key: cord-0847875-r0pszgax authors: Von Kohorn, Isabelle; Stein, Sydney R; Shikani, Beatrix T; Ramos-Benitez, Marcos J; Vannella, Kevin M; Hewitt, Stephen M; Kleiner, David E; Alejo, Julie C; Burbelo, Peter; Cohen, Jeffrey I; Wiedermann, Bernhard L; Chertow, Daniel S title: In Utero SARS-CoV-2 Infection date: 2020-10-22 journal: J Pediatric Infect Dis Soc DOI: 10.1093/jpids/piaa127 sha: 4f6228a57adaadea4cf1795e9435e468152a4b6e doc_id: 847875 cord_uid: r0pszgax Evidence for in utero transmission of SARS-CoV-2 is growing but not definitive. We present a case of neonatal infection that supports in utero transmission of SARS-CoV-2 and provides insight into hematogenous spread from mother to fetus. associated with neonatal infection. We present a case of neonatal infection with viral RNA in cord blood that supports in utero transmission of SARS-CoV-2 and provides insight into hematogenous spread from mother to fetus. This study was exempted by the Institutional Review Board at Holy Cross Hospital. A 34-week gestation, 2414 gram boy was born to a mother diagnosed 14 hours prior to delivery with SARS-CoV-2 infection via positive NP swab, obtained because of cough for one week. The mother sought medical attention for vaginal bleeding and cramping and was found to have thrombocytopenia, transaminitis, and hyperuricemia. She had a history of gestational diabetes controlled by diet and was not hypertensive. The infant was delivered via Cesarean section because of concern for Hemolysis, Elevated Liver enzymes, Low Platelet syndrome, suspicion for systemic COVID-19, and history of prior Cesarean section. During delivery mother wore a non-rebreather face mask and medical personnel wore airborne-level personal protective equipment (PPE) including N-95 masks and face shields. Amniotic fluid was clear. The infant's cord was clamped after 30 seconds, and he received routine care 20 feet from his mother's head. Cord blood was collected immediately using standard procedure. Apgar scores were 7 at 1 minute and 9 at 5 minutes. Physical exam was normal. He was shown to his mother for a few seconds from a distance of 4 feet while she wore a surgical mask. The infant was placed in an incubator maintained outside the delivery room, transported to the adjacent neonatal intensive care unit (NICU), placed in a negative pressure room, and bathed shortly after birth. All NICU personnel wore airborne-level PPE while in contact with the infant. NICU personnel had no contact with the mother until after her hospital discharge, and the mother elected not to breastfeed or provide breast milk. The infant had no contact with family members until day of life (DOL) 7 when his mother had recovered. A c c e p t e d M a n u s c r i p t 5 The infant continued to be asymptomatic with normal laboratory studies (see Supplemental Table) but NP swabs tested positive for SARS-CoV-2 RNA. The infant was discharged on DOL 8 and remained healthy through the first month of life. NP swab for SARS-CoV-2 utilizing the Cepheid Xpert Xpress qualitative test for the presence of the SARS-CoV-2 target envelope protein (E) and nucleocapsid 2 (N2) genes was negative at 24 hours of life and then positive for only N2 at 49 hours of life. Because of concern for falsepositive results, multiple other specimens were tested with Xpert Xpress and a second qualitative test, the Roche Cobas SARS-CoV-2 assay, which utilizes ORF1 and E gene targets (Table) . Qualitative tests indicated an increasing viral burden in the nasopharynx over time based on decreasing cycle threshold (Ct) values. We performed studies to determine the presence of viral RNA in cord blood, infant blood, and infant urine and of antibodies in cord blood. Samples were limited to those collected for clinical care that were still available after NP swab-confirmed infant infection. Blood and urine samples were refrigerated after collection and were subsequently frozen before being processed via the QIAamp Viral RNA Mini kit (Qiagen) according to manufacturer's instructions. To detect the presence of SARS-CoV-2 we utilized the Bio-Rad SARS-CoV-2 ddPCR Kit, a triplex assay for the nucleocapsid genes N1 and N2 with a human RNase P gene (RP), according to manufacturer's instructions. We found SARS-CoV-2 RNA corresponding to genes N1 and N2 in umbilical cord serum and the SARS-CoV-2 gene N2 in DOL 2 urine (Table) . An immunoprecipitation assay was used to detect IgG to SARS-CoV-2 as previously reported. [7] The assay was modified to include anti-human IgM agarose beads [8] to detect IgM to SARS-CoV-2. Known SARS-CoV-2 seronegative and seropositive samples for IgG and IgM antibodies against nucleocapsid and spike proteins were used for assigning seropositive cut-off values and for standardization. Testing of A c c e p t e d M a n u s c r i p t 6 both serum and plasma from the cord blood was seronegative for IgM and IgG antibodies against both proteins. The placenta was fixed in formalin on the day of birth, and several paraffin blocks were prepared for review. Clinical pathology showed no gross abruption. Two weeks after delivery, a larger sample of placenta was transferred to ethanol. We performed both in situ hybridization and ddPCR studies on the placenta. (Figure) The RNAscope assay was negative in placenta, membranes, and cord. Preserved placental tissue was processed via the RNeasy FFPE kit to extract RNA and analyzed via ddPCR. Neither N1 nor N2 gene targets were detected in placental or umbilical cord tissue. This infant most likely acquired SARS-CoV-2 hematogenously from mother at or prior to delivery. All caregivers were aware of mother's COVID-19 diagnosis prior to delivery, and it is extremely unlikely that the infant could have acquired infection from mother via the respiratory route with such brief and distant exposure in the delivery room. While we cannot rule out microscopic maternal blood contamination of cord blood in this or any other delivery, cord blood collection procedures are designed to avoid gross contamination with maternal blood. Microscopic contamination would not explain the RNA levels observed in our patient's cord blood. A c c e p t e d M a n u s c r i p t 7 The scientific community has not agreed upon a case definition for in utero transmission of SARS-CoV-2, although two appealing approaches have been described. [9, 10] This case provides evidence for in utero hematogenous transmission of SARS-CoV-2: we found SARS-CoV-2 RNA in cord blood and in newborn urine and nasopharynx, with evidence for viral replication. We detected SARS-CoV-2 RNA in infant NP secretions on DOL 2, suggesting that virus The absence of anti-SARS-CoV-2 IgG and IgM in cord blood neither supports nor refutes the hypothesis that virus was transmitted in utero. Seroconversion for IgG against the SARS-CoV-2 spike protein generally occurs more than a week after symptom onset in adults. [7] Since the mother experienced symptoms for 7 days before birth, there was likely insufficient time for IgG seroconversion and placental transfer. IgM, which cannot cross the placenta, can be detected in the blood of neonates with infections during the first weeks of life, [11] but the seroconversion interval for neonates exposed to SARS-CoV-2 is unclear. There are several possible reasons we did not find SARS-CoV-2 RNA in placental tissue. First, the mother presented with bleeding indicating possible damage to placental structures, which may have caused direct viral seeding of the cord blood. Second, we did not examine the entire placenta and may have missed an area of local infection. Finally, the placenta was not preserved for evaluation of RNA until approximately 2 weeks after delivery, which may have caused degradation of viral RNA to an undetectable level. Hematogenous spread of SARS-CoV-2 from mother to fetus or newborn has at least two This case adds to the emerging story of in utero transmission of SARS-CoV-2. Study protocols to identify risk factors for in utero transmission of SARS-CoV-2 should be based on a clear case definition and include serial samples with optimal collection and processing. [12] Clinical recommendations should take into account that hematogenous spread is uncommon but possible, that infants may be infected with SARS-CoV-2 with few or no symptoms, and that newborn NP secretions may not contain detectable virus until after 48 hours. M a n u s c r i p t 12 A c c e p t e d M a n u s c r i p t 15 Figure 1 Antibodies in infants born to mothers with COVID-19 pneumonia Severe COVID-19 during pregnancy and possible vertical transmission Probable congenital SARS-CoV-2 infection in a neonate born to a woman with active SARS-CoV-2 infection Transplacental transmission of SARS-CoV-2 infection Neonatal early-onset infection with SARS-CoV-2 in 33 neonates born to mothers with COVID-19 in Wuhan, China SARS-CoV-2 infection of the placenta Sensitivity in detection of antibodies to nucleocapsid and spike proteins of severe acute respiratory syndrome coronavirus 2 in patients with coronavirus disease 2019 A simplified immunoprecipitation method for quantitatively measuring antibody responses in clinical sera samples by using mammalian-produced Renilla luciferase-antigen fusion proteins Classification system and case definition for SARS-CoV-2 infection in pregnant women, fetuses, and neonates Vertical transmission of SARS-CoV-2: what is the optimal definition? Newborn screening for congenital infectious diseases SARS-COV-2 maternal-child transmission: can it occur before delivery and how do we prove it? We thank William A. Meyer III, Ph.D., D(ABMM), MLS(ASCP) CM Quest Diagnostics, Baltimore, MD, for assistance in determining cycle times for patient samples run in his laboratory. We thank Dr. Stefania Pittaluga at the National Cancer Institute for her laboratory's contribution of RNAscope assays and for her insightful comments about the work. A c c e p t e d M a n u s c r i p t A c c e p t e d M a n u s c r i p t 11