Immunoglobulin G Enzyme-linked Immunosorbent Assay Research Articles

The Development of Toxoplasma gondii Recombinant Trivalent Chimeric Proteins as an Alternative to Toxoplasma Lysate Antigen (TLA) in Enzyme-Linked Immunosorbent Assay (ELISA) for the Detection of Immunoglobulin G (IgG) in Small Ruminants.

This study presents an evaluation of seventeen newly produced recombinant trivalent chimeric proteins (containing the same immunodominant fragment of SAG1 and SAG2 of Toxoplasma gondii antigens, and an additional immunodominant fragment of one of the parasite antigens, such as AMA1, GRA1, GRA2, GRA5, GRA6, GRA7, GRA9, LDH2, MAG1, MIC1, MIC3, P35, and ROP1) as a potential alternative to the whole-cell tachyzoite lysate (TLA) used in the detection of infection in small ruminants. These recombinant proteins, obtained by genetic engineering and molecular biology methods, were tested for their reactivity with specific anti-Toxoplasma IgG antibodies contained in serum samples of small ruminants (192 samples of sheep serum and 95 samples of goat serum) using an enzyme-linked immunosorbent assay (ELISA). The reactivity of six recombinant trivalent chimeric proteins (SAG1-SAG2-GRA5, SAG1-SAG2-GRA9, SAG1-SAG2-MIC1, SAG1-SAG2-MIC3, SAG1-SAG2-P35, and SAG1-SAG2-ROP1) with IgG antibodies generated during T. gondii invasion was comparable to the sensitivity of TLA-based IgG ELISA (100%). The obtained results show a strong correlation with the results obtained for TLA. This suggests that these protein preparations may be a potential alternative to TLA used in commercial tests and could be used to develop a cheaper test for the detection of parasite infection in small ruminants.

Accuracy of commercial ELISA and ICT for screening schistosomiasis infections at a low endemicity area in Brazil.

Control interventions recommended by the World Health Organization have successfully resulted in low-intensity schistosomiasis transmission areas. To achieve elimination of transmission, new diagnostic screening tools are needed to overcome less than adequate sensitivity of the currently used Kato-Katz faecal thick smear method. Ideally, in-house serological tests should be avoided due to not having a continuous supply of kits as would be necessary for large population studies. Quality assurance provided by manufacturers and proper performance evaluations are also needed. We evaluated the accuracy of two commercially available serology tests as screening methods for detecting light schistosomiasis infections. Serum samples were collected in 2015 from individuals living in a low-endemicity locality in northeastern Brazil and deposited in a biorepository. We evaluated immunoglobulin G (IgG) and IgM enzyme-linked immunosorbent assays (ELISAs) and an immunochromatographic test (ICT). The Helmintex method was used to define true-positive samples. Overall sensitivity was close to 90% for both the IgG ELISA and ICT, yet specificity was 28% and 18%, respectively. For the IgM ELISA, the values were estimated to be 55% and 43%, respectively. Poor specificity and positive predictive values prevent these tests from being recommended for screening populations in low-intensity schistosomiasis-endemic areas.

Have we attended herd immunity against COVID? A cross-sectional analytical study of seroprevalence among blood donors

Abstract BACKGROUND: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) (COVID-19) is one of the worst health-care problem humankind has faced in the last few centuries. Understanding the population’s antibody responses is crucial for effective decision-making while implementing public health interventions in view of the COVID-19 pandemic. The study was done to know the immune status of healthy donors to COVID-19 infections and to know the duration of persistence of immunoglobulin G (IgG) antibodies with respect to infections and vaccination. AIMS: This study aimed to determine the prevalence of SARS-CoV-2 IgG antibodies in healthy blood donors in our tertiary care center. MATERIALS AND METHODS: This was a cross-sectional analytical study conducted for a period of 1 year in our blood center. Our study had a sample size of 809 healthy blood donors. Samples of the first 81 donors of each month were chosen. Donor details such as previous COVID positivity, symptoms, hospitalization, comorbidities, contact history, and vaccination status were collected using a structured questionnaire. A 5-ml sample was collected and was tested using Qualisa COVID-19 IgG enzyme-linked immunosorbent assay detection kits. The antigens used for coating the kits were recombinant antigens representing SARS-CoV-2 S1 protein. Data were analyzed using SPSS version 21. The prevalence of positive IgG seroprevalence of COVID-19 antibodies was reported using proportion along with 95% confidence interval. Association between all the categorical variables and the presence of seropositive status for IgG antibodies was assessed using the Chi-square test. P <0.05 was considered statistically significant. RESULTS: The seroprevalence of COVID-19 antibodies in our study participants was 82.3%. There is a significant association between COVID-19 infectivity, vaccination, with seroprevalence. There was a seropositivity of 51% among those who were neither infected nor vaccinated, which may be due to the development of herd immunity in our population. CONCLUSIONS: Population who were neither vaccinated nor had a history of infection also had a significant prevalence of antibodies suggesting the development of herd immunity in our community. The present study will add to the knowledge and better understanding of COVID-19 antibodies in the existing literature. Further studies will be required to verify our findings and to know the persistent nature of COVID antibodies.

A third (booster) dose of the inactivated SARS-CoV-2 vaccine elicits immunogenicity and T follicular helper cell responses in people living with HIV.

This study sought to explore the immunogenicity of a booster dose of an inactivated severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) vaccine in people living with human immunodeficiency virus (HIV) and identify the factors affecting the magnitude of anti-SARS-CoV-2 antibody levels. A total of 34 people living with HIV (PLWH) and 34 healthy donors (HD) were administered a booster dose of the same SARS-CoV-2 vaccine. Anti-SARS-CoV-2 antibody and immunoglobulin G (IgG) levels were measured using the SARS-CoV-2 S protein neutralizing antibody Enzyme-Linked Immunosorbent Assay (ELISA) and 2019-nCov IgG Chemiluminescent Immunoassay Microparticles, respectively. Spearman correlation analysis was used to measure the correlation between laboratory markers and neutralizing antibody and IgG levels. Peripheral blood mononuclear cells (PBMCs) were extracted from each subject using density gradient centrifugation and the numbers of memory T and T follicular helper (Tfh) cells were determined using flow cytometry. PLWH had a marked reduction in CD4 and B cell levels that was accompanied by a lower CD4/CD8 T cell ratio. However, those who received a supplementary dose of inactivated SARS-CoV-2 vaccines exhibited antibody positivity rates that were analogous to levels previously observed. The booster vaccine led to a reduction in IgG and neutralizing antibody levels and the amplitude of this decline was substantially higher in the PLWH than HD group. Correlation analyses revealed a strong correlation between neutralizing antibody levels and the count and proportion of CD4 cells. Anti-SARS-CoV-2 IgG antibody levels followed a similar trend. The expression of memory T and Tfh cells was considerably lower in the PLWH than in the HD group. PLWH had an attenuated immune response to a third (booster) administration of an inactivated SARS-CoV-2 vaccine, as shown by lower neutralizing antibody and IgG levels. This could be attributed to the reduced responsiveness of CD4 cells, particularly memory T and cTfh subsets. CD4 and cTfh cells may serve as pivotal markers of enduring and protective antibody levels. Vaccination dose recalibration may be critical for HIV-positive individuals, particularly those with a lower proportion of CD4 and Tfh cells.

Multiplexed bead-based assay for the simultaneous quantification of human serum IgG antibodies to tetanus, diphtheria, pertussis toxin, filamentous hemagglutinin, and pertactin.

Luminex bead-based assays offer multiplexing to test antibodies against multiple antigens simultaneously; however, this requires validation using internationally certified reference standards. Therefore, there is an urgent need to characterize existing reference standards for the standardization of multiplex immunoassays (MIAs). Here, we report the development and validation of an MIA for the simultaneous estimation of levels of human serum immunoglobulin G (IgG) antibodies for pertussis toxin (PT), filamentous hemagglutinin (FHA), pertactin (PRN), diphtheria toxoid (DT), and tetanus toxoid (TT). The MIA was assessed using a panel of human serum samples and WHO reference standards. The WHO reference standards were also studied for suitability in the MIA. Purified antigens (PT, FHA, PRN, DT, and TT) were coupled to the spectrally unique magnetic carboxylated microspheres. The method was validated in accordance with the United States Food and Drug Administration (US FDA), European Medicines Agency (EMA), and the International Committee of Harmonization Multidisciplinary (ICH M10) guidelines, and parameters such as precision, accuracy, dilutional linearity, assay range, robustness, and stability were assessed. Method agreements with commercially available IgG enzyme-linked immunosorbent assay (ELISA) assays were also evaluated. In addition, the study assessed the level of correlation between the IgG levels estimated by the MIA and the cell-based neutralizing antibody assays for PT and DT. We identified that an equimix of WHO international standards (i.e., 06/142, 10/262, and TE-3) afforded the best dynamic range for all the antigens in the MIA. For all five antigens, we observed that the back-fitted recoveries using the four-parameter logistic (4-PL) regression fits ranged between 80% and 120% for all calibration levels, and the percentage coefficient of variation (% CV) was < 20%. In addition, the difference in mean fluorescence intensity (MFI) between the monoplex and multiplex format was < 10% for each antigen, indicating no crosstalk among the beads. The MIA also showed good agreement with conventional and commercially available assays, and a positive correlation (> 0.75) with toxin neutralization assays for PT and DT was observed. The MIA that was calibrated in accordance with WHO reference standards demonstrated increased sensitivity, reproducibility, and high throughput capabilities, allowing for the design of robust studies that evaluate both natural and vaccine-induced immunity.

Prevalence of Barmah Forest Virus, Chikungunya Virus and Ross River Virus Antibodies among Papua New Guinea Military Personnel before 2019.

Barmah Forest virus (BFV), Chikungunya virus (CHIKV) and Ross River virus (RRV) belong to the Alphavirus genus of the family Togaviridae. All three virus infections have been reported in Papua New Guinea (PNG) previously, but the exact prevalence and distribution of these three alphaviruses in PNG has not been established. Sera collected from 204 PNG Military Personnel (PNGMP) study participants in April 2019 was tested for the presence of anti-BFV, anti-CHIKV and anti-RRV immunoglobulin G (IgG) antibodies using commercially available enzyme-linked immunosorbent assay (ELISA) IgG detection kits, as well as for specific neutralizing antibodies (NAb) against individual viruses. Overall, sero-positivity of the sera was anti-BFV IgG 12.3% (25/204), anti-BFV NAb 8.3% (17/204); anti-CHIKV IgG 47.1% (96/204), anti-CHIKV NAb 34.8% (71/204); and anti-RRV IgG 93.1% (190/204), anti-RRV NAb 56.4% (115/204), respectively. Of the 137/204 participants that were Nab-positive for at least one virus, we identified 4 BFV, 40 CHIKV and 73 RRV single infections, and 9 RRV+CHIKV and 11 BFV+RRV double infections. The lower proportion of NAb sero-positive compared to the ELISA IgG sero-positive assay samples suggests that the currently available commercial ELISA detection kits for these three alphaviruses may not be suitable for diagnostic/surveillance purposes in endemic areas such as PNG, due to serological cross-reactivity among these three alphaviruses. Laboratory testing using known positive control sera indicated no cross-neutralization between BFV and RRV; however, some RRV or BFV single infection human sera demonstrated low-level cross-neutralization against CHIKV (the ratio of RRV/CHIKV NAb titers or BFV/CHIKV ≥ 4). Our preliminary results indicate that the majority of PNGMP have previously been exposed to RRV, with mild exposure to CHIKV and low-level exposure to BFV, suggesting that multiple alphaviruses have been circulating among PNGMP. The transmission landscapes of these three alphaviruses across PNG should be prioritized for further investigation, including identification of specific vectors and hosts that mediate human spillover in order to mitigate future outbreaks. Ongoing education regarding precautionary and protective measures are needed to better protect individuals who travel to PNG.

Booster Vaccination Against SARS-CoV-2 Induces Potent Immune Responses in People With Human Immunodeficiency Virus.

People with human immunodeficiency virus (HIV) on antiretroviral therapy (ART) with good CD4 T-cell counts make effective immune responses following vaccination against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). There are few data on longer term responses and the impact of a booster dose. Adults with HIV were enrolled into a single arm open label study. Two doses of ChAdOx1 nCoV-19 were followed 12 months later by a third heterologous vaccine dose. Participants had undetectable viraemia on ART and CD4 counts >350 cells/µL. Immune responses to the ancestral strain and variants of concern were measured by anti-spike immunoglobulin G (IgG) enzyme-linked immunosorbent assay (ELISA), MesoScale Discovery (MSD) anti-spike platform, ACE-2 inhibition, activation induced marker (AIM) assay, and T-cell proliferation. In total, 54 participants received 2 doses of ChAdOx1 nCoV-19. 43 received a third dose (42 with BNT162b2; 1 with mRNA-1273) 1 year after the first dose. After the third dose, total anti-SARS-CoV-2 spike IgG titers (MSD), ACE-2 inhibition, and IgG ELISA results were significantly higher compared to Day 182 titers (P < .0001 for all 3). SARS-CoV-2 specific CD4+ T-cell responses measured by AIM against SARS-CoV-2 S1 and S2 peptide pools were significantly increased after a third vaccine compared to 6 months after a first dose, with significant increases in proliferative CD4+ and CD8+ T-cell responses to SARS-CoV-2 S1 and S2 after boosting. Responses to Alpha, Beta, Gamma, and Delta variants were boosted, although to a lesser extent for Omicron. In PWH receiving a third vaccine dose, there were significant increases in B- and T-cell immunity, including to known variants of concern (VOCs).

Detectable Respiratory Viruses IgM and IgG Antibodies among Children with Acute Respiratory Tract Infections in Owo, Ondo State, Nigeria

Globally, in children, less than 5 years of age, acute respiratory tract infections (ARTIs) are a major cause of morbidity and mortality. Long-lasting immunity is not induced by respiratory infections as reinfection can occur throughout life. This study aimed to determine the seroprevalence of six respiratory viruses specific immunoglobulin M (IgM) and immunoglobulin G (IgG) antibodies in children with ARTIs in Owo, Ondo State. The sera samples of two hundred (200) subjects who consented to participate in the study were collected and tested using serum-specific Enzyme-Linked Immunosorbent Assay (ELISA) kits, anti-immunoglobulin M (IgM) and anti-immunoglobulin G (IgG) antibodies to the influenza A virus (FLU-A), respiratory syncytial virus (RSV), parainfluenza virus (PIV), coronavirus (CoV), rhinovirus (RV) and adenovirus (AdV) respectively (IgM and IgG ELISA Kits; Melsin Medical Co., China). The mean age of the subjects tested was 3.49±1.41. The total IgM seropositivity was detected in 83% of the children with the highest being AdV 91 (45.5%), followed by PIV 89 (44.5%), FLU-A and RV with 88 (44%) respectively, CoV 85 (42.5%), and RSV 80 (40%). The total IgG seropositivity was detected in 87.5% of the children with the highest being PIV 152 (76%), followed by RSV 135 (67.5%), RV 93 (46.5%), AdV 81 (40.5%), CoV and FLU A 76 (38%) respectively. The study revealed the presence of primary and secondary infection of respiratory viruses in Owo and the need for preventive and control measures against respiratory tract viruses are suggested.

Assays for the Evaluation of the Immune Response to Marburg and Ebola Sudan Vaccination-Filovirus Animal Nonclinical Group Anti-Marburg Virus Glycoprotein Immunoglobulin G Enzyme-Linked Immunosorbent Assay and a Pseudovirion Neutralization Assay.

Since the discovery of the Marburg virus (MARV) in 1967 and Ebola virus (EBOV) in 1976, there have been over 40 reported outbreaks of filovirus disease with case fatality rates greater than 50%. This underscores the need for efficacious vaccines against these highly pathogenic filoviruses. Due to the sporadic and unpredictable nature of filovirus outbreaks, such a vaccine would likely need to be vetted through the U.S. Food and Drug Administration (FDA), following the Animal Rule or similar European Medicines Agency (EMA) regulatory pathway. Under the FDA Animal Rule, vaccine-induced immune responses correlating with survival of non-human primates (NHPs), or another well-characterized animal model, following lethal challenge, will need to be bridged for human immune response distributions in clinical trials. A correlate of protection has not yet been identified for the filovirus disease, but antibodies, specifically anti-glycoprotein (GP) antibodies, are believed to be critical in providing protection against the filovirus disease following vaccination and are thus a strong candidate for a correlate of protection. Thus, species-neutral methods capable of the detection and bridging of these antibody immune responses, such as methods to quantify anti-GP immunoglobulin G (IgG)-binding antibodies and neutralizing antibodies, are needed. Reported here is the development and qualification of two Filovirus Animal Nonclinical Group (FANG) anti-GP IgG Enzyme-Linked Immunosorbent Assays (ELISAs) to quantify anti-MARV and anti-Sudan virus (SUDV) IgG antibodies in human and NHP serum samples, as well as the development of pseudovirion neutralization assays (PsVNAs) to quantify MARV- and SUDV-neutralizing antibodies in human and NHP serum samples.

Serum and breastmilk SARS-CoV-2 specific antibodies following BNT162b2 vaccine: prolonged protection from SARS-CoV-2 in newborns and older children

ObjectivesVaccination is the best strategy against COVID-19. We aimed to determine antibodies against SARS-CoV-2 in breastmilk and serum of mothers vaccinated with the mRNA vaccine. MethodsThis prospective study included 18 lactating women vaccinated with the BNT162b2 vaccine. Serum and breastmilk were collected before the first dose (T0), at the second dose (T1), 3 weeks after the second dose (T2), and 6 months after the first dose (T3). Serum anti-SARS-CoV-2 Spike (S) Immunoglobulin G (IgG) and Immunoglobulin A (IgA) were measured using a semi-quantitative enzyme-linked immunosorbent assay (ELISA) and secretory antibody (s) IgG and IgA in breastmilk using quantitative analysis. ResultsWe detected serum anti-S IgG and IgA in all women after vaccination. Specific IgG and IgA were higher at T1, T2, and T3 compared with T0 (P <0.0001). Higher antibody levels were observed at T2 and lower values at T3 versus T2 (P = 0.007). After 6 months, all patients had serum IgG, but three of 18 (16%) had serum IgA. In breastmilk, sIgA was present at T1 and T2 and decreased after 6 months at T3 (P = 0.002). Breastmilk sIgG levels increased at T1 and T2 and peaked at T3 (P = 0.008). ConclusionSecretory antibodies were transmitted through breastmilk until 6 months after anti-COVID-19 mRNA vaccination. Protection of the newborn through breastfeeding needs to be addressed.

The first study on confirmation and risk factors of acute and chronic canine distemper in stray dogs in Wasit Province, Iraq, using enzyme-linked immunosorbent assay and reverse transcription-polymerase chain reaction.

Background and Aim:In Iraq, stray dogs represent a critical population of free-roaming animals, which probably play a role in the transmission of different infections to other animals. Canine distemper is one of the most growing viral threats to carnivores in many countries worldwide, including Iraq. Therefore, this study was aimed to diagnose the disease using serological and molecular assay and the role of risk factors in the spreading infection.Materials and Methods:In all, 158 venous blood samples were collected randomly from stray dogs in rural and sub-urban areas of Iraq from May 2019 to December 2020. The samples were examined serologically using two enzyme-linked immunosorbent assay (ELISA) kits, immunoglobulin G (IgG) and immunoglobulin M (IgM), and molecularly by reverse transcription-polymerase chain reaction (RT-PCR) to detect and confirm chronic and acute infections. To determine the association between infection and various risk factors, the study animals were divided according to their locations, sexes, and ages. The age groups were ≤8 months (puppy), 1-3 years (young), and ≥3 years (old/mature).Results:ELISA result shows that 6.96% and 19.62% of dogs were seropositive for acute and chronic distemper, respectively. The titer of chronic infections (0.421±0.027) was significantly higher (p≤0.025) than that of acute canine distemper (0.337±0.016). On RT-PCR, 8.86% of dogs were found positive for distemper. Using RT-PCR as the gold standard, the sensitivity and specificity of the IgM ELISA kit were 75% and 98.63%, respectively, whereas the positive and negative predictivity were 81.82% and 97.96%, respectively. A significant variation (p<0.05) was observed in the distribution of positive findings among the different epidemiological risk factors. Compared with rural areas, positivity was significantly higher (p<0.05) in sub-urban areas on IgM (26.92%) and IgG (64.15%) ELISA and RT-PCR (34.62%). On IgM ELISA and RT-PCR, no significant differences (p>0.05) were found among the three age groups; however, positivity was significantly higher (p≤0.048) in the ≥3 years group (22.73%) on IgG ELISA. Furthermore, only IgG ELISA showed a significantly higher (p≤0.032) positivity rate in female dogs (25.23%) than in male dogs (7.84%).Conclusion:To the best of our knowledge, this is the first Iraqi study to demonstrate acute and chronic canine distemper in stray dogs, suggesting that the prolonged shedding of virus from positive dogs is a critical point in the epidemiology of the disease. Additional studies in dogs or other carnivores are required to establish baseline data on the prevalence of the disease in Iraq.

Is Immuno-PCR Better than ELISA Test for Detection of Toxoplasma gondii IgG Antibody?

IntroductionIgG antibodies against T. gondii persist for years, and can act as a reliable serological biomarker for the diagnosis of previous exposure to this parasite. Hence, the current investigation was designed to compare diagnostic power of immuno-polymerase chain reaction (iPCR) and enzyme-linked immunosorbent assay (ELISA) methods for detection of T. gondii IgG antibody.MethodsImmunoglobulin M (IgM) and immunoglobulin G (IgG) antibodies against T. gondii were measured by the ELISA method in 81 participants. In addition, detection of acute and chronic toxoplasmosis was performed via the ELISA IgG avidity. The set-up of iPCR was carried out and then, serum IgG of subjects were detected using the iPCR method.ResultsOf 81 samples, 4 (4.9%) and 30 (37%) cases were be found positive for IgM and IgG against T. gondii in the ELISA method, respectively. Moreover, of 81 specimens, 42 (51.9%) and 39 (48.1%) samples had low-avidity IgG and high-avidity IgG by the IgG avidity kit, respectively. While, 59 (72.8%) of 81 samples were detected positive using the iPCR technique. Kappa (κ) value coefficient, between the iPCR and ELISA (for IgG) showed a strong agreement (0.360, p value < 0.001). A value of 0.25 I.U./ml for serum IgG [area under curve (AUC) = 0.720 (CI = 0.613–0.827); p = 0.002] was the cut-off value to differentiating between positive and negative toxoplasmosis (with sensitivity 66.0% and specificity 60.0%).ConclusionOur findings indicated despite a strong agreement shown between iPCR and ELISA methods, the diagnostic power of iPCR technique was more sensitive than ELISA test for detection of T. gondii IgG antibody. However, more complementary investigations are widely needed in this regard.

SARS-CoV-2 IgG Amongst Dental Workers During the COVID-19 Pandemic

ObjectivesSince the SARS-CoV-2 outbreak in 2019, special safety protocols have been introduced in dentistry. Dental professionals were determined to be mostly at risk for contracting the virus due to aerosol-generating procedures used. This preliminary study starts the cycle of the laboratory protocols describing the quality and efficacy of laboratory tests in the SARS-CoV-2 immunoglobulin G (IgG) detection in the serum of asymptomatic dental personnel during the last quarter of 2020. MethodsIgG levels were measured with the use of a semi-quantitative enzyme-linked immunosorbent assay (ELISA) in vitro diagnostic kit in the serum of a study group that consisted of 127 employees of the dental clinic divided into 3 subgroups: SUB1: dentists (n = 67); SUB2: dental assistants, dental hygienists, nurses, laboratory workers (n = 40); SUB3: administrative workers (n = 20). Pearson analysis of results from the questionnaires attached to the study protocol were provided to assure that the results compare to the participants’ impressions about their general health. ResultsPositive ELISA IgG results were found in 6% (n = 4) of the SUB1 group, 7.50% (n =3) of the SUB2 group, and 5% of the SUB3 group. The percentage of participants without work interruption from the beginning of the pandemic was 54% of dentists and 60% of chairside assistants. ConclusionsSerum IgG prevalence with the use of a semi-quantitative test was low, and further research on the biobanked samples should follow to determine the levels of IgG with quantitative methods and/or to evaluate the presence of neutralising antibodies in dental personnel. Because of the low representation of seropositivity studies in this group, it will be crucial to confirm the risk of COVID-19 transmission in dental offices.

IgG Avidity in Samples Collected on Filter Paper: Importance of The Early Diagnosis of Congenital Toxoplasmosis.

The purpose of the present study is to standardize and evaluate the use of the immunoglobulin G (IgG) antibody avidity test on blood samples from newborns collected on filter paper to perform the heel test aiming at its implementation in ongoing programs. Blood samples from newborns were collected on filter paper simultaneously with the heel prick test. All samples were subjected to immunoglobulin M IgM and IgG enzyme-linked immunosorbent assays (ELISA). Peripheral blood was collected again in the traditional way and on filter paper from newborns with high IgG levels (33). Three types of techniques were performed, the standard for measuring IgG in serum, adapted for filter paper and the technique of IgG avidity in serum and on filter paper. The results of the avidity test were classified according to the Rahbari protocol. Among the 177 samples, 17 were collected in duplicate from the same child, 1 of peripheral blood and 1 on filter paper. In this analysis, 1 (5.88%) of the 17 samples collected in duplicate also exhibited low IgG avidity, suggesting congenital infection. In addition, the results obtained from serum and filter paper were in agreement, that is, 16 (94.12%) samples presented high avidity, with 100% agreement between the results obtained from serum and from filter paper. The results of the present study indicate that the avidity test may be another valuable method for the diagnosis of congenital toxoplasmosis in newborns.

Toxoplasmosis in Treatment-Resistant Obsessive-Compulsive Disorder Patients.

The current study was done to evaluate the relationship between T. gondii and obsessive-compulsive disorder (OCD) as well as prevalence rate of toxoplasmosis in treatment-resistant patients with OCD in comparison with treatment-sensitive patients with OCD. A total of 180 subjects were selected, including 90 patients with OCD and 90 control participants. Immunoglobulin G (IgG) and immunoglobulin M (IgM) antibodies against T. gondii were measured by the enzyme-linked immunosorbent assay (ELISA) technique. Detection of acute and chronic toxoplasmosis was carried out using the ELISA IgG avidity. In addition, the presence of this parasite in blood was detected using the nested-polymerase chain reaction (PCR) method. Regarding T. gondii IgG antibodies 42 (46.7%) and 17 (18.9%) cases were detected in the patients and controls, respectively (P = 0.000). Also, 16 (17.8%) and 5 (5.6%) cases were positive for B1 gene in patients and controls, respectively (P = 0.018). The antibodies were found to be related to risk of OCD [OR (95% CI) = 3.71 (1.88-7.30); P < 0.001]. Moreover, out of 90 patients, 35 and 55 cases were resistant and sensitive to treatment, respectively, so that 24 (68.6%) out of 35 and 18 (32.7%) out of 55 were positive for the antibodies (P = 0.01) as well as 11 (31.4%) out of 35 and 5 (9.1%) out of 55 were positive for B1 gene (P = 0.010). The antibodies were also associated with risk of resistance to treatment in patients with OCD [OR (95% CI) = 3.81 (1.42-10.17); P = 0.008]. Our findings showed that toxoplasmosis was more frequent in patients with OCD than the control group. In addition, prevalence rate of toxoplasmosis in treatment-resistant patients with OCD was significantly more than that in treatment-sensitive patients with OCD.

Performance assessment of seven SARS-CoV-2 IgG enzyme-linked immunosorbent assays.

The pandemic of COVID‐19 has caused enormous fatalities worldwide. Serological assays are important for detection of asymptomatic or mild cases of COVID‐19, and sero‐prevalence and vaccine efficacy studies. Here, we evaluated and compared the performance of seven commercially available enzyme‐linked immunosorbent assay (ELISA)s for detection of anti‐severe acute respiratory syndrome corona virus 2 (SARS‐CoV‐2) immunoglobulin G (IgG). The ELISAs were evaluated with a characterized panel of 100 serum samples from qRT‐PCR confirmed COVID‐19 patients, collected 14 days post onset disease, 100 SARS‐CoV‐2 negative samples and compared the results with that of neutralization assay. Results were analysed by creating the receiver operating characteristic curve of all the assays in reference to the neutralization assay. All kits, were found to be suitable for detection of IgG against SARS‐CoV‐2 with high accuracy. The DiaPro COVID‐19 IgG ELISA showed the highest sensitivity (98%) among the kits. The assays demonstrated high sensitivity and specificity in detecting the IgG antibodies against SARS‐CoV‐2. However, the presence of IgG antibodies does not always correspond to neutralizing antibodies. Due to their good accuracy indices, these assays can also aid in tracing mild infections, in cohort studies and in pre‐vaccine evaluations.

Antibodies to Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) in All of Us Research Program Participants, 2 January to 18 March 2020.

BackgroundWith limited severe acute respiratory syndrome coronavirus (SARS-CoV-2) testing capacity in the United States at the start of the epidemic (January–March 2020), testing was focused on symptomatic patients with a travel history throughout February, obscuring the picture of SARS-CoV-2 seeding and community transmission. We sought to identify individuals with SARS-CoV-2 antibodies in the early weeks of the US epidemic.MethodsAll of Us study participants in all 50 US states provided blood specimens during study visits from 2 January to 18 March 2020. Participants were considered seropositive if they tested positive for SARS-CoV-2 immunoglobulin G (IgG) antibodies with the Abbott Architect SARS-CoV-2 IgG enzyme-linked immunosorbent assay (ELISA) and the EUROIMMUN SARS-CoV-2 ELISA in a sequential testing algorithm. The sensitivity and specificity of these ELISAs and the net sensitivity and specificity of the sequential testing algorithm were estimated, along with 95% confidence intervals (CIs).ResultsThe estimated sensitivities of the Abbott and EUROIMMUN assays were 100% (107 of 107 [95% CI: 96.6%–100%]) and 90.7% (97 of 107 [83.5%–95.4%]), respectively, and the estimated specificities were 99.5% (995 of 1000 [98.8%–99.8%]) and 99.7% (997 of 1000 [99.1%–99.9%]), respectively. The net sensitivity and specificity of our sequential testing algorithm were 90.7% (97 of 107 [95% CI: 83.5%–95.4%]) and 100.0% (1000 of 1000 [99.6%–100%]), respectively. Of the 24 079 study participants with blood specimens from 2 January to 18 March 2020, 9 were seropositive, 7 before the first confirmed case in the states of Illinois, Massachusetts, Wisconsin, Pennsylvania, and Mississippi.ConclusionsOur findings identified SARS-CoV-2 infections weeks before the first recognized cases in 5 US states.

Marked Increase in Avidity of SARS-CoV-2 Antibodies 7-8 Months After Infection Is Not Diminished in Old Age.

The kinetics of immunoglobulin G (IgG) avidity maturation during severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) infection obtained from 217 participants of the Ischgl cohort, Austria, was studied 0.5–1.5 months (baseline) and 7–8 months (follow-up) after infection. The IgG avidity assay, using a modified IgG enzyme-linked immunosorbent assay (ELISA) and 5.5 M urea, revealed that old age does not diminish the increase in avidity, detected in all participants positive at both time points, from 18% to 42%. High avidity was associated with a marked residual neutralization capacity in 97.2.% of participants (211/217), which was even higher in the older age group, revealing an important role of avidity assays as easy and cheap surrogate tests for assessing the maturation of the immune system conveying potential protection against further SARS-CoV-2 infections without necessitating expensive and laborious neutralization assays.

Molecular and serological detection of Anaplasma infection in carrier cattle in north India.

Anaplasma marginale infection in cattle (n=216) in the states of Uttar Pradesh and Uttarakhand, North India was screened by microscopy and nested-polymerase chain reaction (PCR). Two recombinant proteins viz. major surface protein (MSP) 5 and MSP2 of A. marginale were expressed in Escherichia coli and their potential in the detection of antibodies to Anaplasma species in the cattle was evaluated by immunoglobulin G-enzyme linked immunosorbent assay (IgG-ELISA). The MSP5 IgG ELISA results were compared with competitive (c) inhibition ELISA. Microscopy being the least sensitive diagnostic test detected 12.0% of animals positive for A. marginale infection while nested-PCR detected 87.9% of these animals as positive for A. marginale infection. The recombinant MSP5 antigen showed positive reactivity in 170/190 nested-PCR confirmed positive animals (sensitivity 89.5%) with specificity of 77.0%. In comparison, the recombinant MSP2 antigen showed lesser sensitivity and specificity of 79.0% and 69.2%, respectively. The cELISA was more sensitive and specific than IgG-ELISA. However, molecular detection by msp5 nested-PCR was highly sensitive and reliable for detection of carrier cattle for Anaplasma infection. The study indicated that a large cattle population (87.9%) was carrier for A. marginale infection in this region of the country.

Diagnosis value of SARS-CoV-2 antigen/antibody combined testing using rapid diagnostic tests at hospital admission.

The implementation of rapid diagnostic tests (RDTs) may enhance the efficiency of severe acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2) testing, as RDTs are widely accessible and easy to use. The aim of this study was to evaluate the performance of a diagnosis strategy based on a combination of antigen and immunoglobulin M (IgM) or immunoglobulin G (IgG) serological RDTs. Plasma and nasopharyngeal samples were collected between 14 March and 11 April 2020 at hospital admission from 45 patients with reverse transcription polymerase chain reaction (RT‐PCR) confirmed COVID‐19 and 20 negative controls. SARS‐CoV‐2 antigen (Ag) was assessed in nasopharyngeal swabs using the Coris Respi‐Strip. For IgM/IgG detection, SureScreen Diagnostics and Szybio Biotech RDTs were used in addition to laboratory assays (Abbott Alinity i SARS‐CoV‐2 IgG and Theradiag COVID‐19 IgM enzyme‐linked immunosorbent assay). Using the Ag RDT, 13 out of 45 (29.0%) specimens tested positive, the sensitivity was 87.0% for cycle threshold (C t) values ≤25% and 0% for C t values greater than 25. IgG detection was associated with high C t values and the amount of time after the onset of symptoms. The profile of isolated IgM on RDTs was more frequently observed during the first and second week after the onset of symptoms. The combination of Ag and IgM/IgG RDTs enabled the detection of up to 84.0% of COVID‐19 confirmed cases at hospital admission. Antigen and antibody‐based RDTs showed suboptimal performances when used alone. However when used in combination, they are able to identify most COVID‐19 patients admitted in an emergency department.