Panel Of Sera Research Articles

Urine and serum metabolomic analysis of endometrial cancer diagnosis and classification based on ultra-performance liquid chromatography mass spectrometry.

This study aimed to reveal the urinary and serum metabolic pattern of endometrial cancer (EC) and establish diagnostic models to identify EC from controls, high-risk from low-risk EC, and type II from type I EC. This study included 146 EC patients (comprising 79 low-risk and 67 high-risk patients, including 124 type I and 22 type II) and 59 controls. The serum and urine samples were analyzed using ultraperformance liquid chromatography mass spectrometry. Analysis was used to elucidate the distinct metabolites and altered metabolic pathways. Receiver operating characteristic (ROC) analyses were employed to discover and validate the potential biomarker models. Serum and urine metabolomes displayed significant differences between EC and controls, with metabolites related to amino acid and nicotinamide metabolisms. The serum and urine panels distinguished these two groups with Area Under the Curve (AUC) of 0.821 and 0.902, respectively. The panel consisting of serum and urine metabolites demonstrated the best predictive ability (AUC = 0.953 and 0.976 in discovering and validation group). In comparing high-risk and low risk EC, differential metabolites were enriched in purine and glutamine metabolism. The AUC values for serum and urine panels were 0.818, and 0.843, respectively. The combined panel exhibited better predictive accuracy (0.881 in discovering group and 0.936 in external validation). In the comparison between type I and type II group, altered folic acid metabolism was identified. The serum, urine and combined panels discriminated these two groups with the AUC of 0.829, 0.913 and 0.922, respectively. The combined urine and serum metabolome effectively revealed the metabolic patterns in EC patients, offering valuable diagnostic models for EC diagnosis and classification.

Autoantibodies, antigen-autoantibody complexes and antigens complement CA125 for early detection of ovarian cancer

BackgroundMultiple antigens, autoantibodies (AAb), and antigen-autoantibody (Ag-AAb) complexes were compared for their ability to complement CA125 for early detection of ovarian cancer.MethodsTwenty six biomarkers were measured in a single panel of sera from women with early stage (I-II) ovarian cancers (n = 64), late stage (III-IV) ovarian cancers (186), benign pelvic masses (200) and from healthy controls (502), and then split randomly (50:50) into a training set to identify the most promising classifier and a validation set to compare its performance to CA125 alone.ResultsEight biomarkers detected ≥ 8% of early stage cases at 98% specificity. A four-biomarker panel including CA125, HE4, HE4 Ag-AAb and osteopontin detected 75% of early stage cancers in the validation set from among healthy controls compared to 62% with CA125 alone (p = 0.003) at 98% specificity. The same panel increased sensitivity for distinguishing early-stage ovarian cancers from benign pelvic masses by 25% (p = 0.0004) at 95% specificity. From 21 autoantibody candidates, 3 AAb (anti-p53, anti-CTAG1 and annt-Il-8) detected 22% of early stage ovarian cancers, potentially lengthening lead time prior to diagnosis.ConclusionA four biomarker panel achieved greater sensitivity at the same specificity for early detection of ovarian cancer than CA125 alone.

A serum panel of three microRNAs may serve as possible biomarkers for kidney renal clear cell carcinoma

BackgroundAlthough non-invasive radiological techniques are widely applied in kidney renal clear cell carcinoma (KIRC) diagnosis, more than 50% of KIRCs are detected incidentally during the diagnostic procedures to identify renal cell carcinoma (RCC). Thus, sensitive and accurate KIRC diagnostic methods are required. Therefore, in this study, we aimed to identify KIRC-associated microRNAs (miRNAs).MethodsThis three-phase study included 224 participants (112 each of patients with KIRC and healthy controls (NCs)). RT-qPCR was used to evaluate miRNA expression in KIRC and NC samples. Receiver operating characteristic (ROC) curves and the area under the ROC curve (AUC) were used to predict the usefulness of serum miRNAs in KIRC diagnosis. In addition, we performed survival and bioinformatics analyses.ResultsWe found that miR-1-3p, miR-129-5p, miR-146b-5p, miR-187-3p, and miR-200a-3p were significantly differentially expressed in patients with KIRC. A panel consisting of three miRNAs (miR-1-3p, miR-129-5p, and miR-146b-5p) had an AUC of 0.895, ranging from 0.848 to 0.942. In addition, using the GEPIA database, we found that the miRNAs were associated with CREB5. According to the survival analysis, miR-146b-5p overexpression was indicative of a poorer prognosis in patients with KIRC.ConclusionsThe identified three-miRNA panel could serve as a non-invasive indicator for KIRC and CREB5 as a potential target gene for KIRC treatment.

Development and evaluation of two rapid lateral flow assays for on-site detection of African swine fever virus.

African swine fever (ASF) is a lethal and highly contagious transboundary animal disease with the potential for rapid international spread. In the absence of a widely available and definitively proven vaccine, rapid and early detection is critical for ASF control. The quick and user-friendly lateral flow assay (LFA) can easily be performed by following simple instructions and is ideal for on-site use. This study describes the development and validation of two LFAs for the rapid detection of ASF virus (ASFV) in pig serum. The highly immunogenic antigens (p30 and p72) of ASFV Georgia 2007/1 (genotype II) were expressed in plants (Nicotiana benthamiana) and were used to immunize BALB/c mice to generate specific monoclonal antibodies (mAbs) against the p30 and p72 proteins. mAbs with the strongest binding ability to each protein were used to develop p30_LFA and p72_LFA for detecting the respective ASFV antigens. The assays were first evaluated using a spike-in test by adding the purified p30 or p72 protein to a serum sample from a healthy donor pig. Further validation of the tests was carried out using serum samples derived from experimentally infected domestic pigs, field domestic pigs, and feral pigs, and the results were compared with those of ASFV real-time PCR. p30_LFA and p72_LFA showed no cross-reaction with common swine viruses and delivered visual results in 15 min. When testing with serially diluted proteins in swine serum samples, analytical sensitivity reached 10 ng/test for p30_LFA and 20 ng/test for p72_LFA. Using real-time PCR as a reference, both assays demonstrated high sensitivity (84.21% for p30_LFA and 100% for p72_LFA) with experimentally ASFV-infected pig sera. Specificity was 100% for both LFAs using a panel of PBS-inoculated domestic pig sera. Excellent specificity was also shown for field domestic pig sera (100% for p30_LFA and 93% for p72_LFA) and feral pig sera (100% for both LFAs). The results obtained in this study suggest that p30_LFA and p72_LFA hold promise as rapid, sensitive, user-friendly, and field-deployable tools for ASF control, particularly in settings with limited laboratory resources.

Antibody reactions of horses against various domains of the EHV-1 receptor-binding protein gD1.

Equid alphaherpesviruses 1 (EHV-1) and 4 (EHV-4) are closely related and both endemic in horses worldwide. Both viruses replicate in the upper respiratory tract, but EHV-1 may additionally lead to abortion and equine herpesvirus myeloencephalopathy (EHM). We focused on antibody responses in horses against the receptor-binding glycoprotein D of EHV-1 (gD1), which shares a 77% amino acid identity with its counterpart in EHV-4 (gD4). Both antigens give rise to cross-reacting antibodies, including neutralizing antibodies. However, immunity against EHV-4 is not considered protective against EHM. While a diagnostic ELISA to discriminate between EHV-1 and EHV-4 infections is available based on type-specific fragments of glycoprotein G (gG1 and gG4, respectively), the type-specific antibody reaction against gD1 has not yet been sufficiently addressed. Starting from the N-terminus of gD1, we developed luciferase immunoprecipitation system (LIPS) assays, using gD1-fragments of increasing size as antigens, i.e. gD1_83 (comprising the first 83 amino acids), gD1_160, gD1_180, and gD1_402 (the full-length molecule). These assays were then used to analyse panels of horse sera from Switzerland (n = 60) and Iceland (n = 50), the latter of which is considered EHV-1 free. We detected only one true negative horse serum from Iceland, whereas all other sera in both panels were seropositive for both gG4 (ELISA) and gD1 (LIPS against gD1_402). In contrast, seropositivity against gG1 was rather rare (35% Swiss sera; 14% Icelandic sera). Therefore, a high percentage of antibodies against gD1 could be attributed to cross-reaction and due to EHV-4 infections. In contrast, the gD1_83 fragment was able to identify sera with type-specific antibodies against gD1. Interestingly, those sera stemmed almost exclusively from vaccinated horses. Although it is uncertain that the N-terminal epitopes of gD1 addressed in this communication are linked to better protection, we suggest that in future vaccine developments, type-common antigens should be avoided, while a broad range of type-specific antigens should be favored.

Harmonization of Lipoprotein(a) Immunoassays Using A Serum Panel Value Assigned with The IFCC-Endorsed Mass Spectrometry-Based Reference Measurement Procedure as A First Step Towards Apolipoprotein Standardization.

Lipoprotein (a) [Lp(a)] is a well-established risk factor for cardiovascular disease independent of low-density lipoprotein-cholesterol (LDL-C). The Lp(a) concentrations were inconsistent between the immunoassays. This study aimed to investigate whether harmonization of Lp(a) measurements can be achieved using a serum panel value assigned with the IFCC-endorsed mass spectrometry-based reference measurement procedure (IFCC-MS-RMP). We measured the Lp(a) concentrations using five Lp(a) immunoassays in 40 panel sera provided by the Centers for Disease Control and Prevention (CDC), and 500 Japanese subjects enrolled in the Bunkyo Health Study. Of the five immunoassays, only the Roche Lp(a) assay was traceable to the WHO-IFCC reference material SRM2B. Lp(a) concentrations in CDC samples were also determined by IFCC-MS-RMP, provisionally calibrated to SRM2B. Lp(a) concentrations were expressed in mass units (mg/dL) for most reagents, but in SI units (nmol/L) for Roche's reagent and IFCC-MS-RMP. In the CDC panel sera, all immunoassays, including Roche's reagent, showed good correlations with IFCC-MS-RMP. In the Bunkyo Health Study samples, all immunoassays showed good correlations with Roche's reagent (rs, 0.986-0.998) although the slopes of the regression lines ranged from 0.292 to 0.579. After recalibration with the CDC's panel sera, Lp(a) results of Bunkyo Health Study samples were converted to the equivalent values determined by the IFCC-MS-RMP, thus resulting in a marked reduction in the intermethod CV among the assays. We achieved harmonization of Lp(a) measurements with five immunoassays using a serum panel value assigned with the IFCC-MS-RMP.

A species-independent lateral flow microarray immunoassay to detect WNV and USUV NS1-specific antibodies in serum

Arboviruses such as West Nile Virus (WNV) and Usutu Virus (USUV) are emerging pathogens that circulate between mosquitoes and birds, occasionally spilling over into humans and horses. Current serological screening methods require access to a well-equipped laboratory and are not currently available for on-site analysis. As a proof of concept, we propose here a species-independent lateral flow microarray immunoassay (LMIA) able to quickly detect and distinguish between WNV Non-Structural 1 (NS1) and USUV NS1-specific antibodies. A double antigen approach was used to test sera collected from humans, horses, European jackdaws (Corvus monedula), and common blackbirds (Turdus merula). Optimization of the concentration of capture antigen spotted on the LMIA membrane and the amount of detection antigen conjugated to detector particles indicated that maximizing both parameters increased assay sensitivity. Upon screening of a larger serum panel, the optimized LMIA showed significantly higher spot intensity for a homologous binding event. Using a Receiver Operating Characteristics (ROC) curve, WNV NS1 LMIA results in humans, horses, and C. monedula showed good correlation when compared to “gold standard” WNV FRNT90. The most optimal derived sensitivity and specificity of the WNV NS1 LMIA relative to corresponding WNV FRNT90-confirmed sera were determined to be 96% and 86%, respectively. While further optimization is required, this study demonstrates the feasibility of developing a species-independent LMIA for on-site analysis of WNV, USUV, and other arboviruses. Such a tool would be useful for the on-site screening and monitoring of relevant species in more remote or low-income regions.

Cross-Reactivity of Human, Wild Boar, and Farm Animal Sera from Pre- and Post-Pandemic Periods with Alpha- and Βeta-Coronaviruses (CoV), including SARS-CoV-2.

Panels of pre- and post-pandemic farm animals, wild boar and human sera, including human sera able to neutralize SARS-CoV-2 in vitro, were tested in serological tests to determine their cross-reactivity with β- and α-CoV originating from farm animals. Sera were tested in neutralization assays with high ascending concentrations (up to 1 × 104 TCID50 units/well) of β-CoV Bovine coronavirus (BCV), SARS-CoV-2, and porcine α-CoV-transmissible gastroenteritis virus (TGEV). In addition, sera were tested for immunostaining of cells infected with β-CoV porcine hemagglutinating encephalomyelitis (PHEV). Testing revealed a significantly higher percentage of BCV neutralization (78%) for sera of humans that had experienced a SARS-CoV-2 infection (SARS-CoV-2 convalescent sera) than was observed for human pre-pandemic sera (37%). Also, 46% of these human SARS-CoV-2 convalescent sera neutralized the highest concentration of BCV (5 × 103 TCID50/well) tested, whereas only 9.6% of the pre-pandemic sera did. Largely similar percentages were observed for staining of PHEV-infected cells by these panels of human sera. Furthermore, post-pandemic sera collected from wild boars living near a densely populated area in The Netherlands also showed a higher percentage (43%) and stronger BCV neutralization than was observed for pre-pandemic sera from this area (21%) and for pre- (28%) and post-pandemic (20%) sera collected from wild boars living in a nature reserve park with limited access for the public. High percentages of BCV neutralization were observed for pre- and post-pandemic sera of cows (100%), pigs (up to 45%), sheep (36%) and rabbits (60%). However, this cross-neutralization was restricted to sera collected from specific herds or farms. TGEV was neutralized only by sera of pigs (68%) and a few wild boar sera (4.6%). None of the BCV and PHEV cross-reacting human pre-pandemic, wild boar and farm animal sera effectively neutralized SARS-CoV-2 in vitro. Preexisting antibodies in human sera effectively neutralized the animal β-CoV BCV in vitro. This cross-neutralization was boosted after humans had experienced a SARS-CoV-2 infection, indicating that SARS-CoV-2 activated a "memory" antibody response against structurally related epitopes expressed on the surface of a broad range of heterologous CoV, including β-CoV isolated from farm animals. Further research is needed to elucidate if a symptomless infection or environmental exposure to SARS-CoV-2 or another β-CoV also triggers such a "memory" antibody response in wild boars and other free-living animals.

Performance of a Differentiation of Infected from Vaccinated Animals (DIVA) Classical Swine Fever Virus (CSFV) Serum and Oral Fluid Erns Antibody AlphaLISA Assay.

Classical swine fever virus (CSFV) is an OIE-listed disease that requires effective surveillance tools for its detection and control. The aim of this study was to develop and evaluate the diagnostic performance of a novel CSFV Erns IgG AlphaLISA for both serum and oral fluid specimens that would likewise be compatible with the use of CSFV E2 DIVA vaccines. Test performance was evaluated using a panel of well-characterized serum (n = 760) and individual (n = 528) or pen-based (n = 30) oral fluid samples from four groups of animals: (1) negative controls (n = 60 pigs); (2) inoculated with ALD strain wild-type CSFV (n = 30 pigs); (3) vaccinated with LOM strain live CSFV vaccine (n = 30 pigs); and (4) vaccinated with live CSFV marker vaccine on commercial farms (n = 120 pigs). At a cutoff of S/P ≥ 0.7, the aggregate estimated diagnostic sensitivities and specificities of the assay were, respectively, 97.4% (95% CI 95.9%, 98.3%) and 100% for serum and 95.4% (95% CI 92.9%, 97.0%) and 100% for oral fluid. The Erns IgG antibody AlphaLISA combined DIVA capability with solid diagnostic performance, rapid turnaround, ease of use, and compatibility with both serum and oral fluid specimens.

Effect of serum panel reactive antibodies on allogeneic hematopoietic stem cell transplantation in pediatric thalassemia patients: A single-center experience.

The aim of this study was to assess the impact of serum panel reactive antibodies (PRA) on the outcomes of allogeneic hematopoietic stem cell transplantation (HSCT) in pediatric thalassemia patients. A total of 73 pediatric patients with thalassemia were included in this single-center study. Pre-transplant PRA levels were evaluated, and the patients were divided into two groups: PRA-negative (group 1; n = 44) and PRA-positive (group 2; n = 29). Patient characteristics, including age, gender, donor type, stem cell source, and HLA compatibility, were analyzed. Transplant outcomes, including engraftment, transfusion requirements, and transplant-related complications, were compared between the two groups. Further subgroup analysis was performed based on MFI values. At the time of transplantation, patients in group 1 were younger than those in group 2 (p = .008). The number of fully matched donors within the family (MSD and MFD) was significantly higher in group 1 (p = .049). Additionally, Rh blood group incompatibility was higher in group 2 (p = .03). There was no statistically significant difference in the engraftment days of neutrophils, platelets, and erythrocytes between the two groups. The frequency of poor graft function and graft failure was higher in the group 2, but there was no statistically significant difference. Post-transplant transfusion requirements for platelets and red blood cells were significantly higher in the group 2 (p < .001). Transplant-related complications such as VOD, PRES, and aGvHD were more common in the group 2, but no statistical significance was detected. Serum PRA in pediatric thalassemia patients may impact the outcomes of HSCT. PRA-positive patients had higher rates of blood product transfusion requirements. Although poor graft function, graft failure, and post-transplant complications were more common in the group 2, statistical significance was not observed. Identifying patients with high PRA levels can assist in optimizing transplant strategies and post-transplant care, leading to improved outcomes for the patients.

Limited value of current and new in silico predicted oocyst-specific proteins of Toxoplasma gondii for source-attributing serology

Toxoplasma gondii is a zoonotic parasite infecting all warm-blooded animals, including humans. The contribution of environmental contamination by T. gondii oocysts to infections is understudied. The aim of the current work was to explore T. gondii serology as a means of attributing the source of infection using a robust stepwise approach. We identified in silico thirty-two promising oocyst-specific antigens from T. gondii ´omics data, recombinantly expressed and purified them and validated whether serology based on these proteins could discriminate oocyst- from tissue cyst-driven experimental infections. For this, three well-characterized serum panels, sampled from 0 to 6 weeks post-infection, from pigs and sheep experimentally infected with T. gondii oocysts or tissue cysts, were used. Candidate proteins were initially screened by Western blot with sera from pigs or sheep, infected for different times, either with oocysts or tissue cysts, as well as non-infected animals. Only the recombinant proteins TgCCp5A and TgSR1 provoked seroconversion upon infection and appeared to discriminate between oocyst- and tissue cyst-driven infections with pig sera. They were subsequently used to develop an enzyme-linked immunosorbent assay test for pigs. Based on this assay and Western blot analyses, a lack of stage specificity and low antigenicity was observed with all pig sera. The same was true for proteins TgERP, TgSporoSAG, TgOWP1 and TgOWP8, previously described as source-attributing antigens, when analyzed using the whole panels of sera. We conclude that there is currently no antigen that allows the discrimination of T. gondii infections acquired from either oocysts or tissue cysts by serological tests. This work provides robust new knowledge that can inform further research and development toward source-attributing T. gondii serology.

Study on identification of a three-microRNA panel in serum for diagnosing neonatal early onset sepsis

Background Comparing with other diseases, early onset sepsis (EOS) is a global health concern in neonatal period for its high morbidity and mortality rates. In recent years, many studies have contributed to the figure out the expression patterns of circulating micro-RNAs (miRNAs) in different diseases and progressions, which could function as diagnostic biomarkers for EOS. The purpose of this study was to analyze the expression patterns of selected miRNAs and evaluate their diagnostic value for early detection and treatment. Methods This was a prospective cross-sectional study conducted from 1 July 2021 to 30 June 2022. We collected surplus peripheral blood and demographic statistics of septic neonates and non-infected neonates during the first 24 h after delivery and obtained 11 candidate miRNAs by literature screening. First, we extracted the candidate miRNAs from the serum of selected neonates and analyzed their expression levels, and then the receiver operating characteristic (ROC) curve was used to select the differentially expressed miRNAs. We analyzed their sensitivity and specificity and obtained the best diagnostic panel. Finally, with the help of differentially expressed miRNAs, we performed gene ontology (GO) enrichment and protein–protein interaction (PPI) analyses by their target genes. Results In patients with EOS, three miRNAs (mir-223-3p, mir-15a-5p, and mir-17-5p) in serum were significantly downregulated, and mir-146a-5p, mir-1-3p, and mir-16-5p were upregulated. The diagnostic value of these miRNAs (miR-15a-5p, AUC = 0.67; miR-223-3p, AUC = 0.72; miR-16-5p, AUC = 0.68; miR‐17‐5p, AUC = 0.70; miR-1-3p, AUC = 0.69; miR‐146a‐5p, AUC = 0.72) was moderate, and the diagnostic panel constructed by miR-15a-5p, miR-223-3p, and miR-16-5p possessed a comparatively higher diagnostic value (AUC = 0.85, sensitivity: 74.6%, specificity: 86%), indicating that their combined application may be a promising biomarker for clinical diagnosis of EOS. According to GO enrichment analysis, most proteins encoded by target genes were located in the cytosol as for cellular component (CC), for molecular function (MF), most proteins acted as regulators in protein binding, and for biological process (BP). Most genes function in positive or negative regulation of transcription from RNA polymerase II promoter, and the top 10 hub genes were CDKN1A, YAP1, CCNE1, CCND1, CKK6, ERBB4, CHEK1, DICER1, VEGFA, and APP by rank degree after PPI construction. Conclusions The three-miRNA panels (miR-15a-5p, miR-223-3p, and miR-16-5p) may be a novel noninvasive biological marker for EOS screening.

The value of a three-microRNA panel in serum for prostate cancer screening.

Globally, prostate cancer is the second most common malignancy in males. Serum microRNAs (miRNAs) may function as non-invasive and innovative biomarkers for various cancers. Our study aimed to determine potential miRNAs for prostate cancer screening. A three-stage study was accomplished to ascertain crucial miRNAs as markers. In the screening stage, we searched PubMed for aberrantly expressed miRNAs relevant to prostate cancer and selected them as candidate miRNAs. In training and validation stages, with serum specimens from 112 prostate cancer patients and 112 healthy controls, expressions of candidate miRNAs were identified through quantitative reverse transcription-polymerase chain reaction. The diagnostic capabilities of miRNAs were determined by receiver operating characteristic curves. Bioinformatic analysis was utilized to explore the function of the critical miRNAs. Expression of six serum miRNAs (miR-34b-3p, miR-556-5p, miR-200c-3p, miR-361-5p, miR-369-3p, miR-485-3p) were significantly altered in prostate cancer patients contrasted with healthy controls. The optimal combination of critical miRNAs is a three-miRNA panel (miR-34b-3p, miR-200c-3p, and miR-361-5p) with good diagnostic capability. FLRT2, KIAA1755, LDB3, and NTRK3 were identified as the potential genes targeted by the three-miRNA panel. The three-miRNA panel may perform as an innovative and promising serum marker for prostate cancer screening.

A comparative study of serological tests used in the diagnosis of Toxoplasma gondii infection in small ruminants evidenced the importance of cross-reactions for harmonizing diagnostic performance

Toxoplasma gondii is a major foodborne zoonotic pathogen that can be transmitted through the consumption of raw or undercooked meat of small ruminants, among others. Serology has been suggested as an epidemiological indicator and several tests are available nowadays. However, there is no comparative study with the most used ones. Therefore, the objective of this study was to develop and validate two in-house tests (Western blot -TgSALUVET WB- and ELISA -TgSALUVET ELISA 2.0-) and perform a comparative study including such tests and four commercial ELISA kits (IDScreen®, PrioCHECK®, Pigtype® and IDEXX). First, a specific pattern of recognition of immunodominant antigens by TgSALUVET WB was determined with serum panels of noninfected sheep and sheep infected with T. gondii or Neospora caninum. Next, TgSALUVET WB was used as a reference to preliminary validate TgSALUVET ELISA 2.0 using sera from sheep and goats naturally infected with T. gondii. Then, the abovementioned sheep serum panels were analyzed by all tests and subjected to TG-ROC analyses and agreement tests, and cross-reactivity with the anti-N. caninum IgGs was studied.All the techniques were accurate enough for the cutoff values initially suggested with all serum panels (Se and Sp ≥ 94%), except for PrioCHECK®, which showed 83% Sp. However, a cutoff readjustment improved their diagnostic performance. Additionally, cross-reactions between anti-N. caninum antibodies and T. gondii antigens were detected with all tests. Thus, a second cutoff readjustment was carried out and the use of both readjusted cutoff values is recommended to obtain comparable data and avoid false-positive results.

Antigenic mapping of the hemagglutinin of the H9 subtype influenza A viruses using sera from Japanese quail (Coturnix c. japonica).

Determining the relevant amino acids involved in antigenic drift on the surface protein hemagglutinin (HA) is critical to understand influenza virus evolution and efficient assessment of vaccine strains relative to current circulating strains. We used antigenic cartography to generate an antigenic map of the H9 hemagglutinin (HA) using sera produced in one of the most relevant minor poultry species, Japanese quail. Key antigenic positions were identified and tested to confirm their impact on the antigenic profile. This work provides a better understanding of the antigenic diversity of the H9 HA as it relates to reactivity to quail sera and will facilitate a rational approach for selecting more efficacious vaccines against poultry-origin H9 influenza viruses in minor poultry species.

Shooting Your Own Foot: A Case Report on Autoimmune Encephalitis Following a Viral Trigger

Aims: The aim of this case report is to highlight the frequently overlooked association between Anti-NMDA Receptor Encephalitis and a preceding Herpes Simplex Viral Encephalitis trigger, the clinical settings under which to suspect this autoimmune disease, the need for appropriate workup to clinch the diagnosis and the necessity of speedy initiation of immunotherapy, in a disease where timely treatment is paramount.&#x0D; Presentation of Case: A middle aged woman presented with a history of fever and syncope, followed by slurring of speech, quadriparesis, seizures and altered sensorium. She was diagnosed with HSV-1 Encephalitis on basis of CSF studies and MRI brain. Despite a timely 28-day course of intravenous acyclovir therapy, she showed incomplete clinical recovery. A serum and CSF autoantibody panel was performed, which clinched a diagnosis of Anti-NMDA Receptor Encephalitis. A course of steroids and IVIg was given and the patient was discharged after showing clinical improvement.&#x0D; Discussion: Anti-NMDA Receptor Encephalitis is an autoimmune encephalitis characterized by complex neuropsychiatric features and presence of IgG antibodies against NR1 subunit of NMDA receptor detectable in CSF and serum. It is associated with various malignancies, chiefly ovarian teratomas, and with HSV-1 viral encephalitis. Diagnosis involves autoantibody detection in CSF or serum and first line treatment is with steroids, IVIg or plasma exchange, and tumour resection.&#x0D; Conclusion: Prompt diagnosis of Anti-NMDA receptor encephalitis is crucial as it enables treatment with timely immunosuppression and tumour resection. This disease must be suspected in adults or children presenting with subacute onset of neuropsychiatric symptoms, with CSF lymphocytic pleocytosis and presence of autoantibodies to NMDA receptor in CSF or serum. Early treatment is associated with good outcomes.

A multispecies competitive nanobody-based ELISA for the detection of antibodies against hepatitis E virus

The hepatitis E virus (HEV) is an emergent zoonotic virus causing viral hepatitis worldwide. Clinically, hepatitis E is not easily distinguished from other types of acute viral hepatitis. There is a need for HEV diagnostic assays to detect and prevent interspecies transmission among susceptible populations. Nanobodies (Nbs) are expressed recombinantly in different systems, produced with high yields, and have superior physicochemical properties compared with conventional antibodies (Ab). Several Nbs against ORF2, the capsid protein and main antigen, were selected and produced in E. coli. Nb39 and Nb74 specifically recognized HEV ORF2 (genotypes 3 and 4). A competitive ELISA (cELISA) was developed and validated using a reference panel of human (n = 86) and swine sera (n = 116) tested in comparison with a commercial kit. The optimal cutoff values determined by ROC analysis were 69.16% (human) and 58.76% (swine); the sensitivity and specificity were high: 97.4% (95% CI 86.5–99.5%) and 95.8% (95% CI 86.0–98.8%) for human vs. 100% (95% CI 93.5–100%) and 98.3% (95% CI 91.0–99.7%) for swine. Further, the cELISA detected total anti-HEV antibodies in wild boar, deer, and mice. To our knowledge, this is the first report of production of Nbs against HEV-3 ORF2 for diagnostic purposes.

Bile salts enhance the susceptibility of the peach allergenic lipid transfer protein, Pru p 3, to in vitro gastrointestinal proteolysis

Sensitisation to the lipid transfer protein Pru p 3 is associated with severe allergic reactions to peach, the proteins stability being thought to play a role in its allergenicity. Lipid binding increases susceptibility of Pru p 3 to digestion and so the impact of bile salts on the in vitro gastrointestinal digestibility of Pru p 3 was investigated and digestion products mapped by SDS-PAGE and mass spectrometry. Bile salts enhanced the digestibility of Pru p 3 resulting in an ensemble of around 100 peptides spanning the protein’s sequence which were linked by disulphide bonds into structures of ~ 5–6 kDa. IgE binding studies with a serum panel from peach allergic subjects showed digestion reduced, but did not abolish, the IgE reactivity of Pru p 3. These data show the importance of including bile salts in vitro digestion systems and emphasise the need to profile of digestion in a manner that allows identification of immunologically relevant disulphide-linked peptide aggregates.

Optimization of the most widely used serological tests for a harmonized diagnosis of Toxoplasma gondii infection in domestic pigs

The intake of Toxoplasma gondii tissue cysts through raw or undercooked pork meat is one of the main infection sources for humans. Thus, surveillance is recommended to control and prevent infection in domestic pigs. However, the lack of comparative studies hampers the updating of their performance and the comparison of seroprevalence data. Therefore, the aim of this study was to develop and validate three in-house tests and accomplish a comparative analysis of the most widely used serological tests employed in pigs. A panel of sera from pigs experimentally infected with either oocysts or tissue cysts from type II and III isolates (n = 158) was used to develop and validate a tachyzoite-based Western blot assay. Then, this technique was used as a reference to develop and preliminary validate a lyophilized tachyzoite-based enzyme-linked immunosorbent assay and an immunofluorescence antibody test. Next, a comparative study of the three in-house tests and three widely used commercial ELISAs (IDScreen®, PrioCHECK™ and Pigtype®) was accomplished with the abovementioned sera together with an additional serum panel of pigs experimentally infected with oocysts from the type II isolate (n = 44) and a panel of naturally infected pigs (n = 244). The results obtained by the majority of the tests were regarded as reference, and data analyses included TG-ROC calculations and agreement tests. Finally, the kinetics of anti-T. gondii IgGs from experimentally infected pigs was analyzed. Excellent sensitivity (Se) and specificity (Sp) values (≥ 93%) and moderate to near perfect agreement (k = 0.63–0.91) were observed using sera from experimental infections without requiring further readjustment, except for PrioCHECK (100% Se, 73% Sp). However, the Se of IDScreen® (87%) and TgSALUVET WB (71%) and the Sp of PrioCHECK (72%) were slightly or notably reduced when sera from naturally infected animals were analyzed, which also influenced the kappa values (k = 0.30–0.91). Cutoff readjustments increased the Se and Sp values to equal to or above 97% for all tests, except for TgSALUVET WB, which can be used as a reference for initial validation of tests, but it is not recommended for routine diagnosis. Seroconversion was recorded from two weeks post-infection by most of the tests, with significantly higher IgG levels in sera from pigs infected with the T. gondii type III vs. type II isolate. Again, differences regarding the test employed were observed. Differences in the diagnostic performance among tests evidenced the need to harmonize serological techniques to obtain comparable and reliable results.

Screening and validation of novel serum panel of microRNA in stratification of prostate cancer.

Owing to the heterogeneous nature of prostate cancer (PCa) and errors in the characterization of the disease, researchers have been trying to unveil molecular biomarkers like microRNA (miRNA) as diagnostic markers. The purpose of our study is to demonstrate the precision of a panel of miRNAs as biomarkers with diagnostic potential for risk stratification. The present study demonstrates the comparative expression profiles of miRNA-141,-1290,-100, and -335 in both tissue and serum, including Benign Prostate Hyperplasia (BPH) and PCa, with healthy volunteers. Firstly, we demonstrate the expression of all miRNAs in the discovery cohort, including metastasis and benign tissue, and later validate their non-invasive diagnostic potential in BPHand PCa with healthy volunteers. MiRNA was isolated from tissue and serum to be quantified by RT-PCR and analyzed for biomarker potential by receiver operating characteristic (ROC) curve analysis, followed by targetome analysis of each miRNA. Among the non-invasive miRNA assessed, it was seen that miRNA 141 (P=0.0003) and miRNA 1290 (P<0.0001) are oncogenic with significantly higher expression, while miRNA 100 (P=0.0002) and miRNA 335 are tumor suppressor, in PCaas compared to controls. While for BPH, miRNA 141 (P=0.003) and miRNA 335 (P=0.0002) were found to be significantly oncogenic and tumor suppressors, respectively. The analysis of the ROC curve of panel miRNAs (miRNA-141,-1290, and -100) portrayed a significant area under the curve with greater sensitivity and specificity. Moreover, in-silico prediction of their respective targetomes represents their extensive involvement in PCa progression and various other cascades that aid in PCa networks. To the best of our knowledge, we are going to report for the first time this panel of miRNA that can be used to accurately and efficiently diagnose BPH and PCa patients from healthy males.