Nasopharyngeal Microbiota Profiling of Pregnant Women With Sars-Cov-2 Infection (preprint)

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.

Maternal outcomes and risk factors for COVID-19 severity among pregnant women.

Pregnant women may be at higher risk of severe complications associated with the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), which may lead to obstetrical complications. We performed a case control study comparing pregnant women with severe coronavirus disease 19 (cases) to pregnant women with a milder form (controls) enrolled in the COVI-Preg international registry cohort between March 24 and July 26, 2020. Risk factors for severity, obstetrical and immediate neonatal outcomes were assessed. A total of 926 pregnant women with a positive test for SARS-CoV-2 were included, among which 92 (9.9%) presented with severe COVID-19 disease. Risk factors for severe maternal outcomes were pulmonary comorbidities [aOR 4.3, 95% CI 1.9-9.5], hypertensive disorders [aOR 2.7, 95% CI 1.0-7.0] and diabetes [aOR2.2, 95% CI 1.1-4.5]. Pregnant women with severe maternal outcomes were at higher risk of caesarean section [70.7% (n = 53/75)], preterm delivery [62.7% (n = 32/51)] and newborns requiring admission to the neonatal intensive care unit [41.3% (n = 31/75)]. In this study, several risk factors for developing severe complications of SARS-CoV-2 infection among pregnant women were identified including pulmonary comorbidities, hypertensive disorders and diabetes. Obstetrical and neonatal outcomes appear to be influenced by the severity of maternal disease.

Antibody Conversion rates to SARS-CoV-2 in Saliva from Children Attending Summer Schools in Barcelona, Spain (preprint)

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.

Multiplex Antibody Analysis of IgM, IgA and IgG to SARS-CoV-2 in Saliva and Serum from Infected Children and their Close Contacts (preprint)

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.

Agreement between commercially available ELISA and in-house Luminex SARS-CoV-2 antibody immunoassays (preprint)

Serological diagnostic of the severe respiratory distress syndrome coronavirus 2 (SARS-CoV-2) is a valuable tool for the determination of immunity and surveillance of exposure to the virus. In the context of an ongoing pandemic, it is essential to externally validate widely used tests to assure correct diagnostics and epidemiological estimations. We evaluated the performance of the COVID-19 ELISA IgG and IgM/A (Vircell, S.L.) against a highly specific and sensitive in-house Luminex immunoassay in a set of samples from pregnant women and cord blood. The agreement between both assays was moderate to high for IgG but low for IgM/A. Considering seropositivity by either IgG and/or IgM/A, the technical performance of the ELISA was highly imbalanced, with 96% sensitivity at the expense of 22% specificity. As for the clinical performance, the negative predictive value reached 87% while the positive predictive value was 51%. Our results stress the need for highly specific and sensitive assays and external validation of diagnostic tests with different sets of samples to avoid the clinical, epidemiological and personal disturbances derived from serological misdiagnosis.

Maternal Outcomes and Risk Factors for Severity Among Pregnant Women With COVID-19: A Case Control Study From the COVI-Preg International Registry (preprint)

Background: Recent evidence suggests that pregnant women might be at higher risk of severe disease associated with the emerging pandemic severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), while exposed fetuses/newborns could suffer from preterm birth, growth restriction and neonatal infections. The magnitude of this increased risk and specific risk factors for severity remains unclear.Methods: We performed a case control study comparing pregnant women with severe coronavirus disease 19 (case) to pregnant women with a milder form (controls) enrolled in COVI-Preg international registry cohort between from March 24 to July 26, 2020. Risk factors for severity, obstetrical, fetal and neonatal outcomes were assessed.Findings: A total of 926 pregnant women with a positive test for SARS-CoV-2 were included, among which 92 (9.9%) presented a severe COVID-19 disease. Risk factors for severe maternal outcomes were pulmonary comorbidities [aOR 4.3, 95% CI 1.9-9.5], hypertensive disorders [aOR 2.7, 95% CI 1.0-7.0] and diabetes [aOR2.2, 95% CI 1.1-4.5]. Pregnant women with severe maternal outcomes were at higher risk of cesarean sections [70.7% (n=53/75)], preterm deliveries [62.7% (n= 32/51)] and newborns requiring admission to the neonatal intensive care unit [41.3% (n=31/75)].Interpretation: Pregnant women, particularly those with associated comorbidities, seem to be at higher risk of severe complications of SARS-CoV-2 infection. Obstetrical and neonatal outcomes appear to be influenced by the severity of maternal disease; complications include cesarean sections, prematurity and neonatal admission to the intensive care unit.Funding Statement: None.Declaration of Interests: The authors declare that they have no conflicts of interest.Ethics Approval Statement: The study was approved by both the Swiss Ethical Board (CER-VD- 2020-00548) and the local ethics boards at each participating center.

Validation and implementation of a direct RT-qPCR method for rapid screening of SARS-CoV-2 infection by using non-invasive saliva samples (preprint)

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.

Validation and Implementation of a Direct RT-qPCR Method for Rapid Screening of SARS-CoV-2 Infection by Using Non-Invasive Saliva Samples (preprint)

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).

Maternal and Obstetrical Outcomes in a Cohort of Pregnant Women Tested for SARS-CoV-2: Interim Results of the COVI-Preg International Registry (preprint)

Background: Pregnant women represent a vulnerable population at higher risk of complications of infectious diseases. Data regarding the consequences of the emerging pandemic severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) during pregnancy are scarce. Recent evidence suggests that pregnant women might be at higher risk of severe disease, while exposed fetuses and newborns could suffer from preterm birth, growth restriction and neonatal infections.Methods: We developed an international web registry to allow structured data collection. Pregnant women at any stage during gestation tested for SARS-CoV-2 infection were enrolled. Maternal, obstetrical and neonatal outcomes were recorded.Findings: 1033 pregnant women tested for SARS-CoV-2 were included, among which 926 tested positive and 107 tested negative. Positive pregnant women were at higher risk of severe maternal outcomes compared to negative women [aRR 5.6, 95% CI 1.4-22.7]. Risk factors for severe maternal outcomes among positive women were pulmonary comorbidities [aOR 4.3, 95% CI 1.9-9.5], hypertensive disorders [aOR 2.7, 95% CI 1.0-7.0] and diabetes [aOR2.2, 95% CI 1.1-4.5]. No difference in term of obstetrical and neonatal outcomes were observed between positive and negative women. Positive pregnant women with severe maternal outcomes were at higher risk of cesarean sections [70.7% (n=53/75)], preterm deliveries [62.7% (n= 32/51)] and newborns requiring admission to the neonatal intensive care unit [41.3% (n=31/75)]. A positive neonatal SARS-CoV-2 test was observed in 2.9% (n=11/384) of newborns with an available test at birth.Interpretation: Pregnant women, particularly those with associated comorbidities, seem to be at higher risk of severe complications of SARS-CoV-2 infection. Preliminary data regarding obstetrical and neonatal outcomes among women with a mild disease are reassuring.Funding Statement: None.Declaration of Interests: The authors declare that we have no conflicts of interest.Ethics Approval Statement: The study was approved by both the Swiss Ethical Board (CER-VD-2020-00548) and the local ethics boards at each participating center.

LOW BIRTH WEIGHT AS A RISK FACTOR FOR SEVERE COVID-19 IN ADULTS (preprint)

The identification of factors predisposing to severe COVID-19 in young adults remains partially characterized. Low birth weight (LBW) alters cardiovascular and lung development and predisposes to adult disease. We hypothesized that LBW is a risk factor for severe COVID-19 in non-elderly subjects. We analyzed a prospective cohort of 397 patients (18-70y) with laboratory-confirmed SARS-CoV-2 infection attended in a tertiary hospital, where 15% required admission to Intensive Care Unit (ICU). Perinatal and current potentially predictive variables were obtained from all patients and LBW was defined as birth weight [≤]2,500 g. Age (adjusted OR (aOR) 1.04 [1-1.07], P=0.012), male sex (aOR 3.39 [1.72-6.67], P<0.001), hypertension (aOR 3.37 [1.69-6.72], P=0.001), and LBW (aOR 3.61 [1.55-8.43], P=0.003) independently predicted admission to ICU. The area under the receiver-operating characteristics curve (AUC) of this model was 0.79 [95% CI, 0.74-0.85], with positive and negative predictive values of 29.1% and 97.6% respectively. Results were reproduced in an independent cohort, from a web-based survey in 1,822 subjects who self-reported laboratory-positive SARS-CoV-2 infection, where 46 patients (2.5%) needed ICU admission (AUC 0.74 [95% CI 0.68-0.81]). LBW seems to be an independent risk factor for severe COVID-19 in non-elderly adults and might improve the performance of risk stratification algorithms.

COVID-19 causing HELLP-like syndrome in pregnancy and role of angiogenic factors for differential diagnosis (preprint)

Importance: The clinical presentation of hemolysis, elevated liver enzymes, and low platelet count (HELLP) syndrome is one of the more severe forms of preeclampsia. COVID-19 infection exhibits signs that are shared with preeclampsia and HELLP syndrome, which may lead to needless interventions and iatrogenic preterm delivery. Objective: We evaluated the prevalence of HELLP-like signs in pregnant women admitted for COVID-19 and the value of angiogenic factors to rule out preeclampsia. Methods: a consecutive series of 27 pregnant women beyond 20 weeks of gestation, with symptomatic COVID-19. Clinical and analytical features were recorded and those cases with signs of HELLP syndrome were tested for sFlt-1/PlGF ratio. Results: Seven patients (25.9%) presented at least one sign of suspected HELLP syndrome, of which 2 (7.4%) were diagnosed clinically with PE because of hypertension and high transaminases and 5 (18.5%) had only elevated transaminases. sFlt-1/PlGF ratio was normal in 6 of 7. Conclusion: Symptomatic COVID-19 may simulate severe preeclampsia in pregnancy. Angiogenic factors may be essential to avoid false diagnosis and needless interventions. These data were presented in a Virtual Symposium on Covid-19 and Pregnancy on 17 April: 2020:(http://medicinafetalbarcelona.org/simposiocovid19/ [Spanish] and https://medicinafetalbarcelona.org/symposiumcovid19/ [English]

SEROPREVALENCE AND CLINICAL SPECTRUM OF SARS-CoV-2 INFECTION IN THE FIRST VERSUS THIRD TRIMESTER OF PREGNANCY (preprint)

Introduction: Case registries of pregnant women diagnosed with coronavirus disease (COVID-19) by polymerase chain reaction (PCR) have reported that the majority experienced mild infection, but up to 9% may require critical care. Most COVID-19 cases published were in the third trimester of pregnancy, which could reflect reporting bias, higher risk of infection or increased disease severity in late pregnancy. Seroprevalence studies may allow reliable estimates of the susceptibility to infection and clinical spectrum since they include asymptomatic and mild infections not tested for PCR. We evaluated the seroprevalence and clinical presentation of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection in pregnant women in the first and third trimester. Methods: The study was approved by the Institutional Review Board at each institution and informed consent was obtained. We recruited 874 consecutive pregnancies attending for first trimester screening (10-16 weeks of gestation, n=372) or delivery (n=502) from April 14 to May 5. All women were interviewed with a structured questionnaire for COVID-19 symptoms two months prior to sampling. SARS-CoV-2 IgG and IgM/IgA antibodies were tested (COVID-19 VIRCLIA Monotest, Vircell Microbiologist, Spain; reported sensitivity 70% IgG and 89% IgM/IgA, and specificity 89% and 99% respectively). Indeterminate results were re-tested (VITROS Immunodiagnostic Products Anti-SARS-CoV2 Total Tests, Ortho Clinical Diagnostics, USA; 100% sensitivity and specificity) and re-classified as positive or negative. Women with COVID-19 were diagnosed and managed according to standard protocols and guidelines3,4. Statistical differences were tested using the {chi}2 test or Student t-test as appropriate (p<0.05). Results: A total of 125 of 874 women (14.3%) were positive for either IgG or IgM/IgA SARS-CoV-2 antibodies, 54/372 (14.5%) in the first and 71/502 (14.1%) in the third trimester. A total of 75/125 (60%) reported no symptoms of COVID-19 in the past 2 months, whereas 44 (35.2%) reported one or more symptoms, of which 31 (24.8%) had at least 3 symptoms or anosmia and 8 (6.4%) dyspnea. Overall, 7 women (5.6%) were admitted for persistent fever despite paracetamol and dyspnea, of which 3 had signs of pneumonia on chest radiography. All 3 had criteria for severity (bilateral chest condensation, respiratory rate>30 and leukopenia) and required oxygen support but not critical care or mechanical ventilation, and they were all discharged well. The rates of symptomatic infection, hospital admission or dyspnea were significantly higher in third trimester women (Table and Figure). Discussion: The 14.3% seroprevalence of SARS-COV-2 in pregnant women in this study was substantially larger than the contemporary rates of PCR positive cases (0.78%) reported for women 20-40y in Barcelona. The data confirm that COVID-19 is asymptomatic in the majority of pregnant women6 and illustrate the value of seroprevalence studies to capture the high proportion of asymptomatic or mild infections. In this study, none of the 125 pregnant women with SARS-CoV-2 infection required critical care as compared to 9% reported in cases diagnosed with PCR. However, the proportion of infections with symptoms or dyspnea was remarkably higher in the third trimester, and these results are in line with COVID-19 registries, reporting that 81% of hospitalized women were in late pregnancy or peripartum. These results provide reassuring information that, even in settings with a high prevalence, SARS-CoV-2 infection in pregnancy mostly presents with asymptomatic or mild clinical forms. The susceptibility to infection seemed to be the same in the first and the third trimesters of gestation. The data further suggest that, as with other respiratory viruses, COVID-19 could be more severe and require increased surveillance in late pregnancy. These findings should be confirmed and extended with larger consecutive prevalence studies in pregnancy.