Moving molecular diagnostics from laboratory to clinical application: a case study using infectious pancreatic necrosis virus serotype A.

AIMS: Using an RT-PCR method for detection of infectious pancreatic necrosis virus (IPNV) in Atlantic salmon as a model, this study examined the optimization and validation required to provide a method suitable for IPNV detection from fish tissue. METHODS AND RESULTS: IPNV-positive Atlantic salmon kidney samples that had been titred or kidney spiked with IPNV were used. The amount of RNA in the reverse transcription (RT), RT denaturation temperature and incubation time, PCR annealing temperature and number of cycles were optimized. The optimized RT-PCR was able to detect IPNV in Atlantic salmon kidney calculated to have a titre of ten infectious units. CONCLUSIONS: Extensive optimization is required to produce a PCR for detection of fish pathogens from methods designed in the laboratory. SIGNIFICANCE AND IMPACT OF THE STUDY: This study demonstrated some of the many variables that should be optimized before a fully validated assay can be claimed and illustrates the extensive validation required to fulfil requirements of the OIE and is of relevance to laboratories carrying out clinical testing.

Canadian Cardiovascular Society commentary on implantable cardioverter defibrillators in Canada: waiting times and access to care issues.

The Canadian Cardiovascular Society is the national professional society for cardiovascular specialists and researchers in Canada. In the spring of 2004, the Canadian Cardiovascular Society Council formed an Access to Care Working Group in an effort to use the best science and information to establish reasonable triage categories and safe wait times for access to common cardiovascular services and procedures. The Working Group has elected to publish a series of commentaries to initiate a structured national discussion on this very important issue. Access to treatment with implantable cardioverter defibrillators is the subject of the present commentary. The prevalence pool of potentially eligible patients is discussed, along with access barriers, regional disparities and waiting times. A maximum recommended waiting time is proposed and the framework for a solution-oriented approach is presented.

Progression to chronic atrial fibrillation after pacing: the Canadian Trial of Physiologic Pacing. CTOPP Investigators.

OBJECTIVES: This study examined the effect of physiologic pacing on the development of chronic atrial fibrillation (CAF) in the Canadian Trial Of Physiologic Pacing (CTOPP). BACKGROUND: The role of physiologic pacing to prevent CAF remains unclear. Small randomized studies have suggested a benefit for patients with sick sinus syndrome. No data from a large randomized trial are available. METHODS: The CTOPP randomized patients undergoing first pacemaker implant to ventricular-based or physiologic pacing (AAI or DDD). Patients who were prospectively found to have persistent atrial fibrillation (AF) lasting greater than or equal to one week were defined as having CAF. Kaplan-Meier plots for the development of CAF were compared by log-rank test. The effect of baseline variables on the benefit of physiologic pacing was evaluated by Cox proportional hazards modeling. RESULTS: Physiologic pacing reduced the development of CAF by 27.1%, from 3.84% per year to 2.8% per year (p = 0.016). Three clinical factors predicted the development of CAF: age > or =74 years (p = 0.057), sinoatrial (SA) node disease (p < 0.001) and prior AF (p < 0.001). Subgroup analysis demonstrated a trend for patients with no history of myocardial infarction or coronary disease (p = 0.09) as well as apparently normal left ventricular function (p = 0.11) to derive greatest benefit. CONCLUSIONS: Physiologic pacing reduces the annual rate of development of chronic AF in patients undergoing first pacemaker implant. Age > or =74 years, SA node disease and prior AF predicted the development of CAF. Patients with structurally normal hearts appear to derive greatest benefits.

New-onset atrial fibrillation: sex differences in presentation, treatment, and outcome.

BACKGROUND: Although sex differences in coronary artery disease have received considerable attention, few studies have dealt with sex differences in the most common sustained cardiac arrhythmia, atrial fibrillation (AF). Differences in presentation and clinical course may dictate different approaches to detection and management. We sought to examine sex-related differences in presentation, treatment, and outcome in patients presenting with new-onset AF. METHODS AND RESULTS: The Canadian Registry of Atrial Fibrillation (CARAF) enrolled subjects at the time of first ECG-confirmed diagnosis of AF. Participants were followed at 3 months, at 1 year, and annually thereafter. Treatment was at the discretion of the patients' physicians and was not directed by CARAF investigators. Baseline and follow-up data collection included a detailed medical history, clinical, ECG, and echocardiographic measures, medication history, and therapeutic interventions. Three hundred thirty-nine women and 560 men were followed for 4.14+/-1.39 years. Compared with men, women were older at the time of presentation, more likely to seek medical advice because of symptoms, and experienced significantly higher heart rates during AF. Compared with older men, older women were half as likely to receive warfarin and twice as likely to receive acetylsalicylic acid. Compared with men on warfarin, women on warfarin were 3.35 times more likely to experience a major bleed. CONCLUSIONS: Anticoagulants are underused in older women with AF relative to older men with AF, despite comparable risk profiles. Women receiving warfarin have a significantly higher risk of major bleeding, suggesting the need for careful monitoring of anticoagulant intensity in women.

Circadian variation of paroxysmal atrial fibrillation. PA3 Investigators. Atrial Pacing Peri-ablation for Prevention of Atrial Fibrillation Trial.

The circadian variation of paroxysmal atrial fibrillation (AF) was studied in 67 patients who received a dual-chamber pacemaker 3 months before a planned atrioventricular node ablation. A distinct circadian variation of AF was observed with 2 time peaks in initiation (1 in the early morning and 1 in the early evening hours), which was modulated by atrial pacing, the duration of AF, and the use of beta-adrenergic blocking agents.

Randomized crossover comparison of DDDR versus VDD pacing after atrioventricular junction ablation for prevention of atrial fibrillation. The atrial pacing peri-ablation for paroxysmal atrial fibrillation (PA (3)) study investigators.

BACKGROUND: Some clinical data suggest that atrial-based pacing prevents paroxysmal atrial fibrillation (AF). This study tested the hypothesis that DDDR pacing compared with VDD pacing prevents AF after atrioventricular (AV) junction ablation. METHODS AND RESULTS: Patients were randomized to DDDR pacing (n=33) or to VDD pacing (n=34) after AV junction ablation and followed every 2 months for 6 months. Patients then crossed over to the alternate pacing mode and were followed for an additional 6 months. Primary analysis included the time to first recurrence of sustained AF (duration >5 minutes), total AF burden, and the development of permanent AF. The time to first episode of AF was similar in the DDDR group (0.37 days, 95% CI 0.1 to 1.3 days) and the VDD pacing group (0.5 days, 95% CI 0.2 to 1.7 days, P=NS). AF burden increased over time in both groups (P<0.01). At the 6-month follow-up, AF burden was 6.93 h/d (95% CI 4. 37 to 10.96 h/d) in the DDDR group and 6.30 h/d (95% CI 3.99 to 9.94 h/d) in the VDD group (P=NS). Twelve (35%) patients in the DDDR group and 11 (32%) patients in the VDD group had permanent AF within 6 months of ablation. Within 1 year of follow-up, 43% of patients had permanent AF. CONCLUSIONS: DDDR pacing compared with VDD pacing does not prevent paroxysmal AF over the long term in patients in the absence of antiarrhythmic drug therapy after total AV junction ablation. Many patients have permanent AF within the first year after ablation.

Clinical trials of pacing mode selection.

Current recommendations in favor of dual-chamber over single-chamber ventricular pacing for patients with sinus node dysfunction or AV conduction disorders were made largely based on observational data and expert opinions. The first randomized pacing mode selection study was relatively small and suggested survival advantage with physiologic pacing only after an extended follow-up duration of 5.5 years. Preliminary results of the first large-scale multicenter randomized pacing mode selection trial revealed only modest reduction in atrial fibrillation without survival advantage after 3 years of physiologic pacing. Two other large-scale multicenter randomized trials comparing physiologic versus ventricular pacing are currently ongoing. They may provide further scientific evidence based on which more objective recommendations can be made with respect to pacing mode selection.

Pacing in right ventricular dysplasia after disconnection surgery.

This report describes a 33-year-old patient with arrhythmogenic right ventricular (RV) dysplasia who had a dual chamber pacemaker implanted at age 23 years for drug-induced bradycardia. Pacing was continued after right ventricular free-wall disconnection (RVFWD) at age 24 years. Her pacemaker was not replaced after battery depletion 7 years later. She presented the following year in severe right-sided heart failure. Her old pacemaker generator was replaced. This was followed by rapid resolution of her clinical failure and return to a full, active, physical lifestyle. This observation suggests the potential benefit of dual chamber pacing in patients with RV dysplasia after RVFWD.

Effects of physiologic pacing versus ventricular pacing on the risk of stroke and death due to cardiovascular causes. Canadian Trial of Physiologic Pacing Investigators.

BACKGROUND: Evidence suggests that physiologic pacing (dual-chamber or atrial) may be superior to single-chamber (ventricular) pacing because it is associated with lower risks of atrial fibrillation, stroke, and death. These benefits have not been evaluated in a large, randomized, controlled trial. METHODS: At 32 Canadian centers, patients without chronic atrial fibrillation who were scheduled for a first implantation of a pacemaker to treat symptomatic bradycardia were eligible for enrollment. We randomly assigned patients to receive either a ventricular pacemaker or a physiologic pacemaker and followed them for an average of three years. The primary outcome was stroke or death due to cardiovascular causes. Secondary outcomes were death from any cause, atrial fibrillation, and hospitalization for heart failure. RESULTS: A total of 1474 patients were randomly assigned to receive a ventricular pacemaker and 1094 to receive a physiologic pacemaker. The annual rate of stroke or death due to cardiovascular causes was 5.5 percent with ventricular pacing, as compared with 4.9 percent with physiologic pacing (reduction in relative risk, 9.4 percent; 95 percent confidence interval, -10.5 to 25.7 percent [the negative value indicates an increase in risk]; P=0.33). The annual rate of atrial fibrillation was significantly lower among the patients in the physiologic-pacing group (5.3 percent) than among those in the ventricular-pacing group (6.6 percent), for a reduction in relative risk of 18.0 percent (95 percent confidence interval, 0.3 to 32.6 percent; P=0.05). The effect on the rate of atrial fibrillation was not apparent until two years after implantation. The observed annual rates of death from all causes and of hospitalization for heart failure were lower among the patients with a physiologic pacemaker than among those with a ventricular pacemaker, but not significantly so (annual rates of death, 6.6 percent with ventricular pacing and 6.3 percent with physiologic pacing; annual rates of hospitalization for heart failure, 3.5 percent and 3.1 percent, respectively). There were significantly more perioperative complications with physiologic pacing than with ventricular pacing (9.0 percent vs. 3.8 percent, P<0.001). CONCLUSIONS: Physiologic pacing provides little benefit over ventricular pacing for the prevention of stroke or death due to cardiovascular causes.

The Canadian Registry of Atrial Fibrillation: a noninterventional follow-up of patients after the first diagnosis of atrial fibrillation.

The Canadian Registry of Atrial Fibrillation (CARAF) is a nondirected, follow-up study of 1,086 patients who are enrolled at 6 centers across Canada at the time of initial electrocardiographically documented diagnosis of atrial fibrillation (AF). Enrollment commenced in 1991 with an intended 10-year follow-up. Comprehensive baseline data, including clinical history, laboratory, and echocardiographic variables were collected. The patients were treated by their own referring physicians and CARAF did not direct their care. Detailed follow-up was performed at 3 months, 1 year, then yearly, with echocardiograms repeated every 2 years. Several studies, which evaluated patient populations, predictors of events, and cardiac structure and functioning, have been performed and are ongoing. Thyroid function was evaluated at baseline, and, of 707 patients evaluated, only 6 patients were found to be hyperthyroid. Symptoms during AF were evaluated and a profile of the types of symptoms and the predictors of symptoms was compiled. Antiarrhythmic drug use is being followed. Sotalol and propafenone were the most commonly used medications, with the use of antiarrhythmic drugs increasing with recurrence of AF. The use of anticoagulants was assessed. The overall use of warfarin was relatively low, but its use increased dramatically with the presence of various risk factors including congestive heart failure, hypertension, and previous stroke. The one risk factor that did not result in increased use of warfarin was hypertension. Therefore, CARAF was able to identify that hypertension appears to be under-recognized and undertreated in its risk for thromboembolic events. CARAF is just now reaching maturity, with the majority of patients having > or=4 years of follow-up. Therefore, extensive investigations are currently under way that will evaluate the baseline characteristics and utilize these as predictors of recurrence of AF, progression to chronicity, and the occurrence of major events such as stroke and death. A very large cohort of patients with serial echocardiograms over 4 years will permit an understanding of the progression of structural and valvular disease. Therefore, CARAF offers a unique opportunity for comprehensive, nondirected follow-up of patients from their initial diagnosis of AF.

Atrial fibrillation: extending basic science to clinical frontiers.

Basic research in atrial fibrillation is advancing with enormous speed, extending the boundaries from research in intact tissues, to cellular electrophysiology and to molecular biology. Never before has the need been greater for a true 'bench to bedside' approach to research. The basic researchers need to understand the potential clinical relevance of their work so that their efforts may be directed to areas of clinical impact. On the other hand, the clinician needs the aid of the basic researcher to help solve some of the vexing clinical problems. Emphasizing the need for this liaison, this paper discusses problems confronted by the clinician and suggests areas of basic research that may help answer the frustration of the clinician in dealing with patients with atrial fibrillation.

Characteristics of restitution kinetics in repolarization of rabbit atrium.

The study was designed to characterize restitution kinetics in atrial repolarization of rabbits and to examine effects of K+ or Ca2+ channel blockers on restitution. Action potentials were recorded from rabbit atrial tissue. Restitution curves of phase I amplitude and action potential duration at 50 and 90% repolarization (APD50, APD90) were defined at a basic cycle length of 0.5 s during control and with interventions. Restitution of phase I amplitude had a monoexponential function with a time constant of 2.8 +/- 0.2 s. The curves of APD50 frequently had a monoexponential function and time constants were 1.8 +/- 0.1 s. Restitution curves of APD90 were biphasic: a descending phase followed by an ascending phase. The blocker of Ito1 (a 4-aminopyridine-sensitive component of the transient outward current), 4-aminopyridine, flattened the restitution curves of phase I amplitude, and APD50 and APD90 curves became monophasic. Sotalol, a selective IKr (a rapid component of the delayed rectifier K+ current) blocker, did not alter curves of phase I amplitude and APD50 but shifted APD90 curves upward. Cadmium, a Ca2+ blocker shifted curves of phase I amplitude and APD50 downward and abolished the ascending phase of APD90 curves. We conclude that kinetics of Ito1 and ICa (calcium current) may account for characteristics of restitution of atrial repolarization in rabbit.

How useful is thyroid function testing in patients with recent-onset atrial fibrillation? The Canadian Registry of Atrial Fibrillation Investigators.

BACKGROUND: Patients with recent-onset atrial fibrillation often undergo routine thyroid function screening to rule out thyroid disease as a cause of atrial fibrillation. METHODS: Patients with recent (< 3 months) onset of documented atrial fibrillation or flutter were enrolled in the Canadian Registry of Atrial Fibrillation from outpatient clinics, emergency departments, and hospital wards across Canada. Seven hundred twenty-six patients underwent baseline thyroid function screening and were assessed for presence of clinical thyroid disease. Serum thyrotropin level (TSH) was measured in 707 patients (97%), and thyroxine level (T4) in 407 patients (56%). RESULTS: A TSH level less than 0.1 mU/L was present in 5 patients (0.7%). A TSH level less than normal but more than 0.1 mU/L was present in 34 patients (4.7%). No patient had definite hypothyroidism (TSH > 20 mU/L), but 56 patients (7.7%) had an elevated TSH level that was less than 20 mU/L. During 1.7 years of follow-up, only 7 patients were found to have clinical hyperthyroidism, and 11 patients (1.5%) had hypothyroidism. Logistic regression analysis showed that palpitations (odds ratio, 4.9; 95% confidence interval, 1.7-14.0) and asymptomatic presentation (odds ratio, 5.5; 95% confidence interval, 1.9-16.2) were risk factors for low TSH level, and increasing age (odds ratio, 1.32 every 10 years; 95% confidence interval, 1.01-1.66) was a risk factor for high TSH level. The positive predictive value of palpitations and asymptomatic presentation for low TSH level were 9% and 8%, respectively. CONCLUSIONS: An abnormal TSH level is common in patients with recent-onset atrial fibrillation. However, clinical thyroid disease is uncommon. Routine TSH screening of patients who have atrial fibrillation has a low yield and may be better applied to those patients at higher risk of having undiagnosed clinical thyroid disease.

pH-dependent actions of 4-aminopyridine on atrial repolarization: effects on the transient outward current.

Effects of extracellular pH (pHo) were examined on the changes in atrial repolarization induced by 4-aminopyridine (4AP), which is a selective blocker of the transient outward potassium channel, I(to). Action potential parameters were measured, using the conventional microelectrode technique, in the absence and presence of 4AP (0.1-3.0 mM) at pHo 6.5, 7.25, and 8.0. Phase 1 amplitude served as an index of I(to). The onset and recovery kinetics of phase 1 amplitude were assessed at a basic cycle length of 0.5 s, and time constants (tau on and tau r) were computed. Both onset and recovery kinetics had monoexponential functions. Tonic blockade was influenced by external pH, and Kd for half block was 0.19, 0.44, and 2.43 mM for pHo 8.0, 7.25, and 6.5, respectively. Phasic block was defined and exhibited cycle length dependence. Phasic block was also modified by external pH with the greatest effect at pHo 8.0. 4AP (0.3 mM) accelerated tau on, 0.62 +/- 0.2, 0.55 +/- 0.1, and 2.0 +/- 0.8 beats for pHo 8.0, 7.25, and 6.5 compared with control 6.8 +/- 1.9, 6.3 +/- 1.9, and 5.1 +/- 1.4 beats. In contrast, 4AP slowed tau r by about 1 s from control value to 1.5 +/- 0.5 s at pHo 6.5, 4.8 +/- 1.5 s at pHo 7.25 (p < 0.05), and 5.7 +/- 2.0 s at pHo 8.0. We conclude that an increase in extracellular pH enhances block of Ito induced by 4AP, whereas low pHo attenuates the block.