M Vähäkuopus

J Snellman

M.Sc.; Jari Jalava

Vuopio

Ph.D.; Tuula Siljander

M.D Outi Lyytikäinen

Ph.D.; Susanna Vähäkuopus

M.Sc.; Marja Snellman

Ph.D.; Jaana Vuopio

Keywords: group A streptococcus, Streptococcus pyogenes, epidemiology, emm typing, outcome, bacteraemia group A streptococcus, Streptococcus pyogenes, epidemiology, emm typing, outcome, 43 bacteraemia 44

Epidemiology, outcome and types of invasive group A streptococcal infections in Finland

Tables: nice to see the rate ratios by sex (table 1). Response: It is true that the seasonal patterns vary by year. We have to remember that our observations are based on very small numbers (3-23 cases per month). During the study, we did not detect any major outbreaks (within time/region/emm type), except for a small increase in cases caused by type emm84 [START_REF] Siljander | Rapid 311 emergence of emm84 among invasive Streptococcus pyogenes infections in Finland[END_REF]. J Clin Microbiol 47:477-480). A showing seasonality by sex per year. We decided to exclude this figure from the manuscript as it seemed too busy to read. The summer peak occurred for males during 6/10 study years but for females only during 2/10 study years. We decided to slightly reformulate our statements in the manuscript as follows:

Please see below Figure

Results (page 5, lines 105-107): "Variation in the seasonal activity of iGAS disease could be identified, with occasional peaks of cases occurring during the midwinter (for both sexes) and midsummer (with male cases dominating; Figure 1a and1b)." Discussion (page 8, lines 198-201): "Seasonal variation in iGAS disease was observed; in addition to a midwinter peak season, there were also occasional peaks during the midsummer. In the northern hemisphere, peaks of cases are generally observed during the winter and spring. There were also sexspecific differences in the peak seasons, with male cases dominating during the summer." Discussion: First paragraph is a bit weak; could be strengthened (this is not necessary but would make for a better read).

Response: We moved the text containing the most interesting points and strengths of the study into this paragraph (page 6 lines 135-141): "This study presents a comprehensive description of the recent epidemiology and outcome of invasive group A streptococcal (Streptococcus pyogenes) disease with information on emm type prevalence in a well-defined population. The main strength of the study is the nationwide population-based surveillance and the good coverage of isolate collection. We describe how the incidence and case fatality of iGAS infections were influenced by changes in the emm type prevalence. Age-and sex-specific differences in the incidence, uncharacteristic seasonal patterns of infections, and aspects of vaccine coverage are also discussed." Authors should briefly discuss/support (if true) consistency in surveillance methods over time. Could the increasing incidence be due to improved surveillance methods? For example, any changes in blood culture practices, any change in completeness of reporting by labs?

Response: It is true that changes in blood culture practises or in the reporting system may have had an effect on the results. The annual blood-culturing rate increased by one-third during 1995-2002 in Finland (Skogberg et al.2008. Epidemiol Infect 136:108-114). Skogberg et al. concluded that "the increase in bloodstream infection rates may have been due to more frequent blood culturing but was not associated with changes in the reporting system [from paper to electronic]or aetiology of blood stream infections".

We added the following sentences in the discussion (page 9, lines 205-207): "There are some limitations to this study. Firstly, some of the increase in the incidence may be due to more frequent blood culturing activity [ref. Skogberg et al. 2008. Epidemiol Infect 136:108-114]. It is also possible but less likely that the incidence would have been influenced by changes in the reporting system, which was established in 1995."

We also detected regional differences in the blood-culturing rates. This was acknowledged in the discussion (page 9, lines 210-211): "Thirdly, differences in blood culture sampling practices between countries may have an effect on the observed incidence [ref. [START_REF] Skogberg | Increase in bloodstream 314 infections in Finland, 1995-2002[END_REF]. Epidemiol Infect 136:108-114]."

In addition, minor modifications were made in the paragraph regarding the limitations of this study to improve the discussion (page 9, lines 205-218).

Good to see comparisons to other European countries since this country is rather small and possibly not representative of a larger geographic area. Why are there two seasonal peaks? Any theories?

Response: Without supporting evidence and data, we could not discuss our theories of the dual peak seasonality in the manuscript. The timing of the winter peak was somewhat earlier but near the expected peak based on data by other countries, and as it occurred for both sexes, we did not specifically question it. However, the summer peak differed from expectations on seasonality of these infections.

Factors associated with the summer peak could be the summer vacation period of Finns with the increased outdoor activities and possible risk behaviour. Alcohol-related diseases have been identified among the most common risk factors for invasive pneumococcal disease (Klemets et al. 2008. BMC Infect Dis 8: 96.) Exposure of the skin to insect bites and minor trauma may lead to skin infections and eventually invasive infections, and the higher temperature and humidity may delay the healing of wounds. Males and females may have different predisposing factors and underlying conditions and behavioural patterns. Males may also have a higher threshold than females for seeking medical care in case of trauma.

We concluded that presumably the predisposing factors and underlying conditions associated with these peak seasons were different. Without data on all these above mentioned factors, we could not draw further conclusions on the reasons for seasonality. The small study size may cause some of the variation in the seasonality, which was discussed in the manuscript (pages 8-9, lines 202-204): "However, the unusual pattern and irregularity of the seasonality observed in this study may partly arise from natural variation due to a reasonably small study size."

Although it has been shown that emm types 1 and 3 are associated with increased mortality than other serotypes after controlling for age and underlying diseases, the authors should include this in their discussion. Although this surveillance does not capture other medical conditions, the authors could also state (if true) that emm 1 CFR is still higher when adjusted for age (for elderly) to strengthen argument.

Response: We agree with the above. However, in addition to the patient age, the underlying conditions of a patient also have a role in the case fatality. Without the clinical data, and with this small a study size, we did not adjust the case fatality of emm1 infections for age. Nevertheless, to discuss this in the manuscript, we added the following sentences (page 8, lines 194-197): "Having an infection by emm type 1 or 3 has been identified as an independent factor associated with death due to iGAS disease [ref. [START_REF] O'loughlin | The 295 epidemiology of invasive group A streptococcal infection and potential vaccine implications: 296 United States, 2000-2004[END_REF] Clin Infect Dis 45:853-62]. Also in our study, the case fatality by emm1 was higher than average, however we did not adjust it for age." Page 7, line 170: add % of 26-valent coverage for US and Japan.

Response: We modified the sentence as follows (page 7, lines 171-173): "The emm types included in the putative 26-valent recombinant vaccine would have covered approximately half of the Finnish isolates in 2004-2007, a notably smaller proportion than has been estimated for the U.S.A. (79%) and Japan (82%)." Any documented support for the idea of strain competition? Couldn't this just reflect changes in general community immunity?

Response: It is true that we do not have sufficient evidence for strain competition because the time of surveillance and strain typing is not long enough to allow for that. We agree that it may be more accurate to stick to the concept of population immunity, whereas strain competition may only be part of that concept. We altered the statement as follows (page 8, lines 177-178): "Such a pattern of alternating type prevalence may be a reflection of changes in the general immunity of the population."

It has been speculated that the fluctuation in the prevalence of types reflects the population's susceptibility but also the prevalence of circulating types (mostly associated with mild infections and carriage of which we lack data) in the community. Regarding the idea of strain competition, the changing prevalence of emm1 and emm28 in Finland is of interest. Please see Figure B below (unpublished data; the coverage of surveillance may differ during the time period). The alternating prevalence of these types might suggest a competitive situation between these two types. Page 8: lines 188-189: where did this 4-fold increase in case fatality come from? These numbers, if true, really need to be presented in the results or tables. This reviewer cannot find the data in the paper and questions the accuracy. Did the CFR reach 32% in 2005? Please check and correct! Or, rewrite this section to be more clear.

Response: We agree that the data was presented only partly which may distract the reader. The fourfold increase in the case fatality is true, but the statement needs more clarity to avoid misunderstanding. We modified the sentences regarding the case fatality as follows:

Results (pages 5-6, lines 122-123): "The case fatality was the lowest in 2004 (3%), increasing to its highest in 2005 (12%), and then declined by 2006 (10%) and 2007 (7%)." Discussion (page 8, lines 191-192): "A sudden four-fold increase in the case fatality occurred from 3% in 2004 to 12% in 2005."

Tables: nice to see the rate ratios by sex (table 1). Response: It is true that the seasonal patterns vary by year. We have to remember that our observations are based on very small numbers (3-23 cases per month). During the study, we did not detect any major outbreaks (within time/region/emm type), except for a small increase in cases caused by type emm84 [START_REF] Siljander | Rapid 311 emergence of emm84 among invasive Streptococcus pyogenes infections in Finland[END_REF]. J Clin Microbiol 47:477-480). A showing seasonality by sex per year. We decided to exclude this figure from the manuscript as it seemed too busy to read. The summer peak occurred for males during 6/10 study years but for females only during 2/10 study years. We decided to slightly reformulate our statements in the manuscript as follows:

Please see below Figure

Results (page 5, lines 105-107): "Variation in the seasonal activity of iGAS disease could be identified, with occasional peaks of cases occurring during the midwinter (for both sexes) and midsummer (with male cases dominating; Figure 1a and1b)."

Discussion (page 8, lines 198-201): "Seasonal variation in iGAS disease was observed; in addition to a midwinter peak season, there were also occasional peaks during the midsummer. In the northern hemisphere, peaks of cases are generally observed during the winter and spring. There were also sex-specific differences in the peak seasons, with male cases dominating during the summer."

Discussion: First paragraph is a bit weak; could be strengthened (this is not necessary but would make for a better read).

Response:

We moved the text containing the most interesting points and strengths of the study into this paragraph (page 6 lines 135-141): "This study presents a comprehensive description of the recent epidemiology and outcome of invasive group A streptococcal (Streptococcus pyogenes) disease with information on emm type prevalence in a well-defined population. The main strength of the study is the nationwide population-based surveillance and the good coverage of isolate collection. We describe how the incidence and case fatality of iGAS infections were influenced by changes in the emm type prevalence. Age-and sex-specific differences in the incidence, uncharacteristic seasonal patterns of infections, and aspects of vaccine coverage are also discussed."

Authors should briefly discuss/support (if true) consistency in surveillance methods over time. Could the increasing incidence be due to improved surveillance methods? For example, any changes in blood culture practices, any change in completeness of reporting by labs? Response: It is true that changes in blood culture practises or in the reporting system may have had an effect on the results. The annual blood-culturing rate increased by one-third during 1995-2002 in Finland (Skogberg et al.2008. Epidemiol Infect 136:108-114). Skogberg et al. concluded that "the increase in bloodstream infection rates may have been due to more frequent blood culturing but was not associated with changes in the reporting system [from paper to electronic]or aetiology of blood stream infections".

We added the following sentences in the discussion (page 9, lines 205-207): "There are some limitations to this study. Firstly, some of the increase in the incidence may be due to more frequent blood culturing activity [ref. Skogberg et al. 2008. Epidemiol Infect 136:108-114]. It is also possible but less likely that the incidence would have been influenced by changes in the reporting system, which was established in 1995."

We also detected regional differences in the blood-culturing rates. This was acknowledged in the discussion (page 9, lines 210-211): "Thirdly, differences in blood culture sampling practices between countries may have an effect on the observed incidence [ref. Skogberg et al. 2008. Epidemiol Infect 136:108-114]."

In addition, minor modifications were made in the paragraph regarding the limitations of this study to improve the discussion (page 9, lines 205-218).

Good to see comparisons to other European countries since this country is rather small and possibly not representative of a larger geographic area. Why are there two seasonal peaks? Any theories?

Response: Without supporting evidence and data, we could not discuss our theories of the dual peak seasonality in the manuscript. The timing of the winter peak was somewhat earlier but near the expected peak based on data by other countries, and as it occurred for both sexes, we did not specifically question it. However, the summer peak differed from expectations on seasonality of these infections.

Factors associated with the summer peak could be the summer vacation period of Finns with the increased outdoor activities and possible risk behaviour. Alcohol-related diseases have been identified among the most common risk factors for invasive pneumococcal disease (Klemets et al.

2008. BMC Infect Dis 8: 96.) Exposure of the skin to insect bites and minor trauma may lead to skin infections and eventually invasive infections, and the higher temperature and humidity may delay the healing of wounds. Males and females may have different predisposing factors and underlying conditions and behavioural patterns. Males may also have a higher threshold than females for seeking medical care in case of trauma.

We concluded that presumably the predisposing factors and underlying conditions associated with these peak seasons were different. Without data on all these above mentioned factors, we could not draw further conclusions on the reasons for seasonality. The small study size may cause some of the variation in the seasonality, which was discussed in the manuscript (pages 8-9, lines 202-204): "However, the unusual pattern and irregularity of the seasonality observed in this study may partly arise from natural variation due to a reasonably small study size."

Although it has been shown that emm types 1 and 3 are associated with increased mortality than other serotypes after controlling for age and underlying diseases, the authors should include this in their discussion. Although this surveillance does not capture other medical conditions, the authors could also state (if true) that emm 1 CFR is still higher when adjusted for age (for elderly) to strengthen argument.

Response:

We agree with the above. However, in addition to the patient age, the underlying conditions of a patient also have a role in the case fatality. Without the clinical data, and with this (79%) and Japan (82%)."

Any documented support for the idea of strain competition? Couldn't this just reflect changes in general community immunity?

Response: It is true that we do not have sufficient evidence for strain competition because the time of surveillance and strain typing is not long enough to allow for that. We agree that it may be more accurate to stick to the concept of population immunity, whereas strain competition may only be part of that concept. We altered the statement as follows (page 8, lines 177-178): "Such a pattern of alternating type prevalence may be a reflection of changes in the general immunity of the population."

It has been speculated that the fluctuation in the prevalence of types reflects the population's susceptibility but also the prevalence of circulating types (mostly associated with mild infections and carriage of which we lack data) in the community. Regarding the idea of strain competition, the changing prevalence of emm1 and emm28 in Finland is of interest. Please see Figure B below (unpublished data; the coverage of surveillance may differ during the time period). The alternating prevalence of these types might suggest a competitive situation between these two types. Response: We agree that the data was presented only partly which may distract the reader. The four-fold increase in the case fatality is true, but the statement needs more clarity to avoid misunderstanding. We modified the sentences regarding the case fatality as follows:

Results 1980s, a change in the epidemiology of severe GAS infections, with an increase in the incidence and 49 severity of disease was documented [START_REF] Schwartz | Changing epidemiology of group A streptococcal 304 infection in the USA[END_REF]. The role and increased pathogenic potential of type M1 in 50 epidemics were of specific interest [START_REF] Martin | Molecular epidemiology of group A streptococcus M type 1 282 infections[END_REF][START_REF] Stromberg | Outbreak of group A streptococcal bacteremia in 316 Sweden: an epidemiologic and clinical study[END_REF]. The incidence of severe GAS disease shows variations 51 over time and geographic region, possibly reflecting the population's susceptibility to particular strains 52 with diverse virulence properties [START_REF] Lamagni | Epidemiology of severe Streptococcus pyogenes disease 271 in Europe[END_REF][START_REF] O'loughlin | The 295 epidemiology of invasive group A streptococcal infection and potential vaccine implications: 296 United States, 2000-2004[END_REF][START_REF] Tyrrell | Invasive group A streptococcal disease in 318 Alberta, Canada (2000 to 2002)[END_REF]. Dynamic changes in the type distribution of GAS may 53 also influence the case fatality of these infections.

54

In Finland, the incidence rate of invasive group A streptococcal (iGAS) disease has also shown 55 fluctuations during this and the previous decade, and an increasing trend has been noticed [START_REF] Siljander | emm 306 typing of invasive T28 group A streptococci, 1995-2004, Finland[END_REF]. In

93

RESULTS

94

Incidence rates. From January 1998 to December 2007, 1318 cases of iGAS (range by year, 100-207)

95

were identified (Table 1). The median age of case-patients was 52 years (range, 0-95 years); 55% were 96 males. The average annual incidence rate of iGAS was 2.5 cases per 100,000 population. The rate was 97 higher in males than in females and in males of adult age groups it increased with age (Table 2). The 98 highest rates were observed among the elderly patients. Males had a significantly higher rate of 99 infection than females especially among persons aged 45-64 years, whereas females had a higher rate 100 than males among persons aged 25-34 years.

101

The annual incidence rate fluctuated (range by year, 1.9-3.9 cases per 100,000 population) but showed Overall, infections by emm1 were significantly associated with higher case fatality (14% vs. 8% with 128 all other types; P<0.02, Table 3). Cases by emm75 and 79 were also associated with higher than 129 average case fatality (14% for both). In contrast, emm28 was associated with lower case fatality (2% 130 vs. all other types, P<0.01) along with type 81 (0%, respectively). 143

The observed incidence rate of 3.9 cases per 100,000 population in 2007 is the highest in Finland since 144 1995. This study together with our earlier research shows that the incidence of iGAS disease has 145 fluctuated since the 1990s but continued its increasing trend [START_REF] Siljander | emm 306 typing of invasive T28 group A streptococci, 1995-2004, Finland[END_REF][START_REF] Siljander | Rapid 311 emergence of emm84 among invasive Streptococcus pyogenes infections in Finland[END_REF]. During 2003-2004, the U.K.,

146

Sweden, Denmark and Finland had the highest rates of infection (range 2.5-3.3 per 100,000) in a 147

European study [START_REF] Lamagni | Epidemiology of severe Streptococcus pyogenes disease 271 in Europe[END_REF]. Since then, indications of an increase in the incidence are seen in Sweden to 4.5 148 cases per 100,000 by 2007 (http://www.smittskyddsinstitutet.se/in-english/statistics/beta-hemolytic-149 group-a-streptococci-gas-invasiv-infection/) and very recently in the U.K. [START_REF] Lamagni | Increase in invasive 276 group A streptococcal infections in England, Wales and Northern Ireland, 2008-9[END_REF]. In contrast, the 150 incidence of iGAS disease in the U.S.A. has remained relatively stable, at an average of 3.5 cases per 151 100,000 during 1995-2004, with a moderate increase to 3.8 in 2007 152 (http://www.cdc.gov/abcs/survreports.htm) [START_REF] O'loughlin | The 295 epidemiology of invasive group A streptococcal infection and potential vaccine implications: 296 United States, 2000-2004[END_REF].

153

The incidence of iGAS disease in Finland generally increased with age as expected, but the rate in the 154 elderly (65 years) still remained at a lower level than has been reported in Sweden, Denmark, the 155 U.K. or the U.S.A. [START_REF] Darenberg | Molecular and clinical characteristics of invasive 253 group A streptococcal infection in Sweden[END_REF][START_REF] Lamagni | 273 Efstratiou A (2008) Severe Streptococcus pyogenes infections, United Kingdom, 2003-2004[END_REF][START_REF] Luca-Harari | 279 Clinical and epidemiological aspects of invasive Streptococcus pyogenes infections in Denmark 280 during 2003 and 2004[END_REF][START_REF] O'loughlin | The 295 epidemiology of invasive group A streptococcal infection and potential vaccine implications: 296 United States, 2000-2004[END_REF]. Males had a higher incidence of iGAS disease compared to females, 156 which has also been noted by some other studies [START_REF] Lamagni | Epidemiology of severe Streptococcus pyogenes disease 271 in Europe[END_REF][START_REF] O'loughlin | The 295 epidemiology of invasive group A streptococcal infection and potential vaccine implications: 296 United States, 2000-2004[END_REF]. Differences could also be identified in the 157 age-and sex-specific rates, with males having significantly more infections than females in the age of 158 45-64 years. In contrast, females had more infections in the age of 25-34 years than males. A similar 159 clear peak in the incidence of females around the same age has been noticed in earlier studies from 160

Denmark and the U.K. [START_REF] Lamagni | 273 Efstratiou A (2008) Severe Streptococcus pyogenes infections, United Kingdom, 2003-2004[END_REF][START_REF] Luca-Harari | 279 Clinical and epidemiological aspects of invasive Streptococcus pyogenes infections in Denmark 280 during 2003 and 2004[END_REF].

161

The characteristic fluctuation in the incidence of iGAS disease is presumably a reflection of changes 162 in the emm type distribution and the population's susceptibility towards particular strains, especially of 163 emerging types [START_REF] Darenberg | Molecular and clinical characteristics of invasive 253 group A streptococcal infection in Sweden[END_REF][START_REF] O'brien | Epidemiology of invasive group A streptococcus 292 disease in the United States, 1995-1999[END_REF][START_REF] O'loughlin | The 295 epidemiology of invasive group A streptococcal infection and potential vaccine implications: 296 United States, 2000-2004[END_REF]. The emm type distribution in Finland underwent major changes during 164 2004-2007 when emm1 replaced emm28 as the most predominant type. The incidence of iGAS disease 165 increased simultaneously with the increasing prevalence of type emm1, as has also been observed by 166 others [START_REF] Stromberg | Outbreak of group A streptococcal bacteremia in 316 Sweden: an epidemiologic and clinical study[END_REF]. However, changes in the prevalence of other types, such as the sudden emergence of 167 emm84, also contributed to the increase [START_REF] Siljander | Rapid 311 emergence of emm84 among invasive Streptococcus pyogenes infections in Finland[END_REF].

168

The observed earlier in 1988-1990 and 1997-1998, and in this study in 2006-2007 [6, 15, 21]. Similarly, 178 the prevalence of type emm28 has peaked in 1993-1995 and 2002-2004 [15, 21]. Such a pattern of 179 alternating type prevalence may be a reflection of changes in the general immunity of the population.

180

Types emm28 and more rarely also emm4 have been known to associate with postpartum infections 181 and puerperal sepsis [START_REF] Chuang | Population-based surveillance for 245 postpartum invasive group a streptococcus infections, 1995-2000[END_REF][START_REF] Colman | The serotypes of Streptococcus 249 pyogenes present in Britain during 1980-1990 and their association with disease[END_REF][START_REF] Tyrrell | Invasive group A streptococcal disease in 318 Alberta, Canada (2000 to 2002)[END_REF]. Also in this study, emm28 infections were found to be specifically 182 concentrated in females of child-bearing age. However, estimations of how many of these cases were 183 pregnancy-related infections are beyond the scope of this study.

184

The

195

In parallel, the proportion of emm28, associated with a low case fatality, decreased. Having an 196 infection by emm type 1 or 3 has been identified as an independent factor associated with death due to 197 iGAS disease [START_REF] O'loughlin | The 295 epidemiology of invasive group A streptococcal infection and potential vaccine implications: 296 United States, 2000-2004[END_REF]. Also in our study, the case fatality by emm1 was higher than average, however we 198 did not adjust it for age.

199

Seasonal variation in iGAS disease was observed; in addition to a midwinter peak season, there were 200 also occasional peaks during the midsummer. In the northern hemisphere, peaks of cases are generally 201 observed during the winter and spring [START_REF] Darenberg | Molecular and clinical characteristics of invasive 253 group A streptococcal infection in Sweden[END_REF][START_REF] Lamagni | Epidemiology of severe Streptococcus pyogenes disease 271 in Europe[END_REF][START_REF] O'loughlin | The 295 epidemiology of invasive group A streptococcal infection and potential vaccine implications: 296 United States, 2000-2004[END_REF][START_REF] Tyrrell | Invasive group A streptococcal disease in 318 Alberta, Canada (2000 to 2002)[END_REF]. There were also sex-specific differences in the 202 peak seasons, with male cases dominating during the summer. These two peak seasons might be 203 associated with different predisposing factors and underlying conditions. However, the unusual pattern 204 and irregularity of the seasonality observed in this study may partly arise from natural variation due to 205 a reasonably small study size.

206

There are some limitations to this study. Firstly, some of the increase in the incidence may be due to 207 more frequent blood culturing activity [START_REF] Skogberg | Increase in bloodstream 314 infections in Finland, 1995-2002[END_REF]. It is also possible but less likely that the incidence would Finnish case definition may partly explain the observed lower incidence rate. Thirdly, differences in 212 blood culture sampling practices between countries may have an effect on the observed incidence [START_REF] Skogberg | Increase in bloodstream 314 infections in Finland, 1995-2002[END_REF].

213

In addition, the observed differences in fatality may arise from the case definition used, the timing of

218

These data would be needed to address reasons behind the observed differences in the age-and sex-219 specific incidence, and such a study has been undertaken within one healthcare district in Finland [START_REF] Rantala | Clinical presentations and 298 epidemiology of beta-haemolytic streptococcal bacteraemia: a population-based study[END_REF].

220

In conclusion, dynamic changes in the emm type prevalence influenced the incidence and case fatality 221 of iGAS disease in Finland. An unusual seasonal pattern with peaks in the midwinter and midsummer 222 was observed. Differences in age-and sex-specific incidence rates and seasonality suggest variation in

  Fig b: how does the seasonality of cases by sex look per year? A large outbreak could distort the findings as shown in Fig b, appearing to be a repeated seasonal difference between men and women that doesn't actually exist each year.

Figure B.

  Figure B.

  Fig b: how does the seasonality of cases by sex look per year? A large outbreak could distort the findings as shown in Fig b, appearing to be a repeated seasonal difference between men and women that doesn't actually exist each year.

  Figure A:

  size, we did not adjust the case fatality of emm1 infections for age. Nevertheless, to discuss this in the manuscript, we added the following sentences (page 8, lines 194-197): "Having an infection by emm type 1 or 3 has been identified as an independent factor associated with death due to iGAS disease [ref.[START_REF] O'loughlin | The 295 epidemiology of invasive group A streptococcal infection and potential vaccine implications: 296 United States, 2000-2004[END_REF] Clin Infect Dis 45:853-62]. Also in our study, the case fatality by emm1 was higher than average, however we did not adjust it for age." Page 7, line 170: add % of 26-valent coverage for US and Japan.Response: We modified the sentence as follows (page 7, lines 171-173): "The emm types included in the putative 26-valent recombinant vaccine would have covered approximately half of the Finnish isolates in 2004-2007, a notably smaller proportion than has been estimated for the U.S.A.

Figure B. Prevalence of types emm1 and emm28 in invasive Streptococcus pyogenes diseasePage 8 :

 8 Figure B.

56 2006 ,

 2006 clinicians contacted the National Institute for Health and Welfare and described suddenly 57 encountering more severe disease manifestations with a poor outcome. These events prompted us to 58 investigate the recent trend and outcome of iGAS infections and emm type distribution in Finland in The national healthcare system of Finland (population 5.3 million) is organised into 20 62 healthcare districts (with catchment populations ranging from 58,000 to 1.5 million), forming five 63 tertiary care districts. Since 1995, all clinical microbiology laboratories have mandatorily notified all 64 isolations of S. pyogenes from blood or cerebrospinal fluid (CSF) to the National Infectious Disease 65 Register (NIDR). With each notification, the following information is transmitted (generally 66 electronically) to the NIDR: date and type of specimen, unique national identity code of the patient 67 (since 2004), date of birth, sex, and place of treatment. Within an interval of three months, 68 notifications concerning the same patient are merged into a single case. The corresponding S.

69

  pyogenes isolates are submitted to the national reference laboratory. 70 88 according to Poisson distribution were calculated. 7-day case fatality was calculated for 2004-2007 89 and for each emm type separately.  2 and Kruskal-Wallis tests were applied where appropriate to test 90 for statistical significance between subgroups and distributions. Differences were considered 91 significant when P<0.05. Data were analyzed with Intercooled Stata 9.1 software for Windows 92 (StataCorp, U.S.) and R statistical software version 2.9.0 (R Development Core Team, Austria).

  102 an increasing trend during the study period. Peaks occurred in 2002 (2.9 cases per 100,000) and in 103 in 2005 (12%), and then declined by 2006 (10%) and 2007 (7%). The case-patients with fatal outcome 125 were older than those who survived (median age, 60 vs. 54 years; P<0.01). No fatalities occurred in 126 patients aged <1 and 15-24 years.

  127

  a comprehensive description of the recent epidemiology and outcome of invasive 137 group A streptococcal (Streptococcus pyogenes) disease with information on emm type prevalence in a 138 well-defined population. The main strength of the study is the nationwide population-based 139 surveillance and the good coverage of isolate collection. We describe how the incidence and case 140 fatality of iGAS infections were influenced by changes in the emm type prevalence. Age-and sex-141 specific differences in the incidence, uncharacteristic seasonal patterns of infections, and aspects of 142 vaccine coverage are also discussed.

175

  Within the last 20 years, a periodic fluctuation of peaks and troughs of the two most common 176 genotypes emm1 and emm28 has occurred in Finland. Peaks in numbers of M/emm1 have been 177

  208 have been influenced by changes in the reporting system, which was established in 1995. Secondly, 209 case definitions of iGAS disease differ between countries and complicate comparisons of incidence 210 rates. Exclusion of non-bacteraemic cases with other sterile site isolations and/or toxic shock by the 211

  214death considered (7/30-day/in-hospital mortality), and the source of information (e.g. population 215 registry vs. hospital records). In this study, the 7-day mortality information obtained from the 216 Population Information System was used. Finally, the surveillance does not include collection of 217 clinical data of disease manifestations, possible risk factors and underlying conditions of patients.

Fig. 1a Fig. 1b

 1a1b Fig. 1a Cases of invasive group A streptococcal disease during 1998-2007 in Finland. Thin line,

outcome and emm types of invasive group A streptococcal infections in Finland 1

  Tuula Siljander 1 , Outi Lyytikäinen 2 , Susanna Vähäkuopus 1 , Marja Snellman 2 , Jari Jalava 3 , Jaana

	Manuscript
	Click here to
	Epidemiology, 2
	3	Vuopio 1
	4
	5	(pages 5-6, lines 122-123): "The case fatality was the lowest in 2004 (3%), increasing to its highest in 2005 (12%), and then declined by 2006 (10%) and 2007 (7%)." 1 Bacteriology Unit, Department of Infectious Disease Surveillance and Control, National Institute for
	6	Discussion (page 8, lines 191-192): "A sudden four-fold increase in the case fatality occurred from 3% in 2004 to 12% in 2005." Health and Welfare, P.O. Box 30, FI-00271, Helsinki, Finland
	7	2 Epidemiologic Surveillance and Response Unit, Department of Infectious Disease Surveillance and
	8	Control, National Institute for Health and Welfare, P.O. Box 30, FI-00271, Helsinki, Finland
	9	3 Antimicrobial Resistance Unit, Department of Infectious Disease Surveillance and Control, National
	10	Institute for Health and Welfare, Kiinamyllynkatu 13, FIN-20521, Turku, Finland
	11
	12	Running title: Invasive group A streptococcus, Finland
	13
	14	Corresponding author: Tuula Siljander, Department of Infectious Disease Surveillance and Control,
	15	National Institute for Health and Welfare, P.O. Box 30, FI-00271, Helsinki, Finland. E-mail:
	16	tuula.siljander@thl.fi, phone: +358 20 610 8558, fax: +358 20 610 8238,
	17
	18
	19	Category:
	20	Word count for abstract: 200
	21	Word count for text: 2347
	22	Number of figures: 1
	23	Number of tables: 3
	24

download Manuscript: Epidemiology_emm_types_iGAS_Finland_rev1.doc Click here to view linked References INTRODUCTION 46

  Streptococcus pyogenes (group A streptococcus, GAS) causes a wide array of infections ranging in 47 severity from mild pharyngitis and skin/soft tissue infections to severe invasive infections. In the late 48

  type distribution of iGAS isolates in Finland has common features with that of other countries but

	169
	170	also some unique characteristics. emm types 1, 28 and 89 are common globally but 75 and 84 are rarer
	171	and have not featured among the five most common emm types elsewhere [4, 5, 7, 12, 17, 26]. The
	172	low prevalence of cases by emm3 is of interest, as it is a common type in many countries, and often
	173	associated with high case fatality [7, 12, 17, 26]. The emm types included in the putative 26-valent
	174	recombinant vaccine would have covered approximately half of the Finnish isolates in 2004-2007, a
		notably smaller proportion than has been estimated for the U.S.A. (79%) and Japan (82%) [7, 14, 17].

  overall erythromycin resistance during 2004-2007 was expectedly low, as the rate has declined

	185
	186	during this decade in Finland
	187	(http://www.ktl.fi/portal/english/projects/fire/finres/finres__antimicrobial_resistance_statistics_/strept
	188	ococcus_pyogenes/). The Finnish iGAS strains remain susceptible to clindamycin. Similar low

resistance rates have been reported for example in Denmark and the U.S.A.

[START_REF] Luca-Harari | 279 Clinical and epidemiological aspects of invasive Streptococcus pyogenes infections in Denmark 280 during 2003 and 2004[END_REF][START_REF] Richter | Macrolide-resistant Streptococcus pyogenes in the United States, 2002-2003[END_REF]

. Tetracycline 189 resistance was associated with certain clones, as has also been found previously, and the resistant 190 strains were mainly of uncommon emm types

[START_REF] Kataja | 266 Clonal spread of group A streptococcus with the new type of erythromycin resistance. Finnish 267 Study Group for Antimicrobial Resistance[END_REF]

.

191

The overall case fatality of iGAS disease in Finland was relatively low at 8%, compared to those 192 reported by some other European countries and the U.S.A.

[START_REF] Darenberg | Molecular and clinical characteristics of invasive 253 group A streptococcal infection in Sweden[END_REF][START_REF] Ekelund | Variations in emm type among group A streptococcal isolates causing invasive or 256 noninvasive infections in a nationwide study[END_REF][START_REF] Lamagni | 273 Efstratiou A (2008) Severe Streptococcus pyogenes infections, United Kingdom, 2003-2004[END_REF][START_REF] O'loughlin | The 295 epidemiology of invasive group A streptococcal infection and potential vaccine implications: 296 United States, 2000-2004[END_REF]

. A sudden four-fold increase 193 in the case fatality occurred from 3% in 2004 to 12% in 2005. During the study, the proportion of 194 infections by types emm1 and emm75 increased, these types being associated with a high case fatality.

Table 1

 1 Annual incidence of invasive group A streptococcal disease and the number of cases and

	330
	331	isolates studied, Finland, 1998-2007
		Year	Rate a of invasive	Number of
			disease	cases (isolates)

Table 3

 3 The number and proportion (% of total number) of most common emm types encountered in 341 the order of overall prevalence, and case fatality (%) by emm type, 2004-2007, Finland.

	342
				Number of isolates (%)				Fatal cases
											(%)
		2004	2005		2006		2007	2004-2007	2004-2007
	emm type	(N=128)	(N=110)	(N=161)	(N=203)	(N=602)	(N=48)
	28	46 (35.9)	22 (20.0)	32	(19.9)	25 (12.3)	125 (20.8)	3	(2)
	1	6 (4.7)	9 (8.2)	26	(16.1)	57 (28.1)	98 (16.3)	14	(14)
	84	0 (0.0)	1 (0.9)	24	(14.9)	32 (15.8)	57	(9.5)	4	(7)
	75	10 (7.8)	17 (15.5)	12	(7.5)	4	(2.0)	43	(7.1)	6	(14)
	89	10 (7.8)	4 (3.6)	11	(6.8)	12	(5.9)	37	(6.1)	2	(5)
	79	10 (7.8)	12 (10.9)	4	(2.5)	2	(1.0)	28	(4.7)	4	(14)
	12	2 (1.6)	6 (5.5)	6	(3.7)	12	(5.9)	26	(4.3)	2	(8)
	81	7 (5.5)	3 (2.7)	5	(3.1)	8	(3.9)	23	(3.8)	0	(0)
	27G	3 (2.3)	1 (0.9)	4	(2.5)	11	(5.4)	19	(3.2)	2	(11)
	other a	34 (26.6)	35 (31.8)	37	(23.0)	40 (19.7)	146 (24.2)	11 b	(7)

a Including isolates from a total of 37 other emm types and nontypable isolates. 343 b Other types causing poor outcome: emm4 and emm110 (2 cases each); emm22, emm59, emm60, emm77, 344 emm85, emm87 and emm112 (1 case each). 345 346

predisposing factors and underlying conditions.


Ángel L Álvarez

Solomon Habtemariam

Malindra Juan-Badaturuge

Caroline Jackson

Francisco Parra

Ángel ; Álvarez

Ángel L Álvarez

Malindra Juan- Badaturuge

In vitro Anti HSV-1 and HSV-2 Activity of Tanacetum vulgare Extracts and Isolated Compounds: An Approach to their Mechanisms of Action

Keywords: Tanacetum vulgare, tansy, antiviral, HSV, ethnomedicine Tanacetum vulgare, tansy, antiviral, HSV, ethnomedicine

published or not. The documents may come   L'archive ouverte pluridisciplinaire

INTRODUCTION

Herpes simplex viruses (HSV) are members of the Herpesviridae family, which are known to cause long-term latent infections with periods of recurring viral replication [START_REF] Whitley | Herpes simplex virus infections[END_REF]. The two common human pathogenic HSVs are type 1 (HSV-1) and type 2 (HSV-2), which cause lesions around the lips, eyes, mucous membrane of the oral cavity and the genitals. Acyclovir (ACV) is the most commonly used chemotherapy agent against HSV-1 and HSV-2, in systemic or topical therapy.

Other clinically relevant drugs include valaciclovir, penciclovir, famciclovir and vidarabine [START_REF] Gupta | Genital herpes[END_REF]). However, these drugs are not always well tolerated and drug-resistant strains are becoming a major problem, especially in immunocompromised patients (e.g. HIV infected patients). Moreover, the incidence of HSV-2 infections (main cause of genital herpes) not only raised in recent years but also have been linked with an increased (three-fold) risk of sexually acquired HIV infection [START_REF] Freeman | Herpes simplex virus 2 infection increases HIV acquisition in men and women: systematic review and meta-analysis of longitudinal studies[END_REF]. Therefore, anti HSV drugs with novel modes of action are urgently needed.

Tanacetum vulgare L. (Asteraceae), commonly known as tansy, is an herbaceous plant growing in temperate Europe and Asia. Various preparations of tansy are known to be used for treating rheumatic pain, skin eruption, and diuretic conditions (Ognyanov & Todorova 1983;Ognyanov et al. 1983;[START_REF] Lahlou | Diuretic activity of the aqueous extracts of Carum carvi and Tanacetum vulgare in normal rats[END_REF]). Tansy has also been reported as an anthelmintic, antihypertensive, stimulant, emmenagogue, carminative, antiseptic, antihypertensive and as an antispasmodic agent [START_REF] Grieve | A Modern Herbal[END_REF][START_REF] Barnes | Herbal medicines: a guide for healthcare professionals[END_REF]. Among the most studied biological activities of tansy are the antioxidant [START_REF] Juan-Badaturuge | Antioxidant principles of Tanacetum vulgare L. aerial parts[END_REF], anti-inflammatory, diuretic and wound-healing effects [START_REF] Williams | The flavonoids of Tanacetum parthenium and T. vulgare and their anti-inflammatory properties[END_REF][START_REF] Xie | Immunomodulatory activity of acidic polysaccharides isolated from Tanacetum vulgare L[END_REF][START_REF] Lahlou | Diuretic activity of the aqueous extracts of Carum carvi and Tanacetum vulgare in normal rats[END_REF][START_REF] Pålsson | Tick repellent substances in the essential oil of Tanacetum vulgare[END_REF].

Antiherpes virus activity of the crude ethyl acetate extract of aerial parts of tansy of Brazilian origin has recently been reported [START_REF] Onozato | Tanacetum vulgare: antiherpes virus activity of crude extract and the purified compound parthenolide[END_REF]). This study further revealed that parthenolide was one of the major anti HSV-1 principles of tansy aerial parts. Our present study on tansy aerial parts, however revealed the presence of known antiviral agents including 3,5-dicaffeoylquinic acid (3,5-DCQA) which was isolated as a major constituent of the plant [START_REF] Juan-Badaturuge | Antioxidant principles of Tanacetum vulgare L. aerial parts[END_REF]. Through a

MATERIALS AND METHODS

Plant material and extraction procedures.

The extraction and isolation of antioxidant compounds from the aerial parts of Tanacetum vulgare were described previously [START_REF] Juan-Badaturuge | Antioxidant principles of Tanacetum vulgare L. aerial parts[END_REF]. Briefly, T.

vulgare from an authenticated source grown at Hadlow Agricultural College medicinal gardens (Hadlow, UK) was harvested during its flowering stage (July 2006). The dried plant material was extracted by cold methanol and fractionated using solvents of ascending polarity: petroleum ether, chloroform, ethyl acetate, butanol and water. Further activity-directed fractionation of the most antioxidant fraction (ethyl acetate) resulted in the isolation of the most abundant constituent 3,5-DCQA together with axillarin and luteolin [START_REF] Juan-Badaturuge | Antioxidant principles of Tanacetum vulgare L. aerial parts[END_REF].

The underground rhizome of the plant was harvested from the same source in March 2009 and allowed to air-dry. A portion of the plant material (1 kg) was soaked in 7.5 L methanol and left in the dark for two weeks. The extract was collected and dried using a rotary evaporator to yield 132 g of the extract residue.

Quantification of parthenolide by HPLC.

An Agilent 1200 series gradient HPLC system composed of a degasser (G1322A), a quaternary pump (G1322A), an auto sampler (G1329A), a thermostat column compartment (G1316A) maintained at 25 o C and a diode array detector (G1315) was used. Solutions of the standard parthenolide (Sigma-Aldrich, Dorset, UK) and various plant extract fractions were made in acetonitrile. Samples (20 µL) were injected onto a reverse phase column (Agilent-Eclipse XDB-C18, 5 µm, 4.6 x 150 mm). The mobile phase was a mixture of acetonitrile (A) and 10 mM potassium phosphate buffer (pH=3.0) (B). The composition of the mobile phase at a flow rate of 1.5 min/ml was maintained at 41% A for the first 8 minutes and then raised to 70% A in 5 minutes. The parthenolide concentrations in the crude extract and fractions were quantified from the standard curves constructed from three-fold standard parthenolide dilutions made from a stock solution of 5 mg/ml. All experiments were repeated at least six times and the amount of parthenolide was calculated based on the peak area at λ = 210 nm using

Viruses and cells.

HSV-1 and HSV-2 clinical isolates were kindly provided by Dr. María Oña (Servicio de Microbiología, Hospital Universitario Central de Asturias. Spain) and propagated in African green monkey kidney (Vero) cells (ECACC No. 84113001). Virus titrations were performed using both the endpoint dilution method described by [START_REF] Reed | A simple method of estimating fifty per cent endpoints[END_REF], and by plaque assay. Vero cells were propagated in Dulbecco-modified Eagle's minimal essential medium (DMEM) (Gibco BRL) supplemented with 10% foetal bovine serum (Sigma) and maintained in DMEM without bovine serum.

Cytotoxicity assay.

Vero cells were seeded into 96-well plates at a density of 2 × 10 4 cells/well, and incubated at 37 ºC in a 5% CO 2 atmosphere for 48-72 h, until 90% or greater confluency of the monolayers was reached. Increasing concentrations of the test extract were added to cells, with a replicate number of six wells per concentration. After a 3days incubation period, a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) solution was added (final concentration 0.5 mg/mL) and the plates were incubated for another 4 h to allow formazan production. The solid precipitate was dissolved with absolute 2-propanol and the absorbance at 570 nm was measured using a 96-well µQuant Spectrophotometer (Biotek Instruments) at 620 nm. The resulting cell viability was calculated as described previously [START_REF] Mosmann | Rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxicity assays[END_REF].

Antiviral activity assay

Ninety-six-well plates containing confluent cell monolayers were preincubated for 1 h with increasing non-cytotoxic concentrations of the extracts. Six replicate wells were used for each analyzed sample. Afterwards, 10 Tissue Culture Infectious Dose 50 (TCID 50 ) of HSV-1 or HSV-2 were added into each well. The plates were incubated at 37 ºC in a 5% CO 2 humidified atmosphere and observed daily for cytopathic effect (CPE) using a light microscope. When CPE was observed in all virus control wells, the percentage of wells with CPE was determined for each treatment concentration. Acyclovir (ACV) at concentrations varying from 0.1 to 50 µg/mL served as positive control. The effect on HSV replication of tansy extracts or purified fractions, added to cell cultures at different times before, during or after infection was investigated as described previously [START_REF] Alvarez | In vitro anti-herpetic activity of an aqueous extract from the plant Phyllanthus orbicularis[END_REF]) with some modifications. The analyzed sample was added to the first series of wells at a fixed non-cytotoxic concentration (100 µg/mL for ethyl acetate and petroleum ether; 500 µg/mL for rhizome extract; 300 µg/mL for DCQA; and 5 µg/mL for parthenolide) and incubated for 1 h. All wells in the plate were then infected with 200 pfu of HSV-1 or HSV-2 and the same concentration of plant sample was added to the second series of wells at the time of infection. After 1 h of adsorption, the medium was removed from each well and fresh medium containing the plant extract was added to the first, the second and the third series of wells corresponding to -1, 0 and 1 h post-infection (hpi). Subsequent additions were performed 2, 4 and 6 hpi. After the last addition of compound the culture medium was replaced by a 1.5% methylcellulose overlay containing an identical concentration of the test extract. The plates were further incubated at 37°C in a 5% CO 2 atmosphere for 96 h, or until the appearance of evident viral lysis plaques in the mock-treated (virus control) wells, observed using a light inverted microscope. The cells were fixed with 500 µL/well 10% formaldehyde solution and stained with 0.5% crystal violet in 20% methanol. For each time of addition the percentage of inhibition (%I) was calculated as follows: %I= (1-number of viral plaques in treated wells/number of viral plaques in virus control) ×100.

Adsorption inhibition assay.

Twelve-well plates containing confluent Vero cells monolayers were incubated for 1 h at 4°C. Increasing non-cytotoxic concentrations of the test extract were added in duplicate and 200 pfu of HSV-1 or HSV-2 were immediately added. The plates were further incubated at 4°C for 3 h to allow virus adsorption. After this treatment, the medium was removed, the cell monolayers were gently washed with DMEM without bovine serum, 1.5% methylcellulose overlay was added to each well and the plates were Separate HSV-1 and HSV-2 suspensions were mixed with identical volumes of the plant extracts or the equivalent quantity of culture medium (negative control) and then incubated at room temperature for 30 min. The residual virus infectivity was investigated as described previously [START_REF] Reed | A simple method of estimating fifty per cent endpoints[END_REF]. Three different concentrations of each plant extract or isolated compounds were assayed.

Statistics.

The concentration of the test solution reducing cell viability by 50% (mean cytotoxic concentration, CC 50 ) and the concentration reducing viral-induced cytopathic effect by 50% (mean effective concentration, EC 50 ) were calculated by regression analysis using dose-response curves (not shown) generated from the experimental data. A selectivity index (SI) was calculated for each extract and virus, by dividing the CC 50 by the EC 50 value. CC 50 and EC 50 values were compared using ANOVA, followed by Tukey's multiple mean comparison tests. EC 50 values from samples that inhibited both viruses were compared using the unpaired Student's t test. All comparisons were performed using the Statistica 6.1 software. Values of p<0.05 were considered indicative of statistical differences.

RESULTS AND DISCUSSION

In order to assess the cytotoxic effects of the tansy extracts, a colorimetric MTT assay was performed after incubating Vero cell cultures with increasing concentrations of the plant samples [START_REF] Mosmann | Rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxicity assays[END_REF]. The CC 50 values obtained (Table 1) correlated well with morphological changes such as cell rounding, presence of cytoplasmic inclusions and loss of monolayer confluence recorded by microscopic examination of treated cultures. These changes became evident from 250 µg/mL of the crude extract, petroleum ether and ethyl acetate fractions; or from 100 or 500 µg/mL for the chloroform fraction or isolated 3,5-DCQA respectively. The water and butanol extracts were innocuous to Vero cells since no morphological alterations were observed during the course of the experiment (data not shown). At the highest concentrations used in antiviral assays for all samples, the mean cell viabilities recorded were close to 100%. The analyses of crude extracts from aerial parts of tansy grown in Britain showed antiviral activity against HSV-1, in agreement with previously published data of tansy of Brazilian origin [START_REF] Onozato | Tanacetum vulgare: antiherpes virus activity of crude extract and the purified compound parthenolide[END_REF]). In the present report we have found that this antiviral effect could be also extended to an HSV-2 clinical isolate (Table 1).

In order to investigate the nature of the active antiviral principles of tansy, five fractions obtained from the crude extract using solvents of ascending polarity (Juan-Badaturuge et al. 2009) were also tested. As shown in Table 1, the petroleum ether and ethyl acetate extracts were active against both herpes virus types with good selectivity indexes. The water and butanol fractions did not show anti-herpetic effects up to 150µg/mL. However it should be noted that, due to their low cytotoxicity in mammalian cells (CC 50 >500 µg/mL), antiviral activity at higher concentrations cannot be ruled out.

Three antioxidant compounds, namely 3,5-DCQA, axillarin and luteolin, previously isolated from the ethyl acetate fraction were also included in antiviral analyses.

Interestingly, the strongest inhibition (Table 1) was recorded for 3,5-DCQA which was isolated as a major constituent of the plant [START_REF] Juan-Badaturuge | Antioxidant principles of Tanacetum vulgare L. aerial parts[END_REF]. Other constituents such as axillarin, showed anti HSV-2 activity with a good selectivity index (Table 1) while luteolin appeared to be toxic to Vero cells and was unable to block HSV-2 replication at non-toxic concentrations (Table 1). The chloroform fraction showed severe cytotoxic effects at low concentrations and its antiviral activity could not be further analyzed.

Recently, a germacrane type sesquiterpene lactone (namely parthenolide) was reported to be one of the major anti HSV-1 active principles in T. vulgare [START_REF] Onozato | Tanacetum vulgare: antiherpes virus activity of crude extract and the purified compound parthenolide[END_REF]).

In the present study, we assessed the cytotoxicity of parthenolide together with its putative antiviral activity against HSV-1 and HSV-2 clinical isolates. This compound showed the highest toxicity (25.2 µg/mL) among all tested samples although the CC 50 value obtained in our study was about 4-times the previously reported (CC 50 =5.5 µg/mL) [START_REF] Onozato | Tanacetum vulgare: antiherpes virus activity of crude extract and the purified compound parthenolide[END_REF]. Surprisingly, our data revealed that purified parthenolide did not inhibit the virus-induced CPE (Table 1) up to a concentration 1.5-fold lower than the CC 50 value (SI<1.5). In order to further investigate the correlation between parthenolide and antiviral/cytotoxic effects, we measured its relative concentration in crude tansy extracts and fractions using HPLC. The analyses gave the following F o r P e e r R e v i e w 9 parthenolide yields: chloroform (2.7% w/w vs the tansy extract), petroleum ether (1.88%), ethyl acetate (0.49%), crude aerial parts (0.33%). Parthenolide could not be detected in n-butanol, water or crude rhizome extracts. Considering that the antiviral effect of the tansy rhizome extract was similar to that of the aerial parts, and that rhizome extract did not contain parthenolide it is reasonable to conclude that the observed anti-HSV activity should be due to some other compound.

Whether the active principles in tansy extracts acted at an intracellular stage of viral replication or directly inactivate HSV virions was investigated through a series of specific experiments including time-of-addition, adsorption inhibition and virucidal assays. In these analyses we investigated the effects of ethyl acetate, petroleum ether and rhizome extracts as well as purified parthenolide and 3,5-DCQA. The studies on reduction of virus-induced lysis plaques as a function of the time of addition showed a very high HSV-1 inhibition (close to 100%) when the ethyl acetate, petroleum ether fraction or 3,5-DCQA were added to cultured cells, up to 6 hpi (Figure 1A). These data suggested that the active compounds acted on late processes of the viral replication cycle. However, some differences were observed using HSV-2. In the case of ethyl acetate extract, a slight drop in HSV-2 inhibition was recorded when this extract was added beyond 1 hpi, suggesting that some immediate-early or early event in HSV-2 replication cycle was the main target of active principle(s) in this sample (Figure 1B). Some differences were also observed for the rhizome extract, because the reduction of HSV-2 virus-induced plaques dropped to 50% (Figure 1B), instead of completely losing its effect as observed for HSV-1 when this sample was added at 2 hpi. Parthenolide did not inhibit HSV-1 or HSV-2 in this assay and virus plaques were observed in all cases in a similar number to those in control wells, no matter the time of addition of this compound.

The ability of T. vulgare extracts to inhibit the attachment of HSV particles to Vero cells was investigated using adsorption inhibition experiments. For this purpose the cell monolayers were pre-incubated with HSV-1 (Figure 2A) or HSV-2 virions (Figure 2B) in the presence of the tansy extracts or purified compounds at 4°C. The number of virus-induced lysis plaques dramatically decreased when adsorption was performed in the presence of ethyl acetate, petroleum ether extracts or purified 3,5-DCQA (Figure 2). Adsorption inhibition ranged from 66.2% to 100% at concentrations between 18.7 µg/mL and 150 µg/mL. No inhibition was observed when parthenolide was used below its CC 50 , at 5 µg/mL or 15 µg/mL (Figure 2). The rhizome extract also inhibited HSV-1 and HSV-2 adsorption by 85.5 and 70.1%, respectively using 500 µg/mL of this extract during virus adsorption (data not shown).

Considering that adsorption inhibition might be due to virion inactivation, we further carried out virucidal assays as described in Materials and Methods. The data indicated that the ethyl acetate extract did not significantly decreased HSV-1 nor HSV-2 infectivity (Figure 3) whereas the petroleum ether extract (≥ 500 µg/mL) showed a virucidal index greater than 2 log10 units, corresponding to the inactivation of more than 99% virions in suspension [START_REF] Hu | Evaluation of new antiviral agents: I. In vitro perspectives[END_REF]. At 1 mg/mL the petroleum ether extract completely inactivated HSV particles (undetectable residual infectious titre). In contrast, mild virucidal effects were recorded for 3,5-DCQA against both herpesviruses at the highest assayed concentration (1 mg/mL), which is consistent with the observed inhibition beyond 6 hpi in the time-of-addition assay (see Figure 1). Neither the rhizome extract nor parthenolide inactivated virions at any tested concentration. Since the rhizome extract was able to inhibit HSV propagation when added to cell cultures before or at the time of infection (Figure 1), it could be suggested that its active constituents could interfere with virus adsorption perhaps by interacting with the cell surface receptors of HSV virions.

A relevant finding of the present study is the observation that the previously described antiviral principle of tansy, parthenolide, showed no anti herpesvirus activity in our study model, neither against an intracellular viral replication event nor as an extracellular virucidal agent. On the other hand, our data pointed out to 3,5-DCQA as a promising antiviral molecule, in agreement to previous studies where HIV-1 integrase inhibition [START_REF] Mcdougall | Dicaffeoylquinic and dicaffeoyltartaric acids are selective inhibitors of human immunodeficiency virus type 1 integrase[END_REF]) and antiherpetic effects were reported [START_REF] Shi | Purification and identification of antiviral components from Laggera pterodonta by high-speed countercurrent chromatography[END_REF]). In addition, other minor constituents of the plant, such as axillarin, observed in the present study are likely to contribute to the antiherpetic effect of tansy. Although the precise molecular targets for tansy extract and active principles remain to be elucidated, the observed virucidal properties suggest their potential use in topical formulations to treat the widespread dermatological lesions caused by HSV. Further research is required to identify additional as yet unknown antiviral principles, in particular from the petroleum ether fractions and the underground rhizome extracts of tansy.

  based antiherpetic activity study, we now reveal that constituents other than parthenolide are responsible for the antiviral activity of tansy.

  software (GraphPad, San Diego, USA). The calculated r 2 values of the standard curves were in the range 0.998 to 0.999.

  finally incubated at 37°C in a 5% CO2 atmosphere for 96 h or until complete development of viral lysis plaques in mock-treated (virus control) wells. The percentage of inhibition (%I) was calculated as follows: %I= (1-number of viral plaques in treated wells/number of viral plaques in virus control) ×100.

  Figure captions.

Figure 1 .

 1 Figure 1. Effect of T. vulgare extracts and compounds on HSV-1 (A) and HSV-2 (B) lysis plaques counts, when added to cell cultures at different times before, at or after infection. ethyl acetate extract, petroleum ether extract, rhizome extract, 3,5-DCQA, parthenolide. Data are presented as the mean of two independent experiments and their standard deviation values.

Figure 2 .

 2 Figure 2. Effect of T. vulgare extracts and compounds on HSV-1 (A) and HSV-2 (B) adsorption to Vero cells. ethyl acetate extract, petroleum ether extract, 3,5-DCQA, parthenolide. Data are presented as the mean of two independent experiments and their standard deviation values.

Figure 3 .

 3 Figure 3. Virucidal activity of T. vulgare extracts and purified compounds against HSV-1 (A) and HSV-2 (B).

  Figure 1 178x295mm (96 x 96 DPI)

Table 1 .

 1 Cytotoxicity and antiviral activity of T. vulgare extracts and purified compounds. Data are shown as the mean values and standard deviations from two independent experiments.

		Cytotoxicity		Antiviral activity
	Extract/ fraction	CC 50 (µg/mL)	HSV-1 CE 50 (µg/mL)	SI	HSV-2 CE 50 (µg/mL)	SI
	Crude aerial part 515.2 ± 10.3	327.0 ± 48.7	1.57	>400	<1.3
	Petroleum ether 235.7 ± 2.316	69.9 ± 7.69	3.37	61.16 ± 0.92	3.85
	Chloroform	56.55 ± 8.451	>50	<1.1	>50	<1.1
	Ethyl acetate	194.2 ± 2.908	95.7 ± 2.81	2.03	59.4 ± 0.13	3.27
	Butanol	>500	>150	ND	>150	ND
	Water	>500	>150	ND	>150	ND
	Rhyzome	1089.4 ± 71.2	295.8 ± 63.5	3.68	500.0 a	2.18
	3,5-DCQA	409.5 ± 10.61	31.1 ± 3.92	13.2	46.99 ± 2.19	8.71
	Axillarin	209.5 a	NA	-	42.7 ± 3.63	4.91
	Luteolin	67.92 ± 10.34	NA	-	>50	<1.3
	Parthenolide	25.17 ± 0.58	>16	<1.5	>16	<1.5
	Acyclovir	>1000	0.943 ± 0.229 >1060 0.977 ± 0.051 >1023

a : value from an individual experiment. ND: not determined. NA: not analyzed.

http://mc.manuscriptcentral.com/ptr Phytotherapy Research

http://mc.manuscriptcentral.com/ptr

ACKNOWLEDGEMENTS

This work was partially funded at FP laboratory by AECID PCI grant D/023290/09. The phytochemical analysis work was supported by HEFCE capability funding.