MSc Sanela Svraka
email: svraka.sanela@rivm.nl

Ed Kuijper

Erwin Duizer

Dennis Bakker

Marion Koopmans

Prof Marion Koopmans Dear

Dr Alex Van Belkum

Koopmans

Clostridium difficile is not associated with outbreaks of viral gastroenteritis in elderly in the Netherlands

Keywords: viral gastroenteritis, norovirus, Clostridium difficile, nursing homes, the Netherlands, elderly viral gastroenteritis, norovirus, Clostridium difficile, nursing 47 homes, the Netherlands, elderly 48 49

The coincidental increase in norovirus outbreaks and Clostridium difficile infection (CDI) raised the question if these events could be related, e.g. by enhancing spread through by diarrhoeal disease outbreaks. Therefore, we studied the prevalence of C. difficile in outbreaks of viral gastroenteritis in nursing homes for elderly and characterized enzyme-immunoassay (EIA) positive stool samples. Stool samples from nursing homes residents (n=752) in 137 outbreaks of viral aetiology were investigated by EIA for the presence of C. difficile toxins. Positive samples were further tested by a cell neutralization cytotoxicity test, a second EIA and culture. Cultured isolates were tested for the presence of toxin genes, the production of toxins, and characterized by 16S rRNA PCR and sequencing. Twenty-four samples (3.2 %) tested positive in the EIA. Of these 24 positive samples, only two were positive by cytotoxicity and 3 by a second EIA. Bacterial culture of 21 available stool samples yielded a toxinogenic C. difficile PCR ribotype 001 in one patient sample only. In conclusion, we found no evidence in this retrospective study for an association between viral gastroenteritis outbreaks and C. difficile. The high rate of false positive EIA samples emphasizes the need for second confirmation tests to diagnose CDI.

Materials and Methods:

-Ideally, the study design would be a matched case-control study. A control group of non-ill patients would have been particularly useful if significant detection of C. difficile was shown to help distinguish from asymptomatic colonization. However, this was not the case and a convenience sample of stools was used. In our study we strived to assess if the C. difficile infection was related to norovirus infection and if coinfections could contribute to the more serious illness. We decided to study patients from residential institutions because they are a high risk group for diarreal diseases and rarely studied for the infections with C. difficile, and year 2006 was chosen because of the increased reporting of C. difficile infections in hospitals in the Netherlands. For that purpose we have included the desired control groups of ill patients who were not infected with noroviruses, but with rotaviruses or of unexplained etiologies (material and methods section, page 4, lines 85-100). A matched group of non-ill patients would complete the study, however this was a retrospective study on patients suspected of viral gastroenteritis from residential institutions for elderly. We were not able to obtain stool specimens from this group from the same period. Furthermore, we should note that we investigated outbreaks of gastroenteritis, meaning that at least 2 patients which were linked in time and space and had two or more episodes of vomiting or diarrhea were reported and tested.

-The methods for viral detection were not described or adequately referenced. We added the reference for detection of viral pathogens in gastroenteritis outbreaks that is used at our institute, we also briefly described the order of testing, see page 4, lines 88-92. We added "Viral detection as been performed as described previously by Svraka et al. [START_REF] Svraka | Etiological role of viruses in outbreaks of acute gastroenteritis in 290 The Netherlands from 1994 through 2005[END_REF], the outbreaks were firstly tested using the assays for noroviruses, all outbreaks that remained negative were further tested using the assays for rotaviruses, adenoviruses, astroviruses, and sapoviruses [START_REF] Svraka | Etiological role of viruses in outbreaks of acute gastroenteritis in 290 The Netherlands from 1994 through 2005[END_REF]." -The major limitation of this study is the length of time between collection of stools and testing for Cdiff (up to 16 months). It is difficult to interpret the low detection of Cdiff in this cohort because there is no literature demonstrating that Cdiff toxins are stable for this long under these conditions. While the authors compared stools stored for 5 months at 4 deg vs -20 deg, the ideal comparison would have been stools stored at 4 deg for this duration of time vs. stools processed within 48 hours. We agree that the ideal comparison would have been comparison of stools that are processed within 48 hours and those stored at 4 degrees, and that this meets clostridium criteria, however in this study we tested stool specimens sent for norovirus detection, and storage conditions for samples suspected of norovirus infections are set on 4ºC. After performing diagnostic assays for viral pathogens causing gastroenteritis, we tested these samples for presence of C. difficile. Because of this we decided to split a cohort of obtained specimens, store them at 4C and -20C and to compare the results. Storage at 4C did not lead to lower number of positive samples, moreover we detected three positive samples in stead of two that were found when stored at -20C. To our knowledge cl inical laboratories freeze stool samples prior to PTAB testing to prevent false negative results. In our study we are dealing with the false positive results obtained using the EIA, which, to our knowledge, has never been published. So, we were expecting that the sensitivity would be lower, but we did not expect that the specificity would change. Furthermore, Borriello et Given the concerns raised in your review, we decided to do an additional experiment. We retracted stored total nucleic acids extracted from the fecal samples on the day (up to three days) of arrival of the stool samples which were included in our study and tested them by a diagnostic real time PCR for Clostridium difficile. We used a real-time PCR as described by van We tested 154 fecal specimens (20% of all samples tested in EIA) that were isolated within three days of arrival, only two of these samples were positive (table I samples 13/8 and 23/18), these samples were found positive in used assays. All other samples were negative. Therefore, we believe that data we present are true and have added this data to the manuscripts as follows: to sections material and methods (Lines 158-165 we added "Evaluation of the approach of Clostridium detection Optimal sample storage conditions differ for viral testing and bacterial testing, and this raised the question whether Clostridium EIA test results could be explained by suboptimal use of the assay. Therefore we validated this approach by testing original total nucleic acids isolated from 154 (20% of all samples tested in EIA) fecal samples within three days of arrival from the stool samples which were included in our study. These extracts had been stored at -80°C. We used a real-time PCR for TcdB gene as described by van den Berg et al. [START_REF] Van Den Berg | Evaluation of real-time PCR and conventional diagnostic methods for the 333 detection of Clostridium difficile-associated diarrhoea in a prospective multicentre 334 study[END_REF][START_REF] Van Den Berg | Rapid 352 diagnosis of toxinogenic Clostridium difficile in faecal samples with internally 353 controlled real-time PCR[END_REF]."), results (lines 218-222 we added "Evaluation of the approach of Clostridium detection Of the 154 total nucleic acids that had been isolated from stool samples within three days of the arrival, the two samples that were cytotoxic and VIDAS positive were positive by tcdB real-time PCR assay (Table I samples 13/8 and23/18). All other samples were negative."), and discussion (lines 248-252 we added "Furthermore, testing of original total nucleic acids isolated from fecal samples within three days of arrival yielded the same positives as the combination of cytotoxicity test and VIDAS. Therefore, we conclude that our findings are not due to degradation of C. difficile toxins [START_REF] Borriello | Evaluation of a 361 commercial enzyme immunoassay kit for the detection of Clostridium difficile toxin 362 A[END_REF][START_REF] Turcios | 364 Reevaluation of epidemiological criteria for identifying outbreaks of acute 365 gastroenteritis due to norovirus: United States, 1998-2000[END_REF].") Results: -Again, it is difficult to interpret the results because the toxin may have degraded under the storage conditions for such a prolonged period of time. See comment mentioned above. We did not find any samples positive in the real-time PCR that were negative when EIA was used, therefore we strongly believe that data we present are true. In result section lines 218-222 we added "Evaluation of the approach of Clostridium detection Of the 154 total nucleic acids that had been isolated from stool samples within three days of the arrival, the two samples that were cytotoxic and VIDAS positive were positive by tcdB real-time PCR assay (Table I samples 13/8 and 23/18). All other samples were negative. ". -Can the authors explain why a number of the cultured bacteria had a positive VIDAS test when the stools were negative for Cdiff by VIDAS? Positive VIDAS test of the cultured bacteria while stools are negative for C. difficile can be explained by used cut off values. We applied same cut off values of VIDAS for in vitro bacterial toxin production as for detection in stool samples (as stated in material and methods section, page 7 line 153-158) while the cut off values probably need to have other (probably higher) cut off value. Moreover, cut off values as presented by the manufacturer are evaluated for the stool specimens and not for cultured bacteria.

Discussion: -The last concluding sentence probably needs to be rephrased because the limitations of the methods are so substantial. The title also needs to be revised because it is misleading since the data are so difficult to interpret.

Based on the additional data provided here, we think that our conclusions are valid. Therefore we have not modified the title. [START_REF] Kuijper | Clostridium difficile ribotype 306 027, toxinotype III, the Netherlands[END_REF][START_REF] Notermans | De epidemiologie van Clostridium difficile PCR-ribotype 027 in Nederland 309 sinds 2005 en de opkomst van andere typen[END_REF]. Antibiotic use 66 has been associated with an increased risk of infection with C. difficile and is 67 responsible for 15% to 25% of all cases of antibiotic-associated diarrhoea in hospitals 68 [START_REF] Levy | Antibiotics and Clostridium 313 difficile diarrhea in the ambulatory care setting[END_REF]. Outbreaks and cases outside hospitals are less frequently recognized but have 69 been described, sometimes without the typical risk factors [START_REF] Hirschhorn | 315 Epidemiology of community-acquired Clostridium difficile-associated diarrhea[END_REF][START_REF]Severe Clostridium difficile-associated disease in populations 318 previously at low risk--four states, 2005[END_REF]. C. difficile outbreaks 70 and cases in nursing homes have been studied less systematically, and although C. 71 difficile type 027 has been found at least in 10 nursing homes in the Netherlands, 72 detailed information about these infections is absent [START_REF] Notermans | De epidemiologie van Clostridium difficile PCR-ribotype 027 in Nederland 309 sinds 2005 en de opkomst van andere typen[END_REF]. suggested in some recent publications [START_REF] Wilcox | Viral gastroenteritis increases the reports of 324 Clostridium difficile infection[END_REF][START_REF] Bignardi | A case of norovirus and 326 Clostridium difficile infection: casual or causal relationship?[END_REF][START_REF] Barrett | Increased 329 detection of Clostridium difficile during a norovirus outbreak[END_REF]. Therefore, we decided to investigate a performed as described previously by Svraka et al. [START_REF] Svraka | Etiological role of viruses in outbreaks of acute gastroenteritis in 290 The Netherlands from 1994 through 2005[END_REF], the outbreaks were firstly 91 tested using the assays for noroviruses, all outbreaks that remained negative were 92 further tested using the assays for rotaviruses, adenoviruses, astroviruses, and 93 sapoviruses [START_REF] Svraka | Etiological role of viruses in outbreaks of acute gastroenteritis in 290 The Netherlands from 1994 through 2005[END_REF]. Of these, 109 were confirmed norovirus outbreaks (644 stool 94 samples), 14 outbreaks were confirmed rotavirus outbreaks (70 samples), and 14 95 outbreaks (38 samples) were of unexplained aetiology, reflecting the typical aetiology 96 of such outbreaks. The outbreaks were reported throughout the year, but with a peak 97 in winter as part of our ongoing surveillance for viral gastroenteritis outbreaks, which 98 explains the high number of NoV outbreaks in this study [START_REF] Svraka | Etiological role of viruses in outbreaks of acute gastroenteritis in 290 The Netherlands from 1994 through 2005[END_REF]. Undiluted stool samples 99 had been stored at 4ºC, as recommended for viral gastroenteritis diarrhoea, before Clostridium suspected colonies were tested further using PCR assays for C. difficile-132 specific GluD targeting GDH gene [START_REF] Zheng | Molecular characterization and antimicrobial susceptibilities of 337 extra-intestinal Clostridium difficile isolates[END_REF], C. difficile-ribotyping [START_REF] Van Den Berg | Coexistence of multiple PCR-ribotype strains of Clostridium 340 difficile in faecal samples limits epidemiological studies[END_REF][START_REF] Bidet | Development of a 343 new PCR-ribotyping method for Clostridium difficile based on ribosomal RNA gene 344 sequencing[END_REF], enterotoxin A 133 (tcdA) [START_REF] Kuijper | Clostridium difficile ribotype 306 027, toxinotype III, the Netherlands[END_REF], cytotoxin B (tcdB) [START_REF] Kato | Identification of toxin A-negative, toxin B-347 positive Clostridium difficile by PCR[END_REF], and 16S rRNA PCR [START_REF] Pille | Detection of bacterial DNA in synovial fluid 350 from horses with infectious synovitis[END_REF]. For this, DNA was 1) was performed on 24 182 stool samples. For three stool samples culture was negative, and in 21 stool samples 183 Clostridium suspected colonies were found. Subsequently, neutralization cytotoxicity 184 assay, and VIDAS assay were performed on 21 stool specimens of whom sufficient 185 amounts of material were available. This yielded two positive samples both by 186 neutralization cytotoxicity assay and VIDAS, and an additional weak positive using 187 VIDAS assay (Table 1).

  al. (Evaluation of a Commercial enzyme immunoassay kit for the detection of C. difficile toxin A, Eur. J. Clin. Microbiol. Infct. Dis Vol. 11, 1992) and J. Freeman and M.H. Wilcox (The effects of storage conditions on viability of C. difficile vegetative cells and spores and toxin activity in human feces, J Clin Path 2003 (56): 126-128) described that the storage for respectively 44 and 56 days at 4ºC had no discernible effect on C. difficile cytotoxin.

  den Berg et al. (van den Berg, R. J., Kuijper, E. J., van Coppenraet, L. E. & Claas, E. C. J. (2006). Rapid diagnosis of toxinogenic Clostridium difficile in faecal samples with internally controlled real-time PCR. Clin Microbiol Infect 12, 184-186.)

  characterized enzyme-immunoassay (EIA) positive stool samples. 32 Stool samples from nursing homes residents (n=752) in 137 outbreaks 33 of viral aetiology were investigated by EIA for the presence of C. difficile 34 toxins. Positive samples were further tested by a cell neutralization cytotoxicity 35 test, a second EIA and culture. Cultured isolates were tested for the presence of 36 toxin genes, the production of toxins, and characterized by 16S rRNA PCR and 37 sequencing. 38 Twenty-four samples (3.2 %) tested positive in the EIA. Of these 24 positive 39 samples, only two were positive by cytotoxicity and 3 by a second EIA. Bacterial 40 culture of 21 available stool samples yielded a toxinogenic C. difficile PCR ribotype 41 001 in one patient sample only. 42 In conclusion, we found no evidence in this retrospective study for an 43 association between viral gastroenteritis outbreaks and C. difficile. The high rate of 44 false positive EIA samples emphasizes the need for second confirmation tests to 45 diagnose CDI. Introduction 50 Outbreaks of acute gastroenteritis (AGE) of suspected viral aetiology are 51 reported to the National Institute for Public Health and The Environment (RIVM) as 52 part of the ongoing viral gastroenteritis outbreak surveillance system. Although these 53 outbreaks are selected based on criteria thought to be highly specific for viral 54 gastroenteritis outbreaks, and despite extensive testing the cause of approximately 55 12% of outbreaks of AGE remains unknown [1]. Of the outbreaks in which a viral 56 pathogen was detected, the vast majority is associated with noroviruses. 57 Since 2002, the epidemiology of noroviruses (NoV) appears to have changed, 58 with the introduction of a novel lineage of the dominant genogroup (G) II.4 viruses. 59 This introduction coincided with increased levels of reporting of NoV outbreaks 60 across Europe and a noticeable peak in mortality in elderly, which coincides with the 61 NoV seasonal peak [2]. Simultaneously, Canada, the USA, and later the United 62 Kingdom (UK), the Netherlands, Belgium, France, and Austria reported the 63 emergence of Clostridium difficile ribotype 027, which appeared to have increased 64 virulence compared to other C. difficile strains [3, 4]. In the Netherlands, this ribotype 65 was detected in 26 hospitals and 10 nursing homes by May 2008

73 4

 73 The coincidental increase in norovirus GII.4 outbreaks and spread of C. 74 difficile ribotype 027 raised the question if these events could be related[START_REF] Lopman | Increase in viral gastroenteritis outbreaks in Europe and epidemic spread of 296 new norovirus variant[END_REF][START_REF] Siebenga | Gastroenteritis 321 caused by norovirus GGII.4, The Netherlands, 1994-2005[END_REF]. In 75 theory, mixed infections of noroviruses and Clostridium difficile 027 could increase 76 the severity of the disease. Furthermore, norovirus induced episodes of vomiting and 77 diarrhoea might trigger or exacerbate C. difficile diarrhoea resulting in increased 78 spread of C. difficile due to multiple episodes of vomiting and diarrhoea as has been 79

  80 possible association of norovirus and rotavirus outbreaks with C. difficile infections, 81 particularly with respect to C. difficile PCR ribotype 027. we tested 752 faecal samples from 137 outbreaks of gastroenteritis 86 (number of stool samples varying from 2 to 25 per outbreak). All outbreaks occurred 87 in 2006 in nursing homes and were selected because the Clostridium difficile 027 88 problem emerged in that period in the Netherlands and these samples provided a 89 snapshot of what is present within elderly population. Viral detection as been 90

6 Stool

 6 100 testing for presence of C. difficile. 101 Diagnostic assays used for screening of samples 102 Screening of 752 outbreak specimens for the presence of Clostridium difficile 103 was done using PTAB enzyme immunoassay (EIA, Premier C. difficile Toxin A&B, 104 Meridian Diagnostics, Inc., Cincinnati, Ohio) on all stool samples. Assay was 105 performed as described by manufacturer. Stool samples had been stored at 4ºC for 106 periods longer than 3-4 days, which is in accordance with recommendations for 107 diagnostics of viral gastroenteritis, but not in accordance to the manufacturer's 108 recommendations, before testing for presence of C. difficile. Consequently, we 109 validated the PTAB EIA assay for use in samples that were stored longer at 4ºC by 110 parallel testing of 96 stool samples that were aliquoted directly after receipt and stored 111 for five months at -20ºC and 4ºC. 112 The PTAB EIA was interpreted visually (within 15 minutes) and by 113 spectrophotometer (within 30 minutes), as described by the manufacturer; 376 114 samples were read both visually and on a Labsystems Multiskan RC 115 spectrophotometer plate reader (Thermo Scientific, Breda, the Netherlands) using dual 116 wavelengths of 450 nm and 450/630 nm. The samples were described as positive if 117 optical density (OD) values of 0.100 or more were measured at wavelength of 118 450/630 nm and/or 0.150 or more at wavelength of 450 nm, as described by 119 manufacturer. There was 100% concordance between reading visually and by 120 spectrophotometer plate reader. The remaining 376 samples were read visually only. 121 Confirmatory testing on PTAB positive samples 122 samples found positive using PTAB EIA assay were cultured, and tested 123 using neutralization cytotoxicity assay, and automated immunoanalyzer VIDAS ® 124 Clostridium difficile A & B (BioMerieux) [14]. 125 For culture, stool samples found positive in PTAB EIA assay were treated 126 with absolute ethanol (96%) prior to inoculation onto Columbia agar containing 127 colistin and nalidixic acid, C. difficile selective agar with cefoxitin, amphotericin B 128 and cycloserine (CLO; bioMerieux), and blood agar. CLO medium was also used to 129 inoculate faecal samples without pre-treatment with ethanol. Inoculated faecal 130 samples were incubated in an anaerobic environment at 37C for 2 days.

  131

134 isolated

 134 from suspected colonies by QiaAMP DNA isolation column (Qiagen, 135 Holden, Germany) according to manufacturers recommendations, including 10 136 minutes incubation at 55C with proteinase K. Final volume of the DNA extracts was 137 200 µL. 138 The neutralization cytotoxicity assay was performed using Vero cells in a 24-139 well format. Faecal samples were diluted in 1:4 Eagle's minimum essential medium 140 (EMEM) containing 5% fetal bovine serum and centrifuged for 10 minutes at 3.000 141 rpm. The supernatant was filtered through a 0.45 µm pore-filter, 150 µL supernatant 142 was mixed with 150 µl EMEM and neutralization of the cytotoxic effect was 143 performed using 150 µL faecal supernatant and 150 µl of 1:25 dilution of specific C.144 difficile antitoxin (Techlab, Blacksburg, USA). These two mixtures were incubated 145 for 30 minutes at room temperature; subsequently 200 µl of each mix was inoculated 146 on Vero cells. The cytotoxic effect was evaluated daily for three days 147 microscopically. 148 Stool samples positive in PTAB EIA were also tested using VIDAS assay. 149 Stool samples were centrifuged for 10 minutes at maximum speed (14.000 rpm) and 150 300 µl of supernatant was used, according to manufacturer's instructions. Samples 151 with OD value lower than 0.13 were described as negative, OD values between 0.13 152 and 0.37 were equivocal, and OD values higher than 0.37 were described as positive. 153 Bacterial isolates cultured from EIA positive faeces samples were also 154 investigated for the production of TcdA and TcdB by VIDAS and EIA. Bacteria were 155 cultured in liquid brain heart infusion (BHI) bouillon medium for four days. 156 Subsequently, the BHI cultures were centrifuged at 3.000 rpm for 10 minutes and 157 supernatant was further investigated. Same cut-off values of EIA and VIDAS were 158 used for in vitro bacterial toxin production as for toxin detection in faeces samples. 159 Evaluation of the approach of Clostridium detection 160 Optimal sample storage conditions differ for viral testing and bacterial testing, and 161 this raised the question whether Clostridium EIA test results could be explained by 162 suboptimal use of the assay. Therefore we validated this approach by testing original 163 total nucleic acids isolated from 154 (20% of all samples tested in EIA) fecal samples 164 within three days of arrival from the stool samples which were included in our study. 165 These extracts had been stored at -80°C. We used a real-time PCR for TcdB gene as 166 described by van den Berg et al. [14, 20] 167 168 Results 169 Testing of stool samples using PTAB EIA For the validation of the use of PTAB EIA on our sample set, we used 96 stool 171 samples that were stored for five months at -20ºC and at 4ºC. Of the samples stored at 172 -20ºC two were positive and of the samples stored at 4ºC three were positive: 173 including the ones that were positive after storage at -20ºC. 174 In total, 24 (3.2 %) of 752 stool samples from outbreaks of acute 175 gastroenteritis in nursing homes in 2006 tested positive in the PTAB EIA assay (Table 176 1). Of the 24 PTAB EIA positive stool samples, 22 were from 17 norovirus outbreaks, 177 one was from a rotavirus outbreak and the other one was from an outbreak of 178 unexplained aetiology. 179 180 Confirmatory testing of PTAB positive stool samples 181 Specialized stool culture for Clostridium spp. (Table

188 189

 188 Analysing of bacterial isolates from the stool samples190 From the 21 Clostridium suspected colonies, which were grown on the CLO 191 medium plates, DNA was extracted and analysed by 16S DNA sequencing and further 192 using a range of assays for characterization of toxin producing Clostridium difficile 193 (

  we investigated whether the emergence of a successful norovirus 227 strain[START_REF] Siebenga | Gastroenteritis 321 caused by norovirus GGII.4, The Netherlands, 1994-2005[END_REF] could coincides with the spread of C. difficile and whether the emergence 228 of C. difficile could explain some unresolved outbreaks of acute gastroenteritis in 229 nursing homes as has been postulated. This was initiated by publications suggesting 230 that such an association may exist[START_REF] Wilcox | Viral gastroenteritis increases the reports of 324 Clostridium difficile infection[END_REF][START_REF] Bignardi | A case of norovirus and 326 Clostridium difficile infection: casual or causal relationship?[END_REF][START_REF] Barrett | Increased 329 detection of Clostridium difficile during a norovirus outbreak[END_REF][START_REF] Martin | 355 Simultaneous control of norovirus and Clostridium difficile outbreaks due to 356 enhanced infection prevention and control measures[END_REF]. We did not find any evidence for such 231 an association in our retrospective study. C. difficile PCR ribotype 001 was found in a 232 single patient in one gastroenteritis outbreak of unexplained aetiology in a nursing 233 home. This type is one of the most frequently circulating and detected types in the 234 Netherlands. 235 However, our study was performed on outbreaks in nursing homes, while 236 other studies describe hospital outbreaks where C. difficile infections are more 237 common [3-5]. Wilcox and co-workers have shown that C. difficile infection rates are 238 higher in closed hospital units which are affected by viral gastroenteritis than in open 239 units where no viral gastroenteritis was detected [11]. Results were not confirmed 240 using C. difficile specific assays, and our data suggest that false positivity may explain 241 previously noted association between norovirus and Clostridium. We were unable to 242 confirm the presence of C. difficile in all but one PTAB EIA positive stool. This is not 243 explained by inability to culture, because other Clostridium species were isolated 244 from 63.3% of all reactive samples. 245 Our study does have some limitations: first, stool samples had been sent by 246 regular mail and stored at 4ºC with a maximum of 16 months. This is in accordance 247 with recommendations for diagnostics of viral gastroenteritis, but unusual for CDI 248 tests. We did however find that the sensitivity of the PTAB EIA was not affected by 249 storage of 5 months at 4C relative to storage at -20C. Furthermore, testing of 250 original total nucleic acids isolated from fecal samples within three days of arrival 251 yielded the same positives as the combination of cytotoxicity test and VIDAS. 252 Therefore, we conclude that our findings are not due to degradation of C. difficile 253 toxins [22, 23]. 254 In addition, this was a retrospective study and stool samples were selected 255 from patients with a suspected viral gastroenteritis [24]. Criteria for viral 256 gastroenteritis differ from those for Clostridium gastroenteritis since symptoms for C. 257 difficile infections are highly variable, ranging from mild diarrhoea to life-threatening 258 colitis, and including watery diarrhoea [25]. 259 Bignardi et al. noted that if a large number of stool specimens are submitted 260 for testing of prevalence of C. difficile during norovirus outbreaks, it is likely that the 261 number of false positive results will increase [12]. The Clostridium species, such as C. 262 disporicum, C. perfringens, C. sordellii, C. boltei, C. butyricum, C. barati, C. 263 subterminale and C. bifermentans, found in this study are bacteria that are commonly 264 found in the gut. These bacterial isolates did react weakly positive, using the cut-off 265 values for the faecal material, by C. difficile PTAB EIA confirming the specificity 266 problem on stool samples stored for a long period at 4C [26-31]. The Premier C. 267 difficile Toxin A & B EIA assay has a positive predictive value (PPV) of 87.4% 268 according to manufacturer's assessment when tested according to manufacturer's 269 recommendations. A prospective multicenter study using the cytotoxicity assay as a 270 gold standard, revealed a PPV value of 50.9% and specificity of 94.3%, indicating 271 that this assay is not highly specific when used for broader testing [14]. A recently 272 performed meta-analysis by the European Study group of Clostridium difficile 273 confirmed the PTAB to have an unacceptable low PPV of 50% at a prevalence rate of 274 5% [32]. 275 A possible explanation for the high rate of false positive PTAB results could 276 be that suboptimal sample storage influenced the tests with weak positive results just 277 nearby the cut off values. Unfortunately, we were unable to test this hypothesis by a 278 comparison of OD values of the false positive samples with OD values of CDI 279 confirmed samples. Nonetheless, we recommend additional confirmatory testing, 280 preferably a cell neutralization cytotoxicity assay, which is reference-testing method 281 for detection, or a molecular detection test including typing, specifically when 282 samples are not sent in and stored according to C. difficile diagnostic criteria. 283 In summary, we found no evidence for an association between spread of 284 norovirus and Clostridium difficile. Previous reports that suggested this association 285 may be explained by false positive PTAB tests. 286 287 Table I. Results on stool samples and characterization of the isolates from the stool samples. These results are obtained for stool samples positive 392 in PTAB EIA and tested further using neutralization cytotoxicity, VIDAS, and culture assays. OD values of VIDAS assay are given between 393 brackets. Results on the isolates cultured from the stool samples by PCR assays for GluD, ribotyping, tcdA, tcdB, and 16S rRNA and 394 sequencing. Additionally, grown cultures were tested using neutralization cytotoxicity assay, PTAB EIA, and VIDAS. OD values of VIDAS and 395 PTAB EIA (measured at 450 nm) are given between brackets.

Table 1 )

 1 . 16S DNA sequencing revealed Lactobacilli in six stool samples (37.5%), C. perfringens, C. sordellii, C. boltei, C. butyricum, C. barati, C. subterminale and C.

	195

194 and different Clostridium species in 15 samples (62.5%): C. difficile, C. disporicum, 196 bifermentans (

Table 1 )

 1 . Using the GluD, ribotyping PCR, tcdA and tcdB assays, one 197 sample was positive, and identified as C. difficile. One isolate of C. disporicum was 198 weak positive for tcdA and one isolate of C. subterminale for tcdB. Evaluation of the approach of Clostridium detection 220 Of the 154 total nucleic acids that had been isolated from stool samples within three 221 days of the arrival, the two samples that were cytotoxic and VIDAS positive were 222 positive by tcdB real-time PCR assay (Table I samples 13/8 and 23/18). All other

	199
	200	One clostridium species from a faecal sample (13/8, Table 1) tested positive
	201	by EIA, cytotoxicity and VIDAS, was identified as Clostridium sordelli, this isolate
	202	was negative for tcdA and tcdB and did not produce a cytotoxin.
	203	The results of in-vitro bacterial toxin production measured by cytotoxicity,
	204	PTAB and VIDAS revealed only strong positive results for one sample that was
	205	identified as C. difficile. Using cut-off values of PTAB and VIDAS as applied on
	206	stool samples, (weak) positive reactions were obtained for 14 samples using PTAB
	207	and 6 samples using VIDAS assay. However, none of these isolates produced
	208	cytotoxins.
	209
	210	Epidemiological data on C. difficile and C. sordellii positive samples
	211	Only one stool sample and the corresponding bacterial isolate were positive in
	212	all assays and proven to be C. difficile type 001 (23/18, Table 1). This sample was
	213	from an outbreak of unexplained aetiology that occurred in November 2006, with
	214	eight cases reported. All eight samples were tested using PTAB EIA, however only
	215	one was positive and tested further in other assays.
	216	The C. sordellii positive stool sample (13/8, Table 1) was from a norovirus
	217	outbreak that affected four persons from a residential institution in an endemic region
	218	for C. difficile in November 2006. The faeces samples of 3 other patients were tested
		for CDI, but were negative.

219

Assays on cultured bacteria

Cytotoxic

In your reaction you advised us to revise our manuscript and to come up with convincing data on the adequacy of the procedure and resubmit before 23-01-2010. We have gone back to total nucleic acid extracts from the original stool samples, made within 3 days of arrival in the laboratory and stored at -80°C. These extracts were tested by PCR targeting the non-repeat region of the tcdB gene. Only the 2 samples that we identified as positives by VIDAS and cytotoxicity assay were PCR positive. We think this provides convincing evidence that the lack of confirmation of PTAB positivity is not due to storage losses. We have added this new data to the manuscript.

Please find in this letter the response to the questions and points raised by the reviewer.

In the revised manuscript we have made the required changes (indicated in red). We hope that our comments meet your expectations and that the manuscript is now acceptable for publication in the European Journal of Clinical Microbiology & Infectious Diseases.

Response to: Clostridium difficile is not associated with outbreaks of viral gastroenteritis in elderly in the Netherlands (EJCMID-D-09-00481Version For that purpose we have included the desired control groups of ill patients who were not infected with noroviruses, but with rotaviruses or of unexplained etiologies (material and methods section, page 4, lines 85-100). A matched group of non-ill patients would complete the study, however this was a retrospective study on patients suspected of viral gastroenteritis from residential institutions for elderly.

We were not able to obtain stool specimens from this group from the same period. Furthermore, we should note that we investigated outbreaks of gastroenteritis, meaning that at least 2 patients which were linked in time and space and had two or more episodes of vomiting or diarrhea were reported and tested.

-The methods for viral detection were not described or adequately referenced.

We added the reference for detection of viral pathogens in gastroenteritis outbreaks

that is used at our institute, we also briefly described the order of testing, see page 4, lines 88-92. We added "Viral detection as been performed as described previously by Svraka et al. [START_REF] Svraka | Etiological role of viruses in outbreaks of acute gastroenteritis in 290 The Netherlands from 1994 through 2005[END_REF], the outbreaks were firstly tested using the assays for noroviruses, all outbreaks that remained negative were further tested using the assays for rotaviruses, adenoviruses, astroviruses, and sapoviruses [START_REF] Svraka | Etiological role of viruses in outbreaks of acute gastroenteritis in 290 The Netherlands from 1994 through 2005[END_REF]."

-The major limitation of this study is the length of time between collection of stools and testing for Cdiff (up to 16 months). It is difficult to interpret the low detection of Cdiff in this cohort because there is no literature demonstrating that Cdiff toxins are stable for this long under these conditions. While the authors compared stools stored for 5 months at 4 deg vs -20 deg, the ideal comparison would have been stools stored at 4 deg for this duration of time vs. stools processed within 48 hours.

We agree that the ideal comparison would have been comparison of stools that are processed within 48 hours and those stored at 4 degrees, and that this meets clostridium criteria, however in this study we tested stool specimens sent for norovirus detection, and storage conditions for samples suspected of norovirus infections are set on 4ºC. After performing diagnostic assays for viral pathogens causing gastroenteritis, we tested these samples for presence of C. difficile. Because of this we decided to split a cohort of obtained specimens, store them at 4C and -20C and to compare the results. Storage at 4C did not lead to lower number of positive samples, moreover we detected three positive samples in stead of two that were found when stored at -20C. To our knowledge clinical laboratories freeze stool samples prior to PTAB testing to prevent false negative results. In our study we are dealing with the false positive results obtained using the EIA, which, to our knowledge, has never been published. So, we were expecting that the sensitivity would be lower, but we did not expect that the specificity would change. Of the 154 total nucleic acids that had been isolated from stool samples within three days of the arrival, the two samples that were cytotoxic and VIDAS positive were positive by tcdB real-time PCR assay (Table I samples 13/8 and 23/18). All other samples were negative."), and discussion (lines 248-252 we added "Furthermore, testing of original total nucleic acids isolated from fecal samples within three days of arrival yielded the same positives as the combination of cytotoxicity test and VIDAS. Therefore, we conclude that our findings are not due to degradation of C. difficile toxins [START_REF] Borriello | Evaluation of a 361 commercial enzyme immunoassay kit for the detection of Clostridium difficile toxin 362 A[END_REF][START_REF] Turcios | 364 Reevaluation of epidemiological criteria for identifying outbreaks of acute 365 gastroenteritis due to norovirus: United States, 1998-2000[END_REF].")

Results:

-Again, it is difficult to interpret the results because the toxin may have degraded under the storage conditions for such a prolonged period of time. See comment mentioned above. We did not find any samples positive in the realtime PCR that were negative when EIA was used, therefore we strongly believe that data we present are true. In result section lines 218-222 we added "Evaluation of the approach of Clostridium detection Of the 154 total nucleic acids that had been isolated from stool samples within three days of the arrival, the two samples that were cytotoxic and VIDAS positive were positive by tcdB real-time PCR assay (Table I samples Discussion: -The last concluding sentence probably needs to be rephrased because the limitations of the methods are so substantial. The title also needs to be revised because it is misleading since the data are so difficult to interpret.

Based on the additional data provided here, we think that our conclusions are valid. Therefore we have not modified the title.


MD Michael Dall
email: mdall@health.sdu.dk

Ove B Schaffalitzky De Muckadell

Annmarie Touborg Lassen

Jesper Hallas

Is there an association between selective serotonin reuptake inhibitor use and uncomplicated peptic ulcers? -A population-based case-control study

Keywords: Peptic ulcer disease < Disease-based, Epidemiology < Topics, Endoscopy < Topics, X SSRI, selective serotonin reuptake inhibitor, Uncomplicated peptic ulcer, UPU, Upper gastrointestinal bleeding, UGIB, proton pump inhibitor

Is there an association between selective serotonin reuptake inhibitor

It is unknown if SSRIs has a direct ulcerogenic effect. The aim of this study was to investigate if there is a possible association between use of SSRIs and uncomplicated peptic ulcers (UPU).

Methods: A population based case-control study was conducted in the county of Funen, Denmark, using local prescription database and patient register. The 4862 cases all had a first diagnosis of UPU from 1995 to 2009. Controls (n=19448), matched for age and sex, were selected by risk-set sampling.

Results:

The adjusted odds ratio (OR) of UPU among current, recent, and past users of SSRIs were 1.50 (95% CI 1.18-1.90), 1.56 (95% CI 0.98-2.49), and 1.32 (95% CI 1.08-1.61). There was no association with tricyclic antidepressants (OR 0.94 (95% CI 0.65-1.35)). The adjusted OR for the SSRI-UPU association was 0.76 (95% CI 0.46-1.25) among users of proton pump inhibitors. Conclusions: Use of SSRI was associated with UPU, possibly by some effect on the healing process. We cannot exclude some effect of residual confounding or bias by frequent physician contact.

Ethics: According to Danish law, neither Ethics Board review nor patient consent is required for register studies.The study was approved by the Danish Data Protection Agency.

Introduction

In the last decade, a number of studies have reported an association between use of selective serotonin reuptake inhibitors (SSRIs) and serious upper gastrointestinal bleeding (UGB) [1][START_REF] De Abajo | Association between selective serotonin reuptake inhibitors and upper gastrointestinal bleeding: a population-based case-control study[END_REF][START_REF] Van Walraven | Inhibition of serotonin reuptake by antidepressants and upper gastrointestinal bleeding in elderly patients: retrospective cohort study[END_REF][START_REF] Dalton | Use of selective serotonin reuptake inhibitors and risk of upper gastrointestinal tract bleeding: a population-based cohort study[END_REF][START_REF] Tata | Does concurrent prescription of selective serotonin reuptake inhibitors and non-steroidal antiinflammatory drugs substantially increase the risk of upper gastrointestinal bleeding?[END_REF][6][START_REF] Opatrny | Gastro-intestinal haemorrhage risks of selective serotonin receptor antagonist therapy: a new look[END_REF][START_REF] Lewis | Moderate and high affinity serotonin reuptake inhibitors increase the risk of upper gastrointestinal toxicity[END_REF][START_REF] De Abajo | Risk of upper gastrointestinal tract bleeding associated with selective serotonin reuptake inhibitors and venlafaxine therapy: interaction with nonsteroidal anti-inflammatory drugs and effect of acid-suppressing agents[END_REF][START_REF] Targownik | Selective serotonin reuptake inhibitors are associated with a modest increase in the risk of upper gastrointestinal bleeding[END_REF] but others have not. [START_REF] Dunn | Association between SSRIs and 168[END_REF][12][13] Some have found an interaction between concurrent use of non-steroidal antiinflammatory drug (NSAID) and SSRI. 1,[START_REF] De Abajo | Association between selective serotonin reuptake inhibitors and upper gastrointestinal bleeding: a population-based case-control study[END_REF][START_REF] Dalton | Use of selective serotonin reuptake inhibitors and risk of upper gastrointestinal tract bleeding: a population-based cohort study[END_REF]6,[START_REF] De Abajo | Risk of upper gastrointestinal tract bleeding associated with selective serotonin reuptake inhibitors and venlafaxine therapy: interaction with nonsteroidal anti-inflammatory drugs and effect of acid-suppressing agents[END_REF]12 The pharmacological mechanism underlying this adverse effect is thought to depend on the release of serotonin in platelets, which is important for their aggregation process. [START_REF] Li | Effects of serotonin on platelet activation in whole blood[END_REF] Platelets are unable to synthesize serotonin, but can acquire it from the circulation by serotonin transporters. [START_REF] Skop | Potential vascular and bleeding complications of treatment with selective serotonin reuptake inhibitors[END_REF] SSRIs inhibit these transporters and will lower the level of serotonin inside the platelets after a short duration of treatment. [START_REF] Javors | Reduction of platelet serotonin content in depressed patients treated with either paroxetine or desipramine[END_REF] It is unknown if processes other than the anti-thrombocytic effect might be involved in the SSRI-induced UGB, and it is unknown if SSRIs has a direct ulcerogenic effect. We undertook this case-control study to assess a possible association between SSRIs and uncomplicated peptic ulcers (UPU). If the anti-aggregatory effect were the sole mechanism involved, we would not expect an association between SSRIs and UPU. To our knowledge, such an association has not been investigated before. until January 1994, ICD-10 thereafter. [START_REF] Gaist | The Danish Prescription Registries[END_REF] system and defined daily doses (DDD) methodology. [START_REF]ATC index with DDDs and guidelines for ATC classification and DDD assignment[END_REF] The indication for treatment and the dosing instruction are not recorded. Drugs not reimbursed and therefore not recorded in the database are over-the-counter drugs and some non-reimbursed prescription drugs, mainly oral contraceptives, hypnotics, sedatives and some antibiotics. [START_REF] Gaist | The Danish Prescription Registries[END_REF] We used the Central Person Register [START_REF] Frank | When an entire country is a cohort[END_REF] to extract the controls and to ensure that all cases and controls were Funen County residents on their index dates and during the previous 365 days before it.

All these data were linked by using the mutual person identifier, the Central Person Registry (CPR)-code, which is shared with virtually all other health related registries in Denmark, thereby allowing record-linkage studies. [START_REF] Frank | When an entire country is a cohort[END_REF] The study was approved by the Danish Data Protection Agency. An ethics committee approval was not required.

Cases and controls

• Cases (n=4862) were patients who had a diagnosis of UPU, either on an in-patient or an out-patient basis on one of the county's hospitals between January the 1 st 1995 and January 1 st 2009. All UPUs were verified by endoscopy. Only the first episode within this time-window was counted. Cases were subdivided into three categories of UPU: Uncomplicated duodenal/gastro duodenal ulcers: ICD8: 53291, 53299, 53391, 53399, ICD10: K26.3, K26.7, K26.9, K27.3, K27.7 and K27.9.

• Uncomplicated gastric ulcers: ICD8: 53191, 53193, 53194, 53196, 53198, 53199, ICD10: K25.3, K25.7 and K25.9.

• Others (anastomosis ulcers): ICD8: 53491, 53499, ICD10: K28.3, K28.7 and K28.9.

Cases were given an index date equivalent to the first registered date of an UPU diagnosis. Controls (n=19448) were matched for age-and sex and sampled by use of a risk set sampling technique. In brief, four controls for each case were randomly selected among those within the county who matched the case with respect to gender and exact birth year. The controls were assigned an index date identical to the admission date of the corresponding case. Both cases and controls were required to have been residents of the Funen County (from January 1 st 2007; the Region of Southern Denmark) for at least one year on the index date. Cases were eligible as control subjects until their first diagnosis of UPU. By this sampling technique, the generated odd ratios will be unbiased estimates of the incidence rate ratios. [START_REF] Rothman | Epidemiology: an introduction[END_REF]

Case validation

We performed a manual review of 500 clinical records randomly chosen among our cases. An UPU was defined as having a mucosal break that was [START_REF] Bernersen | Towards a true prevalence of peptic ulcer: the Sørreisa gastrointestinal disorder study[END_REF] Not all physicians recorded the size of the ulcera in millimetres but almost everybody described it in manners of e.g. the size of a coin or the severity as "stenosis or narrowing of the bulb". We decided to include such examples as valid UPUs. 310/500 (62%) of UPU diagnoses could be verified, and 163/500 (32.6%) did not meet our definition of an UPU. 27/500 (5.4%) could not be accounted for because information was not available in the patient's records or the records were not retrievable.

We tested whether the non-valid cases were different from the valid cases with respect to their SSRI use and found no difference (p=0.69, chi-square)

Exposure definition

All the relevant SSRI doses are available on the Danish market as single tablets. We defined the exposure based on the assumption that the majority of patients would take one tablet per day. This assumption has been validated previously. [START_REF] Rosholm | Are there differences in the use of seelctive serotonin reuptake inhibitors and tricyclic antidepressants? A prescription database study[END_REF] To allow for minor non-compliance and for irregular prescription refills, the exposure period were defined as days equivalent to the number of tablets in the dispensed package plus 20%. The exposure period started the day the prescription was redeemed, and the exposure clock was reset with each new prescription. "Recent exposure" was defined as the first 90 days after the expiry of an exposure period. The following period was defined as "past exposure". We undertook a sensitivity analysis with the grace period assigned to each prescription of 10%, 30% and 40% instead of the 20% used in the primary analysis. Results differed very little from the main analysis (data not shown).

Unless otherwise stated, analyses were based on current exposure, and the reference was person-time never-exposed to SSRI. "SSRI" refers to citalopram (N06AB04), escitalopram (N06AB10), fluoxetine (N06AB03), fluvoxamin (N06AB08), paroxetin (N06AB05) and sertraline (N06AB06).

Data analysis

The crude and adjusted ORs with 95% confidence intervals (CI), when relevant, were calculated by use of conditional logistic regression with adjustment for the potential confounders. Age, gender and calendar year were accounted by the study design.

A number of potential confounders were considered;

From FPAS, we retrieved information on the following diagnosis: previous From OPED we retrieved data on use of the following drugs: NSAIDs (ATC M01A), low dose acetylsalicylic acid (ASA) (ATC B01AC06), oral anticoagulants (ATC B01AA), proton pump inhibitors (PPI) (ATC A02BC), H2 receptor antagonist (ATC A02BA), nitrate vasodilators (ATC C01DA), ever use of antidiabetics (ATC A10), ever use of antihypertensive agents (ATC C03A, C07, C08, C09), ever use of systemic beta-agonists or inhaled anticholinergics (ATC R03C and R03BB), use of disulfiram (ATC N07BB01), statins (ATC C10AA), spironolactone (ATC C03DA01). For all drug exposures except SSRIs, current exposure was defined by the redeeming of a prescription within the past 90 days.

The confounders for the final model were selected by bivariate regression analysis. Potential confounders that changed the OR for SSRI by more than 5%, when added to a model that included only SSRI and case status, were all included. Such confounders were PPI (ATC A02BC), alcohol abuse (ICD8 303; ICD10-F10) and past peptic ulcer (ICD8-533; ICD10-K27). On The dose-response relationship was evaluated both in terms of daily dose taken and cumulative dose before the index date. To account for the difference in potency between SSRI, the Prescribed Daily Dose (PDD) was expressed in units of defined daily doses. [START_REF]ATC index with DDDs and guidelines for ATC classification and DDD assignment[END_REF] Intervals were defined by ≤0.5 DDD/day, 0.5-1.0 DDD/day and ≥1.0 DDD/day. We defined the index date for the cases as being equivalent to the first registered date of an UPU diagnosis. An UPU could have been present a longer period before the endoscopy was performed. We performed a sensitivity analysis by redefining our index date to occur one and two months before the endoscopy, respectively.

The risk associated with current use of SSRI was expressed in absolute terms by using the NNTH principle ("the number of patients needed to be treated for one additional patient to be harmed"). [START_REF] Bjerre | Expressing the magnitude of adverse effects in case-control studies[END_REF] The adjusted odds ratios (OR) and the incidence of UPU in the background population unexposed to SSRI and aged 18 or above (ER unexp ) were used. NNTH is calculated by the formula; NNTH = 1/(ER unexp ×(OR -1)). [START_REF] Bjerre | Expressing the magnitude of adverse effects in case-control studies[END_REF] We chose to exclude persons Regarding the dose-response association we found the highest adjusted OR for ≥ 1.0 DDD per day (1.93 (95% CI 1.05-3.57)). Lower doses were associated with lower risk; the OR was 1.48 (95% CI 1.13-1.94) and 0.96 (95% CI 0.43-2.15) for doses of 0.5-1.00 DDD per day and ≤0.5 DDD per day, respectively. For the duration of SSRI use we found the highest adjusted OR for those current users who had started SSRI within the past 30 days, 3.14 (95% CI 1.20-8.18), while a duration in excess of 180 days was associated with a lower OR, 1.40 (95% CI 1.07-1.82)(table 2).

The association between SSRI and UPU in subgroups of patients is shown in table 3. We found an OR of 2.16 (95% CI 1.44-3.24) in the group of ≥55 and <75 years of age and also an elevated OR (1.71 95% CI 1.04-2.80) in the group below 55 years of age and no significant association in the group above 75 years of age (OR 1.07 95% CI 0.75-1.54). There was no effect modification by gender. There was no association between SSRI and UPU among users of PPI (OR; 0.76 95% CI (0.46 -1.25))

The SSRI-UPU association did not differ by ulcer site. The adjusted estimates for gastric, duodenal or other ulcers were 1.50 (95% CI 1.13-2.00) and 1.43 (95% CI 0.93-2.20), and 1.67 (95% CI 0.12-21.3).

The sensitivity analyses based on redefined index dates showed results, similar to the main analysis (OR; 1.50 (95% CI 1. 18 We found 4538 cases aged 18 or above who were unexposed to SSRI, accrued from 5.203.530 person years of follow-up in our background population. The resulting unexposed event rate (ER unexp ) was 0.87 per 1000 person years. NNTH were calculated to 2300 person years (95% CI 1300-6400).

Discussion

In this large case-control study, patients with current use of SSRI had a moderate risk of being diagnosed with UPU. The increasing OR suggests a dose-response effect and a particularly high risk if the SSRI treatment is newly started. We found no association with current use of TCAs. Patients taking PPI appeared to be protected against SSRI-induced UPU.

The current study has several strengths. It is a true population-based study which has full coverage of admission and prescription data. Secondly we have manually validated approximately 10% of the case material. Thirdly; with the current data we are able to account for a large number of covariates that are potential confounders.

Among the sources of error, we need to consider information bias, selection bias and residual confounding by variables that are not accounted for. Unfortunately, not all UPU diagnoses were valid. Our validity of 61% was on the level of what other researchers have reported [START_REF] Lassen | Eradication of Helicobacter pylori and use of antisecretory drugs: population based cohort study[END_REF] . It is unlikely that the validity of a UPU diagnosis would depend on whether the subject was an SSRI user. Thereby, the inclusion of non-valid cases is a non-differential misclassification bias which would tend to dilute the ORs towards unity. [START_REF] Rothman | Epidemiology: an introduction[END_REF] In other words, the true OR without this bias would have been higher. We have performed a crude sensitivity analysis [START_REF] Rothman | Spreadsheets for the analysis of Epidemiologic Data[END_REF] which showed the OR to be fairly robust over a reasonable range of assumed validity for the diagnoses (data not shown).

It is conceivable that the frequent physician contact entailed in having depression or being treated with SSRIs would lead to endoscopy referral for ulcer-related complaints that would otherwise not come to medical attention. This could generate a spurious SSRI-UPU association. A Norwegian study has showed that if an endoscopy is performed among healthy people, 7.4 % of the males and 4.6 of the women had peptic ulcers. [START_REF] Bernersen | Towards a true prevalence of peptic ulcer: the Sørreisa gastrointestinal disorder study[END_REF] We attempted to address this "frequent physician contact" bias by analysing the association between anti-hypertensive drugs and UPU, as these drugs were thought to entail frequent physician contact as well. The adjusted ORs for ACEinhibitors, calcium antagonists and thiazides, estimated by the same techniques as for SSRIs, were 1.20 (95% CI 1.02-1.42), 1.29 (95% CI 1.07-1.56) and 1.16 (95% CI 0.93-1.45), respectively. We thus cannot exclude As with any observational study, residual confounding is a possibility. Table 1 shows that cases and controls differ substantially in their prevalence of traits that are risk factors or proxies for risk factors for UPU. As they should in an observational study. However, these risk factors will only bias our estimates to the extent that they are unevenly distributed between users and non-users of SSRIs. In our adjustment, we have included all variables that could be demonstrated as having a small confounding effect, and we have added the known strong risk factors for UPU. We believe it is unlikely that there is strong confounding by variables that have been registered. However, the use of a registry-based approach renders our study vulnerable to potential confounders that are not covered by our databases; alcohol, smoking, and use of ulcerogenic over-the-counter (OTC) drugs. Users of SSRI smoke more than others [START_REF] Bak | Selective serotonin reuptake inhibitors and the risk of stroke: A population based casecontrol study[END_REF] , but smoking is by itself only a modest risk factor for UPUs. [START_REF] Kurata | Meta-analysis of risk factors for peptic ulcer. Nonsteroidal antiinflammatory drugs, Helicobacter pylori, and smoking[END_REF] In addition, our estimates were not altered by the inclusion and exclusion of COPD in our model, i.e., a crude proxy for heavy smoking (OR 1.50 vs 1.49). Thus smoking is unlikely to confound our findings substantially. The studies we have been able to locate on alcohol and UPU report no association. [START_REF] Adami | Is duodenal ulcer really a psychosomatic disease? A population-based case-control study[END_REF][START_REF] Friedman | Cigarettes, alcohol, coffee and peptic ulcer[END_REF] Alcohol abuse is a risk factor for bleeding ulcers but the risk is low with moderate to low intake. [START_REF] Kaufman | The risk of acute major upper gastrointestinal bleeding among users of aspirin and ibuprofen at various levels of alcohol consumption[END_REF] We found a SSRI- 3) and we cannot exclude some confounding by this covariate.

High and low dose ASA and some NSAIDs are sold over the counter (OTC), which is not covered by our prescription database. To account for OTC sales, we compared the quantity registered in our database with the available data on total sales. The estimated coverage of our database for high dose ASA, low dose (<= 150 mg) ASA and NSAIDs was 4%, 90.5% and 84.3%, respectively. The gross sales volume of high-dose ASA was low, compared to low-dose ASA and NSAID; 11.0, 65.3 and 37.9 defined daily doses per 1000 inhabitants per day. 33 Although our prescription database thus has captured the major part of ulcerogenic drug use, we cannot exclude some residual confounding.

We also need to consider the indication for SSRIs as a source of confounding. To our knowledge, there is no known association between depression per se and UPU. In addition, we found no increased risk among users of tricyclic antidepressants, which have different pharmacological actions but are largely used for the same indications.

Could our results reflect a direct ulcerative effect by SSRI or is it mediated by its well known effect on the platelets? We have not been able to find any studies that could indicate a genuine ulcerative effect, but the function of the platelets in the process of healing gastric ulcers has been studied by some groups. [START_REF] Wallace | Platelets accelerate gastric ulcer healing through presentation of vascular endothelial growth factor[END_REF] Wallace et al showed in an animal model that the healing process of peptic ulcers was accelerated by vascular endothelial growth factor (VEGF), which is present in the platelets. It has also been shown that plug formation by platelets is inhibited by paroxetine. [START_REF] Hergovich | Paroxetine decreases platelet serotonin storage and platelet function in human beings[END_REF] This is a part of the same process already described by other groups. [START_REF] Li | Effects of serotonin on platelet activation in whole blood[END_REF][START_REF] Skop | Potential vascular and bleeding complications of treatment with selective serotonin reuptake inhibitors[END_REF][START_REF] Wägner | Effects of fluoxetine treatment of platelet H-imipramine binding, 5-HT uptake and 5-HT content in major depressive disorder[END_REF] Wägner et al described that blocking of the serotonin transporters on the surface of the platelets by fluoxetine resulted in lowering of the serotonin concentration inside the platelets. If the platelets already are affected by SSRIs this could induce a possible malfunction which would result in an indirect inhibition of ulcers healing by the lack of VEGF and possibly a delayed plug formation. This would thereby be a feasible mechanism and explanation for the association found between use of SSRIs and UPUs.

In addition we also found the highest adjusted OR for the most recent commencement of SSRI. Platelets are depleted of serotonin [START_REF] Wägner | Effects of fluoxetine treatment of platelet H-imipramine binding, 5-HT uptake and 5-HT content in major depressive disorder[END_REF] in the first week of treatment but we are not aware of any pharmacological explanation for the initial elevated risk for UPUs unless this could be an indication of a direct ulcerogenic effect. Another possible explanation could be a "depletion of susceptibles" phenomenon also observed with NSAIDs and bleeding ulcers; the highly sensitive subjects develop their bleeding early and the rest [START_REF] Moride | Evidence of the depletion of susceptibles effect in non-experimental pharmacoepidemiologic research[END_REF] Finally, we found a higher OR for paroxetine than for the other SSRIs. The reason for this is unknown. Paroxetine has a very high affinity among the SSRIs examined in this study and this could be the reason for the magnitude of the OR that we find. [START_REF] De Abajo | Antidepressants and risk of upper gastrointestinal bleeding[END_REF] However, it should be mentioned that this was not a pre-specified hypothesis, and it should be corroborated by others before any inferences can be made.

Given the strengths and weaknesses of our setting and the direction and strength of known biases, we believe that the association reported here reflects a genuine biological effect of SSRIs. We cannot exclude some effect of residual confounding or bias by frequent physician contact. Presently, we cannot know which underlying pharmacological action is responsible. Its clinical implications are also uncertain; the high NNTH values indicate that this is not something that should be considered routinely when prescribing SSRIs. In table 3 we found that among users of PPI, there is no association between SSRI and UPU. However, there might be a problem in high risk groups where prophylactic use of PPIs might be relevant. Possibly, PPI may be a relevant measure against SSRI-induced UPU. Further studies are needed in this area.

F

3

 3 Conflict of interest:J.H. has received fees for teaching from AstraZeneca, MSD and the Danish Association of the pharmaceutical Industry, and research grants from Novartis, Pfizer and Nycomed. O.SdeM. has received fees for teaching from AstraZeneca.All other authors: none declared.

  : Persons who use serotonin reuptake inhibitors (SSRIs) seems at increased risk of having serious upper gastrointestinal bleeding. In vitro studies have shown that SSRIs inhibit platelet aggregation.

  this study were retrieved from three different sources, the Funen County Patient Administrative System (FPAS) Odense University Pharmaco-epidemiological Database (OPED) and the Danish Central Person Register (CPR). Data on patient contacts were retrieved from FPAS. All Funen County (population 470,000) residents have all their discharges from hospitals registered since 1973 and out-patient contacts since 1989. Diagnoses are encoded by the International Classification of Diseases-8 th version (ICD-8)

  ICD-9 has not been used in Denmark. Since in-patient care is furnished almost exclusively by the national health services, these data sources allow in effect true population based epidemiological studies. Information on reimbursed drug dispensing in the County of Funen has been recorded in the OPED since 1990 and in the Region of Southern Denmark (population 1.2 million) since the 1st of January 2007.Each prescription record includes a person identifier, the date of dispensing, the brand, quantity, and form of the drug. The substances and quantities are according to WHO's anatomical-therapeutic-chemical (ATC)

  with appreciable depth.

  gastric ulcer (ICD8-531; ICD10-K25), duodenal ulcer (ICD8-532; ICD10-K26), gastroduodenal ulcers (ICD8-533; ICD10-K27), gastritis and duodenitis (ICD8-535; ICD10-K29), a diagnosis of diabetes (ICD8-250, ICD10 E10-14), hypertension (ICD8-40, ICD10-I10), congestive heart -427; ICD10-I50), ischemic heart disease or acute myocardial infarction (ICD8 412-414; ICD10 I20-25), cerebral ischemia or stroke (ICD8-431, 433-435; ICD10 I61, I63 and I64 excluding I631 and 641), chronic obstructive pulmonary disease (COPD) (ICD8 490-491; ICD10-J44), any history of alcohol-related disorder (ICD8 303; ICD10-F10, K70.0-K70.9), and psychiatric disorder (ICD8 295-300; ICD10-F20, 30-33).

  we also included low dose ASA (ATC B01AC06), NSAIDS (ATC M01A), corticosteroids (ATC H02AB), Helicobacter eradication[START_REF] Lassen | Eradication of Helicobacter pylori and use of antisecretory drugs: population based cohort study[END_REF] , past UGB (ICD8 53190, 53290, 53390 and 53490; ICD10 K25.4, K26.4, K27.4 and K28.4) and cirrhosis (ICD8 571; ICD10 K70.0-K70.9), as these were established risk factors for UPU.

  of age from this calculation to avoid inclusion of persons who were unlikely to use SSRIs and unlikely to have UPU and who would dilute the NNTH estimates into unrealistically high values.All the analyses were performed using Stata release 10.1.25 Results 4862 cases were identified, corresponding to an incidence rate of 0.65 per 1000 person years in our background population. 2410 were men (49.6%), and 1122 (23.1%) were 75 years or older (Table1). Known risk factors such as a past history of ulcer, use of aspirin or NSAIDs were more prevalent among cases than controls. However, other co-morbidity was also more prevalent among cases, such as hypertension, ischemic heart disease and COPD. The extent of current PPI use was 3055 (62.8%) and 765 (3.9%), among the 4862 cases and 19448 controls respectively. Other characteristics of cases and controls are tabulated in table1.There were 838 (17.2%) and 1956 (10.1%), among the 4862 cases and 19448 controls respectively, who had ever taken SSRI. The adjusted OR of UPU among respectively current, recent and past users were 1.50 (95% CI 1.18-1.90), 1.56 (95% CI 0.98-2.49) and 1.32 (95% CI 1.08-1.61) (table2). The adjusted OR for tricyclic antidepressant (TCA) was 0.94 (95% CI 0.65-1.35).

  the apparent ulcerogenic effect of SSRIs are attributable to frequent physician contact.

19 UPU

 19 association slightly lower among persons with no markers of alcohol abuse (OR 1.38 CI 95% (1.08-1.80) (table

  Adjusted for; low-dose aspirin, proton pump inhibitors, NSAIDS, alcohol abuse, cerebral ischaemia, stroke, corticosteroids, helicobacter eradication, past peptic ulcer, past upper gastrointestinal bleeding and cirrhosis. Abbreviations: CI; 95% confidence interval, SSRI; selective serotonin reuptake inhibitors Adjusted for; low-dose aspirin, proton pump inhibitors, NSAIDS, alcohol abuse, cerebral ischaemia, stroke,corticosteroids, helicobacter eradication, past peptic ulcer, past upper gastrointestinal bleeding and cirrhosis. Abbreviations: CI; 95% confidence interval; SSRI; selective serotonin reuptake inhibitors, ATC; Anatomical Therapeutic Chemical, NSAID; non-steroidal anti-inflammatory drugs, ASA; acetylsalicylic acid, PPI; proton pump inhibitors, #): ACE inhibitors, calcium antagonist, AT2-antagonist, Beta blockers and Thiazids.

	upper gastrointestinal bleeding. SSRIs are no more likely than other Table 1 disease Table 2 SSRI dose: No hypertension or
	use of
	Characteristics of 4862 cases of uncomplicated ulcers and their 19448 -Peptic ulcer 555 (11.4%) 419 (2.2%) <0.0001 Association between exposure to Selective Serotonin Reuptake Inhibitors ≤0-0.5 DDD per 22 / 4024 71 / 17492 1.36 (0.82 -2.26) 0.96 (0.43 -2.15) antihypertensives 139 / 2630 327 / 13088 2.33 (1.82 -2.99) 1.92 (1.28 -2.88) drugs to cause such bleeding. BMJ 2000;320:1405-06. control subjects. -UGB 154 (3.2%) 92 (0.5%) <0.0001 and Uncomplicated Peptic Ulcers day Alcohol abuse or
	o r P F o r P F o r P Wessinger S, Kaplan M, Choi L et al. Increased use of selective serotonin reuptake inhibitors in patients admitted with gastrointestinal 12. haemorrhage: a multicentre retrospective analysis. Aliment Pharmacol Ther. 2006 Apr 1;23(7):937-44. 13. Vidal X, Ibáñez L, Vendrell L et al. Risk of Upper Gastrointestinal Bleeding and the Degree of Serotonin Reuptake Inhibition by F o r P F o r P F o r P F o r P F o r P Cases (n=4862) Controls (n=19448) p-values Age, mean (SD) 60.7 (±16.2) -Ischemic heart disease 643 (13.2%) 1139 (5.9%) Cases Controls Crude Odds Ratio Adjusted Odds 0.5-1.0 DDD per 246 / 4024 508 / 17492 2.17 (1.84 -2.55) 1.48 (1.13 -1.94) ever use of <0.0001 Uncomplicated 95 % CI Ratio 95% CI disulfiram 66 / 188 46 / 358 1.00 (0.25 -4.00) 1.00 (0.06 -15.99) day Exposed/ 60.7 (±16.2) <55 1772 (36.4%) 7088 (36.4%) -Hepatic cirrhosis 61 (1.3%) 71 (0.4%) <0.0001 Peptic Ulcers Unexposed ≥1.0 DDD per day 56 / 4024 78 / 17492 3.24 (2.24 -4.71) No alcohol abuse or 1.93 (1.05 -3.57) ever use of 1.00000 ≥55 & <74 1968 (40.5%) 7872 (40.5%) ≥75 1122 (23.1%) 4488 (23.1%) Men 2410 (49.6%) 9640 (49.6%) 1.00000 Current drug use (ATC code): -Low-dose aspirin (B01AC06) 461 (9.5%) 1050 (5.4%) <0.0001 F o r P -Heart failure 328 (6.7%) 784 (4.0%) -Hypertension 535 (11.0%) 1191 (6.1%) <0.0001 -Stroke 274 (5.6%) 642 (3.3%) -Alcohol related diagnosis# 329 (6.8%) 468 (2.4%) P Antidepressants P P P <0.0001 r SSRI, past use 432 / 4024 1141 / 17492 1.74 (1.54 -1.96) 1.32 (1.08 -1.61) r >180 days 259 / 4024 554 / 17492 2.08 (1.77 -2.44) 1.40 (1.07 -1.82) r r <0.0001 F o SSRI, current use 324 / 4024 657 / 17492 2.19 (1.90 -2.54) 1.50 (1.18 -1.90) SSRI, recent use 82 / 4024 158 / 17492 2.39 (1.80 -3.18) 1.56 (0.98 -2.49) F o 31-90 days 29 / 4024 41 / 17492 3.10 (1.87 -5.14) 1.62 (0.69 -3.82) 91-180 days 20 / 4024 37 / 17492 2.69 (1.51 -4.81) 2.19 (0.78 -6.16) F o o F <0.0001 1.00000 -Renal failure 62 (1.3%) 74 (0.4%) Exposure: 0-30 days 16 / 4024 25 / 17492 2.71 (1.40 -5.25) 3.14 (1.20 -8.18) <0.0001 1.00000 -Diabetes mellitus 309 (6.4%) 784 (4.0%) <0.0001 Exposed/ Unexposed SSRI duration: disulfiram 258 / 3836 611 / 17134 1.94 (1.65 -2.27) 1.39 (1.08 -1.80)
	e e r R e v i e w e e r R e v i e w e e r R e v i e w Antidepressants : A Case-Control Study. Drug Saf. 2008;31(2):159-e e r R e v i e w e e r R e v i e w e e r R e v i e w e e r R e v i e w e e r R e v i e w -Anticoagulants (B01AA) 109 (2.2%) 317 (1.6%) 0.0036 -NSAIDs (M01A) 838 (17.2%) 1819 (9.4%) e e r -Alcohol related prescriptions (disulfiram) 76 (1.6%) 101 (0.5%) <0.0001 e e r substance: Fluoxetine (N06AB03) 24 / 4024 40 / 17492 2.42 (1.43 -4.10) 1.32 (0.58 -3.00) e e r e e r e e r <0.0001 -Systemic corticosteroids (H02AB) 236 (4.9%) 512 (2.6%) <0.0001 -H2RA (A02BA) 977 (20.1%) 332 (1.7%) <0.0001 -PPIs (A02BC) 3055 (62.8%) 765 (3.9%) <0.0001 -Statins (C10AA) 291 (6.0%) 827 (4.3%) <0.0001 -Nitrates (C01DA) 242 (5.0%) 403 (2.1%) <0.0001 R e v i e w -Alcohol-related diagnosis or drug use 344 (7.1%) 513 (2.6%) <0.0001 #: ICD8; 295-300, ICD10; F10, F20, 30-33 and K70.0-K70.9. Unless otherwise indicated, data are shown in numbers. R e v i e w Citalopram (N06AB04) 172 / 4024 365 / 17492 2.05 (1.69 -2.49) 1.38 (1.00 -1.90) Paroxetine (N06AB05) 30 / 4024 62 / 17492 2.54 (1.57 -4.11) 4.86 (2.28 -10.4) Sertraline (N06AB06) 59 / 4024 122 / 17492 2.23 (1.60 -3.10) 1.50 (0.87 -2.58) Fluvoxamine (N06AB08) 1 / 4024 2 / 17492 1.81 (0.16 -20) 4.57 (0.02 -988) Escitalopram (N06AB10) 38 / 4024 66 / 17492 2.61 (1.71 -4.00) 1.06 (0.52 -2.17) R e v i e w R e v i e w R e v i e w
	Tri-cyclic	100 / 3970	325 / 17289	1.35 (1.07 -1.71)	0.94 (0.65 -1.35)
	-TCA (N06AA) antidepressants	100 (2.1%)		325 (1.7%)	0.0665
	-Spironolactone (N06AA)	112 (2.3%)		171 (0.9%)	<0.0001
	(C03DA01)
	History of:
	-Hp eradication	1276 (26.2%)	207 (1.1%)	<0.0001
	-Chronic	289 (5.9%)		659 (3.4%)	<0.0001
	obstructive lung
						24 25 28 29 30 31 32 34