Simon G Edwards
email: sedwards@harper-adams.ac.uk

Nigel P Godley

in wheat with early fungicide applications of prothioconazole

Keywords: Mycotoxins -fusarium, Mycotoxins -trichothecenes Food Types: Cereals and grain Proline®, yield, specific weight, thousand grain weight, fungicide timing, mycotoxins

published or not. The documents may come

Reduction of Fusarium head blight and deoxynivalenol

in wheat with early fungicide applications of prothioconazole

Introduction

Fusarium head blight (FHB) is an important disease of small grain cereals as it results in decreased yield, reduced grain quality (specific weight and thousand grain weight), processing quality and the presence of fusarium mycotoxins in harvested grain [START_REF] Parry | Fusarium ear blight (Scab) in small-grain cereals -a review[END_REF]. The disease can be caused by several pathogens; the dominant ones are Fusarium graminearum (teleomorph Gibberella zeae), F. culmorum, F. poae, F. avenaceum, Microdochium nivale and M. majus. The Fusarium species produce a wide range of mycotoxins, the most important ones in wheat are deoxynivalenol (DON) and zearalenone. Both these mycotoxins are produced by F. graminearum and F. culmorum. Surveys have indicated that fusarium mycotoxins are common contaminants of wheat, however they usually occur at low concentrations [START_REF] Edwards | Fusarium mycotoxin content in UK organic and conventional wheat[END_REF]. High concentrations can occur when weather conditions are conducive, in particular wet weather from flowering to harvest [START_REF] Edwards | Fusarium mycotoxin content in UK organic and conventional wheat[END_REF]. DON causes reduced feed intake, reduced weight gain and vomiting in farm animals (Anon. 2004a). Nausea, vomiting, diarrhoea, abdominal pain, headache, dizziness and fever have been reported when high concentrations of DON were consumed by humans.

The European Commission set legislative limits for the fusarium mycotoxins including DON in cereal grains and cereal-based products intended for human consumption (Anon. 2006c). The maximum limit for DON in unprocessed wheat is 1250 µg kg -1 ; this limit applies to wheat placed on the market for processing for human consumption. Maximum limits are set on unprocessed cereals to avoid highly contaminated cereals entering the food chain and to encourage all measures to minimise fusarium mycotoxin contamination to be taken during the field stages of the production chain. The European Commission has also set guideline limits for fusarium mycotoxins in animal feed (Anon. 2006b). The lowest guidance limits have been set for pig feed owing to the high proportion of cereals in pig feed and their higher sensitivity to fusarium mycotoxins.

The DON guidance value for complimentary and complete feedingstuffs for pigs is 900 µg kg -1 . Numerous studies have been conducted to evaluate the efficacy of fungicides to reduce fusarium head blight and resultant DON in harvested wheat [START_REF] Boyacioglu | Effect of 3 Systemic Fungicides on Deoxynivalenol (Vomitoxin) Production by Fusarium graminearum in Wheat[END_REF][START_REF] Edwards | Quantification of trichothecene-producing Fusarium species in harvested grain by competitive PCR to determine efficacies of fungicides against fusarium head blight of winter wheat[END_REF][START_REF] Ellner | Influence of fungicide treatment on deoxynivalenol content in winter wheat artificially infected with Fusarium culmorum[END_REF][START_REF] Ioos | The effects of fungicides on Fusarium spp. and Microdochium nivale and their associated trichothecene mycotoxins in French naturally-infected cereal grains[END_REF][START_REF] Mennitti | Effect of fungicides on Fusarium head blight and deoxynivalenol content in durum wheat grain[END_REF][START_REF] Simpson | Differential control of head blight pathogens of wheat by fungicides and consequences for mycotoxin contamination of grain[END_REF]). These studies have highlighted that azole fungicides have the best efficacy, although it is important to note that the efficacy reported is highly variable between different azole fungicides and can be variable between experiments [START_REF] Beyer | Quantifying the effects of previous crop, tillage, cultivar and triazole fungicides on the deoxynivalenol content of wheat grain -a review[END_REF]. Tebuconazole is the most tested triazole and has been the industry standard for many years. Metconazole was introduced in 1994, and was

shown to have similar efficacy to tebuconazole [START_REF] Edwards | Quantification of trichothecene-producing Fusarium species in harvested grain by competitive PCR to determine efficacies of fungicides against fusarium head blight of winter wheat[END_REF][START_REF] Ioos | The effects of fungicides on Fusarium spp. and Microdochium nivale and their associated trichothecene mycotoxins in French naturally-infected cereal grains[END_REF]). More recently, prothioconazole was introduced in 2004, and this triazolinthione had the greatest inhibitory activity against F. graminearum in vitro of the fungicides tested [START_REF] Klix | Comparison of the declining triazole sensitivity of Gibberella zeae and increased sensitivity achieved by advances in triazole fungicide development[END_REF]) and has been shown to have high efficacy in field experiments [START_REF] Paul | Efficacy of triazole-based fungicides for Fusarium head blight and deoxynivalenol control in wheat: A multivariate meta-analysis[END_REF].

Field experiments of fungicide efficacy against head blight have been [START_REF] Parry | Fusarium ear blight (Scab) in small-grain cereals -a review[END_REF]. The traditional timing for fungicides to control head blight has been at flowering [START_REF] Zadoks | A decimal code for the growth stages of cereals[END_REF] Growth Stage 59-69) when the infection primarily occurs. This is typically the third fungicide spray for wheat in the UK, designated T3. The early fungicide applications are at first or second node detectable (GS31-32; T1) and flag leaf fully emerged (GS39; T2). As part of a preliminary study (results not shown) using oat-grain-inoculum applied at stem extension (GS30), several fungicide programs with or without prothioconazole at T1

and T2 appeared to reduce head blight and DON. However, the spread of inoculum between plots may have reduced the ability of prothioconazole at T1 and T2 to reduce the disease and DON within these small plot experiments. It was therefore decided to conduct a full factorial design plus/minus prothioconazole at all three timings to maximise the statistical strength of the experiment to detect significant differences between treatments at each timing and any interaction between applications at each timing; and to use guard plots to minimise the spread of Fusarium inoculum between experimental plots.

The aim of these experiments was to measure the efficacy of prothioconazole (150 g ha -1 ) applied as the formulated product Proline® (Bayer CropScience) at three timings (T1, T2 and T3) to reduce fusarium head blight incidence and DON content of harvest grain and to increase yield and grain quality. Proline® (ai prothioconazole 250 g l -1 ) treated plots at three timings: T1 (GS 31; first node detectable), T2 (GS39; flag leaf fully emerged) and T3 (GS65; mid-anthesis).

Material and methods

Experimental design

A
The rate of Proline® applied at each timing was 0.75 of the recommended single dose. Treatments are listed in Table 1. T1 and T2 treatments were fully randomised (whole plot) and T3 treatments were randomised between sub-plots (2 x 12 m). All fungicides were applied in 200 l ha -1 water using an 'AZO' knapsack sprayer with 110 o flat fan nozzles. All guard plots received a robust fungicide regime containing prothioconazole to minimise spread of inoculum between treated plots. Treated plots were mist irrigated for 17 hours each day (05:00-22:00) for five days from 1 day after the T3 fungicide was applied to optimise conditions for FHB infection.

Artificial inoculation

Three isolates each of F. graminearum (Fg75/11, Fg113, Fg2001/169) and F. culmorum (Fc2001/158, Fc2001/152, Fc103) were taken from the culture collection at

Harper Adams University College. Isolates were sub-cultured on fresh potato dextrose agar (PDA, Merck KGaA, Germany) and after 5 days growth at room temperature used to seed 500 ml of potato dextrose broth (PDB, Merck) in 2-litre flasks. Flasks were shaken twice a day by hand for 5 days. One kg of oats were added to 100 ml of deionised water in a 400 x 550 mm autoclave bag, soaked for 1 hour at room temperature and then autoclaved for 1 h at 121°C. One hundred ml of inoculated PDB was used to inoculate each bag of sterilised oat grains. Bags were gently mixed to distribute the inoculum and incubated for 2 weeks at ca. 20°C.

Inoculated oat grains were mixed together to produce a composite inoculum and treated plots were inoculated with 19 g m -2 of oat grain inoculum at GS 30 (stem extension).

Disease assessment

Head blight assessments were completed on all plots at late milk (GS 77). Incidence of FHB was calculated as number of infected heads per square metre based on counts conducted in ten quadrats (33 cm -2 ) within each plot. Data was converted to % FHB incidence based on average number of heads per square metre.

Yield assessment

At maturity, each plot was harvested using a plot combine and the moisture content and total grain yield recorded. Yield was adjusted to tonnes ha -1 at 15% moisture content. One kilogram grain samples were taken for determination of grain quality parameters; thousand grain weight (TGW) and specific weight (SW). Grain samples were then milled (ZM100 mill with 1 mm screen, Retsch UK Ltd, Leeds), mixed in a tumbler mixer and laboratory samples removed for DON analysis.

Statistical analysis

The statistical package used for all data analysis was Genstat (Version 12, Lawes Agricultural Trust, Rothamsted, UK). Percentage FHB incidence was logit transformed and DON data log10 transformed to obtain normally distributed residuals. Data from both experiments was first analysed by split-plot analysis of variance (ANOVA) with year as whole plot and treatment as sub-plot. This identified if there was a significant difference between years, treatments and an interaction between year and treatment. Both experiments were then analysed together using a split plot factorial (T1*T2*T3) ANOVA with a block structure of block nested within year; T1*T2 as whole plots and T3 as sub-plots.

Results

There was no significant difference in TGW between the two field experiments conducted in 2007/08 and 2008/09, for all other parameters measured there was a highly significant difference (p<0.001) between the two experiments (Table 2). For FHB disease incidence the predicted mean for 2008 and 2009 was 0.7 and 15%

respectively. There was a corresponding impact on DON, yield and SW (Table 2).

Treatment differences were highly significant (p<0.001) for all parameters. There was a significant interaction between year and treatment for yield (p=0.029) and SW (p=0.037) but not for FHB incidence, DON or TGW. As the interactions were either not significant or were much less significant than the main effects then the treatment differences were broadly consistent between years. When each experiment was analysed by ANOVA the residual mean squares were similar, it was therefore acceptable to analyse the datasets for both years together (block nested within year) and these results are presented (Table 3). Factorial analysis identified that there was no significant interactions between fungicide timings for any parameter measured. Table 3 shows the p-values and prothioconazole predicted mean values as percentage differences compared to the untreated control for each fungicide application timing.

Each application of prothioconazole significantly (p<0.05) reduced the incidence of FHB at GS 77 and DON at harvest. The most effective timing was T3 with 83% and 57% reduction respectively (Table 3) and the cumulative benefit of three applications of prothioconazole resulted in the greatest observed reductions of 97% and 83% respectively compared to the untreated controls (Figure 1). These values are close to the calculated cumulative reduction based on the individual T1, T2

and T3 reductions presented in Table 2 (96% and 84% respectively).

Overall yield, specific weight (SW) and thousand grain weight (TGW) were as expected considering the severity of FHB in the untreated controls. Factorial analysis identified that all fungicide treatments resulted in a highly significant (p<0.001) increases in yield, SW and TGW, except for SW (p=0.050) and TGW (p=0.005) with a T1 application (Table 3). Again, benefits of prothioconazole were cumulative resulting in greatest increases in yield, SW and TGW from the application of prothioconazole at all three timings. Compared to the untreated control the application of prothioconazole at all three timings resulted in a yield increase of 4.6 ton ha -1 (98%). The greatest contribution to increased yield was from the T2 application (26%).

Compared to the untreated control the application of prothioconazole at all three timings resulted in an increase of SW of 11.4 kg hl -1 (19%) and an increase in TGW of 13.4 g (38%).

Discussion

As part of a preliminary study (results not shown) using oat-grain-inoculum at GS30, several fungicide programs with or without prothioconazole at T1 and T2 appeared to any reduction in head blight inoculum as a result of T1 and T2 fungicide sprays were masked due the dispersal of inoculum between plots during subsequent periods of high rainfall. It was therefore decided to modify the experimental design to include guard plots to minimise spread of inoculum between treated plots. As the inoculum pressure at flowering would be the same for fungicide programs with the same T1 and T2 treatments, the treatments were paired together to provide whole plots treated with combinations of plus/minus prothioconazole at T1 and T2 and subplots plus/minus prothioconazole at T3. This reduced the number of guard plots required and therefore increased the potential size of guard plots for a given experimental area available.

The use of inoculated oat grain as inoculum applied early in the wheat growing season and the use of guard plots to minimise the spread of inoculum between plots allowed the successful identification of the benefit of prothioconazole applied at all three timings. The reduction in head blight and DON was greatest from an application of prothioconazole at T3 and least at T1. Prothioconazole resulted in 83% reduction of head blight and 57% reduction of DON in harvested grains with a single application of three quarter dose at GS65 (T3 timing). This compares well with previous studies, where on average tebuconazole and metconazole applied at full rate, resulted in 58% (n=7 experiments) and 60% (n=24 experiments) DON reduction respectively [START_REF] Beyer | Quantifying the effects of previous crop, tillage, cultivar and triazole fungicides on the deoxynivalenol content of wheat grain -a review[END_REF]. The timing of application is critical at flowering with a drop in fungicide efficacy as the fungicide is applied further away from the timing of infection [START_REF] Pirgozliev | Effect of timing of fungicide application on the development of Fusarium head blight and the accumulation of deoxynivalenol (DON) in winter wheat grain[END_REF].

Two previous studies using controlled environment and glasshouse experiments indicated that early applications of fungicides could reduce FHB and in one study, DON [START_REF] Greenfield | The effect of a range of novel and established fungicides on Fusarium growth and mycotoxin production[END_REF][START_REF] Hutcheon | Fungicide timing and performance for fusarium control in wheat[END_REF].

However, statistical analysis was not presented and the studies were not repeated. In a field experiment on disease control of leaf spot and head blight with natural infection, there was no significant reduction of FHB with tebuconazole applied at GS39 but there was a significant reduction in DON (40%) in one year out of three [START_REF] Wiersma | Evaluation of five fungicide application timings for control of leaf-spot diseases and fusarium head blight in hard red spring wheat[END_REF].

It is not clear how fungicide applications before head emergence could reduce head blight and subsequent DON. For the T1 application the likely mechanism is in the reduction of Fusarium on the young crop, particularly the dead outer leaf sheaths and on surrounding crop debris and soil. At T2, most fungicide is deposited on the upper leaf canopy. At this timing the fungicide may reduce the number of Fusarium spores on the leaf surfaces. There is evidence of natural suppression of FHB from saprophytic microflora [START_REF] Liggitt | The role of saprophytic microflora in the development of Fusarium ear blight of winter wheat caused by Fusarium culmorum[END_REF]; prothioconazole may benefit the competitors of Fusarium spp. due to its high inhibitory activity towards this genus [START_REF] Klix | Comparison of the declining triazole sensitivity of Gibberella zeae and increased sensitivity achieved by advances in triazole fungicide development[END_REF]). As prothioconazole is systemic and the T2 application is applied within 7-14 days of head emergence, there may also be some direct inhibition of infection from prothioconazole translocated to the wheat heads from the T2 application.

In contrast, some strobilurin fungicides have been shown to increase DON when applied during flowering [START_REF] Simpson | Differential control of head blight pathogens of wheat by fungicides and consequences for mycotoxin contamination of grain[END_REF]). This may be due to the disruption of natural suppression of Fusarium spp. by other head blight pathogens (Microdochium spp.) [START_REF] Jennings | Overview of fusarium ear blight in the UK -effect of fungicide treatment on disease control and mycotoxin production[END_REF] and saprophytic microflora [START_REF] Liggitt | The role of saprophytic microflora in the development of Fusarium ear blight of winter wheat caused by Fusarium culmorum[END_REF]. [START_REF] Ellner | Results of long-term field studies into the effect of strobilurin containing fungicides on the production of mycotoxins in several winter wheat varieties[END_REF] reported that applications of some strobilurins before flowering (GS33-55) could also result in an increase in DON.

Increases in grain quality were also greatest after a T3 application of prothioconazole and least from a T1 application. This correlation would indicate that the increase is grain quality was associated with the reduction in head blight. The greatest increase in yield was associated with the T2 application of prothioconazole.

As the greatest control of FHB was from the T3 timing, this would indicate that the control of other pathogens was a major contributing factor to the yield benefit from application of prothioconazole at T2. This is likely to be partially due to the control of foliar pathogens, such as Septoria tritici, on the flag leaf, as this leaf is the predominant source of yield potential in wheat [START_REF] Milne | A model of the effect of fungicides on disease-induced yield loss, for use in wheat disease management decision support systems[END_REF]).

There was a large difference in the severity of head blight between the two years even though the experimental design was unchanged. This is likely to be due to differences in environmental conditions during key crop growth stages for head blight infection. As well as flowering, when both experiments were mist irrigated, key environmental conditions are during the spring when spore production occurs and the week before flowering when spores can be dispersed onto the emerging wheat heads [START_REF] Wolf | Risk assessment models for wheat Fusarium head blight epidemics based on within-season weather data[END_REF]. In late spring of 2009 it was observed that oat grains were covered with large perithecia, indicating that conditions had been conducive for ascospore production by Gibberella zeae. These perithecia were more prolific and larger than observed in the previous year. Two other factors may have increased the disease pressure in 2009. Firstly, in the week before flowering there were 5 days with rainfall greater than 5 mm whereas in 2008 there was no rainfall in the same period. Secondly, the average temperature during mist irrigation in 2009 was 16.3°C compared to 13.4°C in 2008. The optimum temperature for F. graminearum growth is 25°C [START_REF] Brennan | Studies on in vitro growth and pathogenicity of European Fusarium fungi[END_REF]. Under the high disease pressure which occurred in 2009 a higher rate of prothioconazole would be required to reduce the DON concentration at harvest to below the legal limit of 1250 µg kg -1 .

The ideal method to control crop diseases is through host resistance.

Resistance to Fusarium head blight is polygenic and several resistance loci are closely linked to negative agronomic traits [START_REF] Bai | Management and resistance in wheat and barley to Fusarium head blight[END_REF]. Consequently the availability of economically viable varieties with partial resistance to head blight is limited in many wheat growing regions of the world. At least for the short to medium term, fungicides will continue to play a key role in the reduction of fusarium mycotoxins in small grain cereals. This study has provided clear evidence that application of prothioconazole early in fungicide programs (ie before head emergence) can have a significant contribution to reducing head blight and subsequent DON contamination of harvested grain. The benefit of prothioconazole applied at each timing was additive, with control achieved from all three timings was 97% reduction of FHB and 83% reduction of DON. Growers should therefore consider Fusarium-active fungicides within all application timings as part of an integrated control strategy. Earlier fungicide applications will be particularly beneficial when weather conditions at flowering are not conducive to application of a head blight fungicide at the optimum timing. Table 1. Factorial split-plot design for winter wheat FHB field experiment with three spray timings (T1, T2 and T3) when plots were untreated or treated with 150 g ha -1 prothioconazole. T1*T2 combinations were whole plots and T3 treatments were applied to sub-plots.

  field experiment was conducted in 2007/08 and repeated in 2008/09. Experimental plots (4 x 12 m) were separated by guard plots (6 x 12 m). Winter wheat, cv. Solstice was sown and grown according to standard farm practice in Shropshire, UK. The experiments were designed as a split plot randomised block with eight treatments replicated four times. The design was a full factorial design of untreated and 0.6 l ha -1

  using a DON FAST ELISA kit (R-Biopharm Rhone, Glasgow) according to the manufacturer's instructions. Eight grams of flour were extracted in 40 ml deionised water.

  reduce head blight and DON in 2006 but not in 2007. These studies were conducted in a standard small plot (2 x 12 m) randomised block design. The difference observed between 2006 and 2007 may have been due to large differences in rainfall. In 2006, the period from T1 application to end of flowering was relatively dry with only 86 mm of rainfall of which only 8 mm fell during flowering. In the following year over the same period there was 172 mm rainfall, including 64 mm over 3 days during flowering. Rainfall events are known to result in splash dispersal of Fusarium conidia[START_REF] Jenkinson | Splash Dispersal of Conidia of Fusarium-Culmorum and Fusarium-Avenaceum[END_REF] and stimulate release of Gibberella zeae ascospores[START_REF] Paulitz | Diurnal release of ascospores by Gibberella zeae in inoculated wheat plots[END_REF]. It was therefore concluded that in 2007, the high rainfall resulted in

Figure 1 .

 1 Figure 1. Back-transfomed mean percentage Fusarium head blight incidence (A) and DON concentration (B) for winter wheat plots untreated or treated with prothio (prothioconazole; 150 g ha -1 ) at three fungicide application timings; T1, T2 and T3. Replication was two years x four blocks.

http://mc.manuscriptcentral.com/tfac Email: fac@tandf.co.ukFood Additives and Contaminants

Acknowledgements

SGE acknowledges technical support of the Crop and Environment Research Centre, in particular; Matthew Rodenhurst, Fikirini Ramadhani, Samuel Imathiu and Danielle Henderson and the funding of field experiments by Bayer CropScience.

Treatment

Whole plot T1 (GS31) T2 (GS39) T3


David ; Aldred

Naresh Magan
email: n.magan@cranfield.ac.uk

Kalliopi Mylona

Ronald J W Lambert

Prof N Magan

Limiting mycotoxins in stored wheat -a review 5 6 7

Keywords: Mycotoxins -trichothecenes, Mycotoxins -fungi, Antioxidants Food Types: Cereals and grain, Animal feed spoilage fungi, mycotoxins, dry matter losses, boundary models, modified 20 atmospheres, preservatives, biocontrol, post-harvest management

The quality of harvested wheat grain can deteriorate markedly during the post-harvest 27 management stages. The biotic factors such as grain type and ripeness, coupled with the 28 prevailing abiotic factors such as water content and temperature, and also preservative 29 concentration will influence the safe storage life and the level of contamination with 30 mycotoxins such as deoxynivalenol (DON) produced pre-harvest and zearalenone (ZEA) 31 produced post-harvest by Fusarium graminearum and Fusarium poae respectively, 32 ochratoxin (OTA) produced by Penicillium verrucosum post-harvest in cool damp northern 33 European climates, and perhaps T-2 and HT-2 toxins produced by Fusarium langsethiae. 34 This review presents recent data on the relationship between dry matter losses caused by F. 35 graminearum under different environmental regimes (water activities, temperatures) and the 36 level of contamination with DON. This is important as poor post-harvest drying and storage 37 management may exacerbate DON contamination already present pre-harvest. It is thus 38 critical to relate the environmental factors in stored wheat grain during storage, especially of 39 intergranular relative humidity (RH) and temperature, to safe storage periods without 40 spoilage or risk from increased DON contamination. The growth/no growth and DON/no 41 DON (F. graminearum) and OTA/no toxin production (P. verrucosum) have been used to 42 build a model with a simple interface to link the temperature and RH values to the potential 43 risk level which may allow growth or toxin production. This paper also considers the use of 44 modified atmospheres, preservatives and biocontrol to minimise DON and OTA in moist 45 wheat grain. These approaches together with clear monitoring criteria and hygiene could 46 contribute to better post-harvest management of stored temperate cereals and ensure that 47 mycotoxin contamination is minimised during this key phase in the food/feed chain.

 Aldred, 2007). This approach may 114 be beneficial for combining dry matter losses in relation to m.c.s during drying and initial 115 storage with the range of potential risk conditions for OTA and DON, relative to the EU 116 legislative limits. Previous comprehensive respiration data sets in relation to a w and 117 temperature of stored wheat, have been used to develop the possible relationship between the 118 variables. Thus, the results obtained as O 2 d -1 kg -1 dry grain (R) was divided by the incubation 119 temperature ( ) to give units of O 2 d -1 kg -1 dry matter o C -1 . There was a linear relationship 120 (r 2 =0.9594) between R/ and a w (Figure 2, Lacey et al., 1997). This is based on O2 121 consumption, while our new data is based on CO2 production. Thus some conversion will be 122 required to relate our new data to the previous data. However, it may be possible to relate our 123 present data to the risk of exceeding the EU legislative limits for mycotoxigenic fungi. This Measurements can be made within stored grain silos to measure the changes in temperature 142 and relative humidity in the inter-granular air spaces in the grain during drying and storage.

143

This has been done at the top and in the middle of a flat bed store. Generally, any leaks or 144 even small openings via damage to the structure can lead to an increase in temperature and 145 intergranular m.c. This can often occur during the early periods after drying and storage 146 phase (5-10 days) where both these parameters can increase, especially in the top layers 147 which can increase the risk from spoilage and mycotoxin contamination [START_REF] Aldred | Environmental factors affect efficacy of 332 some essential oils and resveratrol to control growth and ochratoxin A production by 333 Penicillium verrucosum and A. westerdijkiae on wheat grain[END_REF]. et al., 2004;[START_REF] Cairns-Fuller | Water, temperature and gas composition 336 interactions affect growth and ochratoxin A production by isolates of Penicillium 337 verrucosum on wheat grain[END_REF]. This data is useful as a guide to conditions 152 where there may be a risk of growth and toxin production. A polynomial equation has been 153 used to model these growth/no growth and toxin/no toxin boundaries. Figure 3 2004). They developed very useful models which relate the potential for exceeding the 170 EU legislative limit for OTA in cereals to the CFUs isolated from grain. They showed that 171 >10 3 CFUS g -1 on a selective medium for P. verrucosum would represent a risk of OTA 172 contamination >5 µg kg -1 stored grain which is the EU legislative limit. They also developed suggest that mycotoxins such as DON, have a spatial structure in the store and thus a regular 290 grid may be more precise than using a random sampling approach. This may not be the case 291 for other mycotoxins and in other commodities and thus further work is imperative to enable 292 more accurate representative samples to be taken and improve the accuracy of such sampling.

294

We believe that there are new technologies which need to be utilised in the future to improve 295 the strategies to minimise mycotoxins in temperate cereals. As has been mentioned earlier the 296 potential for using remote sensing especially in silos linked to post-harvest models of

  a w , and lower at 0.85-0.90 a w and 25 o C (Magan and

  a valuable practical aid if developed for different grains and nuts prone to 125 contamination with different mycotoxins. that grain is effectively dried immediately after harvest. Often heated air drying 131 is used to achieve the target safe moisture content of about 14.5%. However, sometimes in 132 the harvesting season grain is left for short periods of time before drying because of logistical 133 issues. In some European countries ambient air drying is always used. Where heated drying is 134 used the grain is less affected by the prevailing weather conditions, especially in damp 135 autumn seasons in northern Europe. In the UK, where ambient drying is often used, this can 136 result in slow drying with a moist front moving vertically up through the grain bed, especially 137 in flat bed dryers/stores. This can result in grain at the top of the store being rewetted, while 138 grain at the bottom can become over dried. This can lead to layers of mouldy grain with the 139 potential for increased DON or OTA contamination.140 141

7

  We have previously developed boundary layer contour maps for growth and DON and OTA 150 production by F. culmorum and F. graminearum and for P. verrucosum (Hope and Magan, 151 2003; Hope

  shows an 154 example of the data for F.graminearum in relation to minimal growth rates (0.1 mm day -1 ) 155 and for DON production (0.01 ppm).

Figure 3

 3 Figure3shows the simple front end diagram which can be used for this purpose. This would

  also been developed for P.verrucosum and OTA production by Lindblad 169 et al. (

  , chemical preservation systems and biocontrol 180Moist grain, especially for animal feed use is sometimes stored in sealed silos to enable the 181 natural respiration of the grain to increase the CO 2 concentration and inhibit spoilage moulds 182 and the potential for mycotoxin formation post-harvest. However, if there are any leaks or the 183 conditions remain micro-aerophilic then there is potential for both growth and mycotoxin 184 formation. Studies suggest that increasing concentrations of CO 2 to 25 or 50% has little effect 185 on growth or OTA production by P. verrucosum or indeed Aspergillus ochraceus 186 (=Aspergillus westerdijkiae). Spore germination of these two fungi is unaffected, although 187 germ tube extension and growth can be affected by up to 75% CO 2(Cairns-Fuller, 2004; 188[START_REF] Cairns-Fuller | Water, temperature and gas composition 336 interactions affect growth and ochratoxin A production by isolates of Penicillium 337 verrucosum on wheat grain[END_REF]. However, this does not mean that mycotoxin production is 189 significantly affected. While inhibition of OTA did occur when a w and CO 2 were increased, 190 the effect was not synergistic and sometimes inhibition was not to a level below the 191 legislative limits. 192 193 The effect of modified atmosphere on F. culmorum and other species has been examined and 194 showed that they are micro-aerophilic and thus very tolerant of very low O 2 concentrations 195 (Magan & Lacey, 1984). Paster (2000) has reviewed the effect of modified atmospheres on 196 mycotoxigenic and other moulds and this has shown that there is very little information on 197 mycotoxigenic Fusaria. Work has been carried out with Fusarium sporotrichioides and this 198 demonstrated that T-2 toxin was reduced by 80% in the presence of 50% CO 2 /20%O 2 but that 199 added to moist grain, especially for grain destined for animal feed. 203 Commercial products are mainly based on mixtures of salts of aliphatic acids such as 204 propionic, sorbic and benzoic acids. However, these are all fungistats and require effective 205 contact with the product. Thus mixing is very important to ensure that inhibition of spoilage 206 is maintained. Work has been carried out to try and identify alternative compounds which 207 may be used to replace these aliphatic acid based products. Since P. verrucosum often causes 208 problems post-harvest then the use of natural and novel preservatives may have an impact on 209 OTA contamination. Aldred et al. (2008) screened a number of essential oils and antioxidants 210 for control of both growth and OTA production by both P. verrucosum and A. ochraceus.211They found that while essential oils such as clove, cinnamon and thyme were effective in 212 vitro, in situ on grain they were relatively ineffective. This may perhaps be due to binding of 213 the essential oil components to grain constituents. However, they did find that anti-oxidants, 214 such as butylhydroxyanisole (BHA), propyl paraben, and especially resveratrol, were 215 effective on naturally contaminated and grain artificially inoculated with P. verrucosum. 216 217 A range of essential oils (23) and 6 antioxidants were screened for control of growth and 218 DON and NIV production by F. culmorum, DON production by F. graminearum and T-2 and 219 HT-2 by F. langsethiae (Hope, 2004). Of these, three essential oils (bay, clove, cinnamon oil) 220 and two antioxidants (propyl paraben and hydroxymethylanisole) were found to be effective 221 in controlling growth of these species in the range 50-200 ppm at 15 and 25 o C at 0.995 and 222 0.955 a w in vitro on wheat-based media. More detailed studies on stored wheat grain at 15 223 and 25°C and essential oil/antioxidants treatments (100ppm; 500ppm) were carried out. This 224 approach is beneficial if you have the exact grid positions of the samples in three 288 dimensions, it can then be used on any scale. Recent studies by Rivas et al. (2009 a, b) 289

  with the legislative limits could be very useful. There are 298 now many useful molecular-based techniques which could be applied effectively post-299 harvest. For example,Schmidt-Heydt et al. (2000) utilised the expression of the OTA 300 polyketide synthase (otapksPV) to examine temporal colonisation of stored wheat grain of 301 different moisture contents (14-24%) and this was correlated with OTA production analysed 302 by HPLC and populations by CFUs. Thus the RT-PCR approach could be used as a sensitive 303 measure of the quality status with regard to OTA contamination in wheat during 3 months 304 storage. These powerful molecular tools have been applied pre-harvest but not widely post-305 harvest. This is supported by the recent work by Suanthe et al. (2009) who have developed 306 multiplex RT-PCR for detection and quantification of mycotoxigenic Aspergillus, Penicillium 307 and Fusarium species.

  another important potential impact is that environmental change. This may have 310 an impact on the relative importance of different mycotoxins in different geographical 311 regions. This may become important and have a significant impact on which mycotoxins may 312 become important in the next 5-10 years. There is a lack of data on the impact that twice or 313 three times the existing CO 2 concentrations combined with 2-3 o C elevation in temperature 314 might have on colonisation and mycotoxin production. A certain amount of this information 315 can be generated by modelling approaches. However, the potential for stimulation of 316 mycotoxin production is more difficult to predict. In conclusion, while this paper has dealt 317 with post-harvest issues in temperate cereals it has to be seen in the context of a more holistic 318 food chain systems approach with better and more effective management post-harvest being 319 just one component of this.

Figure 1 . 425 Figure 2 .Figure 3 .

 142523 Figure legends

  Table1. Effect of 500 ppm of three essential oils (extracts of bay leaf (Bay), cinnamon (Cin) and clove) and two anti-oxidants (butylhydroxyanisole (BHA), propyl paraben (PP) on (a) deoxynivalenol (DON, ppm) production by F.graminearum at 15, 25 o C and 0.995 and 0.95 water activity, and (b) T-2 and HT-2 (ppm) by F.langsethiae on stored wheat grain at 15, 25 o C and 0.95 water activity. (a) F.graminearum DON (PPM)____________

Figure 1 :Figure 3 :

 13 Figure 1: Magan et al.

  These are very useful practically for the cereal industry. Studies by Jonsson et al. 175 (2000) suggest that the maximum storage time without any visible moulding was probably 176 reduced by 50% if the mc at harvest was decreased by 1-3% (=0.05 a w ) or if the storage 177 temperature was increased by 5 o C, for example in a mild autumn year, in temperate cereals.

	F o r
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173 models of toxin free storage times under different temperature and moisture content 8 conditions.

http://mc.manuscriptcentral.com/tfac Email: fac@tandf.co.ukFood Additives and Contaminants


MB Susan Byrne
email: suabyrne@gmail.com

Cathal Walsh Phd

MSc Catherine Lynch
email: clynch@rcsi.iebedepeter@hotmail.com

MB Peter Bede

MB Marwa Elamin
email: marwaelamin08@gmail.com

Kevin Kenna Bsc

Russell Mclaughlin Bsc

MD Orla Hardiman

Cathal Walsh
email: cathaldwalsh@gmail.com

Russell Mc Laughlin

Prof Hardiman

Kevin Kenna
email: kennak@tcd.iemclaugr@tcd.ieorla@hardiman.net

Russell Mclaughlin

Rate of Familial Amyotrophic Lateral Sclerosis: A Systematic Review and Metaanalysis

Keywords:

Background: The population rate of familial amyotrophic lateral sclerosis (FALS) is frequently reported as 10%. However a systematic review and meta-analysis of the true population-based frequency of FALS has never been performed.

Method: A Medline literature review identified all original articles reporting a rate of FALS. Studies were grouped according to the type of data presented and examined for sources of case ascertainment. A systematic review and meta-analysis of reported rates of FALS was then conducted to facilitate comparison between studies and calculate a pooled rate of FALS.

Results: Thirty-eight papers reported a rate of FALS. Thirty-three papers were included in analysis and the rate of FALS for all studies was 4.6% (95% CI 3.9-5.5).

Restricting the analysis to prospective population-based registry data revealed a rate of 5.1% (95% CI 4.1-6.1). The incidence of FALS was lower in southern Europe.

There was no correlation between rate of FALS and reported SOD1 mutation rates.

Conclusion:

The rate of FALS among prospective population based registries is 5.1% (CI 4.1-6.1%), and not 10% as is often stated. Further detailed prospective population-based studies of familial ALS are required to confirm this rate.

Introduction

Amyotrophic Lateral Sclerosis (ALS) is a neurodegenerative disease, which was first described by Charcot in the mid-nineteenth century. It is predominantly a sporadic disease but a small percentage of cases occur within kindreds. [START_REF] Wechsler | Primary and symptomatic Amyotrophic Lateral Sclerosis: A clinical study of 81 cases[END_REF] In 1955, Kurland et al published a case series of 58 patients in which 10% reported a family history of ALS [START_REF] Kurland | Epidemiologic Investigations of Amyotrophic Lateral Sclerosis. Familial Aggregation indicative of Dominant Inheritance[END_REF] . Although subsequent publications have reported varying rates of FALS, the figure of 10% remains as the accepted population frequency of FALS 3- 7 .

There is no definitive definition for FALS. It is generally accepted that the presence of ALS in either a first or second-degree relative of the index case constitutes the familial form of the disease. Genes known to be associated with FALS include SOD1, TARDP, FUS and ANG [START_REF] Andersen | Good practice in the management of amyotrophic lateral sclerosis: clinical guidelines. An evidence-based review with good practice points. EALSC Working Group[END_REF] . The frequency of SOD1 mutations has been estimated to account for 10-20% of all FALS cases 6 . This estimate is not populationbased and most studies preferentially recruit patients with familial ALS [START_REF] Andersen | Phenotypic heterogeneity in motor neuron disease patients with CuZn-superoxide dismutase mutations in Scandinavia[END_REF][START_REF] Eisen | SOD1 gene mutations in ALS patients from British Columbia, Canada: Clinical features, neurophysiology and ethical issues in management[END_REF] . To date only one population-based estimate of SOD1 frequency has been carried out, reporting a rate of 13.6% [START_REF] Chio | Prevalence of SOD1 mutations in the Italian ALS population[END_REF] . Estimation of the rates of SOD1 related FALS is also confounded by the occurrence of mutations in up to 5% of the sporadic population, some of whom represent familial disease with incomplete penetrance [START_REF] Orwell | Clinical characteristics of SOD1 gene mutation in UK families with ALS[END_REF] . The population frequency of the other known causative genes within populations has not been established.

We performed a systematic review and meta-analysis of all studies that present original data reporting a rate of FALS (i.e. the proportion of familial cases amongst all ALS cases, either in a defined population or in a case series). Analysis of the population-based frequency of FALS was then undertaken and where possible, a geographic comparison was made with the frequency of known SOD1 mutations.

Methods

Systematic search: A Medline literature search was performed to identify all published studies on FALS, in addition to ALS incidence and prevalence studies from 1966 to October 2009. The MeSH terms "ALS", "Amyotrophic Lateral Sclerosis", "FALS", "Familial Amyotrophic Lateral Sclerosis", "familial motor neuron(e) disease", "motor neuron(e) disease", "MND", "incidence", "prevalence" and "mortality" were used. Additional references were sought from cited articles. Where no information was reported on the rate of FALS in a population-based study, the corresponding author was contacted where possible. Unpublished up-to-date data from the Irish ALS prospective population based register was also used.

Eligibility criteria and Data Collection: All studies presenting original data that reported a rate of FALS (i.e. the proportion of familial cases within a defined cohort) were included in the systematic review. Only studies that demonstrated complete enrollment, either in the form of population-based registry or in sequential case series, were analysed. Studies with non-random enrollment or cohorts enriched for familial disease were not included in analysis.

Studies fulfilling inclusion criteria were grouped together according to the type of data presented; 1) Prospective registry based studies that aim to capture all cases within a given geographic region in order to define incidence and prevalence, 2)

Retrospective studies that attempt to capture all cases in a given geographic region with the aim of estimating incidence and prevalence, 3) Prospective cases series, 4) Retrospective case series.

Statistical analysis: In each study the rate of FALS is reported as the number of B y r n e 6 familial cases among all cases of ALS. Statistical analysis was carried out to combine proportions using the Meta function of R. (R Development Core Team (2009). R: A language and environment for statistical computing. R Foundation for Statistical Computing, Vienna, Austria. ISBN 3-900051-07-0, URL http://www.R-project.org.)

The inverse variance method was used to pool proportions. Both Fixed and Random effects models were estimated and confidence intervals of 95% were calculated.

Results

Systematic review: Fifty four epidemiologic studies provided original information on cohorts of patients with ALS 9,10 ,13-66 . A rate of FALS was reported in 38 of these studies 9,10,13-46,63-66, , although in no case was FALS ascertainment the primary objective. . Five studies were not included in analysis [START_REF] Andersen | Phenotypic heterogeneity in motor neuron disease patients with CuZn-superoxide dismutase mutations in Scandinavia[END_REF][START_REF] Eisen | SOD1 gene mutations in ALS patients from British Columbia, Canada: Clinical features, neurophysiology and ethical issues in management[END_REF][START_REF] O'toole | Epidemiology and clinical features of Amyotrophic Lateral Sclerosis in Ireland between 1995 and 2004[END_REF][START_REF] Zoccolella | Signs and symptoms at diagnosis of amyotrophic lateral sclerosis: a population-based study in southern Italy[END_REF][START_REF] Scottish | The Scottish Motor Neuron Disease Register: a prospective study of adult onset motor neuron disease in Scotland. Methodology, demography and clinical features of incident cases in 1989[END_REF] . Of these, two were undertaken to genotype mutations in the ALS population. Both studies exhibited enrollment bias [START_REF] Andersen | Phenotypic heterogeneity in motor neuron disease patients with CuZn-superoxide dismutase mutations in Scandinavia[END_REF][START_REF] Eisen | SOD1 gene mutations in ALS patients from British Columbia, Canada: Clinical features, neurophysiology and ethical issues in management[END_REF] . Three studies reported data on more than one occasion [START_REF] O'toole | Epidemiology and clinical features of Amyotrophic Lateral Sclerosis in Ireland between 1995 and 2004[END_REF][START_REF] Zoccolella | Signs and symptoms at diagnosis of amyotrophic lateral sclerosis: a population-based study in southern Italy[END_REF][START_REF] Scottish | The Scottish Motor Neuron Disease Register: a prospective study of adult onset motor neuron disease in Scotland. Methodology, demography and clinical features of incident cases in 1989[END_REF] and therefore only the most recent study was included in the meta-analysis. Reports from geographical areas with high-incidence clusters (Guam/Kii Peninsula of Japan) were excluded [START_REF] Kihira | Changes in the incidence of Amyotrophic Lateral Sclerosis in Wakayama, Japan[END_REF] .

Of the thirty-three studies analysed , 8 reported incidence data from prospective population-based registers [START_REF] Logroscino | Incidence of amyotrophic lateral sclerosis in southern Italy: a population based study[END_REF][START_REF] Chiò | Epidemiology of ALS in Italy A 10-year prospective population-based study[END_REF][START_REF] Forbes | The incidence of Motor Neuron Disease in Scotland[END_REF][START_REF] Abhinav | Amyotrophic Lateral Sclerosis in South-East England: A Population-Based Study.The South-East England Register for Amyotrophic Lateral Sclerosis (SEALS Registry)[END_REF][START_REF] Mcguire | Incidence of amyotrophic lateral sclerosis in three counties in western Washington state[END_REF][START_REF] Del Aguila | Prognosis in amyotrophic lateral Byrne 13 sclerosis: A population-based study[END_REF][START_REF] Cruz | Physical Trauma and Family History of Neurodegenerative Diseases in Amyotrophic Lateral Sclerosis: A Population-Based Case-Control Study[END_REF][START_REF] Vzquez | Incidence and prevalence of Amyotrophic Lateral Sclerosis in Uruguay: a population-based study[END_REF][START_REF] Norris | Onset, natural history and outcome in idiopathic adult motor neuron disease[END_REF] ; 14 reported retrospective incidence and prevalence data; [START_REF] Murphy | Increasing incidence of ALS in Canterbury, New Zealand: A 22-year study[END_REF][START_REF] Sorenson | Amyotrophic lateral sclerosis in Olmsted County, Minnesota, 1925 to[END_REF][START_REF] Mandrioli | The epidemiology of ALS in Modena, Italy[END_REF][START_REF] Turabelidze | An epidemiologic investigation of amyotrophic lateral sclerosis in Jefferson County, Missouri, 1998-2002[END_REF][START_REF] Hudson | The incidence of Amyotrophic Lateral Sclerosis in southwestern Ontario, Canada[END_REF][START_REF] Murros | Amyotrophic Lateral Sclerosis in Middle-Finland: an epidemiological study[END_REF][START_REF] Forsgren | Epidemilogy of motor neuron disease in northern Sweden[END_REF][START_REF] Lopez-Vega | Motor Neuron Disease in Cantabria[END_REF][START_REF] Giaggheddu | Epidemiologic study of amyotrophic lateral sclerosis in Sardinia, Italy[END_REF][START_REF] Argyriou | Clinical and epidemiological features of motor neuron disease in south-western Greece[END_REF][START_REF] Alcaz | Epidemiological and clinical characteristics of ALS in Belgrade, Yugoslavia[END_REF][START_REF] Fong | Motor Neuron Disease in Hong Kong Chinese: Epidemiology and Clinical Picture[END_REF][START_REF] Hojer-Pedersen | Incidence of prevalence of Motor Neuron Disease in Two Danish Counties[END_REF][START_REF] Tysnes | Epidemiology of amyotrophic lateral sclerosis in Hordaland county, western Norway[END_REF] , 3 reported data from prospective case series of disease progression [START_REF] Haverkamp | Natural history of amyotrophic lateral sclerosis in a database population: Validation of a scoring system and a model for survival prediction[END_REF][START_REF] Rosen | Amyotrophic Lateral Sclerosis: Clinical features and prognosis[END_REF][START_REF] Ringel | The natural history of ALS[END_REF] and 8 reported data from retrospective case series [START_REF] Werneck | A clinical epidemiological study of 251 cases of amyotrophic lateral sclerosis in the south of Brazil[END_REF][START_REF] Li | Comparison of sporadic and familial disease amongst 580 cases of motor neuron disease[END_REF][START_REF] Kurland | Epidemiologic Investigations of Amyotrophic Lateral Sclerosis. Familial Aggregation indicative of Dominant Inheritance[END_REF][START_REF] Sabatelli | Natural history of young-adult amyotrophic lateral sclerosis[END_REF][START_REF] Murray | Motor Neuron Disease in Novia Scotia[END_REF][START_REF] Thjis | Demographic characteristics and prognosis in a Flemish Amyotrophic Lateral Sclerosis population[END_REF][START_REF] Gubbay | Amyotrophic lateral sclerosis. A study of its presentation and prognosis[END_REF][START_REF] Louwerse | Amyotrophic lateral sclerosis: mortality risk during the course of the disease and prognostic factors[END_REF] . Only two studies stated how they defined FALS [START_REF] Giaggheddu | Epidemiologic study of amyotrophic lateral sclerosis in Sardinia, Italy[END_REF][START_REF] Haverkamp | Natural history of amyotrophic lateral sclerosis in a database population: Validation of a scoring system and a model for survival prediction[END_REF] . In total 575 cases of FALS were recorded among 11,221 reported cases of ALS.

The type of study, rate of FALS, source of case ascertainment and case inclusion criteria are outlined in Table 1. .

Meta-Analysis

Pooled analysis of all studies generated a rate of FALS of 4.6% (95% CI 3.9-5.5%).

A degree of heterogeneity was noted between reported rates of FALS in the prospective population based registers available for analysis [START_REF] Logroscino | Incidence of amyotrophic lateral sclerosis in southern Italy: a population based study[END_REF][START_REF] Chiò | Epidemiology of ALS in Italy A 10-year prospective population-based study[END_REF][START_REF] Forbes | The incidence of Motor Neuron Disease in Scotland[END_REF][START_REF] Abhinav | Amyotrophic Lateral Sclerosis in South-East England: A Population-Based Study.The South-East England Register for Amyotrophic Lateral Sclerosis (SEALS Registry)[END_REF][START_REF] Mcguire | Incidence of amyotrophic lateral sclerosis in three counties in western Washington state[END_REF][START_REF] Del Aguila | Prognosis in amyotrophic lateral Byrne 13 sclerosis: A population-based study[END_REF][START_REF] Cruz | Physical Trauma and Family History of Neurodegenerative Diseases in Amyotrophic Lateral Sclerosis: A Population-Based Case-Control Study[END_REF][START_REF] Vzquez | Incidence and prevalence of Amyotrophic Lateral Sclerosis in Uruguay: a population-based study[END_REF][START_REF] Norris | Onset, natural history and outcome in idiopathic adult motor neuron disease[END_REF] . The pooled result for rate of FALS for these studies was 5.1% (95% CI 4.1-6.1).

In the 14 retrospective population based studies, the pooled result for rate of FALS was 3.7% (95% CI 2.9-4.7) [START_REF] Murphy | Increasing incidence of ALS in Canterbury, New Zealand: A 22-year study[END_REF][START_REF] Sorenson | Amyotrophic lateral sclerosis in Olmsted County, Minnesota, 1925 to[END_REF][START_REF] Mandrioli | The epidemiology of ALS in Modena, Italy[END_REF][START_REF] Turabelidze | An epidemiologic investigation of amyotrophic lateral sclerosis in Jefferson County, Missouri, 1998-2002[END_REF][START_REF] Hudson | The incidence of Amyotrophic Lateral Sclerosis in southwestern Ontario, Canada[END_REF][START_REF] Murros | Amyotrophic Lateral Sclerosis in Middle-Finland: an epidemiological study[END_REF][START_REF] Forsgren | Epidemilogy of motor neuron disease in northern Sweden[END_REF][START_REF] Lopez-Vega | Motor Neuron Disease in Cantabria[END_REF][START_REF] Giaggheddu | Epidemiologic study of amyotrophic lateral sclerosis in Sardinia, Italy[END_REF][START_REF] Argyriou | Clinical and epidemiological features of motor neuron disease in south-western Greece[END_REF][START_REF] Alcaz | Epidemiological and clinical characteristics of ALS in Belgrade, Yugoslavia[END_REF][START_REF] Fong | Motor Neuron Disease in Hong Kong Chinese: Epidemiology and Clinical Picture[END_REF][START_REF] Hojer-Pedersen | Incidence of prevalence of Motor Neuron Disease in Two Danish Counties[END_REF][START_REF] Tysnes | Epidemiology of amyotrophic lateral sclerosis in Hordaland county, western Norway[END_REF][START_REF] Werneck | A clinical epidemiological study of 251 cases of amyotrophic lateral sclerosis in the south of Brazil[END_REF] Figure 1 demonstrates a Forrest plot of the results (see figure 1).

The individual rate of FALS for each country in Europe was plotted on a map to determine a possible geographic pattern. (see figure 2) Regions of relatively homogenous genetic origin, such as Ireland and Scotland, (both of which both have large prospective databases of over twelve hundred patients) exhibit almost the same rate of FALS. Lower rates are reported in the more diverse populations of southern Italy and the Balkan Peninsula. Variations in the distribution Byrne 9 of families with SOD1 mutations have also been identified. The population-based rate of SOD1 mutations in Italian FALS patients is 13%, and the rate in SALS in 0.7% [START_REF] Chio | Prevalence of SOD1 mutations in the Italian ALS population[END_REF] . This contrasts sharply with rates of 1.9% in Dutch FALS group and 0.44% in Dutch SALS patients [START_REF] Van Es | Large scale SOD1 mutation screening B y r n e 1 8 provides evidence for genetic heterogeneity in amyotrophic lateral sclerosis[END_REF] . The true population-based rate of SOD1 gene mutations in other countries remains unclear, as the majority of studies performed to date have been neither prospective nor population-based. Moreover, we were unable to identify a geographic correlation between FALS rates and reported SOD1 mutation rates for the same region.

Discussion

Our analysis indicates that the commonly accepted frequency of 10% for FALS represents an overestimation of the dominantly inherited Mendelian form of ALS.

The population-based frequency of 5.1% is likely to represent the most accurate estimation of rate of FALS, as population-based studies include all patients in a defined geographic area in a given period of time. Accordingly, reported rates of FALS of 10% from previous studies are likely to have been biased by ascertainment from populations enriched by FALS cases.

Geographic variation in the rate of FALS in Europe (figure 2) may reflect true population-based differences across different regions. Heterogeneity in the genetic substructure of European populations has been demonstrated recently [START_REF] Novembre | Genes mirror geography within Europe[END_REF] . These geographic differences may occur because of variability in the underlying genetic structure of the European population. However, difficulties in case ascertainment could also account for this difference. Evidence for population-based differences is emerging for SOD1: -a founder effect for the A4V mutations in SOD1 has been B y r n e 1 0 recently identified in the USA [START_REF] Saeed | Age and founder effect of SOD1 A4V mutation causing ALS[END_REF] and this mutation is rare in Europe. Mutations in OPTN seem to be primarily in Japanese populations [START_REF] Maruyama | Mutations of optineurin in amyotrophic lateral sclerosis[END_REF] . The existence of geographic variability for TARDP and FUS has not yet been established. As more causative genes are identified in FALS, detailed analysis of the frequency of various mutations within individual FALS populations will become available.

The meta-analysis is limited by the absence of a clear definition of FALS. While most groups define FALS as the presence of ALS in at least two members of an extended kindred, the degree or relatedness and the size of the extended kindred are rarely considered. Given that the lifetime risk of ALS is 1:450 for females and 1:350 for males [START_REF] Alonso | Incidence and lifetime risk of Motor Neuron Disease in the United Kingdom, a population based study[END_REF] , there is an increasing probability of 2 affected members with sporadic ALS occurring in the same kindred as the size of the kindred increases. Therefore, ensuring segregation of mutations within kindreds is also important, as exemplified by a recent report of two apparent SOD1 kindreds in which the disease did not segregate with a known pathogenic SOD1 mutations [START_REF] Rabe | The epidemiology of CuZn-SOD mutations in Germany: a study of 217 families[END_REF] .

In conclusion, we have demonstrated that the rate of FALS across population-based studies rarely exceeds 5% of all cases of ALS. This contrasts with the generally accepted figure of 10%, which originates from a paper written in 1955 [START_REF] Kurland | Epidemiologic Investigations of Amyotrophic Lateral Sclerosis. Familial Aggregation indicative of Dominant Inheritance[END_REF] .

Careful prospective population based analysis of kindreds using validated criteria for diagnosis of FALS is required to confirm that 5% represents the true populationbased rate of familial ALS, and to determine whether the documented geographic variation in rates of FALS is corroborated.

Table 1 .

 1 Comparison of the type of study, rate of FALS, source of case ascertainment and case

	Byrne 7

Acknowledgements Byrne 11 This work was supported by the IMNDA, IMNDRF and HRB funding.

The Corresponding Author has the right to grant on behalf of all authors and does grant on behalf of all authors, an exclusive licence (or non exclusive for government employees) on a worldwide basis to the BMJ Publishing Group Ltd and its Licensees to permit this article (if accepted) to be published in the Journal of Neurology, Neurosurgery & Psychiatry editions and any other BMJPGL products to exploit all subsidiary rights, as set out in our licence (http://jnnp .bmjjournals.com/ifora/licence.pdf)."

Her group has received unrestricted research grants from Merck Serono, Biogen Idec and Bayer Schering.

Percentage FALS cases reported


Karl-A ˚ke ˚ Jansson
email: karl-ake.jansson@karolinska.se

Paul Blomqvist

Per Svedmark

Fredrik Granath

Erik Buskens

Martin Larsson

Johanna Adami

Á F Granath

Á J Adami

LOCO-MOTOR DISEASES Thoracolumbar vertebral fractures in Sweden: an analysis of 13,496 patients admitted to hospital

Keywords: Case fatality rate, Epidemiology, Mechanism of injury, Population based study, Spine, Vertebral fractures

National Longitudinal data of thoracolumbar fracture incidence, trends or mortality rates are lacking. The correlation between admissions and operations of thoracolumbar vertebral fractures has not been investigated. The aim of our nationwide population-based epidemiological study was to analyse the incidence, admissions, operations, and case fatality rate among patients with thoracolumbar vertebral fractures admitted to hospital in Sweden. The Swedish Hospital Discharge Register (SHDR) and the Cause of Death Register (CDR) were linked to determine the incidence of surgical interventions, trends, characteristics of the patients, and case fatality rate for thoracolumbar vertebral fractures based on comprehensive national data. The annual incidence of thoracolumbar fractures was on average 30 per 100,000 inhabitants and did not change considerably during the study period. Among patients younger than 60 years of age the annual incidence was 13 per 100,000 and was twice as high in men compared to women. The proportion operated on was 15%. In the age-group 60 years and older the majority were women. In this group two percent were operated on. However, males were operated on twice as often as women. The 90-day case-fatality rate after surgery was 1.4%. This information may assist health care providers in health care planning. Moreover, these data can also be used for power calculations when planning future clinical studies.

Introduction

There are few international studies with population based data on the epidemiology of vertebral fractures. Most earlier epidemiological studies were retrospective case series or from single hospitals [START_REF] Chen | A Nationwide epidemiological study of spinal cord injuries in Taiwan from July 1992 to June 1996[END_REF][START_REF] Hu | Epidemiology of incident spinal fracture in a complete population[END_REF]. In previous studies, the annual fracture incidence varied from 23 to 90 per 100,000 inhabitants [START_REF] Chen | A Nationwide epidemiological study of spinal cord injuries in Taiwan from July 1992 to June 1996[END_REF][START_REF] Belmont Pj | Incidence, epidemiology, and occupational outcomes of thoracolumbar fractures among U.S. Army aviators[END_REF][START_REF] Bengner | Changes in incidence and prevalence of vertebral fracture during 30 years[END_REF][START_REF] Cooper | Incidence of clinically diagnosed vertebral fractures: a population-based study in Rochester, Minnesota, 1985-1989[END_REF][START_REF] Kanis | Long term risk of osteporotic fracture in Malmo[END_REF][START_REF] Kannus | Continuously increasing number and incidence of fall-induced, fracture-associated, spinal cord injuries in elderly persons[END_REF][START_REF] Samuelsson | Incidence and risk factor for vertebral fracture in women and men: 25-year follow-up results from the population-based Framingham study[END_REF][START_REF] Van Der Klift | The incidence of verteberal fractures in men and women: the Rotterdam Study[END_REF]. The Framingham cohort study included 5,209 patients with a mean age of 44 years (range 28-62 years) and a prevalence of vertebral fracture of 14% [START_REF] Samuelsson | Incidence and risk factor for vertebral fracture in women and men: 25-year follow-up results from the population-based Framingham study[END_REF].

In Sweden, with a population of 9.2 million, about 2,700 patients with thoracolumbar vertebral fractures are admitted to hospitals annually. Most of these fractures are caused by blunt trauma [START_REF] Kanis | Long term risk of osteporotic fracture in Malmo[END_REF][START_REF] Kannus | Continuously increasing number and incidence of fall-induced, fracture-associated, spinal cord injuries in elderly persons[END_REF]. In younger patients, thoracolumbar vertebral fractures are usually caused by high energy accidents such as falls, or motor vehicle accidents whereas in elderly patients, osteoporosis is the dominant etiology [START_REF] Robertsson | Spinal injury pattern resulting from car and motorcycle accidents[END_REF]. Many patients have permanent functional impairment and pain. Long term reductions in quality of life and ability to work are frequent [START_REF] Robertsson | Spinal injury pattern resulting from car and motorcycle accidents[END_REF][START_REF] Robert | Functional outcomes after surgery for spinal fractures: return to work and activity[END_REF].

Treatment of thoracolumbar vertebral fractures has been debated extensively [START_REF] Denis | Acute thoracolumbar burst fracture in the absence of neurologic deficit: a comparison between operative and non operative treatment[END_REF][START_REF] Glenn | Non-operative management and treatment of spinal injuries[END_REF][START_REF] Hartman | Non-operative treatment of thoracolumbar fractures[END_REF][START_REF] Moller | Nonoperatively treated burst fractures of the thoracic and lumbar spine in adults: a 23-to 41-year follow-up[END_REF][START_REF] Mumford | Thoracolumbar burst fracture: the clinical efficacy and outcome of nonoperative management[END_REF][START_REF] Parker | Successful short-segment instrumentation and fusion for thoracolumbar spine fractures: a consecutive 41/2-year series[END_REF][START_REF] Shen | Nonsurgical treatment of three-column thoracolumbar junction burst fractures without neurologic deficit[END_REF][START_REF] Shen | Nonoperative treatment versus posterior fixation for thoracolumbar junction burst fractures without neurologic deficit[END_REF][START_REF] Vaccaro | The thoracolumbar injury severity score: a proposed treatment algorithm[END_REF][START_REF] Van Der Roer | Management of traumatic thoracolumbar fractures: a systematic review of the literature[END_REF][START_REF] Verlaan | Surgical treatment of traumatic fractures of the thoracic and lumbar spine. A systematic review of the literature on techniques, complications, and outcome[END_REF][START_REF] Wood | Operative compared with nonoperative treatment of a thoracolumbar burst fracture without neurological deficit. A prospective, randomized study[END_REF]. Three comprehensive reviews of unstable traumatic thoracolumbar vertebral fracture management did not find any clear pattern in the relative merits of operative compared to non-operative treatment [START_REF] Glenn | Non-operative management and treatment of spinal injuries[END_REF][START_REF] Van Der Roer | Management of traumatic thoracolumbar fractures: a systematic review of the literature[END_REF][START_REF] Verlaan | Surgical treatment of traumatic fractures of the thoracic and lumbar spine. A systematic review of the literature on techniques, complications, and outcome[END_REF].

To our knowledge, previous studies have not investigated the proportion of patients admitted for thoracolumbar vertebral fractures and operated on. Therefore, the aim of our nationwide population-based epidemiological study was to analyse the incidence, admissions, operations, external causes, and case fatality rate among patients with thoracolumbar vertebral fractures admitted to hospital in Sweden.

Patients and methods

Sweden has a national public healthcare system, based on independent county councils, mainly financed by local taxes.

Since 1964, the Swedish National Board of Health and Welfare has compiled data on hospital discharges in the Swedish Hospital Discharge Register (SHDR) [24]. From 1997 onward all Swedish hospitals participate. Each record contains information about demographics, diagnoses, operations, and administrative data about the healthcare provider and the admission. Each year, about 1.3 million discharges are recorded. The validity of the data in the Register has previously shown to be adequate [START_REF] Nilsson | Relability of the hospital registry. The diagnostic data are better than their reputation[END_REF].

We identified all patients with a primary discharge diagnosis of thoracic or lumbar vertebral fractures, spine dislocations, or disc ruptures in the SHDR (ICD 10 S22.0, S22.1, S23.0, S23.1, S32.0, S32.1, S33.0, S33.1, International Classification of Diseases, 1997) [26]. Surgery in these patients was categorized by procedure codes indicating spine operations (NA* **), and operations on the nervous system (A** **; Nordic Classification of Operations and Major Procedures, 1997) [START_REF]Swedish Classification of Operations and Major Procedures[END_REF]. The standard diagnostic workup was examination of clinical signs and radiological examination.

In order to assess survival, linkage was performed to the Swedish Death Register using the unique personal identity number (PIN) [START_REF]The Swedish Death Register[END_REF]. From this register, information was obtained about underlying cause of death, and date of death. All patients were followed from January 1, 1997 through December 31, 2001 or date of death. Both these registers are maintained by the Centre of Epidemiology, National Board of Health and Welfare, Stockholm Sweden. (WWW.sos.se/epc/epceng.htm).

Age was categorized as 0-19, 20-29 years etc. and 80-years. In the descriptive and multivariate analyses, patients were stratified by age 0-59 years or 60 years and older.

Causes of fractures were reported for 95% of the patients and were grouped into six categories by ICD E-codes (External causes).

All patients operated on during their first admission and, in addition, patients having a first admission without surgery but a second or a later admission with an operation were identified. No readmissions were counted. Indication for surgery was based on the clinicians' evaluations.

Cases fatality rates at 30 and 90 days were calculated at time at risk from the date of admission to the date of death.

The study was approved by the Regional Research Ethics Board (Dnr 04-530/5).

Statistical analyses

Incidence rates of thoracolumbar vertebral fractures and surgical procedures were calculated from the Swedish national and regional population in the year 2000 (Statistics Sweden) [24]. Only patients with complete records were included. Multivariate logistic regression analyses were performed to estimate the probability of surgery considering sex, age, primary diagnosis, external cause of fracture, year of operation, and geographic region. Odds ratios (OR) and the corresponding 95% confidence intervals were calculated.

Results

The study base consisted of 13,496 admitted patients. Of these, a third (34%) was aged 0-59 years (Table 1a) and a majority was men (62%). The most frequent external causes were transport accidents and falls from heights.

Among the 4,530 patients aged 0-59 years, 684 patients (15%) were operated on. Surgery was most frequent in the patients 20-39 years of age. The crude annual proportion operated on decreased over the study years.

The 8,966 patients 60 years or older (Table 1b) constituted two-thirds of the study population (66%), and twothirds were women (68%). More than half of the patients were 80 years or older, the majority with lumbar vertebral fractures, most of them caused by falls at the same level. The crude annual number of patients admitted decreased slightly over the study years.

Operations were uncommon in these older patients (2%). It was four times more frequent in men than in women (4 vs. 1.0%) and most operations were performed in patients 60-69 years of age. The crude annual proportion of patients operated on increased each year.

To accommodate the impact of each variable on the probability of spine surgery, a multivariate analysis was performed for the two age groups (Table 2). In patients 0-59 years of age at surgery, we found no difference between sexes but the probability was significantly higher in the youngest patients from 21 to 39 years of age. Patients with thoracic fractures had a lower probability than those with lumbar fractures. Patients who had fallen from heights or were injured by transport accidents had an increased probability. The probability of surgery was lowest in the western region. There were no changes over time.

In patients 60 years or older, men had a significantly higher probability of an operation than women, and the probability decreased steeply with increasing age. The highest probability was seen in patients who had fallen from heights or suffered from transport accidents. The probability increased significantly during the last 2 years. There were no significant differences between geographical regions. The operations were mainly performed at university or regional hospitals (83% of the admissions), and most patient were discharged from departments of orthopaedic surgery (85%). The median length of stay was 8 days, changing only slightly during the study period.

Among those who were operated the 30 day case fatality rate was 0.7% and the 90-day case fatality rate was 1.4%. Median age for those who died was 64 years (Range 44-83 years). Common underlying causes of deaths were suicides, intoxications and accidents (29%), cardiovascular diseases (27%), and tumours (12%).

The average annual incidence of patients admitted to hospital with thoracolumbar vertebral fractures was 30.4 per 100, 000 person year (Fig. 1). In patients younger than 60 years of age, the incidence was 13 and twice as high in men as in women. The incidence increased steeply with age and in patients 60 years or older, it was higher in women than in men.

The annual incidence decreased slightly from 1997 to 2001 (Table 3). In patients 59 years or younger, the incidence was stable but decreased by 21% in older patients. The incidence was higher in the South-Eastern and Annual incidence of fractures and operations per 100,000 population Northern regions. The annual incidence of operations was 2 per 100,000 person and year with small changes over the study years. The incidence of operations was highest in the Northern region, was twice as high in men as in women, and decreased with increasing age (Fig. 2).

Discussion

In our study the incidence of thoracolumbar fractures was 13 per 100,000 inhabitants per year in patients younger than 60 years of age, and twice as common in men as in women.

Transport accidents and falls from heights were frequent causes. One patient in seven was operated on, and the probability was unchanged over the study period. Patients older than 60 years of age were mainly women, and only two percent were operated on. Males were operated on twice as often as women and the most common accidents were falls on a level. The probability of an operation increased the last two study years. The case fatality rate was very low.

In contrast to our study a Finnish study conducted 1970-1995 predicted the overall fall-induced fracture incidence in the population older than 50 years to be 32 per 100,000 in 2010 [START_REF] Kannus | Continuously increasing number and incidence of fall-induced, fracture-associated, spinal cord injuries in elderly persons[END_REF]. The study design was different comparing patients aged 50 years or older to a reference group of patients aged 20-39 years. The time period differed, 1970-1995, from which they made further prognosis. Another study performed in an earlier time period with long term follow-up, also reports higher incidences [START_REF] Samuelsson | Incidence and risk factor for vertebral fracture in women and men: 25-year follow-up results from the population-based Framingham study[END_REF]. The low incidence figures in our study could be due to the fact that the total incidence of vertebral fractures remains largely underestimated. A population based study showed that only about one-third of vertebral fractures comes to medical attention, whereas the majority remains unnoticed [START_REF] Cooper | Incidence of clinically diagnosed vertebral fractures: a population-based study in Rochester, Minnesota, 1985-1989[END_REF]. However, our study design was unable to detect these asymptomatic osteoporotic fractures. Only large population-based cohort studies which screen all participants would be able to elucidate this aspect. Up to 35% of unreported osteoporotic vertebral fractures coming to attention as incidental findings in imaging studies [START_REF] Woo | Incidental vertebral fractures on multidetector CT images of the chest: prevalence and recognition[END_REF]. This figure reflects the low reporting rates by interpreting radiologists. Bartalena T et al. reported that only 15% of vertebral fractures had been noted in the radiology final report, most likely because of unawareness of their clinical importance [START_REF] Bartalena | Prevalence of thoracolumbar vertebral fractures on multidetector CT: underreporting by radiologists[END_REF].

In general clinical practice, subjective visual diagnostic methods are used. However, these are considered as being not reproducible enough for epidemiological studies [START_REF] Grados | Prevalence of vertebral fractures in French women older than 75 years from the EPIDOS study[END_REF]. Also, usually the more severe fractures tend to come to attention in clinical practice. Mild fractures are also of clinical importance despite being under diagnosed [START_REF] Roux | Mild prevalent and incident vertebral fractures are risk factors for new fractures[END_REF].

Many patients with thoracolumbar vertebral fractures may be treated on an out-patient basis. Richard Hu et al. [START_REF] Hu | Epidemiology of incident spinal fracture in a complete population[END_REF] showed that a large percentage of all osteoporotic fractures were treated as out-patients, In our study we did not capture the patients treated as outpatients. We had only information on the patients discharged from hospitals. Also a few patients who died prior to admission to hospital could be missed. However, in the year 2007 there were only 4 patients out of 91,820 deaths in Sweden who had vertebral fracture as underlying cause of death [24]. These injuries could also be missed in polytrauma patients. However, the vast majority of thoracolumbar vertebral fractures due to high energy trauma are diagnosed and treated as inpatients.

We can only speculate regarding which factors contribute to the decreased incidence in our study. Many accident prevention programs have been launched in several areas, i.e. road safety, leisure time accident prevention training, fall prevention devices in geriatric care and pharmacologic prophylaxis against osteoporosis.

In order to include all potential thoracolumbar spine fractures we include disc ruptures. It is of course questionable if these can be counted as thoracolumbar fractures. However, we found only 8 patients per year (=0.03%) with that diagnose. The reason for including sacral fracture in the study was that in axial trauma an spinopelvic dissociation can arise associated with lumbar spine surgery. This injury should strictly be defined as a pelvic or lumbosacral injury. This leads to a thoracolumbar fracture overestimation of 0.6%.

A major strength of our study is the calculation of population based incidence rate of fractures and surgical During the duration of this study, we observed an increasing incidence of operations among patients older than 60 years. A possible explanation of this could be the introduction of new surgical methods [START_REF] Garfin | New technologies in spine: Kyphoplasty and vertebroplasty for the treatment of painful osteoporotic compression fractures[END_REF].

We found that the probability of surgery was lower among women compared to men. The influence of sex and gender on musculoskeletal health has been reported by Tosi et al. [START_REF] Tosi | Does sex matter in musculoskeletal health? The influence of sex and gender on musculoskeletal health[END_REF] who discussed the biological basis for differences in injury mechanism, pain sensation, drug handling, and healing response that cannot be explained simply by hormone levels.

Patients of male sex, younger age, high-energetic trauma or with lumbar fractures had an increased probability of surgery. In patients with spinal cord injury the mean age is around 30 years and 80% of them are men [35]. This figure indicates that high-risk behaviour is more common among young men. High-energetic trauma is strongly related to more severe vertebral injury and subsequently a higher indication for surgery. Even in patients older than 60 men have high risk behaviour falling from heights or have more transport accidents. Most fracture occurs in the lumbar region (thoracolumbar), whereas the thoracic cage does not protect the spine as well [START_REF] Magerl | A comprehensive classification of thoracic and lumbar injuries[END_REF].

The study also showed signs of regional differences in the incidence of operations. The data may reflect considerable regional differences in treatment strategies among spine surgeons. The optimal treatment of patients with spinal fracture has been debated for years, and numerous studies have been published [START_REF] Mumford | Thoracolumbar burst fracture: the clinical efficacy and outcome of nonoperative management[END_REF][START_REF] Shen | Nonsurgical treatment of three-column thoracolumbar junction burst fractures without neurologic deficit[END_REF][START_REF] Van Der Roer | Management of traumatic thoracolumbar fractures: a systematic review of the literature[END_REF][START_REF] Verlaan | Surgical treatment of traumatic fractures of the thoracic and lumbar spine. A systematic review of the literature on techniques, complications, and outcome[END_REF].

As opposed to common belief in the medical community the study reported low case fatality rates in our surgically treated cohort (90-day 1.4%) [START_REF] Hu | Epidemiology of incident spinal fracture in a complete population[END_REF]. An explanation could be that these patients are healthier than the general population or by advances in the perioperative management. In previous studies the case fatality rate was calculated at time at risk. This loses 1/12 in the tail-end and that would not influence the results.

Unfortunately we did not include co-morbidity or coexisting injuries in our study. Therefore, the relative risk of dying could have been higher in patients who were not operated on. Others have shown that the relative risk of death was very high in young individuals and decreased in both sexes with age [START_REF] Kanis | Excess mortality after hospitalisation for vertebral fracture[END_REF]. The reason for this is not known but may be related to severe trauma injuries or other significant co-morbidity. Our analyses of the case fatality rate was limited to patients operated on and could therefore be a source of bias because very severe co-morbidity could have been a contra-indication for surgical intervention.

Kanis et al. showed excess mortality after hospitalization for all vertebral fractures in Sweden between the years 1987 and 1996 [START_REF] Kanis | Excess mortality after hospitalisation for vertebral fracture[END_REF]. In comparison to our cohort, they included only patients older than 50 years. They reported that the risk of death was markedly increased immediately after the event of the vertebral fracture. After a short period of declining risk, the risk increased with age at a rate which was higher than that of the general population and comparable to the rate that 1 year after a hip fracture. The latter function was assumed to be due to deaths related to co-morbidity and the residuum assumed to be due to the vertebral fracture.

Our study includes all thoracolumbar fractures regardless of the energy of trauma or if patients have fragility fractures i.e. osteoporosis. Unfortunately we could not extract if the patient has osteoporosis. The results have to be interpreted with caution due to the different risk factors in these subgroups (high energy trauma vs. osteoporosis). Geriatric high energy trauma is common and makes this analysis even more complex [START_REF] Lane | Geriatric trauma patients-are they receiving trauma center care?[END_REF]. The type of injuries and outcomes associated with particular mechanisms of injury is therefore important to further study. Performing an evaluation during a longer time period will reflect time trends and the results of preventions of injuries will be possible to study.

In conclusion, our study gives new information on the burden of thoracolumbar vertebral fractures as well as important information about the surgical incidence and differences according to gender and age. This information should be taken into consideration in the planning of health care and when studies are planned.

Open Access This article is distributed under the terms of the Creative Commons Attribution Noncommercial License which permits any noncommercial use, distribution, and reproduction in any medium, provided the original author(s) and source are credited.

Fig. 1

 1 Fig. 1 Thoracolumbar vertebral fractures in Sweden 1997-2001. Age and sex specific incidence per 100,000 population

Fig. 2

 2 Fig. 2 Thoracolumbar vertebral fractures in Sweden 1997-2001. Age and sex specific surgical incidence per 100,000 population

Table 1

 1 Thoracolumbar fractures inSweden 1997Sweden -2001

	Thoracolumbar vertebral fractures in Sweden
						Table 1 continued
		Admissions	Surgery		Admissions	Surgery
		N	% Of total N	% With		N	% Of total N	% With
					surgery					surgery
	A. Patients 0-59 years n = 4,530				Lumbar vertebral fracture 5,583	62.3	115	2.1
	Sex					External cause
	Male	2,801	61.8	426 15.2	Falls same level	5,275	58.8	50	0.9
	Female	1,729	38.2	258 14.9	Falls from heights	710	7.9	53	7.5
	Age (years)					Falls not specified	1,426	15.9	19	1.3
	0-19	624	13.8	68 10.9	Transport accidents	380	4.2	22	5.8
	20-29	932	20.6	174 18.7	Miscellaneous	697	7.8	23	3.3
	30-39	839	18.5	153 18.2	None reported	478	5.3	5	1.0
	40-49	921	20.3	129 14.0	Year
	50-59	1,214	26.8	160 13.2	1997	1,895	21.1	22	1.2
	Diagnosis					1998	1,951	21.8	27	1.4
	Thoracic vertebral	1,971	43.5	236 12.0	1999	1,754	19.6	28	1.6
	fracture					2000	1,760	19.6	46	2.6
	Lumbar vertebral fracture 2,559	56.5	448 17.5	2001	1,606	17.9	49	3.1
	External cause					Region
	Falls same level	712	15.7	75 10.5	Southern	1,749	19.5	36	2.1
	Falls from heights	1,076	23.8	181 16.8	Western	1,661	18.5	31	1.9
	Falls not specified	297	6.6	33 11.1	South-East	934	10.4	11	1.2
	Transport accidents	1,753	38.7	281 16.0	Stockholm	1,545	17.2	38	2.5
	Miscellaneous	489	10.8	79 16.2	Central	1,905	21.2	29	1.5
	None reported	203	4.5	35 17.2	Northern	1,161	12.9	25	2.2
	Year					Missing data	11	0.1	2 18.2
	1997	877	19.4	147 16.8	Total	8,966 100.0	172	1.9
	1998 1999	955 902	21.1 19.9	153 16.0 124 13.7	Characteristics of admissions and operations
	2000	873	19.3	138 15.8
	2001	923	20.4	122 13.2
	Region
	Southern	805	17.8	137 17.0
	Western	775	17.1	93 12.0
	South-East	502	11.1	75 14.9
	Stockholm	798	17.6	149 18.7
	Central	949	20.9	133 14.0
	Northern	686	15.1	96 14.0
	Missing data	15	0.3	1	6.7
	Total	4,530 100.0	684 15.1
	B. Patients 60-years n = 8,966
	Sex
	Male	2,891	32.2	113	3.9
	Female	6,075	67.8	59	1.0
	Age (years)
	60-69	1,311	14.6	91	6.9
	70-79	3,051	34	59	1.9
	80-	4,604	51.3	22	0.5
	Diagnosis
	Thoracic vertebral	3,383	37.7	57	1.7
	fracture

Table 2

 2 Thoracolumbar vertebral fractures in Sweden 1997-2001

		0-59 years	60-years
		OR 95% CI	OR 95% CI
	Sex
	Female	1.00 Ref.	1.00 Ref.
	Male	0.98 (0.82-1.16) 2.42 (1.70-3.45)
	Age (years)
	B 20	1.00 Ref.
	21-29	1.83 (1.34-2.50)
	30-39	1.77 (1.28-2.43)
	40-49	1.31 (0.94-1.81)
	50-59	1.24 (0.90-1.69)
	60-69		1.00 Ref.
	70-79		0.42 (0.30-0.61)
	80-89		0.14 (0.08-0.23)
	Transport accidents	1.44 (1.09-1.90) 2.92 (1.67-5.12)
	Miscellaneous	1.52 (1.07-2.15) 2.71 (1.59-4.60)
	None reported	1.73 (1.10-2.71) 1.11 (0.43-2.88)
	Year
	1997	1.00 Ref.	1.00 Ref.
	1998	0.99 (0.76-1.28) 1.07 (0.59-1.93)
	1999	0.83 (0.63-1.09) 1.37 (0.77-2.44)
	2000	1.02 (0.78-1.32) 2.19 (1.29-3.71)
	2001	1.03 (0.79-1.35) 2.93 (1.73-4.97)
	Region
	Southern	1.00 Ref	1.00 Ref
	Western	0.61 (0.45-0.82) 0.93 (0.56-1.55)
	South-East	0.75 (0.54-1.03) 0.54 (0.26-1.09)
	Stockholm	1.05 (0.81-1.37) 1.21 (0.74-1.96)
	Central	0.74 (0.56-0.97) 0.60 (0.35-1.00)
	Northern	0.73 (0.55-0.98) 0.73 (0.42-1.27)
	Probability of spine surgery Odds ratios (OR) and 95% confidence
	intervals (95% CI)

Diagnosis Lumbar vertebral fracture 1.00 Ref 1.00 Ref Thoracic vertebral fracture 0.65 (0.55-0.77) 0.73 (0.52-1.03) External cause Falls same level 1.00 Ref. 1.00 Ref. Falls from heights 1.59 (1.19-2.14) 3.32 (2.13-5.19) Falls not specified 0.96 (0.62-1.50) 1.36 (0.79-2.37)

Table 3

 3 Thoracolumbar vertebral spine fractures in Sweden 1997-2001

	Age	Fractures			Operations
		0-59	60-	All	0-59	60-	All
	Year
	1997	12.7	97.2	31.3	2.1	1.1	1.9
	1998	13.8	99.7	32.8	2.2	1.4	2.0
	1999	13.1	89.3	30.0	1.8	1.4	1.7
	2000	12.6	89.0	29.6	2.0	2.3	2.1
	2001	13.2	80.5	28.2	1.7	2.4	1.9
	Region
	Southern	12.1	88.2	29.6	2.1	1.8	2.0
	Western	13.3	101.2	32.7	1.6	1.9	1.7
	South-East	13.4	81.6	29.4	2.0	1.0	1.8
	Stockholm	10.6	88.1	25.2	2.0	2.2	2.0
	Central	12.9	85.0	29.7	1.8	1.3	1.7
	Northern	20.1	108.9	41.3	2.8	2.3	2.7