Improving the Sensitivity and the Specificity of HLA-B27 Typing by Replacing Serological Tests with a TaqMan-PCR Assay

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%

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Demonstrating the presence of Ehrlichia canis DNA from different tissues of dogs with suspected subclinical ehrlichiosis

Parasites & Vectors ◽

10.1186/s13071-020-04363-0 ◽

2020 ◽

Vol 13 (1) ◽

Author(s):

Carlos A. Rodríguez-Alarcón ◽

Diana M. Beristain-Ruiz ◽

Angélica Olivares-Muñoz ◽

Andrés Quezada-Casasola ◽

Federico Pérez-Casio ◽

...

Keyword(s):

Bone Marrow ◽

Lymph Node ◽

Cross Reactivity ◽

Ehrlichia Canis ◽

Serological Tests ◽

Blood Samples ◽

Negative Results ◽

Target Organs ◽

Polymerase Chain ◽

Positive Results

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.

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Demonstrating the presence of Ehrlichia canis DNA from different tissues of dogs with negative PCR in blood

10.21203/rs.2.23276/v4 ◽

2020 ◽

Author(s):

Carlos Arturo Rodríguez-Alarcón ◽

Diana M. Beristain-Ruiz ◽

Angélica Olivares-Muñoz ◽

Andrés Quezada-Casasola ◽

Federico Pérez-Casio ◽

...

Keyword(s):

Bone Marrow ◽

Lymph Node ◽

Cross Reactivity ◽

Ehrlichia Canis ◽

Serological Tests ◽

Blood Samples ◽

Negative Results ◽

Target Organs ◽

Polymerase Chain ◽

Positive Results

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.

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False Positive Results in SARS-CoV-2 Serological Tests for Samples From Patients With Chronic Inflammatory Diseases

Frontiers in Immunology ◽

10.3389/fimmu.2021.666114 ◽

2021 ◽

Vol 12 ◽

Author(s):

Nastya Kharlamova ◽

Nicky Dunn ◽

Sahl K. Bedri ◽

Svante Jerling ◽

Malin Almgren ◽

...

Keyword(s):

Lupus Erythematosus ◽

False Positive ◽

Inflammatory Diseases ◽

Serological Tests ◽

Chronic Inflammatory Diseases ◽

Negative Results ◽

False Positive Signal ◽

Lateral Flow Assays ◽

Positive Results ◽

Multiplex Bead

Patients with chronic inflammatory diseases are often treated with immunosuppressants and therefore are of particular concern during the SARS-CoV-2 pandemic. Serological tests will improve our understanding of the infection and immunity in this population, unless they tests give false positive results. The aim of this study was to evaluate the specificity of SARS-Cov-2 serological assays using samples from patients with chronic inflammatory diseases collected prior to April 2019, thus defined as negative. Samples from patients with multiple sclerosis (MS, n=10), rheumatoid arthritis (RA, n=47) with or without rheumatoid factor (RF) and/or anti-cyclic citrullinated peptide antibodies (anti-CCP2) and systemic lupus erythematosus (SLE, n=10) with or without RF, were analyzed for SARS-CoV-2 antibodies using 17 commercially available lateral flow assays (LFA), two ELISA kits and one in-house developed IgG multiplex bead-based assay. Six LFA and the in-house validated IgG assay correctly produced negative results for all samples. However, the majority of assays (n=13), gave false positive signal for samples from patients with RA and SLE. This was most notable in samples from RF positive RA patients. No false positive samples were detected in any assay using samples from patients with MS. Poor specificity of commercial serological assays could possibly be, at least partly, due to interfering antibodies in samples from patients with chronic inflammatory diseases. For these patients, the risk of false positivity should be considered when interpreting results of the SARS-CoV-2 serological assays.

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Identification of etiological agents of selected bacterial and viral infections based on serological tests

Postępy Higieny i Medycyny Doświadczalnej ◽

10.5604/01.3001.0012.8266 ◽

2018 ◽

Vol 72 ◽

pp. 1162-1178

Author(s):

Aleksandra Lewandowicz-Uszyńska ◽

Piotr Naporowski ◽

Gerard Pasternak ◽

Danuta Witkowska

Keyword(s):

False Positive ◽

Cellular Response ◽

Bacterial Infections ◽

Viral Infections ◽

False Negative ◽

Main Role ◽

Serological Tests ◽

Negative Results ◽

False Negative Results ◽

Positive Results

The human immune system’s response to infection is closely related with the type of pathogen. First, a rapid, metabolically inexpensive and non-specific innate immunity is induced, then a specific acquired immunity is activated. In bacterial infections caused by intracellular pathogens, the main role is played by cellular response. In infections caused by bacterial extracellular pathogens, a crucial role is played by antibodies. The clinical symptoms of bacterial and viral infections very often are similar, which is why diagnosing them based only on medical history and physical examination is insufficient. To identify the etiological factors of infections differentiating media, biochemical tests, molecular methods and serological tests are used. The detection of microorganisms or their genetic material can be performed within a short time after the occurrence of an infection. The detection of antibodies is possible only in the appropriate time called the serological window. In a serological diagnostic of infections there are problems with an appropriate interpretation of obtained results. Cross-reactivity can give false positive results for the diagnosis of Chlamydophila pneumonia infection. The problem with the detection of Borrelia burgdorferi infection can be caused by a simultaneous coinfection with different spirochetes, syphilis, mononucleosis or HIV. In serological diagnostics of bacterial infections, the administration of antibiotics to patients before taking serum samples can be responsible for false negative results. Another reason for such results can be a weak humoral response in infected patients. In viral infections, false positive results can be caused by a coinfection of different viruses, especially from the same family or by bacterial or protozoal coinfections or by autoimmune diseases. False-negative results in viral infections often are caused by the early phase of an infection. To properly recognize an etiological factor of infection it is necessary to use an appropriate method, precision of test and collect samples at the appropriate time.

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Serology in COVID-19: Comparison of Two Methods

International Journal of Environmental Research and Public Health ◽

10.3390/ijerph18126497 ◽

2021 ◽

Vol 18 (12) ◽

pp. 6497

Author(s):

Anna Moniuszko-Malinowska ◽

Wojciech Jelski ◽

Justyna Dunaj ◽

Barbara Mroczko ◽

Piotr Czupryna ◽

...

Keyword(s):

Infectious Diseases ◽

Nucleocapsid Protein ◽

Spike Protein ◽

Serological Tests ◽

Negative Results ◽

Positive Results ◽

Test Result

Background: The aim of our study was to examine the performance of two assays in detecting SARS-CoV-2 antibodies. Methods: A total of 127 COVID-19 disease contacts from the Infectious Diseases Department were included. Two serological tests were used: SARS-CoV-2 IgG CMIA on the Alinity system (Abbott) and LIAISON® SARS-CoV-2 S1/S2 IgG CLIA (DiaSorin). Results: The assays exhibited a 96.85% (123/127 patients) test result agreement. In two cases, the positive results obtained by SARS-CoV-2 IgG CMIA on the Alinity system (Abbott) were negative based on the LIAISON® SARS-CoV-2 S1/S2 IgG CLIA (DiaSorin) test, and in two cases, negative results from the LIAISON® SARS-CoV-2 S1/S2 IgG CLIA (DiaSorin) test were positive with the SARS-CoV-2 IgG CMIA on the Alinity system (Abbott). Conclusions: Based on the results of our study, we conclude that in population medicine, the assessments of anti-SARS-CoV-2 antibodies after exposure to SARS-CoV-2 virus based on spike protein or nucleocapsid protein show comparable effectiveness.

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Demonstrating the presence of Ehrlichia canis DNA from different tissues of dogs with suspected subclinical ehrlichiosis

10.21203/rs.2.23276/v2 ◽

2020 ◽

Author(s):

Carlos Arturo Rodríguez-Alarcón ◽

Diana M. Beristain-Ruiz ◽

Angélica Olivares-Muñoz ◽

Andrés Quezada-Casasola ◽

Federico Pérez-Casio ◽

...

Keyword(s):

Bone Marrow ◽

Cross Reactivity ◽

Ehrlichia Canis ◽

Morbidity And Mortality ◽

Blood Samples ◽

Serologic Tests ◽

Negative Results ◽

Target Organs ◽

Positive Results ◽

Different Tissues

Abstract Background: Nowadays, Ehrlichia canis receives more attention because of its great morbidity and mortality in animals. Dogs in the subclinical and chronic phases can be asymptomatic, and serologic 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 negative by PCR in blood. Methods: We evaluated by PCR the prevalence of E. canis in blood, liver, spleen, lymphatic nodules, and bone marrow in 59 recently euthanized 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% yield positive results in tissue biopsies as follows: 63.15% from bone marrow, 52.63% from liver, 47.36% from spleen and 15.78% from lymphatic nodules. In addition, 33% had infection in three tissues (spleen, liver and bone marrow). Conclusions: Our results show prevalence of E. canis in tissue from dogs that were negative by PCR in blood. E. canis DNA in tissue was 30% lower in dogs that tested negative in blood samples by PCR, 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 others tissues.

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SARS-CoV-2 serological tests can generate false positive results for samples from patients with chronic inflammatory diseases

10.1101/2020.11.13.20231076 ◽

2020 ◽

Author(s):

Nastya Kharlamova ◽

Nicky Dunn ◽

Sahl K Bedri ◽

Svante Jerling ◽

Malin Almgren ◽

...

Keyword(s):

Lupus Erythematosus ◽

False Positive ◽

Inflammatory Diseases ◽

Serological Tests ◽

Chronic Inflammatory Diseases ◽

Negative Results ◽

False Positive Signal ◽

Lateral Flow Assays ◽

Positive Results ◽

Multiplex Bead

AbstractObjectivesPatients with chronic inflammatory diseases are often treated with immunosuppressants and therefore are of particular concern during the SARS-CoV-2 pandemic. Serological tests will improve our understanding of the infection and immunity in this population, unless the tests give false positive results. The aim of this study was to evaluate the specificity of SARS-Cov-2 serological assays with samples from patients with chronic inflammatory diseases collected before April 2019, thus defined as negative.MethodsSamples from patients with multiple sclerosis (MS, n=10), rheumatoid arthritis (RA, n=47) with or without rheumatoid factor (RF) and/or anti-cyclic citrullinated peptide antibodies (anti-CCP2) and RF +/- systemic lupus erythematosus (SLE, n=10), were tested with 17 commercially available lateral flow assays (LFA), two ELISA kits and one in-house developed multiplex bead-based assay.ResultsSix LFA and the in-house IgG assay gave the correct negative results for all samples. However, the majority of assays (n=13), gave false positive signal with samples from patients with RA and SLE. This was most notable in RF positive RA samples. MS samples did not give any false positive in any of the assays.ConclusionThe majority of the verified serological assays were sensitive to interfering antibodies in samples from patients with chronic inflammatory diseases and therefore may have poor specificity in this context. For these patients, the risk of false positivity should be considered when interpreting results of the SARS-CoV-2 serological assays.

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Serology in COVID-19 – Comparison of Two Methods

10.21203/rs.3.rs-122628/v1 ◽

2020 ◽

Author(s):

Anna Moniuszko-Malinowska ◽

Wojciech Jelski ◽

Justyna Dunaj ◽

Barbara Mroczko ◽

Piotr Czupryna ◽

...

Keyword(s):

Serological Tests ◽

S Protein ◽

Negative Results ◽

The Difference ◽

Positive Results

Abstract The aim of our study was to investigate the difference in the anti-SARS-CoV-2 antibodies assessment with two various assays. 127 patients exposed to SARS-CoV-2 were included. Two serological tests were used: SARS-CoV-2 IgG CMIA on the Alinity system (ABBOTT) and LIAISON® SARS-CoV-2 S1/S2 IgG CLIA (DiaSorin). Both assays exhibited 96.85% (123/127 patients) overall compatibility. In 2 cases the positive results in N-based test, were negative in S-based test, and in 2 cases negative results in S-based test were positive in N-based test. Based on the results of our study we concluded that the assessment of anti-SARS-CoV-2 antibodies against N and S protein in population medicine shows comparable usefulness of both tests.

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Immunoblot Assay Using Recombinant Antigens as a Supplemental Test To Confirm the Presence of Antibodies to Trypanosoma cruzi

Clinical and Vaccine Immunology ◽

10.1128/cvi.00401-06 ◽

2007 ◽

Vol 14 (4) ◽

pp. 355-361 ◽

Cited By ~ 31

Author(s):

Kevin Y. Cheng ◽

Chi-Deu Chang ◽

Vince A. Salbilla ◽

Louis V. Kirchhoff ◽

David A. Leiby ◽

...

Keyword(s):

Trypanosoma Cruzi ◽

False Positive ◽

Test Results ◽

Serological Tests ◽

Assay Sensitivity ◽

Immunoblot Assay ◽

Recombinant Antigens ◽

Persistent Problem ◽

Negative Results ◽

Positive Results

ABSTRACT The diagnosis of chronic Chagas' disease is generally made by detecting antibodies to Trypanosoma cruzi. Most conventional serological tests are based on lysates of whole parasites or semipurified antigen fractions from T. cruzi epimastigotes grown in culture. The occurrence of inconclusive and false-positive results has been a persistent problem with the conventional assays, and there is no universally accepted gold standard for confirmation of positive test results. We describe here an immunoblot assay for detecting antibodies to T. cruzi in which four chimeric recombinant antigens (rAgs), designated FP3, FP6, FP10, and TcF, are used as target antigens. Each of these rAgs is composed of several antigenically distinct regions and includes repetitive as well as nonrepetitive sequences. Each rAg is coated as a discrete line on a nitrocellulose strip. Assay sensitivity was assessed by testing 345 specimens known to be positive for antibodies to T. cruzi. All 345 of these samples showed two to four reactive test bands in addition to the three on-board control bands that are on each strip. Assay specificity was determined by testing 500 specimens from random U.S. blood donors, all of which gave negative results. Based on the results obtained in this study, we propose the following scheme for interpretation of test results: (i) no bands or a single test band = a negative result; (ii) two or more test bands with at least one band showing intensity of 1+ or higher = a positive result; and (iii) multiple faint test bands (±) = indeterminate result. Based on this scheme, the prototype immunoblot assay showed sensitivity of 100% (n = 345) and specificity of 100% (n = 500). Additionally, all 269 potentially cross-reacting and T. cruzi antibody-negative specimens tested negative in our immunoblot assay. The rAg-based immunoblot assay has potential as a supplemental test for confirming the presence of antibodies to T. cruzi in blood specimens and for identifying false-positive results obtained with other assays.

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