Non-operative management after immune checkpoint inhibitors for early-stage, dMMR/MSI-H gastrointestinal cancers.

Surgery is a standard treatment for early-stage gastrointestinal cancers, often preceded by neoadjuvant chemo(radio)therapy or followed by adjuvant therapy. While leading to cure in a proportion of patients, it has some drawbacks such as intra/post-operative complications, mutilation and life-long functional sequelae. Further to the unprecedented efficacy data from studies of immune checkpoint inhibitors for advanced mismatch repair deficient/microsatellite instable (dMMR/MSI-H) tumours, a strong interest has recently emerged for the investigation of such agents in the neoadjuvant setting. Although limited by the exploratory design and small sample size, trials of neoadjuvant immune checkpoint inhibitors for early-stage dMMR/MSI-H gastrointestinal cancers have consistently reported complete response rates ranging from 70 % to 100 %. As a result, the question has arisen as to whether surgery is still needed or organ-preserving strategies should be offered to this especially immuno-sensitive population. In this article, we discuss the available evidence for neoadjuvant immune checkpoint inhibitors in dMMR/MSI-H gastrointestinal cancers and analyse opportunities and challenges to the implementation of non-operative management approaches in this setting.

Fruquintinib as new treatment option in metastatic colorectal cancer patients: is there an optimal sequence?

INTRODUCTION: Available treatments for colorectal cancer are limited. However, in the last few years several advances and new treatment options became available and expanded the continuum of care in metastatic colorectal cancer (mCRC). AREAS COVERED: Fruquintinib, a tyrosine kinase inhibitor, has been shown to be effective in heavily pretreated mCRC progressing to trifluridine-tipiracil (FTD/TPI) or regorafenib or both. Preclinical studies have shown that fruquintinib inhibits with high selectivity VEGFR 1-2-3, leading to a blockade in angiogenesis process, but also acts, with weak inhibition, on RET, FGFR-1, and c-kit kinases. Fruquintinib demonstrated good efficacy and tolerance in chemorefractory mCRC in two phase III trial: FRESCO and FRESCO 2. These results led to FDA approval of fruquintinib for pretreated mCRC patients who received prior fluoropyrimidine-, oxaliplatin-, and irinotecan-based chemotherapy. EXPERT OPINION: Fruquintinib is a valid therapeutic option for heavily pretreated mCRC patients. However, an optimal sequence of treatments is yet to be defined. In this review, we propose an algorithm for later lines of treatment to integrate fruquintinib as a standard of care together with the new therapeutic combinations that recently showed clinical benefit for chemorefractory mCRC, in both molecularly selected (e.g. KRASG12C or HER2 amplification) and in non-oncogenic driven patients.

Neoadjuvant chemotherapy for early-stage colon cancer.

Surgery with or without adjuvant chemotherapy is the standard treatment for early-stage colon cancer. However, evidence has recently emerged for neoadjuvant chemotherapy, with the results of randomised clinical trials sparking debates within multidisciplinary teams and splitting the gastrointestinal oncology community. Further to a systematic search of the literature, we provide a thorough and in-depth analysis of the findings from these trials, highlighting the advantages and disadvantages of neoadjuvant chemotherapy. We conclude that, while there is a potential value of moving systemic therapy from the post-operative to the pre-operative setting, the available evidence does not justify a shift in the treatment paradigm of early-stage colon cancer, and surgery with or without adjuvant chemotherapy should remain the standard approach for these patients.

Prognostic Value of Circulating Cytokines in Chemorefractory Colorectal Cancer.

Circulating cytokines could be optimal biomarkers for prognostication and management decisions in colorectal cancer (CRC). Chemorefractory CRC patients with available plasma samples were included in this study. In the discovery cohort (n = 85), 182 circulating cytokines were tested with a semi-quantitative multiplex assay, and prognostic cytokines were analyzed in the validation cohort (n = 111) by ELISA. Overall survival (OS) was the primary outcome measure, with the false discovery rate (FDR) method (significance level of <0.01) being used to correct for multiple comparisons. Four cytokines were associated with OS in the discovery cohort: insulin-like growth factor-binding protein 1 (IGFBP-1) (HR 2.1 [95%CI: 1.58-2.79], FDR < 0.001), insulin-like growth factor-binding protein 2 (IGFBP-2) (HR 1.65 [95%CI: 1.28-2.13], FDR = 0.006), serum amyloid A (SAA) (HR 1.84 [95%CI: 1.39-2.43], FDR < 0.001), and angiotensin II (HR 1.65 [95%CI: 1.29-2.1], FDR = 0.006). Of these, IGFBP-1 (HR 2.70 [95%CI: 1.56-4.76], FDR = 0.007) and IGFBP-2 (HR 3.33 [95%CI: 1.64-6.67], FDR = 0.008) were confirmed to be independently associated with OS in the validation cohort. Patients with high concentrations of IGFBP-1 and/or IGFBP-2 had a median OS of 3.0 months as compared with 6.9 months for those with low concentrations of both cytokines (HR 2.44 [95%CI: 1.52-4.0], FDR = 0.002) Validation of circulating IGFBP-1 and IGFBP-2 as independent prognostic biomarkers for chemorefractory CRC in larger, independent series is warranted.

Total Neoadjuvant Treatment for Locally Advanced Rectal Cancer Patients: Where Do We Stand?

The therapeutic landscape in locally advanced rectal cancer (LARC) has undergone a significant paradigm shift in recent years with the rising adoption of total neoadjuvant treatment (TNT). This comprehensive approach entails administering chemotherapy and radiation therapy before surgery, followed by optional adjuvant chemotherapy. To establish and deliver the optimal tailored treatment regimen to the patient, it is crucial to foster collaboration among a multidisciplinary team comprising healthcare professionals from various specialties, including medical oncology, radiation oncology, surgical oncology, radiology, and pathology. This review aims to provide insights into the current state of TNT for LARC and new emerging strategies to identify potential directions for future research and clinical practice, such as circulating tumor-DNA, immunotherapy in mismatch-repair-deficient tumors, and nonoperative management.

Expressions of Type I and III Interferons, Endogenous Retroviruses, TRIM28, and SETDB1 in Children with Respiratory Syncytial Virus Bronchiolitis.

Interferons (IFNs) and IFN-stimulated genes (ISGs) play essential roles for the control of viral infections. Their expression in infants with respiratory syncytial virus (RSV) bronchiolitis is poorly defined. Human endogenous retroviruses (HERVs) represent 8% of our genome and modulate inflammatory and immune reactions. TRIM28 and SETDB1 participate in the epigenetic regulation of genes involved in the immune response, including IFNs and HERVs. No study has explored the expression of HERVs, TRIM28, and SETDB1 during RSV bronchiolitis. We assessed, through a PCR real-time Taqman amplification assay, the transcription levels of six IFN-I ISGs, four IFNλs, the pol genes of HERV-H, -K, and -W families, the env genes of Syncytin (SYN)1 and SYN2, and of TRIM28/SETDB1 in whole blood from 37 children hospitalized for severe RSV bronchiolitis and in healthy children (HC). The expression of most IFN-I ISGs was significantly higher in RSV+ patients than in age-matched HC, but it was inhibited by steroid therapy. The mRNA concentrations of IFN-λs were comparable between patients and age-matched HC. This lack of RSV-driven IFN-III activation may result in the defective protection of the airway mucosal surface leading to severe bronchiolitis. The expression of IFN-III showed a positive correlation with age in HC, that could account for the high susceptibility of young children to viral respiratory tract infections. The transcription levels of every HERV gene were significantly lower in RSV+ patients than in HC, while the expressions of TRIM28/SETDB1 were overlapping. Given the negative impact of HERVs and the positive effects of TRIM28/SETDB1 on innate and adaptive immune responses, the downregulation of the former and the normal expression of the latter may contribute to preserving immune functions against infection.

MW Polyomavirus in diarrheal Italian infants.

BACKGROUND: MXPyV, like MWPyV, was identified in stool samples from children suffering diarrhea in Mexico. In this study, we used a home-made real time PCR to investigate the presence of this novel viruses in stool specimen collected from under-five-year-old children with gastroenteritis. METHODS: A total of 192 fecal specimens previously screened for RV, ADV, NoV, HPeV and SaV, were tested for MWPyV with Taqman real time PCR. RESULTS: The most detected virus was NoV GII (33.8%), followed by RV (21.3%), SaV (10.9%), HPeV (8%), NoV GI (6.7%) and Adv (1%). Real time PCR detected MWPyV in 1/192 (0.5%) patients. CONCLUSIONS: We detected MWPyV in 0.5% of fecal specimens collected from pediatric patients suffering gastroenteritis which is smaller than the previously reported in literature (4.4% in Australia and 12% Mexico).

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.

A Rapid and Specific Real-Time PCR Assay for the Detection of Clinically Relevant Mucorales Species.

Infections triggered by filamentous fungi placed in the order Mucorales, phylum Zygomycota, can cause serious harm to immunocompromised patients. Since there is lack of a standardized PCR (polymerase chain reaction) assay for early diagnosis of this fungal infection, this work was aimed to develop a new PCR assay able to detect the presence of Mucorales genera in clinical specimens. Here, we describe a novel diagnostic TaqMan MGB probe assay for precise and rapid detection of the most common clinical species of Mucorales. Zygomycete-specific oligonucleotides were designed to specifically amplify and bind highly conserved sequences of fungal 28S rRNA gene. Additionally, we succeeded in differentiating Mucorales species (i.e., Rhizopus, Lichtheimia, Mucor, and Rhizomucor) in artificially infected serum samples, suggesting that the quantitative capability of this real-time PCR assay could potentially optimize the diagnosis of mucormycosis.

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.

A new protocol for validation of Chondro, Adipo and Osteo differentiation kit of Cultured Adipose-Derived Stem Cells (ADSC) by real-time rt-QPCR.

Mesenchymal stem cells (MSCs) are multipotent cells, originally derived from the embryonic mesenchyme, and able to differentiate into connective tissues such as bone, fat, cartilage, tendon, and muscle. Furthermore, MSCs derived from adipose tissue ADSC (Adipose derived Stem Cells) show a great potential for degenerative diseases treatment. In this study, we designed a series of experiments based on real-time rt-QPCR to validated a commercially available kit able to explore changes in gene expression under osteogenic, adipogenic and chondrogenic differentiation of human ADSC. Initially, we selected a better indicators of trilineage differentiation by using third passages of cultured ADSC from stromal vascular fraction (SVF) isolated from fresh adipose tissue by enzymatic digestion. On the basis of statistically significant results ACAN, FABP4A and Col11a1 were chosen as indicators of chondrogenic, adipogenic and osteogenic differentiation respectively. An in-vitro aging analysis was then performed to evaluate the ADSC passage with the highest differentiation potential. Total RNA extraction from induced differentiation and controls ADSC from passage 2-6 and relative quantifications of mRNA expression of selected genes were performed according to rt-PCR kits tested. The chondrogenic differentiation test showed equivalent ∆∆Ct values for ACAN detection for cell passages ranging from P3 to P6, proving that they can be considered as equivalent samples for differentiation assays evaluation. For what concerns adipogenic differentiation and FABP4 detection, similar results were observed in all the cell passages tested; on the contrary only passage P6 showed suitable ∆∆Ct values for Col11a1 detection for osteogenic differentiation evaluation. In conclusion, we have validated a suitable real-time rt-QPCR protocol for osteogenic, chondrogenic and adipogenic ADSC differentiation ability evaluation in-vitro.

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.

Analysis of Serum Th2 Cytokines in Infants with Non-IgE Mediated Food Allergy Compared to Healthy Infants.

Background: The aim of this study is to assess the serum values of IL-4, IL-5, IL-10, and IL-13 in a group of infants with non-IgE mediated food allergies treated with a hydrolyzed formula and compare them with a group of healthy peers. Methods: A total of 53 infants aged 1 to 4 months, of which 34 with non-IgE mediated food allergies and 19 healthy infants were enrolled in this study. Infants were eligible if they had gastrointestinal symptoms of food allergy and needed to switch from their initial formula to hydrolyzed formulas with an improvement of symptoms. Controls were fed with either breastmilk or standard formula. Blood samples were taken within one week of a special diet for cases. Interleukinsin in peripheral blood was detected and analyzed using the real-time PCR MAMA method. Fecal calprotectin was evaluated using a quantitative assay. Results: Values of IL-4 and IL-13 were significantly higher in the non-IgE food allergy group compared to the control group (p < 0.05), while IL-5 and IL-10 were significantly lower than the control group (p < 0.05). Fecal calprotectin in the non-IgE food allergy group was significantly higher compared to the control group (p < 0.05). Conclusion: This study provides a theoretical basis that Th2 cytokine expression in infants with a non-IgE mediated food allergy is significantly different than in healthy infants; this finding supports the use of early dietetic treatment with hydrolyzed formulas.

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.

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.

Evaluation of two RNA extraction methods for human Parechovirus detection on pediatric stool specimens.

BACKGROUND: Human Parechovirus (HPeVs), along with human enteroviruses is associated with gastrointestinal and respiratory infections. The aim of this study was to assess the performance characteristics of two nucleic acid extraction for HPeV-RNA quantitation, the RNAlev Extraction Kit associated with Maxwell automated nucleic acid extractor (Promega, Milan, Italy) and RNAzol manually protocol. METHODS: A total of 137 fecal specimens previously routinely screened for Rotavirus and Adenovirus were tested for HPeV virus. RESULTS: Methods 1 and 2 detected HPeV-RNA in 11 and 10 samples, respectively, with a 96.2% concordance. In particular, 124/137 (90.5%) were concordantly negative by both methods; 8/137 (5.8%) concordantly positive. For the 8 specimens that were positive by both tests, the population mean (SD) was 320 (601) copies/mg with method 1 and 1216 (2338) copies/mg with method 2. By referring to the 8 specimens that were concordantly positive, the correlation value between the two methods was not statistically significant (r=0.381 and P=0.3599). Bland-Altman analysis showed that differences between the two methods were within ±1000 copies/mg of the averaged results for all of the tested specimens. CONCLUSIONS: Method 2, being a semi-automated method, provides benefits over a manual method, in terms of turnaround time, less errors and reliability.