Polona J Maver

Boštjan J Kocjan

Katja Seme

Marko Potočnik

Nina Gale

Mario Poljak
email: mario.poljak@mf.uni-lj.si

Prof Mario Poljak

Prevaccination genomic diversity of human papillomavirus genotype 1 11: a study on 63 clinical isolates and 10 full-length genome 2 sequences

Keywords: human papillomaviruses, HPV 11, genomic diversity, genomic variants, complete genome human papillomaviruses, HPV 11, genomic diversity, genomic variants, complete 39 genome

Sequence variations of L1 ORF, long control region (LCR), E6, E5a, and E5b ORFs of 63 Slovenian HPV 11 isolates. At the top, genomic positions containing specific mutations are indicated vertically. Genomic positions without mutations compared to the prototype HPV 11 sequence (GenBank acc. no. M14119) are shaded in grey. Mutations in the form of nucleotide substitutions, insertions and deletions are marked with letters, I and ∆, respectively. The last column (f) indicates the number of isolates belonging to each HPV 11 genomic variant. At the bottom, codon positions and amino acid changes (aa) compared to reference HPV 11 sequence are indicated. Insertions: I1 = C; I2 = CC. 199x144mm (500 x 500 DPI)

b Primer reported by Caparros-Wanderley et al. (1999).

166

The reaction mixture contained up to 500 ng of template DNA, 2.5 µl of Expand Long Template

307

substitutions in these regions on a reduction or loss of binding capacity of neutralizing antibodies 308 has been reported (Ludmerer et al., 1996(Ludmerer et al., , 1997(Ludmerer et al., , 2000)). et al., 2000).

317

Sequence variations within the LCR region could theoretically influence the binding affinity of the

321

to the HPV 11 prototype was found in only 2 of our isolates, while the maximum genomic 322 difference between the sequences of the prototype and the newly identified LCR variants was 6 323 mutations. Of these, a nucleotide substitution at position 7479 and a nucleotide deletion at 324 position 7509 were found to be characteristic for all non-prototypic HPV 11 genomic variants. In 325 addition, the inclusion of two base pairs (GC) following a G at position 7716 in the prototype

326

HPV 11 sequence determined by [START_REF] Heinzel | Variation of human 507 papillomavirus type 6 (HPV-6) and HPV-11 genomes sampled throughout the world. J Clin 535 transformation by a unique isolate of human papillomavirus type 11[END_REF] was confirmed, since it was found in all 327 63 isolates studied. In contrast to the findings of [START_REF] Heinzel | Variation of human 507 papillomavirus type 6 (HPV-6) and HPV-11 genomes sampled throughout the world. J Clin 535 transformation by a unique isolate of human papillomavirus type 11[END_REF], no inserts longer than 2 328 bp were found in the LCR region in any of our isolates.

329

In contrast to the well established roles of high-risk HPV E6 proteins in cellular transformation

333

in a manner similar to that observed for the proteins of high-risk HPVs [START_REF] Oh | Roles of the E6 and E7 proteins in the life cycle of 548 low-risk human papillomavirus type 11[END_REF]. Thus,

334

even a single mutation in the HPV 11 E6 protein sequence can affect dramatically its function 335 [START_REF] Oh | Roles of the E6 and E7 proteins in the life cycle of 548 low-risk human papillomavirus type 11[END_REF]. Among six E6 genomic variants identified in this study, only one (Fig. 1, variant

336

E6-5), represented by two HPV 11 isolates, contained specific amino acid substitutions in the E6

337

protein: I92L (nt 186) and G122E (nt 466). Both mutations identified in our study were different 338 from those previously described as impairing episomal maintenance [START_REF] Oh | Roles of the E6 and E7 proteins in the life cycle of 548 low-risk human papillomavirus type 11[END_REF].

339

Interestingly, I92L and G122E have been described previously in the HPV 11VC genomic 340 variant detected in penile verrucous carcinoma (McGlennen et al., 1992). In cell transformation 341 assays, this genomic variant transformed neonatal rat kidney epithelial cells with an efficiency 342 comparable to HPV 16 (McGlennen et al., 1992). Characterization of the immunologic responses

343

elicited by the experimental therapeutic DNA vaccine that targets the HPV 11 E6 gene, identified

344

E6 aa44-51 peptide as the most immunogenic region for HPV 11 [START_REF] Peng | Characterization of human papillomavirus type 11-specific immune responses in a 552 preclinical model[END_REF]. According to

345

the results of this study, none of the 23 HPV 11 genomic variants contained amino acid changes 346 in that particular region; the efficiency of this candidate vaccine does not therefore seem to be 347 compromised by genomic variation.

348

The HPV E5 proteins are highly hydrophobic proteins found in the membrane compartments of

396

insertion at position 5405 in the HPV 11-2 genomic variant (Fig. 3), which seemed to be a 397 duplication of an adjacent 9 bp segment (nt 5397-5405), resulted in three additional amino acids 398 (AAE). The 9 bp duplication was ''in frame'' in all respects in the DNA, so the protein has part of 399 its sequence repeated. Otherwise, no novel amino acid substitutions were found in L2 protein.

400

The biological and clinical significance of several novel amino acid changes detected in the E1,

401

E2, E4 and L2 genomic regions during full-length sequence comparison of 12 HPV 11 isolates 402 needs to be further investigated.

403

The results of this study allowed for the first time phylogenetic

Figure 4 .

 4 Figure 4. Neighbor-joining algorithm based phylogenetic tree of 10 complete HPV 11 genomic variants determined in this study (HPV 11-1, HPV 11-2, HPV 11-3, HPV 11-4, HPV 11-7, HPV 11-13, HPV 11-14, HPV 11-16, HPV 11-21, and HPV 11-23), prototype HPV 11 isolate (HPV 11-REF) and HPV 11 isolate LZod45 from China. Bootstrap values (%) above 50 are shown (1000 bootstrap replicates for each grouping of a tree). 231x159mm (72 x 72 DPI)

a

  Figure 4. Neighbor-joining algorithm based phylogenetic tree of 10 complete HPV 11 genomic variants determined in this study (HPV 11-1, HPV 11-2, HPV 11-3, HPV 11-4, HPV 11-7, HPV 11-13, HPV 11-14, HPV 11-16, HPV 11-21, and HPV 11-23), prototype HPV 11 isolate (HPV 11-REF) and HPV 11 isolate LZod45 from China. Bootstrap values (%) above 50 are shown (1000 bootstrap replicates for each grouping of a tree). 231x159mm (72 x 72 DPI)

158

  Two overlapping DNA fragments (2900 and 5400 bp), covering the complete HPV 11 genome,159were generated by PCR using two pairs of primers. The 2900 bp fragment was generated using 160 11-L1F-PCR (5'-TTTATTACAGGTTCTGACTTCTATTTGC-3, nt 5686-5713) and 11-E6R-PCR 161 (5'-CCACCTTGTCCACCTCATCTTC-3', nt 650-629) primers and the 5400 bp fragment was 162 generated using 11-E7F-PCR (5'-CGTTGTGTGAAATAGAAAAACTAAAGC-3', nt 430-456) and 163 11-L2R3-PCR (5-' ATACAGGGTTGGGAGGAGGC-3', nt 5822-5803) primers. Both PCR 164 reactions were performed using Expand Long Template PCR System (Roche Diagnostics, 165 Manheim, Germany).

167 buffer 2

 2 with MgCl 2 , 500 µM of dNTPs, 0.75 U of Expand Long Template Enzyme mix, 0.3 µM of 168 each of the gene-specific primers, and water up to 25 µl. The cycling conditions used were 94°C 169 for 2 min, followed by 40 cycles of 94°C for 10 s, 52°C for 30 s and 68°C for 3-5 min. This was 170 followed by a final extension step at 68°C for 7 min and cooling of reaction mixtures to 4°C. were performed using the MEGA4.0.1 program package, as described 181 previously[START_REF] Tamura | MEGA4: Molecular Evolutionary Genetics Analysis 576 (MEGA) software version 4.0[END_REF]. Neighbor-joining, minimal evolution, UPGMA and maximum 182 parsimony algorithms were used to construct the phylogenetic trees. 11 isolates included in the study were amplified successfully and sequenced across 186 the entire L1 (nt 5682-7451), LCR (nt 7236-125), E6 (nt 81-650) and E5a-E5b (nt 3772-4456) 187 genomic regions. The HPV 11 L1, E6, E5a and E5b genomic variants were identified using the 188 prototype HPV 11 genome (GenBank acc. no. M14119) as a standard for comparisons and 189 nucleotide position numbering. For the determination of HPV 11 LCR genomic variants, an LCR 190 sequence of the prototype HPV 11 amended by the inclusion of two base pairs (GC) following a191G at position 7716[START_REF] Heinzel | Variation of human 507 papillomavirus type 6 (HPV-6) and HPV-11 genomes sampled throughout the world. J Clin 535 transformation by a unique isolate of human papillomavirus type 11[END_REF] was used. 11 isolates included in the study, a total of eleven L1 genomic variants, including 195 the prototype L1 sequence, were identified (Fig.1). Five of the 63 (7.9%) isolates corresponded 196 to the prototype L1 sequence. Point mutations were observed in 11 genomic positions, with a 197 maximum genomic distance of 3 nucleotides between the variants and the prototype HPV 11 198 sequence (0.2% of the entire L1 ORF). Nucleotide substitutions altered the amino acid 199 sequence of the L1 protein by a maximum of one amino acid (0.2% of the L1 protein) in four L1 200 genomic variants (L1-2, L1-3, L1-5 and L1-10) represented by 10 isolates (one, one, seven and HPV 11 L1 protein have previously been identified, and the effect of amino acid

318

  different transcription factors and thus affect viral transcription and replication (Sichero and Villa 319 2006). A total of 12 LCR genomic variants were identified in this study, and all clearly separated 320 into prototypic or non-prototypic HPV 11 variant groups. An LCR sequence completely identical

330

  and viral life cycle, the biological functions of low-risk HPV E6 proteins remain largely unclear. It 331 has been shown, however, that HPV 11 E6 and E7 proteins play an important role in the viral by facilitating the episomal maintenance of the viral genomes, and may act

Table 2 :

 2 List of primers used for sequencing the long range PCR product (5400 bp) containing E7, E1, E2, E5 and L2 regions of the HPV 11 genome.

Amplification of HPV 11 L1, LCR, E6, and E5 genomic regions 105

  Germany). The reaction mixture was prepared in 0.2 ml reaction tubes, each containing up to 125 125 ng of template DNA, 2.5 µl of 10 x FastStart High Fidelity Reaction Buffer with 18 mM 126 MgCl 2 , 200 µM of dNTPs, 1.25 U of FastStart High Fidelity Enzyme, 0.4 µM of each of the gene-127 specific primers and water up to 25 µl. The cycling conditions used were 95°C for 5 min,

	47 99	INTRODUCTION from 48 male and 3 female patients suffering from exophytic genital warts on different parts of Full-
	100	Human papillomavirus genotype 11 (HPV 11) is one of the most prevalent HPV genotypes, the anogenital area, 8 isolates were obtained from tissue specimens collected from 6 male and 2
	101	female patients suffering from recurrent laryngeal papillomatosis, and 4 isolates were obtained
	102 128	from cervical swabs collected from women suffering from cervical intraepithelial neoplasia grade
	103 129	1 lesions. followed by 40 cycles of 95°C for 30 s, 52-54°C for 30 s and 72°C for 1 min. This was followed
	104 130	by a final extension step at 72°C for 7 min and cooling of reaction mixtures to 4°C.
	131
	106 107 132	F o F o The genomic diversity of all HPV 11 isolates was investigated within the L1, LCR, E6, E5a, and E5b regions of the HPV 11 genome. A 1770 bp fragment containing the complete L1 ORF (1506 F o Sequencing and identification of HPV 11 genomic variants F o
	108	r r bp) was amplified by PCR using HPV11-L1F (5'-TGTTTTTATTACAGGTTCTGACTTC-3', nt r r
	109	P P 5682-5706) and HPV11-L1R (5'-AAAACATACATACACATTCCACAAA-3', nt 7451-7427) P P
	110 111 112	e e r e e r primers. An 823 bp fragment containing the complete LCR genomic region (758 bp) was amplified by PCR with the primer pair HPV11-LCR-F (5'-CTACAGCCCCCAAACGAA-3', nt 7236-7253) and HPV11-LCR-R (5'-CGTGGAGGCATCTTTACTTTC-3', nt 125-105). A 570 bp e e r e e r
	113 114 115 116 117 142 143	R e v i e w R e v i e w fragment containing the complete E6 ORF was amplified by PCR using the primer pair HPV11-E6F (5'-AAAATTAGCAGACGAGGCATT-3', nt 81-101) and HPV11-E6R (5'-CCACCTTGTCCACCTCATCT-3', nt 650-631). A 685 bp fragment, which contained both E5a and E5b ORF (276 and 225 bp) was PCR amplified using the primer pair HPV11-E5F (5'-AATACCACCCACCATTAGGC-3', nt 3772-3791) and HPV11-E5R (5'-R e v i e w Raleigh, USA) and the sequence of prototype HPV 11 genome (GenBank acc. no. M14119) was R e v i e w
	118 144	TGGCTGACGCACGTTTAC-3', nt 4456-4439). All primers used in the study were designed used as a standard for comparisons and genomic position numbering. The corrected LCR
	119 145	according to the prototype HPV 11 genome sequence (GenBank acc. no. M14119) using sequence of prototype HPV 11 genome was used to determine HPV 11 LCR genomic variants
	146	(Heinzel et al., 1995). In order to minimize PCR and sequencing errors, all isolates that
	147	represented a single HPV 11 genomic variant were amplified and sequenced twice. According to
	148	the presence of specific fixed nucleotide signtures, the genomic variants were grouped into
	149
	150
		5 6 7

Primer3 (http://frodo.wi.mit.edu/) and Netprimer (http://www.premierbiosoft.com) programs. A 120 corrected LCR sequence with the insertion of 2 bp at nt 7717-7718

[START_REF] Heinzel | Variation of human 507 papillomavirus type 6 (HPV-6) and HPV-11 genomes sampled throughout the world. J Clin 535 transformation by a unique isolate of human papillomavirus type 11[END_REF]

was 121 used for designing and genomic position numbering of the LCR specific primers.

122

All PCRs were carried out on the GeneAmp PCR instrument 9700 (PE Applied Biosystems,

The PCR products were analyzed on ready-to-use PCR CheckIT Wide Mini S-2x25 gels 133 (Elchrom Scientific, Zurich, Switzerland) using a 100 or 500 bp DNA ladder (Roche Diagnostics, 134 Manheim, Germany) and purified with a QIAquick PCR purification kit (Qiagen, Hilden, 135 Germany). Concentrations of purified PCR products were estimated on a 0.8% agarose gel 136 using a MassRuler DNA Ladder Low Range (Fermentas, Vilnius, Lithuania) and set to 50 ng/µl.

137

Sequencing of PCR products was performed at Macrogen. (Seoul, Korea) with the same set of 138 primers as those used for PCR and additional sequencing primers that are listed in Table

1

. All 139 sequences were processed and assembled with Vector NTI Advance v11.0 (Invitrogen, 140 Carlsbad, CA). The identification of HPV 11 L1, LCR, E6, E5a, and E5b genomic variants was 141 made with BioEdit Sequence Alignment Editor v7.0.9.0 (North Carolina State University, "prototypic" and "non-prototypic" HPV 11 genomic variant groups.

genome amplification and sequencing of selected HPV 11 isolates 151

	152	Full-length genome sequencing was performed on a total of 10 HPV 11 isolates: 5 isolates were
	153	obtained from anal swabs of male patients suffering from exophytic genital warts (variants HPV
	154	11-1, HPV 11-3, HPV 11-7, HPV 11-13, HPV 11-16), 2 isolates from tissue specimens of male
	155	patients suffering from exophytic genital warts (variants HPV 11-14, HPV 11-21), 2 isolates from
	156	tissue specimens of male patients suffering from laryngeal papillomatosis (variants HPV 11-4,
	157	HPV 11-23) and one isolate from a cervical swab of a female patient with a cervical
		intraepithelial neoplasia grade 1 lesion (variant HPV 11-2).

  A total of 11 L1 genomic variants were

	309
	310	identified in the present study, of which four, represented by 10 HPV 11 isolates, contained
	311	specific amino acid substitutions in L1 protein. The L1 genomic variants had specific amino acid
	312	substitutions (H35Y, L119V, A235S or K347T) at nucleotide positions 5873, 6125, 6473 or 6810,
	313	respectively. The single amino acid change A235S was the most frequent mutation, being found
	314	in 7/63 (11.1%) HPV 11 isolates. The biological and clinical significance of the majority of these
	315	mutations is at present unclear and needs to be investigated further. To the best of our
	316	knowledge, only the K347T is located in a domain responsible for elicitation of a neutralizing
		response to HPV 11 (Ludmerer

  Q46R and T70P detected at positions 3391 and 3462 constituted additional characteristics of 395 five and one non-prototypic HPV 11 genomic variants, respectively. Within L2 ORF, a 9 bp

	385	amino acid substitution R318A were also found in the prototypic HPV 11 isolate LZod45-11 (Wu
	386	et al., 2009), thus in 11 out of 12 HPV 11 isolates for which a full-length sequence is available at
	387	present, we suggest the resequencing of these particular positions in the HPV 11 prototype
	388	clone. Within E2 ORF, no variation was observed in three prototypic HPV 11 genomic variants
	389	studied. In contrast, among seven non-prototypic HPV 11 genomic variants, one contained a
	390	specific amino acid substitution N247T at nucleotide position 3642 and all non-prototypic
	391	variants harbored a specific amino acid substitution K308R at nucleotide position 3645. In E4
	392 393	F o ORF, four nucleotide mutations were detected and all resulted in amino acid exchanges. Amino F o acid substitutions G61E and S78L detected at nucleotide positions 3436 and 3487 were
	394	r r characteristic features of all seven non-prototypic HPV 11 genomic variants, while substitutions
		P P
		e e r e e r
	349 350 351	the host cells. Their physiological roles are not completely understood. It is thought that they have the ability to modulate cell signaling, possess transforming activity, play a role in the R e v i e w R e v i e w
	352	stimulation of cell proliferation and, together, with the HPV E6 proteins induce koilocytosis
	353	(Doorbar et al., 2006; Krawczyk et al., 2008). Unlike the majority of other HPV genotypes, which
	354	have one E5 ORF, HPV 11 and closely related HPV 6 possess two E5 genes: E5a and E5b. It
		has been shown that HPV 11 E5 proteins are involved in transformation (Chen et al., 1994,
		16

  comparison of full-length HPV 11 404 genomes. As shown in Fig. 4, full-length genome comparison of all HPV 11 isolates for which a 405 full-length sequence is available at present: 10 isolates from this study, prototype HPV 11 isolate406and isolate LZod45-11[START_REF] Wu | Detection of HPV types and neutralizing antibodies in Gansu[END_REF], confirmed the existence of two major HPV 11 genomic

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

E5a sequences (FN870498-FN870560), and E5b sequences (FN870561-FN870623).

classified taxonomically in the Alpha papillomavirus genus -species 10 or "HPV 6 species",

Sequencing of the long range PCR products was performed at Macrogen. (Seoul, Korea) with

primers listed in Tables1 and 2. All sequences were edited and assembled with Vector NTI

manner as described above. In addition, the L1-LCR-E6 genomic fragments (2900 bp) were

1996a, 1996b) and stimulation of cell proliferation[START_REF] Tsao | Human papillomavirus type 11 and 16 E5 represses 579 p21 (WafI/SdiI/CipI) gene expression in fibroblasts and keratinocytes[END_REF]. The mutational analysis of

four E5a genomic variants were found, of which three (all non-prototypic variants), differed from

most frequent E5a genomic variant (Fig.1, variant E5a-2) identified in our study contained two

3, variant HPV 11-7) contained a specific amino acid substitution N100T at nucleotide position

Aubin F, Prétet JL, Jacquard AC, Saunier M, Carcopino X, Jaroud F, Pradat P, Soubeyrand B,

Sci (Lond) 110:525-541.

Kocjan BJ, Poljak M, Cimerman M, Gale N, Potočnik M, Bogovac Ž, Seme K. 2009.

McGlennen RC, Ghai J, Ostrow RS, LaBresh K, Schneider JF, Faras AJ. 1992. Cellular

papillomavirus variant lineages, viral persistence, and cervical neoplasia. Cancer Res 70:3159-

specific amino acid substitutions (I28F and V41L) at nucleotide positions 3952 and 3991, 362 respectively. Since these two substitutions were found in the majority (56/63, 88.8%) of HPV 11 363 isolates characterized in this study, it is presumed that they prevail in the naturally occurring


Florence Hunsel

Christine Welle

PhD Anneke Passier

Eugène Puijenbroek

Kees

PharmD Florence P A M Van Hunsel
email: f.vanhunsel@lareb.nl

MSc Christine Van Der Welle

MD, PhD Eugène Van Puijenbroek

MD, PhD Kees Van Grootheest

Keywords: adverse drug reactions (ADRs), pharmacovigilance, patient reporting, consumer reporting, web-based questionnaire adverse drug reactions (ADRs), pharmacovigilance, patient reporting, consumer reporting, web-based questionnaire

Motives for reporting adverse drug reactions by patient-reporters in the Netherlands

Introduction

Direct reporting of adverse drug reactions (ADRs) by patients has been incorporated into the pharmacovigilance systems in a growing number of countries [1]. The knowledge about patient reporting in daily pharmacovigilance has been increasing over the past few years [1][2][3][4].

The Netherlands Pharmacovigilance Centre Lareb is the organization who maintains the spontaneous reporting system for ADRs in the Netherlands, on behalf of the Medicines Evaluation Board. In addition to healthcare professionals and marketing authorization holders, patients have been able to report ADRs directly to Lareb since April 2003.

Patients can report ADRs through an electronic reporting form on the Lareb website. The content of the reporting form is identical to the form for healthcare professionals. Several mandatory fields in this form ensure the completeness of the information before it can be send to the pharmacovigilance centre. In rare cases where it is not possible for a patient to report electronically, a paper reporting form can be sent. Patients' and healthcare professionals' reports are coded according to MedDRA (Medical Dictionary for Regulatory Activities) terminology, individually assessed by trained assessors and stored in the same database. All reporters, both patients and healthcare professionals, receive feedback information about their reported ADR(s) [2].

In 2008, the Netherlands Pharmacovigilance Centre Lareb published their experiences with three years of patient reporting [2]. Patients' reports gave different information compared to healthcare professionals' reports with regard to categories of seriousness and outcome of the reported ADRs [2]. When comparing patients' and healthcare professionals' ADR reports about statins after media attention, patient reports provided additional information about different categories of adverse reactions, about the impact of ADRs on daily life and about the patient-health professional relationship [3]. In Denmark, patients reported different categories of ADRs for different types of medicines, compared to other reporters [4]. This study concluded that consumers should be actively included in systematic drug surveillance systems. Patient's own firsthand reports of their experiences with drugs can capture side effects that clinicians might miss [5,6]. According to Foster et al. 'patient-reporting can be an important source of information about side effects in the context of real-life clinical practice' [7].

Whereas for healthcare professionals the motivations and attitudes towards ADR reporting to a pharmacovigilance centre have been studied extensively [8][9][10][11][12][13][14][15][16][17][18][19][20], the reasons why patients report ADRs are less well known. As Aagaard et al. [4] mentioned consumers' experiences with and perspectives on ADRs should be further studied. A qualitative study with guided interviews with twenty-one patients in the Netherlands was performed to gain insight in motivations of patients to report ADRs to a pharmacovigilance centre [21]. Most patients expressed altruistic motives, but also the severity of the ADR and the need for extra information about the ADR were mentioned as motives for reporting.

The aim of this study is to quantify the reasons and opinions of patients who reported ADRs in the Netherlands to a pharmacovigilance centre. Data from interviews investigating patients' motives for reporting ADR's [21] were used to develop a questionnaire that could be sent to a large group of patient reporters. Questionnaires were web-based and sent with use of the online web service SurveyMonkey [22].

Study population

The target population existed of patients who reported an ADR to the Netherlands Pharmacovigilance Centre Lareb in the period between January 1 st , 2008 and March 1 st , 2009. In the research period a total of 1503 case reports of ADRs were reported by patients. 1440 of these patients had reported by means of the electronic reporting form on the Lareb website, which is the method the pharmacovigilance centre prefers patients to use for reporting. For each of these 1440 reports the email address was selected. Some patients reported several times and therefore e-mail addresses could exist twice or more in the list (but with different report numbers). A final list of 1370 e-mail addresses of patient-reporters remained who were approached.

Questionnaire

A list of all categories of quotes from an earlier qualitative study [21] was created with support of QSR NVivo version 8.0.264.0, a software programme for ordering qualitative data [23]. These categories were rephrased to statements (with rating scales) as questionnaire items. Categories of quotes from the interviews that were much alike were combined in order to keep the time needed for answering the questionnaire limited. The statements were divided into 'Reasons' and 'Opinions'.

The questionnaire items could be rated at a five-point Likert scale (strongly agree to strongly disagree) [24], where the middle position was labeled 'neutral' to reflect a neutral position, and not an inability to answer the question.

The first theme contained statements about reasons to report an ADR. Next to the response options about reasons to report an ADR, an 'Other' option was included along with a free text area for 'please specify'.

The second theme included statements about opinions of patients about reporting an ADR.

Patients were also asked if they would report a possible ADR again in the future.

Table 1.

The questionnaire addressed some demographic questions about gender, age (<18 years, 18-35 year, 36-64 year, >65 years), level of education (primary school, secondary school, vocational

Sending the questionnaire

The web-based survey was tested in a small group of field testers before the survey was sent. The survey was sent on March 23, 2009. Two weeks later (April 6, 2009), a reminder was sent to all non-responders. Collection of responses was finished after one month (April 24, 2009). The link in the invitation e-mail was uniquely tied to the survey and the respondents e-mail address.

Therefore, the message could not be forwarded by respondents, and only one response per e-mail address was allowed. This also implied that random surfers on the Internet could not reach the online survey.

Data analysis

Descriptive statistics gave an overview of the patient characteristics, the reasons to report ADRs and opinions about reporting ADRs.

A Pearson Chi-square (χ 2 ) test was performed to detect significant differences in motives and opinions between patients who where a member of a patient organization or not and differences in answers between men and women. Significance was based on a 2-sided χ 2 -test: p<0.05.

Correlations were calculated to measure possible relationships between two or more statements.

Because the data were measured at the ordinal level (Likert-scale) Spearman's correlation coefficient (r) was used in this analysis. In this study a correlation was considered strong if r > 0.7, moderate if r > 0.4 and < 0.7, and weak if r < 0.4 [25]. Data were analyzed using the

Results

Response

A total number of 1370 potential participants were approached by e-mail, of which 57 emails were undeliverable. 1005 of the remaining 1313 patients filled in the whole questionnaire, which resulted in a 76.5% response rate.

Among the 1005 responses were 6 responses by healthcare professionals whose email addresses had been selected because they had reported an ADR with the use of the patient reporting form in stead of the healthcare professional form, by accident. These responses were not taken into account, which resulted in 999 useful responses for the analysis.

The responses to the questionnaire are shown in Figure 1. The characteristics of the patientreporters are given in Table 2.

Reasons to report an ADR

An overview of the responses on the reasons for reporting adverse drug reactions is given in Table 3.

The main reasons for patients to report ADRs were to share their experiences (89% agreed or strongly agreed), the severity of the reaction (86% agreed or strongly agreed to the statement), worries about their own situation (63.2% agreed or strongly agreed) and the fact the ADR was not mentioned in the patient information leaflet (57.6% agreed or strongly agreed).

Responses to the open question

The open text field in the questionnaire for "other reasons for reporting" was filled in by 228 respondents. The reasons to report an ADR which were most frequently mentioned in this open question are "For other people" (n=33), "Medical practitioner or pharmacist does not acknowledge the problem or take it seriously" (n= 27), "ADR was severe" (n =21) and "Comments on patient information leaflet" (n=20). The other responses to the open question (n=127) could not be grouped together easily because of a large variety of subjects.

The responses to the open question were compared to the statements in the questionnaire. The reason 'medical practitioner or pharmacist doesn't acknowledge the problem or take it seriously' was not included as a statement in this survey. Apparently, the respondents considered this a different reason than the statement 'it was difficult to discuss the adverse drug reaction with my medical practitioner or pharmacist'.

Deleted: Some motives were already mentioned in the statements, but others were new. 93.8% of the patients share the opinion that reporting an ADR can prevent harm to other people and 97.9% believe that reporting contributes to research and knowledge. 90.7% of the patients stated that they felt responsible for reporting an ADR and 92.5% of all patients stated that they will report a possible ADR once again in the future. The opinions about reporting ADRs are shown in Table 4.

There was no statistically significant difference in the answers between men and women for all statements (χ2 tests p > 0.05), except for the motive 'I was worried about my own situation' (χ 2 tests p = 0.022). 70% of the men were worried about their situation in comparison with 59.8% of the women.

Membership of patient organization was compared with the answers on the statements.

There was no statistically significant difference in the answers (agree, neutral, disagree) between members or non-members for all statements (χ2 tests p > 0.05), except for the opinion 'In the future I will report a possible adverse drug reaction once again' (χ 2 tests p = 0.036). 95.5% of the members of a patient organization agreed to this statement compared to 91.3% of non-members.

The correlation coefficients between the reasons for reporting ADRs, between the opinions and between the reasons and opinions were mostly moderate or weak (r > 0.4 < 0.7 or r < 0.4). The highest correlation coefficient (r) was .616 (between Reason 7 'I was angry about the situation' and Reason 8 'I wanted action to be taken').

Findings

This study confirms the findings of the previous qualitative interviews [21]. Patients report ADRs for various reasons. The most important motives for reporting were the reaction being severe, the patients wanted to share their experiences, they were worried about their situation and because the ADR was not mentioned in the patient information leaflet. Furthermore, patients believe that reporting an ADR can prevent harm to other people, that reporting contributes to research and knowledge and most patients stated that they felt responsible for reporting an ADR.

An additional motive for reporting ADRs, derived from the open question, was 'my medical practitioner or pharmacist doesn't acknowledge the problem or take it seriously'. This motive was mentioned during the qualitative interviews [21]. Probably the wording of the statement in the questionnaire about communication with the health care professional (reason 3) was not clear enough in emphasizing the acknowledgement of the medical practitioner or pharmacist instead of the difficulty to discuss the ADR.

The severity of an ADR as a motive for reporting does not necessarily mean that the patient suffered from an ADR that was serious according to international criteria [26]. Medical seriousness may differ from patients' views on what constitutes a serious problem [27].

In 2009, 27.0% of the average Dutch population (age 15 to 65) had a diploma on the highest degree (higher professional education / academic) [28]. In comparison 55% of the participants in this study had a high level of education. The high level of education among the respondents is not surprising considering that this group might be better informed about healthcare in general and the possibility to report ADRs.

Two third of the respondents to the questionnaire were female. This is almost the same as in the whole population of patient-reporters in our research period (1440 in total, 63.6 % women). This is also in line with a previous study by De Langen et al. [2], were it was found that 63% of the patient-reporters were female. Of the Dutch population in 2009, 50.5% was female. This difference in the percentages could be a result of women experiencing a higher incidence of ADRs than men [29] and possibly of women being more eager to report ADRs to a pharmacovigilance centre. However, there were no differences in answers between men and women, except for the answers to reason 'being worried about own situation'.

Elderly people are more at risk of developing an adverse drug reaction [30], so it is important that the motives and opinions of this group are also taken into account. Of the respondents to the questionnaire 14.6% was older than 65 years. Of this group less then 2% of the

Comparison to other studies

To our knowledge, only the Netherlands pharmacovigilance centre has previously published studies about patient's motives for reporting ADRs to a pharmacovigilance centre [31,21]. During the first one-year trial period of patient reporting in the Netherlands, patients were asked why they reported an ADR to the pharmacovigilance centre instead of to their healthcare professional. 23% of the respondents stated that they felt their complaints wouldn't be taken serious elsewhere and 22% had already reported the ADR to a healthcare professional but nothing happened [31]. In a qualitative study with twenty-one patients almost all patients had multiple motives for reporting.

Altruistic motives concerned preventing harm to other patients, making the ADR publicly known, increasing medical knowledge and wanting to improve the patient information leaflet. Personal motives to report an ADR concerned wanting more information about the ADR, indicating that the ADR was too severe not to report, being angry, or wanting confirmation about their ADR [21]. These results have been generalized and quantified in a large group in the present study.

For healthcare professionals several studies have been conducted to investigate their motivations for (not) reporting ADRs to a pharmacovigilance centre [8][9][10][11][12][13][14][15][16][17][18][19][20]. Hasford et al. [12] and Ekman et al. [9] indicated that severity of the reaction, unusual reaction to the reporter and a reaction caused by a new drug were the main reasons to report. In the study by Bäckström et al. [20] three-quarters of the respondents stated that the severity of the reaction was the main factor determining whether a suspected ADR was reported or not. Biriell et al. [16] found the following motives for reporting ADR's; motivation to contribute to medical knowledge, reaction previously unknown to reporter, reaction to new drug, all significant reactions reported, known association between drug and reaction and severity of reaction as positive reasons for physicians and pharmacists to report an ADR. Some of the motives found for healthcare professionals are also important reasons for patients to report like severity of the reaction and wanting to contribute to medical knowledge.

Validity of the study design

The statements in the questionnaire were based upon qualitative interviews with patients to increase the validity of the survey instrument [32]. The response rate to the questionnaire was more than 76.5%, which is high compared to some other studies about motivations for (not) reporting ADRs [9,20]. The high response rate increases the validity of the results found in this study.

There are also some limitations to this study; the study population existed of patients who had already reported an ADR to the pharmacovigilance centre in the Netherlands, which is a selected population. One could assume that patients who are interested in their drug treatment and especially those who have experienced an adverse drug reaction would be more willing to answer this type of questionnaire. We did not include motives and opinions of patients who experienced an ADR but who choose not report this to the pharmacovigilance centre.

Because 96% of our target study population existed of patients who had reported an ADR previously through the online reporting form it was possible to use a web-based questionnaire.

Most people in the Netherlands have access to the internet; in 2009, 91% of the Dutch population younger than 65 years had access, according to Statistics Netherlands [28]. However, selection bias cannot be excluded; in the age category 65 to 75 only 64% of the population had access.

Access to the internet is not specified for age groups older than 75. 91% of people up to 55 years had used internet in the past three months in a survey in 2009. For people aged 55 to 65 years this was 82% and for people aged 65 to 75 years 53% [28]. Patients who did not use the electronic reporting form (n=63) were excluded from this study, it is possible that other motives were present in this group. The pharmacovigilance centre accepts consumers reporting for relatives, for example children with access to the internet could report for an elderly parent.

Use of the internet is also correlated to the level of education [28], which might increase selection bias. The level of education in the Netherlands is higher compared to the average level in the European Union (EU-27 countries) were 19.1% of the population has received education on the highest level [33]. In 2009 on average 65% of the households of the EU-27 countries had access to the internet, which is considerably lower than 90% in the Netherlands [33]. In fact, the percentage of access to the internet in the Netherlands is the highest of all European countries [33]. Therefore the use of a web-bases questionnaire might not be feasible in all countries in the same manner as in the Netherlands.

Conclusions

The main motives for patients to report their adverse drug reactions to a national pharmacovigilance centre were the severity of the ADR and their need to share experiences. Altruistic motives are reflected by the high responses to the statements 'Reporting an adverse drug reaction can prevent harm to other people', 'I felt responsible for reporting an adverse drug reaction' and 'Reporting an adverse drug reaction contributes to research and knowledge'. There is only a small group who feels that they would benefit themselves from reporting an ADR. The high level of response to the questionnaire shows that patients are involved when it comes to adverse drug reactions and also willing to share their motivations for and opinions about the reporting of adverse drug reactions with the pharmacovigilance centre. More than 90% of the patients, who had already reported an ADR once, agree with the statement 'In the future I will report a possible adverse drug reaction once again'.

Conflicts of interest:

Nothing to declare The aim of this study is to quantify the reasons and opinions of patients who reported adverse drug reactions (ADRs) in the Netherlands to a pharmacovigilance centre.

Method

A web-based questionnaire was sent to 1370 patients who had previously reported an ADR to a pharmacovigilance centre. For the analysis descriptive statistics, χ 2 tests and Spearman's correlation coefficients were used.

Results

The response rate was 76.5% after one reminder. The main reasons for patients to report ADRs were to share their experiences (89% agreed or strongly agreed), the severity of the reaction (86% agreed or strongly agreed to the statement), worries about their own situation (63.2% agreed or strongly agreed) and the fact the ADR was not mentioned in the patient information leaflet (57.6% agreed or strongly agreed ).

93.8% of the patients share the opinion that reporting an ADR can prevent harm to other people and 97.9% believe that reporting contributes to research and knowledge. 90.7% of the patients stated that they felt responsible for reporting an ADR and 92.5% of all patients stated that they will report a possible ADR once again in the future.

Conclusion

The main motives for patients to report their ADRs to a pharmacovigilance centre were the severity of the ADR and their need to share experiences. The high level of response to the questionnaire shows that patients are involved when it comes to ADRs and also willing to share their motivations for and opinions about the reporting of ADRs with a pharmacovigilance centre. pharmacovigilance systems in a growing number of countries [1]. The knowledge about patient reporting in daily pharmacovigilance has been increasing over the past few years [1][2][3][4].

The Netherlands Pharmacovigilance Centre Lareb is the organization who maintains the spontaneous reporting system for ADRs in the Netherlands, on behalf of the Medicines Evaluation Board. In addition to healthcare professionals and marketing authorization holders, patients have been able to report ADRs directly to Lareb since April 2003.

Patients can report ADRs through an electronic reporting form on the Lareb website. The content of the reporting form is identical to the form for healthcare professionals. Several mandatory fields in this form ensure the completeness of the information before it can be send to the pharmacovigilance centre. In rare cases where it is not possible for a patient to report electronically, a paper reporting form can be sent. Patients' and healthcare professionals' reports are coded according to MedDRA (Medical Dictionary for Regulatory Activities) terminology, individually assessed by trained assessors and stored in the same database. All reporters, both patients and healthcare professionals, receive feedback information about their reported ADR(s) [2].

In 2008, the Netherlands Pharmacovigilance Centre Lareb published their experiences with three years of patient reporting [2]. Patients' reports gave different information compared to healthcare professionals' reports with regard to categories of seriousness and outcome of the reported ADRs [2]. When comparing patients' and healthcare professionals' ADR reports about statins after media attention, patient reports provided additional information about different categories of adverse reactions, about the impact of ADRs on daily life and about the patient-health professional relationship [3]. In Denmark, patients reported different categories of ADRs for different types of medicines, compared to other reporters [4]. This study concluded that consumers should be actively included in systematic drug surveillance systems. Patient's own firsthand reports of their experiences with drugs can capture side effects that clinicians might miss [5,6]. According to Foster et al. 'patient-reporting can be an important source of information about side effects in the context of real-life clinical practice' [7].

Whereas for healthcare professionals the motivations and attitudes towards ADR reporting to a pharmacovigilance centre have been studied extensively [8][9][10][11][12][13][14][15][16][17][18][19][20], the reasons why patients report ADRs are less well known. As Aagaard et al. [4] mentioned consumers' experiences with and perspectives on ADRs should be further studied. Data from interviews investigating patients' motives for reporting ADR's [21] were used to develop a questionnaire that could be sent to a large group of patient reporters. Questionnaires were web-based and sent with use of the online web service SurveyMonkey [22].

Study population

The target population existed of patients who reported an ADR to the Netherlands Pharmacovigilance Centre Lareb in the period between January 1 st , 2008 and March 1 st , 2009. In the research period a total of 1503 case reports of ADRs were reported by patients. 1440 of these patients had reported by means of the electronic reporting form on the Lareb website, which is the method the pharmacovigilance centre prefers patients to use for reporting. For each of these 1440 reports the email address was selected. Some patients reported several times and therefore e-mail addresses could exist twice or more in the list (but with different report numbers). A final list of 1370 e-mail addresses of patient-reporters remained who were approached.

Questionnaire

A list of all categories of quotes from an earlier qualitative study [21] was created with support of QSR NVivo version 8.0.264.0, a software programme for ordering qualitative data [23]. These categories were rephrased to statements (with rating scales) as questionnaire items. Categories of quotes from the interviews that were much alike were combined in order to keep the time needed for answering the questionnaire limited. The statements were divided into 'Reasons' and 'Opinions'.

The questionnaire items could be rated at a five-point Likert scale (strongly agree to strongly disagree) [24], where the middle position was labeled 'neutral' to reflect a neutral position, and not an inability to answer the question.

The first theme contained statements about reasons to report an ADR. Next to the response options about reasons to report an ADR, an 'Other' option was included along with a free text area for 'please specify'.

The second theme included statements about opinions of patients about reporting an ADR.

Patients were also asked if they would report a possible ADR again in the future.

Table 1.

The questionnaire addressed some demographic questions about gender, age (<18 years, 18-35 year, 36-64 year, >65 years), level of education (primary school, secondary school, vocational Therefore, the message could not be forwarded by respondents, and only one response per e-mail address was allowed. This also implied that random surfers on the Internet could not reach the online survey.

Data analysis

Descriptive statistics gave an overview of the patient characteristics, the reasons to report ADRs and opinions about reporting ADRs.

A Pearson Chi-square (χ 2 ) test was performed to detect significant differences in motives and opinions between patients who where a member of a patient organization or not and differences in answers between men and women. Significance was based on a 2-sided χ 2 -test: p<0.05.

Correlations were calculated to measure possible relationships between two or more statements.

Because the data were measured at the ordinal level (Likert-scale) Spearman's correlation coefficient (r) was used in this analysis. In this study a correlation was considered strong if r > 0.7, moderate if r > 0.4 and < 0.7, and weak if r < 0.4 [25]. Data were analyzed using the

Response

A total number of 1370 potential participants were approached by e-mail, of which 57 emails were undeliverable. 1005 of the remaining 1313 patients filled in the whole questionnaire, which resulted in a 76.5% response rate.

Among the 1005 responses were 6 responses by healthcare professionals whose email addresses had been selected because they had reported an ADR with the use of the patient reporting form in stead of the healthcare professional form, by accident. These responses were not taken into account, which resulted in 999 useful responses for the analysis.

The responses to the questionnaire are shown in Figure 1. The characteristics of the patientreporters are given in Table 2.

Reasons to report an ADR

An overview of the responses on the reasons for reporting adverse drug reactions is given in Table 3.

The main reasons for patients to report ADRs were to share their experiences (89% agreed or strongly agreed), the severity of the reaction (86% agreed or strongly agreed to the statement), worries about their own situation (63.2% agreed or strongly agreed) and the fact the ADR was not mentioned in the patient information leaflet (57.6% agreed or strongly agreed).

Responses to the open question

The open text field in the questionnaire for "other reasons for reporting" was filled in by 228 respondents. The reasons to report an ADR which were most frequently mentioned in this open question are "For other people" (n=33), "Medical practitioner or pharmacist does not acknowledge the problem or take it seriously" (n= 27), "ADR was severe" (n =21) and "Comments on patient information leaflet" (n=20). The other responses to the open question (n=127) could not be grouped together easily because of a large variety of subjects.

The responses to the open question were compared to the statements in the questionnaire. The reason 'medical practitioner or pharmacist doesn't acknowledge the problem or take it seriously'

was not included as a statement in this survey. Apparently, the respondents considered this a different reason than the statement 'it was difficult to discuss the adverse drug reaction with my medical practitioner or pharmacist'. 93.8% of the patients share the opinion that reporting an ADR can prevent harm to other people and 97.9% believe that reporting contributes to research and knowledge. 90.7% of the patients stated that they felt responsible for reporting an ADR and 92.5% of all patients stated that they will report a possible ADR once again in the future. The opinions about reporting ADRs are shown in Table 4.

There was no statistically significant difference in the answers between men and women for all statements (χ2 tests p > 0.05), except for the motive 'I was worried about my own situation' (χ 2 tests p = 0.022). 70% of the men were worried about their situation in comparison with 59.8% of the women.

Membership of patient organization was compared with the answers on the statements.

There was no statistically significant difference in the answers (agree, neutral, disagree) between members or non-members for all statements (χ2 tests p > 0.05), except for the opinion 'In the future I will report a possible adverse drug reaction once again' (χ 2 tests p = 0.036). 95.5% of the members of a patient organization agreed to this statement compared to 91.3% of non-members.

The correlation coefficients between the reasons for reporting ADRs, between the opinions and between the reasons and opinions were mostly moderate or weak (r > 0.4 < 0.7 or r < 0.4). The highest correlation coefficient (r) was .616 (between Reason 7 'I was angry about the situation' and Reason 8 'I wanted action to be taken').

Findings

This study confirms the findings of the previous qualitative interviews [21]. Patients report ADRs for various reasons. The most important motives for reporting were the reaction being severe, the patients wanted to share their experiences, they were worried about their situation and because the ADR was not mentioned in the patient information leaflet. Furthermore, patients believe that reporting an ADR can prevent harm to other people, that reporting contributes to research and knowledge and most patients stated that they felt responsible for reporting an ADR.

An additional motive for reporting ADRs, derived from the open question, was 'my medical practitioner or pharmacist doesn't acknowledge the problem or take it seriously'. This motive was mentioned during the qualitative interviews [21]. Probably the wording of the statement in the questionnaire about communication with the health care professional (reason 3)

was not clear enough in emphasizing the acknowledgement of the medical practitioner or pharmacist instead of the difficulty to discuss the ADR.

The severity of an ADR as a motive for reporting does not necessarily mean that the patient suffered from an ADR that was serious according to international criteria [26]. Medical seriousness may differ from patients' views on what constitutes a serious problem [27].

In 2009, 27.0% of the average Dutch population (age 15 to 65) had a diploma on the highest degree (higher professional education / academic) [28]. In comparison 55% of the participants in this study had a high level of education. The high level of education among the respondents is not surprising considering that this group might be better informed about healthcare in general and the possibility to report ADRs.

Two third of the respondents to the questionnaire were female. This is almost the same as in the whole population of patient-reporters in our research period (1440 in total, 63.6 % women). This is also in line with a previous study by De Langen et al. [2], were it was found that 63% of the patient-reporters were female. Of the Dutch population in 2009, 50.5% was female. This difference in the percentages could be a result of women experiencing a higher incidence of ADRs than men [29] and possibly of women being more eager to report ADRs to a pharmacovigilance centre. However, there were no differences in answers between men and women, except for the answers to reason 'being worried about own situation'.

Elderly people are more at risk of developing an adverse drug reaction [30], so it is important that the motives and opinions of this group are also taken into account. Of the respondents to the questionnaire 14.6% was older than 65 years. Of this group less then 2% of the

Comparison to other studies

To our knowledge, only the Netherlands pharmacovigilance centre has previously published studies about patient's motives for reporting ADRs to a pharmacovigilance centre [31,21]. During the first one-year trial period of patient reporting in the Netherlands, patients were asked why they reported an ADR to the pharmacovigilance centre instead of to their healthcare professional.

23% of the respondents stated that they felt their complaints wouldn't be taken serious elsewhere and 22% had already reported the ADR to a healthcare professional but nothing happened [31]. In a qualitative study with twenty-one patients almost all patients had multiple motives for reporting.

Altruistic motives concerned preventing harm to other patients, making the ADR publicly known, increasing medical knowledge and wanting to improve the patient information leaflet. Personal motives to report an ADR concerned wanting more information about the ADR, indicating that the ADR was too severe not to report, being angry, or wanting confirmation about their ADR [21]. These results have been generalized and quantified in a large group in the present study.

For healthcare professionals several studies have been conducted to investigate their motivations for (not) reporting ADRs to a pharmacovigilance centre [8][9][10][11][12][13][14][15][16][17][18][19][20]. Hasford et al. [12] and Ekman et al. [9] indicated that severity of the reaction, unusual reaction to the reporter and a reaction caused by a new drug were the main reasons to report. In the study by Bäckström et al.

[20] three-quarters of the respondents stated that the severity of the reaction was the main factor determining whether a suspected ADR was reported or not. Biriell et al. [16] found the following motives for reporting ADR's; motivation to contribute to medical knowledge, reaction previously unknown to reporter, reaction to new drug, all significant reactions reported, known association between drug and reaction and severity of reaction as positive reasons for physicians and pharmacists to report an ADR. Some of the motives found for healthcare professionals are also important reasons for patients to report like severity of the reaction and wanting to contribute to medical knowledge.

Validity of the study design

The statements in the questionnaire were based upon qualitative interviews with patients to increase the validity of the survey instrument [32]. The response rate to the questionnaire was more than 76.5%, which is high compared to some other studies about motivations for (not) There are also some limitations to this study; the study population existed of patients who had already reported an ADR to the pharmacovigilance centre in the Netherlands, which is a selected population. One could assume that patients who are interested in their drug treatment and especially those who have experienced an adverse drug reaction would be more willing to answer this type of questionnaire. We did not include motives and opinions of patients who experienced an ADR but who choose not report this to the pharmacovigilance centre.

Because 96% of our target study population existed of patients who had reported an ADR previously through the online reporting form it was possible to use a web-based questionnaire.

Conclusions

The main motives for patients to report their adverse drug reactions to a national pharmacovigilance centre were the severity of the ADR and their need to share experiences.

Altruistic motives are reflected by the high responses to the statements 'Reporting an adverse drug reaction can prevent harm to other people', 'I felt responsible for reporting an adverse drug reaction' and 'Reporting an adverse drug reaction contributes to research and knowledge'. There is only a small group who feels that they would benefit themselves from reporting an ADR. The high level of response to the questionnaire shows that patients are involved when it comes to adverse drug reactions and also willing to share their motivations for and opinions about the reporting of adverse drug reactions with the pharmacovigilance centre. More than 90% of the patients, who had already reported an ADR once, agree with the statement 'In the future I will report a possible adverse drug reaction once again'.

Conflicts of interest:

Nothing to declare

  professional education, academic education), and about possible membership of a patients' association. Patients who are a member of a patient organization might have more knowledge about the reporting of ADRs to a pharmacovigilance centre and also may have other motives or opinions about this subject.

  statistical software program SPSS Statistics, version 17.0. The answers to the open question about the reason to report (the question 'Other, please specify…') were labeled by two researchers independently (FvH and CvdW) and categorized. These responses to the open question were then compared with the responses to the statements in the questionnaire to identify possible new motives for reporting that were not covered by the questionnaire.

Deleted: 6 Deleted: only 25. 2 Deleted:

 62 More women than men responded to the questionnaire. than 80 years. This fairly corresponds to the the average Dutch population in 2009 were 11.2% was aged 65-80 years and 3.8% were older than 80 years[28].

Deleted: 8

 8 Deleted: , however selection bias cannot be excluded.

ReasonsI

  wanted extra information The adverse drug reaction was severe It was difficult to discuss the adverse drug reaction with my medical practitioner or pharmacist The possibility for reporting an adverse drug reaction just exists I wanted to be heard Someone else pointed the possibility for reporting an adverse drug reaction I was angry about the situation I wanted action to be taken I wanted to share my experiences The adverse drug reaction was not mentioned in the patient information leaflet I was worried about my own situation Opinions Reporting an adverse drug reaction can prevent harm to other people I felt responsible for reporting an adverse drug reaction Reporting an adverse drug reaction that is already mentioned in the patient information leaflet is useless I only report an adverse drug reaction if it is serious Reporting an adverse drug reaction contributes to research and knowledge I report an adverse drug reaction if it is not mentioned in the patient information leaflet I benefit from reporting an adverse drug reaction Reporting an adverse drug reaction contributes to improvement of drugs I report an adverse drug reaction if it is unexpected In the future I will report a possible adverse drug

Introduction

  Direct reporting of adverse drug reactions (ADRs) by patients has been incorporated into the

4 A

 4 qualitative study with guided interviews with twenty-one patients in the Netherlands was performed to gain insight in motivations of patients to report ADRs to a pharmacovigilance centre[21]. Most patients expressed altruistic motives, but also the severity of the ADR and the need for extra information about the ADR were mentioned as motives for reporting.The aim of this study is to quantify the reasons and opinions of patients who reported ADRs in the Netherlands to a pharmacovigilance centre.

  professional education, academic education), and about possible membership of a patients' association. Patients who are a member of a patient organization might have more knowledge about the reporting of ADRs to a pharmacovigilance centre and also may have other motives or opinions about this subject.Sending the questionnaireThe web-based survey was tested in a small group of field testers before the survey was sent. The survey was sent onMarch 23, 2009. Two weeks later (April 6, 2009), a reminder was sent to all non-responders. Collection of responses was finished after one month (April 24, 2009). The link in the invitation e-mail was uniquely tied to the survey and the respondents e-mail address.

  statistical software program SPSS Statistics, version 17.0. The answers to the open question about the reason to report (the question 'Other, please specify…') were labeled by two researchers independently (FvH and CvdW) and categorized. These responses to the open question were then compared with the responses to the statements in the questionnaire to identify possible new motives for reporting that were not covered by the questionnaire.

  than 80 years. This fairly corresponds to the the average Dutch population in 2009 were 11.2% was aged 65-80 years and 3.8% were older than 80 years [28].

11 reporting

 11 ADRs[9,20]. The high response rate increases the validity of the results found in this study.

ReasonsI

  wanted extra information The adverse drug reaction was severe It was difficult to discuss the adverse drug reaction with my medical practitioner or pharmacist The possibility for reporting an adverse drug reaction just exists I wanted to be heard Someone else pointed the possibility for reporting an adverse drug reaction I was angry about the situation I wanted action to be taken I wanted to share my experiences The adverse drug reaction was not mentioned in the patient information leaflet I was worried about my own situation Opinions Reporting an adverse drug reaction can prevent harm to other people I felt responsible for reporting an adverse drug reaction Reporting an adverse drug reaction that is already mentioned in the patient information leaflet is useless I only report an adverse drug reaction if it is serious Reporting an adverse drug reaction contributes to research and knowledge I report an adverse drug reaction if it is not mentioned in the patient information leaflet I benefit from reporting an adverse drug reaction Reporting an adverse drug reaction contributes to improvement of drugs I report an adverse drug reaction if it is unexpected In the future I will report a possible adverse drug reaction once again

Table 1

 1 Statements about reasons and opinions about reporting used in the questionnaire

	F o
	r
	P
	e e r
	R e v i e w
	13

Table 2

 2 Respondent characteristics

	Variable	Percentage (N)
	Gender
	Male	33.4 % (334)
	Female	66.6 % (665)
	Age
	<18 years	0.2 % (2)
	18-35 year	16.1 % (161)
	36-64 year	69.1 % (690)
	>65 years	14.6 % (146)
	Education
	Primary school	1.1 % (11)
	Secondary school	10.8 % (108)
	Vocational education	33.0 % (330)
	Higher prof. education	37.1 % (371)
	Academic	17.9 % (179)
	Member of patients association *
	Yes	24.7 % (247)
	No	75.1 % (750)
	* 997 responses, 2 not reported

Table 3

 3 Motives for reporting adverse drug reactions (percentage (frequency)) Bold: most frequently reported.

	Strongly	Agree	Neutral Disagree Strongly
	agree		disagree

Table 4

 4 Opinions about reporting ADRs (percentage (frequency)). Bold: most frequently reported.

	Strongly	Agree	Neutral Disagree Strongly
	agree		disagree

  Most people in the Netherlands have access to the internet; in 2009, 91% of the Dutch population younger than 65 years had access, according to Statistics Netherlands[28]. However, selection bias cannot be excluded; in the age category 65 to 75 only 64% of the population had access.Access to the internet is not specified for age groups older than 75. 91% of people up to 55 years had used internet in the past three months in a survey in 2009. For people aged 55 to 65 years this was 82% and for people aged 65 to 75 years 53%[28]. Patients who did not use the electronic reporting form (n=63) were excluded from this study, it is possible that other motives were present in this group. The pharmacovigilance centre accepts consumers reporting for relatives, for example children with access to the internet could report for an elderly parent.Use of the internet is also correlated to the level of education[28], which might increase selection bias. The level of education in the Netherlands is higher compared to the average level in the European Union (EU-27 countries) were 19.1% of the population has received education on the highest level[33]. In 2009 on average 65% of the households of the EU-27 countries had access to the internet, which is considerably lower than 90% in the Netherlands[33]. In fact, the percentage of access to the internet in the Netherlands is the highest of all European countries[33]. Therefore the use of a web-bases questionnaire might not be feasible in all countries in the same manner as in the Netherlands.

	F o
	r
	P
	e e r
	R e v i e w
	12

Table 1

 1 Sturkenboom MC, van Grootheest K, Kingma HJ, Stricker BH. Adverse drug reaction-related hospitalisations: a nationwide study in The Netherlands. Drug Saf 2006; 29(2):161-168 doi:10.2165/00002018-200629020-00006 31. van Grootheest AC, Passier JL, van Puijenbroek EP (2005) [Direct reporting of side effects by the patient: favourable experience in the first year]. Ned Tijdschr Geneeskd 149:529-33 No DOI found 32. O'Cathain A, Murphy E, Nicholl J (2007) Why, and how, mixed methods research is undertaken in health services research in England: a mixed methods study. BMC.Health Serv.Res 7:85 doi:10.1186/1472-6963-7-85 33. Eurostat.(2010) http://epp.eurostat.ec.europa.eu. (access date 10-06-2010) Flowchart of the respondents to the questionnaire Statements about reasons and opinions about reporting used in the questionnaire

	F o
	r
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	e e r
	R e v i e w
	13

Table 2

 2 Respondent characteristics

	Variable	Percentage (N)
	Gender
	Male	33.4 % (334)
	Female	66.6 % (665)
	Age
	<18 years	0.2 % (2)
	18-35 year	16.1 % (161)
	36-64 year	69.1 % (690)
	>65 years	14.6 % (146)
	Education
	Primary school	1.1 % (11)
	Secondary school	10.8 % (108)
	Vocational education	33.0 % (330)
	Higher prof. education	37.1 % (371)
	Academic	17.9 % (179)
	Member of patients association *
	Yes	24.7 % (247)
	No	75.1 % (750)
	* 997 responses, 2 not reported

Table 3

 3 Motives for reporting adverse drug reactions (percentage (frequency)) Bold: most frequently reported.

	Strongly	Agree	Neutral Disagree Strongly
	agree		disagree

Table 4

 4 Opinions about reporting ADRs (percentage (frequency)). Bold: most frequently reported.

	Strongly	Agree	Neutral Disagree Strongly
	agree		disagree

European Journal of Clinical Pharmacology