After the plates had been washed three times, enzymatic activity was developed by incubation with p-nitrophenyl phosphate (Sigma). for the detection of IgG in serum from CL patients by flow cytometry. To validate the assay we carried out a comparative test (ELISA) commonly used as a diagnostic test for parasitic diseases. To determine cross-reactivity we used serum from patients with Chagas disease, caused by a trypanosome that has several proteins with high homology to those of the genus. We observed that the flow cytometry technique was more sensitive than the ELISA, but, less specific. Our results show that the flow cytometry serologic test can be used to confirm CL cases in transmission areas, however, presence of Chagas disease has to be ruled out in these individuals. Introduction Cutaneous Nafarelin Acetate leishmaniasis (CL) caused by is characterized by the presence of one or more well-delineated ulcerated lesions that is mainly composed of lymphocytes, mononuclear phagocytes and plasma cells [1, 2]. In CL patients the immune response is predominantly mediated by mononuclear cells, which involve mechanisms associated with delayed type hypersensitivity with production of IFN-gamma and TNF [3C5]. This kind of response mediates parasite killing through activation of macrophages and also leads to tissue damage observed in these individuals [5]. The diagnosis of CL is mainly based on clinical observations and skin test; histopathologic or PCR techniques are usually used as confirmatory tests [6C9]. However, due to the low frequency of parasites in lesions of have been detected in CL patients, mainly due to differences in parasitic load, species involved, time since infection and intrinsic host factors [15C18]. Methods to evaluate the humoral immune response are mainly based on serologic surveys using Nafarelin Acetate soluble antigens, recombinant antigens and fixed parasites, such as indirect immunofluorescence, indirect hemaglutination and ELISA. Problems with the analysis of antibody titers by conventional serologic methods to detect infection include cross-reactivity with other species of the Trypanosomatidae family, low sensitivity and lack of association with the presence of active infection [19, 20]. Serological studies based on flow cytometry using polystyrene microspheres coated with soluble antigens constitute a field with growth potential due to the increased sensitivity of this method [21, 22]. In the present study we have developed a serological technique using polystyrene microspheres sensitized with soluble antigen (SLA) for the detection of IgG antibodies in the serum of CL patients by flow cytometry and have compared this with an ELISA test. We show that the flow cytometry-based test has greater sensitivity compared to the ELISA test, though neither test has the capacity to distinguish between samples from and infected individuals. Materials and Methods Patients Participants of this study were from the Corte de Pedra endemic area in Northeastern Brazil, a transmission area where more than 1000 instances are diagnosed per year. The study human population consisted of 27 CL individuals, Nafarelin Acetate 26 household contacts of CL individuals, with evidence of exposure to but without disease, 9 individuals with Chagas disease and 10 healthy subjects living in a non-endemic area. Leishmaniasis patients were diagnosed based ATF3 on medical presentation compatible with cutaneous leishmaniasis, positive Montenegro pores and skin test and parasite isolation. Chagas disease individuals were diagnosed by a serologic test to detect IgG to (Diagnostic Automation, INC, CA, USA). Individuals with evidence of exposure to but without disease were recognized by positive delayed type hypersensitivity (DTHMontenegro pores and skin test), IFN-gamma production to SLA and absence of lesions or history of leishmaniasis. All blood samples were collected before treatment of CL or Chagas disease had been started. To determine level of sensitivity, specificity, positive and negative predictive value we used 2 by 2 contingency furniture containing: true positive; false positive; true negative; false bad (Furniture ?(Furniture1,1, ?,22 and ?and3).3). The number of true Nafarelin Acetate positive, false positive, true bad and false bad individuals from each group analysed are displayed on Furniture ?Furniture22 and ?and3.3. This study was authorized by honest committee of the University or college Hospital in the Federal government University or college of Bahia. Written educated consent was from all participants. Table 1 Representative table and formulas used to calculate diagnostic checks overall performance. IgG was measured by ELISA as follows: highly sensitive microplates (Thermo medical, Waltham, USA) were sensitized with 100l of 20g/ml soluble antigen of and incubated at 4C over night. The plates were then washed five instances with PBS-Tween and incubated with 100 l/well of each individuals serum diluted 1:100 to 1 1:800 in 1x PBS for 1 hour at 37C. After washing three times with PBS-Tween, 100 l/well of anti-human IgG (-chain specific) was added and plates were incubated at 37C for 1.