Short-read, high-throughput sequencing technology cannot identify the chromosomal position of repetitive insertion sequences that typically flank horizontally acquired genes such as bacterial ...virulence genes and antibiotic resistance genes. The MinION nanopore sequencer can produce long sequencing reads on a device similar in size to a USB memory stick. Here we apply a MinION sequencer to resolve the structure and chromosomal insertion site of a composite antibiotic resistance island in Salmonella Typhi Haplotype 58. Nanopore sequencing data from a single 18-h run was used to create a scaffold for an assembly generated from short-read Illumina data. Our results demonstrate the potential of the MinION device in clinical laboratories to fully characterize the epidemic spread of bacterial pathogens.
Most anaplastic lymphoma kinase (ALK)-positive non-small cell lung cancers (NSCLCs) are highly responsive to treatment with ALK tyrosine kinase inhibitors (TKIs). However, patients with these cancers ...invariably relapse, typically within 1 year, because of the development of drug resistance. Herein, we report findings from a series of lung cancer patients (n = 18) with acquired resistance to the ALK TKI crizotinib. In about one-fourth of patients, we identified a diverse array of secondary mutations distributed throughout the ALK TK domain, including new resistance mutations located in the solvent-exposed region of the adenosine triphosphate-binding pocket, as well as amplification of the ALK fusion gene. Next-generation ALK inhibitors, developed to overcome crizotinib resistance, had differing potencies against specific resistance mutations. In addition to secondary ALK mutations and ALK gene amplification, we also identified aberrant activation of other kinases including marked amplification of KIT and increased autophosphorylation of epidermal growth factor receptor in drug-resistant tumors from patients. In a subset of patients, we found evidence of multiple resistance mechanisms developing simultaneously. These results highlight the unique features of TKI resistance in ALK-positive NSCLCs and provide the rationale for pursuing combinatorial therapeutics that are tailored to the precise resistance mechanisms identified in patients who relapse on crizotinib treatment.
Lung cancers harboring mutations in the epidermal growth factor receptor (EGFR) respond to EGFR tyrosine kinase inhibitors, but drug resistance invariably emerges. To elucidate mechanisms of acquired ...drug resistance, we performed systematic genetic and histological analyses of tumor biopsies from 37 patients with drug-resistant non-small cell lung cancers (NSCLCs) carrying EGFR mutations. All drug-resistant tumors retained their original activating EGFR mutations, and some acquired known mechanisms of resistance including the EGFR T790M mutation or MET gene amplification. Some resistant cancers showed unexpected genetic changes including EGFR amplification and mutations in the PIK3CA gene, whereas others underwent a pronounced epithelial-to-mesenchymal transition. Surprisingly, five resistant tumors (14%) transformed from NSCLC into small cell lung cancer (SCLC) and were sensitive to standard SCLC treatments. In three patients, serial biopsies revealed that genetic mechanisms of resistance were lost in the absence of the continued selective pressure of EGFR inhibitor treatment, and such cancers were sensitive to a second round of treatment with EGFR inhibitors. Collectively, these results deepen our understanding of resistance to EGFR inhibitors and underscore the importance of repeatedly assessing cancers throughout the course of the disease.
Three benchtop high-throughput sequencing instruments are now available. The 454 GS Junior (Roche), MiSeq (Illumina) and Ion Torrent PGM (Life Technologies) are laser-printer sized and offer modest ...set-up and running costs. Each instrument can generate data required for a draft bacterial genome sequence in days, making them attractive for identifying and characterizing pathogens in the clinical setting. We compared the performance of these instruments by sequencing an isolate of Escherichia coli O104:H4, which caused an outbreak of food poisoning in Germany in 2011. The MiSeq had the highest throughput per run (1.6 Gb/run, 60 Mb/h) and lowest error rates. The 454 GS Junior generated the longest reads (up to 600 bases) and most contiguous assemblies but had the lowest throughput (70 Mb/run, 9 Mb/h). Run in 100-bp mode, the Ion Torrent PGM had the highest throughput (80–100 Mb/h). Unlike the MiSeq, the Ion Torrent PGM and 454 GS Junior both produced homopolymer-associated indel errors (1.5 and 0.38 errors per 100 bases, respectively).
Background COPD is a recognized risk factor for lung cancer, but studies of coexisting COPD in relation to lung cancer outcomes are limited. We assessed the impact of COPD on overall survival (OS) ...and progression-free survival (PFS) in patients with early-stage non-small cell lung cancer (NSCLC). Methods Patients (N = 902) with early-stage (stage IA-IIB) NSCLC treated with surgical resection were retrospectively analyzed. The association of self-reported, physician-diagnosed COPD with survivals of NSCLC was assessed using the log-rank and Cox regression models, adjusting for age, sex, BMI, smoking, stages, and performance status. Results Among this cohort of patients with NSCLC, 330 cases had physician-diagnosed COPD, and 572 did not have COPD. The 5-year OS in patients with COPD (54.4%) was significantly lower ( P = .0002) than that in patients without COPD (69.0%). The 5-year PFS rates for patients with COPD and without COPD were 50.1% and 60.6%, respectively ( P = .007). Compared with patients without COPD, patients with COPD had increased risk of worse OS (adjusted hazard ratio HRadj = 1.41, P = .002) and PFS (HRadj = 1.67, P = .003). The associations between COPD and worse survival outcomes were stronger in men and in squamous cell carcinoma (SCC). Conclusions Coexisting COPD is associated with worse survival outcomes in patients with early-stage NSCLC, particularly for men and for SCC.
Background Acquired nonmalignant tracheoesophageal fistula in the adult patient develops in a variety of conditions. We have applied surgical closure with success for 35 years. Methods From 1975 to ...1991, 38 patients underwent surgical repair of a tracheoesophageal fistula. A retrospective study of 36 additional patients undergoing surgical repair from 1992 to 2010 was conducted. Results The most common causes were postintubation injury (n = 17, 47%), trauma (n = 6, 17%), prior laryngectomy (n = 6, 17%), and prior esophagectomy (n = 4, 11%). Four patients presented after failing fistula control with an endoluminal stent. The tracheal defect was closed with resection and reconstruction (n = 17, 41%), laryngotracheal resection (n = 5, 12%), membranous tracheal repair (n = 17, 41%), or repair over a tracheal T tube (n = 2, 5%), while esophageal repair consisted of 2-layer closure (n = 31, 78%), 1-layer closure (n = 6, 15%), esophagostomy (n = 1, 3%), end-to-end esophageal anastomosis (n = 1, 3%), or full thickness skin graft reconstruction (n = 1, 3%). The esophageal and tracheal repairs were buttressed by interposing pedicled muscle or omental flaps in all patients. There was 1 postoperative death (3%). Recurrence after repair developed only in fistulas arising after esophagectomy or laryngectomy (n = 4, 11%). Fistula closure was ultimately successful in 34 patients (94%). Twenty-nine patients (83%) resumed oral intake and 25 patients (71%) were breathing without a tracheal appliance. Conclusions Successful closure of benign tracheoesophageal fistula is achieved with several surgical techniques based on buttressed repair and restoration of normal breathing and swallowing. Closure of complex postsurgical fistula may fail. Endoluminal stenting was not found useful.
Salmonella enterica nomenclature has evolved over the past one hundred years into a highly sophisticated naming convention based on the recognition of antigens by specific antibodies. This serotyping ...scheme has led to the definition of over 2500 serovars which are well understood, have standing in nomenclature and, for the majority, biological relevance. Therefore, it is highly desirable for any change in naming convention to maintain backwards compatibility with the information linked to these serovars. The routine use of whole genome sequencing and the well-established link between sequence types and serovars presents an opportunity to update the scheme by incorporating the phylogenetically relevant sequence data whilst preserving the best of serotyping nomenclature. Advantages include: overcoming the variability in antibody preparations; removing the need to use laboratory animals and implementing a truly universal system. However, the issue of trying to reproduce the phenotyping gold standard needs to be relaxed if we are to fully embrace the genomic era. We have used whole genome sequence data from over 46,000 isolates of Salmonella enterica subspecies enterica to define clusters in two stages: Multi Locus Sequence Typing followed by antigen prediction. Sequence type-serotype discrepancies were resolved using core SNP clustering to determine the phylogenetic groups and this was confirmed by overlaying the antigenic prediction onto the core SNP clusters and testing the separation of clusters using cgMLST Hierarchical Clustering. This allowed us to define any major antigenic clusters within an ST-here called the MAC type and written as ST-serovar. Using this method, 99.96% of Salmonella isolates reported in the UK were assigned a MAC type and linked to a serovar name taken from the Kauffmann and White scheme. We propose a change for reporting of Salmonella enterica sub-types using the ST followed by serovar.
Summary Control of typhoid fever relies on clinical information, diagnosis, and an understanding for the epidemiology of the disease. Despite the breadth of work done so far, much is not known about ...the biology of this human-adapted bacterial pathogen and the complexity of the disease in endemic areas, especially those in Africa. The main barriers to control are vaccines that are not immunogenic in very young children and the development of multidrug resistance, which threatens efficacy of antimicrobial chemotherapy. Clinicians, microbiologists, and epidemiologists worldwide need to be familiar with shifting trends in enteric fever. This knowledge is crucial, both to control the disease and to manage cases. Additionally, salmonella serovars that cause human infection can change over time and location. In areas of Asia, multidrug-resistant Salmonella enterica serovar Typhi ( S Typhi) has been the main cause of enteric fever, but now S Typhi is being displaced by infections with drug-resistant S enterica serovar Paratyphi A. New conjugate vaccines are imminent and new treatments have been promised, but the engagement of local medical and public health institutions in endemic areas is needed to allow surveillance and to implement control measures.
Background Idiopathic subglottic stenosis is a rare inflammatory condition affecting the subglottic larynx. We have treated 263 patients (only 2 were male) with this condition. The purpose of this ...study is to determine factors affecting outcome and predisposing to complications. Methods Information was gathered from chart reviews, surveys, and a prospective database. Results Median time from diagnosis to surgery was 24 months. Antinuclear antibodies when measured were positive in 76 patients (47%). Prior tracheal procedures were done in 58 patients (22%), and 184 patients (70%) had prior endoscopic procedures. Resection of the posterior cricoid mucosa with tracheal membranous wall flap was done in 150 patients (57%). Tailored cricoplasty was performed in 105 patients (40%). Extubation in the operating room was achieved in 247 patients (94%). Steroid therapy for edema was required in 63 patients (24%). Anastomotic complications occurred in 30 patients, 17 granulations and 7 subcutaneous air. Twenty-three patients (8.7%) have recurrence (14 mild, 9 recalcitrant) requiring dilation. Risk factors for anastomotic complications and recurrence were edema requiring steroids, use of mitomycin C, and prior tracheostomy, stents, and vocal cord involvement. Follow-up was available for 227 patients. Follow-up survey of 180 patients revealed, on a 10-point scale, effectiveness 9.4, satisfaction 9.4, and symptom improvement 9.4. A normal voice was present in 82 patients (45%); 96 patients (54%) had change in voice; and 121 patients (67%) had difficulty projecting their voice. Conclusions Single-stage reconstructive surgery resulted in 96% good-to-excellent results. Recalcitrant stenosis developed in 4% of patients. Stents, postoperative edema, mitomycin use, and vocal cord involvement are risks for recurrence. Recurrence was related to reactivation of disease in 14 patients and to technical problems in 6 patients.
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