Mutation Detection by Clonal Sequencing of PCR Amplicons and Grouped Read Typing is Applicable to Clinical Diagnostics

We describe a sensitive technique for mutation detection using clonal sequencing. We analyzed DNA extracted from 13 cancer cell lines and 35 tumor samples and applied a novel approach to identify disease‐associated somatic mutations. By matching reads against an index of known variants, noise can be dramatically reduced, enabling the detection and quantification of those variants, even when they are present at less than 1% of the total sequenced population; this is comparable to, or better than, current diagnostic methods. Following the identification or exclusion of known variants, unmatched reads are grouped for BLAST searching to identify novel variants or contaminants. Known variants, novel variants, and contaminants were readily identified in tumor tissue using this approach. Our approach also enables an estimation of the per‐base sequencing error rate, providing a confidence threshold for interpretation of the results in the clinic. This novel approach has immediate applicability to clinical testing for disease‐associated genetic variants.     

The Influence of Triclosan on Biomarkers of Cardiovascular Risk in Patients in the Cardiovascular and Periodontal Study (CAPS): A Randomized Controlled Trial

Background: Triclosan toothpaste is effective in controlling plaque and gingivitis and slowing progression of periodontitis; however, its influence on inflammatory biomarkers of cardiovascular disease (CVD), as well as on kidney and liver function, is unknown. Methods: Patients recruited from the Cardiovascular Unit at Prince Charles Hospital, Brisbane, Australia, were randomized to triclosan (n = 193) or placebo (n = 190) groups and assessed for total cholesterol (TC), high density lipoprotein (HDL) and low density lipoprotein (LDL) cholesterol, triglycerides, C‐reactive protein, erythrocyte sedimentation rate (ESR), hemoglobin, total white cell count (WCC), estimated glomerular filtration rate (eGFR), and liver function enzymes, annually for 5 years. A standard mixed model for each marker included group, sex, age, hypertension, diabetes, periodontal status, statin and anti‐inflammatory drug use, and smoking as covariates. Changes in eGFR, WCC, and ESR were further analyzed using transition modeling. Results: Triclosan toothpaste led to a greater decrease in TC (P = 0.03), LDL cholesterol (P = 0.04), and HDL cholesterol (P = 0.05) than placebo toothpaste. ESR increased at a slower rate in the triclosan group (P ≈ 0.06) and was less likely to increase and more likely to improve in males on statins but not anti‐inflammatory drugs in the triclosan group versus the placebo group. Markov modeling of the binary response for eGFR (greater than or less than/equal to the baseline median value) showed that patients with diabetes in the placebo group were significantly (P ≈ 0.05) more likely to deteriorate than either patients with diabetes in the triclosan group or patients without diabetes in each group. Conclusions: These data suggest that triclosan toothpaste may influence some inflammatory biomarkers of CVD, but not kidney or liver function. However, it is unclear if this influence is clinically significant.     

Living with AIDS in Uganda: a qualitative study of patients’ and families’ experiences following referral to hospice

Background

Globally, the majority of people with HIV/AIDS live in sub-Saharan Africa. While the increasing availability of antiretroviral therapy is improving the outlook for many, its effects are yet to reach all of those in need and patients still present with advanced disease. This paper reports findings from qualitative interviews with patients living with AIDS and their caregivers who were receiving palliative care from Hospice Africa Uganda (HAU). We aimed to understand what motivated patients and their families to seek formal healthcare, whether there were any barriers to help- seeking and how the help and support provided to them by HAU was perceived.

Methods

We invited patients with AIDS and their relatives who were newly referred to HAU to participate in qualitative interviews. Patients and carers were interviewed in their homes approximately four weeks after the patient’s enrolment at HAU. Interviews were translated, transcribed and analysed using narrative and thematic approaches.

Results

Interviews were completed with 22 patients (10 women and 12 men) and 20 family caregivers, nominated by patients. Interviews revealed the extent of suffering patients endured and the strain that family caregivers experienced before help was sought or accessed. Patients reported a wide range of severe physical symptoms. Patients and their relatives reported worries about the disclosure of the AIDS diagnosis and fear of stigma. Profound poverty framed all accounts. Poverty and stigma were, depending on the patient and family situation, both motivators and barriers to help seeking behaviour. Hospice services were perceived to provide essential relief of pain and symptoms, as well as providing rehabilitative support and a sense of caring. The hospice was perceived relieve utter destitution, although it was unable to meet all the expectations that patients had.

Conclusion

Hospice care was highly valued and perceived to effectively manage problems such as pain and other symptoms and to provide rehabilitation. Participants noted a strong sense of being “cared for”. However, poverty and a sense of stigma were widespread. Further research is needed to understand how poverty and stigma can be effectively managed in hospice care for patients for advanced AIDS and their families.

     

Evaluation of the basic erosive wear examination (BEWE) for use in general dental practice

Objective The basic erosive wear examination (BEWE) is a relatively new index proposed for the screening and recording of tooth wear in adults. The aim of this study was to test the validity and reliability of the BEWE.Design Cross sectional study of a sample of 164 adult patients.Setting General dental practice in East Lancashire, UK in 2010.Subjects Patients attending for routine examination or treatment.Main outcome measures By screening patients with the BEWE and comparing the results to the established tooth wear index (TWI) the sensitivity and specificity of the BEWE was established.Results The BEWE predicted moderate to severe wear (BEWE grade 3) with a sensitivity of 48.6% and a specificity of 96.1%, and predicted severe wear with a sensitivity of 90.9% and a specificity of 91.5% (also BEWE score 3). Inter- and intra-examiner reliability for the BEWE were both moderate (κ(w) = 0.43 and 0.57 respectively).Conclusion(s) BEWE scores show a similar distribution to TWI scores and the examination is an effective screening test for severe tooth wear. The moderate levels of examiner reliability suggests the BEWE scores should be interpreted with some caution.     

The Effects of Bariatric Procedures on Bowel Habit

Background

Bariatric procedures are increasingly being used to combat the rising obesity epidemic. The aim of this study was to assess the effect of these interventions on bowel habit.

Methods

We recruited obese adults listed for a bariatric procedure. Demographic data, medical history, medications and anthropometric measurements were recorded. Bowel habit was characterized using a 7-day Bristol Stool Form Scale (BSFS) diary. A validated food frequency questionnaire (FFQ) was used to assess diet.

Results

Twenty-six patients were assessed pre-operatively and at a median of 6.4 months post-operatively. Nineteen had a Roux-en-Y gastric bypass (RYGB), five had a sleeve gastrectomy (SG) and two had an intra-gastric balloon (IGB) with median percentage excess weight loss (% EWL) of 67.9, 52.4 and 31.3 %, respectively. Dietary fibre intake decreased from 24.4 (±12.1) g/day pre-operatively to 17.5 (±7.3) g/day post-operatively (P?=?0.008). Frequency of bowel motions decreased from 8.6 (±3.5) to 5.7 (±3.5) motions/week (P?=?0.001). Mean usual BSFS score decreased (towards firmer stool) from 4.1 (±1.3) pre-operatively to 3.1 (±1.9) post-operatively (P?=?0.016). Constipation increased from 8 to 27 %, but this did not reach statistical significance (P?=?0.125).

Conclusions

Constipation is a common problem after bariatric surgery. The decrease in bowel motion frequency and change towards firmer stools suggest prolonged intestinal transit time after bariatric procedures. Reduction in dietary fibre intake is likely to be a contributory factor.

     

Postoperative Analgesia Due to Sustained-Release Buprenorphine,Sustained-Release Meloxicam,and Carprofen Gel in a Model of Incisional Pain in Rats (Rattus norvegicus)

Postoperative analgesia in laboratory rats is complicated by the frequent handling associated with common analgesic dosing requirements. Here, we evaluated sustained-release buprenorphine (Bup-SR), sustained-release meloxicam (Melox-SR), and carprofen gel (CG) as refinements for postoperative analgesia. The aim of this study was to investigate whether postoperative administration of Bup-SR, Melox-SR, or CG effectively controls behavioral mechanical and thermal hypersensitivity in a rat model of incisional pain. Rats were randomly assigned to 1 of 5 treatment groups: saline, 1 mL/kg SC BID; buprenorphine HCl (Bup HCl), 0.05 mg/kg SC BID; Bup-SR, 1.2 mg/kg SC once; Melox-SR, 4 mg/kg SC once; and CG, 2 oz PO daily. Mechanical and thermal hypersensitivity were tested daily from day–1 through 4. Bup HCl and Bup-SR attenuated mechanical and thermal hypersensitivity on days 1 through 4. Melox-SR and CG attenuated mechanical hypersensitivity–but not thermal hypersensitivity–on days 1 through 4. Plasma concentrations, measured by using UPLC with mass spectrometry, were consistent between both buprenorphine formulations. Gross pathologic examination revealed no signs of toxicity in any group. These findings suggest that postoperative administration of Bup HCl and Bup-SR—but not Melox-SR or CG—effectively attenuates mechanical and thermal hypersensitivity in a rat model of incisional pain.Abbreviations: Bup HCl, buprenorphine HCl; Bup-SR, sustained-release buprenorphine; CG, carprofen gel; Melox-SR, sustained-release meloxicamPostoperative analgesia is a vital aspect of laboratory animal medicine. Investigators have a responsibility to follow an effective and safe pain management protocol for research animals that have undergone surgical procedures. Pain and distress are serious animal welfare concerns that directly affect animal physiology and can result in altered research data.^1,17,30 Continued refinement of pre-, intra-, and postoperative pain management in rodents is necessary to improve animal wellbeing, obtain high-quality research data, and ensure compliance with standards set forth by the Guide for the Care and Use of Laboratory Animals.²¹Many classes of analgesics are available to veterinary practitioners, but in the laboratory setting, the options tend to be simpler and typically involve 1 of 2 drug classes, opioids and NSAID. Buprenorphine HCl (Bup HCl), a partial μ-opioid receptor agonist, has long been the ‘gold standard’ for postoperative analgesia in laboratory animals due to the drug''s prolonged plasma half-life and effective analgesic properties.^15,28 Buprenorphine effectively controls mild to moderate postoperative pain in rodents for 6 to 12 h.¹⁶ Because many rodent surgical procedures might cause pain for at least 48 h, researchers must handle these animals at least twice daily during this time period to readminister buprenorphine. Repeated dosing requires frequent handling of surgically manipulated animals, resulting in handling-associated stress.¹ In addition, handling an animal frequently likely is disruptive to its cagemates and potentially to animals in the same room. Because of their analgesic and antiinflammatory properties, NSAID are often used either in conjunction with or as an alternative to opioids to control pain in laboratory animals.^11,33 Meloxicam and carprofen are 2 NSAID that preferentially inhibit cyclooxygenase 2 and thus prostaglandin synthesis.^10,11 Although generally considered safe, reported side effects of NSAID include gastrointestinal ulceration, altered platelet function, and renal dysfunction.¹¹Novel formulations of opioid and NSAID analgesics have recently been introduced to the veterinary market and include sustained-release injectables,^2,5,14,22 gel-based oral compounds,^6,19 and transdermal patches.^13,18,25,37 Our group previously demonstrated the effectiveness of sustained-release buprenorphine (Bup-SR) in controlling mild to moderate incisional pain in rats.⁷ Another study found that Bup-SR successfully controlled orthopedic surgical pain in rats.¹⁴ These alternative formulations show great potential in decreasing the stress associated with frequent handling and dosing requirements. Many of these products are still considered new in the veterinary market, and few evidence-based recommendations for their use in laboratory animal species are available. The main goal of the current study was to refine postoperative analgesia by using longer-lasting or gel-formulation products. To this end, we investigated whether Bup-SR, sustained-release meloxicam (Melox-SR), or carprofen gel (CG) provided postoperative analgesia in the rat plantar incisional model according to results of behavioral testing. We hypothesized that Bup-SR, Melox-SR, and CG would provide effective postoperative analgesia as evidenced by reduced pain responses in this model.