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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]

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]

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]

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]

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]

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]

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]

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]

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]

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. 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. 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. 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 FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

We used nasopharyngeal swab samples of patients with a symptomatic (n = 82) or asymptomatic (n = 20) coronavirus disease 2019 (COVID-19) diagnosis to assess the ability of antigen detection tests to infer active (potentially transmissible) or inactive (potentially non-transmissible) infection by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Using the subgenomic RNA (sgRNA) as an active replication marker of SARS-CoV-2, 48 (76.2%), 56 (88.9%), and 63 (100%) of 63 samples with sgRNA positive results tested positive with the SD BIOSENSOR STANDARD Q COVID-19 Ag (Standard Q), the SD BIOSENSOR STANDARD F COVID-19 Ag FIA (Standard F), or the Fujirebio LUMIPULSE G SARS-CoV-2 Ag (Lumipulse) assay, respectively. Conversely, 37 (94.9%), 29 (74.4%), and 7 (17.9%) of 39 samples with sgRNA negative results tested negative with Standard Q, Standard F, or Lumipulse, respectively. Stratifying results by the number of days of symptoms before testing revealed that most antigen positive/sgRNA positive results were among samples tested at 2–7 days regardless of the assay used. Conversely, most antigen negative/sgRNA negative results were among samples tested at 16–30 days only when Standard Q or Standard F were used. In conclusion, based on our findings, a negative antigen test, especially with the Lumipulse assay, or a positive antigen test, especially with the Standard F assay, may suggest, respectively, the absence or presence of replication-competent SARS-CoV-2. [ABSTRACT FROM AUTHOR]

PCR on nasopharyngeal exudates, the cornerstone of the detection of SARS-CoV-2, is time-consuming and commonly unavailable at primary health care centres. Detection of viral nucleocapsid antigens using lateral flow point-of-care tests is helpful for the early triage of patients who attend health care facilities. This was a prospective study carried out in clinically suspected cases and close asymptomatic contacts who attended a primary care centre (Madrid, Spain) for SARS-CoV-2 detection. Patients were divided into three 300-patient cohorts (n = 200 symptomatic cases and n = 100 close asymptomatic contacts per cohort). Three antigen detection tests (SGTI-Flex COVID-19 Ag, Panbio COVID-19 Ag Rapid Test Device, and GSD NovaGen SARS-CoV-2 Ag Rapid Test) were used and compared. Paired nasopharyngeal exudates were obtained, one swab for PCR and the other for antigen detection. Each antigen detection test was evaluated on one cohort. All tests showed invariably 100% specificity. Sensitivity was 68.9% (95% CI: 55.7–80; SGTI-Flex), 71.1% (95% CI: 55.6–83.6; Panbio), and 84.6% (95% CI: 72–93.1; NovaGen) in clinically suspected patients and 84.6% (95% CI: 54.5–98.1), 33.3% (95% CI: 11.8–61.6), and 55.6% (95% CI: 30.7–78.4) in close asymptomatic contacts, respectively. Sensitivity was systematically higher in samples yielding positive PCR results with Ct ≤ 20. We found considerable test-to-test antigen detection variations among patients with clinical suspicion of COVID-19 and close asymptomatic contacts. Negative antigen results, regardless of the test used, should be confirmed by PCR. [Display omitted] [ABSTRACT FROM AUTHOR]

• Polyester and FLOQSwabs perform in similar manner when testing for SARS-CoV-2. • Tests using nasal swabs by RT-PCR were more sensitive than tests using tongue swabs for SARS-CoV-2. • Self-collected nasal swabs are an accurate method for diagnosis of SARS-CoV-2. 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. 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. 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. 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. [ABSTRACT FROM AUTHOR]

Simple Summary: Rabbit Haemorrhagic Disease is caused by a virus that affects the liver, the spleen and the lungs of rabbits, causing hepatitis, splenomegaly and haemorrhages. A new genotype of the virus was first reported in France in 2010 and has spread globally since then, replacing most of the circulating former viruses in many countries. The detection of the virus and the differentiation of both genotypes is of crucial importance for disease surveillance. In this article, a rapid test for antigen detection is described and evaluated, providing the first description of a quick and easy-to-use test that allows for the simultaneous detection and differentiation of the genotypes. A total of 136 samples, rabbit liver samples and liver exudates (liquid collected after freeze–thawing) classified as infected and non-infected, were analysed, with good results. These data confirm that the developed rapid test can be used as a reliable diagnostic test for disease surveillance, especially in farms and the field. Rabbit Haemorrhagic Disease Virus 2 (RHDV2, recently named Lagovirus europaeus/GI.2) was first reported in France in 2010 and has spread globally since then, replacing most of the circulating former RHDV (genotype GI.1) in many countries. The detection and differentiation of both genotypes is of crucial importance for the surveillance of the disease. In this article, a duplex lateral flow assay (LFA) for antigen detection is described and evaluated, providing the first description of a quick and easy-to-use test that allows for the simultaneous detection and differentiation of RHDV genotypes GI.1 and GI.2. A panel of GI.1- or GI.2-infected and non-infected rabbit liver samples and liver exudates (136 samples) was analysed, obtaining a total sensitivity of 94.4% and specificity of 100%. These data confirm that the developed duplex LFA can be used as a reliable diagnostic test for RHD surveillance, especially in farms and the field. [ABSTRACT FROM AUTHOR]

Avian influenza (AI) is a disease of birds caused by type A strain of influenza virus. Only type A influenza viruses are known to cause natural infection in birds. This study investigated the presence of avian influenza viruses in pigeons, local chickens, and ducks in randomly selected three Local Government Areas of Enugu North Agricultural Zone of Enugu State, Nigeria, from December 2016 to May 2017. A total of three hundred cloacal swabs were collected from pigeons, local chickens and ducks in Igbo-Eze North, Igbo-Eze South and Nsukka Local Government Areas respectively. The samples were moved to the laboratory in cold chain for laboratory analysis within an hour of collection. The cloacal swab samples were analyzed for AI using rapid antigen detection kit (X/pect H5N1 kit by Thermo Fisher Scientific Remel Inc. Lenexa, KS, USA), the spot and serial dilution haemagglutination (HA) methods. Of the one hundred samples from pigeons tested, 2% and 7% tested positive in rapid antigen detection and spot HA tests, respectively, while none of the samples from local chickens and ducks tested positive in the two tests. The detection of positive samples from pigeon in the two tests is an indication that pigeon is a potential threat to public health in the area because pigeons serve as a reservoir of H5N1 which is zoonotic. [ABSTRACT FROM AUTHOR]

A photochromic immunoassay was built for tumor marker detection based on ZnO/AgI nanophotocatalyst. Frist, ZnO/AgI nanoparticles were synthesized and characterized by scanning electron microscopy (SEM), transmission electron microscopy (TEM), X-ray powder diffraction (XRD), and Fourier transform infrared spectrometry (FTIR). The color development is caused by tetramethyl benzidine (TMB) solution oxidated by ZnO/AgI nanomaterials. The electron transitions in ZnO/AgI nanomaterials are driven by visible light irradiation, generating photogenerated hole and oxidizing TMB to blue solution. Appropriate band width between ZnO and AgI promotes separation of photogenerated electrons and holes and enhances oxidation efficiency. A sandwich-type immunoassay was constructed based on ZnO/AgI nanomaterial as labels. The absorbance at 650 nm of reaction solution is positively correlated with antigen concentration. The developed immunoassay showed good performance for carcinoma embryonic antigen (CEA) detection in the range 0.1–7.0 ng/mL with a detection limit of 65 pg/mL. The photochromic immunoassay also exhibited preferable selectivity, repeatability, and stability. A novel colorimetric immunoassay was constructed based on ZnO/AgI photocatalyst. ZnO/AgI nanomaterials occur electron transitions under visible light irradiation and generate photogenerated hole, which can oxidize TMB to blue solution. Carcinoembryonic antigen in sample was detected sensitively due to the high catalytic efficiency of ZnO/AgI nanomaterials. [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. [ABSTRACT FROM AUTHOR]

Among symptomatic outpatients, subgenomic RNA of severe acute respiratory syndrome coronavirus 2 in nasal midturbinate swab specimens was concordant with antigen detection but remained detectable in 13 (82.1%) of 16 nasopharyngeal swab specimens from antigen-negative persons. Subgenomic RNA in midturbinate swab specimens might be useful for routine diagnostics to identify active virus replication. [ABSTRACT FROM AUTHOR]

Background Resolving the coronavirus disease 2019 (COVID-19) pandemic requires diagnostic testing to determine which individuals are infected with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). The current gold standard is to perform reverse-transcription polymerase chain reaction (PCR) on nasopharyngeal samples. Best-in-class assays demonstrate a limit of detection (LoD) of approximately 100 copies of viral RNA per milliliter of transport media. However, LoDs of currently approved assays vary over 10,000-fold. Assays with higher LoDs will miss infected patients. However, the relative clinical sensitivity of these assays remains unknown. Methods Here we model the clinical sensitivities of assays based on their LoD. Cycle threshold (Ct) values were obtained from 4700 first-time positive patients using the Abbott RealTime SARS-CoV-2 Emergency Use Authorization test. We derived viral loads from Ct based on PCR principles and empiric analysis. A sliding scale relationship for predicting clinical sensitivity was developed from analysis of viral load distribution relative to assay LoD. Results Ct values were reliably repeatable over short time testing windows, providing support for use as a tool to estimate viral load. Viral load was found to be relatively evenly distributed across log10 bins of incremental viral load. Based on these data, each 10-fold increase in LoD is expected to lower assay sensitivity by approximately 13%. Conclusions The assay LoD meaningfully impacts clinical performance of SARS-CoV-2 tests. The highest LoDs on the market will miss a majority of infected patients. Assays should therefore be benchmarked against a universal standard to allow cross-comparison of SARS-CoV-2 detection methods. [ABSTRACT FROM AUTHOR]

Emerging SARS-CoV-2 variants potentially influence the effectiveness of existing laboratory diagnostics. In this study we determined whether the British (20I/501Y.V1) and South-African (20H/501Y.V2) SARS-CoV-2 variants-of-concern (VOC) are detected by an in-house S1-based antigen-detection assay. Analysis was performed in spiked pools of qRT-PCR negative nasopharyngeal swab specimens. The assay, composed of a combination of four monoclonal antibodies, allowed sensitive detection of both the wild-type and the analyzed VOCs, despite the accumulation of several mutations in the variants' S1 region. We suggest that the combination of four monoclonal antibodies, targeting distinct epitopes, maintained both the specificity and the universality of the assay. [ABSTRACT FROM AUTHOR]

Compared to RT-PCR, lower performance of antigen detection assays, including the Lumipulse G SARS-CoV-2 Ag assay, may depend on specific testing scenarios. We tested 594 nasopharyngeal swab samples from individuals with COVID-19 (RT-PCR cycle threshold [Ct] values ≤ 40) or non-COVID-19 (Ct values >40) diagnoses. RT-PCR positive samples were assigned to diagnostic, screening, or monitoring groups of testing. With a limit of detection of 1.2 × 104 SARS-CoV-2 RNA copies/mL, Lumipulse showed positive percent agreement (PPA) of 79.9% (155/194) and negative percent agreement of 99.3% (397/400), whereas PPAs were 100% for samples with Ct values of <18 or 18–<25 and 92.5% for samples with Ct values of 25–<30. By three groups, Lumipulse showed PPA of 87.0% (60/69), 81.1% (43/53), or 72.2% (52/72), respectively, whereas PPA was 100% for samples with Ct values of <18 or 18–<25, and was 94.4, 80.0, or 100% for samples with Ct values of 25–<30, respectively. Additional testing of RT-PCR positive samples for SARS-CoV-2 subgenomic RNA showed that, by three groups, PPA was 63.8% (44/69), 62.3% (33/53), or 33.3% (24/72), respectively. PPAs dropped to 55.6, 20.0, or 41.7% for samples with Ct values of 25–<30, respectively. All 101 samples with a subgenomic RNA positive result had a Lumipulse assay's antigen positive result, whereas only 54 (58.1%) of remaining 93 samples had a Lumipulse assay's antigen positive result. Lumipulse assay was highly sensitive in samples with low RT-PCR Ct values, implying repeated testing to reduce consequences of false-negative results. [ABSTRACT FROM AUTHOR]

The rapid and accurate detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is required to prevent the spread of COVID-19. This study evaluated the utility of two SARS-CoV-2 antigen detection methods. We evaluated two types of antigen detection methods using immunochromatography (Espline) and quantitative chemiluminescent enzyme immunoassay (Lumipulse). RT-PCR was performed as a standard procedure for COVID-19 diagnosis. Lumipulse and RT-PCR were performed for all 486 nasopharyngeal swabs and 136 saliva samples, and the Espline test was performed for 271 nasopharyngeal swabs and 93 saliva samples. The sensitivity and specificity of the Espline test were 10/11 and 260/260 (100%), respectively for the nasopharyngeal swabs and 3/9 and 84/84 (100%), respectively for the saliva samples. High sensitivities for both saliva (8/9) and nasopharyngeal swabs (22/24) were observed in the Lumipulse test. The specificities of the Lumipulse test for nasopharyngeal swabs and saliva samples were 460/462 (99.6%) and 123/127 (96.9%), respectively. The Espline test is not effective for saliva samples but is useful for simple and rapid COVID-19 tests using nasopharyngeal swabs because it does not require special devices. The Lumipulse test is a powerful high-throughput tool for COVID-19 diagnosis because it has high detection performance for nasopharyngeal swabs and saliva samples. [ABSTRACT FROM AUTHOR]

Little knowledge of antigen existence in the pulmonary tuberculosis (PTB) patient serum impeded its development in antigen detection technology, despite its considerable potential. Human ligand proteins and their adsorbent Mycobacterium tuberculosis (M.tb) proteins in the serum of PTB patients were identified using human protein chip (HuProt™) and LC-MS/MS, successively. The monoclonal antibody of ligand proteins, C5orf24, and polyclonal antibody of 9 M.tb proteins were prepared on mice and rabbits which were used to develop a novel enzyme-linked ligand-sorbent assay (ELLSA). The 412 volunteers were divided into the PTB group (n = 250) and the healthy control (n = 162). The PTB group was further divided into ATB (n = 131), LTBI (n = 18), Clinical diagnosis (n = 18), and Suspected (n = 73). All samples were tested by ELLSA to evaluate the diagnostic performance of ELLSA in PTB patients. Nine ligand proteins specific to PTB patients were identified on chips, with Chromosome 5 Open Reading Frame 24 (C5orf24) and kinocilin (KNCN) showing significantly higher signals. Proteomic analysis of the C5orf24-and KNCN-adsorbent protein complexes revealed 10 and 10 of the M.tb proteins, respectively. According to the composition reference of standard, the ELLSA based on C5orf24 ligand demonstrated a higher sensitivity of 69.6% and specificity of 90.18% in ATB patients and had a sensitivity of 64.22% in bacterial negative pulmonary tuberculosis, whereas the sensitivity of MGIT 960 and Xpert M.tb /RIF were 0%, respectively. M.tb proteins in serum can be enriched by ligand proteins and detected by ELLSA which proved to have excellent diagnostic performance for PTB. [ABSTRACT FROM AUTHOR]

• N-protein detection in saliva without any sample treatment. • No waste solution after the saliva analysis. • Miniaturised and easy-to-use disposable device. • Smartphone-assisted electrochemical immunosensor. Herein, we developed a new waste solution-free paper-based electrochemical immunosensor for SARS-CoV-2 detection in saliva, by combining vertical and lateral flow. In detail, the device was constituted of a reservoir containing all reagents for the construction of the immunological chain onto the magnetic beads and a lateral flow holder which contained a polyester-based electrode, a magnet, and an adsorbent pad. The measurement was carried out by adding the saliva sample into the reservoir, followed by the addition of this solution in the hole present in the lateral flow holder. The successive additions of washing buffer and TMB solution in the lateral flow holder allowed the detection of N protein in saliva in the range of 0.06 to 4 µg/mL with a detection limit equal to 30 ng/mL. The analysis of several saliva samples with the sensing tool and the reference method, demonstrated the effectiveness of this device, being able to identify positive patients with high values of CT e.g. 35. This new configuration paves the way for the realization of any magnetic beads-based immunosystem without waste solution production, enlarging the application of paper-based devices. [ABSTRACT FROM AUTHOR]

To prevent the novel coronavirus disease 2019 (COVID-19) pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), it is necessary to perform early identification and isolation of people shedding the infectious virus in biological materials with high viral loads several days prior to symptom onset. Rapid antigen tests for infectious diseases are useful to prevent the pandemic spread in clinical settings. We evaluated a SARS-CoV-2 antigen test, Espline® SARS-CoV-2 reagent, with reverse transcription polymerase chain reaction (RT-PCR) as reference test, using 129 nasopharyngeal swab specimens collected from COVID-19 hospitalized patients or from patients suspected having COVID-19-like symptoms. Out of these, 63 RT-PCR positive and 66 RT-PCR negative specimens were identified. Among 63 RT-PCR positive specimens, 25 were positive in the Espline test. Test sensitivity was estimated based on the 532.4 copies/reaction of SARS-CoV-2 RNA obtained through receiver operating characteristic analysis. When the specimens were classified based on time since symptom onset, Espline test sensitivity were 73.3% and 29.2% in specimens collected before day 9 and after day 10, respectively. Although the overall sensitivity of the Espline® SARS-CoV-2 reagent compared with RT-PCR is less, this antigen test can be useful in identifying people with high risk of virus transmission with high viral loads in order to prevent the pandemic and is useful for diagnosing COVID-19 within 30 min [ABSTRACT FROM AUTHOR]

The diagnosis of blastomycosis and histoplasmosis can be difficult for clinicians who rarely see infections caused by these environmentally restricted dimorphic fungi. Historically, the diagnosis of blastomycosis has been established by culture and sometimes by histopathologic identification. Currently, antigen detection in urine and serum has been shown to aid in the rapid diagnosis of blastomycosis, and newer antibody assays are likely to contribute to our diagnostic capability in the near future. The gold standard for the diagnosis of histoplasmosis has been culture of the organism from involved tissues, aided in some patients by histopathological verification of the typical yeast forms in tissues. Antigen detection has contributed greatly to the ability of clinicians to rapidly establish the diagnosis of histoplasmosis, especially in severely ill and immunocompromised patients, and antibody testing for Histoplasma capsulatum provides important adjunctive diagnostic capability for several forms of both acute and chronic histoplasmosis. For both of these endemic mycoses, novel molecular tests are under active investigation, but remain available in only a few reference laboratories. In this review, we provide a synopsis of diagnostic test options that aid in establishing whether a patient has blastomycosis or histoplasmosis. [ABSTRACT FROM AUTHOR]

Background: Respiratory syncytial virus (RSV) is a major cause of hospitalization in infants. Early detection of RSV can optimize clinical management and minimize use of antibiotics. BinaxNOW RSV (BN) is a rapid antigen detection test that is widely used. We aimed to validate the sensitivity of BN in hospitalized and nonhospitalized infants against the gold standard of molecular diagnosis.Methods: We evaluated the performance of BN in infants with acute respiratory tract infections with different degrees of disease severity. Diagnostic accuracy of BN test results were compared with molecular diagnosis as reference standard.Results: One hundred sixty-two respiratory samples from 148 children from October 2017 to February 2019 were studied. Sixty-six (40.7%) samples tested positive for RSV (30 hospitalizations, 31 medically attended episodes not requiring hospitalization, and 5 nonmedically attended episodes). Five of these samples tested positive with BN, leading to an overall sensitivity of BN of 7.6% (95% confidence interval [CI], 3.3%-16.5%) and a specificity of 100% (95% CI, 96.2%-100%). Sensitivity was low in all subgroups.Conclusions: We found a low sensitivity of BN for point-of-care detection of RSV infection. BinaxNOW RSV should be used and interpreted with caution. [ABSTRACT FROM AUTHOR]

Toxocariasis is a human infection primarily caused by larvae of Toxocara canis from dogs, and also by T. cati from cats. Children have a more significant risk of acquiring the infection due to their closer contact with pets, and greater chances of ingesting soil. Diagnosis of toxocariasis is based on clinical, epidemiological, and serological data. Indirect IgG ELISA is a widely used serodiagnostic method for toxocariasis, with native T. canis TES most commonly used as the antigen. Western blots, using the same antigen, can be used to confirm positive ELISA findings to reduce false-positive results. Improvements in Toxocara serodiagnosis include the use of recombinant TES antigens, simpler and more rapid assay formats, and IgG4 subclass detection. Also, incorporation of recombinant T. cati TES protein increases the diagnostic sensitivity. Development of antigen detection tests using polyclonal and monoclonal antibodies, nanobodies, or aptamers can complement the antibody detection assays, and enhance the effectiveness of the serodiagnosis. [ABSTRACT FROM AUTHOR]

Cystic echinococcosis (CE) or hydatid disease is a parasitic infection caused by Echinococcus granulosus. Early serodiagnosis and continuous monitoring of the disease is very important for medical treatment. Here, we report the detecting of both echinococcus antigen and antibody for the diagnosis of hydatid disease using square wave voltammetry (SWV)‐based immunosensors. The gold electrodes were functionalized using cysteamine/phenylene diisothiocyanate linkers and used for the immunosensors fabrication. The hydatid antigen and antibody immunosensors were constructed by the immobilization of either purified rabbit polyclonal antibody or recombinant antigen B (AgB), respectively on the functionalized gold electrodes surfaces. The detection in both cases was achieved by following the change in the SWV reduction peak current of the ferro/ferricyanide redox couple upon antibody or antigen binding. These immunosensors enabled the detection of echinococcus antigen and antibody within a concentration range of 1 pg.mL−1 to 1 μg.mL−1 with detection limits of 0.4 pg.mL−1 and 0.3 pg.mL−1, respectively. A preliminary application of the developed immunosensor was performed in spiked serum sample showing good recovery percentages ranging from 102 to 110 % for both hydatid antibody and antigen detection. This easy‐to‐use, sensitive, and low cost quantitative method holds great promise for the early diagnosis of hydatid disease and thus, better managements and treatment outcomes. [ABSTRACT FROM AUTHOR]

Background: Several refugee settlements in Bangladesh have provided housing and medical care for the forcibly-displaced Myanmar nationals (FDMN, also known as Rohingya) population. The identification of malaria infection status in the refugee settlements is useful in treating infected persons and in developing malaria prevention recommendations. Assays for Plasmodium antigens and human IgG against Plasmodium parasites can be used as indicators to determine malaria infection status and exposure. Methods: Dried blood spot (DBS) samples (N = 1239) from a household survey performed April–May 2018 in three settlements in Cox's Bazar district, Bangladesh were utilized for a sample population of children from ages 1–14 years of age. The samples were tested using a bead-based multiplex antigen assay for presence of the pan-Plasmodium antigen aldolase as well as Plasmodium falciparum histidine rich protein 2 (HRP2). A bead-based multiplex assay was also used to measure human IgG antibody response to P. falciparum, Plasmodium malariae, and Plasmodium vivax merozoite surface protein 1 antigen (MSP1) isoforms, and P. falciparum antigens LSA1, CSP, and GLURP-R0. Results: There were no detectable Plasmodium antigens in any samples, suggesting no active malaria parasite infections in the tested children. IgG seroprevalence was highest to P. vivax (3.1%), but this was not significantly different from the percentages of children antibody responses to P. falciparum (2.1%) and P. malariae (1.8%). The likelihood of an anti-Plasmodium IgG response increased with age for all three malaria species. Evidence of exposure to any malaria species was highest for children residing 8–10 months in the settlements, and was lower for children arriving before and after this period of time. Conclusions: Absence of Plasmodium antigen in this population provides evidence that children in these three Bangladeshi refugee settlements did not have malaria at time of sampling. Higher rates of anti-malarial IgG carriage from children who were leaving Myanmar during the malaria high-transmission season indicate these migrant populations were likely at increased risk of malaria exposure during their transit. [ABSTRACT FROM AUTHOR]

Summary: Background: Many patients with histoplasmosis are treated with anti‐tubercular therapy (ATT) in tuberculosis endemic regions as diagnosis of histoplasmosis requires invasive sampling. We sought to study the utility of urinary Histoplasma antigen detection test. Methods: Case records of patients with a diagnosis of histoplasmosis prior to (Period A) and after (Period B) introduction of urinary Histoplasma antigen detection test were analysed in this single centre retrospective study. Results: Thirty‐seven patients (18 in Period A, and 19 patients in Period B) were studied. There was nearly a threefold increase in diagnoses (from 0.39 cases to 1.18 cases per month) after the introduction of antigen test. Nine patients (24.3%) were immunocompromised (6 had HIV infection and 3 were on steroids), and 28 (75.6%) were immunocompetent. Empirical ATT had been given to 10 patients prior to histoplasmosis diagnosis. Invasive tissue sampling was required in only two patients in Period B to confirm the diagnosis. Immunocompromised patients were younger, were more likely to have skin and mucosal findings, anaemia and leucopenia as compared to immune‐competent patients. Conclusion: This study emphasises that histoplasmosis cases may be missed and patients may receive ATT unnecessarily. Histoplasma antigen increased the diagnostic yield by almost threefold in our study. [ABSTRACT FROM AUTHOR]

PfHRP2-based rapid diagnostic tests (RDTs), based on the recognition of the Plasmodium falciparum histidine-rich protein 2, are currently the most used tests in malaria detection. Most of the antibodies used in RDTs also detect PfHRP3. However, false-negative results were reported. Significant variation in the pfhrp2 gene could lead to the expression of a modified protein that would no longer be recognized by the antibodies used in PfHRP2-based RDTs. Additionally, parasites lacking the PfHRP2 do not express the protein and are, therefore, not identifiable. This review aims to assess the pfhrp2 and pfhrp3 genetic variation or the prevalence of gene deletion in areas where malaria is endemic and describe its implications on RDT use. Publications of interest were identified using PubMed, Google Scholar and Google. More than 18 types of amino acid repeats were identified from the PfHRP2 sequences. Sequencing analysis revealed high-level genetic variation in the pfhrp2 and pfhrp3 genes (>90% of variation in Madagascar, Nigeria or Senegal) both within and between countries. However, genetic variation of PfHRP2 and PfHRP3 does not seem to be a major cause of false-negative results. The countries that showed the highest proportions of pfhrp2 -negative parasites were Peru (20%–100%) and Guyana (41%) in South America, Ghana (36%) and Rwanda (23%) in Africa. High prevalence of pfhrp2 deletion causes a high rate of false-negatives results. Presence of parasites lacking the pfhrp2 gene may pose a major threat to malaria control programmes because P. falciparum -infected individuals are not diagnosed and properly treated. [ABSTRACT FROM AUTHOR]

Summary: Despite the existence of endemic mycoses in Latin America and the Caribbean, in addition to a large population of patients at risk for invasive mycoses, the capability of medical centres to perform a proper diagnosis in mycology has not been studied in the region. Moreover, availability of antifungal drugs in the region is unknown. Here, we report the results of a survey involving 129 centres in 24 countries. Only 9% of centres would have the potential to apply for the minimum standards in mycology, as determined by the European Confederation of Medical Mycology. There is an urgent need to improve diagnostic conditions in Latin America and the Caribbean, as well as providing access to safer and more efficacious antifungal drugs. [ABSTRACT FROM AUTHOR]

Abstract Cystic echinococcosis (CE) is a neglected zoonotic disease with a worldwide distribution and is a major public health problem in some areas. Diagnosis of CE is mainly based on clinical symptoms, imaging and serological testing, however, improvement in serodiagnosis is still needed. This study was aimed at detecting circulating Echinococcus antigen in CE patients using a lateral flow dipstick (LFD) assay. Three types of hydatid antigens i.e. hydatid cyst fluid (HCF), native antigen B (nAgB) and recombinant antigen B (rAgB) were prepared and polyclonal rabbit antiserum was raised against each antigen. Purified IgG fractions were prepared and a portion was conjugated to gold nanoparticles. After a series of optimizations, a final antigen detection LFD assay was developed using a combination of anti-nAgB-IgG and gold-conjugated anti−HCF-IgG. Evaluation of the assay showed that 27 out of 35 (77%) serum samples from CE patients gave positive results. Meanwhile, the test showed a diagnostic specificity of 82% when tested with sera from 38 healthy individuals and 13 patients with other parasitic diseases. In conclusion, the antigen detection LFD assay seemed to be useful for diagnosis of CE and possibly for post-treatment follow-up, and merit further evaluation studies. We foresee that it may improve serodiagnosis of CE when used in tandem with an antibody detection test. [ABSTRACT FROM AUTHOR]

Background: Detection of Plasmodium antigens provides evidence of malaria infection status and is the basis for most malaria diagnosis.Methods: We developed a sensitive bead-based multiplex assay for laboratory use, which simultaneously detects pan-Plasmodium aldolase (pAldo), pan-Plasmodium lactate dehydrogenase (pLDH), and P. falciparum histidine-rich protein 2 (PfHRP2) antigens. The assay was validated against purified recombinant antigens, monospecies malaria infections, and noninfected blood samples. To test against samples collected in an endemic setting, Angolan outpatient samples (n = 1267) were assayed.Results: Of 466 Angolan samples positive for at least 1 antigen, the most common antigen profiles were PfHRP2+/pAldo+/pLDH+ (167, 36%), PfHRP2+/pAldo-/pLDH- (163, 35%), and PfHRP2+/pAldo+/pLDH- (129, 28%). Antigen profile was predictive of polymerase chain reaction (PCR) positivity and parasite density. Eight Angolan samples (1.7%) had no or very low PfHRP2 but were positive for 1 or both of the other antigens. PCR analysis confirmed 3 (0.6%) were P. ovale infections and 2 (0.4%) represented P. falciparum parasites lacking Pfhrp2 and/or Pfhrp3.Conclusions: These are the first reports of Pfhrp2/3 deletion mutants in Angola. High-throughput multiplex antigen detection can inexpensively screen for low-density P. falciparum, non-falciparum, and Pfhrp2/3-deleted parasites to provide population-level antigen estimates and identify specimens requiring further molecular characterization. [ABSTRACT FROM AUTHOR]

Background: Strep throat is also known as Streptococcal Pharyngitis. It is an infection in back of the throat including tonsils which reasoned by group A Streptococcus (GAS). General signs like fever, red tonsils, sore throat and grow lymph nodes in the neck. Nausea, headache and vomiting may also happen due to Streptococcal Pharyngitis. Objective: The aim of study is to conclude the correctness in discover Group A β-Hemolytic Streptococci (GABHS) through brisk antigen testing evaluate with throat culture methods which are generally used. Materials and Methods: Study Design: Cross-sectional study. Setting: Sir Ganga Ram Hospital Lahore. Period: 1st July 2016 to 31st December 2016. At first throat culture, Streptococcal select agar or sheep blood agar, performed on 192 patients with severe strep throat and after that brisk antigen detection tests, Directigen Group A Strep, was also executed. Statistical investigation contained sensitivity, specificity, positive predictive value, negative predictive value as well as its prevalence. Results: The prevalence of group A β-hemolytic streptococci is 13.54%. Sensitivity is 96.15%, specificity is 95.18%, positive predictive value is 75.76% along with negative predictive value is 99.37% which shows that a very low percentage of patients with Group A β-Hemolytic Streptococci as <1%. Conclusion: This showed that a very low percentage as <1% of patients with Group A β-Hemolytic Streptococci evade findings by brisk screening test methods. [ABSTRACT FROM AUTHOR]

An optical fiber interferometric microprobe for detection of specific proteins is presented in this paper. The microprobe is an all-fiber device, which is based on Michelson interferometer configuration, which allows for detection of protein antigens in an analyzed solution thanks to antibodies immobilized on the sensor surface. The interferometer is made of dual core fiber and has a precisely formed arm length difference, achieved by splicing a fragment of polarization maintaining fiber to one of the cores. An all-fiber configuration of the sensor decreases substantially cross-sensitivities to temperature and deformation in comparison to other optical fiber interferometers. Reported sensor has a sensing region on the tip of the interferometer and therefore may be used for point measurements in medicine. The immunosensor and optical measuring system are designed to utilize the most common broadband light sources that operate at a central wavelength of 1.55 µm. The results show that it is possible to detect a protein antigen present in a solution by using an all-fiber interferometer coated with specific antibodies. The resulting peak shift can reaches 0.6 nm, which is sufficient to be measured by an optical spectrum analyzer or a spectrometer. A model allowing for estimation of the value of lower limit of detection for such sensors has been elaborated. The elaborated detection system may act as a framework for detection of various antigens and thus it can find future applications in medical diagnostics. [ABSTRACT FROM AUTHOR]

Background: Diagnosis of Visceral Leishmaniasis (VL) is still challenging. This review highlighted current status and challenges in the serological diagnosis of VL. Furthermore, the drawback of currently available serological tests and the most recent advancement in the designing and application of these assays for the diagnosis of VL are addressed. Methods: All the published literature cited within PubMed, ISI Web of Science, Google Scholar, Scopus, and IranMedex, regarding the immunodiagnosis of VL in human were sought from 2000 till Mar 2017. The search terms were "visceral leishmaniasis", or "kala-azar" subsequently combined with the search terms "diagnosis", "serodiagnosis", "human", "serological", "antigen detection" or "antibody detection". Data were extracted from literature which fulfilled our eligibility criteria. Results: Direct agglutination test (DAT) and rk39 dipstick have made a great improvement in the serological diagnosis of VL. Besides, other kinesin-related protein including K26, K28, and KE16 provided promisingly diagnostic accuracy in the diagnosis of VL. The Latex Agglutination Test for the diagnosis of VL (KAtex), with moderate sensitivity but high specificity, made a substantial contribution to the field. Moreover, a range of protein antigens has recently been detected in the urine of VL patients with encouraging diagnostic value. Conclusion: The suboptimal diagnostic accuracy of the currently available serological assays for the diagnosis of human VL necessitates further research and development in this field. Outcomes of immunodiagnostic tests based on recombinant antigens are favorable. These proteins might be the most appropriate antigens to be further evaluated and utilized for the diagnosis of human VL. [ABSTRACT FROM AUTHOR]

Objectives Efficient interruption of Ebola virus disease (EVD) transmission chains critically depends on reliable and fast laboratory diagnosis. We evaluated the performance of the EBOLA Virus Antigen Detection K -SeT (EBOLA Ag K -SeT), a new rapid diagnostic antigen test in field settings. Methods The study was conducted in a field laboratory located in Freetown (Sierra Leone) by the Italian National Institute for Infectious Diseases ‘L. Spallanzani’ and the EMERGENCY Onlus NGO. The EBOLA Ag K -SeT was tested on 210 residual plasma samples (EVD prevalence 50%) from patients hospitalized at the EMERGENCY Ebola treatment center in Goderich (Freetown), comparing the results with quantitative real-time PCR. Results Overall, the sensitivity of EBOLA Ag K -SeT was 88.6% (95% confidence interval (CI), 82.5–94.7), and the corresponding specificity was 98.1% (95% CI, 95.5–100.7). The positive and negative predictive values were 97.9% (95% CI, 95.0–100.8) and 89.6% (95% CI, 84–95.2), respectively. The sensitivity strongly increased up to 98.7% (95% CI, 96.1–101.2) for those samples with high virus load (≥6.2 log RNA copies/mL). Conclusions Our results suggest that EBOLA Ag K -SeT could represent a new effective diagnostic tool for EVD, meeting a need for resource-poor settings and rapid diagnosis for individuals with suspected EVD. [ABSTRACT FROM AUTHOR]