Multicenter Evaluation of the Clinical Performance and the Neutralizing Antibody Activity Prediction Properties of 10 High-Throughput Serological Assays Used in Clinical Laboratories.

As the coronavirus disease 2019 (COVID-19) pandemic second wave is emerging, it is of the upmost importance to screen the population immunity in order to keep track of infected individuals. Consequently, immunoassays for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) with high specificity and positive predictive values are needed to obtain an accurate epidemiological picture. As more data accumulate about the immune responses and the kinetics of neutralizing-antibody (nAb) production in SARS-CoV-2-infected individuals, new applications are forecast for serological assays such as nAb activity prediction in convalescent-phase plasma from recovered patients. This multicenter study, involving six hospital centers, determined the baseline clinical performances, reproducibility, and nAb level correlations of 10 commercially available immunoassays. In addition, three lateral-flow chromatography assays were evaluated, as these devices can be used in logistically challenged areas. All assays were evaluated using the same patient panels in duplicate, thus enabling accurate comparison of the tests. Seven immunoassays examined in this study were shown to have excellent specificity (98 to 100%) and good to excellent positive predictive values (82 to 100%) when used in a low (5%)-seroprevalence setting. We observed sensitivities as low as 74% and as high as 95% at ≥15 days after symptom onset. The determination of optimized cutoff values through receiver operating characteristic (ROC) curve analyses had a significant impact on the diagnostic resolution of several enzyme immunoassays by increasing the sensitivity significantly without a large trade-off in specificity. We found that spike-based immunoassays seem to be better correlates of nAb activity. Finally, the results reported here will add to the general knowledge of the interlaboratory reproducibility of clinical performance parameters of immunoassays and provide new evidence about nAb activity prediction.

Molecular surveillance of hepatitis C virus genotypes identifies the emergence of a genotype 4d lineage among men in Quebec, 2001-2017.

BACKGROUND: Molecular phylogenetics are generally used to confirm hepatitis C virus (HCV) transmission events. In addition, the Laboratoire de santé publique du Québec (LSPQ) has been using molecular phylogenetics for surveillance of HCV genotyping since November 2001. OBJECTIVES: To describe the emergence of a specific lineage of HCV genotype 4d (G4d) and its characteristics using molecular phylogenetics as a surveillance tool for identifying HCV strain clustering. METHODS: The LSPQ prospectively applied Sanger sequencing and phylogenetic analysis to determine the HCV genotype on samples collected from November 2001 to December 2017. When a major G4d cluster was identified, demographic information, HIV-infection status and syphilis test results were analyzed. RESULTS: Phylogenetic analyses performed on approximately 22,000 cases identified 122 G4d cases. One major G4d cluster composed of 37 cases was singled out. Two cases were identified in 2010, 10 from 2011-2014 and 25 from 2015-2017. Cases in the cluster were concentrated in two urban health regions. Compared to the other G4d cases, cluster cases were all male (p<0.001) and more likely to be HIV-positive (adjusted risk ratio: 4.4; 95% confidence interval: 2.5-7.9). A positive syphilis test result was observed for 27 (73%) of the cluster cases. The sequences in this cluster and of four outlier cases were located on the same monophyletic lineage as G4d sequences reported in HIV-positive men who have sex with men (MSM) in Europe. CONCLUSION: Molecular phylogenetics enabled the identification and surveillance of ongoing transmission of a specific HCV G4d lineage in HIV-positive and HIV-negative men in Quebec and its cross-continental spread. This information can orient intervention strategies to avoid transmission of HCV in MSM.

Seroprevalence of seven zoonotic infections in Nunavik, Quebec (Canada).

In Nunavik, common practices and food habits such as consumption of raw meat and untreated water place the Inuit at risk for contracting zoonotic diseases. The aim of this study was to determine the seroprevalence of seven zoonotic infections among the permanent residents of Nunavik. The study was conducted in the fall 2004 as part of the Nunavik Health Survey. Blood samples from adults aged 18-74 years (n = 917) were collected and analysed for the presence of antibodies against Trichinella spp., Toxocara canis, Echinococcus granulosus, Brucella spp., Coxiella burnetii, Leptospira spp. and Francisella tularensis. Information on sociodemographic characteristics, traditional activities, drinking water supply and nutrition was gathered using english/inuktitut bilingual questionnaires. The chi-squared test was used to evaluate associations between seropositivity and other measured variables. Statistically significant variables were included in a multivariate logistic regression model to control for confounding factors. Estimated seroprevalences were 8.3% for E. granulosus, 3.9% for T. canis, 5.9% for Leptospira spp. and 18.9% for F. tularensis. Seroprevalence was ≤ 1% for Trichinella spiralis, Brucella spp. and C. burnetii. For most infections, seropositivity tended to increase with age. In multivariate analyses, seroprevalence was positively (i.e. directly) associated with age and residence in the Ungava coast area for F. tularensis; age and residence in the Hudson coast area for T. canis; female gender, lower level of schooling and frequent cleaning of water reservoirs for E. granulosus. No risk factor for Leptospira spp. infection was identified. No associations were detected with regards to food habits or environmental exposures. A small but significant portion of the Nunavik population has serologic evidence of exposure to at least one of the pathogenic microorganisms investigated. Further studies are needed to better understand the mechanisms for transmission of zoonotic infections and their potential reservoirs in Nunavik.

Seroprevalence of Toxoplasma gondii among Nunavik Inuit (Canada).

As a result of their intimate contact with the land and their nutritional habits, the Inuit of Nunavik are considered to be at risk from zoonotic infections. To better understand the risk factors for Toxoplasma gondii infection, a serosurvey was conducted in Nunavik, Québec, in September 2004. A representative sample of the Inuit adult population of Nunavik participated in this cross-sectional study (n = 917). Antibodies (IgG) against T. gondii were detected by immunoassay. Information on sociodemographic characteristics, traditional activities, domestic environment and nutrition was gathered by questionnaire and explored as variables explanatory of seropositive results. Associations found to be statistically significant in univariate analyses were assessed by multivariable logistic regression to control for confounding factors. Almost two thirds (59.8%) of the Inuit of Nunavik were found to be seropositive for T. gondii. In multivariate analyses, risk factors for seropositivity were: increasing age, gender (women > men), lower level of education, consumption of potentially contaminated water (determined by an index of risk from waterborne infections), frequent cleaning of water reservoirs, and consumption of seal meat and feathered game. There was some variation in seroprevalence between the Ungava Bay coast (52.3%) and the Hudson Bay coast (65.6%), the two main regions of Nunavik, but this variation was not significant in the multivariable logistic regression model. This cross-sectional study demonstrated high T. gondii seroprevalence in the Inuit population and revealed that age, gender, schooling and community of residence all influence serostatus in this population. Variables related to drinking water and food choices may also influence the risk of infection. These results raise important questions about T. gondii transmission in Nunavik including possible links between terrestrial and marine cycles.

Measurement of measles virus-specific neutralizing antibodies: evaluation of the syncytium inhibition assay in comparison with the plaque reduction neutralization test.

Plaque reduction neutralization (PRN) is the "gold-standard" for the measurement of measles-specific neutralizing antibodies. However, it is a complicated assay and tends to be operator-dependent. It has been suggested that the simpler syncytium inhibition assay (SIA) can give results comparable to the PRN test. We compared these two assays using 594 serum or plasma samples obtained from children at various times after natural infection, primary measles immunization, and measles revaccination. The results of the two assays correlated well overall (r = .86; p < 0.0001). The strain of challenge virus (wild-type versus vaccine strain) did not significantly influence SIA titers and the assay performed equally well with serum and plasma. PRN titers > or = 120 and > 800 are thought to indicate protection against clinical illness and infection respectively. The equivalent SIA cut-off values using 125 plaque-forming units as the challenge inoculum were > or = 16 and > 128 respectively. At low PRN titers (< 200), the correlation between PRN and SIA values was reasonable (r = 0.60; p < 0.001) when a challenge inoculum of 12.5 plaque-forming units was used. At the lowest PRN titers (< 100), 15% of the samples gave divergent results. These data confirm the utility of the SIA in the determination of measles-specific neutralizing antibodies when antibody titers are high. However, the PRN assay remains the test of choice when maximum sensitivity at low titers is required.

Cloning and characterization of a dextranase gene (dexS) from Streptococcus suis.

A gene (dexS) coding for a Streptococcus suis capsular type 2 dextranase (DexS) was detected in a recombinant gene library constructed in phage lambda ZapII, and its nucleotide sequence was determined. Sequence comparison showed that the dexS gene product had significant similarities with enzymes which hydrolyze glucose polymers. Moreover, conserved amino acids that are suggested to be part of the active site of the glucosidases are also found in DexS. The dexS gene, adjacent to the gene encoding a S. suis IgG-binding protein, encoded a protein of approximately 62 kDa which exhibited DexS activity.

Purification and characterization of a 52-kilodalton immunoglobulin G-binding protein from Streptococcus suis capsular type 2.

We previously reported that group D streptococci exhibited immunoglobulin G (IgG)-binding activity and that a 52-kDa IgG-binding protein was present in all Streptococcus suis strains examined (B. Serhir, R. Higgins, B. Foiry, and M. Jacques, J. Gen. Microbiol. 139:2953-2958, 1993). The objective of the present study was to purify and characterize this protein. Pig IgG were immobilized through their Fab fragments to ECH-Sepharose 4B, and the protein was purified by affinity chromatography. Electron microscopy observations of the purified material showed filamentous structures with a diameter of approximately 4 nm; these structures were not observed when the material was treated with either urea or ethanolamine. Electrophoretic and Western immunoblot analyses showed that the 52-kDa protein constituted the bulk of the recovered material. This protein was stained with either Coomassie brilliant blue or silver nitrate; it reacted with a large variety of mammalian IgG, human IgG (Fc) fragments, human IgA, and other human plasma proteins. The 52-kDa protein exhibited lower IgG-binding affinities than protein A and protein G. However, it was able to compete with protein A and protein G for binding to human IgG. In addition, it bound chicken IgG with high affinity. This last property differentiated the 52-kDa protein of S. suis from the six IgG-binding proteins described to date. The 52-kDa protein displayed similar affinities for untreated and deglycosylated pig IgG. The N-terminal amino acid sequence (SIITDVYAXEVLDSXGNPTLEV) revealed no homology with any bacterial proteins in the Swiss-Prot database. Its isoelectric point of approximately 4.6 and its amino acid composition, rich in aspartic and glutamic acids, showed that it had some similarities with other IgG-binding proteins. In this report, we have purified and characterized a 52-kDa IgG-binding protein from S. suis capsular type 2. Although this protein shares some similarities with other IgG- and/or IgA-binding proteins, it is unique in reacting with chicken IgG.

Detection of immunoglobulin-G-binding proteins in Streptococcus suis.

This study was undertaken to search for the presence of immunoglobulin G (IgG)-binding proteins in Streptococcus suis, an important swine pathogen. Whole bacterial cells were incubated with human or pig IgG conjugated to gold particles and examined by transmission electron microscopy. Cells of some S. suis strains were labelled as were cells of the positive control strain, Staphylococcus aureus Cowan I. Binding of pig and human IgG to five different bacterial species of group D streptococci, to reference strains representing the 29 capsular types of S. suis, and to 12 S. suis capsular type 2 strains was then examined using Western blotting. All strains interacted with pig and human IgG, although the binding profiles were slightly different. A 52 kDa protein was observed in all capsular types of S. suis. This protein, absent in other group D streptococcal species, was observed in all capsular type 2 isolates originating from diseased or clinically healthy pigs, and was shown to bind human IgG-Fc fragments. The IgG-binding activity was also observed in the culture supernatant and was sensitive to proteolysis.

A sandwich enzyme-linked immunosorbent assay for the detection of Streptococcus suis.

A double-antibody sandwich enzyme-linked immunosorbent assay (ELISA) was developed for the detection and the identification of Streptococcus suis capsular types 1, 2, 1/2, 3 and 22. The specificity of this test was first evaluated using reference strains of S. suis capsular types 1 to 28 and 1/2 as well as 15 different bacterial species susceptible to be isolated from swine. The ELISA developed was very specific for capsular types 1, 3 and 22 but it could not discriminate between capsular types 2 and 1/2. In a second study, S. suis isolates from 328, 493, 368 and 76 diseased pigs were used to detect capsular types 1, 2 or 1/2, 3 and 22 respectively. The relative specificity and sensitivity varied between 98% and 100%. The ELISA results were in excellent agreement with the standard techniques (biochemical tests, coagglutination and capsular reaction tests) in detecting both positive and negative strains. Kappa values were 0.80, 0.99, 0.97 and 1.00 for detecting S. suis capsular types 1, 2 or 1/2, 3, and 22 respectively. To evaluate the relative-sensitivity of the test, primary cultures from 73 diseased pigs and tissue samples from 67 diseased pigs were used directly for detecting these capsular types. With primary cultures, the relative specificity and sensitivity (95.9% and 91.6% respectively) remained high and the test was very suitable (Kappa = 0.87). The ELISA using tissue samples gave a good specificity (97.6%), a moderate sensitivity (62.5%) and a low agreement with standard tests (Kappa = 0.64).(ABSTRACT TRUNCATED AT 250 WORDS)