scholarly journals Performance assessment of a multi-epitope chimeric antigen for the serological diagnosis of acute Mayaro fever

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Paloma Napoleão-Pêgo ◽  
Flávia R. G. Carneiro ◽  
Andressa M. Durans ◽  
Larissa R. Gomes ◽  
Carlos M. Morel ◽  
...  
Keyword(s):  
High Performance ◽  
Clinical Symptoms ◽  
Chimeric Protein ◽  
Cross Reactivity ◽  
Effective Control ◽  
Antigenic Determinants ◽  
Gene Encoding ◽  
Accurate Identification ◽  
Serological Tests ◽  
Mayaro Virus

AbstractMayaro virus (MAYV), which causes mayaro fever, is endemic to limited regions of South America that may expand due to the possible involvement of Aedes spp. mosquitoes in its transmission. Its effective control will require the accurate identification of infected individuals, which has been restricted to nucleic acid-based tests due to similarities with other emerging members of the Alphavirus genus of the Togaviridae family; both in structure and clinical symptoms. Serological tests have a more significant potential to expand testing at a reasonable cost, and their performance primarily reflects that of the antigen utilized to capture pathogen-specific antibodies. Here, we describe the assembly of a synthetic gene encoding multiple copies of antigenic determinants mapped from the nsP1, nsP2, E1, and E2 proteins of MAYV that readily expressed as a stable chimeric protein in bacteria. Its serological performance as the target in ELISAs revealed a high accuracy for detecting anti-MAYV IgM antibodies. No cross-reactivity was observed with serum from seropositive individuals for dengue, chikungunya, yellow fever, Zika, and other infectious diseases as well as healthy individuals. Our data suggest that this bioengineered antigen could be used to develop high-performance serological tests for MAYV infections.

scholarly journals Performance assessment of a multi-epitope chimeric antigen for the serological diagnosis of acute Mayaro fever

2021 ◽  
Author(s):  
Paloma Napoleão-Pêgo ◽  
Flávia R. G. Carneiro ◽  
Andressa M. Durans ◽  
Carlos M. Morel ◽  
David W. Provance ◽  
...  
Keyword(s):  
High Performance ◽  
Clinical Symptoms ◽  
Chimeric Protein ◽  
Cross Reactivity ◽  
Effective Control ◽  
Antigenic Determinants ◽  
Serological Tests ◽  
Molecular Approaches ◽  
Mayaro Virus ◽  
Sensitivity Specificity

Abstract Mayaro virus (MAYV) is endemic to limited regions of South America and the possible involvement of Aedes spp. mosquitoes in its transmission increase the risk that its geographical range will reach greater populations. MAYV shares clinical symptoms and structural similarities with other emerging members of the alphavirus family such as Chikungunya (CHIKV), Ross River (RRV), and O'nyong nyong (ONNV) viruses, which complicate diagnosis and is currently restricted to nucleic acid-based tests. Effective control of the spread of these diseases will require the identification of infected individuals as well as the development of preventative and prophylactic therapies. Serological tests have a greater potential to expand testing at a reasonable cost than molecular approaches. Their performance largely reflects that of the antigen utilized to capture pathogen-specific antibodies. Here, we describe the assembly of a synthetic gene coding multiple copies of antigenic determinants mapped from the nsP1, nsP2, E1, and E2 proteins of MAYV that readily produced a stable chimeric protein in bacteria. The serological performance (AUC, sensitivity, specificity, and confidence intervals) of this multi-epitope protein as the target in ELISAs revealed a high diagnostic accuracy for the detection of specific anti-MAYV IgM antibodies. No cross-reactivity was observed with serum from seropositive individuals for CHIKV, yellow fever (vaccinated), dengue, Zika, and other infectious diseases as well as healthy individuals. Our data suggest that this bioengineered antigen could be used to develop high-performance serological tests for MAYV infections.

scholarly journals A High-Performance Multiplex Immunoassay for Serodiagnosis of Flavivirus-Associated Neurological Diseases in Horses

2015 ◽  
Vol 2015 ◽  
pp. 1-13 ◽  
Author(s):  
Cécile Beck ◽  
Philippe Desprès ◽  
Sylvie Paulous ◽  
Jessica Vanhomwegen ◽  
Steeve Lowenski ◽  
...  
Keyword(s):  
High Performance ◽  
Neurological Diseases ◽  
Expression System ◽  
Cross Reactivity ◽  
Encephalitis Virus ◽  
Serological Tests ◽  
Multiplex Immunoassay ◽  
Tick Borne Encephalitis ◽  
Tick Borne Encephalitis Virus ◽  
Negative Controls

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.

scholarly journals GenSeizer: A Multiplex PCR-Based Targeted Gene Sequencing Platform for Rapid and Accurate Identification of Major Mycobacterium Species

2020 ◽  
Author(s):  
Bing Li ◽  
Liyun Xu ◽  
Qi Guo ◽  
Jianhui Chen ◽  
Yanan Zhang ◽  
...  
Keyword(s):  
Multiplex Pcr ◽  
Clinical Symptoms ◽  
Simultaneous Detection ◽  
Gene Sequencing ◽  
Large Data ◽  
Cross Reactivity ◽  
Accurate Identification ◽  
Data Set ◽  
Sequencing Platform ◽  
Targeted Gene Sequencing

Mycobacterium tuberculosis (MTB) and non-tuberculous mycobacteria (NTM) infections often exhibit similar clinical symptoms. Timely and effective treatment relies on the rapid and accurate identification of species and resistance genotypes. In this study, a new platform (GenSeizer), which combines bioinformatics analysis of a large data set and multiplex PCR-based targeted gene sequencing, was developed to identify 10 major Mycobacterium species that cause pulmonary, as well as extrapulmonary, human diseases. Simultaneous detection of certain resistance erm(41) and rrl genotypes in M. abscessus was also feasible. This platform was specific and sensitive, exhibited no cross-reactivity among reference strains and a detection limit of 5 DNA copies or 50 CFU Mycobacterium/ml. In a blinded comparison, GenSeizer and multigene sequencing showed 100% agreement in the ability to identify 88 clinical, Mycobacterium isolates. The resistance genotypes, confirmed by whole genome sequencing of 30 M. abscessus strains, were also correctly identified by GenSeizer 100% of the time. These results indicate that GenSeizer is an efficient, reliable platform for diagnosing major pathogenic Mycobacterium species.

scholarly journals Multiepitope Proteins for the Differential Detection of IgG Antibodies against RBD of the Spike Protein and Non-RBD Regions of SARS-CoV-2

Vaccines ◽  
2021 ◽  
Vol 9 (9) ◽  
pp. 986
Author(s):  
Larissa R. Gomes ◽  
Andressa M. Durans ◽  
Paloma Napoleão-Pêgo ◽  
Jessica A. Waterman ◽  
Mariana S. Freitas ◽  
...  
Keyword(s):  
High Performance ◽  
De Novo ◽  
Chimeric Protein ◽  
Emerging Diseases ◽  
High Reactivity ◽  
Diagnostic Methods ◽  
Spike Protein ◽  
Spot Synthesis ◽  
Serological Tests ◽  
Large Populations

The COVID-19 pandemic has exposed the extent of global connectivity and collective vulnerability to emerging diseases. From its suspected origins in Wuhan, China, it spread to all corners of the world in a matter of months. The absence of high-performance, rapid diagnostic methods that could identify asymptomatic carriers contributed to its worldwide transmission. Serological tests offer numerous benefits compared to other assay platforms to screen large populations. First-generation assays contain targets that represent proteins from SARS-CoV-2. While they could be quickly produced, each actually has a mixture of specific and non-specific epitopes that vary in their reactivity for antibodies. To generate the next generation of the assay, epitopes were identified in three SARS-Cov-2 proteins (S, N, and Orf3a) by SPOT synthesis analysis. After their similarity to other pathogen sequences was analyzed, 11 epitopes outside of the receptor-binding domain (RBD) of the spike protein that showed high reactivity and uniqueness to the virus. These were incorporated into a ß-barrel protein core to create a highly chimeric protein. Another de novo protein was designed that contained only epitopes in the RBD. In-house ELISAs suggest that both multiepitope proteins can serve as targets for high-performance diagnostic tests. Our approach to bioengineer chimeric proteins is highly amenable to other pathogens and immunological uses.

scholarly journals Microbiological Laboratory Diagnosis of Human Brucellosis: An Overview

Pathogens ◽  
2021 ◽  
Vol 10 (12) ◽  
pp. 1623
Author(s):  
Giovanni Di Bonaventura ◽  
Silvia Angeletti ◽  
Andrea Ianni ◽  
Tommasangelo Petitti ◽  
Giovanni Gherardi
Keyword(s):  
Clinical Symptoms ◽  
Early Stage ◽  
Point Of Care ◽  
Laboratory Diagnosis ◽  
Cross Reactivity ◽  
Diagnostic Methods ◽  
Nucleic Acid Amplification ◽  
Human Brucellosis ◽  
Microbiological Analysis ◽  
Serological Tests

Brucella spp. are Gram-negative, non-motile, non-spore-forming, slow-growing, facultative intracellular bacteria causing brucellosis. Brucellosis is an endemic of specific geographic areas and, although underreported, represents the most common zoonotic infection, with an annual global incidence of 500,000 cases among humans. Humans represent an occasional host where the infection is mainly caused by B. melitensis, which is the most virulent; B. abortus; B. suis; and B. canis. A microbiological analysis is crucial to identifying human cases because clinical symptoms of human brucellosis are variable and aspecific. The laboratory diagnosis is based on three different microbiological approaches: (i) direct diagnosis by culture, (ii) indirect diagnosis by serological tests, and (iii) direct rapid diagnosis by molecular PCR-based methods. Despite the established experience with serological tests and highly sensitive nucleic acid amplification tests (NAATs), a culture is still considered the “gold standard” in the laboratory diagnosis of brucellosis due to its clinical and epidemiological relevance. Moreover, the automated BC systems now available have increased the sensitivity of BCs and shortened the time to detection of Brucella species. The main limitations of serological tests are the lack of common interpretative criteria, the suboptimal specificity due to interspecies cross-reactivity, and the low sensitivity during the early stage of disease. Despite that, serological tests remain the main diagnostic tool, especially in endemic areas because they are inexpensive, user friendly, and have high negative predictive value. Promising serological tests based on new synthetic antigens have been recently developed together with novel point-of-care tests without the need for dedicated equipment and expertise. NAATs are rapid tests that can help diagnose brucellosis in a few hours with high sensitivity and specificity. Nevertheless, the interpretation of NAAT-positive results requires attention because it may not necessarily indicate an active infection but rather a low bacterial inoculum, DNA from dead bacteria, or a patient that has recovered. Refined NAATs should be developed, and their performances should be compared with those of commercial and home-made molecular tests before being commercialized for the diagnosis of brucellosis. Here, we review and report the most common and updated microbiological diagnostic methods currently available for the laboratory diagnosis of brucellosis.

scholarly journals Cas12a/Guide RNA-Based Platform for Rapid and Accurate Identification of Major Mycobacterium Species

2019 ◽  
Vol 58 (2) ◽  
Author(s):  
Guohui Xiao ◽  
Xing He ◽  
Su Zhang ◽  
Yaya Liu ◽  
Zhihang Liang ◽  
...  
Keyword(s):  
Clinical Symptoms ◽  
Cost Effective ◽  
Cross Reactivity ◽  
Mycobacterium Abscessus ◽  
Mycobacterium Fortuitum ◽  
Accurate Identification ◽  
Content Type ◽  
Guide Rna ◽  
Promising Tool ◽  
Species Specific

ABSTRACT Mycobacterium tuberculosis infection and nontuberculous mycobacteria (NTM) infections exhibit similar clinical symptoms; however, the therapies for these two types of infections are different. Therefore, the rapid and accurate identification of M. tuberculosis and NTM species is very important for the control of tuberculosis and NTM infections. In the present study, a Cas12a/guide RNA (gRNA)-based platform was developed to identify M. tuberculosis and most NTM species. By designing species-specific gRNA probes targeting the rpoB sequence, a Cas12a/gRNA-based platform successfully identified M. tuberculosis and six major NTM species (Mycobacterium abscessus, Mycobacterium intracellulare, Mycobacterium avium, Mycobacterium kansasii, Mycobacterium gordonae, and Mycobacterium fortuitum) without cross-reactivity. In a blind assessment, a total of 72 out of 73 clinical Mycobacterium isolates were correctly identified, which is consistent with previous rpoB sequencing results. These results suggest that the Cas12a/gRNA-based platform is a promising tool for the rapid, accurate, and cost-effective identification of both M. tuberculosis and NTM species.

Chapter 10: Diagnosis

2019 ◽  
Author(s):  
Gerhard Dobler
Keyword(s):  
Cerebrospinal Fluid ◽  
Viral Encephalitis ◽  
Clinical Symptoms ◽  
Serological Tests ◽  
Specific Antibodies ◽  
Cross Reactions ◽  
Neuro Imaging ◽  
Tbev Infection

• TBE appears with non-characteristic clinical symptoms, which cannot be distinguished from oth-er forms of viral encephalitis or other diseases. • Cerebrospinal fluid and neuro-imaging may give some evidence of TBE, but ultimately cannot confirm the diagnosis. • Thus, proving the diagnosis “TBE” necessarily requires confirmation of TBEV-infection by detec-tion of the virus or by demonstration of specific antibodies from serum and/or cerebrospinal fluid. • During the phase of clinic symptoms from the CNS, the TBEV can only rarely be detected in the cerebrospinal fluid of patients. • Most routinely used serological tests for diagnosing TBE (ELISA, HI, IFA) show cross reactions resulting from either Infection with other flaviviruses or with other flavivirus vaccines.

Chapter 10: Diagnosis

2020 ◽  
Keyword(s):  
Cerebrospinal Fluid ◽  
Viral Encephalitis ◽  
Clinical Symptoms ◽  
Serological Tests ◽  
Specific Antibodies ◽  
Cross Reactions ◽  
Neuro Imaging ◽  
Tbev Infection

TBE appears with non-characteristic clinical symptoms, which cannot be distinguished from other forms of viral encephalitis or other diseases. Cerebrospinal fluid and neuro-imaging may give some evidence of TBE, but ultimately cannot confirm the diagnosis. Thus, proving the diagnosis “TBE” necessarily requires confirmation of TBEV-infection by detection of the virus or by demonstration of specific antibodies from serum and/or cerebrospinal fluid. During the phase of clinic symptoms from the CNS, the TBEV can only rarely be detected in the cerebrospinal fluid of patients. Most routinely used serological tests for diagnosing TBE (ELISA, HI, IFA) show cross reactions resulting from either infection with other flaviviruses or with other flavivirus vaccines.

Validity of a Serological Diagnostic Kit for SARS-CoV-2 Available in Iran

2020 ◽  
Vol 23 (9) ◽  
pp. 629-632
Author(s):  
Hamid Reza Shamsollahi ◽  
Mostafa Amini ◽  
Shaban Alizadeh ◽  
Saharnaz Nedjat ◽  
Ali Akbari-Sari ◽  
...  
Keyword(s):  
Diagnostic Techniques ◽  
Effective Control ◽  
Molecular Technique ◽  
Size Estimation ◽  
Medical Sciences ◽  
Serological Tests ◽  
Standard Case ◽  
Epidemic Size ◽  
Low Sensitivity ◽  
Negative Controls

Background: The Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) epidemic broke out in December 2019 and is now characterized as a pandemic. Effective control of this infectious disease requires access to diagnostic techniques, for both case finding and epidemic size estimation. The molecular technique is routinely used worldwide. Although it is the "standard" case detection and management method, it has its own shortcomings. Thus, some easy-to-use rapid serological tests have been developed. Methods: One hundred and fourteen positive RT-PCR-diagnosed patients were tested by VivaDiag Kit, a brand of rapid serological kits available in hospitals affiliated to Tehran University of Medical Sciences (TUMS), Tehran, Iran. Frozen serum specimens taken from healthy people in summer and fall 2019 were also tested as negative controls. Results: Test sensitivity was 47.9% (95% confidence interval [CI]: 38.8-56.9) for IgM and 47.0% (95% CI: 38.0–56.0) for IgG. There was no difference between IgG and IgM seropositivity except in one case. Specificity was calculated as 99.0% (95% CI: 96.4–99.9) for IgM and of 100.0% (95% CI: 0.98.2–100.0) for IgG. Sensitivity was higher in men and older participants. Conclusion: This test can be used for epidemiological investigations, especially for the estimation of the level of infection in the community, after it is properly corrected for sensitivity and specificity. The low sensitivity could be attributed to the technical limitations of the kit or low levels of antibodies after infection. The different sensitivity in age and sex groups supports the hypothesis that different people show different immune responses to this virus.

Enterovirus D-68 Molecular Virology, Epidemiology, and Treatment: an update and way forward

2020 ◽  
Vol 20 ◽  
Author(s):  
Mahmoud Ahmed Ebada ◽  
Notila Fayed ◽  
Souad Alkanj ◽  
Ahmed Wadaa Allah
Keyword(s):  
Current Knowledge ◽  
Rna Virus ◽  
Neurological Diseases ◽  
Cross Reactivity ◽  
Serological Tests ◽  
Molecular Virology ◽  
Acute Flaccid Myelitis ◽  
The Family ◽  
Pcr Products ◽  
Enterovirus D68

: 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.

Sign in / Sign up

Export Citation Format

Share Document