Sensitivity Of Serological Tests Research Articles

Seroprevalence and risk factor analysis of brucellosis among dairy farmers in Aligarh region, North India: creating awareness of a neglected disease.

Introduction. Brucellosis, a globally distributed zoonotic disease, is caused by the Gram-negative bacteria known as Brucella. Humans acquire infection through direct contact with the blood, urine and placenta of animals, inhalation of dust or aerosols at infected animal farms, and raw milk and meat intake. This study aimed to assess the prevalence of brucellosis in dairy farmers in and around the Aligarh region of North India, to document various clinical signs and symptoms in Brucella-positive individuals, and to create awareness in dairy farmers concerning brucellosis and ways to prevent it. Methods. This was an observational study that included 125 dairy farmers in and around the Aligarh region. Serum samples were taken from this high-risk group after obtaining informed consent. Further, a pre-designed proforma was used to collect information about their knowledge, attitude and practices (KAP) concerning brucellosis and assess the risk factors for the disease. The Rose Bengal test (RBT), serum agglutination test (SAT) and enzyme-linked immunosorbent assay (ELISA) were performed to detect the seroprevalence of brucellosis. Result.Brucella infection was diagnosed in 64 (51.20 %) cases by indirect ELISA (IgM+IgG), 41 (32.8 %) by RBT and 4 (3.2 %) by SAT. Significant clustering of patients was seen in the 20-55 years age group. The most common symptoms in ELISA IgM-positive patients were joint pain (16.07 %), fatigue (14.28 %), anorexia (12.50 %), weight loss (8.92 %), malaise (5.35 %), undulant fever (3.57 %), night sweats (3.57 %) and headache (1.78 %). The findings of this study indicate that ELISA (IgM+IgG) exhibits great sensitivity as compared to SAT and RBT. KAP was very poor among dairy farmers. Conclusion. In India, Brucella is a frequent but severely underreported illness. ELISA is the most sensitive serological test for diagnosing brucellosis. No potential vaccine has yet been introduced for humans against brucellosis. Thus, it is necessary to impart awareness and sensitize high-risk groups concerning brucellosis.

CER818: A Highly Specific and Sensitive HPV L1 High-Risk Serological Lateral Flow Rapid Test for Early Detection of Cervical Cancer and Its Precursor Lesions.

Objective: The objective of the study is to validate a new human papillomavirus (HPV) L1 high-risk specific serological assay in a case-control study. Methods: Serum samples of 138 patients (cervical intraepithelial neoplasia (CIN) 1, 2, and 3 and cervical cancer), 21 vaccinees, and 246 female controls were tested for the presence of HPV L1 high-risk specific antibodies. Results: HPV L1 high-risk antibodies were detected in 100% of the CIN1 and 2, 86.6% of the CIN3 and 82.4% of the cervical cancer cases, 100% of the vaccinees, and 3.9% of the female controls. Area under the curve (AUC) was calculated with 0.91 for controls versus CIN2+, 0.923 for controls versus CIN1+, and 0.968 for controls versus CIN1/2. Conclusion: The HPV L1 high-risk specific serological lateral flow rapid test shows promising data in the field of early detection of HPV high-risk induced cervical cancer and its precursor lesions. This easy-to-use, robust, and affordable approach could offer a chance to reach women in low- or middle-income countries (LMICs) that could not be reached by HPV molecular testing-based cervical cancer screening programs.

Multiplexed on-yeast serological assay for immune escape screening of SARS-CoV-2 variants

The emergence of the SARS-CoV-2 Omicron variant altered patient risk profiles and shifted the trajectory of the COVID-19 pandemic. Therefore, sensitive serological tests capable of analyzing patient IgG responses to multiple variants in parallel are highly desirable. Here, we present an adaptable serological test based on yeast surface display and serum biopanning that characterizes immune profiles against SARS-CoV-2 Wuhan (B lineage), Delta (B.1.617.2 lineage), and Omicron (B.1.1.529 lineage) receptor-binding domain (RBD) variants. We examined IgG titers from 30 serum samples from COVID-19-convalescent and vaccinated cohorts in Switzerland, and assessed the relative affinity of polyclonal serum IgG for RBD domains. We demonstrate that serum IgGs from patients recovered from severe COVID-19 between March-June 2021 bound tightly to both original Wuhan and Delta RBD variants, but failed to recognize Omicron RBDs, representing an affinity loss of >10- to 20-fold. Our yeast immunoassay is easily tailored, expandable and parallelized with newly emerging RBD variants.

Proof of Concept of a Novel Multiepitope Recombinant Protein for the Serodiagnosis of Patients with Chagas Disease.

Chagas disease remains a neglected disease that is considered to be a public health problem. The early diagnosis of cases is important to improve the prognosis of infected patients and prevent transmission. Serological tests are the method of choice for diagnosis. However, two serological tests are currently recommended to confirm positive cases. In this sense, more sensitive and specific serological tests need to be developed to overcome these current diagnosis problems. This study aimed to develop a new recombinant multiepitope protein for the diagnosis of Chagas disease, hereafter named rTC. The rTC was constructed based on amino acid sequences from different combinations of Trypanosoma cruzi antigens in the same polypeptide and tested using an enzyme-linked immunosorbent assay (ELISA) to detect different types of Chagas disease. rTC was able to discriminate between indeterminate (IND) and cardiac (CARD) cases and cross-reactive diseases, as well as healthy samples, with 98.28% sensitivity and 96.67% specificity, respectively. These data suggest that rTC has the potential to be tested in future studies against a larger serological panel for the diagnosis of Chagas disease.

Evolution of Blood Safety in Switzerland over the Last 25 Years for HIV, HCV, HBV and Treponema pallidum.

During the last few decades, efforts to increase the safety of blood and blood products have mainly focused on preventing the viral infections HCV, HIV, HBV and Treponema pallidum. The evolution of these approaches and the achieved increase in safety is shown for the last 25 years in Switzerland. In detail, the prevalences and incidences of the infection disease and the theoretical estimated residual risks (RR) of these blood-borne infections are presented. Prevalences, incidences and, in particular, the RR have decreased considerably over the last 25 years. This was achieved primarily by the adoption of strict criteria for the selection of blood donors, refined questionnaires, the introduction of increasingly sensitive serological screening tests and the implementation of nucleic acid testing (NAT) for these blood-borne pathogens. These NAT assays have significantly shortened the window period between infection and the first detection of the infectious agent in the blood of an infected individual. A form of "real life" comparison or confirmation is provided by the reported lookback procedures (LBP) and the haemovigilance data of the Swiss competent authority, Swissmedic. These data are in agreement, and thus support the very low prevalences, incidences and RR.

Selection, Characterization, Calibration, and Distribution of the U.S. Serology Standard for Anti-SARS-CoV-2 Antibody Detection.

The SARS-CoV-2 pandemic resulted in a demand for highly specific and sensitive serological testing to evaluate seroprevalence and antiviral immune responses to infection and vaccines. Hence, there was an urgent need for a serology standard to harmonize results across different natural history and vaccine studies. The Frederick National Laboratory for Cancer Research (FNLCR) generated a U.S. serology standard for SARS-CoV-2 serology assays and subsequently calibrated it to the WHO international standard (National Institute for Biological Standards and Control [NIBSC] code 20/136) (WHO IS). The development included a collaborative study to evaluate the suitability of the U.S. serology standard as a calibrator for SARS-CoV-2 serology assays. The eight laboratories participating in the study tested a total of 17 assays, which included commercial and in-house-derived binding antibody assays, as well as neutralization assays. Notably, the use of the U.S. serology standard to normalize results led to a reduction in the inter-assay coefficient of variation (CV) for IgM levels (pre-normalization range, 370.6% to 1,026.7%, and post-normalization range, 52.8% to 242.3%) and a reduction in the inter-assay CV for IgG levels (pre-normalization range, 3,416.3% to 6,160.8%, and post-normalization range, 41.6% to 134.6%). The following results were assigned to the U.S. serology standard following calibration against the WHO IS: 246 binding antibody units (BAU)/mL for Spike IgM, 764 BAU/mL for Spike IgG, 1,037 BAU/mL for Nucleocapsid IgM, 681 BAU/mL for Nucleocapsid IgG assays, and 813 neutralizing international units (IU)/mL for neutralization assays. The U.S. serology standard has been made publicly available as a resource to the scientific community around the globe to help harmonize results between laboratories.

Conjugated silver nanoparticles as a diagnostic tool for circulating hydatid antigens

Cystic hydatid disease is one of the most significant worldwide zoonotic diseases. The causative agent is the larval stage of Echinococcus granulosus. The diagnosis of cystic echinococcosis by clinical symptoms and scanning alone is often difficult and confusing. The definite diagnosis needs sensitive and reliable serological tests. This study aimed to evaluate a nano silver-based enzyme linked immunosorbent assay (ELISA) in the detection of circulating hydatid antigen in human serum samples. The study included 66 human serum samples (36 hydatidosis confirmed cases, 15 cases infected with other parasites, and 15 normal subjects as negative control). The circulating protoscolices antigen was assayed by the nano-silver dot ELISA, nano-silver sandwich ELISA and the traditional methods (dot ELISA and sandwich ELISA). Our study revealed that the sensitivity and specificity of the nano-silver dot ELISA were 97.2% and 93.3%, respectively. The sensitivity and specificity of nano-silver sandwich ELISA were 94.4% and 96.7% respectively. The nano-silver-based ELISA showed higher sensitivity, specificity, positive predictive value, negative predictive value, and diagnostic accuracy than the traditional ELISA. In conclusion, the nano-silver-based ELISA can be proposed as a confirmatory test in the diagnosis of cystic echinococcosis.

P128 Chronic pulmonary aspergillosis in a Tuberculosis c enter of Assam—a 2-year study

Poster session 1, September 21, 2022, 12:30 PM - 1:30 PMObjectivesChronic pulmonary aspergillosis is a slow and progressive lung disease that occurs in apparently immune-competent patients. Pulmonary tuberculosis is considered to be a major predisposing factor. Early diagnosis of the condition can improve morbidity and mortality. As the number of studies to detect this disease in India is sparse, this study was undertaken in District Tuberculosis center attached to Assam Medical College and Hospital, Dibrugarh to find out the occurrence of chronic pulmonary aspergillosis in treated pulmonary TB patients.MethodsThis prospective hospital-based cross-sectional study was done in Assam Medical College and Hospital, Dibrugarh from December 2015-November 2017. Every consecutive pulmonary TB case after completion of intensive therapy attending the outpatient department with respiratory symptoms was included in the study. Cases were identified with clinical, serological/mycological, and radiological criteria.ResultsA total of 26 cases of CPA were confirmed out of 211 enrolled cases. Positivity rate of CPA in the enrolled patients was 12.3% (95% CI 8.19-17.51%). The hospital-based occurrence rate was found to be 6/1000 (95% CI 3.8-8.5) in the outdoor attendants in DTC, Dibrugarh. Most of the cases (88.46%) were late cases presenting with aspergilloma. Majority (51.72%) of the aspergilloma cases were detected within 5 years of diagnosis of PTB. Very few (15.38%, n = 4) early cases were detected. The sensitivity of the ELISA test (DRG, Germany) was found to be 72.72% as compared with reference Immunocap method used in this study. A section (37.90%) of the patients with fungal ball was seronegative by Aspergillus fumigatus serological test. Two of them were positive for Aspergillus flavus in sputum culture.ConclusionA serological test to detect Aspergillus flavus IgG may complement the Aspergillus fumigatus IgG test in detecting CPA. CPA is a neglected disease with a high morbidity and mortality. Routine follow-up protocol for the cavitary patients in tuberculosis center by a sensitive serological test for a minimum period of 5 years may be necessary for early detection of cases in PTB high prevalence countries.

The influence of time on the sensitivity of SARS-CoV-2 serological testing

Sensitive serological testing is essential to estimate the proportion of the population exposed or infected with SARS-CoV-2, to guide booster vaccination and to select patients for treatment with anti-SARS-CoV-2 antibodies. The performance of serological tests is usually evaluated at 14–21 days post infection. This approach fails to take account of the important effect of time on test performance after infection or exposure has occurred. We performed parallel serological testing using 4 widely used assays (a multiplexed SARS-CoV-2 Nucleoprotein (N), Spike (S) and Receptor Binding Domain assay from Meso Scale Discovery (MSD), the Roche Elecsys-Nucleoprotein (Roche-N) and Spike (Roche-S) assays and the Abbott Nucleoprotein assay (Abbott-N) on serial positive monthly samples collected as part of the Co-STARs study (www.clinicaltrials.gov, NCT04380896) up to 200 days following infection. Our findings demonstrate the considerable effect of time since symptom onset on the diagnostic sensitivity of different assays. Using a time-to-event analysis, we demonstrated that 50% of the Abbott nucleoprotein assays will give a negative result after 175 days (median survival time 95% CI 168–185 days), compared to the better performance over time of the Roche Elecsys nucleoprotein assay (93% survival probability at 200 days, 95% CI 88–97%). Assays targeting the spike protein showed a lower decline over the follow-up period, both for the MSD spike assay (97% survival probability at 200 days, 95% CI 95–99%) and the Roche Elecsys spike assay (95% survival probability at 200 days, 95% CI 93–97%). The best performing quantitative Roche Elecsys Spike assay showed no evidence of waning Spike antibody titers over the 200-day time course of the study. We have shown that compared to other assays evaluated, the Abbott-N assay fails to detect SARS-CoV-2 antibodies as time passes since infection. In contrast the Roche Elecsys Spike Assay and the MSD assay maintained a high sensitivity for the 200-day duration of the study. These limitations of the Abbott assay should be considered when quantifying the immune correlates of protection or the need for SARS-CoV-2 antibody therapy. The high levels of maintained detectable neutralizing spike antibody titers identified by the quantitative Roche Elecsys assay is encouraging and provides further evidence in support of long-lasting SARS-CoV-2 protection following natural infection.

Fcγ-Receptor-Based Enzyme-Linked Immunosorbent Assays for Sensitive, Specific, and Persistent Detection of Anti-SARS-CoV-2 Nucleocapsid Protein IgG Antibodies in Human Sera.

ABSTRACTSensitive and specific serological tests are mandatory for epidemiological studies evaluating severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) prevalence as well as coronavirus disease 2019 (COVID-19) morbidity and mortality rates. The accuracy of results is challenged by antibody waning after convalescence and by cross-reactivity induced by previous infections with other pathogens. By employing a patented platform technology based on capturing antigen-antibody complexes with a solid-phase-bound Fcγ receptor (FcγR) and truncated nucleocapsid protein as the antigen, two SARS-CoV-2 IgG enzyme-linked immunosorbent assays (ELISAs), featuring different serum and antigen dilutions, were developed. Validation was performed using a serum panel comprising 213 longitudinal samples from 35 COVID-19 patients and a negative-control panel consisting of 790 pre-COVID-19 samples from different regions of the world. While both assays show similar diagnostic sensitivities in the early convalescent phase, ELISA 2 (featuring a higher serum concentration) enables SARS-CoV-2 IgG antibody detection for a significantly longer time postinfection (≥15 months). Correspondingly, analytical sensitivity referenced to indirect immunofluorescence testing (IIFT) is significantly higher for ELISA 2 in samples with a titer of ≤1:640; for high-titer samples, a prozone effect is observed for ELISA 2. The specificities of both ELISAs were excellent not only for pre-COVID-19 serum samples from Europe, Asia, and South America but also for several challenging African sample panels. The SARS-CoV-2 IgG FcγR ELISAs, methodically combining antigen-antibody binding in solution and isotype-specific detection of immune complexes, are valuable tools for seroprevalence studies requiring the (long-term) detection of anti-SARS-CoV-2 IgG antibodies in populations with a challenging immunological background and/or in which spike-protein-based vaccine programs have been rolled out.

Chagas Immunochromatographic Rapid Test in the Serological Diagnosis of Trypanosoma cruzi Infection in Wild and Domestic Canids.

Canis lupus familiaris (domestic dog) represents a reliable sentinel for the occurrence of a well-established transmission cycle of Trypanosoma cruzi among wild mammals in the surroundings and, consequently, where the risk of human infection exists. Serological diagnosis is the chosen method to identify T. cruzi infection in dogs that, in Brazil, rarely present positive parasitological tests. The use of recombinant chimeric parasitic antigens results in a sensitive and specific serological diagnostic test in contrast to the use of crude T. cruzi antigens. Our objective was to evaluate the Chagas/Bio-Manguinhos Lateral Flow Immunochromatographic Rapid Test (Chagas-LFRT) for the diagnosis of T. cruzi infection in domestic dogs and the potential of application of this diagnostic platform to wild canid species. Two recombinant proteins (IBMP-8.1 and IBMP-8.4) that displayed the best performance in the enzyme immunoassay (ELISA) in previous studies were tested in a platform with two diagnostic bands. A panel of 281 dog serum samples was evaluated: 133 positive for T. cruzi by serological diagnosis, including 20 samples with positive blood cultures belonging to different discrete typing units (DTUs); 129 negative samples; and 19 samples from dogs infected by other trypanosomatids: Leishmania infantum, Trypanosoma rangeli, Trypanosoma caninum and Crithidia mellificae, in addition to samples infected by Anaplasma platys, Dirofilaria immitis and Erlichia sp. that were employed to evaluate eventual cross-reactions. We also evaluated the Chagas-LFRT to detect T. cruzi infection in 9 serum samples from six wild canid species. We observed that the intensity pattern of the bands was directly proportional to the serological titer observed in IFAT. The sensitivity was 94%, the specificity was 91% according to the ROC curve, and the defined cutoff was an optical density of 4.8. The agreement obtained was considered substantial by the kappa analysis (84%). From T. cruzi positive hemoculture samples, 88.9% were positive by Chagas-LFRT. The test was efficient in recognizing infections by five of the six T. cruzi DTUs. Cross-reactions were not observed in infections by L. infantum, T. rangeli, T. caninum and D. immitis; however, they were observed in sera of dogs infected by Crithidia mellificae, Anaplasma sp. and Erlichia sp. A strong reaction was observed when serum samples from wild canids were submitted to the Protein A affinity test, confirming its applicability for these species. This test will allow rapid preventive actions in areas with high risk to the emergence of Chagas disease in a safer, reliable, low-cost and immediate manner, without the need for more complex laboratory tests.

Comprehensive mapping of SARS-CoV-2 peptide epitopes for development of a highly sensitive serological test for total and neutralizing antibodies.

Quantification of the anti-SARS-CoV-2 antibody response has proven to be a prominent diagnostic tool during the COVID-19 pandemic. Antibody measurements have aided in the determination of humoral protection following infection or vaccination and will likely be essential for predicting the prevalence of population level immunity over the next several years. Despite widespread use, current tests remain limited in part, because antibody capture is accomplished through the use of complete spike and nucleocapsid proteins that contain significant regions of overlap with common circulating coronaviruses. To address this limitation, a unique epitope display platform utilizing monovalent display and protease-driven capture of peptide epitopes was used to select high affinity peptides. A single round of selection using this strategy with COVID-19 positive patient plasma samples revealed surprising differences and specific patterns in the antigenicity of SARS-CoV-2 proteins, especially the spike protein. Putative epitopes were assayed for specificity with convalescent and control samples, and the individual binding kinetics of peptides were also determined. A subset of prioritized peptides was used to develop an antibody diagnostic assay that showed low cross reactivity while detecting 37% more positive antibody cases than a gold standard FDA EUA test. Finally, a subset of peptides were compared with serum neutralization activity to establish a 2 peptide assay that strongly correlates with neutralization. Together, these data demonstrate a novel phage display method that is capable of comprehensively and rapidly mapping patient viral antibody responses and selecting high affinity public epitopes for the diagnosis of humoral immunity.

Evaluation of in-house ELISA and 11 commercial ELISA kits in the serological diagnosis of COVID-19

Introduction: ELISA-Immunoassays can complement the molecular diagnostic methods, and can be one of the important tools of sero-surveillance and vaccine evaluation. The aim of the presented study was to develop in-house ELISA and evaluate 11 commercial ELISA tests for detection of anti-SARS-CoV-2 antibodies in serum samples collected from COVID patients. Methods: In total, 237 serum samples obtained from 165 people with COVID-19 with RT-PCR confirmed SARS-CoV-2 virus infection were used for the study. The specificity of the developed in-house ELISA kit was tested using 170 serum samples obtained from patients with various bacterial and viral infections. The study used an in-house ELISA and 11 commercial ELISA kits developed by various manufacturers. Results: The presented study showed high sensitivity (81.0%) and specificity (97.2%) of the developed in-house kit in relation to the RT-PCR method. The sensitivity of the inhouse test significantly increased (98.1%) when only convalescents - persons at least 3 weeks after COVID-19 were examined. Commercial ELISA kits most frequently detected IgG antibodies (from 44.9% to 89.4%), especially in samples obtained later in the disease, and the least frequent detection of IgM antibodies (from 4.2% to 42.4%). Conclusions: All the presented ELISA kits may be used in serodiagnosis of COVID-19 however the detection of antibodies in individual tests differed quite significantly and was dependent on the period of the disease, on the class of immunoglobulins and the type of antigen used. The sensitivity of serological tests in the IgG class is clearly higher when examining samples obtained at least 2-3 weeks from the onset of clinical symptoms. Searching for IgA antibodies may be useful mainly in the early phase of the disease while IgM antibodies does not provide significant additional information. In the case of asymptomatic or mild infection, the level of antibodies is low which may be the cause problems with the correct interpretation of epidemiological surveys

Evaluation of rapid, cassette immunochromatographic tests in the serological diagnosis of COVID-19

Introduction: Lateral flow assays (LFIA) are the technology behind low-cost, simple, rapid and portable detection devices popular in biomedicine. Lately, they are very common used in serodiagnosis of SARS-CoV-2 infections. The aim of the presented study was to assess the usefulness of selected LFIA in serological diagnosis of COVID-19. Methods: The usefulness of seven lateral flow assays in the serodiagnosis of COVID-19 was evaluated (VAZYME, DIAGNOSIS, PCL, INGEZIM, BIOSENSOR, ACCU-TELL, NOVAtest). The study used 107 serum samples obtained from 74 individuals with current SARS-CoV-2 infection confirmed by RT-PCR. The ELISA-IgG (Euroimmun) was used as the reference assay for sensitivity and specificity testing. Results: The highest percentage of positive results was obtained when searching for IgG antibodies with the NOVAtest (40.6%) and DIAGNOSIS (39.2%) sets and the lowest detection for the PCL set - 25.5%. In the case of searching for IgM antibodies in all sets, significantly lower percentages of positive results compared to the IgG class were recorded. In general, all lateral flow assays showed low sensitivity in relation to the Euroimmun ELISA-IgG. The DIAGNOSIS kit (64.5%) was characterized by the highest sensitivity, and the PCL kit was the lowest (38.7%). On the other hand, the specificity of all kits was very high, almost 100% in almost all cases. Conclusions: Lateral flow assays due to their low sensitivity are not suitable for quick diagnosis of the current SARS-CoV-2 infections and cannot be an alternative to genetic or even antigen tests. They may be used only to retrospectively test the presence of IgG antibodies. However, a negative results of LFIA in suspected disease or after vaccination should be confirmed by more sensitive serological tests.

Quiste hidatídico cervical primario

Cervical echinococcosis is a rare disease, even in endemic areas. The lesions usually present as painless slow-growing cystic lesion. We report the case of a 19-year-old female patient with a cystic lesion in the central cervical region that was initially interpreted as a thyroglossal duct cyst. The diagnosis should be suspected when the epidemiology is consistent, and ultrasound is the imaging method of choice. The sensitivity of serologic tests is low in extrahepatic locations. Surgery is the treatment of choice and the administration of albendalzole before surgery is indicated. The cyst must be handled with care to avoid rupture and leakage of cyst contents, which can cause anaphylaxis, recurrence and multiple echinococcosis. In patients with high surgical risk, medical treatment alone may be the option.

Obtaining Multiprotein Antigens Of Brucella And Study Of Their Immunoreactivity

Rose-Bengal test, complement fixation test and the agglutination test are mainly used for the diagnosis of animal brucellosis. These tests are characterized by low sensitivity and specificity, which is one of the main reasons for the low effectiveness of measures aimed at eradicating brucellosis. The use of modern highly sensitive serological tests requires the availability of antigens specific to Brucella spp. The aim of the study was to obtain multiproteins of the pathogen by recombinant DNA technology and to study their antigenic properties. In the course of the study, three types of multiproteins were obtained, constructed from diagnostically important peptides that form B.abortus and B.melitensis outer membrane proteins. All target products were synthesized by the producer strain in a form that is authentic to natural proteins, and showed immunogenicity in mouse model. Antibodies produced against the multiproteins were specific for the single proteins of the pathogen's cell wall. The data obtained indicate the need to continue studies to determine the possibility of using multiproteins as an antigen in an enzyme-linked immunosorbent assay to detect anti-Brucella specific antibodies.

Development of in-house ELISAs for the detection of anti-SARS‑CoV‑2 RBD and N IgG and IgM antibodies in biological samples

By the end of year 2019, the new virus SARS-CoV-2 appeared, causing the Coronavirus Disease 2019 (COVID-19), and spread very fast globally. A continuing need for diagnostic tools is a must to contain its spread. Till now, the gold standard method, the reverse transcription polymerase chain reaction (RT-PCR), is the precise procedure to detect the virus. However, SARS-CoV-2 may escape RT-PCR detection for several reasons. The development of well-designed, specific and sensitive serological test like enzyme immunoassay (EIA) is needed. This EIA can stand alone or work side by side with RT-PCR.In this study, we developed several EIAs including plates that are coated with either specially designed SARS-CoV-2 nucleocapsid or surface recombinant proteins. Each protein type can separately detect anti-SARS-CoV-2 IgM or IgG antibodies. For each EIAs, the cut-off value, specificity and sensitivity were determined utilizing RT-PCR confirmed Covid-19 and pre-pandemic healthy and other viruses-infected sera. Also, the receiver operator characteristic (ROC) analysis was performed to define the specificities and sensitivities of the optimized assay. The in-house EIAs were validated by comparing against commercial EIA kits. All in-house EIAs showed high specificity (98–99%) and sensitivity (97.8–98.9%) for the detection of IgG/IgM against RBD and N proteins of SARS-CoV-2.From these results, the developed Anti-RBD and anti-N IgG and IgM antibodies EIAs can be used as a specific and sensitive tool to detect SARS-CoV-2 infection, calculate the burden of disease and case fatality rates.

Role of rapid antibody and ELISA tests in the evaluation of serological response in patients with SARS-CoV-2 PCR positivity.

From 7 to 8 days after the onset of symptoms in COVID-19 infection, the sensitivity of serological tests was found to be higher than that of nucleic acid tests. The aims of this study were to investigate antibody levels in patients with SARS-CoV-2 infection, to examine the relationship between antibody levels and virus load, and to evaluate the performance of 2 different commercial kits. A total of 103 patients with confirmed SARS-CoV-2 infection were included in the study. Antibodies against SARS-CoV-2 in serum samples taken from patients were investigated simultaneously with anti-SARS-CoV-2 IgG and IgA ELISAs (Euroimmun) and COVID-19 (SARS-CoV-2) IgG/IgM (Deep Blue) kits. No positivity was detected with any of the test kits in 18 (17.4%) of the 103 samples. In symptomatic patients, 100% of IgM and IgA tests were found to be positive in the group sampled after 10 days, while 100% of IgG tests were found positive after 20 days. The sensitivity of the Deep Blue COVID-19 IgG antibody kit was calculated as 81.48% and the specificity was 97.96%. While there was no statistically significant difference between the PCR CT and ELISA OD values, a positive correlation was found between the ELISA OD values and the days since the date of symptom initiation. The sensitivity and specificity of the rapid antibody test used in this study were found to be quite high. In conditions where ELISA tests cannot be applied, it is thought that it can give an idea in terms of the presence of antibodies as a simple and fast test. Although ELISA tests are valuable in the diagnosis of COVID-19 during the acute period, they are tests that can be used safely in the diagnosis of previous infections and seroepidemiological studies.

Age- and Sex-Associated Variations in the Sensitivity of Serological Tests Among Individuals Infected With SARS-CoV-2

This cohort study examines the sensitivity of antibody tests to detect previous severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection by time, test, sex, and age.

Monitoring of SARS-COV-2 antibodies in tuberculosis in-patient clinic stuff

Objective. Тo study the features of humoral immunity to SARS-CoV-2 among the medical stuff of an antitubercular hospital in the context of the spread of a new coronavirus infection. Materials and methods. The interim results of a single-center prospective observational study involving 350 employees of a pulmonary tuberculosis hospital that was not repurposed to work with COVID-19 are presented. The results of weekly monitoring of IgM and IgG levels for the period from May 15 to November 28, 2020, were analyzed in comparison with the results of diagnosis of COVID-19 cases (identification of the pathogen in a smear from the oropharynx and nasopharynx, changes in lung tissue according to computed tomography). Results. During the observation period, COVID-19 was detected in 106 people (30.3%). Antibodies to SARS-CoV-2 (IgG and/or IgM) were detected at the start of monitoring in 30 people (8.6%, 95%CI 5.8-12.2%), by the end of the follow – up period-in 95 people (27.1%, 95%CI 22.7- 32.0%). The frequency of detection of antibodies in the patients was 70.8% (95%CI 55.6-88.7%): IgM class in 23 (21.7%), G class in 73 (68.9%). The median time from disease detection to IgM detection was 1.5 weeks. (interquartile range 0-4 weeks), before the discovery of IgG – 4 weeks. (interquartile range 2-5 weeks). At any follow-up period, the chances of IgG production in the protective titer were higher in the case of moderate or severe disease with the development of pneumonia compared to the mild course (OR 2.6, 95%CI 1.1-6.3); the IgG titer was also higher (p < 0.01). «Atypical» variants of the antibody response were identified in individuals with confirmed COVID-19: no antibody formation (31 people), production of class M antibodies only (4 people); long-term persistence of IgG in a titer of less than 30 g/l, long ahead of the disease manifestation (6 people). The sensitivity of serological testing as a method for detecting current and / or transmitted coronavirus infection was 70.8% (95%CI 61.1-79.2%), and the specificity was 91.8% (95%CI 87.6-94.9%). Conclusion. The frequency of detection of antibodies to SARS-CoV-2 among employees of the TB hospital corresponds to the dynamics of the incidence of COVID-19. Virus-specific antibodies were detected in 70.8% of COVID-19 survivors (IgG in 68.9%); in the absolute majority of patients, the antibodies remained in a significant titer (IgG > 10 g/l) for 6 months or more at the start of testing. When the disease occurs without the development of viral pneumonia, the chances of forming a significant and persistent antibody response are significantly lower. Serological testing is a sensitive and highly specific method for detecting a previous COVID-19 disease.