Elisabeth Fredlund

Anna Maria Thim

Ann Gidlund

Siv Brostedt

Marianne Nyberg

Monica Olsen

A-M Thim

Moulds and mycotoxins in rice in Swedish retail

Keywords: basmati rice, aflatoxin, ochratoxin A, jasmine rice, retail, Aspergillus flavus, 22 mould 23

la diffusion de documents scientifiques de niveau recherche, publiés ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

Rice (Oryzae sativa L) is one of the most important staple foods world-wide. Various 2 varieties of rice are cultivated in different parts of the world and some of them are restricted to 3 specific geographical regions, such as basmati rice in Pakistan and India (Bhattacharjee et al. 4 2002) or jasmine rice in Thailand (International Rice Institute, http://www.irri.org/). 5 6

The paddy rice or rough rice is harvested with the hull (or husk) when the water content is 7 approximately 20%. The water content must be further reduced by drying to 13-14% to 8 eliminate microbiological activity during storage. The hull is removed and the remaining 9 brown rice is processed further into various rice products such as whole-grain rice, parboiled 10 rice, polished rice etc. The rice kernels can be of varies sizes and are classified as long-grain 11 rice, medium-grain rice, and short-grain rice. 12 13 During cultivation and subsequent handling of rice, kernels can be contaminated by moulds, 14 which can grow and produce mycotoxins if conditions are favourable. The fungi may later die 15 due to increased temperature or dry periods, but once produced, the stable mycotoxins will 16 remain in the rice. Fungal activity depends on the moisture content and temperature, which 17 can both vary significantly in a silo depending on its design and environmental factors. harvest treatment of rice, including adequate drying and conditions of storage, are crucial 19 factors that will determine storage stability. 20 21 Species of Fusarium have been isolated from newly harvested paddy rice [START_REF] Pitt | The 17 normal mycoflora of commodities from Thailand. 2. Beans, rice, small grains and other 18 commodities[END_REF]22 Pacin et al. 2002) and low levels of Fusarium-toxins have been detected [START_REF] Kim | Natural occurrence of fumonisin B 1 in 23 Korean rice and its processed food by enzyme-linked immunosorbent assay[END_REF]; 23 Park et al. 2005). However, Fusarium-toxins are not considered a risk in rice (EC Regulation 24 856/2005). In stored rice, the fungal flora is different from that in newly harvested rice. 25 Aspergillus spp. are common contaminants of stored rice [START_REF] Kim | Natural occurrence of fumonisin B 1 in 23 Korean rice and its processed food by enzyme-linked immunosorbent assay[END_REF]Park et al. 2005; 1 Sales and Yoshizawa 2005) but species of Alternaria and Penicillium have also been reported 2 [START_REF] Pitt | The 17 normal mycoflora of commodities from Thailand. 2. Beans, rice, small grains and other 18 commodities[END_REF]Park et al. 2005). Several studies have reported detectable levels of aflatoxins 3 and ochratoxin A in rice from different countries including Cuba (Escobar and Regueiro 4 2002), Korea (Park et al. 2005), Malaysia [START_REF] Abdullah | Survey of fungal counts and natural ocurrence of 9 aflatoxins in Malaysian starch-based foods[END_REF] which both concluded that aflatoxins should be treated as carcinogenic food contaminants and 19 the intake should be reduced to levels as low as reasonably achievable. Aflatoxins are 20 genotoxic substances and therefore no tolerable daily intake (TDI) levels have been set for 21 these toxins. However, an intake of one ng per kg bodyweight (bw) and day, corresponding to 22 a life-time cancer risk of one extra cancer case for 10 5 individuals may be considered as an 23 acceptable risk. 24 foods. The ML's in cereals for ochratoxin A is three µg kg -1 , for aflatoxin B 1 two µg kg -1 and 10 for the sum of aflatoxin B 1 , B 2 , G 1 , and G 2 four µg kg -1 . There are no regulated levels for 11 mould in food. However, the presence of aflatoxin-producing species such as Aspergillus 12 flavus, A. parasiticus and A. nomius or the ochratoxin-producing species Penicillium 13 verrucosum, A. ochraceus, A. niger, A. carbonarius, and A. westerdijkiae may indicate the 14 presence of these toxins. 15 16 In 2006, high levels of aflatoxin M 1 was detected in milk in the companies own quality 17 controls. The source of contamination was aflatoxin-contaminated rice bran, a by-product 18 from a rice-mill, which was used as an ingredient in cattle feed in the south of Sweden. In 19 contrast to milk, rice is not routinely controlled for the presence of aflatoxin. Rice has become 20 an important stable food for the Swedish consumers. In 2006, Swedes consumed 5.4 kg rice 21 per person, which can be compared to 9.5 kg pasta per person, which is another important 22 staple food [START_REF] Jordbruksverket | STATISTIK FRÅN JORDBRUKSVERKET Statistikrapport 20[END_REF]. 23 The objective of this survey was to investigate the presence of mould, aflatoxins and 1 ochratoxin A in rice available for purchase in he Swedish retail market. The results were used 2 to estimate the intake of these mycotoxins from rice by Swedish consumers. Rapid semi-3 quantitative methods, such as lateral-flow devices, for the analysis of aflatoxin in various food 4 items (nuts, figs etc.) have been developed. To evaluate whether such methods may be 5 suitable also for screening for aflatoxin in rice, one such method, RIDA®QUICK Aflatoxin 6 (R-Biopharm AG, Darmstadt, Germany) was used in parallel with HPLC analysis. 7

Sampling and sample treatment 2

The majority of rice samples were purchased from retail outlets in the South and Central parts 3 of Sweden in Malmö, Stockholm, Uppsala, and Gothenburg. Rice samples were purchased 4 from large retail stores, small local shops, health shops, shops with organic products and 5 shops with products of mainly ethnical origin. Most samples (73 of 99 samples) were taken 6 according to the alternative sampling plan for official control of mycotoxins in food (EC 7 401/2006). 8 9 Sub-samples (0.5-2 kg) were pooled and mixed for 15 min (IGF 2400/S382, Svea, Linköping, 10 Sweden) and 1 kg of the pooled sample was then ground to fine powder (Retsch GmbH & Co,11 Haan, Tyskland) and stored in room temperature until analysis. The particle size after 12 grinding was below 1.5 mm and approximately 80% of the particles were below 0.3 mm. [START_REF] Frisvad | Important mycotoxins and the fungi which 14 produce them[END_REF] Water-activity (a w ) was analysed in all samples (Aqua Lab, Series 3 TE, Decagon Devices, 14 Inc., Pullman, WA, USA). 15 16

Quantification and identification of mould 17

Ground rice samples (40 g) were diluted (1:10 and 1:100) in 0.1 % peptone water (BD, 18

Becton, Dickinson and Company, Sparks, MD, USA) and poured or spread on 18 % dichloran 19 glycerol agar (DG18; Samson et al. 2004) plates. These were incubated in 25 ± 0.5°C for 20 seven days and colonies were thereafter counted and presented as the number of colony 21 forming units (cfu) per g rice. The level of quantification was one log unit cfu g -1 , which 22 equals 10 cfu g -1 rice. 23 Ground rice samples (50 g) were transferred to glass bottles (500 ml) and mixed with 200 ml 9 of 84 % acetonitrile (HPLC grade) on a shaking table for 30 min. The samples were then 10 filtered (Munktell V150) and six ml were further transferred to a MultiSep AflaZON 226+-11 column (Romer Labs, Germany). Two ml were collected and nitrogen-evaporated to dryness 12 before re-suspension in 300 µl acetonitrile:water:acetic acid (50:450:5). The samples were 13 filtered (0.45 µm) before injection (20 µl) on the HPLC column (C18 3 µm, 100 x 4.6 mm) 14 equipped with a fluorescence detector (excitation 365 nm, emission 450 nm). Derivatisation 15 was performed with a KobraCell. The mobile phase was water:acetonitrile:methanol (15:3:4) 16 supplemented with one mM KBr and 1.4 mM HNO 3 , the flow was 1.0 ml min -1 and column 17 temperature 40°C. The level of quantification (LOQ) was 0.1 µg kg -1 for aflatoxin B 1 , B 2 , G 1 , 18 and G 2 . 19 20

In addition to HPLC analysis, 76 samples were also analysed for aflatoxin with the rapid 21 method RIDA®QUICK Aflatoxin by R-Biopharm AG, Darmstadt, Germany. Ten g of ground [START_REF] Kim | Natural occurrence of fumonisin B 1 in 23 Korean rice and its processed food by enzyme-linked immunosorbent assay[END_REF] rice was mixed with 20 ml of 70 % methanol by vortexing for 3 min. The samples were 23 centrifuged (3400 g) for two min and 50 µl were transferred to eppendorf tubes and mixed 24 with 100 µl mobile solvent supplied with the RIDA®QUICK kit. A hundred µl was applied 25 on the lateral flow strip. Results were read after four (>20 µg kg -1 ), eight (10-20 µg kg -1 ), and 1 16 (4-10 µg kg -1 ) min. The LOQ was four µg aflatoxin kg -1 sample. 2 3

Analysis of ochratoxin A 4

Ground rice samples (50 g) were transferred to glass bottles (500 ml) and mixed with 200 ml 5 of 60 % acetonitrile (HPLC grade) supplemented with NaHCO 3 (0,4 %) on a shaking table for 6 30 min. The samples were then filtered (Munktell V150) and four ml of the sample was 7 mixed with 50 ml 0.01 M PBS (Merck) and further transferred to an immunoaffinity column 8

Ochraprep 50 (R. Biopharm Rhône Ltd.). The column was washed with 20 ml 0.01 M PBS-9 buffer (Merck) and then eluted with three ml methanol:acetic acid (49:1). The elute was 10 nitrogen-evaporated to dryness before re-suspension in 500 µl mobile phase 11 (acetonitrile:water:acetic acid; 50:50:1) and injected (20 µl) on the HPLC column (Spherisorb 12 3 µm, ODS 50 x 4.6 mm) equipped with a fluorescence detector (excitation 333 nm, emission 13 460 nm). The flow was 1.0 ml min -1 and column temperature 30°C. The LOQ for ochratoxin 14 A was 0.1 µg kg -1 . 15 16

Intake estimations 17

The daily intake of aflatoxins from a specific type of food depends on the concentration in the 18 food and the amount of food consumed. To estimate the concentration of aflatoxin in rice 19 mean values from this study were used as no additional data were available. Three different 20 mean values were calculated (mean value for the total number of rice samples, for the basmati 21 rice samples, and for the rice samples purchased in stores with products of mainly ethnical 22 origin). Negative samples were included as half the LOQ, i.e. 0.05 µg kg -1 . This treatment of 23 negative data may result in an overestimation of mycotoxin in rice. The consumption data for rice was based on a consumption study of Swedish consumers 1 performed in 1997-98 [START_REF] Becker | Kostvanor och näringsintag i Sverige -Metod och 18 resultatanalys[END_REF]. This study included adult consumers 2 between 18 and 74 years old from approximately 2000 house holds. To compensate for the 3 100% increase in rice consumption for Swedish consumers reported by FAOSTAT, FAO 4 (Food and Agriculture Organisation of the United Nations) Statistics Division 2007, intake 5 estimations were also performed based on a 100% increase of the consumption data from 6 1997-98 [START_REF] Becker | Kostvanor och näringsintag i Sverige -Metod och 18 resultatanalys[END_REF]. Consumers that originate from other countries than 7

Sweden, such as India, Pakistan, Korea etc., are likely to consume rice more than 1-2 times 8 per week as estimated for the mean consumer in the consumption survey Riksmaten 1997-98 9 [START_REF] Becker | Kostvanor och näringsintag i Sverige -Metod och 18 resultatanalys[END_REF]. These consumers are more likely to consume rice twice a day, 10 seven days a week. Therefore, an estimated intake was also performed on this group of high-11 consumers. Aflatoxin intake estimations are based on results from analysis of aflatoxin in 12 dried rice. However, recent research by Park et al. (2005[START_REF] Park | Effect of pressure cooking on aflatoxin B 1 in rice[END_REF] have shown that the aflatoxin 13 content in rice may be reduced by approximately 30% during traditional cooking and 80% 14 during pressure cooking. It is therefore more likely that the intake is lower in ready-to-eat rice 15 than in dried rice due to the 30% aflatoxin reduction during cooking (Park et al. 2005). [START_REF] Hedayati | Aspergillus flavus: human 17 pathogen, allergen and mycotoxin producer[END_REF] Pressure cooking is rarely used in Swedish homes. 17 18

Results & Discussion 19

Rice samples 20 A total of 99 rice samples were purchased from retail outlets. The majority of rice samples 21 were taken from local shops with products of mainly ethnical origin (56 samples) and samples 22 from larger retail stores (35 samples), but samples were also taken from organic (five 23 samples) and health food shops (three samples). The majority of samples were basmati rice 24 but also rice with higher fibre content, i.e. brown rice or whole-grain rice, jasmine rice and 25 long-grain rice were sampled (Table 1). The group of rice samples with higher fibre content 1 included long-grain rice (nine samples), basmati rice (three samples), jasmine rice (two 2 samples), and short-grain rice (one sample). The group of organic rice samples included 3 basmati rice (three samples), brown basmati rice (one sample), brown short-grain rice (one 4 sample), and brown long-grain rice (four samples). 5 6

Mycotoxin analysis 7

All samples were analysed for aflatoxin B 1 , B 2 , G 1 , G 2 , and ochratoxin A with HPLC. 8

Aflatoxin B 1 was detected in 71% of the basmati rice samples and in 20% of the jasmine rice 9 samples (brown rice included; Table 1). Aflatoxin was not detected in short-or long-grain 10 rice (brown rice included) nor in any of the organically grown rice samples (Table 1). 11

Aflatoxin G 1 , G 2 , and ochratoxin A were not detected in any of the rice samples. 12 13 Seventy-six samples were also analysed for the total amount of aflatoxins with the rapid 14 lateral flow method RIDA®QUICK Aflatoxin (R-Biopharm AG). The RIDA®QUICK 15 method has previously been evaluated for aflatoxin detection in food items such as grain, soy, 16 flour, nuts, and dried fruit and was included in this study to evaluate weather it can also be 17 used as a screening method for aflatoxin in rice exceeding more than four µg kg -1 . Of the 76 18 samples, 11 samples contained more than four µg aflatoxin kg -1 (HPLC, LOQ 0.1 µg kg -1 ). 19

These were identified with RIDA®QUICK as positive (LOQ 4 µg kg -1 ; Figure 1) showing 20 that no positive samples were identified as false negatives. Only one sample containing 0.5 µg 21 aflatoxin kg -1 was falsely identified as above 4 µg aflatoxin kg -1 by RIDA®QUICK. 22

23

The RIDA®QUICK method gave a rough indication of the level of total aflatoxin in the rice 24 samples (Figure 1). The method RIDA®QUICK Aflatoxin may therefore be used as a rapid 25 screening to identify rice samples that contain levels exceeding the European maximum limit 1 for the sum of aflatoxins. However, samples that contain between 2 and 4 µg aflatoxin B 1 kg - 2 1 , thus exceeding the European maximum limit for aflatoxin B 1 of 2µg kg -1 , will be missed in 3 such a screening. 4 5 Fungal species isolated from the rice samples 6 Fungal contamination analysis was performed by dilution plating of the ground rice samples. 7

The level of fungal contamination were generally low (log one to four cfu g -1 rice) and no 8 significant difference could be found between the different types or origin of rice with regard 9 to contamination level and fungal species isolated. Species of Aspergillus were the most 10 frequently isolated genus but also species of Penicillium and Eurotium were common (Table 11 2). The most frequently isolated species was A. candidus, being present in 50% of the 99 rice 12 samples and completely dominating in 22% of the samples (including samples of basmati 13 rice, jasmine rice, long-grain rice, and brown rice). A. candidus has previously been reported 14 to contaminate rice (Park et al. 2005) and is known to contaminate other cereals (Samson et 15 al. 2004). However, this species is not known to produce any regulated mycotoxins. Less 16 frequently found species were A. flavus (21%), A. fumigatus (13%), A. niger (5%), P. 17 polonicum (2%), and P. chrysogenum (2%). Occasional colonies belonging to the genus 18 Wallemia, Cladosporium, Epicoccum, Alternaria, Trichotecium or to the taxonomical group 19 of Zygomycetes were also isolated. A. fumigatus is commonly isolated from food and indoor 20 environments. It does not produce any regulated toxins but may pose an environmental risk to 21 the workers in the rice industry due to its pathogenicity and allergenic properties (Hedayati et 22 al. 2007). 23 No correlation was found between the occurrence of A. flavus and the level of aflatoxin 1 detected in the samples. This is likely due to the long storage period under dry conditions, 2 which eventually leads to the reduction of viable mould count compared to the initial value. In 3 addition to aflatoxin B 1 and B 2 , which are produced by both A. flavus and A. parasiticus, A. 4 parasiticus is also able to produce aflatoxin G 1 and G 2 [START_REF] Frisvad | Important mycotoxins and the fungi which 14 produce them[END_REF]. A. parasiticus 5 was not isolated from the rice samples, nor were aflatoxin G 1 and G 2 detected. This may 6 indicate that A. flavus is the main producer of aflatoxin in rice. 7 8 A. niger was isolated from five samples, however, few A. niger are capable of producing 9 ochratoxin A and the low incidence of potential ochratoxin-producing species correlated well 10 with the absence of ochratoxin A. 11

12

The low level of mould infection as well as the low water-activity (a w ) of the rice samples 13 (0.50±0.08) may indicate that mould growth and mycotoxin production took place before 14 drying and dehulling of the rice kernels. 15 16

Intake and consumer health 17

For genotoxic compounds, such as aflatoxins, the TDI cannot be used as a safety factor as the 18 intake of such substances should be kept as low as reasonably possible. However, a 19 provisional maximum TDI of one ng aflatoxin kg -1 bw day -1 may be used as a guiding value 20 in the risk assessment of aflatoxin from food (JECFA 1998). Rough estimations of the intake 21 of aflatoxin from rice for Swedish consumers showed that high-consumers (defined as the 22 95th percentile), of both women and men, that consume mainly basmati rice and rice bought 23 in shops with products of ethnical origin were in the range of 1.1-2.0 ng aflatoxin kg -1 bw day - 24 1 (Table 3) and that the group of consumers that base all their meals on rice were in the range 25 of 2.3-3.7 ng aflatoxin kg -1 bw day -1 (Table 3). The reduction of aflatoxin during cooking 1 should be considered when discussing aflatoxin intake from rice. Park et al. (2005[START_REF] Park | Effect of pressure cooking on aflatoxin B 1 in rice[END_REF]) 2 showed that the aflatoxin content was reduced by 30% during cooking. The most extreme 3 group of rice consumers is therefore likely to have an intake closer to 1.6-2.6 ng aflatoxin kg -1 4 bw day -1 , which is still above the guiding value of one ng aflatoxin kg -1 bw day -1 , rather than 5 2.3-3.7 ng aflatoxin kg -1 bw day -1 . Aflatoxin intake from more traditional sources of aflatoxin 6 such as nuts, spices, milk, dried fruit and figs have previously been estimated for Swedish 7 consumers to 0.8 ng aflatoxin kg -1 bw day -1 (Thuvander et al. 2001). This study did not 8 include rice. 9 10 To reduce the aflatoxin content in rice, preventive measures such as improved farming 11 systems, post-harvest handling, proper drying and storage must be taken. In addition, 12 intensified internal and official control may be necessary to improve the quality of the rice 13 available on the market. 14 15

Concluding remarks 16

The main sources of aflatoxin from food are traditionally considered to be nuts, maize and 17 dried fruit. However, this study showed that 71% of the basmati rice and 20% of the jasmine 18 rice samples analysed contained detectable levels of aflatoxin. Only few samples of other 19 types of rice were included in this survey and conclusions on the aflatoxin prevalence in other 20 rice than basmati and jasmine rice cannot be drawn. Aflatoxin was not only a common 21 contaminant but also occurred in levels exceeding the EC limit values in 13% of the basmati 22 samples and 20% of the jasmine rice samples. The mould content was generally very low in 23 stored rice and no correlation was found with the content of aflatoxin. Intake estimations 24

  , Sri Lanka (Bandara et al. 5 1991), the Philippines (Sales and Yoshizawa 2005), the United Arab Emirates (Osman et al. 6 1999), India (Toteja et al. 2006), and Côte d'Ivoire (Sangare-Tigori et al. 2006). Rice is a 7 major cereal crop consumed by the European population, yet, reports on the actual mycotoxin 8 content in rice is limited. This was also pointed out by the UK Food Standard Agency, which 9 performed a survey of mycotoxins in rice in 2002 (FSA, 2002). Of the 100 rice samples 10 analysed, none of the samples (including 18 samples of basmati rice) contained mycotoxins 11 over the EC regulated limits. 12 13 Aflatoxins are genotoxic and carcinogenic substances that may induce liver cancer in both 14 humans and animals. Aflatoxins are produced by several species of Aspergillus including A. 15 flavus (aflatoxin B 1 and B 2 ) and A. parasiticus (B 1 , B 2 , G 1 , and G 2 ). A toxicological 16 evaluation of aflatoxins was made by the Joint FAO/WHO Expert Committee of Food 17 Additives (JECFA) in 1998 (WHO 1998) and by EFSA in 2007 (CONTAM Panel 2007), 18

6

  Ochratoxin A is a nephrotoxic agent with immunotoxic, neurotoxic and teratogenic effects at 1 higher dose levels. The toxin is produced by representatives of the two genera Penicillium and 2 Aspergillus genera. Ochratoxin A was evaluated by JECFA in 2001 (WHO 2001) and by 3 EFSA in 2006 (CONTAM Panel 2006). The PTWI for ochratoxin A was set to 100 ng per kg 4 bw by JECFA (WHO 2001) and to 120 ng per kg bw by EFSA (CONTAM Panel 2006). 5 Within the European Union, contamination of mycotoxins in food are regulated by the EC 7 regulation 1881/2006, which regulates the maximum level (ML) of mycotoxins allowed in 8 food, and the EC Regulation 401/2006, which regulates the sampling for mycotoxin control of 9

7

  Colonies of P. verrucosum was identified directly on DG18 by their bright orange-yellow 1 reverse whereas other Penicillium-species were re-inoculated on CREA, Czapek, YES, and 2 CYA and incubated at 25°C for 7 days for further morphological and biochemical 3 characterization (Samson et al. 2004). Colonies of Aspergillus were identified by 4 morphological characteristics (Samson et al. 2004) and potential colonies of A. flavus and A. 5 parasiticus were confirmed on Aspergillus Flavus Parasiticus Agar (AFPA; Pitt et al. 1983). 6 Analysis of aflatoxin B 1 , B 2 , G 1 and G 2 8

3 4 FoodFigure 1 .

 341 Figure captions1

  Fredlund et al. "Moulds and mycotoxins…"Table 3 from 1997 to 2005 [FAOSTAT, FAO (Food and Agriculture Organisation of the United Nations) Statistics Division 2007]. Fredlund et al. "Moulds and mycotoxins... " mc.manuscriptcentral.com/tfac Email: fac@tandf.co.uk Food Additives and Contaminants

Table 1 Table 1 .

 11 Number of samples and aflatoxin (µg kg -1 ) content for each type of rice (n= 99 rice samples). The number of samples exceeding European maximum limits and the number of samples below LOQ (0.1 µg kg -1 ) are also given. The analytical uncertainty was 0.4 µg aflatoxin kg -1 rice (nd=not detected). aflatoxin G 1 and G 2 were not detected in any of the rice samples and are therefore not included in Table1; b rice with higher fibre content excluded; c rice with higher fibre content included; d one jasmin rice, one basmati rice.

	Aflatoxin a (µg kg -1 ),
	interval for positive samples (mean)

a

Table 2 Table 2 .

 22 The most common mould species isolated from the rice samples.Tabell 3. Intake estimations of aflatoxin from rice by different consumer groups (bw=bodyweight). mean value from HPLC analysis of aflatoxin B 1 , B 2 , G 1 , and G 2 . Samples with levels <LOQ were set to 0.05 µg kg -1 , i.e. half the LOQ; b consumption data based on results from Riksmaten 98-97 (Becker and Pearson 1997); c based on consumption data of the 95th percentile; d based on consumption of rice (60 g dried rice per meal) twice a day, seven days a week; e consumption data based on a 100% increase of rice consumption in Sweden

		Food Additives and Contaminants	Page 22 of 25
	Mould species	Isolated from	Level of infection (log cfu g -1 )
		number of samples
		(n=99)
	Aspergillus spp.	63
	A. candidus	49	Dominating flora in 22 rice samples from log 1-1.5 to 3-3.5 cfu g -1
	A. flavus	21	Mostly low levels (log 1-1.5 cfu g -1 ) but dominating in one sample
			(log 3 cfu g -1 )
	A. fumigatus	13	Mostly low levels (log 1-1.5 cfu g -1 ) but dominating in one sample
			(log 2 cfu g -1 )
	A. niger	5	Low levels (log 1-1.5 cfu g -1 )
	Penicillium spp.	38	Mostly low levels (log 1-1.5 cfu g -1 )
	P. polonicum	2	Dominating flora in two samples (log 4.3 and 2.2 cfu g -1 )
	P. chrysogenom 2	Low levels (log 1-1.5 cfu g -1 )
	Eurotium spp.	28	Mostly low levels (log 1-2 cfu g -1 )

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Eva Askar

Giuliano Ramadori

Prof. Dr. rer. nat Sabine Mihm
email: smihm@med.uni-goettingen@de

Toll-like receptor 7 rs179008/Gln11Leu Gene Variants in Chronic Hepatitis C Virus Infection

Keywords: Toll-like receptor 7 (TLR7), single-stranded RNA (ssRNA), hepatitis Toll-like receptor7 (TLR7), single-stranded RNA (ssRNA), hepatitis C 46 virus

24

Hepatitis C virus (HCV) infection affects an estimated 3% of the world's population. The 25 natural outcome of infection and the natural course of disease are highly variable. Sensing of 26 viral single-stranded RNA (ssRNA) by Toll-like receptor 7 (TLR7) is likely involved in early 27 pathogen detection and host response to viral infections. This study analyzed epidemiological 28 and clinical data from 136 patients with HCV infection with regard to rs179008/Gln11Leu, a 29 non-synonymous polymorphism within exon 3 of the X-linked TLR7 gene, the variant allele 30 of which is suggested to code for a functionally impaired protein. Allele-specific transcript 31 quantification (ASTQ) analyses in heterozygous females revealed individual skewed 32 mosaicism in peripheral blood mononuclear cells (PBMCs). Thus, analyses were restricted to 33 homo-and hemizygous individuals. Among the clinical and histological parameters studied, 34 the variant allele T was found to be solely associated with the presence of portal lymphoid 35 aggregates. Whereas hepatic viral load and expression of genes known to be induced in 36 chronic HCV infection were not found to differ in patients with wildtype or variant TLR7 37 rs179008 genotype, significant lower gene expression of interleukin-29 (IL-29)/lambda 1 38 interferon (IFN-λ 1 ) and both of its receptor subunits was found for T homo-and hemizygous 39 patients. Irrespective of the minor differences in disease phenotype including hepatic viral 40 load, natural and alpha interferon (IFN-α)-mediated outcome of infection, and disease activity 41 and progression, the significant differences in hepatic IL-29/IFN-λ 1 and IFN-λ receptor gene 42 expression between TLR7 rs179008 T and A allele patients might have implications for 43 responsiveness to future IFN-λ-based approaches. 44 45

INTRODUCTION 48 49

Chronic infection caused by hepatitis C virus (HCV), an enveloped single-stranded RNA 50 (ssRNA) virus [START_REF] Choo | Isolation of a cDNA clone derived from a blood-borne non-A, non-B viral hepatitis genome[END_REF], develops in 70%-80% of patients [START_REF] Schwabe | Toll-like receptor signaling in the liver[END_REF]. 51

Patients are at a high risk of developing severe disease as liver cirrhosis and hepatocellular 52 carcinoma [START_REF] Schwabe | Toll-like receptor signaling in the liver[END_REF]. Toll-like receptors (TLRs) play a critical role in the innate 53 immune sensing of the invasion of pathogenic microorganisms [START_REF] Akira | Toll-like receptor signalling[END_REF]. 54

Alpha interferon (IFN-α) is an important antiviral cytokine produced principally by 55 plasmacytoid dentritic cells (pDCs), which circulate in the blood at low frequency and even 56 lower in chronic hepatitis C [START_REF] Kanto | Reduced numbers and impaired ability of myeloid and plasmacytoid dendritic cells to polarize T helper cells in chronic hepatitis C virus infection[END_REF], through the stimulation of TLR7 and TLR9 57 [Hornung et al., 2005;Ito et al., 2005]. TLR7 senses unmethylated viral ssRNA [Diebold et 58 al., 2004;[START_REF] Heil | Species-specific recognition of single-stranded RNA via toll-like receptor 7 and 8[END_REF]. The expression of TLR7 in humans is mainly confined to the 59 endosome-lysosome membrane of pDCs (including hepatic pDCs), hepatic natural killer cells 60 [START_REF] Seki | Toll-like receptors and adaptor molecules in liver disease: update[END_REF], and B lymphocytes [START_REF] Hornung | Quantitative expression of toll-like receptor 1-10 mRNA in cellular subsets of human peripheral blood mononuclear cells and sensitivity to CpG oligodeoxynucleotides[END_REF]. When the virus or virus-61 infected apoptotic cells are taken up by phagocytes, viral RNA is released in the highly 62 acidified phagolysosome by degradation enzymes, leading to ssRNA release and recognition 63 by TLR7. Upon TLR7 stimulation, a complex cascade is formed, starting with myeloid 64 differentiation factor 88 (MyD88) and ending with the production of IFN-α/IFN-inducible 65 genes and proinflammatory cytokines through the phosphorylation of interferon regulatory 66 factor 7 and the liberation of nuclear factor-κB, respectively (reviewed in [START_REF] Akira | Pathogen recognition and innate immunity[END_REF]67 Schwabe et al., 2006;[START_REF] Seki | Toll-like receptors and adaptor molecules in liver disease: update[END_REF]). 68

Macrophages overexpressing HCV nonstructural proteins NS3, NS3/4A, NS4B, or NS5A 69 show a strong suppression of TLR-MyD88-dependent signaling pathway. NS5A interacts 70 with MyD88 to prevent cytokine production, such as interleukin-1 (IL-1), IL-6, and beta 71 interferon (IFN-β) in response to TLR7 ligands [START_REF] Abe | Hepatitis C virus nonstructural protein 5A modulates the toll-like receptor-MyD88-dependent signaling pathway in macrophage cell lines[END_REF]. In addition, pDCs from 72 HCV patients have reduced deviation marker (HLA-DR) and IFN-α expression in response to 73 F o r P e e r R e v i e w 4 TLR7 ligand, which is associated with an impaired activation of naive CD4 T cells [Yonkers 74 et al., 2007]. TLR7 has been recently of particular medicinal chemistry interest because its 75 small molecule ligands may serve as immune stimulants by enhancing endogenous IFN-α 76 production and thus, they may complement IFN-α therapy of chronic HCV infection, 77 especially in IFN-α-resistant patients. Horsmans and colleagues have applied a well-tolerated 78 intravenous isatoribine treatment with only few mild to moderate adverse events for one week 79 to chronic hepatitis C patients. It has resulted in viral load reduction regardless of the patients' 80 HCV genotype, an induction of the antiviral immunity marker 2'-, 5'-oligoadenylate 81 synthetase, and an increase in the levels of the gamma interferon (IFN-γ)-inducible protein 10 82 (IP-10) and neopterin, a marker of macrophage activation [START_REF] Horsmans | Isatoribine, an agonist of TLR7, reduces plasma virus concentration in chronic hepatitis C infection[END_REF]. Moreover, 83 a high-affinity ligand of TLR7, namely SM360320, has been found to inhibit HCV replication 84 both through type I IFN production by leukocytes, and direct activation of antiviral 85 mechanisms in infected hepatocytes [START_REF] Lee | Activation of anti-hepatitis C virus responses via Toll-like receptor 7[END_REF]. 86 TLR7 gene is located on the X-chromosome and contains three exons [START_REF] Du | Three novel mammalian toll-like receptors: gene structure, expression, and evolution[END_REF]. 87

Recently, the leucine (Leu) variant encoded by the T allele of the nonsynonymous single 88 nucleotide polymorphism (SNP) rs179008, which is located within TLR7 exon 3 and leads to 89 the replacement of the wild allele A-encoded glutamine (Gln) at codon 11 in the protein 90 (Gln11Leu), has been correlated with higher susceptibility to HCV infection and less chances 91 of response to an IFN-α-based therapy in chronic HCV-infected females [START_REF] Schott | Association of TLR7 single nucleotide polymorphisms with chronic HCV-infection and response to interferon-a-based therapy[END_REF]. 92 Moreover, this variant has been associated with higher viral loads, accelerated progression to 93 advanced immune suppression in human immune deficiency virus (HIV) infection, increased 94 susceptibility to HIV-1 in women, and decreased IFN-α production after stimulation of 95 healthy peripheral blood mononuclear cells (PBMCs) with the TLR7 ligand Imiquimod [Oh et 96 al., 2009]. 97

Taking the X-linked location into account, the present study aimed to investigate the 98 correlation between TLR7 rs179008 genotype and disease parameters in chronic hepatitis C, 99 Center Goettingen (UMG), Germany, between 1993 and2006, 136 with complete data sets 108 (mean age 45.0±12.3, median 44 years, 60 females) were enrolled in epidemiological, 109 biochemical and histological analyses. Chronic infection was proven by detection of HCV-110 specific antibodies and HCV RNA in the patients' sera using a highly sensitive nested PCR over a period of at least six months as described [Mihm et al., 1996a] . Before the start of 112 therapy, liver biopsy procedures were performed and liver disease was confirmed in the 113 course of a defined histological evaluation as described below. Biochemical liver disease 114 parameters, i.e. serum activities of aspartate aminotransferase (AST), alanine 115 aminotransferase (ALT), and gamma-glutamyl transferase (γ-GT) were recorded in parallel. 116

Patients with concomitant non-C viral infections and those with continued alcohol or other 117 drug abuse were excluded. 118 A total of 55 patients (mean age 46.1±12.3, median 45 years, 25 females) were treated with 119 IFN-α 2a (Roferon A; Hoffman-La Roche, Basel, Switzerland) at an initial dose of 6x10 6 IU 3x 120 per week for at least 4 months (mean, 7.7 months; range 4-12 months). Depending on well 121 being and response parameters, both dose and duration were adapted individually. Initial 122 virological response to therapy, which is defined as the elimination of HCV RNA below the 123 limit of detectability during the first 4 months for a period of a least three consecutive months, 124 was analyzed with regard to TLR7 rs179008 genotype. 125

Another group of 44 patients with self-limited HCV infection (mean age 37.0±10.3, median 126 36 years, 14 females) was studied in addition. Spontaneous elimination was assured by the 127 presence of anti-HCV antibodies in the absence of detectable amounts of HCV RNA (for 128 HCV genotyping was performed using the Innolipa HCV II line probe assay (Innogenetics,135 Ghent,Belgium). 136 137

Histological Evaluation 138

Before the start of therapy, liver biopsies were taken from patients for histological evaluation. 139

In brief, sections (5-10 µm) from formalin-fixed and paraffin-embedded liver biopsies were 140 stained with hematoxylin-eosin, trichrome, and Prussian blue. According to Desmet and 141 colleagues, necroinflammatory activity (grading, score 1 to 3) and architectural alterations 142 (staging, score 0 to 4) were scored separately [START_REF] Desmet | Classification of chronic hepatitis: diagnosis, grading and staging[END_REF]. Other lesions typical of 143 hepatitis C such as the degree of steatosis (score 0 to 3), the presence or absence of portal 144 lymphoid aggregates, and the presence or absence of bile duct damage were studied 145 additionally as previously described [START_REF] Mihm | Analysis of histopathological manifestations of chronic hepatitis C virus infection with respect to virus genotype[END_REF]. 146 147

Preparation of PBMCs 148

PBMCs from approximately 30 ml of heparinized peripheral blood samples were prepared by 149 Ficoll density centrifugation using guanidinium isothiocyanate as described [START_REF] Boyum | Separation of lymphocytes, granulocytes, and monocytes from human blood using iodinated density gradient media[END_REF]. 150 151 Isolation of Genomic DNA (gDNA) and Total Cellular RNA 152 gDNA was purified from PBMCs or from 2 ml samples of serum using the QIAamp DNA 153 Mini or Midi Kits, respectively, following the blood and body fluid spin protocol (Qiagen, 154 Hilden, Germany). The concentration and the purity of the DNA isolated from PBMCs were 155 determined photometrically by reading the absorbance levels at 260 nm and 280 nm. The 156 integrity of gDNA was ascertained through electrophoresis using a 0.6% agarose gel. 157

Total cellular RNA was prepared from available freshly isolated PBMCs and homogenized 158 liver tissue samples by CsCl density gradient ultracentrifugation essentially as described 159 [Mihm et al., 1996b]. 160 161

Reverse Transcription 162

To get complementary DNA (cDNA), an amount of 1 µg of total cellular RNA was reverse 163 transcribed by using random hexamers (6 µM) for priming as described previously [Mihm et 164 al., 1996b]. Competitive RT-PCR was applied to quantify mRNA transcripts of HCV, the IFN-α/ß 181 inducible antiviral myxovirus resistance protein-1 gene (MxA), IFN-α and, as a reference 182 gene, albumin, essentially as described [START_REF] Mihm | Interferon type I gene expression in chronic hepatitis C[END_REF], and transcripts of IP-10, the gene 183 encoding IFN-α/ß-inducible p44 [START_REF] Patzwahl | Enhanced expression of interferonregulated genes in the liver of patients with chronic hepatitis C virus infection: detection by suppression-subtractive hybridization[END_REF], IFN-γ [Mihm et al., 1996b]. The 184 relative number of interleukin-29 (IL-29)/lamda 1 interferon (IFN-λ 1 ), IFN-λ receptor subunits 185 (IL-10Rß and IL28Rα), and IFN-α/ß receptor 2 (IFNAR2) mRNA transcripts was calculated 186 by real-time RT-PCR using the sequence detection system ABI prism 7000 following the 187 supplier' instructions (Applied Biosystems, Darmstadt, Germany) as described [Doyle et al., 188 2006;[START_REF] Mihm | Interferon type I gene expression in chronic hepatitis C[END_REF]

202

Genotyping of HCV with Regard to TLR7 rs179008 203

A total of 136 patients with chronic hepatitis C (60 females/76 males) were genotyped for the 204 bi-allelic SNP rs179008/Gln11Leu within exon 3 of the X-linked TLR7 gene (Table I). 205 Genotype distribution in women followed Hardy-Weinberg equilibrium (P = 0.318). The 206 frequency of the minor allele (MAF) was found to be close to that given for Caucasians in 207 public data bases and to be similar in females and males (P = 0.175). 208

ASTQ 210

Due to the X-linked location of TLR7, in females, the random inactivation of one X 211 chromosome leads to cellular mosaicism with two populations of cells differing in the 212 parental origin of the active X [START_REF] Fish | The X-files in immunity: sex-based differences predispose immune responses[END_REF]. In heterozygous females, the presence of two 213 kinds of cells might set up a competition between them [START_REF] Migeon | The role of X inactivation and cellular mosaicism in women's health and sex-specific diseases[END_REF]. Non-random 214 inactivation (skewing) has been implicated for discrete cell populations, e.g. dendritic cells 215 [START_REF] Fish | The X-files in immunity: sex-based differences predispose immune responses[END_REF][START_REF] Migeon | The role of X inactivation and cellular mosaicism in women's health and sex-specific diseases[END_REF]. In order to find out whether heterozygous females should be 216 assigned either to the wildtype, the variant genotype or to be considered as a separate, i.e. true 217 heterozygous group, ASTQ was performed to quantify the relative proportion of A and T 218 allele transcript variants in RNA preparation from freshly isolated PBMCs (Fig. 1). gDNA, a 219 natural source of equal amounts of A and T sequences served as a control. RNA preparations 220 from 3 heterozygous women were found to contain nearly equal numbers of both alleles' 221 transcripts, whereas material from 4 heterozygous females was found to contain an excess of 222 either A or T, 2 women each, respectively (Fig. 1A). 223

Because of the limited amount of available samples, 5 heterozygous females were further 224 identified among a total of 42 healthy blood donors. ASTQ with these 5 samples yielded 225 comparable results (equal expression in 2 samples, an excess of A in 3 samples) (data not 226 shown). 227 Demographic analysis revealed a slight trend of the variant allele carriers to be older than 232 those who carry the wild type allele (47.8±15.0 vs. 42.5±11.2, respectively, P = 0.069). This 233 difference, however, was clearer in males (P = 0.083) (Table I). 234

As expected for a European population, most patients (80.1%) were infected with HCV type-1 235 (including subtypes 1b, 1a and 1a+1b), while 19.9% were infected with HCV non-1 236 (including mainly subtypes 3a, and a minority of 2a and 2b). No significant difference among 237 the distribution of HCV types (or subtypes, data not shown) infections according to patients' 238 TLR7 rs179008 genotype was found (Table I). 239 AST, ALT, and γ-GT serum activities were recorded as indicators of liver injury in chronic 240 hepatitis C. Although T homo-and hemizygotes seemed to have higher AST and ALT but 241 lower γ-GT serum levels than the A counterparts, the proportion of A or T homo-and 242 hemizygote patients among those with markedly elevated transaminase activities, i.e. greater 243 than 2-fold of the upper normal limit, was not found to be significantly different (Table I). 244 245

Genotyping of Individuals with Self-limited HCV Infection 246

The T allele is suggested to confer enhanced susceptibility to chronic HCV infection as MAF 247 has been found to be significantly lower in healthy individuals [START_REF] Schott | Association of TLR7 single nucleotide polymorphisms with chronic HCV-infection and response to interferon-a-based therapy[END_REF]. To 248 address the question whether this enhanced susceptibility is due to a higher incidence of 249 infection or to an impaired capacity to self-eliminate the virus, TLR7 rs179008 genotype 250 distribution of the chronic hepatitis C patients was compared to a group of 44 patients with 251 self-limited HCV infection [START_REF] Wietzke-Braun | Spontaneous elimination of hepatitis C virus infection: a retrospective study on demographic, clinical, and serological correlates[END_REF]. The proportion of T homo-and 252 hemizygous individuals or even of heterozygous female patients was not found to be 253 12 significantly higher in chronic HCV patients (15.4% and 14.7%, respectively) in 254 comparison to patients with self-limited HCV infection (11.4% and 13.6%, respectively) 255 (Table I). 256 257

Hepatitis C Histological Manifestations 258

To investigate whether a functionally impaired TLR7 protein might be related to histological 259 features of chronic hepatitis C, liver biopsy specimens were taken and evaluated histologically 260 with regard to hepatitis activity, fibrosis progression, steatosis stage, portal lymphoid 261 aggregates and bile duct damage (Table II). TLR7 rs179008 genotype distribution showed no 262 association with the first three characteristics. Nevertheless, a higher frequency of the minor 263 allele T among patients with portal lymphoid aggregates was observed (P = 0.013), this 264 difference, however, was only valid in males (P = 0.032) (Table II). Whereas only 30.5% of A 265 carriers were found to have bile duct damage, 52.4% of T carriers did have the lesion (P = 266 0.051) (Table II). 267 Noteworthy, it was reported recently that patients homozygous for the variant allele T of the 268 endotoxin receptor CD14 SNP rs2569190/C-159T have more frequent portal lymphoid 269 aggregates than C carriers [START_REF] Askar | Endotoxin receptor CD14 gene variants and histological features in chronic hepatitis C virus infection[END_REF]. Interestingly, analyzing CD14 C-159T 270 genotype separately in the two genders revealed that the significant association was only 271 confined to males (P = 0.004) (unpublished observation). 272 273

Response to an IFN-α 2a Monotherapy 274

A total of 55 patients were treated with IFN-α2a as described in the patients and methods 275 section. The initial virological response to therapy is defined as the elimination of HCV-RNA 276 below the limit of detectability during the first 4 months for a period of at least 3 consecutive 277 months. Some patients kept undetectable viral RNA levels till completing therapy i.e. end-of-278 treatment response, or even for a period of at least continuous six months after the last dose of 279 13 IFN-α 2a , i.e. sustained virological response. While 22 (63%) of the A carriers responded, at 280 least initially, to the therapy, only 3 (30%) of the T counterparts (and 30% of the heterozygous 281 females) were responders (P = 0.069) (Table III). Similar results were found considering a 282 larger cohort of 145 patients treated with an IFN-α-based therapy (data not shown). 283 284

Hepatic Gene Expression 285

To examine whether a functionally impaired TLR7 protein might be related to hepatic gene 286 expression in chronic HCV infection, innate immunity gene transcripts were quantified in 287 freshly derived liver tissue samples. Data were related both to GAPDH and to albumin as 288 reference genes. When A homo-and hemizygous samples were compared to T homo-and 289 hemizygous samples, no significant difference was found with regard to the amount of hepatic 290 viral RNA (Fig. 2A), or the genes that have been shown to be enhanced in chronic HCV 291 infection when compared to healthy liver tissue as IP-10, p44, MxA, or IFN-γ [Mihm et al., 292 2004] (Fig. 2B). In contrast, T homo-and hemizygotes were found to express significant 293 lower amounts of IL-29/IFN-λ 1 (P = 0.015), IL-10 receptor beta (IL-10Rß) (P = 0.001) and 294 IL-28 receptor alpha (IL-28Rα) (P = 0.003), which constitute the two components of IFN-λ 295 heterodimeric receptor, as well as lower amounts of IFN-α and IFNAR 2 (Fig. 2B). 296 The TLR7 rs179008/Gln11Leu is located in the signal sequence of TLR7, adjacent to the 298 typical basic residues in the N-terminal part of this sequence. Signal peptide degeneracy 299 modulates posttranslational modification, localization, quantity and thus the functionality of 300 the affected protein [START_REF] Hegde | The surprising complexity of signal sequences[END_REF]]. In the studied cohort of 136 chronic 301 hepatitis C patients, no significant association was found between TLR7 rs179008 and any of 302 the epidemiological or biochemical characteristics, inflammation activity (grading) or fibrosis 303 progression (staging). These results are in concordance with previous findings [Schott et al., 304 2007]. Considering the presence of portal lymphoid aggregates, a significant higher frequency 305 of the T hemizygosity was found among male patients. Portal lymphoid aggregates are 306 defined as densely packed collection of small lymphocytes within the portal tract with or 307 without the formation of a germinal center [START_REF] Luo | Clinical significance of portal lymphoid aggregates/follicles in Chinese patients with chronic hepatitis C[END_REF]. Their presence, which is 308 suggested to play an immunological albeit indeterminate role in chronic HCV liver injury 309 similar to the mechanism of autoimmune hepatitis [START_REF] Hino | Analysis of lymphoid follicles in liver of patients with chronic hepatitis C[END_REF][START_REF] Mosnier | The intraportal lymphoid nodule and its environment in chronic active hepatitis C: an immunohistochemical study[END_REF], 310 has been found to be significantly correlated with hepatic inflammatory activity and bile duct 311 damage [START_REF] Askar | Endotoxin receptor CD14 gene variants and histological features in chronic hepatitis C virus infection[END_REF][START_REF] Freni | Focal lymphocytic aggregates in chronic hepatitis C: occurrence, immunohistochemical characterization, and relation to markers of autoimmunity[END_REF][START_REF] Luo | Clinical significance of portal lymphoid aggregates/follicles in Chinese patients with chronic hepatitis C[END_REF]Wong et al., 1996]. 312

Interestingly, the common allele A trended to be low-frequented among patients with bile duct 313 damage (P = 0.051) (Table II). 314

Portal lymphoid aggregates have been recently found to be more frequent among patients 315 homozygote for the T allele of CD14 rs2569190/C-159T [START_REF] Askar | Endotoxin receptor CD14 gene variants and histological features in chronic hepatitis C virus infection[END_REF], this association, 316 however, was valid only in males as it is for TLR7 rs179008 in the present study. Noteworthy, 317 sex itself is neither found to be associated with portal lymphoid aggregates [Askar et al., 318 2009;[START_REF] Luo | Clinical significance of portal lymphoid aggregates/follicles in Chinese patients with chronic hepatitis C[END_REF][START_REF] Mihm | Analysis of histopathological manifestations of chronic hepatitis C virus infection with respect to virus genotype[END_REF], nor with CD14 rs2569190 genotype distribution 319 [START_REF] Askar | Endotoxin receptor CD14 gene variants and histological features in chronic hepatitis C virus infection[END_REF]. Freni et al. described the cellular composition of this manifestation as a 320 core of B cells -which do express TLR7-mixed with many T helper/inducer lymphocytes, and 321 an outer ring prominently formed by T suppressor/cytotoxic lymphocytes, and a rarely 322 15 identifiable germinal center [START_REF] Freni | Focal lymphocytic aggregates in chronic hepatitis C: occurrence, immunohistochemical characterization, and relation to markers of autoimmunity[END_REF]. Taken together, being the first observation 323 of its kind, although its real biological mechanism is still to be found out, replication in an 324 independent larger cohort, and correction of multiple testing are required, TLR7 rs179008 325 might have a role in the formation of portal lymphoid aggregates. 326 TLR7 rs179008 T allele has been found recently to be over-represented and predictive of 327 unfavourable outcome of IFN-α therapy in female patients with chronic HCV infection 328 [START_REF] Schott | Association of TLR7 single nucleotide polymorphisms with chronic HCV-infection and response to interferon-a-based therapy[END_REF]. In the present study, we investigated the distribution of this SNP among 329 patients who spontaneously resolved HCV infection. Comparing the two cohorts with regard 330 to TLR7 rs179008 genotype did not reveal any significant difference (Table I). Unfortunately, 331 this analysis lacks statistical power due to the small cohort of self-limited individuals, yet it 332 did not give any preliminary indication for an altered capacity of resolving the infection 333 spontaneously. Moreover, the observed slight (but still non-significant) trend of the TT/T 334 patients to be non-responders to a mono-or a combined IFN-α-based therapy and to have 335 lower hepatic expression of both IFN-α and IFNAR 2 might confirm -in general -the previous 336 findings of [START_REF] Schott | A Toll-like receptor 7 single nucleotide polymorphism protects from advanced inflammation and fibrosis in male patients with chronic HCV-infection[END_REF] with cautious limitations due to the novel ASTQ analyses in 337 this study that let us omit female heterozygotes from our analyses. 338

The impaired receptor appeared not to affect HCV hepatic viral load, accordingly, no further 339 effect was observed on p44, MxA, IFN-γ or IP-10, genes known to be upregulated in chronic 340 HCV infection in the absence of hepatic type I IFN induction [START_REF] Mihm | Interferon type I gene expression in chronic hepatitis C[END_REF]. The minor 341 allele T, however, was found to be significantly associated with lower hepatic mRNA 342 expression of IL-29/IFN-λ 1 and both IL-10Rß and IL-28Rα (Fig. 2B). This suggests that, 343 rather than being useful in forecasting the current IFN-α-based therapy outcomes, genotyping 344 for TLR7 rs179008 might be predictive for response to IL-29/IFN-λ 1 based therapy 345 approaches [START_REF] Sheppard | IL-28, IL-29 and their class II cytokine receptor IL-28R[END_REF] currently being in phase 2 of clinical development. 346 Three independent genome-wide association studies (GWASs) have reported recently 347 onseveral SNPs in the intergenic region between the genes coding for IL-28A/IFN-λ 2 and IL-348 based therapy [START_REF] Ge | Genetic variation in IL28B predicts hepatitis C treatment-induced viral clearance[END_REF][START_REF] Suppiah | IL28B is associated with response to chronic hepatitis C interferon-alpha and ribavirin therapy[END_REF][START_REF] Tanaka | Genomewide association of IL28B with response to pegylated interferon-alpha and ribavirin therapy for chronic hepatitis C[END_REF] and with 350 spontaneous clearance of HCV [Thomas et al., 2009]. The minor non-responder allele of 351 rs8099917 in IL-28B/IFN-λ 3 gene has been found, moreover, to be associated with lower IL-352 28 mRNA expression in PBMCs [START_REF] Suppiah | IL28B is associated with response to chronic hepatitis C interferon-alpha and ribavirin therapy[END_REF][START_REF] Tanaka | Genomewide association of IL28B with response to pegylated interferon-alpha and ribavirin therapy for chronic hepatitis C[END_REF]. These GWASs 353 have identified as well many SNPs in several genes to be of minor predictability for based therapy outcomes, TLR7, however, not to be among them. 355

Taken together, despite of significant decreased hepatic gene expression in TLR7 rs179008 T 356 compared to A allele patients, that might be due to improper virus sensing and that might 357 affect responsiveness to IL-29/IFN-λ 1 rather than IFN-α, differences in phenotype of disease 358 including hepatic viral load, natural outcome of infection, and disease activity and progression 359 appear to be minor with the exception of the presence of portal lymphoid aggregates in T 360 hemizygous males. Further investigations will elucidate the impact of this polymorphism on 361 responsiveness to endogenous and probably exogenous IFN-λ. 362

  outcome of infection, i.e. chronic versus self-limited course, 100 histological features, and the initial virological response to an IFN-α-based treatment on the 101 one hand, and hepatic expression of innate immunity genes on the other hand. 102 of 144 mainly Caucasian chronic hepatitis C patients who consulted the Liver 106 Unit of the Department of Gastroenterology and Endocrinology at the University Medical 107

  serological description of this cohort please refer to[Wietzke- 129 Braun et al., 2007]). 130The study was approved by the local ethical committee and conformed to the ethical 131 guidelines of the 2000 Declaration of Helsinki. Patients gave their informed consent. 132 133 Determination of HCV Genotype 134

  the TLR7 exon 3-located SNP was performed by the commercially 168 available TaqMan genotyping assay C_2259574_10 (Applied Biosystems, Foster City, CA). 169 Reactions of 10 µl containing 4 ng of PBMCs-derived gDNA-or an aliquot corresponding to 170 6.7 µl serum-were performed in the sequence detection system StepOne-Plus (Applied 171 Biosystems, Darmstadt, Germany) according to the supplier's instructions. 172 173 Allele-Specific Transcript Quantitation (ASTQ) of TLR7 rs179008 Variants 174 Discrimination and quantitation of TLR7 rs179008 transcript variants (A and T) was achieved 175 by applying the commercially available TaqMan genotyping assay C_2259574_10 (Applied 176 Biosystems, Foster City, CA) on cDNA samples (3.2 ng). Heterozygote gDNA and 177 homozygote gDNA and cDNA samples served as controls.

  of these findings we decided to restrict analyses to the comparison between A 228 and T homo-and hemizygous individuals. 229 230 Epidemiological and Biochemical Characteristics 231

  -λ 3 on chromosome 19 to be associated with response outcomes to an IFN-α-349

  all physicians of the Department of Gastroenterology and Endocrinology, 365 UMG, who were involved in patients' care and control for their kind cooperation, PDDr. 366 Stefan Schweyer, Department of Pathology, UMG, for his help in histological evaluation of 367 patients' liver biopsies, and Waltraut Kopp for expert technical assistance. The authors also 368 thank Prof. em. Dr. Gerhard Hunsmann, German Primate Center and the Institute of Virology, 369 Goettingen, Germany, for his interest, supervision and many helpful discussions. E.A. is 370 thankful for her scholarship from Damascus University, Syria. This project was supported by 371 a grant of the Deutsche Forschungsgemeinschaft MI 474/1ASTQ carried out on corresponding gDNA and cDNA samples from 3 representative 377 TLR7 rs179008 heterozygous female patients revealed either nearly equal amounts of both 378 alleles (top), a 2.7-fold excess of the A allele variant (middle), or a 2.6-fold excess of the T 379 allele variant (bottom). Analyses were made in duplicate, therefore the mean ∆ CT is given 380 but one representative amplification plot is shown. 381 (B) ASTQ carried out on one representative pair of corresponding gDNA and cDNA samples 382 from a TLR7 rs179008 homozygote T patient yielded only non-specific signal for the allele A 383 as defined by a 10-fold less fluorescence intensity in the plateau phase at the end of the from liver biopsy specimens taken from homo-and hemizygous patients 392 with chronic hepatitis C was quantified with respect to (A) HCV RNA, (B) p44, MxA, IP-10, 393 IFN-γ and IFN-α in relation to albumin mRNA transcripts by using competitive quantitative 394 RT-PCR, and IL-10Rß, IL-28Rα, IL-29 and IFNAR 2 in relation to GAPDH mRNA by using 395 quantitative real-time RT-PCR assays. Data are given as ratios of the target to reference gene 396 x10 -3 . Medians are indicated by horizontal bars. Levels of significance are given. Similar 397 results were obtained when data were related to β-actin (data not shown). 398

Table I . TLR7 rs179008 Genotype Distribution in Patients with Chronic or Self-limited HCV Infection with Regard to Epidemiological and Biochemical Parameters TLR7 rs179008 genotype

 I

									MAF
					Females		Males
			n	AA	AT	TT	A	T	P
	Patients with chronic HCV infection
	Number (%)		136 34 (56.7)	20 (33.3)	6 (10.0)	61 (80.3)	15 (19.7)	0.175 b	0.267/0.197 a
	Age (Mean±SD)			45.8 ± 10.7 46.6 ± 11.5 50.0 ± 13.6 40.7 ± 11.2 46.9 ± 15.9 0.069 c
	HCV types HCV type-1 n (%) HCV non-1 n (%)		109 28 (53.9) 27 6 (75.0)	19 (36.5) 2 (25.0)	5 (9.6) 0 (0)	47 (82.5) 14 (73.7)	10 (17.5) 5 (26.3)	d 0.449	a 0.279/0.175 0.143/0.263 a
	Biochemical Serum Parameters
	AST (Median, IQR) No. Patients with elevated/normal AST	e	136	23.0, 15-38 18.5, 13.5-31.5 45, 16-80 12/22 6/14 3/3	29, 18-67 36, 18-52 28/33 7/8	0.411 d
	ALT (Median, IQR)			32.5, 22-60 31.5, 20.5-44 58, 22.90 58, 36.5-129 78, 47-92
	No.

Patients with elevated/normal ALT

	e	136	13/21	7/13	3/3	36/25	12/3	0.078 d
	γ-GT (Median, IQR)		19.5, 10-45 19.5, 13.5-41	17, 5-34 39, 23.5-62.5 24, 14-72
	No

. Patients with elevated/normal γ-GT

	e	136	10/24	5/15	2/4	17/44	5/10	0.419

d Patients

with self-limited HCV infection

  χ²-test was applied to compare A with T homo-and hemizygous patients (combined females males).Markedly elevated serum activities of transaminases (> 2-fold of the upper normal limit) were considered. Upper normal limits for females/males, respectively are: 15 U/L/19 U/L for AST, 19 U/L/23 U/L for ALT; and 18 U/L/28 U/L for γ-GT. test was applied to compare TLR7 rs179008 genotype distribution in patients with chronic-and patients with self-limited HCV infection.

	F o r
	P
	Number (%) Age (Mean±SD) MAF is given for females/males, respectively, 44 e 7 (50.0) 39.3 ± 10.4 38.0 ± 13.2 6 (42.9) a e r	1 (7.1) 30	26 (86.7) 36.0 ± 9.9 40.5± 9.7 0.722 4 (13.3) 0.766 f g	0.286/0.133 a
	R
	e
	v i e
	w

b χ²-test was applied to compare MAF between females and males. C Independent samples t-test was applied, the trend was valid only in males (P = 0.083), d e f χ²g Independent samples t-test was applied.

Table II . Histological Manifestations of Chronic Hepatitis C Patients with Regard to TLR7 rs179008 Genotype

 II was applied on A and T homo-and hemizygous patients, to compare mild vs. moderate and severe hepatitis activity, absent, mild vs. moderate and marked fibrosis and cirrhosis, and absent, mild vs. moderate and marked steatosis.

							23
			TLR7 rs179008 genotype
	Histological manifestations	Females n (%)		Males n (%)
		AA	AT	TT	A	T	P a
		Hepatitis activity
	Mild	20 (57.1) 12 (34.3)	3 (8.6)	35 (85.4)	6 (14.6)
	Moderate	13 (59.1)	6 (27.3)	3(13.6)	22 (75.9)	7 (24.1)	0.156
	Severe	1 (33.3)	2 (66.7)	0 (0)	4 (66.7)	2 (33.3)
		Fibrosis
	Absent	5 (55.7)	3 (33.3)	1(11.1)	9 (100.0)	0 (0)
	Mild	18 (56.3) 11 (34.4)	3 (9.4)	29 (80.6)	7 (19.4)
	Modorate	6 (54.6)	4 (36.4)	1 (9.1)	11 (37.3)	4 (26.7)	0.222
	Marked	4 (66.7)	1 (16.7)	1(16.7)	5 (62.5)	3 (37.5)
	Cirrhosis Absent Mild Moderate Marked	1 (50.0) Steatosis 15 (60.0) 12 (50.0) 10 (41.7) 1 (50.0) 7 (28.0) 3 (42.9) 3 (42.9) 4(100.0) 0 (0) o F r	0 (0) 3(12.0) 2 (8.3) 1 (14.3) 0 (0)	7 (87.5) 27 (77.1) 20 (83.3) 9 (75.0) 5 (100.0)	1 (12.5) 8 (22.9) 4 (16.7) 3 (25.0) 0 (0)	0.509
	Absent Present Absent Present	Portal lymphoid aggregates 25 (64.1) 11 (28.2) 3 (7.7) 9 (42.9) 9 (42.9) 3(14.3) P e Bile duct damage 26 (63.4) 12 (29.3) 3 (7.3) 8 (42.1) 8 (42.1) 3(15.8) e r	39 (88.6) 22 (68.8) 40 (85.1) 21 (72.4)	5 (11.4) 10 (31.3) 7 (14.9) 8 (27.6)	b 0.013 0.051
			R
			e
			v i e
				w

a χ²-test b

The difference is valid only in males (P = 0.032).

Table III . Initial Virological Response to an IFN-α 2a Monotherapy in Chronic Hepatitis C Patients with Regard to TLR7 rs179008 Genotype

 III

				TLR7 rs179008 genotype
			Females		Males
		AA	AT	TT	A	T	P
	No. Non-responsive patients (%) 6 (35.3)	7 (41.2)	4 (23.5)	7 (70.0)	3 (30.0)
	No. Responsive patients (%)	4 (50.0)	3 (37.5)	1 (12.5)	18 (90.0)	2 (10.0)	0.069 a
	a χ²-test was applied to compare A with T homo-and hemizygous patients.
		F o r F o r F o r F o r F o r
		P P P P P
			e e e e e
			e r e r e r e r e r
				R R R R R
				e e e e e
				v i e v i e v i e v i e v i e
					w w w w w

Journal of Medical Virology

John Wiley & SonsJournal of Medical Virology


Maria Cecilia Cercato
email: cercato@ifo.it

Luciano Mariani

Amina Vocaturo

Angela Carrone

Irene Terrenato

Giuseppina Morano

Maria Benevolo

Francesca Rollo

Chiara Germelli

Francesca Paolini

M Benevolo #

F Rollo #

A Venuti

Cercato Maria Cecilia

Predictors of Human Papilloma Virus (HPV) infection in Italian women

Keywords: HPV, Cervical cancer, risk factors, sexual behaviour, questionnaire HPV, cervical cancer, epidemiology, risk factors, sexual behaviour, questionnaire

teaching and research institutions in France or abroad, or from public or private research centers.

INTRODUCTION

Cervical cancer (CC) is associated with Human Papillomavirus (HPV) infection, which is transmitted during sexual intercourse [START_REF] Walboomers | Human papillomavirus is a necessary cause of invasive cervical cancer worldwide[END_REF]American Cancer Society, 2008]. CC is the second most common cancer in woman worldwide and is more frequent in socio-economically deprived regions where proper screening and/or vaccination programs are not easily available [World Health Organization, 2006;[START_REF] Almonte | Risk factors for human papillomavirus exposure and co-factors for cervical cancer in Latin America and the Caribbean[END_REF].

Over one hundred HPV genotypes have been identified and classified into lowrisk (LR) (e.g. type 6,11) and high-risk (HR) (e.g. type 16,18,31,33,45) groups, according to their potential oncogenic effects [START_REF] Muñoz | Chapter 1: HPV in the etiology of human cancer[END_REF]]. In the U.S.A., 75% of women aged between 14 and 50 years become infected during their lives, but the great majority of HPV genital infections are transient and are cleared within 2 years by host immune response [World Health Organization, 2006]. Persistent infections with HR-HPV is involved in the pathogenesis of cervical intraepithelial neoplasia and in the progression to cervical cancer [START_REF] Snijders | HPV-mediated cervical carcinogenesis: concepts and clinical implications[END_REF].

HPV DNA prevalence varies by continent and by age: it is high (up to 30%) in young women (<25 years) all over the world except for Asia, decreasing in the middleage group, even though a second rise among in perimenopausal women has been recently observed [START_REF] Almonte | Risk factors for human papillomavirus exposure and co-factors for cervical cancer in Latin America and the Caribbean[END_REF][START_REF] Bosch | Epidemiology and natural history of Human Papilloma Virus infections and type-specific implications in cervical neoplasia[END_REF][START_REF] Smith | Age-specific prevalence of infection with human papillomavirus in females: a global review[END_REF].

Contracting HPV genital infection appears to be associated with increased sexual activity and thus with number of partners; other involved factors are age at the first sexual intercourse, marital status, cigarette smoking and contraceptives use [START_REF] Vaccarella | Sexual behavior, condom use, and human papillomavirus: pooled analysis of the IARC human papillomavirus prevalence surveys[END_REF][START_REF] Almonte | Risk factors for human papillomavirus exposure and co-factors for cervical cancer in Latin America and the Caribbean[END_REF][START_REF] Herbert | Reducing patient risk for human papillomavirus infection and cervical cancer[END_REF]. However epidemiological studies investigating risk factors for HPV infection have not been entirely consistent, due to the differences in population characteristics (demographic 4 growth rate, sexual habits, HPV prevalence) as well as to the potential misclassification involved in the assessment of sexual history [START_REF] Schlecht | Repeatability of sexual history in longitudinal studies on HPV infection and cervical neoplasia: determinants of reporting error at follow-up interviews[END_REF][START_REF] Schroder | Methodological challenges in research on sexual risk behavior: II. Accuracy of self-reports[END_REF].

Despite increasing effort, the burden and natural history of HPV infection is not completely understood and the upcoming mass vaccination will certainly change HPV epidemiology. Therefore baseline (i.e. pre-vaccine) epidemiological investigations on risk factors for infection should be encouraged to provide the basis to implement prophylactic strategies.

The aim of the present study was to investigate predictors of HR-HPV infection in women referred as outpatients to the "Regina Elena" National Cancer Institute and to the University "La Sapienza" in Rome, Italy, for HPV DNA detection and cervical cancer prevention.

MATERIALS AND METHODS

Population and study design

Questionnaire

The questionnaire, to be filled in by the patient, consisted of multiple choice questions addressing: ethnicity, religion, educational level, marital status, gynaecologic and obstetric history, sexual behaviour (age at first intercourse, lifetime number of partners, frequency of intercourse), contraceptive use, alcohol use and smoking. Knowledge about Pap test and frequency of testing was also investigated.

HPV DNA Detection and Genotyping

Cervico-vaginal specimens were collected by cytobrush (Cytyc, Rome, Italy) and Ayre spatula (Cytyc), in 20 mL of PreservCyt solution (Cytyc), stored at 4°C until the use. HR HPV test was performed according to scientific evidence by means of the HC2 technique (Qiagen, Milan, Italy), a semi-quantitative signal-amplified hybridization assay for the chemiluminescent detection of the 13 most common HR HPV types (genotypes: 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59 and 68), as described by the manufacturer [START_REF] Leinonen | Age-specific evaluation of primary human papillomavirus screening vs conventional cytology in a randomized setting[END_REF]. Before the HC2 testing, 4 ml of PreservCyt solution were processed by means of the HC2 Sample Conversion Kit (Qiagen). The positive cut-off (CO) value was considered the average of the positive control samples.

The results were considered positive when the ratio between the specimen's light emission units (RLU) and the chosen positive CO (RLU/CO) was 1 or greater.

HPV DNA genotyping was carried out by using Linear Array HPV Genotyping Test for DNA extraction, as described by the manufacturer. The assay involves the amplification of a 450 bp amplicon, generated by a consensus PGMY primer set, targeting the conserved L1 region [START_REF] Gravitt | Improved amplification of genital human papillomaviruses[END_REF][START_REF] Coutlée | Use of PGMY primers in L1 consensus PCR improves detection of human papillomavirus DNA in genital samples[END_REF]. The detection of amplified products was achieved by the hybridization to a nylon strip, containing two levels of β-globin control probes and 37 LR and HR anogenital HPV types probes (types 6,11,16,18,26,31,33,35,39,40,42,45,51,52,53,54,55,56,58,59,61,62,64,66,67,68,69,70,71,72,73,81,82,83,84,IS39 and CP6180).

Statistical analysis

For the analysis the patients were categorized as "HPV-positive" when at least one of the two basal HPV DNA tests were positive for high risk genotypes and as "HPVnegative" when it tested negative to both tests.

Descriptive statistics were computed for all variables. Continuous data were reported as the mean and standard deviation or the median and range; categorical data were represented by frequencies and percentage values. The Student's T-test was used to compare continuous variables; the Pearson's Chi-Squared and Fisher's Exact test, when appropriate, were used to detect potential differences between categorical variables.

Cohen's Kappa and its relative 95% Confident Interval (95% CI) was used to assess the concordance the between HC2 and Linear Array HPV Genotyping Test in detecting the HPV status. P-values less than 0.05 were considered statistically significant.

Odds ratios (ORs) and their relative 95% CIs were computed through univariate and multivariate logistic regression models in order to evaluate the association between HPV infection and socio-demographic and behavioural variables. Data of some variables were grouped due to the small number. "Primary" and "secondary school" educational levels were grouped together. Furthermore, "marital status" was divided into two categories "never married-separated/divorced" and "married-living together- widow", because, according to the "marriage protection hypothesis", family life is considered to be associated with healthier behaviour and because widowhood is similar to cohabitation in sociologic terms [Contoyannis and Jones, 2004;[START_REF] Sellors | Incidence, clearance and predictors of human papillomavirus infection in women[END_REF].

The number of lifetime sexual partners was categorized into up to three and more than three according to the median value. The multivariate logistic regression model was developed using stepwise regression (forward selection) with predictive variables that were significant in the univariate analyses. The enter and remove limits were p = 0.10 and p = 0.15, respectively The data were analysed by using the SPSS version 11.5 (SPSS, Inc., Chicago, IL).

.

RESULTS

Ninety-four out of the 100 consecutive eligible women submitted to the HPV DNA detection and genotyping tests agreed to complete the questionnaire.

According to the results of detection and genotyping HPV DNA tests 54 (57%) women were classified as "HR-HPV-positive" and 40 (43%) as "HPV-negative". The agreement between the two tests was 91% (Cohen's K: 0.83 95% CI[0.63 -1.00] ).

Overall, twenty-five different genotypes were identified: the most common high-risk types were 16 (28%), 31 (16%), 51 and 52 (14%) (Figure 1). No single low-risk infection was found. Multiple HPV types were detected in 26 (50%) out of the 52 positive women at the Linear Array HPV Genotyping Test. Co-infection by two viruses were found in 25% of the positive cases, by three viruses in 15% and by more than three viruses in 10%. Co-infection by three or more genotypes were more frequent in young (>35 years) woman (33% vs 9%). I shows the characteristics of the population. All except one were Caucasian and the great majority (90%) belonged to the Christian faith (data not shown). The "positive" women were slightly younger but the mean age values did not differ significantly in the two groups. The "positive" women were more frequently "never married-separated/divorced", reported a lower age at their first sexual intercourse (FSI) (17,9 vs 20.3 years), a higher number of lifetime partners (5.3 vs 3.0), smoking habits and alcohol consumption. No differences in educational level, number of pregnancies and of childbirths, frequency of intercourse, and use of estroprogestinic as well as condoms were observed. Almost all women reported to have knowledge of the Pap test and to undergo it regularly or occasionally.

At univariate analysis marital status, age at the FSI, lifetime numbers of sexual partners, past or current smoking habits, years of smoking, number of cigarettes, and regular use of alcohol were all factors predicting HPV infection (Tab.II). Multivariate analysis confirmed that the "never married-separated/divorced" status and a lower age at the first sexual intercourse were independent factors predicting HR-HPV infection (Tab.II). A trend for the association between the infection and having more than three lifetimes partners was also observed. No association was found for other variables included in the logistic regression model.

DISCUSSION

Cervical cancer is the second most common cancer in women worldwide and the estimate of the attributable fraction due to HPV infection range from 90% to 98% [START_REF] Bosch | Chapter 1 : Human papillomavirus and cervical cancerburden and assessment of causality[END_REF]]. HPV 16 and 18 are the most frequent types associated with CC and two prophylactic vaccines against these high-risk variants have been recently introduced in several countries mainly in young women (up to 26 years) [Ault and the FUTURE II Study Group, 2007;[START_REF] Harper | Age for HPV vaccination[END_REF].

This study describes the predictors of HR-HPV infection in a sample of Italian women. The results highlight the behavioural characteristics that identify a "high-risk" profile for HPV infection, providing useful information to widen future prophylactic strategies also in older age-classes.

The high compliance obtained (94%) confirmed the practicable features of the questionnaire employed and showed a positive attitude towards cervical cancer prevention of these women who, once informed about the aim of the study, agreed to disclose information on their sexual behaviour.

The results of the multivariate analysis showed that having a "never marriedseparated/divorced" status and an early age at the FSI are independent risk factors for HR-HPV infection. A trend, although not significant probably for the small size of the population, was also observed for having more than three lifetime partners. These findings are in agreement with previous studies showing that having a "never married" or a "separated/divorced" status, an early age at the FSI or a higher "sexual age" (i.e. years of being sexually active) are all factors associated with HPV infection [START_REF] Almonte | Risk factors for human papillomavirus exposure and co-factors for cervical cancer in Latin America and the Caribbean[END_REF][START_REF] Sellors | Prevalence and predictors of human papillomavirus infection in women in Ontario, Canada. Survey of HPV in Ontario Women (SHOW) Group[END_REF][START_REF] Lenselink | Sexual behaviour and HPV infections in 18 to 29 year old women in the pre-vaccine era in the Netherlands[END_REF][START_REF] Tonon | Human Papillomavirus cervical infection and associated risk factors in a region of Argentina with a high incidence of cervical carcinoma[END_REF]. According to other authors age at the FSI is more likely to be a predictor of the total number of partners in a lifetime than a factor independently associated with the infection [START_REF] Hildesheim | HPV co-factors related to the development of cervical cancer: results from a population-based study in Costa Rica[END_REF][START_REF] Burchell | Chapter 6: Epidemiology and transmission dynamics of genital HPV infection[END_REF].

Sexual activity with changing partners represents an additional well-known predictor of infection [START_REF] Almonte | Risk factors for human papillomavirus exposure and co-factors for cervical cancer in Latin America and the Caribbean[END_REF][START_REF] Herbert | Reducing patient risk for human papillomavirus infection and cervical cancer[END_REF][START_REF] Vaccarella | Sexual behavior, condom use, and human papillomavirus: pooled analysis of the IARC human papillomavirus prevalence surveys[END_REF][START_REF] Chan | Epidemiologic risk profile of infection with different groups of human papillomaviruses[END_REF][START_REF] Sellors | Prevalence and predictors of human papillomavirus infection in women in Ontario, Canada. Survey of HPV in Ontario Women (SHOW) Group[END_REF][START_REF] Sellors | Incidence, clearance and predictors of human papillomavirus infection in women[END_REF][START_REF] Lenselink | Sexual behaviour and HPV infections in 18 to 29 year old women in the pre-vaccine era in the Netherlands[END_REF][START_REF] Shields | A case-control study of risk factors for invasive cervical cancer among U.S. women exposed to oncogenic types of human papillomavirus[END_REF][START_REF] Venuti | Determinants of human papillomavirus types 16 and 18 infections in the lower femal genital tract in an Italian population[END_REF]. A pooled analysis of the IARC HPV surveys of Spain and Africa) reported a significant increased risk of HPV infection (any type) for women with two or more lifetime partners vs one (OR: 1.86; 95%CI: 1.63-2.11) [START_REF] Vaccarella | Sexual behavior, condom use, and human papillomavirus: pooled analysis of the IARC human papillomavirus prevalence surveys[END_REF]. In several case-control and cross sectional studies conducted in countries at different HPV prevalence the lifetime number and the number of sexual partners in the last 6-12 months were strongly associated with the presence of the infection [START_REF] Almonte | Risk factors for human papillomavirus exposure and co-factors for cervical cancer in Latin America and the Caribbean[END_REF][START_REF] Lenselink | Sexual behaviour and HPV infections in 18 to 29 year old women in the pre-vaccine era in the Netherlands[END_REF][START_REF] Winer | Genital human papillomavirus infection: incidence and risk factors in a cohort of female university students[END_REF].

In this study the multivariate analysis showed no association between the risk of infection and the use of oral contraceptives, the use of condom, smoking and alcohol intake. The findings are consistent with the results of other studies, however no conclusive data are available [START_REF] Lenselink | Sexual behaviour and HPV infections in 18 to 29 year old women in the pre-vaccine era in the Netherlands[END_REF][START_REF] Sellors | Incidence, clearance and predictors of human papillomavirus infection in women[END_REF][START_REF] Sellors | Prevalence and predictors of human papillomavirus infection in women in Ontario, Canada. Survey of HPV in Ontario Women (SHOW) Group[END_REF][START_REF] Winer | Genital human papillomavirus infection: incidence and risk factors in a cohort of female university students[END_REF]. In fact, the protective effect of a condom against HPV infection is not well established and does not seem to be as strong as for other sexual transmitted infections because of its transmission by skin-to-skin contact [START_REF] Vaccarella | Sexual behavior, condom use, and human papillomavirus: pooled analysis of the IARC human papillomavirus prevalence surveys[END_REF][START_REF] Burchell | Chapter 6: Epidemiology and transmission dynamics of genital HPV infection[END_REF]. Data on the role of smoking is still debated and some studies show a positive association with current or past smoking habits [START_REF] Sellors | Prevalence and predictors of human papillomavirus infection in women in Ontario, Canada. Survey of HPV in Ontario Women (SHOW) Group[END_REF][START_REF] Winer | Genital human papillomavirus infection: incidence and risk factors in a cohort of female university students[END_REF][START_REF] Chan | Epidemiologic risk profile of infection with different groups of human papillomaviruses[END_REF]. Smoking has been independently associated with an increased risk of cervical cancer and therefore has an acting role as a HPV co-factor in the cancerogenesis process [International Collaboration of Epidemiological studies of [START_REF] Cancer | Cervical cancer and hormonal contraceptives: collaborative reanalysis of individual data for 16,573 women with cervical cancer and 35,509 women without cervical cancer from 24 epidemiological studies[END_REF]. 11 sample that does not allow to investigate the role of a specific HR-HPV type, and, as highlighted by other authors, the potential misclassification involved in reporting sexual history due to the recall biases or to intentional under-reported number of partners [START_REF] Schroder | Methodological challenges in research on sexual risk behavior: II. Accuracy of self-reports[END_REF][START_REF] Schlecht | Repeatability of sexual history in longitudinal studies on HPV infection and cervical neoplasia: determinants of reporting error at follow-up interviews[END_REF]. Finally, previous infections in HPV negative patients were not assessed by serologic markers taking into account the low sensibility of the test as well as the scant relevance of the transient infections in the carcinogenesis [START_REF] Snijders | HPV-mediated cervical carcinogenesis: concepts and clinical implications[END_REF][START_REF] Schiffman | Epidemiologic studies of a necessary causal risk factor: human papillomavirus infection and cervical neoplasia[END_REF] Epidemiology of HPV infection is going to chance because of upcoming mass vaccination. HPV vaccine trials have established that both vaccines (the bivalent against 16,18 and the quadrivalent against 16,18,6,11) produce a potent immunological response and are associated with an efficacy rate close to 100% for at least 5 years in naïve women [START_REF] Villa | Prophylactic quadrivalent human papillomavirus (types 6, 11, 16, and 18) L1 virus-like particle vaccine in young women: a randomised double-blind placebocontrolled multicentre phase II efficacy trial[END_REF][START_REF] Villa | High sustained efficacy of a prophylactic quadrivalent human papillomavirus types 6/11/16/18 L1 virus-like particle vaccine through 5 years of follow-up[END_REF][START_REF] Harper | Efficacy of a bivalent L1 virus-like particle vaccine in prevention of infection with human papillomavirus types 16 and 18 in young women: a randomized controlled trial[END_REF][START_REF] Harper | Sustained efficacy up to 4.5 years of a bivalent L1 virus-like particle vaccine against human papillomavirus types 16 and 18: follow-up from a randomized control trial[END_REF]Ault and the FUTURE II Study Group, 2007;[START_REF] Paavonen | Efficacy of human papillomavirus (HP oncogenic HPV types (PATRICIA): final analysis of a double-blind, randomised study in young women[END_REF]. National regulatory agencies (i.e. Food and Drug Administration, European Medicines Agency) have approved the two commercial products (Gardasil TM and Cervarix TM ) in most countries for use in young women (up to 26 years). Furthermore, because of the evidence of immunobridging also in women up to the age of 55 years, in selected regions there are approvals for women aged >26 years [START_REF] Harper | Age for HPV vaccination[END_REF][START_REF] Schwarz | Immunogenicity and tolerability of an HPV-16/18 AS04-adjuvanted prophylactic cervical cancer vaccine in women aged 15-55 years[END_REF][START_REF] Castellsagué | HPV vaccination against cervical cancer in women above 25 years of age: key considerations and current perspectives[END_REF].

Taking into account the safety and efficacy data available, increasing interventions to immunize women of different ages are expected.

CONCLUSIONS

Prevalence of HR-HPV infection is high in this selected population and is mainly associated to sexual behaviour. These findings contribute to the knowledge of an

  This study was part of a cooperative project conducted from April 2006 to December 2008, supported by the Italian Ministry of Health and coordinated by the "Regina Elena" National Cancer Institute. The study population were women who consecutively attended the Department of Gynaecologic Oncology of the "Regina Elena" National Cancer Institute and of the University "La Sapienza" of Rome, as outpatients for HPV DNA detection and cervical cancer prevention. Exclusion criteria were: age <18 years, positive history for cancer, previous surgical treatment for cervical lesions, presence of immunological disorders, pregnancy or cognitive impairment.Eligible patients were asked to participate and a written informed consent obtained. The study was approved by the Local Medical Ethical Committee.

  At enrolment a baseline questionnaire investigating common risk factors for HPV infection and for cervical cancer was given by the physician just before the clinical examination. A gynaecologist collected clinical data and during the scheduled clinical procedures collected cervicovaginal specimens for HPV DNA analysis to be delivered to the Department of Pathology.

(

  LA Roche Molecular System, Rome, Italy). 250 µl of PreservCyt sample were taken

  women of nine countries (including areas Latino America, South-Eastern Asia,

  The study was not aimed to assess the prevalence of HPV infection. The high prevalence of the HR-HPV DNA infection observed in this population is probably due to the sample selection among patients referred by their general practitioners to two qualified Institutions for Research for HPV detection and cervical cancer screening, and could represent a limitation of the study. Other limitations are the small size of the

Fig. 1 .

 1 Fig. 1. Distribution of HPV genotypes detected by Linear Array Genotyping Test in the Italian women. * CP6108 8x3mm (600 x 600 DPI)

Table I .

 I Characteristics of the 94 patients according to HPV DNA tests results. the sum may not add to the total because of missing values.

	adult population in defining a "high risk" behavioural profile and could be helpful to
	target prophylactic strategies in older woman.	HR-Positive	Negative
	Variable			N* (%)	N* (%)	p-value
	N of patients			54 (57)	40 (43)
	Age
	mean (sd)			35 (10.2)	39 (11.2)	0.112
	Education					0.755
	Primary-Secondary school		15 (60)	10 (40)
	High school			27 (54)	23 (46)
	University			12 (63)	7 (37)
	Marital status					<0.0001
	Married-Living togheter-Widow		13 (33)	26 (67)
	Never married-Separeted/Divorced		40 (74)	14 (26)
	Age at first sexual intercourse				0.003
	mean (sd) Number of partners mean (sd) Number of pregnancies mean (sd) Number of childbirth	F o r F o r F o r F o r F o r F o r F o r F o r F o r F o r F o r	17.9 (2.4) 5.3 (3.8) 1 (1.5)	20.3 (4.9) 3.0 (3.3) 1.3 (1.3)	0.004 0.209 0.080
	media (sd) Estroprogestinic contraceptive use Yes No	P e P e P e P e P e P e P e P e P e P e P e	0.6 (0.8) 33 (63) 18 (47)	1 (1.2) 19 (37) 20 (53)	0.128
	Condom contraceptive use Yes No	e r e r e r e r e r e r e r e r e r e r e r	14 (70) 37 (53)	6 (30) 33(47)	0.172
	N°sexual intercourse Mean (sd) per months Smoking		R R R R R R R R R R 7.9 (6.5) R	5.7 (5.7)	0.105 0.043
	Yes No Age at first cigarette media (sd) Years of smoking media (sd)		e v i e e v i e e v i e e v i e e v i e e v i e e v i e e v i e e v i e e v i e 33 (67) 21 (47) e 16.8 (2.8) 9.7 (11.1) v i e	16 (33) 24 (53) 18.5 (3.0) 4.3 (7.2)	0.072 0.008
	Number of smoked cigarettes media (sd) Alcohol consumption		w w w w w w w w w w 7.3 (7.7) w	3.6 (6.1)	0.014 0.026
	Yes (regular and occasional drinkers)		23 (26)	8 (74)
	No			31 (50)	31 (50)
	Pap Test knowledge					0.178
	Yes			54 (59)	38 (41)
	No			0 (0)	2 (100)
	Pap test frequency					0.930
	Every 1-2 years			44 (58)	32 (42)
	Occasionally			7 (58)	5 (42)
	Never			3 (50)	3 (50)
						12	1 2

*

Table II .

 II Predictors of HR-HPV infection in women: univariate and multivariate logistic regression analysis.

	Variables	Univariate OR	95% CI	p-value Multivariate OR 95% CI p-value
	Age*	0,97	0,93-1,00	0,116
	Education
	Primary-Secondary	0,87	0,26-2,99	0,831
	High School	0,69	0,23-2,03	0,494
	University	1,00	-	-
	Marital Status
	Married/Living together/Widow	1,00	-	<0,0001	1,00	-	0,029
	Never married-Separated/Divorced	5,71	2,32-14,08		3,38	1,14-10,12
	Age at first sexual intercourse*	0,82	0,71-0,95	0,007	0,84	0,71-1,00 0,055
	Number of partners			<0,0001
	≤ 3	1,00	-		1,00	-	0,092
	> 3	5,32	2,09-13,95		2,57	0,86-7,71
	Smoking
	No	1,00	-	0,044	1,00	-	0,131
	Yes	2,36	1,02-5,44		2,24	0,79-6,39
	Alchol consumption			0,029
	No	1,00	-		1,00	-	0,117
	Yes (regular and occasional drinkers)	2,88	1,12-7,41		2,56	0,79-8,33
	Estroprogestinic contraceptive use			0,13
	Yes	1,93	0,82-4,52
	No	1,00	-
	Condom use
	Yes	0,48	0,17-1,39	0,178
	No	1,00	-
	* considered as continuos variables

The Italian Ministry of Health partially funded the study.

Conflict of interest statement

The authors have no conflict of interest to disclose.

AKNOWLEDGMENTS

The authors thank the Italian Ministry of Health that partially funded the study and the Lega Italiana Lotta Tumori (LILT).