Development of a latex microsphere-based lateral flow immunoassay for the diagnosis of schistosomiasis japonica

doi:10.21203/rs.3.rs-4974469/v1

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Development of a latex microsphere-based lateral flow immunoassay for the diagnosis of schistosomiasis japonica

https://doi.org/10.21203/rs.3.rs-4974469/v1

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Background: Zoonotic schistosomiasis, caused by Schistosoma japonicum, remains prevalent in China, the Philippines and small foci of Indonesia. Rapid point-of-care (POC) diagnostic tools are attractive and promising tools for evaluating the efficacy of intervention strategies against schistosomiasis.

Methodology: The diagnostic potential for five recombinant antigens was tested by enzyme-linked immunosorbent assay (ELISA) using sera from individuals with a positive Kato-Katz (KK) result for S. japonicum (n=28) and non-endemic controls (n=12). A latex microsphere (LM)-based lateral flow immunoassay (LFIA) incorporating the recombinant SjSAP4 (rSjSAP4) antigen was developed for the diagnosis of schistosomiasis japonica. The testing conditions including diluent, dilution factor, and reaction time, were optimised for the developed LFIA. Under the optimised conditions, serum samples from individuals living in an S. japonicum endemic area (n=549) and non-endemic controls (n=50) were tested with the established LFIA cassettes. Results were imaged by a smartphone and analyzed by the ImageJ program. An R-value, which was the intensity ratio of the test line vs. control line, was calculated for each strip, converting the results into quantitative data.

Main findings: ELISA assays confirmed that the SjSAP4 was the optimal candidate for serological diagnosis of schistosomiasis japonica. Under the optimised testing conditions, the LM-based LFIA strips showed a sensitivity of 80.6% and a specificity of 98.0% at a cut-off R-value of 0.1031. Meanwhile, the developed LFIA showed a significant positive correlation with the rSjSAP4-ELISA assay (r=0.8269, 95% CI, 0.8127-0.9446; p<0.0001). The schistosomiasis prevalence determined by the LFIA strips was about 1.8 times higher than that obtained with the 6-slide KK procedure.

Conclusions/Significance: The study indicated that the LFIA assay established here represents a POC diagnostic tool that is suitable for on-site screening of human S. japonicum infection, using minimal equipment and adhering to the ASSURED criteria for POCs from the WHO.

Schistosoma japonicum

Schistosomiasis

Point-of-care

Latex microsphere

LFIA

ELISA

Saposin

Schistosomiasis is a severely debilitating and potentially fatal chronic parasitic disease. Caused by helminth parasites of the genus Schistosoma, it infects an estimated 230 million people worldwide [1]. Schistosoma mansoni (Africa and South America), S. haematobium (Africa), and S. japonicum (Asia) are the three major schistosome species infecting humans. Among them, S. japonicum is a zoonotic parasite which can use over 40 mammalian as reservoir hosts [2, 3]. The control of schistosomiasis focuses on reducing disease through periodic, large-scale population preventive treatment with praziquantel, referred to as mass drug administration (MDA) [4]. Both the prevalence and infection intensity of the disease have declined in many endemic areas primarily due to the implementation of MDA as part of national control programs, although other more integrated methods have been used [5]. In China for instance, bovines, major reservoirs of S. japonicum infection, were removed from endemic areas and replaced with tractors [6]; however, this method is not sustainable in the Philippines. The sustainability of schistosomiasis control programs is challenged by multiple facets, such as repeated infection – MDA does not prevent re-infection [1], and climate change – which may extend or change endemic zones [7, 8], and for Asia schistosomiasis, its zoonotic nature.

In the low endemicity areas (LEAs) of schistosomiasis particularly those with low infection intensity there is a need to develop and implement new, cost-effective and accurate diagnostic tools for rapid mapping and monitoring of schistosomiasis [9]. While conventional parasitological diagnostics (e.g., the microscopy-based Kato-Katz (KK) procedure and urine filtration) are labor- and time-intensive, and have limited sensitivity in the detection of schistosomiasis in LEAs, point-of-care (POC) diagnostics are becoming ideal tools for the disease control and elimination. To aid development of POCs the WHO has compiled the ASSURED criteria, which now has been updated as REASSURED [10].

POC diagnostics of schistosomiasis can be mainly divided into three categories: immunological, molecular-based, and mobile phone microscopes [11]. Molecular-based POCs aim to take advantage of rapid DNA extraction (e.g. DNA dipstick) [12] and isothermal nucleic acid amplification methods [e.g. loop-mediated isothermal amplification (LAMP) and recombinase polymerase amplification (RPA)]), and/or CRISPR-based techniques to amplify signals [13, 14]. However while exhibiting some promising results, these are mostly still in the development phase, with cost as a major concern. Mobile phone microscopes are portable and battery-powered devices designed to analyse samples at the point of collection [15], such devices for schistosomiasis have been suggested to be an ideal tool for rapid screening of S. haematobium infection in community- or school-based settings [16, 17]. The immunological POCTs could be further subcategorized into antigen detection (AgD) and antibody detection (AbD)-based tests [11]. AgD-based POC tests for schistosomiasis use specific monoclonal antibodies (mAbs) to capture Schistosoma gut-associated proteoglycan components known as circulating anodic antigens (CAAs) or circulating cathodic antigens (CCAs) [18]. The CAA-targeting POCTs such as POC-CCA and POC-ECO have been used to diagnose S. mansoni infection by testing urine samples [19, 20]. However, the assays suffer some pitfalls, such as cross-reactivity and specificity problems, as well as the issue in reading ‘trace’ results [21, 22]. In addition, the POC-CCA showed limited applicability for the diagnosis of other Schistosoma species [22–24]. The up-converting phosphor-lateral flow CAA (UCP-LF CAA) assay can detect all Schistosoma species quantitatively, exhibiting more accurate diagnostic performance than the POC-CCA [25]. The UCP-LF CAA is currently not available as a commercial test [26], and has equipment and reagent requirements that make it less than ideal as a POC [27]. The AbD-based POCTs of developed for the diagnosis of schistosomiasis typically use crude worm or egg antigens as targets, causing potential cross-reactions with other helminths [11]. Nevertheless, three recombinant antigen-based lateral flow immunoassays (LFIAs) have been developed for the detection of schistosome infections [28, 29].

We previously developed a colloidal gold immunochromatographic assay (GICA) incorporating SjSAP4 with a sensitivity of 83.3% and absolute specificity in the detection of KK positive individuals recruited from Northern Samar, a moderate S. japonicum-endemic area in the Philippines [30]. For developing LFIA, colloidal gold nanoparticles (Au NPs) labelling is the easiest, fastest, and most conventional method to produce conjugates for visualisation and results can be determined by the naked eyes. However, various nanomaterials with characteristic optical properties, such as fluorescent/coloured latex microspheres (LMs), carbon NPs, quantum dots (QDs), and up-converting phosphor/NPs [31–38], are also used as probes for LFIA development. It has been previously reported that the LM-based LFIA cassettes have several advantages over the AuNPs-based cassettes, such as increased sensitivity and stability [39–41]. In this study, we aimed to develop an LM-based LFIA for the diagnosis of schistosomiasis japonica by incorporating rSjSAP4 and assess its performance with sera samples collected from a rural area endemic for S. japonicum in Leyte province, the Philippines.

Ethics

The research ethical approval for this study was obtained the QIMR Berghofer Medical Research Institute (QIMRB) Animal (Project: P3706) and Human Ethics Committees (Projects: P3700 and P3797), the Institutional Review Board of the Research Institute for Tropical Medicine (RITM) (ID No. 2022-16), the Philippines, and the Institutional Review Board, Vector Control Division, Ministry of Health, Uganda (IRB No. VCDREC160). The study was conducted in accordance with the Australian Code for the Care and Use of Animals for Scientific Purposes (8th edition, 2013) and the National Statement on Ethical Conduct in Human Research (July 2018).

Animals and parasites

Experimental S. japonicum infection in a mouse model was performed as previously described [42]. Briefly, S. japonicum cercariae (Philippine strain, SjP) were shed from parasite-infected Oncomelania hupensis snails under light stimulus. Eight-week-old female BALB/c mice (n = 3) were infected percutaneously with 80 ± 5 S. japonicum cercariae. Mice were euthanised at 7 weeks post-infection (p.i.). Blood samples were collected by tail vein cut and serum separated by centrifugation at 4,000 rpm for 10 minutes. Serum samples were collected from naive mice using the same method and served as controls. Serum samples were collected from a New Zealand white rabbit infected with approximately 1,000 S. japonicum cercariae (China strain, SjC) prior to infection and at 6 weeks p.i.

Study cohort, sample collection, processing and storage

During Oct 2022, study participants (aged 2 to 65 years old) living in the endemic village of Ekiran in Leyte province, the Philippines, were consented and enrolled (n = 599). Individuals who have lived in the study area for less than two years were excluded. For each participant, three stool samples were sought on different days. For blood sample collection, 10 ml blood (1 to 2 ml from children aged 2 to 5 years) was collected from each individual. Blood samples were allowed to clot for at least 30 minutes followed by centrifugation at 1,500 g for 10 minutes. The separated serum was aspirated and aliquoted into 2 ml cryotubes, and stored at -80°C. Serum samples (n = 50) collected from a rural area non-endemic for schistosomiasis in Kabale District, Uganda served as negative controls.

Parasitological detection

Two KK slides were prepared for each stool sample. The stool sample was sieved by pressing it through a thin gauze. Approximately 50 mg of the sieved stool was placed on a microscope slide using a volume template. A piece of cut green cellophane soaked in glycerol was placed over the sample and then pressed firmly to spread the faeces. The slides were left for at least 30 minutes and then placed in a slide box for storage. The KK slides were prepared by laboratory staff and read by trained microscopists at RITM, the Philippines.

Recombinant proteins

Four recombinant proteins, rSj23-LHD (GenBank Accession No. XBP28865.1), rSjSP13-V1 (GenBank Accession No. UTN00450.1), rSjSAP4 (GenBank Accession No. UTN00449.1), and rSjSAP5 (GenBank Accession No. XBP28864.1) were produced in a previous study [43]. For producing recombinant protein SjSP13-V2 (GenBank Accession No. UTN00451.1), primers (forward: 5’-AAGGATCCCTTGAAAATTCTGTGTCACC-3’ and reverse: 5’-CTGCTCGAGAATAGTGAATTGAACTAGAAACTTC-3’) were used to amplify a specific region of the target from cDNA isolated from SjP adult worms by PCR. The DNA fragment was cloned into the pET-28a plasmid using BamHI and XhoI digestion sites. After transformation into the competent Escherichia coli strain JM109, the recombinant plasmids were extracted from multiple colonies and sequenced by the Sanger method. One correct plasmid was transformed into the competent E. coli strain BL21 (DE3). Expression of the rSjSP13-V2 was induced by 0.5 mM isopropyl β-D-1-thiogalactopyranoside (IPTG) at 37°C for 4 hr. The rSjSP13-V2 protein was purified under denaturing condition as previously described [43].

Enzyme-linked immunosorbent assay (ELISA)

Indirect ELISA was performed as previously described [43, 44]. Briefly, serum samples were diluted (1:100) with 1% BSA blocking buffer (PBS with 0.05% Tween 20, 1% BSA), then stored at 4°C until use. Wells of the MaxiSorp 96-well plates (Thermo Fisher Scientific, Australia) were coated with 100 µl 1 µg/ml recombinant protein in coating buffer and incubated overnight at 4°C. Plate contents were discarded and the plates were patted dry on tissue paper, then blocking buffer (130 µl) was added to each well and allowed to block for 1 hour at 37°C. Diluted sera (1: 100, 100 µl) were added to their corresponding wells in duplicate. The plates were then incubated at 37°C for 1 hr. The secondary antibody, a mouse monoclonal anti-human IgG Fc specific)-biotin antibody (Sigma-Aldrich Co, MO, USA) was diluted with the blocking buffer and added to each well (1:20,000, 100 µl/well). After incubation at 37°C for 1 hr, the streptavidin-HRP (BD Biosciences, CA, USA) was diluted with the blocking buffer and added to each well (1:10,000,100 µl/well). Plates were then incubated at 37°C for 30 min prior to adding 100 µl of TMB substrate mixture for color development, which was stopped by adding 50 µl of 2M H₂SO₄ to the wells. For each plate, the optical density at a wavelength of 450 nm (OD_450nm) was measured using a microplate reader (PowerWave XS2, BioTek, VT, US). After incubation with human sera, the plates were washed with PBST for 5 times between each step. ELISA cut-off value was calculated as 2.1× of the mean OD_450nm of all the negative controls with outlier removed.

Preparation of LFIA cassettes

An LM-based LFIA cassette was designed (Fig. 1) and then fabricated by Zoonbio Biotechnology (Nanjing, China) as previously described [28], with a modification in the conjugate labelling. In the current cassette, red LMs (300 nm) were used in place of colloidal AuNPs to conjugate rSjSAP4.

LFIA test and analysis

To determine the optimal serum diluent, a pooled serum sample mixed from S. japonicum-infected individuals (n = 3) was diluted 1:10 with five buffers, including PBS, PBS with 0.05% Tween 20, PBS with 1% BSA, 0.9% NaCl, and 2.5% sucrose with 1% Tween-20. To test the LFIA cassette, 50 µl of each diluted serum sample was dispensed into the sample well of the cassette. The strip results were captured by a smartphone after 10 minutes and every five minutes thereafter, till 25 minutes.

To determine the best serum dilution ratio, PBS and 2.5% sucrose with 1% Tween-20 were selected as diluents. Three sera from KK-positive [KK (+)] individuals (n = 3) showing different levels of anti-rSjSAP4 IgG antibodies and one serum sample from a non-endemic control (n = 1) were diluted at different ratios (1:10, 1:100, 1:1000 and 1:10000). Fifty µl of each diluted serum sample was then tested with the LFIA cassette. The results were captured by a smartphone at 20 min after sample loading.

For formal test of the LFIA cassettes, PBS was used as diluent and 1:10 was used as dilution factor. The LFIA results were captured by a smartphone (iPhone 8) at 20 min after sample loading. For result analysis, all images were uploaded to a computer and profile intensity plot of the test images were generated using the Java-based image-processing program ImageJ 1.53 to quantify intensity of developed T and C lines. Quantitative results were produced by introducing an R-value, which was defined as the intensity ratio between the T line vs. C line, for each test.

Statistical analysis

Statistical analyses were performed using GraphPad Prism version 10.2.3 software (GraphPad Software, Inc., San Diego, CA, USA), with the significant p-value set as < 0.05. For ELISA and LFIA tests, a comparison between the KK (+) and control samples was performed using the Mann-Whitney U test. The Kruskal-Wallis test followed by Dunn’s comparison was used to determine the difference in R-values among different groups. Receiver operating characteristic (ROC) curve analysis was performed to measure the overall performance of the developed diagnostics. The cut-off R-value for the LFIA was set as the maximum Youden index. Correlation between the rSjSAP4-ELISA and LFIA results was assessed using the Spearman's rho (ρ). The qualitative agreement between the rSjSAP4-ELISA and LFIA tests was measured by the kappa statistic (https://www.graphpad.com/quickcalcs/kappa1/).

Diagnostic performance of five S. japonicum recombinant antigens determined by indirect ELISA

Indirect ELISA was performed to detect IgG response to five S. japonicum recombinant antigens, including the tegumental-associated protein Sj23-LHD and four saposin proteins SjSP13-V1, SjSP13-V2, SjSAP4, and SjSAP5 (Fig. 2). Mean IgG levels against all except SjSP13-V1 were significantly higher in the KK (+) individuals (n = 28) compared with those from the controls (n = 12). The highest seropositivity among KK (+) individuals was achieved in the rSjSAP4-ELISA with a sensitivity of 85.7%, and with no positivity in non-endemic sera. Both the rSjSP13-V1-ELISA and rSjSAP5-ELISA yielded a high sensitivity of 82%, but three control samples had positive reactions resulting in a specificity of 75% for these assays. In contrast, the rSj23-LHD-ELISA and rSjSP13-V1-ELISA had poor diagnostic performance, detecting only 29% and 21% of infected samples, respectively.

Optimisation of testing parameters for the LM-based LFIA

Three serum samples from KK (+) individuals showing different anti-rSjSAP4 IgG levels in SjSAP4-ELISA were equally pooled. The pooled serum was then diluted 1:10 with five different buffers, including PBS, PBS with 0.05% Tween-20 (PBST), PBS with 1% BSA, 0.9% NaCl, and 2.5% sucrose with 1% Tween-20. Each diluted serum sample was tested with the LFIA cassette, and the results were imaged at different time points and analyzed with the ImageJ program to generate R-value curves over time (Fig. 3A). For all diluents tested, the R-value peaked at 20 minutes after sample loading, with the sample diluted with PBS exhibiting the highest R-value over different time points (Fig. 3A). In terms of the intensity of the T line, the pooled serum sample diluted in the sucrose buffer yielded the highest value.

Two diluents, PBS and 2.5% sucrose with 1% Tween-20, were further tested for optimal dilution factor using sera from three infected individuals showing low, medium and high anti-rSjSAP4 IgG levels, respectively, and a non-endemic control. Using PBS as the diluent, the R-values of infected samples decreased with increasing dilution factor while the non-endemic control serum remained stable (Fig. 3B). Sera with high and medium anti-rSjSAP4 IgG levels were positive up to the highest dilution tested (1:10,000); while the serum with low anti-rSjSAP4 IgG level was positive up to the dilution of 1:100 (Fig. 3B, a test was determined as positive when the R-value was higher than 2.1 times of the R-value of the control sample at the same dilution). When diluted with 2.5% sucrose containing 1% Tween-20, R-values were decreased for the three serum samples with increasing dilution factor (Fig. 3C). Notably, the control serum diluted in the sucrose buffer also exhibited high R-values at dilution of 1:10, 1:100 and 1:1000, with a peak at dilution of 1:1000 (Fig. 3C), indicating a non-specificity issue for the diluent. These results thus indicate that 1:10 is an optimal dilution factor, and PBS is the optimal diluent for testing the developed LFIA strips.

Formal test of the developed LFIA cassette

The LFIA cassettes were further tested with sera from KK (+) (n = 139), KK-negative [KK (-)] individuals (n = 410) and non-endemic controls (n = 50) using the optimised testing conditions (1:10 dilution with PBS). Figure 4A shows LFIA cassettes displaying an increment in R-value from left to right. The difference in R-values between the controls and the target cohort stratified by infection status and/or intensities were assessed. The R-values were significantly higher in subgroups with EPG > 100 (n = 11, p = 0.002), EPG 50–100 (n = 15, p < 0.0001), EPG 10–49 (n = 39, p < 0.0001), and EPG < = 10 (n = 74, p < 0.0001), as well as the KK (-) individuals (n = 410, p = 0.01) compared with the non-endemic controls (n = 50) (Fig. 4B). In the target cohort, all KK (+) subgroups showed a higher R-value compared with the KK (-) group (p = 0.002, p < 0.001, p < 0.001, p < 0.001 for groups with EPG > 100, 50–100, 10–49, and < = 10, respectively) (Fig. 4B). There are no differences in R-value among different KK (+) subgroups.

Diagnostic performance of the developed LFIA cassette

By testing samples from the KK (+) individuals (n = 139) and non-endemic controls (n = 50), the LFIA exhibited a sensitivity of 80.6% and a specificity of 98.0% at a cut-off R-value of 0.1031 (Fig. 5A). The SjSAP4-ELISA tested with the same serum samples showed 82.0% sensitivity and 100.0% specificity (Fig. 5B). ROC curve analysis for the LFIA and rSjSAP4-ELISA determined an area under the ROC Curve (AUC) value of 0.8999 (95% CI, 0.8569 to 0.9428, p < 0.0001) and 0.9593 (95% CI, 0.9334 to 0.9851, p < 0.0001), respectively (Fig. 5C and D). In the whole cohort, 266 and 248 serum samples were tested positive by the LFIA and rSjSAP4-ELISA, respectively (Table 1). The agreement between the LFIA and rSjSAP4-ELISA was substantial with a kappa value of 0.795 (95% CI, 0.745–0.846) (Table 1). Similarly, the R-values from the LFIA tests and OD values from rSjSAP4-ELISA exhibit a significant positive correlation (p < 0.0001) with an r value of 0.8269 (95% CI, 0.7989–0.8513) (Fig. 5E).

Table 1

Results of testing 549 serum samples collected from an endemic area for *S. japonicum* with the LFIA cassettes and rSjSAP4-ELISA assay.
		SjSAP4-ELISA			Kappa index (95% CI)
		Positive	Negative	Total	Kappa index (95% CI)
LFIA	Positive	229	37	266	0.795 (0.745–0.846)
LFIA	Negative	19	264	283
	Total	248	301	549

Applicability of the LFIA cassette across S. japonicum strains and host species, and cross-reactivity test

The developed immunochromatographic cassette was performed on a serum sample from a human subject infected with the China strain of S. japonicum (Fig. 6A), demonstrating its applicability in the detection of SjC infection. Further, the cassettes were tested with serum samples from SjC-infected rabbit (n = 1, 6 weeks pi) and SjP-infected mice (pooled, n = 3, 7 weeks pi), showing an R-value of 3.439 and 0.2384, respectively (Fig. 6A), indicating its compatibility in the detection of S. japonicum infection in other mammalian hosts. In addition, the developed LFIA strips were tested using sera from Uganda individuals infected with hookworm, A. lumbricoides and T. trichuira, and showed no cross-reactivity with these helminth species (Fig. 6B).

S. japonicum remains highly endemic in a number of areas in the Philippines despite decades of control efforts centered on case management and preventative chemotherapy with praziquantel through MDA [6, 45]. Integrated controls with the aim of eliminating the disease in humans, animals and the environment have been initiated in the Philippines by the Department of Health (DOH) through the Schistosomiasis Control and Elimination Program (SCEP), addressing to the complex epidemiology of the disease [46]. One of these crucial efforts is to develop affordable, accurate and field-applicable diagnostics for screening, monitoring and surveillance [46]. The traditional KK stool microscopy, although field-friendly, was proven to be not sufficiently sensitive to identify the true infection status in individuals with low infection schistosome burden [9, 47, 48]. Developing cost-effective and accurate POC diagnostics, which has been given priority in the SCEP Strategic Plan [49], has the potential to meet this need.

The current study aimed to develop an AbD-based LFIA for the diagnosis of S. japonicum infection by targeting recombinant antigens, where the identification of sensitive and specific antigens is a key step. Using ELISA, we evaluated the diagnostic applicability of five S. japonicum recombinant antigens. The present results are in agreement with our previous study involving a cohort in Northern Samar, the Philippines, in terms of sensitivities for the rSjSAP4- and rSjSAP5-ELISAs [43]. For example, the rSjSAP4-ELISA had a sensitivity of 82.0% in this study, close to 84.3% as observed in the previous study [43]. Seropositivity for IgG antibodies against SjSAP5 and SjSP13-V2 was also highly sensitive, identifying > 80% of the infected individuals, but showed positivity in some samples collected from a rural area non-endemic for schistosomiasis, resulting in a specificity of 75% for both antigens, contrast to that the rSjSAP5-ELISA showed a specificity of 96.7% when the serum samples collected from donors living in an urban area not endemic for schistosomiasis were used as controls [43]. However, the rSjSAP4-ELISA retained a high specificity (100%) in this current study as previously observed [43]. These results thus further confirmed rSjSAP4 as the most suitable antigen in the detection of S. japonicum infection among these antigens, with considerable sensitivity and notably no cross-reaction with samples from a rural area non-endemic for schistosomes in Uganda.

We have previously reported the development and assessment of an rSjSAP4-incorporated GICA using AuNPs as the detector probe [28, 30]. Typically, as probes to be conjugated in LFIA, the LMs are more sensitive, specific, stable, and eco-friendly than AuNPs [40, 48, 50]. Here, we developed an LFIA with red LMs instead of AuNPs as the colorimetric detector using a similar format as adopted in the previous developed GICA. By testing a pooled serum sample, optimisation of the test condition for the developed LFIA validates PBS as the optimal diluent based on the observed R-values at different read points, with the optimal read time being 20 minutes as observed peak R-values. Serum sample with low anti-SjSAP4 IgG level remained positive up to a dilution of 1:100. With an optimal cut-off value determined by maximizing the Youden’s index, the LFIA had a sensitivity of 80.6% and specificity of 98.0%, exhibiting a similar diagnostic performance with the previously developed GICA strip, which has a sensitivity of 83.3% and absolute specificity [30]. As expected, the LFIA cassette showed applicability in testing individuals infected with the Chinese strain of S. japonicum as SjSAP4 orthologs between SjP and SjC shared 99% identity.

After sample loading, the LFIA test results must be read within a limited time. There are a number of methods that can be used to read strip results, including the naked eye, reference to an interpretation card [51], chromogenic test reader/analyzer [52, 53], or image processing programs, such as Image Studio Lite [54] and ImageJ [28]. Direct interpretation of results by the naked eye is simple and straightforward but can be subjective due to differences in the reader’s visual acuity and/or training, particularly in the reading of trace results [55]. The use of a colour interpretation card as an auxiliary reference for the result readout converts the assay to be a semi-quantitative test, but still engaging in visual judgment. In contrast, quantification of color intensity by a chromogenic test reader or image processing algorithms can enable the assay to be accurately interpreted. In this study, the LFIA results were captured by a smartphone and further analyzed by the ImageJ [23]. An R-value then was introduced to eliminate the inter-reader variability and convert the assay results into quantitative data, which can minimise the system errors such as the difference in sample absorbance rates. This also enabled us to assess the correlation between the LFIA and rSjSAP4-ELISA results, which showed a significant positive correlation (r = 0.8269), indicating the applicability of the LFIA test as a field-friendly qualitative alternative test to ELISA without the demand of sophisticated equipment.

Currently, there are two major designs used for the development of AbD-based LFIA assays for the diagnosis of schistosomiasis. For the first design, gold-conjugated recombinant streptococcal protein G (rSPG) is used as detector probe on the conjugate pad, while the schistosome-derived antigen and rSPG are employed to form the T and C lines, respectively [56, 57]. For the second design, the gold-conjugated mouse anti-human IgG is sprayed onto the conjugate pad, and the schistosome antigen and goat anti-mouse IgG are used to shape the T and C lines, respectively [29, 51]. The first design can be applied for the detection of schistosomiasis in both humans and domestic animals [57], while the second format can only be used for testing human schistosomiasis. The current LFIA cassette adopted a third format, in which the latex microsphere-labeled rSjSAP4 was used as detector probe on the conjugate pad, while protein G and anti-His tag mAb were immobilized on the T and C lines, respectively (Fig. 1). Theoretically, this format enables to detect anti-rSjSAP4 IgG antibodies across mammalian hosts. Indeed, the developed LFIA showed the applicability in the detection of the S. japonicum infection in rabbit and mouse, however, varied R-values were observed between the two models (Fig. 5A), probably due to the differences in infection intensity and/or binding affinity of protein G with Abs originating from different mammals [58]. More than 40 mammalian animals such as bovines, goats and pigs, serve as reservoir hosts for S. japonicum [1], playing a key role in the transmission of the schistosomiasis japonica. The applicability and reliability of the developed LFIA in the detection of S. japonicum infection in other reservoir hosts, needs to be validated before its field application.

Consistent with previous observations [30, 48], the developed LFIA showed a high concordance with the rSjSAP4-ELISA since the both assays employed the same target antigens. Nevertheless, the results for a portion of samples remains inconsistent between the two methods. A total of 37 (6.74%) serum samples tested positive by the developed LFIA but negative by the rSjSAP4-ELISA, which may be caused by the fact that the LFIA probes all types of Abs against the target antigen, while the rSjSAP4-ELISA detected IgG Ab only. In contrast, 19 (3.46%) serum samples were positive in the rSjSAP4-ELISA, but came out negative by the LFIA test, which may be caused by different formats of rSjSAP4 used in the two tests, i.e. rSjSAP4 merged with a fragment encoded by plasmid vector was used in the ELISA assay, while the fragment has been trimmed from the rSjSAP4 incorporated in the LFIA.

Incorporating crude antigen lysate, such as soluble egg antigen (SEA) or somatic antigen, in the development of LFIA can cause cross-reaction to other parasitic flukes or soil-transmitted helminths [56, 57, 59]. For example, Rodpai et al [51] employed somatic extract from adult S. japonicum as target antigen for the development of an immunochromatographic test (Sj-ICT), which showed cross-reactions with fluke cases, such as Opisthorchiasis viverrini, Clonorchiasis sinensis, and Paragonimiasis heterotremus, and other infection of foodborne helminths. The use of purified recombinant antigens in immunoassays enables reduction of non-specific binding observed when using crude antigens. Previously, it has been demonstrated that the recombinant protein, rSjSAP4 showed no cross-reaction with alveolar echinococcosis and trichinellosis samples [60]. In addition, homologous analysis also predicted a low chance of SjSAP4 to be cross-reactive with other parasitic flukes, such as C. sinensis and Fasciola hepatica, although this still needs to be tested [28]. This study further confirmed that the rSjSAP4-incorprated LFIA cassette also had no cross-reaction with samples from individuals infected with hookworm, A. lumbricoides, and T. trichuira. Nevertheless, it is necessary to examining cross-reactions with other helminths, particularly other trematodes, for the LFIA.

We developed an LM-based LFIA by incorporating rSjSAP4 and assessed its performance for the diagnosis of schistosomiasis japonica in a cohort residing in Barangay Ekiran, Leyte province, the Philippines. Targeting the KK (+) individuals, the developed LFIA had a sensitivity of 80.6% and a specificity of 98.0% using the non-endemic subjects as controls, displaying a similar diagnostic ability of the previously developed GICA targeting the same antigen rSjSAP4. The established LFIA and rSjSAP4-ELISA showed high consistency in the detection of S. japonicum infection. The prevalence of schistosomiasis japonica determined by the methods was about 1.8 times higher than obtained with the KK procedure performed on three stool samples. The developed LFIA can potentially be used a powerful diagnostic tool for screening of S. japonicum infection in humans and animal reservoirs in rural-endemic areas with limited access to equipment and supplies.

Funding

This work was supported by an e-ASIA Joint Research Program with grants from the National Health and Medical Research Council (NHMRC) of Australia (APP2008433), the Philippines Council for Health Research and Development (Grant No. FP 220025) and the Japan Agency for Medical Research and Development (Grant No. JP21jm0210091). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Acknowledgements

We thank all participants who provided clinical samples and acknowledge the hard work and dedication of the local field and clinical staff who collected samples and undertook field studies. We thank Mary Duke (QIMR Berghofer Medical Research Institute) for the maintenance of the S. japonicum life cycle and assistance in animal infection. We thank Prof Jing Xu (National Institute of Parasitic Diseases, Shanghai, China) for gifting the human sera infected with S. japonicum China strain. We thank Fanzhen Mao (Jiangsu Institute of Parasitic Diseases), for providing and testing rabbit sera samples.

Author Contributions

Conceptualization: DPM and PC. Data curation: EJT, CAG, YM, MJ, MI, EA, TGE, MA, and PC. Formal analysis: EJT, CAG, YM, MJ, MI, EA, and PC. Funding acquisition: MJ, YS, and PC. Investigation: EJT, CAG, YM, MJ, MI, EA, DG, MLM, DPM, TGE, MA, YS, MOS, MS, HY, MK, and PC. Methodology: EJT, CAG, YM, KY, MJ, MI, EA, DG, MLM, DPM, TGE, MA, YS, MOS, MS, and PC. Project administration: CAG and PC. Resources: CAG, MJ, MI, EA, DG, MLM, DPM, TGE, MA, and PC. Supervision: PC. Visualization: EJT, YM, and PC. Original draft: EJT, YM, and PC. Review & Editing: CAG, MJ, MI, EA, DG, MLM, TGE, MA, YS, MOS, MS, HY, MK, and PC. The authors read and approved the final manuscript.

Competing interests

The authors declare no conflict of interests.

Availability of data and materials

The original contributions presented in the study are included in the article.

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Development of a latex microsphere-based lateral flow immunoassay for the diagnosis of schistosomiasis japonica

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Abstract

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Introduction

Materials and Methods

Ethics

Animals and parasites

Study cohort, sample collection, processing and storage

Parasitological detection

Recombinant proteins

Enzyme-linked immunosorbent assay (ELISA)

Preparation of LFIA cassettes

LFIA test and analysis

Statistical analysis

Results

Optimisation of testing parameters for the LM-based LFIA

Formal test of the developed LFIA cassette

Diagnostic performance of the developed LFIA cassette

Discussion

Conclusions

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References

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