Pyroptosis: A Promising Mechanism Linking SARS-CoV-2 Infection to Adverse Pregnancy Outcomes.

Pregnancy is characterized by a delicate immune balance; therefore, infectious diseases might increase the risk of adverse pregnancy outcomes (APOs). Here, we hypothesize that pyroptosis, a unique cell death pathway mediated by the NLRP3 inflammasome, could link SARS-CoV-2 infection, inflammation, and APOs. Two blood samples were collected from 231 pregnant women at 11-13 weeks of gestation and in the perinatal period. At each time point, SARS-CoV-2 antibodies and neutralizing antibody titers were measured by ELISA and microneutralization (MN) assays, respectively. Plasmatic NLRP3 was determined by ELISA. Fourteen miRNAs selected for their role in inflammation and/or pregnancy were quantified by qPCR and further investigated by miRNA-gene target analysis. NLRP3 levels were positively associated with nine circulating miRNAs, of which miR-195-5p was increased only in MN+ women (p-value = 0.017). Pre-eclampsia was associated with a decrease in miR-106a-5p (p-value = 0.050). miR-106a-5p (p-value = 0.026) and miR-210-3p (p-value = 0.035) were increased in women with gestational diabetes. Women giving birth to small for gestational age babies had lower miR-106a-5p and miR-21-5p (p-values = 0.001 and 0.036, respectively), and higher miR-155-5p levels (p-value = 0.008). We also observed that neutralizing antibodies and NLRP3 concentrations could affect the association between APOs and miRNAs. Our findings suggest for the first time a possible link between COVID-19, NLRP3-mediated pyroptosis, inflammation, and APOs. Circulating miRNAs might be suitable candidates to gain a comprehensive view of this complex interplay.

Digital Detection of Single Virus Particles by Multi-Spot, Label-Free Imaging Biosensor on Anti-Reflective Glass.

Rapid detection of whole virus particles in biological or environmental samples represents an unmet need for the containment of infectious diseases. Here, an optical device enabling the enumeration of single virion particles binding on antibody or aptamers immobilized on a surface with anti-reflective coating is described. In this regime, nanoparticles adhering to the sensor surface provide localized contributions to the reflected field that become detectable because of their mixing with the interfering waves in the reflection direction. Thus, these settings are exploited to realize a scan-free, label-free, micro-array-type digital assay on a disposable cartridge, in which the virion counting takes place in wide field-of-view imaging. With this approach we could quantify, by enumeration, different variants of SARS-CoV-2 virions interacting with antibodies and aptamers immobilized on different spots. For all tested variants, the aptamers showed larger affinity but lower specificity relative to the antibodies. It is found that the combination of different probes on the same surface enables increasing specificity of detection and dynamic range.

SARS-CoV-2 Seroconversion and Pregnancy Outcomes in a Population of Pregnant Women Recruited in Milan, Italy, between April 2020 and October 2020.

The possible link between SARS-CoV-2 infection and adverse pregnancy outcomes has so far demonstrated heterogeneous results in terms of maternal, fetal, and neonatal complications. We aim to investigate the correlation between SARS-CoV-2 seroconversion and/or neutralization titer and pregnancy outcomes. We analyzed a population of 528 pregnant women followed up from the first trimester of gestation until delivery. For each woman, we collected a first blood sample between 11 and 13 weeks of gestation and a second sample in the perinatal period (between peripartum and puerperium) to assess the presence of SARS-CoV-2 antibodies and/or microneutralization titer (MN titer). Data on pregnancy outcomes (gestational age at delivery, preterm birth before 34 weeks, hypertensive disorders, gestational diabetes, and abnormal fetal growth) were collected. We observed that serologic status per se is not associated with major pregnancy complications. On the contrary, the MN titer was associated with increased odds of gestational diabetes. Although we mainly reported asymptomatic SARS-CoV-2 infections and the absence of severe maternal and neonatal adverse outcomes, SARS-CoV-2 infection might challenge the maternal immune system and explain the moderate increase in adverse outcome odds.

Digital RT-PCR Chip method for detection of SARS-CoV-2 virus.

The "gold standard" method for detection of SARS-CoV-2 is the real time reverse transcription-polymerase chain reaction, but due to pre-analytical and technical limitations, biological samples with low viral load are not sometimes detected. For this purpose a digital RT-PCR method on-chip was developed for detection of the SARS-CoV-2 virus, using two TaqMan™ Assays for quantification of the N Protein (Nucleocapsid) and the S Protein (Spike), and the QuantStudio™ 3D Digital PCR instrument. The method was applied to assess the nasopharyngeal swabs of asymptomatic subjects recruited in the UNICORN Study. The digital RT-PCR method is characterized by a higher sensitivity than the RT-qPCR method, even if performed with the same TaqMan™, and could be a promising tool for SARS-CoV-2 viral load quantification.

Nasopharyngeal Bacterial Microbiota Composition and SARS-CoV-2 IgG Antibody Maintenance in Asymptomatic/Paucisymptomatic Subjects.

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) causes the coronavirus disease 2019 (COVID-19), ranging from asymptomatic conditions to severe/fatal lung injury and multi-organ failure. Growing evidence shows that the nasopharyngeal microbiota composition may predict the severity of respiratory infections and may play a role in the protection from viral entry and the regulation of the immune response to the infection. In the present study, we have characterized the nasopharyngeal bacterial microbiota (BNM) composition and have performed factor analysis in a group of 54 asymptomatic/paucisymptomatic subjects who tested positive for nasopharyngeal swab SARS-CoV-2 RNA and/or showed anti-RBD-IgG positive serology at the enrolment. We investigated whether BNM was associated with SARS-CoV-2 RNA positivity and serum anti-RBD-IgG antibody development/maintenance 20-28 weeks after the enrolment. Shannon's entropy α-diversity index [odds ratio (OR) = 5.75, p = 0.0107] and the BNM Factor1 (OR = 2.64, p = 0.0370) were positively associated with serum anti-RBD-IgG antibody maintenance. The present results suggest that BNM composition may influence the immunological memory against SARS-CoV-2 infections. To the best of our knowledge, this is the first study investigating the link between BNM and specific IgG antibody maintenance. Further studies are needed to unveil the mechanisms through which the BNM influences the adaptive immune response against viral infections.

The Efficacy of the Mineralcorticoid Receptor Antagonist Canrenone in COVID-19 Patients.

BACKGROUND: In COVID-19 patients, aldosterone via angiotensin-converting enzyme-2 deregulation may be responsible for systemic and pulmonary vasoconstriction, inflammation, and oxidative organ damage. AIM: To verify retrospectively the impact of the mineralcorticoid receptor antagonist canrenone i.v. on the need of invasive ventilatory support and/or all-cause in-hospital mortality. METHODS: Sixty-nine consecutive COVID-19 patients, hospitalized for moderate to severe respiratory failure at Fondazione Istituti di Ricovero e Cura a Carattere Scientifico (IRCCS) Ca' Granda Ospedale Maggiore Policlinico of Milan, received two different therapeutic approaches in usual care according to the personal skills and pharmacological management experience of the referral medical team. Group A (n = 39) were given vasodilator agents or renin-angiotensin-aldosterone system (RAAS) inhibitors and group B (n = 30) were given canrenone i.v. RESULTS: Among the 69 consecutive COVID-19 patients, those not receiving canrenone i.v. (group A) had an event-free rate of 51% and a survival rate of 64%. Group B (given a mean dose of 200 mg/q.d. of canrenone for at least two days of continuous administration) showed an event-free rate of 80% with a survival rate of 87%. Kaplan-Meier analysis for composite outcomes and mortality showed log rank statistics of 0.0004 and 0.0052, respectively. CONCLUSIONS: The novelty of our observation relies on the independent positive impact of canrenone on the all-cause mortality and clinical improvement of COVID-19 patients ranging from moderate to severe diseases.

A serological investigation in Southern Italy: was SARS-CoV-2 circulating in late 2019?

In March 2020, the first pandemic caused by a coronavirus was declared by the World Health Organization. Italy was one of the first and most severely affected countries, particularly the northern part of the country. The latest evidence suggests that the virus could have been circulating, at least in Italy, before the first autochthonous SARS-COV-2 case was detected in February 2020. The present study aimed to investigate the presence of antibodies against SARS-CoV-2 in human serum samples collected in the last months of 2019 (September-December) in the Apulia region, Southern Italy. Eight of 455 samples tested proved positive on in-house receptor-binding-domain-based ELISA. Given the month of collection of the positive samples, these findings may indicate early circulation of SARS-CoV-2 in Apulia region in the autumn of 2019. However, it cannot be completely ruled out that the observed sero-reactivity could be an unknown antigen specificity in another virus to which subjects were exposed containing an epitope adventitiously cross-reactive with an epitope of SARS-CoV-2.

Epidemic Preparedness-Leishmania tarentolae as an Easy-to-Handle Tool to Produce Antigens for Viral Diagnosis: Application to COVID-19.

To detect and prevent emerging epidemics, discovery platforms are urgently needed, for the rapid development of diagnostic assays. Molecular diagnostic tests for COVID-19 were developed shortly after the isolation of SARS-CoV-2. However, serological tests based on antiviral antibody detection, revealing previous exposure to the virus, required longer testing phases, due to the need to obtain correctly folded and glycosylated antigens. The delay between the identification of a new virus and the development of reliable serodiagnostic tools limits our readiness to tackle future epidemics. We suggest that the protozoan Leishmania tarentolae can be used as an easy-to-handle microfactory for the rapid production of viral antigens to face emerging epidemics. We engineered L. tarentolae to express the SARS-CoV-2 receptor-binding domain (RBD) and we recorded the ability of the purified RBD antigen to detect SARS-CoV-2 infection in human sera, with a sensitivity and reproducibility comparable to that of a reference antigen produced in human cells. This is the first application of an antigen produced in L. tarentolae for the serodiagnosis of a Coronaviridae infection. On the basis of our results, we propose L. tarentolae as an effective system for viral antigen production, even in countries that lack high-technology cell factories.

HDL in COVID-19 Patients: Evidence from an Italian Cross-Sectional Study.

A number of studies have highlighted important alterations of the lipid profile in COVID-19 patients. Besides the well-known atheroprotective function, HDL displays anti-inflammatory, anti-oxidative, and anti-infectious properties. The aim of this retrospective study was to assess the HDL anti-inflammatory and antioxidant features, by evaluation of HDL-associated Serum amyloid A (SAA) enrichment and HDL-paraoxonase 1 (PON-1) activity, in a cohort of COVID-19 patients hospitalized at the Cardiorespiratory COVID-19 Unit of Fondazione IRCCS Ca' Granda Ospedale Maggiore Policlinico of Milan. COVID-19 patients reached very low levels of HDL-c (mean ± SD: 27.1 ± 9.7 mg/dL) with a marked rise in TG (mean ± SD: 165.9 ± 62.5 mg/dL). Compared to matched-controls, SAA levels were significantly raised in COVID-19 patients at admission. There were no significant differences in the SAA amount between 83 alive and 22 dead patients for all-cause in-hospital mortality. Similar findings were reached in the case of PON-1 activity, with no differences between alive and dead patients for all-cause in-hospital mortality. In conclusion, although not related to the prediction of in-hospital mortality, reduction in HDL-c and the enrichment of SAA in HDL are a mirror of SARS-CoV-2 positivity even at the very early stages of the infection.

Timeline of SARS-CoV-2 Spread in Italy: Results from an Independent Serological Retesting.

The massive emergence of COVID-19 cases in the first phase of pandemic within an extremely short period of time suggest that an undetected earlier circulation of SARS-CoV-2 might have occurred. Given the importance of this evidence, an independent evaluation was recommended by the World Health Organization (WHO) to test a subset of samples selected on the level of positivity in ELISA assays (positive, low positive, negative) detected in our previous study of prepandemic samples collected in Italy. SARS-CoV-2 antibodies were blindly retested by two independent centers in 29 blood samples collected in the prepandemic period in Italy, 29 samples collected one year before and 11 COVID-19 control samples. The methodologies used included IgG-RBD/IgM-RBD ELISA assays, a qualitative micro-neutralization CPE-based assay, a multiplex IgG protein array, an ELISA IgM kit (Wantai), and a plaque-reduction neutralization test. The results suggest the presence of SARS-CoV-2 antibodies in some samples collected in the prepandemic period, with the oldest samples found to be positive for IgM by both laboratories collected on 10 October 2019 (Lombardy), 11 November 2019 (Lombardy) and 5 February 2020 (Lazio), the latter with neutralizing antibodies. The detection of IgM and/or IgG binding and neutralizing antibodies was strongly dependent on the different serological assays and thresholds employed, and they were not detected in control samples collected one year before. These findings, although gathered in a small and selected set of samples, highlight the importance of harmonizing serological assays for testing the spread of the SARS-CoV-2 virus and may contribute to a better understanding of future virus dynamics.

Evaluation and correlation between SARS-CoV-2 neutralizing and binding antibodies in convalescent and vaccinated subjects.

Since the first detection of a novel Coronavirus (SARS-CoV-2) in December 2019 in Wuhan (China), it has become crucial to assess and quantize the human humoral immune response after SARS-CoV-2 natural infection and/or vaccination. Having well standardized and reliable serological assays able to accurately measure the total IgG antibodies response as well as the neutralization dynamics, play a pivotal role for the evaluation of "second" and "third" vaccines generation and in monitoring the effect in case of reinfection in the human population caused by the original strains or new SARS-CoV-2 variants. In the present study we reported that both symptomatic convalescent and vaccinated donors showed the presence of different levels of neutralizing antibodies. In addition, vaccinated subjects presented high levels of anti-S antibodies, whereas the complete absence of anti-N antibodies, whereas convalescent patients presented high levels of both anti-S and anti-N antibodies. The evaluation of the correlation between SARS-CoV-2 neutralizing and binding antibodies in convalescent and vaccinated subjects revealed that the IgG anti-S log-values were significantly higher in the vaccinated group respect to convalescent subjects. In addition, the level of binding antibodies recognizing the S protein shows a positive linear regression when compared to neutralizing titres in both the two groups evaluated.

Characterization of antibody response in asymptomatic and symptomatic SARS-CoV-2 infection.

SARS-CoV-2 pandemic is causing high morbidity and mortality burden worldwide with unprecedented strain on health care systems. To investigate the time course of the antibody response in relation to the outcome we performed a study in hospitalized COVID-19 patients. As comparison we also investigated the time course of the antibody response in SARS-CoV-2 asymptomatic subjects. Study results show that patients produce a strong antibody response to SARS-CoV-2 with high correlation between different viral antigens (spike protein and nucleoprotein) and among antibody classes (IgA, IgG, and IgM and neutralizing antibodies). The antibody peak is reached by 3 weeks from hospital admission followed by a sharp decrease. No difference was observed in any parameter of the antibody classes, including neutralizing antibodies, between subjects who recovered or with fatal outcome. Only few asymptomatic subjects developed antibodies at detectable levels.

Prognostic parameters of in-hospital mortality in COVID-19 patients-An Italian experience.

Background During COVID-19 outbreak, Italy was the first country in Europe to be heavily affected with an intensive care unit mortality of 26%. In order to reduce this percentage, physicians should establish clear and objective criteria to stratify COVID-19 patients at high risk of in-hospital death. Thus, the aim has been to test a large spectrum of variables ranging from clinical evaluation to laboratory biomarkers to identify which parameter would best predict all-cause in-hospital mortality in COVID-19 patients. Design observational study. Results Multivariate Cox regression analysis showed that each 5 years of increase in age corresponded to a hazard ratio (HR) of 1.28 (95% CI 1.00-1.65, P = .050); each increment of 803 ng/L of N-terminal pro-B-type natriuretic peptide (NT-proBNP) corresponded to a HR of 1.24 (95% CI 1.11-1.39, P < .001); each increment of 58 ng/L of interleukin (IL)-6 corresponded to a HR of 1.23 (95% CI 1.09-1.40, P < .001), and each increment of 250 U/L of lactate dehydrogenase (LDH) corresponded to a HR of 1.23 (95% CI 1.10-1.37, P < .001). According to the calculated cut-points for age (≥70 years), NT-proBNP (≥803 ng/L), IL-6 (≥58 ng/L) and LDH (≥371 U/L) when 2 out of these 4 were overcome, the HR was 2.96 (95% CI 1.97-4.45, P < .001). Conclusion In COVID-19 patients, besides age, the evaluation of three biochemical parameters, available in few hours after hospital admission can predict in-hospital mortality regardless of other comorbidities.

SARS-CoV-2 tests in occupational settings: what you look for is what you get.

During the last month of 2019, a new Coronavirus from China started to spread all around the world causing a pandemic emergency still ongoing. The outbreak made imperative the need for diagnostic and screening tests that could identify the current and past infection state of an individual. Occupational medicine is facing a very demanding challenge, as the pandemic set off the need to re-evaluate many aspects of workplace safety. A fundamental role has been played by tests used to diagnose COVID-19 and to isolate infected asymptomatic subjects, with a view to the viral evolution and the emerging variants. However, the need for the urgent set-up of new methods for assessing both new and past infections has resulted in a large number of methods, not always comparable with each other, in terms of laboratory techniques, viral antigens used for detection, and class of antibodies detected. These factors make it difficult to understand the serological test results and their possible application. In this paper, we reviewed the types of assays currently available, to address some key aspects that characterize each technique, and might have an impact on results interpretation.

Detection of IgM, IgG and SARS-CoV-2 RNA among the personnel of the University of Milan, March through May 2020: the UNICORN study.

OBJECTIVES: In Italy, the pandemic of COVID-19 resulted in congestion of hospitals and laboratories and probably determined an underestimation of the number of infected subjects, as the molecular diagnosis of SARS-CoV-2 infection was mainly performed on hospitalised patients. Therefore, limited data are available about the number of asymptomatic/paucisymptomatic subjects in the general population across time. To understand SARS-CoV-2 infection in the general population, we have developed a cross-sectional study (the 'UNIversity against CORoNavirus study') to investigate infection trends in asymptomatic/paucisymptomatic subjects in Milan (Italy), between March and June 2020. PARTICIPANTS: The study population included 2023 subjects asymptomatic at the enrolment. PRIMARY OUTCOME MEASURES: A nasal mid-turbinate swab for the detection of SARS-CoV-2 RNA and blood specimen for testing serum antibodies (immunoglobulin M (IgM) and IgG) were collected. RESULTS: Subjects showing positivity for the SARS-CoV-2 RNA and/or for anti-SARS-CoV-2 Ig is 237 (11.7%). Only 1.2% (n=25) of the total population had a positive nasal swab for SARS-CoV-2 and the large majority (21/25) of them were observed in March. A total of 226 subjects (11%) had IgM (n=19; 0.9%), IgG (n=155; 7.7%) or both (n=52; 2.6%) against SARS-CoV-2. Subjects with a present or past SARS-CoV-2 infection did not differ from other subjects as regards the number of cohabiting family members, travels, fever and upper and lower respiratory infection episodes. CONCLUSIONS: Results from the present study support the hypothesis that the actual spread of the virus in Lombardy was underestimated in the official records. However, as it is not known how long Ig persist, numbers should be taken cautiously.

Bronchiolitis and SARS-CoV-2.

BACKGROUND: It has been speculated that the SARS-CoV-2 was already widespread in western countries before February 2020. METHODS: We gauged this hypothesis by analysing the nasal swab of infants with either bronchiolitis or a non-infectious disease admitted to the Ospedale Maggiore, Milan (one of the first epicentres of SARS-CoV-2 outbreak in Europe) from November 2019. RESULTS: The SARS-CoV-2 RNA was never detected in 218 infants with bronchiolitis (95 females, median age 4.9 months) and 49 infants (22 females, median age 5.6 months) with a non-infectious disease between November 2019 and February 2020. On the contrary, two infants hospitalised for bronchiolitis between March and April 2020 tested positive for SARS-CoV-2. CONCLUSIONS: This study does not support the hypothesis that SARS-CoV-2 was already circulating among infants before the official outbreak of SARS-CoV-2 infection. However, it shows for the first time that SARS-CoV-2 might cause bronchiolitis requiring hospitalisation.

Serological follow-up of SARS-CoV-2 asymptomatic subjects.

SARS-CoV-2 symptoms are non-specific and can range from asymptomatic presentation to severe pneumonia. Asymptomatic subjects carrying SARS-CoV-2 often remain undiagnosed and it is still debated whether they develop immunoglobulins (Ig) and how long they persist. The aim of this study was to investigate the development and persistence of antibodies against SARS-CoV-2 in asymptomatic subjects infected by the virus. This follow-up study was performed on the 31 asymptomatic subjects who presented a positive nasal swab or serology against SARS-CoV-2 (Ig against Spike-RBD) in the first part of the UNICORN study (March 2020) aimed at attesting previous or current contacts with the virus in the personnel of the University of Milan. Eight weeks after the first Ig measure, these subjects were invited to donate a second blood sample for testing serum antibodies (IgM, IgG and total antibodies) and to fill-in a structured questionnaire. About 80% of asymptomatic subjects did not present circulating immunoglobulins against SARS-CoV-2 after 8 weeks from a positive nasal swab against the virus. Moreover, in more than 40% of these subjects, no Ig against SARS-CoV-2 were detected at any time. Finally, about two third of subjects with immunoglobulins at baseline did not present IgG against SARS-CoV-2 after 8 weeks. The majority of subjects who developed an asymptomatic SARS-CoV-2 infection do not present antibodies against the RBD-spike protein after 8 weeks of follow-up. These data should be taken into account for the interpretation of the serological evidences on SARS-CoV-2 that are emerging nowadays.

Unexpected detection of SARS-CoV-2 antibodies in the prepandemic period in Italy.

There are no robust data on the real onset of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection and spread in the prepandemic period worldwide. We investigated the presence of SARS-CoV-2 receptor-binding domain (RBD)-specific antibodies in blood samples of 959 asymptomatic individuals enrolled in a prospective lung cancer screening trial between September 2019 and March 2020 to track the date of onset, frequency, and temporal and geographic variations across the Italian regions. SARS-CoV-2 RBD-specific antibodies were detected in 111 of 959 (11.6%) individuals, starting from September 2019 (14%), with a cluster of positive cases (>30%) in the second week of February 2020 and the highest number (53.2%) in Lombardy. This study shows an unexpected very early circulation of SARS-CoV-2 among asymptomatic individuals in Italy several months before the first patient was identified, and clarifies the onset and spread of the coronavirus disease 2019 (COVID-19) pandemic. Finding SARS-CoV-2 antibodies in asymptomatic people before the COVID-19 outbreak in Italy may reshape the history of pandemic.

The Efficacy of the Mineralcorticoid Receptor Antagonist Canrenone in COVID-19 Patients

Background: In COVID-19 patients, aldosterone via angiotensin-converting enzyme-2 deregulation may be responsible for systemic and pulmonary vasoconstriction, inflammation, and oxidative organ damage. Aim: To verify retrospectively the impact of the mineralcorticoid receptor antagonist canrenone i.v. on the need of invasive ventilatory support and/or all-cause in-hospital mortality. Methods: Sixty-nine consecutive COVID-19 patients, hospitalized for moderate to severe respiratory failure at Fondazione Istituti di Ricovero e Cura a Carattere Scientifico (IRCCS) Ca"Granda Ospedale Maggiore Policlinico of Milan, received two different therapeutic approaches in usual care according to the personal skills and pharmacological management experience of the referral medical team. Group A (n = 39) were given vasodilator agents or renin-angiotensin-aldosterone system (RAAS) inhibitors and group B (n = 30) were given canrenone i.v. Results: Among the 69 consecutive COVID-19 patients, those not receiving canrenone i.v. (group A) had an event-free rate of 51% and a survival rate of 64%. Group B (given a mean dose of 200 mg/q.d. of canrenone for at least two days of continuous administration) showed an event-free rate of 80% with a survival rate of 87%. Kaplan-Meier analysis for composite outcomes and mortality showed log rank statistics of 0.0004 and 0.0052, respectively. Conclusions: The novelty of our observation relies on the independent positive impact of canrenone on the all-cause mortality and clinical improvement of COVID-19 patients ranging from moderate to severe diseases.