Validation and Establishment of the SARS-CoV-2 Lentivirus Surrogate Neutralization Assay as a Prescreening Tool for the Plaque Reduction Neutralization Test.

Neutralization assays are important for understanding and quantifying neutralizing antibody responses toward severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). The SARS-CoV-2 lentivirus surrogate neutralization assay (SCLSNA) can be used in biosafety level 2 (BSL-2) laboratories and has been shown to be a reliable alternative approach to the plaque reduction neutralization test (PRNT). In this study, we optimized and validated the SCLSNA to assess its ability as a comparator and prescreening method to support the PRNT. Comparability between the PRNT and SCLSNA was determined through clinical sensitivity and specificity evaluations. Clinical sensitivity and specificity assays produced acceptable results, with 100% (95% confidence interval [CI], 94% to 100%) specificity and 100% (95% CI, 94% to 100%) sensitivity against ancestral Wuhan spike-pseudotyped lentivirus. The sensitivity and specificity against B.1.1.7 spike-pseudotyped lentivirus were 88.3% (95% CI, 77.8% to 94.2%) and 100% (95% CI, 94% to 100%), respectively. Assay precision measuring intra-assay variability produced acceptable results for high (50% PRNT [PRNT50], 1:≥640), mid (PRNT50, 1:160), and low (PRNT50, 1:40) antibody titer concentration ranges based on the PRNT50, with coefficients of variation (CVs) of 14.21%, 12.47%, and 13.28%, respectively. Intermediate precision indicated acceptable ranges for the high and mid concentrations, with CVs of 15.52% and 16.09%, respectively. However, the low concentration did not meet the acceptance criteria, with a CV of 26.42%. Acceptable ranges were found in the robustness evaluation for both intra-assay and interassay variability. In summary, the validation parameters tested met the acceptance criteria, making the SCLSNA method fit for its intended purpose, which can be used to support the PRNT. IMPORTANCE Neutralization studies play an important role in providing guidance and justification for vaccine administration and helping prevent the spread of diseases. The neutralization data generated in our laboratory have been included in the decision-making process of the National Advisory Committee on Immunization (NACI) in Canada. During the coronavirus 2019 (COVID-19) pandemic, the plaque reduction neutralization test (PRNT) has been the gold standard for determining neutralization of SARS-CoV-2. We validated a SARS-CoV-2 lentivirus surrogate neutralization assay (SCLSNA) as an alternative method to help support the PRNT. The advantages of using the SCLSNA is that it can process more samples, is less tedious to perform, and can be used in laboratories with a lower biosafety level. The use of the SCLSNA can further expand our capabilities to help fulfill the requirements for NACI and other important collaborations.

A case series of inactivated Japanese encephalitis virus vaccination associated with positive West Nile virus blood donor screening nucleic acid tests.

BACKGROUND: West Nile Virus (WNV) is a member of the Japanese Encephalitis (JE) serocomplex within the Flaviviridae family. We report four whole blood donors and one plasma donor with WNV nucleic acid test (NAT)-reactive donations between September 2018 and November 2019, following recent Japanese Encephalitis virus (JEV) vaccination. CASE SERIES: Cases 1 and 4 had reactive WNV NAT donations 1 day after receiving the JEV vaccine. Case 2 had a reactive WNV donation 3 days after receiving the JEV vaccine. Case 3 had a reactive WNV NAT donation 3 days after returning from Arizona and 1 day after receiving the JEV vaccine. Case 5 had a reactive WNV donation the same day as receiving the JEV vaccine. STUDY DESIGN AND METHODS: WNV screening used the Roche cobas WNV nucleic acid test (NAT) (Roche Molecular Systems). Reference testing on WNV-reactive donations was carried out by the National Microbiology Laboratory (NML). JEV vaccine dilutions were also analyzed. RESULTS: Supplemental NAT was negative for WNV and JEV for Cases 1, 3, and 5. Case 2 had a weak amplification curve for one of two JEV NAT targets. Case 4 was JEV NAT-positive, WNV NAT-negative. Serologic testing on donation specimens for Cases 2, 4, and 5 did not support recent or remote WNV infection. JEV vaccine dilutions were detected by both cobas and supplemental NAT. CONCLUSIONS: We recommend implementing a temporary blood donor deferral following a JEV vaccination, if screening utilizes a WNV assay with the capability of detecting other members of the JE serocomplex.

Combining anti-IgM and IgG immunoassays for comprehensive chikungunya virus diagnostic testing.

Chikungunya virus (CHIKV) is a mosquito-borne pathogen that causes CHIKV fever. Definitive diagnosis is crucial for patients experiencing symptoms similar to other arboviral diseases because they can vary in clinical consequences. An increasing number of patients experience long-term rheumatic effects of CHIKV infection, but these cases may not be optimally detected by molecular assays and anti-CHIKV IgM ELISAs (M-ELISAs) used for confirmation and screening, respectively. The subsequent confirmatory serological test, the plaque reduction neutralization test (PRNT), is laborious and time-consuming. In this study, we evaluated a new diagnostic algorithm in which the M-ELISA is conducted in parallel with an anti-CHIKV IgG ELISA (G-ELISA) and observed that the Euroimmun M-ELISA combined with the Euroimmun G-ELISA or the Abcam G-ELISA exhibited excellent sensitivity and specificity for CHIKV. The combinations demonstrated perfect and near perfect inter-rater agreement with the PRNT, respectively, suggesting their potential to be used as alternatives to the confirmatory serological PRNT assay for CHIKV.

Establishment of a comprehensive and high throughput serological algorithm for Zika virus diagnostic testing.

The previous serological algorithm for Zika virus (ZIKV) comprised screening by anti-ZIKV IgM capture ELISA (MAC-ELISA) for samples collected within 3 months postexposure or onset (MPEO). Samples positive by MAC-ELISA and samples collected beyond 3 MPEO were tested by the confirmatory plaque reduction neutralization test (PRNT), which proved laborious and time-consuming during the 2015 outbreak. Thus, we evaluated several ZIKV ELISAs to establish an anti-IgM and anti-IgG combination for use as a screening tool for all samples prior to PRNT confirmation. The MAC-ELISA or InBios-M in combination with the Euroimmun-G demonstrated sensitivities of 99.1% and 97.2%, respectively, and nonflavivirus specificity of 96.0%. Their cross-reactivities were 71.4% and 50.0%, respectively, for sera positive for Dengue virus antibodies. Due to near-perfect interrater agreement with PRNT and excellent detection of samples collected beyond 3 MPEO, these combinations were recommended as a screening protocol in a new high-throughput algorithm with special considerations for ZIKV diagnostics.

Evaluation of the Diasorin Liaison® XL Zika Capture IgM CMIA for Zika virus serological testing.

Due to the increase of Zika virus (ZIKV) transmission throughout the world, many commercial kits have recently become available to aid in laboratory diagnosis of ZIKV infections in clinical samples. Here, we analyze the fully automated Liaison® XL Zika Capture immunoglobulin M (IgM) assay against the recommended IgM-capture enzyme-linked immunosorbent assay.

Evaluation of 5 Commercially Available Zika Virus Immunoassays.

Because of the global spread of Zika virus, accurate and high-throughput diagnostic immunoassays are needed. We compared the sensitivity and specificity of 5 commercially available Zika virus serologic assays to the recommended protocol of Zika virus IgM-capture ELISA and plaque-reduction neutralization tests. Most commercial immunoassays showed low sensitivity, which can be increased.

California Serogroup Virus Infection Associated with Encephalitis and Cognitive Decline, Canada, 2015.

California serogroup (CSG) viruses, such as Jamestown Canyon and snowshoe hare viruses, are mosquitoborne pathogens that cause febrile illness and neurologic disease. Human exposures have been described across Canada, but infections are likely underdiagnosed. We describe a case of neuroinvasive illness in a New Brunswick, Canada, patient infected with a CSG virus.

Seroprevalence of seven zoonotic pathogens in pregnant women from the Caribbean.

Studies examining the prevalence of zoonotic agents in the Caribbean are very limited. The objective of this study was to examine the seroprevalence of seven zoonotic agents among individuals residing on 10 English-speaking Caribbean countries. Sera from healthy, pregnant women were collected from Antigua-Barbuda, Belize, Bermuda, Dominica, Grenada, Jamaica, Montserrat, St. Kitts-Nevis, St. Lucia, and St. Vincent-Grenadines and tested for the presence of IgG antibodies to dengue virus, hepatitis E virus, hantaviruses, leptospiral agents, spotted fever group rickettsiae (SFGR), typhus group rickettsiae (TGR), and Coxiella burnetii (Q fever). The highest seroprevalence values were observed for dengue virus, SFGR, and leptospirosis, although the lowest seroprevalence values were observed for hepatitis E virus, C. burnetii, and TGR. Antibodies to hantaviruses were not detected in any individuals.

Zoonotic infections in communities of the James Bay Cree territory: An overview of seroprevalence.

The Cree communities of James Bay are at risk for contracting infectious diseases transmitted by wildlife. Data from serological testing for a range of zoonotic infections performed in the general population (six communities), or trappers and their spouses (one community), were abstracted from four population-based studies conducted in Cree territory (Quebec) between 2005 and 2009. Evidence of exposure to Trichinella species, Toxoplasma gondii, Toxocara canis, Echinococcus granulosus, Leptospira species, Coxiella burnetii and Francisella tularensis was verified in all communities, whereas antibodies against Sin Nombre virus and California serogroup viruses (Jamestown Canyon and snowshoe hare viruses) were evaluated in three and six communities, respectively. Seroprevalence varied widely among communities: snowshoe hare virus (1% to 42%), F tularensis (14% to 37%), Leptospira species (10% to 27%), Jamestown Canyon virus (9% to 24%), C burnetii (0% to 18%), T gondii (4% to 12%), T canis (0% to 10%), E granulosus (0% to 4%) and Trichinella species (0% to 1%). No subject had serological evidence of Sin Nombre virus exposure. These data suggest that large proportions of the Cree population have been exposed to at least one of the targeted zoonotic agents. The Cree population, particularly those most heavily exposed to fauna, as well as the medical staff living in these regions, should be aware of these diseases. Greater awareness would not only help to decrease exposures but would also increase the chance of appropriate diagnostic testing.

Les communautés cries de la Baie James sont vulnérables aux maladies infectieuses transmises par les animaux sauvages. Les données tirées des tests sérologiques sur une série de zoonoses effectués dans la population générale (six communautés) ou chez les trappeurs et leur conjointe (une communauté) ont été extraites de quatre études en population menées en territoire cri, au Québec, entre 2005 et 2009. Les manifestations d'exposition aux espèces de Trichinella, au Toxoplasma gondii, au Toxocara canis, à l'Echinococcus granulosus, aux espèces de Leptospira, au Coxiella burnetii et au Francisella tularensis ont été vérifiées dans toutes les communautés, tandis que les anticorps contre le virus Sin Nombre et les virus du sérogroupe Californie (virus Jamestown Canyon et snowshoe hare) ont été évalués dans trois et six communautés, respectivement. La séroprévalence variait considérablement selon les communautés, comme suit : virus snowshoe hare (1 % à 42 %), F tularensis (14 % à 37 %), espèces de Leptospira (10 % à 27 %), virus Jamestown Canyon (9 % à 24 %), C burnetii (0 % à 18 %), T gondii (4 % à 12 %), T canis (0 % à 10 %), E granulosus (0 % à 4 %) et espèces de Trichinella (0 % à 1 %). Aucun sujet n'avait de manifestation sérologique d'exposition au virus Sin Nombre. Ces données laissent supposer que de fortes proportions de la population crie ont été exposées à au moins l'un des agents zoonotiques ciblés. La population crie, notamment les peuples les plus exposés aux animaux sauvages, ainsi que le personnel médical qui habite dans ces régions, devrait connaître ces maladies. Une meilleure sensibilisation contribuerait non seulement à réduire les expositions, mais accroîtrait également la possibilité de tests diagnostiques pertinents.

Seroprevalence of 10 zoonotic infections in 2 Canadian Cree communities.

We evaluated the seroprevalence of 10 zoonotic agents among the general population (15 years old and over) of Eastmain and Wemindji, James Bay, Quebec, in 2007. Overall seroprevalence rates were similar between the 2 communities. Nearly half the individuals tested (n = 251; 146 women, 105 men) were seropositive (n = 115) for at least one zoonosis. The highest seroprevalence rates were for Leptospira sp. (23%), Francisella tularensis (17%), and the California serogroup viruses (JC and SSH viruses) (10%). The other zoonoses (Toxoplasma gondii, Coxiella burnetii, Echinococcus granulosus, Toxocara canis, and Trichinella sp.) had seroprevalence rates ≤5%; no exposures were identified to hantaviruses (Sin Nombre virus). Overall, seropositivity was related to age, gender, hunting, and owning a dog. There was no medical history suggestive of overt diseases. Nonetheless, physicians should consider these agents when confronted with difficult or confusing diagnoses. In particular, the bacterial zoonoses should be ruled out in individuals with high or prolonged fever.

Sin Nombre virus shedding patterns in naturally infected deer mice (Peromyscus maniculatus) in relation to duration of infection.

A 2-year capture-mark-recapture study was conducted in southern Manitoba, Canada, to test for an association between the duration of Sin Nombre virus (SNV) infection in deer mice (Peromyscus maniculatus) and virus shedding. Hantavirus-specific IgG antibodies were detected in 22.2% of captured deer mice, and recently infected deer mice were identified based on the detection of low-avidity IgG antibodies. SNV RNA was detected in blood samples from the majority of seropositive deer mice with no significant difference in the association of SNV RNA between the low- and high-avidity groups (57.8% and 52.1%, respectively). A small subset of seropositive mice (11.6%) had detectable SNV RNA in oropharyngeal fluids (OPF) or urine. A greater proportion of deer mice with low-avidity antibodies had SNV RNA in OPF or urine compared with rodents with high-avidity antibodies (21% versus 6.8%, respectively). This is the first study of naturally infected deer mice to provide evidence that recently infected mice are more likely to shed SNV and thus might represent a greater risk of human infection.