of PM10 with cerebrovascular disease and ischemic stroke were not statistically significant. Using different lag periods, we found at lag 0, 1 and 0 to 1 days, the concentrations of PM2.5 were significantly associated with cerebrovascular diseases and ischemic stroke (**Figure 1**). A significant association of PM10 with cerebrovascular diseases and ischemic stroke was detected at lag 0 and 0 to 1 days (**Figure 2**). Results were essentially unchanged when the df for time trend was changed from 6 to 10 (data not shown).

The stratify analyzes by gender and age groups showed that female and the elder people were more likely to be vulnerable to PM2.5 and PM10. However, this did not hold at different lag days (**Table 3**).

#### DISCUSSION

To our knowledge, this is contemporary- study to analyze the association between PM2.5 and PM10 and the mortality by stroke types in the area with a high concentration of PM. Our present study suggested that short-term exposures to ambient PM2.5 and PM10 were associated with increased mortality of cerebrovascular diseases. When stratified by stroke types, we did not find evidence of an association between PM10 exposure and hemorrhagic stroke mortality.

We observed 0.27% (95% CI, 0.12–0.43%) and 0.19% (95% CI, 0.07–0.32%) increase in cerebrovascular diseases mortality with a 10 µg/m3 increase in PM2.5 and PM10, respectively. Due to large population exposure to ambient PM, it was of great benefits, though the effect was small. If the annual mass concentration of PM2.5 achieved the World Health Organization (WHO) Air Quality Guidelines (10 µg/m3 ) (22), about 400 stroke deaths will be avoided yearly in Beijing. The magnitude of the associations was lower than the results from a metaanalysis (1.1%, 95%CI 1.1–1.2% for each 10 µg/m3 increase of PM2.5; 0.3%, 95%CI 0.2 to 0.4% for each 10 µg/m3 increase of PM10) (23). The main reasons for this disparity may be that admission or mortality was to be recorded as an endpoint and most of the studies contained in this meta-analysis were

TABLE 3 | Percentage increase (mean and 95% confidence intervals) of stroke mortality associated with 10 µg/m3 increase of PM2.5 and PM10 using different lag structures by gender and age.

*PM*2.5 *indicates particulate matter* <*2.5*µ*m in aerodynamic diameter, PM*10 *indicates particulate matter* <*10*µ*m in aerodynamic diameter.*

from high-income countries. Additionally, the specific risks were volatile ranging from −2.9 to 31.4%. However, the the impact of PM on mortality in the present study was similar to that at another city in China (0.44%, 95%CI 0.16– 0.72%) (24). This may occur due to the characteristic of air pollution, weather patterns and the economy. Hence, it is reasonable to summarize the effect according to geographical location.

Though ischemic stroke and hemorrhagic stroke share similar risk factors, they are different clinical entities (23, 25). In order to examine whether the effect of PM on different types of stroke was the same, ischemic stroke and hemorrhagic stroke was evaluated respectively. Our data showed that shortterm elevations in PM2.5 increased the risk of death in both ischemic stroke and hemorrhagic stroke. Nevertheless, we found a significant association between ischemic stroke death and PM10 exposure, but not in hemorrhagic stroke. The studies of PM and hemorrhagic stroke were limited, and the outcomes were inconsistent (15, 26, 27). The mechanisms of hemorrhagic stroke and air pollution might be different from that of ischemic stroke and air pollution (23). Several mechanisms had been proposed, including exposure to particulate air pollution may induce inflammation (28), endothelial injury (29), atherosclerosis (30, 31), and can lower cerebral blood flow velocity (32).

There was not well documented referring to whether gender and age were effect modifiers. In the subgroup analysis, we did not find evidence that gender or age can modify the effect of PM on stroke. However, in some previous studies, it was suggested that females and the elderly were more likely to be vulnerable to air pollution (7, 33, 34). For instance, Kan et al. found that the effect of PM10 on total mortality among females was about twice those among males, though the difference was insignificant. Some stated that the gender difference could be partly explained by differences in particulate deposition, airway size (35), inflammatory response (36). Hong et al. found the elderly were more susceptible to PM10 (33). In the elderly, atherosclerosis is the main reason for ischemic stroke (37). Through pro-oxidant and pro-inflammatory effects, particulate matter modulates the progression of atherosclerosis, as a result of increasing the risk of ischemic stroke (38). However, the exact mechanisms of gender and age difference are unclear and deserve further investigation.

Several potential limitations of our study should be considered. First, as an ecological study, population level exposure was used, which may not reflect actual individual level exposure. The measurement error contains three components: the difference between an individual's deviation and the average personal exposure; the difference between the average personal exposure and the true ambient level; and the difference between the measured and the true ambient level. The first and third components are of the Berkson type and they are likely to have a small effect on the risk estimation (39). The effect of the second component may cause substantial bias, however, it tends to bias the results toward to null and underestimates the air pollutant effect (32, 40). Hence, the results should be interpreted with cautions and we cannot make causal inference form this study. Second, misclassification of cause of death may exist due to diagnostic or coding errors. However, the misclassifications seem to be unrelated with the air pollution levels and the errors may reduce the accuracy of the risk estimation (7). Third, because of data limitations, smoking-, drinking-, education- and complication-specific stroke mortality cannot be accessed, which prevented us from further exploring potential modifiers of association between air pollutants and ischemic stroke and hemorrhagic stroke.

In conclusion, PM exposure was associated with cerebrovascular death in Beijing, China. PM2.5 was associated with both ischemic stroke and hemorrhagic stroke death. However, short-term exposure to PM10 increased the risk of death in ischemic stroke but not in hemorrhagic stroke. Our study adds the evidence of the effect of PM on stroke in low-income countries, and it may have implications for public and environmental healthy policies.

# AUTHOR CONTRIBUTIONS

GL and YOW: study design; GL, JW, and ZW: data acquisition; YJ, YP, and RZ: data analysis and interpretation; RZ: drafting; GL, YIW, and YOW: revising and final approval.

#### REFERENCES

### FUNDING

This work was partially funded by Beijing Municipal Commission of Health and Family Planning (2016-1-2041), Beijing Municipal Administration of Hospitals' Sail Plan (XMLX201712), National Key Research and Development Program of the Ministry of Science and Technology of The People's Republic of China (2016YFC0901001, 2016YFC0901002, 2017YFC1307702).

pollution from nitrogen dioxide: a cohort study. Stroke (2012) 43:320–5. doi: 10.1161/strokeaha.111.629246

**Conflict of Interest Statement:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Copyright © 2018 Zhang, Liu, Jiang, Li, Pan, Wang, Wei, Wang and Wang. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

, Calogero Edoardo Cicero1

, Maria Del Carmen Martinez 2

,

,

# Treating People With Epilepsy in Rural Low-Income Countries Is Feasible. Observations and Reflections From a "Real Life Experience" After a Long Lasting Intervention in the Rural Chaco

, Loretta Giuliano1†, Chiara Colli 2

, David Rojo Mayaregua3

Edited by: Ding Ding, Fudan University, China

#### Reviewed by:

Alessandra Nicoletti 1

Sandra Padilla3

\* †

, Estela Vilte3

Ettore Beghi, Istituto Di Ricerche Farmacologiche Mario Negri, Italy Shichuo N. A. LI, Beijing Neurosurgical Institute, Beijing Tiantan Hospital, Capital Medical University, China

#### \*Correspondence:

Alessandra Nicoletti<EMAIL_ADDRESS>
†These authors have contributed equally to this work

#### Specialty section:

This article was submitted to Neuroepidemiology, a section of the journal Frontiers in Neurology

Received: 27 June 2018 Accepted: 21 September 2018 Published: 10 October 2018

#### Citation:

Nicoletti A, Giuliano L, Colli C, Cicero CE, Padilla S, Vilte E, Rojo Mayaregua D, Martinez MDC, Camargo M, Zappia M, Bartoloni A and Crespo Gómez EB (2018) Treating People With Epilepsy in Rural Low-Income Countries Is Feasible. Observations and Reflections From a "Real Life Experience" After a Long Lasting Intervention in the Rural Chaco. Front. Neurol. 9:855. doi: 10.3389/fneur.2018.00855

Mario Camargo4 , Mario Zappia1 , Alessandro Bartoloni 2 and Elizabeth Blanca Crespo Gómez 5 1 Department of Medical and Surgical Sciences and Advanced Technologies "G. F. Ingrassia," Section of Neurosciences,

University of Catania, Catania, Italy, 2 Department of Experimental and Clinical Medicine, Infectious Diseases Unit, University of Florence, Florence, Italy, 3 Center of Anthropological Researches of the Teko Guaraní, Gutierrez, Bolivia, 4 Bolivian League Against Epilepsy, Santa Cruz, Bolivia, 5 Hospital Universitario Hernández Vera, Santa Cruz, Bolivia

Introduction: Epilepsy represents an important public health issue, in particular in low and middle-income countries where significant disparities are present in the care available for patients with epilepsy. Treatment cost and unavailability of drugs represent important barriers in treating people with epilepsy especially in rural setting. Aim of the study was to evaluate, by means of routine data, the current real-life clinical practice in epilepsy in the rural communities of the Plurinational State of Bolivia. Treatment activity followed educational campaigns and an anthropological fieldwork over more than 20 years.

Material and Methods: Medical records of people with epilepsy (PWE) living in the rural communities of the Bolivian Chaco who received antiepileptic drugs (AEDs), from 2012 to 2016, and were followed-up for at least 1 year were analyzed. Treatment delivery and follow up visits were managed by a neurologist with the support of rural health care workers.

Results: From 2012 to 2016, 157 PWE (76 men with a mean age of 24.2 ± 15.7) have been included in the study. Structural epilepsy was the most common type, recorded in 54 cases (34.4%) and the most common reported causes were perinatal factors, present in 11 subjects (20.0%). Almost all patients presented epilepsy with generalized tonic-clonic seizures (91.4%). The most common AED prescribed was phenobarbital followed by carbamazepine. During the follow-up, a dramatic seizures reduction was observed, with 31 subjects (19.7%) being seizures-free at the last follow-up. However, 48 subjects (30.6%) did not assume the medication regularly and 10 interrupted the drug intake. More than 20% of PWE did not receive any financial supports for AEDs. During the follow-up period 10 patients died but only in one case the death was probably caused by epilepsy.

Conclusion: Our study demonstrated that PWE in rural areas of the Bolivian Chaco are willing to seek medical attention and to receive antiepileptic treatment. However, improvement in care is needed to assure compliance to AED treatment, including activity to increase awareness toward epilepsy among community members and health staff of the rural communities and to guarantee the coverage of treatment costs and drug supply.

Keywords: epilepsy, treatment, low-income countries, management, real-life

#### INTRODUCTION

Throughout the world, epilepsy represents an important public health issue, accounting for an estimated 0.7% of the global burden of diseases. It affects approximately 70 million people worldwide of whom the majority live in low and middleincome countries (LMIC) (1). In particular, about 5 million people living in Latin American Countries (LAC) are affected by epilepsy (2). In the Chaco region, which is part of the Plurinational State of Bolivia, it was found a prevalence of lifetime epilepsy of 12.3/1,000 and a prevalence of active epilepsy of 11.1/1,000. In a recent study the life-time prevalence of epilepsy associated with generalized tonic-clonic seizures (GTCS) was found to be 7.2/1,000 and the prevalence of its active form 6.6/1,000 with a crude incidence risk of 55.4/100,000 in the same area (3–5). According to a recent meta-analysis in Latin American countries the treatment gap (TG) is 60.6% (95% CI 45.3–74.9), with high differences between rural (77.8%; 95% CI 67.4–86.8) and urban (26.2%; 95% CI 10.2–46.4) areas (5). In LMIC there are significant disparities in the care available for patients with epilepsy (PWE). Most neurologists work in the urban private sector, where the level of care is similar to that found in developed countries. However, in the poorer urban and rural areas this level of care is rarely available (6). In this latter setting, indeed, general practitioners (GPs) have an important role in providing care and support to PWE. However, in rural areas non-medical health workers such as nurses and community health workers (CHWs) are often the only health care staff present who can diagnose epilepsy with GTCS. As pointed out by recent WHO recommendations, in LMIC settings epilepsy with GTCS, should be recognized and treated at primary care level by trained non-specialist health care providers (7).

Thus, education of primary health workers and other clinicians working in the rural areas represents a key action in the reduction of the treatment gap. Nonetheless treatment cost and unavailability of drugs represent a further important barrier in treating PWE in rural setting (6). Consequently, specific programs for the improvement of knowledge and awareness about epilepsy in these settings are not sufficient if not accompanied by governmental actions aimed to support the treatment cost and drug supply. The reduction of epilepsy TG in the area of Chaco has been the main aim of many different projects performed by our group over the last 20 years in this area (3, 4, 8–12). However, even if a reduction of TG was recorded over the time by the subsequent studies carried-out (3, 4, 13), the real impact of our activity in the clinical practice of PWE has never been evaluated until now.

Aim of the present study was to evaluate, by means of routine data coming from the rural communities of the Bolivian Chaco, the current real-life clinical practice in epilepsy in the rural communities of the Plurinational State of Bolivia.

#### MATERIALS AND METHODS

#### Study Area

The study has been conducted in the Chaco area of the Plurinational State of Bolivia, which is a low-middle income country where about 4 million people live under the "poverty line" (14). In particular in rural areas the access to the health system is still difficult (15). However, a slight economic improvement has been recorded in the last few years, since the approval of the 2016-2020 National Economic and Social Development Plan by the Government of Bolivia aimed at maintaining growth of 5% and reducing poverty (16, 17). Indeed, with the application of policies to improve the economy, the social and health services and thanks to specific programs such as Bono Juana Azurduy, Programa Mi Salud, Ley de Gratuidad, Seguros departamentales, there was an increase in the social security and an improvement of the Bolivian health system (18).

PWE involved in this study come from three Departments of the Plurinatinal State of Bolivia: the Department of Santa Cruz (municipalities of Boyuibe, Cabezas, Cuevo, Lagunillas, Gutierrez, Eiti, Camiri and S. Antonio Parapetí), the Department of Chuquisaca (municipalities of Macharetí and Muyupampa or Villa Vaca Guzmán) and the Department of Tarija (municipality of Villamontes). These Departments are part of the Chaco region (**Figure 1**), that is a subtropical area, inhabited mainly by indigenous Guaraní people. They live in communities that often lack basic services such as running water or electricity, basing their economy on animal husbandry and agriculture.

#### Study Population

About 190,000 people live in the Bolivian Chaco and among them about 80,000 are indigenous Guarani. It is estimated that 49% of the population live in rural areas with 68.9% of the population living in conditions of poverty, the majority of them still presenting unresolved issues such as energy and sanitation supplies1 . In the last 25 years, many

1 OPS/OMS Paraguay - Rostros, Voces y Lugares: El Chaco Sudamericano. Available online at: https://www.paho.org/par/index.php?option=com\_docman& view=document&layout=default&alias=139-rostros-voces-y-lugares-el-chacosudamericano&category\_slug=salud-familiar-y-comunitaria&Itemid=253 (Accessed September 4, 2018).

activities aimed at improving knowledge about epilepsy and reducing TG have been conducted by our group in the rural communities of the Chaco region. (8–14). After the assessment of the prevalence, incidence, mortality and the most frequent causes of epilepsy, the evaluation of the sociocultural dimension of epilepsy and epilepsy-associated stigma among members of the Guaraní communities, many training courses about epilepsy have been organized directed to the health staff and all the members of the communities (3, 4, 8–13, 19). In particular an educational campaign directed to GPs and non-medical health staff has recently been implemented with teaching courses regarding the main causes of epilepsy, epilepsy diagnosis and treatments, first aid, prevention of the secondary forms of epilepsy and psychosocial aspects such as social stigma and discrimination (10, 19).

From 2012 to 2016 a Bolivian neurologist, working in Santa Cruz, participated to the teaching activity, organized in collaboration with the "Convenio de Salud" and the "Escuela Tekove Katu." The "Convenio de Salud," located in Camiri, works through cooperation projects with the University of Florence in Italy and the Pan American Health Organization. Its objective is to improve the health conditions of the indigenous Guaraní population through the development of health projects in primary care. The "Tekove Katu" school, located in Gutierrez, carries out training courses since 1985 and is an integral part of the educational and health system of the Plurinational State of Bolivia2 . The neurologist guaranteed a bimonthly service of neurological visits to PWE living in the rural communities of the Chaco region.

Subjects identified by trained nurses or CHWs, as well as PWE already diagnosed by the GPs working in the rural areas or identified during the different epidemiological surveys, were attended in the main health centers located in the different areas.

When the diagnosis of epilepsy was confirmed, AEDs were prescribed and for PWE already in AED treatment, prescription was confirmed according to the neurologist's judgement.

This clinical activity was not part of a specific scientific programme and clinical data were routinely collected without the aim to be analyzed. Consequently, due to the lack of a standardized data collection, often incomplete information was gathered with a certain amount of missing data.

All medical records of all PWE coming from rural communities who received AEDs treatment and were followedup for at least 1 year were analyzed by two independent observers (LG, CEC). Those patients for whom not sufficient information was available or who underwent only one neurological visit without follow-up have been excluded (**Figure 2**).

Diagnosis of epilepsy was generally performed just on the bases of the clinical history because the majority of cases never underwent EEG recording or neuroimages. On the bases of

2Home - Scuola di Salute Pubblica Tekove Katu. Available online at: http://www. tekovekatu.org/ (Accessed January 30, 2018).

the clinical history, epilepsy was defined according to the ILAE definition and classification proposed in 2017 (20). In particular, according to the causes, epilepsy was classified as "structural" when an imaging abnormality was the likely cause of the patient's seizures; "genetic generalized" when an idiopathic form with a genetic predisposition was suspected on the bases of the clinical history; "of unknown etiology" when the cause of the epilepsy was not yet known; "unknown" when there was not adequate information to classify epilepsy type into a category (20).

Among the causes of structural epilepsy, perinatal factors were defined when there was an evidence of a hypoxic-ischemic encephalopathy (21); traumatic brain injury was defined as an insult to the brain from an external mechanical force, with an associated diminished or altered state of consciousness (22); neurocysticercosis (NCC) was defined according the most recent criteria (23); for the other possible causes we based our diagnosis on the previous clinical report.

#### Data Management and Statistical Analysis

Medical records were revised twice by two different operators (CC, LG) and relevant data were collected in an ad hoc created standardized form. Data were double entered into the database and statistical analysis was performed using the software STATA 12 (version 12.0, College Station, TX). Data cleaning was performed before the data analysis considering range and consistency checks. Quantitative variables were described as mean and standard deviation (SD) while frequencies using percentages. The frequency comparisons were performed using the chi-squared test.

# Ethical Approval

The study has been approved by the Ethical committee of the Bolivian Neurological Society.

# RESULTS

From 2012 to 2016, 215 PWE from the rural communities of the Gran Chaco area underwent a neurological examination and received at least an AED prescription. Of these, 18 were excluded from the analysis because no sufficient information was available, while 40 were excluded due to the lack of a follow-up visit. Finally, 157 PWE (76 men with a mean age of 24.2 ± 15.7) who had had at least 1 year of follow up were included in the analysis. The majority of them came from the areas of Camiri, Gutierrez and Eiti as shown in **Table 1.** Baseline characteristics are shown in **Table 1.**

The mean age at epilepsy onset was 9.6 ± 12.2. Seizure types were classified on the bases of the clinical history which was available in 113 cases (72%). Almost all patients presented epilepsy with GTCS of whom 26 (23.0%) focal to bilateral tonicclonic seizures, while 81 (71.7%) were classified as generalized tonic-clonic. Only 6 (5.3%) presented focal seizures (**Table 2**). Structural epilepsy was the most common type of epilepsy, recorded in 54 cases (34.4%) followed by epilepsy of unknown etiology (22 cases, 14.0%) and genetic generalized epilepsy (21, 13.4%). Nonetheless, for 60 cases (38.2%) epilepsy was defined as unknown (**Table 2**).

Concerning the causes of structural epilepsy, perinatal factors were the most commonly reported, being present in 11 subjects (20.4%), followed by head trauma (11.1%). A diagnosis of meningitis was reported in 5 cases, NCC in 3 while other structural causes such as stroke or cysts were reported in 6 cases. For 22 cases (14.0%) "known" causes were not reported and were then classified as epilepsy of unknown etiology. Clinical characteristics of epilepsy are shown in **Table 2.**

Presence of co-morbidities was reported in 24 cases (15.3%) and the majority (17 subjects; 10.8%) presented developmental delay; however, for 22 cases this information was missing.

Overall, the most common AED prescribed was phenobarbital followed by carbamazepine and by the association of phenobarbital and carbamazepine. AEDs treatment during the follow-up period (2012–2016) is reported in **Figure 3**.

Information about seizure frequency before and after the treatment were available only for 47 patients. Before the AEDs treatment only 13% of cases presented sporadic seizures (2–3 per year) while the majority (55%) presented monthly or weekly seizures and 32% reported to have seizures every day. During the follow-up, a dramatic seizures reduction was observed and, TABLE 1 | Demographic features of the patients.

N, number; M, men.

TABLE 2 | Classification and causes of epilepsy.

N, number.

in particular, 14/47 (30%) were seizure-free during the last year of follow-up, while 19% presented only sporadic seizures, and most of them just when they did not take the AEDs regularly. Frequency of seizures before and after treatment for these patients is reported in **Figure 4**. Of 157 PWE, 31 (19.7) were seizure-free at the last follow-up.

It should be underlined that 48 subjects (30.6%) stated to not assume regularly the medication and that 10 (6.4%) interrupted the drug intake. Of these, 8 (13.8%) interrupted AED treatment due to the absence of seizures, while for the others the most common reason for an irregular intake or interruption was the lack of medication, in part due to economic reasons. The cost of AEDs, in fact, was covered by the Ministry of Health programmes (Mi Salud and Seguro Universal Materno Infantil, SUMI) or by the municipality ("alcaldia") for 21 (13.4%) patients, by the local Strategic Partner Organizations (SPOs) of the Nongovernmental Organization (NGO) "Liliane Fonds" (20.4%) and by the Convenio de Salud (11.5%). At any rate, more than 20% of PWE did not receive any financial supports.

During the follow-up period, 10 (6.4%), of the 157 PWE, died. Of them, 9 died due to causes not directly related to seizures, while only in one case a seizure-related cause was identified. In particular, the causes of death were the followings: severe malnutrition in four cases, malnutrition and pneumonia in two cases, pneumonia in one case, alcohol addiction complications in two cases, severe burn following a fall during a seizure in another case. Five of the 10 subjects suffered from structural epilepsy and six (60%) had also other comorbidities. Among these subjects, three had suspended the treatment, while five declared an irregular intake. The patient for whom a seizurerelated death has been suspected was suffering from epilepsy with GTCS and alcoholism and he had suspended his treatment because of alcohol abuse.

#### DISCUSSION

Epilepsy treatment gap is an important public health problem in LMICs and in particular in the rural areas. Inadequate trained staff, cost of antiepileptic treatment and its unavailability, cultural misbeliefs about epilepsy, use of traditional medicine, and distance from the health posts represent its main causes (6) and appropriate interventions have been suggested to reduce it (24).

Among the suggested interventions, the core actions are represented by training nurses and community health workers, in order to be able to identify people with epilepsy, and training GPs working in the rural communities in order to diagnose and initiate epilepsy treatment (10). Indeed, according to the WHO guidelines, in LMICs settings epilepsy with GTCS should be diagnosed at primary care level by trained non-specialist health care providers (7). Moreover, increasing communities' awareness about epilepsy represents a further important action in order to reduce stigma and improve the adherence to the epilepsy treatment, even if misconceptions about epilepsy and recourse to traditional healers are challenging to change.

During our long-lasting activity in rural Bolivia, in agreement with the ILAE and WHO recommendations, from 1994 up to date we performed several epidemiological and interventional surveys including an anthropological survey, training programs directed to GPs, nurses and CHWs of the rural communities of the Chaco region as well as communities awareness programs (ongoing activity). Training activities were mainly directed to the Knowledge, Attitudes and Practice (KAP) and management of epilepsy with GTCS. The activities conducted by our group during these last 25 years, have surely increased the level of knowledge and awareness about epilepsy in this region as

demonstrated by the drop in the level of treatment gap recorded during different epidemiological survey over the time. Indeed, in our first survey performed in 1994 a treatment gap of about 90% was recorded while in 2010 a reduction of TG to values of about 70% has been found; finally in 2016 a value of TG of 45% has been recorded (3, 4, 13).

Although recognizing the importance of data coming from well-designed epidemiological studies, we believe that it can be worthwhile to evaluate the impact of our activity on the "real life" and not only under experimental conditions. Several trials have already demonstrated the feasibility of the treatment of epilepsy with GTCS under experimental conditions (25), but few data are available regarding the following step, namely the clinical practice in these settings. In experimental conditions, in fact, PWE are strictly monitored, the treatment is guaranteed and often delivered directly in the communities; furthermore, sometimes the costs of the AEDs have been directly covered by the project. However, what happens after the trial? Which is the real adherence to the treatment and the risk of an abrupt interruption? Which are the main causes of an irregular intake? All these important questions can be addressed only by the analysis of routine data, taking into account all the limits related to this kind of study. In fact, routine data collected for the clinical practice have the fault of lacking information with possible missing data for many subjects and many variables. Moreover, we are absolutely aware about the possible selection bias related to the fact that PWE treated and included in the analysis can have different characteristics with respect to those never treated. Nonetheless, from our point of view, despite all the possible limits, important food for thought comes from this study.

As expected on the bases of our activities that, in agreement with the WHO guidelines, have been focused on the management of GTCS, more than 90% of PWE on treatment presented convulsive epilepsy. Concerning the epilepsy causes, it was possible to define the epilepsy type only in a minority of cases, since in 14% of cases epilepsy was defined as of unknown etiology and in 38.2% of patients, epilepsy diagnosis was not possible. This high level of diagnostic uncertainty, with consequent underestimation of the most frequent recognized epilepsy causes, such as NCC (11), is certainly due to the low availability of reliable diagnostic tools in these areas. Treating epilepsy with GTCS in rural communities, regardless of its causes, is feasible as demonstrated by the high frequency of a regular intake and the dramatic seizures reduction. Nonetheless 30% of subjects reported an irregular intake often due to irregular AEDs supply and economic reasons. On the other hand, AEDs adherence does not seem to be influenced by stigma or cultural beliefs. Thus, when we increase knowledge and awareness about epilepsy, the treatment is well accepted and sought by PWE. However, cost and supply still represent an important barrier in these areas. Currently, the costs of AEDs are in part covered by NGOs or other non-governmental institutions. An effort should be made to extend the cost coverage of AEDs by national and departmental health institutions since the cost of the most common AEDs used in Bolivia is very low. In fact, considering the most common drugs used, the annual cost of a treatment with the average dosage used of 100 mg of PB daily would be only about 50 \$ per person a year, as well as the annual treatment with 400 mg of CBZ, which would cost about 95 \$ per person a year. Only 21.6% of cases are currently taken in charge by the municipality and Ministry of Health (Mi Salud and SUMI). Furthermore, the follow-up depends entirely by the volunteer action of local neurologists. It should be underlined that even if the follow-up can be performed by the GPs working in the rural area, in Bolivia, as well as in other countries, phenobarbital, that represents the first line drug, can be prescribed only by neurologists. In this perspective, the presence of a neurologist should be considered as an essential element within a team attending PWE, acting as prescriber and reference specialist for GPs. Programs should consider this presence and the relative costs.

Mortality rate for epilepsy in the Chaco area was 10/1,000 (12) and even if, due to the different study design, a direct comparison cannot be made, the number of deaths recorded in this sample (10 out of 157 PWE) is probably higher than expected. As already reported it is possible that patients seeking treatment and consequently included in the study were the most severe ones, thus leading to a possible selection bias. Indeed, patients who died were affected by more severe forms of disease as demonstrated by a high rate of comorbidities present in 60% of subjects, mainly represented by developmental delay. Moreover, all patients presented a structural epilepsy with only GTCS and with a frequency higher with respect to the rest of the sample (57.15% with weekly or daily seizures before treatment). In agreement with this observation, nine patients died due to causes not directly related to seizures, such as severe malnutrition, pneumonia and alcohol addiction, while only in one case a seizure-related cause was identified (severe burn following a fall during a seizure). Nonetheless, this latter patient interrupted his treatment intake because of alcohol abuse.

We are aware that our study includes only a part of PWE residing in this area. Indeed, considering the total rural population of the study areas (about 75,000 inhabitants according to the 2012 census) and according to the prevalence of active epilepsy associated with GTCS (6.6/1,000) (4) which is the most common type, about 495 PWE are expected in this population. Thus, we can conclude that about 50% of PWE (n = 215) received medical attention and were treated with an AED in this area, percentage that is roughly close to the last TG recorded in the same area (13). Nonetheless, this is not a population survey and, as reported, selection bias is probable. It is possible to hypothesize, in fact, that PWE affected by more severe forms were more prone to seek medical attention and treatment. To these reasons, our data do not allow to reach any conclusion regarding the effective TG. According to our experience, after improving knowledge and awareness in the rural population, PWE are willing to seek medical attention and to receive antiepileptic treatment, and these considerations can be extended to other similar settings.

At this point governmental and departmental actions are urgently needed in order to guarantee an adequate treatment and medical attention in these areas. In particular, local government should strengthen specific training programs for the GPs and CHWs living in the rural area and should support supply of generic, low-cost, first-line AEDs. These actions could lead to a significant improvement of epilepsy care in rural areas.

Furthermore, the national organizations and international official epilepsy institutions should reinforce their actions in order to obtain equal conditions people with epilepsy in the different parts of the world.

# AUTHOR CONTRIBUTIONS

LG and AN contributed to conception and design of the study. SP, EV, DR, and MM contributed to the organization of the project. LG, AN, CC, and CEC organized the database. LG and AN performed the statistical analysis. LG and AN wrote the first draft of the manuscript. CC, CEC, and AB wrote sections of the manuscript. MZ, MC, AB, and EC critically reviewed the manuscript. All authors contributed to manuscript revision, read, and approved the submitted version.

# FUNDING

Financial support for this work was provided by Sanofi Global Health Programs. Sanofi has not been involved in the study design, in data collection, analysis nor interpretation.

# REFERENCES

**Conflict of Interest Statement:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Copyright © 2018 Nicoletti, Giuliano, Colli, Cicero, Padilla, Vilte, Rojo Mayaregua, Martinez, Camargo, Zappia, Bartoloni and Crespo Gómez. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

# Validation of a Questionnaire for Distinguishing X-Linked Dystonia Parkinsonism From Its Mimics

Jose Danilo B. Diestro1 \*, Mark Angelo C. Ang2 , Mark Willy L. Mondia1 and Paul Matthew D. Pasco1

*1 Department of Neurosciences, College of Medicine and Philippine General Hospital, University of the Philippines Manila, Manila, Philippines, 2 Department of Laboratories, College of Medicine and Philippine General Hospital, University of the Philippines Manila, Manila, Philippines*

Objectives: X-linked dystonia parkinsonism (XDP) is a neurodegenerative movement disorder endemic to the island of Panay in the Philippines. We undertook a population-based prevalence study to enumerate all cases of XDP in Panay. We first developed a 4-item questionnaire to distinguish XDP suspects from the general population. In the present study we aimed to revalidate this questionnaire to distinguish XDP from similar conditions so as to give it greater utility in the clinical setting.

#### Edited by:

*Ding Ding, Fudan University, China*

#### Reviewed by:

*Andrea Giordano, Istituto Neurologico Carlo Besta (IRCCS), Italy Tetsuya Asakawa, Fujian University of Traditional Chinese Medicine, China*

> \*Correspondence: *Jose Danilo B. Diestro<EMAIL_ADDRESS>
#### Specialty section:

*This article was submitted to Neuroepidemiology, a section of the journal Frontiers in Neurology*

Received: *27 May 2018* Accepted: *18 September 2018* Published: *15 October 2018*

#### Citation:

*Diestro JDB, Ang MAC, Mondia MWL and Pasco PMD (2018) Validation of a Questionnaire for Distinguishing X-Linked Dystonia Parkinsonism From Its Mimics. Front. Neurol. 9:830. doi: 10.3389/fneur.2018.00830* Patients and Methods: A total of 306 subjects (114 cases and 192 controls) were screened in from the 16 towns and 1 city of Capiz province. Their responses to the previously developed 4-item questionnaire were collected and multivariable logistic regression was performed to develop a predictive model. The accuracy of the model was determined by using it on a subset of patients; then, a scoring system based on the model coefficients was established.

Results: With a cut-off score of 6, the questionnaire had an accuracy of 70.7% (95% CI 0.57-0.82), a sensitivity of 84.6 % (95% CI 0.65-0.96) and a specificity of 59.4 % (95% CI 0.41-0.76). The item on "shuffling of feet" was the strongest predictor in distinguishing XDP from its common mimics.

Conclusion: We were able to revalidate a simple, four-item questionnaire that could distinguish XDP from its common mimics with fair accuracy. The questionnaire along with other clinical features can be used to determine which patients need specialty evaluation and genetic testing to verify a diagnosis of XDP.

Keywords: XDP, dystonia, questionnaire, parkinsonism, genetic, prevalence, epidemiology

# INTRODUCTION

X-linked dystonia parkinsonism (XDP) is a neurodegenerative movement disorder endemic to Panay island in the Philippines, with recessive inheritance and a genetic founder effect. Molecular genetic studies suggest reduced expression of a neural isoform of TAF1 in the striatum of patients (1). The disease initially manifests with focal dystonia, which then progresses to generalized dystonia and overlaps with symptoms of parkinsonism about 5–10 years after onset. Parkinsonism predominates after about 10 years of disease (2). Some XDP patients have may have predominantly parkinsonian symptoms initially (3), causing diagnostic confusion with idiopathic Parkinson

**50**

Disease. Among inherited forms of isolated dystonia, XDP stands out with its overlapping parkinsonian features and X-linked mode of inheritance (4). The disease was brought to the attention of the international scientific community in 1975 when it was presented in the Second Dystonia Meeting held in New York City (5).

A population-based prevalence study was initiated in 2016 in order to do a complete enumeration of all XDP cases in Panay. The first phase involved the development of a 4-item screening questionnaire that can be used by community health workers (CHWs). The second phase involved screening by the CHWs of all the households in the island using this questionnaire. Individuals who were screened in were then evaluated by neurologists and movement disorder specialists. All consenting patients deemed to have probable XDP underwent genetic testing. Only Capiz province in Panay island has undergone all four phases of the study.

The first phase of the prevalence study was accomplished in 2015. A questionnaire with four items was validated to be highly sensitive and specific for screening patients with possible XDP in a general population where XDP is endemic. The questions screened for sustained twisting, repetitive jaw opening and closing, slowness in movement, and feet shuffling (6). Since the questionnaire was designed for screening at-risk individuals in a healthy population, the validity of this tool is unknown in patients with symptoms similar to XDP, such as Parkinson's disease, stroke, and cerebral palsy (6). A questionnaire that can distinguish genetically confirmed XDP from its clinical mimics is therefore lacking.

New diagnostic criteria for another genetic movement disorder, Huntington Disease, have recently been established. The criteria are based on motor scoring systems, genetic testing and historical findings that classify the patients into six strata (7). Similarly, a questionnaire for another neurologic condition, myotonic dystrophy (MD), identified 5 questions predictive of MD among healthy controls and diseases that mimic MD. The study emphasized the importance of such questionnaires especially in neurologic conditions such as MD that may be missed by non-neurologists (8). Common neurologic diseases, such as stroke, migraine, dementia and Parkinson disease also have screening tools used by non-specialists that lead to more definitive investigations (9–12).

The main objective of our study is to develop a weighted questionnaire that will differentiate XDP from other neurological diseases with similar symptoms. Many patients reside in remote areas of Panay, accessible only by long treks on foot and boat rides. General health professionals who are first to see patients with possible XDP will need to know which ones will need specialist assessment. The present questionnaire, if validated, can help direct meager community resources toward diagnosing and treating patients most likely to have XDP.

#### MATERIALS AND METHODS

The study design involved the development of a predictive model using retrospectively obtained data from the ongoing Population-Based Prevalence Study of XDP in Panay. Over a period of 2 months, data was gathered from all the 16 towns and 1 city of Capiz province. The CHWs screened the entire province of about 440,000 adults1 using the previously validated 4-item questionnaire. In brief, the questionnaire was adapted from existing screening tools and tested on a random sample of 64 genetically confirmed and 64 normal healthy individuals (7). Responses to the questionnaire, the clinical diagnoses made by the movement disease specialists and the results of genetic testing provided data for the current study. Multivariable logistic regression was performed on a training subset of the data (n = 248) to create a predictive model and scoring system. The predictive model's performance was evaluated by applying the scoring system on another subset of data (n = 56). Sensitivity, specificity, and accuracy of the predictive model were obtained with 95% CI.

To be included in the dataset, subjects needed to be patients of the ongoing Population Based Prevalence Study of XDP in Panay, screened in by the CHWs in the second phase of the study. The subjects had a "yes" answer to at least one question in the 4-item questionnaire and a final disposition which is either a diagnosis of XDP or an alternative diagnosis, based on results of clinical and genetic testing.

Cases were defined as subjects with at least one positive response to the 4-item questionnaire and a positive genetic test result for XDP with the SVA retrotransposon insertion using a polymerase chain reaction (PCR) based genetic test (13). On the other hand, controls were defined as subjects with at least one positive response to the 4-item questionnaire and one of the following: a negative genetic test result or a more likely alternative diagnosis (in which case genetic testing was no longer done).

The patient's age, sex, and responses to the 4-item questionnaire, final clinical diagnosis and genetic testing results, where available, were tabulated on data collection sheets and subsequently, tabulated in an Excel file (Microsoft Excel for Mac v15.35). Anonymity was maintained by ensuring that only the codes were used in data gathering.

The collected data was randomly partitioned into two: a training set and a test set. The training set (248 subjects: 88 cases and 160 controls) was used to develop a predictive model through multivariable logistic regression analysis, which was appropriate to model the data because the output variable (whether or not a subject has XDP) was based on a set of predictor variables (responses to the screening questions) Each of the four questions in the screening tool was assigned a score based on the raw coefficients of the logistic regression model. A simulated dataset consisting of a list of all the possible combinations of answers to the four questions was used to determine the total score cut-off value. The resulting model was then run on the test set (58 subjects: 26 cases and 32 controls) to estimate its prediction abilities (diagnostic accuracy). The predicted probability cut-off value used in the estimation of the diagnostic accuracy, sensitivity, and specificity was chosen

1Philippine Statistics Authority. Capiz Quickstat.

as the point on the ROC curve that maximizes sensitivity and specificity.

To allow for the predictive model to be used at the point of clinical encounter, a scoring system for the 4-item questionnaire was developed based on the model coefficients of the logistic regression model. All calculations and statistical analysis were done in R version 3.2.4 (www.R-project.org). **Figure 1** provides a graphic summary of the experimental design.

The identity of all the subjects were concealed throughout the process. They were only referred to by their codes. The study obtained approval from the Expanded Health Research Office of the Philippine General Hospital prior to data collection. Also, a memorandum of agreement with the primary author of the Population Based Prevalence Study was signed to allow review of the study's de-identified data.

#### RESULTS

The baseline characteristics of all patients involved in the study is summarized in **Table 1**. Of all those seen by the CHWs during the second phase of the Population Based Prevalence Study, 306 subjects were screened in with an answer of "yes" to at least one item in the 4-item questionnaire. Out of all these patients, 114 had genetic confirmation of XDP, hence were labeled as cases. Of these, five were female. The average age was 53.14 years old (±10.79) with a range of 23 to 77 years old.

On the other hand, 192 either had a negative genetic test or a likelier alternative diagnosis after clinical evaluation, hence were labeled as controls. The diagnoses of the control group composed mainly of other neurologic diseases mimicking XDP are illustrated in **Figure 2**. Cerebral palsy (18.75%) was the most common XDP mimic. Forty one of the 192 patients in the control group also underwent genetic testing and had a negative result. Of the 12 Parkinson Disease patients, all in the control group, 5 underwent genetic testing and tested negative.

Based on the logistic regression model, the question on feet shuffling and taking smaller steps was found to be the single strongest predictor in distinguishing XDP from conditions that mimic it. The receiver operating characteristic (ROC) curve is shown in **Figure 3**. A cut-off probability of 0.179 for a "positive" result of the questionnaire was set, which was determined to be the best cut-off threshold maximizing the model's sensitivity and specificity. The area

TABLE 1 | Baseline Characteristics of Study Subjects.

under the curve (AUC) is 0.770—reflecting a fair ability of the questionnaire to distinguish XDP from its mimics (14).

The final scoring system based on raw coefficients of the logistic regression model is shown in **Table 2**. The total scores for each combination of answers were compared with the predicted probabilities generated by running the model on the simulated dataset. The predicted probabilities of the simulated dataset are shown in **Supplementary Material**. The total score can range from 0 to 13. A score of 6 or more is considered "positive" i.e., the patient has a high likelihood of having XDP.

The over-all accuracy of the model in separating XDP from non XDP individuals in the test set is 71%, which is statistically different from the accuracy that will be achieved if all subjects are classified as XDP without regard to their answer to the questions (no information rate). The model can correctly identify 85% of subjects with XDP as having XDP (sensitivity) and can correctly identify 59% of subjects without XDP as not having XDP (specificity). On the other hand, a positive identification made by the model will be correct 63% of the time (positive predictive value) and a negative identification made by the model will be correct 83% of the time (negative predictive value). The negative predictive value (NPV) of the test, 83%, ensures that most of those excluded by the test are true negatives.

#### DISCUSSION

In this study we attempted to validate a four-item questionnaire originally for use by lay workers to screen a population endemic for XDP, in a group of individuals with signs and symptoms that may be misdiagnosed as XDP by non-specialists. We found that

#### TABLE 2 | Questionnaire items with corresponding weights.

\**Statistically significant (p* < *0.05).*

though highly sensitive and specific in the general population, it was fairly accurate in this subset of XDP mimics.

Experience from the Population-Based Prevalence Study reveals that many patients who were suspected to have XDP could not even be brought to the municipal health centers for screening because of time and financial constraints. This emphasizes the great resources that need to be mobilized to get expert opinion for XDP suspects. Once this study ends, evaluation by a movement disorder specialist and genetic testing will become even more elusive. The questionnaire we developed can be used to screen for patients most likely to have XDP. Identifying this subset of patients will enable a more focused use of resources for those most likely to benefit. The questionnaire can be used by both lay workers and general health professionals.

Up to a quarter to one half of patients with dystonia may be misdiagnosed or undiagnosed (15). Even among neurologists the correct diagnosis of movement disorders may be difficult to make. The rate of accurate diagnosis correlates with the physician's experience—with movement disorder specialists arriving at the right diagnosis more consistentl (16). Unfortunately, in the Philippines, for a population of 100,981,437 (17), there are only about 10 movement disorder specialists with formal training. This questionnaire can be used to assist general health professionals and CHWs in Panay in selecting patients who are most likely to benefit from subspecialty consult and genetic testing.

The developed questionnaire has a fair ability to distinguish XDP from its common mimics with an accuracy of 71% (0.57- 0.82, 95% CI). It has a relatively high negative predictive value of 83% (0.61-0.95, 95% CI), which makes it useful for first line health workers in selecting patients that need to be triaged for further work-up and evaluation in tertiary institutions with neurologists and movement disorder specialists. A patient with a score of 6 or more would likely benefit from further evaluation by a movement disorder specialist and subsequent genetic testing. While a score of less than 6 would identify patients who probably have an alternate diagnosis. The earlier validation study that distinguished XDP patients from the healthy general population only required that any one of the 4 identified questions be positive for the screened individual to move to the next phase of the study (6).

Perhaps the main differential diagnosis for XDP is idiopathic parkinsonism. Multivariable logistic regression analysis reveals that the question on feet shuffling and taking smaller steps has the biggest weight for determining XDP in this population but is not by itself able to distinguish between genetically positive XDP and its mimics. A previous review on the phenomenology of XDP revealed that only 5.7% of XDP patients begin with parkinsonian symptoms, including shuffling of gait, and that only 18.6% are predominantly parkinsonism throughout the course of the illness (5). This study suggests that shuffling of feet and parkinsonism may be more important than previously thought. In contrast, the original validation study for the 4-item questionnaire gave constant jaw opening and closing the largest weight for distinguishing XDP from the general population (6).

A screening questionnaire for Parkinsonism found the following questions most predictive: stiffness and rigidity in legs, tremor and shaking, troublesome arm swing and feet stuck to floor (18). It is interesting to note that two of these items relate to lower extremity mobility as well. This fact emphasizes the need for the other 3 components not found in the PD questionnaire to come up with a scoring system that can distinguish XDP from PD. The original version of the 4-item questionnaire found the question on "shuffling of feet" to have excellent positive and negative clinical utility in differentiating XDP from the normal population (6). It is not surprising then that the same symptom is found to be most important in differentiating XDP from its mimics.

The most interesting aspect of the results perhaps is the 31 clinically diagnosed XDP patients who had a negative genetic test. In this study we used a PCR-based test that detected the presence of the SVA retrotransposon insertion in intron 32 of the TATA binding protein factor 1 (TAF1) in the XDP critical region in Xq13.1 (13). This retrotransposon insertion however, is not the only variant associated with XDP in the XDP haplotype on the X chromosome. Six other variants have been identified: singlenucleotide changes (DSC1, DSC2, DSC3, DSC10, DSC12) and one 48-bp deletion (19), although previous data has shown that all these genetic variants are in tight linkage disequilibrium (i.e., no variant has been described in patients in isolation from the rest).

Theoretically, each of the 31 patients with clinical features consistent with XDP but a negative genetic test could have any of the other 6 genetic variants apart from the SVA retrotransposon insertion; however, a previous study that investigated the genetic features in 166 clinically diagnosed XDP patients found that all 7 disease-associated genetic variants always occurred together with each other in all but 5 individuals. These 5 patients, clinically diagnosed to have XDP, had none of the 7 genetic variants, and were classified as phenocopies (19). The 31 clinically diagnosed XDP patients that were negative for the XDP haplotype on genetic testing may harbor a yet unidentified genetic variation that may be common to the 5 phenocopies in the previous study. Unfortunately, details on the individual phenomenology of the XDP in these 31 patients apart from their answers to the questionnaire were not documented. We recommend that further clinical evaluation be done for these subjects. Features such as age of onset of the disease and initial neurologic manifestations may determine if this subset of patients has a phenotype that is unique from genetically confirmed XDP patients.

Further modifications to the questionnaire can be made that can utilize other questions from an expanded list of screening questions (6). The test-retest reliability of the questionnaire can also be explored in future studies. New questions can be added from focused group discussions involving movement disorder specialists, health workers and patients. A questionnaire with better diagnostic performance (i.e., higher sensitivity and specificity) can thus be produced. Involvement of stakeholders will also improve the acceptability of the questionnaire later. All these can help produce a more accurate and acceptable questionnaire. In addition, a convenient electronic application can also be developed.

Similar to developments in Parkinson Disease research, the principles of objectification, multi-purpose design and simplification should be applied to future assessment tools for XDP (20). Further modification of the tool can include an objective assessment of the pathologic movements of XDP by clinical observation or the use of wearable movement analysis devices. Doing so would make the evaluation adhere to an objective multipurpose design that gathers data for XDP phenomenology research (20).

There are several limitations of this study. The prevalence study only screened those able to understand and respond to a "yes or no" questionnaire-type interview. The new scoring tool was developed using subjects who had already been screenedin using the 4-item XDP screening questionnaire developed in phase 1 of the Prevalence Study. Only the same 4 questions were used to develop the new scoring tool. The questionnaire by itself cannot be used to decide which patients merit genetic testing. A careful evaluation by a movement disorder specialist is needed to elicit clinical clues that cannot be provided by the questionnaire.

All data was only collected through review of the database of an ongoing study. Because not all controls were genetically tested, some patients, especially those thought to have PD, may be XDP patients with predominant parkinsonian symptoms. Besides answers to the four questions no other clues to the patients' clinical features were found; hence, correlations between signs and symptoms to the genetic variant tested for could not be done. Most importantly, XDP patients without any of the currently known 7 mutations may not have been selected by the questionnaire.

# CONCLUSIONS AND RECOMMENDATIONS

We were able to revalidate a simple, four-item questionnaire that can be used to distinguish XDP from its common mimics with fair accuracy. The questionnaire is intended for use by local health workers and general practitioners to determine which patients need specialty evaluation and genetic testing to ascertain or dispute the diagnosis of XDP. Doing so would direct time and finances to the patients most likely to have XDP and need specialty care.

The clinical features of all subjects classified as cases should be more completely characterized. Doing so would contribute greatly to data regarding the phenomenology of XDP. Further genetic profiling should be done on the 31 clinically diagnosed XDP patients with negative genetic testing, as they could be harboring a yet to be discovered genetic mutation associated with XDP. A follow-up study that includes more controls with genetic testing may perhaps improve the accuracy of this questionnaire.

# DATA AVAILABILITY STATEMENT

The raw data supporting the conclusions of this manuscript will be made available by the authors, without undue reservation, to any qualified researcher.

# AUTHOR CONTRIBUTIONS

JD, MM, MA, and PP were all equally responsible for the conception and design, acquisition of data or analysis and interpretation of data. MA designed and carried out the statistical analysis of the data. JD and MM drafted the article while MA and PP took charge of critical revisions for important intellectual content. JD, MM, MA, and PP agreed to be accountable for the article and to ensure that all questions integrity of the article are investigated and resolved.

# FUNDING

Collaborative Center for X-linked Dystonia Parkinsonism (CCXDP) of the Massachusetts General Hospital is responsible for the funding of the Population-based prevalence study of X-Linked Dystonia Parkinsonism (XDP) in Panay Island, an ongoing study whose database our raw data is from.

#### ACKNOWLEDGMENTS

We wish to acknowledge Dr. Roland Dominic Jamora for his review of the study design during the conception of this research. We would also like to thank the neurologists and genetic counselors who were instrumental in the implementation of this study. But most of all we are grateful to the health workers of Panay who bear the daily burden of caring for our XDP brothers and sisters. This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.

# SUPPLEMENTARY MATERIAL

The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fneur. 2018.00830/full#supplementary-material

# REFERENCES

**Conflict of Interest Statement:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Copyright © 2018 Diestro, Ang, Mondia and Pasco. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

# Corrigendum: Validation of a Questionnaire for Distinguishing X-Linked Dystonia Parkinsonism From Its Mimics

Jose Danilo B. Diestro1 \*, Mark Angelo C. Ang2 , Mark Willy L. Mondia1 and Paul Matthew D. Pasco1

*1 Department of Neurosciences, College of Medicine and Philippine General Hospital, University of the Philippines Manila, Manila, Philippines, 2 Department of Laboratories, College of Medicine and Philippine General Hospital, University of the Philippines Manila, Manila, Philippines*

Keywords: XDP, dystonia, questionnaire, parkinsonism, genetic, prevalence, epidemiology

#### **A Corrigendum on**

#### **Validation of a Questionnaire for Distinguishing X-Linked Dystonia Parkinsonism From Its Mimics**

#### Approved by:

*Frontiers in Neurology, Frontiers Media SA, Switzerland*

> \*Correspondence: *Jose Danilo B. Diestro<EMAIL_ADDRESS>
#### Specialty section:

*This article was submitted to Neuroepidemiology, a section of the journal Frontiers in Neurology*

Received: *01 November 2018* Accepted: *02 November 2018* Published: *20 November 2018*

#### Citation:

*Diestro JDB, Ang MAC, Mondia MWL and Pasco PMD (2018) Corrigendum: Validation of a Questionnaire for Distinguishing X-Linked Dystonia Parkinsonism From Its Mimics. Front. Neurol. 9:991. doi: 10.3389/fneur.2018.00991* by Diestro, J. D. B., Ang, M. A. C., Mondia, M. W. L., and Pasco, P. M. D. (2018). Front. Neurol. 9:830. doi: 10.3389/fneur.2018.00830

In the original article, we neglected to include the funder, Collaborative Center for X-linked Dystonia Parkinsonism (CCXDP) of the Massachusetts General Hospital. CCXDP is responsible for the funding of the Population-based prevalence study of X-Linked Dystonia Parkinsonism (XDP) in Panay Island, an ongoing study whose database our raw data is from.

In addition, we neglected to acknowledge the neurologists and genetic counselors who examined the patients of the study. The corrected Acknowledgment appears below:

We wish to acknowledge Dr. Roland Dominic Jamora for his review of the study design during the conception of this research. We would also like to thank the neurologists and genetic counselors who were instrumental in the implementation of this study. But most of all we are grateful to the health workers of Panay who bear the daily burden of caring for our XDP brothers and sisters. This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.

The authors apologize for this error and state that this does not change the scientific conclusions of the article in any way. The original article has been updated.

**Conflict of Interest Statement:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Copyright © 2018 Diestro, Ang, Mondia and Pasco. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

# A Retrospective Analysis of the Clinical Features of Inpatients With Epilepsy in the Ganzi Tibetan Autonomous Prefecture

#### Edited by:

*Ding Ding, Fudan University, China*

#### Reviewed by:

*Chong Tin Chong Tan, University of Malaya, Malaysia Jing-Jane Tsai, National Cheng Kung University Hospital, Taiwan Kheng Seang Lim, University of Malaya, Malaysia*

> \*Correspondence: *Dong Zhou<EMAIL_ADDRESS>
*†These authors have contributed equally to this work*

#### Specialty section:

*This article was submitted to Neuroepidemiology, a section of the journal Frontiers in Neurology*

Received: *11 July 2018* Accepted: *01 October 2018* Published: *30 October 2018*

#### Citation:

*Chen J, Wu X, He Y, Li S, Deng Y, Chen J, Fang W, Zeren Z, Peng J, Li Y, Mu J and Zhou D (2018) A Retrospective Analysis of the Clinical Features of Inpatients With Epilepsy in the Ganzi Tibetan Autonomous Prefecture. Front. Neurol. 9:891. doi: 10.3389/fneur.2018.00891* Jiani Chen1†, Xintong Wu1†, Yongqiao He2 , Sisi Li 1 , Yongyi Deng2 , Jie Chen2 , Wenyu Fang2 , Zhamu Zeren2 , Jianmei Peng2 , Yingjuan Li 2 , Jie Mu1 and Dong Zhou1 \*

*1 Department of Neurology, West China Hospital, Sichuan University, Chengdu, China, 2 Department of Neurology, Ganzi Tibetan Autonomous Prefecture People's Hospital, Kangding, China*

Background: There is limited detailed clinical information for patients with epilepsy in Tibet. This study sought to provide data about the clinical features of epilepsy in the Ganzi Tibetan Autonomous Prefecture to improve strategies for epilepsy prevention and management in this region.

Methods: We reviewed the clinical record of patients with epilepsy in the Neurology Department, Ganzi Tibetan Autonomous Prefecture People's Hospital and compared the clinical features and compared it with control, from West China Hospital in Chengdu.

Results: This retrospective study included 165 patients with epilepsy admitted between January 2015 and February 2018. Majority of patients (97%) in this study had active epilepsy; 28.5% had generalized onset seizures and 68.5% had focal onset seizures. Fifty-four patients had received anti-epileptic drug (AED) treatment prior to hospitalization, however, 38 (70.4%) patients took the medication irregularly. The leading etiology of this cohort was head trauma (20.6%), followed by stroke (10.9%), neurocysticercosis (7.9%), brain hydatidosis (6.7%) and tuberculous infection (5.5%). Compared with in-patients in Chengdu, epilepsy in Ganzi was more frequently caused by infection (OR = 4.216, 95% CI, 2.124–8.367), including neurocysticercosis (OR = 29.301, 95% CI, 1.727–497.167) and brain hydatidosis (OR = 24.637, 95% CI, 1.439–421.670).

Conclusions: These data suggest that the control of cerebral infections, especially parasite infection, is essential for the prevention of epilepsy in the Ganzi Tibetan Autonomous Prefecture. Education of local primary doctors and patients about the literacy of epilepsy will enable better management of epilepsy in this population.

Keywords: cerebral infection, clinical features and atiology, epilepsy, Ganzi Tibetan Autonomous Prefecture, retrospective study

# INTRODUCTION

Epilepsy is one of the most common neurological disorders in the world that affects people of all nations and socioeconomic classes. The Tibetan area, which is on the Qinghai-Tibetan plateau, is ∼4,000 m above sea level. There are very few studies of epilepsy in this area (1, 2). A survey done in one of the counties in Tibet found that the lifetime prevalence of convulsive epilepsy was ∼2.5 per 1,000 with a treatment gap of up to 97%. No EEG or imaging data were available in this study (1). Another study summarized 180 cases of patients with epilepsy (PWE) and reported that the etiology was unknown in 145 cases (80.6%) and no EEG or imaging data were available (2). Detailed clinical information, such as MRI and EEG results in the Tibet area, remains limited.

This study sought to provide data about the clinical features of epilepsy in the Ganzi Tibetan Autonomous Prefecture. Ganzi Tibetan Autonomous Prefecture is on the South-eastern edge of the Qinghai-Tibetan plateau, in the Western part of Sichuan Province. The average altitude is 3,500 meters above sea level. Tibetan is the primary ethnic group. With a population of one million, Ganzi Tibetan Autonomous Prefecture is the second largest inhabited region of Tibetan people in China. Many locals earn their living by farming or yak herding (3).

In this study, clinical records of patients admitted to Ganzi Tibetan Autonomous Prefecture People's Hospital and diagnosed with epilepsy were retrospectively reviewed. The study aimed at providing an updated information on the clinical features of epilepsy in the Tibetan area to help improve strategies for epilepsy prevention and management in this region.

# MATERIALS AND METHODS

# Study Location and Population

The Ganzi Tibetan Autonomous Prefecture People's Hospital is in Kangding City, Sichuan province, the capital of the Ganzi Tibetan Autonomous Prefecture. It is the only hospital equipped with MRI and Video EEG in Ganzi. West China Hospital, a large tertiary referral center in West China, has been sending a neurologist to the Ganzi Tibetan Autonomous Prefecture People's Hospital every year since 2014 to live and work with the local neurologists.

### Study Design

Patients with epilepsy that were admitted to the Neurology Department of Ganzi Tibetan Autonomous Prefecture People's Hospital, between January 2015 and February 2018, were retrospectively and consecutively included in this study. To explore the clinical features of inpatients with epilepsy in Ganzi, demographic and clinical data collected were compared with the data from West China Hospital in Chengdu (control group). Controls' data were retrieved from in-patient epilepsy database of West China Hospital in Chengdu City between June 2017 and February 2018. PWE in Ganzi were identified and matched for gender, age (±2 years) in the controls database. In cases where there were several matched controls, the one with early admission date would be included.

The following definitions were considered: epilepsy is defined according to the 2014 International League Against Epilepsy (ILAE) as two spontaneous seizures with one occurring within the last 6 months, or one seizure with an epileptiform discharge shown by EEG or an enduring relevant lesion shown by imaging (4, 5).

Status epilepticus (SE) is defined according to the 2015 ILAE as a condition resulting from the failure of the mechanisms responsible for seizure termination or initiation of mechanisms which lead to abnormally prolonged seizures. Convulsive SE is defined as more than 5 min of continuous seizure or two or more discrete seizures with incomplete recovery between them (6).

Patients were excluded if they reported symptomatic seizures occurring <2 weeks after the onset of acute cerebral insults and if they lived outside of the Ganzi Tibetan Autonomous Prefecture.

Controls were excluded if they reported symptomatic seizures occurring <2 weeks after the onset of acute cerebral insults.

# Socio-Demographic Status and Clinical Information

Data were collated retrospectively using a structured data collection form by reviewing the medical charts. All patients recruited had been diagnosed with epilepsy by two neurologists, from both the Ganzi Tibetan Autonomous Prefecture People's Hospital and West China Hospital. Differences in opinion were resolved by consensus. Demographic information included gender, age, marriage status, living area, education, job, and ethnicity were collected.

Clinical information was collected from all patients and the control. This included: age at the seizure onset, seizure frequency, and AEDs. The results of EEG and MRI/CT scans were reviewed and recorded. The MRI prescribed was a 1.5-T imaging system (Philips Achieva, Holland & Sonata, Siemens, Germany). Most patients in our clinic underwent neuroimaging as part of routine checks. Local or generalized slow waves in the EEG recording were considered non-specific abnormalities, while local or generalized paroxysms of sharp waves, spike waves, sharp and slow waves, or spike and slow waves were considered epileptiform discharges. Epileptic seizures were classified according to ILAE 2017 classification as focal seizures (focal aware seizures, focal impaired awareness seizures, or focal to bilateral tonic-clonic seizures), generalized seizures (non-motor, other motor or tonic-clonic), and unknown (7). Etiology was defined as the presence of remote brain injury that causes seizure episodes (8). Diagnosis was typically established on the basis of clinical history, past medical records, and laboratory results, and was further confirmed by neuro imaging. Etiology was classified according to ILAE 2017 classification as structural, infectious, genetic, metabolic, immune, and unknown etiology (7, 9). Specific etiologies were also listed, including neurocysticercosis, brain hydatidosis, stroke, head trauma, cerebral tumor, hippocampal sclerosis, cortical dysplasia, unclear intracranial space-occupying lesions, and so on.

The study was approved by the West China Hospital clinical trial and biomedical ethics committee and the ethics committee of the Ganzi Tibetan Autonomous Prefecture People's Hospital.

#### Data Analysis

Descriptive statistics of the patients and controls were conducted. Quantitative data were expressed as the mean ± SD. Qualitative data were summarized as proportions. A comparison of demographic and clinical data between the two groups was done using Student's T-tests/Mann Whitney U test for continuous variables and Chi-squared tests for categorical variables, respectively.

IBM SPSS 23.0 was used to perform all analyses. All of the tests were two-tailed, and p-values <0.05 were considered to be statistically significant.

# RESULTS

### Demographic Data of the Participants

One hundred and sixty-seven PWE were admitted to the Neurology Department of the Ganzi Tibetan Autonomous Prefecture People's Hospital from January 2015 to February 2018. Out of these, two patients were excluded because they were travelers living outside the Ganzi Tibetan Autonomous Prefecture. Hence, 165 patients were included in the study.

Our patient population included 96 (58.2%) men and 69 (41.8%) women. Other demographic features are listed in **Table 1**. The mean patient age was 39.4 ± 16.8 years. Forty-one (24.8%) patients lived in an urban location, and 124 (75.2%) lived in a rural/pastoral location. Most patients were Tibetan (n = 128, 77.6%) or Han Chinese (n = 34, 20.6%). The number of illiterate patients was 104 (63.0%), and 60.6% of the total patients were farmers.

A comparison of the demographic characteristics of the inpatients in Ganzi and gender and age-matched controls in Chengdu was done (**Table 1**). The comparison indicates that there were more patients in Ganzi who lived in rural/pasture areas compared to the controls (75.2 vs. 36.4%, p < 0.001) as well as lower education levels (the illiterate up to 63.0 vs. 1.2%, p < 0.001). There was also a significant difference in job groups (p < 0.001) and ethnic group distribution (77.6% Tibetan in Ganzi patients, p < 0.001). No differences were detected for the age, gender, and marital status.

### Clinical Features

The mean age at the time of seizure onset was 34.5 ± 18.0 years. No significant difference was noted when this was compared with the data from the control group (**Table 2**). In addition, there were more patients newly diagnosed with epilepsy 104 (63.0%) compared to the control group (63.0 vs. 33.3%, p < 0.001). The different seizure types were as follows: focal aware seizures (n = 5, 3.0%); focal impaired awareness seizures (n = 12, 7.3%); focal to bilateral tonic-clonic seizures (n = 96, 58.2%); and generalized tonic-clonic seizures (n = 46, 27.9%). Thirtythree (20%) out of 165 patients were diagnosed with SE, while 2 (1.2%) died following prolonged SE. No significant difference in seizure type fund between groups (focal/generalized/unknown, p = 0.087). Most (98.8%) patients underwent an MRI or CT scan; abnormalities were noted in 102 (61.8%) patients. These were more than those in the control group (61.8 vs. 38.2%, p < 0.001). EEG or video EEG was performed in 51 (30.9%) patients. TABLE 1 | Demographic features of PWEs in the neurology department of Ganzi people's hospital and control group from West China hospital.

\**P* < *0.05.*

*aFisher exact test.*

*b Including housewife.*

These were less than those in the control group (30.9 vs. 91.5%, p < 0.001). The results of the EEG studies revealed that 27 (52.9%) and 19 (37.3%) patients showed epileptiform discharge and normal EEG, respectively.

\**P* < *0.05.*

*aHaldane–Anscombe correction.*

*bFisher exact test.*

The data collected also revealed that 61 (36.9%) patients had been diagnosed with epilepsy prior to hospitalization with 54 of these patients receiving prescriptions for AEDs. However, 38 (70.4%) patients reported irregular use of medication, significantly higher than the control group (70.4 vs. 22.5%, p < 0.001). Moreover, 5 cases of SE were precipitated by nonadherence to the medication regime. The mean duration of status epilepticus before admission was 15.5 h (range 0.5–48). Most patients in this study (97.0%) had active epilepsy (seizure free <1 year) (10).

#### Etiology

The etiological factors of epilepsy in Ganzi are summarized in **Table 3**. With the ILAE 2017 classification, 39.4% had a structural etiology, 24.8% had an infectious etiology, 4.8% had a presumed genetic cause, and 29.7% was classified as unknown.

As to specific causes of epilepsy, the leading cause was head trauma (n = 34, 20.6%), followed by stroke (n = 18, 10.9%), neurocysticercosis (n = 13, 7.9%), brain hydatidosis (n = 11, 6.7%) and tuberculous infection (n = 9, 5.5%). Other etiologies included presumed genetic cause (n = 8, 4.8%), viral infection (n = 6, 3.6%) and unclear intracranial space-occupying lesions (n = 5, 3.0%). Less common etiologies included tumor (n = 3, 1.8%), cortical dysplasia (n = 4, 2.4%), and hippocampal sclerosis (n = 1, 0.6%).

When compared with the control group, PWEs in Ganzi significantly had more frequent infectious etiology (OR = 4.216, 95% CI, 2.124–8.367). And epilepsy in Ganzi was caused more frequently by head trauma (OR = 1.994, 95% CI, 1.084– 3.666), neurocysticercosis (OR = 29.301, 95% CI, 1.727–497.167) and brain hydatidosis (OR = 24.637, 95% CI, 1.439–421.670). Epilepsy in Ganzi was less frequently caused by hippocampus sclerosis (OR = 0.094, 95% CI, 0.012–0.747). The percentage of patients with unknown etiology in Ganzi was lower compared with the control group (OR = 0.532, 95% CI, 0.338–0.838).

### DISCUSSION

The present study focused on the clinical features of epilepsy in the Ganzi Tibetan Autonomous Prefecture, which is currently under-studied. The aim is to obtain clinical information that would lead to improved strategies in prevention, management, education, and treatment of epilepsy in this region.

#### Status Epilepticus

Thirty-three PWE who were admitted to the hospital had SE. Two of them (6.1%) died in the hospital. The reported overall mortality is ∼20% in the first month after SE (11). However, in SE without provoked factors, the reported rate of death is ∼10% (11, 12). In the current study, we excluded acute symptomatic cases from our cohort. For the two fatalities recorded, it was noted that the SE lasted for more than 1 day before proper treatment was initiated. In addition, some people living in the remote county of the Ganzi Autonomous Tibetan Prefecture would require 2 days drive to reach the People's Hospital. This may have been the reason for delayed diagnosis and treatment. The mean duration of SE before admission was 15.5 h (range 0.5– 48). It is therefore important that primary hospital physicians have to provide early diagnosis and control of SE. A survey done on attitudes and management practices of epilepsy among 100 primary practitioners in Ganzi, 2017, revealed that 49% did not know how to treat SE (unpublished data). It is therefore important to train primary practitioners and equip them with skills to control these cases.

\**P* < *0.05.*

*aHaldane–Anscombe correction.*

*bAutoimmune encephalitis.*

*c Included Genetic Generalized Epilepsies.*

# AED, Seizure Control and Medication Compliance

The findings of this study indicate that 63.0% of patients were newly diagnosed. Most of the patients who had been diagnosed with epilepsy previously had received AED treatment prior to hospital admission (54 out of 61 patients). However, 38 (70.4%) out of 54 patients reported irregular treatment. Majority of the of the 165 patients (97.0%), reported having active epilepsy (seizure free <1 year) (10). Previous studies have shown that medication compliance is poor in this region. One study reported that 87.5% of patients were not taking regular treatment (1). The factors that may contribute to non-compliance include: patients' perception of the importance of the medication or lack of regular doctor's appointments (13). Furthermore, many patients turn to traditional Tibetan treatment in the Tibet Autonomous Region (2). A survey on the attitudes toward epilepsy among the general population in Ganzi, 2017, revealed that up to 67% of patients preferred traditional Tibetan medicine because they thought it would work (unpublished data). The current study revealed that 78.2% of patients had mono-therapy prescriptions when discharged from Ganzi People's Hospital. However, follow up was not done to ascertain their compliance to the medication. Medication reminders (14) and educational programmes (13) may increase compliance in these patients.

## Etiological Factors and Prevention of Parasite Infection

Two previous population-based studies from the Tibet Autonomous Region did not conclusively describe the etiology of epilepsy in the patient populations studied. This may be due to the unavailability of EEG and CT/MRI data (1, 2). One study reported that seven and three patients developed epilepsy due to head trauma and neurocysticercosis, respectively (1). Another study (2) identified one patient with head trauma, one with neurocysticercosis, and 35 patients with seizures associated with heavy drinking or alcohol withdrawal. However, about 80% of patients in both studies have unknown etiologies. A strength of the current study is that most patients in our study underwent CT/MRI scans, hence, we were able to identify more etiological factors than previous study. The leading cause in this study was head trauma (20.6%), followed by stroke (10.9%), neurocysticercosis (7.9%), brain hydatidosis (6.7%), and tuberculous infection (5.5%). Head trauma, stroke, cerebral infection are common causes for epilepsy. In the epidemiological survey of epilepsy in Rochester, Minnesota described the following etiological factors, such as cerebrovascular diseases (6%), neurologic deficits from birth (5%), trauma (5%), and infection (4%) (15). Head trauma, intracranial infection, and cerebrovascular diseases were the leading causes of epilepsy in a large population-based study in China in the 1980s (16). In comparison, the inpatient sample in Ganzi Tibetan area had parasite infection of the brain accounting for about 14.6% percent of all epilepsy patinets in this sample. This was higher than the age and gender matched control group. It was also significantly higher when compared with another etiology study in Chengdu (neurocysticercosis: 7.9 vs. 1.3%, OR 6.271, 95% CI 2.808–14.004; Brain hydatidosis: 6.7 vs. 0%, OR 132.864, 95% CI 7.788–2,266.400) (17). The adult of tapeworm live in the human intestine, and the intermediate hosts are pigs or yaks often found in this area. Humans often develop neurocysticercosis when they ingest raw meat containing cysts (18, 19). The adult of Echinococcus live in dogs or Tibetan foxes. Yaks serve as the intermediate hosts. Humans often get infected when they eat food contaminated with echinococcus eggs. Hydatidosis could occur in liver, lung or brain (20). Both cysticercosis and hydatidosis are endemic diseases in Ganzi. One study has shown that 22.5% of the study participants had positive serology positive for cysticercosis in Yajiang County, Ganzi. Half of the seropositive patients were noted to experience seizures (18). Approximately an eighth (12.9%) of screened people were infected with hydatidosis in Shiqu County, Ganzi (21).

In the high pastures, many locals still live a traditionally pastoralism and semi-nomadism lifestyles, keeping dogs for herding animals. The domestic dog appears to be a key risk factor for both cystic and alveolar echinococcosis (20). Rates of echinococcosis infection in domestic dogs have been reported to be 5–15% in Tibetan area (22). Controlling the number of dogs and monthly praziquantel dog dosing programme is essential in preventing echinococcosis (20). Improvement of hygiene and promotion of hand-washing could also help in reducing infections (18).

Human taeniasis is acquired by eating uncooked or poorly cooked pork or beef infected with the larval form of the human taeniid tapeworms (19), there is a habitual consumption of undercooked beef and air-dried raw beef in this Tibetan community. Strategies to educate the locals on proper cooking of meat are important in the prevention of taeniasis (18).

Suprisely, the percentage of patients with unknown etiology was higher in the control group, suggesting that in-patients in Ganzi might have more identifiable seizure etiologies, such as infectious and structural changes. Hydatidosis is a critical and often fatal disease if left untreated (23). This study therefore proposes that all patients in this area with a new diagnosis of epilepsy should undergo a hydatidosis screen test.

The average altitude is 3,500 meters above sea level in Ganzi Tibetan Autonomous Prefecture. Excepted the two study in Tibet Autonomous Region which had been described above (1, 2), epidemiology of epilepsy in high altitude region was few (24), though there have been multiple case reports of seizures occurring in non-epileptic individuals and wellcontrolled epileptic patient at altitude (25–27). Still, the effect of altitude on the seizure threshold has not been studied in depth. This issue need to be explored more in future studies.

# Gender and Age

Our study cohort comprised 58.2% male patients (male: female, 1.39:1). A door-to-door survey of six cities in China in 1987 regarding PWE reported a male-to-female ratio of 1.15:1 (16). An additional population-based survey in the Tibet Autonomous Region reported a male-to-female ratio of 1.73:1 (2). Our study may have a higher number of man than the number that is in the general population; however, our ratio is similar to those observed in previous studies that note a greater number of male patients than female patients.

Mean age of patients was 39.4 ± 16.8 years. The mean onset age was 34.5 ± 18.0. Our age group distribution was similar to that observed in previous population-based surveys in Tibetan area, with over half of the patients in the 15–44 years age group (1, 28). However, the predominant middle-age group in this study is unusual as compared to the bimodal age distribution reported in the literature (15, 28). This may be because many of them had infectious etiology, as the mean onset age of infectious etiology was 34.3 ± 15.1. Besides, Children <14 years of age were directed to the pediatrics department and hence most were not included in this study.

# Semiology

In this cohort 28.5% of patients had generalized onset seizures and 68.5% of patients had focal onset seizures. Generalized tonicclonic seizures and focal to bilateral tonic-clonic seizures were the most commonly observed seizure type. This result is consistent with the findings of previous studies in the Tibet Autonomous Region (1, 2). However, the EEG application rate for epilepsy diagnosis was 30.9% in our study, which is significantly lower than the control group in Chengdu. This may compromise the accuracy of diagnosis, in particular for seizure semiology. Though the EEG rate in the current study is higher than that in previous research conducted in the Tibetan area (1, 2), it would be beneficial to have a much higher EEG rate in future studies.

# Study Limitations

This is a hospital-based cohort and a retrospective study. Children younger than 14 years old, who were typically admitted to the Pediatric Department, were not included. This admission bias and our study results may not apply to the entire population in the Ganzi region. In addition, we found that 30.9% patients underwent EEG for epilepsy diagnosis; therefore, the accuracy of seizure semiology may be reduced. Nonetheless, the present study provides useful imformation for future studies and can guide the development of strategies for managing epilepsy in the Tibetan region.

# CONCLUSIONS

This study suggests that the control of endemic infectious diseases in local regions, such as cysticercosis, and hydatidosis, is important for better prevention of epilepsy. In addition, education about seizures for local primary doctors and patients will enable better management of epilepsy in this population.

# DATA AVAILABILITY STATEMENT

The raw data supporting the conclusions of this manuscript will be made available by the authors, without undue reservation, to any qualified researcher.

# AUTHOR CONTRIBUTIONS

DZ designed this study. JiaC and XW carried out this study; those two authors collected data, analyzed data, and contributed equally to this study. JiaC, YH, YD, JieC, WF, and YL helped in data collecting. SL helped analyzed results. ZZ, JP, and JM helped in EEG data analysis, JiaC wrote the draft and XW revised the final version.

# FUNDING

This research was supported by funding from the International Bureau for Epilepsy 2017: The Education and Control Program of Epilepsy in Tibet Area (Item No. HX-H1711139).

### REFERENCES

#### ACKNOWLEDGMENTS

We thank all doctors in the Neurology Department of Ganzi People's Hospital for their hard work in the world's highest plateaus known as the roof of the world.

**Conflict of Interest Statement:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Copyright © 2018 Chen, Wu, He, Li, Deng, Chen, Fang, Zeren, Peng, Li, Mu and Zhou. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

# The Epidemiological Characteristics of Epilepsy in the Province of Khyber Pakhtunkhwa, Pakistan

Shakir Ullah1,2 \*, Niaz Ali 1 , Adnan Khan3 , Saad Ali 3 and Haleema Rehana Nazish1

1 Pharmacology/Institute of Basic Medical Sciences, Khyber Medical University, Peshawar, Pakistan, 2 Center for Neuroscience, Shantou University Medical College, Shantou, China, 3 Neurology Departments, Lady Reading Hospital Peshawar, Peshawar, Pakistan

Previous studies have shown that Khyber Pakhtunkhwa, Pakistan has a high incidence of epilepsy and a high proportion of low socioeconomic background and high treatment gap. Considering the changes over the past 20 years little is known about the current epidemiological characteristics of epilepsy in Khyber Pakhtunkhwa, Pakistan. The current study was focused to find the impact of various contributing factors on the clinical response to anti-epileptic drugs in the KP population, Pakistan. A total of 315 participants aged 19.1 ± 8.6 years were examined. Mean age of the patients was 18 ± 8.1 year. Epilepsy was high in male patients (64.39%) and urban areas (60.1%). Mostly, 88.6% of patients were belonging to low socioeconomic status background. 42.4% patients have positive family history for epilepsy and 42.8% patients had consanguineous marriages. Middle SES class patients (OR, 2.22 [CI, 0.54–9.1]) were slightly associated with controlled response to CBZ and VPA therapy. Absence seizure (OR, 1.16 [CI, 0.59–2.3]), and Complex partial seizure (OR, 1.29 [CI, 0.58–6.3]) showed good response to CBZ therapy while, Myoclonic seizure (OR, 2.23 [CI, 0.05–8.8]) was responsive to VPA therapy. However, non-compliance (R 2 0.82, P < 0.0001) and nature of seizures (R 2 0.83, P < 0.0001) were associated with the high risk for poor response to both CBZ and VPA therapy. Epilepsy was high in male patients and in urban areas. Most patients were belonging to low socioeconomic status. Non-compliance, low socioeconomic and nature of seizures strongly predict poor clinical response of anti-epileptic drugs therapy.

Keywords: epilepsy, demographics, clinical outcome, social profile, socioeconomic status, types of seizures, carbamazepine, valproic acid

# INTRODUCTION

Epilepsy is a neurological disease, which is characterized by recurrent seizures that may occur suddenly without any provoking factors (1). Approximately 70 million people are suffering from epilepsies throughout the world. Epilepsy contributes 1% burden to global diseases while this contribution is 80% in the developing countries (2). Both developing and developed countries have different health-care priorities according to the need for primary prevention, to the recognition of seizures, and access to sustainable and appropriate therapy. Different approaches are used to properly treat epilepsy in the context of regional resources depend on the prevalence of a disease. Diversities in demographic factors such as gender (3), race (4), age (3), geographic area (5), religion, and culture (5) along with provoking factors are associated with poor responsive epilepsy.

#### Edited by:

Ding Ding, Fudan University, China

#### Reviewed by:

Tomas Mesa, Pontificia Universidad Católica de Chile, Chile Edward Bertram, University of Virginia, United States

> \*Correspondence: Shakir Ullah<EMAIL_ADDRESS>
#### Specialty section:

This article was submitted to Neuroepidemiology, a section of the journal Frontiers in Neurology

Received: 14 July 2018 Accepted: 20 September 2018 Published: 06 November 2018

#### Citation:

Ullah S, Ali N, Khan A, Ali S and Nazish HR (2018) The Epidemiological Characteristics of Epilepsy in the Province of Khyber Pakhtunkhwa, Pakistan. Front. Neurol. 9:845. doi: 10.3389/fneur.2018.00845

**65**

Demographic and clinical factors have a crucial role in the management of epilepsy. False believes, low SES have a very important role in the poor management of epilepsy. Similarly, consanguineous marriages among the positive family history for epilepsy can increase the burden of epilepsy in a society. It is well-known that the incidence of epilepsy is linked to low socioeconomic status, positive family histories of epilepsy, limited access to health care system and environmental factors (6). Reports suggest that socioeconomically deprived people are more susceptible to epilepsy. The association is strongly supported by the facts that the incidence of epilepsy is high in developing countries as compared to developed countries (7). Several other studies also corroborate that socioeconomic status (SES), positive family history and poor compliance has an established association with resistant epilepsy (8–10). Health related quality of life (HRQL) is associated to proper response to AEDs and it has been reported that clinical response may vary across epileptic patients with different clinical, demographic, and socioeconomic variations (11, 12). With regard to clinical variables, seizures types and frequency have been found to be significant predictors of HRQL scores and clinical response (5, 13, 14). It has been found that psycho-social factors is also related to quality of life (QOL) in Korean epilepsy patients. Good QOL is directly related to better clinical response to AEDs and recognition of these factors will lead health professionals to develop different strategies to improve the QOL of these patients (15). Common clinical factors, seizure frequency, is closely associated with poor outcomes, QOL, and mortality (16). As these above variables are associated with epilepsy. Therefore, it has been suggested that demographic factors, gender difference, culture aspects, beliefs in different religion, geographic variations, and SES of an patients with epilepsy may also affect clinical response of a drug and will widen treatment gap. These are the factors in which interventions are very easy and treatment response can be improved which can reduce the treatment gap. Therefore, the purpose of the present study is used to examine the impact of these factors on the clinical outcomes of CBZ and VPA therapy in epileptic in Pakhtun population of Khyber Pakhtunkhwa, Pakistan. It will help us to improve the clinical response to carbamazepine and valproic acid therapy by exploring the impact of demographic and social profile of the patients during treatment.

# MATERIALS AND METHODS

#### Study Design and Protocol

Patients with epilepsy sample size (N = 323) was calculated at 95% CI with SD of 8 and marginal error of 1. Keeping in view 20% drop out total patients included in the study were 315. Patients from Khyber Pakhtunkhwa, Pakistan who visited the Neurology Department of Government Lady Reading Hospital for opting treatment were included in the study on consent, assent, and surrogate assent form. The study was started in August 2014 and completed in April, 2016. The study was executed according to the CONSORT flow chart presented in **Figure 1**.

Ethics Board of the Khyber Medical University, Peshawar approved the study via no: DIR/KMU-EB/AC/000047 dated 04/07/2014. The study was conducted according to Helsinki declarations. The data was collected on a standard questionnaire designed according to the protocol of the WHO which includes all those necessary information to reduce the biases in the study.

Mean dose of carbamazepine was 452 mg/day and valproic acid 859 mg/day and was titrated for better management of epilepsy at scheduled visits.

# Inclusion Criteria

Patients with epilepsy who were suitable candidate for carbamazepine and valproic acid as monotherapy or polypharmacy for first time or newly diagnosed for epilepsy were enrolled in the study. Patients with epilepsy who were willing to participate in the study after explaining the steps and the aim of the study in the context of local language were included in the study upon consent and surrogate consent form. Seizures provocative factors were controlled by proper counselling of the patients and ensure proper clinical response to CBZ and VPA as monotherapy or polypharmacy.

# Exclusion Criteria

Patients with epilepsy were not included in the study who was not suitable candidate for carbamazepine and valproic acid as monotherapy or polypharmacy. Patients were also excluded from the study that was not willing to participate in the study or were suffering from some other co-morbidity. Non-compliant patients were also excluded from the study.

# Assessments of Seizure Characteristics and Socioeconomic Status

Types of epilepsy were diagnosed according to guidelines of International League against Epilepsy (ILAE), witness presentation; family history; EEG report and expert opinion of concern OPD/Ward neurologists. All available information was considered in the classifications of seizures, including the medical record, interviews, EEG, and brain imaging. The SES of patients was are classified as reported by Association American Psychology report 2011 (17). The higher Socioeconomic class will have 50–100 and above, Middle class 15–49, and Low economic class will have below 15 Per capita monthly income limits (Rs.) (17).

#### Measurement of Seizure Control in Patients With Epilepsy

Seizures' control was recorded in the form of reduction of frequencies of seizures using standardized proforma in local language. The numbers of seizures per week were recorded at baseline and these patients were then evaluated for seizure control at 3rd and 6th month of the therapy. The patients with epilepsy compliance to the medication were checked by counting the pills remaining in the strip on scheduled visits. Drug response was determined as either freedom from seizures or reduction in

**Abbreviations:** AEDs, Anti-epileptic drugs; CBZ, Carbamazepine; HRQL, Health related quality of life; ILAE, International League against Epilepsy; QOL, quality of life; SES, Socioeconomic Status; VPA, Valproic Acid.

number of seizure per week after initiation of carbamazepine and valproic acid therapy at 3rd and 6th month.

#### Statistical Analysis

The distribution of age, clinical, and demographic characteristic of epileptic patients were presented in the form of percentage. The Mean ± SD were determined for age. The expected outcome (EXP B) was determined using univariate logistic regression analysis. The prediction of clinical response with respect to all variables was determined using multivariate regression analysis by incorporating step by step variables and to determine the combined as well as individual impact of each variable on clinical outcomes of CBZ and VPA as monotherapy or polypharmacy. SPSS 20 and Stata version 12 was used for analysis.

# RESULTS

# Demographic Features of Epileptic Patients

Mean age of epileptic patients were 19.1 ± 8.6 years (range 1– 42) (**Table 1**). The prevalence of epilepsy was higher in patients having age of 10–20 years. Epilepsy was high in male (64.39%)

TABLE 1 | Age and gender wise distribution of epilepsy in 315 epileptic patients.

TABLE 2 | Demographic and clinical features of epileptic patients.

\*Grading system of educational level.

than female (35.98%) patients in study population (**Table 2**). The percentage of cousin marriage and positive family history of epilepsy was (42.8%) and (42.4%) (**Table 2**). The level of education was very low in these patients. Furthermore, these patients were having low socioeconomic status (SES) (88.63%).

#### Phenomenology

Types of seizures were classified according to the ILAE guidelines and clinical presentation of patients with epilepsy. Generalized tonic clonic seizures was commonly observed 153 (57.9%) during this study in epileptic patients of KP (**Figure 2**). Other types of seizures were Generalized tonic seizures 25 (9.5%), Absence seizures 19 (7.2%), Generalized atonic 8 (3.4%) secondary generalized complex seizures 22 (8.3%), simple partial seizures 9 (3.4%), and complex partial seizures 17 (6.4%) were found in the target population (**Figure 2**).

TABLE 3 | Impact of different factors on clinical response of CBZ and VPA therapy.

The reference category is poor controlled seizure, EXP (B) mean expected value of prediction using univariate regression analysis.

## Impact of Demographic and Clinical Features vs. Response of Anti-epileptic Drugs

The association of different demographic variables with clinical outcome were determined using univariate logistic regression analysis (**Table 3**). It has been found that high education level were slightly associated with controlled response (OR, 1.84 [CI, 0.50–6.7]) to CBZ and VPA as monotherapy or polypharmacy. Similarly, good SES was also associated with good response (OR, 2.22 [CI, 0.54–9.1]) to CBZ and VPA as monotherapy or polypharmacy. Absence seizure (OR, 1.16 [CI, 0.59–2.3]), Complex partial seizure (OR, 1.29 [CI, 0.58–6.3]), and Myoclonic seizure (OR, 2.23 [CI, 0.05–8.8]) showed a good response to CBZ and VPA as monotherapy or polypharmacy (**Table 3**).

Similarly, multivariate regression models were applied to demographic variables to predict clinical response of CBZ and VPA as monotherapy or polypharmacy in epileptic patients. Over all the factors added in Model 9 (R 2 0.82, P < 0.0001) and model 10 (R 2 0.83, P < 0.0001) were found to increase the risk of poor response to AEDs.

#### DISCUSSION

In our study the prevalence rate of epilepsies was high in male patients where most of them were belonging to urban areas. Our observations were in line to Khan et al. (18) that epilepsy was high in male population and urban areas (18). Though we found an inverse figure as reported by Mohammad et al. where epilepsy is more prevalent in female than male patients in Iran (19). We found that epilepsy was common in patients having age group of 10–20 years. These findings were in line with the reports of previous studies conducted in Pakistan and India (20–26). The percentage of consanguineous marriages and positive family history of epilepsy was high in the study population. The SES of most patients observed in our study was very low. Heaney et al. observed that low SES has an association with epilepsy because of low quality of life increased the susceptibility of an individuals to different provoking factors of epilepsy (27). Other studies also show that low SES was associated with an increased risk for epilepsy in individuals with seizures of unknown etiology (28–35). Luengo et al. reported that genetic history of patients with epilepsy have a 2.5 times higher risk for epilepsy in their coming generation (36). GTCS was common among other types of seizures in the target population which is in line with Nowshad et al. observations (18). It has been found that clinical outcome in patients with absence seizures and myoclonic seizures were good compared to other types of seizure. Poor response was also high in non-compliance patients. Regression model showed that poor compliance and nature of seizures significantly associated with poor response to CBZ and VPA as monotherapy or polypharmacy even at optimal adjusted dose of CBZ and VPA as monotherapy or polypharmacy. Thomson et al. (38) stat that compliance has a direct association with seizure response (37, 38). Dragoumi et al. (39) demonstrate that types of seizure also affect clinical outcome in epileptic patients (39). If patients show non-compliance with therapy and do not take their AEDs according to prescription then it leads to poor clinical outcome. Similarly, low SES of patient is also a hurdle in proper compliance and lead to poor clinical response. Low SES also responsible for large treatment gap. Nature of seizure is also a main factor for resistant epilepsy.

#### REFERENCES

## LIMITATIONS AND STRENGTH OF THE STUDY

It is a hospital based study which doesn't show the clear picture of whole society. It has an advantage that this study will provide the platform to conduct a community based study to determine the prevalence and stigmatization of epilepsy.

#### CONCLUSION

Male patients were having a high prevalence (64.39%) of epilepsies where 60.1% were from urban areas. Epilepsy was high in patients with age ranging 11–20 years. Positive family history of epilepsy and consanguineous marriages was the key characteristics of the epileptic patients. Non-compliance and nature of seizures strongly predict poor clinical response of Anti-epileptic drugs therapy.

# AVAILABILITY OF DATA AND MATERIAL

The analyzed dataset during the current study will be provided from the corresponding author on reasonable request.

# AUTHOR CONTRIBUTIONS

SU carried out experimental work as Ph.D. Scholar. Also prepared the 1st draft of manuscript. NA extensively revised the manuscript. He is also designed the study and wrote the research project. AK helped in diagnosis and patients follow up for their seizures control. SA helped in Clinical scoring of epilepsies. HN helped in experimental work. All authors approved the final version of manuscript.

### FUNDING

The authors want to thank the Directorate of Science & Technology, Govt. of Khyber Pakhtunkhwa for providing funds for some of the consumable items (chemicals and plastic wares) involved in the study. Grant No: dirrtt:/S&T/KPK/BT/RP/FS/2013-14 (1592).

# ACKNOWLEDGMENTS

The authors also want to thank AK and then Chief Executive of Govt. Lady Reading Hospital Peshawar for providing opportunity for research work.

**Conflict of Interest Statement:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Copyright © 2018 Ullah, Ali, Khan, Ali and Nazish. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

# High Low-Density Lipoprotein Cholesterol Inversely Relates to Dementia in Community-Dwelling Older Adults: The Shanghai Aging Study

Fen Zhou1,2,3, Wei Deng1,2,3 \*, Ding Ding4,5 \*, Qianhua Zhao4,5, Xiaoniu Liang4,5 , Fei Wang1,2,3, Jianfeng Luo1,2,3, Li Zheng4,5, Qihao Guo4,5 and Zhen Hong4,5

*1 Department of Biostatistics, School of Public Health, Fudan University, Shanghai, China, 2 Key Lab of Health Technology Assessment, National Health and Family Planning Commission of the People's Republic of China (Fudan University), Shanghai, China, 3 Key Laboratory of Public Health Safety of Ministry of Education (Fudan University), Shanghai, China, 4 Institute of Neurology, Huashan Hospital, Fudan University, Shanghai, China, 5 National Clinical Research Center for Aging and Medicine, Huashan Hospital, Fudan University, Shanghai, China*

#### Edited by:

*Alessandra Solari, Istituto Neurologico Carlo Besta (IRCCS), Italy*

#### Reviewed by:

*Maurizio A. Leone, Casa Sollievo della Sofferenza (IRCCS), Italy Tetsuya Asakawa, Fujian University of Traditional Chinese Medicine, China*

#### \*Correspondence:

*Wei Deng<EMAIL_ADDRESS>Ding Ding<EMAIL_ADDRESS>
#### Specialty section:

*This article was submitted to Neuroepidemiology, a section of the journal Frontiers in Neurology*

Received: *02 May 2018* Accepted: *24 October 2018* Published: *12 November 2018*

#### Citation:

*Zhou F, Deng W, Ding D, Zhao Q, Liang X, Wang F, Luo J, Zheng L, Guo Q and Hong Z (2018) High Low-Density Lipoprotein Cholesterol Inversely Relates to Dementia in Community-Dwelling Older Adults: The Shanghai Aging Study. Front. Neurol. 9:952. doi: 10.3389/fneur.2018.00952* Background: The relationship between cholesterol and cognitive function is unclear from the previous studies. This study was conducted to explore this association in older Chinese adults.

Methods: Data were from the Shanghai Aging Study, comprising 3,836 residents aged 50 years or over in an urban community. Diagnoses of dementia and mild cognitive impairment were established according to the fourth edition of diagnostic and statistical manual of mental disorders (DSM-IV) and Petersen criteria. Multivariate logistic regression models, non-matched and propensity score (PS) matched, were used to examine the association between cholesterol levels and cognitive function.

Results: There was a significantly higher proportion of participants with low levels of total cholesterol (TC) and low density lipoprotein cholesterol (LDL-C) in the dementia group than in groups without dementia (*P* < 0.05). High LDL-C level was inversely associated with dementia, with a negative trend in the PS matched model. TC and high density lipoprotein cholesterol (HDL-C) were not significantly related to dementia in either non-matched models or PS matched models.

Conclusion: Our result indicates that high level of LDL-C is inversely associated with dementia. High level of LDL-C may be considered as a potential protective factor against cognition decline.

Keywords: low density lipoprotein cholesterol, cognitive impairment, dementia, population-based study, propensity score matching

# INTRODUCTION

Dementia is a progressive brain disorder accompanied by a deterioration in memory and thinking, and often with a decrease in motivation and emotional and language problems. It has physical, mental, economic, and social effects, not only on the patients but also on their careers, their families and society at large. Worldwide, there are over 50 million prevalent dementia cases, and

**71**

nearly 10 million new cases are diagnosed per year (1). It is estimated that the existing worldwide costs of dementia are US \$818 billion in 2017 and that it will become a trillion-dollar disease by 2018—equivalent to the world's 18th largest economy (2).

The brain contains 25–30% of the total body cholesterol. As one of the most essential components of neurons, cholesterol is of great importance to develop and maintain neuronal plasticity and function (3). Studies from western countries have investigated how cholesterol levels related to cognitive impairment, but have yielded inconsistent and conflicting results. In China, only a few studies have investigated the relationship between cholesterol and cognitive impairment among older community residents. The results were varied due to different study design and target population. A cross-sectional study with 597 participants in southwest China has reported that low (TC) (OR = 0.94) and (LDL-C) (OR = 0.94) were associated with mild cognitive impairment (MCI) after adjusting for age, gender and education (4). Another cross-sectional study involving 2,000 community dwellings in eight longevity areas found that higher cholesterol, including TC (OR = 0.73), LDL-C (OR = 0.82), and (HDL-C) (OR = 0.81), was associated with better cognitive function in the oldest old (5). Furthermore, an inversely U-shaped effect of TC on cognition has been observed in a multicenter study with 1,889 people aged 65 years and over from four rural counties (6).

The Shanghai Aging Study is a population-based cohort study with a design, operational procedures and diagnostic criteria similar to most cohort studies in western countries. The study is intending to examine the prevalence and incidence of dementia and MCI as well as their risk and protective factors (7). We analyzed the baseline data of this cohort to explore the association between cholesterol levels and cognitive function among older community dwellers in urban China.

# METHODS

#### Study Population

Between January 2010 and December 2012, 3,836 residents aged ≥50 years were recruited from Jingansi community in central downtown Shanghai, China. Participants were excluded from the study if they were (1) living in nursing homes or other institutions; (2) experiencing mental deficiency or severe schizophrenia, according to their medical record or diagnoses from neurologists; or (3) having severe impairment of hearing, vision, or verbal such that could not accomplish the neuropsychological evaluation. Detailed recruitment procedures were published previously (8).

This study was approved by the Medical Ethics Committee of Huashan Hospital, Fudan University, Shanghai, China (approval number: HIRB2009-195). All participants and/or their legally acceptable representatives provided signed written informed consent to participate this study.

#### Characteristics and Medical History

Demographic and lifestyle characteristics were collected via a face-to-face questionnaire survey. Data included age, sex, education background, and smoking habits (smoking: regular cigarette smoking more than 1 year). Participants' heights and weights were measured and were used to calculate body mass index (BMI). History of hypertension, diabetes, stroke, and coronary artery disease was collected and confirmed with participants' medical records.

# Laboratory Test

A 2-ml blood sample was collected from each participant by research nurses in the morning after 12 h overnight fasting. Blood samples were sent to the central laboratory in Huashan hospital. Cholesterol profiles were measured from serum by Hitachi 7600 full automatic biochemical analyzer. TC was measured with oxidase method and LDL-C and HDL-C were measured with direct method.

## Neuropsychological and Neurological Assessments

Considering different cultural backgrounds, neuropsychological tests from western countries were translated, adapted and normalized for the Chinese population. Tests applied in our study evaluated global cognition, attention, memory, language, executive function, and spatial construction function of each participant. The tests included: (1) the Mini-Mental State Examination (MMSE); (2) the Conflicting Instructions Task (Go/No Go Task); (3) the Stick Test; (4) the Modified Common Objects Sorting Test; (5) the Auditory Verbal Learning Test; (6) the Modified Fuld Object Memory Evaluation; (7) the Trailmaking test A&B; (8) the Renminbi (official currency of China) Test, translated from the EURO test. Participants with at least 6 years of education were given tests 1 to 5 and 7, whereas those with <6 years of education were given tests 1 to 4 and 6 and 8 (**Figure 1**). Normative data and more details on these tests were reported elsewhere (8).

The motor responses and reflexes of participants were examined by neurologists. The Center for Epidemiologic Studies Depression and Scale (CESD) (9) was used to evaluate the depression status of participants within the previous week and a major depressive episode was defined as CESD ≥ 16. The Clinical Dementia Rating (CDR) (10) and the Lawton and Brody Activity of Daily Living (ADL) (11) were also conducted to assess the cognitive complaints and the ability to perform activities of daily living and physical self-maintenance.

# Diagnoses of Cognitive Function

The consensus diagnosis of cognitive function was made by an expert panel (two neurologists, one neuropsychologist, and one neuro-epidemiologist) on the basis of all available information for each participant, including medical, neuropsychological, and neurological data and where possible computed tomography (CT) and magnetic resonance images (MRI) scans. Dementia was diagnosed according to DSM-IV criteria (12). A diagnosis of MCI, referring to Petersen's criteria (13), was considered only for subjects without dementia.

#### APOE Genotype Assessment

Because the APOE-ε4 variant has been found as the largest known genetic risk factor for Alzheimer's disease in a variety of

ethnic groups (14), we assessed APOE genotype in our cohort. The extraction of DNA from blood or saliva samples and the assay of APOE genotyping through the TaqmanSNP method (15) were conducted in the central laboratory in Huashan hospital. APOE-ε4 positivity was defined by the occurrence of one or two ε4 alleles.

# Statistical Analysis

TC, LDL-C, and HDL-C values were divided into three levels according to tertiles (5): low TC level, <4.87 mmol/L; moderate TC level, 4.87–5.72 mmol/L; high TC level, >5.72 mmol/L; low LDL-C level, <2.9 mmol/L; moderate LDL-C level, 2.9–3.7 mmol/L; high LDL-C level, >3.7 mmol/L; low HDL-C level, <1.17 mmol/L; moderate HDL-C level, 1.17–1.45 mmol/L; and high HDL-C level, >1.45 mmol/L. Differences in characteristics among groups with different cognition status (normal, MCI, or dementia) were evaluated with Kruskal–Wallis test for continuous variables and Cochran–Mantel–Haenszel statistics for categorical variables (16, 17).

Multivariate logistic regression models (non-matched models) were used to examine the association between cholesterol levels and cognition status (MCI or dementia compared with normal). TC, LDL-C, and HDL levels were included in the logistic model as dummy variables, with the low level group used as the reference to estimate the odds ratio (OR) and 95% confidence interval (CI), and with adjustment for covariates such as age, sex, years of education, BMI, depression, diabetes, stroke, hypertension, and APOE-ε4 genotype.

PS methods are being used to reduce the impact of treatment selection bias in the estimation of causal treatment effects using observational data (18). They also can be used to estimate the relationship between any non-randomized factors (19). The PS is the estimated conditional probability of assignment to a particular "treatment" given a series of vector covariates (20). In our study, PS matching was applied to reduce the bias due to nonrandom exposures (cholesterol levels) and potential confounders; the conditional probability of assignment to moderate levels of cholesterol was chosen as the PS. Matching was conducted in a stepwise fashion. First, we matched the PS in the low cholesterol group with those in the moderate cholesterol group, with 0.05 as a caliper (upper limit of the allowed difference in the scores). Subsequently, the scores in the high cholesterol group were matched to the effective matched pair from the first step, and the mean difference between the high cholesterol group and the other two groups was also not allowed to exceed 0.05. Finally, we obtained matched subgroups with a more balanced distribution of covariates. Multivariate logistic regression models (PS matched models) were then performed by using the PS matched subgroups in a sensitivity analysis, with adjustment of the same covariates as in non-matched models.

All analyses were conducted by SAS 9.4 (SAS Institute Inc., Cary, NC, United States). P < 0.05 (two-tailed) were considered statistically significant.

#### RESULTS

The full analysis set of this study comprised 3,467 participants who had completed diagnosis of cognition and measurement of TC, LDL-C, and HDL-C (**Figure 1**). As shown in **Table 1**, the analyzed participants were characterized by old age (mean [SD] = 69.4 [8.0] years old) and relatively high level of education (mean [SD] = 11.7 [4.0] years). Among them, 86 were diagnosed as dementia and 603 were diagnosed as MCI. The characteristics of age, education, MMSE, history of stroke, hypertension, diabetes, depression, and APOE-ε4 allele carrier status were significantly different among the normal, MCI, and dementia groups.

Significant differences in the category distributions of TC and LDL-C were found in groups with different cognition status. The lowest proportion was found in participants with dementia who had high level of TC (26.7%) and LDL-C (23.3%).

After adjusting for age, sex, years of education, APOE-ε4 genotype, BMI, depression, diabetes, hypertension and stroke, the non-matched models indicated that the OR of high LDL-C level (OR [95%CI] = 0.56[0.30, 1.04]) had an upper limit awfully close to the null threshold. A U-shaped relationship between HDL-C levels and ORs was found (P for trend = 0.0084) despite of the non-significant effect of HDL-C on dementia when the low level acted as the reference (**Table 2**). The effects of all the covariates adjusted were showed in the **Supplementary Material**.

The inverse associations were significant between high LDL-C level and dementia, whereas the associations between TC or HDL-C levels and dementia remained non-significant, according to the PS matched models (**Table 2**). In addition, the effect of LDL-C was higher than that in the non-matched

#*Comparison among groups with different diagnoses of cognition.*

\**P-value* < *0.05,* \*\**P-value* < *0.01.*

*MCI, mild cognition impairment; BMI, body mass index; MMSE, Mini-Mental State Examination; APOE-*ε*4 (*+*), ApoE-*ε*4 allele positive; ADL, Activities of Daily Living; TC, total cholesterol; LDL-C, low-density lipoprotein cholesterol; HDL-C, high-density lipoprotein cholesterol.*

*TC: low,* <*4.87 mmol/L; moderate, 4.87–5.72 mmol/L; high,* >*5.72 mmol/L.*