scholarly journals A Bacterial Glycoengineered Antigen for Improved Serodiagnosis of Porcine Brucellosis

2016 ◽  
Vol 54 (6) ◽  
pp. 1448-1455 ◽  
Author(s):  
María E. Cortina ◽  
Rodrigo E. Balzano ◽  
Diego A. Rey Serantes ◽  
Ana J. Caillava ◽  
Sebastián Elena ◽  
...  
Keyword(s):  
Laboratory Diagnosis ◽  
Enzyme Linked Immunosorbent Assay ◽  
Human Brucellosis ◽  
Human Beings ◽  
Serum Samples ◽  
Serological Tests ◽  
Recombinant Glycoprotein ◽  
Cutoff Value ◽  
Content Type ◽  
Porcine Brucellosis

Brucellosis is a highly zoonotic disease that affects animals and human beings.Brucella suisis the etiological agent of porcine brucellosis and one of the major human brucellosis pathogens. Laboratory diagnosis of porcine brucellosis mainly relies on serological tests, and it has been widely demonstrated that serological assays based on the detection of anti O-polysaccharide antibodies are the most sensitive tests. Here, we validate a recombinant glycoprotein antigen, anN-formylperosamine O-polysaccharide–protein conjugate (OAg-AcrA), for diagnosis of porcine brucellosis. An indirect immunoassay based on the detection of anti-O-polysaccharide IgG antibodies was developed coupling OAg-AcrA to enzyme-linked immunosorbent assay plates (glyco-iELISA). To validate the assay, 563 serum samples obtained from experimentally infected and immunized pigs, as well as animals naturally infected withB. suisbiovar 1 or 2, were tested. A receiver operating characteristic (ROC) analysis was performed, and based on this analysis, the optimum cutoff value was 0.56 (relative reactivity), which resulted in a diagnostic sensitivity and specificity of 100% and 99.7%, respectively. A cutoff value of 0.78 resulted in a test sensitivity of 98.4% and a test specificity of 100%. Overall, our results demonstrate that the glyco-iELISA is highly accurate for diagnosis of porcine brucellosis, improving the diagnostic performance of current serological tests. The recombinant glycoprotein OAg-AcrA can be produced in large homogeneous batches in a standardized way, making it an ideal candidate for further validation as a universal antigen for diagnosis of “smooth” brucellosis in animals and humans.

scholarly journals Immunodetection of Fasciola gigantica Circulating Antigen in Sera of Infected Individuals for Laboratory Diagnosis of Human Fascioliasis

2013 ◽  
Vol 20 (10) ◽  
pp. 1569-1577 ◽  
Author(s):  
Abdelfattah M. Attallah ◽  
Faisal A. Bughdadi ◽  
Atef M. El-Shazly ◽  
Hisham Ismail
Keyword(s):  
Laboratory Diagnosis ◽  
Fasciola Gigantica ◽  
Antigen Detection ◽  
Enzyme Linked Immunosorbent Assay ◽  
Target Antigen ◽  
Immunoperoxidase Staining ◽  
Serum Samples ◽  
Content Type ◽  
Human Fascioliasis ◽  
Circulating Antigen

ABSTRACTCurrently, the laboratory diagnosis of human fascioliasis is based on the parasitological examination of parasite eggs in stool specimens and serological detection of specific antibodies in serum samples, which are often unreliable diagnostic approaches. Ideally, a sensitive and specific diagnostic test forFasciolainfection should be based on the detection of circulatingFasciolaantigen, which implies active infection. Here, a 27-kDa-molecular-mass antigen was identified in aFasciola giganticaadult worm antigen preparation, excretory-secretory products, and sera fromF. gigantica-infected individuals, and it was not detected in antigenic extracts of other parasites and sera from noninfected individuals. The target antigen was isolated and partially characterized as a protein. Immunoperoxidase staining located the target epitope within teguments and guts ofF. giganticaadult worms. The performance characteristics of a newly developed enzyme-linked immunosorbent assay (ELISA) based onF. giganticacirculating antigen detection in serum (FgCA-27 ELISA) were investigated using sera of 120 parasitologically diagnosedF. gigantica-infected individuals and 80 noninfected individuals. The area under the receiving operating characteristic (ROC) curve (AUC) for ELISA was significantly high (AUC = 0.961,P< 0.0001) for discriminatingFasciola-infected and noninfected individuals. The developed assay showed high degrees of sensitivity, specificity, and efficiency (>93%), and a significant correlation (r= 0.715,P< 0.0001) between antigen level and parasite egg count was shown. In conclusion, a 27-kDaFasciolaantigen was identified in sera ofF. gigantica-infected individuals. A highly sensitive and specificFasciolaantigen detection assay, FgCA-27 ELISA, was developed for laboratory diagnosis of human fascioliasis.

scholarly journals B-Cell-Specific Peptides of Leptospira interrogans LigA for Diagnosis of Patients with Acute Leptospirosis

2014 ◽  
Vol 21 (3) ◽  
pp. 354-359 ◽  
Author(s):  
Murugesan Kanagavel ◽  
Santhanam Shanmughapriya ◽  
Kumarasamy Anbarasu ◽  
Kalimuthusamy Natarajaseenivasan
Keyword(s):  
Early Diagnosis ◽  
B Cell ◽  
Predictive Value ◽  
Acute Stage ◽  
Antibody Detection ◽  
Enzyme Linked Immunosorbent Assay ◽  
Serum Samples ◽  
Serological Tests ◽  
Content Type

ABSTRACTLeptospirosis is a reemerging infectious disease that is underdiagnosed and under-recognized due to low-sensitivity and cumbersome serological tests. Rapid reliable alternative tests are needed for early diagnosis of the disease. Considering the importance of the pathogenesis-associated leptospiral LigA protein expressedin vivo, we have evaluated its application in the diagnosis of the acute form of leptospirosis. The C-terminal coding sequence ofligA(ligA-C) was cloned into pET15b and expressed inEscherichia coli. Furthermore, the B-cell-specific epitopes were predicted and were synthesized as peptides for evaluation along with recombinant LigA-C. Epitope 1 (VVIENTPGK), with a VaxiJen score of 1.3782, and epitope 2 (TALSVGSSK), with a score of 1.2767, were utilized. A total of 140 serum samples collected from leptospirosis cases during the acute stage of the disease and 138 serum samples collected from normal healthy controls were utilized for evaluation. The sensitivity, specificity, positive predictive value, and negative predictive value were calculated for the recombinant LigA-C-specific IgM enzyme-linked immunosorbent assay (ELISA) and were found to be 92.1%, 97.7%, 92.8%, and 97.5%, respectively. Epitopes 1 and 2 used in the study showed 5.1 to 5.8% increased sensitivity over recombinant LigA-C in single and combination assays for IgM antibody detection. These findings suggest that these peptides may be potential candidates for the early diagnosis of leptospirosis.

scholarly journals Development and Comparative Evaluation of a Plate Enzyme-Linked Immunosorbent Assay Based on Recombinant Outer Membrane Antigens Omp28 and Omp31 for Diagnosis of Human Brucellosis

2013 ◽  
Vol 20 (8) ◽  
pp. 1217-1222 ◽  
Author(s):  
Sapana Tiwari ◽  
Ashu Kumar ◽  
Duraipandian Thavaselvam ◽  
Smita Mangalgi ◽  
Vedika Rathod ◽  
...  
Keyword(s):  
Outer Membrane ◽  
Immunosorbent Assay ◽  
Expression System ◽  
Disease Diagnosis ◽  
Cross Reactivity ◽  
Clinical Samples ◽  
Enzyme Linked Immunosorbent Assay ◽  
Human Brucellosis ◽  
Serological Tests ◽  
Content Type

ABSTRACTBrucellosis is an important zoonotic infectious disease of humans and livestock with worldwide distribution and is caused by bacteria of the genusBrucella. The diagnosis of brucellosis always requires laboratory confirmation by either isolation of pathogens or detection of specific antibodies. The conventional serological tests available for the diagnosis of brucellosis are less specific and show cross-reactivity with other closely related organisms. These tests also necessitate the handling ofBrucellaspecies for antigen preparation. Therefore, there is a need to develop reliable, rapid, and user-friendly systems for disease diagnosis and alternatives to vaccine approaches. Keeping in mind the importance of brucellosis as an emerging infection and the prevalence in India, we carried out the present study to compare the recombinant antigens with the native antigens (cell envelope and sonicated antigen) ofBrucellafor diagnosis of human brucellosis by an indirect plate enzyme-linked immunosorbent assay (ELISA). Recombinant outer membrane protein 28 (rOmp28) and rOmp31 antigens were cloned, expressed, and purified in the bacterial expression system, and the purified proteins were used as antigens. Indirect plate ELISAs were then performed and standardized for comparison of the reactivities of recombinant and native antigens against the 433 clinical samples submitted for brucellosis testing, 15 culture-positive samples, and 20 healthy donor samples. The samples were separated into four groups based on their positivity to rose bengal plate agglutination tests (RBPTs), standard tube agglutination tests (STATs), and 2-mercaptoethanol (2ME) tests. The sensitivities and specificities of all the antigens were calculated, and the rOmp28 antigen was found to be more suitable for the clinical diagnosis of brucellosis than the rOmp31 antigen and native antigens. The rOmp28-based ELISA showed a very high degree of agreement with the conventional agglutination tests and promising results for further use in clinical screening and serodiagnosis of human brucellosis.

scholarly journals Use of a Toxin Neutralization Assay To Characterize the Serologic Response to Adenylate Cyclase Toxin after Infection with Bordetella pertussis

2016 ◽  
Vol 24 (1) ◽  
Author(s):  
Joshua C. Eby ◽  
Mary C. Gray ◽  
Jason M. Warfel ◽  
Tod J. Merkel ◽  
Erik L. Hewlett
Keyword(s):  
Adenylate Cyclase ◽  
Bordetella Pertussis ◽  
Neutralization Assay ◽  
Enzyme Linked Immunosorbent Assay ◽  
Immunologic Response ◽  
Serum Samples ◽  
Content Type ◽  
Adenylate Cyclase Toxin ◽  
Strong Candidate ◽  
Candidate Antigen

ABSTRACT Adenylate cyclase toxin (ACT) is an essential virulence factor of Bordetella pertussis, and antibodies to ACT protect against B. pertussis infection in mice. The toxin is therefore a strong candidate antigen for addition to future acellular pertussis vaccines. In order to characterize the functionality of the immunologic response to ACT after infection, we developed an assay for testing the ability of serum samples from subjects infected with B. pertussis to neutralize ACT-induced cytotoxicity in J774 macrophage cells. Baboons develop neutralizing anti-ACT antibodies following infection with B. pertussis, and all sera from baboons with positive anti-ACT IgG enzyme-linked immunosorbent assay (ELISA) results neutralized ACT cytotoxicity. The toxin neutralization assay (TNA) was positive in some baboon sera in which ELISA remained negative. Of serum samples obtained from humans diagnosed with pertussis by PCR, anti-ACT IgG ELISA was positive in 72%, and TNA was positive in 83%. All samples positive for anti-ACT IgG ELISA were positive by TNA, and none of the samples from humans without pertussis neutralized toxin activity. These findings indicate that antibodies to ACT generated following infection with B. pertussis consistently neutralize toxin-induced cytotoxicity and that TNA can be used to improve understanding of the immunologic response to ACT after infection or vaccination.

scholarly journals Serological Evaluation of Specific-Antibody Levels in Patients Treated for Chronic Chagas' Disease

2007 ◽  
Vol 15 (2) ◽  
pp. 297-302 ◽  
Author(s):  
Olga Sánchez Negrette ◽  
Fernando J. Sánchez Valdéz ◽  
Carlos D. Lacunza ◽  
María Fernanda García Bustos ◽  
María Celia Mora ◽  
...  
Keyword(s):  
Chagas Disease ◽  
Treatment Effectiveness ◽  
Enzyme Linked Immunosorbent Assay ◽  
Serum Samples ◽  
Serological Tests ◽  
Recombinant Antigens ◽  
Antibody Titers ◽  
Laboratory Procedures ◽  
The Individual ◽  
Antibody Levels

ABSTRACT Serological tests are the main laboratory procedures used for diagnosis during the indeterminate and chronic stages of Chagas' disease. A serological regression to negativity is the main criterion used to define parasitological cure in treated patients. The aim of this work was to monitor the individual specificities of antibody levels for 3 years posttreatment in 18 adult patients. Conventional serological techniques (hemagglutination assays and enzyme-linked immunosorbent assay [ELISA]) were modified by using recombinant antigens to detect early markers of treatment effectiveness. For this purpose, serum samples were taken before and during treatment and every 6 months after treatment for at least 3 years. When hemagglutination assays were used, a decrease in antibody levels was observed in only one patient. When ELISA with serum dilutions was used, antibody clearance became much more apparent: in 77.7% (14/18) of the patients, antibody titers became negative with time. This was observed at serum dilutions of 1/320 and occurred between the 6th and the 30th months posttreatment. The immune response and the interval for a serological regression to negativity were different for each patient. For some of the recombinant antigens, only 50% (9/18) of the patients reached the serological regression to negativity. Recombinant antigen 13 might be a good marker of treatment effectiveness, since 66.6% (six of nine) of the patients presented with an early regression to negativity for specific antibodies to this antigen (P = 0.002).

scholarly journals Increased detection of schistosomiasis with Kato-Katz and SWAP-IgG-ELISA in a Northeastern Brazil low-intensity transmission area

2012 ◽  
Vol 45 (4) ◽  
pp. 510-513 ◽  
Author(s):  
Teiliane Rodrigues Carneiro ◽  
Marta Cristhiany Cunha Pinheiro ◽  
Sara Menezes de Oliveira ◽  
Ana Lúcia de Paula Hanemann ◽  
José Ajax Nogueira Queiroz ◽  
...  
Keyword(s):  
Serological Test ◽  
Laboratory Diagnosis ◽  
Elisa Test ◽  
Northeastern Brazil ◽  
Enzyme Linked Immunosorbent Assay ◽  
Serological Tests ◽  
Predictive Values ◽  
Transmission Area ◽  
Elisa Technique ◽  
Endemic Areas

INTRODUCTION: The laboratory diagnosis of schistosomiasis is based mainly on the detection of parasite eggs in stool samples through the Kato-Katz (KK) technique, reading one slide by test. However, a widely known limitation of parasitological methods is reduced sensitivity, particularly in low endemic areas. METHODS: To increase sensitivity, we conducted further slide readings from the same stool sample using the parasitological method associated with a serological test. We used the KK method (three slides) and the IgG anti-Schistosoma mansoni-enzyme-linked immunosorbent assay (ELISA) technique to diagnose schistosomiasis in low endemic areas in the Brazilian State of Ceará. Fecal samples and sera from 250 individuals were analyzed. RESULTS: Sixteen percent and 47.2% of samples were positive in parasitological tests and serological tests, respectively. Parasitological methods showed that 32 (80%) individuals tested positive on the first slide, 6 (15%) on the second slide, and 2 (5%) on the third. The performance of the ELISA test in the diagnosis, using the KK method as diagnostic reference, showed a negative predictive value of 100%, with specificity and positive predictive values of 62.8% and 33.9%, respectively. CONCLUSIONS: In this study, the increase from one to three slides analyzed per sample using the KK technique was shown to be a useful procedure for increasing the diagnostic sensitivity of this technique.

scholarly journals Serodiagnosis of Louse-Borne Relapsing Fever with Glycerophosphodiester Phosphodiesterase (GlpQ) fromBorrelia recurrentis

2000 ◽  
Vol 38 (10) ◽  
pp. 3561-3571 ◽  
Author(s):  
Stephen F. Porcella ◽  
Sandra J. Raffel ◽  
Merry E. Schrumpf ◽  
Martin E. Schriefer ◽  
David T. Dennis ◽  
...  
Keyword(s):  
Serological Test ◽  
Geometric Mean ◽  
Eastern Africa ◽  
Enzyme Linked Immunosorbent Assay ◽  
Relapsing Fever ◽  
Serum Samples ◽  
Serological Tests ◽  
Whole Cell ◽  
Convalescent Phase ◽  
Glycerophosphodiester Phosphodiesterase

Human louse-borne relapsing fever occurs in sporadic outbreaks in central and eastern Africa that are characterized by significant morbidity and mortality. Isolates of the causative agent,Borrelia recurrentis, were obtained from the blood of four patients during a recent epidemic of the disease in southern Sudan. TheglpQ gene, encoding glycerophosphodiester phosphodiesterase, from these isolates was sequenced and compared with the glpQ sequences obtained from other relapsing-fever spirochetes. Previously we showed that GlpQ of Borrelia hermsii is an immunogenic protein with utility as a serological test antigen for discriminating tick-borne relapsing fever from Lyme disease. In the present work, we cloned and expressed theglpQ gene from B. recurrentis and used recombinant GlpQ in serological tests. Acute- and convalescent-phase serum samples obtained from 42 patients with louse-borne relapsing fever were tested with an indirect immunofluorescence assay (IFA) and an enzyme-linked immunosorbent assay (ELISA) that used whole cells ofB. recurrentis and with immunoblotting to whole-cell lysates of the spirochete and Escherichia coli producing recombinant GlpQ. The geometric mean titers of the acute- and convalescent-phase serum samples measured by IFA were 1:83 and 1:575, respectively. The immunoblot analysis identified a high level of reactivity and seroconversion to GlpQ, and the assay was more sensitive than the whole-cell IFA and ELISA using purified, recombinant histidine-tagged GlpQ. Serum antibodies to GlpQ and other antigens persisted for 27 years in one patient. We conclude that assessment of anti-GlpQ antibodies will allow serological confirmation of louse-borne relapsing fever and determination of disease prevalence.

Sero - Prevalence and Risk Factors of Cattle and Human Brucellosis at Seka Chokorsa and Shebe Sonbo Jimma Zone Districts, South West Ethiopia

2019 ◽  
Author(s):  
Ftsum Assefa Tokon ◽  
Benti Deresa Gelalcha ◽  
Teferi Benti Moti ◽  
Redeat Belaneh Alemu ◽  
Hailu Degefu Awash
Keyword(s):  
Public Health ◽  
Risk Factors ◽  
Brucella Abortus ◽  
Enzyme Linked Immunosorbent Assay ◽  
Human Brucellosis ◽  
Bacterial Disease ◽  
Serum Samples ◽  
Public Health Importance ◽  
Herd Level ◽  
Jimma Zone

Abstract Background: Brucellosis is contagious bacterial disease of major socio-economic and public health importance globally and it is also one of the priority zoonotic diseases in Ethiopia. Across-sectional epidemiological study was carried out from April 2017 to April 2018 to estimate the sero prevalence of bovine and human brucellosis and to assess the associated risk factors of brucellosis in Seka Chokorsa and Shebe Sonbo districts of Jimma zone. Results: The overall prevalence of cattle brucella infection at individual and herd level were 5.9% (95%CI: 4.1%-8.1%) and 26.6 (95%CI: 19.1%-35.3%) based on diagnosis using commercial kits of the competitive enzyme-linked Immunosorbent assay (CELISA) from Brucella abortus antibodies. Univariate logistic regression analysis at individual animal level showed that animals from large herd size and households which had a practice of introduction of new animals in their herds were 3.7 and 2.3 times more likely to be seropositive, respectively. The same scenario has been observed at herd level. Molecularly, five Brucella abortus was identified as the species affecting cattle in the study areas. From the total human serum samples tested only one serum was found to be positive for CFT and thus the prevalence is 0.42 %,( 95%CI: 0.01%-2.38%). From 238 respondents, 90% of them drink raw milk and milk products, and 70% of them also eat raw meat. Furthermore, slaughtering, assisting during delivery and poor management of aborted material is a common practice that favor transmission of the pathogen in the area. Conclusions: In conclusion, the results of this study showed that brucellosis is an important and widely distributed disease in cattle in the study areas. The finding of the infection in human indicates the public health importance of brucellosis in the area. The risk behaviors and practices observed suggests the need to design all-inclusive health programmes, such one-health approach aimed at controlling brucellosis spread in the study area.

scholarly journals Mosquito Bite-Induced Controlled Human Malaria Infection withPlasmodium vivaxorP. falciparumGenerates Immune Responses to Homologous and Heterologous Preerythrocytic and Erythrocytic Antigens

2018 ◽  
Vol 87 (3) ◽  
Author(s):  
Cysha E. Hall ◽  
Lisa M. Hagan ◽  
Elke Bergmann-Leitner ◽  
Donna M. Tosh ◽  
Jason W. Bennett ◽  
...  
Keyword(s):  
Malaria Infection ◽  
Surface Protein ◽  
Merozoite Surface Protein ◽  
Similar Proportion ◽  
Enzyme Linked Immunosorbent Assay ◽  
Serum Samples ◽  
Content Type ◽  
Human Malaria ◽  
Before And After ◽  
Controlled Human Malaria Infection

ABSTRACTSeroepidemiological studies on the prevalence of antibodies to malaria antigens are primarily conducted on individuals from regions of endemicity. It is therefore difficult to accurately correlate the antibody responses to the timing and number of prior malaria infections. This study was undertaken to assess the evolution of antibodies to the dominant surface antigens ofPlasmodium vivaxandP. falciparumfollowing controlled human malaria infection (CHMI) in malaria-naive individuals. Serum samples from malaria-naive adults, collected before and after CHMI with eitherP. vivax(n= 18) orP. falciparum(n= 18), were tested for the presence of antibodies to the circumsporozoite protein (CSP) and the 42-kDa fragment of merozoite surface protein 1 (MSP-142) ofP. vivaxandP. falciparumusing an enzyme-linked immunosorbent assay (ELISA). Approximately 1 month following CHMI with eitherP. vivaxorP. falciparum, >60% of subjects seroconverted to homologous CSP and MSP-1. More than 50% of the subjects demonstrated reactivity to heterologous CSP and MSP-142, and a similar proportion of subjects remained seropositive to homologous MSP-142>5 months after CHMI. Computational analysis provides insight into the presence of cross-reactive responses. The presence of long-lived and heterologous reactivity and its functional significance, if any, need to be taken into account while evaluating malaria exposure in field settings.

scholarly journals Subtractive Phage Display Selection from Canine Visceral Leishmaniasis Identifies Novel Epitopes That Mimic Leishmania infantum Antigens with Potential Serodiagnosis Applications

2013 ◽  
Vol 21 (1) ◽  
pp. 96-106 ◽  
Author(s):  
Lourena E. Costa ◽  
Mayara I. S. Lima ◽  
Miguel A. Chávez-Fumagalli ◽  
Daniel Menezes-Souza ◽  
Vivian T. Martins ◽  
...  
Keyword(s):  
Visceral Leishmaniasis ◽  
Phage Display ◽  
Leishmania Infantum ◽  
Cross Reactivity ◽  
Enzyme Linked Immunosorbent Assay ◽  
Serum Samples ◽  
Phage Display Technology ◽  
Predictive Values ◽  
Content Type ◽  
Phage Elisa

ABSTRACTVisceral leishmaniasis (VL) is a zoonotic disease that is endemic to Brazil, where dogs are the main domestic parasite reservoirs, and the percentages of infected dogs living in regions where canine VL (CVL) is endemic have ranged from 10% to 62%. Despite technological advances, some problems have been reported with CVL serodiagnosis. The present study describes a sequential subtractive selection through phage display technology from polyclonal antibodies of negative and positive sera that resulted in the identification of potential bacteriophage-fused peptides that were highly sensitive and specific to antibodies of CVL. A negative selection was performed in which phage clones were adhered to purified IgGs from healthy andTrypanosoma cruzi-infected dogs to eliminate cross-reactive phages. The remaining supernatant nonadhered phages were submitted to positive selection against IgG from the blood serum of dogs that were infected withLeishmania infantum. Phage clones that adhered to purified IgGs from the CVL-infected serum samples were selected. Eighteen clones were identified and their reactivities tested by a phage enzyme-linked immunosorbent assay (phage-ELISA) against the serum samples from infected dogs (n= 31) compared to those from vaccinated dogs (n= 21), experimentally infected dogs with cross-reactive parasites (n= 23), and healthy controls (n= 17). Eight clones presented sensitivity, specificity, and positive and negative predictive values of 100%, and they showed no cross-reactivity withT. cruzi- orEhrlichia canis-infected dogs or with dogs vaccinated with two different commercial CVL vaccines in Brazil. Our study identified eight mimotopes ofL. infantumantigens with 100% accuracy for CVL serodiagnosis. The use of these mimotopes by phage-ELISA proved to be an excellent assay that was reproducible, simple, fast, and inexpensive, and it can be applied in CVL-monitoring programs.

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