Development and evaluation of an indirect ELISA using a multiepitope antigen for the diagnosis of intestinal schistosomiasis

Abstract

The laboratory diagnosis of intestinal schistosomiasis, carried out by detecting parasite eggs in feces, has low sensitivity when applied to individuals with low parasitic load. Serological tests can be more sensitive for the diagnosis of the disease. Therefore, the objective of this work was to develop and evaluate an ELISA-based immunoenzymatic assay, using a Schistosoma mansoni multiepitope antigen (ELISA IgG anti-SmME). For this, the amino acid sequences of S. mansoni cathepsin B and asparaginyl endopeptidase were submitted to the prediction of B cell epitopes and, together with peptide sequences obtained from earlier works, were used in the construction of a minigene. The multiepitope protein was expressed in Escherichia coli and the performance of the ELISA IgG anti-SmME for schistosomiasis was evaluated using serum samples from 107 individuals either egg positive or negative. In addition, 11 samples from individuals with other helminth infections were included. The ELISA IgG anti-SmME showed a sensitivity of 81.1% and a specificity of 46.1%. Further analysis revealed a 77.2% sensitivity in diagnosis of individuals with egg counts of ≤12 epg (eggs per gram feces) and 87.5% for individuals with 13–99 epg. It is worth mentioning that, to our knowledge, this was the first study using a multiepitope recombinant antigen in an ELISA for diagnosis of intestinal schistosomiasis, which demonstrated promising results in the diagnosis of individuals with low parasitic loads.

Keywords: Asparaginyl endopeptidase; Schistosoma mansoni; cathepsin B; diagnosis; low parasitic load; multiepitope antigen; serology.

Graphical abstract

Figure 1.

Flowchart for infection groups and number of serum samples used in the ELISA IgG anti-SmME. In the first phase, the assay performance was evaluated and carried out with a group of 118 serum samples, 53 belonging to the egg-positive (POS) group and 65 from the egg-negative group, according to parasitological techniques. In addition, individuals from the negative group were subdivided into egg-negatives from endemic areas (NegE), egg-negatives from non-endemic areas (NegNE) and egg-negatives infected with soil-transmitted helminths (STH). The second phase consisted of assessing the level of IgG antibodies after PZQ treatment. For this, samples of 12 individuals residing in Brejo do Amparo, plus a subgroup of 8 individuals included in the first phase, totalling 20 individuals, were subjected to the subsequent tests. These individuals participated in the follow-up study at 3, 6 and 12 months post-treatment.

Figure 2.

Production of recombinant P1–P5 antigen. (A) 12% SDS-PAGE with culture lysate before (lane 1) and 4 h after induction with IPTG (lanes 2–3). (B) Western blotting using monoclonal 6x-His tag antibody against recombinant P1–P5 antigen. (C) 12% SDS-PAGE with purified recombinant P1–P5 antigen (lanes 1–2). MW: molecular weight marker [(A) and (B): Precision Plus Protein Dual Color Standards, Bio-Rad Laboratories; (C): Amersham ECL Rainbow Markers, GE Healthcare, Chicago, IL, USA].

Figure 3.

Evaluation of the ELISA IgG anti-SmME for the serological diagnosis of intestinal schistosomiasis. (A) Analysis of the ROC curve of the ELISA IgG anti-SmME, performed with sera from 53 egg-positive samples (POS) group and from 33 Schistosoma mansoni egg-negative individuals from non-endemic areas (NegNE) or with other 11 confirmed soil-transmitted helminth infections (STH). (B) Overall IgG antibody immunoreactivity for the ELISA IgG anti-SmME in the groups of egg-positive and egg-negative participants for intestinal schistosomiasis. The graph was constructed from the mean OD readings of 53 serum samples of egg-positive individuals and 65 egg-negative individuals from endemic (n = 32) and non-endemic areas (n = 22) and from individuals infected with other helminth infections (n = 11). The bars indicate median values, the boxes the 25 and 75% intervals and the whiskers the 5 and 95 percentiles. The red dotted line indicates the calculated cut-off point, according to ROC curve analysis (OD = 0.235). ***P < 0.001.

Figure 4.

Individual and mean IgG antibody immunoreactivity (OD) against P1–P5 antigen in egg-positive participants and subgroups of egg-negative participants. (A) IgG immunoreactivity of antibodies against P1–P5 antigen in egg-positive individuals (POS), individuals from endemic (NegE) and non-endemic areas (NegNE) and in individuals with soil-transmitted helminth infections (STH). The graph was constructed by average of the absorbance readings of 53 S. mansoni egg-positive and 65 S. mansoni egg-negative samples. (B) Individual and mean IgG antibody immunoreactivity in S. mansoni egg-positive participants stratified by parasitic load. Individual S. mansoni parasitic loads expressed as mean epg values considered as very low (⩽12), low (13–99), medium (100–399) and high (⩾400). The number of epg was calculated from 2 Kato–Katz slides of 1 fecal sample. Fecal samples classified as egg negative in 2 Kato–Katz slides, but positive for S. mansoni eggs in other parasitological techniques, were classified as epg <12. Error bars indicate the median and interquartile ranges of 25 and 75% and the red dotted line the cut-off point (0.235). **P < 0.01, ***P < 0.001.

Figure 5.

Kinetics of the IgG antibody measured by ELISA IgG anti-SmME in a group of S. mansoni-infected individuals before (0) and 3, 6 and 12 months after treatment with PZQ. The bars indicate the median and interquartile ranges of 5 and 95% and the dotted line indicates the cut-off point (0.235)