The N and C-terminal deleted variant of the dengue virus NS1 protein is a potential candidate for dengue vaccine development.

NS1 is an elusive dengue protein, involved in viral replication, assembly, pathogenesis, and immune evasion. Its levels in blood plasm are positively related to disease severity like thrombocytopenia, hemorrhage, and vascular leakage. Despite its pathogenic roles, NS1 is being used in various vaccine formulations due to its sequence conservancy, ability to produce protective antibodies and low risk for inducing antibody-dependent enhancement. In this study, we have used bioinformatics tools and reported literature to develop an NS1 variant (dNS1). Molecular docking studies were performed to evaluate the receptor-binding ability of the NS1 and dNS1 with TLR4. NS1 and dNS1 (153 to 312 amino acid region) genes were cloned, expressed and protein was purified followed by refolding. Docking studies showed the binding of NS1 and dNS1 with the TLR4 receptor which suggests that N and C-terminal sequences of NS1 are not critical for receptor binding. Antibodies against NS1 and dNS1 were raised in rabbits and binding affinity of anti-dNS1 anti-NS1 sera was evaluated against both NS1 and dNS1. Similar results were observed through western blotting which highlight that N and C-terminal deletion of NS1 does not compromise the immunogenic potential of dNS1 hence, supports its use in future vaccine formulations as a substitute for NS1.

Acute and prolonged effects of interleukin-33 on cytokines in human cord blood-derived mast cells.

Mast cells are multifaceted cells localized in tissues and possess various surface receptors that allow them to respond to inner and external threat signals. Interleukin-33 (IL-33) is a cytokine released by structural cells in response to parasitic infections, mechanical damage, and cell death. IL-33 can activate mast cells, causing them to release an array of mediators. This study aimed to identify the different cytokines released by human cord blood-derived mast cells (hCBMCs) in response to acute and prolonged stimulation with IL-33. For this purpose, a hCBMC model was established and stimulated with 10 ng and 20 ng of recombinant human IL-33 (rhIL-33) for 6 h and 24 h. Total RNA was hybridized using a high-density oligonucleotide microarray. A multiplex assay was performed to assess the released cytokines. Acute exposure to rhIL-33 increased the expression of IL-1α, IL-1ß, IL-6, and IL-13, whereas prolonged exposure increased the expression of IL-5 and IL-10, and cytokines were detected in the culture supernatant. WebGestalt analysis revealed that rhIL-33 induces pathways and biological processes related to the immune system and the acute inflammatory response. This study demonstrates that rhIL-33 can activate hCBMCs to release pro- and anti-inflammatory cytokines, eliciting distinct acute and prolonged responses unique to hCBMCs.

Veterinary perspectives on the urbanization of leishmaniosis in Morocco.

BACKGROUND: Leishmaniosis caused by Leishmania infantum, L. major and L. tropica is endemic in Morocco. Growing evidence of both human and canine Leishmania infections in urban centres has been reported. Since many forms of the disease are zoonotic, veterinarians play an important role in leishmaniosis control by intervening at the parasite host level. This study aimed to bring together One Health principles to connect canine and feline leishmaniosis epidemiology within urban centres of Morocco (Rabat and Fez) and assess the level of awareness of Moroccan veterinarians about facing this threat. METHODS: A molecular survey was conducted for Leishmania DNA detection in canine (n = 155) and feline (n = 32) whole-blood samples. Three conventional polymerase chain reaction (PCR) protocols were implemented. The first PCR aimed at identifying infected animals by targeting Leishmania spp. kinetoplast minicircle DNA (kDNA). The second and third PCR targeted the Leishmania internal transcribed spacer region (ITS-1) and the Leishmania small subunit ribosomal RNA (SSUrRNA) gene, respectively, aiming at identification of the infecting species after Sanger sequencing-positive amplicons. Total immunoglobulin G (IgG) against Leishmania spp. was evaluated in 125 dogs by enzyme-linked immunosorbent assays (ELISA) using an in-house protocol, including three Leishmania-specific antigens (SPLA, rKDDR and LicTXNPx). Sera from 25 cats were screened for total IgG to Leishmania spp. by an indirect immunofluorescence antibody test (IFAT). An online questionnaire was presented to Moroccan veterinarians addressing their knowledge and practices towards animal leishmaniosis. RESULTS: Overall, 19.4% of the dogs tested positive for Leishmania kDNA and ITS-1 and sequencing revealed infection with L. infantum among PCR-positive dogs. These animals presented a wide range of ELISA seropositivity results (16.7%, 34.9% and 51.6%) according to the tested antigens (rKDDR, SPLA and LicTXNPx, respectively). Use of kDNA-PCR revealed 12.5% cats positive to Leishmania spp. otherwise found to be seronegative by IFAT. CONCLUSIONS: A considerable prevalence of infection was identified in dogs from urban centres of Morocco. Additionally, this is the first report of feline infection with Leishmania spp. in this country and in urban settings. Moroccan veterinarians are aware that animal leishmaniosis is endemic in Morocco, representing a public health threat, and are knowledgeable about canine leishmaniosis diagnosis and treatment.

Microbial infection among SARS-COV-2-infected patients in a COVID-19-dedicated tertiary care hospital of Bangladesh: a cross-sectional study.

Objectives. This study aimed to determine patterns of respiratory, blood-borne and uropathogenic microbial pathogens among SARS-CoV-2-infected patients in a COVID-19-(coronavirus disease 2019) dedicated tertiary care hospital in Dhaka, Bangladesh. Design.This was a cross-sectional study. Setting. In a COVID-19-dedicated tertiary care hospital in Dhaka, Bangladesh, conducted from March to June 2021. Participants. Hospitalized individuals with COVID-19 infection regardless of age or sex. Primary and secondary outcome measures. The percentage of co-infected COVID-19 patients and the characterization of the micro-organisms responsible for co-infection served as the primary outcome measures. Finding any associations between co-infection and age, co-infection and sex and co-infection and comorbidity was the secondary outcome variable. Interventions. Not applicable. Results.Out of 79 patients, 61 % were male, and the mean age was 49.53 years. Co-infection was seen in 7.7 % of patients, out of which 5.1 % of isolates were from urine samples, followed by 2.6 % from blood. Bacteria isolated from urine were Enterococcus (2.6 %), coagulase-negative Staphylococcus (CONS) (1.3 %) and Enterobacter spp. (1.3 %). Pseudomonas spp. was the only organism isolated from blood sample. Mixed growth was found in nasopharyngeal and throat swabs, with the predominant species being Staphylococcus aureus and Streptococcus spp. At the time of data collection, 55.7 % of patients had been given antimicrobials, and 30.4 % of patients had been given a single antimicrobial. HBsAg was positive in 1.3 % of patients and none were anti-hepatitis C or dengue NS1Ag positive. Conclusion. Microbial infection has been seen to be associated with SARS-CoV-2 infections and is of great value in prescribing antimicrobials and reducing fatal outcomes of hospitalized patients.

Production of highly soluble and immuno-reactive recombinant flagellin protein of Clostridium chauvoei.

OBJECTIVE: Flagellin protein, an integral component of flagella, provides motility to several bacterial species and also acts as a candidate antigen in diagnostics and subunit vaccines. The bulk production of flagellin with retention of all conformational epitopes using recombinant protein technology is of paramount importance in the development of pathogen-specific immuno-assays and vaccines. We describe the production of highly soluble and immuno-reactive rFliA(C) protein of Clostridium chauvoei, a causative agent of blackleg or black quarter (BQ) affecting cattle and small ruminants worldwide. The bacterium is known to possess peritrichous flagella that provide motility and also act as a virulence factor with high protective antigenicity. METHODS: Upon sequence and structural analysis, a partial fliA(C) gene from Clostridium chauvoei was cloned and the recombinant mature protein with N- and C- terminal truncation was over-expressed as a His-tagged fusion protein (∼25 kDa) in Escherichia coli. Subsequently, rFliA(C) protein was purified by single-step affinity chromatography and characterized for its immuno-reactivity in laboratory animals, Western blot, and indirect-ELISA format. RESULTS: rFliA(C) was highly soluble and was purified in high quantity and quality. rFliA(C) elicited antigen-specific conformational polyclonal antibodies in rabbit and guinea pig models, as well as anti-Clostridium chauvoei-specific antibodies being specifically detected in BQ-vaccinated and convalescent sera of bovines in Western blot and in indirect-ELISA format. Further, no cross reactivity was noted with antibodies against major bovine diseases (e.g., foot-and-mouth disease, IBR, LSDV, hemorrhagic septicaemia, brucellosis, and leptospirosis). CONCLUSION: The study indicated the production of conformational recombinant flagellin-rFliA(C)-antigen and its potential utility in development of diagnostics for detection of Clostridium chauvoei-specific antibodies in BQ-recovered and/or vaccinated animals.

Analysis of 132 Submandibular Salivary Glands Using the Randox Evidence Investigator and Randox DOA ULTRA WB Array.

Occasionally, obtaining an adequate or acceptable postmortem blood specimen for drug analysis is not possible due to factors such as decomposition, exsanguination, or embalming. Submandibular salivary gland tissue, one of three major types of salivary gland tissue in the oral cavity of humans, has been reported to be a viable alternative postmortem specimen for toxicological testing. In this study, we evaluated the performance of the Randox Evidence Investigator instrument and Randox DOA (Drugs of Abuse) Ultra Whole Blood Array for the semi-quantitative determination of 21 immunoassays in an alternative matrix, submandibular salivary gland tissue. We analyzed 132 submandibular salivary gland tissue specimens and compared the generated results to concomitantly collected postmortem whole blood specimen results. Oxycodone 2, meprobamate, barbiturate, benzodiazepine assay 1, zolpidem, and buprenorphine all showed perfect agreement (Cohen's Kappa Score = 1.00) between the submandibular salivary gland tissue results and the postmortem whole blood results; dextromethorphan, fentanyl, benzoylecgonine, methamphetamine, tricyclic antidepressants, oxycodone 1, and opiate showed an almost perfect agreement (Cohen's Kappa Score = 0.81-0.99); methadone, generic opioids, and amphetamine exhibited substantial agreement (Cohen's Kappa Score = 0.61-0.80). Tramadol demonstrated fair agreement (Cohen's Kappa Score = 0.41-0.60). The lowest measure of agreement was observed with cannabinoids, meeting criteria for slight agreement (Cohen's Kappa Score = 0.01-0.20). An application of the techniques described in this study could be implemented in postmortem toxicology laboratories as well as medical examiners offices to provide preliminary drugs of abuse test results that can be used to direct additional testing. This study highlights the successful integration of a novel specimen matrix and an "off-label" use of an established analytical technique.

Development of two competitive ELISAs based on monoclonal antibodies for the serological detection of Bovine ephemeral fever virus.

Bovine ephemeral fever virus (BEFV) is a member of the genus Ephemerovirus in the family Rhabdoviridae. It is an arthropod-borne virus transmitted by many species of midges and mosquitoes. It can cause severe economic consequences due to losses in milk production and the general condition of cattle and water buffalo. BEF occurs in some tropical, subtropical and warm temperate regions of Africa, Australia, the Middle East and Asia with seasonal outbreaks, but its possible spread to other areas (e.g. Europe) cannot be excluded. Therefore, using and developing rapid diagnostic methods with optimal performance is essential for identifying emerging pathogens and their control. In the present study, we developed two competitive serological ELISAs based on monoclonal antibodies (mAbs), designed by using BEFV inactivated antigen and the BEF recombinant nucleoprotein (N), respectively. A panel of 77 BEF-positive and 338 BEF-negative sera was used to evaluate the two tests. With a diagnostic sensitivity of 97.4 % using the inactivated virus and 98.7 % using the recombinant N, and a diagnostic specificity of 100 % using both antigens, our results suggest that these tests are suitable for the serological diagnosis of BEF.

Further validation of 2 nonstructural protein-specific antibody tests for diagnosis and surveillance of foot-and-mouth disease in the United States.

Foot-and-mouth disease (FMD) is a highly contagious disease of cloven-hoofed animals. FMD poses an economic threat to the livestock industry in the United States. Due to the potential use of vaccines composed of partially purified structural proteins of the FMD virus (FMDV), it is important to test samples from infected and vaccinated animals with a competitive ELISA that detects antibodies against nonstructural proteins (NSPs) of FMDV. Our study extends the diagnostic validation of the Prionics ELISA (Thermo Fisher) and the VMRD ELISA. We used diverse serum sample sets from bovine, porcine, and other cloven-hoofed animals to evaluate the analytical specificity and sensitivity, diagnostic specificity and sensitivity, and differentiation of infected from vaccinated animals (DIVA) per validation guidelines outlined by the World Organisation for Animal Health (WOAH). The 2 tests were analytically 100% accurate. The VMRD test was diagnostically more sensitive than Prionics, but Prionics was diagnostically more specific than the VMRD test. Both tests could tell if animals were infected or vaccinated. Considering these data, both VMRD and Prionics ELISAs can be used for serodetection of FMDV antibodies at the Foreign Animal Disease Diagnostic Laboratory and within the National Animal Health Laboratory Network laboratories.

Buccal DNA global methylation and cognitive performance in stunted children under 5 years of age.

The prevalence of stunting in Indonesian children under five years of age is about 20%. Chronic maternal malnutrition contributes to the risk of stunting by affecting global DNA methylation. In the present study, we aimed to evaluate the levels of 5-methyl-cytosine (5mC), as a surrogate marker of global DNA methylation, in buccal swabs and its potential association with risk of stunting and cognitive performance. The levels of 5mC were measured using the enzyme-linked immunosorbent assay. The Wechsler Preschool and Primary Scale of Intelligence was used to measure cognitive functions. Buccal swab DNA samples and anthropometric data were collected from a total of 231 children aged zero to five years. In this cross-sectional cohort, the prevalence of stunting was 37% in 138 children aged zero to two years and 30% in 93 children aged > two years. The univariable analysis revealed that the levels of 5mC in buccal swab DNA were significantly lower in severely stunted children (median, 2.84; interquartile range [IQR], 2.39-4.62; P-value, 0.0314) and in children of a younger age (median, 2.81; IQR 2.53-4.62, P-value, 0.0001) than in normal (median, 3.75; IQR, 2.80-4.74) and older children (median, 4.01, IQR, 3.39-4.87), respectively. We also found that the average cognitive scores tended to be low in boys and stunted children, although the differences were not statistically significant. Furthermore, levels of 5mC found in buccal and mouthwash DNA were not associated with cognitive scores.

Baculovirus expression and purification of virion core and envelope proteins of goatpox virus to evaluate their diagnostic potential.

Goatpox and sheeppox are highly contagious and economically important viral diseases of small ruminants. Due to the risk they pose to animal health, livestock production, and international trade, capripoxviruses are a considerable threat to the livestock economy. In this study, we expressed two core proteins (A4L and A12L) and one extracellular enveloped virion protein (A33R) of goatpox virus in a baculovirus expression vector system and evaluated their use as diagnostic antigens in ELISA. Full-length A4L, A12L, and A33R genes of the GTPV Uttarkashi strain were amplified, cloned into the pFastBac HT A donor vector, and introduced into DH10Bac cells containing a baculovirus shuttle vector plasmid to generate recombinant bacmids. The recombinant baculoviruses were produced in Sf-21 cells by transfection, and proteins were expressed in TN5 insect cells. The recombinant proteins were analysed by SDS-PAGE and confirmed by western blot, with expected sizes of ~30 kDa, ~31 kDa, and ~32 kDa for A4L, A12L, and A33R, respectively. The recombinant proteins were purified, and the immunoreactivity of the purified proteins was confirmed by western blot using anti-GTPV serum. The antigenic specificity of the expressed proteins as diagnostic antigens was evaluated by testing their reactivity with infected, vaccinated, and negative GTPV/SPPV serum in indirect ELISA, and the A33R-based indirect ELISA was optimized. The diagnostic sensitivity and specificity of the A33R-based indirect ELISA were found to be of 89% and 94% for goats and 98% and 91%, for sheep, respectively. No cross-reactivity was observed with other related viruses. The recombinant-A33R-based indirect ELISA developed in the present study shows that it has potential for the detection of antibodies in GTPV and SPPV infected/vaccinated animals.

Comparison of in-house enzyme-linked immunosorbent assay (ELISA) and six commercial ELISAs for Detection of antibodies to Bordetella pertussis.

Enzyme-linked immunosorbent assays (ELISA) are currently the method of choice for the serodiagnosis of pertussis and play a key role in the diagnosis of pertussis in adolescents and adults, as well as in epidemiological studies. In the present study, the in-house developed indirect ELISA was comparatively evaluated with six commercial kits from various manufacturers. Antipertussis antibodies were measured in 40 serum samples from patients with clinical symptoms of respiratory tract infection, in two WHO standards, and in seven human ECDC control sera. IgA and IgG antibodies were detected at a diagnostically significant level by different ELISA kits of 5.0% to 27.0% and 12.0% to 70.0% of patients' sera, appropriately. The analysis of results carried out with six commercial kits showed only 17.5% consistent results in class IgG (either clearly positive or negative). The average percentage of errors in the level of antibodies determined in the control samples, reference serum samples, differed quite significantly and ranged from 9.5% to 35.4% depending on the kit. This poor correlation of the results obtained on various serological tests intended for the serodiagnosis of pertussis may cause very serious diagnostic problems, especially when examining a serum sample obtained once during the course of the disease.

Selenium nanoparticles effect on foot and mouth disease vaccine in local Awassi breed male lambs.

Objective: The goal of this research was to evaluate where selenium nanoparticles impact the activity of antibodies in immunized lambs with foot and mouth vaccines by modulating the immune system. Materials and Methods: Two groups of lambs of 3-4 months of age were injected with 1 ml of ARRIAH-VAC vaccine intramuscularly in the neck, five Lambs were given selenium nanoparticles (size 100 nm) oral administration of selenium nano dose of 0.1 mg/kg of body mass once every day for sixty days considered as group one (G1) while the other five used as control Group 2 (G2). Results: This resulted in the establishment of an immune response, as evidenced by a rise in antibody titer in the blood using the ELISA test for three serotypes A, O, and Asia 1, when selenium nanoparticles were given orally at a dose of 0.1 mg/kg body weight after immunization, we noticed a significant (p >0:05) selenium nano group increase in IgG response in all immunized groups in contrast to lambs that had only received the foot-and-mouth disease vaccine. Conclusion: We have demonstrated that selenium nanoparticles administered orally significantly enhance immune responses while also increasing body weight.

Detection and seroprevalence of Q fever infection in dairy goats in Besut district, Malaysia.

Objective: This study aimed to investigate the seroprevalence of Q fever and its association with age and gender among Saanen dairy goats in Malaysia. Materials and Methods: One hundred dairy goats (n = 100) aged 6 months to 6 years were randomly selected, and blood samples were collected for serological analysis using the enzyme-linked immunosorbent assay technique. Results: The results revealed a seropositive rate of 70% among the goats, with medium-positive titers being the most common. The prevalence of Q fever varied among different age groups, with higher rates observed in adult goats aged between 5 and 6 years. Gender analysis showed that males had a higher positive rate (p < 0.05) of Q fever compared to females. Conclusion: These findings strongly indicate the presence of Coxiella burnetii in the dairy goat population and highlight the importance of implementing biosecurity measures and control strategies to prevent further transmission of this disease. This research has contributed to a better understanding of Q fever epidemiology and provides insights for effective control and prevention strategies in dairy goat populations.

Advances in the clinical measurement of glucagon: from diagnosis to therapy.

Glucagon has many functions: it promotes glucose production, fatty acid oxidation, thermogenesis, energy consumption, lipolysis, and myocardial contraction, and suppresses lipogenesis, appetite, and gastrointestinal motility. Which of these functions are physiological and which are pharmacological is not fully understood. Although the Mercodia sandwich ELISA provides significantly higher specificity of glucagon measurement than does conventional competitive RIA, it cannot provide accurate plasma glucagon values in the presence of elevated cross-reacting plasma glicentin. This occurs in patients post-pancreatectomy or bariatric surgery and in around 30% of outpatients suspected for glucose intolerance who have not had surgery. Thus, our newly developed sandwich ELISA with higher specificity and higher sensitivity than the Mercodia sandwich ELISA is needed for accurate measurements of plasma glucagon in diabetic patients. It is expected that the new sandwich ELISA will contribute to personalized medicine for diabetes by its use in clinical tests to accurately diagnose the conditions of diabetic patients in order to design better individual treatment strategies. Meanwhile, clinical trials are being conducted worldwide to apply glucagon/GLP-1 receptor dual agonists and glucagon/GLP-1/GIP receptor triagonists to the treatment of obesity, fatty liver, and diabetes. Most clinical trials have shown that both types of drugs have stronger effects on weight reduction, improving fatty liver, and glucose tolerance than do the single GLP-1 receptor agonists. Glucagon is expected to be used as a new diagnostic marker and in a new therapeutic strategy based on a true understanding of its physiological and pharmacological functions.

Establishment and application of PDCoV antigen-specific DAS-ELISA detection method.

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.

A species-independent indirect-ELISA for detection of antibodies to the non-structural protein 2B of foot-and-mouth disease virus.

Diagnostic assays that are able to detect foot-and-mouth disease (FMD) virus infection in the vaccinated population are essential tools in the progressive control pathway for the FMD. However, testing of serum samples using a single diagnostic assay may not completely substantiate freedom from the virus infection. Therefore, viral non-structural proteins (NSPs)-based various serological assays have been developed for the detection of FMD infection. Nevertheless, the NSPs-based ELISAs have been developed in the indirect-ELISA format, thereby necessitating the use of species-specific conjugated secondary-antibodies for the detection of anti-NSP antibodies in various FMD-susceptible species. Therefore, this study presents a novel recombinant 2B-NSP-based indirect ELISA, employing HRP-conjugated protein-A/G detection system which can detect anti-NSPs antibodies from multiple FMD-susceptible species in a single ELISA platform. Recombinant 2B (r2B) protein was expressed as His-SUMO tagged protein in the E. Coli cells and purified using NI-NTA affinity column chromatography. Using the r2B protein and HRP-conjugated protein A/G, an indirect ELISA was developed and validated for the detection of anti-2B antibodies in serum samples collected from multiple FMD-susceptible animal species with known FMD status. Further, a resampling based statistical technique has been reported for determination of optimal cut-off value for the diagnostic assay. Through this technique, the optimal cut-off of 44 percentage of positivity value was determined for the assay. At this optimal cut-off value, the developed diagnostic assay provided diagnostic sensitivity, specificity, and accuracy, positive and negative predictive values (PPV and NPV) of 92.35 %, 98.41 %, 95.21 %, 98.58 %, and 91.67 %, respectively. The assay was validated further by analyzing random serum samples collected across multi-locations in India. The assay can be used as a single platform for testing serum samples from different species of FMDV-susceptible animals and will be useful for NSP-based serosurveillance of FMDV.

Detection of EHDV Antibodies Using Enzyme-Linked Immunosorbent Assay (ELISA).

Enzyme-linked immunosorbent assay (ELISA) is a relatively inexpensive, rapid, and high-throughput diagnostic tool to detect antibodies raised against epizootic hemorrhagic disease virus (EHDV) in ruminant serum. While the presence of EHDV antibodies only confirms prior exposure to the virus, it does not conclusively determine infection status. The c-ELISA can be used in conjunction with other diagnostic tests (e.g., real-time PCR) to reinforce diagnosis of infection or as a surveillance tool to support disease control. The EHDV competition ELISA (c-ELISA) described here is a commercial diagnostic assay, recommended by the World Organisation for Animal Health (WOAH), that detects ruminant antibodies against the highly conserved EHDV structural protein, VP7.

Practical Considerations for the Use of the Rapid AcuStar® ADAMTS13 Activity Assay in the Diagnosis of Acute Thrombotic Thrombocytopenic Purpura (TTP).

Introduction: Conventional practice in the management of acute TTP entails empirical treatment of suspected cases whilst awaiting confirmatory ADAMTS13 deficiency testing. Rapid ADAMTS13 assays offer increased accessibility and rapid diagnostics. The new automated HemosIL AcuStar® ADAMTS13 assay has seen increasing use among UK TTP Specialist Centres alongside the traditional ELISA method to confirm severe ADAMTS13 deficiency. Methods: A multi-centre retrospective case-control study was performed to review patients demonstrating discrepant ADAMTS13 activity results measured using rapid (AcuStar®) and ELISA assays in parallel from September 2019 to December 2021. Cases were compared with a cohort of suspected TTP patients exhibiting no difference in assay results and in relation to their presenting characteristics and pre-test probability of a diagnosis of TTP. Results: Where the clinical index of suspicion for TTP was high at presentation, acute TTP was confirmed using the AcuStar® assay < 0.2 IU/dL and subsequently < 10 IU/dL by ELISA with zero incidence of discrepancy. For patients with low clinical suspicion of acute TTP, a discrepancy between the AcuStar® and ELISA assay results was observed in 2% of cases; 5-10 IU/dL in AcuStar®, confirmed as >20 IU/dL by ELISA. A concurrent cancer diagnosis or sepsis was observed in 40% of discrepant cases. Conclusions: Where acute TTP is strongly suspected, there is a good correlation between the rapid AcuStar® ADAMTS13 assay and the conventional ELISA assay. Where the clinical suspicion of acute TTP is low, caution should be exercised in the interpretation of the ADAMTS13 activity using the AcuStar® assay. Accurate interpretation requires robust ADAMTS13 testing algorithms to be incorporated into diagnostic pathways.

Propagating and banking genetically diverse human sapovirus strains using a human duodenal cell line: investigating antigenic differences between strains.

There are four genogroups and 18 genotypes of human sapoviruses (HuSaVs) responsible for acute gastroenteritis. To comprehend their antigenic and virological differences, it is crucial to obtain viral stocks of the different strains. Previously, we utilized the human duodenum-derived cell line HuTu80, and glycocholate, a conjugated bile acid, to replicate and propagate GI.1, GI.2, and GII.3 HuSaVs (H. Takagi et al., Proc Natl Acad Sci U S A 117:32078-32085, 2020, https://10.1073/pnas.2007310117). First, we investigated the impact of HuTu80 passage number on HuSaV propagation. Second, we demonstrated that taurocholate improved the initial replication success rate and viral RNA levels in fecal specimens relative to glycocholate. By propagating 15 HuSaV genotypes (GI.1-7, GII.1-5, -8, and GV.1-2) and accomplishing preparation of viral stocks containing 1.0 × 109 to 3.4 × 1011 viral genomic copies/mL, we found that all strains required bile acids for replication, with GII.4 showing strict requirements for taurocholate. The deduced VP1 sequences of the viruses during the scale-up of serial passaged virus cultures were either identical or differed by only two amino acids from the original sequences in feces. In addition, we purified virions from nine strains of different genotypes and used them as immunogens for antiserum production. Enzyme-linked immunosorbent assays (ELISAs) using rabbit and guinea pig antisera for each of the 15 strains of different genotypes revealed distinct antigenicity among the propagating viruses across genogroups and differences between genotypes. Acquisition of biobanked viral resources and determination of key culture conditions will be valuable to gain insights into the common mechanisms of HuSaV infection. IMPORTANCE: The control of human sapovirus, which causes acute gastroenteritis in individuals of all ages, is challenging because of its association with outbreaks similar to those caused by human norovirus. The establishment of conditions for efficient viral propagation of various viral strains is essential for understanding the infection mechanism and identifying potential control methods. In this study, two critical factors for human sapovirus propagation in a conventional human duodenal cell line were identified, and 15 strains of different genotypes that differed at the genetic and antigenic levels were isolated and used to prepare virus stocks. The preparation of virus stocks has not been successful for noroviruses, which belong to the same family as sapoviruses. Securing virus stocks of multiple human sapovirus strains represents a significant advance toward establishing a reliable experimental system that does not depend on limited virus-positive fecal material.

Assessing the diagnostic accuracy of serological tests for hepatitis delta virus diagnosis: a systematic review and meta-analysis.

Hepatitis Delta Virus (HDV), a satellite virus of Hepatitis B virus, exacerbates liver damage in affected individuals. Screening for HDV antibodies in HBsAg positive patients is recommended, but the diagnostic accuracy of serological tests remains uncertain. This review aimed to assess the diagnostic accuracy of serological tests for HDV. We searched PubMed, Web of Science, Cochrane Central Register of Controlled Trials, Scopus etc. for relevant studies. Studies measuring the sensitivity and specificity of serological HDV tests against PCR as a reference standard were included. Pooled sensitivity and specificity for each test method and sero-marker were calculated. The review included six studies with 11 study arms, evaluating ARCHITECT immunoassay, EIA, ELISA, QMAC, RIA, and Western Blot test methods targeting Anti-HDV IgG, Total anti-HDV and Anti-HDV IgM. Sensitivities for Anti-HDV IgG, Total Anti-HDV and Anti-HDV IgM, tests were 97.4%, 51.9%, and 62.0%, respectively, with specificities of 95.3%, 80.0%, and 85.0%. Our findings, with its limited number of studies, suggest that HDV serological tests, particularly those identifying Anti IgG exhibit high accuracy and can serve as effective screening tools for HDV.