Case presentation
A 27-year-old woman (gravida 2, para 1) was transported with ambulance to the regional university hospital in gestational week (GW) 34 + 4 due to a three-day history of fever, abdominal pain and reduced fetal movements. She had developed a dry cough one day prior to the admission.
The woman, an immigrant of Turkish descent did not speak Swedish, was slightly overweight (BMI 27 kg/m2) but otherwise healthy. She had normal antenatal controls during the current pregnancy. In 2016, she had a normal vaginal delivery in Turkey which was complicated by postpartum hemorrhage. During the current pregnancy, an obstetric ultrasound at GW 32 + 2 showed a normal fetal weight deviation of +8%7.
At admission, the patient was promptly isolated in a negative pressure room at the delivery unit and standard operating procedures and personal protective equipment (PPE) were used8. A combined nasopharynx (NPH) throat swab for SARS-CoV-2 real time reverse transcriptase quantitative polymerase chain reaction (RT qPCR) was obtained and normal vital parameters (apart from fever 38.3 degrees Celsius) were registered.
The admission cardiotocograph (CTG) test showed reduced baseline variability, absence of accelerations with recurrent prolonged, and late decelerations (Figure 1). In light of the pathological CTG pattern, the obstetric team made the prompt decision to terminate the pregnancy by an immediate-emergency CS. Within eight minutes, the patient was anesthetized and intubated. An uncomplicated CS was performed in an operating theatre with negative pressure in line with the international recommendations for COVID-198. The total blood loss was 200 mL. The amniotic fluid was of normal amount and there were no signs of meconium staining, premature rupture of the amniotic membranes or blood in the amniotic fluid. The placenta was easily detached.
The neonate was immediately transported to a separate room by a designated midwife equipped with sterile clothing and gloves. The neonate showed no initial signs of spontaneous breathing and was ventilated by neonatal staff. A maximum of 80% supplemental oxygen was needed to maintain adequate saturation. At six minutes of age, the neonate established spontaneous breathing and continuous positive airway pressure (5 cm H2O) was maintained for an additional 24 minutes, whereafter further ventilatory support was not needed. At one minute of age, the neonate had an Apgar score of 1 (heart rate = 1, remaining items = 0), at five minutes of age Apgar 4 (heart rate = 2, muscular tonus = 1, reflex irritability = 1, remaining items = 0), and at ten minutes of age Apgar 8 (heart rate = 2, respiratory activity = 2, skin color = 1, muscular tonus = 2, reflex irritability = 1). Validated umbilical cord blood gases9 from both cord artery and vein were retrieved showing a cord arterial pH of 7.20 and lactate 11 mmol/L (Supplementary table 2). The birth weight was appropriate for gestational age. Figure 2 illustrates the timeline of events for mother and child.
Maternal clinical course
After the CS, the mother was isolated in the postpartum ward and the NPH/throat swab taken upon admission returned positive for SARS-CoV-2. Analysis of maternal blood was also RT qPCR positive for SARS-CoV-2 (Table 1). Serology from the day of delivery revealed that the mother was weakly positive for immunoglobulin (Ig) M and negative for IgG (Table 2). Other maternal laboratory tests are shown in Supplementary table 1. Along with lymphocytopenia (0.7 x109/L) and thrombocytopenia (98 x109/L); inflammatory markers including c-reactive protein (36 mg/L), ferritin (340 µmol/L) and lactate dehydrogenase (9.5 µkat/L) were found to be elevated. The clinical condition of the mother improved and she was discharged four days after delivery with thromboprophylaxis (Tinzaparin 4500 IE subcutaneously once daily) for a total six weeks postpartum. A NPH/throat swab taken at day 9 postpartum returned negative for SARS-CoV-2 (Table 1). By day 11 postpartum, the mother was seropositive for anti-SARS-CoV-2 IgM and IgG (Table 2). Breast milk analyzed day 14 postpartum was RT qPCR negative for SARS-CoV-2 (Table 1), and further, at day 35 postpartum, negative for anti-SARS-CoV-2 total immunoglobulin (Table 2).
Infection control measures
According to current national Swedish guidelines for newborns admitted to neonatal ward and with COVID-19 within the family10, a neonate can be cared for together with the parent if the required care and isolation measures are possible to provide. Due to a lack of isolation rooms, the neonate in the current case had no contact with any family member, including the mother, during the first 60 hours of life. Since neither skin-to-skin care nor any other contact with the mother occurred, the neonate was regarded as non-infected, and neonatal staff did not use PPE. In accordance with national guidelines10, the neonate was tested for COVID-19 using a NPH swab 48 hours after delivery. This test returned positive for SARS-CoV-2 (Table 1) and the neonate was then regarded as contagious. Infection control routines were initiated to investigate a potential COVID-19 breakout at the neonatal ward but also to rule out the possibility of postpartum transmission. All staff that had tended to the neonate (n=27) and all nearby patients (n=4) were tested for COVID-19. The NPH/throat swabs for SARS-CoV-2 RT qPCR returned negative in all cases (data not shown). Symptom surveillance in this group was continued for a further 14 days but no COVID-19 positive cases were discovered during this time.
Neonatal clinical course
Isolation was carried out as presented above. The neonate was admitted to the neonatal ward at 30 minutes of age due to transient asphyxia and prematurity. Apart from the initial resuscitation, no ventilatory support or supplementary oxygen was needed. During the first day of life (DOL) blood gas analyses were performed mainly to monitor blood glucose levels (Supplementary table 3). Mild hypoglycemia (2.4 mmol/L) was found at 6 hours of life and it was managed successfully with oral glucose gel and intensified feeding of formula milk via a nasogastric tube.
As the routine RT qPCR test obtained at 48 hours of age returned positive for SARS-CoV-2 and since the clinical condition of the neonate was stable, the neonate was transferred and united with the mother at the postpartum ward isolation room at DOL 3 (60 hours after birth). Breastfeeding was thereafter initiated and the neonate did not receive any breastmilk before this time point. When the mother was later discharged from the postpartum ward at DOL 4, the neonate was transferred to an isolation room at the pediatric infectious disease ward. The mother was still present and breastfeeding was continued. During the entire hospitalization period, the neonate displayed no upper respiratory tract symptoms nor any signs of neonatal COVID-19. At DOL 5, it was observed that the neonate was undernourished and clinical examination revealed moderate signs of hypovolemia, slight ventilatory distress and a mild systolic heart murmur. Pulmonary auscultatory findings were normal. A chest X-ray revealed no abnormalities (Supplementary figure 1). Blood tests including coagulation, liver and hematological analyses were found to be normal for the gestational age. C-reactive protein was not elevated (Supplementary table 3). Following intensified breastfeeding, the hypovolemia, heart murmur and respiratory distress subsided by DOL 7. The neonate was discharged at DOL 15 and was being fully breastfed. A follow-up control at DOL 35 confirmed that the neonate had no respiratory or feeding problems. The physical examination was normal apart from a small, left-sided, asymptomatic, inguinal hernia and additional blood tests revealed no abnormalities (Supplementary table 3).
Regarding the neonatal COVID-19 infection, repeated RT qPCR analyses showed the lowest CT-value at DOL 5 where after a gradual increase was seen. By DOL 20, SARS-CoV-2 was not detectable in NPH or throat swabs (Table 1). Further, serology revealed that the neonate was anti-SARS-CoV-2 IgG negative at DOL 7 (IgM not analysed due to lack of material). At DOL 14, IgM was positive and IgG still negative and at DOL 20, the neonate was both IgM and IgG seropositive (Table 2).
Viral genome sequencing
To determine the genetic clade and to fully investigate the viral genetic similarities, virus isolates from the mother (NPH/throat swab obtained on the day of delivery), and neonate (NPH swab obtained at 48 hours of age, labelled DOL 2, and further at DOL 5) as well as from placental tissue, were sent to the Public Health Agency of Sweden for whole-genome sequencing. Next-generation sequencing of samples produced several full length 29 903bp, SARS-CoV-2 genomes, all belonging to the genetic clade 20B/GR/B.1.111. All four sequences showed high identity, which is illustrated in a phylogenetic tree (Supplementary figure 2). Further sequencing data analysis identified 12 variant positions in the sequences from isolates of the mother and placenta compared to the SARS-CoV-2 reference genome (NC_045512). These variants were also present in the sequences of the neonate isolates (Table 3). Notably, an additional variant, A107G, was identified in the neonate samples but only present in 67 and 80%, respectively, of the sequences.
Placental pathology
The placenta was easily detached from the uterus during the CS. The remaining umbilical cord stump had a central insertion, was 9 cm long with a diameter of 1 × 1.5 cm and contained three vessels. The membranes had normal color without signs of meconium staining. The trimmed weight of the placental disc was 342 grams, within the 10th to 90th percentile for GW 34+0 to 34+612. At gross sectioning, fibrin depositions were evident as glistening white-grey-pink confluent lesions, encompassing approximately 50% of the total placental volume (Figure 3 A).
Microscopic examination confirmed the presence of confluent intervillous fibrin depositions accompanied by denudation of the villi from trophoblasts and syncytiotrophoblasts with dislocated syncytiotrophoblasts visible in the fibrin (Figure 3 B-C). There were multiple regions of dense intervillous infiltrates of neutrophilic granulocytes and macrophages (Figure 3 D). The areas devoid of intervillous fibrin depositions frequently showed chorangiosis (Figure 3 E). Immunohistochemistry confirmed that the inflammatory cell component of the intervillisotis was dominated by myeloperoxidase positive granulocytes and CD68 positive macrophages with sparse amounts of CD3 and CD20 positive lymphocytes (Figure 3 F-G).
Immunohistochemical detection of SARS-CoV-2 nucleoprotein was strongly positive in the cytoplasm and nucleus of villous cytotrophoblasts and syncytiotrophoblasts in areas with intervillositis and fibrin depositions, with some positive staining in the villous stromal cells (Figure 3 H-J). In contrast, SARS-CoV-2 nucleoprotein staining was focal or absent in most but not all areas devoid of intervillositis (Figure 3 K-L). Additionally, presence of ribonucleic acid (RNA) virus was confirmed in both cytotrophoblasts and syncytiotrophoblasts by in-situ staining for double stranded RNA (Figure 3 M). There were no signs of villitis or inflammation in the membranes or umbilical cord. Immunohistochemistry for SARS-CoV-2 nucleoprotein was absent or showed faint signal in the amniotic membranes and the fetal chorionic vessels.
In summary, three main histopathological features were found; I) massive perivillous fibrin deposition, II) acute intervillositis in areas with strong positivity for SARS-CoV-2 and III) chorangiosis in the areas less affected by infection and inflammation.