MSc, RPT Katarina Mikaelsson
email: katarina.mikaelsson@ltu.se

MSc, RPT Kristina Eliasson

PhD (professor Jack Lysholm

PhD (professor Lars Nyberg

PhD, RPT. Peter Michaelson

Physical capacity in physically active and non-active adolescents

Keywords: adolescents, balance, cardiovascular capacity, muscular capacity, physical activity

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Physical activity contributes to several positive effects on physical capacity and both physical and mental health [START_REF] Blair | Is physical activity or physical fitness more important in defining health benefits?[END_REF][START_REF] Hallal | Adolescent physical activity and health: a systematic review[END_REF]. For children and young people, all types of physical activity and exercise are essential to create conditions for the body to adapt and to cope with both current and future physical and mental demands and are therefore important properties for children's physical, mental and social development [START_REF] Malina | Physical activity and training: effects on stature and the adolescent growth spurt[END_REF]). This is one reason why physical education and health are part of the core curriculum for all programs in the Swedish upper secondary school. Due to this, Swedish adolescents would not be likely to be completely physically non-active; nevertheless several of them are less active than what is considered to be necessary to maintain good health (Swedish National Institute of Public Health 2009).

The three main health components of physical capacity are: cardio-respiratory capacity, muscular capacity and mobility [START_REF] Ortega | Physical fitness in childhood and adolescence: a powerful marker of health[END_REF]. Between 18 and 20 years of age, the cardio-respiratory capacity peaks in both sexes [START_REF] Rogol | Growth and pubertal development in children and adolescents: effects of diet and physical activity[END_REF][START_REF] Rutenfranz | Maximal aerobic power affected by maturation and body growth during childhood and adolescence[END_REF][START_REF] Wilmore | Physiology of sport and exercise[END_REF][START_REF] Zaichkowsky | Physical, motor, and fitness development in children and adolescents[END_REF][START_REF] Åstrand | Textbook of work Physiology[END_REF]. The development of muscle mass occurs mainly during puberty, but between the ages of 20 to 30 years the maximum muscle strength in men increases, while women's growth is less (Åstrand 1997).

Most studies show that the degree of physical activity has decreased among Swedish children and adolescents over the past decades and that the number of sedentary young people has increased [START_REF] Bratteby | A 7-day activity diary for assessment of daily energy expenditure validated by the doubly labelled water method in adolescents[END_REF][START_REF] Ekblom | Health-related fitness in Swedish adolescents between 1987 and 2001[END_REF][START_REF] Engström | Barn och ungdomars idrottsvanor i förändring.(Swe: Children and young people´s sporting habits in change)[END_REF]). As physical activity has decreased, physical capacity has shown similar pattern [START_REF] Ekblom | Health-related fitness in Swedish adolescents between 1987 and 2001[END_REF][START_REF] Westerstahl | Secular trends in body dimensions and physical fitness among adolescents in Sweden from 1974 to 1995[END_REF]. [START_REF] Ekblom | Health-related fitness in Swedish adolescents between 1987 and 2001[END_REF] present how the physical capacity changed between 1987 and 2001 for children of ages of 10, 13 and 16 years. The study showed a significant reduction in the cardio-respiratory capacity of 16-years old boys from 1987 to 2001, while it remained unchanged in girls. The study also showed that the muscular capacity was reduced in 2001 as compared to 1987 in both sexes [START_REF] Ekblom | Health-related fitness in Swedish adolescents between 1987 and 2001[END_REF]). [START_REF] Malina | Physical activity and fitness: pathways from childhood to adulthood[END_REF] has shown that children and adolescents who are physically inactive will remain inactive as adults. This is in many ways alarming, as physical inactivity as well as impaired cardiovascular and muscular capacity has a strong relationship with most health related problems [START_REF] Pate | Age-related change in physical activity in adolescent girls[END_REF][START_REF] Pedersen | Evidence for prescribing exercise as therapy in chronic disease[END_REF]).

Many countries have adapted guidelines for physical activity. The recommendation of

Swedish Institute for Public Health is: "All individuals should, preferably every day, be physically active for a total of at least 30 minutes. The intensity should be at least moderate, such as brisk walking. Additional health effect can be obtained if in addition you increase the daily volume or intensity" (Swedish National Institute of Public Health 2009). Sweden and other Nordic countries have a common recommendation for children and young people of at least 60 minutes of physical activity per day and that activity should include both moderate and high intensity (Swedish National Institute of Public Health 2009). This is consistent with the World Health Organization (WHO) recommendations for children, which reads: "Each day school-aged youth should accumulate at least 60 minutes of moderate-to vigorousintensity physical activity to ensure healthy development. This can provide young people with important physical, mental and social health benefits." [START_REF] Strong | Evidence based physical activity for school-age youth[END_REF]World Health Organisation 2009). In a large Swedish study the physical activity habits of 14 to 15 year old adolescents in the Stockholm region 2000-2001 were examined. The study showed that 71% of young people met with the recommendations regarding 60 minutes of daily physical activity. There was an obvious sex difference; more girls than boys did not reach the recommended level [START_REF] Rasmussen | Fysisk aktivitet, matvanor, övervikt och självkänsla bland ungdomar 8Swe: Physical activity, diet, obesity and self-esteem among young people)[END_REF].Other studies have also shown that girls are less physically active [START_REF] Pate | Compliance with Physical Activity Guidelines Prevalence in a Population of Children and Youth[END_REF][START_REF] Sallis | A review of correlates of physical activity of children and adolescents[END_REF].

Previous studies regarding physical activity and its relation to physical capacity were targeted at either children, young people of 15-16 years of age, or adults. To our knowledge, there are no studies regarding physical capacity in the last year in the upper secondary school (i.e. 18 years old) and as oxygen uptake peaks at this age it would be of great interest to examine potential differences in capacity between active and non-active individuals. Also, this age group is about to leave school and move into adulthood. Research has shown that the level of physical activity decreases after high school, particularly among females [START_REF] Nelson | Longitudinal and secular trends in physical activity and sedentary behavior during adolescence[END_REF].

Altogether, this age-group is at a turning point in life. Previous research have shown a tendency to a reduced physical activity which reinforces the individual's habits of inactivity and therefore contributes to a subsequent physical inactivity [START_REF] Malina | Physical activity and fitness: pathways from childhood to adulthood[END_REF].

The aim of this study was to investigate differences in physical capacity in physically active and non-active males and females among graduates of upper secondary school.

METHODS

Participants

Participants were recruited on two different occasions. In total, 99 persons participated, of which 38 were females and 61 were males. All participants were between 18-20 years old.

In the first test occasion participants were recruited through a questionnaire survey on physical activity among third grade students from upper secondary school in three different municipalities Boden (Björknäs), Luleå (Luleå gymnasieby) and Piteå (Strömbacka) (results not yet published). Each participant of the questionnaire survey was also asked whether they were interested to participate in a forthcoming study of either physical capacity and/or interview as part of a larger data collection. In total 62 adolescents accepted to participate, out of 884 who answered the questionnaire. These 62 adolescents conducted the tests of physical capacity during spring 2007 in three different cities in northern Sweden.

After the first data collection, there was a skewed distribution of participants in the study, with the majority being physically active. So, to obtain more equal group sizes of physically active and non-active a second round of data collection was conducted. The recruitment of participants for the second data collection took place at one of the upper secondary school involved in the first data collection, Björknäs school at Boden. Students in grade three of the vocational programs were personally asked if they wanted to participate in a study of physical capacity. The reason for the choice of the vocational programs was that studies have shown that more students at these programs are physically non-active compared to those participating in the more theoretical programs [START_REF] Sollerhed | Low physical capacity among adolescents in practical education[END_REF][START_REF] Westerstahl | Low physical activity among adolescents in practical education[END_REF]. A total number of 143 students were invited to participate and 37 agreed. All of them performed their testing within the next few days.

Assessments

The physical tests were selected in order to provide reliable and valid measures of the aerobic capacity, the muscular strength of the upper and lower body and balance. The test battery has previously been used in studies with similar aims, designs and study population [START_REF] Ekblom | Secular trends of physical fitness in Swedish adults[END_REF][START_REF] Ekblom | Physical performance and body mass index in Swedish children and adolescents[END_REF][START_REF] Westerstahl | Secular trends in body dimensions and physical fitness among adolescents in Sweden from 1974 to 1995[END_REF]. The tests were easy to perform without demanding technical equipment, making them advantageous to use and easy to replicate in clinical practice.

Physical activity

Physical activity was determined using the responses of the International Physical Activity Questionnaire (IPAQ) short form (International Physical Activity Questionnaire 2007) IPAQ has been tested for validity and reliability in adults and adolescents [START_REF] Craig | International physical activity questionnaire: 12-country reliability and validity[END_REF]. A small modification was made for the purpose of this study, whereas respondents were asked to rate their usual activity during an ordinary week in the past six month, instead of just the last seven days. This was made because we wanted to describe activity habits rather than the most recent activity performance, and because of the sensitivity of the relatively small study sample for temporary variations due to sickness and other aleatoric reasons.

Based on responses regarding physical activity on the IPAQ's vigorous and moderate levels, participants were characterised dichotomously as physically active or non-active. To be characterised as physically active, a respondent should have reached the WHO recommendation of physical activity for adolescents (World Health Organisation 2009) at least a total of 60 minutes per day and 7 days per week. Of the 38 females, 22 (58 %) were characterised as physically active and 16 (42 %) as non-active. Among the 61 males, there were 40 (66 %) physical active and 21 (34 %) physically non-active, adding up to 99 participants.

Anthropometric measurement

Prior to the physical testing, the participants' height and weight were measured. Height was measured with the participant standing against the wall using a Stadio meter (Seca Gmbh & Co.Ka. Germany). The weight in kilograms to the nearest decimal was measured on a digital scale (Seca Gmbh & Co.Ka. Germany). During the entire test procedure the participants were wearing light workout clothes and were barefooted. Height, weight and Body Mass Index (BMI) [START_REF] Cole | Establishing a standard definition for child overweight and obesity worldwide: international survey[END_REF] are presented in Table 1.

Insert Table 1 about here

Aerobic capacity

The aerobic capacity was determined by a sub maximal cycle ergometer test (Åstrand, 1997) performed on Monark Ergomedic 826E. Prior to the beginning of test, the bicycles were calibrated. Cadenza was set to 50 rpm, guided by a metronome during the test. Heart rate was registered with the Polar heart rate watch (Polar Electro Oy, Kempele, Finland). The participants pulse was recorded every minute as well as their respiratory effort, and their perceived effort in the legs using the BORG scale [START_REF] Borg | Phychophysical bases of preceived exertion[END_REF]. The achieved steady state pulse rate at the given workload at the end of the test was converted to the oxygen consumption using Åstrands -Ryhming monogram (Åstrand 1997).

Upper extremity muscle strength

Muscle strength of the upper extremity was assessed using push-ups and grip strength.

Push-ups are a test recommended as a reliable method for measurement of muscle strength in the arms [START_REF] Ryman Augustsson | Gender differences and reliability of selected physical performance in young woman and men[END_REF]. In order to standardize the "push-ups", the hands were placed on a line with an optional distance between the hands. The depth of the push-ups was controlled by a 15 cm cone placed under the sternum, and which had to be touched during the trial for the push-up to be counted as correctly performed. The frequency of pushups was 30 beats per minute, which was guided by a metronome. The numbers of correctly performed push ups was then noted. Either three incorrectly carried out push ups or exhaustion terminated the test.

Hand grip strength was measured with a grip dynamometer, Grip D, UH 5401 Eleiko Sport (Takei & Co LTS, Tokyo, Japan). The method of measurement is tested for reliability [START_REF] Watanabe | The short-term reliability of grip strength measurement and the effects of posture and grip span[END_REF]. Before the test, the size of the grip was adjusted in accordance with the size of participants' hand. The test was carried out with the participant standing with the arm hanging alongside holding the dynamometer in the hand. Three trials were performed for each hand, switching between hands for each trial and the best value for each hand was recorded. A mean value was calculated from the best value from the left and right hand and used in the data analyses.

Lower extremity muscle strength

The strength of lower extremity was measured with a vertical jump. The participants were instructed to jump as high as possible with a plyometric flexion of the hip and knee downwards, followed by an extension of the hip and knee and a simultaneous flexion of the shoulders while performing the jump. The landing, to be counted as a correct jump, should be performed with stretched wrists followed by a bending of the knees inside a 150 cm box outlined on the floor. Three correct jumps were performed of which the best result was used in data analyses. To determine the height of the jump a micro Musclelab Power + was used, which measures time spent in the air that is converted to jump height in centimetres. The method has been tested for validity and reliability [START_REF] Markovic | Reliability and factorial validity of squat and countermovement jump tests[END_REF][START_REF] Suni | Healthrelated fitness test battery for adults: aspects of reliability[END_REF].

Trunk muscle strength

The strength of the trunk muscles was measured by the participants performing sit-ups lying on a mat with knees and hips flexed at a 90 degrees angle with the lower parts of the legs resting on a psoas pad. The arms were crossed over the chest with each hand resting on the opposite shoulder. The accepted height of each sit-up was determined in relation to the height of each participant as follows: Stature ≤ 160 cm had to lift enough for a line at 45 -centimetre distance from the buttocks to become visible, length ≤ 175 cm to a 50 cm marker, length ≤ 190 cm to a 55 cm marker and length > 190 cm to a 60-cm marker. The sit-ups were conducted at a rate of 50 beats per minute, guided by a metronome. The test leader counted the numbers of correct sit-ups with a hand calculator. After three incorrectly performed situps the test was terminated. Reliability of the test has been demonstrated [START_REF] Ryman Augustsson | Gender differences and reliability of selected physical performance in young woman and men[END_REF].

Balance

The test for balance was performed by letting the participant stand barefooted with one foot at a time on a 3 cm wide, 5 cm high and 20 cm long list for one minute. Each time the participants lost their balance, and touched the ground with (usually) the other foot, the attempt was registered as unsuccessful. The total number of unsuccessful attempts during one minute was recorded, and the mean number between the right and the left foot was calculated and used in the data analyses. This balance test has been tested for reliability [START_REF] Tsigilis | Test-retest reliability of the Eurofit test battery administered to university students[END_REF].

Procedure

All measurements were performed following strict protocols at three different test sites. First, body height and weight were measured. After that the physical tests were made in the following order: Åstrands cycle ergometer test, push-ups, vertical jump, sit-ups, grip strength and balance. In between the tests the participants completed a survey about their physical activity habits (IPAQ). Three assessors were involved in each participant's test session. One performed the cycle ergometer test while the other two performed the physical tests, in order to achieve a correct test procedure and quantification of the performance while simultaneously encouraging the participant. One of the assessors took part in all test sessions.

In order to avoid that the participants feeling uncomfortable, test situations were adapted so that the performance could only be observed by the assessors involved. Participants were dressed in comfortable wear of their own choice. Altogether the test session took about 60 minutes for each participant.

Data analyses

Data processing was done using Statistical Package for Social Science (SPSS) version 17.0.

As there are substantial differences in physical capacity between males and females (Ortega et al. 2008;[START_REF] Rogol | Growth and pubertal development in children and adolescents: effects of diet and physical activity[END_REF][START_REF] Viljanen | Strength characteristics of a healthy urban adult population[END_REF][START_REF] Åstrand | Textbook of work Physiology[END_REF] the analyses were performed separately. By use of box plot analyses, extreme outliers (defined as values exceeding the distance between median and quartile values more than three times) were identified and excluded from further analyses. This happened only for the sit-ups performance of one female. By the use of the Student's independent sample t-test, hypotheses regarding differences in aerobic capacity, push-ups, grip strength, vertical jump, sit-ups and balance between those classified as physically active or non-active were tested, separately for females and males. Levene's test for equality of variances (SPSS v. 17) was used to decide the proper t-test algorithm for each analysis. A p-value lower than 0.05 was considered as indicating statistical significance.

Ethical approval

The study is approved by the Regional Ethical Review Board in Umeå, Sweden (3202/2006).

RESULTS

The results from the tests of physical capacity for the physically active and non-active females and males are presented separately in Tables 2 and3. In some cases data were missing for particular tests because of occasional physical symptoms such as neck pain and ankle strains.

Aerobic capacity differed significantly between physically active and non-active participants in both sexes with a difference of 20 % for both males (p = 0.002) and females (p = 0.016) (Tables 2 and3). Regarding strength measures, the numbers of push ups showed a significant difference (p < 0.001) for males, with the physically active males performing 30 % more push ups compared to the physically non-active males (Table 3). Also for sit-ups, there was a significant difference (p = 0.01) between the active and non-active males (a 49 % difference) (Table 3). No other statistically significant differences were found between the physically active and non-active males or females. The performance in the balance test did not differ significantly between the active and non-active of either of the sexes. Among the males, there was a non-significant tendency (p = 0.08) towards a lower capacity among the more active participants.

Insert Tables 2 about here

Insert Tables 3 about here non-existent if it is carried out correctly. On the contrary, there is much support for positive effects from muscle strength training for children and adolescents regarding, for instance, sports injury prevention [START_REF] Abernethy | Strategies to prevent injury in adolescent sports: a systematic review[END_REF]), motor learning [START_REF] Falk | The effect of resistance and martial arts training in 6-to 8-year-old boys[END_REF] bone mineralization (Wang 2007) and obesity and body composition [START_REF] Benson | The effect of high-intensity progressive resistance training on adiposity in children: a randomized controlled trial[END_REF].

There were no significant differences in the balance test between the active and non-active groups. In fact, there was even a tendency for the active males to have poorer results than the non-active ones. The results are inconsistent with a study by [START_REF] Oddsson | Bra eller dålig balans -vad är det vi mäter? (Swe: Good or bad balance-what is it we measure?[END_REF] who found a positive association between performance in a balance test and activity level.

The lack of differences between active and non-active students in the majority of the strength and balance tests may theoretically be due to the fact that the test activities were not sufficiently challenging for strength and balance in these age groups. However, most of these tests or similar ones have been used in other studies including similar groups of test persons [START_REF] Ekblom | Secular trends of physical fitness in Swedish adults[END_REF][START_REF] Westerstahl | Secular trends in body dimensions and physical fitness among adolescents in Sweden from 1974 to 1995[END_REF]. Many of the tests are included in the Eurofit battery of physical tests [START_REF] Adam | Eurofit. Handbook for Eurofit test of physical fitness. Rome: Council of Europe, Committee for the Development of Sport Andersson[END_REF]. Moreover, they are easy to perform without demanding technical equipment. Another possibility is that the size of the cohort was too small to obtain statistical power for all the test activities.

The recruitment of participants for this study was performed simultaneously as the participants filled in a questionnaire regarding physical activity habits (results not yet published). Retrospectively, we acknowledge the fact that this design of recruitment was not the most efficient way as the response rate was low. The question of participation in future studies was one of many items in the questionnaire, and a more specific recruitment procedure would have been more appropriate. A major concern during data collection was a skewing of the sample with respect to that only physical active adolescent volunteered to participate, which was the case after the initial data collection. So, despite of the methodological problem with an additional sampling of participants, to achieve a fairly representative group of upper secondary school graduates, students at programs known to be less physically active [START_REF] Sollerhed | Low physical capacity among adolescents in practical education[END_REF][START_REF] Westerstahl | Low physical activity among adolescents in practical education[END_REF] were specially invited for the second round of data collection. This resulted in a more even distribution of physically active and non-active adolescents.

Concerning the estimate of amount of physical activity, which was derived from the IPAQ questionnaire, studies have shown that IPAQ can overestimate self-reported time spent in physical activity [START_REF] Ekelund | Criterion-related validity of the last 7-day, short form of the International Physical Activity Questionnaire in Swedish adults[END_REF]. This could potentially bias the categorisation, overestimating the percentage of physically active participants. When interpreting the results, however, it seems that we have achieved a valid categorisation of physically active and inactive participants, since there were statistically significant differences in measures most likely to be influenced by physical activity. Our modification of the IPAQ, asking the respondent to refer to a usual week instead of the last seven days was made because of the relatively small sample where reference to the last seven days would imply a greater sensitivity to temporary activity restrictions due to for example temporary sickness, injuries or examination in school, as pointed out by participants in a larger questionnaire survey (results not yet published).

Regarding the classification of participants as active and non-active, WHO recommendations for physical activity both for adults (from age 19 and up) as well as for children and young people (up to age 18), might apply to our sample (World Health Organisation 2009). We chose to use the recommendation for children and young people since 72% of participants were 18 years old, and as the aim was to study graduates from upper secondary school, which implies that they have not adapted to adulthood. The WHO recommendation for amount of physical activity as health promoting factor are widely spread and acknowledge in modern societies. For that reason it seemed natural to choose this recommendation when classifying participants as physically active or not.

Based on comparisons regarding body height and weight data from the Swedish National Service Administration (2007), the males in our study did not differ from the national population at the same age. The females however were significantly shorter and lighter (Swedish National Service Administration 2007). Aerobic capacity test values were compared with population data from LIV 90 (national sampling of physical capacity, physical activity and health) [START_REF] Engström | Livsstil-Prestation-Hälsa LIV 90: Rapport 1[END_REF]. This revealed that the males in our sample did not differ from a male population aged 20-24; however, the females had significantly higher aerobic capacity. Compared with data from studies performed on 16 year old adolescents in the Stockholm area [START_REF] Ekblom | Physical performance and body mass index in Swedish children and adolescents[END_REF][START_REF] Westerstahl | Secular trends in body dimensions and physical fitness among adolescents in Sweden from 1974 to 1995[END_REF], the participants in our study seemed to fit in quite well. Regarding grip strength, vertical jump, and balance, the 95% confidence intervals of test results in our study enclosed the reported mean values of the studies mentioned above. Also in this comparison, the females seemed to have a higher aerobic capacity, while the males seemed comparable [START_REF] Ekblom | Physical performance and body mass index in Swedish children and adolescents[END_REF]. Altogether, it appears that the female participants of our sample were somewhat more fit regarding aerobic capacity than what could be expected from a national population, while the male were representative.

Important reliability aspects of this study are that all test procedures followed a strict protocol and that test leaders were careful to try to give the same verbal encouragement to all participants. It has been shown that the way encouragement is given to the participant influences his or her test performance (Andersson et al. 2004). Further, the test leaders were unaware of which activity group the participants belonged to.

In conclusion, aerobic capacity differed significantly between physically active and nonactive males and females among upper secondary school graduates. The activity level did not seem have the same association with muscular fitness and not at all with balance.

The results of the study emphasize the promotion of physical activity in childhood and adolescence, as there are links between both activity and aerobic capacity in adolescence and later life, and in addition to health. The differences in aerobic capacity and some of the strength tests between active and non-active students are not negligible, but are likely to influence the future physical capacity and level of activity. For further research, the relation between muscle strength and different forms of physical activity, especially in females, should be assessed in more well-powered studies. It would also be of importance to establish health-related threshold values for muscular strength and balance.

Table 1 .

 1 Gender, activity level, height, body weight and BMI reported by mean (m), standard deviation (SD) and min-max values.

			Female			Male
		Active (n = 22)	Non-active (n = 16)	Active (n = 40)	Non-active (n = 21)
		Mean (SD)	Min-max	Mean (SD)	Min-max	Mean (SD)	Min-max	Mean (SD)	Min-max
	Height (cm)	166 (4.8)	155-173	165 (5.2)	156-174	180 (6.6)	165-193	180 (6.3)	169-191
	Weight (kg)	60.5 (6.0)	50-76	59 (9.4)	46-90	77 (10.3)	54-103	76 (14.4)	57-114
	BMI (kgxm -2 )	22 (2.0)	18-25	22 (3.4)	19-33	23 (2.5)	19-29	24 (5.1)	19-38

Table 2 .

 2 Differences between physically active and physically non-active females aged 18-20 in aerobic capacity, muscle strength and balance.

			Active (n = 22)	Non-active (n = 16)
		n Mean (SD)	Min -max	n Mean (SD) Min -max	p
	Aerobic capacity (l/min) 22	3.0 (0.6)	1.7 -4.2	16 2.5 (0.3)	2.0 -3.3	0.016
	Push-ups (no.)	22 21.4 (12.0)	0 -49	16 15.4 (11.8)	2 -35	0.131
	Grip strength (kg)	22	30.0 (4.7)	21.1 -40.9	16 29.2 (3.0)	22.1 -33.4	0.505
	Vertical jump (cm)	20	28.9 (5.6)	15 -37	15 28.3 (5.5)	20 -39	0.757
	Sit-ups (no.)	21 41.7 (23.5)	0 -96	16 44.8 (23.9)	0 -98	0.696
	Balance (no. of failures) 22	4.3 (3.6)	0 -14	16 5.0 (3.4)	0 -13	0.559

Table 3 .

 3 Differences between physically active and physically non-active males aged 18-20 in aerobic capacity, muscle strength and balance.

			Active (n=40)	Non-active (n=21)
		n Mean (SD)	Min -max	n Mean (SD) Min -max	p
	Aerobic capacity (l/min) 40	3.6 (0.7)	2.2 -5.0	20 3.0 (0.6)	2.0 -4.3	0.002
	Push-ups (no.)	40	37.1 (9.0)	20 -60	20 28.5 (7.0)	14 -39	0.000
	Grip strength (kg)	40	47.8 (7.5)	33.4 -66.4	21 48.9 (5.7)	39.6 -65.6	0.539
	Vertical jump (cm)	37	42.4 (5.5)	32 -55	21 40.9 (7.5)	19 -54	0.374
	Sit-ups (no.)	35 59.2 (30.2)	23 -136	21 39.6 (19.4)	15 -88	0.010
	Balance (no. of failures) 40	4.4 (3.9)	0 -16.5	21 2.8 (2.5)	0 -8	0.080

ACKNOWLEDGEMENTS

We thank the participants for taking part in this study. We will also thank Winternet in Boden, and Step In in Piteå, Sweden, for providing us with staff and test facilities. Financial support was provided from the Department of Health Science, Luleå University of Technology, and the Swedish National Centre for Research in Sports CIF 30/05.

DISCUSSION

The result showed significant differences between physically active and non-active male and female adolescents regarding aerobic capacity. The activity level did not seem to have strong association with muscular fitness and not at all with balance. Among males, performance in push-ups and sit-ups were better for the active students, something which was not seen among the females.

Concerning adults, knowledge about relationship between health's related outcomes as aerobic capacities and physical activity is immense for example [START_REF] Hasselstrom | Physical fitness and physical activity during adolescence as predictors of cardiovascular disease risk in young adulthood[END_REF]; U.S Department of Health and Human services 1996), yet as to our knowledge, the literature is sparsely regarding studies on adolescents on the brink of adulthood. As knowledge is lacking, we consider that our findings that physically non-active young male and females have significant lower aerobic capacity should be valued as important. The adolescents at this age are about to leave school, and their physical activity level will no longer be supported by regular physical education, but only dependent on the their own choices. Children and adolescents who are physically non-active tend to remain so as adults [START_REF] Malina | Tracking of physical activity and physical fitness across the lifespan[END_REF][START_REF] Ruiz | Traditional and novel cardivascular risk factors in school-agd children: a call for the further development of public health strategies with emphasis on fitness[END_REF]. Furthermore, aerobic capacity in children and adolescents appear to be linked to the cardio respiratory profile later in life [START_REF] Mesa | The importance of cardiorespiratory fitness for healthy matabolic traits in children and adolescents The AVEA study[END_REF][START_REF] Ruiz | Traditional and novel cardivascular risk factors in school-agd children: a call for the further development of public health strategies with emphasis on fitness[END_REF]) and a low aerobic capacity in late adolescence is associated with increased risk of cardiovascular disease later on [START_REF] Hagstromer | Concurrent validity of a modified version of the International Physical Activity Questionnaire (IPAQ-A) in European adolescents: The HELENA Study[END_REF][START_REF] Twisk | The relationship between physical fitness and physical activity during adolescence and cardiovascular disease risk factors at adult age. The Amsterdam Growth and Health Longitudinal Study[END_REF].

Compared to the difference in aerobic capacity related to activity, the relation between muscle strength and activity is more unclear and in some cases contradictory. Besides the results for push-ups and sit-ups among the males, which were significantly better for the active students compared to the non-active ones, no significant differences were found for all the other strength tests. [START_REF] Viljanen | Strength characteristics of a healthy urban adult population[END_REF] found a difference in vertical jump related to activity; however, this was studied in an older and more non-active population (Viljanen et al.1991).

Traditionally, there has been a conservative attitude against introducing strength training for children and young adolescents. However, there is no evidence supporting such a cautious attitude. [START_REF] Rians | Strength training for prepubescent males: is it safe?[END_REF] conducted a study, which specifically examined possible risks associated with strength training in children. The results showed no adverse effect on any of the investigated parameters (damage to bones, muscles, possible impact on growth, mobility and motor function). The authors concluded that the risk of injury with strength training is

CONFLICT OF INTEREST

The authors declare that they have no conflict interest.


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Hormonal Deficiencies During and After Puumala Hantavirus

Keywords: Puumala hantavirus, hemorrhagic fever with renal syndrome, hypogonadism, hypopituitarism, hypothyroidism Hormonal Deficiencies During and After Puumala Hantavirus Infection Puumala hantavirus, hemorrhagic fever with renal syndrome, hypogonadism, hypopituitarism, hypothyroidism

Purpose: Previous reports have described panhypopituitarism associated with severe cases of hemorrhagic fever with renal syndrome (HFRS), but the prevalence of hormonal deficiencies after nephropathia epidemica (NE), a milder form of HFRS, has not been studied. This study was conducted to determine the prevalence of hormonal defects in patients with acute NE and during long-term follow-up. Methods: Fifty-four patients with serologically confirmed acute NE were examined by serum hormonal measurements during the acute NE, after 3 months, and after 1 to 10 (median 5) years. Results: Thirty out of 54 (56%) patients had abnormalities of the gonadal and/or thyroid axis during the acute NE. After a median follow-up of 5 years, 9 (17%) patients were diagnosed with a chronic, overt hormonal deficit: hypopituitarism was found in 5 patients and primary hypothyroidism in 5. In addition, chronic subclinical testicular failure was found in 5 men. High creatinine levels and inflammatory markers during NE associated with the acute central hormone deficiencies, but not with the chronic ones. Conclusions: Hormonal defects are common during acute NE and surprisingly many patients develop chronic hormonal deficiencies after NE. The occurrence of long-term hormonal defects cannot be predicted by the severity of acute NE.

Response to Reviewers: Ms. No.

during the follow-up. Since the clinical diagnoses of hormonal deficiencies are now shown in Table 3 (in the revised version), we have omitted the corresponding text from Results, Hormonal deficiencies: "Of these 9 patients, 2 were diagnosed with isolated central hypogonadism, 2 had multiple pituitary hormone deficiencies, 2 had primary hypothyroidism only, one had primary hypothyroidism and primary hypogonadism, one was diagnosed with isolated central hypogonadism and peripheral hypothyroidism, and one with primary hypothyroidism and a possible GH deficiency (undetectable GH level and IGF-1 6.5 nmol/l; reference range 10-29 nmol/l)." In order to further clarify the results, we have also referred to the patients described in Table 3 (e.g. "patient 1, table 3") in the revised text: Results, page 8, Hormonal deficiencies, lines 6 and 12, as well as in Results, Imaging studies, page 9, the first and the third line). We have omitted the following text from Results, Imaging studies: "Testo 11.6-10.8-8.1-7.4 nmol/l, LH 1.6-1.4-2.0-2.6 U/l, fT4 12.5-11.8-10.4-10.4 pmol/l, and TSH 1.2-1.1-1.0-0.9 mU/l, measured in the acute phase, and after 1 year, 5 and 9 years, respectively", since the same values are now shown in Table 3 (patient 2) of the revised version.

B.

The reviewer correctly points out that in the original manuscript (Results, the second paragraph) the expression "Table 1 shows that serum median cortisol and PRL levels increased and Testo concentration decreased during the acute phase of NE" is misleading. We have now revised the sentence in page 7, the second line, as follows: "Table 1 shows that serum median cortisol and PRL levels were higher and Testo concentrations lower during the acute phase of NE". Accordingly, we have revised the Discussion, the second line, as follows: "Serum cortisol and PRL levels were higher and serum Testo concentrations lower during acute NE compared to the corresponding values after 3 months".

C.

The reviewer is right that it would have been valuable to include patients with other viral infections as controls, as we have clearly stated in the discussion (the penultimate paragraph). The findings of the present study give rise to controlled studies in the future. We were not quite sure if the reviewer wished for a table of the limitations of this study, but we did not make any, since there are already four tables in the manuscript.

Minor point: We have added a symbol ">" and a parenthesis to the page 6, Results, line 5: "Acute impairment of renal function (maximum plasma creatinine concentration measured during hospital stay > 100 μmol/l) was observed in 78 % of the patients, and 3 needed transient dialysis therapy".

Point-by-point responses to the comments by Reviewer 2

The diagnosis of PUUV infection is based on serological tests, detection of PUUV IgM or low avidity of PUUV IgG. In Finland, PUUV is the only hantavirus that causes HFRS. We have now added a more detailed description of the serological test in the revised manuscript on page 4, line 2: "The original patient group consisted of 70 prospectively collected consecutive patients, hospitalized due to serologically verified acute PUUV infection at Tampere University Hospital, Finland, during the years 1997 to 1999. The specific serological diagnosis was based on an immunoglobulin M-capture enzyme immunoassay and PUUV Sotkamo strain full-length N protein expressed by using the baculovirus system in Sf9 insect cells [START_REF] Vapalahti | Antigenic properties and diagnostic potential of Puumala virus nucleocapsid protein expressed in insect cells[END_REF]. In comparison with various other protocols the assay showed optimal sensitivity and specificity [START_REF] Sjölander | Evaluation of serological methods for diagnosis of Puumala hantavirus infection (nephropathia epidemica)[END_REF]". We have also added a new reference 19: Sjölander, K.B., Elgh, F., [START_REF] Sjölander | Evaluation of serological methods for diagnosis of Puumala hantavirus infection (nephropathia epidemica)[END_REF] Evaluation of serological methods for diagnosis of Puumala hantavirus infection (nephropathia epidemica). J Clin Microbiol 35, 3264-3268.

Introduction

Nephropathia epidemica (NE) is a form of hemorrhagic fever with renal syndrome (HFRS), caused by Puumala hantavirus (PUUV) [START_REF] Vapalahti | Hantavirus infections in Europe[END_REF]. PUUV along with its host, the bank vole (Myodes glareolus), is found all over Europe, excluding the Mediterranean region. In Finland, 1000-3000 serologically verified cases occur annually and the average PUUV seroprevalence in the population is 5 %. Other hantaviruses causing HFRS include Hantaan (HTNV), Dobrava (DOBV), Saaremaa, and Seoul viruses, while several others found in Americas cause hantavirus cardiopulmonary syndrome [START_REF] Vapalahti | Hantavirus infections in Europe[END_REF]. NE is clinically characterized by fever, headache, nausea, backache and abdominal pain, as well as transient renal insufficiency [START_REF] Lähdevirta | Nephropathia epidemica in Finland. A clinical histological and epidemiological study[END_REF][START_REF] Mustonen | Nephropathia epidemica in Finland: a retrospective study of 126 cases[END_REF][START_REF] Settergren | Clinical characteristics of nephropathia epidemica in Sweden: prospective study of 74 cases[END_REF]. Hemorrhages are rare. Complete recovery from NE is the usual outcome, but a convalescent phase with fatigue may last for months [START_REF] Lähdevirta | Nephropathia epidemica in Finland. A clinical histological and epidemiological study[END_REF][START_REF] Mustonen | Nephropathia epidemica in Finland: a retrospective study of 126 cases[END_REF][START_REF] Settergren | Clinical characteristics of nephropathia epidemica in Sweden: prospective study of 74 cases[END_REF]. Genetic susceptibility seems to determine the severity of NE, as individuals with the HLA B8 DRB1*0301 haplotype have been shown to suffer from a severe form of the disease [START_REF] Mustonen | Genetic susceptibility to severe course of nephropathia epidemica caused by Puumala hantavirus[END_REF], while the HLA B27 allele correlates with a mild clinical course [START_REF] Mustonen | Association of HLA B27 with benign clinical course of nephropathia epidemica caused by Puumala hantavirus[END_REF].

There are case reports of hypophyseal hemorrhage and panhypopituitarism in patients with acute NE [START_REF] Forslund | Complications of nephropathia epidemica: three cases[END_REF][START_REF] Hautala | Hypophyseal hemorrhage and panhypopituitarism during Puumala Virus Infection: Magnetic Resonance Imaging and detection of viral antigen in the hypophysis[END_REF][START_REF] Sane | Hypopituitarism and hepatitis as complications of nephropathia epidemica[END_REF][START_REF] Settergren | A case of nephropathia epidemica associated with panhypopituitarism and nephrotic syndrome[END_REF][START_REF] Valtonen | Four fatal cases of nephropathia epidemica[END_REF]. Postmortem studies of patients with HTNV-induced HFRS, as well as in the few fatal cases of NE, have revealed hemorrhage and necrosis of the pituitary gland in 50-100 % of the autopsies [START_REF] Valtonen | Four fatal cases of nephropathia epidemica[END_REF][START_REF] Hullinghorst | Pathology of epidemic hemorrhagic fever[END_REF][START_REF] Lukes | The pathology of thirty-nine fatal cases of epidemic hemorrhagic fever[END_REF]. PUUV antigen has been detected in hypophyseal neuroendocrine and endothelial cells of a patient with fatal NE [START_REF] Hautala | Hypophyseal hemorrhage and panhypopituitarism during Puumala Virus Infection: Magnetic Resonance Imaging and detection of viral antigen in the hypophysis[END_REF]. In a recent retrospective Serbian study of 60 adults with a previous HFRS, a high prevalence of hypopituitarism was identified after recovery from HFRS [START_REF] Stojanovic | High risk of hypopituitarism in patients who recovered from hemorrhagic fever with renal syndrome[END_REF]. Immunological factors including tumor necrosis factor alpha (TNF-α) and interleukin-6 (IL-6) have been suggested to be involved in the pathogenesis of NE [START_REF] Kanerva | Pathogenesis of Puumala and other hantavirus infections[END_REF]. Cytokines produced in inflammatory foci may also cause changes in the endocrine system, including activation of the hypothalamicpituitary-adrenal (HPA) axis [START_REF] Imura | Endocrine-paracrine interaction in communication between the immune and endocrine systems. Activation of the hypothalamic-pituitary-adrenal axis in inflammation[END_REF][START_REF] Turnbull | Regulation of the hypothalamic-pituitary-adrenal axis by cytokines: actions and mechanisms of action[END_REF].

In the present study we examined the prevalence of hormonal abnormalities in hospital-treated patients with NE, during both the acute illness and long-term follow-up. We also studied whether the hormonal abnormalities associated with NE were related to the clinical severity of the acute illness, the intensity of the cytokine response (plasma IL-6 and TNF-α levels), or the immunogenotype of the patient (HLA-B8, -DR3 and -B27 alleles).

Materials and Methods

Subjects

The original patient group consisted of 70 prospectively collected consecutive patients, hospitalized due to serologically verified acute PUUV infection at Tampere University Hospital, Finland, during the years 1997 to 1999. The specific serological diagnosis was based on an immunoglobulin M-capture enzyme immunoassay and PUUV Sotkamo strain full-length N protein expressed by using the baculovirus system in Sf9 insect cells [START_REF] Vapalahti | Antigenic properties and diagnostic potential of Puumala virus nucleocapsid protein expressed in insect cells[END_REF].

In comparison with various other protocols the assay showed optimal sensitivity and specificity [START_REF] Sjölander | Evaluation of serological methods for diagnosis of Puumala hantavirus infection (nephropathia epidemica)[END_REF]. Fifty-four of the patients attended the 12-month follow-up visit, and they comprised the present study population. The median patient age was 42 years (range 15-70 years), and 37 (69 %) of them were male. Fourteen patients had previous diseases: hypertension in four patients, hypothyroidism in two, as well as ankylosing spondylarthritis, bronchial asthma, aortic valve disease, coeliac disease, sequel of renal tuberculosis, operated cancer of the vocal cords, operated meningeoma and osteoporosis in one each. All the patients continued their regular medications during the study, including two patients with L-thyroxine substitution for hypothyroidism. None of the patients received corticosteroid therapy, or any other drug that could possibly affect plasma hormone concentrations, except for the two patients on L-thyroxine substitution therapy. One female patient was breast-feeding during acute NE and her estradiol (E2), follicle-stimulating hormone (FSH) and prolactin (PRL) values were excluded from the analyses. The Ethics Committee of Tampere University Hospital approved the study and every patient gave a written informed consent to participate.

Study protocol

All the patients were examined during their hospital stay. Detailed medical histories were obtained, and a careful physical examination was performed. All blood specimens were obtained between 0730-0930 h in the morning.

Blood samples for plasma creatinine, C-reactive protein (CRP), IL-6, TNF-, and blood cell counts, as well as 24-h urinary protein excretion were collected on three consecutive mornings after hospital admission. The first blood sample for hormonal analyses was obtained on the first morning of hospital care, and the second sample was taken on the third morning. The highest or the lowest value of each variable measured during hospitalization was designated as the maximum or the minimum value, respectively. All the serum samples for hormone analyses were stored at -70 C until analyzed.

The patients were scheduled to an outpatient examination 3 and 12 months after the acute disease.

Forty-nine patients attended the 3-month visit (74 to 146 days, median 102 days after admission), and 54 the 12month visit. The last prearranged follow-up visit was organized in 2004 (4 to 7 years, median 5 years after NE), and 38 patients participated. All serum hormonal levels were analyzed in 2007. Due to the hormonal results, we invited six patients to an additional visit in 2008 (9 to 10 years, median 10 years after NE), and they underwent physical, laboratory and imaging studies, as appropriate (see Methods).

Methods

Plasma creatinine and CRP concentration, blood cell counts, and 24-h urinary protein excretion were determined by standard laboratory methods. Plasma IL-6 and TNF- concentrations were determined using enzyme-linked immunosorbent assays (PeliKine Compact human IL-6 and TNF- kits, Red Cross Blood Transfusion Service, Amsterdam, The Netherlands). The analyses of the alleles HLA-B8, -DR3 and -B27 were performed as described elsewhere [START_REF] Westman | An HLA-DR typing protocol using group-specific PCR-amplification followed by restriction enzyme digests[END_REF].

Serum free thyroxine (fT4) and thyrotropin (TSH) were measured by a chemiluminescent microparticle immunoassay (Abbott Architect i2000 system, Abbott Laboratories, IL, USA), cortisol, PRL, luteinizing hormone (LH), and FSH by fluoroimmunoassay (1235 AutoDELFIA, Wallac Ltd, Turku, Finland), and E2 as well as testosterone (Testo) by radioimmunoassay (1277 Gammamaster, Wallac Ltd). The six patients invited to an additional visit in 2008 were also studied for serum levels of growth hormone (GH) by timeresolved fluoroimmunoassay (AutoDELFIA hGH, Wallac Ltd), insulin-like growth factor 1 (IGF-1) by immunoluminometric assay (DiaSorin Liaison IGF1, Saluggia, Italy), antibodies against thyroid peroxidase (TPOAb) by immunoluminometric assay (Abbott Architect Anti-TPO, Wiesbaden, Germany), as well as for adrenocortical function by standard 250 g ACTH stimulation test.

The reference ranges were 9.0-19.0 pmol/l for fT4, 0.4-4.0 mU/l for TSH, 180-680 nmol/l for cortisol, < 450 mU/l for PRL in male patients, < 600 mU/l in premenopausal women, and < 280 mU/l in postmenopausal women. The male reference ranges were 10.4-34.6 nmol/l for Testo, and 0.7-6.7 U/l for LH. As the phase of menstrual cycle was not known, E2 below 0.1 nmol/l was regarded as a hypogonadal value for a premenopausal woman. GH reference range was 0-11.5 mU/l, and the age-specific reference values for IGF-1 were 19-65 nmol/l at 18-20 y, 15-45 nmol/l at 21-30 y, 14-36 nmol/l at 31-50 y, 10-29 nmol/l at 51-70 y and 8-23 nmol/l over 70 years of age. TPOAb reference range was < 6 U/ml. Cortisol response exceeding 550 nmol/l was considered normal in the ACTH test.

Peri-and postmenopausal women were not analyzed for defects of the gonadal axis, as they were hypogonadal by definition. However, their FSH and E2 levels were included in the analysis of hormonal alterations between the acute illness and 3 months afterwards. Chronic, overt hypogonadism was defined as Testo (in men) or E2 (in premenopausal women) repeatedly below the reference range one year or more after the acute NE. Chronic, subclinical/compensated testicular failure was diagnosed in a male patient with chronically elevated LH but Testo within the reference range. Chronic, overt hypothyroidism was defined as fT4 repeatedly below 9.0 pmol/l and/or TSH repeatedly above 4.0 mU/l in a patient with chronic thyroiditis, goiter, and/or symptoms suggestive of hypothyroidism [START_REF]American Association of Clinical Endocrinologists Medical Guidelines for Clinical Practice for the Evaluation and Treatment of Hyperthyroidism and Hypothyroidism[END_REF].

Thyroid ultrasound imaging (Vivid I, GEMS Ultrasound, Tirat Carmel, Israel) was performed by the same investigator (P.J.) on the six patients attending the additional visit in 2008. The hypothalamic-pituitary region was studied by magnetic resonance imaging (MRI, MAGNETOM Trio 3-Tesla scanner, Siemens, Erlangen, Germany) in the patients showing distinct hormonal deficits of central origin.

Statistics

To describe the data, medians (ranges) were given for continuous variables and percentages for categorical variables. Comparisons between the groups were made with Mann-Whitney U-test for continuous variables, while Pearson chi-square test or Fisher's exact test was used for categorical data. Wilcoxon signed-rank test was used to evaluate changes in serum hormone levels between the acute phase of NE and 3 months afterwards, and between 3 months and 1 year or 5 years, respectively. Correlations were calculated by means of Spearman´s rank correlation coefficient. A p-value of less than 0.05 in two-sided testing was considered statistically significant.

Statistical analyses were performed using SPSS for Windows version 7.5 (SPSS Inc., Chicago, IL, USA).

Results

Clinical and laboratory findings during acute NE

The clinical picture was typical of acute NE in all patients. All had fever lasting for a median of 5 days (range 2 to 14 days). Ten (19 %) received empirical antibiotics, but in none of them could a bacterial infection be confirmed. Acute impairment of renal function (maximum plasma creatinine concentration measured during hospital stay > 100 μmol/l) was observed in 78 % of the patients, and 3 needed transient dialysis therapy.

Thrombocytopenia (blood platelet count < 150 x 10 9 /l) was present in all patients, but none had major bleeding complications. Hypotension (systolic blood pressure < 90 mmHg) was observed on admission in 3 patients, and was corrected by fluid infusion.

Serum hormone levels during the acute phase of NE

Table 1 shows that serum median cortisol and PRL levels were higher and Testo concentrations lower during the acute phase of NE, compared to the corresponding values after 3 months. Serum median LH level did not change. There were, however, remarkable individual differences in the direction and magnitude of serum Testo and LH alterations (Figure 1). Serum TSH level was slightly higher and FSH concentration lower during the acute phase than after 3 months (Table 1). There were no differences between the median hormone levels (cortisol, PRL, fT4, TSH, Testo, LH, E2 and FSH) measured at 3 months compared to the levels measured after 1 or 5 years (data not shown).

Serum cortisol concentration during acute NE correlated positively with maximum plasma creatinine and IL-6 levels (r=0.352, p=0.009, and r=0.319, p=0.019, respectively), with blood leukocyte count (r=0.370, p=0.006), and with serum PRL level (r=0.402, p=0.003). Similarly, serum PRL level correlated with creatinine and IL-6 levels (r=0.511, p<0.001, and r=0.338, p=0.012, respectively), and with blood leukocyte count (r=0.421, p=0.002).

Eighteen out of 37 (49 %) men presented with a low Testo level during the acute phase of NE. Fifteen of them had a coincident serum LH level within the reference range, and three had an increased LH level. In addition, 5 (14 %) men had an elevated LH concentration together with a Testo level within the reference range.

None of the eight male patients with acute primary hypogonadism (elevated LH) complained of testicular pain.

The minimum Testo concentration correlated inversely with age and leukocytosis (r=-0.341, p=0.039, and r=-0.493, p=0.002, respectively), and with maximum creatinine, IL-6, CRP and PRL levels (r=-0.437, p=0.007; r=-0.411, p=0.011; r=-0.352, p=0.032, r=-363, p=0.027, respectively), but not with body mass index (BMI) (r=-0.009, p=0.957). Three out of six premenopausal women (aged 16, 21 and 37 years) presented with low E2 and FSH levels during the acute phase of NE (E2 range 0.04-0.05 nmol/l, and FSH range 0.08-0.8 U/l).

Two patients had a low serum fT4 level together with a low-normal TSH level during the acute phase.

Five out of 54 (9%) patients presented with an increased serum TSH level, together with fT4 within the lownormal range; one of them had a previously diagnosed primary hypothyroidism, and she used L-thyroxine substitution throughout the study. Serum fT4 and TSH levels measured during acute NE did not correlate with age, BMI, plasma creatinine, CRP, or cytokine levels (data not shown).

Altogether 30 out of 54 (56 %) patients had abnormalities of the gonadal and/or thyroid axis during the acute phase of NE. Central hormonal defects were found in 19 patients, while 13 patients had a primary deficit. As shown in Table 2, the patients with central hormonal deficiencies had a higher plasma creatinine concentration, and blood leukocyte count than the patients with normal hormone levels.

There were no differences in the clinical picture or basic laboratory findings during acute NE between the patients with primary hormonal deficiencies and those with normal hormone levels (data not shown). Neither did the cytokine levels or HLA-B8, -DR3 and -B27 alleles predict the acute primary hormone deficits.

Hormonal deficiencies one to ten years after NE

During a median follow-up of 5 years, 9 out of 54 (17 %) NE patients presented with chronic, overt hormonal deficits. Table 3 shows the serial plasma hormone levels of these patients. The clinical diagnoses of overt hormonal deficiencies were based both on the hormone measurements and on the clinical signs and symptoms of the patients.

One of the 2 patients with hormone levels suggestive of combined pituitary hormone defects, a 43year old male patient (patient 1, table 3), presented with low testosterone levels and declining fT4 levels within the low reference range during acute NE, as well as after 3 and 12 months (Testo 7.1-7.8-7.6 nmol/l, LH 2.8-2.1-1.8 U/l, fT4 11.9-10.6-10.4 pmol/l, and TSH 2.8-3.8-1.9 mU/l, respectively). He died for an unknown reason soon after his 1-year follow-up visit. The hormone levels, indicating central hypogonadism and possible hypothyroidism, were analyzed only after his death. The overt hormonal deficiencies of the other patients, as well, had gone unnoticed until the patients were invited to the additional visit in 2008. Only one female patient had been started on L-thyroxine, due to overt hypothyroidism diagnosed 9 years after the acute NE (patient 8, table 3). There were no differences in the age or gender distribution, clinical picture or basic laboratory findings during acute NE between those 9 patients who presented with chronic hormone deficiencies after NE and those with normal hormone levels (Table 4). None of the 3 patients who were hypotensive on admission developed chronic hormonal deficiencies. The prevalence of HLA-B8, -DR3 and -B27 alleles did not differ between the groups (data not shown).

Imaging studies on the additional visit in 2008

Thyroid ultrasound examination showed diffuse or patchy hypoechogenicity, pertinent with thyroiditis, in the five patients with primary hypothyroidism, while TPOAb were measurable in only 2 of them.

A sellar MRI scan was performed during the additional visit in 2008 in two men whose symptoms and hormone levels suggested central hormonal defects. One had an apparent GH deficiency (patient 5, table 3), and the other was a 41-year-old patient who developed a marked secondary hypogonadism and a probable secondary hypothyroidism (patient 2, table 3). The MRI scans showed no pathology of the hypothalamic-pituitary region in either of them.

Discussion

To our knowledge, this is the first study on hormonal alterations occurring during and after NE. Serum cortisol and PRL levels were higher and serum Testo concentrations lower during acute NE compared to the corresponding values after 3 months. These acute hormonal alterations were related to the severity of acute renal insufficiency and inflammation. After one to ten years, surprisingly many patients suffered from chronic hormonal deficits of either central or peripheral origin. The chronic defects could not be predicted by renal insufficiency, degree of inflammation, or other markers of the clinical severity of acute NE, or by the host genetic factors studied.

Approximately half of the men and premenopausal women presented with low serum gonadal hormone levels during the acute phase of NE. Serum Testo levels in general increased during a 3-month followup, while concomitant LH levels remained stable, indicating that the acute hypogonadism was transient and of central origin in most cases. The degree of hypothalamic-pituitary-gonadal axis suppression, as well as the increase in plasma cortisol production, are known to be related to the severity of acute illness [START_REF] Jurney | Spectrum of serum cortisol response to ACTH in ICU patients. Correlation with degree of illness and mortality[END_REF][START_REF] Spratt | Reproductive axis suppression in acute illness is related to disease severity[END_REF]. Also in our patients with acute NE, low Testo levels and high cortisol and PRL levels were related to the degree of inflammation and the severity of acute renal failure. As the kidneys are involved in the elimination of PRL and corticosteroids [START_REF] Bauer | The kidney is the main site of prolactin elimination in patients with liver disease[END_REF][START_REF] Whitworth | The kidney is the major site of cortisone production in man[END_REF], the acute impairment of renal function during NE may have resulted in diminished clearance of PRL and cortisol, contributing to their increased serum levels.

We also found eight male patients with acute primary hypogonadism during PUUV infection. Three out of these 8 acute testicular failures developed into chronic, compensated ones, i.e., Testo levels remained within the normal range, while LH levels were chronically elevated. In addition, two cases of chronic, compensated hypogonadism developed during the follow-up, without testicular dysfunction during the acute NE.

There are no previous reports of clinical orchitis or primary hypogonadism in hantaviral diseases. However, the possibility remains that PUUV-infection may have contributed to the pathogenesis of acute and chronic primary hypogonadism, which were over-represented [START_REF] Gray | Age, disease, and changing sex hormone levels in middle-aged men: results of the Massachusetts Male Aging Study[END_REF][START_REF] Härkönen | The polymorphic androgen receptor gene CAG repeat, pituitary-testicular function and andropausal symptoms in ageing men[END_REF] in this relatively young group of patients (median age in males 39 years, the youngest patient with chronic hypogonadism 30 years).

Primary hypothyroidism was remarkably frequent in our patients with NE. Two patients had been diagnosed with hypothyroidism prior to NE, and 5 more diagnoses of primary hypothyroidism were made during the follow-up. The overall prevalence of primary hypothyroidism thus mounted up to 24 % in the female patients and 8% in the males, which are about three-fold prevalences compared to those recently reported for similar age groups in the general population [START_REF] Bjoro | Prevalence of thyroid disease, thyroid dysfunction and thyroid peroxidase antibodies in a large, unselected population. The Health Study of Nord-Trondelag (HUNT)[END_REF][START_REF] Hollowell | Serum TSH, T(4), and thyroid antibodies in the United States population (1988 to 1994): National Health and Nutrition Examination Survey (NHANES III)[END_REF][START_REF] Vanderpump | The epidemiology of thyroid diseases[END_REF]. All of the patients diagnosed with primary hypothyroidism in the present study showed a hypoechogenic structure of the thyroid on ultrasound examination, suggesting chronic thyroiditis [START_REF] Hegedus | Thyroid ultrasound[END_REF][START_REF] Mcgahan | Diagnostic ultrasound[END_REF]. TPOAb were present in only 2 of the 5 "new" cases of hypothyroidism, which may indicate that in the other 3 cases, the thyroiditis was not of autoimmune origin, but was possibly induced by the PUUV infection. The primary thyroid, or gonadal, defects did not relate to age, gender, or the degree of renal insufficiency or inflammation during acute NE. A high level of vigilance for possible signs of thyroid or gonadal dysfunction seems warranted in patients with a history of previous NE.

Seventeen percent of the present patients were diagnosed with a chronic, overt hormonal deficit. In a recent retrospective Serbian study the prevalence of any endocrine deficiency 2 years after HFRS was 18 % [START_REF] Stojanovic | High risk of hypopituitarism in patients who recovered from hemorrhagic fever with renal syndrome[END_REF].

It is noteworthy that the patients in the Serbian study had a more severe HFRS than our patients. In the present study, only three patients needed dialysis treatment, while 57 % of the Serbian patients were dialyzed. PUUV and DOBV coexist in the Balkan area, and induce HFRS with significant differences in severity [START_REF] Avsic-Zupanc | Hemorrhagic fever with renal syndrome in the Dolenjska region of Slovenia--a 10-year survey[END_REF][START_REF] Markotic | Characteristics of Puumala and Dobrava infections in Croatia[END_REF].

DOBV-infected patients have more frequently suffered from acute renal failure requiring dialysis treatment, shock, severe thrombocytopenia, and hemorrhagic complications than patients with PUUV [START_REF] Avsic-Zupanc | Hemorrhagic fever with renal syndrome in the Dolenjska region of Slovenia--a 10-year survey[END_REF][START_REF] Markotic | Characteristics of Puumala and Dobrava infections in Croatia[END_REF]. In the present study, chronic hormonal defects could not be predicted by the severity of acute NE. Hence, it is worth noticing that chronic hormonal deficits might occur even after a relatively mild hantaviral infection.

Previous case reports of severe HFRS have described hypophyseal hemorrhage and panhypopituitarism associated with PUUV and HTNV infections [START_REF] Forslund | Complications of nephropathia epidemica: three cases[END_REF][START_REF] Hautala | Hypophyseal hemorrhage and panhypopituitarism during Puumala Virus Infection: Magnetic Resonance Imaging and detection of viral antigen in the hypophysis[END_REF][START_REF] Sane | Hypopituitarism and hepatitis as complications of nephropathia epidemica[END_REF][START_REF] Settergren | A case of nephropathia epidemica associated with panhypopituitarism and nephrotic syndrome[END_REF][START_REF] Valtonen | Four fatal cases of nephropathia epidemica[END_REF][START_REF] Kim | A case of torsade de pointes associated with hypopituitarism due to hemorrhagic fever with renal syndrome[END_REF][START_REF] Park | Delayed onset of diuresis in a patient with acute renal failure due to hemorrhagic fever with renal syndrome who also developed anterior hypopituitarism[END_REF][START_REF] Pekic | Hypopituitarism as a late complication of hemorrhagic fever[END_REF][START_REF] Suh | Pituitary hemorrhage as a complication of hantaviral disease[END_REF]. The development of hypophyseal hemorrhage and subsequent panhypopituitarism has varied from acute to subacute. The mechanisms of pituitary hemorrhage are unknown, although hypovolemic shock and thrombocytopenia may contribute to the pathogenesis. In the present patients, thrombocytopenia or hypotension during acute NE did not correlate with either acute-phase or long-term hormonal abnormalities. Neither were there any MRI findings pertinent with previous hypophyseal or hypothalamic hemorrhage in the patients with central hormonal defects.

It is likely that the secondary hormone deficiencies found in the present series of NE patients developed by mechanisms other than an acute hemorrhagic disruption of the pituitary. Headache, vertigo, visual disturbances, and other signs of central nervous system involvement occur frequently in NE [START_REF] Lähdevirta | Nephropathia epidemica in Finland. A clinical histological and epidemiological study[END_REF][START_REF] Mustonen | Nephropathia epidemica in Finland: a retrospective study of 126 cases[END_REF][START_REF] Ahlm | Central nervous system and ophthalmic involvement in nephropathia epidemica (European type of haemorrhagic fever with renal syndrome)[END_REF][START_REF] Alexeyev | Neurological manifestations of hemorrhagic fever with renal syndrome caused by Puumala virus: review of 811 cases[END_REF], and cases of PUUV-induced meningoencephalitis have been reported [START_REF] Lähdevirta | Nephropathia epidemica in Finland. A clinical histological and epidemiological study[END_REF][START_REF] Mustonen | Nephropathia epidemica in Finland: a retrospective study of 126 cases[END_REF][START_REF] Bergmann | Encephalitis due to a hantavirus infection[END_REF][START_REF] Launes | Nephropathia epidemica encephalitis[END_REF]. Presence of PUUV RNA in cerebrospinal fluid in a patient with acute NE has been confirmed [START_REF] Mähönen | Puumala virus RNA in cerebrospinal fluid in a patient with uncomplicated nephropathia epidemica[END_REF]. It has recently been shown that infections of the central nervous system may cause transient or permanent hypothalamic and/or pituitary dysfunction [START_REF] Schaefer | Hypothalamic-pituitary insufficiency following infectious diseases of the central nervous system[END_REF]. Viral meningoencephalitis or hypophysitis may also have contributed to the central hormone defects frequently found in our patients.

An important feature in hantaviral infections is a universally increased capillary permeability, causing tissue edema and hypotension [START_REF] Vapalahti | Hantavirus infections in Europe[END_REF]. Immunological factors including overproduction of pro-inflammatory cytokines during acute infection are probably involved in the pathogenesis, and might result in immune-mediated damage to the endothelial cells [START_REF] Kanerva | Pathogenesis of Puumala and other hantavirus infections[END_REF]. The endothelial damage and increased vascular permeability may have been involved in the pathogenesis of central and peripheral hormonal defects of the NE patients. A tight interaction between the immune and endocrine systems, mainly through the HPA axis [START_REF] Imura | Endocrine-paracrine interaction in communication between the immune and endocrine systems. Activation of the hypothalamic-pituitary-adrenal axis in inflammation[END_REF][START_REF] Turnbull | Regulation of the hypothalamic-pituitary-adrenal axis by cytokines: actions and mechanisms of action[END_REF], is also likely to play a central role in the hormonal alterations, at least during acute NE.

Our study has some limitations. First, due to the retrospective nature of the hormonal analyses we did not have complete data on the possible signs and symptoms related to hormonal alterations during and after NE. This may have resulted in underestimation of the hormonal sequelae of NE, as milder forms of pituitary failure cannot be diagnosed by means of basal hormone levels alone. Stimulatory tests of the GH-IGF-1 axis or the HPA axis could have revealed more subtle hormonal defects, over and above the prevalence of overt hormonal failures reported here. In addition, it would have been valuable to include patients with other viral infections as controls. These limitations, however, should be set against the strengths of investigating a prospectively gathered group of consecutive patients both during the acute phase of NE and repeatedly during the long-term follow-up.

The findings of the present study give rise to prospective, controlled studies to verify the prevalence of hormonal defects after hantaviral diseases, and to clarify the pathogenesis of these hormonal alterations.

In summary, hormonal alterations were common during acute NE, and the acute hormone deficits of central origin correlated with the clinical severity of NE. Chronic hormonal defects, however, could not be predicted by the severity of acute NE, and chronic deficits emerged even after a relatively mild course of NE.

Patients with a history of NE should be readily investigated for hormonal deficiencies, in view of the high prevalence of chronic central and peripheral hormone defects in these patients. The reference ranges were 10.4-34.6 nmol/l for testosterone, and 0.7-6.7 U/l for LH in men. An E2 level below 0.1 nmol/l was considered hypogonadal in premenopausal women, as the phase of menstrual cycle was not known. Peri-and postmenopausal women were not analyzed for gonadal defects, as they were hypogonadal by definition.

The reference range for fT4 was 9.0-19.0 pmol/l, and for TSH 0.4-4.0 mU/l. The Mann-Whitney U-test was used for continuous variables; Pearson chi-square test was used for gender.

Abbreviations: M = male, F = female, IL = interleukin, TNF-α = tumor necrosis factor alpha, min = minimum, max = maximum.

Legend to Figure 1. A. According to the reviewer's advice, we have now added two new tables (Tables 3 and4)

to clarify the presentation of the observations. Table 3 shows the clinical diagnoses and serial plasma hormone levels in those 9 NE patients who presented with chronic, overt hormonal deficits. In B. The reviewer correctly points out that in the original manuscript (Results, the second paragraph) the expression "Table 1 shows that serum median cortisol and PRL levels increased and Testo concentration decreased during the acute phase of NE" is misleading.

We have now revised the sentence in page 7, the second line, as follows: "Table 1 shows that serum median cortisol and PRL levels were higher and Testo concentrations lower during the acute phase of NE". C. The reviewer is right that it would have been valuable to include patients with other viral infections as controls, as we have clearly stated in the discussion (the penultimate paragraph). The findings of the present study give rise to controlled studies in the future. We

were not quite sure if the reviewer wished for a table of the limitations of this study, but we did not make any, since there are already four tables in the manuscript.

Minor point: We have added a symbol ">" and a parenthesis to the page 6, Results, line 5: "Acute impairment of renal function (maximum plasma creatinine concentration measured during hospital stay > 100 μmol/l) was observed in 78 % of the patients, and 3 needed transient dialysis therapy".

Point-by-point responses to the comments by Reviewer 2

The diagnosis of PUUV infection is based on serological tests, detection of PUUV IgM or low avidity of PUUV IgG. In Finland, PUUV is the only hantavirus that causes HFRS. We have now added a more detailed description of the serological test in the revised manuscript on page 4, line 2:

"The original patient group consisted of 70 prospectively collected consecutive patients, hospitalized due to serologically verified acute PUUV infection at Tampere University Hospital, Finland, during the years 1997 to 1999. The specific serological diagnosis was based on an immunoglobulin M-capture enzyme immunoassay and PUUV Sotkamo strain full-length N protein expressed by using the baculovirus system in Sf9 insect cells [START_REF] Vapalahti | Antigenic properties and diagnostic potential of Puumala virus nucleocapsid protein expressed in insect cells[END_REF]. In comparison with various other protocols the assay showed optimal sensitivity and specificity [START_REF] Sjölander | Evaluation of serological methods for diagnosis of Puumala hantavirus infection (nephropathia epidemica)[END_REF]". We have also added a new

4 9 . 0 - 11 . 5 -

 90115 16.1 PHT CHG=central hypogonadism, PHT=primary hypothyroidism, E2=estradiol, LH=luteinizing hormone, FSH=follicle-stimulating hormone, fT4=free thyroxine, TSH=thyrotropin. * only the hormone levels measured during acute NE and 1 year later are shown for the two patients, whose follow-up ended at the 1-year visit ** and a possible growth hormone (GH) deficiency: an undetectable GH level and insulin-like growth factor-1 6,5 nmol/l (reference range 10-29 nmol/l) *** on L-thyroxine treatment

Figure

  Figure shows serum testosterone (A) and luteinizing hormone (LH) (B) levels measured in 37 men during acute NE and after 3 months. Testosterone levels were below the reference range in half of the men during acute NE, while LH levels were within the reference range in the majority, indicating that the hypogonadism was mostly of central origin. The reference ranges were 10.4-34.6 nmol/l for testosterone (vertical line showing lower reference limit) and 0.7-6.7 U/l for LH (vertical line showing higher reference limit).

Figure

  Figure Click here to download high resolution image

Manuscript Click here to

  download Authors' Response to Reviewers' Comments: Itemized_responses_17_03_2010.doc Accordingly, we have revised the Discussion, the second line, as follows: "Serum cortisol and PRL levels were higher and serum Testo concentrations lower during acute NE compared to the corresponding values after 3 months".

reference 19 :

 19 Sjölander, K.B., Elgh, F., Kallio-Kokko, H et al. (1997) Evaluation of serological methods for diagnosis of Puumala hantavirus infection (nephropathia epidemica). J Clin Microbiol 35, 3264-3268.

Table 1 .

 1 Serum hormone levels during acute NE, and after 3 months.

		Acute phase a	At 3 months	p
		Median Range	Median Range
	All patients (n=54):
	Cortisol (nmol/l)	520	267 -1157 361	163 -606	<0.001*
	Prolactin (mU/l) b	266	32 -1364	131	46 -550	<0.001*
	Thyrotropin (mU/l)	1.9	0.1 -9.0	1.7	0.02 -7.6	0.011*
	Free thyroxine (pmol/l)	12.2	7.5 -18.1	12.8	10.1 -17.4 0.526
	Males (n=37):
	Testosterone (nmol/l)	9.9	1.1 -27.0	17.3	7.8 -25.4	<0.001*
	Luteinizing hormone (U/l)	4.0	0.9 -18.0	3.6	2.0 -7.9	0.399
	Females (n=16) b :
	Follicle-stimulating hormone (U/l) 4.7	0.6 -40.4	23.7	3.0 -73.2	0.001*
	Estradiol (nmol/l)	0.07	0.02 -0.42 0.11	0.04 -0.49 0.263

a the highest or the lowest serum concentration of each hormone measured during hospitalization, as appropriate. b One patient was breast-feeding during acute NE and her E2, FSH and PRL values were excluded from the analyses (see Subjects and Methods).*Statistically significant difference between hormone levels measured in the acute phase and 3 months later (Wilcoxon signed-rank test).

Table 2 .

 2 Clinical and laboratory findings of the 19 patients diagnosed with central hormonal defects during the acute phase of NE, compared to those of the 23 patients with normal hormone levels.

	Finding	Central hormonal defects	Normal hormone levels	p
		n=19		n=23
		Median	Range	Median	Range
	Age (years)	41	17 -62	41	24 -70	0.909
	Gender (M/F)	15/4		13/10		0.125
	Body mass index (kg/m 2 )	25	19 -37	26	19 -42	0.456
	Creatinine max (µmol/l)	368	79 -749	208	76 -878	0.029*
	Leukocytes max (10 9 /l)	13.4	4.4 -22.8	9.2	6.7 -23.4	0.033*
	Platelets min (10 9 /l)	62	13 -127	53	18 -136	0.869
	C-reactive protein max (mg/l)	78	22 -213	67	27 -136	0.791
	Plasma IL-6 max (pg/ml)	19.6	5.9 -40.7	13.5	3.3 -59.1	0.052
	Plasma TNF-α max (pg/ml)	3.4	1.4 -26.9	3.8	1.4 -37.4	0. 693
	Proteinuria max (g/day)	1.8	0.2 -9.5	2.0	0.3 -17.8	0.990
	Duration of hospital stay (days)	8	5 -13	7	3 -11	0.134
	*Statistically significant difference between patients with acute central hormone deficits and those with
	normal hormone levels. The Mann-Whitney U-test was used for continuous variables; Pearson chi-square test
	was used for gender. Abbreviations: M = male, F = female, IL = interleukin, TNF-α = tumor necrosis factor
	alpha, min = minimum, max = maximum.

Table 3 .

 3 Serial plasma hormone levels of 9 NE patients who presented with chronic, overt hormonal deficits during a median follow-up of 5 years. The hormone levels presented in the table were measured during the acute phase of NE (first value in each series) and on the follow-up visits 1 to 10 years later (2 nd and 3 rd value of each series).

	Abnormal values are printed in bold.
	Patient	Gender / age	Duration	Testosterone in males	LH in males and	fT4 (pmol/l)	TSH (mU/l)	Clinical
		during acute	of follow-	and E2 in females	FSH in females (U/l)			diagnosis
		NE (years)	up (years)	(nmol/l)
	1	M/43	1 *	7.1 -7.6	2.8 -1.8	11.9 -10.4	2.8 -1.9	CHG
	2	M/41	9	11.6 -8.1 -7.4	1.6 -2.0 -2.6	12.5 -10.4 -10.4	1.2 -1.0 -0.9	CHG
	3	M/59	5	1.9 -11.5 -7.6	1.7 -3.6 -2.3	11.0 -11.4 -12.1	1.3 -1.7 -1.5	CHG
	4	M/52	1 *	7.9 -9.8	2.5 -2.8	14.5 -13.3	2.3 -1.5	CHG
	5	M/42	9	11.3 -15.7 -21.9	18.0 -5.5 -6.8	11.2 -13.9 -13.4	1.9 -4.3 -5.0	PHT **
	6	M/58	9	12.5 -15.3 -10.7	6.9 -6.7 -9.2	14.0 -12.8 -12.0	3.7 -4.3 -5.7	PHT
	7	M/60	7	1.1 -19.2 -21.1	2.8 -5.0 -4.0	9.4 -10.9 -11.7	1.7 -5.8 -9.9	PHT
	8	F/17	9	0.05 -0.07 -<0.06	0.6 -2.8 -0.4	16.2 -11.5 -12.5	3.5 -2.7 -6.6 ***	CHG, PHT
	9	F/47	9	0.05 -<0.02 -<0.06	2.6 -41.5 -39.8	9.5 -9.9 -9.

Table 4 .

 4 Clinical and laboratory findings during the acute phase of NE in the 9 patients who developed chronic hormonal defects after NE, compared to those in the 45 patients whose hormone levels remained normal during the follow-up.

	Finding	Chronic hormonal defects	No chronic defects	p
		n=9		n=45
		Median	Range	Median	Range
	Age (years)	47	17 -60	39	15 -70	0.194
	Gender (M/F)	7/2		30/15		0.512
	Body mass index (kg/m 2 )	26	21 -37	25	19 -42	0.347
	Creatinine max (µmol/l)	380	98 -725	213	76 -878	0.190
	Leukocytes max (10 9 /l)	11.2	5.7 -20.4	10.3	4.4 -23.4	0.926
	Platelets min (10 9 /l)	54	17 -139	62	3 -136	0.898
	C-reactive protein max (mg/l)	72	25 -213	67	20 -136	0.423
	Plasma IL-6 max (pg/ml)	19.9	5.0 -43.0	14.2	3.3 -59.1	0.102
	Plasma TNF-α max (pg/ml)	2.8	1.4 -8.7	3.7	1.4 -37.4	0.267
	Proteinuria max (g/day)	0.9	0.3 -3.9	2.0	0.1 -17.8	0.114
	Duration of hospital stay (days)	6	5 -13	7	3 -12	0.833

Table 4 ,

 4 we show the acute-phase clinical and laboratory findings of the 9 patients, who developed chronic hormonal defects after NE, compared to those 45 patients, whose hormone levels remained normal during the follow-up. Since the clinical diagnoses of hormonal deficiencies are now shown in Table3(in the revised version), we have omitted the corresponding text from Results, Hormonal deficiencies: "Of these 9 In order to further clarify the results, we have also referred to the patients described in Table 3 (e.g. "patient 1, table 3") in the revised text: Results, page 8, Hormonal deficiencies, lines 6 and 12, as well as in Results, Imaging studies, page 9, the first and the third line). We have omitted the following text from Results, Imaging studies: "Testo 11.6-10.8-8.1-7.4 nmol/l, LH 1.6-1.4-2.0-2.6 U/l, fT4 12.5-11.8-10.4-10.4 pmol/l, and TSH 1.2-1.1-1.0-0.9 mU/l, measured in the acute phase, and after 1 year, 5 and 9 years, respectively", since the same values are now shown in Table3(patient 2) of the revised version.

patients, 2 were diagnosed with isolated central hypogonadism, 2 had multiple pituitary hormone deficiencies, 2 had primary hypothyroidism only, one had primary hypothyroidism and primary hypogonadism, one was diagnosed with isolated central hypogonadism and peripheral hypothyroidism, and one with primary hypothyroidism and a possible GH deficiency (undetectable GH level and IGF-1 6.5 nmol/l; reference range 10-29 nmol/l)."

Acknowledgements: There is no conflict of interest and nothing to declare. This work was supported by the Competitive Research Funding of the Pirkanmaa Hospital District, by the Finnish Kidney Foundation and by the European Commission Project "Diagnostics and control of rodent-borne viral zoonoses in Europe" (QLK2-CT-2002-01358). We thank Jukka Partanen for the analyses of HLA alleles, and Esko Väyrynen for language revision. The skilful technical assistance of Heidi Hällström, Katriina Ylinikkilä and Mirja Ikonen is greatly appreciated.