Karabük is a city in the western Black Sea region located at latitude 40.08°N and longitude 32.04°E. The city is at an altitude of 280 m and covers an area of 760 km². Most of its surface is covered by trees. In winter it is cold and snowy while in summer it is hot, and the mean annual rainfall is 780 mm. The population of Karabük is highly homogeneous, comprising mainly Turkish people, and there is little migration. Most people are laborers, with most households including a worker at the local iron-and-steel factory or a retiree from that factory. Farmers constitute a small proportion of the population. The mean gross domestic product (GDP) per capita in Karabük was US$ 5,800 in 2014, and the mean duration of education is 6.7 years. There are 6 hospitals and 234 specialist doctors in the province. There is easy access to hospitals, specialists, and magnetic resonance imaging (MRI) devices. Karabük was built in the 1930s as the seat of the iron and steel industry in Turkey. There are also chemical plants that produce sulfuric acid and phosphates, and the Zonguldak coal fields are nearby.

One of the main steel producers in Turkey, named Kardemir (Karabük Iron and Steel Works), is located in Karabük. It has been in operation continuously since being constructed in 1937, and is the main source of local air pollution. According to the 2014 census, the population of the city center was 132,658.1415 The population pyramid for Karabük is shown in Fig. 1.

Akçakoca is a small city located 100 km from Karabük and at the same latitude, and the racial and geographical characteristics of the two cities are similar. Akçakoca was chosen for inclusion in this study because it does not have any factories. Akçakoca is a coastal town in the western Black Sea region located at latitude 41.05°N and longitude 31.07°E. It has a 35-km-long coastline along the Black Sea and covers an area of 462 km². Two-fifths of its surface is covered by trees, most of which are nut trees. In winter it is rainy while in summer it is mild, and the mean annual rainfall is 990 mm. Like Karabük, the population of Akçakoca is highly homogenous, comprising mainly Turkish people and with little migration. Most of the population grows plant nuts, while some of the inhabitants are fishermen. The mean GDP per capita in Akçakoca was US$ 6,500 in 2014 and the mean duration of education is 6.4 years. There are 8 hospitals and 312 specialist doctors in the province, with these facilities reachable within 20 minutes by all of the populace. There is easy access to hospitals, specialists, and MRI devices. Few local tourist visit Akçakoca in the summer, and there are no officially reports of air pollution causing health problems. According to the 2014 census, 24,161 people lived in Akçakoca.1516 The population pyramid for Akçakoca is shown in Fig. 2. Geographical locations of Karabük and Akçakoca in Turkey map are shown in Fig. 3.

We used validated screening forms that we had prepared for our prevalence study conducted in Maltepe, Istanbul in 2002.5 The questionnaires comprised 26 questions in 2 sections. The first section assessed knowledge about MS, while the addressed MS symptoms. The questions enquired about the presence of present and past symptoms, motor and sensory dysfunction, walking ability, sphincter control, sleep, balance disturbances, ataxia, depression, increased fatigability, and memory disturbances. The teams conducting the field interviews received practical training. The interviewers were informed about the steps of the procedure that were to be followed; that is, which doors to knock on, what to do when nobody is home, the methods of intervention, how to present the questions, and how to fill in the forms.

Based on the population of Karabük city center being 132,658,15 the required sample was calculated as 29,790 for a confidence level of 95% and a margin of error of 0.5%. We therefore visited every fourth household. The houses to be visited were determined beforehand, and in cases where no one was at home, the same house would be visited later. The study was conducted in Karabük between April 1 and 30, 2014.

Based on a population of 24,161 for Akçakoca,15 the required sample was calculated as 14,833 for a confidence level of 95% and a margin of error of 0.5%. We therefore visited every second household. The houses to be visited were determined beforehand, and in cases where no one was at home, the same house would be visited later. The study was conducted in Akçakoca between May 25 and June 24, 2015.

In order to encourage participation in both cities, an announcement was made at a public health center and at a council meeting 1 week prior to the study commencement to inform the citizens.

Four teams of six persons each (a neurology assistant, a nurse, and four interviewers per team) were established in order to obtain reliable and precise data with a correct provisional diagnosis. The teams were provided with both the theoretical and practical training by neurologists and public health specialists for 2 days on aspects. The teams conducted their visits between 8:30 a.m. and 5:00 p.m. both on weekdays and over the weekend.

Interviews with children or elderly where there were communication difficulties were performed with the help of the persons responsible for their care. The field study lasted approximately 3 weeks.

The field study comprised two phases:

1) The questions were first asked directly and the questionnaires were completed. Persons who answered affirmatively to one or more of the questions in the section about MS symptoms were examined by the neurology assistant in the team.

2) Persons who showed signs of having MS were subsequently re-examined by an experienced neurologist. Those unable to come to their local hospital were examined in their homes.

The previous medical results of patients including their MRI images were studied. All laboratory and examination findings were recorded. If needed, further investigations (e.g. immunological screening or MRI) were conducted at a local hospital. The McDonald Criteria were used for diagnosing of MS.17 Kurtzke's Expanded Disability State Score (EDSS) was used to assess the level of neurological impairment.18 The medical records of patients diagnosed with MS were checked at the local hospital and compared with the screening results.

SPSS software (version 11.5, SPSS Inc., Chicago, IL, USA) was used for calculating frequency distributions and percentages. Minitab statistical software (version 16, SPSS Inc.) was used for calculating the Poisson confidence intervals and comparing prevalence rates.

In total, 29,586 inhabitants of Karabük were visited, and 28,127 answered the questionnaires (1,459 declined to participate). Neurological examinations revealed suspected MS in 31 people. One female patient was diagnosed by our team. She was 22 years old and had experienced two clinical attacks: the first was optic neuritis and in the second she had sensorial and autonomic symptoms. Her EDSS was 2.5. MRI was performed in Karabük State Hospital: cranial MRI revealed five or six T2-weighted hyperintense periventricular lesions, and her spinal MRI findings were normal.

In total, 27 patients were diagnosed with clinically definite multiple sclerosis (CDMS). Other suspected cases were diagnosed with migraine, dementia, normal pressure hydrocephalus, and posttraumatic encephalomalacia. There were 17 patients diagnosed with relapsing-remitting multiple sclerosis (RRMS), 7 with secondary progressive multiple sclerosis (SPMS), and 3 with primary progressive MS. The female/male ratio was 1.2 (15 females). The patients were aged 39.5±10.2 years (mean±SD), their age at disease onset was 31.1±9.6 years, and the EDSS was 3.37±1.70. Nineteen patients were receiving immunomodulatory treatments and two had a family history of MS.

Cerebrospinal fluid (CSF) examinations were performed on 18 patients, of which 12 had a positive oligoclonal band (OCB). Visual evoked potentials (VEP) were measured in 16 patients, and 12 showed prolonged P100 latency. Somatosensory evoked potentials were measured in 12 patients of whom 5 displayed abnormal results. Brainstem auditory evoked potentials (BAEP) were measured in 12 patients, with abnormal results found in 6.

Cranial and spinal MRI was performed in 27 patients. All of them exhibited demyelinating lesions in the brain that fulfilled the 2010 McDonald Criteria. Two patients had cervical spinal demyelinating lesions; these patients were negative for the aquaporin-4 antibody.

The raw prevalence rate of MS in Karabük was 95.9/100,000 and it was 107.1/100,000 after age standardization.

Table 1 presents air pollution data (PM₁₀, SO₂, and NO₂) from the National Air Pollution Monitoring Network System for Karabük city and also the World Health Organization (WHO) normal threshold values. The trace-element values in the area surrounding the factory as well as in the control area 30 km away are listed in Table 2.

In total, 13,793 inhabitants of Akçakoca were visited, and 12,992 completed the screening form (803 declined to participate). Neurological examinations revealed suspected MS in ten people. A 15-year-old female patient had blurred vision and a urinary emergency. Her complaint had begun 1 month previously. Her brain MRI findings were normal, and she was diagnosed with optic neuritis. She was not diagnosed with MS because she did not fulfill the 2010 McDonald Criteria. The other suspected three cases were diagnosed with Chiari I malformation, migraine, and Behçet's disease; they were also not diagnosed with MS.

Six patients were diagnosed with CDMS. One patient was male (female/male ratio of 5.0), and four patients had RRMS while two had SPMS. The patients were aged 47.5±9.9 years, they had a disease duration of 17.8±7.7 years, their age at disease onset was 32.2±4.5 years, and the EDSS was 4.4±4.0. Two patients were taking immunomodulatory drugs.

The CSF was analyzed in two patients, which produced normal findings. VEP, BAEP, and aquaporin-4 antibody results were not obtainable.

Cranial MRI was performed in all of the patients, which revealed multiple demyelinating lesions in all of them. Their cranial MRI findings supported an MS diagnosis according to the 2010 McDonald Criteria. Cervical MRI was performed in two patients, for which the findings were normal.

The raw prevalence rate of MS in Akçakoca was 46.1/100,000, it was found to be 41.5/100,000 after age standardization.

In Karabük and Akçakoca collectively, 43,379 people were visited and 41,119 of them completed the questionnaire form (the other 2,262 persons declined to take part in the study). CDMS was diagnosed in 33 people. Their demographical and clinical characteristics are presented in Table 3, their initial symptoms of are shown in Table 4, and their laboratory findings are summarized in Table 5.

One of the most important strengths of this study was its design as a door-to-door study with a high participation rate. No hospital records are kept in outpatient hospital clinics in Turkey, except in university and teaching hospitals. The initial diagnosis in outpatients is mostly made based on the immediate clinical findings without any further investigations. In addition to this, outpatients are mostly seen at their next visit in another institution or in private offices, where medical records are unreliable. These factors mean that the results from a prevalence study using hospital records are unlikely to be highly reliable. This study was carried out with assistance from a local neurologist. All but one of the patients were diagnosed by at least two neurologists and at two medical centers; only one patient was diagnosed by us. Another strength was the use of a detailed questionnaire that covered all MS symptoms, and all patients who answered yes to at least one question were examined twice.

This study was subject to several limitations. First of all, the symptoms were reported by the patients themselves, which could have underestimated the prevalence of MS. Another limitation is that other diseases that mimic MS such as neuromyelitis optica spectrum disorder and neurodegenerative diseases can be mistaken for MS. It may be impossible to exclude some of these diseases by applying the McDonald Criteria. Another limitation was that fewer people were screened in Akçakoca.

Furthermore, due to lack of data, other environmental confounding factors that may affect the etiology of MS were not investigated in this study: urbanization, smoking habits, working practices, vitamin D levels, nutrition, vaccinations, viral infections such as the Epstein-Barr virus, cytomegalovirus, and varicella-zoster virus. There may also have been some other unknown environmental factors that should have been taken into consideration.

In conclusion, the significant difference in the prevalence rate of MS between Karabük and Akçakoca suggests that air pollutants affect the pathogenesis of MS. However, larger epidemiological studies are needed to confirm this hypothesis.