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.

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.

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.

Chronic HCV Infection Is Associated with Overexpression of Human Endogenous Retroviruses that Persists after Drug-Induced Viral Clearance.

Chronic hepatitis C virus (HCV) infection is associated with several hepatic and extrahepatic complications, including cancers and autoimmune disorders, whose frequency is reduced but not abolished after drug-induced viral clearance. The causes of these complications and of their persistence are ill-defined. Human endogenous retroviruses (HERVs) are remnants of ancestral infections and constitute 8% of the human genome. Most HERV elements are inactive, but some are transcribed. HERV overexpression is associated with many cancers and autoimmune diseases with a putative pathogenetic role. Several viral infections trigger HERV activation, but there are no studies on HCV-infected subjects. We assessed, through a PCR real-time amplification assay, the transcription levels of the pol genes of HERV-H, -K, and -W, and of their repressor TRIM28 in white blood cells (WBCs) of vertically infected children, both before and after therapy with direct-acting antivirals (DAAs). The results documented significantly higher expressions of HERV-H-pol and HERV-K-pol, not of HERV-W-pol, in HCV-infected subjects as compared to age-matched controls. HERV RNA levels remained unchanged after DAA-driven viral clearance. No significant variations in transcription levels of TRIM28 were observed in infected subjects. Our findings demonstrate HERV-H-pol and HERV-K-pol overexpression in subjects with chronic HCV infection, without variations after a positive response to DAAs; this might justify their predisposition to cancers and autoimmune disorders that persist after a DAA-induced resolution of viremia.

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.

CD27 mRNA expression in mycosis fungoides.

BACKGROUND: The etiopathogenesis of MF remains obscure. CD27 is a member of the tumor necrosis factor receptor superfamily (TNFRS) that regulates lymphocyte function. Expression of CD27 protein and mRNA has been reported in B-cell lymphomas and adult T-cell leukemia/lymphoma. In this study, we examined the expression of CD27 in the skin of MF patients by real time PCR. The amount of CD27 was measured in MF patients and healthy controls. METHODS: A total of 98 skin biopsies were analyzed: 12 obtained from healthy donors and 86 obtained cryostatic sections OCT-embedded affected by MF. Relative quantification of mRNA CD27 expression was achieved by means of TaqMan (Thermo Fisher Scientific, Waltham, MA, USA) amplification and normalization to glyceraldehyde-3-phosphate dehydrogenase (GAPDH). RESULTS: Housekeeping gene was detectable in all skin samples and there is no difference between healthy control and MF P value =0.1564. CD27 mRNA sequences were found in 3 of 12 (25%) of skin obtained from healthy donors and in 59 of 86 (68%) of skin obtained from cryostatic sections OCT-embedded affected by MF. The χ2 statistic with Yates correction is 6.8413 and the P value is 0.0089. When we compared the CD27 expression in MF and controls the RQ analysis showed a value of 9.12±14.13. A RQ of 9.12 means that this gene is 9.12 times more expressed in MF skin samples then in the healthy skin samples. No difference was observed in the MF clustered by stages. CONCLUSIONS: Our findings indicates that CD27 can be used as diagnostic/prognostic markers, and whether anti-CD27 antibodies can be used in therapy.

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.

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.

Modulation of human endogenous retroviruses -H, -W and -K transcription by microbes.

The human endogenous retroviruses (HERVs) are endogenous retroviruses that are inserted into the germ cell DNA of humans over 30 million years ago. Using real-time RT-PCR we describe HERV modulation by commensal microbes in the human gut. Infants, exclusively or predominant breast milk feeding, less than 12 weeks of age, during bacteria gut colonization, were assessed for eligibility. Our data demonstrate that the colonization with commensal microbes, in particular, Bifidobacterium spp., of the gut causes modulation of HERVs.