of VL because of its high sensitivity and its automation as part of large laboratory routines that allow the testing of large numbers of samples. IgG ELISAs were adapted for use with different antigens, which gives them the advantage in terms of sensitivity and specificity (Singh, 2006), but which also leads to the problem of CIC interference.
Many infectious diseases result in the formation of antigen-IgG immune complexes that worsen the chronic disease state, mainly through renal glomerular disease. These diseases may be caused by bacteria, such as Mycobacterium tuberculosis, Mycobacterium leprae, Legionella pneumophila, Salmonella sp. and Shigella sp. (Halstead et al., 2010); viruses, such as dengue virus (Koraka et al., 2003) and HIV (Lewis et al., 1995); fungi (Durkin et al., 2009); or parasites, such as other Leishmania spp. or Schistossoma spp. (Carvalho et al., 1983). Longstanding VL in humans or experimental animals also presents with secondary amyloidosis and vascular disease, both of which can be attributed to CIC (de Vallière et al., 2009).
Recently, some studies have attempted to elucidate the role of immune complexes in the pathology of VL. However, most of these studies focus either on the importance of CIC as a marker of disease progression (Miles et al., 2005), or on the mechanisms of the disease itself, particularly addressing the role of cytokines and the humoral immune response (Elshafie et al., 2007). Quantification assays for CIC are few and remain rare without routine use because most require expensive sample preparation (Evans and Pearson, 1988). Thus, the role of CIC in serological testing used for the diagnosis and detection of IgG antibodies to Leishmania is not well established.
Here, we describe the development of an easy immunoassay designed to detect CIC using a modified ELISA and pH shock to promote the dissociation of immune complexes present in VL samples obtained by experimental and natural infection.
Section snippets
Parasites and animals
L. (L.) chagasi (MHOM/BR/1972/LD) was maintained in vivo through the experimental infection of Syrian hamsters (Mesocricetus auratus) by i.p. injection of amastigotes obtained from the spleens of previously infected hamsters. All hamsters and New Zealand rabbits used in the experiments were supplied the animal breeding facility of Faculdade de Medicina, Universidade de São Paulo, were maintained at the animal facility of Laboratório de Protozoologia at Instituto de Medicina Tropical de São
Experimental infection
Overall, 91 hamsters were successfully infected with L. (L.) chagasi amastigotes, presenting with a typical progressive infection and visceral disease as determined by the parasite load in the spleen; 17 non-infected controls were also used, resulting in 108 hamster samples. Disease progression was determined by sacrificing the hamsters at different stages of infection. Spleen parasite burden was determined as described in the methods, and blood was collected for ELISA. The parasitological and
Discussion
In this study, we devised and standardized an ELISA method using a dissociation step for immune complexes that allowed the detection of anti-Leishmania IgG antibody-antigen complexes that were otherwise non-detectable in cELISA. Our procedure involves the acid dissociation of IgG–antigen complexes followed by the formation of new associations with excess antigen in solid phase, thus minimizing soluble antigen interference in a simple, quick ELISA. We used acid as a chaotropic reagent for the
Acknowledgements
We gratefully thank the patients and staff from the Adolfo Lutz Institute and the Araguaína (TO) public laboratory for supplying serum samples. We also gratefully thank M.D. Laurenti for helpful discussions and the constructive reviewer staff of Acta Tropica for the careful review. The technical assistance of Roselaine P.A. Cardoso was also extremely important. C.A. Carvalho was a fellow of CAPES, and this work formed a part of his M.Sc. dissertation in Tropical Medicine. H.F. Andrade Jr. was a
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