Perinatal outcomes after regional analgesia during labour.

BACKGROUND: Regional analgesia is a common and effective form of in-labour analgesia. However, there are concerns whether it is associated with adverse maternal and neonatal outcomes. AIMS: To examine the association between regional analgesia and maternal and neonatal outcomes. MATERIALS AND METHODS: A retrospective population-based cohort study of singleton term births in Victoria, Australia, between 2014 and 2020. Women who received regional analgesia were compared with women who did not. Multivariable logistic and linear regressions were used. RESULTS: There were 107 013 women who received regional analgesia and 214 416 women who did not. Compared to women who did not receive regional analgesia, regional analgesia was associated with an increased risk of instrumental birth (adjusted odds ratio (aOR) = 3.59, 95% CI: 3.52-3.67), caesarean section (aOR = 2.30, 95% CI: 2.24-2.35), longer duration of the second stage of labour (ß coefficient = 26.6 min, 95% CI: 26.3-27.0), Apgar score below seven at five minutes (aOR = 1.30, 95% CI: 1.21-1.39), need for neonatal resuscitation (aOR = 1.44, 95% CI: 1.40-1.48), need for formula in hospital (aOR = 1.68, 95% CI: 1.65-1.72), and the last feed before discharge not exclusively from the breast (aOR = 1.59, 95% CI: 1.56-1.62). CONCLUSION: Regional analgesia use in labour was associated with adverse maternal and neonatal outcomes. These findings may add to the risk-benefit discussion regarding regional analgesia for pain relief and highlight the importance of shared decision-making. Further large prospective studies and randomised controlled trials will be useful.

Induction of labor and cesarean birth in lower-risk nulliparous women at term: A retrospective cohort study.

OBJECTIVE: To evaluate whether induction of labor (IOL) is associated with cesarean birth (CB) and perinatal mortality in uncomplicated first births at term compared with expectant management outside the confines of a randomized controlled trial. METHODS: Population-based retrospective cohort study of all births in Victoria, Australia, from 2010 to 2018 (n = 640,191). Preliminary analysis compared IOL at 37 weeks with expectant management at that gestational age and beyond for uncomplicated pregnancies. Similar comparisons were made for IOL at 38, 39, 40, and 41 weeks of gestation and expectant management. The primary analysis repeated these comparisons, limiting the population to nulliparous women with uncomplicated pregnancies and excluding those with a medical indication for IOL. We compared perinatal mortality between groups using Chi-square tests and multivariable logistic regression for all other comparisons. Adjusted odds ratios and 99% confidence intervals were reported. p < 0.01 denoted statistical significance. RESULTS: Among nulliparous, uncomplicated pregnancies at ≥37 weeks of gestation in Victoria, IOL increased from 24.6% in 2010 to 30.0% in 2018 (p < 0.001). In contrast to the preliminary analysis, the primary analysis showed that IOL in lower-risk nulliparous women was associated with increased odds of CB when performed at 38 (aOR 1.23(1.13-1.32)), 39 (aOR 1.31(1.23-1.40)), 40 (aOR 1.42(1.35-1.50)), and 41 weeks of gestation (aOR 1.43(1.35-1.51)). Perinatal mortality was rare in both groups and non-significantly lower in the induced group at most gestations. DISCUSSION: For lower-risk nulliparous women, the odds of CB increased with IOL from 38 weeks of gestation, along with decreased odds of perinatal mortality at 41 weeks only.

Objective measures of smoking and caffeine intake and the risk of adverse pregnancy outcomes.

BACKGROUND: In pregnancy, women are encouraged to cease smoking and limit caffeine intake. We employed objective definitions of smoking and caffeine exposure to assess their association with adverse outcomes. METHODS: We conducted a case cohort study within the Pregnancy Outcome Prediction study to analyse maternal serum metabolomics in samples from 12, 20, 28 and 36 weeks of gestational age. Objective smoking status was defined based on detectable cotinine levels at each time point and objective caffeine exposure was based on tertiles of paraxanthine levels at each time point. We used logistic and linear regression to examine the association between cotinine, paraxanthine and the risk of pre-eclampsia, spontaneous pre-term birth (sPTB), fetal growth restriction (FGR), gestational diabetes mellitus and birthweight. RESULTS: There were 914 and 915 women in the smoking and caffeine analyses, respectively. Compared with no exposure to smoking, consistent exposure to smoking was associated with an increased risk of sPTB [adjusted odds ratio (aOR) = 2.58, 95% CI: 1.14 to 5.85)] and FGR (aOR = 4.07, 95% CI: 2.14 to 7.74) and lower birthweight (ß = -387 g, 95% CI: -622 g to -153 g). On univariate analysis, consistently high levels of paraxanthine were associated with an increased risk of FGR but that association attenuated when adjusting for maternal characteristics and objective-but not self-reported-smoking status. CONCLUSIONS: Based on objective data, consistent exposure to smoking throughout pregnancy was strongly associated with sPTB and FGR. High levels of paraxanthine were not independently associated with any of the studied outcomes and were confounded by smoking.

Elective induction of labour at full-term gestations and childhood school outcomes.

AIM: To explore the association between induction of labour at full-term gestations in low-risk nulliparous women and childhood school outcomes. METHODS: A retrospective whole-of-population cohort study linking perinatal data to educational test scores at grades 3, 5 and 7 in Victoria, Australia. Low-risk nulliparous women with singleton pregnancies induced at 39 and 40 weeks without a medical indication were compared to those expectantly managed from that week of gestation. Multivariable logistic regressions were used as well as generalised estimating equations on longitudinal data. RESULTS: At 39 weeks, there were 3687 and 103 164 infants in the induction and expectant arms, respectively. At 40 weeks' gestation, there were 7914 and 70 280 infants, respectively. Infants born to nulliparous women induced at 39 weeks' gestation had significantly poorer educational outcomes at grade 3 (adjusted odds ratio (aOR) = 1.39, 95% confidence interval (CI): 1.13-1.70) but not grades 5 (aOR = 1.05, 95% CI: 0.84-1.33) and 7 (aOR = 1.07, 95% CI: 0.81-1.40) compared to those expectantly managed. Infants born to nulliparous women induced at 40 weeks had comparable educational outcomes at grade 3 (aOR = 1.06, 95% CI: 0.90-1.25) but poorer educational outcomes at grades 5 (aOR = 1.23, 95% CI: 1.05-1.43) and 7 (aOR = 1.23, 95% CI: 1.03-1.47) compared to those expectantly managed. CONCLUSIONS: There were inconsistent associations between elective induction of labour at full-term gestations in low-risk nulliparous women and impaired childhood school outcomes.

Childhood school outcomes for infants born to women with hypertensive disorders during pregnancy.

BACKGROUND: To investigate the childhood school outcomes for infants born to women with hypertensive disorders during pregnancy. STUDY DESIGN: A retrospective population-based cohort study linking perinatal data from 2003 to 2013 to developmental scores at preparatory school and educational scores at school grades 3, 5, and 7 in Victoria, Australia. Exposures of interest were the presence of hypertensive disorders during pregnancy and iatrogenic delivery for preeclampsia. Multivariable logistic regression and generalised estimating equation models were employed. RESULTS: In total, 682,386 births ≥32 weeks' gestation were linked to 175,665 child developmental results and 412,834 with at least one educational result. Compared to infants born to women without a hypertensive disorder, infants born to women with a hypertensive disorder had no increased risk of poorer developmental outcomes at school entry but a significantly increased risk of poorer educational outcomes across grades 3, 5, and 7. Compared to infants born to women without preeclampsia, infants born to women iatrogenically delivered for preeclampsia had no increased risk of poorer developmental outcomes (aOR = 1.12, 95 % CI: 0.98-1.28) but a significantly increased risk of poorer educational outcomes at grades 3 (aOR = 1.23, 95 % CI: 1.09-1.38), 5 (aOR = 1.27, 95 % CI: 1.13-1.43), and 7 (aOR = 1.24, 95 % CI: 1.09-1.43). CONCLUSION: The presence of maternal hypertension in pregnancy, particularly where preeclampsia was the indication for iatrogenic delivery, is associated with impaired school educational outcomes.

The association between intrapartum interventions and immediate and ongoing breastfeeding outcomes: an Australian retrospective population-based cohort study.

BACKGROUND: The use of intrapartum interventions is becoming increasingly common globally. Interventions during birth, including caesarean section (CS), epidural analgesia and synthetic oxytocin infusion, can be important in optimizing obstetric care, but have the potential to impact breastfeeding. This study aimed to identify whether women who have certain intrapartum interventions have greater odds of unfavourable breastfeeding outcomes, both the immediate post-partum period and in the months after birth. METHODS: This was a population-based cohort study of singleton livebirths at ≥37 weeks' gestation between 2010 and 2018 in Victoria, Australia using routinely-collected state-wide data from the Victorian Perinatal Data Collection (VPDC) and the Child Development Information System (CDIS). The interventions included were pre-labour CS, in-labour CS, epidural analgesia, and synthetic oxytocin infusion (augmentation and/or induction of labour). Outcomes were formula supplementation in hospital, method of last feed before hospital discharge and breastfeeding status at 3-months and 6-months. Descriptive statistics and multivariable logistic regression models adjusting for potential confounders were employed. RESULTS: In total, 599,191 women initiated breastfeeding. In-labour CS (aOR 1.96, 95%CI 1.93,1.99), pre-labour CS (aOR 1.75, 95%CI 1.72,1.77), epidural analgesia (aOR 1.45, 95%CI 1.43,1.47) and synthetic oxytocin infusion (aOR 1.24, 95%CI 1.22,1.26) increased the odds of formula supplementation in hospital. Long-term breastfeeding data was available for 105,599 infants. In-labour CS (aOR 0.79, 95%CI 0.76,0.83), pre-labour CS (aOR 0.73, 95%CI 0.71,0.76), epidural analgesia (aOR 0.77, 95%CI 0.75,0.80) and synthetic oxytocin infusion (aOR 0.89, 95%CI 0.86-0.92) decreased the odds of exclusive breastfeeding at 3-months post-partum, which was similar at 6-months. There was a dose-response effect between number of interventions received and odds of each unfavourable breastfeeding outcome. CONCLUSION: Common intrapartum interventions are associated with less favourable breastfeeding outcomes, both in hospital and in the months after birth. This confirms the importance of only undertaking interventions when necessary. When interventions are used intrapartum, an assessment and identification of women at increased risk of early discontinuation of breastfeeding has to be performed. Targeted breastfeeding support for women who have intrapartum interventions, when they wish to breastfeed, is important.

The 5-minute Apgar score and childhood school outcomes.

AIM: To examine the association between Apgar score at 5 min and childhood developmental and educational outcome. METHODS: A population-based data linkage study of births ≥37 weeks' gestation linked to developmental outcomes at preparatory school and educational outcomes at school grades 3, 5 and 7 in Victoria, Australia. Multivariable logistic regressions and generalised estimating equations were used. RESULTS: There were 167,126 singleton infants with developmental results and 392,933 singleton infants with at least one educational result. There was an inverse relationship between Apgar score at 5 min and poor developmental and educational outcomes, with the worst outcomes among Apgar scores of 0-3. Apgar scores of 7, 8 and 9 were all associated with poorer developmental outcomes (aOR = 1.31, 95% CI: 1.12-1.54; aOR = 1.17, 95% CI: 1.05-1.29; aOR = 1.08, 95% CI: 1.02-1.13 respectively), while Apgar scores of 7 and 8 were associated with poorer educational outcomes at grades 3, 5, and 7. With progression through grades 3, 5, and 7, the extent of the difference in educational outcomes diminished (e.g. for Apgar scores of 0-3: aOR = 3.33, 95% CI: 1.85-6.00 in grade 3 and aOR = 1.49, 95% CI: 0.75-2.96 in grade 7). CONCLUSION: Apgar scores below 10 at 5 min are associated with poorer developmental and educational outcomes in school.

A Standardized Diagnostic Pathway for Suspected Appendicitis in Children Reduces Unnecessary Imaging.

Ultrasound (US) for the diagnosis of acute appendicitis is often nondiagnostic, and additional imaging is required. A standardized approach may reduce unnecessary imaging. Methods: We retrospectively analyzed all patients who had imaging for appendicitis in our emergency department in 2017 and evaluated patient characteristics associated with nondiagnostic US. Using these results, we developed a pediatric appendicitis score (PAS)-based imaging pathway and compared imaging trends prepathway and postpathway implementation. Results: A total of 971 patients received imaging for suspected appendicitis prepathway in 2017. Female sex, obesity, and low/intermediate PAS were significantly associated with nondiagnostic US, but not magnetic resonance imaging (MRI) (P < 0.0001). Nearly one-third of patients received multiple imaging studies (US followed by MRI/computed tomography). As low/intermediate PAS was most strongly associated with a nondiagnostic US on multivariate analysis, we developed a PAS-based imaging stewardship pathway to eliminate imaging in low-PAS patients and reduce the number of patients with an intermediate PAS who received multiple imaging studies by obtaining an MRI as the first-line study. After implementation, only 22 low-PAS patients received imaging (compared with 238 preimplementation), and the proportion of intermediate-PAS patients receiving multiple imaging studies decreased from 31.4% to 13% (P < 0.0001). The cost of imaging per 100 patients increased from $24,255 to $31,082. Conclusion: A PAS-based imaging stewardship pathway reduces unnecessary imaging for suspected appendicitis.

Hypertensive disorders in pregnancy - Trends over eight years: A population-based cohort study.

OBJECTIVE: To describe the incidence and trends of hypertensive disorders of pregnancy and adverse pregnancy outcomes in recent years in Victoria, Australia. DESIGN: Retrospective population-based cohort study, 2010 to 2017. SETTING: State of Victoria, Australia. PARTICIPANTS: Population-based cohort study. MAIN OUTCOME MEASURES: Incidence of hypertensive disorders and its subtypes over time. Composite of major adverse maternal and perinatal outcome. RESULTS: The incidence of hypertensive disorders (n = 36,406/614,524 pregnancies with 624,193 births) and all its subtypes has been stable, (n = 4,192/73,235 = 5.7% in 2010 to 4,601/78,576 = 5.9% in 2017). Compared to no hypertension, hypertensive disorders were associated with medically-initiated birth (aOR 4.70 [4.56, 4.84]), caesarean section (aOR 1.46 [1.43, 1.50]), placental abruption (aOR 1.94 [1.69, 2.22]), maternal intensive care or high-dependency unit admission (aOR 6.80 [6.45, 7.17]), composite of major adverse maternal outcome (aOR 3.87 [3.70, 4.04]), and composite of major adverse perinatal outcome (aOR 1.63 [1.56, 1.70]). The worst maternal and perinatal outcomes were among women with superimposed and early preterm preeclampsia. CONCLUSION: The incidence of all hypertensive disorders in pregnancy has remained stable over time. Early-onset preeclampsia and superimposed preeclampsia were most strongly associated with adverse pregnancy outcomes.

Routine first trimester combined screening for preterm preeclampsia in Australia: A multicenter clinical implementation cohort study.

OBJECTIVE: To assess pregnancy outcomes following first trimester combined screening for preterm preeclampsia in Australia. METHODS: We compared pregnancy outcomes of women with singleton pregnancies who underwent first trimester combined preeclampsia screening with the Fetal Medicine Foundation algorithm between 2014 and 2017 in Melbourne and Sydney, Australia, with those from women who received standard care. The primary outcomes were preterm preeclampsia and screening performance. Effect estimates were presented as risk ratios with 95% confidence intervals. RESULTS: A total of 29 618 women underwent combined screening and 301 566 women received standard care. Women who had combined screening were less likely to have preeclampsia, preterm birth, small neonates, and low Apgar scores than the general population. Women with high-risk results (≥1 in 100) were more likely to develop preterm preeclampsia (2.1% vs. 0.7%, risk ratio [RR] 3.04, 95% CI 2.46-3.77), while low-risk women (risk <1 in 100) had lower rates of preterm preeclampsia (0.2% vs. 0.7%, RR 0.26, 95% CI 0.19-0.35) and other pregnancy complications. Screening detected 65.2% (95% CI 56.4-73.2%) of all preterm preeclampsia cases, with improved performance after adjustment for treatment effect. CONCLUSIONS: First trimester screening for preeclampsia in clinical practice identified a population at high risk of adverse pregnancy outcomes and low-risk women who may be suitable for less intensive antenatal care.

Neointimal Hyperplasia after Carotid Transection and Anastomosis Surgery is Associated with Degradation of Decorin and Platelet Derived Growth Factor Signaling.

OBJECTIVE: Intimal hyperplasia (IH) is the expansion of the vascular intimal region after intervention, which can lead to stenosis and eventual failure of vascular grafts or interventional procedures such as angioplasty or stent placement. Our goals were to investigate the development of IH in a rabbit open surgical model and to evaluate the associated pathophysiological processes involving decorin and the platelet derived growth factor-BB / platelet derived growth factor receptor-ß / mitogen activated protein kinase (PDGF/PDGFR-ß/MAPK) pathway. METHODS: We conducted carotid transection and primary anastomosis on five New Zealand White rabbits to induce IH and examined the associated pathophysiological changes. Tissue was obtained for histological and protein analysis on post-operative day 21 using the contralateral vessel as a control. Intimal medial thickness (IMT) was calculated to measure IH and compared with the unoperated side. Western blot analysis was performed on tissue lysates to determine the expression of decorin core protein, PDGF-BB, PDGFR-ß, and phosphorylated-MAPK (ph-MAPK). Immunofluorescence microscopy was used to assess tissue distribution of matrix metalloproteinase-2 (MMP-2) and phosphorylated-PDGFR-ß (ph-PDGFR-ß). RESULTS: Bilateral carotid arteries were harvested on postoperative day 21. We compared the IMT in operated with unoperated specimens. IMT was significantly elevated in operated arteries vs. unoperated arteries in all 5 animals (148.6 µm +/- 9.09 vs. 103.40 µm +/- 7.08; 135.2 µm +/- 8.30 vs. 92.40 µm +/- 2.35; 203.1 µm +/- 30.23 vs.104.00 µm +/- 4.52; 236.2 µm +/- 27.22 vs. 141.50 µm +/- 9.95; 226.9 µm +/- 11.12 vs. 98.8 µm +/- 3.78). Western blot analysis revealed degradation of decorin protein in the operated tissue, including loss of a 50 kDa band and the appearance of a cleaved fragment at 10 kDa. Decorin and MMP-2 were observed, via immunofluorescence microscopy, in the neointima of the operated vessels. Western blot analysis also revealed increased PDGF-BB, PDGFR-ß, and ph-MAPK levels in operated tissue. Immunofluorescent staining for ph-PDGFR-ß primarily localized to the neointima, indicating increased signaling through PDGF in this region. CONCLUSION: Carotid transection and primary reanastomosis in rabbits induced IH that was associated with MMP-2 activation, degradation of decorin, and activation of the PDGF/PDGFR-ß /MAPK pathway. The findings in this study should lead to further mechanistic evaluation of these pathways to better understand the potential to modify the intimal hyperplastic response to surgery.

Preventing harm: A balance measure for improving the detection of fetal growth restriction.

BACKGROUND: Increasing the detection of fetal growth restriction (FGR), while reducing stillbirth, also leads to unnecessary early intervention, and associated morbidity, for normally grown babies who are incorrectly suspected of FGR. AIMS: We sought to design a balance measure that addresses the specificity of FGR detection. METHODS: A retrospective cohort study on all singleton births ≥32 weeks gestation in 2016 and 2017 in Victoria. We compared two balance measures for the detection of FGR, defined as the proportion of all babies iatrogenically delivered before 39 weeks gestation for suspected FGR that had a birthweight ≥10th centile (balance measure 1) or ≥25th centile (balance measure 2). Hospital level performance on each balance measure was derived and compared to an existing performance measure for severe FGR detection in Victoria. RESULTS: Of the 38 hospitals analysed, 12 (32%) had a favourable performance on an existing indicator of FGR detection, seven (18%) hospitals had a favourable performance on balance measure 1, and 15 (39%) had a favourable performance on balance measure 2. There was a moderate correlation between hospital performance on the existing indicator and on balance measure 1 (r = 0.447, P = 0.005) but not balance measure 2 (r = -0.063, P = 0.71). There was no difference in perinatal mortality between high performing hospitals and low performing hospitals. CONCLUSION: Introducing a balance measure into routine reporting may bring greater awareness to the unintended harm associated with increased detection of FGR.

Does detection of fetal growth restriction improve neonatal outcomes?

AIM: Timely delivery of fetal growth restriction (FGR) is a balance between avoiding stillbirth and minimising prematurity. We sought to assess the neonatal outcomes for babies suspected of FGR, both true and false positives. METHODS: This population cohort study examined all singleton births in Victoria, Australia from 2000 to 2017 (n = 1 231 415). Neonatal morbidities associated with neonatal intensive care unit (NICU) admission were assessed for babies born ≥32 weeks' with severe FGR (<3rd centile) and babies with birthweight ≥10th centile who were iatrogenically delivered for suspected FGR. RESULTS: Babies with severe FGR iatrogenically delivered for suspected FGR were more likely to require NICU admission than babies with severe FGR who were not detected (3.0% vs. 1.1%, P < 0.001). After adjusting for potential confounders, the odds of NICU admission were increased (adjusted odds ratio (aOR) = 3.00, 95% confidence interval = 2.45-3.67; P < 0.001). Rates of NICU admission were also higher in ≥10th centile babies iatrogenically delivered for suspected FGR than for ≥10th centile babies who entered labour spontaneously (1.8% vs. 0.5%, P < 0.001). After adjustments, the odds of NICU admission were increased (aOR = 3.91, 95% confidence interval = 3.40-4.49; P < 0.001). NICU admissions were associated with morbidities related to iatrogenic prematurity. CONCLUSIONS: Detection and planned delivery of FGR reduces stillbirth but may be associated with increased neonatal morbidity related to iatrogenic prematurity.

Institution-initiated text messaging can reduce unplanned emergency department visits after appendectomy.

BACKGROUND/PURPOSE: The purpose of this study was to reduce unplanned Emergency Department (ED) visits for minor complaints in children after appendectomy through proactive institution-driven communication and utilization of telehealth resources. METHODS: We developed a text messaging system to initiate communication with parents of postappendectomy patients and connect them with a telehealth visit or a phone call with a surgical provider as needed. Using descriptive statistics, chi square, and statistical process control analytics, we compared rates of postoperative ED visits for the 8 months pre- and post-implementation of the messaging system and summarized the feedback we received from patients. RESULTS: A total of 791 laparoscopic appendectomies were performed in two institutions (preintervention = 382, post-intervention = 409). The postoperative ED visit rate decreased from 5.8% preimplementation to 2.4% post-implementation (p = 0.02). Over one-fifth of families messaged (21.6%) had questions in the postoperative period. The majority expressed interest in a video visit (52.5%), while some preferred to speak with the surgeon's office (25%). Over 90% of respondents found the system helpful, and 4.9% opted out. CONCLUSION: Implementation of a hospital-initiated text messaging system has the potential to reduce ED visits in the immediate postoperative period after appendectomy. This system can be scaled to include different surgeries across multiple disciplines. LEVEL OF EVIDENCE: III. TYPE OF STUDY: Clinical Retrospective Pre/Post Intervention Study.

Netrin G1 Promotes Pancreatic Tumorigenesis through Cancer-Associated Fibroblast-Driven Nutritional Support and Immunosuppression.

Pancreatic ductal adenocarcinoma (PDAC) has a poor 5-year survival rate and lacks effective therapeutics. Therefore, it is of paramount importance to identify new targets. Using multiplex data from patient tissue, three-dimensional coculturing in vitro assays, and orthotopic murine models, we identified Netrin G1 (NetG1) as a promoter of PDAC tumorigenesis. We found that NetG1+ cancer-associated fibroblasts (CAF) support PDAC survival, through a NetG1-mediated effect on glutamate/glutamine metabolism. Also, NetG1+ CAFs are intrinsically immunosuppressive and inhibit natural killer cell-mediated killing of tumor cells. These protumor functions are controlled by a signaling circuit downstream of NetG1, which is comprised of AKT/4E-BP1, p38/FRA1, vesicular glutamate transporter 1, and glutamine synthetase. Finally, blocking NetG1 with a neutralizing antibody stunts in vivo tumorigenesis, suggesting NetG1 as potential target in PDAC. SIGNIFICANCE: This study demonstrates the feasibility of targeting a fibroblastic protein, NetG1, which can limit PDAC tumorigenesis in vivo by reverting the protumorigenic properties of CAFs. Moreover, inhibition of metabolic proteins in CAFs altered their immunosuppressive capacity, linking metabolism with immunomodulatory function.See related commentary by Sherman, p. 230.This article is highlighted in the In This Issue feature, p. 211.

Laboratory Testing Methods for Novel Severe Acute Respiratory Syndrome-Coronavirus-2 (SARS-CoV-2).

Following the first reports of coronavirus disease-19 (COVID-19) by China to the World Health Organization (WHO) on 31st December 2019, more than 4,302,774 novel severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) cases have been reported by authorities in 212 countries and territories by 12th May 2020. The outbreak and spread of COVID-19 worldwide, highlights the critical need for developing rapid and accurate diagnostic testing methods for emerging human coronavirus (CoV) infections. Testing is crucial to track the spread of disease during a pandemic, and to swiftly permit public health interventions including isolation, quarantine, and appropriate clinical management of afflicted individuals. The key components of viral diagnostic tests are (1) collection of the appropriate sample (blood, nasal swab, and throat swab), (2) availability of the genetic and proteomic sequences of the novel virus for analysis, and (3) rapid and accurate laboratory testing methods. The current gold standard for the molecular diagnosis of SARS-CoV-2 infection is the real-time reverse transcriptase-polymerase chain reaction (RT-PCR) for the qualitative and quantitative detection of viral nucleic acids. Other relevant laboratory methods include enzyme-linked immunoassays (EIA) for viral antibody and antigen detection, and serum viral neutralization (SVN) assays for antibody neutralization determination. The challenges faced in developing a diagnostic test for a novel pathogen are the ability to measure low viral loads for early detection, to provide low or no cross-reactivity with other viral strains and to deliver results rapidly. Several point-of-care molecular devices are currently being integrated for fast and accurate diagnosis of SARS-CoV-2 infections. This review discusses the current laboratory methods available to test for coronaviruses by focusing on the present COVID-19 outbreak.

ZBP1/DAI Drives RIPK3-Mediated Cell Death Induced by IFNs in the Absence of RIPK1.

Receptor-interacting protein kinase 1 (RIPK1) regulates cell fate and proinflammatory signaling downstream of multiple innate immune pathways, including those initiated by TNF-α, TLR ligands, and IFNs. Genetic ablation of Ripk1 results in perinatal lethality arising from both RIPK3-mediated necroptosis and FADD/caspase-8-driven apoptosis. IFNs are thought to contribute to the lethality of Ripk1-deficient mice by activating inopportune cell death during parturition, but how IFNs activate cell death in the absence of RIPK1 is not understood. In this study, we show that Z-form nucleic acid binding protein 1 (ZBP1; also known as DAI) drives IFN-stimulated cell death in settings of RIPK1 deficiency. IFN-activated Jak/STAT signaling induces robust expression of ZBP1, which complexes with RIPK3 in the absence of RIPK1 to trigger RIPK3-driven pathways of caspase-8-mediated apoptosis and MLKL-driven necroptosis. In vivo, deletion of either Zbp1 or core IFN signaling components prolong viability of Ripk1-/- mice for up to 3 mo beyond parturition. Together, these studies implicate ZBP1 as the dominant activator of IFN-driven RIPK3 activation and perinatal lethality in the absence of RIPK1.

Analysis of Cytokine- and Influenza A Virus-Driven RIPK3 Necrosome Formation.

In multicellular organisms, regulated cell death plays a vital role in development, adult tissue homeostasis, and clearance of damaged or infected cells. Necroptosis is one such form of regulated cell death, characterized by its reliance on receptor-interacting protein kinase 3 (RIPK3). Once activated, RIPK3 nucleates a protein complex, termed the "necrosome," which includes the adaptors RIPK1 and FADD, and the effector protein MLKL. From the necrosome, RIPK3 phosphorylates MLKL to drive necroptosis, and can also induce RIPK1/FADD-mediated apoptosis, via caspase-8. Assembly of the necrosome thus serves as a useful readout of RIPK3 activation. In this chapter, we describe molecular methods for examining necrosome activation in response to the cytokines TNF-α, IFN-ß, and IFN-γ, and upon infection with influenza A virus (IAV).

Host functions used by hepatitis B virus to complete its life cycle: Implications for developing host-targeting agents to treat chronic hepatitis B.

Similar to other mammalian viruses, the life cycle of hepatitis B virus (HBV) is heavily dependent upon and regulated by cellular (host) functions. These cellular functions can be generally placed in to two categories: (a) intrinsic host restriction factors and innate defenses, which must be evaded or repressed by the virus; and (b) gene products that provide functions necessary for the virus to complete its life cycle. Some of these functions may apply to all viruses, but some may be specific to HBV. In certain cases, the virus may depend upon the host function much more than does the host itself. Knowing which host functions regulate the different steps of a virus' life cycle, can lead to new antiviral targets and help in developing novel treatment strategies, in addition to improving a fundamental understanding of viral pathogenesis. Therefore, in this review we will discuss known host factors which influence key steps of HBV life cycle, and further elucidate therapeutic interventions targeting host-HBV interactions.