贵阳市412例腹泻病例空肠弯曲菌的检测分析

目的建立常规的空肠弯曲菌检测方法,了解贵阳市腹泻人群空肠弯曲菌的感染状况。方法采用简单生化及多重PCR方法对贵阳市412例腹泻人群进行空肠弯曲菌检测。结果 412份腹泻标本中,7份空肠弯曲菌阳性,平均检出率1.70%。1岁以下婴幼儿感染率与其它人群差异无统计学意义(P>0.05)。男女患者感染率差异无统计学意义(P>0.05)。粘液便空肠弯曲菌检出率(7.69%)显著高于水样便(0.60%)(P<0.01)。结论利用简单生化及多重PCR方法能很好地检出空肠弯曲菌;贵阳市腹泻人群中存在空肠弯曲菌的感染,且主要感染婴幼儿,需加强婴幼儿腹泻中空肠弯曲菌感染的监测。     

登革病毒Taqman双重荧光PCR分型研究

目的建立鉴定登革病毒型别的双重实时Taqman PCR反应体系,以准确快速鉴定登革病毒型别。方法根据GenBank上已发表的登革病毒四个型别的全基因序列,进行对比分析,分别设计登革病毒的四个型别引物和探针,登革I、III型探针用FAM-TAMARA标记,登革II、IV型探针用JOE-TAMARA标记。经过条件优化后,建立检测登革病毒I/II型和III/IV型的两套双重实时荧光RT-PCR方法,扩增四型登革病毒RNA、登革病毒阴性样本和登革病毒RNA稀释样本,检测方法的特异性、重复性和检测限性。结果通过设计筛选序列和优化反应条件,建立登革病毒I、II型和登革病毒III、IV型的双重荧光PCR反应体系,通过试验证明,所建立的方法具有良好的特异性、重复性和检测限性,能准确快速地对登革病毒进行分型。结论建立了一种快速双重荧光PCR方法能同时对登革病毒进行分型和鉴定。     

HLA-genotyping of clinical specimens using Ion Torrent-based NGS

We have evaluated and validated the NXType™ workflow (One Lambda, Inc.) and the accompanying TypeStream™ software on the Ion Torrent Next Generation Sequencing (NGS) platform using a comprehensive testing panel. The panel consisted of 285 genomic DNA (gDNA) samples derived from four major ethnic populations and contained 59 PT samples and 226 clinical specimens. The total number of alleles from the six loci interrogated by NGS was 3420. This validation panel provided a wide range of HLA sequence variations including many rare alleles, new variants and homozygous alleles. The NXType™ system (reagents and software) was able to correctly genotype the vast majority of these specimens. The concordance rate between SBT-derived genotypes and those generated by TypeStream™ auto-analysis ranged from 99.5% to 99.8% for the HLA-A, B, C, DRB1 and DQB1 loci, and was 98.9% for HLA-DPB1. A strategy for data review was developed that would allow correction of most of the few remaining typing errors. The entire NGS workflow from gDNA amplification to genotype assignment could be completed within 3 working days. Through this validation study, the limitations and shortcomings of the platform, specific assay system, and software algorithm were also revealed for further evaluation and improvement.     

Accuracy of comprehensive PCR analysis of nasopharyngeal and oropharyngeal swabs for CT-scan-confirmed pneumonia in elderly patients: a prospective cohort study

ObjectivesWe aimed to assess the accuracy of PCR detection of viruses and bacteria on nasopharyngeal and oropharyngeal swabs (NPS) for the diagnosis of pneumonia in elderly individuals.MethodsWe included consecutive hospitalized elderly individuals suspected of having pneumonia. At inclusion, NPS were collected from all participants and tested by PCR for the presence of viral and bacterial respiratory pathogens (index test, defined as comprehensive molecular testing). Routine diagnostic tests (blood and sputum culture, urine antigen detection) were also performed. The reference standard was the presence of pneumonia on a low-dose CT scan as assessed by two independent expert radiologists.ResultsThe diagnosis of pneumonia was confirmed in 127 of 199 (64%) included patients (mean age 83 years, community-acquired pneumonia in 105 (83%)). A pathogen was identified by comprehensive molecular testing in 114 patients (57%) and by routine methods in 22 (11%). Comprehensive molecular testing was positive for viruses in 62 patients (31%) and for bacteria in 73 (37%). The sensitivity and specificity were 61% (95% CI 53%–69%) and 50% (95% CI 39%–61%) for comprehensive molecular testing, and 14% (95% CI 82%–21%) and 94% (95% CI 86%–98%) for routine testing, respectively. Positive likelihood ratio was 2.55 for routine methods and 1.23 for comprehensive molecular testing.ConclusionComprehensive molecular testing of NPS increases the number of pathogens detected compared with routine methods, but results are poorly predictive of the presence of pneumonia. Hence, comprehensive molecular testing is unlikely to impact clinical decision-making (NCT02467192).Clinical Trials RegistrationNCT02467192.     

Characterisation of nosocomial and community-acquired influenza in a large university hospital during two consecutive influenza seasons

BackgroundNosocomial influenza is increasingly recognized as an important public health threat causing considerable morbidity and mortality each year. However, data on nosocomial influenza is usually collected during outbreaks only and clinical information of nosocomial influenza is sparsely available.ObjectivesTo systematically analyse the distribution of nosocomial and community-acquired influenza and epidemiological characteristics in a tertiary care unit in two consecutive seasons.Study designA retrospective observational study was conducted to identify and characterise cases of nosocomial and community-acquired influenza at Freiburg University hospital from 1 January 2013 to 30 April 2014. A validated multiplex RT-PCR to detect influenza virus and other respiratory pathogens was used throughout. Clinical information was retrieved from the hospital-based information system.ResultsOverall, 218 patients with laboratory-confirmed influenza were included (179 in the first, 39 patients in the second season). A rate of 20% of nosocomial influenza was observed throughout. A fatal outcome was recorded for 9% of nosocomial cases, which were mainly associated with influenza virus A(H1N1)pdm09. Nosocomial influenza occurred in all age groups, but fatalities were only observed in patients ≥18 years. Patients with nosocomial influenza were significantly older, underwent therapy for blood malignancies and immunosuppressive regimens more frequently, and received solid organ transplantation more often compared to community-acquired patients.ConclusionsDespite the different distribution of virus subtypes and epidemiological properties between both influenza seasons, the rate of nosocomial cases remained similar. Systematic detection and targeted prevention measures seem mandatory to minimize nosocomial influenza.     

An observational study of antibody responses to a primary or subsequent pertussis booster vaccination in Australian healthcare workers

Adult pertussis vaccination is increasingly recommended to control pertussis in the community. However, there is little data on the duration and kinetics of immunity to pertussis boosters in adults. We compared IgG responses to vaccination with a tetanus, low-dose diphtheria, low-dose acellular pertussis (Tdap) booster at 1 week, 1 month and 1 year post-vaccination in whole-cell (wP)-primed Australian paediatric healthcare workers who had received an adult Tdap booster 5–12 years previously, to those who received their first Tdap booster.Tdap vaccination was well tolerated in both groups. Previously boosted adults had significantly higher pre-vaccination IgG concentrations for all vaccine-antigens, and more were seropositive for pertussis toxin (PT)-specific IgG (≥ 5 IU/mL) (69.5%; 95% confidence interval (CI) 59.5–79.5) than adults in the naïve group (45.2%; 95% CI 32.8-57.5). Tdap vaccination significantly increased IgG responses 1 month post-vaccination in both groups. This increase was more rapid in previously boosted than in naïve adults, with geometric mean fold-increases in PT-IgG at 1 week post vaccination of 3.6 (95% CI 2.9–4.3) and 2.6 (95% CI 2.2–3.2), respectively. Antibody waning between 1 month and 1 year post-vaccination was similar between groups for IgG specific to PT and filamentous haemagglutinin (FHA), but was faster for IgG against pertactin (PRN) in the naïve group (GMC ratio 0.36; 95% CI 0.31–0.42) than the previously boosted group (GMC ratio 0.45; 95% CI 0.39–0.50). At baseline, all but one adult had protective IgG titres against tetanus toxin (TT) (≥ 0.1 IU/mL), and 75.6% in the previously boosted and 61.3% in the naïve group had protective IgG titres against diphtheria toxoid (DT) of ≥ 0.1 IU/mL.This study shows that pertussis immune memory is maintained up to 12 years after Tdap vaccination in wP-primed Australian adults. There was no evidence that pertussis immune responses waned faster after a booster dose. These findings support current recommendations of repeating Tdap booster vaccination in paediatric healthcare workers at least every 10 years. Clinical trials registry: ACTRN12615001262594.     

Evaluation of a Dual ALK/ROS1 Fluorescent In Situ Hybridization Test in Non–Small-cell Lung Cancer

Background

Several therapeutics targets have emerged to treat patients with non–small-cell lung carcinoma (NSCLC), with numerous biomarkers available to test for treatment choices. Minimum tumor wastage is necessary to permit the analysis of every potentially relevant target. Searching for targetable ALK and ROS1 rearrangements is now mandatory in NSCLC. In the present study, we evaluated the performance of a dual ALK/ROS1 fluorescent in situ hybridization (FISH) probe that concurrently analyzed the 2 oncogenes on a same FISH slide.

Applications of eTag trade mark assay platform to systems biology approaches in molecular oncology and toxicology studies

We have developed a universal eTag trade mark multiplex assay platform that can be uniquely applied to survey the molecule profiles of biologic systems in sub-global large-scale analyses. The effectiveness of eTag trade mark assays when applied to focused system biology studies in molecular oncology and predictive toxicology is herein described while reviewing the current methods commonly used. The multi-analyte and multi-parameter assay approach for parallel analysis will form the basis of an emerging paradigm of multiplexed molecular profiling for signaling pathway networks and various aspects of drug development processes.