scholarly journals Identification of Immunogenic Candidate for New Serological Tests for Brucella melitensis by a Proteomic Approach

2021 ◽  
Vol 15 (1) ◽  
pp. 92-97
Author(s):  
Valentina Paci ◽  
Pierina Visciano ◽  
Ivanka Krasteva ◽  
Tiziana Di Febo ◽  
Fabrizia Perletta ◽  
...  

Background: The diagnosis of brucellosis by serological tests is based on antigen suspensions derived from smooth lipopolysaccharide extracts, which can give false positive results linked to cross-reactivity with other Gram-negative microorganisms, especially Yersinia enterocolitica O:9 and Escherichia coli O157:H7. Objective: The objective of the present study was the characterization by proteomic analysis of specific immunogenic proteins not associated with smooth lipopolysaccharide to improve the diagnostic tests used in the ovine brucellosis eradication programs. Methods: The serum from a sheep positive to Brucella melitensis was treated to eliminate all antibodies against such lipopolysaccharide and highlight the reaction towards the immunoreactive proteins in Western Blotting. Results: The immunoreactive bands were identified by nLC-MS/MS and through bioinformatic tools, it was possible to select 12 potential candidates as protein antigens specific for Brucella melitensis. Conclusion: The detection of new antigens not subjected to cross-reactivity with other Gram-negative microorganisms can offer an additional tool for the serological diagnosis of such disease.

2020 ◽  
Vol 13 (1) ◽  
Author(s):  
Carlos A. Rodríguez-Alarcón ◽  
Diana M. Beristain-Ruiz ◽  
Angélica Olivares-Muñoz ◽  
Andrés Quezada-Casasola ◽  
Federico Pérez-Casio ◽  
...  

Abstract Background Nowadays, Ehrlichia canis receives increasing attention because of its great morbidity and mortality in animals. Dogs in the subclinical and chronic phases can be asymptomatic, and serological tests show cross-reactivity and fail to differentiate between current and past infections. Moreover, there could be low parasitaemia, and E. canis might be found only in target organs, hence causing results to be negative by polymerase chain reaction (PCR) on blood samples. Methods We evaluated by PCR the prevalence of E. canis in blood, liver, spleen, lymph node and bone marrow samples of 59 recently euthanised dogs that had ticks but were clinically healthy. Results In total, 52.55% of the blood PCRs for E. canis were negative, yet 61.30% yielded positive results from tissue biopsies and were as follows: 63.15% from bone marrow; 52.63% from liver; 47.36% from spleen; and 15.78% from lymph node. In addition, 33% had infection in three tissues (spleen, liver and bone marrow). Conclusions Our results show the prevalence of E. canis from tissues of dogs that were negative by blood PCR. Ehrlichia canis DNA in tissue was 30% lower in dogs that tested negative in PCR of blood samples compared to those that were positive. However, it must be taken into account that some dogs with negative results were positive for E. canis in other tissues.


2017 ◽  
Vol 56 (3) ◽  
Author(s):  
Lakshmanane Premkumar ◽  
Matthew Collins ◽  
Stephen Graham ◽  
Guei-Jiun Alice Liou ◽  
Cesar A. Lopez ◽  
...  

ABSTRACT Zika virus (ZIKV) is an emerging flavivirus that can cause birth defects and neurologic complications. Molecular tests are effective for diagnosing acute ZIKV infection, although the majority of infections produce no symptoms at all or present after the narrow window in which molecular diagnostics are dependable. Serology is a reliable method for detecting infections after the viremic period; however, most serological assays have limited specificity due to cross-reactive antibodies elicited by flavivirus infections. Since ZIKV and dengue virus (DENV) widely cocirculate, distinguishing ZIKV infection from DENV infection is particularly important for diagnosing individual cases or for surveillance to coordinate public health responses. Flaviviruses also elicit type-specific antibodies directed to non-cross-reactive epitopes of the infecting virus; such epitopes are attractive targets for the design of antigens for development of serological tests with greater specificity. Guided by comparative epitope modeling of the ZIKV envelope protein, we designed two recombinant antigens displaying unique antigenic regions on domain I (Z-EDI) and domain III (Z-EDIII) of the ZIKV envelope protein. Both the Z-EDI and Z-EDIII antigens consistently detected ZIKV-specific IgG in ZIKV-immune sera but not cross-reactive IgG in DENV-immune sera in late convalescence (>12 weeks postinfection). In contrast, during early convalescence (2 to 12 weeks postinfection), secondary DENV-immune sera and some primary DENV-immune sera cross-reacted with the Z-EDI and Z-EDIII antigens. Analysis of sequential samples from DENV-immune individuals demonstrated that Z-EDIII cross-reactivity peaked in early convalescence and declined steeply over time. The Z-EDIII antigen has much potential as a diagnostic antigen for population-level surveillance and for detecting past infections in patients.


2016 ◽  
Author(s):  
Jinhee Yi ◽  
Kelsey Herring ◽  
Timothy C. Sanchez ◽  
Srinivas Iyer ◽  
Joshua K. Stone ◽  
...  

AbstractBurkholderia pseudomallei is the causative agent of the melioidosis and is endemic to Southeast Asia and northern Australia. There is no available vaccine and accurate diagnosis is difficult, time-consuming and labor intensive. Early diagnosis is an important part of successful treatment and current serological tests are inadequate and based upon multiple antigens. Identifying specific immunogenic proteins which are highly seroreactive may yield potential diagnostic targets for detecting antibodies and antigens specific to melioidosis. We have used 2D gel electrophoresis and Western blotting analysis to analyze protein antigenicity of whole cell lysates extracted from four B. pseudomallei strains and the sera from the specific infected humans. We found a total of 135 immunogenic proteins, 62 of which we were able to identify to a specific gene by mass-spectrometry. Results from the Western blotting of each strain’s proteins and the corresponding patient serum reveal between 30 – 40% serum x strain specific immunogenic proteins. In most cases, these differences exist despite the fact that the genes encoding these proteins were present among all four B. pseudomallei strains. Eight particular proteins were immunogenic in all four strain x serum combinations and could represent novel diagnostic and vaccine subunit targets.


Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 4720-4720
Author(s):  
Xiaoduan Weng ◽  
Louise Robin ◽  
Marie-lise Audet ◽  
Linda Hébert ◽  
Ginette Lapointe ◽  
...  

Abstract HLA-B27 is a strong diagnostic biomarker as it is found in 90– 95% of ankylosing spondylitis. Routinely, the false-positive results generated by cross-reactivity of HLA-B27 monoclonal antibodies (mAbs) with some other type of HLA-B antigens are problematic. The present study aims at improving the sensitivity and specificity of typing for HLA-B27 by using a more accurate DNA assay. A real time sequence specific primer polymerase chain reaction (PCR-SSP) is performed by using MGB TaqMan oligoprobe and an ABI PRISM™ 7500 sequence detection system. The TaqMan PCR-SSP assay is compared with Immunophenotyping by flow cytometric antigen assay (BD HLA-B27-Kit, clone GS145.2). The results are finally confirmed by sequencing (ABI PRISM ® 3100). As summarised in the table, three methods are identical for clearly positive and negative results. There is 90% concordance for borderline negative results obtained by immunophenotyping with TaqMan PCR-SSC and/or sequencing. However, only 15.4% of borderline positives obtained by immunophenotyping are confirmed as true HLA-B27 positives by sequencing as well as TaqMan PCR-SSC. TaqMan PCR-SSP DNA assay completely correlates with sequencing (gold standard) with 100% PPV and NPV, compared to a PPV of 15% and a NPV of 98% for borderline results by immunophenotyping. Conclusion: when compared with flow cytometric antigen assay, typing HLA-B27 by TaqMan PCR-SSP clearly demonstrates an advantage to better establish the genotype of the patient. Specifically, there is a marked difference for ambitious positive results from immunophenotyping. Moreover, compared to immunophenotyping, there is no need of fresh samples, can test a larger number of samples concomitantly, and reduces technical time as well as cost. N = 52 Immunophenotyping TaqMan PCR-SSP Sequencing Immuno vs Sequencing TaqMan PCR-SSP vs Sequencing Positive 11 11 11 100% 100% Negative 17 17 17 100% 100% Positive (borderline by Immunophenotyping) 13 2 2 15% 100% Negative (borderline ) by Immunophenotyping) 10 9 9 98% 100%


2020 ◽  
Author(s):  
Carlos Arturo Rodríguez-Alarcón ◽  
Diana M. Beristain-Ruiz ◽  
Angélica Olivares-Muñoz ◽  
Andrés Quezada-Casasola ◽  
Federico Pérez-Casio ◽  
...  

Abstract Background: Nowadays, Ehrlichia canis receives increasing attention because of its great morbidity and mortality in animals. Dogs in the subclinical and chronic phases can be asymptomatic, and serological tests show cross-reactivity and fail to differentiate between current and past infections. Moreover, there could be low parasitaemia, and E. canis might be found only in target organs, hence causing results to be negative by polymerase chain reaction (PCR) on blood samples.Methods: We evaluated by PCR the prevalence of E. canis in blood, liver, spleen, lymph node and bone marrow samples of 59 recently euthanised dogs that had ticks but were clinically healthy.Results: In total, 52.55% of the blood PCRs for E. canis were negative, yet 61.30% yielded positive results from tissue biopsies and were as follows: 63.15% from bone marrow; 52.63% from liver; 47.36% from spleen; and 15.78% from lymph node. In addition, 33% had infection in three tissues (spleen, liver and bone marrow).Conclusions: Our results show the prevalence of E. canis from tissues of dogs that were negative by blood PCR. Ehrlichia canis DNA in tissue was 30% lower in dogs that tested negative in PCR of blood samples compared to those that were positive. However, it must be taken into account that some dogs with negative results were positive for E. canis in other tissues.


2020 ◽  
Author(s):  
Himadri Nath ◽  
Abinash Mallick ◽  
Subrata Roy ◽  
Soumi Sukla ◽  
Subhajit Biswas

The world is going through the scourge of COVID-19 pandemic since last several months. The pandemic appears to be less severe in highly Dengue endemic countries. Furthermore, COVID-19 in two elderly patients (with no evidence of Dengue virus (DV) infection) elicited antibodies that gave false-positive results in DV serological tests. We anticipated that SARS-CoV-2 and DV share antigenic similarity and performed molecular docking studies. Our computational modelling studies predicted that human anti-DV antibodies can indeed, bind to RBD of SARS-CoV-2 Spike protein. Some of these interactions can also potentially intercept human ACE2 receptor binding to RBM. Our computational analysis showed that m396 Ab (against SARS-CoV-1) did not dock with RBM of SARS-CoV-2, a fact already proven experimentally. This confirmed reliability and robustness of our approach. So, immunological memory to DV in endemic countries is thwarting COVID-19. Available Dengue vaccines can be repurposed against SARS-CoV-2 in DV non-endemic countries until approved vaccines/ antivirals become available against COVID-19. Based on the observations that COVID-19 and Dengue severity maps do not tend to overlap and the fact that serological cross-reactivity has been reported for COVID-19 antibodies with Dengue antigen (s), together with results from our computational studies, it is imperative that serology-based diagnosis should be complemented with NAT/virus antigen detection-based tests for definitive diagnosis of either disease in regions where both of these viruses are now co-existent. Furthermore, we still do not know whether antibodies to SARS-CoV-2 will hinder/ameliorate DV infections by binding to DV particles and reduce dengue incidences in the future or, augment DV infection and severity by means of antibody-dependent enhancement (ADE).


Author(s):  
Mahmoud Ahmed Ebada ◽  
Notila Fayed ◽  
Souad Alkanj ◽  
Ahmed Wadaa Allah

: Enterovirus D68 (EV-D68) is a single-stranded positive-sense RNA virus, and it is one of the family Picornaviridae. Except for EV-D68, the family Picornaviridae has been illustrated in literature. EV-D68 was first discovered and isolated in California, USA, in 1962. EV-D68 has resulted in respiratory disorders’ outbreaks among children worldwide, and it has been detected in cases of various neurological diseases such as acute flaccid myelitis (AFM). A recent study documented a higher number of EV-D68 cases associated with AFM in Europe in 2016 compared to the 2014 outbreak. EV-D68 is mainly diagnosed by quantitative PCR, and there is an affirmative strategy for EV-D68 detection by using pan-EV PCR on the untranslated region and/or the VP1 or VP2, followed by sequencing of the PCR products. Serological tests are limited due to cross-reactivity of the antigens between the different serotypes. Many antiviral drugs for EV-D68 have been evaluated, and showed promising results. In our review, we discuss the current knowledge about EV-D68 and its role in the development of AFM.


2021 ◽  
Vol 22 (1) ◽  
pp. 429
Author(s):  
Luca Bini ◽  
Domitille Schvartz ◽  
Chiara Carnemolla ◽  
Roberta Besio ◽  
Nadia Garibaldi ◽  
...  

Osteogenesis imperfecta (OI) is a heritable disorder that mainly affects the skeleton. The inheritance is mostly autosomal dominant and associated to mutations in one of the two genes, COL1A1 and COL1A2, encoding for the type I collagen α chains. According to more than 1500 described mutation sites and to outcome spanning from very mild cases to perinatal-lethality, OI is characterized by a wide genotype/phenotype heterogeneity. In order to identify common affected molecular-pathways and disease biomarkers in OI probands with different mutations and lethal or surviving phenotypes, primary fibroblasts from dominant OI patients, carrying COL1A1 or COL1A2 defects, were investigated by applying a Tandem Mass Tag labeling-Liquid Chromatography-Tandem Mass Spectrometry (TMT LC-MS/MS) proteomics approach and bioinformatic tools for comparative protein-abundance profiling. While no difference in α1 or α2 abundance was detected among lethal (type II) and not-lethal (type III) OI patients, 17 proteins, with key effects on matrix structure and organization, cell signaling, and cell and tissue development and differentiation, were significantly different between type II and type III OI patients. Among them, some non–collagenous extracellular matrix (ECM) proteins (e.g., decorin and fibrillin-1) and proteins modulating cytoskeleton (e.g., nestin and palladin) directly correlate to the severity of the disease. Their defective presence may define proband-failure in balancing aberrances related to mutant collagen.


2015 ◽  
Vol 2015 ◽  
pp. 1-13 ◽  
Author(s):  
Cécile Beck ◽  
Philippe Desprès ◽  
Sylvie Paulous ◽  
Jessica Vanhomwegen ◽  
Steeve Lowenski ◽  
...  

West Nile virus (WNV), Japanese encephalitis virus (JEV), and tick-borne encephalitis virus (TBEV) are flaviviruses responsible for severe neuroinvasive infections in humans and horses. The confirmation of flavivirus infections is mostly based on rapid serological tests such as enzyme-linked immunosorbent assays (ELISAs). These tests suffer from poor specificity, mainly due to antigenic cross-reactivity among flavivirus members. Robust diagnosis therefore needs to be validated through virus neutralisation tests (VNTs) which are time-consuming and require BSL3 facilities. The flavivirus envelope (E) glycoprotein ectodomain is composed of three domains (D) named DI, DII, and DIII, with EDIII containing virus-specific epitopes. In order to improve the serological differentiation of flavivirus infections, the recombinant soluble ectodomain of WNV E (WNV.sE) and EDIIIs (rEDIIIs) of WNV, JEV, and TBEV were synthesised using theDrosophilaS2 expression system. Purified antigens were covalently bonded to fluorescent beads. The microspheres coupled to WNV.sE or rEDIIIs were assayed with about 300 equine immune sera from natural and experimental flavivirus infections and 172 nonimmune equine sera as negative controls. rEDIII-coupled microspheres captured specific antibodies against WNV, TBEV, or JEV in positive horse sera. This innovative multiplex immunoassay is a powerful alternative to ELISAs and VNTs for veterinary diagnosis of flavivirus-related diseases.


1977 ◽  
Vol 6 (3) ◽  
pp. 274-279
Author(s):  
Omar O. Barriga

Six diethylaminoethyl-cellulose fractions of a larval Trichinella spiralis extract, an Ascaris suum extract, and a nonrelated protein were used for cutaneous tests in guinea pigs with 8-, 14-, and 73-day-old T. spiralis infections, in guinea pigs with 13-day-old A. suum infections, and in normal guinea pigs. A selected T. spiralis fraction was used in hemagglutination (HA) tests with sera of 8 T. spiralis -infected rabbits, 41 sera of trichinellosis patients positive by bentonite agglutination tests, and 50 sera of clinically healthy persons. Immediate-type cutaneous reactions revealed extensive cross-reactivity between both parasites, although the establishment of conventional limits for considering a reaction positive allowed the specific diagnosis of acute or chronic trichinellosis with different fractions. Delayed-type reactions were specific with all fractions except one, and different fractions reacted during either the acute or the chronic phase of trichinellosis. HA detected anti- Trichinella antibodies in all the rabbits 9 to 10 days postinfection, in all trichinellosis patients, and in none of the healthy people. Correlation between HA and bentonite agglutination titers and other considerations suggest that HA with the selected fraction detects early antibodies. HA inhibition tests with A. suum extract suggest lack of HA cross-reactivity between the A. suum - and T. spiralis -selected fractions. The use of different fractions in diverse tests for clinical or epidemiological studies is suggested.


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