Performance characteristics of next-generation sequencing for the detection of antimicrobial resistance determinants in Escherichia coli genomes and metagenomes

Short-read sequencing can provide detection of multiple genomic determinants of antimicrobial resistance from single bacterial genomes and metagenomic samples. Despite its increasing application in human, animal, and environmental microbiology, including human clinical trials, the performance of short-read Illumina sequencing for antimicrobial resistance gene (ARG) detection, including resistance-conferring single nucleotide polymorphisms (SNPs), has not been systematically characterized. Using paired-end 2 x 150 bp (base pair) Illumina sequencing and an assembly-based method for ARG prediction, we determined sensitivity, positive predictive value (PPV), and sequencing depths required for ARG detection in an Escherichia coli isolate of sequence type (ST) 38 spiked into a synthetic microbial community at varying abundances. Approximately 300,000 reads or 15x genome coverage was sufficient to detect ARGs in E. coli ST38, with comparable sensitivity and PPV to ~100x genome coverage. Using metagenome assembly of mixed microbial communities, ARG detection at E. coli relative abundances of 1% would require assembly of approximately 30 million reads to achieve 15x target coverage. The minimum sequencing depths were validated using public data sets of 948 E. coli genomes and 10 metagenomic rectal swab samples. A read-based approach using k-mer alignment (KMA) for ARG prediction did not substantially improve minimum sequencing depths for ARG detection compared to assembly of the E. coli ST38 genome or the combined metagenomic samples. Analysis of sequencing depths from recent studies assessing ARG content in metagenomic samples demonstrated that sequencing depths had a median estimated detection frequency of 84% (interquartile range: 30%-92%) for a relative abundance of 1%. IMPORTANCE Systematically determining Illumina sequencing performance characteristics for detection of ARGs in metagenomic samples is essential to inform study design and appraisal of human, animal, and environmental metagenomic antimicrobial resistance studies. In this study, we quantified the performance characteristics of ARG detection in E. coli genomes and metagenomes and established a benchmark of ~15x coverage for ARG detection for E. coli in metagenomes. We demonstrate that for low relative abundances, sequencing depths of ~30 million reads or more may be required for adequate sensitivity for many applications

Is there a role for postoperative radiotherapy following open partial laryngectomy when prognostic factors on the pathological specimen are unfavourable? A survey of head and neck surgical/radiation oncologists

Our aim was to survey the opinions of Italian radiation and ENT oncologists regarding the role of postoperative radiotherapy (PRT) and the appropriate dose to be given to patients with remnant larynx (RL) after open partial laryngectomy (OPL). The radio-oncologists (RO s) of the Italian Radiation-Oncologist Association (AIRO) and the ENTs of the Head-Neck Oncology Society (AIOCC-IHNS) were contacted through a SurveyMonkey online interface questionnaire. There were 148 usable responses. The majority of RO s recommended PRT in the case of positive/close margins (R(+)/Rclose) or in the case of initial involvement of thyroid cartilage (pT3tci). In the same cases, ENTs prefer a "watch and wait" policy (w&w). Both disciplines recommended w&w in the case of negative margins (R(-)). Finally, the majority of RO s recommended irradiating RL with 62-66 Gy in R(+), with 56-66 Gy (61.4%) in Rclose and with 56-60 Gy (34%) in pT3tci. In Conclusion, OPL raises new considerations about PRT

Molecular Characteristics of Extended-Spectrum Beta-Lactamases and qnr Determinants in Enterobacter Species from Japan

The incidence of extended-spectrum β-lactamases (ESBLs) has been increasing worldwide, but screening criteria for detection of ESBLs are not standardized for AmpC-producing Enterobacteriaceae such as Enterobacter species. In this study, we investigated the prevalence of ESBLs and/or AmpC β-lactamases in Japanese clinical isolates of Enterobacter spp. and the association of plasmid-mediated quinolone resistance (PMQR) determinants with ESBL producers. A total of 364 clinical isolates of Enterobacter spp. collected throughout Japan between November 2009 and January 2010 were studied. ESBL-producing strains were assessed by the CLSI confirmatory test and the boronic acid disk test. PCR and sequencing were performed to detect CTX-M, TEM, and SHV type ESBLs and PMQR determinants. For ESBL-producing Enterobacter spp., pulsed-field gel electrophoresis (PFGE) was performed using XbaI restriction enzyme. Of the 364 isolates, 22 (6.0%) were ESBL producers. Seven isolates of Enterobacter cloacae produced CTX-M-3, followed by two isolates producing SHV-12. Two isolates of Enterobacter aerogenes produced CTX-M-2. Of the 22 ESBL producers, 21 had the AmpC enzyme, and six met the criteria for ESBL production in the boronic acid test. We found a significant association of qnrS with CTX-M-3-producing E. cloacae. The 11 ESBL-producing Enterobacter spp. possessing blaCTX-M, blaSHV, or blaTEM were divided into six unique PFGE types. This is the first report about the prevalence of qnr determinants among ESBL-producing Enterobacter spp. from Japan. Our results suggest that ESBL-producing Enterobacter spp. with qnr determinants are spreading in Japan

Comparative Evaluation of a Chromogenic Agar Medium, the Modified Hodge Test, and a Battery of Meropenem-Inhibitor Discs for Detection of Carbapenemase Activity in Enterobacteriaceae▿

Three phenotypic methods (modified Hodge test, chromogenic agar, and meropenem discs combined with specific inhibitors) used for the detection of carbapenemase activity were tested on a panel of characterized Enterobacteriaceae expressing various β-lactamase mechanisms. Overall, the meropenem-plus-inhibitor approach was more sensitive and specific than the other methods, despite its limitation of being unable to detect class D carbapenemases

Facial basal cell carcinomas treated with hypo-fractionated radiotherapy: A retrospective analysis in 117 elderly patients

A study on the treatment of facial basal cell carcinoma of the elderly with hypo-fractotionated radiotherap

Radiotherapy alone or with concomitant daily low-dose carboplatin in locally advanced, unresectable head and neck cancer: definitive results of a phase III study with a follow-up period of up to ten years.

AIM AND BACKGROUND: Radiotherapy is the conventional treatment for locally advanced inoperable head and neck squamous cell carcinoma. However, the poor therapeutic results justify the development of radiochemotherapy combinations. In an attempt to improve local control and survival in patients with stage III and IV unresectable head and neck squamous cell carcinoma and based on the results of our previous dose escalation study, we undertook a prospective multicentric randomized trial. MATERIALS AND METHODS: From November 1992 through December 1995, a total of 164 patients were randomized to receive radiotherapy alone (arm I) or combined (arm II) with daily low-dose carboplatin. RESULTS: The 3, 5 and 10-year local-regional recurrence-free survival rates were better in arm II (21.7%, 15.1% and 15.1%, respectively) than in arm I (15%, 10.7% and 10.7%), but without statistical significance (P = 0.11). The 3, 5 and 10-year disease-free survival rates showed the same positive trend for arm II (16%, 6.8% and 6.8% vs. 9%, 5.5% and 5.5%, in arm I, respectively), again without statistical significance (P = 0.09). Instead, a statistical advantage was found in overall survival rates at 3, 5 and 10-years (28.9%, 9% and 5.5% in arm II and 11.1%, 6.9% and 6.9% in arm I, respectively) (P = 0.02). The 3, 5 and 10-year local-regional recurrence-free survival rates in stage IV disease were statistically better in arm II (21.5%, 15.9% and 15.9%) than in arm I (12.8%, 7.7% and 7.7%, respectively) (P = 0.04). CONCLUSIONS: Long-term results in both treatment arms of the trial appear less positive than most published series. However, our findings do not exclude that carboplatin may be beneficial, but the benefit in local control must be lower than the 15% assumed to dimension the trial