Detection of Iodine Deficiency Disorders (Goiter and Hypothyroidism) in School-Children Living in Endemic Mountainous Regions, After the Implementation of Universal Salt Iodization

Iodine and thyroid hormones are indispensable for somatic growth and development of sev‐ eral organs and systems in the fetus and infant. Their most important action is on the devel‐ opment of central nervous system in the critical period of life: from the fetal life up to the third year of age (Dobbing & Sands, 1973; Delong, 1989; Delange, 2000; Koibuchi & Chin, 2000). Thyroid hormone primary is involved in myelination and neuronal-glial cell differen‐ tiation (Bernal, 2005), brain maturation, and is crucial in the development and maintenance of normal physiological processes (Joffe & Sokolov, 1994, Neale et al., 2007).


Physiological aspects
Iodine and thyroid hormones are indispensable for somatic growth and development of several organs and systems in the fetus and infant. Their most important action is on the development of central nervous system in the critical period of life: from the fetal life up to the third year of age (Dobbing & Sands, 1973;Delong, 1989;Delange, 2000;Koibuchi & Chin, 2000). Thyroid hormone primary is involved in myelination and neuronal-glial cell differentiation (Bernal, 2005), brain maturation, and is crucial in the development and maintenance of normal physiological processes (Joffe & Sokolov, 1994, Neale et al., 2007.

Etiology and pathophysiology of Iodine Deficiency Disorders (IDD)
Insufficient dietary iodine intake is the most important etiological factor of disorders caused by iodine deficiency, but goitrogens (perchlorates, thiocyanates), physiological periods with high requirement of iodine (puberty, pregnancy, lactating period), increased urinary iodine excretion (nephrosis syndrome), high thyroxine binding globuline level (hyperestrogenism, oral contraceptives), lack of selenium, latent thyroid enzyme defects and autoimmune thyroid processes may contribute as well (Brook et al., 2008;Delange & Dunn, 2005). Insufficient iodine intake leads to reduced thyroid hormone production, and all the consequences of iodine deficiency.
In children iodine deficiency causes goiter and reduced growth velocity as well. During time diffuse goiters can transform into uni-or multinodular goiters. These nodules can become autonomous, inducing hyperthyroidism, especially after administration of iodine.

Clinical aspects -High-risk populational groups
Iodine deficiency induces a large spectrum of organic and functional consequences grouped under the general heading of iodine deficiency disorders (IDD) (Hetzel, 1983). IDD reflect this public health problem more relevantly than the term "goiter". This group of disturbances is characterised by all of the ill-effects of iodine deficiency specific to different physiological stages (fetus, newborn, infant, schoolchild, adolescent, adult, especially pregnant woman) (Hetzel, 1983;Stanbury, et al., 1998;Lauberg et al., 2000;de Benoist et al., 2004;Delange & Dunn, 2005), that can be prevented by adequate intake of iodine.
The population groups being at the highest risk to develop IDD and severe consequences are fetuses, children under 2 years of age, pregnant and lactating women. The most devastating outcome of IDD is increased perinatal mortality and mental retardation. In infants iodine deficiency is the most frequent cause of preventable mental damage worldwide .
In school-age children and adolescents the main clinical manifestations are diffuse endemic goiter, subclinical thyroid dysfunctions (mainly hypothyroidism and rarely hyperthyroidism), and impaired mental function, retarded physical development as the impact of iodine deficiency on the central nervous system. The iodine-deficient thyroid gland in children is highly susceptible to nuclear radiation (Hetzel, 1983 In moderate iodine-deficient regions hypothyroidism may appear in children (Brook et al., 2008), at the same time delayed neurodevelopment with defective neuromotor and cognitive ability were also met (Vermiglio et al., 1990;Bleichrodt & Born, 1994;Pop et al., 2003).
Clinically euthyroid school-children in iodine-deficient regions have subtle or overt neuropsychointellectual deficits compared to iodine-sufficient children in the same ethnic, demographic, nutritional and socioeconomic system (Vermiglio et al., 1990;Fenzi et al., 1990). The (UIE) was reduced in 2/3 of studied individuals, detecting very low levels in pregnant women (Goldner, 2005).
Mureş County is a 6,700 square km large hilly-mountainous region in Transylvanian Basin, located in the center of Romania, populated with about 600,000 inhabitants. Similar investigations were performed here in 1950's (Cornea, 1957), which were continued until the '80 years, some of the results being published (Vasilescu et al., 1986, Hetzel et al., 1987. After that IDD survey by modern methods (UIE and thyroid ultrasound volumetry) were recommended. In 1998-1999 school-children from localities near the superior and middle hydrographical basin of the river Mureş, including Târgu Mureş (the capital city of Mureş County) were screened. The results showed mild iodine deficiency in most rural localities, moderate iodine deficiency in some villages, and normal iodine state in Târgu Mureş (Balázs et al., 1998(Balázs et al., /a,2000. Our survey targeting neonatal and maternal screening, performed during 2001-2006, investigated the impact of universal salt iodization on the iodine status of these high-risk populations, and it showed that Mureş County is a moderately mild iodine-deficient area (Kun et al., 2003, Kun, 2006.

Official strategies to eliminate iodine deficiency disorders worldwide
In 1990 the heads of States and Governments and other senior officials on the occasion of the World Summit for Children assumed a solemn obligation to eliminate the iodine deficiency disorders. In 2002 an international agreement for the long-term elimination of IDD until 2005 was accepted at the special Session of UNO dedicated to childhood health. The World Health Organization criteria to eliminate IDD through universal iodization of alimentary salt were the use of iodized salt at least in 90% of households, adequate iodine-concentration of salt (20-40mg/kg), and the implementation in practice of these measurements at least within two years. Reviewing the IDD status in Europe, a marked improvement in the status of iodine nutrition was observed, especially in the central parts of the continent (Delange, 2002;Vitti et al., 2003, Gerasimov, 2002).

National strategy on the elimination of IDD in Romania
The legislative measures for the elimination of endemic goiter were controlled for decades by the 637/1955 and the 1056/1962 Governmental Decisions, which ordered among others the distribution of potassium iodide (KI) tablets in school-age children. Thus, legislation on salt iodization did exist, but being not enforced it could not eradicate goiter. So, several Romanian counties, especially hilly-mountainous and rural regions remained iodine deficient according to the above presented data. In 1995 a new governmental order (No. 779/1995) proposed to review the prevention measures of IDD. In 1997 the 21 National Program of the Romanian Ministry of Health intentioned to reduce the frequency of IDD with at least 10% during the following 5 years. Romania has been taken part at the European strategy to eliminate iodine deficiency in Europe on the basis of the WHO criteria from 2002. Extended surveys were performed in Braşov County, Banat (in the central and the western region of the country, respectively, both being parts of the Thyromobil project), Moldova (the eastern ter-ritory) and Dobrogea (south-eastern region), as well as Bucharest, the capital of the country (Simescu, 1999). The nationwide surveys conducted in 2002 have shown that non-iodized salt was still present in Romanian households: 31% of the households in urban and 37% in rural localities (Government of Romania, 2005).
In the frame of the general strategy the following measurements were taken: a governmental decision (No. 586/5 June 2002) was adopted regarding universal salt iodization (mean KIO 3 content of salt 34±8.5mg/kg − i.e. 25.5-42.5mg/kg, higher than previously); the use of iodized alimentary salt has become mandatory since 2003, and the compulsory iodization of salt used in baking industry was decided in 2004. In addition, the National Committee for universal salt iodization and IDD elimination (with multisectorial participation) was founded in 2004; in the same year the National Strategy on the elimination of IDD during 2004-2012 was elaborated and adopted by governmental decision (Government of Romania, 2005). In 2005 the National Strategy on the elimination of IDD was founded in the Institute of Public Health Bucureşti. Consequently, the use of iodized salt in households increased to 96% in 2004 (compared to 53% in 2002) according to data furnished by the Institute of Mother and Child's Protection, but on the other hand iodine content of alimentary salt proved to be insufficient (63%). Therefore, iodine supplementation was necessary, and 10% of school-children received iodine tablets, prescribed by general practitioners. Consequently the iodine-supplementation of school-children and pregnant women has improved considerably after the first 2 years of obligatory use of iodized alimentary salt, the urinary iodine excretion (UIE) becoming almost normal. With all the efforts IDD was still persisting in 2005, requiring enforced monitoring system of iodized salt production and consumption, strengthen the health promotion network etc. (Goldner, 2005).

Indicators to assess baseline IDD status and to monitor and evaluate the IDD control programs
Individual evaluation of IDD is based on clinical exam (physical examination to determine thyroid size and signs of thyroid dysfunctions, inclusion the case into populational group at risk for IDD, psychoneurosomatic assessment of children and adolescents), as well as laboratory and imagistical findings (UIE, TSH, FT 4 , FT 3 , radioiodine uptake, thyroid ultrasound and scintigraphy, fine-needle aspiration biopsy of thyroid nodules).
The epidemiological evaluation of a geoclimatic area includes the determination of iodine content of the water and soil, and the assessment of different types of IDD in the population by indicators of iodine status at baseline and during the salt iodization program (impact indicators: median UIE, goiter frequency and high TSH levels) and by indicators evaluating the degree of successfullness and sustainability of the salt iodization programs (sustainability indicators).
Indicators used to assess the iodine status of school-age children are median UIE, the prevalence of goiter determined by inspection/palpation or ultrasound, and the level of thyroglobulin (Tg).

Urinary iodine excretion (UIE)
is a sensitive tool to indicate the present iodine status, being useful to evaluate the recent changes of iodine intake in the target population (Gorstein, 2001 The frequency of goiter (total goiter prevalence -TGP) reflects the population's history of iodine nutrition but not its present iodine status, because thyroid size becomes normal for months or years after the correction of iodine deficiency. This indicator is useful to assess the severity of IDD at baseline and the long-term impact of control  The screening of thyroid volume assessed by inspection and palpation may furnish subjective bias in as much as 30-40% of the cases (Delange, 1994;Gutekunst & Teichert, 1993;WHO et al., 1994;Vitti et al., 1994), being imprecise mostly for small goiters. Therefore, thyroid ultrasound (US) is a far better method to detect goiter in population, especially in children (Gutekunst & Teichert, 1993). It determines more accurately and objectively the thyroid size, but there is no agreement on reference values. The upper normal limit of thyroid volume in children living in normal iodine-supply regions was elaborated (Delange et al., 1997). In the following years the reevaluation of previous measurements and the standardization of values in iodine-deficient regions were suggest-ed, because the values provided on the basis of data collected in Europe (Delange et al., 1997) were overestimated by 30% (Zimmermann et al., 2001). Reliable new values have been provided in iodine-sufficient school-children (Zimmermann et al., 2004).
A geographical region is defined as iodine-deficient if the median UIE is below 100μg/L or the prevalence of goiter is higher than 5% among school-age children (aged 6 to 12 years) (WHO et al., 2001).
The serum TSH level as an indicator of iodine status is particularly used in neonatal screening programs. A region is considered iodine-deficient if the frequency of serum TSH>5mIU/L is higher than 3% (WHO et al., 1994;. In school-children the level of TSH measured together with free-thyroxine (FT 4 ) determines the thyroid dysfunction (subclinical or overt hypothyroidism) which has appeared in the context of iodine deficiency.
The major goal of IDD elimination programs is to ensure the sustained elimination of iodine deficiency. The sustainability is evaluated by median UIE, besides other sustainability indicators (the proportion of households consuming adequately iodized salt, and programmatic indicators, such as the effectiveness of public health authorities and salt industry to monitor and control the whole procession). Sustained elimination of iodine deficiency is ensured if the proportion of target population with UIE<100μg/L is under 50%, and of those with UIE<50μg/L is below 20% (WHO et al., 2001).

Iodine requirement, IDD prophylaxis in school-age children
The human body needs very small amount of iodine (in average 200μg/day), but the intake should be continuous. The recommended daily iodine intake varies by references. In 2001 the WHO/UNICEF/ICCIDD recommended 120μg/day iodine for school-children (6 to 12 years) and 150 μg for individuals above 12 years, but with these amounts the recommended UIE of 100-200μg/L was not obtained, reaching only 55-80μg/L. In addition to table salt, iodization of animal food and bottled table water can contribute to iodine intake (Andersson et al., 2007;Szybinski, 2011).

Current iodine status worldwide
Iodine deficiency continues to be a major public health problem in many parts of the world. World statistics show that 1.6 billion people are at risk of being affected by the reduced iodine in their diet, 50 million children are suffering of IDD and every year 100.000 children are born with cretenism worldwide. In 2002 a number of 14 countries of Western and Central Europe have reached a normal status of iodine nutrition, 3 countries were close to iodine sufficiency, but 13 countries, including Romania did remain with persisting IDD (Delange, 2002 In the European Union only 44% of population live in iodine sufficient areas (Andersson, 2011). The experience accumulated thus far has shown that the prevention and control of iodine deficiency requires monitoring to be sustainable.
Endemic cretinism as the most severe complication of IDD was practically eradicated as late as the second half of the 20 th century by prophylactic programs conducted worldwide, but serious consequences of IDD are persisting and currently iodine deficiency still remained a major populational health problem.
The iodine status of Mureş County has not been evaluated since 2000. Similarly, the impact of universal salt iodization program initiated in 2003 was not studied in Mureş County. In 2006 we proposed to evaluate the outcome of universal salt iodization in school-age children from iodine-deficient regions of Mureş County, after the legislative changes targeting the improvement of iodine status in Romania were initiated.

Objectives
Our primary objective was to evaluate the impact of universal salt iodization (with increased KIO 3 -content) at school-age children living in formerly iodine-deficient areas from Mures County (mainly from rural mountainous regions). Secondly, we proposed to assess the frequency and grade of endemic goiter, as well as of thyroid dysfunctions in children from these well known iodine-deficient regions. Additionaly, we intended to diagnose other etiological factors of goiter and hypothyroidism, such as juvenile chronic autoimmune thyroiditis (Hashimoto's disease).

Material and methods
Material. The target population was school-age children, having 8-14 years of age of both sexes, living in mountainous rural localities of Mureş County known as iodine deficient regions in 1999-2000, before the implementation of universal salt iodization.
In The endocrinologists arrived to schools located in the mentioned geographical areas, and the children of classes between Vth-VIIIth were examined at the school's health room. This screening activity was previously planed, thus the children were told not to eat on the morning of investigations. Methods. Data recording was completed with initials, age, gender, hight, weight, physical examination, laboratory and imagistic information for every child. In all cases physical examination and thyroid ultrasonography were performed, then urine and blood samples were collected in order to measure UIE, the level of TSH (thyrotropin), free-thyroxine (FT 4 ) and anti-thyroid peroxidase antibodies (TPO-Ab).
Samples of drinking water and alimentary salt accesible in the local food marts in every locality were collected in order to measure their iodine content.
Firstly family and personal history were taken, than physical examination and thyroid ultrasonography were performed.
Family history of thyroid dysfunctions, goiter, or any known thyroid disease of parents and other relatives was clarified. In the case of 82 children we could take a valuable familial anamnesis. Personal history of thyroid disease also was registered.
Physical examination consisted of general examination of organs and segments of the body, focusing on the possible signs and symptoms of thyroid disorders.
Anthropometric parameters were determined. Hight and weight were measured and compared to the standard, being calculated the height-SDS and weight-SDS. Normal height and weight charts of Prader for both sexes from Switzerland reported in 1989 were used, as these are accepted for children living in Romania and similar charts adapted for the Romanian population are not available.
We distributed the casuistry in cohorts with/without hypothyroidism, with/without iodine deficiency, and the mean height and weight of the group was calculated, as well as the data between groups were compared by statistical means.
Local examination of the anterior and lateral cervical regions including the regional lymph nodes represents an important part of the investigation. Inspection and palpation of thyroid gland provided information about the size, consistency and surface of the thyroid, as well as the existence of palpable thyroid nodules and cervical lymph node enlargement. Goiter was clinically defined according to the WHO criteria in 1960 for the classification of thyroid size (Perez et al., 1960). Goiter was diagnosed if its size was grade 1a (palpable thyroid lobes larger than the terminal phalanges of the subject's thumbs), grade 1b (visible with the extended neck), grade 2 (visible with the head in normal position, but the goiter does not extends beyond the medial edge of the sternocleidomastoidian muscles) or grade 3 (visible at a distance and it extends beyond the previous limits) ( Perez et al., 1960). Nodules in the thyroid that is otherwise not enlarged fall into grade 1 category (Delange & Dunn, 2005). The inspection and palpation of the anterior and lateral cervical regions were effectuated at all school-age children included in our study.
Thyroid ultrasound (US) was performed in all cases by a portable instrument (Sono Ace 600 − SA-600) using a 7.5-MHz linear transducer. The volume of thyroid lobes was measured and the total thyroid volume was calculated. The structure of thyroid tissue and the presence/absence of thyroid nodules were also investigated.
The volume of each thyroid lobe was calculated by the formula: V = lenght × width × depth × 0.479 (all values expressed in cm), than they were added together to obtain the total volume of the gland. The isthmus was ignored unless a nodule was present. In our study the volume of lobes was calculated automatically by the computer of the US equipment.
We The measured thyroid volumes were also adjusted to the body surface area (BSA) and the obtained data were evaluated according to the upper normal limits (Table 5).
Body surface area was calculated using a variant of Dubois formula: After evaluation of thyroid volume for every children, the frequency and the grade of goiter was determined in the cohort. Intellectual capacity of 59 children was evaluated by school performances provided by the school's teaching and medical stuff. School performance spectrum was distributed into four subgroups: very good, good, mediocre and low capacity.
In order to measure urinary iodine excretion (UIE), the 24-hours urine was collected, than the whole urine quantity was mixed and a 50mL sample was retained and transported to the laboratory. Urinary iodine concentration was determined with the colorimetric procedure After obtaining absolute urinary iodine levels for individuals, mean and median UIE values were calculated for our studied groups.
Urinary iodine excretion measurement expressed in μg/L refers to the median value of the UIE calculated for the target population. We estimated the impact of universal salt iodization to eliminate iodine deficiency at school-age children by the interpretation of UIE based on WHO criteria ( Table 1).
The assessment of thyroid function was also performed. Serum TSH, FT 4 and TPO-Ab levels were determined from 5 mL total blood collected by venipuncture into heparinized tubes.
Serum TSH and free-T 4 levels were measured from venous blood in the morning in all children included in the study. Third generation ECLIA (electrochemiluminescence immunoassay) was applied at the Central Laboratory of Emergency Clinical Hospital Mureş County. Normal range for TSH was considered between 0.27-4.2mIU/L and for FT 4 between 0.932-1.71ng/dL. We evaluated the presence and the severity of hypothyroidism, being diagnosed overt primary hypothyroidism in case of high TSH with reduced FT 4 , and subclinical form if TSH was increased and FT 4 normal or at the lower normal limit.
The level of anti-thyroid peroxidase antibodies (TPO-Ab) was measured in every schoolchildren. Values under 50IU/mL were considered normal. TPO-Ab above 50IU/mL indicates the presence of juvenile form of Hashimoto's thyroiditis.
Iodine content of drinking water in each village in question was measured. Water samples were collected from the main fountains used by the majority of inhabitants in every localities, and from the water supply network in the school, respectively. These samples were processed by the Laboratory Unit Mureş of the Romanian Academic Research Institute. The iodine-concentration of water under 50μg/L was considered low.
Iodine content of alimentary salt available in the local food marts was also determined. According to the legislative measures the iodine content of the salt must be between 25.5-42.5mg/kg of potassium iodate (KIO 3 ).
The collected data were systematised and statistically processed. The parameters between groups were compared statistically with T-Student test and Chi-square-test. Homologue parameters between the groups were considered statistically different, if P-value<0.05.

Results
Anthropometric parameters of the 135 school-age children living in the three studied mountainous villages (Caşva, Glăjărie, Ibăneşti,) show similar distribution (  Table 9. BSA-related minimum, maximum, median, and mean thyroid volume measured by ultrasound in the investigated school-children  The frequency of goiter assessed by physical examination and ultrasound were compared, and distributed regarding to gender. The overall goiter frequency provided by ultrasound was significantly higher in girls than in boys (28% vs. 14.1%). Thyroid enlargement was almost similar in both sexes in the subgroup from Caşva, twice and more than three times higher in girls from Glăjărie and Ibăneşti, respectively, although stratified data according to localities show non-significant differences between the subgroups (

Urinary iodine excretion measurement
In 2006, at about 2-2.5 years after the implementation in practice of universal salt iodization the mean UIE of the 135 children living in the three mentioned villages from Gurghiu Valley was 85.37±60.05μg/L. The median UIE of 74.88μg/L indicates globally a mild iodine deficiency, but stratified data show large interindividual variations within children.   In Caşva the mean TSH was normal (2.34±1.00mIU/L). Nine (16.3%) children had primary hypothyroidism. In Glăjărie the mean TSH and FT 4 were also normal (2.12±0.93mIU/L and 1.08±0.13, respectively), and 5 school-children (16.6%) were detected with hypothyroidism (4 mild overt and 1 subclinical form). In Ibăneşti 10 subjects (20%) were diagnosed with thyroid insufficiency (6 mild overt and 4 subclinical).
We did not detect any case of hyperthyroidism.
TPO-Ab was negative (<50IU/mL) in all 135 cases, which means that juvenile chronic autoimmune thyroiditis was not met in this cohort, and hypothyroidism was not caused by this thyroid disease, rather it was induced by iodine deficiency.
We did not found considerable or significant differences of somatic development in children with or without iodine-deficiency, with or without hypothyroidism.
Intellectual performances of 59 children were recorded, and than we distributed them into two groups: one (1. subgroup) with children having mediocre and low learning capacity (32 cases), and the other (2. subgroup) consisting of individuals with good or very good performances (27 cases). The goiter frequency based on ultrasound was 22.5% in the first, and 17.8% in the second group (P-value >0.05). We compared the mean UIE, TSH and FT 4 between the subgroups of children with different school performances, but we did not found significant differences, excepting a tendency of reducing school performances with the gradually increasing values of TSH and decreasing levels of FT 4 (Figure 3.).
Our results recorded in 2006 show that the water sample from Caşva contains 3.8μg/L iodine, and the iodized alimentary salt 16.02mg/kg (which was lower than the ordered concentration of 34±8.5mg/kg). In Glăjărie the iodine content of the water from the central fountain was 1.0μg/L, from the parish yard 1.9μg/L, in the running water from the school 0.45μg/L and from the medical unit 4.2 μg/L. The iodine content of water from Ibăneşti was 0.6μg/L and of the iodized alimentary salt 51.64mg/kg (which was higher than the allowed upper normal concentration).  In the present work physical examination shows an owerall goiter frequency of 51.9%, prevailing small goiters (grade Ia) in all studied villages (more than half of goiters), large goiters being very rare (grade II only in 6.7% of all cohort). Comparing the data of the two series we could not found any improvement in the overall frequency of goiter (45.28% in 1999 vs. 51.1% in 2006), but we must take into account, that in the previous study children came from several localities (including Târgu Mureş, the capital city of Mureş County) with different iodine status (mild or moderate iodine deficient, as well as with normal iodine supply). A considerable number of the enrolled children were from Târgu Mureş, a city which proved to be without iodine deficiency. In the present work we focused on mountainous villages, known before as iodine deficient regions, some of them (Caşva and Glăjărie) known partly isolated in socioeconomical and geographical point of view.
In our study physical examination of thyroid gland shows variable goiter frequency in the three localities: it is the highest in Ibăneşti (62.0%) vs. the other two localities (46.7% in Glăjărie and 43.6% in Caşva). This result is not in concordance with the fact that Ibăneşti is the largest among the three localities, with better socioeconomical status and lesser isolated. An explanation might reside in the differences in age, gender and pubertal stage of the children. In Ibăneşti the mean chronological age of school-age children enrolled in the study is higher (12.05 years vs. 11.51 and 11.80 years in the other two localities), and the distribution between sexes is almost 1:1 (differing evidently from boys:girls-ratio of 2:1 in Glăjărie and 3:2 in Caşva). Taking into account that the girls from Ibăneşti were older, between 12-14-years, the pubertal stage was higher in this group. Balázs et al. (1998) have reported the increased incidence of grade Ib and II goiter in children with pubertal onset compared to those being in prepubertal stage, as pubertal onset is an important facilitating factor for goiter development. This is in concordance with our data, too, related to gender. The overall goiter frequency provided by ultrasound was significantly higher in girls than in boys (28% vs. 14.1%): although goiter appeared almost with the same frequency in both sexes from Caşva (14.2% in girls and 17.6% in boys), but it was more than 3-times more frequent in girls than in boys living in Ibăneşti (42.3% vs. 12.5%). Euthyroid pubertal goiter is especially frequent in adolescents, because iodine metabolism is accelerated during this period of life (Delange & Ermans, 1967).
Than we compared our data obtained in 2006 to the results recorded during April-June 1999 at school-age children living in Deda, a mountainous rural locality, presumed as iodine deficient area. In that study 36.22% of the children had goiter, mainly of small size, without considerable differences between the sexes (Balázs et al., 2000/a) -the goiter being assessed by inspection/palpation. This result is similar to that in the present study (40.2% in children with age between 6-12 years), without any significant differences between the groups. It must be emphasized, that the lack of gender differences may be attributed to inspection/ palpation method for the diagnosis of goiter. Taking into account the subjectivity and bias of physical examination of thyroid gland, especially in young children, as this method of evaluation estimates goiter presence with an error as much as 30-40%, our interpretation was based mainly on thyroid ultrasound.
The objective determination of thyroid volume by ultrasound provided the exact size of the thyroid, however it was difficult to chose the adequate standards for the normal thyroid volume fitting to the goiter estimation in our region. The references published during the last 2-3 decades provide very variable standards for the upper normal limit of thyroid utrasound volumetry adjusted for age, gender and BSA.
We evaluated firstly the age-and BSA-adjusted goiter frequency compared to the reference values provided by the Thyromobile survey accross Europe (Delange et al., 1997), and thereafter we reevaluated the results according to parameters published by Zimmermann (2004), but we obtained very different results (Figure 1.). After all we chose the standards provided by a team of endocrinologists from Iaşi performed on a cohort living in a formerly iodine deficient Romanian region (Vulpoi et al., 2002;Zbranca et al., 2008). Thus, goiter frequency in our cohort assessed by ultrasound was 20%, with a considerable difference between the sexes (28% in girls and 14.1% in boys).
In the study of Balázs et al. (2000/a) thyroid ultrasonography was also performed in 83 children living in Târgu Mureş. The mean thyroid volume determined by ultrasound among these children was 5.22±1.51cm 3 , adjusted to a mean body surface area of 1.23±0.14 m 2 . Goiter evaluated by ultrasound was detected in 33.73% of the children, mainly small goiters (grade Ia), predominantly in girls (about 2/3 in girls and 1/3 in boys). In 1998/99 the majority of rural localities situated on the superior and middle hydrographical basin of the river Mureş were mildly iodine-deficient areas, the rest being moderately deficient. The mean UIE was 100.22μg/L, but with a high interindividual variation: SD ± 73.37 (Balázs et al., 1998 not attained yet the most important criteria of normal iodine-supply conditions, i.e. the maximal value of 50% UIE <100μg/L (the 68.1% is still above of this limit with 18.1%); similarly, in our present study the proportion of UIE <50μg/L is 30.3%, for normal iodine-supply it must be ≤20%. These results show that the universal salt iodization program could not be ensured at an optimal level, being not sustained continuously.
The iodine status of the studied localities from Mureş County in 2006 was evaluated by the following indicators: median UIE for the current iodine status, frequency of goiter assessed by ultrasound for the history of iodine nutrition of the previous months-years, and the frequency of UIE concentrations below 100μg/L and below 50μg/L for the sustainability of universal salt iodization program (Table 12.).   (Stănescu, 2005). Although the median UIE became almost normal (105 in urban and 100μg/L in rural localities), a large interindividual variation were found, reflected by the SD of ±60μg/L (Stănescu, 2005). Important differences between iodine status of rural and urban environment were observed. Mild iodine deficiency was recorded in 33.5% of the population, 11% presented moderate and 2.4% severe forms (Stănescu, 2005), although 96.3% of the housholds used iodized salt. These results could derive from the facts that more than half of the families used salt with inadequate iodine content (under 15mg iodine/kg salt), 3/4 of the families consumed iodized salt but in an inadequate manner (adding salt before and during cooking) which reduces the content of iodine; the frequency of iodized salt consumption is higher in families with the higher educational level of the mother.

Interpretation of indicators
In conclusion, on the basis of the indicators for iodine status the mountainous villages located in Mureş Conty, known as mild/moderate iodine deficient regions in 1999, remained in the same iodine deficiency category during 2006, however the absolute values of indicators were considerably, some even significantly improved. The indicators situated before at the lower limit of mild/moderate iodine deficiency interval, after-wise were shifted to the middle zone and upper limit of this range. The universal salt iodization had a beneficial impact on iodine deficient rural regions, as expected, but its sustainability must be maintained, i.e. the effectiveness of public health authorities and salt industry to monitor and control the whole procession must be consolidated. The elimination strategy of IDD in mountainous rural areas remains a very important public health program, as the iodine content of water and soil is very low here. Water samples taken from the main water-provision points from every village showed that the iodine content of drinking water was very low in every locality, as expected in mountainous rural regions. Thus general measures to increase the iodine content of the water should be applied. The iodine content of the running water measured in 1998/99 and 2006 was very variable (the mean value being X=10.15μg/L, SD±9.85 − Balázs et al., 1998), but in all cases the iodine concentration was much more under the accepted value (levels above 50μg/L being considered normal). Iodine content of alimentary salt found at the market of the three villages was in some cases under the prescribed level of 34±8mg/kg. The usage of iodized alimentary salt in households reached 96% in 2004 compared to 53% in the previous years (Goldner, 2005;Stănescu, 2005), but the alimentary salt was insufficient iodized (in 63% of cases, Goldner, 2005). A proportion of 74.5% of the sample of salt tested which was used in the market and for the panification had a standard iodine content of 15-25 mg/kg (Goldner, 2005), which was under the prescribed concentration. In spite of existent legislation to mandatory sale of iodized table salt on the market, about 12% of population consumes not iodized salt (Nanu, 2005), especially in rural environment.
The persistence of moderate iodine deficiency in some zones of Romania was attributed to the insufficient iodization level of the salt and waist during storage, to the uncontrolled selling of industrial salt as alimentary salt, to the lack of control measures in import of salt, to the inadequate monitoring system and the lack of efficient measures to realize entirely the governmental decision, as well as the resistency of alimentary industry (e.g. meat processing industry) to utilize iodized salt (Goldner, 2005).
We must remark the presence of hypothyroidism related to iodine deficiency in 17.7% of the investigated school-children, TPO-Ab being normal in all of them. Although, all cases of hypothyroidism were mildly overt or subclinical forms (15 and 9, respectively), we must take into account that even mild iodine and thyroid hormone deficiencies conduct to psychoneurological damages, the characteristical complication of latent deficiency being AD-HD syndrome in developed countries. Chronic iodine deficiency leads to suboptimal intellectual development. Recent studies performed in the European countries, known with iodine prevention programs initiated decades ago, and thougth that iodine deficiency was eliminated, sustaine surprisingly the presence of mild iodine deficiency in many countries and implication of this in school performances, psychological and neurological status. These mild deficits probably results from transient hypothyroidism during the first 2-years of life, i.e. during the critical period of brain development (Delange, 2002). Besides iodine deficiency, an increase in iodine intake can also lead to a small but significant increase in the incidence of hypothyroidism, probably attributable to autoregulation of thyroid function.
In conclusion, the rural mountainous zones of Mureş County known before as moderate/ mild iodine-deficient areas, became mild-deficient, due to the new measures of iodine prevention. In these areas universal salt iodization is not sufficient, being necessary to apply periodically special prophylactic measures (iodine tablets), too, primarily to prepubertal and pubertal children.

Conclusions
This work underlines considerable improvement of iodine nutrition in mountainous rural regions of Mureş County after the implementation in practice of universal salt iodization in Romania since 2003, although these areas remained mildly (some moderately) iodine deficient at about 2.5-3 years after the IDD elimination strategy was intitiated. On one hand the prophylactic measures with higher iodine content of alimentary salt (KIO 3 25.5-42.5mg/kg) might not be sufficient for the full correction of iodine deficiency in these isolated mountainous rural regions, on the other hand special attention is required to monitorize continuously the market and reaching programs, as well as the sustainability of the prophylactic program must be ensured. In these areas special measures, i.e. iodine supplementation to the highrisk populational groups are periodically required in addition to universal salt iodization, especially as iodine deficiency occured with hypothyroidism in 17.7% of school-children.
The elimination of mild-moderate iodine deficiency in school-age children living in mountainous rural localities presumes special measures by the daily administration of 0.1-0.2 mg iodine tablets.