Detection and quantitation of two cucurbit criniviruses in mixed infection by real-time RT-PCR

Cucurbit chlorotic yellows virus (CCYV) and Cucurbit yellow stunting disorder virus (CYSDV) are whitefly-transmitted criniviruses infecting cucurbit crops inducing similar symptoms. Single and multiplex RT-PCR protocols were developed and evaluated on cucurbit samples collected from commercial greenhouses. Primers and probes were designed from the highly conserved heat shock protein 70 homolog (Hsp70h) gene. Conventional RT-PCR and multiplex RT-PCR assays showed high specificity and suitability for routine screening. TaqMan-based quantitative real-time RT-PCR (RT-qPCR) protocols were also developed for the detection and quantitation of both viruses occurring in single or mixed infection. The assays proved to be highly specific with no cross amplification. RT-qPCR assays showed a 100–1000 times improved sensitivity over conventional RT-PCR. Virus titers in mixed infections were compared to singly infected plants by RT-qPCR. CYSDV and CCYV titers decreased in double infected plants. This paper reports highly specific conventional RT-PCR and quantitative real-time PCR assays for detection, quantitation and differentiation between two closely related cucurbit-infecting criniviruses.     

Comparison of bleeding risk scores in patients with atrial fibrillation: insights from the RE‐LY trial

Background

Oral anticoagulation is the mainstay of stroke prevention in atrial fibrillation (AF), but must be balanced against the associated bleeding risk. Several risk scores have been proposed for prediction of bleeding events in patients with AF.

Objectives

To compare the performance of contemporary clinical bleeding risk scores in 18 113 patients with AF randomized to dabigatran 110 mg, 150 mg or warfarin in the RE‐LY trial.

Methods

HAS‐BLED, ORBIT, ATRIA and HEMORR₂HAGES bleeding risk scores were calculated based on clinical information at baseline. All major bleeding events were centrally adjudicated.

Results

There were 1182 (6.5%) major bleeding events during a median follow‐up of 2.0 years. For all the four schemes, high‐risk subgroups had higher risk of major bleeding (all P  < 0.001). The ORBIT score showed the best discrimination with c‐indices of 0.66, 0.66 and 0.62, respectively, for major, life‐threatening and intracranial bleeding, which were significantly better than for the HAS‐BLED score (difference in c‐indices: 0.050, 0.053 and 0.048, respectively, all P  < 0.05). The ORBIT score also showed the best calibration compared with previous data. Significant treatment interactions between the bleeding scores and the risk of major bleeding with dabigatran 150 mg BD versus warfarin were found for the ORBIT (P  = 0.0019), ATRIA (P  < 0.001) and HEMORR₂HAGES (P  < 0.001) scores. HAS‐BLED score showed a nonsignificant trend for interaction (P  = 0.0607).

Conclusions

Amongst the current clinical bleeding risk scores, the ORBIT score demonstrated the best discrimination and calibration. All the scores demonstrated, to a variable extent, an interaction with bleeding risk associated with dabigatran or warfarin.

     

Randomized trial of candesartan cilexetil in the treatment of patients with congestive heart failure and a history of intolerance to angiotensin-converting enzyme inhibitors

BACKGROUND: Many patients with congestive heart failure do not receive the benefits of angiotensin-converting enzyme (ACE) inhibitors because of intolerance. We sought to determine the tolerability of an angiotensin II receptor blocker, candesartan cilexetil, among patients considered intolerant of ACE inhibitors. METHODS: Patients with CHF, left ventricular ejection fraction less than 35%, and history of discontinuing an ACE inhibitor because of intolerance underwent double-blind randomization in a 2:1 ratio to receive candesartan (n = 179) or a placebo (n = 91). The initial dosage of candesartan was 4 mg/d; the dosage was increased to 16 mg/d if the drug was tolerated. A history of intolerance of ACE inhibitor was attributed to cough (67% of patients), hypotension (15%), or renal dysfunction (11%). RESULTS: The study drug was continued for 12 weeks by 82.7% of patients who received candesartan versus 86.8% of patients who received the placebo. This 4.1% greater discontinuation rate with active therapy was not significant; the 95% confidence interval ranged from 4.8% more discontinuation with placebo to 13% more with candesartan. Titration to the 16-mg target dose was possible for 69% of patients who received candesartan versus 84% of those who received the placebo. Frequencies of death and morbidity were not significantly different between the candesartan and placebo groups (death 3.4% and 3.3%, worsening heart failure 8.4% and 13.2%, myocardial infarction 2.8% and 5.5%, all-cause hospitalization 12.8% and 18.7%, and death or hospitalization for heart failure 11.7% and 14.3%). CONCLUSIONS: Candesartan was well tolerated by this population. The effect of candesartan on major clinical end points, including death, remains to be determined.     

Physical inactivity in patients with rheumatoid arthritis: data from twenty-one countries in a cross-sectional, international study

OBJECTIVE: Regular physical activity is associated with decreased morbidity and mortality. Traditionally, patients with rheumatoid arthritis (RA) have been advised to limit physical exercise. We studied the prevalence of physical activity and associations with demographic and disease-related variables in patients with RA from 21 countries. METHODS: The Questionnaires in Standard Monitoring of Patients with Rheumatoid Arthritis (QUEST-RA) is a cross-sectional study that includes a self-report questionnaire and clinical assessment of nonselected consecutive outpatients with RA who are receiving usual clinical care. Frequency of physical exercise (>or=30 minutes with at least some shortness of breath, sweating) is queried with 4 response options: >or=3 times weekly, 1-2 times weekly, 1-2 times monthly, and no exercise. RESULTS: Between January 2005 and April 2007, a total of 5,235 patients from 58 sites in 21 countries were enrolled in QUEST-RA: 79% were women, >90% were white, mean age was 57 years, and mean disease duration was 11.6 years. Only 13.8% of all patients reported physical exercise>or=3 times weekly. The majority of the patients were physically inactive with no regular weekly exercise: >80% in 7 countries, 60-80% in 12 countries, and 45% and 29% in 2 countries, respectively. Physical inactivity was associated with female sex, older age, lower education, obesity, comorbidity, low functional capacity, and higher levels of disease activity, pain, and fatigue. CONCLUSION: In many countries, a low proportion of patients with RA exercise. These data may alert rheumatologists to motivate their patients to increase physical activity levels.