1, Kotzamani

NEUROLOGY/2011/428276

Appendix e-1

e- Results

In determining the diagnostic delay (the time from the appearance of the first symptoms attributed to MS to the disease’s diagnosis), we took into consideration the fact that the first MRI was installed and became operational on Crete in 1993. As shown in table e-2A and e-2B, the diagnostic delay became considerably shorter after 1993. These results are consistent with those obtained elsewhere (e1). Prior to 1993, the residents of Crete were usually referred to diagnostic centers in Athens in order to have their MRIs performed.

To test the effect of living in either an urban or rural setting on the variables studied, we analyzed our data on MS patients and controls taking into consideration the current place of residence. As shown in table e-3A, these comparisons for urban residents, yielded results that were similar of those obtained using the entire MS and control sample. These comparisons for rural residents only yielded statistically significant differences for smoking, alcohol use and sunbathing (table e-3B).

While the present study included all MS patients with established residency on Crete for at least one year prior to the disease onset, MS can exist subclinically for several years prior to the appearance of the first symptoms. Hence, to avoid including MS patients whose disease might have started while living outside of Crete, we performed additional sensitivity analyses taking into account MS patients and controls who lived on Crete for at least 15 years prior to the disease onset. The new analysis yielded data (table e-4), which were consistent with those obtained for the entire MS and control sample, except that differences in contraceptive use and age at first childbirth did not reach significance.

e- Supplementary Data on Crete

Crete: geography and climate

The island of Crete is situated in the southwest part of Greece, lying approximately 160 km south of the Greek mainland (latitude, 35°15'N; longitude, 25°00' E). It is the largest of the Greek islands and the fifth largest island in the Mediterranean Sea, covering an area of 8,336 km², which is 6.3% of the total area of Greece. The island has a long coastline with numerous beaches and is rugged and mountainous, with few valleys cutting deep passages through the mountains. The climate is warm, during most months of the year, with long periods of sunshine. It has been estimated that there are 7.75 hours of sunshine per day on the average, ranging from 4 hours per day in December to 12 hours per day in June; the mean annual temperature is 20oC, ranging from average 12oC in December to 27o C in August.

Population and economy

The population is Caucasian and its genetic makeup has remained rather stable during the past decades. During the study period, the population has increased from 502.191 in 1981 to 601.131 in 2001 (Greek National Statistical Service, 2001 national census). Females number 296.729 and males number 304.402. The economy of Crete, which was mainly based on farming, began to change visibly during the 1970s. While an emphasis remains on farming and stock breeding, due to the climate and terrain of the island, there has been an observable expansion in its service industries (mainly tourism-related).

Health care services

In Greece, health insurance is essentially universal, as it is provided by several certified health organizations run by the State. From the administrative point of view, Crete is divided in 4 prefectures (Heraklion, Chania, Lasithion and Rethymnon). With respect to the delivery of health care to the rural population on Crete, the Department of Social Medicine of the University of Crete has found this to be generally better than that of other regions of Greece (e2). That study (e2) reported that primary care to the rural population is provided by 157 State run rural Medical Services and 17 Health Centers located throughout the four prefectures of the island. Over the past 20 years, public transportation has expanded throughout Crete, permitting individuals with functional impairments and older persons to have better access to health services. A variety of services are provided to the rural population, including free health care, transportation to community health centers, home care and social support. Regarding hospital health care, each of the four prefectures of Crete has at least one State hospital that employs one or more Neurologists. Even the most remote villages are no more than 35 miles (about 60 Km) from a State Hospital. In Greece regulations require that medical care (including medications) for patients with chronic diseases such as MS be provided by State hospitals. As they are no private hospitals on Crete with a Neurology service, all MS patients living on the island are followed by the State hospitals regardless of whether they are living in urban centers or in the countryside. Also, it should be noted that, in Greece, people can see a medical specialist without needing a referral from a general practitioner.

Health condition, immunization, social and environmental changes

Over the past three decades, the population of Crete underwent profound socio-economical changes as indicated below. From the health point of view, improvement in sanitary conditions, including the use of electric refrigerators and home detergents, has changed the epidemiology of bacterial, parasitic and viral diseases both in the countryside and the urban centres alike. Childhood mortality and life expectancy for at least the past three decades have been comparable to those of Northern Europe. During this time, childhood immunization, based on Northern European and American standards, has become universally available to the population of the island. Historically, mass immunization against diphtheria, tetanus and pertussis began in the early 1950s. Vaccination against the polio virus began in the early ‘1960s. MMR was introduced in the late 1970s, vaccination against haemophilus in the late ‘1980s and obligatory infant HBV vaccination in 1998. Also, since 2001 immunization against meningococcal group C oligosaccharides conjugate has been widely applied in children and adolescents.

Smoking, which was once largely limited to males, has become quite common among females even in their teens. The use of synthetic materials, organic dye staffs, cosmetics and devices producing electromagnetic fields has been widespread. The use of a wide variety of fertilizers, pesticides and other plant protection products spread to the farming regions of the island in the late 1970s. Chlorination, fluorination and ozonation of the water supply have become widely available. There has been substantial improvement in living standards both in the cities and in the countryside, with the per capita income, that was once a small fraction of the mean European average, now approaching that of developed Northern European Regions. Literacy rates have increased steadily and changes in lifestyle, such as getting married at an older age and pre-marital relations, are common practices among the population of the island. Construction and pollution from car emissions have increased steadily during this time.

Diet

Concerning food consumption, substantial changes have occurred in the dietary habits of the Cretan population during the past decades, with a gradual abandonment of the traditional Cretan-Mediterranean diet along with the increasing use of food additives. Comparison of the nutrient intake of the Cretan population in 60s to that of 90s indicates a significant shift toward higher saturated fat and lower mono-unsaturated fat. Also meat consumption has increased from 35 gr to 150 gr per day whereas vegetable consumption has decreased from 600-700 gr to 300 gr per day. While in the past most food was produced locally (nearly all dairy products were from goat or sheep milk), recent decades have seen an increase in food imports, particularly of animal products.

e- Discussion

Most of the changes related to urbanization are reflected in our population of MS patients when compared to the preceding generations. Over 70% of our MS patients were born to parents that originated from the villages of Crete, with a sizable number of these patients having relocated during their childhood years, or later on in life, from a rural setting to urban centers. Study of the MS families’ migration pattern, revealed that while over 85% of the grandparents of MS patients were rural residents, about 40% of their children (parents of our MS patients) and only 25% of their grandchildren (our MS patients) remained in a rural setting. As such, one of the most remarkable findings of this study is that a transition to urbanization over a course of three generations occurred concurrently with a marked rise in MS frequency in this population. Living in an urban setting was associated with a higher disease frequency among females whose work was professional, technical, administrative, managerial or clerical. In contrast, MS was less frequent in the countryside in which no major shift in the gender distribution of MS occurred over the past three decades. As such, male gender, living in a countryside setting and working in agriculture and farming may be protective.

Compared to the parental generation, which showed lower disease rates, our MS patients had fewer children and a substantially higher proportion of female smokers. In our MS population more than 50% of female patients were smokers prior to disease onset. This frequency is higher than that of female controls (36.5%) and three times greater than that of the mothers of our MS patients (17%; p<0.001). Also the frequency of smoking among our female controls (36.5%) was similar to that reported for the female population of Crete, 33-38% of which are reported to be smokers. On the contrary, the percentage of male smokers among our MS patients has not changed significantly as compared to males of the parental generation (fathers of our MS patients). However, our male MS patients tended to smoke more often (64.9%) than our male controls (46.9%) as well as the corresponding male segment (55%) of the general population of Crete.

e- References

e1. R.A. Marrie, MD, MS; G. Cutter, PhD; T. Tyry, PhD; O. Hadjimichael, MPH; D. Campagnolo, MD and T. Vollmer, MD: Changes in the ascertainment of multiple sclerosis. Neurology 2005;65:1066–1070.

e2. Filalithis Α. Tsakos G. and Koutis AD. A study of the infrastructure and operation of health care services between 1994 and 1999 in the Region of Crete. Report to the Health Ministry of Greece, August 2000.

e3. Koutis AD, Isacsson A, Lindholm LH, Lionis CD, Svenninger K, Fioretos M. Use of primary health care in Spili, Crete, and in Dalby, Sweden. Scand J Prim Health Care 1991;9:297-302.

e- Questionnaire. The questionnaire (translated from the Greek) answered by MS patients and controls

Identification Data / Dietary habits (frequency per week) / Immunizations
Name / Oil (olive, animal, vetetable) / Measles-Mumps-Rubella
Surname / Fruits / Diphtheria-Pertussis-Tetanus
Date of birth / Green vegetables (fresh /cooked) / Poliomyelitis
Phone number / Strictly vegetarian / Hepatitis A
Address / Bread / Hepatitis B
Full name of father / Meat (pork, veal, chicken, lamb) / Other
Full name of mother / Fish / Infectious Diseases
Residence (rural or urban) / Eggs / Measles
Childhood (up to year 15) / Yoghurt / Mumps
At MS onset / Milk / Rubella
Current residence / Pasteurized cow / Chickenpox
Level of education / Fresh goat / Acute gastroenteritis
Occupation / Childhood / Frequent childhood diarrhea
Number of siblings / At MS onset / Infectious Mononucleosis
Twin siblings / Adulthood / Intestinal parasitosis
Family history of MS / Alcohol (frequency per week)
Age at menarche / Water (tap/ bottle)
Age at first childbirth
Use of contraceptives / Medical History
Smoking / Diabetes
Before the onset of MS / Thyroid disease
Current / Hypertension
Pack years / Heterozygosity for β-thalassemia
Traveling abroad / Polycystic ovary syndrome
Sunbathing / Chronic bowel disease
Use of sun protection / Liver disease
Use of vitamin supplements / Acne
In-house pets