COVID-19 in pregnancy may significantly affect fetal growth

Objective. Whilst most cases of COVID-19 in pregnancy evolve uneventfully, some others have a poor outcome, such as preterm birth and HDP. The effect of COVID-19 on fetal growth still has to be addressed and appears controversial. Our study aims to focus on fetal growth velocity across the trimesters in pregnancy affected with COVID-19. Materials and Methods. This is a multicentric prospective observational study on data from COVID-19 pregnancies referred to the centers of Careggi University Hospital and S. Stefano Hospital in Prato from 2020 to 2022, included in the local branch of ItOSS surveillance. Fetal growth was evaluated across the three trimesters of pregnancy by abdominal circumference (AC) and expected fetal weight (EFW). Both AC, and EFW plus birthweight were used to calculate growth velocity (Vannuccini et al., 2017). Results. Data from a cohort of 211 cases was collected. The majority (80%) of COVID-19 cases occurred in the third trimester. Of note, the percentage of gestational diabetes (12.3%) and preterm births (7.1%) was comparable to the general population. Mean birthweight was 3259 +/- 509 g. The percentage of cesarean sections was acceptable (13.7%). Concerning the velocity of growth, AC decreased from the second to third trimester to reach values < 10th centile in 24% of cases and < 5th percentile in 17% of cases, even in presence of 2% of hypertensive disorders (HDP). Fetal growth restriction according to Gordjin et al. (2015) was antenatally identified in 4.7% of cases. Fetal growth > 95th centile occurred in 5.7% of cases. At birth, the cumulative percentage of small for gestational age newborns defined as birth weight < 2500 g resulted 5.2%. A significant percentage of newborns required NICU assistance (7.8%). Conclusions. Regardless of the association and prevalence of preterm birth, SGA and HDP in pregnant women, fetal growth appears to be affected by COVID-19 with a higher incidence of impaired growth velocity compared to the general population.

Third level care experience during SARS-CoV-2 pandemic

Objective. As the SARS-CoV-2 Pandemic has widely changed pregnancy experience and assessment, the inpatient and outpatient services have had to be re-organized. Since March 2020, Careggi University Hospital (CUH) has provided a dedicated COVID-pathway: spaces for women with unknown swab status and a COVID-19 ward delivery room. The aim of this study is to analyze the inpatient and outpatient COVID-19 related activities in CUH. Materials and Methods. We prospectively collected data from consecutive COVID-19 pregnancies referred from 2020 to 2022, included in the local branch of the ItOSS surveillance. All patients experienced COVID-19 in pregnancy at various stages of severity and gestational ages. Results. From March 2020 to June 2022, 165 COVID-19 deliveries occurred (169 newborns), while 16 pregnant positive women were admitted without delivering. A single emergency C-section (CS) was performed because of Sars-CoV-2 related ARDS, 15 women experienced serious maternal morbidity and 5 needed ECMO. A single maternal death occurred four months after delivery (C-section). Considering ECMO supported cases during pregnancy or postpartum, the first one tested positive for COVID-19 during the second trimester. She developed ARDS and required ECMO for 38 days. She was discharged in good general conditions and a CS at term was performed following obstetric indication. The second patient developed COVID-19-related ARDS at 28 weeks of gestation and experienced a precipitous vaginal delivery at 31 weeks+6 days of gestation while on ECMO. She was discharged 1 month later in good general conditions. The third patient was an obese (BMI 38) 43-year-old woman who had performed an IVF with embryo donation;she tested positive at 38 weeks+2 days of gestation. A CS was performed because of the worsening of her condition. After the delivery she was admitted in ICU and she underwent ECMO. She died 143 days after the CS by sepsis and multiple organ failure (MOF). For all these pregnancies neonatal outcomes were positive. No perinatal death occurred and only one baby tested positive for SARS-CoV-2 infection at nasal swab sampling (case 3). The anesthesiology team performed neuroassial analgesia intrapartum in all the positive women who needed/requested it. Monoclonal Antibodies (mAbs) have been widely used to treat mild to moderate COVID-19 outpatients (NIH and RCOG recommendations) at risk for developing severe disease. Regarding this specifical therapy, an essential role in the management of the pregnant outpatient was played by the Infectious Disease Department. All patients above 28 weeks requiring hospitalization received LMWH prophylaxis, which was administrated under 28 weeks only in presence of additional risk factors (obesity, IVF, etc.). All new mothers received a ten days LMWH prophylaxis. On the outpatient side, we performed 22 teleconsultations, 43 obstetric ultrasounds (including I trimester screening), 90 obstetric checks with clinical evaluation and home therapy management, 32 fetal monitoring and 47 naso-pharingeal swabs. Conclusions. At Careggi Hospital Maternal Department an extensive re-organization of inpatient and outpatient services has been performed in order to guarantee good practice and management of all pregnant women during the SARS-CoV-2 pandemic. This was only possible thanks to a wide multidisciplinary group which enhanced every professional.

Oral Epithelial Cells Expressing Low or Undetectable Levels of Human Angiotensin-Converting Enzyme 2 are Susceptible SARS-CoV-2Virus Infection in vitro (preprint)

The oral cavity is thought to be one of the portals for SARS-CoV-2 entry. Because there is limited evidence of active oral infection by SARS-CoV-2 viruses, we assessed the capacity of SARS-CoV-2 to infect and replicate in oral epithelial cells. Oral gingival epithelial cells (hTERT TIGKs), salivary gland epithelial cells (A-253), and oral buccal epithelial cells (TR146), which occupy different regions of the oral cavity, were challenged with replication competent SARS-CoV-2 viruses and with pseudo-typed viruses expressing SARS-CoV-2 spike proteins. All oral epithelial cells expressing undetectable or low levels of human angiotensin-converting enzyme 2 (hACE2) but high levels of the alternative receptor CD147 were susceptible to SARS-CoV-2 infection. Viral dynamics in hTERT TIGKs were different from those in A-253 and TR146 cells. For example, levels of viral transcripts were sustained in hTERT TIGKs but were significantly decreased in A-253 and TR146 cells at day 3 after infection. Analysis of oral epithelial cells infected by replication competent SARS-CoV-2 viruses expressing GFP showed that the signals of GFP and SARS-CoV-2 mRNAs were not evenly distributed. Taken together, our results demonstrated oral epithelial cells were susceptible to SARS-CoV-2 viruses despite of low or undetectable levels of hACE2, suggesting that alternative receptors contribute to SARS-CoV-2 infection and may be considered for development of future vaccines and therapeutics.

Beyond emergency politics: Carl Schmitt's substantive constitutionalism

This article problematizes the recent comeback of the exceptionalist jargon as it is conjured by both critics and sympathizers. While in the last decades governments across the globe had recourse to emergency measures to cope with far-reaching emergencies, from terrorism to the COVID-19 pandemic, the received view has it that political power takes advantage of states of emergency as they put themselves in the position to circumvent constitutional limitations. Carl Schmitt is claimed to be the major advocate of this conception of emergency politics in that he elaborated on the concept of the state of exception as the heart of the state political power. This article contends that the received view is doubly wrong. First, soon after his espousal of exceptionalism, Schmitt realized that emergency legislation is an ineffective and costly governmental device that should be transitory and is as unstable as the crisis it is meant to overcome. Second, the received view neglects how Schmitt came to his model of ultraconservative substantive constitutionalism as he maintained that the main task of politics is to protect the normative life of a limited set of state-sponsored institutions as well as the substantive contents they produce. © The Author(s) 2023.

Development of a novel human CD147 knock-in NSG mouse model to test SARS-CoV-2 viral infection.

BACKGROUND: An animal model that can mimic the SARS-CoV-2 infection in humans is critical to understanding the rapidly evolving SARS-CoV-2 virus and for development of prophylactic and therapeutic strategies to combat emerging mutants. Studies show that the spike proteins of SARS-CoV and SARS-CoV-2 bind to human angiotensin-converting enzyme 2 (hACE2, a well-recognized, functional receptor for SARS-CoV and SARS-CoV-2) to mediate viral entry. Several hACE2 transgenic (hACE2Tg) mouse models are being widely used, which are clearly invaluable. However, the hACE2Tg mouse model cannot fully explain: (1) low expression of ACE2 observed in human lung and heart, but lung or heart failure occurs frequently in severe COVID-19 patients; (2) low expression of ACE2 on immune cells, but lymphocytopenia occurs frequently in COVID-19 patients; and (3) hACE2Tg mice do not mimic the natural course of SARS-CoV-2 infection in humans. Moreover, one of most outstanding features of coronavirus infection is the diversity of receptor usage, which includes the newly proposed human CD147 (hCD147) as a possible co-receptor for SARS-CoV-2 entry. It is still debatable whether CD147 can serve as a functional receptor for SARS-CoV-2 infection or entry. RESULTS: Here we successfully generated a hCD147 knock-in mouse model (hCD147KI) in the NOD-scid IL2Rgammanull (NSG) background. In this hCD147KI-NSG mouse model, the hCD147 genetic sequence was placed downstream of the endogenous mouse promoter for mouse CD147 (mCD147), which creates an in vivo model that may better recapitulate physiological expression of hCD147 proteins at the molecular level compared to the existing and well-studied K18-hACE2-B6 (JAX) model. In addition, the hCD147KI-NSG mouse model allows further study of SARS-CoV-2 in the immunodeficiency condition which may assist our understanding of this virus in the context of high-risk populations in immunosuppressed states. Our data show (1) the human CD147 protein is expressed in various organs (including bronchiolar epithelial cells) in hCD147KI-NSG mice by immunohistochemical staining and flow cytometry; (2) hCD147KI-NSG mice are marginally sensitive to SARS-CoV-2 infection compared to WT-NSG littermates characterized by increased viral copies by qRT-PCR and moderate body weight decline compared to baseline; (3) a significant increase in leukocytes in the lungs of hCD147KI-NSG mice, compared to infected WT-NSG mice. CONCLUSIONS: hCD147KI-NSG mice are more sensitive to COVID-19 infection compared to WT-NSG mice. The hCD147KI-NSG mouse model can serve as an additional animal model for further interrogation whether CD147 serve as an independent functional receptor or accessory receptor for SARS-CoV-2 entry and immune responses.

Multiplex PCR Assays for Identifying all Major Severe Acute Respiratory Syndrome Coronavirus 2 Variants.

Variants of concern (VOC) of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), including alpha, beta, gamma, delta, and omicron, threaten to prolong the pandemic, leading to more global morbidity and mortality. Genome sequencing is the mainstay of tracking the evolution of the virus, but is costly, slow, and not easily accessible. Multiplex quantitative RT-PCR assays for SARS-CoV-2 have been developed that identify all VOCs as well as other mutations of interest in the viral genome, nine mutations in total, using single-nucleotide discriminating molecular beacons. The presented variant molecular beacon assays showed a limit of detection of 50 copies of viral RNA, with 100% specificity. Twenty-six SARS-CoV-2-positive patient samples were blinded and tested using a two-tube assay. When testing patient samples, the assay was in full agreement with results from deep sequencing with a sensitivity and specificity of 100% (26 of 26). We have used our design methodology to rapidly design an assay that detects the new omicron variant. This omicron assay was used to accurately identify this variant in 17 of 33 additional patient samples. These quantitative RT-PCR assays identify all currently circulating VOCs of SARS-CoV-2, as well as other important mutations in the spike protein coding sequence. These assays can be easily implemented on broadly available five-color thermal cyclers and will help track the spread of these variants.

Development of a Novel Human CD147 Knock-in NSG Mouse Model to Test SARS-CoV-2 Viral Infection (preprint)

Background: An animal model that can mimic the SARS-CoV-2 infection in humans is critical to understanding the rapidly evolving SARS-CoV-2 virus and for development of prophylactic and therapeutic strategies to combat emerging mutants. Studies show that the spike proteins of SARS-CoV and SARS-CoV-2 bind to human angiotensin-converting enzyme 2 (hACE2, a well-recognized, functional receptor for SARS-CoV and SARS-CoV-2) to mediate viral entry. Several hACE2 transgenic (hACE2Tg) mouse models are being widely used, which are clearly invaluable. However, the hACE2Tg mouse model cannot fully explain: 1) low expression of ACE2 observed in human lung and heart, but lung or heart failure occurs frequently in severe COVID-19 patients; 2) low expression of ACE2 on immune cells, but lymphocytopenia occurs frequently in COVID-19 patients; and 3) hACE2Tg mice do not mimic the natural course of SARS-CoV-2 infection in humans. Moreover, one of most outstanding features of coronavirus infection is the diversity of receptor usage, which includes the newly proposed human CD147 (hCD147) as a possible co-receptor for SARS-CoV-2 entry. It is still debatable whether CD147 can serve as a functional receptor for SARS-CoV-2 infection or entry.Results: Here we successfully generated a hCD147 knock-in mouse model (hCD147KI) in the NOD-scid IL2Rgammanull (NSG) background. In this hCD147KI-NSG mouse model, the hCD147 genetic sequence was placed downstream of the endogenous mouse promoter for mouse CD147 (mCD147), which creates an in vivo model that may better recapitulate physiological expression of hCD147 proteins at the molecular level compared to the existing and well-studied K18-hACE2-B6 (JAX) model. In addition, the hCD147KI-NSG mouse model allows further study of SARS-CoV-2 in the immunodeficiency condition which may assist our understanding of this virus in the context of high-risk populations in immunosuppressed states. Our data show 1) the human CD147 protein is expressed in various organs (including bronchiolar epithelial cells) in hCD147KI-NSG mice by immunohistochemical staining and flow cytometry; 2) hCD147KI-NSG mice are marginally sensitive to SARS-CoV-2 infection compared to WT-NSG littermates characterized by increased viral copies by qRT-PCR and moderate body weight decline compared to baseline; 3) a significant increase in leukocytes in the lungs of hCD147KI-NSG mice, compared to infected WT-NSG mice.Conclusions: hCD147KI-NSG mice are more sensitive to COVID-19 infection compared to WT-NSG mice. The hCD147KI-NSG mouse model can serve as an additional animal model for further interrogation whether CD147 serve as an independent functional receptor or accessory receptor for SARS-CoV-2 entry and immune responses.

A Multiplex PCR Assay for Identifying All Major SARS-CoV-2 Variants (preprint)

Background Variants of Concern (VOC) of SARS-CoV-2, including the Alpha, Beta, Gamma, and Delta, threaten to prolong the pandemic leading to more global morbidity and mortality. Genome sequencing is the mainstay of tracking the development and evolution of the virus, but is costly, slow, and not easily accessible. Methods A multiplex qRT-PCR assay for SARS-CoV-2 was developed, which identifies all VOC as well as other mutations of interest in the viral genome, eight mutations total, using single nucleotide discriminating molecular beacons in a two-tube assay. The sensitivity and specificity of the assay was tested using in vitro-transcribed targets. Twenty-six SARS-CoV-2 positive patient samples were blinded, then tested using this assay and compared with deep sequencing results. Findings The presented variant molecular beacon assay showed high accuracy when testing in vitro-transcribed targets, down to a limit of detection of five copies of the viral RNA, with 100% specificity. When testing patient samples, the assay was in full agreement with results from deep sequencing with a sensitivity and specificity of 100% (26/26). Using this accurate genotyping, the SARS-CoV-2 samples were classified as the appropriate variants, and of the twenty-six samples two were identified as VOC Alpha, eight as VOC Delta, and two as Epsilon. Interpretation We have developed a qRT-PCR assay for the identification of currently circulating VOC of SARS-CoV-2 as well as other important mutations in its Spike protein coding sequence. This assay can be easily implemented on broadly available five-color thermal cyclers and will help track the spread of these variants.

Clinical impact of COVID-19 on patients with cancer (CCC19): a cohort study.

BACKGROUND: Data on patients with COVID-19 who have cancer are lacking. Here we characterise the outcomes of a cohort of patients with cancer and COVID-19 and identify potential prognostic factors for mortality and severe illness. METHODS: In this cohort study, we collected de-identified data on patients with active or previous malignancy, aged 18 years and older, with confirmed severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection from the USA, Canada, and Spain from the COVID-19 and Cancer Consortium (CCC19) database for whom baseline data were added between March 17 and April 16, 2020. We collected data on baseline clinical conditions, medications, cancer diagnosis and treatment, and COVID-19 disease course. The primary endpoint was all-cause mortality within 30 days of diagnosis of COVID-19. We assessed the association between the outcome and potential prognostic variables using logistic regression analyses, partially adjusted for age, sex, smoking status, and obesity. This study is registered with ClinicalTrials.gov, NCT04354701, and is ongoing. FINDINGS: Of 1035 records entered into the CCC19 database during the study period, 928 patients met inclusion criteria for our analysis. Median age was 66 years (IQR 57-76), 279 (30%) were aged 75 years or older, and 468 (50%) patients were male. The most prevalent malignancies were breast (191 [21%]) and prostate (152 [16%]). 366 (39%) patients were on active anticancer treatment, and 396 (43%) had active (measurable) cancer. At analysis (May 7, 2020), 121 (13%) patients had died. In logistic regression analysis, independent factors associated with increased 30-day mortality, after partial adjustment, were: increased age (per 10 years; partially adjusted odds ratio 1·84, 95% CI 1·53-2·21), male sex (1·63, 1·07-2·48), smoking status (former smoker vs never smoked: 1·60, 1·03-2·47), number of comorbidities (two vs none: 4·50, 1·33-15·28), Eastern Cooperative Oncology Group performance status of 2 or higher (status of 2 vs 0 or 1: 3·89, 2·11-7·18), active cancer (progressing vs remission: 5·20, 2·77-9·77), and receipt of azithromycin plus hydroxychloroquine (vs treatment with neither: 2·93, 1·79-4·79; confounding by indication cannot be excluded). Compared with residence in the US-Northeast, residence in Canada (0·24, 0·07-0·84) or the US-Midwest (0·50, 0·28-0·90) were associated with decreased 30-day all-cause mortality. Race and ethnicity, obesity status, cancer type, type of anticancer therapy, and recent surgery were not associated with mortality. INTERPRETATION: Among patients with cancer and COVID-19, 30-day all-cause mortality was high and associated with general risk factors and risk factors unique to patients with cancer. Longer follow-up is needed to better understand the effect of COVID-19 on outcomes in patients with cancer, including the ability to continue specific cancer treatments. FUNDING: American Cancer Society, National Institutes of Health, and Hope Foundation for Cancer Research.