Detection of occult paroxysmal atrial fibrilation by implantable long-term electrocardiographic monitoring in cryptogenic stroke and transient ischemic attack population: a study protocol for prospective matched cohort study

Background Cardio-embolic etiology is the most frequently predicted cause of cryptogenic stroke/TIA. Detection of occult paroxysmal atrial fibrillation is crucial for selection of appropriate medication. Methods Enrolment of eligible cryptogenic stroke and TIA patients began in 2014 and will continue until 2018. The patients undergo long-term (12 months) ECG monitoring (implantable loop recorder) and testing for PITX2 (chromosome 4q25) and ZFHX3 (chromosome 16q22) gene mutations. There will be an appropriate control group of age- and sex-matched healthy volunteers. To analyse the results descriptive statistics, statistical tests for group differences, and correlation analyses will be used. Discussion In our study we are focusing on a possible correlation between detection of atrial fibrillation by an implantable ECG recorder, and PITX2 and/or ZFHX3 gene mutations in cryptogenic stroke/TIA patients. A correlation could lead to implementation of this genomic approach to cryptogenic stroke/TIA diagnostics and management. The results will be published in 2018. Trial registration ClinicalTrials.gov: NCT02216370.


Background
Stroke has consistently been the 2 nd leading cause of death in the age group of 60 plus worldwide [1]. Recently, it has become the 4 th leading cause of death and the 1 st leading cause of serious long-term disability in the USA [2]. Accurate definition of stroke etiology is crucial for the most effective management of the disease.
Cryptogenic stroke (CS), according to the TOAST classification, is defined as an ischemic stroke with undeterminable etiology, despite extensive evaluation [3]. Stroke might be cryptogenic for several reasons: 1) evaluation is not carried out at the time of event; 2) investigation is not sufficiently extensive; or 3) there are other unknown causes. Therefore the proportion of CS depends on the speed and wider-than-routine range of diagnostic evaluation. Epidemiological observational trials using standardized stroke etiological classification report 20-40 %, in some cases up to 50 % incidence of unrevealed causes [4][5][6].
Cardio-embolic stroke is the most frequently predicted cause of CS in studies (58 %) [7,8]. The most frequent cause of cerebral cardio-embolism, as well as the most common cardiac arrhythmia, is atrial fibrilation (AF). The incidence of stroke in people with non-valvular AF is estimated to be 5 times higher than in people without AF [9]. It has been suggested that paroxysmal AF (PAF) is more prevalent than chronic AF in stroke and TIA patients, and they both appear to carry very similar stroke risk [10].
Episodes of PAF may reoccur with variable frequency and their detection has been made possible by ECG monitoring. Short and usually asymptomatic presentations of PAF (also occult PAF) may remain undetected by routine arrhythmia screening methods. Incidence of newly identified PAF varies widely depending on the type of cardiac monitoring devices, the delay in monitoring after stroke onset and the monitoring duration. Detection rate of PAF with external ECG monitoring in patients after CS has been recorded between 5 and 20 % [11][12][13]. An insertable cardiac monitor (ICM) automatically provides non-stop long-term ECG recording with very high sensitivity and specificity for PAF detection. Studies using ICM have suggested a detection rate of approximately 25 % [14][15][16].
In our study we are focusing on a possible correlation between detection of atrial fibrillation by implantable ECG loop, and PITX2 and/or ZFHX3 gene mutations in cryptogenic stroke/TIA patients. A correlation could lead to implementation of this genomic approach to cryptogenic stroke/TIA diagnostics and management [17,18].

Objective and endpoints
The objective of our study is to determine the proportion of patients with CS or cryptogenic TIA (CTIA) that have underlying PAF in comparison with control group. PAF is defined as an episode of irregular heart rhythm without detectable P waves lasting more than 30 s. Primary endpoints are the first documented PAF episode after CS or CTIA and incidence of PITX2 and ZFHX3 genes mutation. Secondary endpoints include recurrent stroke or TIA, new ischemic lesions found by brain imaging (CT and/or MRI), the size of left atrium (TTE/ TEE), and relevant treatment changes (e.g. oral anticoagulant drugs). The follow-up of patient and control groups will last 12 months.

Study participants
The study will analyse data from at least 100 patients with CS or CTIA admitted to the Department of Neurology in Nitra between 2014 and 2018 as well as at least 30 ageand sex-matched volunteers with no known vascular diesease in the control group. The study protocol has been approved by the Ethics Committee of the Faculty Hospital in Nitra (Slovak Republic). A written informed consent will be obtained from all study participants or their legal representatives. To analyse the data, descriptive statistics, statistical tests for differences, and correlation analyses will be used. This prospective and observational study is registered at www.clinicaltrials.gov with study identifier NCT02216370.

Discussion
Unrecognized PAF and the consequent lack of prophylactic anticoagulant treatment may result in significant morbidity first presenting as a heart failure, stroke, systemic embolism, and occasionally death. It is generally accepted that intermittent ECG monitoring (Holter ECG) has low sensitivity to identify patients with PAF.  [28]. Finding a new algorithm for detecting PAF in standard clinical practice should be of great interest as it would allow early indication of anticoagulant therapy in stroke/TIA patients. Additional information that would help to characterize stroke subtype could be provided by genetic data. Study of gene polymorphisms associated with an increased risk of AF suggests that subtle alterations in developmental factors, cell signalling, extracellular matrix regulation, and ion channels function are involved in arrhythmia pathogenesis. Large genome-wide association studies (GWAS) have found that the genetic markers close to the PITX2 gene on chromosome 4q25 and to the ZFHX3 gene on 16q22 are associated with both AF and cardio-embolic stroke [17,18]. The PITX2 gene plays a critical role in development of left atrium and pulmonary vein myocardium. This particular region has been implicated to initiate and maintain AF. The expression patterns of the ZFHX3 gene in human cardiac and pulmonary tissue are not clear, nor are the mechanisms by which variants in this gene may predispose to AF.

Conclusion
Cardioembolic TIA and stroke cause high rate disability and mortality worldwide. As cardio-embolic etiology is responsible for up to 58 % of CS, appropriate highresolution cardiac monitoring plays a crucial role in arrhythmias detection after CTIA/CS. Early PAF diagnosis allows to start immediate anticoagulation therapy in order to reduce TIA/stroke recurrence. The expression profile of the selected genes may be very useful in selecting high-risk PAF individuals who could then undergo extensive ECG monitoring with no history of cerebrovascular disease. Competing interests RH reports research grants from Medtronic. The other authors declare that they have no competing interests.
Authors' contributions AP: literature search; AP: manuscript writing; AP, EK, ŠS, MB, RH, PB: study design; AP, JH, PV, AA, data collection. All authors read and approved the final manuscript.