Search Results (26)

Toxoplasma gondii is a globally significant zoonotic pathogen responsible for severe parasitic diseases in humans and animals. This study aimed to design, develop, and evaluate a novel immunochromatographic test (ICT) using a recombinant MIC2-MIC3 fusion protein (rMIC2-MIC3) for detecting specific antibodies against T. gondii. The ICT demonstrated exceptional sensitivity, capable of detecting T. gondii-specific antibodies in sera diluted up to 1:8. Specificity evaluation confirmed no cross-reactivity with antibodies against other parasites, such as Neospora caninum, Cryptosporidium suis, Eimeria tenella, and Sarcocystis tenella. Stability tests revealed the test strips maintained full functionality after 12 weeks of storage at 24 °C. The coincidence rate of the colloidal gold test strips prepared in this study with a commercial ELISA kit was 94.59%. Comparisons with advanced serodiagnostic tools, such as chimeric antigen-based ELISAs and recombinant protein diagnostics, further highlighted its robustness and applicability. These findings underscore the potential of the rMIC2-MIC3-based ICT as a reliable, economical, and accessible diagnostic tool for toxoplasmosis in veterinary and human medicine. Full article

Feline leukemia virus (FeLV) and Feline immunodeficiency virus (FIV) are globally prevalent retroviral pathogens that pose significant health risks to domestic cats. This study aimed to compare the diagnostic performance of two point-of-care—the immunochromatographic assay (ICA) and the RNase hybridization-assisted amplification (RHAM) test kit—against reverse transcription quantitative polymerase chain reaction (RT-qPCR), the current gold standard for FeLV and FIV detection. For FeLV detection, ICA demonstrated a sensitivity of 86.89%, specificity of 96.55%, accuracy of 90.00%, and precision of 98.15%, while for FIV detection, the assay showed a sensitivity of 75.86%, specificity of 88.52%, accuracy of 84.44%, and precision of 75.86%. In contrast, the RHAM test exhibited superior performance, with FeLV detection sensitivity of 93.44%, specificity of 98.28%, accuracy of 94.44%, and precision of 98.28%. For FIV detection, RHAM demonstrated a sensitivity of 75.86%, specificity of 100%, accuracy of 92.22%, and precision of 100%. Additionally, the RHAM assay significantly reduced detection time compared to RT-qPCR, enabling expedited clinical decision-making, alleviating laboratory workload, and lowering diagnostic costs. These benefits are particularly relevant in veterinary settings with limited access to PCR-based diagnostics, where the RHAM assay represents a rapid, reliable, and resource-efficient alternative for FeLV and FIV detection. Full article

Background: The alarming increase in carbapenemase-producing Enterobacterales is a matter of grave public health concern. The most ubiquitous carbapenemases, Klebsiella pneumoniae carbapenemase (KPC)-, New Delhi metallo-β-lactamase (NDM)-, and oxacillinase (OXA-48)-like enzymes, belong to the Ambler molecular classes A, B, and D, respectively. KPC- and OXA-48-like enzymes have a serine-based hydrolytic mechanism, while NDMs are metallo-β-lactamases that contain zinc in the active site. For the judicious use of reserve drugs and promoting antimicrobial stewardship, timely detection of carbapenemases is essential. While molecular tools are the gold standard for the detection of these enzymes, many laboratories have limited access to them. This study focused on evaluating in-house tools and commercial phenotypic tests for the detection of OXA-48-, KPC-, and NDM-like enzymes in K. pneumoniae, the predominant extremely drug-resistant pathogen in Oman. Methods: In total, 80 GeneXpert/PCR-confirmed (40 OXA-48 and 20 KPC and NDM each) and 37 whole-genome-sequenced (25 OXA-232 and 6 KPC-2, plus NDM-1 and NDM-5) K. pneumoniae were subjected to screening by temocillin (30 μg disk) (MAST Diagnostica, Germany) and D71C (MASTDISCS^®). Isolates resistant to temocillin (<11 mm) and D71C were subjected to four tests: an in-house tool (OXA-48 disk test) and three commercial phenotypic tests: (i) the MASTDISCS^® Combi (D72C) (MAST Group Ltd., Bootle, UK); (ii) the MASTDISCS^® Combi (D73C) (MAST Group Ltd., UK); and (iii) an immunochromatographic assay (ICT), which is the KPC/IMP/NDM/VIM/OXA-48 Combo test kit (Medomics, China), for the detection of OXA-48-, KPC-, and NDM-like carbapenemases. Results: Temocillin exhibited good sensitivity and specificity (100% and 97.50%) compared to D71C (70% and 100%). Among the confirmatory tests, the in-house OXA-48 disk test had 92.50% sensitivity and 100% specificity, while the commercial MAST DISC tests D72C, D73C, and ICT had 97.50%, 95.00%, and 100% sensitivity and 100%, 91.67%, and 95% specificity, respectively. Conclusions: The temocillin disk test is a good screening tool. With high sensitivity and specificity, ease of performance, short turnaround time, and low cost, we recommend the ICT format for routine diagnostic use. In resource-constrained centers, the OXA-48 disk test is an excellent alternative with high sensitivity and specificity. Full article

Porcine circovirus type 2 (PCV2) is the main and primary causative agent of Postweaning Multisystemic Wasting Syndrome (PMWS). To date, immunoperoxidase monolayer assay (IPMA), indirect immunofluorescent assay (IFA), and enzyme linked immunosorbent assay (ELISA) are the most commonly diagnostic methods for detecting PCV2 antigens. However, these methods require specialized equipment and technical expertise and are suitable for laboratory use only. This study aims to develop an immunochromatographic strip and a magnetic chemiluminescence immunoassay for the detection of PCV2 antigens. The recombinant protein was constructed using a prokaryotic expression system, and the polyclonal antibody was obtained by animal experiments. Polystyrene microspheres are used as solid phase carriers to covalently bind to the amino groups of proteins to form immunoprobes. Monodisperse beads are covalently bound to antigens or antibodies as solid phases to bind antibodies or antigens in the liquid phase in a superior manner, thereby capturing and separating antigens and antibodies in the liquid phase. The immunochromatographic strip is qualitative detection method, this method can detect PCV2a strain, PCV2b strain, and PCV2d strain. The immunochromatographic strip had minimum detection limits of 10^2.89TCID₅₀/0.1 mL, 10^3.19TCID₅₀/0.1 mL, and 10^3.49TCID₅₀/0.1 mL for PCV2a/LG, PCV2b/SH, and PCV2d/JH. The results of testing PEDV (CV777 strain), PRV (HB2000 strain), CSFV (WH-09 strain), PRRS (JXA1-R strain), PPV (WH-1 strain), and ASFV (SD strain) were negative. The agreement between the immunochromatographic strip and the ELISA kit was 93.33% (140/150) and the Kappa was 0.866 (Kappa > 0.81). On the premise of ensuring sensitivity, the most important feature of the immunochromatographic strip is that this method can save time when testing; results can be obtained within 5 to 10 min. Magnetic chemiluminescence immunoassay is quantitative detection method; this method can detect PCV2 Cap proteins in swine serum, the linear range of this method was 0.25 ng/mL to 32 ng/mL and R² of the standard curve was 0.9993. The limit of detection (LOD) is 0.051 ng/mL and the limit of quantitation (LOQ) is 0.068 ng/mL. The agreement between the magnetic chemiluminescence immunoassay and the ELISA kit (test PCV2 Cap proteins) was 97.14% (68/70). This method took less than 30 min to achieve results, which is less than the ELISA kit. The results of this study show that immunochromatographic strip and magnetic chemiluminescence immunoassay for PCV2 antigens had great sensitivity and specificity, which lays the foundation for PCV2 clinical detection. Full article

Antibiotic-resistant bacteria are usually found in food-producing animals worldwide. Ciprofloxacin, an antibiotic, can lead to antibiotic residues in food products, posing health risks to consumers and contributing to the development of antimicrobial resistance. Foodborne illnesses occur when adequate attention is not paid to food hygiene and safety, raising the potential for resistant bacteria to spread to humans through the food chain. This study aims to determine the presence of antibiotic-resistant organism contamination and ciprofloxacin residue in raw pork and chicken. Forty-three pork and 33 chicken meat samples were collected from fresh markets in Chiang Mai, Thailand. Antibiotic-resistant organisms were detected by microbial culture and identified by MALDITOF-MS. The antimicrobial sensitivity tests were used to confirm antibiotic resistance. The ciprofloxacin was detected by using an immunochromatographic-based test kit for screening. The results found Extended Spectrum β-lactamase (ESBL)-producing E. coli and K. pneumoniae were detected at 46.51% and 9.30% in pork and 69.70% and 6.06% in chicken meat samples, respectively. Moreover, ciprofloxacin residues were detected in nine samples (11.84%). Based on this study’s findings, the people who are involved in the food chain must be concerned about food safety and food hygiene. Full article

In this study, a novel rapid immunochromatographic (IC) test for African swine fever virus (ASFV) antibodies is presented. An immunochromatographic test (IC) is a detection technique that combines membrane chromatography with immunolabeling. This approach saves time for antibody preparation, resulting in a shorter production cycle. p54 is an important structural protein of African swine fever, and an ideal protein for serotype diagnosis. Gold nanoparticles are attached to the ASFV p54-Fc fusion protein, and the ASFV-specific antigen p54 and Staphylococcus aureus protein A (SPA) are labeled on a nitrocellulose membrane, at positions T and C, respectively. We developed a SPA double sandwich IC test strip, and assessed its feasibility using ASFV p54 and p54-Fc fusion proteins as antigens. ASFV p54 and p54-Fc fusion proteins were expressed and purified. A sandwich cross-flow detection method for p54, which is the primary structural protein of ASFV, was established, using colloidal gold conjugation. Our method can detect ASFV antibodies in field serum samples in about 15 min using a portable colloidal gold detector, demonstrating high specificity and sensitivity (1:320), and the coincidence rate was 98% using a commercial ELISA kit. The dilution of the serum sample can be determined by substituting the absorbance (T-line) interpreted by portable devices into the calibration curve function formula of an African swine fever virus standard serum. In summary, our method is rapid, cost-effective, precise, and highly selective. Additionally, it introduces a new approach for constructing IC test strips using SPA protein without antibody preparation, making it a reliable on-site antibody test for ASFV. Full article

Milk is the most consumed liquid food in the world due to its high nutritional value and relatively low cost, characteristics that make it vulnerable to adulteration. One of the most common types of milk adulteration involves the undeclared addition of cow’s milk to milk from other mammalian species, such as goats, sheep, buffalo or donkeys. The incidence of such adulteration not only causes a crisis in terms of commercial market and consumer uncertainty but also poses a risk to public health, as allergies can be triggered by proteins in undeclared cow’s milk. In this study, a specific qualitative touchdown (TD) PCR method was developed to detect the undeclared addition of cow’s milk in goat and sheep milk based on the discrimination of the peak areas of the melting curves after the modification of bovine-specific primers. The developed methodology has high specificity for the DNA templates of other species, such as buffalos and donkeys, and is able to identify the presence of cow’s milk down to 1%. Repeatability was tested at low bovine concentrations of 5% and 1% and resulted in %RSD values of 1.53–2.04 for the goat–cow assay and 2.49–7.16 for the sheep–cow assay, respectively. The application of this method to commercial goat milk samples indicated a high percentage of noncompliance in terms of labeling (50%), while a comparison of the results to rapid immunochromatographic and ELISA kits validated the excellent sensitivity and applicability of the proposed PCR methodology that was able to trace more adulterated samples. The developed assays offer the advantage of multiple detection in a single run, resulting in a cost- and time-efficient method. Future studies will focus on the applicability of these assays in dairy products such as cheese and yogurt. Full article

Body fluid identification is fundamental in forensic science as it links a specific biological source to a genetic profile, thus providing critical clues for crime scene reconstruction. Blood is one of the most common body fluids found on the crime scene, and several strategies have been developed for its identification in recent decades. Usually, after a preliminary (or presumptive) test to determine the presence of blood (both human and non-human), a confirmatory test is needed to prove that the sample is human blood. Out of the confirmatory tests, immunochromatographic (IC) assays are the most commonly and widely used. This work gives a review of the use of commercial kits specifically developed to detect human hemoglobin or glycophorin A (a surface protein of human red cells) in forensics. Claimed sensitivity varies broadly (ranging from 0.06 to 75 nanoliters of fresh blood), but different values (as low as 0.002 nL) were found during validation procedures. Specificities are high, and the possibility of cross-reaction (with the risk of false-positive results) is so low that it can be considered negligible. False-negative results, however, can be found due to the so-called “hook effect” as well as to the target degradation/modification, which interferes with the Ag-Ab binding. In addition, the chemical compositions of the presumptive test, detergents, and washing can also promote false negative outcomes in peculiar situations. Although IC assays are rapid, inexpensive, specific, and easy to use even on the crime scene, their major limitation is represented by the destructive approach required by this kind of confirmatory test. Since the final goal of the forensic investigation is the genetic typing of a bloodstain, we will describe the strategies developed for IC assays of faint stains as well as the strategies adopted to ensure that exactly the same sample undergoes human blood identification and DNA typing. Full article

Lateral flow immunochromatographic (LFI) tests are widely used in both biomedical and forensic sciences for different applications. In forensic sciences, their main use is to detect body fluids at crime scenes. However, there are situations in which the amount of potential biological evidence is so low that DNA extraction is favored with respect to the identification of body fluids. Here, an efficient and quick protocol is presented to integrate the detection of body fluids through LFI with DNA extraction from a sample swab and buffer, providing a complete characterization of the biological evidence. This protocol is a modification of a general DNA extraction silica-based kit, whose main application is for blood and tissues. Thus, it could be carried out in different settings (forensic labs, hospitals, other testing labs) without the necessity of buying a specific kit for swabs. The validation of this protocol is supported by the results presented here and previous publications from our group, obtaining DNA in good quantity and with good quality. This proves the potential application of the protocol in both forensic scenarios, to fully characterize biological evidence, and biomedical settings, to molecularly confirm the results of LFI tests. Full article

The immunodiagnostic assessment of dogs suspected of having immune-mediated hemolytic anemia (IMHA) is based on persistent autoagglutination of erythrocytes (after three saline washes), marked spherocytosis, and a positive direct antiglobulin (Coombs’) test (DAT). However, the value of using the indirect antiglobulin test (IAT) for the detection of anti-erythrocytic autoantibodies in serum from dogs suspected of having IMHA is unclear. To evaluate the IAT, leftover serum samples from a large cohort of 94 dogs suspected of having IMHA and for which DAT results were known were incubated with DAT− erythrocytes, and five IAT techniques were performed (in part with different reagents and temperatures): microtiter plate (MICRO), microcapillary, laboratory gel column, gel minitube kit (GEL KIT), and immunochromatographic strip kit. Two IAT techniques (MICRO at 37 °C and GEL KIT with rabbit anti-dog polyvalent reagent) detected autoantibodies against erythrocytes in serum from 53% and 57% of DAT+ dogs, respectively, while other IATs performed less well. Moreover, while the analytic specificity of the IAT methods compared to the DAT ranged from 96–100%, the sensitivity range was only 9–57%. Thus, we still recommend DAT for diagnosis and monitoring of IMHA in dogs but conclude that a positive IAT result may aid diagnostically when serum is available, but fresh red blood cells are not. Full article

The COVID-19 pandemic caused by SARS-CoV-2 remains a serious health concern worldwide due to outbreaks of SARS-CoV-2 variants that can escape vaccine-acquired immunity and infect and transmit more efficiently. Therefore, an appropriate testing method for COVID-19 is essential for effective infection control and the prevention of local outbreaks. Compared to reverse-transcription polymerase chain reaction (RT-PCR) tests, antigen tests are used for simple point-of-care testing, enabling the identification of viral infections. In this study, we tested the clinical usefulness of the FUJIFILM COVID-19 Ag test, an antigen test based on silver amplification and immunochromatographic technology. The FUJIFILM COVID-19 Ag test was shown to detect a lower viral concentration as compared to other conventional kits without significant performance loss in detecting prevalent SARS-CoV-2 variants. We tested nasopharyngeal and nasal swabs from a single patient during two different epidemic periods dominated by various SARS-CoV-2 variants. We observed that the sensitivity of the FUJIFILM COVID-19 Ag test was 95.7% and 85.7% in nasopharyngeal and nasal swabs, respectively. These results suggest that the FUJIFILM COVID-19 Ag test is highly sensitive and applicable when RT-PCR testing is unavailable. Furthermore, these results indicate that high-frequency testing using nasal swab specimens may be a valuable screening strategy. Full article

This study established a portable and ultrasensitive detection method based on recombinase polymerase amplification (RPA) combined with high-sensitivity multilayer quantum dot (MQD)-based immunochromatographic assay (ICA) to detect the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). The RPA-MQD-based ICA method is reported for the first time and has the following advantages: (i) RPA is free from the constraints of instruments and can be promoted in point-of-care testing (POCT) scenarios, (ii) fluorescence ICA enhances the portability of detection operation so that the entire operation time is controlled within 1 h, and (iii) compared with common colorimetric-based RPA-ICA, the proposed assay used MQD to provide strong and quantifiable fluorescence signal, thus enhancing the detection sensitivity. With this strategy, the proposed RPA-MQD-based ICA can amplify and detect the SARS-CoV-2 nucleic acid on-site with a sensitivity of 2 copies/reaction, which is comparable to the sensitivity of commercial reverse transcription quantitative polymerase chain reaction (RT-qPCR) kits. Moreover, the designed primers did not cross-react with other common respiratory viruses, including adenovirus, influenza virus A, and influenza virus B, suggesting high specificity. Thus, the established portable method can sensitively detect SARS-CoV-2 nucleic acid without relying on equipment, having good application prospects in SARS-CoV-2 detection scenarios under non-lab conditions. Full article

Chronic human liver fluke infections caused by Opisthorchis viverrini and Clonorchis sinensis can last for decades and cause liver and biliary diseases, including life-threatening pathology prior to cholangiocarcinoma (CCA). CCA generally has a poor prognosis. Serological diagnosis can support parasitological examination in diagnosing disease and screening for the risk of CCA. Here, we present an improved and innovative lateral flow immunochromatographic test (ICT) kit that uses whole-blood samples (WBS) rather than serum to diagnose human opisthorchiasis, which also successfully diagnosed human clonorchiasis. This ICT includes a soluble worm extract of O. viverrini adults and colloidal-gold-labeled conjugates of the IgG antibody to evaluate the diagnostic values with simulated WBS (n = 347). Simulated WBS were obtained by the spiking infection sera with red blood cells. The diagnostic sensitivity, specificity, positive and negative predictive values, and accuracy for detecting opisthorchiasis were 95.5%, 87.0%, 80.5%, 97.2%, and 90.1%, respectively. For clonorchiasis, these findings were 85.7%, 87.0%, 53.6%, 97.2%, and 86.8%, respectively. Combined for both diseases, they were 93.2%, 87.0%, 84.0%, 94.6%, and 89.6%, respectively. The ICT kit can possibly replace the ICT platforms for antibody detection in serum samples in field surveys in remote areas where sophisticated equipment is not available. Full article

The transmission of the SARS-CoV-2 virus, which causes COVID-19, has been documented worldwide. However, the evidence of the extent to which transmission has occurred in different countries is still to be established. Understanding the magnitude and distribution of SARS-CoV-2 through seroprevalence studies is important in designing control and preventive strategies in communities. This study investigated the seropositivity of the SARS-CoV-2 virus antibodies in the communities of three different districts in the Mwanza region, Tanzania. A household cross-sectional survey was conducted in September 2021 using the modified African Centre for Disease and Prevention (ACDC) survey protocol. A blood sample was obtained from one member of each of the selected households who consented to take part in the survey. Immunochromatographic rapid test kits were used to detect IgM and IgG SARS-CoV-2 antibodies, followed by descriptive data analysis. Overall, 805 participants were enrolled in the study with a median age of 35 (interquartile range (IQR):27–47) years. The overall SARS-CoV-2 seropositivity was 50.4% (95%CI: 46.9–53.8%). The IgG and IgM seropositivity of the SARS-CoV-2 antibodies was 49.3% and 7.2%, respectively, with 6.1% being both IgG and IgM seropositive. A history of runny nose (aOR: 1.84, 95%CI: 1.03–3.5, p = 0.036), loss of taste (aOR: 1.84, 95%CI: 1.12–4.48, p = 0.023), and living in Ukerewe (aOR: 3.55, 95%CI: 1.68–7.47, p = 0.001) and Magu (aOR: 2.89, 95%CI: 1.34–6.25, p= 0.007) were all independently associated with SARS-CoV-2 IgM seropositivity. Out of the studied factors, living in the Ukerewe district was independently associated with IgG seropositivity (aOR 1.29, CI 1.08–1.54, p = 0.004). Twenty months after the first case of COVID-19 in Tanzania, about half of the studied population in Mwanza was seropositive for SARS-CoV-2. Full article

Globally, point-of-care testing (POCT) is the most preferable on-site technique for disease detection and includes a rapid diagnostic test (RDT) and fluorescent immunochromatographic strip test (FICT). The testing kits are generally insufficient in terms of signal enhancement, which is a major drawback of this approach. Sensitive and timely on-site POCT methods with high signal enhancement are therefore essential for the accurate diagnosis of infectious diseases. Herein, we prepare cysteamine-gold coated carboxylated europium chelated nanoparticle (Cys Au-EuNPs)-mediated POCT for the detection of the H5N1 avian influenza virus (AIV). Commercial nanoparticles were used for comparison. The spectral characteristics, surface morphologies, functional groups, surface charge and stability of the Cys AuNPs, EuNPs, and Cys Au-EuNPs were confirmed by UV-visible spectrophotometry, fluorescence spectrometry, transmission electron microscope with Selected area electron diffraction (TEM-SAED), Fourier-transform infrared spectroscopy (FTIR) and zeta potential analysis. The particle size distribution revealed an average size of ~130 ± 0.66 nm for the Cys Au-EuNPs. The Cys Au-EuNP-mediated RDT (colorimetric analysis) and FICT kit revealed a limit of detection (LOD) of 10 HAU/mL and 2.5 HAU/mL, respectively, for H5N1 under different titer conditions. The obtained LOD is eight-fold that of commercial nanoparticle conjugates. The photo luminance (PL) stability of ~3% the Cys Au-EuNPs conjugates that was obtained under UV light irradiation differs considerably from that of the commercial nanoparticle conjugates. Overall, the developed Cys Au-EuNPs-mediated dual-mode POCT kit can be used as an effective nanocomposite for the development of on-site monitoring systems for infectious disease surveillance. Full article