key: cord-0910112-yjtjmeis
authors: Milbak, Julie; Holten, Victoria M. F.; Axelsson, Paul Bryde; Bendix, Jane Marie; Aabakke, Anna J. M.; Nielsen, Lene; Friis, Martin Barfred; Jensen, Claus A. J.; Løkkegaard, Ellen Christine Leth; Olsen, Tina Elisabeth; Rode, Line; Clausen, Tine Dalsgaard
title: A prospective cohort study of confirmed severe acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2) infection during pregnancy evaluating SARS‐CoV‐2 antibodies in maternal and umbilical cord blood and SARS‐CoV‐2 in vaginal swabs
date: 2021-11-01
journal: Acta Obstet Gynecol Scand
DOI: 10.1111/aogs.14274
sha: 06417e341ee1a97d6e4a624e098cb7be53c1915c
doc_id: 910112
cord_uid: yjtjmeis

INTRODUCTION: Evidence about the consequences of severe acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2) infection in pregnancy is rapidly increasing; however, data on antibody response and risk of transmission during pregnancy and delivery are still limited. The aim of this study was to evaluate if SARS‐CoV‐2 is detectable in vaginal swabs and whether antibodies against SARS‐CoV‐2 are present in maternal and umbilical cord blood of pregnant women with confirmed SARS‐CoV‐2. MATERIAL AND METHODS: A single‐unit prospective cohort study in Denmark including pregnant women with SARS‐CoV‐2 infection confirmed by a pharyngeal swab between August 20, 2020, and March 1, 2021, who gave birth during the same period. All patients admitted to the maternity ward and antepartum clinic were screened for SARS‐CoV‐2 infection. A maternal blood sample and vaginal swabs were collected at inclusion. If included antepartum, these samples were repeated intrapartum when an umbilical cord blood sample was also collected. Swabs were analyzed for SARS‐CoV‐2 and blood samples were analyzed for SARS‐CoV‐2 total antibodies. Placental and neonatal swabs as well as placental histopathological examinations were performed on clinical indications. RESULTS: We included 28 women, of whom four had serious maternal or fetal outcomes including one case of neonatal death. Within the first 8 days after confirmed SARS‐CoV‐2 infection, SARS‐CoV‐2 was detectable in two vaginal swabs (2/28) and SARS‐CoV‐2 antibodies were detected in 1 of 13 women. From 16 days after confirmed infection, antibodies were observed in 19 of 21 of women. Antibodies in cord blood were not detected during the first 16 days after confirmed infection (n = 7). However, from 26 days, antibodies were present in 16 of 17 cord blood samples of seropositive mothers. Placental examination in two cases of severe fetal outcomes preceded by reduced fetal movements revealed SARS‐CoV‐2 in swabs and severe histopathological abnormalities. CONCLUSIONS: SARS‐CoV‐2 was detected in only 2 of 28 vaginal swabs within 8 days after confirmed infection in pregnant women. Our data suggest that maternal seroconversion occurs between days 8 and 16, whereas antibodies in cord blood of seropositive mothers were present in the majority from 26 days after confirmed infection. Additional data are needed regarding timing of seroconversion for the mother and appearance of antibodies in cord blood.

Whether transmission of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) from mother to child can occur during maternal infection in pregnancy is still unclear. 1 The presence of antibodies in fetal blood might offer protection of the fetus and child against coronavirus disease 2019 (COVID- 19) during pregnancy, vaginal delivery, and in the neonatal period. One study showed that the SARS-CoV-2 antibody titer remained stable from infection in the first trimester until delivery. 8 Other studies have examined the presence of SARS-CoV-2 antibodies at delivery in blood samples from the umbilical cord or the offspring of mothers with COVID-19. Some found anti-SARS-CoV-2 immunoglobulin G (IgG), others found both immunoglobulin M (IgM) and IgG. 3, 9, 10 As IgG is the only antibody class that significantly crosses the placenta, findings of IgM in cord blood indicate in utero infection, whereas IgM in neonates could be caused by either in utero infection or postnatal infection. Additionally, the time span between maternal confirmed SARS-CoV-2 and the presence of antibodies in umbilical cord has been explored. Findings were ambiguous, as in one case IgG was found in cord blood already 1 day after expected maternal infection, which is an unusually fast IgG response. 3 Overall, previous studies may indicate that the risk of mother-to-child transmission is increased if the woman delivers less than 1 week from onset of infection.

The objective of this study was to investigate if SARS-CoV-2 is detectable in vaginal swabs of pregnant women with confirmed SARS-CoV-2 infection and to study the presence and timing of SARS-CoV-2 antibodies in maternal and umbilical cord blood.

We conducted a prospective cohort study entitled the "CareMum COVID-19 study" from August 20, 2020 to March 1, 2021 at the

Hospital-North Zealand. The hospital has approximately 4000 annual deliveries.

We included pregnant women with confirmed SARS-CoV-2 infection based on a pharyngeal swab positive for SARS-CoV-2. The women had been tested at a public or private test center or at a routine test when entering the hospital for admission. During the

The Research Foundation at North Zealand Hospital, Hillerod and Ferring Pharmaceuticals funded the study. The Department of Clinical Biochemistry at North Zealand Hospital, Hillerod and Rigshospitalet Glostrup covered the expenses for analysis of blood samples. SARS-CoV-2 infection, SARS-CoV-2 was detectable in two vaginal swabs (2/28) and SARS-CoV-2 antibodies were detected in 1 of 13 women. From 16 days after confirmed infection, antibodies were observed in 19 of 21 of women. Antibodies in cord blood were not detected during the first 16 days after confirmed infection (n = 7).

However, from 26 days, antibodies were present in 16 of 17 cord blood samples of seropositive mothers. Placental examination in two cases of severe fetal outcomes preceded by reduced fetal movements revealed SARS-CoV-2 in swabs and severe histopathological abnormalities.

Conclusions: SARS-CoV-2 was detected in only 2 of 28 vaginal swabs within 8 days after confirmed infection in pregnant women. Our data suggest that maternal seroconversion occurs between days 8 and 16, whereas antibodies in cord blood of seropositive mothers were present in the majority from 26 days after confirmed infection.

Additional data are needed regarding timing of seroconversion for the mother and appearance of antibodies in cord blood. do not yet protect the neonate. Data are needed regarding seroconversion for the mother and appearance of antibodies in cord blood. study period, the hospital had a protocol for universal screening of all patients admitted to the maternity ward or the antepartum clinic regardless of symptoms. In case of an elective cesarean section the swab was performed the day before delivery. Women were eligible if they were to deliver within the study period and were able to give written and oral informed consent in English or Danish.

Inclusion took place either antepartum at the antenatal clinic or intrapartum when women were admitted for delivery. Vaginal swabs and maternal blood samples were collected from all participants at the time of inclusion. If included antepartum, the samples were repeated intrapartum, when an umbilical cord blood sample was also taken ( Figure 1 ). Intrapartum, two vaginal swabs were taken-the first during the initial vaginal examination and the second during the active phase of delivery or immediately after. Vaginal swabs were stored at −20℃ until analysis (0-30 days, median 5 days). The maternal blood sample was taken just before or immediately after delivery, and the umbilical cord blood sample was collected immediately after birth. Blood samples were stored at −80℃ until analysis.

In some cases, swabs from the neonate and the placenta, as well as a placental histopathological examination and one fetal autopsy were performed on clinical indications (Supporting Information   Table S1 ). Placental swabs were performed from both the maternal and the fetal side, and neonatal swabs from the axillary fold, nasopharynx, and oropharynx. These samples were not part of the study protocol and therefore not systematically sampled, but results are described when present. Pharyngeal swabs as well as vaginal and placental swabs were analyzed by reverse transcription polymerase chain reaction (RT-PCR) as part of the routine diagnostics (see Supporting Information Appendix S1, for further information).

Total SARS-CoV-2 antibodies (including IgG and IgM) in maternal and cord blood samples were analyzed using a qualitative (reactive/ non-reactive) biochemical assay, the VITROS Immunodiagnostic Product Anti-SARS-CoV-2 Total (CoV2T), developed by Ortho-Clinical Diagnostics. A test result (S/C = signal for test sample/signal at cut-off value) of 1.00 or more is considered reactive (ie, positive for antibodies) and a result below 1.00 is considered non-reactive (ie negative for antibodies). The assay has been shown to have a specificity of more than 99% and a sensitivity of 95.3%. 11 We obtained demographic and clinical data of participants as well as non-participants in the "CareMum COVID-19 study" from the Danish "COVID-19 in pregnancy" database, which contains information based on medical records on all women with confirmed SARS-CoV-2 infection during pregnancy in Denmark, as described elsewhere. 12 Case completeness of the database was secured by a retrospective registry linkage to national databases containing information on positive SARS-CoV-2 tests. Furthermore, participants were asked to complete a questionnaire about COVID-19 symptoms at the time of inclusion.

Data were analyzed using SPSS version 27 (SPSS Inc.). Categorical variables are presented as number with percentage and continuous variables as mean with standard deviation or median with interquartile range as appropriate.

The Regional Committee on Health Research Ethics in the capital region of Denmark approved the study on May 3, 2020 with an additional protocol approved November 17, 2020 (H-20028002).

Written informed consent was collected from all participants.

Demographic and clinical data were extracted from the prospective national observational study "COVID-19 in pregnancy", approved by the Danish Patient Safety Authority on April 24, 2020 (reg. no.

Zealand on March 23, 2020 (reg. no. REG022-2020).

We included 28 of 36 (77.8%) women with confirmed SARS-CoV-2 infection during pregnancy in the study period. Only four women tested positive by routine screening when admitted to the maternity ward for labor, of whom one was asymptomatic. All other women were tested positive elsewhere. Non-participants (n = 8, F I G U R E 1 Flowchart of participants and sampled data. Data sampled antepartum included a maternal blood sample and two vaginal swabs. Data sampled intrapartum included a maternal blood sample as well as an umbilical cord blood sample and two vaginal swabs. In some cases, other samples were performed on clinical indications. a One woman (participant 28) had three consecutive samples (both vaginal swabs and blood samples) performed on day 1, 16, and 37 after confirmed SARS-CoV-2 infection resulting in a total of 8 vaginal swabs and 8 maternal blood samples Table 1 ) were women with confirmed SARS-CoV-2 infection who gave birth within the study period, but who were not included in this study for various reasons, eg did not understand Danish/English, did not want to participate or were not asked because of busyness ( Figure 1 ). Twenty-two participants (78.6%) were included intrapartum (Table 2) , and six participants were included and examined antepartum with samples repeated intrapartum (Table 3) In an emergency cesarean section, two vaginal swabs were performed: at an initial vaginal examination and immediately before/after the emergency cesarean section, respectively. In a vaginal delivery, two vaginal swabs were performed: one before an initial vaginal examination and one during the active phase of delivery or immediately after, respectively.

b

Vaginal swabs performed 1 day postpartum.

c Only one vaginal swab was performed due to E-CS.

Pt Data sampled antepartum

to data sampled antepartum If planned cesarean section, the vaginal swabs were performed immediately before and after CS. If vaginal delivery, two vaginal swabs were performed, one before an initial vaginal examination and one during the active phase of delivery or immediately after, respectively.

after confirmed SARS-CoV-2 infection. The test results (S/C) of antibodies in maternal and cord blood at delivery were highly correlated (Supporting Information Figure S1 , R 2 = 0.64, p < 0.001). There was a positive correlation between the test results (S/C) for antibodies in cord blood and the number of days from confirmed SARS-CoV-2 infection to delivery (Supporting Information Figure S2 , R 2 = 0.43, p = 0.002), whereas the correlation between the test result (S/C) for antibodies in maternal blood and the number of days between confirmed SARS-CoV-2 infection and delivery was not statistically significant (Supporting Information Figure S2 , R 2 = 0.20, p = 0.054).

Cord blood samples from three children of 19 seropositive mothers were without detectable antibodies (15.8%), but the remaining 16 cord blood samples had detectable antibodies. Figure   S4 ). SARS-CoV-2 was detected in the vaginal swab at delivery and in swabs from both the fetal and maternal sides of the placenta.

Neonatal swabs were SARS-CoV-2 negative. The mother tested positive for SARS-CoV-2 antibodies but umbilical cord blood tested negative. Blood and urine culture at the time of delivery were negative.

Altogether, SARS-CoV-2 positive swabs from the placenta and placental examinations with findings of severe fibrin deposit indicate the involvement of SARS-CoV-2 infection as part of the pathogenesis. By reference to a systematic classification system, the above-mentioned cases could be classified as "probable congenital infections" because the virus was detected by PCR in the placental swabs from the fetal side of the placenta in neonates delivered by cesarean section before rupture of membranes. 13 Besides the two severe neonatal cases described, few maternal complications were found (Table 1) . Swabs from the neonates were taken in two cases (participant 3 and 5, Supporting Information Table S1 ), neither was positive for SARS-CoV-2. No children had confirmed SARS-CoV-2 infection after birth.

In this prospective cohort study, we found SARS-CoV-2 in 2 of 28

vaginal swabs up to 8 days after confirmed SARS-CoV-2 infection.

SARS-CoV-2 total antibodies were detected in maternal blood samples from day 8 and in the majority from 16 days after confirmed infection. In cord blood, antibodies were detected from 26 days.

Reduced fetal movements combined with abnormal CTG and placental findings associated with fetal asphyxia were observed in two cases.

Our study indicates that the neonate is not protected against Table 2 ) had positive antibodies, but no antibodies were detected in cord blood 16 days after confirmed maternal infection.

In accordance with our study, there was a positive correlation between the levels of maternal and cord-blood IgG. 14 In our study, two highlight that reduced fetal movements should always be handled with concern and that women should seek immediate care at any such occurrence. 4, 7 The study has several strengths, including the prospective design. The study also has limitations. We included 77.8% of eligible women and had a small sample size, although larger than some previous studies examining vaginal swabs. 17, 18 As a result of the small sample size, the study includes a limited number of blood samples, covering only fragments of the period after confirmation of the SARS-CoV-2 infection. We can therefore not conclude on the exact timing of seroconversion, but only estimate a time span for seroconversion as well as the appearance of antibodies in cord blood.

Most vaginal swabs were kept at −20°C until analysis, possibly causing a deterioration of SARS-CoV-2 RNA during storage, decreasing our ability to detect SARS-CoV-2 in the vaginal samples.

However, a previous study found that SARS-CoV-2 RNA was stable when stored at −20℃ for up to 84 days. 28 Lack of infection status from the participants' partners is also a limitation, as it is not fully evaluated whether SARS-CoV-2 can be transmitted through semen and cause detection of SARS-CoV-2 in the vagina. 20 We measured total SARS-CoV-2 antibodies and therefore were not able to discriminate between IgG and IgM. However, for an individual, specific SARS-CoV-2 IgG/IgM assays may be difficult to interpret because of a significant variation in the IgM and IgG antibody response to SARS-CoV-2. Hence, measuring total antibodies improves sensitivity. 29, 30 The assay used in this study targets the spike protein, and the presence of spike protein antibodies is indicative of either previous infection or vaccination. 31 In this study none of the women had In large cohort studies, the overall risk of severe neonatal outcomes as well as neonatal infection related to maternal SARS-CoV-2 infection seems low, however, a recent paper reports increased rates of fetal death, preterm birth, preeclampsia, emergency cesarean delivery, and other adverse maternal and neonatal outcomes. 

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