Serological Diagnostic Tests Research Articles

Diagnostic Tests for SARS COV-2: Application, Challenges and Recommendations

Background: The recent pandemic of human coronavirus infections, at the beginning of the 21st century, have shown the prominent roles of rapid and accurate diagnostic technologies to contain the emerging and re-emerging infections. To control such a large-scale crisis, early identification and prevention of infection are essential. Prompt identification of cases relies on the accurate diagnostic technologies. Before analysis, appropriate sample collection is essential for making a precise molecular diagnosis of SARS-COV-2 (Severe Acute Respiratory Syndrome Coronavirus-2). To prevent further spread of infection and obtain consistent results, proper preventive measures must be taken into consideration to keep laboratory staff safe. Despite the development of specific and sensitive point-of-care tests and serological immunoassays which are considered supplementary methods for diagnosing SARS-COV-2, RT-PCR (Reverse-Transcriptase Polymerase-Chain-Reaction) remains the gold standard performed on respiratory specimens. Upon completion of the sample analysis, testing results must be interpreted using both molecular and serological findings. Finally, random-access, integrated devices available for the purpose of care with scalable capacities will facilitate the rapid and accurate diagnosis and monitoring of Severe Acute Respiratory Syndrome Corona Virus-2 (SARS CoV-2) infections. Objectives: This review covers the types of sample collection, current diagnostic techniques for COVID-19 and challenges in their application Methods: A literature search was performed on the lines for a narrative review, but including features of systematic review methodology (PRISMA flow diagram). Two authors searched three electronic databases for literature review, i.e., PubMed, HINARI, and Google Scholar using Mesh-terms, specimen types or molecular diagnostic tests or serological tests in link with COVID-19 or SARS CoV-2, with different combinations, between March 21, 2020 and March 8, 2023. Conclusion: To control the current pandemic of COVID-19 and reduce its burden on healthcare systems around the world, the capacity of diagnostic tests should be improved. Rapid molecular diagnostic tests that can be fast, reliable, cost-effective, available, sensitive, and specific and point of care, must be urgently developed, produced, and widely distributed all over the globe.

Radiological Imaging Results in HIV-Infected Patients in Mogadishu-Somalia: A Four-Year Retrospective Review

Somalia, which is one of the countries that has been trying to integrate with the rest of the world in terms of many socio-economic parameters, including the field of health in recent years, still faces some important health problems. One of these problems is the difficulties encountered in the early diagnosis and treatment of patients with human immunodeficiency virus (HIV) infection, primarily due to social discrimination and limited treatment access. In this study, it was aimed to examine the findings detected in the radiological imaging examinations requested during the first examination in patients who were diagnosed with HIV for the first time by detecting HIV seropositivity in tests performed for different clinical reasons, and to determine the pathologies caused by advanced disease in patients who did not receive treatment. The study includes 235 patients who were diagnosed for the first time out of 269 patients with HIV confirmed by two different serological diagnostic tests during a previous comprehensive seroepidemiological study, and 14 patients with a suspicious test result for HIV infection but the presence of infection cannot be definitively proven in the absence of molecular diagnostic methods. In retrospective examinations, it was determined that 117 (49.8%) and 13 (92.9%) patients in both groups had radiological imaging (magnetic resonance imaging-MRI, computed tomography-CT, ultrasonography, and chest X-ray), respectively. While 16 of 117 HIV-infected patients had non-specific radiological findings and 53 had normal radiological findings, at least one radiological imaging of 48 patients revealed abnormal radiological imaging findings that may be associated with HIV infection. In these 48 patients, pneumonia findings in thorax CT and chest X-ray examinations, and encephalitis-related findings in patients with central nervous system involvement were the most common anomalies. In addition of these, other abnormal radiological findings included mass lesions compatible with neoplasia, lesions compatible with metastasis, lymphadenopathies, hepatosplenomegaly, and lesions indicating spleen involvement. The presence of similar findings (lesions compatible with pneumonia, encephalitis, and neoplasia) in 9 of 13 patients whose HIV infection could not be confirmed indicates that radiological tests can make significant contributions to clinical evaluations in the management of patients with suspicious laboratory results. The data we present from a region where significant obstacles are encountered in the diagnosis and follow-up of HIV infections due to the fear of discrimination (stigma) of the patients and the lack of molecular tests draw attention to the fact that many patients have advanced lesions when they are diagnosed, and this situation will have negative effects in the patient management and treatment process.

Evaluation of ten (10) SARS-CoV-2 rapid serological tests in comparison with WANTAI SARS-CoV-2 ab ELISA in Burkina Faso, West Africa

BackgroundThe aim of this study was to evaluate the performance of ten (10) SARS-CoV-2 serological rapid diagnostic tests in comparison with the WANTAI SARS-CoV-2 Ab ELISA test in a laboratory setting.Materials and methodsTen (10) SARS-CoV-2 serological rapid diagnostic tests (RDTs) for SARS-CoV-2 IgG/IgM were evaluated with two (2) groups of plasma tested positive for one and negative for the other with the WANTAI SARS-CoV-2 Ab ELISA. The diagnostic performance of the SARS-CoV-2 serological RDTs and their agreement with the reference test were calculated with their 95% confidence intervals.ResultsThe sensitivity of serological RDTs ranged from 27.39 to 61.67% and the specificity from 93.33 to 100% compared to WANTAI SARS-CoV-2 Ab ELISA test. Of all the tests, two tests (STANDARD Q COVID-19 IgM/IgG Combo SD BIOSENSOR and COVID-19 IgG/IgM Rapid Test (Zhejiang Orient Gene Biotech Co., Ltd)) had a sensitivity greater than 50%. In addition, all ten tests had specificity greater than or equal to 93.33% each. The concordance between RDTs and WANTAI SARS-CoV-2 Ab ELISA test ranged from 0.25 to 0.61.ConclusionThe SARS-CoV-2 serological RDTs evaluated show low and variable sensitivities compared to the WANTAI SARS-CoV-2 Ab ELISA test, with however a good specificity. These finding may have implications for the interpretation and comparison of COVID-19 seroprevalence studies depending on the type of test used.

Autoimmune psychosis: a review of diagnostic and treatment guidelines

IntroductionOver this decade, there has been progressive growth and evolution of the concept of autoimmune encephalitis. However, international consensus overly focus on major neurological signs, while discarding some attenuated presentations, sometimes with just psychiatric manifestations. It was only very recently that a new concept arouse from this disorder, named autoimmune psychosis, which can mimic schizophrenia. Unfortunately, there is still a lack of a structured approach of psychotic patients to cover this disorder. This has numerous implications, namely on management and prognosis of these patients. Not only, these patients have an increased risk of neuroleptic malignant syndrome, but it is also important to intervene early in the course of disease.ObjectivesTo conduct a review of the diagnostic and treatment guideliens of autoimmune psychosisMethodsThe authors conducted a non-systematic review, by resorting to the pubmed database, on the concept of autoimmune psychosis and updated proposals of diagnostic orienting lines.ResultsRecently, in 2016, Graus et al proposed diagnostic criteria for possible autoimmune encephalitis, in which the authors acknowledge subacute onset of psychiatric symptoms as a possible clinical manifestation. The authors accept normal diagnostic tests, provided that new neurological focal findings exist. Since then, there have been described a list of signs/symptoms, which should raise suspicion for this diagnostic on psychiatric patients, so called red flags. In accordance to diagnostic guidelines for autoimmune psychosis, defined by Pollak et, the presence of this symptoms should lead clinicians to perform diagnostic exams, as MRI, eletroencephalogram and blood serological tests and lumbar puncture. However, others criticize this initial lineup arguing that some patients could be missed, because they do not have any neurological signs, and so they propose new diagnostic criteria.ConclusionsAutoimmune psychosis represents an attenuated clinical form of autoimmune encephalitis, although demanding the same medication and prompt initiation of treatment as other autoimmune encephalitis. It is important to acknowledge that there are patients who are seronegative and that some of the diagnostic exams mentioned have sometimes limited availability. As acknowledged by Guasp et al, there are patients with first psychotic episodes that have an autoimmune etiology, but because of the lack of neurological signs, could potentially be missed of treatment. So, it is important to establish formal diagnostic guidelines for this disorder, namely orienting lines for first psychotic episodes, which is the most commson psychiatric manifestation. This also enlightens the need for neurologic and psychiatric cooperation for these patients.Disclosure of InterestNone Declared

A comprehensive review and clinical guide to molecular and serological diagnostic tests and future development: In vitro diagnostic testing for COVID-19

Abstract COVID-19 is a contagious syndrome caused by SARS Coronavirus 2 (SARS-CoV-2) that requires rapid diagnostic testing to identify and manage in the affected persons, characterize epidemiology, and promptly make public health decisions and manage the virus present in the affected person and promptly make public health decisions by characterizing the epidemiology. Technical problems, especially contamination occurring during manual real-time polymerase chain reaction (RT-PCR), can result in false-positive NAAT results. In some cases, RNA detection technology and antigen testing are alternatives to RT-PCR. Sequencing is vital for tracking the SARS-CoV-2 genome’s evolution, while antibody testing is beneficial for epidemiology. SARS-CoV-2 testing can be made safer, faster, and easier without losing accuracy. Continued technological advancements, including smartphone integration, will help in the current epidemic and prepare for the next. Nanotechnology-enabled progress in the health sector has aided disease and pandemic management at an early stage. These nanotechnology-based analytical tools can be used to quickly diagnose COVID-19. The SPOT system is used to diagnose the coronavirus quickly, sensibly, accurately, and with portability. The SPOT assay consists of RT-LAMP, followed by pfAgo-based target sequence detection. In addition, SPOT system was used to detect both positive and negative SARS-CoV-2 samples. This combination of speed, precision, sensitivity, and mobility will allow for cost-effective and high-volume COVID-19 testing.

Serological Cross-Reactivity in Zoonotic Flaviviral Infections of Medical Importance.

Flaviviruses are enveloped RNA viruses from the family Flaviviridae that comprise many important human pathogenic arboviruses such as Yellow Fever, Dengue, and Zika viruses. Because they belong to the same genus, these viruses show sequence and structural homology among them, which results in serological cross-reactivity. Upon infection, the immune system produces both species-specific and cross-reactive antibodies, and depending on the virus, in a successive flavivirus infection, cross-reactive antibodies either enhance protection or exacerbate the disease-the latter usually due to antibody-dependent enhancement. These antigenic relationships between different flaviviruses that lead to serological cross-reactivity make them difficult to be identified through serological methods, especially when it comes to successive flavivirus infections. We present here an overview of the main structural, epidemiological, and immunological aspects of flaviviruses, highlighting the role of neutralizing antibodies in fighting viral infections and in the "original antigenic sin" problem. Finally, we draw attention to the importance of developing a rapid serological diagnostic test for flaviviruses with high sensitivity and specificity, especially when considering that cross-reactive immunity can influence the outcome of these infections.

Изучение генетической вариабельности выделенных изолятов вируса лейкоза крупного рогатого скота в Белгородской области

Abstract. The purpose of the research Was to study genetic diversity of bovine leukemia virus isolates obtained in the Belgorod Region using restriction fragment length polymorphism method (RFLP). Scientific novelty. Bovine leukosis is one of the most common chronic infectious diseases of cattle in many countries of the world, which causes significant economic losses in the livestock industry. The typing of bovine leukemia virus (BLV), the study of its genetic structure, the evaluation of the mutation vector and a more detailed disclosure of the biological properties of the pathogen represent fundamental and applied value. Methods. The object of the research was 3-4-year-old cows infected with leukemia virus (n = 10), identified by serological methods in disadvantaged dairy farms. The immunodiffusion (ID) test, hematologic studies, PCR, genotyping, statistical processing of obtained data were conducted. Results. Conducted hematological studies determined the stage of the leukemic process in each animal. The target env fragment of the BLV gene (444 bp) was amplificated by 2-stage nested PCR, and this region was genotyped for all studied leukemia virus isolates using the restriction fragment length polymorphism (RFLP) method. In the course of the work, specific regions of the BLV env (gp51) gene, 970 bp long, were also obtained. We have given a primary assessment of the genetic diversity of BLV with the establishment of a genetic group (Belgian genotype according to RFLP). In the course of the work, specific regions of the env gp51 BLV gene, 444 bp long, were obtained. These fragments will be used for further DNA sequencing followed by phylogenetic analysis and determination of amino acid changes in the structure of the surface glycoprotein (gp51) of the bovine leukemia virus. Monitoring studies of BLV genotypes and the study of antigenic changes in the pathogen will allow timely development of the latest means of controlling and restricting the spread of bovine leukosis and improvement of diagnostic serological and PCR test systems.

Designing of novel chimeric PvpA-pMGA protein of Mycoplasma gallisepticum, applicable for indirect ELISA

BackgroundMycoplasma gallisepticum is the primary agent of chronic respiratory disease in chickens creating important economic losses in poultry industry. pMGA and pvpA genes encode major surface proteins in M. gallisepticum containing pathogenic, antigenic, and immune evasion characteristics. The objective of the present study was to design, express, and purify the recombinant chimeric PvpA-pMGA protein from M.gallisepticum for using in serological diagnostic test. MethodsAntigenic regions of PvpA and pMGA proteins were predicted for designing chimeric pvpA-pMGA gene construct. The codon optimized sequence was cloned into the expression vector pET32a+ and transformed into the Escherichia coli strain BL21 (DE3). The pET32a-PvpA-pMGA recombinant plasmid was expressed and confirmed by SDS-PAGE and immunoblotting. PvpA-pMGA recombinant protein (20μg and 50μg), ts-11 vaccine strain, and S6 strain that formulated by montanide adjuvant and two control groups (PBS and adjuvant) were injected subcutaneously to six groups of chickens. ResultsHigh yield of protein was purified amount 138 mg/L by affinity batch formation method. Indirect ELISA showed the levels of antibodies in rPvpA-pMGA was significantly higher than ts-11 and S6 groups (p<0.05). The results indicated that antigen-specific response was successfully elicited by the rpMGA-PvpA in chickens. The result of the ELISA with sera collected from ts-11 and S6 groups showed that indirect PvpA-pMGA-ELISA is appropriate candidate for detection of specific antibodies against M. gallisepticum with 100% sensitivity and specificity. ConclusionsThe rPvpA-pMGA is a highly candidate immunogenic protein which induced high amount of humoral immune response. Novel rPvpA-pMGA protein could be useful for evaluation of antibody level in vaccinated poultry flocks. Highlights•This is first study to present chimeric protein rPvpA-pMGA as a candidate antigen in ELISA.•High-yield 138 mg/L purification of recombinant protein.•rPvpA-pMGA is a reliable immunogenic protein which induced high amount of humoral immune response.•rPvpA-pMGA showed high performance for evaluation of antibody level in vaccinated poultry flocks.

SARSPLEX: Multiplex Serological ELISA with a Holistic Approach.

Currently, there are over 602 million severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) cases and 6.4 million COVID-19 disease-related deaths worldwide. With ambitious vaccine strategies, reliable and accurate serological testing is needed to monitor the dynamics of the novel coronavirus pandemic and community immunity. We set out to improve serological testing of the immune response against SARS-CoV-2. We hypothesize that by multiplexing the serological diagnostic test kit (SARSPLEX) and screening for three antibodies, an even more robust diagnostic can be developed. A total of 293 sera were analyzed for IgM, IgG, or IgA immune reactions to the subunit 1 spike glycoprotein and the nucleocapsid protein in a standardized ELISA platform. Testing IgM, IgG, and IgA demonstrated high positive and negative agreements compared to RT-PCR and serology reference tests. Comparison with the pre-2019-CoV (n = 102) samples highlighted the specificity of this test kit and indicated that no unspecific binding, even with the summer flu patients (n = 44), was detected. In addition, SARSPLEX demonstrated to be a valuable occupational surveillance tool used in a functional medicine facility. With increased and broader testing, SARSPLEX will be a valuable tool in monitoring immunity and aid in prioritizing access to the SARS-CoV-2 vaccine for high-risk patients.

Prevalence of Lyme Carditis in Patients with Atrioventricular Blocks.

Infections with Borrelia may cause cardiac conduction system abnormalities, including atrioventricular blocks (AVBs). Therefore, we aimed to identify patients in whom Lyme carditis (LC) could be considered as the initial diagnosis among consecutive subjects who were referred for implantation of a permanent pacemaker due to symptomatic AVBs. To date, such a systematic evaluation has not been reported yet. Validation of the Suspicious Index for Lyme Carditis (SILC) in our study population was considered as an additional goal. We investigated consecutive patients with AVB admitted to our department for a pacemaker implantation. Serological diagnostic tests against Borrelia burgdorferi sensu lato (Bbsl) were performed in those with no obvious cardiac causes of AVB. The final study population consisted of 130 patients (80 M, mean age 67.4 ± 17.6). Lyme carditis was assumed as the initial diagnosis in 16 patients (12%) based on ABV and IgM Bbsl seropositivity. The patients with LC were younger and more frequently manifested constitutional symptoms of infection and fluctuating AVB. The highest prognostic value for identification of LC patients was obtained for the modified SILC, which included the following parameters: (1) age lower than 75 years; (2) risky outdoor activity and living in the countryside; (3) tick bite; (4) constitutional symptoms of Lyme disease; (5) erythema migrans; (6) male sex and (7) fluctuating atrioventricular block. We concluded that diagnostics for LC should be routinely considered in patients with advanced AVB. Modified SILC may identify the patients at risk of LC.

A Promising Tool in Serological Diagnosis: Current Research Progress of Antigenic Epitopes in Infectious Diseases.

Infectious diseases, caused by various pathogens in the clinic, threaten the safety of human life, are harmful to physical and mental health, and also increase economic burdens on society. Infections are a complex mechanism of interaction between pathogenic microorganisms and their host. Identification of the causative agent of the infection is vital for the diagnosis and treatment of diseases. Etiological laboratory diagnostic tests are therefore essential to identify pathogens. However, due to its rapidity and automation, the serological diagnostic test is among the methods of great significance for the diagnosis of infections with the basis of detecting antigens or antibodies in body fluids clinically. Epitopes, as a special chemical group that determines the specificity of antigens and the basic unit of inducing immune responses, play an important role in the study of immune responses. Identifying the epitopes of a pathogen may contribute to the development of a vaccine to prevent disease, the diagnosis of the corresponding disease, and the determination of different stages of the disease. Moreover, both the preparation of neutralizing antibodies based on useful epitopes and the assembly of several associated epitopes can be used in the treatment of disease. Epitopes can be divided into B cell epitopes and T cell epitopes; B cell epitopes stimulate the body to produce antibodies and are therefore commonly used as targets for the design of serological diagnostic experiments. Meanwhile, epitopes can fall into two possible categories: linear and conformational. This article reviews the role of B cell epitopes in the clinical diagnosis of infectious diseases.

Humanization and expression of IgG and IgM antibodies in plants as potential diagnostic reagents for Valley Fever.

Monoclonal antibodies (mAbs) are important proteins used in many life science applications, from diagnostics to therapeutics. High demand for mAbs for different applications urges the development of rapid and reliable recombinant production platforms. Plants provide a quick and inexpensive system for producing recombinant mAbs. Moreover, when paired with an established platform for mAb discovery, plants can easily be tailored to produce mAbs of different isotypes against the same target. Here, we demonstrate that a hybridoma-generated mouse mAb against chitinase 1 (CTS1), an antigen from Coccidioides spp., can be biologically engineered for use with serologic diagnostic test kits for coccidioidomycosis (Valley Fever) using plant expression. The original mouse IgG was modified and recombinantly produced in glycoengineered Nicotiana benthamiana plants via transient expression as IgG and IgM isotypes with human kappa, gamma, and mu constant regions. The two mAb isotypes produced in plants were shown to maintain target antigen recognition to CTS1 using similar reagents as the Food and Drug Administration (FDA)-approved Valley Fever diagnostic kits. As none of the currently approved kits provide antibody dilution controls, humanization of antibodies that bind to CTS1, a major component of the diagnostic antigen preparation, may provide a solution to the lack of consistently reactive antibody controls for Valley Fever diagnosis. Furthermore, our work provides a foundation for reproducible and consistent production of recombinant mAbs engineered to have a specific isotype for use in diagnostic assays.

Clinical Performance of Real-Time Polymerase Chain Reaction for Strongyloides stercoralis Compared with Serology in a Nonendemic Setting.

Strongyloides stercoralis is a nematode endemic to subtropical and tropical regions that may cause asymptomatic carriage, peripheral eosinophilia, cutaneous, gastrointestinal, and pulmonary disease, or hyperinfection syndrome. Conventional diagnostic methods for strongyloidiasis include feces microscopy and culture, with low sensitivity in chronic infection due to the low helminth burden, and serology, which may be prone to false-negative results with immunocompromise and false-positive results with other infections and immunological disorders. We evaluated a laboratory-developed real-time polymerase chain reaction (RT-PCR), detecting the 18S SSU ribosomal RNA gene, compared with conventional diagnostic methods, using serology via ELISA as the gold-standard. The population studied included tertiary hospital inpatients and outpatients residing in a nonendemic area. Seven hundred fifty unfixed stool specimens submitted sequentially between 2014 and 2018 were tested for S. stercoralis via microscopy and RT-PCR. Agar plate culture (APC), Harada-Mori culture (HMC), and ELISA were performed in conjunction with 141, 135, and 177 of the specimens, respectively. RT-PCR yielded 13 positive and 730 negative results, with inhibition in seven specimens. ELISA yielded 53 positive, 18 equivocal, and 106 negative results. Results for direct diagnostic methods obtained after treatment with ivermectin were excluded from the performance analysis. Compared with ELISA, RT-PCR, microscopy, APC, and HMC exhibited sensitivities of 38%, 6%, 3%, and 0%, respectively, and specificities of 100%. Given the low sensitivities commensurate with testing a population with remote infection and thus low parasite burden, we recommend a combination of serological and molecular diagnostic testing to achieve the best balance of sensitivity and specificity.

Comparison of parasitological, serological and molecular diagnostic tests for detection of subclinical Trypanosomosis in two organised dairy herds

Trypanosomosis also known as ‘Surra’ caused by Trypanosoma evansi is of significant economic importance in livestock production. Diagnosis of subclinical infection and carrier state of disease is far difficult. Therefore, the present study was conducted to compare parasitological, serological and molecular diagnostic tests for detecting subclinical trypanosomosis in two dairy herds of Murrah buffalo (n=27) and crossbred cattle (n=22) in Hisar district of Haryana. Peripheral blood samples collected from all the animals were subjected to blood smear examination and DNA isolation for molecular detection by PCR, and like-wise serum samples were collected and subjected to latex agglutination test (TE-LAT) for T. evansi antigen detection. PCR products from positive samples were purified and sequenced for confirmation of T. evansi DNA. None of the animals in the two herds was found positive for T. evansi parasites by microscopic examination of thick blood smears. In buffalo farm, 29.62% samples each were found moderately and weak positive by TE-LAT, whereas PCR assay test yielded 40.74% samples positive for T. evansi DNA. In cattle farm, TE-LAT showed 18.18%, 31.89% and 9.09% samples to be strong, moderate and weak positive, whereas 50% samples were found PCR-positive for T. evansi DNA. Agreement among TE-LAT and PCR assay, as expressed by the kappa value, was found to be poor. To conclude, each test has its own merits and limitations. While TE-LAT is suitable for screening purpose, PCR assay confirms parasite in blood. For clinical diagnosis, results of laboratory diagnostic tests must, however, be correlated clinically before initiating the therapy.

First Results of an External Quality Assessment (EQA) Scheme for Molecular, Serological and Antigenic Diagnostic Test for SARS-CoV-2 Detection in Lombardy Region (Northern Italy), 2020-2022.

For diagnosing SARS-CoV-2 infection and for monitoring its spread, the implementation of external quality assessment (EQA) schemes is mandatory to assess and ensure a standard quality according to national and international guidelines. Here, we present the results of the 2020, 2021, 2022 EQA schemes in Lombardy region for assessing the quality of the diagnostic laboratories involved in SARS-CoV-2 diagnosis. In the framework of the Quality Assurance Programs (QAPs), the routinely EQA schemes are managed by the regional reference centre for diagnostic laboratories quality (RRC-EQA) of the Lombardy region and are carried out by all the diagnostic laboratories. Three EQA programs were organized: (1) EQA of SARS-CoV-2 nucleic acid detection; (2) EQA of anti-SARS-CoV-2-antibody testing; (3) EQA of SARS-CoV-2 direct antigens detection. The percentage of concordance of 1938 molecular tests carried out within the SARS-CoV-2 nucleic acid detection EQA was 97.7%. The overall concordance of 1875 tests carried out within the anti-SARS-CoV-2 antibody EQA was 93.9% (79.6% for IgM). The overall concordance of 1495 tests carried out within the SARS-CoV-2 direct antigens detection EQA was 85% and it was negatively impacted by the results obtained by the analysis of weak positive samples. In conclusion, the EQA schemes for assessing the accuracy of SARS-CoV-2 diagnosis in the Lombardy region highlighted a suitable reproducibility and reliability of diagnostic assays, despite the heterogeneous landscape of SARS-CoV-2 tests and methods. Laboratory testing based on the detection of viral RNA in respiratory samples can be considered the gold standard for SARS-CoV-2 diagnosis.

Development of HEK-293 Cell Lines Constitutively Expressing Flaviviral Antigens for Use in Diagnostics.

ABSTRACTFlaviviruses are important human pathogens worldwide. Diagnostic testing for these viruses is difficult because many of the pathogens require specialized biocontainment. To address this issue, we generated 39 virus-like particle (VLP)- and nonstructural protein 1 (NS1)-secreting stable cell lines in HEK-293 cells of 13 different flaviviruses, including dengue, yellow fever, Japanese encephalitis, West Nile, St. Louis encephalitis, Zika, Rocio, Ilheus, Usutu, and Powassan viruses. Antigen secretion was stable for at least 10 cell passages, as measured by enzyme-linked immunosorbent assays and immunofluorescence assays. Thirty-five cell lines (90%) had stable antigen expression over 10 passages, with three of these cell lines (7%) increasing in antigen expression and one cell line (3%) decreasing in antigen expression. Antigen secretion in the HEK-293 cell lines was higher than in previously developed COS-1 cell line counterparts. These antigens can replace current antigens derived from live or inactivated virus for safer use in diagnostic testing.IMPORTANCE Serological diagnostic testing for flaviviral infections is hindered by the need for specialized biocontainment for preparation of reagents and assay implementation. The use of previously developed COS-1 cell lines secreting noninfectious recombinant viral antigen is limited due to diminished antigen secretion over time. Here, we describe the generation of 39 flaviviral virus-like particle (VLP)- and nonstructural protein 1 (NS1)-secreting stable cell lines in HEK-293 cells representing 13 medically important flaviviruses. Antigen production was more stable and statistically higher in these newly developed cell lines than in their COS-1 cell line counterparts. The use of these cell lines for production of flaviviral antigens will expand serological diagnostic testing of flaviviruses worldwide.

Development of highly specific peptibody against Porcine epidemic diarrhea virus

Abstract Porcine epidemic diarrhea virus (PEDV) is an etiological agent of porcine epidemic diarrhea (PED), causing dehydration, vomiting, and acute watery diarrhea. Because it has a high mortality rate in neonatal piglets, PEDV is one of the critical pathogens affecting the swine industry. Since PEDV has shown rapid spreading in herds of swine, early diagnosis of this virus is very important to control the disease. Serological diagnosis test is a simple, rapid method for detecting PEDV infected pigs, and the development of PEDV-specific antibodies is essential for an accurate serological diagnostic test. Because conventional methods of development of monoclonal antibodies are time and hand-consuming work, recombinant antibodies are considered as an alternative strategy for the development of target-specific monoclonal antibodies. Peptibody is one of the promising recombinant antibodies that target-specific peptide is fused with the Fc domain of immunoglobulin G. The peptibody which have a specificity of peptides and immune function of Fc region as advantages have developed for therapeutic and diagnostic purpose. Here, we generated a PEDV-specific peptibody (PEDV-PB) that is fused with PEDV-specific peptide and mouse IgG2b Fc region. PEDV-specific peptide sequences were discovered by random phage display library and PEDV-PB was produced from Chinese hamster ovary (CHO) cells. PEDV-PB showed highly specific binding in several serological tests for PEDV, such as western blotting, immunofluorescence assay, and enzyme-linked immunosorbent assay. Our results suggest that PEDV-PB is very useful for the development of a rapid detection kit for PEDV.

The role and diagnostic accuracy of serology for COVID-19

BackgroundThe role and performance of various serological tests for the diagnosis of COVID-19 are unclear. This study aimed to evaluate the performance of seven commercially available serological assays for SARS-CoV-2 antibodies by testing COVID-19 cases and controls.MethodsAdult patients with fever for > 5 days, admitted to a tertiary-care teaching hospital in South India, were enrolled prospectively between June and December 2020. SARS-CoV-2 RT-PCR confirmed patients were classified as cases, and patients with febrile illness with laboratory-confirmed alternative diagnosis and healthy participants were controls. All participants were tested with SCoV-2 Detect™ IgM ELISA kit and SCoV-2 Detect™ IgG ELISA kit (InBios International, Seattle, USA) (Inbios), SARS-CoV-2 Total and SARS-CoV-2 IgG (Siemens Healthcare Diagnostics Inc., Tarrytown, USA) (Siemens), Roche Elecsys® Anti-SARS-CoV-2 (Roche Diagnostics, Rotkreuz, Switzerland) (Roche), Abbott SARS-CoV-2 IgG (Abbott Diagnostics, IL, USA) (Abbott), and Liaison® SARS-CoV-2 S1/S2 IgG (DiaSorinS.p.A., Saluggia, Italy) (Liaison). The sensitivities, specificities, positive predictive values (PPV), negative predictive values (NPV), and accuracies were compared.ResultsThere were 303 participants: 153 cases and 150 controls. ELISA detecting anti-S protein antibody was more sensitive (88.9% for IgG and 86.3% for IgM) than the CLIAs (82.4% for total antibodies and 76.5–85.6% for IgG). Among CLIAs, Roche IgG was most sensitive (85.6%) followed by Abbott (83%) and Liaison (83%). Abbot had the best PPV (88.8%) and was more specific (89.3%) than Liaison (82%) and Roche (82%). Siemens IgG was less sensitive (76.5%) than Siemens Total (82.4%). The specificity of all the serological assays was modest (75–90%). Antibody test positivity increased with the duration of illness reaching 90% after 10 days of illness. When cases were compared against pre-pandemic controls, the IgG gave excellent specificity (98–100%). For seroprevalence studies, InBios IgG had the best accuracy (90.8%) with 88.9% sensitivity and 97.6% specificity.ConclusionThe serological assays are important adjuncts for the diagnosis of COVID-19 in patients with persistent symptoms, especially in the second week of illness. The value of serological diagnostic tests is limited in the first week of illness and they provide additional value in seroprevalence studies. The diagnostic accuracy of the ELISA and CLIA platforms were comparable.

Assessing Tick-Borne Disease Risk and Surveillance: Toward a Multi-Modal Approach to Diagnostic Positioning and Prediction.

The true extent of tick-borne disease (TBD) incidence and risk among humans is largely unknown, posing significant public health challenges. This study offers an exploratory analysis of a multimodal dataset and is part of a larger ongoing project to determine if entomological data, canine serological reports, self-reported human tick bite encounters (TBEs), and/or associated TBD diagnoses can serve as proxies for human disease risk. Focusing on the United States (U.S.), it characterizes self-reported TBD diagnoses (specifically, anaplasmosis, ehrlichiosis, and Lyme disease), co-infections, and their frequency and distribution across U.S. counties in relation to the presence of other factors related to TBD risk. Survey data was used to construct a list of TBEs localizable to individual U.S. counties. National data regarding these counties—namely the presence of official Lyme Disease (LD) case reports from the Centers for Disease Control and Prevention, as well as the tick vectors I. scapularis and I. pacificus within a given county—were then linked with survey-reported TBEs, tabulated by diagnosis (including co-infections), to determine the distribution of county-level endpoints across diagnostic categories. In addition, data on the presence of positive serological diagnostic tests conducted in canines were considered due to their potential utility as a proxy for TBD and TBE risk. The final dataset contained 249 TBEs localized to a total of 144 counties across 30 states. Diagnostic categories included respondents with LD (n = 70) and those with anaplasmosis and ehrlichiosis diagnoses and co-infections (n < 20 per diagnostic category). TBEs also were indicated by respondents who did not report TBD diagnoses, with some indicating uncertainty. The distribution of respondent-reported TBEs varied between canine TBDs, with LD-positive respondents reporting noticeably larger proportions of TBEs in counties with canine LD and smaller proportions in counties with canine anaplasmosis, compared to respondents without an LD diagnosis; a notional logistic regression suggests these differences may be significant (canine LD: Odds Ratio [OR] = 6.04, p = 0.026) (canine anaplasmosis: OR = 0.50, p = 0.095). These results suggest that certain widely available diagnostic TBD data in animals (in this case, domesticated dogs) may be sensitive to differences in human TBD risk factors and thus may have utility as proxies in future research. In the absence of an available standardized, unified, and national TBD database, such proxies, along with relevant surveys and reports, may provide a much-needed working solution for scientists and clinicians studying TBDs.

Performance of Severe Acute Respiratory Syndrome Coronavirus 2 Serological Diagnostic Tests and Antibody Kinetics in Coronavirus Disease 2019 Patients.

Serological testing is recommended to support the detection of undiagnosed coronavirus disease 2019 (COVID-19) cases. However, the performance of serological assays has not been sufficiently evaluated. Hence, the performance of six severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) binding antibody assays [three chemiluminescence (CLIAs) and three lateral flow immunoassays (LFIAs)] and a surrogate virus neutralization test (sVNT) was analyzed in a total of 988 serum samples comprising 389 COVID-19-positives and 599 COVID-19-negatives. The overall diagnostic sensitivities of CLIAs and LFIAs ranged from 54.2 to 56.6% and from 56.3 to 64.3%, respectively. The overall diagnostic specificities of CLIAs and LFIAs ranged from 98.2 to 99.8% and from 97.3 to 99.0%, respectively. In the symptomatic group (n = 321), the positivity rate increased by over 80% in all assays > 14 days after symptom onset. In the asymptomatic group (n = 68), the positivity rate increased by over 80% in all assays > 21 days after initial RT-PCR detection. In LFIAs, negatively interpreted trace bands accounted for the changes in test performance. Most false-positive results were weak or trace reactions and showed negative results in additional sVNT. For six binding antibody assays, the overall agreement percentages ranged from 91.0 to 97.8%. The median inhibition activity of sVNT was significantly higher in the symptomatic group than in the asymptomatic group (50.0% vs. 29.2%; p < 0.0001). The median times to seropositivity in the symptomatic group were 9.7 days for CLIA-IgG, 9.2 and 9.8 days for two CLIAs-Total (IgM + IgG), 7.7 days for LFIA-IgM, 9.2 days for LFIA-IgG, and 8.8 days for sVNT-IgG, respectively. There was a strong positive correlation between the quantitative results of the four binding antibody assays and sVNT with Spearman ρ-values ranging from 0.746 to 0.854. In particular, when using LFIAs, we recommend using more objective interpretable assays or establishing a band interpretation system for each laboratory, accompanied by observer training. We also anticipate that sVNT will play an essential role in SARS-CoV-2 antibody testing and become the practical routine neutralizing antibody assay.