ABSTRACT
Background: We aimed to characterize the role of the nasopharyngeal microbiota in pregnant women with and without SARS-CoV-2 infection.Methods: Pregnant women were enrolled from a multicenter prospective population-based cohort (March-June 2020 in Barcelona, Spain) in which the status of SARS-CoV-2 infection was determined by nasopharyngeal RT–PCR and antibodies in peripheral blood. DNA was extracted from nasopharyngeal swab samples, and the V3-V4 region of the 16S rRNA of bacteria was amplified using region-specific primers. The differential abundance of taxa was tested, and alpha/beta diversity was evaluated.Results: Among 76 women, 38 were classified as positive and 38 as negative for SARS-CoV-2 infection. All positive women were diagnosed by antibodies, and 14 (37%) also had a positive RT–PCR. SARS-CoV-2 infection altered the overall composition of the nasopharyngeal microbiota (p=0.001), with a higher relative abundance of the Tenericutes and Bacteroidetes phyla and a higher abundance of the Prevotellaceae family. Infected women presented a different pattern of microbiota profiling due to beta diversity and higher richness (observed ASV<0.001) and evenness (Shannon index<0.001) at alpha diversity. These changes persisted after acute infection, as revealed by negative RT–PCR but positive antibodies, suggesting a long-lasting effect of SARS-CoV-2 in the nasopharyngeal microbiota. No significant differences were reported in mild vs. severe cases, suggesting a role of the SARS-CoV-2 infection itself but not on its severity.Conclusion: This is the first study on nasopharyngeal microbiota during pregnancy. SARS-CoV-2 infection altered the overall structure and diversity of the nasopharyngeal microbiota profile, and this effect seems to persist after the acute moment of the infection.
Subject(s)
COVID-19ABSTRACT
Surveillance tools to estimate infection rates in young populations are essential to guide recommendations for school reopening and management during viral epidemics. Ideally, field-deployable non-invasive, sensitive techniques are required to detect low viral load exposures among asymptomatic children. We determined SARS-CoV-2 antibody conversion by high-throughput Luminex assays in saliva samples collected weekly in 1,509 children and 396 adults in 22 Summer schools and 2 pre-schools in 27 venues in Barcelona, Spain, from June 29th to July 31st 2020, between the first and second COVID-19 pandemic waves. Saliva antibody conversion defined as [≥]4-fold increase in IgM, IgA and/or IgG levels to SARS-CoV-2 antigens between two visits over a 5-week period was 3.22% (49/1518), or 2.36% if accounting for potentially cross-reactive antibodies, six times higher than the cumulative infection rate (0.53%) by weekly saliva RT-PCR screening. IgG conversion was higher in adults (2.94%, 11/374) than children (1.31%, 15/1144) (p=0.035), IgG and IgA levels moderately increased with age, and antibodies were higher in females. Most antibody converters increased both IgG and IgA antibodies but some augmented either IgG or IgA, with a faster decay over time for IgA than IgG. Nucleocapsid rather than spike was the main antigen target. Anti-spike antibodies were significantly higher in individuals not reporting symptoms than symptomatic individuals, suggesting a protective role against COVID-19. To conclude, saliva antibody profiling including three isotypes and multiplexing antigens is a useful and more user-friendly tool for screening pediatric populations to determine SARS-CoV-2 exposure and guide public health policies during pandemics.
Subject(s)
COVID-19ABSTRACT
COVID-19 affects children to a lesser extent than adults but they can still get infected and transmit SARS-CoV-2 to their contacts. Field deployable non-invasive sensitive diagnostic techniques are needed to evaluate the infectivity dynamics of the coronavirus in pediatric populations and guide public health interventions. We evaluated the utility of high-throughput Luminex-based assays applied to saliva samples to quantify IgM, IgA and IgG antibodies against five SARS-CoV-2 spike (S) and nucleocapsid (N) antigens in the context of a contacts and infectivity longitudinal study. We compared the antibody levels obtained in saliva versus serum/plasma samples from a group of children and adults tested weekly by RT-PCR over 35 days and diagnosed as positive (n=58), and a group of children and adults who consistently tested negative over the follow up period (n=61), in the Summer of 2020 in Barcelona, Spain. Antibody levels in saliva samples from individuals with confirmed RT-PCR diagnosis of SARS-CoV-2 infection were significantly higher than in negative individuals and correlated with those measured in sera/plasmas. Higher levels of anti-S IgG were found in asymptomatic individuals that could indicate protection against disease in infected individuals. Higher anti-S IgG and IgM levels in serum/plasma and saliva, respectively, in infected children compared to infected adults could also be related to stronger clinical immunity in them. Among infected children, males had higher levels of saliva IgG to N and RBD than females. Despite overall correlation, individual clustering analysis suggested that responses that may not be detected in blood could be patent in saliva, and vice versa, and therefore that both measurements are complementary. In addition to serum/plasma, measurement of SARS-CoV-2-specific saliva antibodies should be considered as a complementary non-invasive assay to better estimate the percentage of individuals who have experienced coronavirus infection. Saliva antibody detection could allow determining COVID-19 prevalence in pediatric populations, alternative to bleeding or nasal swab, and serological diagnosis following vaccination.
Subject(s)
COVID-19ABSTRACT
Objective To validate and implement an optimized screening method for detection of SARS-CoV-2 RNA combining use of self-collected raw saliva samples, single-step heat-treated virus inactivation and RNA extraction, and direct RT-qPCR. Design Study conducted in three successive phases including: i) method analytical validation against standard RT-qPCR in saliva samples; ii), method diagnostic validation against standard RT-qPCR in nasopharyngeal samples; and iii), method implementation through pilot screening in a reference hospital. Setting Sant Joan de Deu University Hospital (Barcelona, Spain). Participants Phase 2, a prospective cohort of asymptomatic teenagers and young adult players and staff in a youth sports academy followed up during 9 to 12 weeks; Phase 3, asymptomatic health workers, students, aid volunteers, and other staff of the setting. Main outcome measures Method diagnostic sensitivity and specificity. Method performance in a pilot screening. Results Diagnostic validation included 173 participants. At week 0, all saliva and nasopharyngeal samples were negative. In the following weeks, standard RT-qPCR yielded 23 positive results in nasopharyngeal samples. Paired saliva specimens yielded 22 positive and one inconclusive result. Method diagnostic sensitivity and specificity values were 95.7% (95% CI, 79.0-99.2%) and 100.0% (95% CI, 98.6-100.0 %), respectively. A total of 2,709 participants engaged in the pilot screening, with a high rate of participation (83.4% among health workers). Only 17 (0.6%) of saliva samples self-collected by participants in an unsupervised manner were invalid. Saliva was positive in 24 (0.9%) out of 2,692 valid specimens and inconclusive in 27 (1.0%). All 24 saliva-positive and 4 saliva-inconclusive participants were positive by standard RT-PCR in nasopharyngeal samples. Use of a high throughput system allowed fast screening workflow (up to 384 samples in <2 hours). Conclusion Direct RT-qPCT on self-collected raw saliva is a simple, rapid, and accurate method with potential to be scaled up for enhanced SARS-CoV-2 community-wide screening.
Subject(s)
COVID-19ABSTRACT
Background: There is an urgent need to curb COVID-19 pandemic through early identification of asymptomatic but infectious cases. We aimed to validate and implement an optimized screening method for detection of SARS-CoV-2 RNA combining use of self-collected raw saliva samples, single-step heat-treated virus inactivation and RNA extraction, and direct RT-qPCR.Methods: The study was conducted in Sant Joan de Déu University Hospital (Barcelona, Spain), including: i) method analytical validation against standard RT-qPCR in saliva samples; ii) method diagnostic validation against standard RT-qPCR in nasopharyngeal samples using a prospective cohort of asymptomatic teenagers and young adult players and staff in a youth sports academy; and iii) method implementation through pilot screening of asymptomatic health workers, students, and aid volunteers in the study site.Findings: The direct method had comparable performance to standard RT-qPCR in saliva and high intra- and inter-assay precision. Diagnostic validation included saliva and nasopharyngeal samples serially obtained from 173 participants during 9-12 weeks. At week 0, all saliva and nasopharyngeal samples were negative. In the following weeks, standard RT-qPCR yielded 23 positive results in nasopharyngeal samples. Paired saliva specimens yielded 22 (95·7%) positive and one inconclusive result. A total of 2,709 participants engaged in the pilot screening, with a high rate of participation (83·4% among health workers). Only 17 (0·6%) of saliva samples self-collected by participants were invalid. Saliva was positive in 24 (0·9%) out of 2,692 valid specimens and inconclusive in 27 (1·0%). All 24 saliva-positive and 4 saliva-inconclusive participants were positive by standard RT-PCR in nasopharyngeal samples. Use of a high throughput system allowed fast screening workflow (up to 384 samples in <2 hours).Interpretation: Direct RT-qPCT on self-collected raw saliva is a simple, rapid, and accurate method with potential to be scaled up for enhanced SARS-CoV-2 community-wide screening.Funding:This work was supported by the Kids Corona Project promoted by SJDH, which received donations from Stavros Niarchos Foundation and Banco Santander.Declaration of Interests: CMA reports past grants to her organization from BioMèrieux, Roche Diagnostics, Qiagen, BioFire Diagnostics, Alere, and Genomica, outside the submitted work and personal fees from BioMèrieux, Roche Diagnostics, and Qiagen for presentations in satellite symposiums outside the submitted work. PB reports personal fees from Roche Diagnostics for a presentation in a satellite symposium outside the submitted work. The rest of authors declare no conflicts of interest.Ethics Approval Statement: The study was approved by the Ethics Commitee of SJDH prior to the beginning of activities (ref. PIC-240-20). Use of samples collected from participants in the “Kids Corona Study of SARS-CoV-2 transmission at Football Club Barcelona Academy “La Masia” for the present and future studies was covered in the informed consent process and approval of that study (ref. PIC-200-20).