Serotype Antigens Research Articles

Serotype-Specific Dengue Virus IgG Assay Using EDIII-Based Recombinant Proteins and Its Application in an Endemic Population in Northeast Brazil.

Dengue is the most prevalent arboviral disease globally, with Brazil currently experiencing a significant rise in cases. Dengue virus (DENV) typically co-circulates with other clinically and antigenically similar flaviviruses, such as Zika virus (ZIKV). The clinical diagnosis is difficult and accurate serological analysis represents an unmet challenge. Traditionally, serological analysis of DENV infection focuses on the acute phase via detection of NS1 or IgM. Developing IgG DENV-specific assays has been defiant due to the co-circulation of four antigenically distinct serotypes and a strong cross-reactivity with other arboviruses, particularly with ZIKV. The goal of this study was to produce recombinant domain III of the Envelope (EDIII) proteins of each DENV serotype and ZIKV and evaluate the ability to detect specific IgG antibodies. The antigens were tested on the ELISA platform to measure DENV-specific IgG in mice and patients infected with ZIKV and DENV. The assay differentiated serotype-specific IgG responses in susceptible mice (AG129) experimentally infected with DENV or ZIKV. In addition, the test demonstrated a robust performance achieving 87.8% sensitivity and 91.4% specificity when tested against 648 well-characterized sera collected from humans infected with DENV and/or ZIKV. The test was further applied to serum samples collected from 318 healthy individuals from an endemic region in Northwest/Central Brazil, without a previous diagnosis of DENV, and revealed that 65% of the samples reacted with at least one DENV serotype antigen, including 123 monotypic samples (88 for DENV-1) and 90 samples reacting with multiple antigens. Collectively, these results indicate that the IgG DENV-EDIII ELISA is a valuable tool for assessing the serological status of populations in endemic areas, particularly in regions where other flaviviruses, particularly ZIKV, co-circulate and offer support to establish public health policies against the disease.

An investigation of group and subtype diversity and distribution of porcine rotaviruses in Canadian suckling piglets with diarrhea, 2019-2023

Objective: To determine the frequency of detection and group diversity of rotavirus (RV) A, B, and C, and G (glycoprotein antigen) serotype (based on viral protein 7 [VP7] gene analysis) infecting suckling piglets with diarrhea in Canadian farms. Materials and methods: Canadian swine veterinarians submitted 1117 enteric samples from suckling piglets between July 2019 and December 2023 to the University of Guelph Animal Health Laboratory for RV group identification and VP7 sequencing for subtyping. Analysis of the VP7 sequence from 837 samples was performed using the Animal Health Sequivity Dashboard (Merck & Co, Inc) and descriptive statistics. Results: Rotavirus A, B, and C were present in 40.7%, 12.5%, and 46.8% of samples, respectively. The most common RV identified was RVC G6, present in 296 samples, followed by RVA G9 in 205 samples. A single RV group was involved in 444 cases (72.3%), while in 170 cases (27.7%), more than one RV group/subtype was detected. Eighteen subtypes were identified by sequencing the VP7 protein (5 RVA, 9 RVB, and 4 RVC). Implications: Rotavirus protection for suckling piglets comes from colostrum and milk. Knowing which RV group is causing diarrhea is important since vaccination does not generate cross-protection among groups. Using molecular diagnostic testing, it is possible to identify the specific group and subtype of RV circulating on the premises and decide the best treatment strategy for the disease.

Molecular Characterization of Escherichia coli Causing Urinary Tract Infections Through Next-Generation Sequencing: A Comprehensive Analysis of Serotypes, Sequence Types, and Antimicrobial and Virulence Genes.

Introduction An enormous increase in antimicrobial resistance (AMR) among bacteria isolated from human clinical specimens contributed to treatment failures. Increased surveillance through next-generation sequencing (NGS) or whole genome sequencing (WGS) could facilitate the study of the epidemiology of drug-resistant bacterial strains, resistance genes, and other virulence determinants they are potentially carrying. Methods This study included 30 Escherichia coli (E. coli) isolates obtained from patients suffering from urinary tract infections (UTIs) attending Prathima Institute of Medical Sciences, Karimnagar, India. All bacterial isolates were identified, and antimicrobial susceptibility patterns were determined through conventional microbiological techniques and confirmed by automated systems. All the isolates were investigated using NGS to identify genes coding for resistance, such as extended-spectrum beta-lactamases (ESBLs), metallo-beta-lactamases, and virulence genes. Multilocus sequence typing (MLST) was used to understand the prevalent strain types, and serotyping was carried out to evaluate the type of O (cell wall antigen) and H (flagellar antigen) serotypes carried by the isolates. Results The conventional antimicrobial susceptibility testing revealed that 15 (50%) isolates were resistant to imipenem (IPM), 10 (33.33%) were resistant to amikacin (AK), 13 (43.33%) were resistant to piperacillin-tazobactam (PTZ), 17 (56.66%) were resistant to cephalosporins, and 14 (46.66%) were resistant to nitrofurantoin (NIT). Among the isolates, 26 (86.66%) had revealed the presence of multiple antibiotic-resistant genes with evidence of at least one gene coding for beta-lactamase resistance. There was a high prevalence of blaCTX-M(19/30, 63.33%) genes, followed by blaTEMand blaOXA-1. The blaNDM-5 gene was found in three isolates (3/30, 10%). The virulence genes identified in the present study wereiutA, sat, iss, and papC, among others. The E. coli serotype found predominantly belonged to O25:H4 (5, 16.66%), followed by O102:H6 (4, 13.33%). A total of 16 MLST variants were identified among the examined samples. Of the MLST-based sequence types (STs) identified, ST-131 (7, 23.33%) was the predominant one, followed by ST-167 (3, 10%)and ST-12 (3, 10%). Conclusions The study results demonstrated that the E. coli strains isolated from patients suffering from UTIs potentially carried antimicrobial resistanceand virulence genes and belonged to different strain types based on MLST. Careful evaluation of bacterial strains using molecular analyses such as NGS could facilitate an improved understanding of bacterial antibiotic resistance and its virulence potential. This could enable physicians to choose appropriate antimicrobial agents and contribute to better patient management, thereby preventing the emergence and spread of drug-resistant bacteria.

Evaluation of specific chicken IgY antibody value developing diagnostic capture antibody ELISA kit against Foot and Mouth disease.

The most preferred method for the detection of foot-and-mouth disease (FMD) viral antigen and identification of viral serotype is the enzyme-linked immunosorbent assay (ELISA). Diagnostic tests with high sensitivity are necessary both to distinguish infected vaccinated animals and execute disease control programs for the identification of the carrier animals. The current strategies for the detection of FMD virus are mainly based on the capture antibody (sandwich) ELISA test. The usage of laying pullets as an animal bioreactor for the production of specific egg yolk antibodies (IgY) has increased in recent years due to its high yield, affinity, low price, and quick production turnover. The present study aimed to produce a concentrated and purified IgY polyclonal antibody to design a capture antibody ELISA kit against the FMD virus (FMDV) serotype A. At first, laying hens were immunized with inactivated FMDV serotype virus, and then, on days 14, 21, and 28 following vaccination, the eggs and sera were collected. Afterward, the IgY polyclonal antibodies were extracted and purified from the chicken egg yolk using a polyethylene glycol 6000-ethanol precipitation procedure. Extracts were filtered, purified by ion exchange chromatography, and dialyzed. The purified IgY concentration, estimated by Bradford assay, confirmed its presence by SDS-PAGE and Western blot and also its specific immune reaction by Ouchterlony double immunodiffusion and Dot blot tests. Moreover, for achieving the optimum concentration of antigen/antibody (sera) in sandwich ELISA, a checkerboardtitrationtest was set up based on indirect ELISA results. Eventually, 119 previously confirmed samples (including 80 positive and 39 negative) by both real-time polymerase chain reaction (quantitative PCR, qPCR) and a commercial ELISA kit were used for evaluation of the sensitivity and accuracy of our developed Capture antibody ELISA kit. In this manner, the sensitivity and specificity of our designed kit were 100% and 98%, respectively. Accordingly, the present developed capture ELISA kit based on IgY had high sensitivity and specificity for FMD virus detection and it could be used in the future for both commercial detecting and serotyping applications.

Clinical and Antigen Serotype O Confirmation of Foot and Mouth Disease (FMD) Virus Outbreak in Punjab Spillover from Domestic Livestock

Foot and Mouth Disease (FMD) is one of the most highly contagious viral diseases affecting cloven-hoofed domestic and wild animals. This study described clinical investigations of FMD outbreaks (n=13) with serotype O confirmation in seven districts of Punjab from October 2023 to April 2024. Samples from sick animals and post-mortem examinations were collected from FMD suspected animals for laboratory diagnosis. The investigation included clinical signs and lesions, blood profiles, presence of other infectious pathogens, differential diagnosis, and the cause of death in the area. A total of 13 outbreaks in farm ruminants were confirmed, detecting FMDV serotype O. Outbreaks were reported in Ludhiana, Barnala, Gurdaspur, Bathinda, Faridkot, Mansa, and Amritsar districts. Animals were diagnosed to be positive for FMDV antigen using Sandwich ELISA. A total of 692 animals, including both cattle and buffalo, were affected in these districts, with a mortality rate of 24.42% (169/692) in young animals and heifers. The most severely affected districts were Bathinda, Barnala, and Faridkot, that experienced high mortality rates, leading to significant economic losses in these areas. Histopathologically samples revealed muscle degeneration and diffuse lymphocytic infiltration, and lymphocytic myocarditis in the heart, which is characteristic of the cardiac form of FMD. Serotype O FMDV, primarily circulating in the study area, was identified as the cause of the outbreaks in farm ruminants under investigation. The local animal health authorities should take steps to minimize the spread of the disease by being prepared for risk factors, particularly animal movement and human activities linked to an outbreak and the need for improved vaccination coverage and enhanced biosecurity measures to control the spread of FMD in the region.

Clinical trial results of an associated vaccine against cattle clostridiosis and escherichiosis

In veterinary practice, gastrointestinal diseases of newborn calves, caused by the association of pathogenic bacteria, in particular Clostridium perfringens and Escherichia coli, are often observed. The results of our research have shown that in the Volga Federal District of the Russian Federation, anaerobic enterotoxemia in newborn calves is most often caused by Clostridium perfringens serotypes A, C, D. For the specific prevention of this form of pathology, an associated vaccine has been developed containing inactivated antigens of Clostridium perfringens serotypes A, C, D and Escherichia coli, which produce adhesion factors K99 and A20. Clinical trials of the effectiveness of the vaccine on cattle were carried out in 3 large agricultural enterprises of the Republic of Tatarstan for the production of milk, permanently dysfunctional for clostridiosis and escherichiosis of newborn calves. Disease prevention began with the immunization of pregnant cows and heifers 60 days before the expected calving. The animals were vaccinated twice with an interval of 14 days at a dose of 10 cm3. Calves obtained from immunized cows were vaccinated twice subcutaneously at a dose of 3 cm3 at the age of 18- 20 days. A total of 3156 cows and heifers were vaccinated in three dairy complexes. It was found that the associated vaccine is harmless for deepwalled cows and heifers, does not cause post-vaccination complications. Immunization of animals in the last months of pregnancy contributes to the accumulation of specific antibodies in colostrum, the intake of which provides passive protection of the newborn calf from infection with Cl. perfringens and E. coli bacteria.

ASSOCIATION OF TLR 5 AND ESCHERICHIA COLI FLIC POLYMORPHISMS WITH RECURRENT URINARY TRACT INFECTIONS IN WOMEN

This study was aimed to investigate the role of some Toll-like receptor 5 (TLR5) and E. coli fliC gene polymorphisms with increased risk to recurrent urinary tract infections (rUTI). From 180 specimens (blood and urine) were collected from women of different age, 60 of them serve as control while 120 had rUTI symptoms. After culturing of urine specimens, 43 (35.8%) were identified as E. coli isolates. Four SNPs were identified when amplified and sequenced of TLR5 include rs5744168, rs775385356, rs2072493 and rs5744174. Twelve flagellar antigen serotypes were obtained from fliC sequence of 28 isolates. By using Expasy and Clustal Omega programs, the FliC proteins of H-serotypes arranged in different lengths ranging between 324-634 residues. The N-terminal and C-terminal were conserved region, in contrast, the central region was variable poorly preserved. The results also showed list of conserved amino acids in both FliC termini included L89, Q90,R91, L95, Q98, N101 and E115 in N- terminus in all studied serotypes. Further, the N277 residue that reported its central role for TLR5 interaction were found in some serotypes. Indeed, it was no distinct relation between genotypes or allele frequencies with fliC polymorphisms found in E. coli isolated from rUTI.

The Association of Class I and II Human Leukocyte Antigen Serotypes With End-Stage Kidney Disease Due to Membranoproliferative Glomerulonephritis and Dense Deposit Disease

Membranoproliferative glomerulonephritis (MPGN), encompassing several distinct diseases, is a rare but significant cause of kidney failure in the United States. The potential etiologies of MPGN are unclear, but prior studies have suggested dysregulation of the alternative complement pathway and, recently, autoimmunity as potential mechanisms driving MPGN pathogenesis. In this study, we examined HLA associations with end-stage kidney disease (ESKD) due to MPGN and dense deposit disease (DDD) in a large racially and ethnically diverse US-based cohort. Case-control study. Using US Renal Data System (USRDS) and United Network for Organ Sharing (UNOS) data, we identified 3,424 patients with kidney failure due to MPGN and 263 due to DDD. We matched patients to kidney donor controls on designated race and ethnicity in a 1:15 ratio. 58 class I and II HLA serotypes. Case-control status. For each disease cohort, univariable and multivariable logistic regression analyses were used to investigate associations between the disease and 58 HLA serotypes. In subgroup analyses, we investigated HLA associations in White and Black patients. We also studied antiglomerular basement membrane (anti-GBM) nephritis as a positive-control outcome. We applied a Bonferroni correction to account for multiple comparisons. Eighteen serotypes were significantly associated with the odds of having MPGN in univariable analyses, with DR17 having the strongest association (odds ratio [OR], 1.55 [95% CI, 1.44-1.68], P=4.33e-28). No significant associations were found between any HLA serotype and DDD. Designated race-specific analyses showed comparable findings. We recapitulated known HLA associations in anti-GBM nephritis. Reliance on HLA serotypes (rather than genotype), lack of biopsy-confirmed diagnoses. HLA-DR17 is associated with ESKD due to MPGN in a racially and ethnically diverse cohort. The strength of association was similar in White and Black patients, suggesting a role in the pathogenesis of MPGN. No HLA associations were observed in patients with DDD. Prior studies have suggested dysregulation of the alternative complement pathway as a potential etiology of membranoproliferative glomerulonephritis (MPGN), but recent evidence from a British White population has implicated an autoimmune mechanism in MPGN pathogenesis. We investigated HLA associations between MPGN and dense deposit disease (DDD) in a large racially and ethnically diverse cohort of patients. We found that HLA-DR17 is associated with end-stage kidney disease (ESKD) due to MPGN in both White and Black patients. By contrast, no significant HLA associations with ESKD due to DDD were identified. These results suggest a role for autoimmunity in some cases of MPGN and highlight differences in the disease etiology of MPGN compared with DDD.

A 20-Year Study of Capsular Polysaccharide Seroepidemiology, Susceptibility Profiles, and Virulence Determinants of Klebsiella pneumoniae from Bacteremia Patients in Taiwan.

In this study, we selected bacteremic Klebsiella pneumoniae isolates from the Taiwan Surveillance of Antimicrobial Resistance program. A total of 521 isolates were collected over a period of 2 decades, including 121 from 1998, 197 from 2008, and 203 from 2018. Seroepidemiology showed that the top five capsular polysaccharide types were serotypes K1, K2, K20, K54, and K62, constituting 48.5% of the total isolates, and the respective ratios at each time point have remained similar over the past 2 decades. The antibacterial susceptibility tests showed that K1, K2, K20, and K54 were susceptible to most antibiotics, while K62 was relatively resistant compared to other typeable and nontypeable strains. In addition, six virulence-associated genes, clbA, entB, iroN, rmpA, iutA, and iucA, were predominant in K1 and K2 isolates of K. pneumoniae. In conclusion, serotypes K1, K2, K20, K54, and K62 of K. pneumoniae are the most prevalent serotypes and carry more virulence determinants in bacteremia patients, which may indicate their invasiveness. If further serotype-specific vaccine development is performed, these five serotypes should be included. Since the antibiotic susceptibility profiles were stable over a long duration, empirical treatment may be predicted according to serotype if rapid diagnosis from direct clinical specimens is available, such as PCR or antigen serotyping for serotype K1 and K2. IMPORTANCE This is the first nationwide study to examine the seroepidemiology of Klebsiella pneumoniae using blood culture isolates collected over a period of 20 years. The study found that the prevalence of serotypes remained consistent over the 20-year period, with high-prevalence serotypes associated with invasive types. Nontypeable isolates had fewer virulence determinants than other serotypes. With the exception of serotype K62, the other high-prevalence serotypes were highly susceptible to antibiotics. If rapid diagnosis using direct clinical specimens, such as PCR or antigen serotyping, is available, empirical treatment can be predicted based on serotype, particularly for K1 and K2. The results of this seroepidemiology study could also help the development of future capsule polysaccharide vaccines.

Detection of dengue virus serotype 4 in Sudan.

Dengue virus infection is spreading globally and most parts of Sudan have witnessed repeated dengue outbreaks, with the detection of DENV-1, DENV-2 and DENV-3 serotypes. In this report we describe the dengue fever outbreaks that occurred in eastern Sudan (Kassala and Port Sudan cities) from August to November 2019. We enrolled 79 (29.8%) suspected cases from Kassala and 186 (70.2%) from Port Sudan who presented with fever. The participants were medically examined and their clinical signs recorded. Blood samples were collected for complete blood count, detection of anti-dengue virus IgM, detection of NS1 dengue antigen and identification of the virus serotype using RT-PCR. The main clinical presentations were fever, abdominal pain, joint pain and vomiting, and thrombocytopenia was the main laboratory finding. One hundred and twenty-five blood samples tested positive for the anti-dengue IgM antibody, and 145 were positive for the NS1 antigen. Using RT-PCR, we identified 35 (24%) infections with DENV-2, 100 (69%) with DENV-3 and 10 (7%) with DENV-4 serotypes. We identified multiple serotypes - DENV-2, DENV-3 and DENV-4 - as the causes of the outbreak. The presence of DENV-4 serotype was documented for the first time in Sudan.

Characterization of invasive meningococcal disease case isolates in Atlantic Canada, 2014 to 2020: spatial-temporal variations of clones and predicted meningococcal B vaccine coverage.

Introduction. Invasive meningococcal disease (IMD) caused by Neisseria meningitidis may show temporal and geographical changes in both the epidemiology and the characteristics of the strains involved.Gap statement. A study that examined invasive N. meningitidis causing IMD in Atlantic Canada from 2009 to 2013 was published in 2014. Data from subsequent years have not been described.Aim. This study examined the molecular epidemiology of IMD in four Atlantic Provinces of Canada as well as potential serogroup B (MenB) vaccine coverage.Methods. Individual IMD case isolates recovered from 2014 to 2020 were analysed for serotype and serosubtype antigens as well as by whole-genome sequencing (WGS) for prediction of potential MenB vaccine coverage.Results. Of the 56 IMD isolates, 42, 8, 5 and 1 were MenB, serogroup Y, serogroup W (MenW) and serogroup C, respectively. Geographical differences in the distribution of MenB clones revealed concentration of sequence type (ST)-269 clonal complex (cc) and ST-60 cc in Newfoundland and Labrador, while ST-41/44 cc (particularly ST-154) was predominantly found in New Brunswick and Nova Scotia. Core genome multi-locus sequence typing (cgMLST) also separated the New Brunswick and Nova Scotia ST-154 isolates into two clusters, with differences in their nhba and penA alleles. Furthermore, cgMLST also separated the ST-269 cc isolates in Atlantic Canada into the ST-1611 and the ST-269/ST-8924 clusters, with the latter showing high similarity to the ST-269 that first emerged in the Province of Quebec. Genetic Meningococcal Antigen Typing System showed that 54.8 % of MenB were predicted to be covered by the MenB vaccine Bexsero, with a further 38.1 % potentially covered by virtue of the presence of genes that encoded factor H-binding protein variant 1 proteins. Meningococcal deduced vaccine antigen reactivity predicted from WGS data showed that 95.3 % of MenB were covered by Trumenba. Four cases of IMD due to MenW ST-11 cc were also identified, with the first case found in 2018.Conclusions. This study provided evidence concerning the dynamics of N. meningitidis strains causing IMD in Atlantic Canada, with both geographical and temporal differences found. MenB vaccine appeared to provide good coverage of MenB IMD, especially towards the predominant strain of ST-154.

Spin-Enhanced Lateral Flow Immunoassay for High-Sensitivity Detection of Nonstructural Protein NS1 Serotypes of the Dengue Virus.

Dengue fever is a global mosquito-borne viral infectious disease that has, in recent years, rapidly spread to almost all regions of the world. Lack of vaccination and directed treatment makes detection at the infection's early stages extremely important for disease prevention and clinical care. In this paper, we developed a rapid and highly sensitive dengue detection tool using a novel platform of diagnosis, called spin-enhanced lateral flow immunoassay (SELFIA) with a fluorescent nanodiamond (FND) as a reporter. Taking advantage of the unique magneto-optical properties of negatively charged nitrogen-vacancy centers in the FND, the SELFIA platform utilizes alternating electromagnetic fields to modulate signals from FND's fluorescence to provide sensitive and specific results. With sandwich SELFIA, we could efficiently detect all four dengue non-structural protein (NS1) serotypes (DV1, DV2, DV3, and DV4). The lowest detection concentration of the dengue NS1 antigens varied from 0.1 to 1.3 ng/mL, which is among the lowest limits of detection to date. The FND-based SELFIA technique is up to 500 and 5000 times more sensitive than carbon black and conventional gold nanoparticles, respectively. By using different anti-NS1 antibodies, we could differentiate the NS1 antigen serotypes contained in the tested samples via three simultaneous assays. Proposed SELFIA allows for both qualitative and quantitative differentiation between different NS1 protein serotypes, which will assist in the development of a highly sensitive and specific detection platform for dengue screening that has the potential to detect the disease at its early stages, especially in high-risk and limited-resource areas.

Foot-and-mouth disease status in India during the second decade of the twenty-first century (2011-2020).

Foot-and-mouth disease (FMD) is a major disease of livestock in India and causes huge economic losses. The formal FMD control program started in 2003–04 in selected districts and was gradually expanded. The present study provides a descriptive review of the FMD outbreaks, prevalent serotypes, and genetic and antigenic features of the FMD virus (FMDV) that circulated in the country between 2011 and 2020. FMD outbreaks were regularly reported in cloven-hoofed domestic livestock and wildlife, with three serotypes including O, A, and Asia1. During the study period, a total of 2226 FMD outbreaks were documented and serotypes confirmed. FMDV serotype O dominated the outbreak scenario, accounting for about 92% of all outbreaks, followed by Asia1 (5% of all outbreaks) and A (3% of all outbreaks). Two major epidemics of FMD on an unprecedented scale during the years 2013 and 2018 by serotype O were recorded. The spatial distribution of FMD was characterized by a larger number of outbreaks in the southern region of the country. In an annual-scale analysis, 2020 was the year with the lowest outbreaks, and 2013 was the year with the highest. The month-scale analysis showed that outbreaks were reported throughout the year, with the highest numbers between October and March. The emergence of three major lineages (O/ME-SA/Ind2001d, O/ME-SA/Ind2001e, and O/ME-SA/Ind2018) of serotype O was observed during the period. In the cases of serotype A and Asia1, the appearance of at least one novel lineage/genetic group, including A/G-18/non-deletion/2019 and Asia1/Group-IX, was documented. While serotype A showed the advent of antigenic variants, serotypes O and Asia1 did not show any antigenic diversity. It was noticed during the course of an outbreak that animal movement contributes significantly to disease transmission. Except for 2018, when numerous FMD outbreaks were recorded, the number of annual outbreaks reported after 2016 has been lower than in the first half of the decade, probably due to mass vaccination and COVID-19 pandemic-linked movement restrictions. Even during outbreaks, disease symptoms in ruminant populations, including cattle, were found to be less severe. Regular six-monthly immunization certainly has a positive impact on the reduction of disease burden and should be followed without fail and delay, along with intensive disease surveillance.

Atorvastatin attenuates NS1 (Non-structural protein-1) of dengue type-2 serotype-induced expressions of matrix metalloproteinases in HL-60 cells, differentiated to neutrophils: Implications for the immunopathogenesis of dengue viral disease

BackgroundThe dengue is a vector borne viral infection in humans. Bite of mosquito infected with a dengue virus transmits the disease. The neutrophils support more to the innate immune response by switching to infected tissues and triggering immunomodulatory mechanisms including the release of proteases and host defence peptides. MethodsCell viability by MTT and trypan blue dye exclusion assay, bright field microscopy for assessment of cell morphology, cytokines measurements by ELISA, estimation of protein by Bradford assay were done. Assessments of matrix metalloproteinase genes mRNA expressions were done using real-time PCR. ResultsIn the present study, we have for the first time unveiled that, NS1 antigen of dengue type-2 serotype, induce and stimulate the neutrophils cells to express high levels of matrix metalloproteases. NS1 exposure of HL-60 cells differentiated to neutrophils affected cell morphology and in 24 h of exposure. We have demonstrated that, the NS1 antigen has induced MMP-2, MMP-14 and MMP-9 expressions in neutrophils in a 24hrs exposure time. NS1 exposure has also further upregulated MMP-1, MMP-13, and MMP-8 expressions in neutrophils in a 24hrs exposure time. Notably, treatment with atorvastatin concentrations downregulated the expression profile of the all matrix metalloprotease significantly. Importantly, NS1 antigen has significantly increased the IL-6, IL-13 release by the HL,60 cells which was reversed by atorvastatin. On the other hand, NS1 exposure enhanced the mRNA expressions of VEGF-A and VEGF-D which was reversed by atorvastatin. However, we found that, NS1 exposure reduced the mRNA expressions profile of VEGF-C, which was reversed by atorvastatin. ConclusionIn conclusion, we report that, neutrophils associated matrix metalloprotease are involved in the pathogenesis of dengue viral disease. VEGF growth factors may also be released by the neutrophils which may subsequently participate in the endothelial dysfunctions leading to dengue shock syndrome.

Cross-Serotype Reactivity of ELISAs Used to Detect Antibodies to the Structural Proteins of Foot-and-Mouth Disease Virus.

Antibodies to the foot-and-mouth disease virus (FMDV) capsid induced by infection or vaccination can provide serotype-specific protection and be measured using virus neutralization tests and viral structural-protein (SP-)ELISAs. Separate tests are needed for each serotype, but cross-serotype reactions complicate serotyping. In this study, inter-serotypic responses were quantified for five SP-ELISA formats by testing 294 monovalent mainly bovine sera collected following infection, vaccination, or vaccination and infection with one of five serotypes of FMDV. Over half of the samples, representing all three immunization categories, scored positive for at least one heterologous serotype and some scored positive for all serotypes tested. A comparative approach to identifying the strongest reaction amongst serotypes O, A and Asia 1 improved the accuracy of serotyping to 73–100% depending on the serotype and test system, but this method will be undermined where animals have been infected and/or vaccinated with multiple FMDV serotypes. Preliminary studies with stabilized recombinant capsid antigens of serotypes O and A that do not expose internal epitopes showed reduced cross-reactivity, supporting the hypothesis that capsid integrity can affect the serotype-specificity of the SP-ELISAs. The residual cross-reactivity associated with capsid surface epitopes was consistent with the evidence of cross-serotype virus neutralization.

Development of Novel Dengue NS1 Multiplex Lateral Flow Immunoassay to Differentiate Serotypes in Serum of Acute Phase Patients and Infected Mosquitoes

Dengue is among the most rapidly spreading arboviral disease in the world. A low-cost, easy to use point-of-care diagnostic tool for the detection and differentiation of dengue virus serotypes could improve clinical management, disease prevention, epidemiological surveillance, and outbreak monitoring, particularly in regions where multiple serotypes co-circulate. Despite widespread deployment, no commercial dengue antigen diagnostic test has proven effective in differentiating among dengue virus serotypes. In the current study, we first established mAb pairs and developed a multiplex lateral flow immunoassay for the simultaneous detection of the dengue viral NS1 antigen and identification of serotype. The proposed system, called Dengue serotype NS1 Multiplex LFIA, provides high sensitivity and specificity. In testing for JEV, ZIKV, YFV, WNV, and CHIKV, the multiplex LFIA gave no indication of cross- reactivity with cell culture supernatants of other flaviviruses or chikungunya virus. In analyzing 187 samples from patients suspected of dengue infection, the detection sensitivity for serotype D1 to D4 was 90.0%, 88.24%, 82.61%, and 83.33% and serotype specificity was 98.74%, 96.13%, 99.39%, and 97.04%, respectively. Our multiplex LFIA can also identify mono- and co-infection of different serotype of dengue viruses in mosquitoes. The proposed Multiplex LFIA provides a simple tool for the rapid detection of dengue serotypes and in the differential diagnosis of fever patients in regions where medical resources are limited and/or multiple DENVs co-circulate.

Botulinum Toxin Type A Immunogenicity across Multiple Indications: An Overview Systematic Review.

Botulinum toxin type A has been used to treat a wide array of neurologic, medical, and aesthetic indications. Several factors contribute to the formation of neutralizing antibodies, such as shorter intervals of treatment, higher dosage, amounts of antigenic proteins, serotypes, and storage of formulations. This overview followed the Cochrane guideline for overview reviews. The AMSTAR-2 (revised version of A Measurement Tool to Assess Systematic Reviews) tool was used for the critical appraisal of the selected systematic reviews. Five systematic reviews consisting of 203 studies (17,815 patients) were included, and their AMSTAR-2 scores were low to critically poor. There was high heterogeneity between the studies. Across the clinical indications, neutralizing antibody prevalence was significantly higher in dystonia, spasticity, and urologic conditions, and nil to insignificant in hyperhidrosis and aesthetic indications. The overall rate for the neutralizing antibody formation across three different formulations, abobotulinumtoxinA, incobotulinumtoxinA, and onabotulinumtoxinA, was 1 to 2.1 percent, with no significant difference between them. Although there is debate on the prevalence rate across the different botulinum toxin type A formulations in individual systematic reviews, the overall frequency of the development of neutralizing antibodies and the immunogenicity of abobotulinumtoxinA, incobotulinumtoxinA, and onabotulinumtoxinA remain low to insignificant. Properly designed comparative trials are required to explore the difference in the prevalence of neutralizing antibodies across the commercially available botulinum toxin type A products. Such studies should also examine the relevance of neutralizing antibody titer to clinical responsiveness and nonresponse.

Quantity of the antigens of Streptococcus mutans serotype e and Candida albicans and its correlation with the salivary flow rate in early childhood caries.

Background:Streptococcus mutans involved in caries pathogenesis is classified into four serotypes, namely serotypes c, e, f, and k. Candida albicans can be found in the plaque of children with early childhood caries (ECC). Aims: The aim of this study was to analyze the quantity of the antigens of S. mutans serotype e and C. albicans and its correlation with the salivary flow rate in ECC.Materials and Methods:The antigen quantities of caries plaque samples and caries-free were determined using an enzyme-linked immunoassay with 450-nm optical density.Results:There was a significant difference between the quantity of S. mutans serotype e and C. albicans antigens in each salivary flow rate category (P < 0.05). The relationship between the antigen quantity of S. mutans serotype e and C. albicans was r = 0.624 (P > 0.05) for caries plaque samples and r = 0.628 (P > 0.05) for caries-free samples.Conclusion:the antigen quantities of S. mutans serotype e and C. albicans and the salivary flow rate might correlate to the pathogenesis of ECC.

Update on Juvenile Spondyloarthritis.

Spondyloarthritis (SpA) is a blanket term encompassing entities such as enthesitis-related arthritis, nonradiographic axial SpA, and ankylosing spondylitis. These diseases share many clinical features, including a predilection for inflammation of the entheses and the sacroiliac joints. The nomenclature is based on the evolution of the classification of the disease and the age of the patient. SpA has a prevalence of approximately 1% of the population of the United States, with 10% to 20% of patients experiencing the onset during childhood. Children with onset of arthritis before age 16 years are classified as having juvenile idiopathic arthritis. Children with enthesitis and/or sacroiliitis are further classified as belonging to the enthesitis-related arthritis subtype of juvenile idiopathic arthritis. The initial manifestations can be subtle and will usually include a peripheral pattern of arthritis and enthesitis. It may take several years for axial disease to develop in children. Except for an association with the human leukocyte antigen (HLA-B27) serotype, there are no laboratory markers for the disease, and the radiographic findings are often negative. A careful clinical evaluation for evidence of inflammation in the entheses and the joints and a search for comorbidities are required. Magnetic resonance imaging facilitates the early detection of sacroiliitis, an important feature that may be clinically silent. Because recent studies indicate that earlier introduction of therapy can help achieve better outcomes, rapid identification and treatment of children with SpA is essential.

HLA-B54 is an independent risk factor for pneumonia in Japanese patients with interstitial lung disease: A multicenter retrospective cohort study

PurposePneumonia is a major cause of respiratory-related hospitalization and an important prognostic factor in patients with chronic interstitial lung disease (ILD). However, the relationship between the incidence of pneumonia and human leukocyte antigen (HLA) serotype has not been fully elucidated. Therefore, this study aimed to determine if there is a relationship between HLA serotype and the incidence of pneumonia in Japanese patients with ILD. MethodsThe medical records of patients with ILD treated at any of three centers in Japan were reviewed to determine their HLA-A and HLA-B serotypes. The characteristics of patients with and without pneumonia were compared. Cox regression analysis was performed to identify risk factors for pneumonia and death in these patients. ResultsOne hundred and forty-four patients with ILD (pneumonia group, n = 27; non-pneumonia group, n = 117) and complete HLA serology data available were included. HLA-B54 positivity was significantly more common in the pneumonia group than in the non-pneumonia group (37.0% vs. 15.4%, p = 0.010). HLA-B54 positivity was also a significant risk factor for pneumonia (hazard ratio [HR] 4.166, 95% confidence interval [CI] 1.862–9.320, p = 0.001) and death (HR 4.050, 95% CI 1.581–10.374, p = 0.004) in patients with ILD. Furthermore, HLA-B54 positivity was a significant risk factor for pneumonia (HR 3.964, 95% CI 1.392–11.090, p = 0.010) and death (HR 8.131, 95% CI 1.763–37.494, p = 0.007) in patients with idiopathic pulmonary fibrosis. ConclusionHLA-B54 positivity was a significant risk factor for pneumonia and death in patients with ILD, including those with idiopathic pulmonary fibrosis.