Professor Robin C Spiller
email: robin.spiller@nottingham.ac.uk

David J Humes

Eugene Campbell

Margaret Hastings

Keith R Neal

George E Dukes

Peter J Whorwell

symptom severity and consulting behaviour in patients with irritable bowel syndrome and symptomatic diverticular disease Running head: PHQ12 SS, IBS, Diverticular disease and consulting behaviour

Keywords: Irritable bowel syndrome < Disease-based, Diverticular disease < Disease-based, Symptom score or index < Topics, Functional GI diseases Irritable bowel, somatisation, anxiety, depression, diverticular disease, diagnosis, sensitivity, specificity PHQ15 Patient Health Questionnaire 15, IBS irritable bowel syndrome, DD diverticular disease, ASYMPDD asymptomatic diverticulosis, SYMPDD symptomatic diverticular disease, IBSSS irritable bowel syndrome symptom severity scale

Introduction

When assessing patients with abdominal pain and disturbed bowel habit associated with normal physical findings, it is recommended to assess psychological variables [START_REF] Spiller | Guidelines on the irritable bowel syndrome: mechanisms and practical management[END_REF]. Many will turn out to have important psychological abnormalities and addressing theses has been shown to improve management and outcomes [START_REF] Owens | The irritable bowel syndrome: long-term prognosis and the physician-patient interaction[END_REF]. Previous studies have tended to emphasise that IBS patients are significantly more anxious and depressed than healthy controls [START_REF] Hu | Anxiety but not depression determines health care-seeking behaviour in Chinese patients with dyspepsia and irritable bowel syndrome: a population-based study[END_REF][START_REF] Whitehead | Systematic review of the comorbidity of irritable bowel syndrome with other disorders: what are the causes and implications?[END_REF][START_REF] Longstreth | Characteristics of patients with irritable bowel syndrome recruited from three sources: implications for clinical trials[END_REF][START_REF] Addolorato | Anxiety and depression: a common feature of health care seeking patients with irritable bowel syndrome and food allergy[END_REF] and have a higher incidence of previous psychiatric disease [START_REF] Irwin | Comorbidity of posttraumatic stress disorder and irritable bowel syndrome[END_REF][START_REF] Walker | Psychiatric diagnoses, sexual and physical victimization, and disability in patients with irritable bowel syndrome or inflammatory bowel disease[END_REF].

Although this is true, anxiety is a major driver of any consultation (9,10) so it is perhaps not surprising that anxiety is not a useful aid in differential diagnosis since it fails to clearly distinguish IBS patients from patients with other gastrointestinal diseases such as colon cancer, coeliac disease , peptic ulcer [START_REF] Van Kerkhoven | Anxiety, depression and psychotropic medication use in patients with persistent upper and lower gastrointestinal symptoms[END_REF], inflammatory bowel disease [START_REF] Addolorato | Inflammatory bowel disease: a study of the association between anxiety and depression, physical morbidity, and nutritional status[END_REF] or indeed most general medical conditions [START_REF] Addolorato | State and trait anxiety and depression in patients affected by gastrointestinal diseases: psychometric evaluation of 1641 patients referred to an internal medicine outpatient setting[END_REF] [START_REF] Esler | Levels of anxiety in colonic disorders[END_REF].

There is increasing recognition of the importance of somatisation in determining health care utilisation in IBS [START_REF] North | The presentation of irritable bowel syndrome in the context of somatization disorder[END_REF]. Somatisation with multiple somatic complaints is also important in understanding mechanisms of disease since IBS patients can be usefully subdivided into those with low somatisation, in whom there is a predominantly GI cause for their symptoms and those with high somatisation, in which a central cause, associated with visceral hypersensitivity, predominates [START_REF] Whitehead | Systematic review of the comorbidity of irritable bowel syndrome with other disorders: What are the causes and implications?[END_REF][START_REF] Guthrie | Cluster analysis of symptoms and health seeking behaviour differentiates subgroups of patients with severe irritable bowel syndrome[END_REF]. Patients also feel these symptoms are important as recently reported in a survey of 755 IBS patients which showed that non-GI pains were one of the factors along with pain, illness belief and defaecatory difficulties which contribute to patient-assessed severity [START_REF] Spiegel | Predictors of patientassessed illness severity in irritable bowel syndrome[END_REF].

While somatisation meeting the criteria laid down in the Diagnostic and Statistical

Manual for psychiatric disease (DSM-IV) is uncommon, being found in only 1-2% of primary care patients, a lesser condition of multiple unexplained somatic symptoms, the Physical Symptom Disorder as detected by the Patient Health Questionnaire 15 [START_REF] Kroenke | Physical symptom disorder: a simpler diagnostic category for somatization-spectrum conditions[END_REF] has an incidence of 10-15% in patients consulting in primary care [START_REF] Escobar | Abridged somatization: a study in primary care[END_REF]. The Patient symptoms including somatic symptoms such as back ache, limb pain and chest pain, cardiovascular system symptoms such as palpitations and breathlessness as well as three gastrointestinal (GI) symptoms, namely abdominal pain, nausea and disordered bowel function, together with sexual dysfunction, lethargy and headaches (Table 1) [START_REF] Kroenke | The PHQ-15: validity of a new measure for evaluating the severity of somatic symptoms[END_REF].

Clinical experience with this instrument in primary care, internal medicine and gynaecology clinics shows it correlates well with symptom severity across a range of conditions [START_REF] Kroenke | The PHQ-15: validity of a new measure for evaluating the severity of somatic symptoms[END_REF]. Our own experience using this instrument in our clinic over the last 4 years suggested that IBS patients score highly, even when it is modified by excluding the 3 GI specific questions, a scale we call the PHQ-12. We believe that this is useful because it detects a sensitisation to painful and other somatic non-GI stimuli which is an important feature of many IBS patients [START_REF] Zhou | Central and peripheral hypersensitivity in the irritable bowel syndrome[END_REF][START_REF] Verne | Hypersensitivity to visceral and cutaneous pain in the irritable bowel syndrome[END_REF].

The aim of this study, therefore, was to assess the value of the PHQ-12 score not only in separating out IBS from other patients but also in predicting both gastrointestinal symptom severity and patient consulting behaviour.

As a disease control with disturbed bowel habit and pain we also included a group of older patients with diverticulosis, some of whom were asymptomatic (ASYMPDD) who provided age-matched controls for those who were symptomatic (SYMPDD).

F

IBS patients

We recruited 148 IBS with diarrhoea (D-IBS) and 132 IBS with constipation (C-IBS) and 39 patients with a history compatible with post-infective IBS (PI-IBS) from our outpatient clinics but excluded those with mixed bowel habits [START_REF] Longstreth | Functional bowel disorders[END_REF]. These had all completed full negative evaluation for other diseases in the Gastroenterology Out-Patient Clinic and were then invited to take part in the study. As recruitment of IBS patients continued we also recruited a total of 151 age and gender matched healthy volunteers.

Diverticulosis patients

We also recruited 296 patients with diverticulosis as a disease control. These patients were identified from our database which includes patients being seen in Nottingham University Hospital NHS Trust with a diagnosis of diverticulosis made either by radiological or endoscopic means as well as from the membership of the National Diverticulitis support group. Of these 113 were asymptomatic without any abdominal pain or discomfort [ASYMPDD] and 173 had abdominal pain or discomfort [SYMPDD]).

Questionnaires

All IBS patients completed the IBS Severity Score questionnaire (IBSS), a simple 5 point scale which uses visual analogue scales to express severity of pain, bloating, as well as dissatisfaction with bowel habit and how much IBS symptoms interference with life [START_REF] Francis | The irritable bowel severity scoring system: a simple method of monitoring irritable bowel syndrome and its progress[END_REF].

We also used the Hospital Anxiety and Depression Scale (HAD) [START_REF] Zigmond | The hospital anxiety and depression scale[END_REF]

Results

Demographics and bowel symptoms (Table 2)

In accordance with our planned age and sex matching there are no differences in age and gender between our healthy volunteers (HV) and the IBS groups except that C-IBS were slightly more likely to be female (93%) compared to PI-IBS (70%) and D-IBS (67.2%), Chi squared p<0.001. Symptomatic diverticular patients (SYMPDD) were likewise female predominant (78%) while in the asymptomatic DD patient group (ASYMPDD) gender was more even with only 56% female (Chi squared p<0.0001). The diverticular patients as expected were significantly older than the IBS and HV groups.

As required by the IBS classification used there were significant differences in bowel frequency and the proportion of days with loose and hard stool in the IBS subgroups.

The diverticular patients had significantly more days with both loose and hard stool than healthy volunteers, even those patients not troubled by abdominal pain. SYMDD had fewer days pain that both CIBS and DIBS, both p<0.001. By definition ASYMDD did not report recurrent abdominal pain.

Somatisation scores

The most striking difference was the clear separation of the 3 groups of IBS patients by the PHQ-12 SS scores (Figure 1, Table 3 and Supplementary Figure 1) from both healthy volunteers, and from the ASYMPDD group.

A receiver-operating curve comparing all IBS patients with HV (Figure 2) gave an ROC score of 0.88. A cut-off of >6 was chosen to give the best combination of sensitivity 66.4% and specificity 94.7%. This gave an excellent Positive Likelihood Ratio of 13.2, with a Negative Likelihood Ratio of 0.3.

Using the cut off of >6 (

Performance of anxiety and depression scores

Although anxiety and depression did differ between the IBS groups and healthy volunteers there was a greater overlap than for PHQ-12 SS (Table 3 &4). The ROC area under the curve for anxiety was less than for the PHQ-12 SS at 0.8 and, using the standard cut off of >7, sensitivity was 72.4% and specificity 74.8% for all IBS versus HV. The PLH was less good than for the PHQ-12 SS at 2.9 and a NLR of 0.4. The ROC area for depression was similar at 0.8 but using the standard cut off of >7 gave a sensitivity of only 36% with specificity of 93.4% (PLR 5.5 and a NLR 0.7). Thus the PHQ-12 SS score was approximately three times as likely to be abnormally high in IBS than depression and 1.3X as likely to be elevated compared to anxiety.

As Table 4 We believe that a high PHQ-12 SS indicates heightened awareness of bodily symptoms.

Earlier studies suggested that visceral hypersensitivity(VH) is a significant component in at least 50% of IBS patients who experience a range of stimuli such as rectal distension or colonic contraction as more painful than normal subjects [START_REF] Mertz | Altered rectal perception is a biological marker of patients with irritable bowel syndrome[END_REF][START_REF] Azpiroz | Mechanisms of hypersensitivity in IBS and functional disorders[END_REF][START_REF] Azpiroz | Mechanisms of hypersensitivity in IBS and functional disorders[END_REF][START_REF] Camilleri | Testing the sensitivity hypothesis in practice: tools and methods, assumptions and pitfalls[END_REF]. These studies suggested that VH did not extend to somatic pain, however more recently the overlap of IBS with fibromyalgia has been recognised and there have been some major advances in understanding the neurological basis of VH which may reflect defective antinociceptive systems [START_REF] Coffin | Alteration of the spinal modulation of nociceptive processing in patients with irritable bowel syndrome[END_REF][START_REF] Wilder-Smith | Brain functional magnetic resonance imaging of rectal pain and activation of endogenous inhibitory mechanisms in irritable bowel syndrome patient subgroups and healthy controls[END_REF][START_REF] Tracey | Importance of anti-and pro-nociceptive mechanisms in human disease[END_REF]. The descending inhibitory pathways, which are normally activated to reduce sensation from the viscera, appear defective in IBS. Some of the most effective treatments in IBS, the tricyclic antidepressants, have been shown to produce changes in brain activation suggesting they may restore this anti-nociceptive activity [START_REF] Morgan | Amitriptyline reduces rectal pain related activation of the anterior cingulate cortex in patients with irritable bowel syndrome[END_REF]. A similar defect may underlie fibromyalgia [START_REF] Bradley | Use of neuroimaging to understand abnormal pain sensitivity in fibromyalgia[END_REF]. Failure to inhibit noxious signals may be a more generalised defect and clinically IBS patients are known to consult frequently not only for bowel symptoms but also for non GI symptoms(38) (39) [START_REF] Whorwell | Non-colonic features of irritable bowel syndrome[END_REF] and also receive more treatments such as antibiotics for non GI complaints [START_REF] Mendall | Antibiotic use, childhood affluence and irritable bowel syndrome (IBS)[END_REF].

Excessive awareness of signals from many parts of the body other than the gut are detected by the PHQ-12 SS scale which provides a rapid and useful clinical assessment. One of the authors, (RS) has been using this for over 4 years in his clinic and this study confirms the clinical impression that this is more useful than anxiety and depression scales which are elevated in patients with organic as well as functional disease [START_REF] Boulton-Jones | Significance of anxiety in IBS outpatients: relation to bowel symptoms and final diagnosis[END_REF].

One reason why the PHQ-12 SS more clearly separates IBS from the other groups is that it may be a way of detecting generalised hypersensitivity, which is a key feature in at least half of IBS patients and seems to predict the more severe symptoms [START_REF] Posserud | Altered rectal perception in irritable bowel syndrome is associated with symptom severity[END_REF].

The recognition that multiple somatic complaints are frequently seen in IBS patients is also of considerable importance from a management point of view. This is because they significantly add to the burden of illness as well as leading to inappropriate referral and even unnecessary treatment [START_REF] Prior | Gynaecological consultation in patients with the irritable bowel syndrome[END_REF][START_REF] Prior | Irritable bowel syndrome in the gynecological clinic. Survey of 798 new referrals[END_REF][START_REF] Francis | High prevalence of irritable bowel syndrome in patients attending urological outpatient departments[END_REF]. Thus patients with IBS are over-represented in gynaecological and urological clinics and it has been shown that they are also subjected to an excess of surgical procedures [START_REF] Longstreth | Irritable bowel syndrome and surgery: a multivariable analysis[END_REF] . These non-GI complaints are also a major cause of concern to patients themselves who often fear that other disease is being overlooked. It has been shown that these symptoms can be just as intrusive as the gastroenterological symptoms [START_REF] Maxton | Ranking of symptoms by patients with the irritable bowel syndrome[END_REF] . Furthermore these features have diagnostic utility

as it has been shown that the more non-colonic symptoms of which a patient complains, the more secure the diagnosis of IBS becomes [START_REF] Maxton | More accurate diagnosis of irritable bowel syndrome by the use of 'non-colonic' symptomatology[END_REF] .

Although both IBS and DD patients suffer abdominal pain and disturbed bowel habit, there are important differences, including the structurally abnormal bowel and the effect of advancing age which increases the prevalence of symptomatic diverticular disease but reduces IBS prevalence. Our previous studies show that using the Rome II criteria only a minority (15%) of SYMDD patients meet Rome criteria for IBS largely because their pain was not relieved by defecation nor closely linked to stool consistency or frequency. A recent survey in Olmstead county found a somewhat higher prevalence but even so only 29% of patients with diverticulosis meet the Rome II criteria for IBS compared to 13% in the population overall [START_REF] Jung | Diarrhea-predominant irritable bowel syndrome is associated with diverticular disease: a population-based study[END_REF]. PHQ-12 SS scores in both asymptomatic and symptomatic patients with diverticulosis in the current study were higher than healthy controls but lower compared to IBS patients despite their greater age and their having a similar Completing the PHQ-12 SS takes only a few minutes and we believe is a valuable addition to the clinical assessment of patients . Not only is the physician gaining useful data, but the patient becomes re-assured that their medical attendants are familiar with these additional features of their condition, which can be so disabling.

Future studies should assess the value of these scores when combined with alarm symptoms and the Rome criteria in reducing the need for unnecessary investigations in this important group of patients.

F

METHODS

Participants 3

The study population: The inclusion and exclusion criteria, setting and locations where data were collected. The reference standard and its rationale. 6 8

Technical specifications of material and methods involved including how and when measurements were taken, and/or cite references for index tests and reference standard. Table 2 16

The number of participants satisfying the criteria for inclusion who did or did not undergo the index tests and/or the reference standard; describe why participants failed to undergo either test (a flow diagram is strongly recommended).

All did

Test results 17 Time-interval between the index tests and the reference standard, and any treatment administered in between.

simultaneo us 18

Distribution of severity of disease (define criteria) in those with the target condition; other diagnoses in participants without the target condition.

4 Health

 4 Questionnaire 15 is an instrument which records the bothersomeness of 15

  habit as judged by the number of days per week with hard or loose stools. This discrepancy supports the idea that the symptoms in at least some DD patients are related to the associated peripheral abnormality, namely muscular hypertrophy and altered innervation[START_REF] Simpson | Post inflammatory damage to the enteric nervous system in diverticular disease and its relationship to symptoms[END_REF] which is absent in IBS, where symptoms are mainly driven by central abnormalities of sensory processing and interpretation. The higher PHQ-12 SS score in the small subgroup with a history of fever, pain lasting >24hr and prescription of antibiotics is intriguing. It may represent a group with somatisation who seek and get more medical attention but alternatively it may indicate that recurrent pain from acute diverticulitis leads to generalised hypersensitivity. Only a prospective study with objective rather than the current subjective criteria for diagnosing diverticulitis could answer this question.

  2 HV= healthy volunteer, PI-IBS= post infectious IBS, IBS-D= IBS with diarrhoea, IBS-C= IBS with constipation, SYMPDD= symptomatic diverticular disease, ASYMPDD= asymptomatic diverticulosis. † ANOVA Kruskal-Wallis test except Age which being normally distributed was analysed using ANOVA Age =Age in years Mean ±SEM BM / Day = Bowel movements per day Median (range) Days /wk loose BM = Days per week with loose bowel movements Median(range) Days/wk hard BM =Days per week hard bowel movements Median (range) Difference from HV *** p<0.001, ** p<0.01 * p<0.05. a P<0.0001 versus IBS b p<0.0001 versus D-IBS c p<0.0001 versus SYMPDD d p<0.05 versus SYMPDD e p<0.0001 versus SYMPDD f p<0.001 versus D-IBS and C-IBS

Figure 1 Figure 2 Figure 1

 121 Figure 1 Somatisation (PHQ12) scores in IBS and Diverticular disease (DD) compared to healthy volunteers Box and whiskers plot showing median and interquartile ranges together with extreme values

5 4 5 5 5 6

 555 Participant recruitment: Was recruitment based on presenting symptoms, results from previous tests, or the fact that the participants had received the index tests or the reference standard? Participant sampling: Was the study population a consecutive series of participants defined by the selection criteria in item 3 and 4? If not, specify how participants were further selected. Data collection: Was data collection planned before the index test and reference standard were performed (prospective study) or after (retrospective study)? Crossection al study Test methods 7

9 9

 9 Definition of and rationale for the units, cut-offs and/or categories of the results of the index tests and the reference standard.

9 10

 9 The number, training and expertise of the persons executing and reading the index tests and the reference standard. Self administere d 11 Whether or not the readers of the index tests and reference standard were blind (masked) to the results of the other test and describe any other clinical information available to the readers. NA Statistical methods 12 Methods for calculating or comparing measures of diagnostic accuracy, and the statistical methods used to quantify uncertainty (e.g. 95% confidence intervals). ROC 13 Methods for calculating test reproducibility, if done. Not done RESULTS Participants 14 When study was performed, including beginning and end dates of recruitment. characteristics of the study population (at least information on age, gender, spectrum of presenting symptoms).

  Subjects and MethodsThis study took place at Nottingham University Hospital NHS Trust, Nottingham and the Wythenshawe Hospital, South Manchester between July 2006 and September 2008. The IBS group , who all met the Rome II IBS criteria[START_REF] Thompson | Functional bowel disorders and functional abdominal pain[END_REF] were part of a larger IBS genetics and biomarker study approved by the Nottingham Research Ethics Committee which also approved the diverticular patient survey.

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  previous 6 months. As this was part of a larger genetic study, only Caucasians were included to avoid heterogeneity due to racial differences. Subjects in the diverticulosis group were not subjected to systematic screening for this study but had already been investigated for their original presenting complaint. Any with a diagnosis of bowel cancer or other serious gastrointestinal disease were excluded.

	F o r according to the Rome II criteria (29) which was the current gold standard at the time of starting the study. In addition, they also filled out the Patient Health Questionnaire 15 (PHQ-15) (Table 1) which asks about the bothersomeness of a range of symptoms (21). Since the PHQ15 contains three questions directly related to gastrointestinal symptoms (constipation or loose bowels, abdominal pain and nausea or indigestion) which would automatically give a high score we calculated a PHQ-12 SS score in which these 3 items were excluded. All diverticulosis patients also completed the bowel symptom questionnaire as well as the HAD and the full PHQ-15. Healthy volunteers also completed the same questionnaires except the IBSSS. The modified Talley Bowel Symptom Questionnaire was used to exclude any who met IBS criteria. F o r SS, age, anxiety and depression to predict IBSS in IBS groups only. PHQ-12 SS, age, anxiety and depression were also used to predict doctor visits and bloating both in the entire group and also within each patient group. Initially we used binary logistic regression to predict dichotomous outcomes (IBSS above 296, abnormal values of anxiety, depression, presence of bloating, doctor visits >2/year) and then used linear regression to derive predictive equations to help understand the relative importance of each variable for the various outcomes of interest (IBSS, doctor visits, PHQ12, bloating). Standard receiver-operator curves were constructed to determine the optimum cut-off of the PHQ-12 SS for distinguishing IBS from healthy volunteers and to compare the efficacy of the different scores in discriminating IBS from HV. We also calculated the F o r gastroenteritis within the F o r
	P e e r Data Analysis and Statistics P P P All data was entered from the original paper questionnaires into Excel spread sheets and e e e verified by two individuals to check for transcribing errors. Data was then imported into SPSS Version 15 for analysis. e r e r e r
	R e v i e All parameters were assessed for abnormality of distribution. Age, IBS severity score, R R R PHQ-12 SS and HAD were normally distributed while bowel symptoms were not. e e e Normally distributed data is expressed as mean ± standard error of the mean and differences between groups were assessed by an initial overall analysis of variance (ANOVA) followed by post doc comparisons using the Tukey test. Correlation was v i e v i e v i e
	w performed using the Pearson correlation coefficient. Non parametric data is presented in w w w
	the text as median (range). The Kruskal-Wallis nonparametric analysis of variance was
	used to assess overall group differences and post hoc comparisons were evaluated using
	Dunn's multiple comparison test.
	Approximately 3% of data was missing owing to undecipherable or missing responses,
	but the numbers on which each assessment is made is indicated in the text. Multivariate
	analysis was also undertaken to assess the relative importance of the factors shown in
	univariate analysis to be significantly associated with outcomes of interest, namely IBSS,
	doctor visits and PHQ12. Backward logistic regression was used including initially PHQ-12
	6 7 8

together with a modified Talley Bowel Symptom Questionnaire (28) allowing the diagnosis of IBS Positive and Negative Likelihood ratios which respectively indicate by how much the probability of having IBS is increased by having an elevated score or how much it decreases if the score is normal. These measures have the advantage over positive and negative predictive values of being independent of the proportion of IBS in the initial sample. It is generally considered that to be useful a diagnostic test should have a PLR in excess of 2, with a value >10 being considered excellent, thus the tissue transglutaminase test has a PLR of 11. Similarly to be useful the Negative Likelihood

Ratio should be <0.2, again the tissue transglutaminase test has a NLR of 0.09. As will be seen by these criteria the PHQ-12 SS performs well.

Inclusion and Exclusion Criteria

All subjects in the IBS study underwent a screening medical examination including a general physical examination and screening blood tests including full blood count, serum calcium and endomysial antibodies to exclude underlying diseases. In the course of the three year study, two supposed IBS patients were shown to have coeliac disease and excluded but no other conditions were identified.

Exclusion criteria were serious concomitant disease, bleeding or clotting disorders, previous gastrointestinal surgery (other than appendectomy or cholecystectomy), history of alcohol or drug dependence and pregnancy. Healthy volunteers were excluded if they had IBS symptoms or had

Table 4

 4

	) 67% of all IBS patients considered as a single group
	had abnormally elevated values compared with only 54.8% of the SYMPDD patients. As
	the supplementary figure shows most of the ASYMPDD had values within the normal
	range.

  Within the entire study group PHQ-12 SS correlated with both anxiety r= 0.54 and depression r=0.55 p<0.001 n=583. Multiple logistic regression in all subjects also showed that depression and anxiety and female gender were all significant independent predictors of having a PHQ-12 SS score greater than the cutoff of >6, p for all variables <0.0001. Linear regression showed that for each unit rise in anxiety score, PHQ-12 SS rose 0.29, and 0.39 for each unit rise in depression score.Females scored on average 6.8 ±0.2 points, significantly greater than males who scored 5.1±0.2, p<0.001. PHQ-12 SS score fell slightly with increasing age but this was not significant, p=0.52.

	increased IBSS 0.31 SD again showing that PHQ12 is a more important predictor of Discussion
	IBSSS than depression.
	This is the first study using the modified PHQ15 to evaluate a large group of patients
	Predicting visits to doctors: role of age, PHQ12, anxiety and depression with gastrointestinal symptoms. After excluding the three items which cover
	IBS patients reported visiting their doctor for their abdominal symptoms an average of gastrointestinal symptoms, we found that the modified scale, which we have called the
	2.2(0.2) times in the previous year, significantly more that the ASYMPDD patients PHQ-12 SS, clearly distinguishes IBS patients from healthy volunteers. Although the the
	0.3(0.1) and SYMPDD patients 1.4(0.2) p<0.0001 and 0.002 respectively. sensitivity of PHQ-12 SS for identifying IBS in general, using a cut off of >6, was only
	Considering the IBS patients separately, GP visits correlated with negatively with age 66%, the specificity was 95%, which would be very helpful in a clinical setting such as
	F o r (r=-0.19) p=0.001, and positively with PHQ-12 SS (r=0.17), p=0.002 anxiety (r=0.12), p=0.03 and depression, (r=0.18) p=0.001, n=310. Gender did not significantly influence F o r Predicting bloating and abnormal bowel function 86% of IBS patients reported being troubled by abdominal distension or bloating r o F outpatients in which IBS is very common. Since in our study IBS accounted for about 2/3
	P P significantly more than ASYMPDD (24%) and SYMPDD (68%), both p<0.001. PHQ-12 SS P
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	R R R
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	w w w	41 versus 0.29 and
	0.34, all p<0.00, n=309). Binary logistic regression showed PHQ-12 SS, lower age and female (16/19) and significantly younger than the other DD patients being 61.6±2.8, Predicting symptoms in diverticular disease
	depression were all independent predictors of IBSS > 296, p<0.000, 0.001 and 0.01 p<0.001. These patients reported visiting their doctor more at 2.2(0.3) visits/year than Binary logistic regression showed both gender, anxiety and PHQ-12 SS were significant
	respectively. Predictably anxiety correlated with depression and was not an independent the remaining SYMPDD excluding these 19 who visited 0.8(0.2) times or ASYMPDD (p<0.001) independent predictors of having symptoms with each 1 unit rise in Anxiety
	predictor of IBSSS. Multiple regression showed that each 1 point rise in PHQ-12 SS or patients 0.3(0.1) visits, p< 0.01 and 0.0001 respectively. They had significantly higher or PHQ-12 SS score increasing the risk of having symptoms 19% and 11% respectively,
	depression score increased IBSS by 8.3 and 7.1 points respectively while each additional PHQ-12 SS scores at 10.3±1.1 compared to the remaining SYMPDD patients as a whole while females were at 2.3 fold increased risk compared to males
	7.1±0.3, p<0.01 and the ASYMPDD at 5.0 ± 0.3, p<0.001
	Predicting PHQ-12 SS
	11 12 13

shows 54.8% SYMPDD had abnormal PHQ-12 SS compared with just 26.6 % in ASYMPDD, Chi-squared 27.0 versus SYMPDD, p<0.001.

Similarly abnormal anxiety/ depression was seen in 71.7%/ 39.3% of the SYMPDD patients significantly greater that the 38.9%/ 23.0% of the ASYMPDD group, Chisquared 27.3 / 8.2, p<0.001/ 0.01 respectively .

Predicting IBS symptom severity score (IBSSS)

Restricting analysis just to IBS patients showed that PHQ-12 SS correlated more strongly with IBS severity scale (IBSSS) than anxiety or depression (r= 0.decade of age reduced the score 13 points. Put another way each 1 standard deviation (SD) rise in depression increased IBSS 0.28 SD while each 1 SD rise in PHQ-12 SS the number of visits. Binary logistic regression using age, PHQ-12 SS, anxiety and depression to predict 2 or more doctor visits per year showed that neither gender nor anxiety nor depression were independent predictors but that age and PHQ-12 SS were.

Multiple linear regression showed that visits increased 0.23 /year for each unit rise in PHQ-12 SS and fell 0.28 visits / year for each advancing decade of life.

The pattern in diverticular disease was slightly different with only PHQ-12 SS showing an independent effect on doctor visits on binary logistic regression. Multiple regression showed that visits increased 0.17 /year for each unit rise in PHQ-12 SS.

Within the diverticulosis there were 19 patients with more severe symptoms. This group reported prolonged periods of pain lasting > 24 hours with fever for which they had sort medical attention and received antibiotic treatment. These 19 were predominantly scores correlated more strongly than anxiety or depression with days with bloating (r= 0.48 versus r=0.43 and 0.33, all p<0.000, n=582). Multiple logistic regression showed that advancing age and male gender were independently protective, Odds ratio for being troubled by bloating 0.99 (p<0.03) and 0.62 (p<0.000), while anxiety and PHQ-12 SS showed a modest adverse effect, Odd's Ratio 1.1 and 1.2, both p<0.000. Advancing years also reduced the incidence of loose stools with every decade of age reducing the number of days with loose stool on average 0.4 days/ week, p<0.001. Male gender in contrast significantly increased the frequency by 1.1 days/ week, p<0.001. of the group we would expect similar performance in a clinical setting and a normal PHQ-12 SS score would be against a diagnosis of IBS and prompt further tests.

Table 1 :

 1 Whorwell has acted as an advisory board member for Rotta Research, Proctor and Gamble, Danone Research, Astellas Pharma, Ironwood Pharmaceuticals, Almirall Pharma and Movetis and received research grants from Danone and Rotta Research The main IBS study was part of a larger study of markers in IBS funded by an investigator initiated educational grant from GlaxoSmithKline which paid the salaries of EC, MGH & M H. The study design, analysis, interpretation and write up of the current report was the sole responsibility of the principle investigator. The diverticular patient study was funded by a joint Royal College of Surgeons / Age Concern Training Fellowship PHQ12 Questionnaire The PHQ15 questionnaire asks about the bothersomeness of each symptom, scored 0= Not bothered at all, 1 = bothered a little, 2 =bothered a lot.

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Table 3 :

 3 Somatisation (PHQ12), anxiety and depression scores in healthy volunteers and patients with IBS or Diverticulosis

		ANOV	HV	PI-IBS D-IBS	C-IBS	ASYM	SYMP
		A					PDD	DD
		p=
	N		151	39	148	132	113	173
	PHQ12	0.000	3.2±	8.2±0	8.0±0	8.4±0	5.0±0	7.4
			0.2	.3 ***	.3***	.3***	.3*** a	±0.3*
								** b
	Anxiety 0.000	4.8±	8.3±0	8.6±0	9.7±0	5.6±0	8.9±0
			0.2	.6	.3	.4	.3 c	.3*** b
				***	***	***
	Depres	0.000	2.1±	5.1±0	5.7±0	5.6±0	4.4±0	5.8.
	sion		0.2	.6	.3	.3	.3 ***g	±0.3
				***	***	***		*** b
	Difference from HV * p<0.05, ** p<0.01, *** p<0.001 a p<0.001 versus all IBS and
	symptomatic DD b p<0.001 versus ASYMPDD c p<0.002 versus PI-IBS and p<0.001
	versus IBS-C, IBS-D and SYMPDD

Table 4 :

 4 Percentage of subjects with abnormal values for somatisation , anxiety or depression

		Chi-	HV	PI-IBS IBS-D	IBS-C	ASYM	SYMP
		squar					PDD	DD
		ed
		p=
	PHQ12	0.000 5.3	71.8	65.5	67.4	26.6	54.8 a
				***	***	***
	Anxiety	0.000 25.2 69.2	70.3	75.8	38.9	71.7 a
				***	***	***
	Depressi	0.000 6.6	30.8	40.5	32.6	23.0	39.3 b
	on			***	***	***
	*** p<0.001 versus healthy volunteers a p<0.001 versus ASYMPDD b p<0.01 versus
	ASYMPDD
	% IBS patients with abnormal PHQ12, Anxiety and Depression were significantly greater
	than HV while %SYMPDD were significantly greater than ASYMPDD for all three
	variables.

  There are very few high values in controls. The range in all patient groups is wide but values do correlate with doctor visits and symptom severity in IBS as assessed by the IBSSSS. Most ASYMPDD are within the normal range.

	STARD checklist for reporting of studies of diagnostic accuracy
			(version January 2003)
	Section and Topic	Item		On page #
		#
	TITLE/ABSTRACT/	1	Identify the article as a study of diagnostic accuracy (recommend MeSH	2
	KEYWORDS		heading 'sensitivity and specificity').
	INTRODUCTION	2	State the research questions or study aims, such as estimating diagnostic
			accuracy or comparing accuracy between tests or across participant
			groups.
		F o r
			P
			e
			e r
			R
			e
			v i e
			w

Table 2 19

 2 A cross tabulation of the results of the index tests (including indeterminate and missing results) by the results of the reference standard; for continuous results, the distribution of the test results by the results of the reference standard.

	NA

Table 4 22

 4 How indeterminate results, missing data and outliers of the index tests were handled.

	F o r
	P
	e
	e r
	R
	e
	v i e
	w

NA 23

Estimates of variability of diagnostic accuracy between subgroups of participants, readers or centers, if done.

Acknowledgement: The authors would like to thank Marguerite Richards and Emma Bradley for their assistance with patient recruitment, data entry and validation. Statement of interests Professor Spiller has acted as an advisory board member for Norgene, McNeil, Albireo, Movetis and Takeda and received research grants from McNeil and Johnson & Johnson. David J Humes None Eugene Campbell None Margaret Hastings None Keith R. Neal None George E. Dukes Employee of GSK


Vera E Valkhoff
email: v.valkhoff@erasmusmc.nl

Geert W T Jong

Eva Marianne Van Soest

Ernst J Kuipers

Miriam C Sturkenboom

Erasmus Mc

Geert W 't Jong

C J M Sturkenboom

use of clopidogrel and proton pump inhibitors

Keywords:

Risk of recurrent myocardial infarction with the concomitant use of clopidogrel and proton pump

inhibitors.

Boehringer, Yamanouchi, and Altana, none of which are related to the subject of this study.

6

M. Sturkenboom has been consultant to Pfizer, Servier and Lundbeck on issues not related to 7 this manuscript. E. Kuipers has served as a speaker and advisory board member for 8 AstraZeneca. readmitted for MI. Among clopidogrel users, current PPI use was associated with an increased 11 risk of recurrent MI (OR:1.62, 95%CI:1.15-2.27) when compared to no PPI use, but not when 12 compared to past PPI use (OR:0.95, 95%CI:0.38-2.41). Among clopidogrel-non-users, current Clopidogrel is an oral antiplatelet agent commonly used in addition to aspirin to 2 reduce cardiovascular (CV) events. Clopidogrel is converted in the liver from an inactive pro-3 drug to its active metabolite, that binds irreversible to P2Y12 adenosine diphosphate (ADP) 4 receptors on the platelet surface, thereby preventing platelet aggregation [START_REF] Beitelshees | Clopidogrel pharmacogenetics: promising steps towards patient 2 care? Arteriosclerosis, thrombosis, and vascular biology[END_REF] .

5

Clopidogrel conversion is catalyzed by several cytochrome P450 (CYP) enzymes, of 6 which CYP2C19 is the most important. Patients with loss-of-function polymorphisms in the 7 gene encoding for CYP2C19 have lower levels of the active metabolite and have reduced 8 platelet inhibition during clopidogrel treatment. This results in higher rates of acute 9 myocardial infarction (MI) 2, 3 . In contrast, rapid metabolizers of clopidogrel (with CYP2C19 10 variants leading to increased enzyme activity) have a higher rate of clopidogrel activation and 11 more efficient platelet inhibition 4 .

12 Proton pump inhibitors (PPIs) are routinely co-administered with clopidogrel to 13 prevent upper gastrointestinal (UGI) bleeding [5][6][START_REF] Ray | Outcomes with concurrent use of clopidogrel and 17 proton-pump inhibitors: a cohort study[END_REF] , which is in line with expert consensus 14 guidelines 8 . PPIs are also metabolized by CYP2C19, and can competitively bind to its catalytic site. Therefore, PPIs are potentially hindering the conversion of clopidogrel to its 16 active metabolite. Both the European Medicines Agency (EMA) and the US Food and Drug 17 Administration (FDA) recently advised against the concurrent use of PPIs and clopidogrel in 18 the absence of a strong indication 9, 10 , because several pharmacodynamic [11][12][START_REF] O'donoghue | Pharmacodynamic effect and clinical 12 efficacy of clopidogrel and prasugrel with or without a proton-pump inhibitor: an analysis of two 13 randomised trials[END_REF][14] and clinical outcome studies [15][START_REF] Juurlink | A population-based study of the drug interaction between 20 proton pump inhibitors and clopidogrel[END_REF][START_REF] Pezalla | Initial assessment of clinical impact of a drug interaction 22 between clopidogrel and proton pump inhibitors (Letter)[END_REF][18][START_REF] Rassen | Cardiovascular outcomes and mortality in 6 patients using clopidogrel with proton pump inhibitors after percutaneous coronary intervention or 7 acute coronary syndrome[END_REF][20] suggest a detrimental interaction between clopidogrel and PPIs.

20

Evidence on the association between cardiovascular events and co-administration of

6

Other studies failed to show an interaction between the use of PPIs and clopidogrel 2, 6, Clopidogrel use was classified at the index date as either "no" use (no use or use at 14 least 30 days before index date), "past" use (end of prescription between seven and 30 days 15 before the index date), or "current" use (use at index date, or end of prescription less than 16 seven days before index date). Because half-time values of PPIs are short (a few hours), the current, past, and no use of PPIs was defined differently: "no" use (no use or use at least 30 18 days before index date), "past" use (end of prescription between three and 30 days before

2

The study is not subject to immortal time bias as the outcome under study could be assessed 3 equally for cases and controls after the 30-or 90-day period.

1

The initial study population comprised 27,513 patients who were admitted to the 2 hospital with an acute MI during follow-up. After exclusion of patients of whom not one year 3 of valid data before baseline MI was available, the final cohort comprised 23,655 patients.

4

The mean age was 64.7 (± standard deviation (SD) 13.2) years and 15,897 (67.2%) were 5 male. The median follow-up time after discharge was 42.6 months (interquartile range (IQR) 6 16.8 to 71.7 months). Co-morbidity at baseline was substantial: 4.4% of patients had been 7 hospitalized for cancer, 3.3% for cardiac dysrhythmia, and 2.8% for cerebrovascular disease.

8

Prevalence of diabetes mellitus was 21.6%. The median number of distinct prescribed drugs 9 in the one year before cohort entry was 5 (IQR 2 to 9 prescriptions) (Table 1; column 2). At 10 baseline, 10.5% of the patients used proton pump inhibitors (PPIs) and 315 patients (1.3%) used clopidogrel.

12 Figure 1 shows that prescription rates of both PPIs and clopidogrel changed over time 13 between January 1st, 1999 and December 31st, 2008, illustrating the increased use of the 14 study drugs over a decade.

15

Within the study cohort, 1,247 patients were readmitted to the hospital with an acute 16 MI. The risk of a recurrent MI at least 30 days after discharge was 2.8% (95% CI: 2.6% -17 3.0%) in the first year, and 10.3% (95% CI: 9.1% -11.5%) in 10 years.

18

Of the cases with a recurrent MI, 1,224 (98.2%) could be matched to at least one control without a recurrent MI. Characteristics of cases and controls (in the unexposed group 20 to PPIs) are depicted in Table 1 (Columns 3 and 4). As expected, cases were more likely to 4 1; columns 5 and 6). Patients using PPIs were older, were more frequently male, had been 5 hospitalized more frequently prior to cohort entry for several co-morbid conditions, and more 6 frequently used cardiovascular medications at baseline. 17 [START_REF] Rassen | Cardiovascular outcomes and mortality in 6 patients using clopidogrel with proton pump inhibitors after percutaneous coronary intervention or 7 acute coronary syndrome[END_REF] , summary disease risk scores (probability of short term mortality) [START_REF] Juurlink | A population-based study of the drug interaction between 20 proton pump inhibitors and clopidogrel[END_REF] , or conventional 1 propensity scores [START_REF] O'donoghue | Pharmacodynamic effect and clinical 12 efficacy of clopidogrel and prasugrel with or without a proton-pump inhibitor: an analysis of two 13 randomised trials[END_REF]20 can be applied. Table 5 provides an overview of techniques used in 2 other observational studies on this topic to deal with confounding by indication.

3

Our study further confirms the relevance of confounding, illustrated by the fact that 4 PPI users had more co-morbidity and used more co-medication than PPI non-users at baseline 5 and also by the results of the analysis in clopidogrel non-users. The increased risk for 6 recurrent MI with current PPI use (OR 1.38, 95% CI: 1.18-1.61) compared to no PPI use in 7 the absence of clopidogrel therapy provides further support for this. Another explanation 8 would be that PPIs have a harmful effect irrespective of clopidogrel status. We are aware of 9 two studies that conducted similar analysis in clopidogrel non-users which also showed 10 significantly elevated risks with PPI use (adjusted OR 1.29 and 1.55) compared to PPI non-11 use 23,[START_REF] Charlot | Proton-pump inhibitors are associated with 12 increased cardiovascular risk independent of clopidogrel use[END_REF] . In two other observational studies however, no significant increase in CV events was 12 shown in patients prescribed PPIs without clopidogrel 15, 32 .

13

We addressed the confounding issue by all possible design and adjustments measures, This study adds to the existing literature because we revealed the difficulties to deal 9 with confounding by indication in observational studies and we adjusted for confounding in 10 the most optimal way by applying past PPI use as the reference category. Furthermore, few 11 other studies could account for the use of low-dose aspirin as important risk factor for 12 recurrent MI [START_REF] Rassen | Cardiovascular outcomes and mortality in 6 patients using clopidogrel with proton pump inhibitors after percutaneous coronary intervention or 7 acute coronary syndrome[END_REF]20 . We avoided immortal time bias by assigning exposure to the category it 13 belongs to and not using follow-up time to define exposure. Immortal time bias alludes to the 14 fact that the outcome under study cannot be assessed when follow-up time is used to define 15 the exposure status. We used a time varying exposure assessment whereas some other cohort 16 studies applied a fixed exposure status [START_REF] O'donoghue | Pharmacodynamic effect and clinical 12 efficacy of clopidogrel and prasugrel with or without a proton-pump inhibitor: an analysis of two 13 randomised trials[END_REF]20 , which leads to misclassification of exposure at 17 the index date (e.g. patients were defined at baseline as PPI non-users irrespective of changes 18 in PPI use during follow-up). In addition, we used an European database whereas many published studies so far were conducted in North-America. None of the PPIs were available 20 over the counter during the study period and we have illustrated that there was no difference 21 in the risk of recurrent MI between esomeprazole, omeprazole, and pantoprazole.

22

There are several important limitations to this analysis. First, we were unable to study

Mean age (SD) 64.7

13 PPI

 13 use was associated with an increased risk of recurrent MI (OR:1.38, 95%CI:1.18-1.61) 14 when compared to no PPI use 15 Conclusions: The apparent association between recurrent MI and use of PPIs with clopidogrel 16 depends on the design and is affected by confounding by indication. The association is not present when (un)measured confounding is addressed by design. Deleted: , but not when compared to past PPI use (OR:1.22, 95%CI:0.79-1.88).

21

  PPIs with clopidogrel remains inconclusive. Observational studies show a small increased risk 22 of recurrent MI in patients using PPI -clopidogrel combination therapy compared to those 23 alone. Estimated relative risks vary from 0.92 to 1.93 2, 7, 13, 15-20 . These 1 results are likely influenced by confounding by indication. Confounding by indication is 2 introduced when more severely ill patients with a worse prognosis are more likely to receive 3 PPIs than healthier patients. Furthermore, some published observational studies may have 4 suffered from immortal time bias 15,21 . Both biases could distort the studied association in 5 either directions.

7 7 ,

 7 13, 19, 22, 23 . Recently, results of a post hoc analysis of a randomized trial revealed no 8 association between PPI use and the risk of the primary CV endpoint for patients treated with 9 clopidogrel or the novel thienopyridine prasugrel (a prodrug also requiring metabolization 10 through CYP enzymes)[START_REF] O'donoghue | Pharmacodynamic effect and clinical 12 efficacy of clopidogrel and prasugrel with or without a proton-pump inhibitor: an analysis of two 13 randomised trials[END_REF] . The main limitation of this randomized trial was that use of a PPI was not randomized. Preliminary analysis (pre-specified sample size and follow-up time not 12 reached) of the unpublished Clopidogrel and the Optimization of Gastrointestinal Events 13 (COGENT) trial, a randomized double-blind trial of omeprazole 20 milligram (mg) vs. 14 placebo in patients taking dual therapy (clopidogrel and aspirin) demonstrated no significant difference in CV events between both study arms (hazard ratio = 1.02, 95% CI: 0.70-1.51) 6 . 16 Because of the potential clinical consequences for a large patient group at risk for both 17 recurrent CV as well as UGI events, residual uncertainty about this potential drug-drug 18 interaction should be minimized. Therefore, we conducted a nested case-control study using data from the PHARMO Record Linkage System (RLS) (1999-2008) to quantify the 20 association between use of PPIs and recurrent MI in the absence or presence of clopidogrel, 21 while addressing the issues of both study design (avoidance of immortal time bias) and 22 residual confounding (using past exposure to PPIs as the reference category and propensity 23 score-based adjustments).

6 7 8 1 between2 13 The

 67113 based nested case-control study(1999-2008) was conducted within a 4 cohort of patients admitted for acute MI during the study period.5SettingThe study was conducted using data from the PHARMO RLS. This system comprises 8 drug-dispensing records mostly from community pharmacies and hospital discharge records 9 of more than three million inhabitants of 50 demographically defined areas in the 10 Netherlands. For all participants, the computerized drug-dispensing histories contain data concerning the name of the dispensed drug, dispensing date, dispensed amount, prescribed 12 dosing regimens, and the legend duration of use (prescription length). All drugs are coded 13 according to the Anatomical Therapeutic Chemical (ATC) classification 24 . The hospital 14 records include detailed information concerning discharge diagnosis, procedures, dates of 15 hospital admission and discharge, discharge destination (or death in the hospital before 16 discharge). Diagnoses are consistently classified according to the International Classification 17 of Diseases, Ninth Revision, Clinical Modification (ICD-9-CM) during the entire study 18period. For a detailed description of the database we refer to a previous publication[START_REF] Herings | Pharmaco-morbidity linkage: a feasibility study 22 comparing morbidity in two pharmacy based exposure cohorts[END_REF] .The study cohort included all patients in the PHARMO RLS who were hospitalized January 1st, 1999 and December 31st, 2008 for a primary diagnosis of acute MI.Cohort entry was date of discharge from hospital of an acute MI, registered as a primary 3 discharge diagnosis code 410 according to ICD-9-CM. Secondary diagnoses were not queried, 4 because it is likely that prevalent MI events (co-morbidity) are recoded as secondary 5 diagnosis. Because of the dynamic study population, patients were required to have had one 6 prescription filled at least one year preceding the date of cohort entry (i.e. one year of valid 7 database history) in order to allow uniform assessment of the presence of co-morbidity and 8 confounding factors. Patients were followed from cohort entry until diagnosis of recurrent MI, 9 last filled prescription, or the end of the study period (December 31st, 2008), whichever came study outcome was hospitalization for a subsequent acute MI, registered as 14 primary diagnosis ICD-9-CM code 410. Only the first encountered recurrent acute MI during 15 follow-up was included in the analysis. The date of rehospitalization for the MI was used as 16 index date. To avoid misclassification of the exposure, we required a 30-day period between 17 discharge from baseline MI and recurrent MI, since outpatient time was needed to observe 18 filling of outpatient prescriptions. In a sensitivity analysis, we used the requirement of a 90day period between discharge from baseline MI and recurrent MI.

9

  For each case readmitted for acute MI, controls were randomly selected from the 1 cohort, matched on gender, age (same year of birth), being at risk of a recurrent MI, and 2 calendar time by means of incidence density sampling 26 . Only those controls were considered 3 to be at risk of developing a recurrent MI when they had a baseline acute MI ≥ 30 days prior 4 to the index date.

7

 7 Drug exposure data were obtained from outpatient drug dispensing files of community 8 pharmacies, as recorded in the PHARMO RLS. The exposures of interest were clopidogrel 9 and PPIs. PPIs included in the analyses were omeprazole, pantoprazole, lansoprazole, 10 rabeprazole, and esomeprazole. Duration of use was obtained by dividing the total amount of 11 dispensed units by the prescribed amount per day. We calculated the end of each prescription 12 by adding the duration to the dispensing date.

13

 13

19

  index date), and "current" use (use at index date, or end of prescription less than three days 20 before index date). To avoid protophatic bias, only prescriptions dispensed at least one day 21 before index date were considered "current" use. In sensitivity analyses, the definition of past 22 PPI use (which was the reference) was adjusted to use between three to 90 days or between 14 23 Deleted: (i.e. having had a baseline acute MI ≥ 30 days prior to the index date) preceding the index date. In the latter case the definition of "current" PPI 1 switched to use at index date, or final prescription date less than 14 days before index date.

62 7 12 1 (

 71 At baseline, we considered the following factors as potential covariates: percutaneous 7 transluminal coronary artery angiography (PTCA) within 30 days of baseline MI, prior 8 hospitalization for cardiogenic shock, congestive heart failure, cancer, cerebrovascular 9 disease, acute renal failure, chronic renal failure, cardiac dysrhythmia (including atrial 10 fibrillation), or upper gastrointestinal (UGI) ulcer. The covariate diabetes mellitus was a 11 combination of prior hospitalization for diabetes mellitus and anti-diabetic drug use. As a proxy for (the degree of) comorbidity, we aggregated all prescriptions per ATC-code in the 13 one year preceding cohort entry and we refer to this 'score' as the number of different 14 prescriptions. Additionally, we considered use of commonly prescribed cardiovascular medications (anti-hypertensive drugs, low-dose acetylsalicylic acid, β-adrenergic antagonists, 16 digoxin, and lipid lowering drugs) or NSAIDs at baseline. We defined comorbidity and 17 medication at baseline rather than at recurrent MI to avoid adjustment on intermediates in the 18 pathway from exposure to outcome. Medications inducing or inhibiting cytochrome P450 19 2C19 or cytochrome P450 3A4 (directly affecting levels of clopidogrel and PPIs) 27 , as well 20 as standard secondary prevention medications such as lipid lowering drugs and antihypertensive agents were assessed at the index date. Furthermore, days of follow-up (number 22 of days between date of discharge of baseline MI and index date) and length of hospital stay 23 for the baseline MI were also considered as covariates. Descriptive analyses were performed to describe the cohort with regard to baseline 3 characteristics. Prescription rates for PPIs and clopidogrel for the cohort were calculated per 4 calendar month to assess prescription time trends. Survival rates were estimated using the 5 Kaplan-Meier method and evaluated the probability of recurrent MI free survival during 6 follow-up.Baseline characteristics were compared between cases with a recurrent MI and 8 controls without recurrent MI in patients unexposed to PPIs and between all PPI users and PPI 9 non-users in patients without recurrent MI using univariate conditional logistic regression 10 analyses. We applied conditional logistic regression analysis to estimate matched and adjusted 11 odds ratios (OR) and 95% confidence intervals (95% CI) for the association between recurrent 12 MI and exposure to PPI. The analyses were performed in two subgroups: 1) current 13 clopidogrel users, and 2) clopidogrel non-users to examine whether there was a true drug-drug 14 interaction between clopidogrel and PPIs. The reference group were patients with no prescription of PPIs in the previous month (non-PPI users). A second analysis that more 16 effectively deals with confounding by indication was performed applying past PPI users as the 17 reference category. In the adjusted model we included all factors that changed the unadjusted 18 odds ratio with more than 10 percent. To test the robustness of our finding, we examined the effect of extension of the past PPI exposure window on the observed associations as a 20 sensitivity analysis. Finally, we estimated the OR for current use of omeprazole and 21 esomeprazole, using current pantoprazole as the reference group.Because the use of PPIs was not randomly assigned, we developed a propensity score summary exposure risk score). The propensity score represents the likelihood of exposure to 2 a PPI for each individual patient28 . The propensity score for use of PPI was developed using 3 logistic regression modeling. The log odds of the probability that a patient received a PPI at 4 any point of time during follow-up was modeled as a function of all previously described 5 baseline covariates and was included in our dataset as a separate confounder. The PPI 6 propensity score was only included in the adjusted analysis when it changed the odds ratio 7 with more than 10 percent, unless we adjusted for individual covariates, to avoid double 8 adjustment for the same covariate. 9 Statistical significance was assumed for two-sided p-values <0.05. All statistical 10 analyses were performed using SPSS software version 15.0 (SPSS inc, Chicago, Ill).

21 have

 21 been hospitalized for co-morbidities associated with an increased risk of recurrent MI, 22 such as congestive heart failure and diabetes mellitus. At baseline, cases used several types of frequently, for instance angiotensin-converting-enzyme (ACE) 1 inhibitors and lipid lowering drugs. To understand whether selective prescribing (channeling) 2 of PPIs to persons at increased risk of recurrent MI occurred, we assessed the presence of risk 3 factors in PPI users and PPI non-users (in patients who did not develop a recurrent MI) (Table

7 1

 1 Among current clopidogrel users, a significant association was observed between 8 current PPI use and recurrent MI when compared to PPI non-use (OR: 1.62, 95% CI: 1.15-9 2.27). When applying past PPI use as the reference category rather than PPI non-use, we 10 found no association between recurrent MI and current use of PPI (OR: 0.95, 95% CI: 0.38-11 2.41) (Table2). Repeated analyses with variations in the definition of past PPI use (3 to 90 12 days or 14 to 90 days) did not affect the study findings appreciably; OR: 1.15, 95% CI: 0.51-13 2.61, and OR: 1.34, 95% CI: 0.50-3.62, respectively. 14 Among clopidogrel non-users, current use of PPI was associated with recurrent MI when compared to PPI non-use (OR:1.38, 95% CI: 1.18-1.61). When applying past PPI users 16 as the reference category, a weak association between recurrent MI and current use of PPI 17 was found (OR: 1.22, 95% CI: 0.79-1.88), although the latter's confidence interval cross the 18 null, which was likely due to insufficient power (Table3).The analysis by type of PPI among current clopidogrel users showed that pantoprazole 21 was most frequently used, followed by omeprazole and esomeprazole. Lansoprazole and 22 rabeprazole were rarely used. Using current pantoprazole as the reference category, current 23 Deleted: control group Deleted: Among clopidogrel non-users, current use of PPI was associated with recurrent MI when compared to PPI nonuse (OR:1.38, 95% CI: 1.18-1.61). When applying past PPI users as the reference category, no Deleted: association between recurrent MI and current use of PPI was found (OR: 1.22, 95% CI: 0.79-1.88) (Table3) and of esomeprazole were not associated with an increased risk of 1 recurrent MI (respectively, OR: 1.07, 95 % CI: 0.58-1.99 and OR: 0.83, 95 % CI: 0.40-1.69, This population-based cohort study showed that the association between clopidogrel -2 PPI co-therapy and risk of recurrent MI is highly affected by confounding by indication 29 , 3 which may explain the contrasting results in the literature. To illustrate the problem of 4 confounding we compared current PPI users to none or past PPI users in the absence of 5 clopidogrel. To deal with the problem of confounding by indication, we compared current use 6 of clopidogrel plus current use of PPI not only to current clopidogrel without PPIs, but also to 7 current clopidogrel use plus past use of PPIs, which reduces confounding by indication. When 8 current PPI use was compared to past PPI use the association disappeared, suggesting that the 9 observed association between current PPI use and recurrent MI when PPI non-use was the 10 reference, may have been the result of residual confounding. 11 Our findings are in line both with other observational studies showing an increased 12 risk among PPI users when compared to PPI non-users 15-20 , and those that did not find such 13 an association if better control of confounding was applied 6, 13, 19, 30 . In observational studies 14 or post-hoc analyses in randomized controlled trials, the use of PPI is by definition not 15 randomly assigned, most likely leading to confounding by indication. It is arguable whether 16 adequate adjustment for this harmful type of confounding is possible since much of the 17 confounding will be subtle and unmeasurable. Design choices and progressive adjustment 18 techniques can be used to avoid residual confounding, as we have shown. By careful selection 19 of the study design one can match cases and controls on important covariates, including 20 calendar time. We illustrated the increased use of the study drugs over a decade (Figure 1), 21 and thereby the importance of matching on index date (thus calendar time) as potential 22 confounder. Additionally, one can use a so-called 'active comparator' as reference, such as 23 past users of PPIs instead of none users. For adjustment high-dimensional propensity scores

14

  but still the comparison against none-use of PPI seemed confounded. Applying past PPI use as active comparator changed the association from increased to no effect and was the most 16 powerful approach to deal with channeling of PPIs, more than adjustment for the propensity 17 score. The propensity score did not have large explanatory power in this study due to the fact 18 that relatively little clinical information was available to construct the score.19Our study is internally valid since we used a population-based design, so selection bias 20 was unlikely as all cases and controls came from the same source population. The same 21 pertains to information bias, because data were gathered prospectively without knowledge of 22 the hypothesis studied. The method to control for confounding was discussed previously. The 23 database is proven valid for research as the diagnoses are labeled with ICD-9-CM codes in 24 official coding personnel from the national registry of hospitalization 1 discharge records and linked to pharmacies with complete information on outpatient 2 dispensing. In the Netherlands patients usually go to one pharmacy because of billing 3 purposes (invoices being directly sent to insurance without the need to pre-pay by patient) and 4 medication surveillance. The exposure drugs, clopidrogel and PPIs, are dispensed through 5 regular pharmacies. With regard to external validity, the individuals captured in the database 6 are representative for the entire population of the Netherlands in terms of age, gender, socioeconomic background, morbidity and mortality, drug use, and geographic distribution.

  8

23 1 Table 1 :

 11 actual drug utilization and adherence to dispensed drugs, because only dispensing data were database instead of more reliable proxies for drug use.. Second, using the past 1 PPI group as reference, we may compare current PPI users to past PPI users who had not 2 been fully adherent to the prescribed PPI and therefore still had medication available and were 3 actually also current PPI users at the time of the recurrent MI. To address this point, we 4 conducted several sensitivity analyses varying the time window to define past PPI use and this 5 did not affect the estimates. Additionally, although the internal validity may be jeopardized by 6 non-compliance of the patient, this is unlikely to have affected the comparison between active 7 compounds (e.g. omeprazole/esomeprazole compared to pantoprazole). Third, since most 8 persons continue PPIs once started, applying past PPI use as reference reduces study power 9 substantially (resulting in wide confidence intervals). Finally, residual confounding remained 10 due to lack of data on important cardiac risk factors, such as smoking status, lipoproteins, or 11 type of coronary stents, which was likely providing an overestimation of the effect measure 12 and this was illustrated when applying PPI non-use as the reference. 13 In conclusion, this study provides an unique angle on the association between 14 clopidogrel -PPI co-therapy and risk of recurrent MI by using several techniques to deal with confounding by indication and other methodological issues. Among clopidogrel users, current 16 use of PPIs was associated with an increased risk of recurrent MI when compared to PPI nonuse, but not when compared to past PPI use, which shows the magnitude of the confounding 18 by indication that is present. In clopidogrel-non-users, current PPI use was also associated 19 with an increased risk of recurrent MI when compared to PPI non-use. This study thus 20 demonstrates that previously reported associations between PPI-clopidogrel co-therapy and 21 risks of recurrent MI are influenced by confounding. It further demonstrates that such 22 confounding can be (at least partly) circumvented by careful selection of study design and 23 conventional adjustments techniques. Further work should concentrate on well-conducted 24Deleted: omeprazole and pantoprazole were available over the counter since respectively February 2004 and November 2007. We could not control for over the counter use of medication. However, since PPIs are only being reimbursed when on prescription, it is unlikely that we missed much (long-term) to dissolve the confusion around this controversial drug-drug Characteristics of cohort patients (column 1), of those readmitted to the hospital with 2 MI (cases) versus their controls (column 2 and 3), and of PPI users versus non-PPI users in 3 the case-control setting (column 4 and 5).

- 31 Figure 1 : 1 2

 3111 Figure 1: Prescriptions of PPI and clopidogrel in MI cohort per month.

Table 2 :

 2 Association between current clopidogrel plus current PPI and recurrent MI compared 1 to clopidogrel without PPI and to clopidogrel with past PPI.

	22

** Index date=date of recurrent MI. Only available for the case-control set.

Table 3 :

 3 Association between current PPI use and recurrent MI compared to no PPI and to 1 past PPI use in the absence of clopidogrel.

	2
			Cases	Controls	OR matched	OR adjusted
			n=1,224	n=153,967	(95% CI)	(95% CI)
		No clopidogrel / No PPI	766	110,002	1 (ref)	1 (ref)
		No clopidogrel / Current PPI	237	23,335	1.56 (1.34-1.81) 1.38 (1.18-1.61)#
		Other*	221	20,630
		No clopidogrel / Past PPI	25	2,941	1 (ref)	1 (ref)
		No clopidogrel / Current PPI	237	23,335	1.22 (0.79-1.88) 1.22 (0.79-1.88)χ
		Other**	962	127,691
	3	*Current or past clopidogrel, or past PPI use.
	4	**Current or past clopidogrel, or no PPI use.
	5	# Adjusted for total number of prescriptions one year prior to baseline MI.
		χ No adjustments.

Table 4 :

 4 Association between current clopidogrel plus specific current PPI use and recurrent

	1
	2	MI compared to clopidogrel with current pantoprazole.
			Cases	Controls	OR matched	OR adjusted **
			n=1,224	n=153,967	(95% CI)	(95% CI)
	Current clopidogrel + pantoprazole 36	2,271	1 (ref)	1 (ref)
	Current clopidogrel + omeprazole	26	1,339	1.03 (0.57-1.84) 1.07 (0.58-1.99)
	Current clopidogrel+ esomeprazole 13	827	0.96 (0.48-1.92) 0.83 (0.40-1.69)
	Current clopidogrel + lanso-or	2	186	-	-
	rabeprazole
	Other*		1,147	149,344
		Combinations of PPIs were excluded.

3 *No or past clopidogrel, or no or past PPI use. 4 **Adjusted for follow-up time in days and propensity score.

Table 5 :

 5 Summary of used methods to deal with confounding in observational studies 1 assessing the effect of PPI on CV events in clopidogrel users, excluding restrictions in study

	2
	3	design.
	4
	Authors Study type	Study population Outcome	Methods to deal with confounding
	Simon		Prospective	Patients with	Death, MI, stroke -Multivariable adjustments
	et al. 2		cohort	acute MI		-Propensity analysis for CYP2C19
						loss-of-function-alleles which was used
						to match 5 controls for each patient with
						2 variant alleles
	Bhatt et	Double-blind	Patients with	MI, stroke,	-Randomization
	al. 6		randomized trial	ACS undergoing	CABG, PCI, CV
				coronary stent	death
				placement
	Ray et		Retrospective	Patients	serious CV	-Multivariable adjustments
	al. 7		cohort study	hospitalized for	disease	-Propensity score for the use of PPI
				MI, coronary	((non)fatal MI,	was converted in deciles and included
				artery	stroke, other CV	as variable in model
				revascularization,	death).
				or unstable AP
				on clopidogrel
	O'Dono	Retrospective	Patients with	CV death, MI,	-Multivariable adjustments
	ghue et	cohort within	ACS undergoing	stroke	-Propensity score for the use of PPI and