Dr Alexander Ford

Nicole C Suares

Nicholas J Talley

Alexander ; Ford

Alexander C Ford

Meta-analysis: Epidemiology of Non-cardiac Chest Pain in the Community

Keywords: Functional GI diseases < Disease-based, GERD or GORD < Diseasebased, Non-cardiac chest pain < Disease-based, Oesophagus GERD gastro-oesophageal reflux disease GI gastrointestinal IBS irritable bowel syndrome Non-cardiac chest pain Prevalence Gastro-oesophageal reflux disease Meta-analysis

Background: Prevalence of, and risk factors for, non-cardiac chest pain in the community have not been well studied.

Aims: To conduct a systematic review and meta-analysis to examine these issues.

Methods: MEDLINE, EMBASE, and EMBASE Classic were searched (up to March 2011) to identify population-based studies reporting prevalence of non-cardiac chest pain in adults (≥15 years) according to self-report, questionnaire, or specific symptom-based criteria. Prevalence of non-cardiac chest pain was extracted for all studies, and according to study location and certain other characteristics including presence or absence of gastro-oesophageal reflux disease (GERD) symptoms, where reported. Pooled prevalence overall, as well as odds ratios (OR), with 95% confidence intervals (CIs) were calculated.

Results: Of 18 papers evaluated, 16 reported prevalence of non-cardiac chest pain in 14 separate populations, containing 24 849 subjects. Pooled prevalence of non-cardiac chest pain in all studies was 13% (95% CI 9%-16%). The prevalence of non-cardiac chest pain was higher in Australian studies, and in studies using a questionnaire to define its presence, compared with those using Rome I or II criteria. Prevalence was no different in females versus males (OR 0.99; 95% CI 0.82-1.20). The prevalence was markedly higher in subjects who also reported GERD (OR 4.71;, and increased according to frequency of GERD symptoms.

Conclusions: Pooled prevalence of non-cardiac chest pain in the community was 13%, but there were few studies. Rates did not appear to differ according to gender or age. Presence of GERD was strongly associated with non-cardiac chest pain.

The pathophysiological mechanisms behind symptom generation remain poorly understood. Proposed mechanisms include visceral hypersensitivity, [START_REF] Richter | Abnormal sensory perception in patients with esophageal chest pain[END_REF] abnormal pain processing, (4) altered autonomic activity, [START_REF] Tougas | Cardiac autonomic function and oesophageal acid sensitivity in patients with non-cardiac chest pain[END_REF] and sustained oesophageal contractions, [START_REF] Balaban | Sustained esophageal contraction: A marker of esophageal chest pain identified by intraluminal ultrasonography[END_REF] while the role of oesophageal dysmotility remains unclear. [START_REF] Katz | Esophageal testing of patients with noncardiac chest pain or dysphagia. Results of three years experience with 1161 patients[END_REF] Potential risk factors for non-cardiac chest pain include obesity, family history of gastro-oesophageal reflux disease (GERD), smoking, analgesic use, and symptoms of GERD, [START_REF] Eslick | Non-cardiac chest pain: Prevalence, risk factors, impact, and consulting -a population-based study[END_REF][START_REF] Wise | Risk factors for non-cardiac chest pain in the community[END_REF][START_REF] Ruigomez | Chest pain without established ischaemic heart disease in primary care patients: Associated comorbidities and mortality[END_REF] although data are sparse.

There have been several cross-sectional surveys that have reported the prevalence of non-cardiac chest pain in the community, (8;11-13) but the prevalence of non-cardiac chest pain according to geographical location has not been wellstudied to date. Nor has any single study synthesised all the available evidence to examine potential risk factors for non-cardiac chest pain, the relationship between the disorder and symptoms of GERD, or the degree of overlap between other functional GI disorders and non-cardiac chest pain. Systematic analysis of studies that report these types of data is important to allow physicians consulting with patients complaining of these symptoms to provide more precise estimates of the prevalence and risk factors for the condition, as well as identify areas where further research is needed. We have therefore conducted a systematic review and meta-analysis of the prevalence of non-cardiac chest pain in the community to examine these issues. 1.

F

Studies on non-cardiac chest pain were identified using the search terms: chest pain (both as a medical subject heading (MeSH) and free text term), as well as noncardiac, non cardiac, non-cardiac, or functional as free text terms. These were combined with the set operator AND with studies identified with the search term prevalence as both a MeSH and free text term. There were no language restrictions.

All abstracts identified by the search were evaluated for appropriateness to the study question, and all potentially relevant papers were obtained and assessed in detail. A recursive search of the literature was conducted using the bibliographies of all eligible studies. Foreign language papers were translated where required. Studies were assessed independently by two investigators, using pre-designed eligibility forms, according to the eligibility criteria. All disagreements were resolved by consensus.

Data Synthesis and Statistical Analysis

The proportion of individuals with non-cardiac chest pain in each study was combined to give a pooled prevalence of non-cardiac chest pain for all studies.

Heterogeneity between studies was assessed using the I 2 statistic with a cut off of 50%, [START_REF] Higgins | Measuring inconsistency in meta-analyses[END_REF] and the χ 2 test with a P value < 0.

RESULTS

The search strategy identified 2076 citations (Figure 1). From these we identified 18 papers that appeared to be relevant to the study question. Of these, 16

studies, reporting prevalence of non-cardiac chest pain in 14 separate adult study populations, were eligible for inclusion. (8;11-13;20-31) Agreement between investigators for assessment of study eligibility was good (kappa statistic = 0.64).

Detailed characteristics of all included studies are provided in Table 2. Most studies were cross-sectional surveys, but one was a case-control study conducted among diabetic patients and non-diabetic controls from the general population. [START_REF] Mjornheim | Gastrointestinal symptoms in type 1 diabetic patients, as compared to the general population[END_REF] For the purposes of the present analysis only data for the non-diabetic controls were extracted from this study. None of the identified studies reported the prevalence of non-cardiac chest pain according to the presence or absence of other functional GI disorders.

Prevalence of Non-cardiac Chest Pain in All Studies

The pooled prevalence of non-cardiac chest pain in all 14 studies containing 24 849 participants using the primary definition for non-cardiac chest pain in each study, was 13.0% (95% CI 9.0% to 16.0%), with statistically significant heterogeneity between studies (I 2 = 98.6%, P < 0.001). Nine studies excluded cardiac causes of chest pain from their definition of non-cardiac chest pain. (8;11-13;20;23-25;29) Seven of these used self-report of a cardiac cause by the participant in the symptom questionnaire, (11;13;20;23-25;29)

Global Prevalence of Non-cardiac Chest Pain

Seven of the studies were conducted in North America or Europe.

(11;13;25;26;29-31) There were no identified studies conducted in South Asia, Africa, or Central America, and only a few studies from other regions. The pooled prevalence of non-cardiac chest pain according to geographical location of the study is provided in Table 3. This was similar for the four geographical regions where more than one study had been conducted, with the exception of Australian studies, where the prevalence was 16.0%. The prevalence in the study from Turkey was 19.5%, [START_REF] Bor | Gastroesophageal reflux in a low-income region in Turkey[END_REF] whilst that in the Argentinean survey was 24.1%. ( 23)

Prevalence of Non-cardiac Chest Pain According to Criteria Used to Define its

Presence

Eleven studies used a questionnaire to define the presence of non-cardiac chest pain. (8;11-13;20;22-24;26;29;30) Three studies used the Rome II criteria, (21;27;31) two used the Rome I criteria, (25;28) and no identified studies used the Rome III criteria. The pooled prevalence of non-cardiac chest pain according to the various criteria used to define its presence is provided in Table 4. Pooled prevalence was lower when either the Rome I or II criteria were used to define its presence, with a prevalence of 9.0% and 2.7% respectively. chest pain in the community, and ensure our results were applicable to the general population. We performed subgroup analyses according to study location, criteria used to define non-cardiac chest pain, and other study characteristics in order to assess the impact of these on pooled prevalence. Finally, we were able to extract data in order to study the relationship between GERD and non-cardiac chest pain.

F

Limitations of the study, as with any systematic review and meta-analysis, arise from the studies that were eligible and available for inclusion. Our search identified only 16 studies, conducted in 14 separate adult populations, emphasizing the relative paucity of population-based data concerning the prevalence of non-cardiac chest pain, when compared to other functional GI disorders such as dyspepsia, irritable bowel syndrome (IBS), and chronic idiopathic constipation. When calculating pooled prevalence, there was a notable absence of studies conducted in certain geographical regions, making an accurate assessment of true global prevalence difficult. There were few studies reporting the effect of age or socioeconomic status on prevalence of non-cardiac chest pain, and no studies reported the degree of overlap with the other functional GI disorders, such as IBS. In addition, there was heterogeneity between study results, which was not explained by any of the subgroup analyses we conducted. Finally, the differences in definitions of non-cardiac chest pain used in the identified studies may limit the usefulness of the pooled estimate of prevalence.

There have been no previous systematic reviews published that have examined the epidemiology of non-cardiac chest pain in the community. This emphasises the need for a definitive study to identify and summarise all available evidence in this field. One previous systematic review reported that evidence from three populationbased studies supported a link between non-cardiac chest pain and GERD. However, there has not been any formal meta-analysis conducted, to our knowledge, which has synthesised all available evidence to quantify this relationship. Our observation that non-cardiac chest pain was reported almost five times more frequently by those who met symptom criteria for GERD, and that the magnitude of this association increased with a higher frequency of symptoms, suggest that there is considerable overlap between the two conditions and possibly a causal connection.

Physiological reflux of acid may therefore play a role in the generation of symptoms in individuals with non-cardiac chest pain. This theory is supported by a study that used wireless pH monitoring in patients with non-cardiac chest pain, and demonstrated that extended monitoring time led to higher rates of detection of GERD.

(33) A meta-analysis of randomised controlled trials of proton pump inhibitors (PPIs) in non-cardiac chest pain suggested that acid suppression is superior to placebo in terms of improvement in symptoms, with a number needed to treat of only three. [START_REF] Cremonini | Diagnostic and therapeutic use of proton pump inhibitors in non-cardiac chest pain: A metaanalysis[END_REF] Furthermore, in a recent study using ambulatory pH monitoring indicators of GERD, including acid exposure time, evidence of relevant acid reflux was present in almost two-thirds of non-cardiac chest pain patients, and these predicted a response to medical therapy. ( 35) Some investigators have demonstrated that acid exposure reduces pain thresholds in the oesophagus in healthy volunteers, [START_REF] Hu | Intraesophageal acid perfusion sensitizes the esophagus to mechanical distension: A Barostat study[END_REF] and that this effect is exaggerated in patients with non-cardiac chest pain, [START_REF] Sarkar | Contribution of central sensitisation to the development of non-cardiac chest pain[END_REF] 41) However, more studies replicating these data are required in order to confirm this association. This systematic review and meta-analysis has also identified other areas of need for future research. Only a minority of the studies we identified were designed with prevalence of non-cardiac chest pain as the primary outcome of interest, and hence risk factors for the condition have been poorly

assessed. In addition, as yet, no single study has reported the prevalence of chest pain using the Rome III criteria, and this needs to be addressed.

In conclusion, this systematic review and meta-analysis has demonstrated a global prevalence of non-cardiac chest pain of 13%. Rates were higher according to a questionnaire-based diagnosis compared with the Rome I or II criteria. Unlike most of the other functional GI disorders, there was no significant difference in prevalence according to gender. Finally, the presence of non-cardiac chest pain seemed to be strongly associated with GERD symptoms, suggesting common pathophysiological mechanisms.

STATEMENTS OF INTEREST

Authors' Declaration of Personal Interests:

Alexander C Ford: none. Nicole C Suares: none. Nicholas J Talley: none.

Declaration of Funding Interests:

None. We agreed with the reviewer that these two conditions are not synonymous, and therefore we amended the term "functional chest pain" to "non-cardiac chest pain" throughout the paper. This modification necessitated some further minor changes to the Introduction and Discussion sections of the paper. We are very grateful to the reviewer for this suggestion to improve our paper. We added a sentence to the Discussion section of the paper reporting data from these references on page 15, lines 17 to 20: "Some investigators have demonstrated that acid exposure reduces pain thresholds in the oesophagus in healthy volunteers, ( We thank the reviewer for performing this check. No response required. We agreed with the reviewer that a rationale for the conduct of the meta-analysis was We agreed with the reviewer. We added this point as a further limitation of our study in the Discussion section on page 14, lines 18 to 20: "Finally, the differences in definitions of functional chest pain used in the identified studies may limit the usefulness of the pooled estimate of prevalence."

F

On

Synthesis of results 21 Present results of each meta-analysis done, including confidence intervals and measures of consistency.

9-12

Risk of bias across studies 22 Present results of any assessment of risk of bias across studies (see Item 15).

9-12

Additional analysis 23 Give results of additional analyses, if done (e.g., sensitivity or subgroup analyses, meta-regression [see Item 16]).

10-12

DISCUSSION

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

Summary of evidence 24 Summarize the main findings including the strength of evidence for each main outcome; consider their relevance to key groups (e.g., healthcare providers, users, and policy makers).

13-16

Limitations 25 Discuss limitations at study and outcome level (e.g., risk of bias), and at review-level (e.g., incomplete retrieval of identified research, reporting bias).

  chest pain is a disorder thought to relate to the gastrointestinal (GI) tract, characterised by episodes of unexplained chest pain that are potentially of oesophageal origin. Patients with non-cardiac chest pain report impairments in quality of life and activities of daily living that are of a similar magnitude to those with chest pain caused by structural coronary disease, (1) and rates of healthcare seeking and work absenteeism are also comparable. (2) Severity of symptoms and anxiety about the underlying cause appear to drive consultation behaviour.[START_REF] Eslick | Non-cardiac chest pain: Predictors of health care seeking, the types of health care professional consulted, work absenteeism and interruption of daily activities[END_REF]

Table 4 .

 4 A; not applicable, too few studies to assess heterogeneity F o r P e e r R e v i e w Ford et al. Pooled Prevalence of Non-cardiac Chest Pain According to Criteria Used to Define its Presence.

1 . 2 . 9 . 3 .

 1293 the other hand, the studies chosen in the discussion to explain the role of GERD in the generation of symptoms in patients with functional chest pain were probably not the best for this purpose. It would be better to include some other references showing that acid exposure can sensitize the esophagus contributing to the development of visceral hypersensitivity. The authors may probably find useful to include some of the following references: Hu WH, Martin CJ, Talley NJ. Intraesophageal acid perfusion sensitizes the esophagus to mechanical distension: a Barostat study. Am J Gastroenterol. Sarkar S, Aziz Q, Woolf CJ et al. Contribution of central sensitisation to the development of non-cardiac chest pain. Lancet 2000;356:1154-Sarkar S, Thompson DG, Woolf CJ et al. Patients with chest pain and occult gastroesophageal reflux demonstrate visceral pain hypersensitivity which may be partially responsive to acid suppression. Am J Gastroenterol. 2004;99(10):1998-2006.

  effect is exaggerated in patients with non-cardiac chest pain,[START_REF] Sarkar | Patients with chest pain and occult gastroesophageal reflux demonstrate visceral pain hypersensitivity which may be partially responsive to acid suppression[END_REF] and may be attenuated by PPIs.[START_REF] Ford | Systematic review and meta-analysis of the prevalence of irritable bowel syndrome in individuals with dyspepsia[END_REF]" the data and references for this manuscript.I have not identified any previous meta-analyses addressing the epidemiology of functional chest pain. I have not identified any further studies that may be eligible for inclusion.I have checked the extracted data and references which are all correct.

1 .

 1 written systematic review has been well executed and is the first summation of data related to the prevalence of functional chest pain. I have a few minor concerns, more based on principle than on the author's analyses: It is not clear to this reviewer that there is a pressing need to estimate the prevalence of functional chest pain. Perhaps the background could make a stronger argument as to why such an estimate is needed.

2 . 3 .

 23 required and added a sentence to the Introduction section on page 4, lines 23 to 25, and page 5, lines 1 to 2: "Systematic analysis of studies that report these types of data is important to allow physicians consulting with patients complaining of these symptoms to provide more precise estimates of the prevalence and risk factors for the condition, as well as identify areas where further research is needed." The strong association between GERD and functional chest pain challenges the definition of such discomfort as "functional". At the end of the day, I'm not sure what the phenomenon being captured in this study represents, as most of the prevalent cases are reflux-associated and hence part of a GERD presentation. Please see our response to reviewer one's first comment above. There is significant heterogeneity among studies in relation to the inclusion/exclusion of cardiac and reflux-related symptoms. The lack of a standard definition of functional chest pain makes the pooled estimate less meaningful.

14 Conclusions 26 16 FUNDINGFunding 27 16 Page

 1426162716 Provide a general interpretation of the results in the context of other evidence, and implications for future research.13-Describe sources of funding for the systematic review and other support (e.g., supply of data); role of funders for the systematic review.

  and the remaining two studies used both a physician's diagnosis of ischaemic heart disease and the Rose angina questionnaire. Ford et al. 10 of 33 was 17.0% (95% CI 13.0% to 21.0%), compared with 6.0% (95% CI 3.0% to 11.0%) in those that did not exclude cardiac causes.

	F o r
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(8;12)

The pooled prevalence in studies that excluded cardiac causes of chest pain

Prevalence of Non-cardiac Chest Pain According to Gender, Age, and Socioeconomic Status

	Ford et al. Ford et al.	12 of 33 14 of 33
	symptoms of GERD was 31.0% (95% CI 27.0% to 36.0%), compared with 9.0% DISCUSSION
	(95% CI 6.0% to 12.0%) in 5828 subjects without. The OR for non-cardiac chest pain
	There were seven studies that reported the prevalence of non-cardiac chest in those with typical symptoms of GERD was 4.71 (95% CI 3.32 to 6.70) (Figure 3), This systematic review and meta-analysis has demonstrated a pooled
	pain according to the gender of participants. (8;12;13;21;25;27;31) Overall, the with significant heterogeneity between studies (I 2 = 86.6%, P < 0.001), but no prevalence for non-cardiac chest pain in the community of 13%, with a similar
	pooled prevalence in 7340 women was 9.7% (95% CI 5.0% to 15.7%), compared with evidence of funnel plot asymmetry (Egger test, P = 0.28). prevalence across the geographical regions studied, although slightly higher in
	9.7% (95% CI 4.7% to 16.1%) in 6773 men. The OR for non-cardiac chest pain in Five studies reported the prevalence of non-cardiac chest pain according to Australian studies. Prevalence appeared lower when the Rome I or II criteria were
	women, compared with men, was 0.99 (95% CI 0.82 to 1.20) (Figure 2), with frequency of typical GERD symptoms. (13;20;23;24;29) The pooled prevalence of used to define non-cardiac chest pain, compared with a questionnaire-based diagnosis.
	o r borderline heterogeneity between studies (I 2 = 54.7%, P = 0.04), but no evidence of F o r non-cardiac chest pain in 4278 subjects with no GERD symptoms was 8.0% (95% CI 5.0% to 12.0%), compared with 27.0% (95% CI 22.0% to 32.0%) in 1699 individuals with occasional GERD symptoms, and 36.0% (95% CI 31.0% to 41.0%) in 918 with F o r There were no studies examining prevalence according to the most recent Rome III F o r
	P P P P
	e e e e
	e r e r e r e r
	R R R R
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	v i e v i e v i e v i e
	w w w w
	Strengths of this study include our exhaustive literature search, which included
	a recursive search of the literature, as well as electronic databases. In addition,
	eligibility assessment and data extraction were performed independently by two
	investigators, with all discrepancies resolved by consensus. We only considered

funnel plot asymmetry (Egger test, P = 0.99).

Data concerning prevalence of non-cardiac chest pain according to age and socioeconomic status were scarce. Only four studies reporting prevalence according to age.

(8;13;22;25)

However, due to different age bands used to report prevalence of non-cardiac chest pain, data available for pooling were limited. Two studies provided data according to age less than 45 years, or 45 years or more.

(22;25)

The prevalence of non-cardiac chest pain in those aged 45 years or more was not significantly different to those aged less than 45 years (OR 0.85; 95% CI 0.39 to 1.86). There was only one study that reported the prevalence of non-cardiac chest pain according to socioeconomic status.

[START_REF] Wong | Population based study of noncardiac chest pain in southern Chinese: Prevalence, psychosocial factors and health care utilization[END_REF]

This demonstrated a declining prevalence with increasing monthly income, from 33% in those with no income to only 9% in those individuals with a monthly salary of more than US$3200.

Prevalence of Non-cardiac Chest Pain According to Presence of GERD

There were six studies, containing 9104 subjects, which collected data on both typical GERD symptoms and presence of non-cardiac chest pain.

(12;13;20;23;24;29)

The pooled prevalence of non-cardiac chest pain in 3276 individuals with typical frequent GERD symptoms. The ORs for non-cardiac chest pain in those with occasional or frequent GERD symptoms compared with those without were 4.20 (95% CI 2.59 to 6.82) (Figure

4

) and 6.37 (95% CI 4.08 to 9.96) (Figure

5

) respectively, with statistically significant heterogeneity between studies in both analyses, but no evidence of funnel plot asymmetry. criteria. Perhaps unexpectedly, prevalence was also lower in studies that did not exclude possible cardiac causes of chest pain from their definition using self-report, a physician opinion, or validated questionnaire. The presence of non-cardiac chest pain did not appear to be affected by gender or age, although studies reporting prevalence according to the latter were sparse, whilst prevalence declined with increasing income in one study.

[START_REF] Wong | Population based study of noncardiac chest pain in southern Chinese: Prevalence, psychosocial factors and health care utilization[END_REF]

Finally, there was a significant association between GERD and non-cardiac chest pain, with an almost five-fold increase in the odds of non-cardiac chest pain in those reporting symptoms compatible with GERD. The magnitude of this association increased with the frequency of GERD symptoms, with a greater than six-fold increase in odds of non-cardiac chest pain in those with frequent GERD symptoms, suggesting a gradient of effect. population-based studies, and excluded convenience samples and studies conducted in primary care or GI clinics, in order not to overestimate the prevalence of non-cardiac

FIGURES Figure 1. Flow Diagram of Assessment of Studies Identified in the Systematic Review and Meta-analysis.

	Studies identified in literature
	search (n = 2076)
	o r F o r F o r F o r F o r F o r F o r F o r	Excluded (title and abstract revealed not appropriate) (n = 2058)
	P P P P P P P P
	e e e e e e e Studies retrieved for evaluation e
	e r e r e r e r e r e r e r (n = 18) e r
	R R R R R R R R
	e e e e e e e e	Excluded because duplicate
	v i e v i e v i e v i e v i e v i e v i e v i e	publication (n = 2)
		w w w w w w w w
	Population-based studies
	reporting prevalence of non-
	cardiac chest pain (n = 16)

Figure 2. Pooled Odds Ratio for Non-cardiac Chest Pain in Women Compared with Men.

	Ford et al.				25 of 33
	Figure 3.
			Odds ratio meta-analysis plot [random effects]
	Drossman 1993				1.12 (0.95, 1.32)
	Thompson 2002				1.27 (0.51, 3.26)
	Eslick 2003				1.06 (0.75, 1.50)
	Wong 2004				0.67 (0.53, 0.87)
	Jeong 2008 Rey 2006 Boyce 2006		F o r		0.87 (0.32, 2.27) 1.05 (0.82, 1.35) 1.26 (0.62, 2.56)
	combined [random]	0.2	0.5 P odds ratio (95% confidence interval) 1 2 e	5	0.99 (0.82, 1.20)
			e r
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Pooled Odds Ratio for Non-cardiac Chest Pain in Individuals with Typical Symptoms of GERD Compared with Those Without.

	Ford et al.			26 of 33
	Figure 4.
			Odds ratio meta-analysis plot [random effects]
	Locke 1997			5.68 (4.10, 7.98)
	Wong 2003			3.86 (3.07, 4.84)
	Bor 2005			3.79 (2.46, 5.87)
	Chiocca 2005			3.38 (2.29, 5.08)
	Rey 2006 Cho 2005		F o r	3.29 (2.56, 4.23) 13.04 (8.40, 20.30)
	combined [random]	2	5 P 10 odds ratio (95% confidence interval) e	4.71 (3.32, 6.70) 100
			e r
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			e
			v i e
			w

Pooled Odds Ratio for Non-cardiac Chest Pain in Individuals with Occasional Symptoms of GERD Compared with Those Without.

			Odds ratio meta-analysis plot [random effects]
	Locke 1997				5.14 (3.64, 7.35)
	Bor 2005				3.00 (1.71, 5.17)
	Chiocca 2005				2.86 (1.86, 4.43)
	Rey 2006 Cho 2005		F o r	2.78 (2.08, 3.69) 11.04 (6.81, 17.86)
	combined [random]	1	2	5 P odds ratio (95% confidence interval) 10 e	4.20 (2.59, 6.82) 100
				e r
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Figure 5. Pooled Odds Ratio for Non-cardiac Chest Pain in Individuals with Frequent Symptoms of GERD Compared with Those Without.

			Odds ratio meta-analysis plot [random effects]
	Locke 1997				6.80 (4.63, 10.03)
	Bor 2005				4.60 (2.77, 7.61)
	Chiocca 2005				4.36 (2.75, 6.95)
	Rey 2006 Cho 2005		F o r	4.57 (3.23, 6.42) 21.23 (10.58, 41.81)
	combined [random]	2	5	10 P odds ratio (95% confidence interval) e	6.37 (4.08, 9.96) 100
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Table 1 :

 1 Eligibility Criteria

	Cohort studies, case-control studies, cross-sectional surveys, or randomised controlled
	trials
	Recruited adults (>90% of participants aged ≥15 years)
	Participants recruited from the general population / community*
	Reported prevalence of non-cardiac chest pain (according to self-report, questionnaire
	data, specific diagnostic criteria †, or a physician's opinion)
	Sample size of ≥ 50 participants

*Convenience samples excluded †Rome I, II, or III criteria F o r P e e r R e v i e w

Table 2 . Characteristics of Included Studies.

 2

	Study	Country	Method of data	Criteria used to	Method used to exclude cardiac	Sample	Number with
			collection	define non-cardiac	causes of chest pain	size	non-cardiac
				chest pain			chest pain (%)
	Ruth 1991	Sweden	Postal questionnaire Questionnaire-defined	Self-report on questionnaire	328	40 (12.2)
	(11)
	Drossman	USA	Postal	Rome I	Self-report on questionnaire	5430	693 (12.8)
	1993 (25)		questionnaire*
	Isolauri 1995	Finland	Postal questionnaire Questionnaire-defined	Not excluded	1700	244 (14.4)
	(26)
	Locke 1997	USA	Postal	Questionnaire-defined	Self-report on questionnaire	1511	337 (22.3)
	(29)		questionnaire*

Comments for Transmission to the Authors

Overall, this meta-analysis by Ford et al is a good study.

We thank the reviewer for their kind comments. No response required.

Most of the limitations, such as the inclusion of only 16 studies, have been correctly addressed by the authors in the discussion

We thank the reviewer for their kind comments. No response required.

However, I have an important concern about the use of the term functional chest pain as synonymous of non-cardiac chest pain. The definition of functional chest

pain requires the exclusion of esophageal and non esophageal disorders. While the term non cardiac chest pain denotes exclusively that the heart is not the origin of the pain and can include patients with a variety of conditions such as GERD. Indeed, most of the studies included in this meta-analysis, with some exceptions, were designed to assess the prevalence of non cardiac chest pain not the prevalence of functional chest pain. The increased association found between GERD and functional chest pain can therefore be explained by the fact that the authors are including patients with GERD related NCCP in the analysis.

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

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

Data collection process 10 Describe method of data extraction from reports (e.g., piloted forms, independently, in duplicate) and any processes for obtaining and confirming data from investigators.

7

Data items 11 List and define all variables for which data were sought (e.g., PICOS, funding sources) and any assumptions and simplifications made.

7 and 8

Risk of bias in individual studies

12 Describe methods used for assessing risk of bias of individual studies (including specification of whether this was done at the study or outcome level), and how this information is to be used in any data synthesis.

and 8

Summary measures 13 State the principal summary measures (e.g., risk ratio, difference in means).

and 8

Synthesis of results 14 Describe the methods of handling data and combining results of studies, if done, including measures of consistency (e.g., I

2 ) for each meta-analysis.

and 8

Risk of bias across studies 15 Specify any assessment of risk of bias that may affect the cumulative evidence (e.g., publication bias, selective reporting within studies).

and 8

Additional analyses 16 Describe methods of additional analyses (e.g., sensitivity or subgroup analyses, meta-regression), if done, indicating which were pre-specified.

7 and 8

RESULTS

Study selection 17 Give numbers of studies screened, assessed for eligibility, and included in the review, with reasons for exclusions at each stage, ideally with a flow diagram.

and 24

Study characteristics 18 For each study, present characteristics for which data were extracted (e.g., study size, PICOS, follow-up period) and provide the citations.

30-32

Risk of bias within studies 19 Present data on risk of bias of each study and, if available, any outcome level assessment (see item 12).

30-32

Results of individual studies 20 For all outcomes considered (benefits or harms), present, for each study: (a) simple summary data for each intervention group (b) effect estimates and confidence intervals, ideally with a forest plot.


Verhamme

MD Kmc

Pharm D Mcjm

Dr Katia Verhamme
email: k.verhamme@erasmusmc.nl

Molewaterplein

Study designs in Paediatric Pharmacoepidemiology

Keywords: Pharmacoepidemiology, children, safety, effectiveness, case-control, cohort

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Introduction

When doctors prescribe medicines to children, they want these drugs to be effective and safe. So far, few randomized controlled trials (RCTs) have been conducted in children meaning that most of these drugs have only been licensed for use in adults. [START_REF] Who | Promoting safety of drugs in children[END_REF] Data from adults are extrapolated to children although we know that children are "no small adults". Instructions on dosing and route of administration for use in children are derived from the data on adults, although pharmacological and pharmacokinetic studies have shown that children metabolize drugs differently. [START_REF] Kearns | Developmental pharmacology--drug disposition, action, and therapy in infants and children[END_REF] Recently, the importance for the need of safe and effective drugs in children has been recognized and this has lead to several initiatives. The new EU paediatric regulation specifies the need for a Paediatric Investigation Plan as a mandatory part for each new licence application and the potential for a Paediatric Use Marketing Authorisation (PUMA) for older off-patent drugs if studies on the efficacy and safety of these older drugs in children will be conducted. [3] Equally, in the US, paediatric research is stimulated via the Pediatric Research Equity Act. [START_REF]Paediatric drug development[END_REF] In addition, several networks have been established to promote the safety and efficacy of drugs in children. [START_REF] Emea | Medicines for children -EU paediatric network[END_REF] Demonstration of efficacy is best achieved using RCTs either against placebo or against the current standard of care. The evaluation of safety is often much more complex. Although some of the safety information can be provided by RCTs, they are often limited in terms of sample size and in length of follow-up. If the incidence of an adverse event is very rare or if the adverse event only arises in the long term, this potential safety issue will not be detected through RCTs. In addition, RCTs often use strict in and exclusion criteria meaning that children with specific co-morbidity will be excluded from the trial and that the information of safety and efficacy cannot be generalized to the whole paediatric population. The EMEA list of Paediatric Needs recognizes the need for long term safety studies in children. [START_REF] Emea | Inventory of paediatric needs[END_REF] By definition, these types of studies require a long term follow-up and large sample size to detect rare events. This implicates that paediatric drug safety can best be answered by pharmacoepidemiological studies. Within this article, we provide an overview on the various study designs that can be used in pharmacoepidemiology and where relevant focus on issues related to paediatric research.

Methods

A PubMed search was conducted on the following MESH term: "Pharmacoepidemiology" with as limits: English, all infant: birth-23 months, all child: 0-18 years, newborn: birth -1 month, infant: 1-23 months, preschool child: 2 -5 years, child: 6-12 years and adolescent: [START_REF] Hippisley-Cox | Risk of myocardial infarction in patients taking cyclooxygenase-2 inhibitors or conventional non-steroidal anti-inflammatory drugs: population based nested case-control analysis[END_REF][START_REF] Johnsen | Risk of hospitalization for myocardial infarction among users of rofecoxib, celecoxib, and other NSAIDs: a population-based case-control study[END_REF][START_REF] Levesque | The risk for myocardial infarction with cyclooxygenase-2 inhibitors: a population study of elderly adults[END_REF][START_REF] Ray | COX-2 selective non-steroidal anti-inflammatory drugs and risk of serious coronary heart disease[END_REF][START_REF] Solomon | Relationship between selective cyclooxygenase-2 inhibitors and acute myocardial infarction in older adults[END_REF][START_REF] Torrence | Understanding Epidemiology[END_REF] years. This search generated 142 hits (search date May 2009). All abstracts were reviewed and if considered to be relevant -meaning containing information on study designs in pharmacoepidemiology, ideally also touching upon pharmacoepidemiology in children-, the full article was consulted. Apart from PubMed, handbooks on pharmacoepidemiology were as well consulted namely the following: "Textbook of pharmacoepidemiology" by Strom and Kummel and "Pharmacoepidemiology and Therapeutic Risk Management" by Hartzema et al. [START_REF] Hartzema | Pharmaco-epidemiology and Therapeutic Risk Management[END_REF][START_REF] Strom | Textbook of pharmacoepidemiology[END_REF] In addition, the authors elaborated on methods that were used to study the safety and efficacy of drugs in children within the scope of the TEDDY (Taskforce in Europe for the Drug Development of the Young) network. [9] The authors in no way intended to conduct a systematic review on methods in pharmacoepidemiology, meaning that certain papers might have been left out based on the decision of the authors.

What is pharmacoepidemiology?

Epidemiology is the study of the frequency, distribution and determinants of a disease and goes back to ancient times. [START_REF] Hartzema | Pharmaco-epidemiology and Therapeutic Risk Management[END_REF] Pharmacoepidemiology is the study of the use and the effects of drugs in large numbers of people. Pharmacoepidemiology implements the basic principles of chronic disease epidemiology in its research methods. In addition, pharmacoepidemiology integrates the pharmacology of a drug in the concept of the different study designs. Pharmacoepidemiology thus bridges between epidemiology and pharmacology. [START_REF] Strom | Textbook of pharmacoepidemiology[END_REF] Awareness of the importance of the safety of a drug and the need of drug regulation is relatively new and goes back to the beginning of the 20 th century. Pharmacoepidemiology mainly got his boost in 1961 where growing concerns on the safety of a drug were triggered after the thalidomide disaster. [START_REF] Taussig | A study of the German outbreak of phocomelia. The thalidomide syndrome[END_REF] Thalidomide was marketed as a mild hypnotic and often used in pregnant women for the symptomatic relief of morning-sickness. Shortly after marketing, a dramatic increase in the occurrence of phocomelia (absence of limbs or part of limbs) was observerd although phocomelia was known to be a rare birth defect. After pharmacoepidemiological investigation, the increase of phocomelia was found to be associated with in utero exposure to thalidomide.

Pharmacoepidemiology is mainly applied to drugs that have already been registered

(postmarketing drug surveillance) but in recent years, the scope of pharmacoepidemiology has broadened considerably. [START_REF] Strom | Textbook of pharmacoepidemiology[END_REF]

Importance of pharmacoepidemiology

As already alluded on in the introduction, it is difficult to study the safety of a drug through RCTs as these trials often use stringent inand exclusion criteria, have limited sample size and short term follow-up. Especially if adverse events are uncommon, if they only appear after long term follow-up or if they are unique to high risk populations, the chance of detecting them within RCTs is low. Pharmacoepidemiology can contribute information on the safety and effectiveness of a drug that is not available from premarketing studies. [START_REF] Strom | Textbook of pharmacoepidemiology[END_REF] A well known example of a pharmacoepidemiological study which was crucial in the detection of a safety issue is the case control study on the association between the use of diethylstilbestrol for the prevention of spontaneous abortion in pregnant women and the occurrence of vaginal cancer in their offspring. [START_REF] Herbst | Adenocarcinoma of the vagina. Association of maternal stilbestrol therapy with tumor appearance in young women[END_REF] As this event only occurred after long term, namely in the female offspring from adolescence on, this signal would never have been picked up by clinical trials.

More recent examples of safety signals that were picked up through observational research are the cohort studies on the relation between the use of cyclooxygenase2-selective non steroidal anti-inflammatory drugs and the risk of myocardial infarction. [START_REF] Graham | Risk of acute myocardial infarction and sudden cardiac death in patients treated with cyclo-oxygenase 2 selective and non-selective non-steroidal anti-inflammatory drugs: nested case-control study[END_REF][START_REF] Hippisley-Cox | Risk of myocardial infarction in patients taking cyclooxygenase-2 inhibitors or conventional non-steroidal anti-inflammatory drugs: population based nested case-control analysis[END_REF][START_REF] Johnsen | Risk of hospitalization for myocardial infarction among users of rofecoxib, celecoxib, and other NSAIDs: a population-based case-control study[END_REF][START_REF] Levesque | The risk for myocardial infarction with cyclooxygenase-2 inhibitors: a population study of elderly adults[END_REF][START_REF] Ray | COX-2 selective non-steroidal anti-inflammatory drugs and risk of serious coronary heart disease[END_REF][START_REF] Solomon | Relationship between selective cyclooxygenase-2 inhibitors and acute myocardial infarction in older adults[END_REF]

Different study designs within pharmacoepidemiology

Different study designs can be applied within pharmacoepidemiology, all with their own specific indications, advantages and disadvantages. Within this article we provide an overview on the different study designs and if available give examples on studies conducted in children.

Although the RCT is considered to be the gold standard to study the association between drug exposure and the outcome of interest, it will not be discussed in this review as this paper only discusses study designs related to observational research.

Case reports

Case reports are reports of individual patients who experience some kind of unexpected event while taking a particular drug. Generally, no definitive inference about causality can be made, although individual reports have other uses, including alerting doctors to the possible existence of a reaction (signal generation) and helping characterize the reaction. [START_REF] Strom | Textbook of pharmacoepidemiology[END_REF][START_REF] Torrence | Understanding Epidemiology[END_REF] Examples of case reports are the reports on tardive dyskinesia observed in children treated with haloperidol for Tourette syndrome. [START_REF] Caine | Gilles de la Tourette's syndrome, tardive dyskinesia, and psychosis in an adolescent[END_REF][START_REF] Mizrahi | Haloperidol-induced tardive dyskinesia in a child with Gilles de la Tourette's disease[END_REF][START_REF] Petty | Haloperidol-induced tardive dyskinesia in a 10-year-old girl[END_REF] Also the numerous case reports on serotonin syndrome, a potentially life-threatening condition, in children being treated with selective serotonin reuptake inhibitors triggered the need to study the safety of antidepressants in children. [START_REF] Likasitwattanakul | Serotonin syndrome: a case report[END_REF][START_REF] Phan | Serotonin syndrome following a single 50 mg dose of sertraline in a child[END_REF] 2. Case series Case series are collections of individual case reports who have had the same exposure or who have presented with the same outcome. Often they are collections of case reports over a fairly short period of time. Although data in case series are more numerous than in case reports, here as well, they cannot be used to test for the presence of causality as there is the lack of an appropriate comparison group. [START_REF] Strom | Textbook of pharmacoepidemiology[END_REF][START_REF] Torrence | Understanding Epidemiology[END_REF] Examples of case series in children are the case series on the association between the use of dextromethorphan and serotonin syndrome. [START_REF] Ziaee | Side effects of dextromethorphan abuse, a case series[END_REF] Although case series cannot investigate causality, they are important to detect potential safety signals, which need to be tested with more rigorous study designs such as case control or cohort studies.

Cross sectional studies

Cross-sectional studies measure both the exposure and disease in an individual or population at the same specific point in time. These studies can also explore the role of risk factors in the cause of a disease. Because an individual's exposure and disease status are examined at the same point in time, it is impossible to say which came first. For this reason, no conclusion about risk or cause can be made. Cross sectional studies offer a quick, easy and inexpensive method of exploring the relationship between exposures and disease. In addition, cross sectional studies can provide information on the prevalence of a disease in a population as well as other variables of interest (e.g. other concomitant diseases, use of concomitant drugs). Data used in cross-sectional studies are often collected through surveys. Surveys can be done by varies ways namely self-administered, mailed, interviews (phone or face-to face)

or via the computer. There are two crucial methodological issues when conducting a survey namely the questionnaire design (open vs. closed questions) and the sampling strategy to get a representative sample of the population of interest. [START_REF] Strom | Textbook of pharmacoepidemiology[END_REF][START_REF] Torrence | Understanding Epidemiology[END_REF] An example of a cross sectional study conducted in children is the study by Lin et al on the home environmental risk factors and the child's asthma status. In this study, households with children 1-17 age were requested to complete a questionnaire on home environmental risk factors and in addition were questioned about the child's asthma status. [START_REF] Lin | Self-reported home environmental risk factors for childhood asthma: a crosssectional study of children in Buffalo, New York[END_REF] Another example is the study by Liberman et al, who studied the prevalence of antihypertensive, antidiabetic and lipid lowering drugs in a population of children aged between 6 -18 years using data from a prescription claims data in the US. [START_REF] Liberman | Prevalence of antihypertensive, antidiabetic, and dyslipidemic prescription medication use among children and adolescents[END_REF]The method used in this study was somewhat different from a classical cross sectional study as the prevalence of use was estimated on the 1 st day of each month for a total of 30 consecutive months. This is called a serial cross sectional study.

Cohort study

A cohort study is a study where a group of people with a particular disease or taking a particular drug are followed up for the occurrence of an outcome of interest. [START_REF] Strom | Textbook of pharmacoepidemiology[END_REF][START_REF] Torrence | Understanding Epidemiology[END_REF][START_REF] Ashby | Establishing causality in the assessment of safety of medicines for children[END_REF][START_REF] Luo | Role of pharmacoepidemiology in evaluating prescription drug safety in pediatrics[END_REF] This outcome of interest will be a potential side effect in the case of safety studies. Cohort studies are generally used to compare exposed to unexposed patients, although they can also be used to compare one exposure to another. From the perspective of timing, data can be collected prospectively or retrospectively. As exposed and unexposed individuals are followed over time, incidence rates can be calculated within a cohort study which enables estimation of risk such as the relative and attributable risk.

Cohort studies have as big advantage that they can study rare exposures, can study multiple outcomes and that time to event analysis is possible. Disadvantages of cohort studies are that they are lengthy and expensive, may require very large samples, are not suitable for rare diseases or for diseases with long latency. The first two arguments do not longer hold when automated databases are as datasource, as all required information is already collected and available in the database at the time the study starts.

The principles of cohort studies can best be explained by some examples. van Staa et al conducted a retrospective cohort study using data from the GPRD (general practice research database) in the UK to study whether the use of inhaled corticosteroids (ICS) is associated with an increased risk of fractures. [START_REF] Van Staa | Are inhaled corticosteroids associated with an increased risk of fracture in children?[END_REF] The cohort consisted of a group of children treated with ICS and an asthma reference group of children being treated with bronchodilators excluding ICS. These patients were followed over time until the occurrence of a fracture or the end of the study. No association between the use of ICS and an increased risk of fractures could be identified. Sturkenboom et al. conducted a cohort study on the association between the use of niflumic acid and the incidence of mucocutaneous reactions in children. [START_REF] Sturkenboom | Incidence of mucocutaneous reactions in children treated with niflumic acid, other nonsteroidal antiinflammatory drugs, or nonopioid analgesics[END_REF] Here as well, date from a primary care database, namely the Pedianet database in Italy was used.

The cohort consisted of 3 groups of children namely children being treated with niflumic acid, other NSAIDs or acetaminophen. The children were followed until the occurrence of a mucocutaneous reaction. No association between the use of niflumic acid and the mucocutaneous reaction could be found in comparison to other NSAIDs or acetaminophen.

Although cohort studies are often used to study the association between the exposure of interest and a specific outcome, they can also be used to study drug utilization patterns over time. In addition, use of specific drugs might be used as a proxy for the prevalence of the underlying disease in children (e.g. use of anti-asthmatic drugs as proxy for prevalence of asthma). [START_REF] Furu | Use of anti-asthmatic medications as a proxy for prevalence of asthma in children and adolescents in Norway: a nationwide prescription database analysis[END_REF] Numerous studies on drug use in children have been done so far. [START_REF] Fox | Building pharmacoepidemiological capacity to monitor psychotropic drug use among children enrolled in Medicaid[END_REF][START_REF] Hugtenburg | Increased psychotropic drug consumption by children in the Netherlands during 1995-2001 is caused by increased use of methylphenidate by boys[END_REF][START_REF] Thrane | A one-year population-based study of drug prescriptions for Danish children[END_REF] As part of the TEDDY project, we investigated the prevalence of drug use in children from 2000 until 2005 using 3 primary care databases in the UK, The Netherlands and Italy. [START_REF] Sturkenboom | Drug use in children: cohort study in three European countries[END_REF] Especially anti-infective, dermatological and respiratory drugs were widely prescribed for all age categories. The prevalence of drug use was the highest for the youngest age categories (age < 2 years)

Case control studies

Case control studies are traditionally designed to investigate causes of rare diseases or to investigate multiple exposures. The cases are compared to healthy controls (not having the outcome) to determine the association between the possible risk factors and the disease under study. Fig 3 In pharmacoepidemiology, the cases are patients who have had a particular adverse event. The controls are drawn from those who do not have the adverse event but otherwise come from a similar population. Case control studies obtain their information on drug exposure retrospectively. Information on past exposure is obtained by abstracting medical records or by administering questionnaires or interviews. As such, case control studies might be subject to limitations in the validity of retrospectively collected exposure information (recall bias). [START_REF] Strom | Textbook of pharmacoepidemiology[END_REF] This bias does not hold when using prescription or pharmacy dispensing databases where information on drug prescription or drug dispensing is automatically collected in the database. Another potential limitation is the risk of selection bias where selection of cases and control might be triggered by knowledge of previous drug exposure. For this reason, validation of cases and sampling of controls should ideally be done by researchers blinded to the individual's drug exposure. As the case control studies do not provide information on the incidence rate of a disease in the exposed and unexposed individuals, relative risks cannot be calculated. Generally, the odds ratio is reported which is a close estimate of the relative risk when the disease is rare.

One of the most famous case control studies is the study by Herbst et al who studied the association between the use of diethylstilbestrol (DES) in pregnant women for the prevention of spontaneous abortion and the risk of vaginal cancer in the off-spring. [START_REF] Herbst | Adenocarcinoma of the vagina. Association of maternal stilbestrol therapy with tumor appearance in young women[END_REF] This study included only 8 cases for which 4 age matched controls per case were selectedthis small sample size number was sufficient to reach statistical significance, as the association between the exposure and the outcome was very strong (seven of the 8 cases, but none of the 32 matched controls had been prenatally exposed to DES.

Nested case control study

A specific type of a case control study is the nested case control study, whereby a case control approach is embedded within an established cohort. This approach guarantees that cases and controls represent random samples from the same study base which might be a weakness in the standard case control approach. Mikaelof et al conducted a case control study to study the association between the use of NSAIDs and the risk of skin and soft tissue complications in patients with varicella disease. [START_REF] Mikaeloff | Nonsteroidal anti-inflammatory drug use and the risk of severe skin and soft tissue complications in patients with varicella or zoster disease[END_REF] The cohort consisted of children diagnosed with varicella. Within this cohort, cases of skin and soft tissue complications were identified. For each case, up to 10 controls were sampled from the same cohort, matched on age and practice. The study showed a 4-fold increased risk of skin or soft tissue complications in children treated with NSAIDs.

Databases for pharmacoepidemiological research:

Spontaneous Reporting Database

Health care providers are encouraged to report each suspicion of an adverse drug reaction (ADR) to their national monitoring system. Data from these national monitoring centers are pooled in the WHO VigiBase database. Although the spontaneous reporting system is prone to biases such as selective and underreporting and in addition lacks denominator data such as the total number of users, the system has been proven to be valid for safety signal generation. [START_REF] Lindquist | The WHO Programme for International Drug Monitoring, its database, and the technical support of the Uppsala Monitoring Center[END_REF] Automated databases

As the incidence of adverse events is often low (incidence rates < 1/10 000) and might only arise in the long term, huge sample sizes with considerate follow-up time are needed to conduct pharmacoepidemiological research. This explains why in recent years, pharmacoepidemiological research is mainly conducted using computerized health care data.

The ideal database would include records from both in and outpatient care, emergency care, all laboratory and radiological tests as well as data on prescribed and over the counter medication. [START_REF] Strom | Textbook of pharmacoepidemiology[END_REF] In practice, based on the existing health policies, claims databases are used in the US and health care databases in Europe. Health care databases consist of pharmacy databases, primary care databases and hospital databases. The biggest advantage of these databases is their very large sample size and the relatively inexpensive use. The major weakness is the uncertain validity of diagnosis data which especially holds for the claims databases. [START_REF] Hartzema | Pharmaco-epidemiology and Therapeutic Risk Management[END_REF] Neubert et al, made an inventory of all databases available for pediatric research in Europe. A total of 25 data sources from 12 European countries were identified of which the majority is suitable for paediatric pharmacoepidemiological research. [START_REF] Neubert | Databases for pediatric medicine research in Europe--assessment and critical appraisal[END_REF] Although the size of the inidiviudal automated databases is quite large, the study might still be underpowered to detect very rare side effects (e.g.1/100.000) or side effects that only occur long term after drug exposure. The power can further be increased by combining the data from the different country specific databases into one analysis set -this is nowadays one of the new developments within the field of pharmacoepidemiological research. Within one country or region, linkage of various databases might as well be appropriate, not to increase the sample size, but to collect all available data on one single individual. Primary care databases for instance might lack information on pathology data, hospital admission and hospital drug use. Linking all the available databases through a patient unique key provides the researcher with all necessary data. This principle of database linkage is common practice in the studies on the potential teratogenic effects of a drug where a prescription database is linked to a pharmacy database and a birth registry.

Items to consider when conducting pharmacoepidemiology in children:

Off label and unlicensed drug use in children

Medicines are frequently prescribed to children without being approved for the paediatric population which makes them off-label. [START_REF] Neubert | Defining off-label and unlicensed use of medicines for children: results of a Delphi survey[END_REF] Studies have shown that the proportion of offlabel use in children might vary between 10 to 30% depending on the population being studied and the definitions that had been used. [START_REF] Chalumeau | An example of hospital-based pharmacoepidemiology in paediatrics: tolerance to fluoroquinolones[END_REF][START_REF] Gw | Unlicensed and off label prescription of drugs to children: population based cohort study[END_REF][START_REF] Mcintyre | Unlicensed and off label prescribing of drugs in general practice[END_REF] As part of the TEDDY network, a consensus was reached on the definition of off label use being the following: "all use of a marketed drug not detailed in the summary of product characteristics including therapeutic indication, use in age-subsets, appropriate strength, pharmaceutical form and route of administration". [START_REF] Neubert | Defining off-label and unlicensed use of medicines for children: results of a Delphi survey[END_REF] Research on the association between off-label status and the risk of specific side effects is scarce. [START_REF]Evidence of harm from off label or unlicensed medicines in children[END_REF] Age and maturation

As maturation plays a crucial role in the pharmacology of a drug, it is important to check whether the risk estimates between the drug exposure and the adverse drug reaction fluctuates by age. In paediatric drug research, it is recommended not only to study the effect of a drug in the paediatric population as a whole (0 -18 years), but as well to repeat the analysis within different age categories eventually the ones suggested by the International Conference on Harmonisation. [START_REF] Emea | ICH Topic E 11: Clinical Investigation of Medicinal Products in the Paediatric Population[END_REF] Gender Sex differences in drug use have been reported in children. [START_REF] Fox | Building pharmacoepidemiological capacity to monitor psychotropic drug use among children enrolled in Medicaid[END_REF][START_REF] Sturkenboom | Drug use in children: cohort study in three European countries[END_REF]. Especially prior to puberty, the prevalence rate of drug use is higher in boys compared to girls. It is unclear whether this can be explained by a gender difference in the incidence of the disease or whether symptoms are perceived differently by parents or health care professionals. For certain diseases such as asthma, it is known that the prevalence is higher in boys. Under influence of hormones around puberty, the incidence rate of asthma changes and becomes less prominent in boys. [START_REF] Almqvist | Impact of gender on asthma in childhood and adolescence: a GA2LEN review[END_REF] As gender might modify the association between the drug and the outcome of interest, it is recommendable to conduct additional analysis, stratifying according to gender.

Pharmacovigilance during pregnancy:

Safety disasters -such as phocomelia in children born from women who used thalidomide during pregnancy for the treatment of morning sickness and the high risk of vaginal cancer in the offspring of mothers treated with DES for the prevention of spontaneous abortionstressed the importance of the safety of a drug when used during pregnancy. [START_REF] Taussig | A study of the German outbreak of phocomelia. The thalidomide syndrome[END_REF][START_REF] Herbst | Adenocarcinoma of the vagina. Association of maternal stilbestrol therapy with tumor appearance in young women[END_REF] As pregnant women are often excluded from clinical trials, the potential of teratogenesis is often uncertain when new drugs are introduced to the market. Record linkage studies are important to study the potential teratogenic effects of a drug. In this type of studies, the prescription database is linked to a pregnancyoutcome database. This type of study is described in the paper by Colvin et al, where an Australian prescription database is linked to a database with all pregnancies, the birth and death registration database and finally the birth defect registry.

They found that women who were dispensed a drug during pregnancy were 14% more likely to have a registered birth defect. [START_REF] Colvin | Pharmacovigilance in pregnancy using population-based linked datasets[END_REF] Choosing the optimal pharmacoepidemiological study design

The choice of the most appropriate study design to be used will be driven by the type of research question. Among the different study designs in pharmacoepidemiological research, cohort and case control studies have the highest level of hierarchy to establish causality, especially if all efforts are taken to reduce the risk of bias and confounding. Case reports and case series have the lowest level of hierarchy to establish causality as they do not have comparator groups.

Case records, case series as well as the analysis of spontaneous ADR reporting are used to detect safety signals. Cross-sectional studies are used to study the association between the exposure and the ADR, however, as exposure and ADRs are assessed at the same point in time, they cannot be used to study causal associations. The causal association between the exposure and the outcome is tested by means of a case control or cohort study.

A case control study will be conducted in case of rare events, the need to study multiple exposures and if results need to be readily available. The risk of bias and confounding is higher compared to cohort studies although this argument no longer holds when using automated databases. Cohort studies will be used in case of low exposure and if one is interested in multiple outcomes. Cohort studies are more expensive and time-consuming especially as data need to be collected during follow-up, unless cohort studies are designed using data from automated databases.

Conclusion

There is a growing consensus on the need of safe and effective medicines in children which has led to a change in the regulation of new medicines both in the US and Europe. In addition, numerous other initiatives have been taken to promote the safety of medicines in children. Among the list of paediatric needs as established by the EMEA's Working Party, there is the need to study the long term safety effects of drugs frequently used in children. [START_REF] Emea | Inventory of paediatric needs[END_REF] These types of research questions can best be answered by observational studies, especially as the domain of pharmacoepidemiology nowadays has access to automated databases with large sample size and long term follow-up. Very few observational studies have been done so far, but this will probably change in the coming years as a result of the growing attention on the need of safe drugs in children. As observational research will become more and more important, it is crucial to further built on competence to guarantee well conducted research and the potential for further development.

Fig 1 and Fig 2

 2 In prospective studies, data will be collected over time whereas in a retrospective cohort study, data have already been collected for other purposes.

Fig 1 :Fig 2 :Fig 3 :

 123 Fig 1: Prospective cohort study

by various pharmaceutical companies and received unconditional research grants from Pfizer, Merck, Johnson&Johnson, Amgen, Roche, GSK, Boehringer, Yamanouchi and Altana.