To evaluate the water compartment antibiotic-resistance contamination rates, 11 wells, five streams, and four treatment plants located in the Oltrepò Pavese area were screened for the presence of third generation cephalosporins resistant Gram-negative bacteria. Enterobacteriaceae were also characterized for the Extended-Spectrum-β-Lactamases (ESBLs), carbapenemases, and mcr-1 genes presence. From December 2014 to November 2015, 246 water samples were filtered, plated on Plate Count Agar, MacConkey Agar, and MacConkey Agar with cefotaxime. Isolates were species identified using AutoSCAN-4-System and ESBLs, carbapenemases, and colistin resistance determinants were characterized by PCR, sequencing, and microarray. Plasmid conjugative transfer experiments, PCR-based Replicon typing, Pulsed-Field Gel Electrophoresis, Multi-Locus-Sequence-Typing, and in-silico plasmid characterization were performed. A total of 132 enterobacteria isolates grew on MacConkey agar with cefotaxime: 82 (62.1%) were obtained from streams, 41 (31.1%) from treatment plants, and 9 (6.8%) from wells. Thirty out of 132 (22.7%) isolates, mainly belonging to Escherichia coli (n = 15) species, showed a synergic effect with piperacillin-tazobactam. A single ESBL gene of blaCTX−M-type was identified in 19/30 isolates. In further two E. coli strains, a blaCTX−M−1 gene co-existed with a blaSHV-type ESBL determinant. A blaSHV−12 gene was detected in two isolates of E. coli (n = 1) and Klebsiella oxytoca (n = 1), while any ESBL determinant was ascertained in seven Yersinia enterocolitica strains. A blaDHA-type gene was detected in a cefoxitin resistant Y. enterocolitica from a stream. Interestingly, two Klebsiella pneumoniae strains of ST307 and ST258, collected from a well and a wastewater treatment plant, resulted KPC-2, and KPC-3 producers, respectively. Moreover, we report the first detection of mcr-1.2 ST10 E. coli on a conjugative IncX4 plasmid (33.303 bp in size) from a stream of Oltrepò Pavese (Northern Italy). Both ESBLs E. coli and ESBLs/carbapenemase-producing K. pneumoniae strains showed clonal heterogeneity by Pulsed-Field Gel Electrophoresis and Multi-Locus-Sequence-Typing. During one-year study and taking in account the whole Gram-negative bacterial population, an average percentage of cefotaxime resistance of 69, 32, and 10.3% has been obtained for the wastewater treatment plants, streams, and wells, respectively. These results, of concern for public health, highlight the need to improve hygienic measures to reduce the load of discharged bacteria with emerging resistance mechanisms.

This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

In recent years, the influence of nutrition on the health and growth of children has become increasingly important. The relevance of nutrition is even greater for children who are facing cancer. Malnutrition, within the context of undernutrition and overnutrition, may impact not only the effectiveness of treatments and outcomes, but also the quality of life for patients and their families. In this article, we review nutritional assessment methods for children with cancer, focusing on the specific characteristics of this population and analyze the efficacy of nutritional interventions, which include enteral, parenteral, and nutritional education. From our analysis, two important conclusions emerged: i) there is a need to focus our attention on the nutritional status and the body composition of oncologic children, since these factors have a relevant impact on clinical outcomes during treatment as well as after their conclusion; ii) the support of skilled clinical nutrition personnel would be extremely helpful for the global management of these patients.

Background Human cytomegalovirus (HCMV) is the leading infectious cause of congenital disabilities. We designed a prospective study to investigate the rate, outcome and risk factors of congenital cytomegalovirus infection (cCMV) in neonates born to immune women, and the potential need and effectiveness of hygiene recommendations in this population. Methods The study (NCT03973359) was composed of 2 sequential parts: an epidemiology (Part 1) and a prevention (Part 2) study. Performance of Part 2 depended upon a cCMV rate > 0.4%. Women enrolled in Part 1 did not receive hygiene recommendations. Newborns were screened by HCMV DNA testing in saliva and cCMV was confirmed by urine testing. Results Saliva swabs were positive for HCMV DNA in 45/9661 newborns and cCMV was confirmed in 18 cases. The rate of cCMV was 0.19% (95% CI: 0.11-0.29%), and three out of 18 infants with cCMV had symptoms of CMV at birth. Age, nationality, occupation and contact with children were similar between mothers of infected and non-infected newborns. Twin pregnancy (OR: 7.2; 95% CI 1.7-32.2; p = 0.037) and maternal medical conditions (OR: 3.9; 95% CI: 1.5-10.1; p = 0.003) appeared associated with cCMV. Given the rate of cCMV lower than expected, the prevention part of the study was cancelled. Conclusion Newborns from women with preconception immunity have a low rate of cCMV, which appears to be mostly due to reactivation of the latent virus. Therefore, serological screening in childbearing age would be pivotal to identify HCMV-seropositive women, whose newborns have a low risk of cCMV.

Background: Strain-specific antibodies to human cytomegalovirus (HCMV) glycoproteins B and H (gB and gH) have been proposed as a potential diagnostic tool for identifying reinfection. We investigated genotype-specific IgG antibody responses in parallel with defining the gB and gH genotypes of the infecting viral strains. Methods: Subjects with primary (n = 20) or non-primary (n = 25) HCMV infection were studied. The seven gB (gB1-7) and two gH (gH1-2) genotypes were determined by real-time PCR and whole viral genome sequencing, and genotype-specific IgG antibodies were measured by a peptide-based enzyme-linked immunosorbent assay (ELISA). Results: Among subjects with primary infection, 73% (n = 8) infected by gB1-HCMV and 63% (n = 5) infected by gB2/3-HCMV had genotype-specific IgG antibodies to gB (gB2 and gB3 are similar in the region tested). Peptides from the rarer gB4-gB7 genotypes had nonspecific antibody responses. All subjects infected by gH1-HCMV and 86% (n = 6/7) infected by gH2-HCMV developed genotype-specific responses. Among women with non-primary infection, gB and gH genotype-specific IgG antibodies were detected in 40% (n = 10) and 80% (n = 20) of subjects, respectively. Conclusions: Peptide-based ELISA is capable of detecting primary genotype-specific IgG responses to HCMV gB and gH, and could be adopted for identifying reinfections. However, about half of the subjects did not have genotype-specific IgG antibodies to gB.

Each patient with a monoclonal gammopathy has a unique monoclonal (M) protein, whose sequence can be used as a tumoral fingerprint to

Introduction In patients affected by monoclonal gammopathies, tumoral B cells or plasma cells secrete a monoclonal antibody (termed M protein), which can be used to track the presence of the tumor itself. Moreover, the M protein can directly cause potentially life-threatening organ damage, which is dictated by the specific, patient's unique clonal light and/or heavy chain, as in patients affected by immunoglobulin light chain (AL) amyloidosis. Yet, the current paradigm in the diagnosis and management of these conditions treats the M protein as a simple tumor biomarker to be identified/quantified. Patients' specific M protein sequences remain mostly undefined and molecular mechanisms underlying M-protein related clinical manifestations are largely obscure. Methods By combining the unbiased amplification of expressed immunoglobulin genes with long-read, single molecule real-time DNA sequencing and bioinformatics analyses, we have established a method to identify the full-length sequence of the variable region of expressed immunoglobulin genes and to rank the obtained sequences based on their relative abundance, thus enabling the identification of the full-length variable sequence of M protein genes from a high number of patients analysed in parallel. Results The assay, which we termed Single Molecule Real-Time Sequencing of the M protein (SMaRT M-Seq), has undergone an extensive technical validation. Sequencing of contrived bone marrow samples generated through serial dilutions of plasma cell lines into control bone marrow, as well as sequencing of bona fide bone marrow samples from AL patients and comparison with gold-standard techniques of immunoglobulin gene sequencing showed: 100% sequence-accuracy at the individual base-pair level; High repeatability (CV<0.8% for sequencing of pentaplicates) in defining the molecular clonal size (i.e. the fraction of total immunoglobulin sequences coinciding with the clonal sequence); A high sensitivity in identifying clonal immunoglobulin sequences (10 -3 when employing low-coverage sequencing on multiple, pooled samples). Noteworthy, SMaRT M Seq was applied to a cohort of 86 consecutive patients with AL amyloidosis (17 κ and 69 λ; median BMPC infiltration 9%, IQR 6-13%; median dFLC 176 mg/L, IQR 75-370 mg/L), including cases with small clonal burden and M protein which was undetectable with conventional M protein studies. A full-length sequence of the variable region of the clonal light chain was obtained in all patients (median molecular clonal size of 88.3%, IQR: 70.7 - 93%). The most common κ germline genes were IGKV1-33 and IGKV4-01 (24% each of the 17 κ AL patients), and the most common λ germline genes were IGLV6-57 (26% of the 69 λ AL patients), IGLV2-14 (17%), IGLV3-01 (17%) and IGLV1-44 (10%). The most frequent λ and κ germline genes together (IGLV6-57, IGLV2-14, IGLV3-01, IGLV1-44, IGKV1-33 and IGKV4-01) accounted for 66% of all the clones. Germline gene usage correlated with selected clinical features. Sequence information was then exploited to improve mass spectrometry-based amyloid typing on fat pad aspirates and to enable the sensitive detection of clonotypic sequences using short-read DNA sequencing of the involved light chain isotype (up to 10 -7 dilution). Conclusions We have established SMaRT M-Seq as a novel valuable assay to reliably identify the full-length variable sequence of M proteins. SMaRT M-Seq has undergone extensive technical validation, showing high accuracy, repeatability and sensitivity. The latter is determined by the number of reads analyzed per sample. This is in turn dictated by the sequencing output of the employed sequencing platform, and by the number of pooled samples analyzed in a given sequencing round, thus proving to be scalable. Even when analyzing multiple samples on a sequencing platform with low sequencing output, the achieved sensitivity of SMaRT M-Seq significantly exceeds the requirements for the identification of clonal B cells/plasma cells in patients with AL amyloidosis. Sequencing disease-associated M proteins from large cohorts of patients has the potential to uncover molecular mechanisms of M protein-related clinical manifestations which have remained largely unexplored so far, and could enable approaches of personalized medicine for the sensitive detection of patients' specific M proteins at diagnosis and after anti-clonal therapy. Disclosures Milani: Celgene: Other: Travel support; Janssen-Cilag: Honoraria. Fazio: Janseen: Honoraria. Petrucci: GSK: Honoraria, Other: Advisory Board; Amgen: Honoraria, Other: Advisory Board; Takeda: Honoraria, Other: Advisory Board; BMS: Honoraria, Other: Advisory Board; Janssen-Cilag: Honoraria, Other: Advisory Board; Celgene: Honoraria, Other: Advisory Board; Karyopharm: Honoraria, Other: Advisory Board. Palladini: Pfizer: Honoraria; Siemens: Honoraria; Janssen Global Services: Honoraria, Other: advisory board fees. Nuvolone: Janssen-Cilag: Honoraria; Oncopeptides, Inc.: Research Funding.

Acute myeloid leukemia (AML) with nucleophosmin (NPM1) genetic mutations is the most common subtype in adult patients. Refractory or relapsed disease in unfit patients or after allogeneic hematopoietic stem cell transplantation (allo-HSCT) has a poor prognosis. NPM1-mutated protein, stably expressed on tumor cells but not on normal tissues, may serve as an ideal target for NPM1-mutated AML immunotherapy. The study aim was to investigate the feasibility of producing mutated-NPM1-specific cytotoxic T cells (CTLs) suitable for somatic cell therapy to prevent or treat hematologic relapse in patients with NPM1-mutated AML. T cells were expanded or primed from patient or donor peripheral blood mononuclear cells by NPM1-mutated protein-derived peptides, and tested for leukemia antigen-targeted cytotoxic activity, cytokine production and hematopoietic precursor inhibitory effect. We found that mutated-NPM1-specific CTLs, displaying specific cytokine production and high-level cytotoxicity against patients’ leukemia blasts, and limited inhibitory activity in clonogenic assays, could be obtained from both patients and donors. The polyfunctional mutated-NPM1-specific CTLs included both CD8+ and CD4+ T cells endowed with strong lytic capacity. Our results suggest that mutated-NPM1-targeted CTLs may be a useful therapeutic option to control low-tumor burden relapse following conventional chemotherapy in older NPM1-mutated AML patients or eradicate persistent MRD after HSCT.