Genotyping and Molecular Characterization of VP6 and NSP4 Genes of Unusual Rotavirus Group A Isolated from Children with Acute Gastroenteritis.

Group A rotavirus (RVA), which causes acute gastroenteritis (AGE) in children worldwide, is categorized mainly based on VP7 (genotype G) and VP4 (genotype P) genes. Genotypes that circulate at <1% are considered unusual. Important genes also include VP6 (genotype I) and NSP4 (genotype E). VP6 establishes the group and affects immunogenicity, while NSP4, as an enterotoxin, is responsible for the clinical symptoms. The aim of this study was to genotype the VP6 and NSP4 genes and molecularly characterize the NSP4 and VP6 genes of unusual RVA. Unusual RVA strains extracted from fecal samples of children ≤16 years with AGE were genotyped in VP6 and NSP4 genes with Sanger sequencing. In a 15-year period (2007-2021), 54.8% (34/62) of unusual RVA were successfully I and E genotyped. Three different I and E genotypes were identified; I2 (73.5%, 25/34) and E2 (35.3%, 12/34) were the most common. E3 genotype was detected from 2017 onwards. The uncommon combination of I2-E3 was found in 26.5% (9/34) of the strains and G3-P[9]-I2-E3 remained the most frequent G-P-I-E combination (20.6%, 7/34). Children infected with RVA E2 strains had a statistically higher frequency of dehydration (50%) than those infected with RVA E3 strains (p = 0.019). Multiple substitutions were detected in NSP4, but their functional effect remains unknown. The result indicates the genetic diversity of RVA strains. Continuous surveillance of the RVA based on the whole genome will provide better knowledge of its evolution.

Kinetics of SARS-CoV-2 Spike Antibodies after the Second and Third Dose of the BNT162b2 COVID-19 Vaccine and Association with Epidemiological Characteristics and Breakthrough Infection in a Cohort Study of Healthcare Workers.

To prospectively study the kinetics of immune responses after immunization with the BNT162b2 mRNA COVID-19 vaccine and their association with epidemiological parameters and breakthrough infection (BI), we measured total (TAbs-WT) and neutralizing antibodies against wild-type (NAbs-WT) and Omicron (NAbs-O) SARS-CoV-2 spike proteins in healthcare workers (HCWs) after the second (4 and 8 months) and third dose (1 and 8 months). Vaccinated HCWs (n = 486), with a median age (IQR) of 49 years (38-56), were included in this prospective cohort study. BI was observed 4 and 8 months after the second dose in 8/486 (1.6%) and 15/486 (3.1%) HCWs, respectively, and 1 and 8 months after the third dose in 17/486 (3.5%) and 152/486 (31.3%) HCWs, respectively. A comparison of immune responses 1 month after the third dose in vaccinated HCWs without a BI or with a BI in the next 7 months did not detect any statistically significant differences in the TAbs-WT (median (IQR): 16,611.0 (13,011.0) U/mL vs. 17,572.5 (14,501.0) U/mL, p = 0.529) and NAbs-WT (median (IQR): 96.5% (1.7) vs. 96.7% (1.9), p = 0.555). After infection, HCWs with a BI had significantly increased TAbs-WT levels at all time points compared to healthy HCWs. The findings of the present study indicate that antibody levels after three doses of the BNT162b2 vaccine are not directly associated with the possibility of a BI.

Epidemiological study of unusual rotavirus strains and molecular characterization of emerging P[14] strains isolated from children with acute gastroenteritis during a 15-year period.

Rotavirus group A (RVA) is characterized by molecular and epidemiological diversity. To date, 42 G and 58 P RVA genotypes have been identified, some of which, like P[14], have a zoonotic origin. In this study, we describe the epidemiology of unusual RVA genotypes and the molecular characteristics of P[14] strains. Fecal samples from children ≤ 16 years of age with acute gastroenteritis (AGE) who were hospitalized during 2007-2021 in Greece were tested for RVA by immunochromatography. Positive RVA samples were G and P genotyped, and part of the VP7 and VP4 genes were sequenced by the Sanger method. Epidemiological data were also recorded. Phylogenetic analysis of P[14] was performed using MEGA 11 software. Sixty-two (1.4%) out of 4427 children with RVA AGE were infected with an unusual G (G6/G8/G10) or P (P[6]/P[9]/P[10]/P[11]/P[14]) genotype. Their median (IQR) age was 18.7 (37.3) months, and 67.7% (42/62) were males. None of the children were vaccinated against RVA. P[9] (28/62; 45.2%) was the most common unusual genotype, followed by P[14] (12/62; 19.4%). In the last two years, during the period of the COVID-19 pandemic, an emergence of P[14] was observed (5/12, 41.6%) after an 8-year absence. The highest prevalence of P[14] infection was seen in the spring (91.7%). The combinations G8P[14] (41.7%), G6P[14] (41.7%), and G4P[14] (16.6%) were also detected. Phylogenetic analysis showed a potential evolutionary relationship of three human RVA P[14] strains to a fox strain from Croatia. These findings suggest a possible zoonotic origin of P[14] and interspecies transmission between nondomestic animals and humans, which may lead to new RVA genotypes with unknown severity.

SARS-CoV-2 seroepidemiology in paediatric population during Delta and Omicron predominance.

Limited prospective severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) data in children regarding the impact of Omicron variant in seropositivity have been reported. We investigated SARS-CoV-2 seropositivity in children between 1 September 2021 and 30 April 2022, representing Delta and Omicron predominance periods. Serum samples from children admitted to the major tertiary Greek paediatric hospital for any cause, except for COVID-19, were randomly collected and tested for SARS-CoV-2 natural infection antibodies against nucleocapsid antigen (Elecsys® Anti-SARS-CoV-2 reagent). A total of 506/1312 (38.6%) seropositive children (0-16 years) were detected (males: 261/506(51.6%); median age (IQR): 95.2 months(24-144)). Seropositivity rates (%) increased from Delta to Omicron period from 29.7% to 48.5% (P-value<0.0001). Seropositivity increased for all age groups, except for the age group of 0-1 year (P-value:0.914). The highest seropositivity rate was detected in April 2022 (52.6%) and reached 73.9% specifically for the age group 12-16 years. No significant differences were detected in seropositivity with respect to gender, origin, or hospitalisation status. Median (IQR) antibody titres were higher in the Omicron vs. Delta period in all age groups, especially in 12-16 years [32.2 COI (7-77.1) vs. 11.4 COI(2.8-50.2), P-value:0.009). During Omicron variant period increased SARS-CoV-2 seropositivity was detected in paediatric population, especially in adolescents, implicating either increased transmissibility or reinfection rates.

Immunogenicity of the COVID-19 BNT162b2 vaccine in adolescents and young adults with cystic fibrosis.

Data regarding immunogenicity of SARS-CoV-2 BNT162b2 vaccine in cystic fibrosis (CF) patients are limited. We prospectively measured total (TAbs-RBD; U/ml) and neutralizing (NAbs-RBD; %) antibodies of SARS-CoV-2 spike-receptor binding domain (RBD) protein in 33 CF patients and 66 healthy controls with median age (IQR): 19.6 (17.6-24.3) years and 31 (29-36) years, respectively and investigated possible associations with epidemiological and clinical parameters. Compared to healthy controls, CF patients had higher levels of TAbs-RBD and NAbs-RBD after both doses (P-value < 0.001). One month after the second dose, CF patients and controls had TAbs-RBD: median (IQR): 3396 (2443) and 1452 (1231) U/ml, respectively. Similarly, the NAbs-RBD (%) were: 97.30 (1.00) and 95.70 (3.71) %, respectively. CF patients also had fewer local and systemic adverse events (AEs) (P-value < 0.001). Among CF patients, no significant differences in immunogenicity were detected regarding the phenotype, genotype, medications, or severity of the disease. BNT162b2 vaccine was immunogenic with limited reactogenicity in CF patients regardless of the phenotype or severity of disease.

Seroepidemiology of SARS-CoV-2 in pediatric population during a 16-month period prior to vaccination.

Limited prospective serosurveillance data in children regarding severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection have been reported. We prospectively investigated SARS-CoV-2 seropositivity in children during a 16-month period of the coronavirus disease 2019 (COVID-19) pandemic, including the four waves of the pandemic, before SARS-CoV-2 adolescents' vaccination. Serum samples from children admitted to the major tertiary Greek pediatric hospital for any cause, except for COVID-19 infection, were randomly collected from 05/2020 to 08/2021. The study period was divided into four 4-month periods representing relevant epidemic waves. Total SARS-CoV-2 antibodies for nucleocapsid protein were determined using the Elecsys® Anti-SARS-CoV-2 reagent. A total of 3099 children (0-16 years) were included in the study. A total of 344 (11.1%) seropositive children were detected (males: 205 [59.5%]; median age [interquartile range [IQR]]: 3 years [0.6-10]). Seropositivity rates (%) increased during the four 4-month periods: 1.4%, 8.6%, 17.2%, and 17.6%, respectively. A correlation of seropositivity rates in children with new diagnosed SARS-CoV-2 cases in the community was detected. No significant differences were detected between males and females. Seropositivity was significantly higher in hospitalized than in nonhospitalized children and in non-Greek compared to Greek children (p < 0.001). The lowest seropositivity rate before school opening (9/2021) was detected in the age groups 6-12 years (14.4%) and 12-16 years (16.1%). However, compared with the other age groups, the lowest median antibody titers were observed in children 0-1 year (median [IQR]: 13.9 cut-off index: [4.5-53.9] [p < 0.001]). Although the seropositivity of children was related to the community epidemic waves, the exposure was limited. Low seropositivity rates in school-age children support the need for SARS-CoV-2 immunization.

SARS-CoV-2 seroepidemiological study in healthcare workers and discordant results using seven different diagnostic methods.

The aim of the study was to access the SARS-CoV-2 antibody seroprevalence in healthcare workers (HCWs) of a tertiary pediatric hospital after the first wave of the pandemic and to compare the results among seven commercially available antibody detection assays, including chemiluminescence (CMIA), electroluminescence (ECLIA), Εnzyme-Linked Immunosorbent Assay (ELISA), and rapid immunochromatography (RIC). SARS-CoV-2 antibody detection was performed in serum samples of 1216 HCWs, using a reference CMIA assay and 8/1216 (0.66%) were detected positive. Positive serum samples were further tested with other assays; however, only one sample was positive by all tests. The rest 7 cases were negative with ECLIA and ELISA and gave discordant results with RIC test. Six months later, new serum samples of seropositive HCWs were analyzed with the same 7 tests, with inconsistent results again. Identification of reliable SARS-CoV-2 antibody tests is important to determine the actual number of past infections, the duration of antibodies, and guide public health decisions.

Rotavirus epidemiology and genotype distribution in hospitalised children, Greece, 2008 to 2020: A prospective multicentre study.

BackgroundTwo rotavirus (RV) vaccines were licensed in Greece in late 2006 and included in the national immunisation programme in 2012.AimTo study the epidemiology and genotype distribution of RV in children during the post-vaccination period and assess the impact of increased vaccination coverage.MethodsIn a prospective multicentre hospital-based study, hospitalised children (≤ 16 years) with an RV-positive faecal sample were recruited. Epidemiological and genotyping analyses were performed; periods of low (2008-12) and moderate (2012-20) RV vaccination coverage were compared. Statistical analysis was performed with a chi-squared or Mann-Whitney U test and logistic regression.ResultsA total of 3,874 children (55.6% male; n = 2,153) with median age of 1.4 years (IQR: 0.5-3.3) were studied during 2008-20. Most RV-infected children were aged ≤ 3 years (72.2%) and hospitalised during December-May (69.1%). Common RV genotypes (G1P[8], G2P[4], G3P[8], G4P[8], G9P[8], G12P[8]) were detected in 92.2% of samples; G-P combinations with prevalence above 1% were G4P[8] (44.1%), G1P[8] (25.4%), G2P[4] (14.9%), G9P[8] (3.5%), G12P[8] (2.2%), G3P[8] (2.1%), other (4.3%) and mixed (3.5%). Of all samples, 97.6% were homotypic or partially heterotypic to vaccines' genotypes. With moderate vaccination coverage, the seasonal peak was detected earlier, children were older and partially or fully heterotypic genotypes were increased (p < 0.001).ConclusionsIn the era of moderate RV vaccination coverage in Greece, epidemiology of RV in hospitalised children seemed to change. However, most circulating genotypes remain homotypic or partially heterotypic to RV vaccines. Continuous epidemiological surveillance and genotyping are important to monitor possible changes arising from RV vaccines' implementation.

Association of clinical and epidemiological characteristics with COVID-19 BNT162b2 mRNA vaccine short-term adverse reactions in healthcare workers.

INTRODUCTION: The aim of the study was to investigate the prevalence and severity of adverse reactions (ARs) after immunization of healthcare workers (HCWs) with BNT162b2 vaccine and to associate them with clinical and epidemiological characteristics. METHODS: A form containing demographic and clinical data as well as ARs after both doses of the vaccine was completed, and statistical association analysis was performed. RESULTS: A total of 502 HCWs (females 78.3%) with mean age (±SD) 48.17 years (±12.97) participated. After the first dose, 404 (80.5%) HCWs reported at least one local AR (LAR) and 366 (72.9%) after the second dose (p-value=0.004). After the first dose, 121 (24.1%) HCWs reported at least one systemic AR (SAR) and 275 (54.8%) after the second dose (p-value<0.0001).In the logistic regression analysis, there was no association of gender or medical history of underlying disease with LARs. There was a negative association of age with the cumulative score (CS) of LARs (OR: 0.82, 95% CI: 0.69-0.96) after the first dose. Females had a positive association with CS of SARs following both doses (OR, 95% CI: 2.57, 1.39-4.73 and 2.71, 1.76-4.19, respectively). Age was negatively associated with CS of SARs (OR: 0.66, 95% CI: 0.57-0.76) after the second dose. Severe ARs included Bell's palsy (1) and tinnitus with temporary hearing loss (1). CONCLUSION: The administration of the BNT162b2 vaccine in our HCWs cohort had a good safety profile with the most common ARs being self-limited. An increasing rate of SARs following the second vaccine dose was noticed. Rare but severe possible ARs should be further investigated.

Association of total and neutralizing SARS-CoV-2 spike -receptor binding domain antibodies with epidemiological and clinical characteristics after immunization with the 1st and 2nd doses of the BNT162b2 vaccine.

BACKGROUND: Data regarding the association of antibody levels elicited after immunization with the BNT162b2 mRNA COVID-19 vaccine with epidemiological and clinical parameters are limited. METHODS: We prospectively measured the total (TAbs-RBD) and the neutralizing antibodies (NAbs-RBD) against the receptor binding domain (RBD) of SARS-CoV-2 spike protein in a cohort of 268 Healthcare workers before immunization, 20 days after the 1st dose and 30 days after the 2nd dose of the BNT162b2 vaccine. A statistical analysis for possible association of antibodies' levels with epidemiological and clinical parameters was performed. RESULTS: The mean age (±SD) of the participants was 45.45 years (±11.93) (range: 24-70 years) and 211 (79.9%) were females. Statistically significant differences were detected regarding both TAbs-RBD and NAbs-RBD between the first and second doses of the vaccine (P < 0.001). The median (IQR) percentage (%) of NAbs-RBD after the 1st dose was 51.07% (31.60%) and after the 2nd dose 95.31% (3.70%) (P < 0.001). The correlation between the TAbs-RBD and NAbs-RBD was after the 1st dose, Spearman's, rho: 0.861 (P < 0.001) and after the 2nd dose rho: 0.989 (P < 0.001). Twenty days after the 1st dose, 56/264 (21.2%) of the participants had low levels of NAbs-RBD, while one month after the 2nd dose all of them had protective levels of NAbs-RBD. After the 2nd vaccine dose, a statistically significant negative association of TAbs-RBD was detected for age (P < 0.001), smoking (P = 0.011), and immunosuppressive medications (P < 0.001), while a positive association was detected for BMI (P = 0.004) and systemic adverse events after immunization (P = 0.001). CONCLUSION: A significant correlation of TAbs-RBD and NAbs-RBD was detected after both vaccine doses. Older age, smoking, and immunosuppressive medications negatively affected the final antibody level after SARS-CoV-2 immunization. Our findings emphasize the significance of the 2nd vaccine dose especially in the older age groups.