Development and assessment of a novel gold immunochromatographic assay for the diagnosis of schistosomiasis japonica

Abstract

Background: The neglected zoonosis, schistosomiasis japonica, remains a major public health problem in the Philippines. The current study aims to develop a novel gold immunochromatographic assay (GICA) and evaluate its performance in the detection of Schistosoma japonicum infection.

Methods: A GICA strip incorporating a S. japonicum saposin protein, SjSAP4 was developed. For each GICA strip test, diluted serum sample (50 µl) was loaded and strips were scanned after 10 min to convert the results into images. ImageJ was used to calculate an R value, which was defined as the signal intensity of the test line divided by the signal intensity of the control line within the cassette. After determination of optimal serum dilution and diluent, the GICA assay was evaluated with sera collected from non-endemic controls (n = 20) and individuals living in schistosomiasis-endemic areas of the Philippines (n = 60), including 40 Kato Katz (KK)-positive participants and 20 subjects confirmed as KK-negative and faecal droplet digital PCR assay (F_ddPCR)-negative at a dilution of 1:20. An ELISA assay evaluating IgG levels against SjSAP4 was also performed on the same panel of sera.

Results: Phosphate-buffered saline (PBS) and 0.9% NaCl were determined as optimal dilution buffer for the GICA assay. The strips tested with serial dilutions of a pooled serum sample from KK-positive individuals (n = 3) suggested that a relatively wide range of dilutions (from 1:10 to 1:320) can be applied for the test. Using the non-endemic donors as controls, the GICA strip showed a sensitivity of 95.0% and absolute specificity; while using the KK-negative and F_ddPCR-negative subjects as controls, the immunochromatographic assay had a sensitivity of 85.0% and a specificity of 80.0%. The SjSAP4-incorperated GICA displayed a high concordance with the SjSAP4-ELISA assay.

Conclusions: The developed GICA assay exhibited a similar diagnostic performance with that of the SjSAP4-ELISA assay, yet the former can be performed by local personnel with minimal training with no requirement for specialised equipment. The GICA assay established here represents a rapid, easy-to-use, accurate and field-friendly diagnostic tool for the on-site surveillance/screening of S. japonicum infection.

Keywords: ELISA; GICA strip; Schistosoma japonicum; lateral flow immunochromatographic test; point-of-care (POC); rapid diagnosis; schistosomiasis; surveillance.

Figure 1

Schematic illustration of the rSjSAP4-incorporated GICA strip. The gold-rSjSAP4 conjugate is added to the conjugate pad. The recombinant protein G and a monoclonal mouse anti-His tag antibody is immobilized on the test (T) and control (C) line, respectively, on the NC membrane. Once serum samples containing specific anti-SjSAP4 antibodies or non-specific antibodies loaded onto the sample well, the conjugated anti-SjSAP4 antibody complexes and non-specific antibodies are captured by the protein G immobilized on the ‘T’ line. The gold-rSjSAP4 conjugate and conjugated anti-SjSAP4 antibody complexes are captured by the anti-His tag antibody on the ‘C’ line. A positive result is indicated by the appearance of pinkish red bands on both the ‘T’ and ‘C’ lines. A negative result is indicated by the appearance of only a single pinkish red band on the ‘C’ line. A test is determined as invalid if no bands appear on both ‘T’ and ‘C’ lines or if only one band appears on the ‘T’ line, and thus needs to be re-tested.

Figure 2

Detection limit of the GICA assay on pooled sera. (A) The GICA strips were tested with a pooled serum sample from KK-positive individuals (n = 3) with an EPG of 13, 14, and 20, respectively, at a series of dilutions (from 1:5 to 1:40960). C, Control line; T, Test line; S, Sample well; NC, a pooled serum sample from non-endemic controls (n = 3) tested at a dilution of 1:5. (B) R values of the GICA assay testing a pooled serum sample from KK (+) individuals (n = 3) at a series of dilutions. Dotted line: cut-off value determined as 2.1 times the mean R values of the pooled serum sample from non-endemic controls. The test was repeated in triplicate. p values were calculated using the Student’s t-test (ns, no significant difference; *p < 0.05; **p < 0.001; ***p < 0.001).

Figure 3

Determination of the optimal dilution buffer for the GICA assay. (A) Four dilution buffers, 1) PBS, 2) 0.9% NaCl, 3) 1% PBST, and 4) 2.5% sucrose containing 1% Tween 20, were selected to determine the optimal serum diluents. For each buffer, a pooled serum sample from three KK (+) individuals was tested in triplicate at a dilution of 1:40. (B) R value analysis revealed a significant impaired binding the gold-rSjSAP4 conjugate with antibodies in pooled serum samples when using PBST and 2.5% sucrose containing 1% Tween 20 as the dilution buffer compared with PBS. Data are represented as the mean ± SD from three different assays p values were calculated using the Student’s t-test (ns, no significance; *p < 0.05; **p < 0.01).

Figure 4

The performance of the SjSAP4-incorpated GICA and SjSAP4-ELISA assay in the diagnosis human schistosomiasis japonica. (A, B) Scatter plot showing the R values of the GICA assay (serum dilution 1:20) and OD values of the SjSPA4-ELISA assay (serum dilution 1:250) in testing serum samples from KK (+) individuals (n = 40), KK (-) and F_ddPCR (-) individuals (n = 20), and the non-endemic controls (n =20). Significance was analyzed by the Student’s t-test (****, p < 0.0001; *, p < 0.05). Gray dotted line: cut-off value determined using non-endemic group as control; Magenta dashed line: cut-off value determined using the KK (-) and F_ddPCR (-) group as control. (C) Correlation between the R values of the GICA strip and OD values determined by the SjSAP4-ELISA assay (n = 80) using Pearson’s correlation coefficient.

Figure 5

Stability assessment of the GICA strips. (A) Eight serum samples (one from healthy control (#1) and 7 from KK-positive subjects (#2 – #8)) diluted with PBS at 1:20 were used to assess the stability of the GICA strips stored at 25 for 12 months; (B) R values and read results for the eight serum samples tested with freshly produced GICA strips and those sealed and stored at 25°C for 12 months, respectively.