Document Type

Article

Publication Date

2019

DOI

10.1371/journal.pntd.0006974

Publication Title

PLOS Neglected Tropical Diseases

Volume

13

Issue

3

Pages

e0006974 (1-27)

Abstract

Background

Despite decades of use 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 needs to be better diagnostic tools to detect low-intensity infections in low-endemic areas in Brazil. The rationale for development of new diagnostic tools is that the current standard test Kato-Katz (KK) is not sensitive enough to detect low-intensity infections in low-endemic areas. In order to develop new diagnostic tools, we employed a proteomics approach to identify biomarkers associated with schistosome-specific immune responses in hopes of developing sensitive and specific new methods for immunodiagnosis.

Methods and findings

Immunoproteomic analyses were performed on egg extracts of Schistosoma mansoni using pooled sera from infected or non-infected individuals from a low-endemic area of Brazil. Cross reactivity with other soil-transmitted helminths (STH) was determined using pooled sera from individuals uniquely infected with different helminths. Using this approach, we identified 23 targets recognized by schistosome acute and chronic sera samples. To identify immunoreactive targets that were likely glycan epitopes, we compared these targets to the immunoreactivity of spots treated with sodium metaperiodate oxidation of egg extract. This treatment yielded 12/23 spots maintaining immunoreactivity, suggesting that they were protein epitopes. From these 12 spots, 11 spots cross-reacted with sera from individuals infected with other STH and 10 spots cross-reacted with the negative control group. Spot number 5 was exclusively immunoreactive with sera from S. mansoni-infected groups in native and deglycosylated conditions and corresponds to Major Egg Antigen (MEA). We expressed MEA as a recombinant protein and showed a similar recognition pattern to that of the native protein via western blot. IgG-ELISA gave a sensitivity of 87.10% and specificity of 89.09% represented by area under the ROC curve of 0.95. IgG-ELISA performed better than the conventional KK (2 slides), identifying 56/64 cases harboring 1–10 eggs per gram of feces that were undiagnosed by KK parasitological technique.

Conclusions

The 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 MEA can be a useful tool combined with other techniques in low-endemic areas to determine the true prevalence of schistosome infection that is underestimated by the KK method. Further, to overcome the complexity of ELISA in the field, a second generation of antibody-based rapid diagnostic tests (RDT) can be developed.

Comments

(Author Summary) Schistosomiasis remains a serious global public health problem. Detecting parasite eggs in patient stool samples using the KK method is the standard diagnostic recommended by the World Health Organization (WHO) for infection by S. mansoni. As a result of intensive control strategies, many previously high-endemic areas are now considered low-endemic areas and the KK 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 a plan from the WHO focusing on elimination of disease as a public health problem. Successful diagnoses and treatment of infected individuals may result in eradication of low-burden transmitters and consequently contribute to interruption of disease transmission. In this regard, immunological techniques have proven to be more sensitive and promising for identifying low-intensity infections where KK may be negative. 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 with acute and chronic infections to identify new target antigens via proteomic screening. Using these approaches, we initially identified 12 different egg proteins in S. mansoni-infected individuals (acute and chronic phase). A single antigen, identified as MEA, was shown to be highly specific as this antigen was not recognized by sera from negative patients or patients infected with other STH. The recombinant MEA protein functioned in an ELISA as a highly sensitive and specific antigen to detect patient IgG-antibodies. Recombinant MEA performed significantly better to detect low-intensity infections (1 egg per gram of feces) than the KK method using 2 slides. Therefore, we were able to use a proteomic screening approach to identify a potential new candidate antigen for development of far more sensitive diagnostic assays. Further diagnostic assays employing the MEA could be useful tools on their own or in combination with other methods for diagnosis of schistosome infection in populations living in extreme low-intensity endemic areas of Brazil.

Rights

© 2019 Silva-Moraes et al.

This is an open access article distributed under the terms of the Creative Commons Attribution 4.0 International (CC BY 4.0) License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Data Availability

Article states: "All relevant data are within the manuscript."

Original Publication Citation

Silva-Moraes, V., Shollenberger, L. M., Castro-Borges, W., Rabello, A. L. T., Harn, D. A., Medeiros, L. C. S., Jeremias, W. d. J., Siqueira, L. M. V., Pereira, C. S. S., Pedrosa, M. L. C., Almeida, N. B. F., Almeida, A., Lambertucci, J. R., Carneiro, N. F. d. F., Coelho, P. M. Z., & Grenfell, R. F. Q. (2019). Serological proteomic screening and evaluation of a recombinant egg antigen for the diagnosis of low-intensity Schistosoma mansoni infections in endemic area in Brazil. PLOS Neglected Tropical Diseases, 13(3), 1-27, Article e0006974. https://doi.org/10.1371/journal.pntd.0006974

ORCID

0000-0002-0943-0838 (Shollenberger)

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