Rotavirus quantification in children with acute gastroenteritis.

INTRODUCTION: Most intractable diarrheas are treated with antibiotics, irrespective of the causative agent. This study aims to quantified Rotavirus with taqman real-time PCR in fecal samples of children with acute gastroenteritis in accordance with the program of reduction of drug-resistance and use of antibiotics. METHODS: A total of 190 fecal specimens were collected from under-five-year-old children with acute gastroenteritis in pediatric Hospital Regina Margherita of Turin in Italy in 2017. A total of 38 out of 67 (56.7%) episodes of acute gastroenteritis were associated with Rotavirus genomic detection with multiplex commercial kit. RESULTS: All fecal specimens were tested for the presence of RV and other GE viruses. The most commonly detected virus was norovirus (41%), astrovirus (15.8%), human bocavirus (8.9%), sapovirus (7.9%), human parechovirus (5.8%), rhinovirus (4.2%), and adenovirus (1%). In 66 out of 190 (34.7%) Rotavirus were detectable with the median viral load 7.2E+11±60E+11genomes/mg fecal specimens. DISCUSSION/CONCLUSIONS: Our results showed that RV was present in around 34.7% of children hospitalized for AGE, a rate similar to those reported in previous studies conducted elsewhere which were in the range of 33-75%. Our protocol are able to quantify the absolute number of viral particle/mg of feces. The clinical utility of quantitative molecular assays, such as Rotavirus viral load, will be markedly improved.

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.

Functional study of TNF-α as a promoter of polymorphisms in psoriasis.

BACKGROUND: TNF-α is an important mediator in the pathogenesis of psoriasis and polymorphisms influence its transcription and could be implicated in psoriasis risk and modify certain aspects of disease, such as age at onset of psoriasis vulgaris and disease severity. Six TNF-α single nucleotide polymorphisms (SNPs) in promoter region has been identified and studied but with discordant results. The aim of this study was to evaluate whether the polymorphisms in TNF-α (-238 [rs361525], -308 [rs1800629], -857 [rs1799724], -1031 [rs1799964]) are associated with severity, itch, early onset or response to drug therapy in psoriasis in Caucasian Italian patients. METHODS: Fifty-eight psoriasis patients from Turin PSOCARE, 23 with psoriasis vulgaris and 35 with psoriatic arthritis were studied. Ready-to-use master mix for allelic discrimination of rs1800629, rs361525 and rs1799964 respectively. RESULTS: Our data showed a significant association between the -857(G) variant and both VAS-itch (P=0.03) and VAS-pain index (P=0.006), OR=0.2 (0.04-0.98) and OR=0.12 (0.02-0.59). No significant association between the genotypes or alleles of TNF-α SNPs has been observed with other clinic-pathologic parameters or etanercept response. CONCLUSIONS: Our data suggest that -857 CC genotype could be involved in pain and itch severity in psoriasis patients.