Characteristics of RNA Stabilizer RNApro for Peripheral Blood Collection.

Peripheral blood is the most practical tissue for human immune system gene expression profiling because it is easily accessible, whereas the site of primary infection in certain diseases may not be easily accessible. Due to the ex vivo instability of RNA transcripts, a key challenge in the gene expression analysis of blood samples is the rapid sample handling and stabilization of the mRNA by adding an RNA preservative (PAXgeneTM Blood RNA Tubes, TempusTM Blood RNA tubes, RNAlater Stabilization Reagent, RNAgard® Blood Tubes). BioMole (Turin, Italy) has developed a novel blood stabilizer, called RNApro, in which RNA is stabilized during phlebotomy and sample storage. In this study, RNApro performance intended as RNA yield, integrity, and stability was evaluated. Our results show that blood samples stored at -80 °C and re-extracted after 7 years show no differences in terms of quantity, quality, and amplificability. The samples in the RNAlater stabilization solution can be stored at room temperature for up to one week or at 4 °C for up to one month. Similar results can also be observed for PAXgene tubes, Tempus tubes, and RNAgard tubes. In agreement with these data, the RNApro stabilization solution preserves the RNA from degradation for up to 1 month at 4 °C and 1 week at room temperature. RNApro can be stored indifferently at -80, -20, 4 °C, or room temperature for up to 2 months after, and then could be stored at -80 °C for up to seven years. In summary, our study is the first to analyze the performance of an RNA stabilizer called RNApro. We can conclude that several studies have shown significant differences in gene expression analysis when the sample was preserved in different RNA stabilizers. Therefore, it is desirable to standardize the method of nucleic acid conservation when comparing data from transcriptomic analyses.

Nasopharyngeal and Peripheral Blood Type II Interferon Signature Evaluation in Infants during Respiratory Syncytial Virus Infection.

Background and Objectives: In this study, we applied one-step real time rt-PCR technology type II INF signature to blood and nasopharyngeal (NPS) swabs of acute early recovery children < 1 years hospitalized for bronchiolitis with laboratory-confirmed RSV infection. Materials and Methods: A prospective observational case-control study was conducted in 2021-2022. The study took place in Children Hospital "Regina Margherita", Torino Italy. The study included 66 infants, of which 30 patients were hospitalized for bronchiolitis due to RSV infection and 36 age-matched controls. Inclusion criteria included a positive RSV test for infants with bronchiolitis. We collected peripheral blood and nasopharyngeal swabs for relative quantification of type II Interferon signature by One-Step Multiplex PCR real time. Results: IFN levels were downregulated in the peripheral blood of bronchiolitis patients; these data were not confirmed in the nasopharyngeal swab. There was no correlation between NPS and the type II IFN score in peripheral blood. Conclusions: our study shows for the first time that type II IFN score was significant reduced in peripheral blood of infants with bronchiolitis by RSV compared to age-matched healthy controls; in the NPS swab this resulted downregulation was not statistically significant and the type II IFN score in the NPS swab can be used as marker of resolution of infection or improvement of clinical conditions.

Impact of the COVID-19 pandemic on Italian students' mental health and relationships: differences between adolescent males and females.

BACKGROUND: The negative effects of the COVID-19 pandemic on the mental health of young people have been reported, often with possible differences between the sexes. This study explores the impact of the COVID-19 pandemic on the differences in mental health status and healthy behaviors between adolescent males and females. METHODS: An online questionnaire exploring sociodemographic characteristics, living conditions, mental health, behaviors, and relationships during the implementation of the COVID-19 social restriction measures, was administered to 16-18 years old high school students living in the metropolitan area of Turin. The World Health Organization-5 Well-Being Index (WHO-5) questionnaire was used to evaluate self-perceived psychological health status. Descriptive analyses of the sample were carried out by sex. Chi-squared test and the associated P value were evaluated. The spatial sign method was used to assess multivariate differences by sex. RESULTS: Seventy-two Italian high school students completed the questionnaire. Sixty-eight percent of the students reported psychological problems, more often females (86% vs. 50%). Those living with multiple cohabitants and in dwellings with less personal space reported lower well-being. Females were also more likely to report stress when talking about COVID-19, difficulties in concentration and in falling asleep, daytime sleepiness, less physical activity, and concerns about losing control of eating. Finally, females spent more time with family members and, compared to males, found it harder to spend time and share experiences with friends and schoolmates. CONCLUSIONS: The pandemic has greatly impacted adolescents in terms of their mental well-being, routines, relationships with friends, and sleeping and eating patterns. Females reported more stress, boredom, bad mood, eating concerns, reduced relationships with friends, and possible difficulty in falling asleep than did males. These issues should be addressed when developing and promoting support programs, particularly at school.

Children with Chronic Immune Thrombocytopenia Exhibit High Expression of Human Endogenous Retroviruses TRIM28 and SETDB1.

Chronic immune thrombocytopenia (CITP) is an autoimmune disease whose underlying biologic mechanisms remain elusive. Human endogenous retroviruses (HERVs) derive from ancestral infections and constitute about 8% of our genome. A wealth of clinical and experimental studies highlights their pivotal pathogenetic role in autoimmune diseases. Epigenetic mechanisms, such as those modulated by TRIM28 and SETDB1, are involved in HERV activation and regulation of immune response. We assessed, through a polymerase chain reaction real-time Taqman amplification assay, the transcription levels of pol genes of HERV-H, HERV-K, and HERV-W; env genes of Syncytin (SYN)1, SYN2, and HERV-W; as well as TRIM28 and SETDB1 in whole blood from 34 children with CITP and age-matched healthy controls (HC). The transcriptional levels of all HERV sequences, with the exception of HERV-W-env, were significantly enhanced in children with CITP as compared to HC. Patients on eltrombopag treatment exhibited lower expression of SYN1, SYN2, and HERV-W-env as compared to untreated patients. The mRNA concentrations of TRIM28 and SETDB1 were significantly higher and were positively correlated with those of HERVs in CITP patients. The over-expressions of HERVs and TRIM28/SETDB1 and their positive correlations in patients with CITP are suggestive clues of their contribution to the pathogenesis of the disease and support innovative interventions to inhibit HERV and TRIM28/SETDB1 expressions in patients unresponsive to standard therapies.

Peripheral Blood and Nasopharyngeal Swab MiRNA-155 Expression in Infants with Respiratory Syncytial Virus Infection.

INTRODUCTION: MicroRNA (miR) 155 has been implicated in the regulation of innate and adaptive immunity as well as antiviral responses, but its role during respiratory syncytial virus (RSV) infections is not known. The objective of this study was to investigate the expression of miR-155 using pharyngeal swabs and peripheral blood in infants with RSV infection and uninfected controls. METHODS: A prospective age-matched study was conducted in primary care in Torino from 1 August 2018 to 31 January 2020. We enrolled 66 subjects, 29 of them patients with RSV infection and 37 age-matched uninfected controls, and collected pharyngeal swabs and peripheral blood in order to assess miR-155 expression with real-time stem-loop-TaqMan real-time PCR. RESULTS: The data show that there is no correlation between pharyngeal swabs and peripheral blood with respect to miR-155 expression. The 1/ΔCq miR-155 expression levels in throat swabs in RSV bronchiolitis patients and healthy controls were 0.19 ± 0.11 and 0.21 ± 0.09, respectively, and were not significantly different between healthy controls and bronchiolitis (p = 0.8414). In the peripheral blood, miR-155 levels were higher than those of healthy control subjects: 0.1 ± 0.013 and 0.09 ± 0.0007, respectively; p = 0.0002. DISCUSSION: Our data provide evidence that miR-155 expression is higher in peripheral blood during RSV infection but not in swabs. This difference in the timing of sample recruitment could explain the differences obtained in the results; miR-155 activation is probably only assessable in the very early stages of infection in the swab and remains visible for longer in the blood. New investigations are needed in order to clarify whether the miR-155 expression in swabs can be influenced by different stages of virus disease of infants.

Pregnancy Is Associated with Impaired Transcription of Human Endogenous Retroviruses and of TRIM28 and SETDB1, Particularly in Mothers Affected by Multiple Sclerosis.

Accumulating evidence highlights the pathogenetic role of human endogenous retroviruses (HERVs) in eliciting and maintaining multiple sclerosis (MS). Epigenetic mechanisms, such as those regulated by TRIM 28 and SETDB1, are implicated in HERV activation and in neuroinflammatory disorders, including MS. Pregnancy markedly improves the course of MS, but no study explored the expressions of HERVs and of TRIM28 and SETDB1 during gestation. Using a polymerase chain reaction real-time Taqman amplification assay, we assessed and compared the transcriptional levels of pol genes of HERV-H, HERV-K, HERV-W; of env genes of Syncytin (SYN)1, SYN2, and multiple sclerosis associated retrovirus (MSRV); and of TRIM28 and SETDB1 in peripheral blood and placenta from 20 mothers affected by MS; from 27 healthy mothers, in cord blood from their neonates; and in blood from healthy women of child-bearing age. The HERV mRNA levels were significantly lower in pregnant than in nonpregnant women. Expressions of all HERVs were downregulated in the chorion and in the decidua basalis of MS mothers compared to healthy mothers. The former also showed lower mRNA levels of HERV-K-pol and of SYN1, SYN2, and MSRV in peripheral blood. Significantly lower expressions of TRIM28 and SETDB1 also emerged in pregnant vs. nonpregnant women and in blood, chorion, and decidua of mothers with MS vs. healthy mothers. In contrast, HERV and TRIM28/SETDB1 expressions were comparable between their neonates. These results show that gestation is characterized by impaired expressions of HERVs and TRIM28/SETDB1, particularly in mothers with MS. Given the beneficial effects of pregnancy on MS and the wealth of data suggesting the putative contribution of HERVs and epigenetic processes in the pathogenesis of the disease, our findings may further support innovative therapeutic interventions to block HERV activation and to control aberrant epigenetic pathways in MS-affected patients.

Prevalence of human Cosavirus and Saffold virus in young children with gastroenteritis in Northern Italy.

BACKGROUND: Gastroenteritis is a common disease in children, characterized by diarrhea, vomiting, abdominal pain, and fever. Human Cosavirus (HCoSV) and Saffold virus (SAFV) both have a worldwide distribution. Both viruses have been detected in the stools of patients with acute gastroenteritis in several countries. METHODS: In order to provide more insights into the epidemiology of enteric viruses that are not included usually in routine diagnostic tests, cases of childhood sporadic gastroenteritis of unknown etiology requiring hospital admission in Turin, Italy, during December 2014 to November 2015, were screened for HCoSV and SAFV. RESULTS: A total of 1 out of 164 (0.6%) episodes of acute gastroenteritis were associated with SAFV genomic detection. Among the 1 SAFV-positive cases, 1 were also positive for Adenovirus. The patient positive for SAFV do not present diarrheal episodes but vomiting. HCoSV was not detected in any of the samples. CONCLUSIONS: In conclusion, this study presents the current epidemiological data regarding the two viruses, HCoSV and SAFV, circulating in pediatric patients admitted to hospital with acute gastroenteritis in Turin, Italy.

Human Bocavirus in children with acute gastroenteritis in Piedmont, Italy.

BACKGROUND: Gastroenteritis is a common disease in children, characterized by diarrhea, vomiting, abdominal pain, and fever. Co-detection of human Bocavirus (HBoV) with other gastroenteric viruses was reported a lot in patients with acute gastroenteritis. METHODS: This paper presents the real-time RT-PCR Taqman assay for the detection and quantification of HBoV for clinical fecal samples collected from hospitalized children with acute gastroenteritis in Piedmont. RESULTS: All fecal specimens were tested for the presence of HBoV with specific primers and probe. A total of 17 out of 123 (13.92%) episodes of acute gastroenteritis were associated with HBoV genomic detection with median viral load 6864.75±19784.79 genomes/mg fecal specimens. Among the 17 HBoV-positive cases, 11 were also positive for other viral pathogens, including Rotavirus (N.=2), astrovirus (N.=1), norovirus GII (N.=6), norovirus GI (N.=2). Two cases were positive for more than one virus including norovirus GII and norovirus GI (N.=1) and Rotavirus, sapovirus and astrovirus (N.=1). A higher detection of HBoV infections was observed in winter, and peaking in February. CONCLUSIONS: Although HBoV is suspected to be responsible for gastroenteritis in children, our data showed that this association was uncertain since no difference was observed in term of viral load in the group with single infection of HBoV and group of coinfections with other viral agent.

Quantification of fecal adenovirus viral load and correlation with Vesikari Score in children with acute gastroenteritis.

BACKGROUND: Human adenoviruses (HAdVs) are an important cause of acute respiratory tract infections, conjunctivitis, hemorrhagic cystitis, and gastroenteritis. In addition to enteric serotypes 40 and 41, some serotypes belonging to subgroups A, B, and C have also been implicated to be etiological agents of gastroenteritis among infants and young children. The Vesikari Scoring System (VSS) is the severity scale that was originally developed to evaluate the effectiveness and efficacy of rotavirus vaccines on 20 points. The aim of this study was to evaluate and compare the diagnostic value of the VSS with HAdVs genome quantification in fecal samples collected from hospitalized children with acute gastroenteritis. METHODS: A total of 137 fecal specimens (69 male and 68 female) were tested for HAdVs. The samples were collected from under-five-year-old children with acute gastroenteritis in pediatric Hospital Regina Margherita of Turin in Italy. RESULTS: A total of 69 out of 137 (50.3%) samples were associated with HAdV genomic detection with a mean viral load of 1.08×1011±9.02×1011 genomes/mg fecal specimens. The samples were grouped on the basis of Mild VSS and Moderate VSS and the HAdV viral load was calculated in the two groups. No statistical differences were observed between two groups (P=0.6123 calculated by Mann-Whitney Test). CONCLUSIONS: Our results did not show a difference in mean viral load between the group with mild VVS and moderate VVS.

Mir-155 expression is downregulated in hematopoietic stem cell transplantation patients with Epstein-Barr virus infection.

BACKGROUND: MicroRNAs (miRNAs) are a class of short length double strand genome encoded RNAs that are produced to repress post-transcriptionally the expression of cellular mRNAs. 2578 unique mature miRNAs are currently annotated in the human genome and participate in the regulation of multiple events, such as cellular proliferation or apoptosis. The over-expression of miR-155 of cellular origin might play a key role in the life cycle of EBV. In this study 24 pediatric patients undergoing HSCT seropositive and seronegative to EBV were enrolled. Thirty-one peripheral blood samples were collected from these patients. The mir-155 expression profile has been evaluated by a stem-loop Real Time PCR in all these conditions. METHODS: Of 24 patients, 4 were seronegative to EBV and EBV negative to PCR (Group I), 10 were seropositive to EBV and EBV negative to PCR (Group II) and 10 were seropositive to EBV and EBV positive to PCR (Group III). RESULTS: Based on relative quantification, the mir-155 expression was compared among the groups. The comparison between HSCT patients without EBV infection seronegative to EBV (Group I) showed higher levels of mir-155 expression than patients seropositive to EBV (P=0.1419). The mir-155 expression levels in seronegative to EBV were not significantly different compared with the patients seropositive to EBV (P=0.6504). The mir-155 expression levels in seropositive to EBV without and with EBV infection (positive viral load), were not significantly (P=0.7667). Also, when we evaluated the mir-155 expression levels comparing all EBV negative patients with an active EBV infection, we did not observe a statistically significant difference (P=0.9782). CONCLUSIONS: Our results are controversial, showing a higher production of mir-155 levels during EBV infection.

Lack of detection of HPyV12 DNA using real-time PCR in Italian infants with diarrhea.

BACKGROUND: HPyV12 was found in organs of the digestive tract, in particular the liver but also in colon, rectum and feces. Until now, the prevalence of HPyV12 is not well characterized. METHODS: In this study, we investigate the presence of this novel polyomavirus DNA in stool specimens collected from under-five-year-old children with gastroenteritis compared to healthy infants. A total of 190 fecal specimens previously screened for rotavirus (RV) and adenovirus (ADV) and 80 fecal samples from healthy infants, were tested for HPyV12 DNA using a home-made real time PCR. All fecal specimens were tested for the presence of HPyV12 with specific primers and probes. RESULTS: None of 190 (0%) episodes of acute gastroenteritis was associated with HPyV12. We did not detect HPyV12 DNA in any of 80 control subjects, as well. CONCLUSIONS: Our study represents a pilot study aiming to clarify the current epidemiological pattern in pediatric Italian patients regarding the novel and rare HPyV12. Based on our negative data and the recent observations reported in literature, doubts remain on human tropism of the HPyV12 and epidemiology: these issues need further investigations.

Enhanced Expression of Human Endogenous Retroviruses, TRIM28 and SETDB1 in Autism Spectrum Disorder.

Human endogenous retroviruses (HERVs) are relics of ancestral infections and represent 8% of the human genome. They are no longer infectious, but their activation has been associated with several disorders, including neuropsychiatric conditions. Enhanced expression of HERV-K and HERV-H envelope genes has been found in the blood of autism spectrum disorder (ASD) patients, but no information is available on syncytin 1 (SYN1), SYN2, and multiple sclerosis-associated retrovirus (MSRV), which are thought to be implicated in brain development and immune responses. HERV activation is regulated by TRIM28 and SETDB1, which are part of the epigenetic mechanisms that organize the chromatin architecture in response to external stimuli and are involved in neural cell differentiation and brain inflammation. We assessed, through a PCR realtime Taqman amplification assay, the transcription levels of pol genes of HERV-H, -K, and -W families, of env genes of SYN1, SYN2, and MSRV, as well as of TRIM28 and SETDB1 in the blood of 33 ASD children (28 males, median 3.8 years, 25-75% interquartile range 3.0-6.0 y) and healthy controls (HC). Significantly higher expressions of TRIM28 and SETDB1, as well as of all the HERV genes tested, except for HERV-W-pol, were found in ASD, as compared with HC. Positive correlations were observed between the mRNA levels of TRIM28 or SETDB1 and every HERV gene in ASD patients, but not in HC. Overexpression of TRIM28/SETDB1 and several HERVs in children with ASD and the positive correlations between their transcriptional levels suggest that these may be main players in pathogenetic mechanisms leading to ASD.

Assessment of interferon gamma and indoleamine 2,3-dioxygenase 1 analysis during respiratory syncytial virus infection in infants in Italy: an observational case-control study.

OBJECTIVES: The aim of this study was to measure interferon gamma (IFN-γ) and indoleamine 2,3-dioxygenase 1 (IDO1) values in the White blood cells of infants during respiratory tract infections and to compare these with healthy age-matched controls. DESIGN: This was a prospective, observational case-control study conducted in 2019-2020. SETTING: The study took place at Regina Margherita Children's Hospital, Turin, Italy. PARTICIPANTS: The study comprised 63 infants, including 26 patients hospitalised for bronchiolitis due to a respiratory syncytial virus (RSV) infection and 37 age-matched controls. The inclusion criteria included a positive RSV test for an infant with bronchiolitis. METHODS: We collected peripheral blood and measured the relative quantification of messenger RNA (mRNA) expression of IFN-γ and IDO1 with TaqMan real-time PCR amplification. The data were collected on the first day of admission. RESULTS: The mean age of the 26 patients with RSV bronchiolitis (53.8% female) was 85 (9-346) days when they were admitted to the hospital. Their mean gestational age at birth was 38 weeks and their mean birth weight was 3100 (2780-3730) g. The expression of IFN-γ was significantly reduced in patients with bronchiolitis RSV compared with healthy controls (p=0.0132). However, there was no significant difference between the two groups when the IDO1 mRNA expression values in their WCC were measured (p=0.0642). CONCLUSION: Our findings did not clarify whether IDO1 expression was related to the early stage of the disease or to the young age of the infants. The data provide evidence that IFN-γ was significantly reduced in infants with bronchiolitis due to RSV, compared with age-matched healthy controls, but the IDO1 was not different. New investigations that focus on subjects infected with RSV at different stages of infancy would help to clarify whether IDO1 expression can be related to age.

Enhanced expression of endogenous retroviruses and of TRIM28 and SETDB1 in children with food allergy.

BACKGROUND: Human endogenous retroviruses (HERVs) represent 8% of our genome. They originate from ancestral infections and although no longer contagious they can regulate transcription of adjacent cellular genes, produce viral RNAs sensed as non-self by pattern recognition receptors, and encode viral proteins, such as Syncytin (SYN) 1 and 2, that exhibit potent immunomodulatory properties. Based on this, HERVs have been studied and proposed as relevant cofactors in several chronic inflammatory and immune-mediated diseases. HERV transcription is regulated by host TRIM28 and SET domain bifurcated histone lysine methyltransferase 1 (SETDB1), which in turn exert crucial regulatory functions on the host immune system. No studies explored the expression of HERVs, TRIM28, and SETDB1 in allergic patients. METHODS: We assessed, through a polymerase chain reaction real time Taqman amplification assay, the transcription levels of pol genes of HERV-H, HERV-K, HERV-W, and of env genes of SYN1 and SYN2, as well as of TRIM28 and SETDB1 in whole blood from 32 children with IgE-mediated food allergy, 19 with food protein-induced enterocolitis syndrome (FPIES), and in healthy control children. RESULTS: The expression levels of pol genes of HERV-H, -K, and -W were significantly enhanced in patients with IgE-mediated FA or FPIES as compared to control subjects, while the mRNA concentrations of SYN1 and SYN2 were comparable in each group of children. Both TRIM28 and SETDB1 mRNA levels were significantly higher in allergic patients. CONCLUSIONS: Given the influence of HERVs and of TRIM28 and SETDB1 on innate and adaptive immune responses, their transcriptional activation in children with food allergies suggest that they might play important roles in the development of these diseases.

Evaluation of the FCGR2B polymorphism in children with immune thrombocytopenia.

BACKGROUND: Childhood immune thrombocytopenia (ITP) is an immune-mediated disease characterized by an isolated low platelet count. Pathogenesis of ITP is complex but many patients have platelet specific autoantibodies leading to accelerated clearance of opsonized platelets by Fc-gamma receptor (FcγR) bearing phagocytes, particularly in the spleen. In humans, there are three main types of Fcγ receptors: high-affinity FcγRI and low-affinity FcγRII and FcγRIII. About FcγRII, genetic variation of FCGR2B is associated with response to IVIg treatment in patients with Kawasaki disease and ITP. OBJECTIVE: We used a TaqMAMA genotyping assay for detection of rs1050501 FCGR2B polymorphism in children with chronic ITP. STUDY DESIGN: A SNP rs1050501 (GenBank access number NG_023318.1, Homo sapiens Fc fragment of IgG receptor IIb (FCGR2B)) on chromosome 1 showing a T/C transition in position 15894 on FCGRB2 gene was chosen in this study. A series of experiments was conducted to evaluate the performance of the FCGR2B-MAMAPCR real time on a QuantStudio™ 5 Real-Time PCR System as compared to the 7500 Real-Time PCR System. RESULTS: Background noise, Genotypes discrimination, Variability, Allele and genotype frequencies and concordance were obtained. About clinical validation, all 60 samples collected from chronic ITP patients were analyzed. We found 53 on the 60 patients (88.4%) were homozygotes (52 TT and 1 CC) and 7/60 (11.6%) heterozygotes (TC). CONCLUSIONS: The ability of the FCGR2B-MAMAPCR real time to detect rs1050501 FCGR2B polymorphism in children with chronic ITP on the QuantStudio™ 5 System is comparable to that on the 7500 System.

Macrophage Receptor With Collagenous Structure Polymorphism and Recurrent Respiratory Infections and Wheezing During Infancy: A 5-Years Follow-Up Study.

Background: Recurrent wheezing is a common clinical manifestation in childhood, and respiratory syncytial virus infection is a well-known risk factor. However, the genetic background favoring the development of recurrent wheezing is not fully understood. A possible role of macrophage receptor with collagenous gene (MARCO) polymorphism has been recently proposed. Objective: To investigate a correlation between MARCO rs1318645 polymorphisms and susceptibility to recurrent wheezing during childhood. Methods: We prospectively recruited 116 infants, of which 58 with respiratory syncytial virus bronchiolitis and 58 controls hospitalized at Regina Margherita Children's Hospital, Turin, Italy, between November 2014 and April 2015. All subjects were investigated for MARCO rs1318645 polymorphisms in the first period of life. Genotyping of rs1318645 was carried out by TaqMan mismatch amplification mutation assay real-time polymerase chain reaction procedure. Subjects were then enrolled in a 5-year follow-up study to monitor the occurrence of wheezing and respiratory infections. Results: The analysis of MARCO rs1318645 of allelic frequencies shows an increasingly significant risk to develop recurrent infection (p = 0.00065) and recurrent wheezing (p = 0.000084) with a wild-type C allele compared with a G allele. No correlation was found between wheezing and past respiratory syncytial virus infection (p = 0.057) and for a history of atopy in the family (p = 0.859). Conclusion: Our finding showed that subjects with C allelic MARCO rs1318645 polymorphism are at higher risk for recurrent infection and wheezing episodes during the first 5 years of life. Future studies of genetic associations should also consider other types of polymorphisms.

COVID-19 in Children: Expressions of Type I/II/III Interferons, TRIM28, SETDB1, and Endogenous Retroviruses in Mild and Severe Cases.

Children with the new coronavirus disease 2019 (COVID-19) have milder symptoms and a better prognosis than adult patients. Several investigations assessed type I, II, and III interferon (IFN) signatures in SARS-CoV-2 infected adults, however no data are available for pediatric patients. TRIM28 and SETDB1 regulate the transcription of multiple genes involved in the immune response as well as of human endogenous retroviruses (HERVs). Exogenous viral infections can trigger the activation of HERVs, which in turn can induce inflammatory and immune reactions. Despite the potential cross-talks between SARS-CoV-2 infection and TRIM28, SETDB1, and HERVs, information on their expressions in COVID-19 patients is lacking. We assessed, through a PCR real time Taqman amplification assay, the transcription levels of six IFN-I stimulated genes, IFN-II and three of its sensitive genes, three IFN-lIIs, as well as of TRIM28, SETDB1, pol genes of HERV-H, -K, and -W families, and of env genes of Syncytin (SYN)1, SYN2, and multiple sclerosis-associated retrovirus (MRSV) in peripheral blood from COVID-19 children and in control uninfected subjects. Higher expression levels of IFN-I and IFN-II inducible genes were observed in 36 COVID-19 children with mild or moderate disease as compared to uninfected controls, whereas their concentrations decreased in 17 children with severe disease and in 11 with multisystem inflammatory syndrome (MIS-C). Similar findings were found for the expression of TRIM-28, SETDB1, and every HERV gene. Positive correlations emerged between the transcriptional levels of type I and II IFNs, TRIM28, SETDB1, and HERVs in COVID-19 patients. IFN-III expressions were comparable in each group of subjects. This preserved induction of IFN-λs could contribute to the better control of the infection in children as compared to adults, in whom IFN-III deficiency has been reported. The upregulation of IFN-I, IFN-II, TRIM28, SETDB1, and HERVs in children with mild symptoms, their declines in severe cases or with MIS-C, and the positive correlations of their transcription in SARS-CoV-2-infected children suggest that they may play important roles in conditioning the evolution of the infection.

TaqMan real time PCR for the Detection of the Gilbert's Syndrome Markers UGT1A1*28; UGT1A1*36 and UGT1A1*37.

Gilbert's syndrome is characterized by mild unconjugated hyperbilirubinemia. The key of this disease is a diminished activity of UDP-glucuronosyltransferase 1A1 (UGT1A1). TA insertion into the TATA box promoter region of the UGT1A1 gene on chromosome 2 is the genetic basis of Gilbert's syndrome (UGT1A1*28). An extra TA insert leads to eight (TA)8 repeats (UGT1A1*37) resulting in a further reduction of glucuronidation activity. A variant lacking one TA repeat (TA)5 (UGT1A1*36) has been identified. (TA)8 repeats (UGT1A1*37) and (TA)5 (UGT1A1*36) have been detected in Africans (frequency up to 0.07 and 0.08 respectively). We present a real time PCR method for genotyping the UGT1A1 (TA)n polymorphism (UGT1A1*28, UGT1A1*36, UGT1A1*37) using Taqman PCR on 7500 and cfx96 Real-Time PCR System. We present a real time PCR method for genotyping the UGT1A1 (TA)n polymorphism (UGT1A1*28, UGT1A1*36, UGT1A1*37) using Taqman PCR. About clinical validation, all 53 samples collected from patients referred for suspected Gilbert's syndrome were analyzed. We found 21 on the 53 patients (39.6%) were homozygotes (UGT1A1-TATA (TA)6) and referred as wild-type, 13 on the 53 patients (24.5%) were homozygotes (UGT1A1-TATA (TA)7) and referred as mutated, 1 on the 53 patients (1.9%) were homozygotes (UGT1A1-TATA (TA)8) and referred as mutated, 1 on the 53 patients (1.9%) were heterozygotes (UGT1A1-TATA (TA)7/8) and referred as mutated, 1 on the 53 patients (1.9%) were heterozygotes (UGT1A1-TATA (TA)5/6) and referred as mutated, and 16 on the 53 patients (30.2%) were heterozygotes (UGT1A1-TATA (TA)6/7). None were homozygotes UGT1A1-TATA (TA)5, homozygotes UGT1A1-TATA (TA)8, or heterozygotes with (TA)5 or (TA)8 alleles. The newly described technique represents a valid alternative method to sequencing, mainly due to its rapidity, easiness, and minor costs.

Comparison of methods for isolating fungal DNA.

OBJECTIVES: The main aim of this work was to compare the methods of DNA isolation in the moulds of genus Mucorales with special regard to the amount and purity of the DNA acquired. The acquired DNA was then amplified by specific real-time PCR. DESIGN: Five DNA extraction procedures were carried out in a Class 2 Biosafety cabinet in a dedicated room with suitable biosafety precautions and appropriate biowaste disposal methods. A total of 6 Mucorales clinical strains were used. RESULTS: From the viewpoint of concentration and purity, methods A shown abundant amount of fungal DNA whereas methods E report a pure fungal DNA with R260/280 of 1.7 near the optimal 1.8. The DNA quantity reach statistically difference at ANOVA test with p value 0.0005. CONCLUSION: Overall, the E method was the most efficient method in the extraction of DNA from fungal cultures compared to the other methods considering time, cost, technical expertise, and instrumentation. Use of this assay will allow researchers to obtain DNA from fungi quickly for use in molecular assays.

Bufavirus, Cosavirus, and Salivirus in Diarrheal Italian Infants.

Three newly discovered viruses have been recently described in diarrheal patients: Cosavirus (CosV) and Salivirus (SalV), 2 picornaviruses, and bufavirus (BuV), a parvovirus. The detection rate and the role of these viruses remain to be established in acute gastroenteritis (AGE) in diarrheal Italian infants. From November 2016 to November 2017, stool samples were collected from 160 children <5 years old suffering from AGE and attending the Children's Hospital in Turin, Italy. During the study period, 1 (0.5%) sample was positive for 1 of the 3 investigated viruses: 0 (0%) CosV, 1 (0.5%) SalV, and 0 (0%) BuV, whereas 42 (26.0%) children were infected with rotavirus and 2 (1%) with adenovirus. No mixed infections involving the 3 viruses were found. Although these viruses are suspected to be responsible for AGE in children, our data showed that this association was uncertain. Therefore, further studies with large cohorts of healthy and diarrheal children will be needed to evaluate their clinical role in AGE.