Qatar Foundation Annual Research Conference Proceedings Volume 2016 Issue 1

Introduction

Most of neurological disorders are network-based diseases. The networks associated with these diseases usually involve spatially disturbed brain regions. Thus efforts were recently evolving from identifying pathological “zones” toward identifying “networks”. In a very recent review, Fornito and colleagues revealed that the identification of alterations in brain networks is one of the most promising paradigms in brain disorders research (Fornito and Bullmore, 2014; Fornito et al., 2015). So far, approaches based on graph theory have characterized the brain networks as sets of nodes connected by edges (Bullmore and Sporns, 2009). Once the nodes (brain regions) and edges (functional/structural connections between regions) are defined from neuroimaging technique, methods based on graph theory may be used to describe the topological properties of the identified networks. This network-based analysis has been largely used to investigate normal (Bressler and Menon, 2010) and pathological (Fornito et al., 2015) brain activities from numerous modalities. It has been used in many clinical applications such as Alzheimer's disease (He et al., 2008; Lo et al., 2010; Mallio et al., 2015; Stam et al., 2007), schizophrenia (Fornito et al., 2011; Liu et al., 2008; van den Heuvel et al., 2013) and autism (Guye et al., 2010; Li et al., 2014). However, a precise tracking of the spatiotemporal dynamics of large-scale pathological networks is still an unsolved issue (Hutchison et al., 2013). The only noninvasive technique that provides the sufficient temporal resolution to track dynamics of brain activity at millisecond scale is the Electro/Magneto encephalogram (EEG/MEG).

Methods

Therefore, the focus of our work is to develop advanced methods to, noninvasively, identify dynamics of functional brain networks using dense EEG signals (256 electrodes). The key advantage of our method, called ‘EEG source connectivity’ (Fig. 1), is the possibility of identifying functional brain networks with excellent temporal (∼ 1 ms) and spatial resolutions (Hassan et al., 2015a; Hassan et al., 2014; Hassan et al., 2015b). The EEG source connectivity involves two main steps: i) the reconstruction of regional time series by solving the EEG inverse problem and ii) the estimation of the functional connectivity using phase synchronization among oscillations present in the time-courses of reconstructed regional time series. The method was originally developed to track dynamics of functional brain networks during short time cognitive activity such as picture naming task ( < 1 second) (Hassan et al., 2015a). In this abstract, we show the first results of applying EEG source connectivity method to two neurological disorders: Epilepsy and Parkinson's disease. Figure 1: Illustrative structure of the proposed method. Dense EEG were used to record data from patients (and healthy control). Structural MRI images will be also recorded, segmented and then anatomically parcellated into a desired number of brain regions (regions of interest). The functional connectivity was then computed giving rise to high-resolution functional brain networks. These networks (graphs) will be finally characterized (quantified) using approach from graph theory to characterize the brain networks (regions and sub regions) involved in the analyzed pathology.

Results

First, in the context of epilepsy, data were recorded from epileptic patients who underwent a full pre-surgical evaluation for drug-resistant partial epilepsy. The patients had a comprehensive evaluation including detailed history and neurological examination, neuropsychological testing, structural MRI, standard 32-channels (Micromed) as well as High-Resolution 256-channels (EGI, Electrical Geodesic Inc.) scalp EEG with video recordings and intracerebral EEG recordings (SEEG). We applied our method to identify epileptogenic networks from the scalp dense-EEG. We also estimated the functional network from depth-EEG. The results revealed a very good matching between networks identified from noninvasive EEG and those obtained using the intra-cerebral electrodes (ground truth). We were able, for the first time from scalp recordings, to identify the network of brain regions (nodes) involved in the generation of the seizure as selected by the epileptologist. Second, we recently applied the EEG source connectivity method to detect alterations in functional networks involved in cognitive impairments. Dense-EEG (128 electrodes) were recorded during task-free paradigm (resting state, eye closed) to produce whole-brain functional connectivity networks in patients with different cognitive phenotypes (Dujardin et al., 2013): 1) cognitively intact patients, 2) patients with mild cognitive impairment and 3) patients with very severe cognitive impairment. Functional connectivity was assessed between each pair of 68 brain regions in 124 patients at different EEG frequency bands. Network measures were realized at global level topology, region-wise connectivity and edge-wise connectivity. Using Network Based Statistics (NBS) (Zalesky et al., 2010), results showed significant differences at alpha band (7–13 Hz) between all groups. The quantification of the networks showed that most of significant alterations (decreased connections) were frontotemporal (functional connections between frontal and temporal lobes) which perfectly match with previous findings of cognitive impairments in patient with neurodegenerative disorders (Pievani et al., 2011).

Discussion

We consider that the identification of pathological brain networks from noninvasive EEG recordings is a topic of great interest. The results of the EEG source connectivity methods on cognitive task (Hassan et al., 2015a) as well on neurological disorders such as epilepsy and Parkinson's disease as presented here, may open new perspectives in the clinical use of such approach. These network dysfunctions may be specific to the clinical syndromes and, in Parkinson's disease and frontotemporal dementia for instance, network disruption may track the pattern of pathological alterations. We speculate that our findings might have practical repercussions for diagnostic purpose, allowing earlier detection of neurodegenerative diseases and tracking of disease development.

Acknowledgment

This work has received a French government support granted to the CominLabs excellence laboratory and managed by the National Research Agency in the “Investing for the Future” program under reference ANR-10-LABX-07-01.

References

Bressler, S.L., Menon, V., 2010. Large-scale brain networks in cognition: emerging methods and principles. Trends in cognitive sciences 14, 277–290.

Bullmore, E., Sporns, O., 2009. Complex brain networks: graph theoretical analysis of structural and functional systems. Nature Reviews Neuroscience 10, 186–198.

Dujardin, K., Leentjens, A.F., Langlois, C., Moonen, A.J., Duits, A.A., Carette, A.S., Duhamel, A., 2013. The spectrum of cognitive disorders in Parkinson's disease: A data-driven approach. Movement Disorders 28, 183–189.

Fornito, A., Bullmore, E.T., 2014. Connectomics: a new paradigm for understanding brain disease. European Neuropsychopharmacology.

Fornito, A., Yoon, J., Zalesky, A., Bullmore, E.T., Carter, C.S., 2011. General and specific functional connectivity disturbances in first-episode schizophrenia during cognitive control performance. Biological psychiatry 70, 64–72.

Fornito, A., Zalesky, A., Breakspear, M., 2015. The connectomics of brain disorders. Nature Reviews Neuroscience 16, 159–172.

Guye, M., Bettus, G., Bartolomei, F., Cozzone, P.J., 2010. Graph theoretical analysis of structural and functional connectivity MRI in normal and pathological brain networks. Magnetic Resonance Materials in Physics, Biology and Medicine 23, 409–421.

Hassan, M., Benquet, P., Biraben, A., Berrou, C., Dufor, O., Wendling, F., 2015a. Dynamic reorganization of functional brain networks during picture naming. Cortex 73, 276–288.

Hassan, M., Dufor, O., Merlet, I., Berrou, C., Wendling, F., 2014. EEG Source Connectivity Analysis: From Dense Array Recordings to Brain Networks. PloS one 9, e105041.

Hassan, M., Shamas, M., Khalil, M., El Falou, W., Wendling, F., 2015b. EEGNET: An Open Source Tool for Analyzing and Visualizing M/EEG Connectome. PloS one 10, e0138297.

He, Y., Chen, Z., Evans, A., 2008. Structural insights into aberrant topological patterns of large-scale cortical networks in Alzheimer's disease. The Journal of neuroscience 28, 4756–4766.

Hutchison, R.M., Womelsdorf, T., Allen, E.A., Bandettini, P.A., Calhoun, V.D., Corbetta, M., Della Penna, S., Duyn, J.H., Glover, G.H., Gonzalez-Castillo, J., 2013. Dynamic functional connectivity: promise, issues, and interpretations. NeuroImage 80, 360–378.

Li, H., Xue, Z., Ellmore, T.M., Frye, R.E., Wong, S.T., 2014. Network-based analysis reveals stronger local diffusion-based connectivity and different correlations with oral language skills in brains of children with high functioning autism spectrum disorders. Human brain mapping 35, 396–413.

Liu, Y., Liang, M., Zhou, Y., He, Y., Hao, Y., Song, M., Yu, C., Liu, H., Liu, Z., Jiang, T., 2008. Disrupted small-world networks in schizophrenia. Brain 131, 945–961.

Lo, C.-Y., Wang, P.-N., Chou, K.-H., Wang, J., He, Y., Lin, C.-P., 2010. Diffusion tensor tractography reveals abnormal topological organization in structural cortical networks in Alzheimer's disease. The Journal of neuroscience 30, 16876–16885.

Mallio, C.A., Schmidt, R., Reus, M.A., Vernieri, F., Quintiliani, L., Curcio, G., Beomonte Zobel, B., Quattrocchi, C.C., den Heuvel, M.P., 2015. Epicentral Disruption of Structural Connectivity in Alzheimer's Disease. CNS Neuroscience & Therapeutics.

Pievani, M., de Haan, W., Wu, T., Seeley, W.W., Frisoni, G.B., 2011. Functional network disruption in the degenerative dementias. The Lancet Neurology 10, 829–843.

Stam, C., Jones, B., Nolte, G., Breakspear, M., Scheltens, P., 2007. Small-world networks and functional connectivity in Alzheimer's disease. Cerebral Cortex 17, 92–99.

van den Heuvel, M.P., Sporns, O., Collin, G., Scheewe, T., Mandl, R.C., Cahn, W., Goñi, J., Pol, H.E.H., Kahn, R.S., 2013. Abnormal rich club organization and functional brain dynamics in schizophrenia. JAMA psychiatry 70, 783–792.

Zalesky, A., Fornito, A., Bullmore, E.T., 2010. Network-based statistic: identifying differences in brain networks. NeuroImage 53, 1197–1207.

Background

There has been some anecdotal evidence linking certain infectious pathogens (such as Chlamydia pneumoniae) with stroke. C. pneumoniae infection may be one of several contributing factors for the development of inflammation of blood vessels and atherosclerosis. Furthermore, a number of inflammatory markers (such as plasma CRP, fibrinogen, IL-6, IL-1ra, ESR, WCC) have recently been linked with acute ischemic stroke. However, their value in predicting short and long-term prognosis has been questioned and deemed to be not useful in defining outcomes due to the lack of evidence and cost implications.

Aims

The aims of this study are to (i) investigate the presence of C. pneumoniae in both echogenic and echolucent carotid plaques; and (ii) explore their role in the destabilization of the plaque, leading to embolization and cerebrovascular events.

Methods

(i) Study design: A prospective study involving carotid plaque specimens collected from routine endarterectomy surgical operations. Carotid plaques were removed en bloc during surgery to preserve the entire plaque structure. (ii) Carotid plaque samples: Twenty specimens of carotid atherosclerotic plaques (ten predominantly echolucent, Types I & II and ten predominantly echogenic Types III & IV) were used. The non-invasive ultrasonic appearance (i.e. echogenicity or echolucency) and the quantitative grading of these plaques were determined pre-operatively using duplex scanning aided by computerized measurements according to the criteria adopted by Gray-Weale et al. (1988). (iii) Immunohistochemistry: Levels of C. pneumoniae, Matrix Metalloproteinase-3 (MMP3), Nitric Oxide Synthase (NOS), Superoxide Dismutase (SOD) enzymes in echogenic (fibrous) and echolucent (lipid-laden) atherosclerotic carotid plaques were determined using immuno-histochemical and Western blotting techniques. (iv) Histological methods: Haematoxylin-Eosin stain was performed to stain the slides that will be used for morphological studies by light microscopy. For Laser Scanning Confocal Microscopy (LSCM), slides were stained with saturated aqueous fluorescein for a few minutes and then dehydrated, cleared and mounted in DPX. Carotid plaque specimens were first examined by light microscopy (at magnification 10, 40 times) to characterize the following histological features: necrosis, calcification, fibrous cap, erosion or ulceration, hemorrhage, fibrous tissue and lipid content. Sections from the same specimens were then analyzed using LSCM; slides were viewed with 488 excitation and 515 LP emission. The presence or absence as well as the pattern of distribution of these markers (proteins) were correlated with the histological and preoperative ultrasonic appearances of the plaques.

Results

Ultrasound imaging of carotid plaques revealed 10 with echolucent (lipid-laden/unstable) properties and 10 with echogenic (fibrous/stable) features. Intense immune-reactivity for C pneumoniae was observed in two thirds of echolucent plaques, clustering near endothelial cells of the intimal region, and in the neo-endothelial regions of the specimens. By contrast, low level and scattered C pneumoniae immune-reactivity was observed in echogenic plaques. MMP-3 (stromelysin) level was higher in echolucent than in echogenic plaques. High levels of this enzyme were observed near regions of ulceration, necrosis and in areas where the fibrous cap was thin or torn. Isoforms of NOS enzymes were seen in all carotid plaques irrespective of intra-plaque features however, levels of inducible NOS (NOS-II) were higher in echolucent than in echogenic plaques. Higher levels of immune-reactive SOD were also observed in plaques with higher degree of stenosis (more than 75%–80% measured by ultrasound scan). No direct relationship was evident between the neurological features experienced by the study group and the immuno-histochemical findings. There was however a relationship between the morphology of the plaque and the immuno-histochemical results. Both bright-field microscopy and Laser Scanning Confocal Microscopy (LSCM) were used to generate 3D images of surgically removed carotid plaques. 3D imaging of carotid plaques, using LSCM showed that most specimens were predominately composed of lipid material, comprising necrotic core of amorphous debris and cholesterol clefts, with varying degrees of fibrous tissue present in all plaques. Regions of actual fibrous cap disruption and some ulceration were also seen. Fraying of the fibrous cap was notable with fibrous cap erosion and exposure of underlying necrotic core to lumen. The extent of breaks in the fibrous cap varied with each plaque. Evidence of carotid plaque vulnerability (to rupture) was demonstrated by reduced fibrous cap thickness, a large lipid-necrotic core, and increased inflammatory cells infiltrate with evidence of cracking.

Discussion

Although the findings of this study are suggestive that C. pneumoniae may be involved in the worsening and destabilization of the carotid atherosclerotic plaque, more evidence is needed to ascertain whether C. pneumoniae infection may be an independent, modifiable risk factor for ischemic stroke. Results of this study also showed that MMP3 or its pro-enzyme may play an important role in digesting fibrous tissue of the plaque, leading to thinning or tearing of the fibrous cap. This might result in subsequent destabilization of the plaque, leading to embolization and cerebrovascular events. Furthermore, increased level of SOD expression in carotid plaques might be linked to the degree of stenosis of the affected carotid artery. Enzymes identified in this study may have been influenced by (i) inflammatory conditions prevailing in these plaques (e.g., C. pneumoniae might be responsible for triggering cascades of inflammatory events leading to activation of these enzymes), or (ii) shear stress which might be responsible for stimulation of these enzymes. The 3D geometry of carotid plaque also showed evidence of shearing stresses, resulting from fluid flow which might contribute to the degree of instability of the plaque. This is a complex process which might also include the mechanical activation of enzymes such as matrix metalloproteinases, nitric oxide synthases and Superoxide Dismutase. The behavior of these enzymes in response to fluctuations in the velocity field in both echogenic and echolucent plaques is yet to be investigated. Possible activation of these enzymes by fluid dynamical instabilities and the implications of such activation on the destabilization and progression of atherosclerotic plaques require further investigation. Once the role of 3D geometry and microrheology on plaque stability has been investigated and quantified, more opportunities will be available for translating the results into clinical applications.

Conclusion

There is a need for stronger evidence to assist in the diagnosis, treatment, and secondary prevention of stroke in patients in whom an infectious cause for stroke is probable. It has been suggested that mechanically induced or flow-sensitive enzymes contain positive and negative shear stress response elements in their encoding genes and can be stimulated or suppressed according to the nature of blood flow. However, it is still not possible to show any definite link between specific enzyme(s) and cerebrovascular events. Such an association is probably multifactorial and mediated by a combination of the degree of stenosis, plaque morphology, dynamic and biological factors. We are currently investigating the link between certain inflammatory markers and cerebral blood flow and cerebral auto-regulation in patients presenting with Transient Ischemic Attack (TIA or mini stroke) for the first time to explore further the role of inflammation in stroke. This study has just started; it involves 200 patients recruited from Qatar and UK. One of its outcomes will be to correlate impaired cerebral auto-regulation with a range of pro-inflammatory and anti-inflammatory markers in first time TIA patients. In doing so, we will study the profile, rather than just the level, of these inflammatory markers in each patient, taking into account the time lapse between onset of symptoms and the collection of blood specimen (for cytokines analysis) from each patient. “Part of this study was made possible by grant NPRP 6 – 565 – 3 – 141 from the Qatar National Research Fund (a member of Qatar Foundation). The statements made herein are solely the responsibility of the author[s].”

Background

Worldwide, cerebrovascular disease (CVD) is the leading disease-related cause of chronic disability; the second most common cause of death and dementia; and a significant burden to patients, caregivers, and healthcare systems. Major risk factors for stroke include diabetes, hypertension, smoking and dyslipidemia which are highly prevalent in the gulf region. Indeed in Qatar, the incidence of cerebrovascular disease is increasing and most patients have major vascular risk factors. In data from the HMC Stroke Registry ∼71% of patients presenting with stroke had diabetes and indeed diabetes was undiagnosed at the time of presentation in 35% of patients. Current imaging techniques to detect early sub-clinical cerebral damage and hence those at risk of stroke includes computerized tomography (CT) and magnetic resonance imaging (MRI). MRI in particular can detect features of small vessel disease such as accumulating silent infarcts, cerebral microbleeds [CMB], periventricular white matter hyperintensity and perivascular spaces which all predict a higher risk of stroke. However, these abnormalities provide a measure of vascular and not neuronal integrity and it is not feasible or practical to undertake MRI simply to identify those at risk of stroke. There is therefore an unmet need for a rapid, non-invasive, sensitive, cost-effective, reproducible and easily imaging biomarker for neuronal damage in subjects at risk of stroke. We have pioneered the technique of corneal confocal microscopy (CCM). Our studies (NIH (5RO1-NS46259-03, R01DK077903-01A1 NINDS), JDRF (17-2008-1031, 8-2008-362)) have shown that corneal confocal microscopy (CCM) can quantify early axonal damage in diabetic neuropathy (1), reliably (2), with high sensitivity and specificity (3). Furthermore, we have shown that subjects with impaired glucose tolerance (IGT) and other vascular risk factors also show corneal nerve loss using CCM (4). Furthermore, these abnormalities improve with an improvement in blood pressure, lipids and HbA1c, all risk factors for stroke (5) and with change in glucose tolerance (6). We have recently published a large normative reference database (7) and developed an automated image analysis algorithm to enable rapid and objective quantification of corneal nerve morphology (8). We therefore hypothesized that CCM may identify corneal nerve loss driven by the common vascular risk factors which may lead to stroke and thereby provide a surrogate for cerebral neuronal loss. Hence, CCM may allow us to identify subjects who may be at high risk of developing stroke and enable risk stratification and targeted risk factor intervention in these individuals.

Aim

We undertook CCM in a cohort of patients admitted to HMC with a clinical and radiological diagnosis of stroke.

Methods

17 stroke patients (age: 53.13 ± 9.09, years) and 15 age-matched healthy control participants (age: 55.30 ± 8.98, years) underwent corneal nerve assessment using CCM (Heidelberg HRT III) to quantify corneal nerve fiber density (CNFD)(no./mm²), corneal nerve fiber branch density (CNFBD)(no./mm²), corneal nerve fiber nerve length (CNFL)(mm/mm²) and corneal nerve fiber tortuosity (TC) and metabolic testing. Stroke patients underwent detailed neurological assessment to define the severity of the stroke using a National Institute of Health Stroke Scale (NIHSS), 3D Carotid Doppler and 3 Tesla MRI. Stroke patients were stratified into different groups for analysis: Normoglycemia (HbA1c <  5.7) (age: 57 ± 8.12, years, n = 5) v dysglycemia (HbA1c ≥  5.7), n = 12); neurological disability: NIHSS <  4 (n = 9) v NIHSS ≥  4 (n = 8), Pathology on MRI: <  10 silent infarcts n = 5 v > 10 silent infarcts (n = 4), patients with periventricular white matter hyperintensity (n = 11) v no periventricular white matter hyperintensity (n = 4).

Results

There was significant difference in HbA1c (8.51 ± 2.35; 5.72 ± 0.36, %, p <  0.001) between stroke patients with dysglycemia and healthy control participants and patients with dysglycemia compared to normoglycemia (8.51 ± 2.35; 5.18 ± 0.35, %, p <  0.009). There was no difference in the total cholesterol (4.96 ± 1.62, 5.34 ± 0.80, mmol/L, p <  0.457), triglycerides (1.32 ± 0.35, 1.64 ± 0.48, mmol/L, p <  0.118), HDL (1.73 ± 1.57, 1.44 ± 0.47, mmol/L, p <  0.527), LDL (2.92 ± 1.42, 3.16 ± 0.69, mmol/L, p <  0.593), hypertension (hypertensive/normal tension, 1/11, 0/15, p <  0.255), height (172.00 ± 4.24, 169.24 ± 9.85, cm, p <  0.708) weight (88.50 ± 13.44, 81.51 ± 13.37, kg, p <  0.500) or BMI (30.00 ± 2.83, 28.47 ± 4.44, Kg/m2, p <  0.649) between stroke patients and control subjects. There was a significant reduction in CNFD (30.47 ± 5.76; 39.50 ± 8.49; p <  0.043) but no change in CNBD (117.50 ± 25.99; 100.54 ± 37.30; p <  0.365), CNFL (29.49 ± 2.65; 27.45 ± 5.56; p <  0.447) or CNFT (19.27 ± 5.07; 15.32 ± 3.94; p <  0.091) in the stroke patients with NGT as compared to the healthy control participants. There was a significant reduction in CNFD (30.17 ± 5.68; 39.06 ± 8.36; p <  0.004) and increase in CNBD (127.71 ± 34.09; 97.80 ± 37.49; p <  0.042) and CNFT (18.57 ± 4.36; 14.93 ± 4.08; p <  0.035) but no change in CNFL (27.06 ± 4.76; 26.97 ± 5.67; p <  0.965) in stroke patients with dysglycemia as compared to the healthy control participants. There was no difference in CNFD (30.17 ± 5.68; 30.47 ± 5.76; p <  0.921), CNBD (127.71 ± 34.09; 117.50 ± 25.99; p <  0.559), CNFT (18.57 ± 4.36; 19.27 ± 5.07; p <  0.777) or CNFL (27.06 ± 4.76; 29.49 ± 2.65; p <  0.306) between stroke patients with and without dysglycemia. There was no significant difference in CNFD (30.36 ± 5.26; 30.14 ± 6.17; p <  0.941), CNBD (123.46 ± 32.58; 126.12 ± 32.37; p <  0.869), CNFT (19.26 ± 5.30; 18.22 ± 3.48; p <  0.645) or CNFL (27.05 ± 4.38; 28.60 ± 4.38; p <  0.479) in stroke patients with NIHSS <  4 compared to NIHSS ≥  4. There was no significant difference in CNFD (30.52 ± 5.27; 32.16 ± 7.73; p <  0.715), CNBD (120.00 ± 39.76; 134.96 ± 43.98; p <  0.609), CNFT (15.71 ± 2.55; 17.61 ± 4.59; p <  0.453) or CNFL (26.54 ± 5.43; 28.43 ± 4.90; p <  0.606) in stroke patients with silent infarcts <  10 vs ≥  10. There was no significant difference in CNFD (30.18 ± 5.15; 30.08 ± 6.00; p <  0.973), CNBD (127.20 ± 28.08; 121.35 ± 45.78; p <  0.766), CNFT (20.36 ± 4.53; 15.15 ± 2.56; p <  0.051) or CNFL (28.91 ± 2.95; 25.50 ± 5.67; p <  0.144) in stroke patients with white matter ischemia compared to stroke patients without white matter ischemia.

Conclusion

Corneal confocal microscopy identifies greater corneal nerve fibre abnormalities in patients admitted with stroke, which is present in patients with and without dysglycemia. It may therefore reflect the consequence of vascular risk factors independent of glycemic status. However, the extent of corneal nerve fibre pathology does not differ in relation to the severity of neurological disability or extent of pathology on MRI. Larger, longitudinal follow up studies are required to determine the prognostic ability and utility of CCM as a surrogate imaging biomarker for stratifying patients at risk of stroke.

References

1. Petropoulos IN, Alam U, Fadavi H, Asghar O, Green P, Ponirakis G, et al. Corneal nerve loss detected with corneal confocal microscopy is symmetrical and related to the severity of diabetic polyneuropathy. Diabetes care. 2013;36(11):3646–51.

2. Petropoulos IN, Manzoor T, Morgan P, Fadavi H, Asghar O, Alam U, et al. Repeatability of in vivo corneal confocal microscopy to quantify corneal nerve morphology. Cornea. 2013;32(5):e83–e9.

3. Petropoulos IN, Alam U, Fadavi H, Marshall A, Asghar O, Dabbah MA, et al. Rapid automated diagnosis of diabetic peripheral neuropathy with in vivo corneal confocal microscopy. Investigative ophthalmology & visual science. 2014;55(4):2071–8.

4. Asghar O, Petropoulos IN, Alam U, Jones W, Jeziorska M, Marshall A, et al. Corneal confocal microscopy detects neuropathy in subjects with impaired glucose tolerance. Diabetes care. 2014;37(9):2643–6.

5. Tavakoli M, Kallinikos P, Iqbal A, Herbert A, Fadavi H, Efron N, et al. Corneal confocal microscopy detects improvement in corneal nerve morphology with an improvement in risk factors for diabetic neuropathy. Diabetic medicine: a journal of the British Diabetic Association. 2011;28(10):1261–7.

6. Azmi S, Ferdousi M, Petropoulos IN, Ponirakis G, Alam U, Fadavi H, et al. Corneal Confocal Microscopy Identifies Small-Fiber Neuropathy in Subjects With Impaired Glucose Tolerance Who Develop Type 2 Diabetes. Diabetes care. 2015;38(8):1502–8.

7. Tavakoli M, Ferdousi M, Petropoulos IN, Morris J, Pritchard N, Zhivov A, et al. Normative values for corneal nerve morphology assessed using corneal confocal microscopy: A multinational normative data set. Diabetes care. 2015;38(5):838–43.

8. Dabbah M, Graham J, Petropoulos I, Tavakoli M, Malik R. Automatic analysis of diabetic peripheral neuropathy using multi-scale quantitative morphology of nerve fibres in corneal confocal microscopy imaging. Medical image analysis. 2011;15(5):738–47.

Background

Road Traffic Injuries (RTIs) are the leading cause of death in Qatar1, but the epidemiology of these injuries in the infant (0–1 years) and toddler (2–4 years) [IAT] population has not been reported. This study aimed to document and analyze the epidemiology of RTIs in IATs of Qatar and make recommendations for targeted and age-specific recommendations to improve road safety for this population.

Methods

A retrospective analysis of data on child RTIs and RTI deaths admitted to the Hamad Medical Corporation [HMC] Trauma Center or Mortuary in Doha, Qatar, from 2008–2014, was conducted. Temporal trends in the nature of RTI's and RTI deaths, road user types and mortality rates were calculated and analyzed for the years that age-group population size was available.

Results

There were 189 severe RTIs and 15 RTI deaths during the study period. Males made up 80% of the injured and 60% of fatalities. The average age of the injured was 3 years and for fatalities was 2.8 years. Pedestrians [53%] and unrestrained passengers [43%] made up the majority of the injured. There have been steady declines in severe RTI and RTI death rates from 2008 to 2012 [25.9 to 22.2 RTIs per 100,000 and 0.9 to 4.0 RTI deaths per 100,000], but these rates are still two times higher than those for IAT in other high-income countries [HICs] like the United States and Germany2.

Conclusions

RTIs and RTI death rates in IAT in Qatar have been declining but proven programs for improved safety of child pedestrians and passengers must be implemented if it is to approximate those in other HICs. This includes programs around child restraint use and improving pedestrian environments and practices/supervision for IAT.

Background

Patient counseling is a term used in the field of pharmacy to describe the communication that takes place between a pharmacist and a patient in regards to his or her medication therapy. Patient counseling is deemed to be the professional responsibility of pharmacists. Patients require education on the proper use of medications to improve therapeutic outcomes and avoid treatment failure. 1 Pharmacists are in an excellent position to provide this type of education and counseling to patients, and are readily accessible to the public as a first point of contact for patients with medical inquiries. 2 Several studies have shown that counseling provided by pharmacists can prevent medication related problems, improve patient compliance with medications, and contribute to positive therapeutic outcomes. 3, 4 Patient counseling practices among Arab countries are not clearly identified in literature or mandated by governing authorities or agencies. The reason may be the low expectation by the public of the pharmacist who for so long had confined their role to filling and selling medications. Many studies have identified various aspects such as the busyness of the pharmacies and the education, age, and attitude of the pharmacists as barriers for the provision of counseling. 4 The assessment of these barriers in addition to patient characteristics will assist in further evaluating and understanding pharmacist's counseling practices in community pharmacies. Since patient counseling is an essential element of patient care, investigating the current state of patient counseling practices in Qatar is necessary. One of the methods that help to gain an accurate assessment of pharmacist's counseling practices is to observe pharmacists in their natural environment without prior knowledge to the minimize the Hawthorne effect. 5 Therefore, to lessen the potential Hawthorne effect (change in someone's behavior due to the knowledge that s/he is being observed), this research project will utilize simulated patients (also known as mystery shopper) to examine the counseling practices of community pharmacists in Qatar. The objectives of this research project are to evaluate the quality of patient counseling practices by community pharmacists in Qatar and to determine if an association exists between the content of patient counseling and the characteristics of community pharmacists, patients, and community pharmacies.

Methods

This is an observational cross sectional study to evaluate the community pharmacist's counseling practices in Qatar using simulated patients. Two patient scenarios were created to assess the counseling provided by the pharmacist to the patients with diabetes and asthma. Two simulated patients of Arab decent fluent in Arabic and two simulated patients of non-Arab decent fluent in English were recruited for this study. Each simulated patient was randomly assigned to either the diabetes or asthma scenario. Each simulated patient entered the community pharmacy and observed the number of customers to assess for busyness of the pharmacy. The simulated patient then asked to speak to the pharmacist and requested their assigned medication. Prompting questions were initiated by the patient after each pharmacist was provided with the opportunity to counseling the patient. The simulated patient then purchased the medication to authenticate the interaction and left the pharmacy. Immediately following the interaction, the simulated patient completed a form that assessed the pharmacist's counseling ability. Once completed, the simulated patient returned to the same the pharmacist to explain the purpose of the study. Consent was obtained from the pharmacist and a survey written in English or Arabic was administered only to those who provided consent. The written survey included questions related to pharmacist demographics (gender, age, native language, pharmacy degree received, country of pharmacy education, graduation year, and practice experience), pharmacist's perception on patient counseling (barriers and importance), and pharmacy characteristics (number of staff, availability of a counseling area, and busyness of the pharmacy).

Results

One hundred and twenty-nine pharmacists consented to participate in this study. The majority of pharmacists were young male pharmacists with at least 2 years of practice experience in Qatar. Most pharmacists have received their pharmacy degree from India and Egypt. Most of the community pharmacies had one pharmacist per shift and no private patient counseling area. In the diabetes scenario (n = 64), the male simulated patient received significantly better counseling from the pharmacist when compared to the female simulated patient. The pharmacist's knowledge regarding diabetes was considered poor with over 60% of pharmacists referring the patient to the physician. For the asthma scenario (n = 65), the male simulated patient received significantly better counseling from the pharmacist compared to the female simulated patients. Only 6% of the pharmacists were able to properly demonstrate the correct inhaler technique to patients. However, more than 50% of the pharmacists were are able educate the simulated patient on the role of the asthma medications. Overall, the pharmacist's gender, age, native language, pharmacy degree, country of pharmacy education, and years of practice experience did not seem to have an effect on the quality of counseling provided to patients. Better counseling was provided by the community pharmacists to the simulated patients when the pharmacy was not busy. Not having a private counseling area in the pharmacy was a significant barrier leading to lower counseling practices among community pharmacists. Some other barriers related to patient counseling identified by pharmacist include time, no access to patient medication records, and patients not interested in the counseling provided by the pharmacist.

Conclusion

The findings from this study suggest the current counseling practices among community pharmacists in Qatar is substandard. Development of continuing education programs for practicing pharmacists to enhance their communication skills and knowledge to improve counseling practices is strongly advocated. These educational programs will need to be tailored to meet the needs of the pharmacists to address the fundamental communication skills that each pharmacist should possess for practice. In addition, pharmacy laws and regulations need to be updated to meet the changes in the pharmacy profession from dispensing to a patient centered care focus. Community pharmacies need to be equipped with a counseling area, mandatory drug information resources, and the ability to maintain medication profiles for each patient. Pharmacy regulations should also consider mandating all pharmacists to provide counseling for each patient.

References

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Background

Peripheral intravenous catheters (PIVs) are invasive catheters that may endure risks of clinical complications affecting health care outcomes and patient satisfaction. Patients requiring frequent PIV insertions due to an extended length of stay of one week or more would undergo multiple PIV pricks during their course of hospitalization. The combined factors of multiple PIV insertions and extended hospital stay make chronic patients especially at a higher risk for phlebitis and subsequently infection No studies in the literature review discussed the phlebitis rates and risks when toxic medications were infused using a peripheral catheter. The aim of this study was to determine the pattern and incidence of PIV complications among patients admitted to a tertiary care military health care facility in central region of Kingdom of Saudi Arabia. This was achieved through the following objectives: (1) Estimation of the cumulative incidence and incidence density for major PIV complications (catheter occlusion, dislodgment, infiltration, and phlebitis) during a period of 1 month, (2) Identification of significant risk factors for PIV complications such as patient related characteristics (gender, age, co-morbidities) or catheter related characteristics (catheter size, dressing, site of insertion, type of infusion, dwell times), and (3) Determination of the time line for the occurrence of various PIV complications.

Methods

An observational prospective cohort study investigated PIV pattern and complications among 359 adults with 842 PIV catheters, admitted to various wards at KAMC with a total of 2505 catheter days. Data on patient characteristics (age, gender, and health complaints) and PIV characteristics (Catheter size, duration, insertion site, nature of PIV infusate, and type of dressing) was collected. PIV catheters-related clinical outcomes (Pain, Phlebitis, leaking and others) were recorded on 12–hour intervals, using the Visual Inspection Phlebitis (VIP) scale. Phlebitis was defined as the presence of two or more signs of pain, tenderness, warmth, erythema, swelling, or a palpable cord, with or without purulent drainage from the catheter insertion site. Infusion Phlebitis scale can range from (0), indicating no symptoms of phlebitis, to (5), with signs of purulent drainage, redness, and a palpable cord greater than 3 inches. Infiltration was defined as permeation of intravenous fluid into the interstitial compartment, causing swelling of the tissue around the site of the catheter. All PIV catheters were changed for a score of 2 or more, determined by the presence of a cold/warmth skin region around the insertion site, pain, redness, and/or edema extending from 1 to 2 inches above the PIV site. Incidence density (DI) and cumulative incidence (CI) of complications were calculated. Regression analyses were applied to determine the significant predictors of complications. Significance limits were set at P < 0.05.

Results

One half of all patients were 65 years and above, with no significant sex difference. The majority of patients (76.88%) had medical chief admission complaints, whereas (51.53%) were admitted in cardiac wards. Complicated catheters were found in 141(39.3%) patients, with 273 complications (32.4/100 catheters), 190 complicated catheters (CI =  22.56/100 catheters & DI =  75.84/1000 catheter days). Phlebitis ranked first among complications, with a CI of (17.58%), followed by pain (7.60%), leaking (3.92%) and dislodgement (2.38%), and extravasations and occlusion (0.48% each). When the analysis was based on failure per 1000 device days, and after adjusting for all possible confounders, female gender remained as a significant predictor of higher incidence of overall complications (p = 0.00001), phlebitis (p = 0.000003) as well as other complications (p = 0.0.0003). Insertion in fore/upper arm was a significant predictor of both phlebitis (0.024) and other complications (0.049), while medical infusion was a significant predictor of phlebitis (0.02) and overall complications (0.006).

Conclusion

Incidence of complications in this study is comparable with figures in previous studies, however, better insertion techniques may be sought to lower the incidences of PIV complications, thus extending their onset beyond day 3. Changing catheters is recommended when clinically indicated rather than routinely post 72 hours of insertion which in return minimizes the frequency of insertions per patient and subsequently complications.

Network querying algorithms provide computational means to identify conserved network modules in large-scale biological networks that are similar to known functional modules, such as pathways or molecular complexes. Two main challenges for network querying algorithms are the high computational complexity of detecting potential isomorphisms between graphs and ensuring the biological significance of the query results. In this work, we propose a novel network querying algorithm that can enhance the biological significance of the query results through the use of a context-sensitive random walk (CSRW) model. In order to identify conserved subnetwork regions in the target network that are similar to a given query network, the algorithm estimates the node correspondence between the query and the target networks based on the CSRW model. Inspired by the pair hidden Markov model (pair-HMM) that has been widely used in comparative sequence analysis, the CSRW model can effectively capture the similarities between graphs by explicitly accounting for potentially inserted and deleted network nodes. Based on the estimated CSRW node correspondence scores, our algorithm first identifies high-scoring regions (referred to as the seed networks) in the target network that bear considerable similarity with (part of) the query. The seed networks are further extended by maximally minimizing the network conductance, which can effectively identify nearby nodes (e.g., proteins) that may share similar functions with other nodes in the current seed. Finally, the query result is pruned by removing irrelevant nodes based on an extension reward score, thereby improving the consistency of the final query result. Through extensive numerical simulations based on 938 real biological complexes, we show that our proposed algorithm yields accurate query results with enhanced biological significance.

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Introduction

The frequent outbreaks of novel or emerging infectious diseases are alarming. There is a need for urgent and appropriate intervention that can be adapted quickly for such diseases. Middle East respiratory syndrome coronavirus (MERS-CoV) previously known as novel CoV is a viral infection that causes severe acute respiratory illness. MERS-CoV is a respiratory pathogen and contagious that is contracted via close contact with infected subject. It appears to have been transmitted from camels to humans, recent studies have revealed an association among the virus found in humans and that found in camels (Drosten, et al., 2014). The epidemic that started in 2012 in the Kingdom of Saudi Arabia has spread to many countries, mostly in the Middle East and North Africa, and more recently South Korea. The new epidemic started when the virus crossed from an infected individual who had recently traveled to the Middle East and subsequently began spreading between people via direct contact while in some cases the infection crossed from animal to human. This has exposed the general populace and especially the healthcare workers to greater risk. Similar to the reaction during other infection outbreaks, such as SARS and Ebola virus, many countries have issued travel restrictions and advisories to the affected countries because of fear of international spread of the disease. The rate at which the outbreak is evolving cannot be overemphasized and the geographical extension of its spread is widening. Neighborhoods constitute a key determinant of socioeconomic disparities and health habits, and therefore there is a high risk that people at geographical proximity to the infection are particularly vulnerable. Additionally, people with preexisting chronic medical conditions are more prone to being infected by the illness or developing a severe case resulting in fatality (Assiri, et al., 2013). Strong links between healthcare facilities and the outbreak of the disease has been found in Jeddah, where the majority of patients were in contact with other patients or health care facilities (Oboho, et al., 2015). Elsewhere, the transmission of MERS-CoV in household contacts revealed that an outcome of approximately 5% as the rate of secondary transmission occurred at home (Assiri, et al., 2013). In spite of the many research conducted on MERS-CoV, very few have investigated the effect of ecological factors on the transmission of the disease. Moreover, very little is known on the possibility of airborne transmission of the virus and discussion is still on going on this issue. Airborne transmission of the pathogen may also occur through dust and movement of people, equipment and animals within infected countries which may contribute to the spread of the virus. Additionally, the effect of alternating temperature highs and lows may imply seasonality that could alter the transmission and survivability of the virus. Objectives During infectious disease outbreaks, transmission of disease may form networks of infected individuals because of the way virus crossed from infected individual to susceptible individual. See figure 1a for a subset of the infection network for MERS disease used in this study. The infected individuals may form network of individuals connected by source of infection (contact) as direct or indirection connection. The study intends to (1) explore and investigate the relationship between the intensity of the disease in a given region and environmental variables, (2) We sought to evaluate the risk factors of and trends in mortality as a result of MERS-CoV infection in a large cohort of patients between June 6, 2012 and May 19, 2015. Use covariate adjustment to correctly assess the disease-risk factor associated with the survival outcome, (3) It also aims to understand the dynamic pattern of the disease over different temporal and spatial scales. A functional spatial time series method of estimating the death rate in the outbreaks will be formulated. Spatial time series analysis of disease outbreaks are versatile tools for studying and understanding transmission and spread of a disease and may be useful in the different frontiers of its upsurge and in the possibility of its containment or eradication. And lastly, (3) a flexible dependence model that reflect the relationship among infected individuals in the same network.

Figure 1: (Top) Network for subset of the MERS data. The numbers represent the identification number of the patient while the arrow represents the infection direction. (Bottom) Showing the patients that were infected by patient 27.

Figure 2: Number of patients infected by each main contact (patient ID). Preliminary Exploratory Analysis This study is based on a retrospective analysis of the Middle East respiratory syndrome coronavirus (MERS-CoV) outbreak in the Arabian Peninsula between June 6, 2012 and May 19, 2015.

Table 1 presents the summary of data used in this study. Of the 958 cases recorded during the study period more than 90% (866) of the disease incidence occurred in the Kingdom of Saudi Arabia while Kuwait has the least incidence of 3. Seventeen percent of those infected with MERS disease were heath care worker. The fatality for the disease is about 0.35 among the total population. The mean age of the infected persons was 50.22 ± 18.37 years, with large percentage (55%) reported that they had some kind of comorbidity. Spatial analyses of disease outbreaks are versatile tools for studying and understanding transmission and spread of a disease.

Figure 3 shows the spread of MERS disease across the Arabian Peninsula within the study period. Majority of the disease incidence occurred in KSA and closed countries.

Table 1. Summary of the MERS data Country No. of Patients No. of Male/female No. with comorbidity Age at incidence % of HCW % fatal Mean/median mean SD KSA 866 565/284 469 49.66/50 49.66 ± 18.54 153 302 UAE 70 50/18 49 54/56 54 ± 15.33 9 30 Qatar 12 9/3 5 59.1/58 59.1 ± 15.99 1 3 Kuwait 3 1/2 3 54/43 54 ± 27.22 0 1 Oman 7 7/0 2 56.28/60 56.28 ± 17.54 1 3 Total 958 632/307 528 50.22/50.5 50.22 ± 18.37 164 339

Hearing loss is a common sensory deficit that can affect all levels of society. The two age groups where hearing loss is most likely to be identified is in newborns and in the elderly. Newborn screens have been very effective in finding individuals affected by hearing loss. The general hearing loss incidence in newborns is 0.2%, with roughly 50% of all cases being hereditary. A recent study in Qatar found that the incidence of hearing loss in newborns was as high as 5.2% 1, 25 times higher than the general global incidence. Much of this increase can be attributed to hereditary forms of hearing loss, as the rate of consanguinity in Qatar is high 1,2. The elderly are the other population group where hearing loss is often identified. Globally, the hearing loss incidence for people over 50 is around 40%, with men showing a higher incidence than women 3,4. In Qatar, 66% of the Qatari population over the age of 50 suffers hearing loss, compared to ∼35% for the non-Qatari populations 5. The gender distribution of hearing loss within the Qatari population is also different from the global trend, with the incidence being 75% for women and 59% percent for men5. Therefore, the incidence of hearing loss is dramatically higher in the Qatari population than it is globally, and presents an important health issue that should be addressed. We have previously shown that the outer nuclear membrane protein Nesprin 4 is essential for hearing in humans and in mice. Nesprin 4 is expressed in the sensory outer hair cells (OHCs) of the cochlea and is important for the proper nuclear positioning and survival of these cells. In the absence of functional Nesprin 4, the nuclei are apically mispositioned and the OHCs die. OHCs act as cochlear amplifiers, augmenting sound signals by up to 100-fold6. This amplification is achieved, at least in part, through electromotility, a somatic cell motility that causes cell length changes in response to depolarization or hyperpolarization of the cell. Electromotility is mediated by SLC26A5/prestin, a membrane protein enriched in the lateral membranes of OHCs7. Prestin-mediated electromotility develops postnatally in mice, with full activity occurring at 12–14 days after birth. We observed that the majority of OHCs in mice lacking Nesprin 4 die around this time and predicted that the mislocalized nucleus was incompatible with electromotility. To test this hypothesis we generated double-null mice for Nesprin 4 and prestin and examined the fate of OHCs in these animals. We found that loss of prestin rescues the loss of OHCs in Nesprin 4-null animals, indicating that while a mispositioned nucleus is incompatible with electromotility, it does not affect cell viability independent of mechanical stress. Others have previously shown that animals heterozygous for the prestin allele exhibit a reduced electromotility. We predicted that a reduction in electromotility should yield a partial rescue of OHCs in Nesprin 4-null animals. When we examine the Nesprin 4-null/prestin heterozygous animals, we did indeed observe a partial rescue of OHCs. Together, these results indicate that proper nuclear positioning mediated by Nesprin 4 in OHCs is essential for these cells to survive the mechanical stress of electromotility.

References

1. Bener, A., EIHakeem, A. A. M. & Abdulhadi, K. Is there any association between consanguinity and hearing loss. Int J Pediatr Otorhinolaryngol 69, 327–333 (2005).

2. Girotto, G. et al. Consanguinity and Hereditary Hearing Loss in Qatar. Hum Hered 77, 175–182 (2014).

3. Roberts, A. Sensorineural Hearing Loss. Synopsis of Causation, Ministry of Defence 1–30 (2008).

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5. Bener, A., Salahaldin, A. H., Darwish, S. M., Al-Hamaq, A. & Gansan, L. Association between hearing loss and type 2 diabetes mellitus in elderly people in a newly developed society. Biomed Res 19, 187–195 (2008).

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