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Antimicrobial resistance is a worldwide problem, and resistance in Enterobacteriaceae, particularly Escherichia coli and Klebsiella pneumoniae, is a critical threat to human health. Inappropriate and unnecessary use of antibiotics in human health care is the most common cause for the development and spread of antimicrobial resistance. In this work, we retrospectively analyzed the antimicrobial data of E. coli strains isolated from midstream urinary samples over a 20-year period (2000-2019). The aim was to provide useful information to clinicians to prescribe a more appropriate empirical antibiotic therapy. A total of 30,955 unique E. coli isolates from positive midstream urine samples of inpatients (1,198) and outpatients (29,757) were collected. Except for carbapenems, over time all the antibiotics tested showed increasing resistance rates in both groups (p < 0.0001). On the other hand, fosfomycin and nitrofurantoin presented significant decreasing trends in resistance rate (p < 0.05). There were significant increases in extended-spectrum β-lactamases- and multidrug resistance positive isolates starting in 2000 (p < 0.0001), with similar results in both groups. Ciprofloxacin, gentamicin, trimethoprim/sulfamethoxazole, and third-generation cephalosporin resistances significantly increased with increasing age (p < 0.0001). Collectively, E. coli resistance rates severely increased during the study period, except for fosfomycin and nitrofurantoin. The need of monitoring studies about antibiotic nonsusceptibilities at local and regional levels are necessary to enhance the focus on antimicrobial stewardship, to reduce antimicrobial consumption and to detect alarming resistance mechanisms.

Antimicrobial resistance is a worldwide problem, and resistance in Enterobacteriaceae, particularly

The correlation of clinical, radiological and laboratory findings of patients at admission in the Emergency Department (ED) with clinical severity and risk of mortality was investigated. Adult coronavirus disease 2019 (COVID-19) patients hospitalized in March 2020 in Desio Hospital, Lombardy, were retrospectively included in the study, and categorized in terms of disease severity and adverse outcome. Out of the 175 patients enrolled, 79% presented one or more comorbidities, with cardiovascular disease being the most frequent (62%). More than half of the patients showed lymphocytopenia and 20% thrombocytopenia. The patients in the severe group presented higher absolute neutrophil count (ANC), C-reactive protein (CRP), AST, LDH, procalcitonin (PCT) and BUN values compared to the non-severe group (p 106.08 µmol/L (OR = 2.87; 95% CI 1.04–7.92) and creatine kinase > 2.90 µkat/L (OR = 3.80; 95% CI 1.31–10.9) were observed on admission (p

Establishing whole exome sequencing (WES) in an accredited clinical diagnostic space is challenging. The validation (as opposed to verification) of an approach that will lead to clinical reports requires adhering to international guidelines and recommendations and developing a robust analytical pipeline that can scale due to the increasing clinical demand for comprehensive gene screening. This chapter will present a step-wise approach to WES validation that any laboratory can follow. The focus will be on highlighting the pivotal technical issues that must be addressed in validating WES and the analytical tools and QC metrics that must be considered before implementing WES in a clinical environment.

Next generation DNA sequencing (NGS) has the potential to improve the diagnostic and prognostic utility of newborn screening programmes. This study assesses the feasibility of automating NGS on dried blood spot (DBS) DNA in a United Kingdom National Health Service (UK NHS) laboratory. An NGS panel targeting the entire coding sequence of five genes relevant to disorders currently screened for in newborns in the UK was validated on DBS DNA. An automated process for DNA extraction, NGS and bioinformatics analysis was developed. The process was tested on DBS to determine feasibility, turnaround time and cost. The analytical sensitivity of the assay was 100% and analytical specificity was 99.96%, with a mean 99.5% concordance of variant calls between DBS and venous blood samples in regions with &ge, 30&times, coverage (96.8% across all regions, all variant calls were single nucleotide variants (SNVs), with indel performance not assessed). The pipeline enabled processing of up to 1000 samples a week with a turnaround time of four days from receipt of sample to reporting. This study concluded that it is feasible to automate targeted NGS on routine DBS samples in a UK NHS laboratory setting, but it may not currently be cost effective as a first line test.

Clostridium like species, particularly Clostridium perfrigens, are the second most common causes of human anaerobic infections, including myonecrosis and bacteremia. Clostridium paraputrificum is an infrequent isolate, which has been identified in only 1% of reported cases of clostridial infections. We herein report a rare case of C. paraputrificum bacteremia in a 78-year-old Caucasian man diagnosed with an intestinal carcinoma and liver neoplastic lesions. The isolate was susceptible to chloramphenicol, meropenem, metronidazole, vancomycin, and resistant to clindamycin and penicillin, and the patient was successfully treated with metronidazole. Malignancy and inflammatory bowel diseases are often associated with clostridial bacteremia, which cannot be neglected.

There was no family history of neuromuscular disease. Examination revealed weakness particularly in the lower limbs, hyperrefl exia, wasting and fas-ciculation. There was no sensory or cognitive impair-ment. A diagnosis of ALS was made following full neurological investigation. She died from respiratory failure 31 months after symptom onset. Pathological material was obtained from the Sheffi eld Brain Tissue Bank. Ethics committee approval and written consent was obtained. Genomic DNA was extracted from cerebellar material and a C9orf72 expansion of 16 GGGGCC repeats was identifi ed by PCR analysis (Figure 1A) and Southern hybridization (8) (Figure 1B). Single nucleotide polymorphisms (SNPs) at rs3849942 and rs2814707 are highly associated with the 9p21 risk allele (1). Genotyping showed that our patient was heterozygous for the minor, or risk, allele at both positions. This is highly suggestive that her intermediate length expan-sion had occurred on the background of the 9p21 risk haplotype. Taqman allelic discrimination assay on an ABI 7900HT Real-Time PCR system was used for genotyping SNPs. Pre-designed primers and probes were purchased from Applied Biosystems (Foster City, USA). Histology revealed loss of lower motor neurons in the medulla and spinal cord with Bunina bodies in residual neurons. The motor cortex had detectable superfi cial cortical vacuolation. Immunohistochemis-try revealed p62 and TDP-43-positive neuronal and glial cytoplasmic inclusions in motor cortex, brain-stem and spinal cord. C9orf72 disease is associated with an abundance of ‘ star-shaped ’ ubiquitylated, TDP-43 negative inclusions in extramotor areas (6)

Background Crossover in the pathogenic mechanisms of amyotrophic lateral sclerosis (ALS) and multiple sclerosis (MS) has been described but is poorly understood. A GGGGCC hexanucleotide repeat expansion of C9ORF72 has recently been identified in a significant proportion of patients with ALS. Methods In approximately 650 patients diagnosed with ALS from the North of England we identified seven patients who initially presented with MS. DNA obtained from five patients with MS-ALS and 215 patients with MS alone was screened for the C9ORF72 expansion. Post-mortem material was examined from two patients with MS-ALS. Gene expression profiling was performed on lymphoblastoid cells and levels of CXCL10 were measured in cerebrospinal fluid (CSF) from patients with ALS with and without the C9ORF72 expansion and controls. Results Concurrence of MS and ALS is higher than expected in our population. The C9ORF72 expansion was identified in 80% of patients with MS-ALS but not in those with MS alone. In the presence of preceding MS, C9ORF72 -ALS was more rapidly progressive. MetaCore analysis identified alteration of the NF-кB pathway in C9ORF72 -ALS and non- C9ORF72 -ALS. NF-кB activation is associated with increased expression of the neuroprotective cytokine CXCL10 but, in C9ORF72 -ALS, CXCL10 is downregulated and CSF levels are reduced. Conclusions We propose that MS-associated neuroinflammation may affect penetrance and progression of the C9ORF72 expansion. In particular, the NF-кB pathway is activated in MS and appears to be dysfunctional in C9ORF72 -ALS. Aberrant downregulation of CXCL10 may explain the predisposition of C9ORF72 expansion carriers to develop ALS in the context of MS and NF-кB activation, and offers a potential therapeutic target.

EGFR mutation testing of tumor samples is routinely performed to predict sensitivity to treatment with tyrosine kinase inhibitors for patients with non-small cell lung cancer. At least 9 different methodologies are employed in UK laboratories, and the aim of this study was to compare the sensitivity of different methods for the detection of EGFR mutations. Participating laboratories were sent coded samples with varying mutation loads (from 0% to 15%) to be tested for the p.Leu858Arg (p.L858R) missense mutation and c.2235_2249del exon 19 deletion. The p.L858R mutation and deletions within exon 19 of the EGFR gene account for ∼90% of mutation-positive cases. The 11 laboratories used their standard testing method(s) and submitted 15 sets of results for the p.L858R samples and 10 for the exon 19 deletion. The p.Leu858Arg (p.L858R) mutation was detected at levels between 1% and 7.5% by Sanger sequencing, pyrosequencing, real-time polymerase chain reaction (PCR), amplification refractory mutation system, and capillary electrophoresis single-strand conformation analysis. The c.2235_2249del mutation was detected at 1% to 5% by fragment size analysis, Sanger sequencing or real-time PCR. A mutation was detected in 24/25 (96%) of the samples tested which contained 5% mutated DNA. The 1% sensitivity claimed for commercial real-time PCR-targeted EGFR tests was achieved and our results show greater sensitivity for the Sanger sequencing and pyrosequencing screening methods compared to the 10% to 20% detection levels cited on clinical diagnostic reports. We conclude that multiple methodologies are suitable for the detection of acquired EGFR mutations.

Objective: To determine the histopathologic bases for the observed incidence of parkinsonism in families with C9ORF72 expansions, which typically cause amyotrophic lateral sclerosis (ALS) and/or frontotemporal dementia. Methods: DNA was extracted from 377 brains with the histopathologic diagnosis of idiopathic Parkinson disease or related disorders and analyzed for C9ORF72 expansions. α-Synuclein and p62 immunohistochemistry of the substantia nigra (SN) was undertaken in brains of 17 ALS cases with ( C9ORF72 +) and 51 without ( C9ORF72 −) the C9ORF72 expansion. Results: Only 1 of 338 cases with pathologically confirmed idiopathic Parkinson disease had a C9ORF72 expansion. Similarly, only 1 of 17 C9ORF72 + brains displayed features suggestive of α-synucleinopathy. In contrast, p62-positive, TDP-43–negative neuronal cytoplasmic inclusions within the SN were considerably more frequent in C9ORF72 + brain tissue than in the C9ORF72 − brains ( p = 0.005). Furthermore, there was a more marked loss of dopaminergic neurons in the SN of C9ORF72 + ALS brains than C9ORF72 − ALS brains ( p = 0.029). Conclusions: SN involvement is common in C9ORF72 + ALS but can be clearly distinguished from Parkinson disease–related mechanisms by the presence of p62-positive inclusions and the absence of α-synuclein–positive Lewy bodies or Lewy neurites.

Intronic expansion of the GGGGCC hexanucleotide repeat within the C9ORF72 gene causes frontotemporal dementia and amyotrophic lateral sclerosis/motor neuron disease in both familial and sporadic cases. Initial reports indicate that this variant within the frontotemporal dementia/amyotrophic lateral sclerosis spectrum is associated with transactive response DNA binding protein (TDP-43) proteinopathy. The amyotrophic lateral sclerosis/motor neuron disease phenotype is not yet well characterized. We report the clinical and pathological phenotypes associated with pathogenic C9ORF72 mutations in a cohort of 563 cases from Northern England, including 63 with a family history of amyotrophic lateral sclerosis. One hundred and fifty-eight cases from the cohort (21 familial, 137 sporadic) were post-mortem brain and spinal cord donors. We screened DNA for the C9ORF72 mutation, reviewed clinical case histories and undertook pathological evaluation of brain and spinal cord. Control DNA samples (n = 361) from the same population were also screened. The C9ORF72 intronic expansion was present in 62 cases [11% of the cohort; 27/63 (43%) familial, 35/500 (7%) cases with sporadic amyotrophic lateral sclerosis/motor neuron disease]. Disease duration was significantly shorter in cases with C9ORF72-related amyotrophic lateral sclerosis (30.5 months) compared with non-C9ORF72 amyotrophic lateral sclerosis/motor neuron disease (36.3 months, P

Background An intronic GGGGCC hexanucleotide repeat expansion in C9orf72 is the most common genetic cause of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). Thirty repeats is the suggested pathogenic cut-off. However, the 9p21 risk haplotype has been associated with C9orf72 expansions >7 repeats, leading to the description of 7–24 repeats as intermediate. The pathogenicity of intermediate repeat lengths is unknown. Studies have suggested phenotypic similarities between patients with intermediate length expansions and those with larger repeat lengths, although given the broad phenotypic spectrum of ALS this is not conclusive. Previously, there has been no pathological characterisation of an intermediate expansion patient. Materials and Methods Pathological material was obtained from the Sheffield Brain Tissue Bank. Genomic DNA was extracted from cerebellar material and reverse primed PCR and Southern hybridisation was conducted to size the expansion. Immunohistochemistry and RNA fluorescence in-situ hybridisation (FISH) was used to screen for pathology typical of C9orf72-ALS. Results Repeat-primed PCR and Southern hybridisation revealed a repeat expansion size of 16 repeats in a sporadic ALS (SALS) patient. Neuropathological analysis demonstrated loss of motor neurons and immunohistochemistry revealed coarse neuronal skein-like inclusions identified by ubiquitin and P62 immunostaining. Pathology characteristic of C9orf72-disease, including extramotor neuronal inclusions, RNA foci and neuronal inclusions containing poly-(Gly-Ala)- dipeptide repeat protein, were absent in this patient. Conclusion A patient with an intermediate C9orf72 expansion of 16 repeats did not show the characteristic C9orf72 pathology suggesting that, in this case, the disease process was distinct from C9orf72-ALS, and the intermediate expansion is an incidental finding. This is consistent with current toxic gain-of-function theories of C9orf72-ALS. The 9p21 risk haplotype may predispose to expansion of the GGGGCC repeat locus, but we suggest that this must occur to some length larger than 16 repeats to cause the pathology characteristic of the C9orf72 subtype of ALS.

Background Crossover between neurodegenerative diseases is described but poorly understood. Hexanucleotide repeat expansions of C9ORF72 are a major cause of motorneuron disease (MND)/frontotemporal dementia but we and others have observed clinical heterogeneity within C9ORF72 -positive probands and their families, including a high incidence of parkinsonism. We aimed to determine whether C9ORF72 expansions are an upstream cause of clinically and pathologically distinct neurodegenerative diseases. This would have significant implications for neurodegeneration research. Methods In cohorts of patients with clinical parkinsonism (n=518), multiple sclerosis (MS) (n=215), and MND (n=563) we screened DNA for the C9ORF72 expansion, reviewed clinical histories, and undertook pathological evaluation of brain tissue where available. Findings We identified the C9ORF72 expansion in one patient with clinical parkinsonism (0·2%), 23 patients with MND (13·7%), and none of the patients with MS. The C9ORF72 positive parkinsonian patient had a family history of MND and displayed pathology consistent with MND with C9ORF72 expansion in addition to α-synucleinopathy. Two further patients with MND were identified with α-synucleinopathy: one with the C9ORF72 expansion, the other without. Of five MND patients who initially presented with MS, four (80%) were positive for the C9ORF72 expansion. C9ORF72 -MND is more rapidly progressive in the presence of preceding MS. Pathological examination of MND patients with C9ORF72 expansions revealed p62 positive, TDP-43 negative neuronal cytoplasmic inclusions in frontal cortex, hippocampus, and substantia nigra, which were relatively absent in MND patients without C9ORF72 expansions. Interpretation C9ORF72 expansions are not a major cause of either classic Parkinson's disease with α-synucleinopathy or MS. MS appears to increase the penetrance of the C9ORF72 expansion and exaggerate the severity. We suggest that p62 positive, TDP-43 negative neuronal cytoplasmic inclusions within the substantia nigra account for the association between C9ORF72 expansions and parkinsonism. Moreover we suggest that the distribution of these inclusions determines the clinical heterogeneity of C9ORF72 disease. Funding UK Medical Research Council.