Development and Validation of an Enzyme Immunoassay for Detection and Quantification of SARS-CoV-2 Salivary IgA and IgG.

Oral fluids offer a noninvasive sampling method for the detection of Abs. Quantification of IgA and IgG Abs in saliva allows studies of the mucosal and systemic immune response after natural infection or vaccination. We developed and validated an enzyme immunoassay (EIA) to detect and quantify salivary IgA and IgG Abs against the prefusion-stabilized form of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike protein expressed in suspension-adapted HEK-293 cells. Normalization against total Ab isotype was performed to account for specimen differences, such as collection time and sample volume. Saliva samples collected from 187 SARS-CoV-2 confirmed cases enrolled in 2 cohorts and 373 prepandemic saliva samples were tested. The sensitivity of both EIAs was high (IgA, 95.5%; IgG, 89.7%) without compromising specificity (IgA, 99%; IgG, 97%). No cross-reactivity with endemic coronaviruses was observed. The limit of detection for SARS-CoV-2 salivary IgA and IgG assays were 1.98 ng/ml and 0.30 ng/ml, respectively. Salivary IgA and IgG Abs were detected earlier in patients with mild COVID-19 symptoms than in severe cases. However, severe cases showed higher salivary Ab titers than those with a mild infection. Salivary IgA titers quickly decreased after 6 wk in mild cases but remained detectable until at least week 10 in severe cases. Salivary IgG titers remained high for all patients, regardless of disease severity. In conclusion, EIAs for both IgA and IgG had high specificity and sensitivity for the confirmation of current or recent SARS-CoV-2 infections and evaluation of the IgA and IgG immune response.

Secretor Status Strongly Influences the Incidence of Symptomatic Norovirus Infection in a Genotype-Dependent Manner in a Nicaraguan Birth Cohort.

BACKGROUND: The role of histo-blood group on the burden and severity of norovirus gastroenteritis in young infants has not been well documented. METHODS: Norovirus gastroenteritis was assessed in 443 Nicaraguan children followed from birth until 3 years of age. Stool samples were tested for norovirus by reverse-transcription quantitative polymerase chain reaction (RT-qPCR), and histo-blood group antigens (HBGAs) were determined by phenotyping of saliva and blood. Hazard ratios and predictors of norovirus acute gastroenteritis (AGE) outcome stratified by HBGA were estimated using Cox proportional hazards models. RESULTS: Of 1353 AGE episodes experienced by children, 229 (17%) tested positive for norovirus with an overall incidence of 21.9/100 child-years. Secretor children were infected as early as 2 months of age and had a higher incidence of norovirus GII compared to nonsecretor children (15.4 vs 4.1/100 child-years, P = .006). Furthermore, all GII.4 AGE episodes occurred in secretor children. Children infected with GI (adjusted odds ratio [aOR], 0.09 [95% confidence interval {CI}, .02-.33]) or non-GII.4 viruses (aOR, 0.2 [95% CI, .07-.6]) were less likely to have severe AGE compared to GII.4-infected children. CONCLUSIONS: Secretor status in children strongly influences the incidence of symptomatic norovirus infection in a genogroup or genotype-dependent manner and provides evidence that clinical severity in children depends on norovirus genotypes.

Humoral and Mucosal Immune Responses to Human Norovirus in the Elderly.

BACKGROUND: Most information on mucosal and systemic immune response to norovirus infection is derived from human challenge studies, birth cohort studies, or vaccine trials in healthy adults. However, few data are available on immune responses to norovirus in the elderly. METHODS: To study the mucosal and systemic immune response against norovirus, 43 long-term care facilities were enrolled prospectively in 2010-2014. Baseline saliva samples from 17 facilities, cases and controls up to day 84 from 10 outbreaks, as well as acute and convalescent sera were collected. RESULTS: Norovirus-specific immunoglobulin A (IgA) levels in baseline saliva samples were low and increased in both symptomatic patients and asymptomatic shedders at day 5 after onset during outbreaks. Receiver operating characteristics analysis correctly assigned prior norovirus infection in 23 (92%) of 25 participants. Cases and asymptomatic shedders showed seroconversion for IgG (80%), IgA (78%), and blockade antibodies (87%). Salivary IgA levels strongly correlated with increased convalescent serum IgA titers and blockade antibodies. CONCLUSIONS: Salivary IgA levels strongly correlated with serum IgA titers and blockade antibodies and remained elevated 3 months after a norovirus outbreak. A single salivary sample collected on day 14 could be used to identify recent infection in a suspected outbreak or to monitor population salivary IgA.

Minimally Invasive Saliva Testing to Monitor Norovirus Infection in Community Settings.

BACKGROUND: Norovirus is a leading cause of acute gastroenteritis worldwide. Routine norovirus diagnosis requires stool collection. The goal of this study was to develop and validate a noninvasive method to diagnose norovirus to complement stool diagnostics and to facilitate studies on transmission. METHODS: A multiplex immunoassay to measure salivary immunoglobulin G (IgG) responses to 5 common norovirus genotypes (GI.1, GII.2, GII.4, GII.6, and GII.17) was developed. The assay was validated using acute and convalescent saliva samples collected from Peruvian children <5 years of age with polymerase chain reaction (PCR)-diagnosed norovirus infections (n = 175) and controls (n = 32). The assay sensitivity and specificity were calculated to determine infection status based on fold rise of salivary norovirus genotype-specific IgG using norovirus genotype from stool as reference. RESULTS: The salivary assay detected recent norovirus infections and correctly assigned the infecting genotype. Sensitivity was 71% and specificity was 96% across the evaluated genotypes compared to PCR-diagnosed norovirus infection. CONCLUSIONS: This saliva-based assay will be a useful tool to monitor norovirus transmission in high-risk settings such as daycare centers or hospitals. Cross-reactivity is limited between the tested genotypes, which represent the most commonly circulating genotypes.

Norovirus Infection and Disease in an Ecuadorian Birth Cohort: Association of Certain Norovirus Genotypes With Host FUT2 Secretor Status.

BACKGROUND: Although norovirus is the most common cause of gastroenteritis, there are few data on the community incidence of infection/disease or the patterns of acquired immunity or innate resistance to norovirus. METHODS: We followed a community-based birth cohort of 194 children in Ecuador with the aim to estimate (1) the incidence of norovirus gastroenteritis from birth to age 3 years, (2) the protective effect of norovirus infection against subsequent infection/disease, and (3) the association of infection and disease with FUT2 secretor status. RESULTS: Over the 3-year period, we detected a mean of 2.26 diarrheal episodes per child (range, 0-12 episodes). Norovirus was detected in 260 samples (18%) but was not found more frequently in diarrheal samples (79 of 438 [18%]), compared with diarrhea-free samples (181 of 1016 [18%]; P = .919). A total of 66% of children had at least 1 norovirus infection during the first 3 years of life, and 40% of children had 2 infections. Previous norovirus infections were not associated with the risk of subsequent infection. All genogroup II, genotype 4 (GII.4) infections were among secretor-positive children (P < .001), but higher rates of non-GII.4 infections were found in secretor-negative children (relative risk, 0.56; P = .029). CONCLUSIONS: GII.4 infections were uniquely detected in secretor-positive children, while non-GII.4 infections were more often found in secretor-negative children.

Evaluation of a New Environmental Sampling Protocol for Detection of Human Norovirus on Inanimate Surfaces.

Inanimate surfaces are regarded as key vehicles for the spread of human norovirus during outbreaks. ISO method 15216 involves the use of cotton swabs for environmental sampling from food surfaces and fomites for the detection of norovirus genogroup I (GI) and GII. We evaluated the effects of the virus drying time (1, 8, 24, or 48 h), swab material (cotton, polyester, rayon, macrofoam, or an antistatic wipe), surface (stainless steel or a toilet seat), and area of the swabbed surface (25.8 cm(2) to 645.0 cm(2)) on the recovery of human norovirus. Macrofoam swabs produced the highest rate of recovery of norovirus from surfaces as large as 645 cm(2). The rates of recovery ranged from 2.2 to 36.0% for virus seeded on stainless-steel coupons (645.0 cm(2)) to 1.2 to 33.6% for toilet seat surfaces (700 cm(2)), with detection limits of 3.5 log10 and 4.0 log10 RNA copies. We used macrofoam swabs to collect environmental samples from several case cabins and common areas of a cruise ship where passengers had reported viral gastroenteritis symptoms. Seventeen (18.5%) of 92 samples tested positive for norovirus GII, and 4 samples could be sequenced and had identical GII.1 sequences. The viral loads of the swab samples from the cabins of the sick passengers ranged from 80 to 31,217 RNA copies, compared with 16 to 113 RNA copies for swab samples from public spaces. In conclusion, our swab protocol for norovirus may be a useful tool for outbreak investigations when no clinical samples are available to confirm the etiology.

Descriptive evaluation of antibody responses to severe acute respiratory coronavirus virus 2 (SARS-CoV-2) infection in plasma and gingival crevicular fluid in a nursing home cohort-Arkansas, June-August 2020.

OBJECTIVE: To characterize and compare severe acute respiratory coronavirus virus 2 (SARS-CoV-2)-specific immune responses in plasma and gingival crevicular fluid (GCF) from nursing home residents during and after natural infection. DESIGN: Prospective cohort. SETTING: Nursing home. PARTICIPANTS: SARS-CoV-2-infected nursing home residents. METHODS: A convenience sample of 14 SARS-CoV-2-infected nursing home residents, enrolled 4-13 days after real-time reverse transcription polymerase chain reaction diagnosis, were followed for 42 days. After diagnosis, plasma SARS-CoV-2-specific pan-Immunoglobulin (Ig), IgG, IgA, IgM, and neutralizing antibodies were measured at 5 time points, and GCF SARS-CoV-2-specific IgG and IgA were measured at 4 time points. RESULTS: All participants demonstrated immune responses to SARS-CoV-2 infection. Among 12 phlebotomized participants, plasma was positive for pan-Ig and IgG in all 12 participants. Neutralizing antibodies were positive in 11 participants; IgM was positive in 10 participants, and IgA was positive in 9 participants. Among 14 participants with GCF specimens, GCF was positive for IgG in 13 participants and for IgA in 12 participants. Immunoglobulin responses in plasma and GCF had similar kinetics; median times to peak antibody response were similar across specimen types (4 weeks for IgG; 3 weeks for IgA). Participants with pan-Ig, IgG, and IgA detected in plasma and GCF IgG remained positive throughout this evaluation, 46-55 days after diagnosis. All participants were viral-culture negative by the first detection of antibodies. CONCLUSIONS: Nursing home residents had detectable SARS-CoV-2 antibodies in plasma and GCF after infection. Kinetics of antibodies detected in GCF mirrored those from plasma. Noninvasive GCF may be useful for detecting and monitoring immunologic responses in populations unable or unwilling to be phlebotomized.

Self-assembly of the recombinant capsid protein of a swine norovirus into virus-like particles and evaluation of monoclonal antibodies cross-reactive with a human strain from genogroup II.

Noroviruses (NoVs) are responsible for the majority of gastroenteritis outbreaks in humans. Recently, NoV strains which are genetically closely related to human genogroup II (GII) NoVs have been detected in fecal specimens from swine. These findings have raised concern about the possible role of pigs as reservoirs for NoVs that could infect humans. To better understand the epidemiology of swine NoVs in both the swine and the human populations, rapid immunoassays are needed. In this study, baculovirus recombinants were generated to express the capsid gene of a swine NoV GII genotype 11 (GII.11) strain which self-assembled into virus-like particles (VLPs). Subsequently, the purified VLPs were used to evoke monoclonal antibodies (MAbs) in mice. A panel of eight promising MAbs was obtained and evaluated for their ability to bind to heterologous VLPs, denaturated antigens, and truncated capsid proteins. The MAbs could be classified into two groups: two MAbs that recognized linear epitopes located at the amino-terminal half (shell domain) of the swine NoV GII.11 VLPs and that cross-reacted with human GII.4 NoV VLPs. The other six MAbs bound to conformational epitopes and did not cross-react with the human GII.4 VLPs. To our knowledge, this is the first report on the characterization of MAbs against swine NoVs. The swine NoV VLPs and the MAbs described here may be further used for the design of diagnostic reagents that could help increase our knowledge of the prevalence of NoV infections in pigs and the possible role of pigs as reservoirs for NoVs.