The global spread of the severe acute respiratory coronavirus (SARS-CoV-2) has confronted the scientific community with a health emergency. The anatomical, physiological, and immunological changes that accompany pregnancy may increase the susceptibility of pregnant women to infections; However, the effects of SARS-CoV2 infection on pregnant women, fetuses, and newborns remain uncertain.1–3
Three mechanisms of vertical transmission have been described: intrauterine transmission, intrapartum transmission, when the infection occurs during childbirth, and childbirth and postnatal transmission, when it occurs through respiratory secretions or other infections through breastfeeding or through contact with other caregivers. infected.4,5
To date, it is not clear whether vertical transmission of SARS-CoV-2 is possible, as conflicting data have been published in the literature.6,7 The rate of perinatal transmission of SARS-CoV-2 is variable, with the most series around 2-5%,8–10 although there are publications that report it up to 12%,11 with transmission being mainly postnatal.12
Although studies that evaluate vertical transmission are mostly epidemiological,11,12 there is not enough evidence of viral transmission in biological samples through microbiological techniques.
The main objective of the authors was to describe the transmission of SARS-CoV-2 infection in infants exposed intrauterinely by analyzing the viral load in biological samples from mothers and newborns. The secondary objective was to describe the epidemiology and clinical characteristics of infected pregnant women and the obstetric and perinatal history of their newborns.
| Materials and methods |
Prospective, observational, descriptive and multicenter study in 13 Spanish hospitals included in the GEStational and NEOnatal (GESNEO)-COVID cohort, which includes RECLIP (Spanish Network of Pediatric Clinical Trials).
> Population and study period
Pregnant women with microbiologically confirmed SARSCoV-2 infection during any trimester of pregnancy or childbirth and their newborns were included in the study. Diagnosis of infection was made by performing reverse transcription-polymerase chain reaction (RT-PCR) testing on nasopharyngeal swabs. Pregnant women with positive IgG serological test results were not included in this analysis. Patients were included in the study between March 15, 2020 and November 30, 2020.
> Epidemiological and clinical variables
Demographic and clinical information, including comorbidities and obstetric history, was collected from the pregnant women. For SARS-CoV-2 infection, the time of diagnosis, clinical presentation, need for treatment, imaging tests and hospitalization for infection were specified.
Perinatal and delivery clinical data, anthropometric data, type of feeding and comorbidities were collected during the neonatal period of the newborns.
Although there may be slight variations depending on the protocol of each participating center, asymptomatic newborns whose mothers presented an adequate clinical status were kept in joint isolation in the obstetrics ward. If a newborn required hospitalization, it was carried out in an isolation room until the result of the RT-PCR.
Newborns were classified according to the transmission mechanism described by Blumberg et al.4
Subsequently, two groups were created to compare baseline maternal demographic and clinical characteristics and neonatal outcomes, according to the infection status of the newborns. Group 1 consisted of uninfected newborns and their mothers, and group 2 consisted of infected newborns (intrauterine, intrapartum, or early postnatal infection) and their mothers.
> Microbiological samples: collection, conservation and processing
In pregnant women, nasopharyngeal swabs for SARS-CoV-2 RT-PCR were obtained at diagnosis and at delivery.
At delivery, maternal and placental blood samples were collected for RT-PCR, as well as umbilical cord blood samples. The samples were initially frozen and archived in the Microbiology Service of the Gregorio Marañón General University Hospital until analysis.
In addition, placenta samples were collected in formalin for subsequent immunohistochemical analysis. Nasopharyngeal swabs for RT-PCR were performed on newborns in the first 24 to 48 hours after delivery. In those with a positive result, a second test was performed immediately to confirm the results. RT-PCR of nasopharyngeal swabs was also performed in all neonates on day 14 of life. Urine and meconium samples were collected during the first 48 hours of life.
In pregnant women who were breastfeeding, breast milk samples were collected by hand or with breast pumps after adequate breast hygiene.
Biological samples in the viral transport media were analyzed for the presence of SARS-CoV-2 RNA by RT-PCR to detect the N gene and the ORF1a1b gene (TaqPath Multiplex, Thermo Fisher).
> Statistical analysis
Continuous variables are described as medians and interquartile ranges (IQR), and categorical variables as absolute frequencies and percentages. For the comparison of categorical variables, the χ2 test or Fisher’s exact test was used, as appropriate, and for continuous variables, the Wilcoxon rank sum test was used, with a p < 0.05, which was considered statistically significant. Data were analyzed using StataCorp. 2019. Stata Statistical Software: Version 16. College Station, TX: StataCorp LLC.
> Ethical considerations
The study was approved by the Clinical Research Ethics Committee of the Gregorio Marañón General University Hospital (IRB Code 00006051) and all participating centers. Informed consent was obtained from the mothers or legal guardians of the newborns.
| Results |
The evolution of 174 pregnant women with SARS-CoV-2 infection during pregnancy and 177 newborns (171 singletons, 3 twins) were detailed.
> Microbiological data
At the time of delivery, 39% of pregnant women had an acute infection (RT-PCR positive, IgG negative), 30% had a recent infection (RT-PCR positive, IgG positive), and 31% had a past infection. (RT-PCR negative, IgG positive with RT-PCR positive during pregnancy).
A total of 115 maternal blood samples and 81 placenta samples were collected for RT-PCR, which revealed only 1 case of viral load in the blood and placenta sample of 1 pregnant woman. These samples belonged to a 33-year-old pregnant woman of Latin American origin with acute infection at the time of delivery, whose mild clinical symptoms (fever, headache and catarrhal symptoms) began 48 hours before delivery. The newborn remained asymptomatic and all samples collected (nasopharyngeal swab, umbilical cord blood, urine, meconium, and breast milk) were negative for SARS-CoV-2.
Immunohistochemical analyzes of all placental samples for SARS-CoV-2 (16) were negative.
Seventy-nine breast milk samples were analyzed and no viral loads were detected in any of the samples.
All RT-PCR results for umbilical cord blood and newborn blood samples (64) were negative. The viral load was detected in 3 newborn urine samples and 3 meconium samples. All cases were newborns with acute SARS-CoV-2 infection diagnosed by positive RT-PCR of a nasopharyngeal swab in their first 48 hours of life.
> Characteristics of newborns with COVID-19 and transmission mechanism
All newborns (177) were analyzed by RT-PCR of nasopharyngeal swabs in the first 24 to 48 hours after delivery and at 14 days of life. A total of 159 newborns had negative RT-PCR results; therefore, they were considered uninfected newborns. Twelve infants were identified with positive RT-PCR results in nasopharyngeal swabs.
According to the Blumberg classification, infection of newborns 1-3 was classified as a result of intrauterine transmission, 4-9 of intrapartum or early postnatal transmission, and 10-12 of contamination of nasopharyngeal secretions or transient viremia. After excluding cases of contamination by nasopharyngeal secretions or transient viremia, 5.1% (9) of newborns were diagnosed with SARS-CoV-2 infection in the neonatal period, 1.7% (3) had contracted it intrauterinely and 3.4% (6) had contracted it intrapartum or early postnatally.
All were born to mothers who were severely infected at the time of delivery. None of the pregnant women had viral load in maternal blood or placenta samples.
Only 2 of 9 infected newborns presented symptoms, both with respiratory distress that evolved satisfactorily during follow-up.
> Comparison of maternal and neonatal characteristics according to newborn infection rates
There were no differences in the demographic and clinical characteristics of pregnant women compared to newborns. No differences were found in the history or perinatal history of the newborns.
Compared with uninfected newborns, infected newborns did not develop more symptoms and did not have higher rates of admission to the neonatal intensive care unit.
Infected newborns were fed more frequently with a combination of formula and breast milk compared to uninfected newborns, with no differences in artificial formula feeding rates.
| Discussion |
In this large cohort of infants born to mothers with SARSCoV-2 infection during pregnancy, 5.1% had neonatal infections, with infections most common in the early postnatal period. No viral loads were found in any of the umbilical cord blood samples from infected newborns, placenta or breast milk collected.
The vertical transmission of SARS-CoV-2 remains highly debated today, with some studies showing controversial results, and the majority of published studies are case reports or retrospective investigations.13 In this cohort of newborns exposed to SARS -CoV-2 during pregnancy, 5.1% had neonatal infections, a result similar to that of other previously published studies.8–10
In a systematic review and meta-analysis by Di Toro et al,14 11 neonates out of 275 tested positive for SARS-CoV-2 (5%). Comparable results were described by Kotylar et al.15 in their systematic review and meta-analysis, with an infection rate of 3.2% and 48 positive neonates out of 936 neonates. Furthermore, they differentiated between studies from China and Europe, with higher rates of vertical transmission reported in European studies than in Chinese studies (4.9% in the UK vs. 2% in China).16
Classifying newborns according to the transmission mechanism is difficult4 due to the great heterogeneity in the definitions of vertical transmission. One of the first classifications was the one proposed by Blumberg et al., 4 which was used in this study. Subsequently, new classifications were published, including that of the World Health Organization (WHO),5 which were updated in February 2021.
There is growing interest in establishing whether intrauterine transmission is possible. In this study and according to the Blumberg classification, the positivity of the nasopharyngeal swabs by RT-PCR in the first 24 hours of life and its subsequent persistence allows the infection of the newborn to be classified as intrauterine transmission. This differs from the WHO classification, where a positive sterile sample in the first 24 to 48 hours of life (umbilical cord blood, placenta, amniotic fluid, bronchoalveolar lavage or cerebrospinal fluid) is required for "confirmed" transmission, with only "possible" transmission identified from nasopharyngeal exudate samples.
In this series, no amniotic or placental fluid samples were obtained from any of the newborns classified as having intrauterine transmission, and only in 1 case was RT-PCR performed on umbilical cord blood, which was negative. According to the WHO classification, the 3 newborns should be categorized as having “possible” intrauterine transmission.
The second potential transmission mechanism is intrapartum or early postnatal transmission. Blumberg and collaborators grouped these two mechanisms, while the WHO classification differentiates between intrapartum and early postnatal transmission according to the time of microbiological testing: a positive test for intrapartum transmission occurs between 24 and 48 hours of life, requiring a negative test in the first 24 hours, and early postnatal transmission is defined when it occurs 48 hours after birth.
The narrow timeline separating the 2 mechanisms makes it extremely difficult to tell them apart. In both cases, the diagnosis can be established from nasopharyngeal samples, without the strict need for positive sterile samples.
Six newborns with intrapartum or early postnatal transmission were included in this study. Five had a negative diagnostic test in the first 48 hours of life, with positive RT-PCR at the follow-up visit at 2 weeks of life, corresponding to early postnatal transmission according to the WHO classification.
The sixth case involved a newborn whose mother tested positive by RT-PCR in the first 24 hours postpartum. The newborn’s RT-PCR results were positive at 48 hours of age. According to the WHO classification, this case would be classified as “possible” early postnatal transmission in the absence of a previous negative diagnostic test.
The last transmission mechanism proposed by Blumberg et al., contamination by nasopharyngeal secretions or transient viremia, was attributed to 3 newborns in this sample. The WHO classification defines these cases as undetermined within intrauterine transmission. However, in most cases, the first nasopharyngeal swab of the newborn was performed after skin-to-skin contact with the mother and joint isolation in the same room. The lack of viral persistence means that these newborns cannot be considered infected, so they were not included in the subsequent analysis.
The clinical-epidemiological characteristics of the pregnant women in this series were similar to those reported in other studies 9, 17, and there were no differences when comparing them according to neonatal infection status.
All mothers of infected newborns had an acute infection at the time of delivery, suggesting that transmission occurs primarily late in the gestation period and during delivery and that there are no maternal risk factors that contribute to transmission to newborns. born.
In this sample, neither the severity of maternal infection nor the presence of symptoms was related to neonatal infection. In any case, due to the small number of infected newborns, these conclusions must be taken with caution. To our knowledge, no prospective study has described the presence of certain maternal characteristics that predispose newborns to infection.
In this cohort, no differences were found in gestational age, type of delivery, symptoms and need for hospitalization between infected and non-infected newborns. The most common neonatal symptoms of COVID-19 described in the literature are tachypnea, regurgitation of milk, cough, vomiting and fever.18
In the present study, of the 9 infected newborns, only 2 presented symptoms (22.2%): transient tachypnea and respiratory distress syndrome, prematurity. Due to the high frequency of these symptoms in neonatal units, it is not possible to ensure that this clinical presentation is due to SARS-CoV-2 infection. None of the infected newborns presented fever or digestive symptoms.
A systematic review published by Mirbeik et al., 8 including 17 articles with microbiological data, found no evidence of SARSCoV-2 in placenta, umbilical cord blood, or breast milk samples. On the other hand, a recent systematic review and meta-analysis published in 2021 found 1 umbilical cord blood sample and 2 placenta samples positive for SARS-CoV-2.
In this cohort, SARS-CoV-2 viral loads were only found in 1 blood sample and 1 placenta sample, both from a pregnant woman with acute infection at the time of delivery. As these were samples from a pregnant woman with mild symptoms, and because vertical transmission to the newborn was not subsequently observed, contamination may have occurred in the analysis of the samples.
In a study by Elbow et al.19 in which 62 maternal and umbilical cord blood samples from newborns and 44 placentas from infected mothers were analyzed, no evidence of SARS-CoV-2 RNA was found.
SARS-CoV-2 was not detected in any of the placenta samples. Several studies analyzed the anatomopathological alterations in the placenta of pregnant women with COVID-19, without finding significant differences compared to uninfected pregnant women.20,21 A study by Levitan et al. 20 found no evidence of the virus when immunohistochemically analyzing placental samples.
At the beginning of the pandemic, there was great controversy over whether breastfeeding should be allowed because it was unknown if it could be a possible route of transmission of the virus. In accordance with the recommendations of the Spanish Society of Neonatology, 22 the WHO23 and the American Academy of Pediatrics, 24 in this cohort of Spanish hospitals, breastfeeding was maintained in infected mothers following the relevant hygienic measures, including the use of masks. maternal contact with the newborn and through hand and breast hygiene.
The authors found no viral loads in breast milk samples. To date, several studies have evaluated the presence of the virus in breast milk, with controversial results.25, 26 Additionally, these studies had a small sample size. Grob et al27 were among the first to find viral RNA in serial milk samples from an infected mother.
Some studies with a larger number of patients, such as that of Pace et al., 28 in which samples from 18 women were studied, did not find viral RNA in serial milk samples from infected mothers.
In this cohort, infected newborns were exclusively breastfed less frequently, with possible loss of the long-term benefits of breastmilk, although these results should be interpreted with caution due to the small sample size.
There are studies published in adult patients29 and pediatric populations30 where the virus was isolated in urine and feces samples, and the viral load can even be maintained in fecal excretions for weeks after infection. In this sample, the virus was detected by RT-PCR in urine and meconium of 4/9 (44.4%) newborns with infection confirmed by PCR in nasopharyngeal exudate.
One of the main limitations of this study is the heterogeneity in patient recruitment; At the beginning of the pandemic, only pregnant women with symptoms were tested by RT-PCR, resulting in a loss of asymptomatic pregnant women who potentially could have been included in the first month of the study. Additionally, blood, placenta, or milk samples were not available from all dyads included in the study. Furthermore, this study only included live newborns; therefore, no information is available on whether infection during pregnancy could cause spontaneous abortion or intrauterine fetal death.
Strengths of this study include its large sample size and multicenter nature. Another notable feature is that this study allowed us to understand the natural history of SARS-CoV-2 infection without the influence of vaccination status, as was done at the beginning of the pandemic.
| Conclusions |
In this large prospective study, intrauterine transmission of SARS-CoV-2 was possible, although rare, and early postnatal transmission through direct contact with infected persons occurred more frequently.
The majority of infected newborns remain asymptomatic or present mild symptoms with good subsequent evolution during follow-up. No maternal epidemiological characteristics were found that predisposed to neonatal infection, although it was observed that the infected newborns were from mothers with acute infection at the time of delivery.
The presence of viremia in maternal and placental blood samples was anecdotal in this cohort, and the virus was not found in umbilical cord blood or newborn blood samples.
The possibility of viral transmission through breast milk is unlikely, since no viral load was detected in the samples studied; Therefore, breastfeeding is not contraindicated in cases of infection.
Finally, the virus can be detected in urine and meconium samples from infected newborns.
| Comment |
This was a prospective, observational, multicenter study that evaluated the vertical transmission of SARS-CoV-2, a condition that still remains controversial.
Neonates exposed to SARS-CoV-2 were studied by PCR of nasopharyngeal swabs within the first 24 to 48 hours of and 14 days of life.
In total, 5.1% were considered to have SARS-CoV-2 infection in the neonatal period, 1.7% intrauterine, and 3.4% intrapartum or early postnatal. It was concluded that intrauterine transmission of SARS-CoV-2 is possible, although rare, with early postnatal transmission being more common.
The majority of infected newborns remained asymptomatic or presented mild symptoms that progressed favorably.
No maternal characteristics were found to predispose infants to neonatal infection. Although there was no presence of SARS-CoV2 in umbilical cord blood or breast milk samples, viral load was detected in urine and meconium samples from infected newborns.
