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Porcine epidemic diarrhea (PED), a contagious intestinal disease caused by the porcine epidemic diarrhea virus (PEDV), has caused significant economic losses to the global pig farming industry due to its rapid course and spread and its high mortality among piglets. In this study, we prepared rabbit polyclonal antibody and monoclonal antibody 6C12 against the PEDV nucleocapsid (N) protein using the conserved and antigenic PEDV N protein as an immunogen. A double-antibody sandwich quantitative enzyme-linked immunosorbent assay (DAS-qELISA) was established to detect PEDV using rabbit polyclonal antibodies as capture antibodies and horseradish peroxidase (HRP)-labeled 6C12 as the detection antibody. Using DAS-qELISA, recombinant PEDV N protein, and virus titer detection limits were approximately 0.05 ng/mL and 103.02 50% tissue culture infective dose per mL (TCID50/mL), respectively. There was no cross-reactivity with porcine reproductive and respiratory syndrome virus (PRRSV), porcine rotavirus (PoRV), porcine pseudorabies virus (PRV), porcine deltacoronavirus (PDCoV), or porcine circovirus (PCV). The reproducibility of DAS-qELISA was verified, and the coefficient of variation (CV) for intra- and inter-batch replicates was less than 10%, indicating good reproducibility. When testing anal swab samples from PEDV-infected piglets using DAS-qELISA, the coincidence rate was 92.55% with a kappa value of 0.85 when using reverse transcription-polymerase chain reaction (RT-PCR) and 94.29% with a kappa value of 0.88 when using PEDV antigen detection test strips, demonstrating the reliability of the method. These findings provide fundamental material support for both fundamental and practical studies on PEDV and offer a crucial diagnostic tool for clinical applications. Key points: • A new anti-PEDV N protein monoclonal antibody strain was prepared • Establishment of a more sensitive double antibody sandwich quantitative ELISA • DAS-qELISA was found to be useful for controlling the PEDV spread [ABSTRACT FROM AUTHOR]

Recently, concern has been raised about the spread of human mpox virus, and the demand for rapid detection reagents for mpox virus has increased. This study aims to establish a time‐resolved fluorescence immunochromatography (TRFICO) method for qualitative/quantitative detection of mpox virus. A double‐antibody sandwich TRFICO method was optimized and established using mpox recombinant fusion antigen and its paired monoclonal antibody. The test performance of the method was evaluated using mpox fusion antigen and control serum, including sensitivity, linearity range, specificity, precision, and reference interval. We successfully established a TRFICO method for qualitative/quantitative detection of mpox antigen, its linearity range 0–100 ng/mL, analytical sensitivity 0.017 ng/mL, and reference intervals greater than 0.045 ng/mL. No cross‐reaction was detected with various poxvirus and clinical negative controls, with good specificity. All average recoveries of the intra‐ and inter‐batch ranged from 81.33% to 97.83%, and all CVs were below 10%. Additionally, the TRFICO strips can be stably stored at 37°C for 7 days without significant changes in the fluorescence intensity. This TRFICO method, with high sensitivity, linearity range, specificity, precision, and stability with 16‐min detection time, provides a new option for qualitative/quantitative and convenient testing of mpox virus. [ABSTRACT FROM AUTHOR]

A simple and rapid system based on Raman nanosphere (R-Sphere) immunochromatography was developed in this study for the simultaneous detection of Influenza A, B virus antigens on a single test line (T-line). Two types of R-Sphere with different characteristic Raman spectrum were used as the signal source, which were labeled with monoclonal antibodies against FluA, FluB (tracer antibodies), respectively. A mixture of antibodies containing anti-FluA monoclonal antibody and anti-FluB monoclonal antibody (capture antibody) was sprayed on a single T-line and goat anti-chicken IgY antibody was coated as a C-line, and the antigen solution with known concentration was detected by the strip of lateral flow immunochromatography based on surface-enhanced Raman spectroscopy (SERS). The T-line was scanned with a Raman spectrometer and SERS signals were collected. Simultaneous specific recognition and detection of FluA and FluB were achieved on a single T-line by analyzing the SERS signals. The findings indicated that the test system could identify FluA and FluB in a qualitative manner in just 15 minutes, with a minimum detection threshold of 0.25 ng ml−1, excellent consistency, and specificity. There was no interference with the other four respiratory pathogens, and it exhibited 8 times greater sensitivity compared to the colloidal gold test strip method. The assay system is rapid, sensitive, and does not require repetitive sample pretreatment steps and two viruses can be detected simultaneously on a single T-line by titrating one sample, which improves detection efficiency, and provide a reference for developing multiplexed detection techniques for other respiratory viruses. [ABSTRACT FROM AUTHOR]

Abstract Background Porcine deltacoronavirus (PDCoV) is a swine enteropathogenic coronavirus that affects young pigs, causing vomiting, acute diarrhea, dehydration, and even death. There is growing evidence that PDCoV can undergo cross-species as well as zoonotic transmissions. Due to the frequent outbreaks of this deadly virus, early detection is essential for effective prevention and control. Therefore, developing a more convenient and reliable method for PDCoV detection is the need of the hour. Results This study utilized a high-affinity monoclonal antibody as the capture antibody and a horseradish peroxidase labeled polyclonal antibody as the detection antibody to develop an enzyme-linked immunosorbent assay (DAS-ELSA) for PDCoV detection.Both antibodies target the PDCoV nucleocapsid (N) protein. The findings of this study revealed that DAS-ELISA was highly specific to PDCoV and did not cross-react with other viruses to cause swine diarrhea. The limit of detection of the virus titer using this method was 103 TCID50/mL of PDCoV particles. The results of a parallel analysis of 239 known pig samples revealed a coincidence rate of 97.07% (κ = 0.922) using DAS-ELISA and reverse transcriptase PCR (RT-PCR). The DAS-ELISA was used to measure the one-step growth curve of PDCoV in LLC-PK cells and the tissue distribution of PDCoV in infected piglets. The study found that the DAS-ELISA was comparable in accuracy to the TCID50 method while measuring the one-step growth curve. Furthermore, the tissue distribution measured by DAS-ELISA was also consistent with the qRT-PCR method. Conclusion The developed DAS-ELISA method can be conveniently used for the early clinical detection of PDCoV infection in pigs, and it may also serve as an alternative method for laboratory testing of PDCoV.

Background: Porcine deltacoronavirus (PDCoV) is a swine enteropathogenic coronavirus that affects young pigs, causing vomiting, acute diarrhea, dehydration, and even death. There is growing evidence that PDCoV can undergo cross-species as well as zoonotic transmissions. Due to the frequent outbreaks of this deadly virus, early detection is essential for effective prevention and control. Therefore, developing a more convenient and reliable method for PDCoV detection is the need of the hour. Results: This study utilized a high-affinity monoclonal antibody as the capture antibody and a horseradish peroxidase labeled polyclonal antibody as the detection antibody to develop an enzyme-linked immunosorbent assay (DAS-ELSA) for PDCoV detection.Both antibodies target the PDCoV nucleocapsid (N) protein. The findings of this study revealed that DAS-ELISA was highly specific to PDCoV and did not cross-react with other viruses to cause swine diarrhea. The limit of detection of the virus titer using this method was 103 TCID50/mL of PDCoV particles. The results of a parallel analysis of 239 known pig samples revealed a coincidence rate of 97.07% (κ = 0.922) using DAS-ELISA and reverse transcriptase PCR (RT-PCR). The DAS-ELISA was used to measure the one-step growth curve of PDCoV in LLC-PK cells and the tissue distribution of PDCoV in infected piglets. The study found that the DAS-ELISA was comparable in accuracy to the TCID50 method while measuring the one-step growth curve. Furthermore, the tissue distribution measured by DAS-ELISA was also consistent with the qRT-PCR method. Conclusion: The developed DAS-ELISA method can be conveniently used for the early clinical detection of PDCoV infection in pigs, and it may also serve as an alternative method for laboratory testing of PDCoV. [ABSTRACT FROM AUTHOR]

Introduction: Rapid identification of infected individuals through viral RNA or antigen detection followed by effective personal isolation is usually the most effective way to prevent the spread of a newly emerging virus. Large-scale detection involves mass specimen collection and transportation. For biosafety reasons, denaturing viral transport medium has been extensively used during the SARS-CoV-2 pandemic. However, the high concentrations of guanidinium isothiocyanate (GITC) in such media have raised issues around sufficient GITC supply and laboratory safety. Moreover, there is a lack of denaturing transport media compatible with SARS-CoV-2 RNA and antigen detection. Methods: Here, we tested whether supplementing media containing low concentrations of GITC with ammonium sulfate (AS) would affect the throatswab detection of SARS-CoV-2 or a viral inactivation assay targeting coronavirus and other enveloped and non-enveloped viruses. The effect of adding AS to the media on RNA stability and its compatibility with SARS-CoV-2 antigen detection were also tested. Results and discussion: We found that adding AS to the denaturing transport media reduced the need for high levels of GITC, improved SARS-COV-2 RNA detection without compromising virus inactivation, and enabled the denaturing transport media compatible with SARS-CoV-2 antigen detection. [ABSTRACT FROM AUTHOR]

Despite several decades of mass drug administration and elimination-related activities, human onchocerciasis still represents a major parasitic threat in endemic regions. Among the challenges encountered by the elimination program is the lack of a suitable diagnostic tool that is accurate and non-invasive. Currently used methods are either invasive or not suitable for monitoring large numbers of patients. Herein, we describe the identification and characterization of Onchocerca volvulus heat shock protein 70 (OvHSP70) as a novel diagnostic biomarker for human onchocerciasis, which can directly be detected in urine samples of infected patients. This nematode-specific antigen was identified through LC-MS after differential SDS-PAGE using urine-derived protein extracts from O. volvulus-infected patients in Cameroon. Polyclonal antibodies generated in rabbits after cloning and expression of OvHSP70 in Escherichia coli reliably differentiated between urine samples from infected- and uninfected patients in a hypoendemic area of human onchocerciasis. These results provide an excellent basis for further development of a non-invasive and scalable diagnostic assay for human onchocerciasis using urine samples. Such a urine-based diagnostic assay will be of major importance for the elimination program of human onchcerciasis in endemic countries. [ABSTRACT FROM AUTHOR]

Controlling the spread of pathogen requires an efficient and accurate diagnosis. Compared with nucleic acid and antibody detection, antigen assays are more convenient to meet clinical diagnostic needs. However, antigen detection is often difficult to achieve high sensitivity in a limited time. In this work, a novel aptasensing method was designed for the purpose of SARS-CoV-2 antigen detection, using a dumbbell padlock probe-mediated circle-to-circle amplification (C2CA) approach. A sandwich complex of antibody-antigen-aptamer is first formed on the magnetic beads. Afterwards, the signal is amplified by a C2CA reaction involving two tandem rolling circle amplifications. Without special instruments or nanomaterials, a detection limit of 575 fg/mL for S1 protein can be achieved in less than 2 h. In the case of the spike pseudovirus SARS-CoV-2 in artificial saliva, the detection limit is 272 TU/μL, which is much lower than average viral load in patients. Therefore, our method provides a timely, efficient and accurate approach for the clinical diagnosis of SARS-CoV-2. It also opens up the application of C2CA in aptamer sensing and antigen detection. [ABSTRACT FROM AUTHOR]

Hepatitis E virus (HEV) causes acute hepatitis in humans, which can progress to chronicity in immunosuppressed individuals. Almost all reported HEV infections are caused by Paslahepevirus balayani genotypes 1–4. The structural ORF2 protein is the major antigen detected in the blood of HEV-infected individuals. ELISA assays to detect IgM antibodies to HEV are the first-line diagnostic tests; however, they showed variable performance with frequently discordant results. A qualitative HEV antigen (ORF2) ELISA is currently available for research use. Here, we report a novel quantitative sandwich ELISA to measure HEV ORF2 protein in 3 matrix types. An optimal pair of capture and detection antibodies was selected among 12 unique combinations tested. A sandwich ELISA protocol was developed using these mAbs and biotin–streptavidin technology. The protocol was further optimized to quantify ORF2 antigen in different matrices by interpolating from a standard curve with a linear range of 3.17 to 50.8 femtomoles/mL. Using this method, ORF2 protein was detected in the cell culture medium of Huh7 cells as early as 2–3 days after transfection with HEV genome RNA and in a medium of human hepatocytes infected with HEV. ORF2 antigen was readily detected in the first 2 weeks post-HEV infection in gerbil sera. In immunosuppressed gerbils, ORF2 was detected up to 6 weeks, and the levels were significantly higher between 3 and 6 weeks post-infection. HEV ORF2 antigen levels showed a strong positive correlation with HEV RNA levels in both cell culture medium and gerbil sera. Our novel sandwich ELISA detected at least 7.3 femtomoles/mL ORF2 protein in human plasma spiked with cell culture propagated HEV and detected ORF2 protein in human plasma samples that tested positive for HEV RNA but negative for anti-HEV antibodies. Further, the assay was nonreactive, with negative human plasma, and HBV or HCV-positive human plasma demonstrating specificity. Overall, our ORF2 antigen ELISA will be useful for quantifying ORF2 antigen in cell culture medium, gerbil serum, and human plasma. Further studies are warranted to evaluate its utility in HEV clinical diagnosis. [ABSTRACT FROM AUTHOR]

African swine fever (ASF) is a contagious disease of wild boar and domestic pigs notifiable to the World Organisation for Animal Health due to its high socio-economic impact. ASF is caused by the complex ASF virus (ASFV), and it can present different clinical manifestations that can be confused with other diseases; for this reason, laboratory testing is necessary for the proper diagnosis of clinically suspected animals. Despite the efforts put into it over decades, no treatment or safe vaccine is globally available, and disease control is based on early diagnosis and the implementation of strict biosecurity measures. In this context, rapid tests have the potential to accelerate and facilitate the identification of infected animals by giving fast on-site results. In this work, we improved the available point-of-care assays for the diagnosis of the disease by the development of a more specific antigen test and a more sensitive antibody test. This antibody detection test allowed for the earlier detection of infected animals than two commercial indirect ELISAs (statistically significant). Moreover, we developed a combined dual rapid test, unifying, in the same cassette, an antigen detection strip and an antibody detection strip. In this study, we confirmed that this combo approach is a useful tool for implementing rapid tests in the field since it increases the percentage of positive samples detected, even when PCR turns negative, while maintaining a good specificity. [ABSTRACT FROM AUTHOR]

Lateral flow assay (LFA) has firmly established itself as a prominent technique for qualitative assessments. However, its potential in quantitative analysis remains limited. In this study, we adapted the laser-assisted absorption measurement of LFA for quantitative detection. LFA assay relies on immobilization of colloidal gold nanoparticles conjugated to the target analyte on the test strip. The abundance of these gold nanoparticles can be quantified by absorption measurement, allowing to determine the concentration of analytes. Rigorous experimental validation of the laser-assisted technology was performed by testing a cohort of 20 patients for hepatitis B surface antigen (HBsAg). Notably, our results show that the laser-assisted LFA and the gold standard technique like enzyme-linked immunosorbent assay are similar statistically.

Background: This study investigated the IgG-specific humoral immune responses against specific antigen-like whole-cell antigen (WCA), outer membrane protein (OMP), periplasmic protein (PP), and cytoplasmic protein (CP) during the acute and subacute stages of Brucella abortus biotype 1 infection in Sprague Dawley (SD) rats. Materials and Methods: The intraperitoneal method was used to experimentally infect forty-four 6- to 8-week-old SD rats with 1 × 109 colony-forming units (CFUs) of B. abortus biotype 1. Following inoculation, the rat was serially sampled for serum at 0, 3, 7, 14, 21, 28, 35, 42, 60, 90, and 120 days. The IgG-specific immune responses and recognition of immunodominant antigens in WCA, OMP, PP, and CP of B. abortus were assessed by indirect enzyme-linked immunosorbent assay (IELISA) and western blot (WB) assay using infected rat sera. Results: The IgG antibody response was detectable at 3 days after infection. The peak serum IgG antibody titers were recorded against CP and PP at 28 days after infection. The highest serum IgG antibody titers were recorded at 42 days after infection against WCA and 90 days after infection only against OMP. WB assay revealed a wide array of protein bands between molecular weight of 13 and 95 kDa for WCA, 13 and 95 kDa for OMP, 15 and 65 kDa for PP, and 12 and 85 kDa for CP. Proteins bands of 10, 13, 20, 24, 46, and 76 kDa for WCA; 28, 35, 39, 85, and 95 for OMP; 20, 30, 40, 43, 46, and 65 kDa for PP, and 12, 23, 68, and 85 for CP were intensely recognized. Conclusion: Data of this study indicated that WCA, CP, and PP of B. abortus could be useful for diagnosis of acute and subacute brucellosis in SD rat model. OMP of B. abortus could be useful for differential diagnosis of subacute brucellosis. [ABSTRACT FROM AUTHOR]

Foot-and-mouth disease (FMD) is endemic in India with a majority of outbreaks caused by FMD virus (FMDV) serotype O. In the present study a panel of eight (2F9, 2G10, 3B9, 3H5, 4C8, 4D6, 4G10 and 5B6) mouse monoclonal antibodies (MAbs) were developed against FMDV serotype O Indian vaccine strain, O/IND/R2/75 via hybridoma systems. The MAbs generated were FMDV/O specific without cross-reactivity against FMDV type A and Asia 1. All the MAbs were identified as IgG1/kappa type. Out of eight, three MAbs (3B9, 3H5 and 4G10) demonstrated virus neutralizing activity. The reactivity of all MAbs increased with heat treated (@560C) serotype O antigen compared to untreated antigen in sandwich ELISA indicating that their binding epitopes are linear. Six MAbs (except 2F9 and 4D6) reacted with recombinant P1 protein of homologous virus in an indirect ELISA among which only MAb 3B9 bound to VP1. MAb profiling of 37 serotype O field viruses isolated between the years 1962 and 2021 demonstrated antigenic similarity between field isolates and reference vaccine strain. MAbs 5B6 and 4C8 consistently reacted with all 37 isolates. In indirect immunofluorescence assay MAb 5B6 bound well with FMDV/O antigen. Finally, a sandwich ELISA was successfully developed using rabbit polyclonal anti-FMDV/O serum and MAb 5B6 for detection of FMDV/O antigen in clinical samples (n = 649). The new assay exhibited 100% and 98.89% diagnostic sensitivity and specificity respectively compared to traditional polyclonal antibody-based sandwich ELISA suggesting that the MAb-based ELISA developed here could be an effective method for detection of FMDV serotype O. [ABSTRACT FROM AUTHOR]

Background: WHO recommends the use of COVID-19 antigen rapid diagnostic tests (Ag-RDT) with at least 80 % sensitivity and 97 % specificity. In the era of Omicron variants, we sought to ascertain the performance of the INDICAID™ Ag-RDT compared to real-time PCR (RT-PCR) as the gold standard. Methods: A laboratory-based study was conducted among consenting individuals tested for COVID-19 at the virology laboratory of the Chantal BIYA International Reference Centre, Yaoundé-Cameron. The samples were processed by INDICAID™ Ag-RDT and DaAn Gene real-time PCR according to the manufacturer's instructions, and PCR-results were interpreted as per cycle thresholds (CT). The sensitivity, specificity, positive and negative predictive values (PPV and NVP) of INDICAID™ Ag-RDT were evaluated according to PCR CT-values. Results: A total of 565 nasopharyngeal swabs were collected from participants (median age [IQR]: 40 [31–75]; M/F sex-ratio was 1.2 and 380 were vaccinated). Following PCR, overall COVID-19 positivity was 5.66 %. For CT < 37, INDICAID™ Ag-RDT sensitivity was 21.9 % (95%CI: [8.3–39.9]), specificity 100 % (95%CI: [99.3–100]); PPV 100 % (95%CI: [59.0–100]), NPV 95.5 % (95%CI: [93.4–97.1]) and kappa = 0.34 (95%CI: [0.19–0.35]). For CT < 25, sensitivity was 100 % (95%CI: [47.8–100.0]), specificity 99.6 % (95%CI: [98.7–99.9]); PPV 94.4 % (95%CI: [51.7–100]), NPV 100 % (95%CI: [99.3–100]) and kappa = 0.83 (95%CI: [0.6–1.0]). COVID-19 sequences generated were all Omicron BA.1 subvariants. Conclusion: For patients infected with high viral loads (CT < 25), INDICAID™ Ag-RDT has high intrinsic (sensitivity and specificity) and extrinsic (predictive values) performances for COVID-19 diagnosis. Due to its simplicity and short turnaround time, INDICAID™ Ag-RDT is, therefore a reliable tool to prevent the spread of COVID-19 at community level in the current era of Omicron subvariants.

Abstract The outbreak of the monkeypox virus (MPXV) worldwide in 2022 highlights the need for a rapid and low-cost MPXV detection tool for effectively monitoring and controlling monkeypox disease. In this study, we developed a flexible lateral flow immunoassay (LFIA) with strong colorimetric and enhanced fluorescence dual-signal output for the rapid, on-site, and highly sensitive detection of the MPXV antigen in different scenarios. A multilayered SiO2-Au core dual-quantum dot (QD) shell nanocomposite (named SiO2-Au/DQD), which consists of a large SiO2 core (~ 200 nm), one layer of density-controlled gold nanoparticles (AuNPs, 20 nm), and thousands of small QDs, was fabricated instead of a traditional colorimetric nanotag (i.e., AuNPs) and a fluorescent nanotag (QD nanobead) to simultaneously provide good stability, strong colorimetric ability and superior fluorescence intensity. With the dual-signal output LFIA, we achieved the specific screening of the MPXV antigen (A29L) in 15 min, with detection limits of 0.5 and 0.0021 ng/mL for the colorimetric and fluorometric modes, respectively. Moreover, the colorimetric mode of SiO2-Au/DQD-LFIA exhibits the same sensitivity as the traditional AuNP- LFIA, whereas the overall sensitivity of this method on the basis of the fluorescent signal can achieve 238- and 3.3-fold improvements in sensitivity for MPXV compared with the AuNP-based LFIA and ELISA methods, respectively, indicating the powerful performance and good versatility of the dual-signal method in the point-of-care testing of the MPXV.

The unprecedented public health and economic impact of the coronavirus disease 2019 (COVID-19) pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection has been met with an equally unprecedented scientific response. Sensitive point-of-care methods to detect SARS-CoV-2 antigens in clinical specimens are urgently required for the rapid screening of individuals with viral infection. Here, we developed an upconversion nanoparticle-based lateral flow immunochromatographic assay (UCNP-LFIA) for the high-sensitivity detection of SARS-CoV-2 nucleocapsid (N) protein. A pair of rabbit SARS-CoV-2 N-specific monoclonal antibodies was conjugated to UCNPs, and the prepared UCNPs were then deposited into the LFIA test strips for detecting and capturing the N protein. Under the test conditions, the limit of detection (LOD) of UCNP-LFIA for the N protein was 3.59 pg/mL, with a linear range of 0.01–100 ng/mL. Compared with that of the current colloidal gold-based LFIA strips, the LOD of the UCNP-LFIA-based method was increased by 100-fold. The antigen recovery rate of the developed method in the simulated pharyngeal swab samples ranged from 91.1 to 117.3%. Furthermore, compared with the reverse transcription-polymerase chain reaction, the developed UCNP-LFIA method showed a sensitivity of 94.73% for 19 patients with COVID-19. Thus, the newly established platform could serve as a promising and convenient fluorescent immunological sensing approach for the efficient screening and diagnosis of COVID-19.

Nucleocapsid protein (NP) is one of the structural proteins of SARS-CoV-2 which is stable, well-conserved, highly immunogenic, and abundantly expressed due to the host's adaptive immune response, making it a promising antigenic biomarker for the early and rapid identification and diagnosis of SARS-CoV-2. Traditional antigen analytical methods with NP as the detection marker often have insufficient sensitivity. To achieve rapid and highly sensitive detection of NP, we constructed a novel single-molecule (digital) fluorescence-linked immunosorbent assay (FLISA) based on streptavidin-modified transparent 96-well microplates. Streptavidin was immobilized on the microplate under optimized conditions with a 15 mM carbonate buffer solution (pH 9.6) as the coating solution, biotinylated antibodies conjugated with streptavidin as capture probes, and carboxylated fluorescent microsphere-conjugated monoclonal antibodies (FMs-mAbs) as fluorescent probes. Individual sandwich immunolabeled complexes of the SARS-CoV-2 diagnostic marker NP were detected and counted though wide-field inverted fluorescence microscopy (1.1 × 1.4 mm2). FLISA had a linear detection range of 0.2 pg/mL to 200 ng/mL and a limit of detection (LOD) of 0.73 fg/mL and 8 fg/mL for NP in phosphate buffer saline and spiked nasal swab samples, respectively. The sensitivity was much higher than commercial antigen detection kits, providing wide detection prospects in future clinical diagnosis, environmental monitoring, and other fields. [ABSTRACT FROM AUTHOR]

Varicella-zoster virus (VZV) is a highly infectious DNA virus that can cause varicella (chickenpox) and herpes zoster (HZ). A simple, sensitive and specific detection method is desirable for the VZV infection. In this study, VZV gE protein, expressed in CHO cells, was used to immunize BALB/c mice for the generation of monoclonal antibodies (mAbs). For the first time, we developed a colloidal gold-based immunochromatographic strip for rapid detection of VZV using a pair of mAbs against gE protein. The limit of detection (LOD) of the strip was 30 ng mL−1 of purified VZV gE antigen, and it could specifically test VZV without cross-reactivity with Enterovirus 71 (EV-71), Herpes simplex virus 1 (HSV-1) and Herpes simplex virus 2 (HSV-2). The coincidence rate between the strip and commercial real-time PCR diagnostic kit was 100% using vesicle as the clinical sample. Our strip provided a technical support for rapid and specific detection of VZV. • 10 monoclonal antibodies against VZV gE were generated with high affinity. • For the first time, the colloidal gold-based immunochromatographic strip was developed for rapid detection of VZV. • The strip could specifically detect VZV without cross-reactivity with EV-71, HSV-1 and HSV-2. • The coincidence rate between the strip and PCR diagnostic kit was 100% using vesicle as the clinical sample. [ABSTRACT FROM AUTHOR]

Despite several decades of mass drug administration and elimination-related activities, human onchocerciasis still represents a major parasitic threat in endemic regions. Among the challenges encountered by the elimination program is the lack of a suitable diagnostic tool that is accurate and non-invasive. Currently used methods are either invasive or not suitable for monitoring large numbers of patients. Herein, we describe the identification and characterization of Onchocerca volvulus heat shock protein 70 (OvHSP70) as a novel diagnostic biomarker for human onchocerciasis, which can directly be detected in urine samples of infected patients. This nematode-specific antigen was identified through LC-MS after differential SDS-PAGE using urine-derived protein extracts from O. volvulus-infected patients in Cameroon. Polyclonal antibodies generated in rabbits after cloning and expression of OvHSP70 in Escherichia coli reliably differentiated between urine samples from infected- and uninfected patients in a hypoendemic area of human onchocerciasis. These results provide an excellent basis for further development of a non-invasive and scalable diagnostic assay for human onchocerciasis using urine samples. Such a urine-based diagnostic assay will be of major importance for the elimination program of human onchcerciasis in endemic countries.

African swine fever (ASF) is a contagious disease of wild boar and domestic pigs notifiable to the World Organisation for Animal Health due to its high socio-economic impact. ASF is caused by the complex ASF virus (ASFV), and it can present different clinical manifestations that can be confused with other diseases; for this reason, laboratory testing is necessary for the proper diagnosis of clinically suspected animals. Despite the efforts put into it over decades, no treatment or safe vaccine is globally available, and disease control is based on early diagnosis and the implementation of strict biosecurity measures. In this context, rapid tests have the potential to accelerate and facilitate the identification of infected animals by giving fast on-site results. In this work, we improved the available point-of-care assays for the diagnosis of the disease by the development of a more specific antigen test and a more sensitive antibody test. This antibody detection test allowed for the earlier detection of infected animals than two commercial indirect ELISAs (statistically significant). Moreover, we developed a combined dual rapid test, unifying, in the same cassette, an antigen detection strip and an antibody detection strip. In this study, we confirmed that this combo approach is a useful tool for implementing rapid tests in the field since it increases the percentage of positive samples detected, even when PCR turns negative, while maintaining a good specificity.

Cryptosporidium spp. is an apicomplexan protozoan parasite associated with gastroenteritis in humans. In 2018, Spain showed 1511 confirmed cases, with a growing trend since 2014. Despite this fact, Cryptosporidium spp. is not usually routinely examined when a parasitological study is ordered, although accurate diagnosis is fundamental to prevent the spread of the illness. The main objectives of the present work is to demonstrate the circulation and to study the epidemiology of cryptosporidiosis in patients who were being tested for the presence of Cryptosporidium spp. parasites in the faeces in the Metropolitan North Area of Barcelona, Maresme, and Vallés Occidental using a two-step algorithm. The stool samples were analysed using the Cryptosporidium/Giardia spp. immunochromatographic test; the positive samples were visualised under a microscope using auramine staining. The proportion of Cryptosporidium spp. cases was around 2% in the studied patients, with a pronounced seasonal incidence peak in late summer–early autumn. In our cohort, weight loss was the main symptom related to confirmed cases. The mean age of confirmed patients was 19 years old, and they were younger than the unconfirmed group. Cryptosporidium spp. is one of the parasites that currently circulate in many areas in Europe. Prevalence must be taken into account for active searching. [ABSTRACT FROM AUTHOR]

In the context of the coronavirus disease 2019 (COVID-19) pandemic, a rapid and reliable point-of-care test is an essential tool for controlling the spread of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). In particular, an immunochromatography test (ICT) that uses saliva specimens for rapid antigen detection not only reduces the risk of secondary infections but also reduces the burden on medical personnel. The newly developed salivary antigen test kit "Inspecter Kowa® SARS-CoV-2" is an ICT to which saliva specimens can be directly applied. We evaluated its usefulness in comparison with reverse transcription quantitative PCR (RT-qPCR) and the Espline® SARS-CoV-2 Kit for the detection of SARS-CoV-2 using nasopharyngeal swab specimens. In this study, 140 patients with suspected symptomatic COVID-19 who visited our hospital were enrolled, and nasopharyngeal swab and saliva specimens were collected after they consented to participate in the study. Inspector Kowa SARS-CoV-2 was positive in 45 of 61 (73.8%) saliva that were positive by RT-qPCR and the Espline® SARS-CoV-2 Kit was also positive in 56 of 60 (93.3%) Np swabs that were positive by RT-qPCR. Good antigen detection was achieved by ICT with saliva and nasopharyngeal swab specimens when viral load was ≥105 copies/mL, whereas detection sensitivity was low when viral load was <105 copies/mL, especially in saliva specimens. This ICT for the detection of SARS-CoV-2 salivary antigen is an attractive tool that does not require specialized equipment and allows patients to perform the entire process from sample collection to self-diagnose and to reduce the burden on medical care during a pandemic. [ABSTRACT FROM AUTHOR]

African swine fever (ASF) is a viral disease of swine with a huge impact due to its high mortality. Lately, the disease has actively spread around the world, affecting new areas from which it had been eradicated long ago. To date, ASF control is carried out by the implementation of strict biosecurity measures such as the early identification of infected animals. In this work, two fluorescent rapid tests were developed to improve the sensitivity of point-of-care diagnosis of ASF. For antigen (Ag) detection in blood, a double-antibody sandwich fluorescent lateral flow assay (LFA) was developed, employing a newly developed recombinant antibody to the VP72 of the virus. To complement the diagnosis, a double-recognition fluorescent LFA was developed using the VP72 for the detection of specific antibodies (Ab) in sera or blood. Both assays statistically improved the detection of the disease when compared to the commercial colorimetric assays INgezim® ASFV CROM Ag and INgezim® PPA CROM Anticuerpo, respectively, with higher statistical significance between 11 and 39 days post-infection. From the observation of results, it can be concluded that the combination of both Ag-LFA and Ab-LFA assays would facilitate the identification of infected animals, regardless of post-infection time. [ABSTRACT FROM AUTHOR]

Angiostrongylus cantonensis is the major etiological nematode parasite causing eosinophilic meningitis and/or eosinophilic meningoencephalitis in humans. The rapid global spread of Angiostrongylus cantonensis and the emerging occurrence of the infection have exposed the shortcomings of traditional/conventional diagnostics. This has spurred efforts to develop faster, simpler and more scalable platforms that can be decentralized for point-of-need laboratory testing. By far, the point-of-care immunoassays such as the lateral flow assay (LFA) are the best-placed. In this work, a LFA in the form of an immunochromatographic test device (designated AcAgQuickDx), based on the detection of a circulating Angiostrongylus cantonensis-derived antigen, was established using anti-31 kDa Angiostrongylus cantonensis antibody as the capture reagent and anti-Angiostrongylus cantonensis polyclonal antibody as the indicator reagent. The AcAgQuickDx was evaluated for its diagnostic potential with a total of 20 cerebrospinal fluids (CSF) and 105 serum samples from patients with angiostrongyliasis and other clinically related parasitic diseases, as well as serum samples from normal healthy subjects. Three of the ten CSF samples from serologically confirmed angiostrongyliasis cases and two of the five suspected cases with negative anti-Angiostrongylus cantonensis antibodies showed a positive AcAgQuickDx reaction. Likewise, the AcAgQuickDx was able to detect Angiostrongylus cantonensis specific antigens in four serum samples of the 27 serologically confirmed angiostrongyliasis cases. No positive reaction by AcAgQuickDx was observed in any of the CSF (n = 5) and serum (n = 43) samples with other parasitic infections, or the normal healthy controls (n = 35). The AcAgQuickDx enabled the rapid detection of active/acute Angiostrongylus cantonensis infection. It is easy to use, can be transported at room temperature and does not require refrigeration for long-term stability over a wide range of climate. It can supplement existing diagnostic tests for neuroangiostrongyliasis under clinical or field environments, particularly in remote and resource-poor areas. [ABSTRACT FROM AUTHOR]

Abstract Background The prevalence of Dirofilaria immitis infection in dogs is increasing globally and spreading into new areas. Prevalence of dirofilariosis in the state of Queensland, Australia, was as high as 90% before the introduction of macrocyclic lactones. Limited research on prevalence of D. immitis infection in dogs in Queensland has been reported in the last 30 years. Antigen testing is the most common method for detection of dirofilariosis but its accuracy is reduced by antigen getting trapped (blocked antigen) in immune complexes (ICs). The objectives of this research were to determine the prevalence of D. immitis infection in dogs from two geographical areas (Brisbane and Townsville) in Queensland, to determine the extent to which blocked antigen affects the validity of antigen testing, and to explore whether this was associated with microfilaraemia, location, age or sex. Methods Blood samples from Brisbane (sub-tropical climate) and Townsville (tropical climate) shelter dogs were evaluated for the presence of D. immitis antigen before (conventional antigen testing, CAT) and after dissociation of ICs by heat treatment (antigen testing after heat treatment, ATHT), using a commercially available test. Microfilariae were detected using modified Knott’s test (MKT). Test proportions were compared with McNemar’s test and the association between antigen test-discordant results (positive for antigen after dissociation of ICs) and microfilaraemia, location, sex and age was modelled using logistic regression. Results Dirofilaria immitis prevalence in dogs from Townsville (22% by CAT, 32.1% by ATHT and 16.7% by MKT) was significantly higher than in dogs from Brisbane (1.1% by CAT and MKT and 1.7% by ATHT) $$(P< 0.001)$$ ( P < 0.001 ) . Dissociation of ICs allowed detection of significantly more D. immitis infected dogs than either conventional antigen testing or microfilariae detection, or the combined antigen and microfilariae detection $$(P< 0.001)$$ ( P < 0.001 ) . The odds of dogs being positive for antigen after dissociation of ICs were significantly higher for microfilaraemic, 3–4-year-old female dogs from Townsville. Conclusions The high prevalence of infection with D. immitis in dogs from Townsville poses a health risk for local susceptible host species, including humans. Dissociation of ICs increases antigen detection and should be considered in dogs suspected of D. immitis infection but negative on routine testing. Graphical Abstract

Qualitative SARS-CoV-2 antigen assays based on immunochromatography are useful for mass diagnosis of COVID-19, even though their sensitivity is poor in comparison with RT-PCR assays. In addition, quantitative assays could improve antigenic test performance and allow testing with different specimens. Using quantitative assays, we tested 26 patients for viral RNA and N-antigen in respiratory samples, plasma and urine. This allowed us to compare the kinetics between the three compartments and to compare RNA and antigen concentrations in each. Our results showed the presence of N-antigen in respiratory (15/15, 100%), plasma (26/59, 44%) and urine (14/54, 28.9%) samples, whereas RNA was only detected in respiratory (15/15, 100%) and plasma (12/60, 20%) samples. We detected N-antigen in urine and plasma samples until the day 9 and day 13 post-inclusion, respectively. The antigen concentration was found to correlate with RNA levels in respiratory (p < 0.001) and plasma samples (p < 0.001). Finally, urinary antigen levels correlated with plasma levels (p < 0.001). Urine N-antigen detection could be part of the strategy for the late diagnosis and prognostic evaluation of COVID-19, given the ease and painlessness of sampling and the duration of antigen excretion in this biological compartment. [ABSTRACT FROM AUTHOR]

Swine acute diarrhea syndrome coronavirus (SADS-CoV) is an emerging swine enteric alphacoronavirus that can cause acute diarrhea, vomiting, dehydration, and death of newborn piglets. In this study, we developed a double-antibody sandwich quantitative enzyme-linked immunosorbent assay (DAS-qELISA) for detection of SADS-CoV by using an anti-SADS-CoV N protein rabbit polyclonal antibody (PAb) and a specific monoclonal antibody (MAb) 6E8 against the SADS-CoV N protein. The PAb was used as the capture antibodies and HRP-labeled 6E8 as the detector antibody. The detection limit of the developed DAS-qELISA assay was 1 ng/mL of purified antigen and 101.08TCID50/mL of SADS-CoV, respectively. Specificity assays showed that the developed DAS-qELISA has no cross-reactivity with other swine enteric coronaviruses, such as porcine epidemic diarrhea virus (PEDV), transmissible gastroenteritis virus (TGEV), and porcine deltacoronavirus (PDCoV). Three-day-old piglets were challenged with SADS-CoV and collected anal swab samples which were screened for the presence of SADS-CoV by using DAS-qELISA and reverse transcriptase PCR (RT-PCR). The coincidence rate of the DAS-qELISA and RT-PCR was 93.93%, and the kappa value was 0.85, indicating that DAS-qELISA is a reliable method for applying antigen detection of clinical samples. Key points: • The first double-antibody sandwich quantitative enzyme-linked immunosorbent assay for detection SADS-CoV infection. • The custom ELISA is useful for controlling the SADS-CoV spread. [ABSTRACT FROM AUTHOR]

Various comorbidities associated with COVID-19 add up in severity of the disease and obviously prolonged the time for viral clearance. This study investigated a novel ultrasensitive MAGLUMI® SARS-CoV-2 Ag chemiluminescent immunoassay assay (MAG-CLIA) for diagnosis and monitoring the infectivity of COVID-19 patients with comorbid conditions during the pandemic of 2022 Shanghai. Analytical performances of the MAG-CLIA were evaluated, including precision, limit of quantitation, linearity and specificity. Nasopharyngeal specimens from 232 hospitalized patients who were SARS-CoV-2 RT-qPCR positive and from 477 healthy donors were included. The longitudinal studies were performed by monitoring antigen concentrations alongside with RT-qPCR results in 14 COVID-19 comorbid participants for up to 22 days. The critical antigen concentration in determining virus infectivity was evaluated at the reference cycle threshold (Ct) of 35. COVID-19 patients were well-identified using an optimal threshold of 0.64 ng/L antigen concentration, with sensitivity and specificity of 95.7% (95% CI: 92.2–97.9%) and 98.3% (95% CI: 96.7–99.3%), respectively, while the Wondfo LFT exhibited those of 34.9% (95% CI: 28.8–41.4%) and 100% (95% CI: 99.23–100%), respectively. The sensitivity of MAG-CLIA remained 91.46% (95% CI: 83.14–95.8%) for the samples with Ct values between 35 and 40. Close dynamic consistence was observed between MAG-CLIA and viral load time series in the longitudinal studies. The critical value of 8.82 ng/L antigen showed adequate sensitivity and specificity in evaluating the infectivity of hospitalized convalescent patients with comorbidities. The MAG-CLIA SARS-CoV-2 Ag detection is an effective and alternative approach for rapid diagnosis and enables us to evaluate the infectivity of hospitalized convalescent patients with comorbidities. [ABSTRACT FROM AUTHOR]

Objectives: Different methods for detecting Plasmodium parasite infection or exposure are available, but a systematic comparison of all these methodologies to predict malaria infection is lacking. Understanding the characteristics of respective tests is helpful in choosing the most appropriate tests for epidemiological or research purposes. Methods: We performed microscopy, rapid diagnostic tests (RDTs), and polymerase chain reaction (PCR) for 496 patients presenting with febrile illness in Dakar, Senegal, in 2015. Blood samples had laboratory multiplex assays performed for Immunoglobin G serology and detection of histidine-rich protein 2 (HRP2) antigen. Sensitivity (Se) and specificity (Sp) for different tests were calculated using PCR as the gold standard for detecting active infection. Modeling through latent class analysis compared each test to a modeled gold standard for Se/Sp estimates. Results: Against PCR, Se/Sp were 95.2%/93.7% for RDT, 90.4%/100.0% for microscopy, and 97.9%/48.1% for laboratory HRP2 detection. Compared with the modeled gold standard, Se of microscopy was 93.5% and Se of RDT, PCR, and laboratory HRP2 detection were all greater than 99%. Se/Sp of Immunoglobin G serology were substantially lower for detecting active infection. Conclusions: Compared with single tests, a combinatorial latent class analysis approach of multiple biomarkers for detecting malaria infection from patient samples provides greater sensitivity and specificity for epidemiological estimates and research objectives.

Abstract Background Viral antigen detection test is the most common method used to detect viruses in the field rapidly. However, due to the low sensitivity, it can only be used as an auxiliary diagnosis method for virus infection. Improving sensitivity is crucial for developing more accurate viral antigen tests. Nano luciferase (Nluc) is a sensitive reporter that has not been used in virus detection. Results In this study, we produced an intracellularly Nluc labeled detection antibody (Nluc-ch2C5) and evaluated its ability to improve the detection sensitivity of respiratory syndrome coronavirus 2 (SARS-CoV-2) antigens. Compared with the traditional horse-radish peroxidase (HRP) labeled antibody (HRP-ch2C5), Nluc-ch2C5 was 41 times more sensitive for inactivated SARS-CoV-2 virus by sandwich chemiluminescence ELISA. Then we applied Nluc-ch2C5 to establish an automatic magnet chemiluminescence immune assay (AMCA) for the SARS-CoV-2 viral spike protein, the limit of detection was 68 pfu/reaction. The clinical sensitivity and specificity reached 75% (24/32) and 100% (48/48) using 32 PCR-positive and 48 PCR-negative swab samples for clinical evaluation, which is more sensitive than the commercial ELSA kit and colloid gold strip kit. Conclusions Here, monoclonal antibody ch2C5 served as a model antibody and the SARS-CoV-2 served as a model pathogen. The Nluc labeled detecting antibody (Nluc-ch2C5) significantly improved the detection sensitivity of SARS-CoV-2 antigen. This labeling principle applies to other viral infections, so this labeling and test format could be expected to play an important role in detecting other virus antigens.

Background: The prevalence of Dirofilaria immitis infection in dogs is increasing globally and spreading into new areas. Prevalence of dirofilariosis in the state of Queensland, Australia, was as high as 90% before the introduction of macrocyclic lactones. Limited research on prevalence of D. immitis infection in dogs in Queensland has been reported in the last 30 years. Antigen testing is the most common method for detection of dirofilariosis but its accuracy is reduced by antigen getting trapped (blocked antigen) in immune complexes (ICs). The objectives of this research were to determine the prevalence of D. immitis infection in dogs from two geographical areas (Brisbane and Townsville) in Queensland, to determine the extent to which blocked antigen affects the validity of antigen testing, and to explore whether this was associated with microfilaraemia, location, age or sex. Methods: Blood samples from Brisbane (sub-tropical climate) and Townsville (tropical climate) shelter dogs were evaluated for the presence of D. immitis antigen before (conventional antigen testing, CAT) and after dissociation of ICs by heat treatment (antigen testing after heat treatment, ATHT), using a commercially available test. Microfilariae were detected using modified Knott's test (MKT). Test proportions were compared with McNemar's test and the association between antigen test-discordant results (positive for antigen after dissociation of ICs) and microfilaraemia, location, sex and age was modelled using logistic regression. Results: Dirofilaria immitis prevalence in dogs from Townsville (22% by CAT, 32.1% by ATHT and 16.7% by MKT) was significantly higher than in dogs from Brisbane (1.1% by CAT and MKT and 1.7% by ATHT) (P < 0.001) . Dissociation of ICs allowed detection of significantly more D. immitis infected dogs than either conventional antigen testing or microfilariae detection, or the combined antigen and microfilariae detection (P < 0.001) . The odds of dogs being positive for antigen after dissociation of ICs were significantly higher for microfilaraemic, 3–4-year-old female dogs from Townsville. Conclusions: The high prevalence of infection with D. immitis in dogs from Townsville poses a health risk for local susceptible host species, including humans. Dissociation of ICs increases antigen detection and should be considered in dogs suspected of D. immitis infection but negative on routine testing. [ABSTRACT FROM AUTHOR]

The spread, prevention and control of novel coronavirus infection and the potential risks and uncertainties of novel coronavirus transmission from donor to recipient have brought serious impacts and great challenges to organ donation and transplantation. There is increasing evidence that the use of non-pulmonary organs (kidney, liver and heart) from novel coronavirus infected donors carries a low risk of transmission, regardless of whether they were symptomatic at the time of acquisition. Delaying organ donation after the death of those who are positive for novel coronavirus antigen or nucleic acid testing, and then waiting until turns negative, will result in the discarding of a significant number of organs that are medically suitable for transplantation. In order to maximally meet the demand for transplantation in patients with end-stage organ failure, Branch of Organ Transplantation of Chinese Medical Association organized relevant experts formulated the “Expert consensus on organ donation from patients infected with novel coronavirus in China” after citizen’ s death by taking into account the epidemic situation of novel coronavirus infection in China and the clinical practice of organ donation and transplantation, and by referring to relevant research results and clinical research evidence at home and abroad. It aims to provide recommendations and references for the procurement and application of donor organs from patients infected with novel coronavirus. [ABSTRACT FROM AUTHOR]

Simple Summary: Untreated and stray dogs and cats, in particular, play an important role in contaminating the environment with important zoonotic parasites. Archive faecal samples collected between 2016–2019 from dogs (n = 789) and cats (n = 241) were examined using the IDEXX Fecal DxTM and SNAPTM Giardia antigen assays for the detection of Toxocara, hookworms, Trichuris and Giardia infections. Giardia duodenalis was the most common parasite (26%) detected in the dogs, followed by ascarids (17.6%) and hookworms (5.3%). Trichuris vulpis was only detected in 1 dog. Ascarids (23.2%) was the most common parasite detected in the cats, followed by Giardia (12.9%) and hookworms (2.9%). This study shows a high prevalence of parasite infection in untreated and stray dogs and cats in the greater Dublin area in Ireland. Since they live in synanthropic conditions and can roam over vast distances, they can contaminate public areas and pose a risk to both humans and owned pets that utilise these spaces. It is therefore important to raise public awareness and increase the knowledge on zoonotic parasites. Endoparasites of dogs and cats, play an important role in both veterinary medicine and public health. Untreated and stray dogs and cats, in particular, play an important role in contaminating the environment with important zoonotic parasites. Thus, the aim of this study was to estimate the prevalence of intestinal parasites in stray dogs and cats using highly sensitive and specific copro-antigen tests. Archive faecal samples from previous surveys conducted between 2016–2019 from dogs (n = 789) and cats (n = 241) were included in this study. The IDEXX Fecal Dx™ antigen panel was used for the detection of Toxocara, hookworms, Trichuris and the SNAP™ Giardia antigen assay was used for the detection of Giardia infection. Giardia duodenalis was the most common parasite (26%, n = 205) detected in the dogs, followed by ascarids (17.6%, n = 139) and hookworms (5.3%, n = 42). Trichuris vulpis was only detected in 1 dog. Ascarids (23.2%, n = 56) was the most common parasite detected in the cats, followed by Giardia (12.9%, n = 31) and hookworms (n = 7, 2.9%). No whipworms were detected in cats. Overall, there was little difference in the positivity between sexes in both dogs and cats. However, in terms of age, adolescent dogs (<3 years) and kittens (<1 year) had the highest parasite prevalence overall, with G. duodenalis and ascarids being the most prevalent. This study shows a high prevalence of parasite infection in untreated and stray dogs and cats in the greater Dublin area in Ireland. Since they live in synanthropic conditions and can roam over vast distances they can contaminate public areas and pose a risk to both humans and owned pets that utilise these spaces. It is therefore important to raise public awareness and increase the knowledge on zoonotic parasites. [ABSTRACT FROM AUTHOR]

Coproantigen immunoassays (IDEXX Fecal Dx® antigen tests) were evaluated for their ability to identify Toxocara cati and Ancylostoma tubaeforme infections in cats and Uncinaria stenocephala infection in dogs. Five cats were experimentally infected with 500 embryonated eggs of T. cati, eight cats with 500 third-stage larvae (L3) of A. tubaeforme and seven dogs with 500 L3 of U. stenocephala. In addition to the three coproantigen tests, the course of infection was monitored by a combined sedimentation-flotation method with ZnSO4 as flotation medium (specific gravity: 1.28–1.30) and a modified McMaster method in case of copromicroscopically positive samples. Eggs of T. cati were first observed between 28 and 54 days post infection (dpi) in four of the five infected cats. In these four cats, positive roundworm coproantigen signals were obtained between 16 and 44 dpi. Positive coproantigen signal always preceded egg observations, but the interval varied between 6 and 30 days. Hookworm-specific positive coproantigen signals were detected in seven of the eight A. tubaeforme infected cats between 10 and 52 dpi, while consecutive egg excretion was observed in three cats between day 26 and 54 pi. Of these three, coproantigen signal preceded egg observation by 12 to 24 days. Four cats had positive coproantigen results in the absence of egg excretion, and one cat never achieved a positive result for egg or coproantigen. In six of seven U. stenocephala infected dogs, infection was confirmed by copromicroscopy between 16 and 24 dpi as well as for hookworm coproantigen between 10 and 14 dpi. Coproantigen signal was detected prior to egg observation by 2 to 14 days. No cross-reactions between the roundworm, hookworm und whipworm tests occurred in study animals. The results of this study demonstrate the ability of the coproantigen tests to detect the common roundworm and hookworm infections in cats and U. stenocephala infections in dogs as well as the ability to detect the prepatent stage of infection. [ABSTRACT FROM AUTHOR]

A novel automated mariPOC SARS‐CoV‐2 antigen test was evaluated in a Health Care Center Laboratory among symptomatic and asymptomatic individuals seeking SARS‐CoV‐2 testing. According to the national testing strategy, reverse transcription polymerase chain reaction (RT‐PCR) was used as a reference method. A total of 962 subjects were included in this study, 4.8% (46/962) of their samples were SARS‐CoV‐2 RT‐PCR‐positive, and 87% (40/46) of these were from symptomatics. Among the symptomatics, the overall sensitivity of the mariPOC SARS‐CoV‐2 test was 82.5% (33/40), though the sensitivity increased to 97.1% (33/34) in samples with a Ct < 30. The mariPOC SARS‐CoV‐2 test detected two of six PCR‐positive samples among the asymptomatics, four cases that remained antigen test negative had Ct values between 28 and 36. The specificity of the mariPOC SARS‐CoV‐2 test was 100% (916/916). The evaluation showed that the mariPOC SARS‐CoV‐2 rapid antigen test is very sensitive and specific for the detection of individuals who most probably are contagious. [ABSTRACT FROM AUTHOR]

Infections by the parasite E. histolytica are increasing in HIV-infected individuals. Interleukin (IL-10) plays an important role in maintaining the mucosal barrier. Therefore, the seroprevalence of E. histolytica was investigated in relation to the IL-10 serum concentration among HIV- infected patients. A total of 647 blood samples were collected from asymptomatic HIV-infected patients. The Entamoeba histolytica antigen (GALNAC lectin) and serum antibodies were assessed using specific ELISAs (TECHLAB, Virginia, USA). IL10 blood levels were measured using a commercial ELISA test, and the results were analyzed using parametric and non-parametric statistical tests. The Gal/GALNAC lectin was detected in only 0.5% (3/647) of individuals, and the antibodies against E. histolytica were detected in 65.2% (422/647) of the samples. A significant increase in IL-10 levels was found in 68.1% of patients who were sero-negative for E. histolytica antibodies compared to patients who were sero-positive. There is a high level of exposure to E. histolytica among HIV patients in South Africa, although the prevalence of amoebic liver abscesses might be low. This study revealed that elevated levels of IL-10 might be associated with a reduced risk of amebiasis. [ABSTRACT FROM AUTHOR]

Classical Swine Fever (CSF) is a highly infectious and fatal disease caused by Classical Swine Fever Virus (CSFV). It is recognized as one of the most serious viral diseases in pig industry. Since CSF was first reported in 1810, CSF has caused significant economic losses to the pig industry in the world and continues to threaten global pork production and human food security. CSF was listed as one of the most important notifiable infectious diseases by the World Organization for Animal Health (OIE), and it was listed as "Class I infectious diseases" in China. At present, CSF is clinically manifested complicated and changeably as acute form with high mortality or subacute type, chronic type, recessive type and continuous infection type with variable mortality, and often infected combined with various diseases. Vaccination is still one of the main means to prevent and control CSF in most countries, especially in developing countries. The development and application of various laboratory diagnostic methods and clinical detection techniques for CSFV antigens and antibodies play a very important role in the prevention and control of CSF. With the rapid development of biotechnology, more diagnostic techniques and new-type vaccines for CSFV have been developed and approved. CSFV antigen and antibody diagnostic techniques, and the development and prospects of live attenuated and new-type CSFV vaccines such as subunit and vector vaccines are reviewed in order to provide references for better prevention and control of CSF.

Objectives: This study assesses and compares the performance of different swab types and specimen collection sites for SARS-CoV-2 testing, to reference standard real-time reverse transcriptase-polymerase chain reaction (RT-PCR) and viral culture. Methods: Symptomatic adults with COVID-19 who visited routine COVID-19 testing sites used spun polyester and FLOQSwabs to self-collect specimens from the anterior nares and tongue. We evaluated the self-collected specimen from anterior nares and tongue swabs for the nucleocapsid (N) or spike (S) antigen of SARS-CoV-2 by RT-PCR and then compared these results with results from RT-PCR and viral cultures from nurse-collected nasopharyngeal swabs. Results: Diagnostic sensitivity was highest for RT-PCR testing conducted using specimens from the anterior nares collected on FLOQSwabs (84%; 95% CI 68-94%) and spun polyester swabs (82%; 95% CI 66-92%), compared to RT-PCR tests conducted using specimens from nasopharyngeal swabs. Relative to viral culture from nasopharyngeal swabs, diagnostic sensitivities were higher for RT-PCR and antigen testing of anterior nares swabs (91-100%) than that of tongue swabs (18-81%). Antigen testing of anterior nares swabs had higher sensitivities against viral culture (91%) than against nasopharyngeal RT-PCR (38-70%). All investigational tests had high specificity compared with nasopharyngeal RT-PCR. Spun polyester swabs are equally effective as FLOQSwabs for anterior nasal RT-PCR testing. Conclusions: We found that anterior nares specimens were more sensitive than tongue swab specimens or antigen testing for detecting SARS-CoV-2 by RT-PCR. Thus, self-collected anterior nares specimens may represent an alternative method for diagnostic SARS-CoV-2 testing in some settings.

Researchers recently have been working on a sensitive, accurate, and easily accessible way to detect coronaviruses. Traditional polymerase chain reaction detection technology has high sensitivity and specificity, but there are some problems, such as a complicated detection process, false negatives caused by gene mutation of coronavirus, and easy infection of operators. Antigen detection, which does not require gene amplification and other preprocessing, can be operated by non-medical staff for rapid detection. However, its detection sensitivity requires high reliability and further improvement. Accordingly, a biosensor based on luminescent resonance energy transfer (LRET) from upconversion nanoparticles (UCNPs) to gold nanoparticles (AuNPs) and antigen detection has been developed for rapid and sensitive detection of N protein of SARS-CoV-2. By adjusting the reaction conditions, LRET can be realized by making the spectral overlap between the luminescence of UCNPs and the absorption band of AuNPs, which can effectively avoid the generation of biological background light and the interference of other signals in the test process. In addition, the thiol-ene click reaction was used to link Thiol-PEG-COOH to UCNPs to improve its biocompatibility. The antigen recognition of the coronavirus based on the as-proposed detection method can be further realized on the test paper. A linear response is obtained ranging from 10 ng mL−1 to 1280 ng mL−1, and the detection limit is around 0.80 ng mL−1 with a detection time of around 10 minutes, which has a wide range of potential future. This fast and sensitive biosensor for coronavirus detection can be further reflected in the test paper and is promising for practical applications. • A biosensor was developed for fast detection of coronavirus based on LRET mechanism. • The linear response ranges of the test paper is from 10 ng mL–1 to 1280 ng mL–1. • The blank limit of detection is 0.199 ng mL–1 with a detection time of around 10 min. [ABSTRACT FROM AUTHOR]

In December 2019, the World Health Organization (WHO) reported that China had accumulated pneumonia of unclear etiology in Wuhan. According to WHO recommendations, in strictly defined situations, antigen tests can be implemented into the diagnostic algorithm to reduce the number of molecular tests performed and support the rapid identification and treatment of COVID-19 patients. According to WHO recommendations, the antigen test for diagnostic use should have a sensitivity of ≥ 80% and a specificity of ≥ 97% compared to molecular tests (NAAT). Based on the comparative analysis, the sensitivity and specificity of the SARS-CoV-2 Antigen ELISA test were determined concerning the RT-PCR reference method. The sensitivity of the SARS-CoV-2 Antigen ELISA was 100% (51/51) and the specificity was 98.84%. The obtained data demonstrate that the analyzed antigen test meets both the WHO and the Ministry of Health criteria.

Antigen detection provides particularly valuable information for medical diagnoses; however, the current detection methods are less sensitive and accurate than nucleic acid analysis. The combination of CRISPR/Cas12a and aptamers provides a new detection paradigm, but sensitive sensing and stable amplification in antigen detection remain challenging. Here, we present a PCR-free multiple trigger dsDNA tandem-based signal amplification strategy and a de novo designed dual aptamer synergistic sensing strategy. Integration of these two strategies endowed the CRISPR/Cas12a and aptamer-based method with ultra-sensitive, fast, and stable antigen detection. In a demonstration of this method, the limit of detection was at the single virus level (0.17 fM, approximately two copies/μL) in SARS-CoV-2 antigen nucleocapsid protein analysis of saliva or serum samples. The entire procedure required only 20 min. Given our system's simplicity and modular setup, we believe that it could be adapted reasonably easily for general applications in CRISPR/Cas12a-aptamer-based detection.

ObjectiveTo evaluate the efficiency of dual test of severe acute respiratory disease coronavirus 2 (SARS-CoV-2) antigen and nucleic acid among freight truck drivers and workers passing through expressway toll gates. MethodsDual tests of SARS-CoV-2 antigen and nucleic acid were conducted among 48 734 freight truck drivers and workers passing through 8 expressway toll gates in Ningbo city from provinces other than Zhejiang province during the period of March 20 – April 17, 2022. The test results were statistically analyzed. ResultsTotally 9 infections of SARS-CoV-2 Omicron variant were detected; of which, 6 were diagnosed clinically as mild coronavirus disease 2019 (COVID-19) case and 3 as asymptomatic infection. No subsequent COVID-19 case was confirmed. For the SARS-CoV-2 antigen test, the sensitivity and specificity were 55.5% (5/9) and 99.9% (48 717/48 725); the positive and negative predictive value were 38.5% (5/13) and 99.9% (48 717/48 721), respectively. For multiplex fluorescence PCR test of SARS-CoV-2 nucleic acid among the participants with positive SARS-CoV-2 antigen result, the Ct values of ORF1ab and N gene M (25th percentile, 75th percentile) were 19.0 (17.1, 21.5) and 19.0 (16.6, 22.5); while, the Ct values of the participants with negative result was significantly higher than those of the participants with positive SARS-CoV-2 antigen result (P < 0.05). ConclusionIn this study, the sensitivity of SARS-CoV-2 antigen test is low but the specificity of the test is high for the detection of SARS-CoV-2 infection. The results suggest that dual test of SARS-CoV-2 antigen and nucleic acid should be implemented to reduce the risk of imported case-induced local COVID-19 epidemic.

Abundant immunological assays currently exist for detecting pathogens and identifying infected individuals, making detection of diseases at early stages integral to preventing their spread, together with the consequent emergence of global health crises. Lateral flow immunoassay (LFIA) is a test characterized by simplicity, low cost, and quick results. Furthermore, LFIA testing does not need well-trained individuals or laboratory settings. Therefore, it has been serving as an attractive tool that has been extensively used during the ongoing COVID-19 pandemic. Here, the LFIA strip's available formats, reporter systems, components, and preparation are discussed. Moreover, this review provides an overview of the current LFIAs in detecting infectious viral antigens and humoral responses to viral infections. [ABSTRACT FROM AUTHOR]

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Post-kala-azar dermal leishmaniasis (PKDL) is a skin condition that occurs in a small percentage of people who have been cured of visceral leishmaniasis (VL), and contributes to transmission of VL. The rK39 rapid test cannot decisively diagnose PKDL due to presence of antileishmanial antibodies from past VL episodes. CL Detect™ Rapid Test, an in-vitro diagnostic test that detects Leishmania antigen peroxidoxin, was assessed for diagnosing PKDL. The CL Detect RDT had 73.3% sensitivity and 100% specificity in the study. The test can be used as a primary screening tool to monitor PKDL in endemic regions and identify active Leishmania infection., (© The Author(s) 2024. Published by Oxford University Press on behalf of Infectious Diseases Society of America. All rights reserved. For commercial re-use, please contact reprints@oup.com for reprints and translation rights for reprints. All other permissions can be obtained through our RightsLink service via the Permissions link on the article page on our site—for further information please contact journals.permissions@oup.com.)