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Background: Acute gastroenteritis is one of the major causes of morbidity and mortality in young children worldwide. Among these, rotavirus, norovirus, and adenovirus have been reported as the primary viral pathogens associated with the disease. Rapid diagnosis of viral pathogens is crucial when diarrhea outbreaks occur to ensure the timely administration of appropriate treatment and control measures. Methods: We evaluated three immunochromatographic test kits designed for the detection of norovirus, rotavirus, and adenovirus in 71 stool specimens collected from children with diarrhea who visited clinics in Japan. The first kit is a triplex immunochromatographic test kit designed for simultaneous detections of norovirus, rotavirus, and adenovirus on a single strip (this kit was referred to as IC-A). The other two immunochromatographic test kits are a dual detection kit for rotavirus and adenovirus, and a single detection kit for norovirus (IC-B). The RT-PCR/PCR was used as the gold standard method. Results: The results revealed that both IC-A and IC-B kits exhibited the same level of sensitivity of detection for rotavirus (72.7%) and adenovirus (22.7%), although the detection rate was lower than that of the RT-PCR/PCR method. However, there was a slight difference in the sensitivity of detection for norovirus between IC-A and IC-B, at 86.7% and 93.3%, respectively. The sensitivity of detection for adenovirus of both kits was relatively lower than those of RT-PCR method. This could be due to low viral load of adenovirus in clinical specimens below the detection limit of IC-A and IC-B kits. However, both immunochromatographic test kits (IC-A and IC-B) exhibited 100% specificity for norovirus, rotavirus, and adenovirus. Conclusions: The triplex immunochromatographic test kit (IC-A) designed for simultaneous detection of norovirus, rotavirus, and adenovirus has been proved to be more practical and convenient than the use of single or dual detection kits with more or less the same sensitivity and specificity of detections.

Abstract Background Equine piroplasmosis is caused by two tick-borne protozoan parasites, Theileria equi and Babesia caballi,, which are clinically relevant in susceptible horses, donkeys, and mules. Moreover, equine piroplasmosis significantly constrains international trading and equestrian events. Rapidly diagnosing both parasites in carrier animals is essential for implementing effective control measures. Here, a rapid immunochromatographic test for the simultaneous detection of antibodies to T. equi and B. caballi was evaluated using samples from horses and donkeys collected in Greece, Israel, and Italy. The results were compared with an improved competitive enzyme-linked immunosorbent assay (cELISA) for detecting antibodies to both parasites using the same panel of samples. Methods Blood samples were collected from 255 horses and donkeys. The panel consisted of 129 horses sampled at four locations in northern Greece, 105 donkeys sampled at four locations in Sicily, and 21 horses sampled at two locations in Israel. The rapid test and the cELISA were performed according to the manufacturer’s instructions, and the results were subjected to a statistical analysis to determine the sensitivity and specificity of both tests and their association. Results The immunochromatographic test provided a result within 15 min and can be performed in the field, detecting both pathogens simultaneously. The overall coincidence rate between the rapid test and the cELISA for detecting antibodies against T. equi was 93% and 92.9% for B. caballi. The rapid test’s sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV) for T. equi were above 91.5%. Sixteen samples were positive for both parasites in the rapid test and eight in the cELISA. Either test had no significant association between T. equi and B. caballi detection. The detection rates of both parasites were significantly higher in Italy than in Greece or Israel and in donkeys than in horses. The agreement for T. equi between the results of both tests was high in Greece (93.8%) and Italy (95.2%) and moderate in Israel (76.2%). For B. caballi, the specificity and NPV of the rapid test were high (94.2% and 98.3%, respectively), although the sensitivity and PPV were moderate (69.2% and 39.1%, respectively) due to the small sample size. However, for B. caballi, the sensitivity was higher with the rapid test. Conclusions The rapid test detected T. equi and B. caballi simultaneously in the field, potentially replacing laborious cELISA testing and is recommended for import/export purposes. The test can also be helpful for the differential diagnosis of clinical cases, since seropositivity may rule out equine piroplasmosis since it does not indicate current or active infection. Graphical Abstract

Paragonimiasis is a harmful food-borne zoonosis caused by lung flukes of the genus Paragonimus. The disease is found on most continents, several million people are at risk of infection, and it is a re-emerging disease in developing countries. The gold standard for diagnosis of pulmonary paragonimiasis requires the finding of eggs in sputa and/or fecal samples. In ectopic paragonimiasis cases, eggs are typically not seen, and supportive information is required such as a history of eating freshwater crabs or crayfishes, radiographic findings and immunological tests. Here, we developed a proof of concept based on lateral flow assay, an immunochromatographic test kit, named the paragonimiasis whole-blood test kit, for detection of specific IgG antibody in simulated whole-blood samples (WBSs) using worm excretory-secretory antigens to diagnose human paragonimiasis. The laboratory diagnostic values of this kit were compared with the detected IgG in serum samples. In simulated WBSs, the diagnostic sensitivity and specificity were 97.8 % and 96.1 %, respectively, while for serum samples, these values were 100.0 % and 94.8 %, respectively. The comparative IgG antibody detections whether a result was positive or negative between simulated WBSs and serum samples did not differ significantly with a concordance of 97.8 % in laboratory conditions using a circumscribed set of samples. The tool is fast and easy to use. The next step involves observing and evaluating native whole blood samples and using specific recombinant antigens need to be evaluated for support diagnosis of paragonimiasis caused by P. heterotremus, P. westermani and P. miyazakii at the bedside or at local and remote hospitals with limited facilities. It will also be valuable for epidemiological surveys in Asia where paragonimiasis is endemic.

Background: The currently available methods for the detection of Streptococcus pneumoniae are labour-intensive, and involve time-consuming steps needing skilled technicians to perform the assays. Therefore, we developed a rapid lateral flow based immunochromatographic test strip for detection of S. pneumoniae using pneumolysin using monoclonal antibody capture approach. Methodology − The strip was designed by assembling a sample pad, conjugation pad, nitrocellulose membrane and adsorbent pad supported by a plastic backing card. Optimization of control spot was done by using three different concentrations of monoclonal antibody (0.1 µg, 0.25 g and 0.5 µg). Monoclonal antibody coupled with gold nanoparticles was immobilized on conjugate pad. Recombinant pneumolysin at three different concentrations (10 ng/ml, 50 ng/ml and 100 ng/ml) was used. Diluted pneumolysin was poured onto the sample pad which migrated through the conjugate pad containing gold antibody conjugate and the antigen–antibody complex migrated to the test spot to bind to the polyclonal antibody. The remaining free antibody bound to the control spot to visualize a control spot. Results: Control spot was optimized at 0.5 µg of mouse IgG. Of three different concentrations of Pneumolysin, the spot was best visualized at 50 ng/ml Pneumolysin concentration. Conclusion: Our ICT based device could detect a commercially available recombinant Pneumolysin of S. pneumoniae. Future study is directed to validate the strip for detection of Pneumolysin using biological fluids of patients (particularly children) infected with S. pneumoniae.

Aflatoxin B1 (AFB1) is highly toxic and difficult to prevent. It is mainly produced by fungi and exists in plants and animals and is classified by the World Health Organization as a class I carcinogen, posing a serious threat to human and animal health. Therefore, it is important to establish an efficient, sensitive, and on-site detection method for AFB1 to protect human health. The immunochromatographic test strip method is simple, sensitive, and can achieve real-time detection. However, traditional immunochromatographic test strips have low sensitivity due to their relatively weak optical properties. In this study, Nb-G8 was biotinylated using a chemical method. Two sizes of gold nanoflowers (AuNFs) were prepared and combined with biotinylated G8 and streptavidin to form two types of probes. These probes were sprayed on gold standard pads and expanded pads, respectively, to enhance the signals through the high affinity interaction between streptavidin and biotin. Under the optimal experimental conditions, the half maximal inhibitory concentration (IC50) of this method was 5.0 ng/mL and the limit of detection (IC10) was 0.03 ng/mL, which increased the sensitivity of the test strip by four-fold compared with that of the traditional biotinylated nanoantibody immunochromatography test strip and had a wider detection range. In conclusion, the use of a high-affinity amplification signal between biotin and streptavidin is a valuable method for the detection of aflatoxin.

Background: Scrub typhus is prevalent in many districts of South Bengal throughout the year where an average temperature of 20–35°C, which contributes to the spread of Leptotrombidium deliense. However, its diagnosis remains complicated by the lack of readily available and validated assays, the non-specificity of clinical symptoms on admission, and even non-availability of the pathognomonic eschar in most of the cases. Aims and Objectives: This study was carried out to evaluate the rapid immunochromatographic test (RICT) for early detection of scrub typhus for using it as an early diagnostic tool at the field level. Materials and Methods: This cross-sectional study in which 181 serum samples from clinically suspected cases (after excluding dengue, malaria, Japanese encephalitis, and typhoid fever) collected over 13 months were processed for the detection of immunoglobulin M (IgM) antibodies for scrub typhus by enzyme-linked immunosorbent assay (ELISA) and rapid test. Results: Considering IgM ELISA for scrub typhus as the gold standard, the sensitivity, specificity, positive predictive value, and negative predictive value for RICT were found to be 100%, 86.87%, 50%, and 100%, respectively. Conclusion: RICT is a simple, rapid, and reliable assay for diagnosis of scrub typhus, capable of providing accurate results quickly and is highly suitable for field deployment in remote areas with limited medical support.

Summary Brucellosis, caused by Gram-negative Brucella, spreads in human and animal populations through contact with infected animals and products. Developing a rapid and sensitive detection technology for pathogen is crucial to reduce the risk of this disease transmitting between animal populations and to humans. We produced a monoclonal antibody LPS-6B5, which shows high affinity to LPS and limited cross-reactivity with other bacteria. Based on LPS-6B5, a colloidal gold immunochromatographic assay (GICA) was developed which demonstrates high sensitivity and specificity in detecting cultured B. melitensis, B. abortus and B. suis. The Gold Immunochromatographic Assay (GICA) strips exhibited the most sensitive detection limits, with a value of 7.8125 × 105 CFU/mL for Brucella melitensis, surpassing the sensitivity levels observed for Brucella abortus and Brucella suis. It is also suitable for clinical and field samples, providing a cost-effective and user-friendly alternative to traditional methods.

Objectives: To evaluate the diagnostic performance of different brands of immunochromatographic test (ICT) reagents for Chlamydia trachomatis using homogenized samples to provide a reference for reagent quality control. Methods: Eight commercially available ICT reagents were evaluated, of which three used the latex method and five used the colloidal gold method. Analytical performance evaluation using a pure culture broth of C. trachomatis, as well as clinical application validation using cervical epithelial cell samples acquired from the research subjects, were conducted. The concentration of C. trachomatis was quantified using a nucleic acid amplification test. Results: The limit of detection (LOD) of different ICT reagents in the analytical performance evaluation varied from 9.5 × 103 to 1 × 105 IFU/mL, and only one reagent met the LOD specified in the manufacturer's instructions. Likewise, only one reagent in the clinical application validation achieved the analytical LOD, four reagents were 2.1–4.2-fold of the analytical LODs, and three reagents failed to detect positive results in clinical samples. Conclusions: The diagnostic performance of different methods and different brands of ICT reagents in clinical practice was different from the manufacturer's instructions and the results of laboratory evaluation. The diagnostic performance of reagents should be evaluated before they are actually used in clinical practice.

BackgroundIt is essential to consider a practical antibody test to successfully implement marker vaccines and validate vaccination efficacy against classical swine fever virus (CSFV). The test should include a serological antibody assay, combined with a tool for differentiating infected from vaccinated animals (DIVA). The immunochromatographic test strip (ICS) has been exclusively designed for detecting CSFV E2 antibodies while lacking in detecting Erns antibodies, which can be employed and satisfy DIVA strategy. This study developed a novel ICS for detecting CSFV E2/Erns dual-antibody. The effectiveness of ICS in evaluating the DIVA capability of two novel chimeric pestivirus vaccine candidates was assessed.MethodsRecombinant E2 or Erns protein was transiently expressed in the plant benthamiana using Agrobacterium tumefaciens. ICS was subsequently assembled, and goat anti-rabbit IgG and recombinant CSFV E2 or Erns protein were plated onto the nitrocellulose membrane as control and test lines, respectively. The sensitivity and specificity of ICS were evaluated using sera with different neutralizing antibody titers or positive for antibodies against CSFV and other pestiviruses. The coincidence rates for detecting E2 and Erns antibodies between ICS and commercial enzyme-linked immunosorbent assay (ELISA) kits were also computed. ICS performance for DIVA capability was evaluated using sera from pigs vaccinated with conventional vaccine or chimeric vaccine candidates.ResultsE2 and Erns proteins were successfully expressed in N. benthamiana-produced recombinant proteins. ICS demonstrated high sensitivity in identifying CSFV E2 and Erns antibodies, even at the low neutralizing antibody titers. No cross-reactivity with antibodies from other pestiviruses was confirmed using ICS. There were high agreement rates of 93.0 and 96.5% between ICS and two commercial ELISA kits for E2 antibody testing. ICS also achieved strong coincidence rates of 92.9 and 89.3% with two ELISA kits for Erns antibody detection. ICS confirmed the absence of CSFV Erns-specific antibodies in sera from pigs vaccinated with chimeric vaccine candidates.ConclusionE2 and Erns proteins derived from the plant showed great potential and can be used to engineer a CSFV E2/Erns dual-antibody ICS. The ICS was also highly sensitive and specific for detecting CSFV E2 and Erns antibodies. Significantly, ICS can fulfill the DIVA concept by incorporating chimeric vaccine candidates.

Introduction: Hepatitis B Virus (HBV) infection remains a significant public health concern globally, necessitating accurate and timely diagnostic methods. Immunochromatographic Tests (ICTs) and Electrochemiluminescence Assays (ECLIAs) are widely used assays for HBV detection due to their rapidity and cost-effectiveness. However, their diagnostic performance should be evaluated to ascertain their reliability. Aim: To detect the presence of Hepatitis B Surface Antigen (HBsAg) in the selected samples using ICT and ECLIA and to compare it with HBV Deoxyribonucleic Acid (DNA) using a molecular assay. Materials and Methods: A cross-sectional study was done with serum samples collected from patients visiting the hospital over a period of six months with prior ethical clearance. Serum samples were obtained from 57 patients suspected of HBV infection. The results of ICT, ECLIA, and HBV DNA viral load (by Polymerase Chain Reaction (PCR)) were cross-tabulated and assessed for differences in diagnostic sensitivity. The positivity and correlation of the ICT and ECLIA with PCR were estimated. All statistical analyses were performed using the R programming language. Results: Out of 57 samples, 53 (92.98%) tested positive in the ICT card test, and 54 (94.74%) were positive in the ECLIA method. McNemar’s test showed that the sensitivity of ICT and ECLIA differed significantly compared to HBV DNA PCR. There was a significant positive correlation between ECLIA and HBV-DNA PCR (Spearman correlation, r-value=0.28, p-value=0.035). Conclusion: The findings suggest that in settings where accurate diagnosis is critical, particularly for screening and monitoring treatment efficacy, molecular assays remain the preferred choice despite their higher cost and complexity. However, in resource-limited settings, ECLIAs can still play a valuable role in HBV screening programs.

Cancer is one of the major public health challenges in the world, which is characterized by rapid progression and high mortality. Immunotherapy, represented by PD-1 monoclonal antibody, has significantly improved the efficacy of malignant tumors and has become one of the most popular immunotherapy methods at present. Therefore, there is an increasing demand for novel detection methods for PD-1 monoclonal antibodies. The aim of this work was to establish a rapid, simple, and sensitive immunochromatographic test strip (ICTS) based on the AuNPs enlargement for both visual and instrumental detection of the PD-1 monoclonal antibody concentration. The mixed solution of NH2OH·HCl and HAuCl4 was used as an enhancement solution to lower the detection limit and achieve higher sensitivity. A test strip reader was used to construct a visualized quantitative detection standard curve for the PD-1 monoclonal antibody concentration. The LOD was 1.58 ng/mL through a triple signal-to-noise ratio. The detection time was within 10 min. The constructed test strips can rapidly, accurately, and efficiently detect the concentration of PD-1 monoclonal antibody in real samples.

This article explores the use of the Hb Bart's immunochromatographic test as a screening tool for alpha thalassemia in a diverse population in Northern Europe. Alpha thalassemia is a hereditary disorder that is commonly found in malaria-prone regions, but has become more prevalent in non-endemic areas due to historical colonization, migration, and interethnic marriages. The study compared the immunochromatographic test to other molecular tests and found that it was effective in identifying different types of alpha thalassemia syndromes. The authors suggest that the immunochromatographic test could potentially replace the Hb H inclusion test in screening algorithms for alpha thalassemia. [Extracted from the article]

Background: Electro-chemiluminescence immunoassay (ECLIA), enzyme-linked immunosorbent assay (ELISA), and rapid immunochromatographic test (RICT) are currently commonly used for the detection of hepatitis B surface antigen (HBsAg). Although a few studies have been performed to compare the efficiency of these three different serological assays, but there are still many grey areas where clear guidelines are required to give the reports as reactive or nonreactive. Materials and Methods: We compared three different methods of detecting HBsAg: RICT, ELISA and ECLIA. The aim of this cross-sectional study was to analyze the performance of these tests and evaluate the cut off index (COI) of ECLIA to be considered as positive. Some of the representative samples that showed a discrepancy between ELISA and ECLIA were confirmed by nucleic acid amplification test (NAAT). Results: A total of 3846 samples were included in the study, of which 259 (6.73%) were positive by ECLIA. Of these 259 samples, 68 were positive by both ECLIA and ELISA and had COI of >5 in ECLIA, whereas 191 were positive by ECLIA only (COI between 0.9 and 5). Hence, the concordance rates of the ECLIA and ELISA in detecting serum HBsAg was 26.25% while the same for ELISA and RICT was 31.57%. Four representative samples which were positive by ECLIA and ELISA (i.e., COI >5) were positive with NAAT. However, one sample which was negative by ELISA and positive by ECLIA (i.e., COI

A novel photothermal immunochromatographic test strip (PITS) with tannic acid (TA) modified cobalt sulfide (CoS) nanospheres (CoS@TA) as immuno-probe element was developed for the detection of aflatoxin B 1 (AFB 1 ). CoS nanospheres (CoS NPs) with excellent photothermal conversion efficiency (η = 47.5 %) was synthesized and skillfully chelated with TA to improve its dispersion, biocompatibility, and chromatographic properties. After modification, the CoS@TA coupled with monoclonal antibody (mAb) against AFB 1 (CoS@TA-mAb) by simple physical adsorption. The CoS@TA based PITS achieved highly sensitive detection of AFB 1 with the limit of detection in photothermal signal (photothermal-LOD) of 0.00503 μg/L, which was 19.88-fold higher than the LOD in visual signal (visual-LOD, 0.1 μg/L). The application of TA in the modification of CoS provided ideas to improve the properties of functional nanomaterials such as dispersion and biocompatibility, and the application of CoS@TA in PITS construction laid a methodological foundation for further improving the detection sensitivity of trace targets., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

A sensitive dual immunochromatographic test strip (dual-ICTS) was developed to detect malachite green (MG) and its metabolite, leucomalachite green (LMG), using two types of gold nanoparticles: round-shaped (red) and star-shaped (blue). The detection limits were determined to be 0.221 μg L -1 for MG and 0.214 μg L -1 for LMG, respectively. The dual-ICTS provided a cut-off value of 1.8 μg L -1 for MG and LMG detection. The dual-ICTS successfully detected MG and LMG in food samples, with recovery rates ranging from 86 % to 116 %. The dual-ICTS was evaluated by correlation analysis between the proposed assay and the well-established enzyme-linked immunosorbent assay in the MG and LMG detection. This is the first report on the development of the ICTS that can detect both MG and LMG at the same time within only 5 min, making it a sensitive and rapid tool for on-site detection., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024. Published by Elsevier Ltd.)

ABSTRACT Brucellosis is an important bacterial disease of global distribution with zoonotic potencial. Serological tests used in Brazil for diagnosis of bovine brucellosis, including the Rose Bengal test (RBT), 2-mercaptoethanol (2-ME), fluorescent polarization (FPA), and complement fixation (FC), are based on the smooth lipopolysaccharide antigen (S-LPS) of Brucella abortus. The aim of this study was to evaluate a recombinant BP26 protein used as antigen in a rapid lateral flow immunochromatographic assay (rBP26-LFIA) for serological diagnosis of bovine brucellosis. Analytical performance of rBP26-LFIA was evaluated in positive and negative bovine serum samples previously characterized by RBT and 2-ME. Estimates of analytical sensitivity and specificity were 73.91% and 97.14%, respectively. Bovine sera reactive to Neospora, Trypanosoma vivax or Leptospira were used to assess specificity because these diseases are commonly associated with abortion in cattle. In addition to a possible cross-reaction induced by commercial vaccines against Leptospira in serological tests for bovine brucellosis using S-LPS as an antigen. In conclusion, rBP26-LFIA, with its current standardization, had good analytical performance. However, a future evaluation of diagnostic performance by rBP26-LFIA with samples from regions with known prevalence is necessary for its recommendation for use in the Brazilian program for the control and eradication of bovine brucellosis.