Rapid and highly sensitive qualitative real-time assay for detection of respiratory syncytial virus A and B using NASBA and molecular beacon technology

The performance of a sensitive and specific qualitative respiratory syncytial virus (RSV) assay based on NASBA technology and real-time molecular beacon detection is presented. Very low detection limits for both RSV A and RSV B were determined: 95% detection hit-rate of 95 and 47 copies/input in isolation for RSV A and RSV B, respectively. RSV was detected in a wide variety of clinical samples including respiratory swabs, nasopharyngeal aspirates (NPA), bronchoalveolar lavages (BAL), endotracheal secretions, and sputum samples. In total 779 clinical samples were tested and a valid result was obtained for 765 (RSV NASBA assay), 765 (cell culture), and 529 (rapid direct immunofluorescence testing (IF)) samples. Of these samples, 229 (RSV NASBA assay), 61 (cell culture), and 122 (IF) samples were positive for RSV. In addition, 106 samples were reported as RSV negative using the NOW RSV assay (Binax). Subsequent testing using the RSV NASBA assay demonstrated that 32 (30%) of these samples were RSV positive. The RSV NASBA assay includes a homologous internal control, which offers a high degree of standardization and quality control. When the RSV NASBA assay was performed on the NucliSens EasyQ platform (bioMérieux), test results of 48 sample extracts were obtained in less than 2h.     

Planning and managing a complex multisite study in the age of technology

A 2‐year long, multisite research study that evaluated cardiopulmonary resuscitation skill decay among nursing students was conducted at 10 schools of nursing across the United States. The study was conducted in two phases and required carefully timed sessions for skill performance. Multisite studies in nursing education need to be carefully planned. Time delays should be anticipated with processes and Institutional Review Board protocols across sites. All team members were trained and consistently supported during the entire study. While challenges and obstacles were identified, innovative solutions were implemented that assisted the research team to successfully complete the study. The use of new and existing technology allowed the team to surmount many of the challenges encountered in this study. The purpose of this article is to describe the logistics, processes, challenges, and lessons learned related to conducting a complex multisite study.     

Double-strand breaks associated with repetitive DNA can reshape the genome

Ionizing radiation is an established source of chromosome aberrations (CAs). Although double-strand breaks (DSBs) are implicated in radiation-induced and other CAs, the underlying mechanisms are poorly understood. Here, we show that, although the vast majority of randomly induced DSBs in G₂ diploid yeast cells are repaired efficiently through homologous recombination (HR) between sister chromatids or homologous chromosomes, ≈2% of all DSBs give rise to CAs. Complete molecular analysis of the genome revealed that nearly all of the CAs resulted from HR between nonallelic repetitive elements, primarily Ty retrotransposons. Nonhomologous end-joining (NHEJ) accounted for few, if any, of the CAs. We conclude that only those DSBs that fall at the 3–5% of the genome composed of repetitive DNA elements are efficient at generating rearrangements with dispersed small repeats across the genome, whereas DSBs in unique sequences are confined to recombinational repair between the large regions of homology contained in sister chromatids or homologous chromosomes. Because repeat-associated DSBs can efficiently lead to CAs and reshape the genome, they could be a rich source of evolutionary change.     

蜂胶对成纤维细胞、单核巨噬细胞系基质金属蛋白酶MMP-9表达的抑制作用

目的探讨蜂胶对糖尿病伤口愈合的作用及其机制。方法采用荧光电泳法,检测不同葡萄糖浓度（5、25mmol／L）和不同蜂胶浓度（10、50、100、200μg／ml）处理的体外培养的人成纤维细胞及单核巨噬细胞系的基质金属蛋白酶MMP-9蛋白的表达。结果高浓度葡萄糖（25mmol／L）状态下,MMP-9表达增加,但与低浓度（5mmol／L）比较,差异无统计学意义。蜂胶可显著降低MMP-9的表达,并呈剂量依赖性（P〈．05）。结论高糖能增加单核巨噬细胞系及成纤维细胞MMP-9的表达,可能会影响糖尿病患者伤口的愈合。蜂胶可抑制这些细胞MMP-9的表达,有益于细胞基质的积聚,从而有益于糖尿病患者伤口的愈合。     

Test–retest repeatability of [18F]Flortaucipir PET in Alzheimer’s disease and cognitively normal individuals

The aim of this study was to investigate the test–retest (TRT) repeatability of various parametric quantification methods for [¹⁸F]Flortaucipir positron emission tomography (PET). We included eight subjects with dementia or mild cognitive impairment due to Alzheimer’s disease and six cognitively normal subjects. All underwent two 130-min dynamic [¹⁸F]Flortaucipir PET scans within 3 ± 1 weeks. Data were analyzed using reference region models receptor parametric mapping (RPM), simplified reference tissue method 2 (SRTM2) and reference logan (RLogan), as well as standardized uptake value ratios (SUVr, time intervals 40–60, 80–100 and 110–130 min post-injection) with cerebellar gray matter as reference region. We obtained distribution volume ratio or SUVr, first for all brain regions and then in three tau-specific regions-of-interest (ROIs). TRT repeatability (%) was defined as |retest–test|/(average (test + retest)) × 100. For all methods and across ROIs, TRT repeatability ranged from (median (IQR)) 0.84% (0.68–2.15) to 6.84% (2.99–11.50). TRT repeatability was good for all reference methods used, although semi-quantitative models (i.e. SUVr) performed marginally worse than quantitative models, for instance TRT repeatability of RPM: 1.98% (0.78–3.58) vs. SUVr_80–100: 3.05% (1.28–5.52), p < 0.001. Furthermore, for SUVr_80–100 and SUVr_110–130, with higher average SUVr, more variation was observed. In conclusion, while TRT repeatability was good for all models used, quantitative methods performed slightly better than semi-quantitative methods.     

Lateralized and focal clinical, EEG, and FLAIR MRI abnormalities in Creutzfeldt-Jakob disease.

OBJECTIVE: Creutzfeldt-Jakob disease (CJD) is a rapidly progressive fatal prion disorder with typical clinical findings of dementia, motor dysfunction, and myoclonus and characteristic electroencephalographic (EEG) findings of bilateral synchronous periodic sharp waves. Advances in neuroimaging capabilities with diffusion-weighted and fluid-attenuated inversion recovery (FLAIR) magnetic resonance imaging (MRI) techniques have shown promise in the non-invasive diagnosis of CJD. This series illustrates the correlation between the lateralized and focal clinical, EEG, and MRI FLAIR sequence abnormalities in 8 patients (7 men and one woman 55-73 years old) with CJD. METHODS: A case series of 8 patients, evaluated at Mayo Clinic, who had a history of rapidly progressive lateralized or focal neurologic dysfunction and laboratory findings consistent with CJD between 1996 and 1999 were identified. EEG, MRI of the head with FLAIR sequence, and cerebrospinal fluid studies were performed in all patients. RESULTS: Mean time to death from symptom onset was 4 months. Symptoms were lateralized to the left hemisphere in 5 patients and to the right hemisphere in two. One patient showed bilateral occipital lobe involvement. In all patients, the EEG showed lateralized or focal periodic sharp waves that colocalized with clinical cerebral dysfunction. FLAIR MRI images revealed increased signal in the cortical ribbon and deep gray matter corresponding to the lateralized clinical and EEG findings in 7 patients. The other patient had bilateral occipital increased signal on FLAIR MRI. CONCLUSIONS: CJD may present with lateralized or focal cortical syndromes with colocalizing EEG and MRI findings. With the appropriate clinical history and laboratory evaluation, the corresponding areas of increased signal on FLAIR MRI provide supportive evidence of the disease. SIGNIFICANCE: CJD can sometimes present with more focal or lateralized clinical findings, and the colocalizing EEG and MRI findings can help make or confirm the diagnosis of CJD.     

Urinary C-peptide creatinine ratio to detect absolute insulin deficiency in type 2 diabetes

BackgroundNational Institute for Health and Clinical Excellence guidelines (CG87) recommend neutral protamine hagedorn (NPH) insulin for the provision of basal insulin in type 2 diabetes, but use of analogue insulin is as much as 40%. Where residual endogenous insulin secretory capacity is present there is no evidence that analogue insulins provide any additional benefit over human insulins, and they come at an expensive premium. Anecdotally, however, there is a reluctance to switch people back to NPH insulin, partly because of a perceived risk of pancreatic failure and potential ketosis. Urinary C-peptide creatinine ratio (UCPCR) has been validated as a method for evaluating residual endogenous insulin secretion in type 1 and type 2 diabetes, with a UCPCR of no more than 0·2 nmol/mmol suggestive of absolute insulin deficiency. We aimed to evaluate the prevalence of true insulin deficiency among patients with type 2 diabetes with UCPCR, and confirm findings with the gold standard mixed meal tolerance test (MMTT).Methods191 insulin-treated patients with a clinical diagnosis of type 2 diabetes (diagnosed at or after age 45 years and who did not start insulin within the first year of diagnosis) collected a 2-h post-prandial urine sample for UCPCR measurement. Nine patients from two subgroups (UCPCR ≤0·2 nmol/mmol and UCPCR >0·2) completed a standard MMTT.Findings11 (5·8%) of 191 patients had two consistent UCPCRs of less than or equal to 0·2 nmol/mmol. Nine were able to do the MMTT, of whom five were confirmed to have absolute insulin deficiency (stimulated serum c-peptide <0·2 nmol/L). Three of these five patients were glutamic acid decarboxylase antibody-negative. Nine of nine patients with UCPCR of more than 0·2 nmol/L had confirmed endogenous insulin secretion in their MMTT. Those with insulin deficiency had a shorter time to starting insulin (median 2·5 years [IQR 1·5–3·0] vs 6·0 [3·0–10·75], p=0·005) and lower body-mass index (25 kg/m² vs 29, p=0·04) but no other significant differences in clinical characteristics.InterpretationWe have demonstrated a very low prevalence of true pancreatic failure in this population of insulin-treated patients with type 2 diabetes. This requires further exploration by comparison of a population being treated with NPH insulin with one on analogue insulin, and then determining whether UCPCR could act as a clinical decision support tool to safely switch from analogue insulin to NPH insulin.FundingNational Institute for Health Research.     

Drug addiction in sickle cell disease: case report

There has been widespread speculation that patients with sickle cell disease (SCD) may become drug dependent if their painful crisis is treated with narcotics. However, there has been no scientific evidence to support this assertion. Paradoxically, individuals suffering from sickle cell disease who are not adequately treated may develop an addiction to narcotics due to self-medication to treat their pain. In this article, we describe a 38-year-old African American woman who became addicted to cocaine due to self-medication of her sickle cell pain with cocaine.     

Establishing a laparoscopic gastric bypass program

OBJECTIVE: To evaluate the outcomes for laparoscopic Roux-en-Y gastric bypass in a newly developed bariatric surgery program. METHODS: A prospective analysis of the initial 100 patients who underwent laparoscopic Roux-en-Y gastric bypass at a community based teaching hospital between December 2000 and October 2001 was performed. Study endpoints included operative time, early (less than 7 days) and late complication rates, hospital length of stay (LOS), time to initiation of oral diet, and percentage of excess body weight loss. RESULTS: There were 91 women and 9 men with a mean age of 39 years (range 21 to 57). Mean preoperative weight was 126.4 kg (range 92.3 kg to 214 kg), with a mean preoperative body mass index of 47.4 (range 37.3 to 75.7). Ninety (90%) patients had at least one significant medical comorbidity (median = 6 per patient). Mean operative time was 2.4 hours (range 1.0 to 6.5; 3.0 hours for the first 50 patients and 1.8 hours for the last 50 patients). Three patients required conversion to open gastric bypass, all due to equipment failure (two harmonic scalpel failures and one linear stapler malfunction). Seven patients had early complications, 3 with anastomotic hemorrhage requiring transfusion, 3 with intestinal leaks requiring reoperation, and 1 with transected nasogastric tube. Eleven patients had late complications: 5 patients with small bowel obstruction, all due to herniation through the transverse colon mesentery (these occurred early in the series, prompting a change in technique, with no subsequent occurrences), 3 with gastrojejunostomy strictures requiring endoscopic dilation, 2 superficial wound infections, and 1 port-site incisional hernia. No complications occurred in the last 40 patients. No deaths occurred. Median hospital stay was 2 days (range 1 to 37); 3 days in the first 50 cases and 1 day in the last 50 cases. The median number of days to the start of an oral diet was 1 day. Average excess body weight loss was 34% (median follow-up 4 months). CONCLUSIONS: Laparoscopic Roux-en-Y gastric bypass is a technically challenging procedure that can be safely integrated into a bariatric treatment program with good results. Improved outcomes, shorter operative times, and fewer complications are associated with increased surgical experience.