Microsphere-Based IgM and IgG Avidity Assays for Human Parvovirus B19, Human Cytomegalovirus, and Toxoplasma gondii

Human parvovirus B19 (here B19), human cytomegalovirus (HCMV), and Toxoplasma gondii infections during pregnancy can lead to severe complications. While traditional diagnosis of infections is mostly confined to one pathogen at a time, a multiplex array is a feasible alternative to improve diagnostic management and cost-efficiency. In the present study, for these three pathogens, we developed microsphere-based suspension immunoassays (SIAs) in multiplex and monoplex formats for the detection of antimicrobial IgM antibodies as well as corresponding chaotrope-based IgG avidity SIAs. We determined the diagnostic performances of the SIAs versus in-house and commercial reference assays using a panel of 318 serum samples from well-characterized clinical cohorts. All the newly developed assays exhibited excellent performance compared to the corresponding high-quality reference methods. The positive and negative percent agreements of the IgM SIAs in comparison with reference methods were 95 to 100% and 98 to 100%, and those of the IgG avidity SIAs were 92 to 100% and 95 to 100%, respectively. Kappa efficiency values between the SIAs and the corresponding reference assays were 0.91 to 1. Furthermore, with another panel comprising 391 clinical samples from individuals with primary infection by B19, HCMV, or T. gondii, the IgM SIAs were highly sensitive for the detection of acute infections, and the IgG avidity SIAs were highly specific for the separation of primary infections from past immunity. Altogether, the strategy of IgM multiplex screening followed by IgG avidity reflex testing can provide high-throughput and accurate means for the detection and stage determination of B19, HCMV, and T. gondii infections.IMPORTANCE Human parvovirus B19, human cytomegalovirus, and Toxoplasma gondii are ubiquitous pathogens. Their infections are often asymptomatic or mild in the general population yet may be transmitted from mother to fetus during pregnancy. Maternal infections by these pathogens can cause severe complications to the fetus or congenital abnormalities. As a rule, the risk of maternal transmission is critically related to the infection time; hence, it is important to determine when a pregnant woman has acquired the infection. In this study, we developed new diagnostic approaches for the timing of infections by three pathogens. All the new assays appeared to be highly sensitive and specific, providing powerful tools for medical diagnosis.Peer reviewe

Kinetics of Neutralizing Antibodies of COVID-19 Patients Tested Using Clinical D614G, B.1.1.7, and B 1.351 Isolates in Microneutralization Assays

Increasing evidence suggests that some newly emerged SARS-CoV-2 variants of concern (VoCs) resist neutralization by antibodies elicited by the early-pandemic wild-type virus. We applied neutralization tests to paired recoveree sera (n = 38) using clinical isolates representing the first wave (D614G), VoC1, and VoC2 lineages (B.1.1.7 and B 1.351). Neutralizing antibodies inhibited contemporary and VoC1 lineages, whereas inhibition of VoC2 was reduced 8-fold, with 50% of sera failing to show neutralization. These results provide evidence for the increased potential of VoC2 to reinfect previously SARS-CoV-infected individuals. The kinetics of NAbs in different patients showed similar decline against all variants, with generally low initial anti-B.1.351 responses becoming undetectable, but with anti-B.1.1.7 NAbs remaining detectable (>20) for months after acute infection

Kinetics of Neutralizing Antibodies of COVID-19 Patients Tested Using Clinical D614G, B.1.1.7, and B 1.351 Isolates in Microneutralization Assays

Increasing evidence suggests that some newly emerged SARS-CoV-2 variants of concern (VoCs) resist neutralization by antibodies elicited by the early-pandemic wild-type virus. We applied neutralization tests to paired recoveree sera (n = 38) using clinical isolates representing the first wave (D614G), VoC1, and VoC2 lineages (B.1.1.7 and B 1.351). Neutralizing antibodies inhibited contemporary and VoC1 lineages, whereas inhibition of VoC2 was reduced 8-fold, with 50% of sera failing to show neutralization. These results provide evidence for the increased potential of VoC2 to reinfect previously SARS-CoV-infected individuals. The kinetics of NAbs in different patients showed similar decline against all variants, with generally low initial anti-B.1.351 responses becoming undetectable, but with anti-B.1.1.7 NAbs remaining detectable (>20) for months after acute infection

Kinetics of Neutralizing Antibodies of COVID-19 Patients Tested Using Clinical D614G, B.1.1.7, and B 1.351 Isolates in Microneutralization Assays

Increasing evidence suggests that some newly emerged SARS-CoV-2 variants of concern (VoCs) resist neutralization by antibodies elicited by the early-pandemic wild-type virus. We applied neutralization tests to paired recoveree sera (n = 38) using clinical isolates representing the first wave (D614G), VoC1, and VoC2 lineages (B.1.1.7 and B 1.351). Neutralizing antibodies inhibited contemporary and VoC1 lineages, whereas inhibition of VoC2 was reduced 8-fold, with 50% of sera failing to show neutralization. These results provide evidence for the increased potential of VoC2 to reinfect previously SARS-CoV-infected individuals. The kinetics of NAbs in different patients showed similar decline against all variants, with generally low initial anti-B.1.351 responses becoming undetectable, but with anti-B.1.1.7 NAbs remaining detectable (>20) for months after acute infection

Blood donation screening for hepatitis B virus core antibodies: The importance of confirmatory testing and initial implication for rare blood donor groups

Background and ObjectivesExclusion of blood donors with hepatitis B virus (HBV) core antibodies (anti-HBc) prevents transfusion-transmitted HBV infection but can lead to significant donor loss. As isolated anti-HBc positivity does not always indicate true past HBV infection, we have investigated the effectiveness of confirmatory anti-HBc testing and the representation of rare blood groups in anti-HBc-positive donors.Materials and MethodsThree hundred ninety-seven HBV surface antigen-negative and anti-HBc initially reactive blood donor samples were tested by five different anti-HBc assays.ResultsEighty percentage of samples reactive in Architect anti-HBc assay were positive by the Murex assay and anti-HBc neutralization. Eleven out of 397 samples showed discordant results in supplementary testing from the Murex confirmatory test result, and five remained undetermined following extensive serological testing. Thirty-eight percentage of anti-HBc-positive donors identified as minority ethnic groups compared with 11% representation in anti-HBc-negative donors (p < 0.0001); the frequency of the Ro blood group in anti-HBc-positive donors was 18 times higher in non-white ethnic groups.ConclusionUsing two anti-HBc assays effectively enabled the identification of HBV-exposed and potentially infectious donors, their deferral and potential clinical follow-up. However, the exclusion of confirmed anti-HBc-positive donors will still impact the supply of rare blood such as Ro

A Protein L -Based Immunodiagnostic Approach Utilizing Time-Resolved Forster Resonance Energy Transfer

Peer reviewe

Time-Resolved FRET -Based Approach for Antibody Detection - A New Serodiagnostic Concept

Peer reviewe

Comparison of approaches for IgG avidity calculation and a new highly sensitive and specific one with broad dynamic range

Monien virusinfektioiden sekä lisäksi joidenkin muiden mikrobi-infektioiden diagnostiikassa IgG-aviditeetti on tärkeässä roolissa. Tällä vasta-ainemittauksella voidaan tunnistaa taudinaiheuttajien ensi-infektioita sekä arvioida aikaa, joka infektion alun ja mittaushetken välillä on kulunut. Nämä tiedot puolestaan ovat välttämättömiä esimerkiksi monien raskauden aikaisten infektiotautien diagnostiikassa ja hoidossa. Kliinisessä käytössä IgG-aviditeetti mitataan usein epäsuorasti ja mittaustulokset joudutaan näin muuttamaan numeeriseksi aviditeettiarvoksi. Tätä laskennallista toimenpidettä varten on julkaistu useita eri menetelmiä, mutta näitä ei juurikaan ole suoraan verrattu toisiinsa. Niin ikään ei ole selvyyttä siitä tuottavatko monimutkaisemmat ja työläämmät menetelmät laadukkaampia tuloksia. Tässä työssä vertaamme toisiinsa erinäisiä aviditeettilaskentamenetelmiä ja esittelemme kliiniseen käyttöön uuden, tarkan ja herkän, menetelmän. Entsyymi-immuunimääritys tulokset (EIA-absorbanssit) 135:stä parvorokkovirus-IgG-positiivisesta seeruminäytteestä tutkittiin rinnakkain sekä uudella että kahdella verrokkimenetelmällä (Avidity1.2-ohjelmisto perustuu luotettavimpana pidettyyn mittausperiaatteeseen ja aviditeetti-indeksi (AI) puolestaan on laajalti käytössä yksinkertaisuutensa vuoksi). Kun näytelaimennokset valittiin sopivien EIA-absorbanssien perusteella, ei menetelmien välillä havaittu eroa (receiver operating characteristic-testin kuvaajan alapuolinen pinta-ala (AUC) akuutin ensi-infektion ja vanhan immuniteetin erottamisessa toisistaan oli 0,969–0,971). Kun näytteet tukittiin uudestaan joko matalammalla tai korkeammalla absorbanssitasolla, aviditeettitulos (matala, raja-arvoinen tai korkea) muuttui keskimäärin uudella menetelmällä 3,7 %:ssa näytteistä. Vastaava muutos oli Avidity1.2:n osalta 5,6 % ja AI:n osalta 28 %. Uuden menetelmän suorituskyky vahvistettiin lisäksi kattavilla sytomegalovirus-, Toxoplasma gondii-, vihurirokkovirus- ja Epstein-Barr virusseerumiaineistoilla (AUC 0,990-1.000). Tällä uudella menetelmällä IgG-aviditeetti voidaan määrittää jopa matalista IgG-pitoisuuksista. Suosittu AI-menetelmä puolestaan on vaihtoehtona näytteille, joiden IgG-pitoisuus on riittävän korkea.In diagnosis of many viral and some other microbial infections, measurement of IgG avidity is a globally emerging approach for identifying primary infections and estimating the time of clinical onset. While many calculation methods have been introduced for conversion of the raw data into a numerical avidity value, little information exists on their comparison and whether the complex and laborious ones show superior performance. We here compare diverse approaches for avidity calculation, and introduce for clinical use a new, highly sensitive and specific one. Enzyme immune assay (EIA) absorbance data from 135 parvovirus B19 IgG-positive sera were analyzed in parallel with the new and two reference methods (Avidity1.2 software representing gold standard methodology; avidity index (AI) widely used due to its simplicity). When sample dilutions were selected on the basis of optimal EIA absorbances, all three approaches performed equally (receiver operating characteristic area-under-curve, AUC, discriminating primary infection from past immunity 0.969-0.971). When the samples were reanalyzed at either lower or higher absorbance levels, avidity status (low, borderline or high) was altered, on the average, in 3.7 % of the samples with the new method, in 5.6 % with Avidity1.2 and in 28 % with AI. The new approach was further validated with extensive serum panels for cytomegalovirus, Toxoplasma gondii, rubella and Epstein–Barr virus (AUC 0.990-1.000). It allows for accurate measurement of antimicrobial IgG avidity even from samples with low IgG level, while the globally popular AI approach provides an alternative for samples with sufficiently high IgG level

Comparison of approaches for IgG avidity calculation and a new highly sensitive and specific method with broad dynamic range

Background: Antimicrobial IgG avidity is measured in the diagnosis of infectious disease, for dating of primary infection or immunization. It is generally determined by either of two approaches, termed here the avidity index (AI) or end-point ratio (EPR), which differ in complexity and workload. While several variants of these approaches have been introduced, little comparative information exists on their clinical utility. Methods: This study was performed to systematically compare the performances of these approaches and to design a new sensitive and specific calculation method, for easy implementation in the laboratory. The avidities obtained by AI, EPR, and the newly developed approach were compared, across parvovirus B19, cytomegalovirus, Toxoplasma gondii, rubella virus, and Epstein-Barr virus panels comprising 460 sera from individuals with a recent primary infection or long-term immunity. Results: With optimal IgG concentrations, all approaches performed equally, appropriately discriminating primary infections from past immunity (area under the receiver operating characteristic curve (AUC) 0.93-0.94). However, at lower IgG concentrations, the avidity status (low, borderline, high) changed in 17% of samples using AI (AUC 0.88), as opposed to 4% using EPR (AUC 0.91) and 6% using the new method (AUC 0.93). Conclusions: The new method measures IgG avidity accurately, in a broad range of IgG levels, while the popular AI approach calls for a sufficiently high antibody concentration. (c) 2021 The Authors. Published by Elsevier Ltd on behalf of International Society for Infectious Diseases. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).Peer reviewe

Evolutionarily conserved exon definition interactions with U11 snRNP mediate alternative splicing regulation on U11–48K and U11/U12–65K genes

<div><p>Many splicing regulators bind to their own pre-mRNAs to induce alternative splicing that leads to formation of unstable mRNA isoforms. This provides an autoregulatory feedback mechanism that regulates the cellular homeostasis of these factors. We have described such an autoregulatory mechanism for two core protein components, U11–48K and U11/U12–65K, of the U12-dependent spliceosome. This regulatory system uses an atypical splicing enhancer element termed USSE (U11 snRNP-binding splicing enhancer), which contains two U12-type consensus 5′ splice sites (5′ss). Evolutionary analysis of the USSE element from a large number of animal and plant species indicate that USSE sequence must be located 25–50 nt downstream from the target 3′ splice site (3′ss). Together with functional evidence showing a loss of USSE activity when this distance is reduced and a requirement for RS-domain of U11–35K protein for 3′ss activation, our data suggests that U11 snRNP bound to USSE uses exon definition interactions for regulating alternative splicing. However, unlike standard exon definition where the 5′ss bound by U1 or U11 will be subsequently activated for splicing, the USSE element functions similarly as an exonic splicing enhancer and is involved only in upstream splice site activation but does not function as a splicing donor. Additionally, our evolutionary and functional data suggests that the function of the 5′ss duplication within the USSE elements is to allow binding of two U11/U12 di-snRNPs that stabilize each others' binding through putative mutual interactions.</p></div