ABSTRACT
Background: mRNA vaccines elicit a durable humoral response to SARS-CoV-2 in adults, whereas evidence in children is lacking. This study aimed to evaluate the early and long-term immunological response after the BNT162b2 vaccine in children with or without a previous SARS-CoV-2 infection. Method(s): In a multicenter, prospective, observational study we profiled the immune response to the BNT162b2 vaccine in children aged 5-11 years attending the Pediatric Departments at the University of Padua and Bambino Gesu Children's Hospital in Rome (Italy). Forty-four healthy children (HC), 20 immune compromised (IC), and 18 children who previously developed MIS-C (MIS-C) were included in the study. Blood samples were collected pre-, 1, and 6 months after a 2-doses vaccination schedule. Neutralizing antibodies (NAbs) and anti-S-RBD IgG titers were analyzed through Plaque Reduction Neutralization Test (PRNT) and chemiluminescent immune-enzymatic assay (CLIA), respectively. B and T cell phenotypes were analyzed by flow cytometry. Geometric mean titers (GMTs) and 95% confidence intervals and median and interquartile range (IQR) of variables were evaluated according to pre-existing confirmed COVID-19. Result(s): Eighty-two children were studied;60 with a molecular-documented previous COVID-19 (Group A) and 22 without previous infection defined as the absence of antigen-specific antibodies before the vaccination (Group B). Overall, in Group A we observed higher NAbs GMTs, anti-S-RBD titers, and T- and B-reg cells than in Group A, at both 1 and 6 mo after vaccination (table);Nabs against the parental virus resulted to be greater in Group A than in Group B by a factor of 18 and 11, at 1 and 6 mo after vaccination, respectively. Both Groups recorded a decrease in antibody titers of approximately 50-70% between 1 and 6 months. A significant difference for Omicron NAbs (p=0.02) and anti-S-RBD (p=0.07) titers decay was observed between Group A and B;in contrast, Parental NAbs titers appeared to have similar trends in the 2 groups (p=0.47). Comparable antibody titers at 1 and 6 mo. (p=0.37) were detected across the three categories of HC, IC, and MIS-C (table). Conclusion(s): mRNA vaccination triggers a higher humoral response in children with a previous history of COVID-19, regardless of the immune deficiency or previous MIS-C, at least up to 6 mo, providing insight into boosting preexisting immunity with mRNA vaccines.
ABSTRACT
Introduction: SARS-CoV-2 immune-response is mediated by both humoral and cellular immunity. However, since Ab levels wane faster than SARS-CoV-2 specific T cells immunity, cellular immunity represents an important factor for COVID-19 immune defence. Determining immunoreactivity of SARS-CoV-2 specific T cells is of clinical relevance in transplant recipients or patients treated with immunomodulant therapy. SARS-CoV-2 specific T cells assays are currently based on ELISA, whilst rapid tests are pivotal for real-time patients' evaluation. In this study, a novel direct real-time PCR (dRT-PCR) targeting mRNA of CXCL10 for measuring SARS-CoV-2 specific T cells, was tested and evaluated. Method(s): A total of 104 healthcare workers, with two or three doses of homologous (Pfizer/BioNTech, n = 82) or heterologous (Pfizer/BioNTech and Vaxzevria or Moderna, n = 22) vaccinations were asked to collect a blood (Li-He) sample. Blood was stimulated overnight with SARS-CoV-2 spike peptides (S-peptide) or treated with non-stimulating substance. Stimulated/treated samples were diluted in Buffer A, mixed with dqTACT MS then loaded into the cartridge. The analysis was performed using SCV2 T Activation kit, bCube and bApp (Hyris srl, Lodi, Italy), equipped by an automatic result interpretation based on artificial intelligence. For a subgroup of 49 samples, IFN-y releases to SARS-CoV-2 spike peptides were tested by Quant-T-Cell SARS-CoV-2 and ELISA (Euroimmune, Lubeck, Germany). Result(s): Seventy-nine (75.9%) and 25 (24.1%) were females and males, respectively. Twenty-nine subjects were previously infected by SARS-CoV-2. Overall mean age (+/- SD) was 45.9+/-13.3 years. At qualitative analyses, 97 subjects (93.2%) resulted reactive to S-peptides, 3 (2.8%) were borderline and 4 were negative (3.8%). These negatives had their third vaccinal dose in December/November 2021. Previous infected individuals presented reactivity to S-peptides, with the exception of one subject with resulted reactive also in the untreated sample. Samples tested with both dRT-PCR and ELISA perfectly agreed (100%) with both methods. At quantitative analyses, between-assay correlation was 0.32 (p<0.001). Conclusion(s): Hyris dRT-PCR appeared accurate for determining presence or absence of immunoreactivity of SARS-CoV-2 specific T cell, especially when rapid analyses are required, such as for organ transplantation.
ABSTRACT
Background: The Severe Acute Respiratory Syndrome Coronavirus 2 related (SARS-CoV-2) has rapidly spread originating into the fifth documented pandemic wave. Dried blood spot (DBS) provides an alternative method to the venous blood samples to determine Ab, presenting several advantages, including the practicability, especially in infants, and the possibility to test a higher number of samples in a limited time. The main goal of this study was to measure seroprevalence of anti-SARS-CoV-2 IgG Ab in newborns, using DBS collected from January 2020 to December 2021. Method(s): Anti-SARS-CoV-2 IgG levels were determined using DBS by Anti-SARS-CoV-2 QuantiVac IgG ELISA assay (Euroimmune, Lubeck, Germany). Result(s): Preliminary analyses included 515 DBS from newborns, 54% males and 46% females, collected 2-3 days after birth. Overall, mean IgG levels, although below the positive threshold (>= 35.2 kBAU/L), were significantly higher in 2021 (Feb/21 and Mar/21), with respect to 2020 (Feb/20: 8.6+/-3.5, n=99 vs Feb/21: 28.1+/-42.8, n=40;Mar/20: 11.6+/-5.1, n=94 -Mar/21: 16.8+/-11.6, n=39, mean kBAU/L +/- DS, p<0.005). Moreover, an increased number of positive samples were found in 2021 (4/44 Gen/21;8/40 Feb/21;2/39 Mar/21). As expected, a trend of increase around 5% in DBS tested positive for anti-SARSCoV-2 IgG were found in December 2021, where IgG were above the positive threshold in 41.54% of DBS (450.926+/-873.291, mean kBAU/L +/- DS, n=65). Conclusion(s): In these preliminary data, newborn DBS seems to reflect population-wide infection rates during the studied periods. This suggests a potential role for DBS in COVID-19 surveillance, especially in infants and in areas where viral testing is limited.
ABSTRACT
Introduction: external Quality Assessment (EQA) is a valuable tool to monitor and improve the analytical performances of clinical laboratories. To guarantee suitable results also during the COVID-19 pandemic, EQA scheme providers have implemented specific schemes assessing different SARS-CoV-2 diagnostic systems. This study aims to describe the results collected in an experimental EQA scheme for molecular diagnostic of SARS-CoV-2 managed by INSTAND eV with the collaboration of the Centre of Biomedical Research for Quality in Laboratory Medicine for Veneto Region Laboratories. Methods: the qualitative EQA results collected in three surveys (two in 2020 and one in 2021) for 18 samples total, have been summarized to identify the percentage of laboratory results per sample. Control samples were provided by NationalesKonsiliarlaboratorium fur Coronaviren of Berlin. Results: even though the average of the participating laboratories strongly decreased between surveys, a good agreement was found among results (95% to 99.8%). A totally of 0.2% - 4% of incorrect results and 0% - 1.1% of indeterminate results were reported. In addition, the sequencing analysis and the point mutations analysis, included in the last analyzed survey, revealed a good agreement between participating laboratories with an overall score from 74.8% to 89.6% for the sequencing and from 90.65% to 95.33% for the point mutations, respectively. Conclusions: the EQA programs are a fundamental quality assurance tool to evaluate the laboratory performance and to appreciate the State-of-the-Art of the different diagnostic systems used by participating laboratories. The need for an EQA scheme for every test performed in the laboratory is mandatory to guarantee patient safety.
ABSTRACT
Background: Understanding the long-term kinetics of the immune response against SARS-CoV-2 infection is crucial in guiding public health policies and optimizing of vaccination strategies. While it is known that SARS-CoV-2 specific antibodies may persist in adults 12 months after infection, data are lacking in the pediatric population. We herein describe the long-term immune response in children following SARS-CoV-2 infection. Methods: Single-centre, prospective observational study analyzing family clusters of COVID-19 attending the Pediatric Department, University of Padua (Italy). Confirmed COVID-19 infection was defined by positive SARS-CoV-2 PCR and/or IgG serology. All patients with confirmed infection at enrolment underwent serological follow-up at 1-4, 5-10, and >10 months after infection. Plasma was analyzed to quantify anti-SARS-CoV-2 S-RBD IgG, by chemiluminescent immunoassay, performed on MAGLUMI™2000 Plus (Snibe Diagnostics). IgG title >4.3 kBAU/L was considered positive. Results: Among 902 subjects (252 COVID-19 family clusters), 698 had confirmed COVID-19, including 352 children/older siblings aged 8.6 ±5.1 years, and 346 parents aged 42.5 ±7.1 years;of those, 96.5% cases had asymptomatic/mild COVID-19. Children showed significantly higher S-RBD IgG titers than older subjects across all follow-up time points, with an overall mean S-RBD IgG titer <3 years of age five-fold higher than adults (282.3 [139-516.6] kBAU/L vs 56.7 [24.6-136.9] kBAU/L, p<0.001) (Table). The longitudinal analysis of 60 subjects sampled at least twice during follow-up demonstrated the persistence of antibodies up to 10 months from infection in all age classes. Subjects >6 years of age showed a significant progressive decline of the S-RBD IgG titer from the first serological follow-up. While, in younger children antibodies remained stable at 5-10 months of follow-up (p=0.0625), with a subsequent significant decline afterwards (p<0.001). Conclusion: In our unique family cluster cohort, we confirmed the different kinetics of the COVID-19 humoral response across several age groups of asymptomatic/mild COVID-19 cases in our family-cluster cohort. Children presented with higher S-RBD IgG titer at every time point up to 10 months of follow-up. Children less than 3 years demonstrated a more intense long-term resilience of their immune response, which started to decline significantly only after ten months from infection.
ABSTRACT
Although reverse real-time PCR (rRT-PCR) remains the gold standard for detecting SARS-CoV-2, high tests demanding has overwhelmed molecular laboratory capacities in all countries around the world, especially during early pandemics. During the second wave, the validation of SARS-CoV-2 antigen rapid diagnostic tests (RDT) has substantially changed testing strategies globally, since results were available within 30 min, reducing turnaround time and therefore exposure risk. Recently, validated self-tests for SARS-CoV-2 based on the nasopharyngeal swab (NPS) or saliva have prompted for the empowerment of the general population in the fight against the spread of infectious. Swabbing is a complex task requiring training and competency assessment, and thus they are performed by trained nurses or physicians. The complexity of NPS, coupled with a lack of a standard swabbing practice may contribute to a high number of false-negative results for SARS-CoV-2. SARS-CoV-2 rRT-PCR false-negative results have been reported to be as high as 41% and several reports exist of patients negative to NPS, who are subsequently positive on repeat testing [1]. Differently, the false-positive ratio for the SARS-CoV-2 molecular test is expected to be very low, since PCR design is mostly unaffected by false-positive results. Recently, Tsang et al. compared the diagnostic performance of different clinical specimens, including nasopharyngeal, nasal, throat, and oropharyngeal swabs and saliva and they found that using NPS as the gold standard, moderate sensitivities were achieved by saliva (85%, 75-93) and nasal swabs (86%, 77- 93) and a much lower sensitivity by throat swabs (68%, 35- 94). The Authors concluded that saliva and nasal swabs are clinically acceptable alternatives to commonly used nasopharyngeal swabs. Saliva is a matrix elective for selfcollection, and molecular testing is reliable but require laboratory instrumentation to be performed. Indeed, antigen determination on salivary samples is still under debate [2]. Most of the errors occur in the preanalytical phase, with relatively few analytical and post-analytical errors. Some issues arising during the pre-analytical phase of SARS-CoV- 2 diagnostics regards: the time of swab, swabbing practice, sample handling and conservation and RNA extraction. NPS should be taken at the time of symptom onset when the highest viral load occurs in COVID-19, thus not the day immediately before (and not too far from) possible close contact with positive subjects. Sample handling and storage were only partially a limiting factor when samples are kept a 4 °C and processed within 5 days [2]. Differently, sample preparation is a crucial factor for antigen testing, and centrifuged vs non-centrifuged samples give discordant results. In conclusion, self-testing could be of aid in the screening programs for reducing viral spread, but other alternatives are possible, such as self-collection of samples with analytical tests performed in clinical laboratories. These required the optimization of pre-analytical steps to reduce the impact on results.
ABSTRACT
Background: External Quality Assessment (EQA) is a valuable tool to monitor and improve the analytical performances of clinical laboratories. During the COVID-19 pandemic, the number of kits to detect the infection and the number of tested samples intensified to satisfy the test request. To guarantee suitable results, EQA scheme providers have implemented specific schemes assessing different SARS-CoV-2 diagnostic systems. This study aims to describe the results collected in an experimental EQA scheme for molecular diagnostic of SARS-CoV-2 managed by INSTAND e.V with the collaboration of the Centre of Biomedical Research for Quality in Laboratory Medicine for Veneto Region Laboratories. Methods: The qualitative EQA results collected, two surveys in 2020 and one in 2021, for 18 samples total, have been summarized to identify the percentage of laboratory results per sample. Control samples included SARS-CoV-2 or other seasonal coronaviruses (MERSCoV, HCoV 229E, HCoV OC43) provided by Nationales Konsiliarlaboratorium fur Coronaviren of Berlin. SARSCoV-2 Variants of Concern (VOCs) were included only in the 2021 survey. Results: The average of the participating laboratories strongly decreased between the first survey of 2020 (927) and the last analysed survey, March 2021 (594). The main analytical procedures used, in the first, second and third survey respectively were CEPHAID kits (11.6%, 12% and 11.7%), in-house produced assays (10.4, 6.2 and 5%), SEEGENE kits (8.5%, 8.1% and 7.9%), ROCHE Diagnostics (8.3%, 8.5% and 6.9%) and ALTONA Diagnostics kits (6.1, 6.2 and 4.5%). A good agreement was found among laboratories results, with an overall range from 95% to 99.8%. Furthermore, generally from 0.2% to 2.9% of incorrect results and 0% to 1.1% of indeterminate results were reported. Conclusions: The EQA programs are a fundamental quality assurance tool to evaluate the laboratory performance and know the State-of-the-Art diagnostic systems used by participating laboratories. The need for an EQA scheme for every test performed in the laboratory is mandatory to guarantee patient safety.
ABSTRACT
Background and Aim: Salivary SARS-CoV-2 Ab determination could be suitable for monitoring the viral spread and vaccination efficacy, especially in pediatric patients. We investigated N/S1-RBD IgG antibody levels in salivary samples of infectious-naïve vaccinated subjects and of COVID-19 patients, further comparing levels with serum anti-SARS-CoV-2 S-RBD IgG. Methods: A total of 72 subjects were enrolled at the Padova University Hospital: 36 COVID-19 patients and 36 health care workers (HCW), who underwent a complete vaccination campaign with BNT162b2 (BioNTech/Pfizer). All collected a salivary sample, using Salivette (Sarstedt, Nümbrecht Germany). For 9 HCW, salivary samples were collected at three different times within the same day (before breakfast, at 10 am, and after lunch). A serum sample was also collected for all individuals. Time post symptoms onset or time from the first vaccine were also recorded. Salivary COVID-19 N/S1 RBD (sal-IgG) ELISA (RayBiotech, GA, USA) and anti-SARS-CoV-2 S-RBD IgG Ab (ser-IgG) (Snibe Diagnostics, Shenzhen, China) were used for determining IgG Ab. Results: Subjects' mean age (±sd) was 35.8±18.2 yrs. Age significantly differed (p<0.001) from COVID-19 patients [29.7±17.3 yrs] and HCW [47.1±12.9 yrs]. Positive sal-IgG were found in 70/72 (97.2%) samples;in sera, 71/72 (98.6%) samples were positive to ser-IgG. The sal-IgG median levels differed from COVID-19 to vaccinated HCW, being in salivary samples 0.21 kAU/L and 0.8 kAU/L (p =0.030), respectively;median levels for ser-IgG in COVID-19 and vaccinated HCW were 135 kBAU/L and 940 kBAU/L, respectively (p<0.001). Salivary IgG levels were not influenced by time post-symptom onset or time post-vaccination, both on vaccinated HCW (rho= -0.147, p=0.402) and COVID-19 subjects (rho=0.0267, p=0.986). Ser-IgG levels was not influenced by the time post-symptom onset for COVID-19 subjects (rho=0.102, p=0.419), while a strong significant correlation was found with time post-vaccination in HCW (rho=-0.6292, p<0.001). Sal-IgG levels were notinfluenced by the daytime of collection (rho=0.148, p=0.373). Passing-Bablok regressions showed that sar- IgG and ser-IgG comparability was assessable only when ser-IgG values were divided by 1000, being slope and intercept 0.068 (95%CI: 0.069-0.341) and 0.221 (95%CI:- 0.097 to 0.786), respectively. Conclusions: Salivary IgG is efficiently detectable both in COVID-19 and in vaccinated individuals and analyses appeared to be not influenced by the daytime of collection. The analyses performed showed that, overall, sal-IgG were lower than ser-IgG, and thus comparability with serum levels needs to be better explored.