Pregnancy and neonatal outcomes of COVID-19: The PAN-COVID study

Objective To assess perinatal outcomes for pregnancies affected by suspected or confirmed SARS-CoV-2 infection. Methods Prospective, web-based registry. Pregnant women were invited to participate if they had suspected or confirmed SARS-CoV-2 infection between 1st January 2020 and 31st March 2021 to assess the impact of infection on maternal and perinatal outcomes including miscarriage, stillbirth, fetal growth restriction, pre-term birth and transmission to the infant. Results Between April 2020 and March 2021, the study recruited 8239 participants who had suspected or confirmed SARs-CoV-2 infection episodes in pregnancy between January 2020 and March 2021. Maternal death affected 14/8197 (0.2%) participants, 176/8187 (2.2%) of participants required ventilatory support. Pre-eclampsia affected 389/8189 (4.8%) participants, eclampsia was reported in 40/ 8024 (0.5%) of all participants. Stillbirth affected 35/8187 (0.4 %) participants. In participants delivering within 2 weeks of delivery 21/2686 (0.8 %) were affected by stillbirth compared with 8/4596 (0.2 %) delivering ≥ 2 weeks after infection (95 % CI 0.3–1.0). SGA affected 744/7696 (9.3 %) of livebirths, FGR affected 360/8175 (4.4 %) of all pregnancies. Pre-term birth occurred in 922/8066 (11.5%), the majority of these were indicated pre-term births, 220/7987 (2.8%) participants experienced spontaneous pre-term births. Early neonatal deaths affected 11/8050 livebirths. Of all neonates, 80/7993 (1.0%) tested positive for SARS-CoV-2. Conclusions Infection was associated with indicated pre-term birth, most commonly for fetal compromise. The overall proportions of women affected by SGA and FGR were not higher than expected, however there was the proportion affected by stillbirth in participants delivering within 2 weeks of infection was significantly higher than those delivering ≥ 2 weeks after infection. We suggest that clinicians’ threshold for delivery should be low if there are concerns with fetal movements or fetal heart rate monitoring in the time around infection

Neurovirulence of H5N1 infection in ferrets is mediated by multifocal replication in distinct permissive neuronal cell regions.

Highly pathogenic avian influenza A (HPAI), subtype H5N1, remains an emergent threat to the human population. While respiratory disease is a hallmark of influenza infection, H5N1 has a high incidence of neurological sequelae in many animal species and sporadically in humans. We elucidate the temporal/spatial infection of H5N1 in the brain of ferrets following a low dose, intranasal infection of two HPAI strains of varying neurovirulence and lethality. A/Vietnam/1203/2004 (VN1203) induced mortality in 100% of infected ferrets while A/Hong Kong/483/1997 (HK483) induced lethality in only 20% of ferrets, with death occurring significantly later following infection. Neurological signs were prominent in VN1203 infection, but not HK483, with seizures observed three days post challenge and torticollis or paresis at later time points. VN1203 and HK483 replication kinetics were similar in primary differentiated ferret nasal turbinate cells, and similar viral titers were measured in the nasal turbinates of infected ferrets. Pulmonary viral titers were not different between strains and pathological findings in the lungs were similar in severity. VN1203 replicated to high titers in the olfactory bulb, cerebral cortex, and brain stem; whereas HK483 was not recovered in these tissues. VN1203 was identified adjacent to and within the olfactory nerve tract, and multifocal infection was observed throughout the frontal cortex and cerebrum. VN1203 was also detected throughout the cerebellum, specifically in Purkinje cells and regions that coordinate voluntary movements. These findings suggest the increased lethality of VN1203 in ferrets is due to increased replication in brain regions important in higher order function and explains the neurological signs observed during H5N1 neurovirulence

VN1203 was more lethal than HK483 in ferrets despite similar weight loss in non-survivors.

<p><b>A.</b> Survival of ferrets intranasally challenged with allantoic fluid (N = 2), A/Vietnam/1203/2004 (VN1203) (N = 10) or A/Hong Kong/483/1997 (HK483) N = 10, ***, p<.0001. <b>B.</b> Mean ± SEM of bodyweight of ferrets instilled with VN1203 or HK483. N = 8 HK483 survivors, N = 2 HK483 non-survivors, N = 10 VN1203 non-survivors, N = 2 control.</p

VN1203 induced significant pathology despite similar nasal turbinate titers in ferrets infected with either virus.

<p><b>A.</b> H&E stained section a primary differentiated ferret nasal turbinate cell monolayer. <b>B.</b> Primary differentiated ferret nasal turbinate cells were infected with VN1203 or HK483 at an MOI of 0.001. Mean ± SEM of N = 4 samples per virus at each time point. <b>C.</b> Viral titers of homogenized nasal turbinates were graphed as mean ± SEM for VN1203 or HK483 infected ferrets. <b>D–I</b>. Representative samples of nasal turbinate tissue of ferrets instilled with allantoic fluid (<b>D,G</b>), HK483 (<b>E,H</b>), or VN1203 (<b>F,I</b>). <b>D–F</b> were stained with H&E, <b>G–I</b> were stained with anti-avian influenza NP and counterstained with Luxol fast blue. <b>F.</b> # indicates inflammatory cells infiltrating the underlying stroma. <b>H,I.</b> Arrows point to H5N1 infected cells. Scale bars: <b>A.</b> 50 µm; <b>D–F</b>, 300 µm; <b>G–I</b>, 100 µm.</p

VN1203 infection resulted in multifocal infection in the cerebral cortex of ferrets.

<p>Representative sections of the cerebrum of ferrets intranasally instilled with VN1203 (<b>A, C–E</b>) and HK483 (<b>B</b>). Viral antigen was detected with IMGENEX-5187A for avian influenza A NP and visualized with DAB. Brown stain indicates the presence of virus. Counterstained with Luxol fast blue. Scale bars represent 10 mm. cc: corpus collosum, ic: internal capsule, ot: optical tract, h: hippocampus, lv: lateral ventricle, oc: olfactory cortex, cp: cerebral peduncle.</p

VN1203 infected and replicated in Purkinje cells and deep cerebellar nuclei of the cerebellum.

<p>Representative sections of the cerebellum of ferrets instilled with allantoic fluid (<b>A,B,G,H</b>), HK483 (<b>C,D,I,J</b>), VN1203 (<b>E,F,K,L</b>). Viral antigen was detected with IMGENEX 5187-A for avian influenza A NP and visualized with DAB. Brown stain indicates the presence of virus. Counterstained with Luxol fast blue. (<b>B–F</b>) higher magnification of boxes in A–E respectively. H) White arrow indicates molecular layer, black arrow indicates granule layer, black arrowhead indicates a Purkinje cell, white arrowhead indicates Purkinje cell axons. <b>L</b>) Arrow indicates infected granule cell and arrowhead indicates infected Purkinje cell. Scale bars represent: <b>A,C,E,G,I,K</b>) 2 mm; <b>B,D,F,H,J,L</b>) 300 µm.</p

Viral titers and histopathology were similar in the lung of H5N1 infected ferrets.

<p><b>A.</b> Viral titers and <b>B.</b> viral RNA was measured from four sections of the lung per ferret. <b>A,B</b>) Data represents the mean ± SEM from four lung regions of N = 1, VN1203 4 dpi, N = 3, VN1203 6 dpi, N = 6, HK483 4, 6, and 8 dpi. Dorsal view of ferret lung following intranasal instillation with allantoic fluid (<b>C</b>), HK483 (<b>D</b>), VN1203 (<b>E</b>). Lung sections stained with H&E of ferrets instilled with allantoic fluid (<b>F</b>), HK483 (<b>G</b>), VN1203 (<b>H</b>). <b>F–H</b>) scale bars = 600 µm.</p

Clinical observations in ferrets post-challenge with VN1203 or HK483.

<p>Number of ferrets with clinical symptoms (# with symptoms/total # in group).</p

VN1203 infection in the ferret brain was multifocal and evident in the olfactory tract.

<p>Representative sections from the frontal lobe of ferrets intranasally instilled with allantoic fluid (<b>A,D</b>), on: olfactory nucleus, rf: rhinal fissure, HK483 (<b>B,E</b>) or VN1203 (<b>C,F</b>) were stained for viral antigen with IMGENEX 5187-A for avian influenza A NP and visualized with DAB. Brown stain indicates the presence of virus. Counterstained with Luxol fast blue. Scale bars: <b>A–C</b>) 5 mm, <b>D–F</b>) 300 µm.</p