Serological proteomic screening and evaluation of a recombinant egg antigen for the diagnosis of low-intensity infections in endemic area in Brazil

Abstract

BackgroundDespite decades of employment of control programs, schistosomiasis remains a global public health problem. To further reduce prevalence and intensity of infection, or to achieve the goal of elimination in low-endemic areas, there need to be better diagnostic tools to detect low intensity infections in low-endemic areas as Brazil. The rationale for development of new diagnostic tools is because in low-endemic settings, the standard Kato-Katz diagnostic method loses its sensitivity and misses low intensity infections. To develop new diagnostic tools, we employed a proteomics approach search for biomarkers associated with schistosome-specific immune responses to develop sensitive and specific new methods for immunodiagnosis.\n\nMethods and findingsImmunoproteomic analysis was performed on an egg extract of Schistosoma mansoni using pooled sera from infected or non-infected individuals from a low-endemic area of Brazil. Cross reactivity with other helminth parasites was determined using pooled sera from individuals infected with different parasitic helminths. Using this approach we identified 23 spots recognized by schistosome acute and chronic sera samples. To identify immunoreactive spots that are likely glycan epitopes, we compared immunoreactivity of spots treated by sodium metaperiodate oxidation of egg extract. This treatment yielded 12/23 spots maintaining immunoreactivity, suggesting they are protein epitopes. From these 12 spots, 11 spots cross-reacted with sera from infection with other helminths and 10 spots cross-reacted with the negative control group. Spot number 5 was exclusively immunoreactive with sera from schistosome-infected groups in native and deglycosylated conditions and corresponds to a Major Egg Antigen. We expressed the Major egg antigen as a recombinant protein and showed by western blot a similar recognition pattern of that of the native protein. IgG-ELISA gave a sensitivity of 87.10% and specificity of 89.09% represented by area under ROC curve of 0.95. IgG-ELISA performed better than the conventional K-K (2 slides) identifying 56/64 cases harboring 1-10 egg per gram of feces that were undiagnosed by K-K parasitological technique.\n\nConclusionsThe serological proteome approach was able to identify a new diagnostic candidate. The recombinant egg antigen provided good performance in IgG-ELISA to detect individuals with extreme low-intensity infections (1 egg per gram of feces). Therefore, the IgG-ELISA using this newly identified recombinant major egg antigen can be a useful tool to be combined with other techniques in low-endemic areas to determine the true prevalence of schistosome infection that is underestimated by Kato-Katz method. Further, to overcome the complexity of ELISA in the field, a second-generation of antibody-based rapid diagnostic tests (RDT) can be developed.\n\nAuthor SummarySchistosomiasis remains a serious global public health problem. Detecting parasite eggs in patient stool samples using the Kato-Katz (KK) method is the standard diagnostic recommended by World Health Organization (WHO) for infection by Schistosoma mansoni. As a result of intensive control strategies, many previously high-endemic areas are now considered low-endemic areas and the K-K method does not function well in low-endemic areas and therefore cannot be considered the gold standard. Thus, a new emphasis on strategies to accurately diagnose low-intensity infections was outlined in plan from WHO focusing on elimination of disease as a public health problem. Successful diagnoses and treatment of the majority of infected individuals may result in elimination of a sufficient number of low-burden transmitters and consequently, in the interruption of the disease transmission. In this regard, immunological techniques have proven to be more sensitive and promising for identifying infection in low-intensity of infection positive individuals with negative K-K results. The identification of antigens is the initial step for developing new immunodiagnostic assays. In this study, we used sets of pooled human sera samples from controls to acute and chronic infections to narrow down the number of new candidate antigens via proteomic screening. Using these approaches we initially identified 12 different egg proteins in schistosome-infected individuals (acute and chronic phase). A single antigen identified as Major Egg Antigen was shown to be highly specific as this antigen was not recognized by sera from negative or patients infected with other helminths. The recombinant Major Egg Antigen protein functioned in ELISA as a highly sensitive and specific antigen to detect patient IgG-antibodies. Recombinant Major Egg Antigen performed significantly better to detect low-intensity infections (1 egg per gram of feces) than the K-K method using 2 slides. Therefore, using a proteomic screening approach we were able to identify a potential new candidate antigen for development of far more sensitive diagnostic assays. Further diagnostic assays employing the Major Egg Antigen could be a useful tool on its own or in combination with other methods for diagnosis of schistosome infection in populations living in extreme low-intensity endemic areas of Brazil.