Immunochromatographic Method Research Articles

Dengue Virus Detection in Dengue Hemorrhagic Fever Patients Using the NS1 Antigen-Based Test with Immunochromatography and SDS-Page Methods in the Regional General Hospital of Kendari City

Dengue Hemorrhagic Fever is one of the diseases that widely damages people; this disease is always found in tropical and subtropical regions, especially in Southeast Asia, Africa, and North Amerika [1]

AMELIORATIVE POTENTIALS OF DICHLOROMETHANE EXTRACT OF Musa paradisiaca BRACT IN INDOMETHACIN ADMINISTERED WISTAR RATS

This study aimed to ascertain the ameliorative potentials of dichloromethane extract of Musa paradisiaca bract in Wistar rats administered indomethacin. The bracts were processed and extracted using dichloromethane and the bioactive components in sample were analysed using gas chromatography-mass spectrometry (GC-MS) methods. Forty five male Wistar rats (120-180g) in five groups of nine rats each were used for in-vivo analysis. A single oral dose of 40mg/kg body weight indomethacin was administered to study groups and faecal occult blood tests (FOB) of animals were carried out using immunochromatographic sandwich methods. Study groups were treated with 200mg, 150mg and 100mg/kg bodyweight of extract for three weeks. The animals were sacrificed and selected biochemical indices, hematological parameters and histopathology of kidney, stomach and liver were determined. GC-MS analyses revealed eleven bioactive compounds; fatty acids, amides and amines in the sample analysed. FOB tests were positive (+) at week 0 and week one, then negative (-) at weeks two and three in all treated groups. Significant dose-dependent increases (p≤ 0.05) in packed cell volume, hemoglobin and red blood count was observed in treated groups (38.80 ± 0.15%, 12.93 ± 0.05g/dl and 5.85 ± 0.05x109/L) respectively compared to untreated control (21.70 ± 0.12%, 7.24 ± 0.04g/dl, and 3.75 ± 0.05x109/L). Serum levels of Aspartate aminotransferase, Alanine aminotransferase, Alkaline phosphatase, creatinine and urea showed a dose-dependent decrease significantly at (p≤ 0.05) in treated groups, compared to untreated control. Kidney, stomach and liver tissues in treated groups showed no cellular degeneration after three weeks of treatment, compared to control. This study revealed that the dichloromethane extract of M. paradisiaca bract showed dose-dependent ameliorative potentials and non-impairment of hepatorenal function in Wistar rats administered indomethacin.

Quality control and mycotoxin levels in food in the Palestinian market

ABSTRACT A total of 51 food samples (18 rice, 17 wheat flour, and 16 nuts) were randomly collected from different shops and analysed for the presence of total aflatoxins, ochratoxin A and fumonisins, using a lateral flow competitive immunochromatographic assay method. Mycological contamination (total aerobic and fungi count), colour index (L*a*b*) and moisture content were also evaluated. Sensory characteristics for mycotoxins contaminated and uncontaminated samples of rice and wheat flour were evaluated by 40 panellists. Forty-five per cent of all samples were positive for aflatoxins as the most occurred mycotoxins, with a range of 2–8.1 μg/kg. Fungal counts were not significantly different between contaminated and uncontaminated rice and wheat flour samples. In addition, panellists were able to discriminate between contaminated and uncontaminated rice samples by detection of mouldy flavour, spoilage symptoms, earthy flavour, and the degree of freshness.

About quantitative determination of the D-dimer in the blood by immunochromatographic method.

The paper presents the results of the development of a technology for the quantitative determination of D-dimer in blood with an immunochromatographic (LFIA) kit of reagents «LFIA-D-dimer» and instrument accounting of the results. Registration and processing of the digitized indicator of the intensity of staining of the LFIA-test strip using the LFIA-analyzer software allows quantifying the D-dimer content in the sample (in ng DDU/ml). The effectiveness of the proposed approach was evaluated on 258 clinical samples examined in the LFIA with visual and instrument accounting of the results, in comparison with the indicators of D-dimer determination in ELISA. The high reproducibility of the digitized LFIA results was shown - the coefficient of variation (CV) for samples in the range of 100-300 ng DDU/ml (near-threshold in relation to pathological values) was 2.5-5.1%; a tendency to increase CV with a further increase in the concentration of D-dimer was traced. A high correlation of the digitized LFIA result with the research data in the ELISA has been established, which makes it possible to recommend the technology for widespread use in urgent medicine.

Seroprevalence of Hepatitis B Virus using HBV-5 Rapid Panel Test and Associated Factors amongst Pregnant Women Attending Antenatal Care in Garoua, North-Cameroon

Background: In Cameroon, prevalence of Hepatitis B Virus (HBV) is high and varies from different places in general population and vulnerable persons such as pregnant women. We performed this survey to determine seroprevalence of HBV using HBV-5 Rapid panel test and associated factors amongst pregnant women attending antenatal care (ANC) in Garoua. Methods: This was a cross-sectional study conducted from February, 15th to April, 15th, 2016 amongst 102 pregnant women attending ANC in city of Garoua. Data were obtained using a structured questionnaire by interview. The blood samples were collected and tested by the immuno-chromatographic panel method (OnSite HBV-5 Rapid Panel Test) for the detection of HBV biomarkers. Statistical analyses were performed by EPI InfoTM version 7 software, with P<0.05 considered significant. Results: Overall seroprevalence of HBV (HBsAg) was 10.78% (11/102) and the other HBV biomarkers were 15.68% (16/102), 9.80 (10/102) and 26.47% (27/102) for anti-HBsAb, anti-HBeAb and total anti-HBcAb respectively. According to general information, marital status (P=0.001) was statistically associated with HBsAg seroprevalence. Bivariate analysis logistic regression recorded that, scarification (OR= 30.10; 95% CI 6.55-138.15; P=0.00000) and piercing or tattoo (OR= 11.80; 95% CI 2.77-50.18; P=0.00008) were statistically associated with seroprevalence of HBsAg. Conclusion: Seroprevalence of HBV biomarkers is high amongst pregnant women attending ANC in Garoua. Associated factors such as scarification and piercing or tattoo were associated with HBV infection. The awareness of pregnant women about vaccination in routine against HBV were necessary to prevent the transmission of Mother-to-Child Transmission (MTCT).

Prevalence of Hepatitis C in Dialysis Patients in Khyber Teaching Hospital Peshawar

Introduction: The hepatitis C virus (HCV) is a Flaviviridae-related virus that causes a systemic illness that can be transmitted. HCV is a blood-borne infection that primarily affects the liver. HCV diagnosis that is accurate and timely is critical in significantly reducing the risk of morbidity and mortality. The goal of this study was to find out the Hepatitis C in chronic renal failure patients who are on hemodialysis. Method: This descriptive cross-sectional study was carried out in the Dialysis Unit of the Khyber Teaching Hospital (KTH) Peshawar's Department of Nephrology. From September to December 2017, this study took place over four months. The study included a total of 114 patients, regardless of their age or gender. Hepatitis C was detected in whole blood samples collected from 114 healthy people using an immunochromatography method. The data was analyzed using SPSS v.22. Result: The Immunochromatography technique for HCV was used to analyze 114 blood samples from recruited individuals. Male patients were 52.6% of the total, while female patients made up 47.4%. The study found that 24.56% of male patients were positive, while 28.07% of female patients were positive. Overall, the technique's sensitivity and specificity were 83.1% and 72.7%, respectively. Conclusion: It is concluded that the HCV rapid testing is still sensitive and specific for detecting hepatitis C virus, it should be used in routine clinical settings for early hepatitis C virus diagnosis when advanced diagnostic options are restricted. Keywords: HCV, Hepatitis C virus, Nephrology, Dialysis

Development of a Rapid Gold Nanoparticle Immunochromatographic Strip Based on the Nanobody for Detecting 2,4-DichloRophenoxyacetic Acid.

2,4-Dichlorophenoxyacetic acid (2,4-D) is a systemic conductive herbicide widely used across the world. With the large-scale and continuous use of 2,4-D, its possible harm to the environment and non-target organisms has attracted increasing attention, and the construction of a stable rapid on-site detection method is particularly important. In order to achieve on-site rapid detection of 2,4-D, we developed a gold nanoparticle immunochromatographic strip method with the visual elimination value was 50 ng/mL, and a quantitative detection limit of 11 ng/mL based on a nanobody. By combing with the color snap, the immunochromatographic strip could quantitatively analyze the amounts of 2,4-D. Meanwhile, a colorimetric card based on the true color of the test strips was developed for the qualitative analysis of 2,4-D on-site. The samples (water, fruits and vegetables) with and without 2,4-D were detected by the immunochromatographic strips, and the results showed the accuracy and reliability. Thus, this assay is a rapid and simple on-site analytical tool to detect and quantify 2,4-D levels in environmental samples, and the analytical results can be obtained in about ten minutes. In addition, the nanobody technology used in this study provides an inexhaustible supply of a relatively stable antibodies that can be archived as a nanobody, plasmid or even its sequence.

Comparative assessment of the results of detection of СagА-positive strains of Helicobacter pylori by molecular-genetic and immunochromatographic methods in different biological materials.

A comparative analysis of the detection of CagA-positive strains of H. pylori by immunochromatographic and molecular genetic methods was carried out. We used H. pylori strains isolated from individuals with diseases of the gastrointestinal tract. The immunochromatographic method was implemented using a developed experimental model of an immunochromatographic test system for detecting the H. pylori CagA protein in various biological materials. Determination of the pathogenicity gene cagA of H. pylori was carried out using the «Helikopol SA» test system («Litekh», Russia). The assessment of the comparability of the results of detecting CagA-positive strains of H. pylori was carried out using statistical methods: Monte-Carlo, calculation of the chi-square test (χ2) and Kendall's τ-b and Somer's d coefficients. Statistical analysis was performed using the software packages «Microsoft Office Excel», «Statistica 10.0», «WinBUGS 1.4.0.» The study showed the absence of a statistically significant difference and the presence of a direct strong correlation between the results of detecting CagA-positive strains by molecular genetic and immunochromatographic methods, which indicates that these methods provide similar results in identifying highly pathogenic strains of H. pylori.

Determining the Prevalence of Norovirus Infection in Diarrheal Patients and Evaluation of Different Diagnostic Methods

Norovirus is one of the viruses that cause gastroenteritis in humans, characterized by symptoms of vomiting and diarrhea. The prevalence of norovirus, known as the leading cause of epidemic gastroenteritis, is remarkable in sporadic cases. Easy-to-apply diagnostic methods based on antigen detection such as enzyme immunoassay (EIA) and immunochromatography (ICG) used to diagnose norovirus infections generally have high specificity rates but lower sensitivity rates that can change according to the conditions. In this study it was aimed to determine the prevalence of norovirus and other gastroenteritis causative viruses in diarrheal patients and determine the sensitivity and specificity rates of EIA and ICG methods in sporadic cases by the chosen gold standard molecular reference method real-time reverse transcriptase polymerase chain reaction (rRT-PCR). In this study, 184 stool samples that met the study criteria and sent to İstanbul University İstanbul Faculty of Medicine Medical Microbiology Laboratory for routine bacteriological culture between January-July 2018 were included. All samples were evaluated with diagnostic kits BD MAX Enteric Viral Panel (Becton Dickinson, Canada) for rRT-PCR, RIDASCREEN Norovirus 3rd Generation (C1401) (R-Biopharm, Germany) for EIA, RIDAQUICK Norovirus Test (N1402) (R-Biopharm, Germany) for ICG, according to the manufacturer instructions. In terms of the presence of norovirus in 184 stool samples, 7 (3.8%) positive results were obtained by EIA method, 8 (4.3%) by ICG method, and 14 (7.6%) by rRT-PCR method. By accepting the rRT-PCR as a reference method, the sensitivity and specificity of the EIA method were determined as 50% and 100% and of the ICG method as 57% and 100%, respectively. The numbers and percentages of positivity for rotavirus, sapovirus, astrovirus and adenovirus, including coinfections were 30 (16.3%), 5 (2.7%), 2 (1%), 1 (0.5%), respectively. In this study, it was determined that norovirus, alone or together with other viral agents is frequently detected in patients with diarrhea and there was no difference in the frequency between age groups and genders. This causative agent which is not routinely investigated in our country should be considered when evaluating patients with diarrhea and/or vomiting. It seems that EIA and ICG methods are easy to apply, have high specificity but have limited sensitivity in sporadic cases in the diagnosis of norovirus. It is thought that the detection of the true frequency of norovirus and high sensitivity rates can only be achieved by preferring rRT-PCR in the diagnosis. It would be useful for the laboratories to choose the method to be used in the diagnosis of norovirus according to the characteristics of the cases and diagnostic methods.

An ultrasensitive colloidal gold immunosensor to simultaneously detect 12 beta (2)-adrenergic agonists

In this study, we first prepared a selective monoclonal antibody against 12 beta (2)-adrenergic agonists (Salbutamol, Clenbuterol, Brombuterol, Clenpenterol, Mabuterol, Carbuterol, Cimbuterol, Mapenterol, Pirbuterol, Terbutaline, Cimaterol, and Clenproperol). Then three haptens were designed and derived, among which, haptenS3 used the amino group of the salbutamol analog to derive a carboxyl group containing a spacer, which is unique to this study. The half-maximal inhibitory concentration (IC50) values were 0.35 ng/mL (Salbutamol), 0.42 ng/mL (Clenbuterol), 0.78 ng/mL (Brombuterol), 0.88 ng/mL (Clenpenterol), 1.34 ng/mL (Mabuterol), 1.38 ng/mL (Carbuterol), 1.71 ng/mL (Cimbuterol), 2.24 ng/mL (Mapenterol), 2.25 ng/mL (Pirbuterol), 2.27 ng/mL (Terbutaline), 3.49 ng/mL (Cimaterol), and 4.89 ng/mL (Clenproperol). We further developed a monoclonal antibody-based colloidal gold immunochromatographic test strip for screening and detecting 12 beta (2)-adrenergic agonists in swine urine and lamb samples. The immunochromatographic method developed in this study is a suitable tool for the on-site rapid detection and screening of beta (2)-adrenergic agonists in swine urine and lamb samples.

A nanomaterial-free and thionine labeling-based lateral flow immunoassay for rapid and visual detection of the transgenic CP4-EPSPS protein

Nanomaterial-based lateral flow immunoassays (LFIAs) have been widely used for the on-site detection of genetically modified components. However, the practical applications are often limited by the complex matrix, such as in red samples. In this study, a thionine (Thi) labeling-based LFIA was developed for the first time to detect CP4-EPSPS protein. The optimal labeling concentration of Thi was 0.5 mg/mL, and the antibody could be rapidly coupled to Thi in 10 min. The visual limit of detection (vLOD) levels for transgenic soybean, sugar beet, and cotton containing the CP4-EPSPS protein reached 0.05%, 0.1%, and 0.1%, respectively, and had no interference from other proteins. After storage at 4 °C for three months, the LFIA sensitivity remained unchanged and showed good stability. This method could be used to screen and detect a variety of transgenic crops containing the CP4-EPSPS protein, and the results were consistent with the current standard assay. This study pioneered the development of an immunochromatographic method using Thi as a marker and applied it to the detection of the CP4-EPSPS protein in herbicide-tolerant transgenic crops. This provides a new method for the rapid immunoassay of Thi as a dye and has good prospects for practical application.

Ultrasensitive lateral flow immunoassay for staphylococcal enterotoxin B using nanosized fluorescent metal-organic frameworks.

Owing to their outstanding optical properties and superior physical/chemical stability, dye-doped fluorescent nanoparticles (NPs) are growing exponentially as signal labels of immunochromatographic lateral flow immunoassay (LFA) for the detection of various analytes. However, the key challenge in the design of these fluorescent NPs is to confine the fluorophores inside NPs at extreme concentrations, at which dyes tend to self-quench resulting from the formation of non-fluorescent aggregates. Looking for other advantageous nanomaterials, we propose for the first time the use of a nanosized fluorescent metal-organic framework (nanoMOF) in LFA for the detection of staphylococcal enterotoxin B (SEB) as a model analyte. Featured by the chromophore assembly, the nanoMOF exhibited a high dye loading (∼60%) and strong fluorescence intensity, which was due to the reduced self-quenching of dyes in a variety of MOF matrices. The strong green fluorescence intensity of the nanoMOF gives a high contrast against the background of the strips and the sensitivity reflected by photoluminescence was improved by the enhanced antenna effect. Furthermore, due to the high surface area for antibody stemming, the limit of detection (LOD) of the MOF based LFA for SEB detection was as low as 0.025 ng mL-1. The compatibility of the MOF based LFA with dairy samples and its stability under long-term storage conditions were also demonstrated. The integration of a nanoMOF into LFA to detect toxins could inspire the utilization of such nanomaterial-based labels in similar immunochromatographic testing methods to improve their performance.

LATERAL FLOW TECHNIQUE FOR THE DETERMINATION OF DIBUTYL PHTHALATE

An immunochromatographic method for the rapid determination of dibutyl phthalate has been developed. For this purpose, the competitive immunochemical interaction of monoclonal antibodies labeled with gold nanoparticles with a hapten-protein conjugate and a free analyte in various media was characterized. An immunochromatographic test system has been tested to detect and evaluate the content of dibutyl phthalate in natural waters.

The early detection of type 1 diabetes mellitus and latent autoimmune diabetes in adults (LADA) through rapid test reverse-flow immunochromatography for glutamic acid decarboxylase 65 kDa (GAD65)

BackgroundDiabetes mellitus (DM) is a chronic and costly disease that has become a primary concern worldwide. Type 1 diabetes mellitus is categorized as an autoimmune disease, which results in islet cell apoptosis and insulin-dependent. GAD65 is known as a potential marker of impaired pancreatic β cell function that appears in the initial phase of type 1 DM and latent autoimmune diabetes in adults (LADA). This study aimed to develop a novel rapid test of anti-GAD65 autoantibodies in human serum samples. MethodsWe have developed a rapid test for anti-GAD65 autoantibodies in this assay based on the reverse-flow immunochromatography method. Human recombinant-protein antigen for GAD65 was attached as the control line over the nitrocellulose membrane. On the other side, the goat anti-mouse immunoglobulin G (IgG) was coated on the same membrane as a control line. The positive result for GAD65 was confirmed by a colloidal gold signal on the strip. Our novel assay analyzed 276 healthy subjects and 51 type 1 diabetes individuals serum samples from several ethnicities in Indonesia for this study. ResultsAmong the 276 healthy samples, 225 samples were identified as positive for anti-GAD65 autoantibodies, while 51 samples were negative. Interestingly, the positive results for anti-GAD65 autoantibodies were linear to the decreasing of high-density lipoprotein (HDL) levels and inversely associated with triglyceride levels. A significant correlation with age was observed in all groups. The sensitivity and specificity test proved that this kit has higher accuracy (AUC value = 0.960). ConclusionThe significant advantages of our rapid test for anti-GAD65 autoantibodies provide higher sensitivity, specificity, and stability compared to previous commercial kits. Therefore, it could be proposed as the future clinical diagnostic kit for patient management of type 1 DM.

ASSESSMENT OF IL-33, TNF- α AND LEUKOTREINS B4 IN PATIENTS SERUM WITH ENTERIC FEVER

Salmonella typhoid and paratyphoid are transmitted mainly by the fecal-oral route. This study was designed to find the correlation between Salmonella and IgM, IgG, and the levels of interleukin-33, TNF-α, and LTB 4. The study was carried out from March 2020 to January 2021 for the detection of Salmonellosis in 100 suspected patients with age group ranging from 17 - 69 years, who attended Baghdad teaching hospitals that had been examined and defined as suspected cases by a specialized physician with the recording of clinical manifestation. The diagnosis was done by immunochromatography method, a blood sample was taken from the patient as well as other 30 healthy controls matching in age and gender. The study included measurement of the level of interleukin -33, Tumor necrosis Factor α, and Leukotreins B4 level in sera of patients and healthy control. The results indicated that anti –salmonella IgM positive in 54 cases, anti- salmonella IgG positive in 46 cases, and 18 positive cases with both IgM and IgG. The Level of interleukin 33, Tumor necrosis Factor – α increased significantly while the serum Leukotreins B4 level decreased significantly in patients sera as compared with healthy control.

Qualitative evaluation of the new rapid point-of-care antigen Afias Covid-19/Flu Ag Combo assay

Background: An interim guidance document by the WHO (World Health Organization) suggests the use of rapid diagnostic tests based on antigen detection as an alternative to the real-time PCR test for the diagnosis of SARS-CoV2 infection, when the molecular RT-PCR test is not available, or the turnaround time is excessive, precluding its clinical and/or public health usefulness. Rapid antigenic tests are recommended when a minimum of 80% sensitivity and 97% specificity are ensured. Methods: Here we employ a new assay for screening applications based on lateral-flow immunofluorescence assay, with microfluidic technology (Boditech AFIAS COVID-19/Flu Ag Combo) on a point-of-care analyzer AFIAS-6 (BoditechMed. Inc.) and compare it with a reference molecular method and an alternative screening immunochromatographic method (Boditech AFIAS COVID-19 Ag). Our study was carried out on stored UTM (at -20°C) samples of patients admitted to Di Venere Hospital of Bari that were already tested with molecular methods. Results: The new AFIAS COVID-19 Flu/Ag Combo test reached a clinical sensitivity of 92% on positive samples with Ct< 30, and a clinical specificity of 97.9% on negative samples. Sensitivity is higher than the reference immunochromatographic test AFIAS COVID-19/Ag (92.0% vs 88.0%) while specificity remains unchanged (97.9% vs 98.0%). In addition the new AFIAS Combo test confirm the same negative predictive value (NPV, 95.9%) of the rapid reference test (AFIAS COVID 19-Ag) and an agreement with the molecular test of 95.9% (Cohen's k = 0.908). Conclusions: Considering its qualitative improvement, rapidity and ease of use we suggests AFIAS Combo test as a valid alternative to the reference lateral flow test (AFIAS COVID-19 Ag) and an adequate screening test. Keywords: SARS-CoV2; COVID-19; POCT; diagnosis; screening; immunoassay; rapid antigen test

Preparation of highly specific monoclonal antibodies against SARS-CoV-2 nucleocapsid protein and the preliminary development of antigen detection test strips.

The coronavirus disease 2019 (COVID‐19) is outbreaking all over the world. To help fight this disease, it is necessary to establish an effective and rapid detection method. The nucleocapsid (N) protein of severe acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2) is involved in viral replication, assembly, and immune regulation and plays an important role in the viral life cycle. Moreover, the N protein also could be a diagnostic factor and potential drug target. Therefore, by synthesizing the N gene sequence of SARS‐CoV‐2, constructing the pET‐28a (+)‐N recombinant plasmid, we expressed the N protein in Escherichia coli and obtained 15 monoclonal antibody (mAbs) against SARS‐CoV‐2‐N protein by the hybridomas and ascites, then an immunochromatographic test strip method detecting N antigen was established. In this study, we obtained 14 high‐titer and high‐specificity monoclonal antibodies, and the test strips exclusively react with the SARS‐CoV‐2‐N protein and no cross‐reactivity with other coronavirus and also recognize the recombinant N protein of Delta (B.1.617.2) variant. These mAbs can be used for the early and rapid diagnosis of SARS‐CoV‐2 infection through serological antigen.

Construction of a TRFIC strip for rapid and sensitive detection of Ralstoniasolanacearum

The development of a sensitive and rapid screening method for Ralstonia solanacearum is critical for the control of tobacco wilt. In the present study, tissue homogenates of three tobacco varieties (Honda, Yunnan 87 and K326) with different resistance to R. solanacearum, were individually used as additives to the bacteria culture medium. The changes in R. solanacearum secretome were investigated and one of the most abundant secretary proteins with increased expression, polygalacturonase (PG), was selected as a marker for R. solanacearum identification. Then PG gene was cloned into E. coli, and the expressed protein was used as the immunogen to develop monoclonal antibodies. Subsequently, the monoclonal antibody against PG was coupled with synthesized polystyrene microspheres, and a rapid test strip system was developed for the detection of R. solanacearum based on time-resolved fluorescent immunochromatographic (TRFIC) method. Under optimal conditions, the detection limit of the strips could reach 72 cells/mL; while it was 422 cells/mL with a linear range from 4 × 102 to 5.12 × 104 cells/mL when testing tobacco samples, which is 1000 times lower than that of colloidal gold-labeled strips. Notably, no cross-reactivity was observed with nine tobacco-related pathogens. Finally, this TRFIC strips was applied to detect R. solanacearum existed in the tobacco and soils of fields with or without bacterial wilt. The results demonstrated that this TRFIC strips could distinguish the difference in bacterial concentration existed in tobacco and soil between the two fields. In summary, this test strip is suitable for sensitive, quick screening of R. solanacearum in tobacco.

Graphene-based hybrid electrical-electrochemical point-of-care device for serologic COVID-19 diagnosis

The outbreak of COVID-19 pandemics highlighted the need of sensitive, selective, and easy-to-handle biosensing devices. In the contemporary scenario, point-of-care devices for mass testing and infection mapping within a population have proven themselves as of primordial importance. Here, we introduce a graphene-based Electrical-Electrochemical Vertical Device (EEVD) point-of-care biosensor, strategically engineered for serologic COVID-19 diagnosis. EEVD uses serologic IgG quantifications on SARS-CoV-2 Receptor Binding Domain (RBD) bioconjugate immobilized onto device surface. EEVD combines graphene basal plane with high charge carrier mobility, high conductivity, low intrinsic resistance, and interfacial sensitivity to capacitance alterations. EEVD application was carried out in real human serum samples. Since EEVD is a miniaturized device, it requires just 40 μL of sample for a point-of-care COVID-19 infections detection. When compared to serologic assays such ELISA and other immunochromatographic methods, EEVD presents some advantages such as time of analyses (15 min), sample preparation, and a LOD of 1.0 pg mL-1. We glimpse that EEVD meets the principles of robustness and accuracy, desirable analytic parameters for assays destined to pandemics control strategies.

1251. Comparative Evaluation of Phenotypic Synergy Tests vs. Resist-4 O.K.N.V® and NG Test Carba 5® Lateral Flow Immunoassays for the Detection and Differentiation of Carbapenemases in Enterobacterales and Pseudomonas aeruginosa

BackgroundThe spread of carbapenem resistant Pseudomonas aeruginosa and carbapenemase-producing Enterobacterales (CPE) has had a great impact on morbidity and mortality. COVID-19 pandemic has favoured the selection of these microorganisms because of the excessive and prolonged use of broad-spectrum antibiotics and the outbreaks related to patient transfer between hospitals and inadequate use of personal protective equipment. Therefore, detection is considered essential for their control. Our aim was to compare conventional phenotypic synergy tests and two lateral flow immunoassays for detecting carbapenemases in Enterobacterales and P. aeruginosa.MethodsWe analysed 100 carbapenem-resistant Gram-negative bacilli isolates, 80 Enterobacterales and 20 Pseudomonas aeruginosa, (86 isolates producing KPC, NDM, OXA-48, IMP and VIM carbapenemases and 14 non-carbapenemase-producing isolates). We performed a modified Hodge test, boronic acid and ethylenediaminetetraacetic acid (EDTA) synergy tests, and two lateral flow immunoassays: RESIST-4 O.K.N.V (Coris Bioconcept®) and NG Test Carba 5® (NG Biotech®).ResultsIn total, 76 KPC, 7 VIM, 1 NDM, 1 OXA-48 and 1 isolate coproducing KPC + NDM enzymes were included. The concordance of different methods estimated by Kappa index was 0.432 (Standard error: 0.117), thus showing a high variability with the synergy tests with boronic acid and EDTA and reporting 16 false negatives that were detected by the two immunochromatographic methods. Co-production was only detected using immunoassays. Figure 1. Errors in synergy tests. 1) KPC-producing K. pneumoniae not detected with boronic acid and EDTA (1a). Positive result adjusting distance to 5 mm (1b) and positive result by NG Test Carba 5® and RESIST-4 O.K.N.V® immunoassays (1c). 2) VIM -producing P. aeruginosa with inconclusive EDTA result (2a), Positive result adjusting distance to 5 mm (2b), confirmation of VIM by immunoassay (2c). 3) K. pneumoniae with KPC and NDM co-production without synergistic effect with boronic acid or EDTA (3a), positivity for both enzymes by immunoassay (3b). 4) Impact of distance between disks: False negative for serine-carbapenemases with 15 mm (4a) positive result at 10 mm (4b). Differences in boronic acid tests for serin-carbapenemases at 10 mm, 5 mm and 0 mm distance (4c, 4d). Differences in synergy between imipenem, meropenem and ertapenem discs at 0 mm distance (4e).Table 2. Results of synergy methods and immunoassay tests for Enterobacterales and Pseudomonas aeruginosa MHTModified Hodge Test, APB: boronic acid synergy; EDTA: EDTA synergy; Pos: positive; Neg: negative. KPC (Klebsiella pneumoniae Carbapenemase), VIM (Verona integron-mediated metallo- β-lactamase), NDM (New Delhi metallo-β-lactamase), OXA (oxacillinase-48-like carbapenemase (OXA-48))ConclusionConventional phenotypic synergy tests with boronic acid and EDTA used for detecting carbapenemases are suboptimal and their routine use should be reconsidered. They depend on the degree of enzyme expression and the distance between disks. Lateral flow immunoassay tests are a rapid and cost-effective tool to detect and differentiate carbapenemases, improving clinical outcomes through targeted therapy and promoting infection prevention measures. Disclosures Diego Josa, Msc, ALIFAX (Speaker’s Bureau) German Esparza, n/a, Biomerieux (Consultant)Pfizer (Speaker’s Bureau) Luis Reyes, n/a, MSD (Speaker’s Bureau)