Home-made lateral flow test strip versus POC-CCA assay for detection of active schistosomiasis in Egypt

Home-made lateral flow test strip versus POC-CCA assay for detection of active schistosomiasis in Egypt | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Home-made lateral flow test strip versus POC-CCA assay for detection of active schistosomiasis in Egypt Manal Kamel, Faten Salah, Zeinab Demerdash, Sara Maher, Hanan El-Baz, and 5 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4535875/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 02 Oct, 2024 Read the published version in Acta Parasitologica → Version 1 posted 9 You are reading this latest preprint version Abstract For years, the Kato-Katz (KK) technique has been considered the gold standard for diagnosing schistosomiasis. The aim of this study was to compare the effectiveness of our previously developed gold nanoparticle-based lateral flow test strip (AuNPs-LFTS) for diagnosing active Schistosoma mansoni with that of the commercially available point-of-care Circulating Cathodic Antigen detection (POC-CCA) kit. In this study, we collected sixty positive and twenty negative urine samples from patients in endemic hot spots in the Nile Delta, as well as from patients visiting the internal medicine clinic at Theodor Bilharz Research Institute (TBRI). We produced monoclonal antibodies (MAbs) against S. mansoni soluble egg antigen (SEA) from cloned hybridoma cells (4D/1D). These MAbs were conjugated with gold and mesoporous silica nanoparticles, and used to develop the LFTS. The LFTS demonstrated a limit of detection (LoD) of 3 ng/ml. The sensitivity and specificity of the developed LFTS were found to be 96.7% and 95%, respectively, compared to 85% and 90% for the POC-CCA detection kit. The cases were divided into groups based on egg count in the stool, categorized as light, moderate, and heavy infections. The sensitivity of the LFTS in the group with light infection was higher than that of the POC-CCA. When using the KK technique (eggs per gram of stool sample [EPG]) as the reference test, the kappa value for the nano-based strips was 0.902, compared to 0.672 for the CCA strips, indicating an almost perfect agreement between KK and our developed LFTS. These results confirm the reliability and effectiveness of the LFTS compared to commercially available kits for rapid, sensitive, and early diagnosis of schistosomiasis. However, it is recommended to conduct further assessments of the developed strip on a larger scale with a broader range of cases before considering its introduction to local or international markets Schistosomiasis monoclonal antibodies Nanoparticles LFTS CCA Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Introduction Schistosomiasis is one of the major neglected health problems in Africa, affecting nearly 220.8 million people in 78 countries worldwide, necessitating preventive treatment. Early diagnosis is crucial for effective control programs and preventive treatment to reduce and prevent morbidity [ 1 ]. The Kato-Katz (KK) method is considered the gold standard for diagnosing schistosome eggs in stool samples. However, it has several limitations [ 2 – 4 ]. Despite years of research, there has been a continuous pursuit to find a rapid and sensitive one-step diagnostic test for schistosomiasis as an alternative to conventional microscopic detection methods. Since 1995, researchers at Theodor Bilharz Research Institute (TBRI) in Cairo, Egypt, have developed a panel of monoclonal antibodies using hybridoma technology against various Schistosoma antigens [ 5 , 6 ]. Initially, monoclonal antibodies were employed in a sandwich enzyme-linked immunosorbent assay (ELISA) to detect circulating antigens in urine and serum samples as diagnostic probes for early active infections. While this method improved the sensitivity and specificity of diagnosis and overcame the drawbacks of previous conventional methods, it was not practical for routine screening due to its multiple steps, long reaction time, and limited suitability to well-equipped laboratories [ 7 , 8 ]. Recently, there has been growing interest in the use of immune-strip assays as an alternative method, which offer a one-step, user-friendly approach with a visual endpoint [ 3 , 9 ]. The detection of circulating cathodic antigen (CCA) using urine strips has been widely applied for routine S. mansoni infection detection [ 10 ]. In 2004, van Dam et al. reported that CCA detects a specific parasite gut carbohydrate antigen regurgitated by adult worms, which circulates in the bloodstream, is eliminated by the kidneys, and can be detected in the urine of infected individuals [ 11 ]. However, ELISA still demonstrated higher sensitivity than CCA, resulting in multiple false positive results. Many studies have focused on improving CCA, and researchers have explored the use of colloidal gold nanoparticles (AuNPs) to enhance the sensitivity of conventional immune-strip assays, enabling rapid and cost-effective detection of infectious agents compared to current available technologies [ 12 – 15 ]. In 2016, Kamel et al. found that loading monoclonal antibodies onto gold nanoparticles increased the specificity and sensitivity of monoclonal antibody-based sandwich ELISA to 100% and 98.7%, respectively [ 16 ]. Gold nanoparticles are widely used as they can be easily functionalized to produce different colors depending on their size, aggregation state, and shape, making them excellent candidates for colorimetric biosensor development [ 17 , 18 ]. Additionally, mesoporous silica nanoparticles (MSN) of the mobile crystalline material (MCM)-41 type offer efficient immobilization of proteins on nitrocellulose membranes, ensuring good efficiency, immune reactivity, and stability of the immobilized protein [ 19 ]. In 2019, Kamel et al. developed a convenient and sensitive gold nanoparticle-based lateral flow test strip (LFTS) assay for rapid detection of soluble egg antigen (SEA) from S. mansoni in serum and urine samples of schistosomiasis-infected patients, utilizing colloidal AuNPs and MSN [ 20 ]. After large-scale production, purification, and characterization of the produced monoclonal antibodies (4D/1D), the LFTS was constructed using gold-conjugated detector monoclonal antibodies and MCM-41-conjugated capture monoclonal antibodies for immobilization on the nitrocellulose membrane. Through optimization and standardization of working conditions, we tested the developed strip using serum and urine samples from infected patients. Our previous results were very promising, with a specificity of 97.5% and sensitivity of 98.3% in urine. Study design and sample collection This study aims to compare the effectiveness of the developed LFTS with the commercially available POC-CCA kit for the rapid detection of S. mansoni SEA in urine samples of infected patients as a validation step for large-scale application in routine diagnosis. Samples were collected from endemic hotspots in the Nile Delta, specifically Elkhamseeny and Sandala villages in Kafr Elsheikh Governorate. All patients were above 18 years old. Three hundred (300) stool samples were screened using the KK technique for S. mansoni infection. Only 60 cases tested positive and were included in this study, along with 20 negative cases used as a control group. The included cases were sex-matched and aged from 12 to 30 years old. The positive cases were subdivided according to the number of eggs per gram (EPG) into the light infection group (12 cases) (< 50 EPG), moderate infection group (11 cases) (51–100 EPG), and heavy infection group (37 cases) (≥ 100 EPG). Urine samples from all included subjects were collected, centrifuged for 5 minutes at 2000 rpm, and then the supernatant was collected and stored at -20°C until used Methods Production of S.mansoni SEA-mAbs Hybridoma cells secreting mAbs (4D/1D), which were raised against S. mansoni SEA, were developed and cryopreserved at the Immunology Department of TBRI in Cairo, Egypt. The mAbs (4D/1D) were characterized as IgG1 kappa-type light chain antibodies that recognize repetitive epitopes on SEA, allowing their use as both antigen-capturing and detecting antibodies in sandwich assays. The reactivity of the hybridoma cells against SEA was checked by indirect ELISA after revival and propagation in a culture medium. Large-scale production of mAbs was maintained by injecting hybridoma cells (2x106/ml) intraperitoneally into BALB/c mice to develop ascitic fluid. The produced mAbs were then purified by ammonium sulfate precipitation according to Nowotny [ 21 ], followed by treatment with caprylic acid according to McKinney and Parkinson [ 22 ]. Preparation of nanoparticles conjugated mAbs Purified mAbs were passively conjugated with AuNPs according to Tanaka et al. In brief, 30 µg (12 µL) of mAbs solution (2.5 mg/mL) was diluted with KH2PO4 solution (5 mM) in ultra-pure water (200 µL) at pH 7.5. The diluted solution was added to AuNPs (1.8 µL, 20 nm) and immediately mixed, then left to rest for 20 minutes at room temperature (RT). A blocking step was performed using 200 µL of 10% bovine serum albumin (BSA w/v), followed by centrifugation at 4°C for 10 minutes at 8000 g. Pulse sonication for a few seconds was applied, and the conjugated mAb-AuNPs were added to 0.05% and 20 mM Tris-HCl buffer, 2 mL of preserving solution (pH 8.2, 1% [w/v] BSA), and then stored at 4°C until used [ 12 ]. The conjugation step of mAbs with MCM-41 type silica nanoparticles was performed according to Omidfar et al. In brief, 1 mg of MCM-41 was dispersed in 1 mL of phosphate buffer solution (PBS) (pH 7.2, 0.1 M). Then, 181.5 µL of the MCM-41 solution was added to 18.1 µL (45.3 µg) of mAb (4D/1D) (2.5 mg/mL) in a 4:1 ratio. The mixture solution was left at 4°C overnight. A blocking step for non-specific binding sites was performed by adding 200 µL of 10% BSA (w/v) in PBS (0.1 M) at pH 7.2. The solution was then centrifuged at 4°C for 4 minutes, the supernatant was discarded, and the sediment pellet was dispersed in PBS (pH 7.2, 0.1 M). The MCM-41-mAb conjugate was stored at 4°C before use [ 23 ]. Fabrication of the AuNPs-LFTS Fabrication of the LFTS was conducted according to our previous work by Kamel et al. In brief, the test strip is composed of a nitrocellulose membrane, sample pad, and absorbent pad. Several optimization trials were performed before determining the standard conditions of the test [ 20 ]. Test line and control line solutions were prepared and standardized. For this patch, the optimal concentration of the capture conjugate (MCM-41-mAbs) on the test line was found to be 5 µL/strip. A control line solution (anti-mouse IgG, 2 mg/mL) was also prepared and dispensed on the nitrocellulose membrane (3 µL/strip). The nitrocellulose membrane was dried at room temperature for 1 hour, then immersed in a 50 mM boric acid buffer (skim milk [0.5% [w/v], pH 8.5) for 25 minutes to block nonspecific adsorption. Finally, the membrane was cut into specific sizes (0.5 cm wide, 6 cm long), and some strips were placed into plastic housings. All strips were stored in a plastic bag at 37°C until use. The urine sample was mixed with the detector probe (AuNPs-mAbs) in an external tube at a ratio of 10:1 (50 µL of urine sample to 5 µL of AuNPs-mAbs) (Fig. 1 )." Application of sandwich based LFTS Urine samples from the studied groups were subjected to the LFTS assay to detect the presence of circulating S. mansoni antigen. All urine samples were also assayed in parallel using 96-well microtiter plates. Briefly, 5 µL of the AuNPs-mAbs conjugate (detector probe) and 50 µL of each urine sample were mixed in separate wells. The test strip's sample pad was then immersed in the well, allowing the mixture to be absorbed and moved by capillary force, forming a complex with the MCM-41-mAbs employed as the capture probe on the test line. In the presence of the antigen, a red color will appear. The control line will turn red after binding with the anti-IgG antibody to any unbound free conjugates. The test results were obtained after 10 minutes. The test result is considered invalid if no red color appears on the control line, regardless of the color on the test line. Application of POC-CCA A point-of-care circulating cathodic antigen detection kit (Rapid Medical Diagnostics, Pretoria, South Africa, RSA) was used to test urine samples from the studied groups for the presence of circulating S. mansoni antigen. Briefly, 100 µL of urine (equivalent to 2 drops) were transferred to the test cassette by gently squeezing the pipette. The sample was completely absorbed into the specimen pad within the circular well, and then 1 drop of buffer was added. The test results were read after 20 minutes. The intensity of the red color on the produced test line for both strips was visually assessed using a gel documentation system (Gel Doc XR+, Biorad, USA). The results were then analyzed using Image Lab software version 3. Statistical analysis Statistical analyses were performed by using the analytical software package IBM-SPSS (version 23). Characteristics of LFTS strip and CCA assays were tested by using receiver operating characteristic curve (ROC). CCA strip and nano-based strip were compared to the EPG test (reference test) based on the following accuracy measure: specificity, sensitivity and Cohen’s kappa statistic (κ) of agreement. According to Kolmogorov-Smirnov test, the data of the intensities of CCA strip and LFTS were not normally distributed. Accordingly, non-parametric tests were used to statistically analyze the data. The association in results between reference test and the immuno-strip assays was detected by Chi-square test. Statistical differences in the intensities between positive and negative groups were compared by using Mann-Whitney U test. Results Kato-Katz technique and infection intensity Based on the KK technique, the level of infection intensity was classified as light (12 cases), moderate (11cases), or heavy (37 case), with thresholds set at < 50 EPG, 51–100 EPG, and ≥ 100 EPG, respectively. Comparative accuracy measures of LFTS and POC-CCA strip Upon application of both assays to detect CSA (target antigen) in urine samples of subjected groups, we observed that 58 out of 60 positive KK confirmed cases were found positive by LFTS while 19 out of 20 of the control group were confirmed negative demonstrating a sensitivity and specificity of 96.7% and 95% respectively. For the POC-CCA assay, 51 out of 60 S.mansoni infected cases were positive while 18 out of 20 control cases were negative exhibiting sensitivity and specificity of 85% and 90% respectively. Furthermore, the kappa value of LFTS with EPG (as a reference method) was 0.902, reflecting an almost perfect agreement between the two assays. On the other hand, POC-CCA strips showed a substantial agreement with EPG where the kappa value was o.672 ( Table 1 ) . Gel documentation system was used to quantitative measure the color intensity of the test line for both assays, where the intensity is directly proportional to the concentration of the antigen in the tested samples. Intensities were displayed as a volume x10 5 using Image Lab software. The receiver operating characteristic (ROC) curve was used for the detection of intensities of both LFTS and CCA strips using the same urine samples with EPG as a reference test is displayed in Fig. 2 . According to ROC analysis, in CCA strips, the area under the curve (AUC) was found 0.968, with an optimal cut-off value of 0.73. On the other hand, AUC in LFTS strips was 0.998, with a 0.68 optimal cutoff value indicating that both strips have a high ability to discriminate the positive from negative cases. However, the sensitivity and specificity of the LFTS strip were always higher than that of the CCA strip, using EPG as the reference test. Moreover, the color intensity of the test line in the positive cases and their accuracy measures using both strips are displayed in Fig. 3 , Tables 2 & 3 . Using LFTS, only 2 out of 60 cases were negative and belonged to the light infection group (L). In contrast, 9 out of 60 cases showed negative results when using CCA, encompassing the light (L), moderate (M), and heavy (H) infection subgroups. The sensitivity of LFTS was higher than that of CCA across all infected subgroups, including L, M, and H infections, with a specificity of 100% in the M and H infection groups and 90% in the L infection group. In comparison, the CCA test exhibited specificity of 82%, 82%, and 95% for L, M, and H infections, respectively. Table 1 The accuracy measures of LFTS and CCA strips for CSA detection in urine samples using EPG as a reference test Strips Strip/ EPG (-/-) Strip/ EPG (+/+) Specificity Sensitivity Kappa value χ2 df p-value LFTS 19/20 58/60 95% 96.7% 0.902 65.4 1 0.000 CCA 18/20 51/60 90% 85% 0.672 37.7 1 0.000 Figure. 3 Determination of the visual detection limit of LFTS by using different concentrations of SEA (starting from 500 ng/ml down to 3 ng/ml). The LFTS had a detection limit of 3 ng/ml for SEA. Table 2 Color intensities of positive S. mansoni cases using LFTS and CCA LFTS Color intensity CCA Color intensity Negative Positive Negative Positive Number of cases 2 58 9 51 Total (60) 0.74 ± 0.01 2.37 ± 0.10 0.74 ± 0.02 1.70 ± 0.09 *** Subgroups L (n = 12) 0.74 ± 0.01 (2) 1.64 ± 0.10 (10) 0.75 ± 0.02 (4) 1.03 ± 0.07 (8) * M (n = 11) --- 1.67 ± 0.12 (11) 0.72 ± 0.03 (4) 1.06 ± 0.04 (7) * H (n = 37) --- 2.78 ± 0.10 (37) 0.79 ± 0.00 (1) 1.97 ± 0.10 (36) *** *: significant differences at p < 0.05 ***: p < 0.000, as compared to LFTS. Table 3 Sensitivity and specificity of CCA and LFTS for each subgroup. CCA LFTS L M H L M H Sensitivity 0.80 0.78 0.95 0.91 0.92 0.97 Specificity 0.82 0.82 0.95 0.90 1.00 1.00 Distribution of intensities using LFTS and POC-CCA Strip: The distribution of color intensities using LFTS and CCA strips is shown in Figs. 4 , 5 , and 6 . Both strips were able to differentiate between positive and negative results without any overlapping areas (p < 0.000) (Fig. 4 ). The median value of the LFTS positive test line intensity was significantly higher than that of CCA (p < 0.004), indicating higher resolution and better visual interpretation of the LFTS assay (Fig. 5 ). In cases of low, moderate, and heavy infections, the intensity values measured by the LFTS were consistently higher than those of CCA (Fig. 6 ). When positive samples were tested on both strips, the color intensity on the test band of our developed LFTS was found to be stronger than that on CCA using the same samples (Fig. 7 ). Discussion Despite significant efforts, schistosomiasis remains a considerable health issue in certain countries, particularly in Africa [ 24 , 25 ]. Many authors have mentioned that the sensitivity of the KK method is low, leading to false negative results due to various factors such as low parasitic burden, recent or chronic infections, post-treatment situations, or day-to-day variability in egg excretion in the host's stool [ 27 , 28 , 29 ]. Therefore, immunological methods have recently emerged as a promising alternative, as they rely on direct antigen detection even before egg detection in the stool. The development of point-of-care (POC) tests for accurate screening is essential [ 8 ]. In our previous research, we successfully developed lateral flow tests (LFTS) for the rapid detection of S. mansoni antigen in urine samples. In this study, our objective was to assess and compare the effectiveness of our developed LFTS with the point-of-care circulating cathodic antigen (POC-CCA) kit. Samples (urine and stool) were collected from hot spot areas in Egypt, with the age range of the patients included in this study being between 12 and 30 years old, encompassing school-age children and young adults. This age group is of particular concern due to the concentration of the disease in children and economically active individuals [ 26 , 29 ]. When both assays were tested on the same positive and negative cases, our developed LFTS exhibited a sensitivity and specificity of 96.7% and 95%, respectively, compared to the CCA strips that showed 85% and 90%, respectively. These results align with various other research works that have discussed the lower sensitivity of the POC-CCA assay [ 30 , 31 ]. Furthermore, in 2021, Bezerra et al. reported that the sensitivity and specificity of POC-CCA varied according to the prevalence and intensity of the infection, and that the diagnostic accuracy of the CCA assay was diminished, resulting in a large number of false negative results in low-intensity infections [ 26 ]. Van Dam et al. also reported the lower sensitivity of POC-CCA in cases of light schistosomiasis infection [ 11 ]. Additionally, different research works have mentioned the cross-reactivity of the CCA assay, which contributes to its false positive results [ 10 , 32 ]. In the current study, while 20% (12 cases out of 60) had a light infection (< 50 EPG) as determined by the KK method, only 2 cases (16.6%) showed negative results by the developed LFTS, compared to 4 cases (33.3%) that showed negative results by POC-CCA, which is consistent with other studies reporting high rates of false-negative results (low sensitivity) ranging from 55.6–58.3% and specificity from 76.9–78.4% [ 33 – 35 ]. Van Dam et al. reported the failure of POC-CCA to identify light infections in their study [ 11 ]. These results highlight the priority of using our developed LFTS for early diagnosis of schistosomiasis, as previously demonstrated by Kamel et al. [ 20 ]. There were 3 false positive results (out of 20), with two (0.4%) being positive by CCA and one (0.2%) by LFTS. Furthermore, the intensity of the test line band, which is directly proportional to the concentration of the antigen in the sample, was consistently higher in the LFTS than in the CCA strip. As shown in our results, the color intensities observed in positive cases were always higher in the LFTS, even with low infection, compared to those of POC-CCA. This difference was statistically significant, as indicated by the elevated median values of color intensity in the LFTS (p = 0.004). Both assays (LFTS & CCA) showed agreement with the intensity of infection (EPG), as reflected by the kappa value. For LFTS, the kappa value was 0.902, indicating an almost perfect agreement between the two assays, while for the CCA assay, it was 0.672. These findings are in line with the work of Ferreira et al. and Bezerra et al., who reported poor agreement (0.146 and 0.37, respectively) between the POC-CCA assay and the Kato-Katz technique [ 35 , 26 ]. In conclusion, based on the results obtained from the current study, the use of our developed LFTS, with its higher sensitivity and specificity, is recommended in Egypt as a reliable, rapid, and easy-to-perform point-of-care test to improve the diagnosis of active schistosomiasis, especially in cases of light infection, and to assist in infection control. However, additional assessment of the kit using a larger number of cases is encouraged before applying it forroutine diagnosis and screening studies. Abbreviations KK Kato-Katz LFTS lateral flow test strip POC-CCA point-of-care Circulating Cathodic Antigen detection EPG Eggs per gram of stool sample TBRI Theodor Bilharz Research Institute ELISA Enzyme-linked immunosorbent assay FP False positive AuNPs Gold nanoparticles MAbs Monoclonal antibodies MCM Mobile crystalline material MSN Mesoporous silica nanoparticles RT Room tempreture BSA Bovine serum albumin PBS phosphate buffer solution ROC Receiver operating characteristic AUC Area under the curve L Light M Moderate H Heavy LFIAs lateral flow immune assays CSA circulating Schistosoma mansoni antigen HRP horse raddish peroxidase OPD O-phenylene diamine dihdrochloride 10 mg (OPD) SEA S. mansoni soluble egg antigen Gel Doc XR+ gel documentation system NIH National Institutes of Health Declarations a-Ethical approval and consent to participate This study was reviewed and approved by both the Research Ethics Committee, Faculty of Medicine,Ain Shams University, study protocol (FMASU MS248/ 2020), and the ethics committee of Theodor Bilhariz Research Institute (TBRI, No. 05/09/16) . Each participant in the current study provided full medical history and informed consent. All relevant guidelines and regulations concerning animal’s manipulation as immunization of target antigens, myeloma and plasma cells fusion procedure and large scale production of monoclonal antibody were carried out. b- Consent for publication : All the authors have approved the manuscript and agree with submission to journal. c- Availability of data and materials: The datasets used and/or analyzed during the current study available from the corresponding author on reasonable request. d- Competing interests: The authors declare that they have no competing interests. e- Funding : No specific grant from any funding agency was received in the current research . f- Author contributions Z.D. and M.K. contributed to Conceptualization. F.S., S.M., A.S., and S.H. contributed to Methodology. S.M,D.A. contributed to Software work. H.B. and A.S. contributed to Validation. M.K. and S.M. contributed to Formal Analysis. Z.D. and D.A. contributed to the Investigation. H.B. and A.S. contributed to Resources. D.A.,S.M., and A.S. contributed to Writing the original draft. Z.D., M.K. and D.A. contributed to Writing-Review& Editing. All authors involved in the acquisition, analysis and interpretation of data. All authors read critically revised and approved the manuscript. References WHO. Report of the First Meeting of the WHO Diagnostic Technical Advisory Group for Neglected Tropical Diseases; World Health Organization: Geneva, Switzerland, 2019. Katz N, Chaves A, Pellegrino J. A simple device for quantitative stool thick-smear technique in Schistosomiasis mansoni. Rev Inst Med Trop Sao Paulo. 1972 Nov-Dec;14(6):397-400. PMID: 4675644. Elbasheir MM, Karti IA, Elamin EM. Evaluation of a rapid diagnostic test for Schistosoma mansoni infection based on the detection of circulating cathodic antigen in urine in Central Sudan. 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Development of new lateral-flow immunochromatographic strip using colloidal gold and mesoporous silica nanoparticles for rapid diagnosis of active schistosomiasis. Asian Pacific Journal of Tropical Biomedicine. 2019 Aug 1;9(8):315-22. Nowotny A. Basic exercises in immunochemistry. Berlin-Heidelberg-New York: Springer; 1969. McKinney MM, Parkinson A. A simple, non-chromatographic procedure to purify immunoglobulins from serum and ascites fluid. J Immunol Methods. 1987 Feb 11;96(2):271-8. doi: 10.1016/0022-1759(87)90324-3. PMID: 3805742. Omidfar K, Khorsand B, Larijani B. Development of a new sensitive immunostrip assay based on mesoporous silica and colloidal Au nanoparticles. Mol Biol Rep. 2012 Feb;39(2):1253-9. doi: 10.1007/s11033-011-0856-5. Epub 2011 May 21. PMID: 21603853. Wilson RA. The Problem with Diagnosis of Intestinal Schistosomiasis. EBioMedicine. 2017 Nov;25:16-17. doi: 10.1016/j.ebiom.2017.10.004. Epub 2017 Oct 3. PMID: 29033139; PMCID: PMC5704051. Mudenda J, Hamooya BM, Tembo S, Halwindi H, Siwila J, Phiri MM. Diagnostic accuracy of Schistosoma immunochromatographic IgG/IgM rapid test in the detection of schistosomiasis in Zambia. The Journal of Basic and Applied Zoology. 2022 Dec;83(1):1-8. Bezerra DF, Pinheiro MCC, Barbosa L, Viana AG, Fujiwara RT, Bezerra FSM. Diagnostic comparison of stool exam and point-of-care circulating cathodic antigen (POC-CCA) test for schistosomiasis mansoni diagnosis in a high endemicity area in northeastern Brazil. Parasitology. 2021 Apr;148(4):420-426. doi: 10.1017/S0031182020002164. Epub 2020 Nov 16 Yu JM, de Vlas SJ, Jiang QW, Gryseels B. Comparison of the Kato-Katz technique, hatching test and indirect hemagglutination assay (IHA) for the diagnosis of Schistosoma japonicum infection in China. Parasitol Int. 2007 Mar;56(1):45-9. doi: 10.1016/j.parint.2006.11.002. Epub 2006 Dec 21. PMID: 17188018. Kongs A, Marks G, Verlé P, Van der Stuyft P. The unreliability of the Kato-Katz technique limits its usefulness for evaluating S. mansoni infections. Trop Med Int Health. 2001 Mar;6(3):163-9. doi: 10.1046/j.1365-3156.2001.00687.x. PMID: 11299032. Nascimento GL, de Oliveira MR. Severe forms of schistosomiasis mansoni: epidemiologic and economic impact in Brazil, 2010. Trans R Soc Trop Med Hyg. 2014 Jan;108(1):29-36. doi: 10.1093/trstmh/trt109. Epub 2013 Dec 4. PMID: 24310377. Danso-Appiah A, Minton J, Boamah D, Otchere J, Asmah RH, Rodgers M, Bosompem KM, Eusebi P, De Vlas SJ. Accuracy of point-of-care testing for circulatory cathodic antigen in the detection of schistosome infection: systematic review and meta-analysis. Bull World Health Organ. 2016 Jul 1;94(7):522-533A. doi: 10.2471/BLT.15.158741. Epub 2016 Apr 22. PMID: 27429491; PMCID: PMC4933137. Beltrame A, Guerriero M, Angheben A, Gobbi F, Requena-Mendez A, Zammarchi L, Formenti F, Perandin F, Buonfrate D, Bisoffi Z. Accuracy of parasitological and immunological tests for the screening of human schistosomiasis in immigrants and refugees from African countries: An approach with Latent Class Analysis. PLoS Negl Trop Dis. 2017 Jun 5;11(6):e0005593. doi: 10.1371/journal.pntd.0005593. PMID: 28582412; PMCID: PMC5472324. Coulibaly JT, N'Gbesso YK, Knopp S, N'Guessan NA, Silué KD, van Dam GJ, N'Goran EK, Utzinger J. Accuracy of urine circulating cathodic antigen test for the diagnosis of Schistosoma mansoni in preschool-aged children before and after treatment. PLoS Negl Trop Dis. 2013;7(3):e2109. doi: 10.1371/journal.pntd.0002109. Epub 2013 Mar 21. PMID: 23556011; PMCID: PMC3605147. Ashton RA, Stewart BT, Petty N, Lado M, Finn T, Brooker S, Kolaczinski JH. Accuracy of circulating cathodic antigen tests for rapid mapping of Schistosoma mansoni and S. haematobium infections in Southern Sudan. Trop Med Int Health. 2011 Sep;16(9):1099-103. doi: 10.1111/j.1365-3156.2011.02815.x. Epub 2011 Jun 21. PMID: 21692957 Kittur N, Castleman JD, Campbell CH, King CH, Colley DG. Comparison of Schistosoma mansoni Prevalence and Intensity of Infection, as Determined by the Circulating Cathodic Antigen Urine Assay or by the Kato-Katz Fecal Assay: A Systematic Review. Am J Trop Med Hyg. 2016 Mar;94(3):605-610. doi: 10.4269/ajtmh.15-0725. Epub 2016 Jan 11. PMID: 26755565; PMCID: PMC4775897. Ferreira FT, Fidelis TA, Pereira TA, Otoni A, Queiroz LC, Amâncio FF, Antunes CM, Lambertucci JR. Sensitivity and specificity of the circulating cathodic antigen rapid urine test in the diagnosis of Schistosomiasis mansoni infection and evaluation of morbidity in a low- endemic area in Brazil. Rev Soc Bras Med Trop. 2017 May-Jun;50(3):358-364. doi: 10.1590/0037-8682-0423-2016. PMID: 28700054. Additional Declarations No competing interests reported. Supplementary Files Onlinefloatimage1.png Graphical abstract Cite Share Download PDF Status: Published Journal Publication published 02 Oct, 2024 Read the published version in Acta Parasitologica → Version 1 posted Editorial decision: Revision requested 25 Jul, 2024 Reviews received at journal 19 Jul, 2024 Reviews received at journal 14 Jul, 2024 Reviewers agreed at journal 28 Jun, 2024 Reviewers agreed at journal 27 Jun, 2024 Reviewers invited by journal 10 Jun, 2024 Editor assigned by journal 06 Jun, 2024 Submission checks completed at journal 06 Jun, 2024 First submitted to journal 05 Jun, 2024 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-4535875","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":314827733,"identity":"a4f205b7-a217-4d04-8208-41e6340989ee","order_by":0,"name":"Manal Kamel","email":"","orcid":"","institution":"Theodor Bilharz Research Institute","correspondingAuthor":false,"prefix":"","firstName":"Manal","middleName":"","lastName":"Kamel","suffix":""},{"id":314827734,"identity":"d53ffe25-ea47-46ff-a89c-262e84ace0ad","order_by":1,"name":"Faten Salah","email":"","orcid":"","institution":"Theodor Bilharz Research Institute","correspondingAuthor":false,"prefix":"","firstName":"Faten","middleName":"","lastName":"Salah","suffix":""},{"id":314827735,"identity":"493eb011-40fc-4e34-b3b1-c32c268b7f78","order_by":2,"name":"Zeinab Demerdash","email":"","orcid":"","institution":"Theodor Bilharz Research Institute","correspondingAuthor":false,"prefix":"","firstName":"Zeinab","middleName":"","lastName":"Demerdash","suffix":""},{"id":314827736,"identity":"e621ccaa-09d1-496d-87ca-bf3688159b61","order_by":3,"name":"Sara Maher","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA4UlEQVRIiWNgGAWjYNCCiv/1/OwNQIaBBbFazjAnSPYcAGmRIFIHYwtzgsGMBBCTCC3y7WcffrrZwJZnIPn86oYfBRIM/O3dCXi1GJxJN5bO3cFTbC6dU3azB+gwiTNnN+DXwpDGIJ17RoJx5+yctBs8QC0GErn4tcj3P2P+ndtmwLjh5pm0m3+I0cJwI41NOrctIXHDDfZjt4myxeDGMzbrnDMHjCV7cthuyxhI8BD0i3x/GvPtnIoDcvzsx5/dfPPHRo6/vZeAwxCAxwBMEqscBNgfkKJ6FIyCUTAKRhAAAFa2SLMm6w74AAAAAElFTkSuQmCC","orcid":"","institution":"Theodor Bilharz Research Institute","correspondingAuthor":true,"prefix":"","firstName":"Sara","middleName":"","lastName":"Maher","suffix":""},{"id":314827737,"identity":"3a00feee-ae6c-4d68-9ba1-d2cc3ba5eee2","order_by":4,"name":"Hanan El-Baz","email":"","orcid":"","institution":"Theodor Bilharz Research Institute","correspondingAuthor":false,"prefix":"","firstName":"Hanan","middleName":"","lastName":"El-Baz","suffix":""},{"id":314827738,"identity":"d9ba8ad0-7181-43c6-ac87-131e95fb8340","order_by":5,"name":"Nahla Yousef","email":"","orcid":"","institution":"Ain Shams University","correspondingAuthor":false,"prefix":"","firstName":"Nahla","middleName":"","lastName":"Yousef","suffix":""},{"id":314827739,"identity":"4d088086-6374-462c-9b4a-7a1f95a0d2a4","order_by":6,"name":"Rania Abu-Shady","email":"","orcid":"","institution":"Ain Shams University","correspondingAuthor":false,"prefix":"","firstName":"Rania","middleName":"","lastName":"Abu-Shady","suffix":""},{"id":314827740,"identity":"da7bcbcd-dbea-4711-af84-151f79af27cb","order_by":7,"name":"Amany Rezk","email":"","orcid":"","institution":"Theodor Bilharz Research Institute","correspondingAuthor":false,"prefix":"","firstName":"Amany","middleName":"","lastName":"Rezk","suffix":""},{"id":314827741,"identity":"75ac7d03-6c67-4183-bb87-6b287765b410","order_by":8,"name":"Salwa Hassan","email":"","orcid":"","institution":"Theodor Bilharz Research Institute","correspondingAuthor":false,"prefix":"","firstName":"Salwa","middleName":"","lastName":"Hassan","suffix":""},{"id":314827742,"identity":"7e2d2b90-7c5f-44a0-80cc-d6fb806500b0","order_by":9,"name":"Doaa Abdel Aziz","email":"","orcid":"","institution":"Ain Shams University","correspondingAuthor":false,"prefix":"","firstName":"Doaa","middleName":"Abdel","lastName":"Aziz","suffix":""}],"badges":[],"createdAt":"2024-06-05 18:51:19","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4535875/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4535875/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1007/s11686-024-00917-9","type":"published","date":"2024-10-02T15:57:11+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":59051267,"identity":"3547e793-628b-40a8-b952-6cd05d10d7e7","added_by":"auto","created_at":"2024-06-25 20:04:52","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":378439,"visible":true,"origin":"","legend":"\u003cp\u003eIllustration for the principle of the sandwich LFTS for detection of active \u003cem\u003eS.mansoni\u003c/em\u003e. In a vial, AuNps-mAb was mixed with urine sample containing circulating \u003cem\u003eSchistosoma mansoni \u003c/em\u003eantigen (CSA), then it was migrated through the nitrocellulose membrane and captured by MCM-41-mAb at the test line forming a distinct red color [20].\u003c/p\u003e","description":"","filename":"floatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-4535875/v1/f54e756f154329d6827a7670.png"},{"id":59052214,"identity":"20d01771-b19e-4f8c-886d-2abd5980d34d","added_by":"auto","created_at":"2024-06-25 20:12:52","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":188414,"visible":true,"origin":"","legend":"\u003cp\u003eROC curve of LFTS and CCA strip intensities using EPG as the reference test. The best cutoff values for LFTS and CCA strip are 0.68 and 0.73, respectively.\u003c/p\u003e","description":"","filename":"floatimage3.png","url":"https://assets-eu.researchsquare.com/files/rs-4535875/v1/9269fad83001f409ebb93866.png"},{"id":59051270,"identity":"6f5b81f2-93de-4108-8e2c-990fb3821efc","added_by":"auto","created_at":"2024-06-25 20:04:52","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":172981,"visible":true,"origin":"","legend":"\u003cp\u003eDetermination of the visual detection limit of LFTS by using different concentrations of SEA (starting from 500 ng/ml down to 3 ng/ml). The LFTS had a detection limit of 3 ng/ml for SEA.\u003c/p\u003e","description":"","filename":"floatimage4.png","url":"https://assets-eu.researchsquare.com/files/rs-4535875/v1/1bc64d6839705d53602babdf.png"},{"id":59051266,"identity":"86d1464a-53b4-4819-9093-1eac0ee8a765","added_by":"auto","created_at":"2024-06-25 20:04:52","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":332337,"visible":true,"origin":"","legend":"\u003cp\u003eDistribution of color intensities of CCA strip (A) and LFTS (B) in urine samples. P\u0026lt;0.000: Significant difference was found according to the Mann-Whitney test\u003c/p\u003e","description":"","filename":"floatimage5.png","url":"https://assets-eu.researchsquare.com/files/rs-4535875/v1/ef5d9999051dfc9157f6d752.png"},{"id":59051272,"identity":"46e35d78-d072-4e1d-9f4e-c81344a091ac","added_by":"auto","created_at":"2024-06-25 20:04:52","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":378184,"visible":true,"origin":"","legend":"\u003cp\u003eDistribution of color intensities of CCA strip and LFTS in urine samples. Compared to the CCA strip, the median value of intensity of the LFTS was significantly elevated (p=0.004).\u003c/p\u003e","description":"","filename":"floatimage6.png","url":"https://assets-eu.researchsquare.com/files/rs-4535875/v1/f91233adbe35d2efc3020efd.png"},{"id":59052213,"identity":"6ec49c74-7966-4536-84d7-35fe3f03375c","added_by":"auto","created_at":"2024-06-25 20:12:52","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":17819,"visible":true,"origin":"","legend":"\u003cp\u003eDistribution of color intensities of CCA strip and LFTS in different levels of infection. The intensity values by the LFTS were always significantly higher than those of CCA.\u003c/p\u003e","description":"","filename":"6.png","url":"https://assets-eu.researchsquare.com/files/rs-4535875/v1/0aba3a4d6a2a1e7bab030f06.png"},{"id":59051271,"identity":"218cd214-9238-4863-a435-f333ab3d8935","added_by":"auto","created_at":"2024-06-25 20:04:52","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":570669,"visible":true,"origin":"","legend":"\u003cp\u003eComparison between CCA (A) and LFTS (B), some positive samples were tested by applying the same samples on both strips.\u003c/p\u003e","description":"","filename":"floatimage8.png","url":"https://assets-eu.researchsquare.com/files/rs-4535875/v1/a79d0b61a098bf6792a29d7a.png"},{"id":66097044,"identity":"874df8b7-e8c5-458f-bf04-40a8baa2e657","added_by":"auto","created_at":"2024-10-07 16:12:58","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":3360881,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4535875/v1/beb9f135-cfa0-4eeb-b548-cc0be95369f1.pdf"},{"id":59051269,"identity":"4d23c23f-0d9d-4b7b-be89-6d608403ef70","added_by":"auto","created_at":"2024-06-25 20:04:52","extension":"png","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":184428,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eGraphical abstract\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"Onlinefloatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-4535875/v1/d996b7e4761bba74058f4865.png"}],"financialInterests":"No competing interests reported.","formattedTitle":"Home-made lateral flow test strip versus POC-CCA assay for detection of active schistosomiasis in Egypt","fulltext":[{"header":"Introduction","content":"\u003cp\u003eSchistosomiasis is one of the major neglected health problems in Africa, affecting nearly 220.8\u0026nbsp;million people in 78 countries worldwide, necessitating preventive treatment. Early diagnosis is crucial for effective control programs and preventive treatment to reduce and prevent morbidity [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. The Kato-Katz (KK) method is considered the gold standard for diagnosing schistosome eggs in stool samples. However, it has several limitations [\u003cspan additionalcitationids=\"CR3\" citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. Despite years of research, there has been a continuous pursuit to find a rapid and sensitive one-step diagnostic test for schistosomiasis as an alternative to conventional microscopic detection methods. Since 1995, researchers at Theodor Bilharz Research Institute (TBRI) in Cairo, Egypt, have developed a panel of monoclonal antibodies using hybridoma technology against various Schistosoma antigens [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. Initially, monoclonal antibodies were employed in a sandwich enzyme-linked immunosorbent assay (ELISA) to detect circulating antigens in urine and serum samples as diagnostic probes for early active infections. While this method improved the sensitivity and specificity of diagnosis and overcame the drawbacks of previous conventional methods, it was not practical for routine screening due to its multiple steps, long reaction time, and limited suitability to well-equipped laboratories [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eRecently, there has been growing interest in the use of immune-strip assays as an alternative method, which offer a one-step, user-friendly approach with a visual endpoint [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. The detection of circulating cathodic antigen (CCA) using urine strips has been widely applied for routine S. mansoni infection detection [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. In 2004, van Dam et al. reported that CCA detects a specific parasite gut carbohydrate antigen regurgitated by adult worms, which circulates in the bloodstream, is eliminated by the kidneys, and can be detected in the urine of infected individuals [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. However, ELISA still demonstrated higher sensitivity than CCA, resulting in multiple false positive results. Many studies have focused on improving CCA, and researchers have explored the use of colloidal gold nanoparticles (AuNPs) to enhance the sensitivity of conventional immune-strip assays, enabling rapid and cost-effective detection of infectious agents compared to current available technologies [\u003cspan additionalcitationids=\"CR13 CR14\" citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. In 2016, Kamel et al. found that loading monoclonal antibodies onto gold nanoparticles increased the specificity and sensitivity of monoclonal antibody-based sandwich ELISA to 100% and 98.7%, respectively [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. Gold nanoparticles are widely used as they can be easily functionalized to produce different colors depending on their size, aggregation state, and shape, making them excellent candidates for colorimetric biosensor development [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e, \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. Additionally, mesoporous silica nanoparticles (MSN) of the mobile crystalline material (MCM)-41 type offer efficient immobilization of proteins on nitrocellulose membranes, ensuring good efficiency, immune reactivity, and stability of the immobilized protein [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eIn 2019, Kamel et al. developed a convenient and sensitive gold nanoparticle-based lateral flow test strip (LFTS) assay for rapid detection of soluble egg antigen (SEA) from S. mansoni in serum and urine samples of schistosomiasis-infected patients, utilizing colloidal AuNPs and MSN [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. After large-scale production, purification, and characterization of the produced monoclonal antibodies (4D/1D), the LFTS was constructed using gold-conjugated detector monoclonal antibodies and MCM-41-conjugated capture monoclonal antibodies for immobilization on the nitrocellulose membrane. Through optimization and standardization of working conditions, we tested the developed strip using serum and urine samples from infected patients. Our previous results were very promising, with a specificity of 97.5% and sensitivity of 98.3% in urine.\u003c/p\u003e\n\u003ch3\u003eStudy design and sample collection\u003c/h3\u003e\n\u003cp\u003eThis study aims to compare the effectiveness of the developed LFTS with the commercially available POC-CCA kit for the rapid detection of S. mansoni SEA in urine samples of infected patients as a validation step for large-scale application in routine diagnosis. Samples were collected from endemic hotspots in the Nile Delta, specifically Elkhamseeny and Sandala villages in Kafr Elsheikh Governorate. All patients were above 18 years old. Three hundred (300) stool samples were screened using the KK technique for S. mansoni infection. Only 60 cases tested positive and were included in this study, along with 20 negative cases used as a control group. The included cases were sex-matched and aged from 12 to 30 years old. The positive cases were subdivided according to the number of eggs per gram (EPG) into the light infection group (12 cases) (\u0026lt;\u0026thinsp;50 EPG), moderate infection group (11 cases) (51\u0026ndash;100 EPG), and heavy infection group (37 cases) (\u0026ge;\u0026thinsp;100 EPG). Urine samples from all included subjects were collected, centrifuged for 5 minutes at 2000 rpm, and then the supernatant was collected and stored at -20\u0026deg;C until used\u003c/p\u003e"},{"header":"Methods","content":"\u003cdiv id=\"Sec4\" class=\"Section2\"\u003e\n \u003ch2\u003eProduction of S.mansoni SEA-mAbs\u003c/h2\u003e\n \u003cp\u003eHybridoma cells secreting mAbs (4D/1D), which were raised against S. mansoni SEA, were developed and cryopreserved at the Immunology Department of TBRI in Cairo, Egypt. The mAbs (4D/1D) were characterized as IgG1 kappa-type light chain antibodies that recognize repetitive epitopes on SEA, allowing their use as both antigen-capturing and detecting antibodies in sandwich assays. The reactivity of the hybridoma cells against SEA was checked by indirect ELISA after revival and propagation in a culture medium. Large-scale production of mAbs was maintained by injecting hybridoma cells (2x106/ml) intraperitoneally into BALB/c mice to develop ascitic fluid. The produced mAbs were then purified by ammonium sulfate precipitation according to Nowotny [\u003cspan class=\"CitationRef\"\u003e21\u003c/span\u003e], followed by treatment with caprylic acid according to McKinney and Parkinson [\u003cspan class=\"CitationRef\"\u003e22\u003c/span\u003e].\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec5\" class=\"Section2\"\u003e\n \u003ch2\u003ePreparation of nanoparticles conjugated mAbs\u003c/h2\u003e\n \u003cp\u003ePurified mAbs were passively conjugated with AuNPs according to Tanaka et al. In brief, 30 \u0026micro;g (12 \u0026micro;L) of mAbs solution (2.5 mg/mL) was diluted with KH2PO4 solution (5 mM) in ultra-pure water (200 \u0026micro;L) at pH 7.5. The diluted solution was added to AuNPs (1.8 \u0026micro;L, 20 nm) and immediately mixed, then left to rest for 20 minutes at room temperature (RT). A blocking step was performed using 200 \u0026micro;L of 10% bovine serum albumin (BSA w/v), followed by centrifugation at 4\u0026deg;C for 10 minutes at 8000 g. Pulse sonication for a few seconds was applied, and the conjugated mAb-AuNPs were added to 0.05% and 20 mM Tris-HCl buffer, 2 mL of preserving solution (pH 8.2, 1% [w/v] BSA), and then stored at 4\u0026deg;C until used [\u003cspan class=\"CitationRef\"\u003e12\u003c/span\u003e]. The conjugation step of mAbs with MCM-41 type silica nanoparticles was performed according to Omidfar et al. In brief, 1 mg of MCM-41 was dispersed in 1 mL of phosphate buffer solution (PBS) (pH 7.2, 0.1 M). Then, 181.5 \u0026micro;L of the MCM-41 solution was added to 18.1 \u0026micro;L (45.3 \u0026micro;g) of mAb (4D/1D) (2.5 mg/mL) in a 4:1 ratio. The mixture solution was left at 4\u0026deg;C overnight. A blocking step for non-specific binding sites was performed by adding 200 \u0026micro;L of 10% BSA (w/v) in PBS (0.1 M) at pH 7.2. The solution was then centrifuged at 4\u0026deg;C for 4 minutes, the supernatant was discarded, and the sediment pellet was dispersed in PBS (pH 7.2, 0.1 M). The MCM-41-mAb conjugate was stored at 4\u0026deg;C before use [\u003cspan class=\"CitationRef\"\u003e23\u003c/span\u003e].\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec6\" class=\"Section2\"\u003e\n \u003ch2\u003eFabrication of the AuNPs-LFTS\u003c/h2\u003e\n \u003cp\u003eFabrication of the LFTS was conducted according to our previous work by Kamel et al. In brief, the test strip is composed of a nitrocellulose membrane, sample pad, and absorbent pad. Several optimization trials were performed before determining the standard conditions of the test [\u003cspan class=\"CitationRef\"\u003e20\u003c/span\u003e]. Test line and control line solutions were prepared and standardized. For this patch, the optimal concentration of the capture conjugate (MCM-41-mAbs) on the test line was found to be 5 \u0026micro;L/strip. A control line solution (anti-mouse IgG, 2 mg/mL) was also prepared and dispensed on the nitrocellulose membrane (3 \u0026micro;L/strip). The nitrocellulose membrane was dried at room temperature for 1 hour, then immersed in a 50 mM boric acid buffer (skim milk [0.5% [w/v], pH 8.5) for 25 minutes to block nonspecific adsorption. Finally, the membrane was cut into specific sizes (0.5 cm wide, 6 cm long), and some strips were placed into plastic housings. All strips were stored in a plastic bag at 37\u0026deg;C until use. The urine sample was mixed with the detector probe (AuNPs-mAbs) in an external tube at a ratio of 10:1 (50 \u0026micro;L of urine sample to 5 \u0026micro;L of AuNPs-mAbs) (Fig. \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e).\u0026quot;\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec7\" class=\"Section2\"\u003e\n \u003ch2\u003eApplication of sandwich based LFTS\u003c/h2\u003e\n \u003cp\u003eUrine samples from the studied groups were subjected to the LFTS assay to detect the presence of circulating S. mansoni antigen. All urine samples were also assayed in parallel using 96-well microtiter plates. Briefly, 5 \u0026micro;L of the AuNPs-mAbs conjugate (detector probe) and 50 \u0026micro;L of each urine sample were mixed in separate wells. The test strip\u0026apos;s sample pad was then immersed in the well, allowing the mixture to be absorbed and moved by capillary force, forming a complex with the MCM-41-mAbs employed as the capture probe on the test line. In the presence of the antigen, a red color will appear. The control line will turn red after binding with the anti-IgG antibody to any unbound free conjugates. The test results were obtained after 10 minutes. The test result is considered invalid if no red color appears on the control line, regardless of the color on the test line.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e\n \u003ch2\u003eApplication of POC-CCA\u003c/h2\u003e\n \u003cp\u003eA point-of-care circulating cathodic antigen detection kit (Rapid Medical Diagnostics, Pretoria, South Africa, RSA) was used to test urine samples from the studied groups for the presence of circulating S. mansoni antigen. Briefly, 100 \u0026micro;L of urine (equivalent to 2 drops) were transferred to the test cassette by gently squeezing the pipette. The sample was completely absorbed into the specimen pad within the circular well, and then 1 drop of buffer was added. The test results were read after 20 minutes. The intensity of the red color on the produced test line for both strips was visually assessed using a gel documentation system (Gel Doc XR+, Biorad, USA). The results were then analyzed using Image Lab software version 3.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec9\" class=\"Section2\"\u003e\n \u003ch2\u003eStatistical analysis\u003c/h2\u003e\n \u003cp\u003eStatistical analyses were performed by using the analytical software package IBM-SPSS (version 23). Characteristics of LFTS strip and CCA assays were tested by using receiver operating characteristic curve (ROC). CCA strip and nano-based strip were compared to the EPG test (reference test) based on the following accuracy measure: specificity, sensitivity and Cohen\u0026rsquo;s kappa statistic (\u0026kappa;) of agreement. According to Kolmogorov-Smirnov test, the data of the intensities of CCA strip and LFTS were not normally distributed. Accordingly, non-parametric tests were used to statistically analyze the data. The association in results between reference test and the immuno-strip assays was detected by Chi-square test. Statistical differences in the intensities between positive and negative groups were compared by using Mann-Whitney U test.\u003c/p\u003e\n\u003c/div\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec11\"\u003e\n \u003ch2\u003eKato-Katz technique and infection intensity\u003c/h2\u003e\n \u003cp\u003eBased on the KK technique, the level of infection intensity was classified as light (12 cases), moderate (11cases), or heavy (37 case), with thresholds set at \u0026lt;\u0026thinsp;50 EPG, 51\u0026ndash;100 EPG, and \u0026ge;\u0026thinsp;100 EPG, respectively.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec12\"\u003e\n \u003ch2\u003eComparative accuracy measures of LFTS and POC-CCA strip\u003c/h2\u003e\n \u003cp\u003eUpon application of both assays to detect CSA (target antigen) in urine samples of subjected groups, we observed that 58 out of 60 positive KK confirmed cases were found positive by LFTS while 19 out of 20 of the control group were confirmed negative demonstrating a sensitivity and specificity of 96.7% and 95% respectively. For the POC-CCA assay, 51 out of 60 \u003cem\u003eS.mansoni\u003c/em\u003e infected cases were positive while 18 out of 20 control cases were negative exhibiting sensitivity and specificity of 85% and 90% respectively. Furthermore, the kappa value of LFTS with EPG (as a reference method) was 0.902, reflecting an almost perfect agreement between the two assays. On the other hand, POC-CCA strips showed a substantial agreement with EPG where the kappa value was o.672 \u003cstrong\u003e(\u003c/strong\u003eTable\u0026nbsp;\u003cspan\u003e1\u003c/span\u003e\u003cstrong\u003e)\u003c/strong\u003e.\u003c/p\u003e\n \u003cp\u003eGel documentation system was used to quantitative measure the color intensity of the test line for both assays, where the intensity is directly proportional to the concentration of the antigen in the tested samples. Intensities were displayed as a volume x10\u003csup\u003e5\u003c/sup\u003e using Image Lab software. The receiver operating characteristic (ROC) curve was used for the detection of intensities of both LFTS and CCA strips using the same urine samples with EPG as a reference test is displayed in Fig.\u0026nbsp;\u003cspan\u003e2\u003c/span\u003e. According to ROC analysis, in CCA strips, the area under the curve (AUC) was found 0.968, with an optimal cut-off value of 0.73. On the other hand, AUC in LFTS strips was 0.998, with a 0.68 optimal cutoff value indicating that both strips have a high ability to discriminate the positive from negative cases. However, the sensitivity and specificity of the LFTS strip were always higher than that of the CCA strip, using EPG as the reference test. Moreover, the color intensity of the test line in the positive cases and their accuracy measures using both strips are displayed in \u003cstrong\u003eFig.\u0026nbsp;3\u003c/strong\u003e, Tables\u0026nbsp;\u003cspan\u003e2\u003c/span\u003e \u0026amp; \u003cspan\u003e3\u003c/span\u003e. Using LFTS, only 2 out of 60 cases were negative and belonged to the light infection group (L). In contrast, 9 out of 60 cases showed negative results when using CCA, encompassing the light (L), moderate (M), and heavy (H) infection subgroups. The sensitivity of LFTS was higher than that of CCA across all infected subgroups, including L, M, and H infections, with a specificity of 100% in the M and H infection groups and 90% in the L infection group. In comparison, the CCA test exhibited specificity of 82%, 82%, and 95% for L, M, and H infections, respectively.\u003c/p\u003e\n \u003cdiv\u003e\n \u003ctable id=\"Tab1\" border=\"1\"\u003e\n \u003ccaption language=\"En\"\u003e\n \u003cdiv\u003eTable 1\u003c/div\u003e\n \u003cdiv\u003e\n \u003cp\u003eThe accuracy measures of LFTS and CCA strips for CSA detection in urine samples using EPG as a reference test\u003c/p\u003e\n \u003c/div\u003e\n \u003c/caption\u003e\n \u003ccolgroup cols=\"9\"\u003e\u003c/colgroup\u003e\n \u003cthead\u003e\n \u003ctr\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eStrips\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eStrip/ EPG\u003c/p\u003e\n \u003cp\u003e(-/-)\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eStrip/ EPG\u003c/p\u003e\n \u003cp\u003e(+/+)\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eSpecificity\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eSensitivity\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eKappa value\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003e\u0026chi;2\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003edf\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003ep-value\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003c/thead\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eLFTS\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e19/20\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e58/60\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e95%\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e96.7%\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.902\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e65.4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.000\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eCCA\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e18/20\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e51/60\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e90%\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e85%\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.672\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e37.7\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.000\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n \u003c/div\u003e\n \u003cdiv\u003e\n \u003c/div\u003e\n \u003cp\u003e\u003cstrong\u003eFigure. 3\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003eDetermination of the visual detection limit of LFTS by using different concentrations of SEA (starting from 500 ng/ml down to 3 ng/ml). The LFTS had a detection limit of 3 ng/ml for SEA.\u003c/p\u003e\n \u003cdiv\u003e\n \u003ctable id=\"Tab2\" border=\"1\"\u003e\n \u003ccaption language=\"En\"\u003e\n \u003cdiv\u003eTable 2\u003c/div\u003e\n \u003cdiv\u003e\n \u003cp\u003eColor intensities of positive \u003cem\u003eS. mansoni\u003c/em\u003e cases using LFTS and CCA\u003c/p\u003e\n \u003c/div\u003e\n \u003c/caption\u003e\n \u003ccolgroup cols=\"5\"\u003e\u003c/colgroup\u003e\n \u003cthead\u003e\n \u003ctr\u003e\n \u003cth align=\"left\"\u003e\u0026nbsp;\u003c/th\u003e\n \u003cth align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003eLFTS Color intensity\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003eCCA Color intensity\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003c/thead\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eNegative\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003ePositive\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eNegative\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003ePositive\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eNumber of cases\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e58\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e9\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e51\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eTotal (60)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.74\u0026thinsp;\u0026plusmn;\u0026thinsp;0.01\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e2.37\u0026thinsp;\u0026plusmn;\u0026thinsp;0.10\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.74\u0026thinsp;\u0026plusmn;\u0026thinsp;0.02\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.70\u0026thinsp;\u0026plusmn;\u0026thinsp;0.09\u003csup\u003e***\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eSubgroups\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eL (n\u0026thinsp;=\u0026thinsp;12)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.74\u0026thinsp;\u0026plusmn;\u0026thinsp;0.01 (2)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.64\u0026thinsp;\u0026plusmn;\u0026thinsp;0.10 (10)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.75\u0026thinsp;\u0026plusmn;\u0026thinsp;0.02 (4)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.03\u0026thinsp;\u0026plusmn;\u0026thinsp;0.07 (8)\u003csup\u003e*\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eM (n\u0026thinsp;=\u0026thinsp;11)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e---\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.67\u0026thinsp;\u0026plusmn;\u0026thinsp;0.12 (11)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.72\u0026thinsp;\u0026plusmn;\u0026thinsp;0.03 (4)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.06\u0026thinsp;\u0026plusmn;\u0026thinsp;0.04 (7)\u003csup\u003e*\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eH (n\u0026thinsp;=\u0026thinsp;37)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e---\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e2.78\u0026thinsp;\u0026plusmn;\u0026thinsp;0.10 (37)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.79\u0026thinsp;\u0026plusmn;\u0026thinsp;0.00 (1)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.97\u0026thinsp;\u0026plusmn;\u0026thinsp;0.10 (36)\u003csup\u003e***\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003ctfoot\u003e\n \u003ctr\u003e\n \u003ctd colspan=\"5\"\u003e*: significant differences at p\u0026thinsp;\u0026lt;\u0026thinsp;0.05\u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tfoot\u003e\n \u003c/table\u003e\n \u003c/div\u003e\n \u003cp\u003e***: p\u0026thinsp;\u0026lt;\u0026thinsp;0.000, as compared to LFTS.\u003c/p\u003e\n \u003cdiv\u003e\n \u003ctable id=\"Tab3\" border=\"1\"\u003e\n \u003ccaption language=\"En\"\u003e\n \u003cdiv\u003eTable 3\u003c/div\u003e\n \u003cdiv\u003e\n \u003cp\u003eSensitivity and specificity of CCA and LFTS for each subgroup.\u003c/p\u003e\n \u003c/div\u003e\n \u003c/caption\u003e\n \u003ccolgroup cols=\"7\"\u003e\u003c/colgroup\u003e\n \u003cthead\u003e\n \u003ctr\u003e\n \u003cth align=\"left\" rowspan=\"2\"\u003e\u0026nbsp;\u003c/th\u003e\n \u003cth align=\"left\" colspan=\"3\"\u003e\n \u003cp\u003eCCA\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\" colspan=\"3\"\u003e\n \u003cp\u003eLFTS\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eL\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eM\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eH\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eL\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eM\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eH\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003c/thead\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eSensitivity\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.80\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.78\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.95\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.91\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.92\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.97\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eSpecificity\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.82\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.82\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.95\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.90\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e1.00\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e1.00\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n \u003c/div\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec13\"\u003e\n \u003ch2\u003eDistribution of intensities using LFTS and POC-CCA Strip:\u003c/h2\u003e\n \u003cp\u003eThe distribution of color intensities using LFTS and CCA strips is shown in Figs.\u0026nbsp;\u003cspan\u003e4\u003c/span\u003e, \u003cspan\u003e5\u003c/span\u003e, and \u003cspan\u003e6\u003c/span\u003e. Both strips were able to differentiate between positive and negative results without any overlapping areas (p\u0026thinsp;\u0026lt;\u0026thinsp;0.000) (Fig.\u0026nbsp;\u003cspan\u003e4\u003c/span\u003e). The median value of the LFTS positive test line intensity was significantly higher than that of CCA (p\u0026thinsp;\u0026lt;\u0026thinsp;0.004), indicating higher resolution and better visual interpretation of the LFTS assay (Fig.\u0026nbsp;\u003cspan\u003e5\u003c/span\u003e). In cases of low, moderate, and heavy infections, the intensity values measured by the LFTS were consistently higher than those of CCA (Fig.\u0026nbsp;\u003cspan\u003e6\u003c/span\u003e). When positive samples were tested on both strips, the color intensity on the test band of our developed LFTS was found to be stronger than that on CCA using the same samples (Fig.\u0026nbsp;\u003cspan\u003e7\u003c/span\u003e).\u003c/p\u003e\n\u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eDespite significant efforts, schistosomiasis remains a considerable health issue in certain countries, particularly in Africa [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e, \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. Many authors have mentioned that the sensitivity of the KK method is low, leading to false negative results due to various factors such as low parasitic burden, recent or chronic infections, post-treatment situations, or day-to-day variability in egg excretion in the host's stool [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e, \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e, \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. Therefore, immunological methods have recently emerged as a promising alternative, as they rely on direct antigen detection even before egg detection in the stool. The development of point-of-care (POC) tests for accurate screening is essential [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eIn our previous research, we successfully developed lateral flow tests (LFTS) for the rapid detection of S. mansoni antigen in urine samples. In this study, our objective was to assess and compare the effectiveness of our developed LFTS with the point-of-care circulating cathodic antigen (POC-CCA) kit. Samples (urine and stool) were collected from hot spot areas in Egypt, with the age range of the patients included in this study being between 12 and 30 years old, encompassing school-age children and young adults. This age group is of particular concern due to the concentration of the disease in children and economically active individuals [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e, \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eWhen both assays were tested on the same positive and negative cases, our developed LFTS exhibited a sensitivity and specificity of 96.7% and 95%, respectively, compared to the CCA strips that showed 85% and 90%, respectively. These results align with various other research works that have discussed the lower sensitivity of the POC-CCA assay [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e, \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]. Furthermore, in 2021, Bezerra et al. reported that the sensitivity and specificity of POC-CCA varied according to the prevalence and intensity of the infection, and that the diagnostic accuracy of the CCA assay was diminished, resulting in a large number of false negative results in low-intensity infections [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. Van Dam et al. also reported the lower sensitivity of POC-CCA in cases of light schistosomiasis infection [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. Additionally, different research works have mentioned the cross-reactivity of the CCA assay, which contributes to its false positive results [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e, \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eIn the current study, while 20% (12 cases out of 60) had a light infection (\u0026lt;\u0026thinsp;50 EPG) as determined by the KK method, only 2 cases (16.6%) showed negative results by the developed LFTS, compared to 4 cases (33.3%) that showed negative results by POC-CCA, which is consistent with other studies reporting high rates of false-negative results (low sensitivity) ranging from 55.6\u0026ndash;58.3% and specificity from 76.9\u0026ndash;78.4% [\u003cspan additionalcitationids=\"CR34\" citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e]. Van Dam et al. reported the failure of POC-CCA to identify light infections in their study [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. These results highlight the priority of using our developed LFTS for early diagnosis of schistosomiasis, as previously demonstrated by Kamel et al. [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. There were 3 false positive results (out of 20), with two (0.4%) being positive by CCA and one (0.2%) by LFTS. Furthermore, the intensity of the test line band, which is directly proportional to the concentration of the antigen in the sample, was consistently higher in the LFTS than in the CCA strip. As shown in our results, the color intensities observed in positive cases were always higher in the LFTS, even with low infection, compared to those of POC-CCA. This difference was statistically significant, as indicated by the elevated median values of color intensity in the LFTS (p\u0026thinsp;=\u0026thinsp;0.004).\u003c/p\u003e \u003cp\u003eBoth assays (LFTS \u0026amp; CCA) showed agreement with the intensity of infection (EPG), as reflected by the kappa value. For LFTS, the kappa value was 0.902, indicating an almost perfect agreement between the two assays, while for the CCA assay, it was 0.672. These findings are in line with the work of Ferreira et al. and Bezerra et al., who reported poor agreement (0.146 and 0.37, respectively) between the POC-CCA assay and the Kato-Katz technique [\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e, \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eIn conclusion, based on the results obtained from the current study, the use of our developed LFTS, with its higher sensitivity and specificity, is recommended in Egypt as a reliable, rapid, and easy-to-perform point-of-care test to improve the diagnosis of active schistosomiasis, especially in cases of light infection, and to assist in infection control. However, additional assessment of the kit using a larger number of cases is encouraged before applying it forroutine diagnosis and screening studies.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003ctable border=\"0\" cellspacing=\"0\" cellpadding=\"0\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd width=\"27.058823529411764%\" valign=\"top\"\u003e\n \u003cp\u003eKK\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"72.94117647058823%\" valign=\"top\"\u003e\n \u003cp\u003eKato-Katz\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"27.058823529411764%\" valign=\"top\"\u003e\n \u003cp\u003eLFTS\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"72.94117647058823%\" valign=\"top\"\u003e\n \u003cp\u003elateral flow test strip\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"27.058823529411764%\" valign=\"top\"\u003e\n \u003cp\u003ePOC-CCA\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"72.94117647058823%\" valign=\"top\"\u003e\n \u003cp\u003epoint-of-care Circulating Cathodic Antigen detection\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"27.058823529411764%\" valign=\"top\"\u003e\n \u003cp\u003eEPG\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"72.94117647058823%\" valign=\"top\"\u003e\n \u003cp\u003eEggs per gram of stool sample\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"27.058823529411764%\" valign=\"top\"\u003e\n \u003cp\u003eTBRI\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"72.94117647058823%\" valign=\"top\"\u003e\n \u003cp\u003eTheodor Bilharz Research Institute\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"27.058823529411764%\" valign=\"top\"\u003e\n \u003cp\u003eELISA\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"72.94117647058823%\" valign=\"top\"\u003e\n \u003cp\u003eEnzyme-linked immunosorbent assay\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"27.058823529411764%\" valign=\"top\"\u003e\n \u003cp\u003eFP\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"72.94117647058823%\" valign=\"top\"\u003e\n \u003cp\u003eFalse positive\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"27.058823529411764%\" valign=\"top\"\u003e\n \u003cp\u003eAuNPs\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"72.94117647058823%\" valign=\"top\"\u003e\n \u003cp\u003eGold nanoparticles\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"27.058823529411764%\" valign=\"top\"\u003e\n \u003cp\u003eMAbs\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"72.94117647058823%\" valign=\"top\"\u003e\n \u003cp\u003eMonoclonal antibodies\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"27.058823529411764%\" valign=\"top\"\u003e\n \u003cp\u003eMCM\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"72.94117647058823%\" valign=\"top\"\u003e\n \u003cp\u003eMobile crystalline material\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"27.058823529411764%\" valign=\"top\"\u003e\n \u003cp\u003eMSN\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"72.94117647058823%\" valign=\"top\"\u003e\n \u003cp\u003eMesoporous silica nanoparticles\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"27.058823529411764%\" valign=\"top\"\u003e\n \u003cp\u003eRT\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"72.94117647058823%\" valign=\"top\"\u003e\n \u003cp\u003e\u0026nbsp;Room tempreture\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"27.058823529411764%\" valign=\"top\"\u003e\n \u003cp\u003eBSA\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"72.94117647058823%\" valign=\"top\"\u003e\n \u003cp\u003e\u0026nbsp;Bovine serum albumin\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"27.058823529411764%\" valign=\"top\"\u003e\n \u003cp\u003ePBS\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"72.94117647058823%\" valign=\"top\"\u003e\n \u003cp\u003ephosphate buffer solution\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"27.058823529411764%\" valign=\"top\"\u003e\n \u003cp\u003eROC\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"72.94117647058823%\" valign=\"top\"\u003e\n \u003cp\u003eReceiver operating characteristic\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"27.058823529411764%\" valign=\"top\"\u003e\n \u003cp\u003eAUC\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"72.94117647058823%\" valign=\"top\"\u003e\n \u003cp\u003eArea under the curve\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"27.058823529411764%\" valign=\"top\"\u003e\n \u003cp\u003eL\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"72.94117647058823%\" valign=\"top\"\u003e\n \u003cp\u003eLight\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"27.058823529411764%\" valign=\"top\"\u003e\n \u003cp\u003eM\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"72.94117647058823%\" valign=\"top\"\u003e\n \u003cp\u003eModerate\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"27.058823529411764%\" valign=\"top\"\u003e\n \u003cp\u003eH\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"72.94117647058823%\" valign=\"top\"\u003e\n \u003cp\u003eHeavy\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"27.058823529411764%\" valign=\"top\"\u003e\n \u003cp\u003eLFIAs\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"72.94117647058823%\" valign=\"top\"\u003e\n \u003cp\u003elateral flow immune assays\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"27.058823529411764%\" valign=\"top\"\u003e\n \u003cp\u003eCSA\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"72.94117647058823%\" valign=\"top\"\u003e\n \u003cp\u003ecirculating Schistosoma mansoni antigen\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"27.058823529411764%\" valign=\"top\"\u003e\n \u003cp\u003eHRP\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"72.94117647058823%\" valign=\"top\"\u003e\n \u003cp\u003ehorse raddish peroxidase\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"27.058823529411764%\" valign=\"top\"\u003e\n \u003cp\u003eOPD\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"72.94117647058823%\" valign=\"top\"\u003e\n \u003cp\u003eO-phenylene diamine dihdrochloride 10 mg (OPD)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"27.058823529411764%\" valign=\"top\"\u003e\n \u003cp\u003eSEA\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"72.94117647058823%\" valign=\"top\"\u003e\n \u003cp\u003eS. mansoni soluble egg antigen\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"27.058823529411764%\" valign=\"top\"\u003e\n \u003cp\u003eGel Doc XR+\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"72.94117647058823%\" valign=\"top\"\u003e\n \u003cp\u003egel documentation system\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"27.058823529411764%\" valign=\"top\"\u003e\n \u003cp\u003eNIH\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"72.94117647058823%\" valign=\"top\"\u003e\n \u003cp\u003eNational Institutes of Health\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003ea-Ethical approval and consent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study was reviewed and approved by both the Research Ethics Committee, Faculty of Medicine,Ain Shams University, study protocol (FMASU MS248/ 2020), and \u0026nbsp;the ethics committee of Theodor Bilhariz Research Institute (TBRI, No. 05/09/16)\u003cstrong\u003e.\u0026nbsp;\u003c/strong\u003eEach participant in the current study provided full medical history and informed consent. All relevant guidelines and regulations concerning animal\u0026rsquo;s manipulation as immunization of target antigens, myeloma and plasma cells fusion procedure and large scale production of monoclonal antibody were carried out.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eb- Consent for publication\u003c/strong\u003e\u003cspan dir=\"RTL\"\u003e:\u003c/span\u003e All the authors have approved the manuscript and agree with submission to journal.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ec- Availability of data and materials:\u0026nbsp;\u003c/strong\u003eThe datasets used and/or analyzed during the current study available from the corresponding author on reasonable request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ed- Competing interests:\u0026nbsp;\u003c/strong\u003eThe authors declare that they have no competing interests.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ee- Funding\u003c/strong\u003e\u003cstrong\u003e:\u0026nbsp;\u003c/strong\u003eNo specific grant from any funding agency \u0026nbsp;was received in the current research\u0026nbsp;.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ef- Author contributions\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eZ.D. and M.K. contributed to \u0026nbsp; Conceptualization. F.S., S.M., A.S., and S.H. contributed to Methodology. S.M,D.A. contributed to Software work. H.B. and A.S. contributed to \u0026nbsp;Validation. \u0026nbsp;M.K. and S.M. contributed to \u0026nbsp;Formal Analysis. \u0026nbsp;Z.D. and D.A. contributed to the Investigation. H.B. and A.S. contributed to Resources. D.A.,S.M., and A.S. contributed to \u0026nbsp;Writing the original draft. Z.D., M.K. and D.A. contributed to Writing-Review\u0026amp; Editing. \u0026nbsp;All authors involved in the acquisition, analysis and interpretation of data. All authors read critically revised and approved the manuscript.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eWHO. Report of the First Meeting of the WHO Diagnostic Technical Advisory Group for Neglected Tropical Diseases; World Health Organization: Geneva, Switzerland, 2019.\u003c/li\u003e\n\u003cli\u003eKatz N, Chaves A, Pellegrino J. 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Comparison of the Kato-Katz technique, hatching test and indirect hemagglutination assay (IHA) for the diagnosis of Schistosoma japonicum infection in China. Parasitol Int. 2007 Mar;56(1):45-9. doi: 10.1016/j.parint.2006.11.002. Epub 2006 Dec 21. PMID: 17188018.\u003c/li\u003e\n\u003cli\u003eKongs A, Marks G, Verl\u0026eacute; P, Van der Stuyft P. The unreliability of the Kato-Katz technique limits its usefulness for evaluating S. mansoni infections. Trop Med Int Health. 2001 Mar;6(3):163-9. doi: 10.1046/j.1365-3156.2001.00687.x. PMID: 11299032.\u003c/li\u003e\n\u003cli\u003eNascimento GL, de Oliveira MR. Severe forms of schistosomiasis mansoni: epidemiologic and economic impact in Brazil, 2010. Trans R Soc Trop Med Hyg. 2014 Jan;108(1):29-36. doi: 10.1093/trstmh/trt109. Epub 2013 Dec 4. PMID: 24310377.\u003c/li\u003e\n\u003cli\u003eDanso-Appiah A, Minton J, Boamah D, Otchere J, Asmah RH, Rodgers M, Bosompem KM, Eusebi P, De Vlas SJ. 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Accuracy of urine circulating cathodic antigen test for the diagnosis of Schistosoma mansoni in preschool-aged children before and after treatment. PLoS Negl Trop Dis. 2013;7(3):e2109. doi: 10.1371/journal.pntd.0002109. Epub 2013 Mar 21. PMID: 23556011; PMCID: PMC3605147.\u003c/li\u003e\n\u003cli\u003eAshton RA, Stewart BT, Petty N, Lado M, Finn T, Brooker S, Kolaczinski JH. Accuracy of circulating cathodic antigen tests for rapid mapping of Schistosoma mansoni and S. haematobium infections in Southern Sudan. Trop Med Int Health. 2011 Sep;16(9):1099-103. doi: 10.1111/j.1365-3156.2011.02815.x. Epub 2011 Jun 21. PMID: 21692957\u003c/li\u003e\n\u003cli\u003eKittur N, Castleman JD, Campbell CH, King CH, Colley DG. Comparison of Schistosoma mansoni Prevalence and Intensity of Infection, as Determined by the Circulating Cathodic Antigen Urine Assay or by the Kato-Katz Fecal Assay: A Systematic Review. Am J Trop Med Hyg. 2016 Mar;94(3):605-610. doi: 10.4269/ajtmh.15-0725. Epub 2016 Jan 11. PMID: 26755565; PMCID: PMC4775897.\u003c/li\u003e\n\u003cli\u003eFerreira FT, Fidelis TA, Pereira TA, Otoni A, Queiroz LC, Am\u0026acirc;ncio FF, Antunes CM, Lambertucci JR. Sensitivity and specificity of the circulating cathodic antigen rapid urine test in the diagnosis of Schistosomiasis mansoni infection and evaluation of morbidity in a low- endemic area in Brazil. Rev Soc Bras Med Trop. 2017 May-Jun;50(3):358-364. doi: 10.1590/0037-8682-0423-2016. PMID: 28700054.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"acta-parasitologica","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"actp","sideBox":"Learn more about [Acta Parasitologica](http://link.springer.com/journal/11686)","snPcode":"11686","submissionUrl":"https://submission.springernature.com/new-submission/11686/3","title":"Acta Parasitologica","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"Schistosomiasis, monoclonal antibodies, Nanoparticles, LFTS, CCA","lastPublishedDoi":"10.21203/rs.3.rs-4535875/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4535875/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eFor years, the Kato-Katz (KK) technique has been considered the gold standard for diagnosing schistosomiasis. The aim of this study was to compare the effectiveness of our previously developed gold nanoparticle-based lateral flow test strip (AuNPs-LFTS) for diagnosing active Schistosoma mansoni with that of the commercially available point-of-care Circulating Cathodic Antigen detection (POC-CCA) kit. In this study, we collected sixty positive and twenty negative urine samples from patients in endemic hot spots in the Nile Delta, as well as from patients visiting the internal medicine clinic at Theodor Bilharz Research Institute (TBRI). We produced monoclonal antibodies (MAbs) against \u003cem\u003eS. mansoni\u003c/em\u003e soluble egg antigen (SEA) from cloned hybridoma cells (4D/1D). These MAbs were conjugated with gold and mesoporous silica nanoparticles, and used to develop the LFTS. The LFTS demonstrated a limit of detection (LoD) of 3 ng/ml. The sensitivity and specificity of the developed LFTS were found to be 96.7% and 95%, respectively, compared to 85% and 90% for the POC-CCA detection kit. The cases were divided into groups based on egg count in the stool, categorized as light, moderate, and heavy infections. The sensitivity of the LFTS in the group with light infection was higher than that of the POC-CCA. When using the KK technique (eggs per gram of stool sample [EPG]) as the reference test, the kappa value for the nano-based strips was 0.902, compared to 0.672 for the CCA strips, indicating an almost perfect agreement between KK and our developed LFTS. These results confirm the reliability and effectiveness of the LFTS compared to commercially available kits for rapid, sensitive, and early diagnosis of schistosomiasis. However, it is recommended to conduct further assessments of the developed strip on a larger scale with a broader range of cases before considering its introduction to local or international markets\u003c/p\u003e","manuscriptTitle":"Home-made lateral flow test strip versus POC-CCA assay for detection of active schistosomiasis in Egypt","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-06-25 20:04:47","doi":"10.21203/rs.3.rs-4535875/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2024-07-25T10:41:40+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-07-19T07:14:22+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-07-14T04:58:11+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"3740937486228275199583555634663474286","date":"2024-06-28T10:14:30+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"21740146051729481659503328274507097121","date":"2024-06-27T20:21:17+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2024-06-10T10:52:49+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2024-06-06T06:14:56+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2024-06-06T06:13:07+00:00","index":"","fulltext":""},{"type":"submitted","content":"Acta Parasitologica","date":"2024-06-05T18:49:07+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"acta-parasitologica","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"actp","sideBox":"Learn more about [Acta Parasitologica](http://link.springer.com/journal/11686)","snPcode":"11686","submissionUrl":"https://submission.springernature.com/new-submission/11686/3","title":"Acta Parasitologica","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"a603c32e-2369-4049-a2c6-efd96f9900d8","owner":[],"postedDate":"June 25th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2024-10-07T16:05:59+00:00","versionOfRecord":{"articleIdentity":"rs-4535875","link":"https://doi.org/10.1007/s11686-024-00917-9","journal":{"identity":"acta-parasitologica","isVorOnly":false,"title":"Acta Parasitologica"},"publishedOn":"2024-10-02 15:57:11","publishedOnDateReadable":"October 2nd, 2024"},"versionCreatedAt":"2024-06-25 20:04:47","video":"","vorDoi":"10.1007/s11686-024-00917-9","vorDoiUrl":"https://doi.org/10.1007/s11686-024-00917-9","workflowStages":[]},"version":"v1","identity":"rs-4535875","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4535875","identity":"rs-4535875","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}