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Background: Severe COVID-19 is less common in children than in adults. Increasing evidence show that distinct immune-pathological changes can persist weeks or months after SARS-CoV2 infection, leading to Long COVID (LC). We investigated the systemic type I/III interferon (IFN-I/III) and inflammation response in peripheral blood mononuclear cells (PBMCs) of children with and without LC symptoms. Method(s): Blood samples were collected from children attending Umberto I hospital of Rome, within 3-6 months after a SARS-CoV-2 positive test and from control children. RNA was extracted from PBMCs for determining the levels of IFN-I (IFN-Alpha2, -Beta, -Epsilon and -Omega), IFN-III (IFN-Lambda1-3), NLRP3 and IL-1beta genes, and miR-141 expression by quantitative RealTime-PCR assays, normalized to housekeeping GUS gene and RNU6B, respectively. Result(s): 28 participants (M 12.5y SD 3.0) with LC symptoms, 28 participants (M 11.8y SD 3.0) without LC symptoms and 18 children who've never had SARS-CoV- 2 infection (M 10.5y SD 3.1) were enrolled. Comparing the three study groups, we found reduced levels of IFN-Lambda1, IFN-Lambda2 and IFN-Lambda3 (p=0.006, p< 0.001, p=0.012, respectively;Kruskal-Wallis (KW) test) mRNA in patients who have had SARS-CoV-2 infection as opposed to control group, whereas transcript levels of IFN-Epsilon (p= 0.019;KW test) were increased in the former with respect to the latter group;as well, remaining IFN-I genes analyzed showed a tendency to be up-regulated. As far as NLRP3 and IL-1beta levels was concerned, these genes were increased in LC patients (p< 0.001 for both genes;KW test). Additionally, miR-141, which has been reported to regulate inflammasome response, was overexpressed in LC patients (p< 0.001;Mann-Whitney test). Conclusion(s): These results showed a decreased levels of IFN-III mRNAs and an overexpression of IFN-Epsilon in children after 3-6 months of SARS-CoV-2 infection regardless of development of LC symptoms, suggesting that SARSCoV- 2 could have caused dysregulation of IFN response through unknown mechanisms (e.g. epigenetic modifications). Also, we found an overexpression of miR-141, NLRP3 and IL-1beta mRNAs in LC patients, indicating that a prolonged activation of inflammasome pathways could be associated with the development of LC symptoms.
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Background: Children generally develop a mild disease after SARS-CoV-2 infection;it has been shown (Loske J al., 2021) that higher basal expression of relevant pattern recognition receptors may result in a stronger early innate antiviral than in adults. However, how the early interferon (IFN) response differs from that in adults is not fully characterized. Hence, we aimed to investigate the expression of several IFN-related genes in nasopharyngeal (NP) cells from children and adults with asymptomatic or mild COVID-19, not requiring hospitalization. Methods: Children and adults attending emergency departments (ED) of Sapienza University Hospital, to perform SARS-CoV-2 molecular tests, were enrolled from November 2020 to February 2021, after informed consent was obtained. RNA from residual NP swabs was purified and 200 ng were reverse transcribed. Gene expression of genes coding for type I and III IFNs and for the well-known markers of IFNs' activation, ISG15 and ISG56, was measured by exonuclease-based Real time PCR assays with relative quantification to the invariant gene GUS (the 2-ΔCt method). Results: Residual NP cells from a total of 132 children and adults were included in the study;56 had SARS-CoV-2 positive results and 76 resulted negative. The expression of all tested genes showed a moderate significant inverse correlation with age, with the exception of ISG15. Participants were further stratified in age groups (< 16;16-35;36-65 years) resulting in: 25 SARS-CoV-2 negative and 26-positive children;14 SARS-CoV-2 negative and 16-positive young adults and 37 SARS-CoV-2 negative and 14-positive adults. In SARS-CoV-2 negative samples, higher levels of all study genes were found in children, while significantly decreasing in young and elderly adults. Among SARS-CoV-2 positive samples, those from children showed significantly higher levels of type I IFNs and of IFN lambda2 whereas ISG15 was far more elevated in adults. Moreover, levels of all type I IFNs, and of IFN lambda2, were significantly higher in individuals with no symptoms (65% of children and 44% of the young adults), whereas ISG15 was elevated in those with a mild COVID-19. Conclusion: The higher baseline expression of IFN-related genes in children may prompt a quicker activation of the IFN response after SARS-CoV-2 infection and contribute to effective control of viral replication;the higher ISG activation in adults may be caused by the inflammatory response and associated to COVID-19 symptoms.
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Background: Children generally develop asymptomatic or mild COVID-19 disease, despite the exact mechanisms that protect them from severity are yet to be defined. Since humoral response to SARS-CoV-2 infection in children is still poorly investigated, we aimed to analyze circulating levels of anti-Spike IgA and IgG in pediatric population up to 8 months after SARS-CoV-2 infection, to delineate whether COVID-19 outcomes could impact on antibody (Ab) levels. Methods: A total of 115 COVID-19 young patients (mean age: 11.5 years, range 1-19 years) were enrolled between October 2020 and March 2021. All cases were confirmed SARS-CoV-2 infection by use of a diagnostic molecular assay on nasopharyngeal swabs. Circulating anti-SARS-CoV-2 IgG and IgA were measured using ELISA assays at one-month (T1), two-month (T2) and eight-month (T3) follow-up blood samples of young partecipants. Results: Longitudinal observation of COVID-19 children showed a decreased circulating level of IgA at T2 and T3 respectively compared to T1 (p<0.001). Persistent levels of anti-Spike IgG were observed at least two-month post infection but they significantly decreased at T3 (p<0.001). Stratifying children in two age-classes (1-9 and 10-19 years old) we found significantly higher levels of IgA in younger children at T1, T2 and T3 than in children older than 10 years old (p=0.012;p=0.041;p=0.036, respectively). Differently, younger children had a significantly higher level of IgG at T2 (p=0.029) and at T3 (p=0.049), but not at T1. Stratifying children based on the presence or absence of SARS-CoV-2 correlated symptoms or on the basis of underlying diseases, we did not observe differences in blood levels of IgA and IgG in all time points analyzed. Conclusion: Our longitudinal data indicated that younger children are characterized by an elevated peak of early IgA and are also defined by a robust induction of IgG, with respect to the older. These results contrast with what is common in SARS-CoV-2 infection in adults that elicit higher levels of polyfunctional Abs in severe disease. If confirmed in larger groups, these data would suggest that pediatric patients that usually have an efficient control of SARS-CoV-2 infections without inflammation would also elicit a humoral immune response protective from reinfections.
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Background: Evidence suggest that critically ill COVID-19 patients have an impairment of IFN-I response. This defect in antiviral mechanisms is explained in some patients by the presence of anti IFN-alpha neutralizing autoantibodies (NAbs). However, whether NAbs fluctuate longitudinally during COVID-19, and what are their specificity toward IFN-I subtypes and consequences on the IFN response remain elusive. Methods: Binding antibodies (BAbs) to IFN-alpha and IFN-beta were screened in serum samples (n=360) of COVID-19 patients using ELISA assays. All serum samples containing BAbs were processed to investigate NAbs using antiviral bioassay. Respiratory samples (n=17) were also included for the NAbs analysis. Transcript levels of IFN-alpha, IFN-beta, IFN-omega and IFN stimulated genes (ISGs) were analyzed through RT/Real Time PCR. Results: Results showed that 16.94% (61/360) of COVID-19 patients had circulating BAbs against IFN-alpha and IFN-beta. Further, 21% (13/61) of critically ill subjects had NAbs with a variable titer against all the IFN-alpha subtypes (70-71680 TRU/ml) while only 1 patient had anti IFN-beta NAbs. About 70% of these serum samples showed cross reactivity to IFN-omega at different extent (27-106667 TRU/ml). Longitudinal evaluation at different time points after hospitalization indicate the persistence of high NAbs titer throughout the time. NAbs to IFN-alpha (10-20 TRU/ml) were also detected in 17.64% of respiratory samples. Patients with NAbs had severe disease and exhibited alterations in the levels of many hematological indicators [(white blood cells, neutrophils, platelets, neutrophils to lymphocytes ratio, platelets to lymphocytes ratio, D-dimer, C-reactive protein and lactate dehydrogenase;p<0.05)]. Transcriptomic analysis indicated that levels of IFN genes were lower in NAbs patients than in healthy donors (p≤0.05). However, only the ISGs levels were reduced compared to those found in the NAbs negative patients. Of note, expression of ISGs, was abolished during hospitalization in all patients with persistent high titer of NAbs. Conclusion: Our finding demonstrate that NAbs with a broad specificity to IFN-I can be found in blood and respiratory samples from severe COVID-19 patients. NAbs detection was associated with a defective IFN response and with an increased levels of markers of disease severity.
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Convalescent plasma (CP) therapy might be effective in patients with haematological malignanciesand B-cell depletion. We report a single-centre experience of COVID-19 patients with non-Hodgkinlymphoma and absence of B-cells as a consequence of anti-CD20 therapy successfully treated withCP from October 2020 to May 2021. CP was given in the presence of pneumonia with respiratoryfailure despite standard treatment and consisted of three infusions on an alternate-day basis. A reviewof the current literature on this topic was also performed. Six patients were identified (medianage 59.5 years (range 50-73)). The last anti-CD20 drug administration occurred 60 days before infection(range 0-360). CP was administered after a median of 51 days (range 9-120) from SARS-CoV-2diagnosis, with an early improvement in all but one subject. We suggest a possible clinical benefitof convalescent CP treatment in COVID-19 patients with haematological malignancies and B-celldepletion having persistent/recurrent pneumonia.
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PURPOSE: Due to relevant repercussions on reproductive medicine, we aimed to evaluate feasibility of RT-PCR as a detection method of SARS-CoV-2 RNA in seminal fluid. METHODS: A qualitative determination of the RT-PCR assays in semen was performed through different approaches: (1) efficiency of RNA extraction from sperm and seminal plasma was determined using PRM1 and PRM2 mRNA and a heterologous system as control; (2) samples obtained by diluting viral preparation from a SARS-CoV-2 panel (virus cultured in Vero E6 cell lines) were tested; (3) viral presence in different fractions of seminal fluid (whole sample, seminal plasma and post-centrifugation pellet) was evaluated. Semen samples from mild and recovered COVID-19 subjects were collected by patients referring to the Infectious Disease Department of the Policlinico Umberto I Hospital - "Sapienza" University of Rome. Control subjects were recruited at the Laboratory of Seminology-Sperm Bank "Loredana Gandini'' of the same hospital. RESULTS: The control panel using viral preparations diluted in saline and seminal fluid showed the capability to detect viral RNA presence with Ct values depending on the initial viral concentration. All tested semen samples were negative for SARS-CoV-2, regardless of the nasopharyngeal swab result or seminal fluid fraction. CONCLUSION: These preliminary data show that RT-PCR for SARS-CoV-2 RNA testing appears to be a feasible method for the molecular diagnosis of SARS-CoV-2 in seminal fluid, supported by results of the control panel. The ability to detect SARS-CoV-2 in semen is extremely important for reproductive medicine, especially in assisted reproductive technology and sperm cryopreservation.
Subject(s)
COVID-19/diagnosis , Pathology, Molecular/methods , Semen/virology , Adult , Animals , Chlorocebus aethiops , Feasibility Studies , Humans , Male , RNA, Messenger/chemistry , RNA, Viral/chemistry , Real-Time Polymerase Chain Reaction , Reproductive Techniques , Vero CellsABSTRACT
Aims An outbreak of coronavirus disease 19 (COVID-19) has altered the dynamic of endoscopic practices. Many guidelines, questionnaires have been published addressing service resumption during the pandemic. Curious about the situation indifferent endoscopic units across the globe, the study was designed to evaluate different aspects of practice resumptionworldwide and their adherence to guidelines. Methods An online questionnaire was created and distributed by national/regional representatives and societies. Redcapplatform was used as the interface;afterwards, Microsoft Excel 2016 and Prism 5 were utilized for data analysis. Results From a total of 307 responses from 47 countries/regions was collected, 290 valid answers were analyzed. Almosthalf (47 %) were in post-peak period by August, 2020. Many units were not designated to be COVID-oriented facility. About15.5 % of centers remained unrecovered, mainly in North and South America;those were recovered, training was still withheld significantly. Nevertheless, opened centers kept safety measurements strictly. Patient load was decreased by 37 %,but waiting list was increased 0-25 %. Among many surveillance methods, body temperature, PCR and chest CT were themost common. 74.8 % increased post-procedural disinfection time and 68.2 % increase in per-case inspection were noted.PPE usage was implemented highly and shortage of these posed as one of the resumption barriers. Post-procedural patientsurveillance was not reinforced. Conclusions The study represented real-time global endoscopic service's adaptation to COVID-19 pandemic. Previouslypublished barriers upon practice resumption remained. Despite Delphi consensus' emphasis on post-procedural surveillance, application was not widely reinforced, raising concerns in disease control.
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Background: A severe SARS-CoV-2 related immunopathology may be the driver cause underlying the deleterious clinical manifestations observed in COVID-19 patients. To identify possible tissue-specific immune responses patterns, a compartmental immunophenotyping analysis of CD4+ and CD8+ T lymphocytes and IFN response has been performed in SARS-CoV-2 infected subjects with acute respiratory distress syndrome. Methods: Bronchoalveolar lavage (BAL) and Peripheral Blood Mononuclear Cells (PBMC) samples were collected from 13 SARS-CoV-2 infected subjects (9 males and 4 females) consecutively admitted to intensive care unit (ICU) of Policlinico Umberto I, Sapienza University Hospital in Rome (Italy). The frequencies of CD4+, CD8+ T lymphocytes and those expressing immune activation markers (CD38, HLADR), naïve, central memory (CMEM), and effector memory (TEM) T cell subsets were evaluated in both anatomical sites by multiparametric flow cytometry. Gene expression levels of Interferon regulatory factor 7 (IRF7) and the Interferon Stimulated Gene 15 (ISG15) were evaluated in BAL and PBMC by Real-time PCR. Results: Critically SARS-CoV-2 infected patients exhibited a lung compartmentalization of CD8+ T cells (p=0.003), with a lower CD4/CD8 ratio in BAL compared to blood district (p<0.01). However, higher frequencies of CD8+ T cells were recorded in PBMC of female SARS-CoV-2 infected patients (p=0.04) and the same trend was observed in the lung compartment. By contrast, a trend of increasing CD4+ T cells frequencies was observed in BAL samples of male patients, as opposed to blood compartment. Additionally, an increased expression of immune activation markers CD38 and HLADR has been detected in BAL CD8+ T cells (p<0.01) as well as in blood CD4+ T cells (p=0.03). An increased frequency of CD4+ and CD8+ TEM cells has been documented in BAL of SARS-CoV-2 infected patients (p<0.05), as opposed to higher frequencies of CD4+ and CD8+ TCM cells recorded in the blood compartment (p<0.01). Notably, higher levels of ISG15 and IRF7 found in BAL were inversely associated to activated CD8+ T cell frequencies in the lung compartment compared to blood district (ISG15: r=-0.570, p<0.05) (IRF7: r=-0.683, p=0.01). Conclusion: Our findings provide new insight into a distinct T cells profile and IFN genes expression in the lung and in the blood compartment of SARS-CoV-2 infected patients, that might be highly relevant for the clinical course of COVID-19.
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INTRODUCTION: The recent appearance of SARS-CoV-2 in Wuhan in 2019 has started a pandemic which has involved over a million people worldwide. A matter of debate is the possible viral detection in different body fluids than respiratory droplets. Thus, we evaluated the possible presence of SARS-CoV-2 in semen and urine samples of a volunteer with confirmed COVID-19. MATERIALS AND METHODS: A 31-year-old man with fever, myalgia, anosmia, and ageusia was tested and found positive for SARS-CoV-2 through a pharyngeal swab. Eight days after he provided semen and urine samples in which viral RNA presence was measured using a Real time RT PCR system (RealStar SARS-CoV-2 RT-PCR, Altona Diagnostics) targeting E and S viral genes. RESULTS AND DISCUSSION: Semen and urine samples search for SARS-CoV-2 RNA was negative. Although this should be interpreted cautiously, it may be possible that either the viral clearance kinetics in these matrices matches the progressive clinical recovery of the patient or that the virus was never present in these fluids at the time of the laboratory diagnosis.