Diagnóstico de onicomicosis. La utilidad del test rápido de detección antigénica en comparación con el cultivo.

BACKGROUND: Ringworm is very prevalent in our environment and is frequently observed in daily practice. Diagnostic confirmation depends on techniques that are not always accessible (PCR), with highly variable sensitivity depending on the observer (direct microscopy) or that offer a delayed result (culture, histopathology). Recently, a rapid test for the antigenic detection of dermatophytes has been developed, based on immunochromatography, called Diafactory®. This diagnostic tool could be useful in the diagnosis of ringworm, allowing early initiation of treatment and a reduction in consultation visits. OBJECTIVE: Determine sensitivity and specificity of the rapid antigen detection test in comparison with culture. MATERIAL AND METHODS: During a period of a year, 333 nail samples were collected from patients with suspected onychomycosis. The rapid test and culture are performed simultaneously on each sample. Those with a positive antigenic test result begin treatment early. The rest of the patients receive appointments for serial cultures and subsequent medical consultation to evaluate the results. RESULTS: The sensitivity and specificity of the rapid antigen detection test compared to culture is 97.2% and 80.7% respectively. CONCLUSION: The effectiveness of the rapid antigen detection test is comparable to culture for the detection of dermatophytes in nail samples. It is a quick and simple diagnostic technique that allows a reduction in the number of patient visits to the hospital, as well as an early start of treatment.

Rapid screening of prednisolone acetate adulterants in health foods using colloidal gold immunochromatographic assay.

In this study, a rapid detection method utilizing colloidal gold immunochromatography (CG-ICA) was developed for the detection of illegally added prednisone acetate in health foods. Initially, the preparation conditions of colloidal gold solution were optimized. The optimal potassium carbonate dosage, antibody diluent type, antibody dosage, probe labeling time, blocking time and BSA dosage were determined. Technical analysis was performed to ensure that the established CG-ICA exhibited satisfactory color development and inhibition rates. Under optimized conditions, the cut-off value of CG-ICA was 250 µg/kg. The assay demonstrated a sensitivity of 100 %, a false positive rate of 8 %, and a false negative rate of 0, indicating high specificity for prednisone acetate. The results obtained from testing actual samples were consistent with those obtained using LC-MS/MS, thereby verifying the reliability of the developed method. This method offers robust support for the rapid detection of illegally added prednisone acetate in health foods.

Establishment of highly sensitive lateral flow immunochromatographic strips for quinclorac detection utilizing signal amplification nanoparticles.

Highly selective herbicide quinclorac (Qui) is a type of quinoline carboxylic acid hormone herbicide, which has the characteristics of long half-life and difficulty for degradation, causing high risk to the environmental safety. In this study, anti-Qui 8A3 monoclonal antibody (mAb) with good specificity and high affinity (3.89 × 109 L/mol) was prepared, and two kinds of lateral flow immunochromatographic strips (LFICS) including nano-flower nanoparticles (AuNF) - and latex microsphere (LM)- based LFICS were established based on the antibody and signal amplification. The linear range of the AuNF- and LM- based LFICS were 5.31-345.48 ng/mL and 2.52-257.92 ng/mL, respectively. The limit of detection (LOD) of the AuNF- and LM- based LFICS were determined to be 5.31 ng/mL and 2.52 ng/mL, respectively. In summary, the developed LFICS using AuNF and LM as signal amplification reporters exhibited excellent sensitivity and provided the rapid on-site screening of Qui and other analytes in food safety field.

Preparation and preliminary application of fluorescent microsphere test strips for feline parvovirus antibodies.

This study introduces a novel diagnostic modality for the detection of feline panleukopenia virus (FPV) antibodies in feline serum by using fluorescent microsphere immunochromatographic test strips (FM-ICTS). Leveraging the inherent specificity of antigen-antibody interactions, the FM-ICTS approach demonstrates considerable potential for efficient and accurate FPV antibody detection within a short timeframe. The FM-ICTS method demonstrates strong diagnostic performance, with consistent accuracy and stability over time. PBS buffer dilution enables detection across the range of FPV antibody haemagglutination inhibition (HI) titres in both healthy and immunized or infected cats. A high correlation (R² = 0.9733) between the T/C ratio and FPV antibody titres confirms the method's effectiveness in quantifying these titres. Clinical validation with 84 samples supports its reliability by matching results with HI assays. Additionally, stability tests show that the test strips maintain performance during storage, with a coefficient of variation (CV) below 12% over three months at 25℃. This innovative FM-ICTS framework emerges as a promising avenue for expedient and dependable disease diagnosis within the realm of veterinary science, offering implications for timely disease management and surveillance.

Detection of sow pregnancy in day-20 urine samples using monoclonal antibody against synthesized porcine early pregnancy factor: Preliminary results.

Early diagnosis of pregnancy is directly related to cost-effective livestock production. We produced a rat monoclonal antibody (mAb) against synthesized porcine early pregnancy factor (pEPF) using conventional hybridoma technology and used it as a tool for the detection of early pregnancy in Duroc sows. The rat pEPF-mAb showed reactivity to uterine tissues of pregnant sows 20 or 30 days post-mating (day 0 defined as the day of mating) and non-pregnant sows (confirmed signs of estrus) in western blotting. Immunohistochemical analysis confirmed that pEPF was located in the stromal and grand epithelial tissues of pregnant sows 20 or 30 days post-mating. In the enzyme-linked immunosorbent assay, pEPF expression in urine and blood showed similar results, with the highest expression observed in pregnant sows 20 days post-mating, whereas there was no significant difference in expression levels between non-pregnant sows and pregnant sows 30 days post-mating. The pEPF-mAb-based pregnancy diagnostic kit can be applied to pig urine samples non-invasively collected at 20 days post-mating with 70 % accuracy. Further improvements to the kit's diagnostic performance may lead to substantial benefits for the swine industry, facilitating more efficient and accurate reproductive management.

Monoclonal antibody based colloidal gold immunochromatographic assay for the visual and rapid screening of profenofos.

Profenofos (PFF) is a commonly used organophosphorus insecticide that requires strict monitoring due to its potential environmental, ecological, and human health risks originating from residues in soil and water systems, as well as accumulation in crops. In this study, a novel monoclonal antibody (mAb) specific to PFF was prepared for the first time and the recognition mechanism was investigated through molecular simulation. Subsequently, a mAb-based colloidal gold immunochromatographic assay (GICA) was developed for the rapid screening of PFF in fruit and vegetable samples. The mAb exhibited an IC50 value of 12.9 ng/mL, and limit of detection (LOD) of 4.6 ng/mL, respectively in indirect competitive immunosorbent enzyme-linked immunosorbent assay (ic-ELISA). After optimization, the developed GICA exhibited a visual limit of detection (vLOD) of 20 ng/mL and a quantitative of detection (qLOD) of 5.2 ng/mL, with a linear range from 10.0 to 83.8 ng/mL. Good correlation was observed between the results of GICA and standard Gas Chromatography-Tandem Mass Spectrometry (GC-MS/MS) in matrix and recovery test. The developed GICA can be used for rapid sample detection within 15 min, which is an excellent tool for screening PFF in foods and environmental samples.

Development of quantum dot-based immunochromatographic strip for detection of antibodies against ASFV pp62.

ASFV is the only known double-stranded insect-borne DNA virus, which can rapidly infect domestic pigs and wild boars with ticks as transmission medium. Since it was first discovered in 1921, it quickly spread to all parts of the world and brought huge economic losses to the pig industry all over the world. At present, there is still no safe and effective vaccine for ASFV. Here, we developed a quantum-dot labeled antibody test strip for the detection of antibodies against ASFV pp62. The pp62 protein was labeled with quantum dots, and the antibody test strip was developed uses it in a detection mode of labeled antigen-SPA interceptor-monoclonal antibody quality control. The test strip showed high sensitivity, the positive detection limit of the strip was 1: 106 by continuous multiple dilution using the positive standard serum of ASFV antibody as reference. The test strip showed good specificity, and there was no cross reaction with other swine diseases virus (PCV2, PRRSV, CSFV, PPV). Using the detection results of commercialized kit for African swine fever virus as reference, 80 ASFV antibody negative serum and 4 different ASFV antibody positive serum were detected using the ASFV pp62 quantum-dot labeled antibody test strip. The results were consistent with the commercial kit. This study provides a new detection method for the prevention and control of African swine fever.

Diagnostic Performances of an in-House Immunochromatography Test Based on the Monoclonal Antibody 18B7 to Glucuronoxylomannan for Clinical Suspected Cryptococcosis: a Large-Scale Prototype Evaluation in Northern Thailand.

OBJECTIVE: Cryptococcosis predominantly presents as a meningoencephalitis in Thailand. Early and expeditious diagnosis is essential for reducing both mortality and morbidity associated with cryptococcal meningitis. We aim to define and establish the diagnostic performances between the benchmark commercially available diagnostic kit (CrAg® LFA) and the large-scale prototype of an inexpensive in-house immunochromatographic test (ICT) based on monoclonal antibody (MAb) 18B7. METHODS: We have developed the large-scale prototype for the rapid detection of cryptococcal polysaccharide antigens by utilizing a single antibody sandwich ICT format employing MAb 18B7, which is highly specific to Cryptococcus neoformans glucuronoxylomannan (GXM) antigens. An in-house MAb18B7 ICT was manufactured in accordance with industry standards under the control of the International Organization for Standardization (ISO) 13485. RESULTS: The diagnostic sensitivity, specificity, and accuracy for the in-house MAb 18B7 ICT were 99.10%, 97.61%, and 97.83%, respectively. The agreement kappa (κ) coefficient was 0.968 based on the retrospective evaluation of 580 specimens from patients living in northern Thailand with clinically suspected cryptococcosis. CONCLUSION: The data suggest that this in-house MAb 18B7 ICT will be highly beneficial for addressing the issue of cryptococcal infection in Thailand. Moreover, it is anticipated that this inexpensive ICT can play a pivotal role in various global strategies aimed at eradicating cryptococcal meningitis among individuals living with HIV by 2030.

Fluorescence immunochromatographic detection of antibodies to the p72 protein of African swine fever virus.

The African swine fever virus (ASFV), a highly contagious pathogen responsible for African swine fever (ASF), causes significant economic losses in the global pork industry. Due to its large and complex structure, ASFV remains refractory to commercial vaccine development, necessitating the creation of rapid, sensitive, and specific diagnostic tools for disease control. In this study, quantum dots were conjugated to ASFV p72 protein to establish a fluorescent immunochromatographic assay for detecting ASFV-specific antibodies. The assay test strips contained four adjacent pads arranged sequentially: a sample-application pad, a pad containing mobile antigen-probe conjugate, a nitrocellulose readout pad featuring a test line containing immobilised staphylococcal protein A and a control line containing immobilised monoclonal antibodies against the ASFV p72 protein, and an absorbent pad driving the directional flow of liquid via capillary action. The resulting fluorescence immunochromatographic assay demonstrated highly sensitive and specific ASFV antibody detection in under 15 min. Specificity testing showed no cross-reactivity with serum antibodies against other viruses and sensitivity surpassing that of commercial ASFV antibody colloidal gold immunochromatographic test strips. This novel approach offers rapid detection, excellent specificity, and high sensitivity, and supports the future development of fluorescent immunochromatographic test strips for ASFV antibody detection.

Establishment of an immunoassay for detection of nonylphenols in surfactant oilfield chemicals based on computer-aided design.

Nonylphenols (NPs) are confirmed endocrine disruptors that are banned in many countries due to correlations with human cancers. NPs pollution in surfactant oilfield chemicals (OFCs) has become an important environmental safety issue. It is significant to establish a simple, accurate and low-cost method for detection of NPs in OFCs. In this research, computer-aided molecular design technology was utilized to design NPs haptens. High affinity monoclonal antibodies against NPs were obtained using a matrix effect-enhanced screening method, with an IC50 value of 183.01 ng/mL. A colloidal gold immunochromatography assay (ICA) for detection of NPs enabled rapid on-site detection of large volumes of OFCs. Under optimal conditions, the limit of detection was 0.72-1.82 mg/kg, with a detection range of 4.49-191.28 mg/kg. The recovery was 84 %-104 %, with coefficients of variation < 13 %. As confirmed by high-performance liquid chromatography of natural positive OFCs samples, the proposed colloidal gold ICA demonstrated accuracy and reliability, with potential for fast and economical on field test.

Development of a new accurate lateral flow immunoassay for diagnosis of human leptospirosis.

PURPOSE: The current diagnostic methods for leptospirosis diagnosis are technically complex and expensive, with limited applicability to specialized laboratories. Furthermore, they lack diagnostic accuracy in the acute stage of the disease, which coincides with a period when antibiotics are highly effective. New simple and accurate tests are mandatory to decentralize and improve diagnosis. Here, we introduced a new lateral flow immunoassay (Lepto-LF) for human leptospirosis. METHODS: We conducted a double-blinded assay using 104 serum samples from patients with confirmed or discarded diagnosis for leptospirosis. The diagnostic performance of Lepto-LF was estimated across different ranges of days from onset of symptoms (dpo), considering the diagnostic algorithm as reference standard. Additionally, it was compared with the screening methods enzyme-linked immunosorbent assay (IgM-ELISA) and the slide agglutination test using temperature-resistant antigen (SATR). RESULTS: Lepto-LF exhibited perfect diagnostic performance with a Youden´s index J = 1 from 6 dpo in the acute phase. IgM-ELISA gave slightly lower accuracy with J = 0.91 and 95.5% of both sensitivity and specificity; while SATR showed a markedly inferior yield (J = 0.41, sensitivity = 95.5%, specificity = 45.5%). The performances remained consistent in the convalescence phase of the disease (> 10 dpo). CONCLUSION: Lepto-LF was found to be a reliable test for simple, rapid and early diagnosis of leptospirosis, resulting a promising tool for decentralizing leptospirosis diagnosis and enabling timely treatment of patients. In addition, Lepto-LF may be employed as confirmatory test, especially in remote areas and vulnerable contexts where the standard MAT is not available.

Establishment of a multi-line immunochromatography based on magnetic nanoparticles for simultaneous screening of multiple biomarkers.

Biomarkers screening is a benefit approach for early diagnosis of major diseases. In this study, magnetic nanoparticles (MNPs) have been utilized as labels to establish a multi-line immunochromatography (MNP-MLIC) for simultaneous detection of carcinoembryonic antigen (CEA), carbohydrate antigen 199 (CA 19-9), and alpha-fetoprotein (AFP) in a single serum sample. Under the optimal parameters, the three biomarkers can be rapidly and simultaneously qualitative screening within 15 min by naked eye. As for quantitative detection, the MNP-MLIC test strips were precisely positioned and captured by a smartphone, and signals on the test and control lines were extracted by ImageJ software. The signal ratio of test and control lines has been calculated and used to plot quantitative standard curves with the logarithmic concentration, of which the correlation coefficients are more than 0.99, and the limit of detection for CEA, CA 19-9, and AFP were 0.60 ng/mL, 1.21 U/mL, and 0.93 ng/mL, respectively. The recoveries of blank serum were 75.0 ~ 112.5% with the relative standard deviation ranging from 2.5 to 15.3%, and the specificity investigation demonstrated that the MNP-MLIC is highly specific to the three biomarkers. In conclusion, the developed MNP-MLIC offers a rapid, simple, accurate, and highly specific method for simultaneously detecting multiple biomarkers in serum samples, which provides an efficient and accurate approach for the early diagnosis of diseases.

Comparison of SensPERT® Leishmania rapid test with two other immunochromatographic tests for the diagnosis of canine visceral leishmaniasis in Brazil.

Background and Aim: In urban environments, dogs serve as the primary reservoir for visceral leishmaniasis (VL). Rapidly diagnosing canine VL through tests enables early treatment and a favorable prognosis. This study aimed to assess the diagnostic performance of the SensPERT® Leishmania test kit (Dechra®), Alere® Leishmaniasis Ac test kit, and the rapid test dual path platform (TR-DPP®) Bio-Manguinhos in detecting VL. Materials and Methods: 30 serum samples from reactive VL dogs and 30 serum samples from healthy dogs were employed for assessing the sensitivity and specificity variation between SensPERT® Leishmania test kit, Alere® Leishmaniasis Ac test kit, and rapid test dual platform - TR-DPP®. Results: The SensPERT® Leishmania test outperformed Alere® and TR-DPP® in terms of sensitivity, specificity, positive and negative predictive values and demonstrated near-perfect concordance with Alere® and substantial concurrence with TR-DPP®. Conclusion: The SensPERT® Leishmania rapid test proved to be a promising test in the detection of VL in dogs.

A paper-based lateral flow immunochromatographic sensor for the detection of tricyclazole in rice.

Tricyclazole is commonly used to prevent rice blast to meet the carbohydrate intake needs of half of the global population, and a large number of toxicological reports indicate that monitoring of tricyclazole is necessary. Here, we analyzed the structure of tricyclazole and designed different hapten derivatization strategies to prepare a high-performance monoclonal antibody (half inhibition concentration of 1.61 ng/mL), and then a lateral flow immunochromatographic sensor based on gold nanoparticles for the detection of tricyclazole in rice, with a limit of detection of 6.74 µg/kg and 13.58 µg/kg in polished and brown rice, respectively. The recoveries in rice were in the range of 84.6-107.4%, no complex pretreatment was required for comparison with LC-MS/MS, and the comparative analysis demonstrated that our method had good accuracy and precision. Therefore, the developed lateral flow immunochromatographic analysis was a reliable and rapid means for the on-site analysis of tricyclazole in rice.

Hybrid nanoflower-based electrochemical lateral flow immunoassay for Escherichia coli O157 detection.

An electrochemical biosensor has been developed for detection of Escherichia coli O157 by integrating lateral flow with screen-printed electrodes. The screen-printed electrodes were attached under the lateral flow detection line, and organic-inorganic nanoflowers prepared from E. coli O157-specific antibodies as an organic component were attached to the lateral flow detection line. In the presence of E. coli O157, an organic-inorganic nanoflower-E. coli O157-antimicrobial peptide-labelled ferrocene sandwich structure is formed on the lateral flow detection line. Differential pulse voltammetry is applied using a smartphone-based device to monitor ferrocene on the detection line. The resulting electrochemical biosensor could specifically detect E. coli O157 with a limit of detection of 25 colony-forming units mL-1. Through substitution of antibodies of organic components in organic-inorganic nanoflowers, biosensors have great potential for the detection of other pathogens in biomedical research and clinical diagnosis.

Epitope-Shared Hapten Enhancing Antibody Polyreactivity for Simultaneous Immunochromatography of Oxyphenisatin Adulterants.

Given the significant threat posed by oxyphenisatin adulterants (OPHs) in weight-loss foods, simultaneous analysis of the OPHs is necessary. Herein, four novel haptens based on the general epitope shared among the OPHs were raised by computer-aided chemical modeling prediction, with the expectation of eliciting antibody responses targeting three of the OPHs. One obtained monoclonal antibody (mAb) showed maximal half-inhibitory concentration (IC50) of 0.40-12.11 ng/mL for OPHs. The key interaction forces responsible for the corecognition of the OPHs were revealed by the intrinsic molecular mechanism. The developed immunochromatography (ICA) indicated a detection capability for screening (CCß) for OPHs estimated to be 5-600 ng/g in jelly, candy tablets, and oral liquid. Furthermore, the analysis of 15 real samples by our method showed a good correlation with liquid chromatography-tandem mass spectrometry (LC-MS/MS). Our research not only presented a rapid approach for identifying OPHs adulteration but also proposed an effective hapten prediction strategy to enhance antibody polyreactivity.

Tris stabilized AuNPs based lateral flow immunochromatography for the simultaneous detection of porcine epidemic diarrhea virus and rotavirus on-site.

Porcine epidemic diarrhea virus (PEDV) and rotavirus has posed a significant threat to the pig industry annually across different nations, resulting in huge economic losses. The frequent co-infection of these two viruses in clinical settings complicates the process of differential diagnoses. Rapid and accurate detection of PEDV and rotavirus is in great demand for timely diarrhea disease prevention and control. In this study, tris stabilized AuNPs were prepared and a sensitive lateral flow immunoassay (LFIA) sensor was developed for the simultaneous and rapid detection of PEDV and rotavirus on site. After the system optimization, the established LFIA can simultaneously identify PEDV and rotavirus with limits of detection (LOD) of 1.25 × 103 TCID50 mL-1 and 3.13 × 102 pg mL-1, respectively. When applying for clinical samples, the LFIA show a concordance of 95 % and 100 % to reverse transcript polymerase chain reaction (RT-PCR) for PEDV and rotavirus respectively. Therefore, this LFIA can qualitatively detect PEDV and rotavirus in 18 min with high sensitivity and accuracy without any sophisticated equipment and operation, making it a promising candidate for the early diagnosis of PEDV or/and rotavirus diarrhea on site.

Preparation of Hybrid Magnetic Nanoparticles for Sensitive and Rapid Detection of Phorate Residue in Celery Using SERS Immunochromatography Assay.

Extensive use of pesticides in agricultural production has been causing serious health threats to humans and animals. Among them, phorate is a highly toxic organophosphorus insecticide that has been widely used in planting. Due to its harmful effects on human and animal health, it has been restricted for use in many countries. Analytical methods for the rapid and sensitive detection of phorate residues in agricultural products are urgently needed. In this study, a new method was developed by combining surface-enhanced Raman spectroscopy (SERS) and immunochromatography assay (ICA). Hybrid magnetic Fe3O4@Au@DTNB-Ab nanoprobes were prepared by modifying and growing Au nanoseeds on an Fe3O4 core. SERS activity of the nanoprobe was optimized by adjusting the concentration of the Au precursor. A rapid and sensitive assay was established by replacing the traditional colloidal gold-based ICA with hybrid SERS nanoprobes for SERS-ICA. After optimizing parameters including coating antibody concentrations and the composition and pH of the buffer solution, the limit of detection (LOD) for phorate could reach 1 ng/mL, with a linear range of 5~100 ng/mL. This LOD is remarkably lower than the maximum residue limit in vegetables and fruits set by the Chinese government. The feasibility of this method was further examined by conducting a spiking test with celery as the real sample. The result demonstrated that this method could serve as a promising platform for rapid and sensitive detection of phorate in agricultural products.

General hapten skeleton motivated duplex-immunoassay for emergent bisoxatin adulterants in slimming foods.

Adulterating hazardous bisoxatin (BSO) and bisoxatin acetate (BSOA) in slimming foods poses a threat to public health. A rapid synchronous detection method is urgently needed. Herein, the precise design of four novel haptens based on the general skeleton of BSO and BSOA was driven by computer-chemical visualization strategy, which was used to raise monoclonal antibody (mAb) toward both target compounds. The generated mAb 1F1 recognized BSO and BSOA with maximal half-inhibitory concentration (IC50) of 0.26 and 16.85 ng/mL, respectively. The molecular mechanism governing the duplex-recognition of mAb was elucidated by homology modeling and molecular docking. Finally, an immunochromatography (ICA) was developed for identifying BSO and BSOA, demonstrating a detection capability for screening (CCß) estimated to be 10-500 ng/g in candy tablets, jellies, and oral liquids. This study provides a robust approach for determining adulteration in food and offers insights into hapten design to improve antibody recognition spectrum.

Development and application of a colloidal-gold immunochromatographic strip for detecting Getah virus antibodies.

Getah virus (GETV) is a re-emerging mosquito-borne alphavirus that is highly pathogenic, mainly to pigs and horses. There are no vaccines or treatments available for GETV in swine in China. Therefore, the development of a simple, rapid, specific, and sensitive serological assay for GETV antibodies is essential for the prevention and control of GETV. Current antibody monitoring methods are time-consuming, expensive, and dependent on specialized instrumentation, and these features are not conducive to rapid detection in clinical samples. To address these problem, we developed immunochromatographic test strips (ICTS) using eukaryotically expressed soluble recombinant p62-E1 protein of GETV as a labelled antigen, which has good detection sensitivity and no cross-reactivity with other common porcine virus-positive sera. The ICTS is highly compatible with IFA and ELISA and can be stored for 1 month at 37 °C and for at least 3 months at room temperature. Hence, p62-E1-based ICTS is a rapid, accurate, and convenient method for rapid on-site detection of GETV antibodies. KEY POINTS: • We established a rapid antibody detection method that can monitor GETV infection • We developed colloidal gold test strips with high sensitivity and specificity • The development of colloidal gold test strips will aid in the field serologic detection of GETV.