Fig 1.
Schematic illustration of the LM-based LFIA cassette for the detection of S. japonicum infection.
The cassette implements a design that incorporates the LM-labelled rSjSAP4 in the conjugate pad, instead of on the test (T) line as in a typical AbD-based LFIA. As a positive serum sample flows into the conjugate pad, specific anti-rSjSAP4 Abs present in the sample bind the LM-conjugated rSjSAP4 to form immunocomplexes, which can be further captured by the protein G immobilised on the T line. On the control (C) line, unbound LM-labelled rSjSAP4 protein is captured by a monoclonal Ab specific to the histidine tag. A positive reaction is determined by the development of both reddish T and C lines. If the C line does not appear, the test is considered invalid and must be repeated.
Fig 2.
IgG antibody levels against five S. japonicum recombinant antigens in sera from KK (+) individuals and non-endemic controls.
Statistical significance between the KK (+) (n = 28) and control (n = 12) samples was determined using the Mann–Whitney U test (ns–no significant difference; ***, p < 0.001; ****, p < 0.0001). Dashed lines correspond to OD450nm cut-off value calculated as 2.1× the mean OD450nm of control samples with outlier datasets removed.
Fig 3.
Determination of optimal testing parameters for the developed LFIA.
(A) Three serum samples from S. japonicum-infected individuals with different anti-rSjSAP4 IgG levels as per the OD values from the rSjSAP4-ELISA, were equally pooled. The pooled serum was diluted with five different diluents (1:10) and tested with the LFIA cassettes. #1, PBS; #2, PBS with 0.05% Tween-20 (PBST); 3#, PBS with 1% BSA; 4#, 0.9% NaCl; #5, 2.5% sucrose with 1% Tween-20. The results captured at 10, 15, 20 and 25 min after sample loading are presented. The LFIA cassettes were tested with (B) PBS- or (C) the sucrose diluent-diluted serum samples from KK (+) individuals (n = 3) with different anti-rSjSAP4 IgG levels and non-endemic controls (n = 3) over different dilution factors. Panels A-C, data are presented as mean ± SEM from two independent analyses. Panels B and C, the results obtained at 20 min after sample loading are presented. High, medium, and low anti-rSjSAP4 IgG levels were determined by the rSjSAP4-ELISA.
Fig 4.
T/C ratio analysis for the developed LFIA cassettes.
(A) Frequency of individuals (those from a barangay endemic for S. japonicum, n = 549) with different levels of T/C ratio. (B) Representative LFIA cassettes showing different levels of T/C ratio. Lanes 1–6, cassettes displaying a T/C ratio that falls in the ranges of 0–0.1, 0.1–0.5, 0.5–1, 1–5, 5–10, and >10, respectively. C, control line; T, test line; S, sample well. (C) Truncated violin plots showing the distribution of T/C ratio in the different KK (+) subgroups (EPG >100, n = 11; EPG 51–100, n = 15; EPG 11–50, n = 39; EPG 1–10, n = 74), KK (-) individuals (n = 410) and non-endemic controls (n = 50). The horizontal black dashed lines indicate median values, and the horizontal black dotted lines represent the interquartile range (IQR) of the data. p values were calculated using the Kruskal–Wallis test followed by Dunn’s comparison. The results obtained at 25 min after sample loading are presented. EPG, eggs per gram of faeces.
Fig 5.
Diagnostic performance of the developed LFIA cassette.
Truncated violin plots showing (A) the T/C ratios from the LFIA test and (B) the OD values from the rSjSAP4-ELISA performed on the serum samples from the KK (+) individuals (n = 139) and non-endemic controls (n = 50). The blue dashed line corresponds to the cut-off values. Statistical significance was determined using the Mann–Whitney U test. ROC curve analysis showing the performance of (C) the LFIA cassette and (D) rSjSAP4-ELISA in discriminating KK (+) individuals from controls. (E) Correlation between the LFIA and rSjSAP4-ELISA was assessed using Spearman’s correlation coefficient (p < 0.0001). The results captured at 25 min after sample loading are presented for the developed LFIA.
Table 1.
Agreement analysis for the LFIA and rSjSAP4-ELISA tests performed on serum samples (n = 549) collected from a barangay endemic for S. japonicum.
Fig 6.
Applicability of the developed LFIA strip in the detection of different S. japonicum strains and cross-reactivity analysis of the immunochromatographic assay.
(A) LFIA cassettes were used to detect S. japonicum infection across SjC and SjP strains. (B) Potential cross-reactivity with other helminth infections was assessed for the established LFIA cassette using the serum samples from individuals infected with hookworm, A. lumbricoides or T. trichuira. C, control line; T, test line; S, sample well.