IgM Assays Research Articles

Longitudinal evaluation of the Abbott ARCHITECT SARS-CoV-2 IgM and IgG assays in a pediatric population

BackgroundClinical laboratory testing has been an essential part of COVID-19 management. Serology can provide valuable information regarding a patient’s exposure to virus, and may have a larger role to play as vaccines becomes available. Limited data is available on the serological response in pediatric patients. Here we investigate the use of one manufacturer’s commercial assays for detecting IgM and IgG in an exclusively pediatric population. MethodsAbbott SARS-CoV-2 IgM and IgG assays were performed on an Abbott ARCHITECT i1000. For specificity studies, we tested 78 patient specimens collected before the COVID-19 pandemic, and 66 specimens from patients who tested negative for SARS-CoV-2 nucleic acid amplification test (NAAT) during the COVID-19 pandemic. For sensitivity we tested 181 specimens from 41 patients with a positive NAAT result. Precision data was acquired for 20 days. ResultsFor IgM, the highest qualitative positive agreement with molecular results was observed to be 15–30 days after a positive NAAT result or after symptom onset. For IgG, the highest positive agreement was 31–60 days after a positive NAAT result or 61–90 days after the start of symptoms. IgM started to decline 30 days after NAAT results and faded by 90 days. IgG started to decrease 60 days after a positive NAAT result. ConclusionThe Abbott IgM and IgG assays have negative agreements of 98.7–100% relative to NAAT results. The IgM and IgG levels assayed by these methods start to decline months after positive molecular results and onset of symptoms in a pediatric population.

Recent Advances in the Evaluation of Serological Assays for the Diagnosis of SARS-CoV-2 Infection and COVID-19.

Introduction: Few data on the diagnostic performance of serological tests for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection are currently available. We evaluated sensitivity and specificity of five different widely used commercial serological assays for the detection of SARS-CoV-2–specific IgG, IgM, and IgA antibodies using reverse transcriptase-PCR assay in nasopharyngeal swab as reference standard test.Methods: A total of 337 plasma samples collected in the period April–June 2020 from SARS-CoV-2 RT-PCR positive (n = 207) and negative (n = 130) subjects were investigated by one point-of-care lateral flow immunochromatographic assay (LFIA IgG and IgM, Technogenetics) and four fully automated assays: two chemiluminescence immunoassays (CLIA-iFlash IgG and IgM, Shenzhen YHLO Biotech and CLIA-LIAISON® XL IgG, DiaSorin), one electrochemiluminescence immunoassay (ECLIA-Elecsys® total predominant IgG, Roche), and one enzyme-linked immunosorbent assay (ELISA IgA, Euroimmune).Results: The overall sensitivity of all IgG serological assays was >80% and the specificity was >97%. The sensitivity of IgG assays was lower within 2 weeks from the onset of symptoms ranging from 70.8 to 80%. The LFIA and CLIA-iFlash IgM showed an overall low sensitivity of 47.6 and 54.6%, while the specificity was 98.5 and 96.2%, respectively. The ELISA IgA yielded a sensitivity of 84.3% and specificity of 81.7%. However, the ELISA IgA result was indeterminate in 11.7% of cases.Conclusions: IgG serological assays seem to be a reliable tool for the retrospective diagnosis of SARS-CoV-2 infection. IgM assays seem to have a low sensitivity and IgA assay is limited by a substantial rate of indeterminate results.

Administration of high titer convalescent anti-SARS-CoV-2 plasma: From donor selection to monitoring recipient outcomes

Early in the SARS-CoV-2 pandemic, convalescent plasma (CP) therapy was proposed as a treatment for severely ill patients. We conducted a CP treatment protocol under the Mayo Clinic Extended Access Program at University Hospital Brooklyn (UHB). Potential donors were screened with a lateral flow assay (LFA) for IgM and IgG antibodies against the SARS-CoV-2 S1 receptor-binding domain (RBD). Volunteers that were LFA positive were tested with an ELISA to measure IgG titers against the RBD. Subjects with titers of at least 1:1024 were selected to donate. Most donors with positive LFA had acceptable titers and were eligible to donate. Out of 171 volunteers, only 65 tested positive in the LFA (38.0%), and 55 (32.2%) had titers of at least 1:1024. Before our donation program started, 31 CP units were procured from the New York Blood Center (NYBC). Among the 31 CP units that were obtained from the NYBC, 25 units (80.6%) were positive in the LFA but only 12 units (38.7%) had titers of at least 1:1024. CP was administered to 28 hospitalized COVID-19 patients. Patients who received low titer CP, high titer CP and patients who did not receive CP were followed for 45 days after presentation. Severe adverse events were not associated with CP transfusion. Death was a less frequent outcome for patients that received high titer CP (>1:1024) 38.6% mortality, than patients that received low titer CP (≤1:1024) 77.8% mortality.

Comparison of diagnostic accuracy for eight SARS-CoV-2 serological assays.

IntroductionSevere acute respiratory syndrome coronavirus 2 (SARS-CoV-2) serological tests have been suggested as an additional diagnostic tool in highly suspected cases with a negative molecular test and determination of seroprevalence in population. We compared the diagnostic performance of eight commercial serological assays for IgA, IgM, and IgG antibodies to the SARS-CoV-2 virus.Materials and methodsThe comparison study was performed on a total of 76 serum samples: 30 SARS-CoV-2 polymerase chain reaction (PCR)-negative and 46 SARS-CoV-2 PCR-positive patients with asymptomatic to severe disease and symptoms duration from 3-30 days. The study included: three rapid lateral flow immunochromatographic assays (LFIC), two enzyme-linked immunosorbent assays (ELISA), and three chemiluminescence immunoassays (CLIA).ResultsAgreement between IgM assays were minimal to moderate (kappa 0.26 to 0.63) and for IgG moderate to excellent (kappa 0.72 to 0.92). Sensitivities improved with > 10 days of symptoms and were: 30% to 89% for IgM; 89% to 100% for IgG; 96% for IgA; 100% for IgA/IgM combination; 96% for total antibodies. Overall specificities were: 90% to 100% for IgM; 85% to 100% for IgG; 90% for IgA; 70% for IgA/IgM combination; 100% for total antibodies. Diagnostic accuracy for IgG ELISA and CIA assays were excellent (AUC ≥ 0.90), without significant difference. IgA showed significantly better diagnostic accuracy than IgM (P < 0.001).ConclusionThere is high variability between IgM assays independently of the assay format, while IgG assays showed moderate to perfect agreement. The appropriate time for testing is crucial for the proper immunity investigation.

Specificity testing by point prevalence as a simple assessment strategy using the Roche Elecsys® anti-SARS-CoV-2 immunoassay

BackgroundThe detection of antibodies to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is mandatory for the diagnosis, retrospective assessment of disease progression, and correct evaluation of the current infection situation in the population. Many such assays have been launched by various manufacturers. Unfortunately, the new US Food and Drug Administration emergency use regulations have resulted in a situation where laboratories have to perform their own validation studies but many of these laboratories do not have the biobank needed to conduct the studies. MethodsWe introduce a method that allows institutions to quickly perform a verification study in a low-prevalence infection situation. As proof of concept, we used the Roche Elecsys® anti-SARS-CoV-2 electrochemiluminescence immunoassay and an SAP-based hospital information system. The Shenzhen YHLO Biotech IgM and IgG assay targeting other surface patterns was used as a confirmatory test. ResultsThe Roche assay demonstrated a limit of detection of 0.069 cutoff index and successfully passed the performance validation according to Clinical and Laboratory Standards Institute EP15-A3. The study population of 627 inpatients has a median age of 64 years, and approximately 13% of the group were under intensive care at the respective time point. All patients included tested negative for SARS-CoV-2 infection by quantitative reverse transcription polymerase chain reaction (cobas® 6800, Roche, Mannheim, Germany). Only one false-positive result was obtained, resulting in a specificity for the Roche Elecsys anti-SARS-CoV-2 test of 99.84% and a negative predictive value of 99.98%. ConclusionsThe anonymized use of residual material enables quick evaluation of anti-SARS-CoV-2 immunoassays, as shown in this work with the Roche Elecsys assay. Comparison of the control population with economic data makes it possible to validate the sampling set and therefore to determine diagnostic specificity. By use of the approach chosen, it was shown that the Roche test achieved very good results in terms of diagnostic specificity, reproducibility, and limit of detection.

Evaluation of Antibody Response in Symptomatic and Asymptomatic COVID-19 Patients and Diagnostic Assessment of New IgM/IgG ELISA Kits.

This study aims to study the immune response and evaluate the performances of four new IgM and five IgG enzyme-linked immunosorbent assay (ELISA) kits for detecting anti-severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) antibodies against different antigens in symptomatic and asymptomatic coronavirus disease 2019 (COVID-19) patients. A total of 291 samples collected from symptomatic and asymptomatic RT–PCR-confirmed patients were used to evaluate the ELISA kits’ performance (EDI, AnshLabs, DiaPro, NovaLisa, and Lionex). The sensitivity was measured at three different time-intervals post symptoms onset or positive SARS-CoV-2 RT–PCR test (≤14, 14–30, >30 days). The specificity was investigated using 119 pre-pandemic serum samples. The sensitivity of all IgM kits gradually decreased with time, ranging from 48.7% (EDI)–66.4% (Lionex) at ≤14 days, 29.1% (NovaLisa)–61.8% (Lionex) at 14–30 days, and 6.0% (AnshLabs)–47.9% (Lionex) at >30 days. The sensitivity of IgG kits increased with time, peaking in the latest interval (>30 days) at 96.6% (Lionex). Specificity of IgM ranged from 88.2% (Lionex)–99.2% (EDI), while IgG ranged from 75.6% (DiaPro)–98.3% (Lionex). Among all RT–PCR-positive patients, 23 samples (7.9%) were seronegative by all IgG kits, of which only seven samples (30.4%) had detectable IgM antibodies. IgM assays have variable and low sensitivity, thus considered a poor marker for COVID-19 diagnosis. IgG assays can miss at least 8% of RT–PCR-positive cases.

Dynamics of Hepatitis E Virus (HEV) Antibodies and Development of a Multifactorial Model To Improve the Diagnosis of HEV Infection in Resource-Limited Settings.

Serological markers are important for the diagnosis of hepatitis E virus (HEV) infection. This study aims to compare the diagnostic performance of the anti-HEV IgM and the HEV antigen (Ag) assays and establish a multifactorial model to improve the diagnosis of current HEV infection when HEV RNA detection is not available. A total of 809 serum samples, including 325 anti-HEV IgM-positive and 484 anti-HEV IgM-negative samples, were tested for HEV RNA. The anti-HEV IgM assay had very high sensitivity (99.4%) but moderate accuracy (79.2%) and specificity (74.3%). By retrospective follow-up of 58 patients with sequential samples (n = 143) tested for anti-HEV antibodies, we found anti-HEV IgM remained positive for more than 10 months in some HEV-infected patients, when HEV RNA was already undetectable; thus, decision solely based on anti-HEV IgM may lead to misdiagnosis. In contrast, the HEV Ag assay had very high specificity (100%). However, the detection efficiency of HEV Ag greatly diminished when the HEV RNA level was low or the anti-HEV IgG level was high. By logistic regression, a model integrating anti-HEV IgM, alanine aminotransferase, and HEV Ag was proposed, and the cutoff value was determined based on the testing results of the 143 sequential samples. The model was further evaluated with 67 randomly selected IgM-positive samples from single-visit patients. Overall, the model outperformed the anti-HEV IgM or the HEV Ag assay in the diagnosis of current HEV infection (sensitivity/specificity/accuracy, 89.5%/95.2%/91.9%). The area under the receiver operating characteristics curve of the model was greater than 0.97.

Anti-SARS-CoV-2 IgM improves clinical sensitivity early in disease course

IntroductionCoronavirus disease 2019 (COVID-19), caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), is diagnosed by molecular-based detection of SARS-CoV-2 RNA. Serologic testing detects antibodies specific to SARS-CoV-2 and IgM specifically may serve as an adjunct test to PCR early in disease. We evaluated the Abbott anti-SARS-CoV-2 IgM and IgG assays along with DiaSorin anti-SARS-CoV-2 IgG and Roche anti-SARS-CoV-2 Total. MethodsSpecimens from 175 PCR-positive patients and 107 control specimens were analyzed using Abbott IgM and IgG, DiaSorin IgG, and Roche Total (IgA, IgG, IgM) assays. Sensitivity, specificity, cross-reactivity, concordance between assays, trends over time, positive predictive value (PPV), and negative predictive value (NPV) were determined. ResultsAbbott IgM sensitivity was 63.6% at 0 days post-PCR positivity, 76.5% at 1-5d, 76.3% at 6-14d, 85.2% at 15-30d, and 63.6% at > 30d. All assays exhibited highest sensitivity 15-30d post-PCR positivity (83.3–85.2%). Combining Abbott IgM and IgG improved sensitivity by 22.7% compared to IgG alone when tested 0d post-PCR positivity. All assays had a specificity of 100% and only Abbott IgG exhibited cross-reactivity (anti-dsDNA). Cohen’s kappa varied between 0.86 and 0.93. Time to seroconversion from PCR positivity was lowest for Abbott IgM and highest for Abbott IgG. NPV was highest for Abbott IgM < 14 days post-PCR positivity and Abbott IgG ≥ 14 days. ConclusionThe Abbott IgM assay exhibited the earliest response and greatest signal in most patients evaluated for serial sampling and had the highest NPV < 14 days post-PCR positivity, suggesting its potential utility as an adjunct test to PCR early in disease course.

Seroprevalence of SARS-CoV-2 Immunoglobulins in Pregnant Women and Neonatal Cord Blood from a Highly Impacted Region

Background: The Severe Acute Respiratory Syndrome coronavirus 2 (SARS-CoV-2) is the cause of the Coronavirus Diseases of 2019 (COVID-19). This virus has been responsible for the global pandemic of 2020 and 2021 with over 107 million confirmed cases and over 2.3 million deaths. An important population that deserves meticulous consideration during the COVID-19 pandemic is pregnant woman; however, in many places in the world there is inadequate screening for SARS-COV-2 during pregnancy. We aimed to determine the impact of maternal and neonatal cord blood SARS-COV-2 antibodies and placental transfer ratios in a region with a low screening plan. Methods: We performed a blind study in Babahoyo, Ecuador; one of the SARS-CoV-2epicenters in South America. Blood samples were collected from 100 serum samples collected from pregnant women at the time of delivery along with corresponding neonatal cord blood. SARS-CoV-2 IgG specific antibodies were determined using a enzyme linked immunosorbent assay (ELISA) for IgG and IgM SARS-CoV-2-specific nucleocapsid and spike antigens (DiaPro, Milano, Italy Cat number COV19G.CE). Findings: We observed 32% of pregnant women to be serological positive in areas lacking an appropriate screening program. We also show data that suggests an efficient passive immunization of the fetus to SARS-CoV-2 during pregnancy. Interpretation: There is a high incidence of SARS-CoV-2 infection during pregnancy. The best prevention for protecting the mother from infection is vaccination, which, will provide the fetus and neonate with a strong protection against SARS-CoV-2 infection through passive placental transfer of SARS-CoV-2 specific antibodies. This is especially important because newborns are highly susceptible to many viral infections. Funding: NIAID 1R01AI145829-01 Declaration of Interest: None to declare. Ethical Approval: This study followed the Strengthening the Reporting of Observational Studies in Epidemiology (STROBE) reporting guidelines. This was retrospective study done with samples sent for tests evaluation that were originally not related to this study. A consent waiver was approved since the samples were not collected for the study and the samples used were defined as dischargeable material.

Comparison of SARS-CoV-2 IgM and IgG seroconversion profiles among hospitalized patients in two US cities

The clinical and public health utility of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) serologic testing requires a better understanding of the dynamics of the humoral response to infection. To track seroconversion of IgG and IgM antibodies in patients with SARS-CoV-2 infection and its association with patient and clinical factors and outcomes. Residual patient specimens were analyzed on the Abbott ARCHITECT i2000 instrument using the Abbott SARS-CoV-2 IgG assay and prototype SARS-CoV-2 IgM assay. Age, sex, comorbidities, symptom onset date, mortality, and specimen collection date were obtained from electronic medical records. Three hundred fifty-nine longitudinal samples were collected from 89 hospitalized patients 0 to 82 days postsymptom onset. Of all, 51.7% of the patients developed IgG and IgM antibodies simultaneously; 32.8% seroconverted for IgM before IgG. On average, patients seroconverted for IgG by 8 days and for IgM by 7 days postsymptom onset. All patients achieved IgG seropositivity by 19 days and IgM seropositivity by 17 days. Median time to IgG and IgM seroconversion was prolonged and initial levels of IgG were lower in immunocompromised patients and patients <65 years of age compared to immune competent patients and those ≥65 years of age. Immunocompromised patients also had persistently lower levels of IgM that peaked on day 17.6 and decreased thereafter compared to immune competent patients. IgM seroconversion in patients who died reached significantly higher levels later after symptom onset than in those who recovered. SARS-CoV-2 infected patients have similar time to seroconversion for IgG and IgM. However, differences in immune status and age alter time to seroconversion. These results may help guide serologic testing application in COVID-19 management.

Seroprevalence of COVID-19 infection among healthcare professionals in Central India using SARS-CoV-2 antibody test

The diagnosis and monitoring of SARS-CoV-2 infection (COVID-19 illness) are based on two different types of tests: (i) RT-PCR (reverse-transcription polymerase chain reaction) test that detects the presence of viral RNA, and (ii) antibody test that detects antibodies to SAR-CoV-2. Studies have demonstrated that antibody testing is useful for the identification of asymptomatic or subclinical infection of SARS-CoV-2 among close contacts with COVID-19 patients, including the HCP.This study presents the first SARS-CoV-2 seroprevalence study among 307 HCP in Central India. This study also evaluates the risk of exposure of the HCP of different roles/departments to COVID-19 infection by the SARS-CoV-2 antibody seroconversion rate. A cross-sectional study was conducted using serum obtained from 307 HCP, who were on duty in the hospitals in Indore City of Central India during the COVID-19 pandemic from March till June 2020. Siemens Healthineers COV2T assay that detects total antibody (including IgM and IgG) against the S1-RBD (receptor binding domain of spike protein subunit 1) antigen was selected to be used in this study due to its robust sensitivity and specificity, compared to IgG or IgM assay alone Overall, the prevalence of COVID-19 infection among HCP in Central India hospital is 7.82% (n=24/307) as evaluated using SARS-CoV-2 total antibody test. Of the 307 HCP. Seropositivity rate varies widely by professions/departments ranging from intensivists (66.7%), general surgeons (25%), ophthalmologist (20%), pediatrician (20%), anesthetist (12.5%), radiologist (10%), general physician (9.7%) to gynaecologist (4.9%). This seroprevalence study, along with other studies, highlights the importance of SARS-CoV-2 antibody in seroprevalence and epidemiology studies which may subsequently guide the policy-making in implementing an effective infection control strategy to curb SARS-CoV-2 transmission in the hospital settings; on top of the use as aid-in-diagnosis, contact-tracing, pre and post vaccination screening and evaluation of convalescent plasma therapy.

Persistence of IgM Antibodies after Vaccination with Live Attenuated Japanese Encephalitis Vaccine.

.Japanese encephalitis (JE) is a vaccine-preventable, mosquito-borne disease. Substantial progress with JE control in Asia has been made during the past decade, with most endemic countries now having JE vaccination programs, commonly using live attenuated SA14-14-2 JE vaccine (trade name CD-JEV). If a child develops encephalitis during the weeks to months following CD-JEV vaccination and anti-JE virus IgM (JE IgM) antibody is detected in serum, the question arises if this is JE virus infection indicating vaccine failure, or persistent JE IgM antibody postvaccination. To better understand JE IgM seropositivity following vaccination, sera from 268 children from a previous CD-JEV study were tested by two different JE IgM assays to determine JE IgM antibody frequency on days 28, 180, and 365 postvaccination. With the CDC JE IgM antibody capture ELISA (MAC-ELISA), 110 children (41%) had JE IgM positive or equivocal results on their day 28 sample, and eight (3%) and two (1%) had positive or equivocal results on day 180 and day 365 samples, respectively. With the InBios JE Detect™ MAC-ELISA (Seattle, WA), 118 (44%) children had positive or equivocal results on day 28 sample, and three (1%) and one (0.4%) had positive or equivocal results on day 180 and day 365 samples, respectively. Our results indicate that more than 40% children vaccinated with CD-JEV can have JE IgM antibodies in their serum at 1 month postvaccination but JE IgM antibody is rare by 6 months. These data will help healthcare workers assess the likelihood that JE IgM antibodies in the serum of a child with encephalitis after vaccination are vaccine related.

Temporal Course of SARS-CoV-2 Antibody Positivity in Patients with COVID-19 following the First Clinical Presentation.

Knowledge of the sensitivities of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) antibody tests beyond 35 days after the clinical onset of COVID-19 is insufficient. We aimed to describe positivity rate of SARS-CoV-2 assays employing three different measurement principles over a prolonged period. Two hundred sixty-eight samples from 180 symptomatic patients with COVID-19 and a reverse transcription polymerase chain reaction (RT-PCR) test followed by serological investigation of SARS-CoV-2 antibodies were included. We conducted three chemiluminescence (including electrochemiluminescence assay (ECLIA)), four enzyme-linked immunosorbent assay (ELISA), and one lateral flow immunoassay (LFIA) test formats. Positivity rates, as well as positive (PPVs) and negative predictive values (NPVs), were calculated for each week after the first clinical presentation for COVID-19. Furthermore, combinations of tests were assessed within an orthogonal testing approach employing two independent assays and predictive values were calculated. Heat maps were constructed to graphically illustrate operational test characteristics. During a follow-up period of more than 9 weeks, chemiluminescence assays and one ELISA IgG test showed stable positivity rates after the third week. With the exception of ECLIA, the PPVs of the other chemiluminescence assays were ≥95% for COVID-19 only after the second week. ELISA and LFIA had somewhat lower PPVs. IgM exhibited insufficient predictive characteristics. An orthogonal testing approach provided PPVs ≥ 95% for patients with a moderate pretest probability (e.g., symptomatic patients), even for tests with a low single test performance. After the second week, NPVs of all but IgM assays were ≥95% for patients with low to moderate pretest probability. The confirmation of negative results using an orthogonal algorithm with another assay provided lower NPVs than the single assays. When interpreting results from SARS-CoV-2 tests, the pretest probability, time of blood draw, and assay characteristics must be carefully considered. An orthogonal testing approach increases the accuracy of positive, but not negative, predictions.

Differences of SARS-CoV-2 serological test performance between hospitalized and outpatient COVID-19 cases

BackgroundSerological severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) antibody assays differ in the target antigen specificity, e.g. of antibodies directed against the viral spike or the nucleocapsid protein, and in the spectrum of detected immunoglobulins. The aim of the study was to evaluate the performance of two different routinely used immunoassays in hospitalized and outpatient COVID-19 cases. MethodsThe test characteristics of commercially available spike1 protein-based serological assays (Euroimmun, EI-assays), determining IgA or IgG and nucleocapsid-based assays (Virotech, VT-assays) determining IgA, IgM or IgG were compared in 139 controls and 116 hospitalized and outpatient COVID-19 cases. ResultsHospitalized COVID-19 patients (n = 51; 115 samples) showed significantly higher concentrations of antibodies against SARS-CoV-2 and differed from outpatient cases (n = 65) by higher age, higher disease severity scores and earlier follow up blood sampling. Sensitivity of the two IgG assays was comparable in hospitalized patients tested ≥ 14 days (EI-assay: 88%, CI95% 67.6–99.9; VT-assay: 96%, CI95% 77.7–99.8). In outpatient COVID-19 cases sensitivity was significantly lower in the VT-assay (86.2%, CI95% 74.8–93.1) compared with the EI-assay (98.5%, CI95% 90.6–99.9). Assays for IgA and IgM demonstrated a lack of specificity or sensitivity. ConclusionsOur results indicate that SARS-CoV-2 serological assays may need to be optimized to produce reliable results in outpatient COVID-19 cases who are low or even asymptomatic. Assays for IgA and IgM have limited diagnostic performance and do not prove an additional value for population-based screening approaches.

Diagnostic biologique de l’infection à Sars-CoV-2 : stratégies et interprétation des résultats

In response to the emergence of Sars-CoV-2 responsible for a global pandemic, a large number of diagnostic tests have been developed and brought to market in a very short period of time. RT-PCR on nasopharyngeal swab samples is the reference method for the diagnosis and screening of Sars-CoV-2 infection, but the tests developed are highly variable in terms of sensitivity and turnaround time. Antigenic tests generally have lower sensitivity but have the advantage of simpler and faster implementation. In front of Covid-19 symptoms with negative RT-PCR results, serology can be recommended with IgM and IgG assays. Serology is also a relevant tool for epidemiological studies. However, it is important to remember that the level of anti-Sars-Cov-2 antibodies decreases over time and can therefore impact the results of sero-epidemiological studies. Despite the need to respond rapidly to an urgent diagnostic need, it remains essential to validate the methods chosen on a well-characterized panel of samples, as the performance of certain tests is sometimes unsatisfactory to ensure a reliable diagnosis.

An ultra-high-density protein microarray for high throughput single-tier serological detection of Lyme disease

Current serological immunoassays have inherent limitations for certain infectious diseases such as Lyme disease, a bacterial infection caused by Borrelia burgdorferi in North America. Here we report a novel method of manufacturing high-density multiplexed protein microarrays with the capacity to detect low levels of antibodies accurately from small blood volumes in a fully automated system. A panel of multiple serological markers for Lyme disease are measured using a protein microarray system, Lyme Immunochip, in a single step but interpreted adhering to the standard two-tiered testing algorithm (enzyme immunoassay followed by Western blot). Furthermore, an enhanced IgM assay was supplemented to improve the test’s detection sensitivity for early Lyme disease. With a training cohort (n = 40) and a blinded validation cohort (n = 90) acquired from CDC, the Lyme Immunochip identified a higher proportion of Lyme disease patients than the two-tiered testing (82.4% vs 70.6% in the training set, 66.7% vs 60.0% in the validation set, respectively). Additionally, the Immunochip improved sensitivity to 100% while having a lower specificity of 95.2% using a set of investigational antigens which are being further evaluated with a large cohort of blinded samples from the CDC and Columbia University. This universal microarray platform provides an unprecedented opportunity to resolve a broad range of issues with diagnostic tests, including multiplexing, workflow simplicity, and reduced turnaround time and cost.

Comparison of Zika virus inactivation methods for reagent production and disinfection methods

Zika virus (ZIKV) infection remains a public health concern necessitating demand for long-term virus production for diagnostic assays and R&D activities. Inactivated virus constitutes an important component of the Trioplex rRT-PCR assay and serological IgM assay (MAC-ELISA). The aim of our study is to establish standard methods of ZIKV inactivation while maintaining antigenicity and RNA integrity. We tested viral supernatants by four different inactivation methods: 1. Heat inactivation at 56 °C and 60 °C; 2. Gamma-Irradiation; 3. Chemical inactivation by Beta-propiolactone (BPL) and 4. Fast-acting commercial disinfecting agents. Effectivity was measured by cytopathic effect (CPE) and plaque assay. RNA stability and antigenicity were measured by RT-PCR and MAC-ELISA, respectively. Results: Heat inactivation: Low titer samples, incubated at 56 °C for 2 h, showed neither CPE or plaques compared to high titer supernatants that required 2.5 h. Inactivation occurred at 60 °C for 60 min with all virus titers. Gamma irradiation: Samples irradiated at ≥3 Mrad for low virus concentrations and ≥5Mrad for high virus titer completely inactivated virus. Chemical Inactivation: Neither CPE nor plaques were observed with ≥0.045 % BPL inactivation of ZIKV. Disinfectant: Treatment of viral supernatants with Micro-Chem Plus™, inactivated virus in 2 min, whereas, Ethanol (70 %) and STERIS Coverage® Spray TB inactivated the virus in 5 min.

Comparative analysis of three laboratory based serological assays for SARS-CoV-2 in an Australian cohort

Many unanswered questions remain regarding the role of SARS-CoV-2 serological assays in this unfolding COVID-19 pandemic. These include their utility for the diagnosis of acute SARS-CoV-2 infection, past infection or exposure, correlation with immunity and the effective duration of immunity. This study examined the performance of three laboratory based serological assays, EUROIMMUN Anti-SARS-CoV-2 IgA/IgG, MAGLUMI 2000 Plus 2019-nCov IgM/IgG and EDI Novel Coronavirus (COVID-19) IgM/IgG immunoassays.We evaluated 138 samples from a reference non-infected population and 71 samples from a cohort of 37 patients with SARS-CoV-2 confirmed positive by RT-PCR. The samples were collected at various intervals of 0–45 days post symptoms onset (PSO).Specificity and sensitivity of these assays was 60.9%/71.4% (IgA) and 94.2%/63.3% (IgG) for EUROIMMUN; 98.5%/18.4% (IgM) and 97.8%/53.1% (IgG) for MAGLUMI; and 94.9%/22.5% (IgM) and 93.5%/57.1% (IgG) for EDI, respectively. When samples collected ≥14 days PSO were considered, the sensitivities were 100.0 and 100.0%; 31.0 and 82.8%; 34.5 and 57.1%, respectively. Using estimated population prevalence of 0.1, 1, and 10%, the positive predictive value of all assays remained low.The EUROIMMUN Anti-SARS-CoV-2 IgA lacked specificity for acute diagnosis and all IgM assays offered poor diagnostic utility. Seroconversion can be delayed although all patients had seroconverted at 28 days in our cohort with the EUROIMMUN Anti-SARS-CoV-2 IgG. Despite this, with specificity of only 94% this assay would not be satisfactory for seroprevalence studies in the general Australian population given this is likely to be currently <1%.

Comparison of Nucleic Acid Amplification and IgM Tests for the Diagnosis of Mycoplasma pneumoniae Infection in Children During a Recent Korean Outbreak

In the absence of standardized methods for Mycoplasma pneumoniae detection, we evaluated the diagnostic value of polymerase chain reaction (PCR) and IgM assays for detecting M. pneumoniae infection in children during a recent Korean outbreak. The diagnostic performances of PCR and IgM assays for M. pneumoniae in 1,109 clinical specimens were evaluated by the Japanese Respiratory Society (JRS) scoring system as an interim reference standard. The level of agreement between both tests was fair. As analyzed by the JRS scoring system, the sensitivity of PCR was 45.2% in the group aged <5 years, 86.8% in the group aged 5 years to 10 years group, and 72.2% in the group aged 10 years to 18 years; the sensitivity of the IgM assay was 66.8%, 71.4%, and 55.6% in each group, respectively. The sensitivity of PCR is relatively low but is superior to that of IgM assays such that diagnostic performance can be improved by both test methods in patients aged <5 years.

Analytical and clinical evaluation of new automated chemiluminescent immunoassays for the detection of IgG and IgM anti-Bartonella henselae antibodies

Serological diagnosis of Bartonella henselae infection mainly rely on microscopic immunofluorescence assays (IFA), which are however time-consuming and poorly standardized. The aim of the study was to assess the use of the new fully automated VirClia® chemiluminescent immunoassays for the detection of IgG and IgM anti-B. henselae antibodies. Eighty-one patients with a well-defined B. henselae infection as well as 80 patients with an alternative disease were included. The VirClia® IgG antibody assay showed a sensitivity of 79.0% and a specificity of 93.8% for the diagnosis of B. henselae infection. For the VirClia® IgM assay, results were more conflicting with a sensitivity of 42.0% and a specificity of 98.2% to predict IFA IgM results. In 11 additional patients with uninterpretable IFA due to autoimmune antibodies, VirClia® assays were able to deliver valuable quantitative results. The VirClia® IgG assay shows good analytical and clinical performances and could be easily integrated in the diagnostic workflow of B. henselae infection.