J Antimicrob Chemother 2012; 67: 1540 – 1546
doi:10.1093/jac/dks073 Advance Access publication 7 March 2012
Perceptions and attitudes of French general practitioners towards
rapid antigen diagnostic tests in acute pharyngitis using a randomized
case vignette study
Céline Pulcini1–3*, Laure Pauvif3–5, Alain Paraponaris3–5, Pierre Verger3–5 and Bruno Ventelou3–6
1
Centre Hospitalier Universitaire de Nice, Service d’Infectiologie, Nice, France; 2Université Nice-Sophia-Antipolis, Faculté de Médecine de
Nice, Nice, France; 3INSERM, U912 (SESSTIM), Marseille, France; 4Université d’Aix-Marseille, IRD, UMR-S912, Marseille, France; 5ORS PACA,
Observatoire Régional de la Santé Provence –Alpes –Côte d’Azur, Marseille, France; 6CNRS, U6579 (greqam), Marseille, France
*Corresponding author. Centre Hospitalier Universitaire de Nice, Service d’Infectiologie, Hôpital l’Archet 1, 151 Route Saint Antoine de Ginestière,
BP 3079, 06202 Nice cedex 3, France. Tel: +33-(0)4-92-03-54-61; Fax: +33-(0)4-92-03-90-66; E-mail: [email protected]
Received 30 October 2011; returned 27 January 2012; revised 7 February 2012; accepted 11 February 2012
Objectives: This study had three objectives: (i) to assess the use of rapid antigen diagnostic tests (RADTs) and
their impact on the antibiotic prescribing behaviour of general practitioners (GPs) for acute pharyngitis; (ii) to
study the barriers to the use of RADTs; and (iii) to identify GPs’ characteristics associated with non-compliance
with French guidelines.
Methods: We conducted a cross-sectional survey of a representative sample of 369 self-employed GPs in southeastern France using a randomized case vignette study.
Results: The availability of an RADT allowed a 44% relative reduction in the rate of antibiotic prescriptions. Of
GPs for whom the test was available, 34% did not use an RADT in our acute pharyngitis vignette and 13% of
those who used the test prescribed an antibiotic despite a negative RADT result. Non-compliance with French
guidelines (i.e. not using an RADT and/or prescribing an antibiotic despite a negative RADT result) was independently associated with the following factors: less reading of medical journals, less benefits/risks discussion with
patients about vaccinations and higher perception that clinical examination was sufficient to prescribe antibiotics. The three main declared barriers to RADT use were: time to perform the test, patient expectations regarding antibiotics and the perception that clinical examination was sufficient to decide to prescribe an antibiotic.
Conclusions: RADTs are a useful but not sufficient tool to reduce antibiotic prescribing in general practice. The
results of this study increase understanding of the factors underlying clinical decision making for acute pharyngitis and may contribute to the development of interventions to improve practice.
Keywords: antibiotic prescription, barriers, medical practice, primary care, survey
Introduction
Acute pharyngitis/tonsillitis (AP) is a leading cause of antibiotic
prescriptions in the outpatient setting, with a high proportion of
unnecessary prescriptions.1 Reducing these prescriptions is
important to curb bacterial resistance. The benefit of antibiotics
in AP is modest and is limited to group A streptococcal infections, which cause only 10% –15% of AP cases in adults and
15% –30% in children; the majority of AP infections are
caused by viruses and do not require antibiotics.1,2 Near-patient
tests are an attractive way to help physicians reduce their
antibiotic prescriptions.3 Rapid antigen diagnostic tests
(RADTs) detecting group A streptococcal infections are recommended by the US, Finnish and French guidelines in order to
reduce antibiotic prescriptions.4 In France, RADTs are recommended in all cases of AP in children ≥3 years old, and in
adults presenting with a score ≥2 using the modified Centor
criteria.5 Antibiotics are recommended only in proven group A
streptococcal infections, in order to slightly shorten the clinical
evolution, relieve symptoms, limit the spread of Streptococcus
pyogenes and prevent suppurative complications and acute
rheumatic fever. This is quite different from other guidelines
(for example in Belgium, the Netherlands, England and Scotland), where AP is considered a self-limiting disease, and antibiotics are not recommended except in high-risk and very ill
patients.4 Appropriate testing for children with AP is nonetheless included as a quality indicator by the US National Committee for Quality Assurance (http://www.ncqa.org/). In France,
# The Author 2012. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy. All rights reserved.
For Permissions, please e-mail: [email protected]
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Rapid antigen diagnostic tests in acute pharyngitis
RADTs have been freely distributed to all French general practitioners (GPs) since 2002 as part of a national campaign to
improve and reduce antibiotic use.6 – 8 The sensitivity of the
RADTs used in France has been assessed by the French
Health Products Safety Agency, and it ranges between 92%
and 97%.9 These tests have proven to be a cost-effective strategy,10 provided that clinicians follow guidelines, since a negative result can avoid unnecessary antibiotic prescriptions. A
prospective before/after study conducted in two French
regions in 1998– 99 showed a high utilization rate of these
RADTs (93%) and a dramatic decrease (from 83% to 43%) in
the rate of antibiotic prescriptions for AP, after implementation
of training sessions regarding guidelines and the use of
RADTs.11 However, more recent studies conducted in the USA
and in France have suggested that RADTs are underused in
patients presenting with AP: they are performed in only half
of the patients.12 – 14 This could explain why antibioticprescribing levels decreased only modestly for tonsillitis (from
93% to 87% between 1984 and 2009) in France.8 Knowledge
of the barriers to RADT use could help us understand the
reasons of this underuse, but these barriers have not been
widely studied.12,13,15
Our survey using a randomized case vignette study aimed at
assessing: (i) the use of RADTs and their impact on the antibiotic
prescribing behaviour in a representative sample of GPs in southeastern France; (ii) the barriers to the use of RADTs; and (iii) GPs’
characteristics associated with non-compliance with French
guidelines.
Procedure and questionnaire
The survey was conducted among GPs by professional investigators with
computer-assisted telephone interviewing. The questionnaire was pilottested for clarity, length and face validity with 10 GPs.
Respondents were randomly allocated by the computer to two different clinical scenarios, depending on availability (or not) of an RADT for the
diagnosis of a typical AP vignette in a 10-year-old presenting with three
Centor criteria (see the Supplementary data available at JAC Online). They
were also asked questions about RADT use, antibiotic prescribing and barriers to RADT use. Potential barriers to RADT use were identified using a
literature review.3,12,13,16
The questionnaire also collected data on individual and occupational
characteristics of respondents: gender, age, solo/group practice and
training practice (i.e. being in charge of junior doctors in training at the
surgery). GPs were also surveyed on the following topics: vaccinations, organization of the surgery (availability of a computer, of the internet and
of a secretary), workload, mode of training [continuing medical education (CME) and reading medical journals] and mode of practice (discussion of clinical cases with colleagues, visits from pharmaceutical
representatives). In parallel with the survey, observational data on the
GP activity in 2010 were obtained from the Social Security exhaustive reimbursement database that includes the total number of consultations
and home visits per year for each GP. The survey was approved by the National Data Protection Authority [Commission Nationale Informatique et
Libertés (CNIL)], which is responsible for ethical issues and the protection
of individual data collected in France.
Statistical analysis
Descriptive analysis
Methods
Participants
The present study was part of the second cross-sectional survey conducted under the framework of the French Regional Panel of General
Practices. This panel survey was initiated in 2010 with the objective
to study the medical practice of self-employed GPs in the Provence–
Alpes– Côte d’Azur region, which had a population of 4.88 million in
2008 [French National Institute for Statistics and Economic Studies
data (INSEE)]. GPs were selected using stratified random sampling
from the ADELI (‘Automatisation des Listes’) database of the Ministry
of Health, which contains exhaustive information on the activity of
French physicians. The ADELI database was stratified for the location
of the general practice (urban, suburban or rural area), gender, age
(,49, 49 –56 or .56) and volume of activity (,Q1, Q1–Q3 or .Q3).
Since French GPs work on a fee-for-service basis, participants received
a compensation equivalent to two consultation fees for their participation in each survey. Of the 1108 GPs invited to participate in the French
Regional Panel of General Practices in 2010, 67 (6%) could not be contacted and 134 (12%) were not eligible [GPs practicing exclusively in
hospitals or long-term care facilities, GPs practicing exclusively alternative medicine (such as homeopathy or acupuncture) and GPs planning
to move out of their present region in the following 6 months]. Of the
907 remaining physicians, 444 (49%) agreed to participate. GPs who
refused did not differ from participants according to gender, age or
volume of activity, but they were less likely to practice in a rural area
(P¼0.035). Lack of time was their main reason for refusal. The
results presented in this study are based on the 369 GPs who participated in the cross-sectional survey conducted between January and
March 2011 [response rate, 369/444 (83%)].
In order to correct potential bias in participants, a computing method to
weight the sample was used to obtain a representative database of the
regional GP population regarding age, gender, location of practice and
volume of activity. All presented percentages are weighted results.
Factors associated with a practice not compliant with
the guidelines
To identify the factors associated with non-compliance with French
guidelines, we studied the GPs randomly allocated to the second scenario
(RADT available). The main outcome variable was GP compliance with
French guidelines, with a compliant attitude being defined by two characteristics: (i) use of the RADT; and (ii) absence of antibiotic prescription
given that the result of the test was negative. Univariate explanatory variables (P≤0.15) were introduced in a backward multivariate logistic model
(P≤ 0.05) to identify independent predictive factors associated with the
compliance. The model was adjusted for the variables used to stratify
the sample (volume of activity, location of general practice, gender and
age). To measure the importance of the variables for the outcome, the
Akaike information criteria (AIC) allowed us to compare 2k models (k is
the number of explanatory variables) with the model having the lowest
AIC value (model averaging). Weights were calculated for each of the
2k models.17 The Akaike weights for each model that contained the parameter of interest were summed. To interpret the results, a scale was
used: an importance weight (x) of 0.50≤ x, 0.75, 0.75≤ x, 0.95,
0.95 ≤x ,0.99 or x ≥0.99 indicated ‘weak’, ‘positive’, ‘strong’ or ‘very
strong’ importance, respectively.18
Data were analysed using SAS 9.2 (SAS Institute, Cary, NC, USA) and
R version 2.13.0 (http://www.r-project.org/). All reported P values were
two-tailed, and a P value ,0.05 was considered to be significant.
1541
Pulcini et al.
AB prescribed
n = 112/182 (61.6%)
NO (1st scenario)
N = 184
RADT available
YES
n = 122/184 (66.2%)
YES (2nd scenario)
N = 185
RADT results
negative
Do you perform
an RADT?
NO
n = 62/184 (33.8%)
AB prescribed
n = 15/120 (12.6%)
AB prescribed
n = 48/62 (77.3%)
Figure 1. Flow chart showing the results of the two scenarios regarding the pharyngitis vignette. There are missing data, since some GPs did not
answer all questions. AB, antibiotic.
Results
Table 1. Perceptions of an the barriers that limit or could limit GP use of
an RADT (369 GPs)
Clinical scenarios
A total of 369 GPs participated in the survey and were presented
with a typical AP vignette in a 10-year-old boy (see the Supplementary data for details). In the first scenario (RADT not available) 112/182 (61.6%) GPs prescribed an antibiotic. In the
second scenario (RADT available) 107/184 GPs (58.0%) complied
with the French guidelines, since they used the RADT and did not
prescribe any antibiotic given that the result of the test was
negative. Overall, the availability of the RADT allowed a 26.9%
absolute decrease and a 43.7% relative decrease in the rate of
antibiotic
prescriptions
(112/182 ¼ 61.6%
versus
63/
182¼ 34.7%). These results are summarized in Figure 1.
For the 175 GPs who prescribed an antibiotic (in both scenarios), the principal antibiotics prescribed were: amoxicillin
(72.8%), non-specified penicillins (10.3%), macrolides (7.0%) or
cephalosporins (4.0%).
Potential declared barriers limiting the use of an RADT
n/N a(%)
Doing the test takes time
Some patients absolutely want an antibiotic therapy
I feel that clinical examination is sufficient to decide to
prescribe an antibiotic
I have doubts regarding the validity of the results of
this test
Some patients refuse the test
Doing the test is technically difficult
The administrative paperwork to get the tests is too
tedious
I do not know how to obtain these rapid diagnostic
tests
242/362 (66.9)
228/363 (62.7)
186/362 (51.5)
116/354 (32.8)
73/350 (20.8)
65/358 (18.1)
40/356 (11.4)
24/365 (6.7)
a
GPs who responded to the question.
Perceptions of barriers to the use of an RADT
Of 367 GPs who responded, 315 (85.8%) declared that they
usually had RADTs in their surgeries. Of these, 151 (47.8%)
declared that they used these tests often or always in cases of
AP in children over 3 years old. The declared barriers limiting
the use of an RADT are presented in Table 1.
Factors associated with a practice not compliant with
the guidelines
We focused on the 185 GPs allocated to the second scenario.
The results are presented in Tables 2 and 3. The perception
that clinical examination was sufficient to decide to prescribe
an antibiotic for AP was the most important factor independently
associated with non-compliance.
Discussion
Of 185 GPs who had access to an RADT, 34% did not use it in our
AP vignette, and of those that did, 13% prescribed an antibiotic
despite a negative RADT result. The availability of an RADT
allowed a 44% relative reduction in the rate of antibiotic prescription. The three main declared barriers to RADT use were:
time to perform the test, patient/parent expectations regarding
1542
antibiotics and the perception that clinical examination was sufficient to prescribe an antibiotic.
We surveyed a representative sample of GPs practicing in a
large French region (5 million inhabitants). Our randomizedcontrolled design used a clinical vignette to measure the
quality of physician practice, since vignettes have been shown
to be a valid tool for this purpose.19,20 Our study has some limitations. Firstly, our findings might not be applicable to other settings, and they refer to GPs’ declared attitudes, not to what they
do in practice; however, they are very similar to published results
that have assessed GP clinical practices, some of which had
included self-employed GPs. Secondly, even if GPs who refused
to participate in this study did not differ from participants
according to gender, age or volume of activity, they might
have differed in their perceptions and attitudes concerning
RADT use and antibiotic prescription in AP; however, this topic
was not mentioned to GPs before they were asked to participate
in the panel, and our findings are in accordance with other
studies. Finally, we focused on the GPs’ characteristics, not on
the patients or the patient– doctor relationship.
In our study, 86% of GPs declared that they usually have
RADTs in their surgeries, which is in line with the 84% – 88%
rates found in the literature.13,15 When an RADT was available
to GPs in our vignette, 34% of GPs did not perform an RADT.
JAC
Rapid antigen diagnostic tests in acute pharyngitis
Table 2. Factors associated with a practice not compliant with the guidelines, regarding the second scenario (RADT available and negative result),
univariate and multivariate logistic models (N ¼181 GPs for the multivariate analysis, missing data for 4 GPs); GPs performing the RADT and not
prescribing an antibiotic were compared with those who did not
Not compliant
with the
guidelines
Variables and variable levelsa
nb
%c
Unadjusted OR
(95% CI)
GPs’ characteristics
Practice type
solo
group
47
30
47.1
35.8
reference
0.63 (0.35– 1.13)
P value
0.12
Number of half-day periods spent in CME or evaluation of practice on ID
topics in the past year
Number of hours spent reading medical journals in the past weeke
≤1
.1
Adjusted OR
(95% CI)d
P value
1.03 (1.01– 1.05)
46
31
52.0
32.6
—
0.003 1.03 (1.01–1.06)
0.002
0.007
0.005
reference
0.45 (0.25– 0.80)
In general, do you discuss the benefits and risks of vaccination with your
patients?
very often or often
sometimes, rarely or never
57
20
37.3
65.0
reference
3.12 (1.40– 6.92)
Perceived barriers to the use of RADTs for AP
Clinical examination is sufficient to decide to prescribe an antibiotic
yes
no
55
21
25.0
56.2
reference
0.26 (0.14– 0.49)
Doing the test takes time
yes
no
55
18
32.2
44.5
reference
0.59 (0.30– 1.16)
reference
0.36 (0.18–0.74)
0.005
0.006
reference
3.72 (1.45–9.53)
,0.001
,0.001
reference
0.22 (0.11–0.47)
0.13
—
ID, infectious diseases.
Only variables associated with non-compliance with the guidelines at the P≤ 0.15 level in the univariate analysis are shown.
b
Non-weighted.
c
Weighted.
d
Adjusted ORs were also controlled for location of general practice, gender, age and volume of activity used to stratify the sample; these variables
were not significant in multivariate analyses (P¼0.54, P¼0.94, P¼0.48 and P¼0.51, respectively). We observed 77.3% pair concordance.
e
Continuous variables were also significantly associated with non-compliance with the guidelines in the multivariate logistic model and were dichotomized at the median value for presentation in the final model.
a
This high prevalence of RADT underuse has also been reported in
the literature (7% to 54%).11,12,14,21
The proportion of GPs prescribing an antibiotic when the RADT
result was negative (13%) is in the lower half of the range
of other studies’ results (3%–40%), perhaps because we
measured a declared attitude in a vignette, and not the real
practice.11 – 13,15,21 – 26 An American study published in 2007
reported a very low prevalence rate of antibiotic prescriptions
in cases of negative RADT result (0.95%), but prescriptions
were made by nurse practitioners and physician assistant staff,
with adherence to AP guidelines clearly targeted as an indicator
of clinical quality.27
Few interventional studies assessing the impact of RADTs on
antibiotic use have been published. Two were monocentric randomized controlled trials (Greece28 and Canada),29 one was a
cluster randomized controlled trial in primary care centres in
Spain26 and two were before-and-after uncontrolled studies
(France11 and the USA).30 Only one, to the best of our knowledge,
included children with AP consulting their GPs.11 Overall, the
impact of RADTs was a relative reduction in antibiotic prescriptions, ranging from 32% to 61%. In our study, the availability
of an RADT allowed a 44% relative reduction of antibiotic prescription, which is quite low compared with that in the literature,
since the result of the test was negative in our typical vignette,
whereas all cases of AP (viral and bacterial) were included in
the literature.
In our study, non-compliance with the guidelines (i.e. either
no RADT performed or an antibiotic prescribed despite a negative RADT result) was noted in 42% of the GPs. Adherence was
no better in younger doctors, a worrying fact that has also
been noted by others.13 Putting a high value on clinical examination to decide when to prescribe antibiotics was an independent factor in non-compliance, a finding that has also
been reported in the literature.13 The reading of medical
1543
Pulcini et al.
Table 3. Relative importance of the variables for the outcome (practice
not compliant with the guidelines), for the second scenario (RADT
available; N¼178 GPs)
Variable
Clinical examination is
sufficient to decide to
prescribe an antibiotic
Number of hours spent
reading medical journals
in the past week
In general, do you discuss
the benefits and risks of
vaccination with your
patients?
Practice type
Number of half-day periods
spent in CME or
evaluation of practice on
ID topics in the past year
Doing the test takes time
Rank
Importance
weight
Interpretation
1
0.9995
very strong evidence
2
0.9431
positive evidence
3
0.9304
positive evidence
4
5
0.4879
0.2484
no evidence
no evidence
6
0.2458
no evidence
ID, infectious diseases.
journals was associated with increased compliance to guidelines, suggesting that active learning is beneficial. Finally, GPs
more prone to discuss benefits and risks of vaccination with
their patients were more likely to comply with the guidelines,
possibly because they are more at ease discussing with their
patients the absence of an antibiotic prescription in cases of
negative RADT results.31
Table 1 shows that obtaining the tests was not a frequently
reported barrier to their use in our study. This is probably the
result of the large national campaign conducted since 2002
by the National Health Insurance, with RADTs freely distributed
to all GPs asking for them. A recent French survey noted that if
RADTs were to be no longer distributed without cost, only 48%
of the GPs would continue to use them.12 Similarly, an American survey reported lack of reimbursement and cost as
major barriers to the use of RADTs in AP among GPs and
paediatricians.15
The time taken to perform the test was the barrier most frequently reported by the GPs (67%). This barrier was reported by
only 3%–4% of the surveyed training-practice GPs in two French
studies.12,13 The second most frequently cited barrier (63% of
GPs) was patient (or parental) expectation regarding antibiotic
prescription. This has been widely described in the literature.15,21,31,32 The third most frequently reported barrier (52%
of GPs) was the high value put on clinical examination in deciding
whether to prescribe an antibiotic, a finding also found by
others.12,13 However, all guidelines concur that clinical findings
do not allow a reliable distinction to be made between viral
and bacterial AP. It is of utmost importance to address this misconception, paricularly since this barrier is the only one independently associated with non-compliance to guidelines in our
study, with the highest importance weight (model averaging,
Table 3).
1544
One-third of GPs expressed doubts concerning the validity of
RADT results. This may explain why some GPs prescribed antibiotics even if the RADT result was negative, since of the GPs
allocated to the second scenario, 43% prescribed an antibiotic
and expressed doubts regarding the validity of RADTs, whereas
29% prescribed an antibiotic while not perceiving validity as a
barrier (x2 ¼ 3.56, P ¼ 0.059, n¼ 176). The use of high-sensitivity
RADTs without back-up cultures for negative test results is
recommended by the French guidelines for children and adults,
provided that the patient does not present any risk factor for
acute rheumatic fever. This recommendation is consistent with
the US 2002 guidelines that state that a negative RADT result
for a child or adolescent should be confirmed by a throat
culture, unless the physician has ascertained in his/her practice
that the RADT being used is comparable in sensitivity to a
throat culture.1 However, the reported sensitivities of the RADT
currently used in France, Streptatestw (Dectrapharm, Strasbourg,
France), differ quite widely in the literature: from 79% in a study
conducted in the UK to 95% in the study performed by the
French Health Products Safety Agency.9,33 This finding deserves
further investigation. Furthermore, guidelines on the management of AP are very different from one country to another,4
and this lack of harmonization could contribute to the doubts
expressed by GPs regarding the validity of RADTs.31 A uniform approach to the management of AP would be of help for clinicians
who face this issue in everyday practice.
In conclusion, the results of this study increase our understanding of the factors underlying clinical decision making and antibiotic
prescribing. Limiting antibiotic overuse will likely continue to
require focus on many aspects of clinical practice rather than on
any single factor (education of the GPs and the public, reduction
of the perceived barriers, development of more specific clinical
scores etc.).31 We suggest the following to improve GP management of AP in France: (i) the National Health Insurance could automatically send RADTs to GP surgeries; and (ii) a national campaign,
targeting the physicians and the public, is necessary to convince
people of the validity of RADTs and the inaccuracy of clinical examination in distinguishing between viral and bacterial AP. Implementing quality indicators on the management of AP could
hasten the process, as in the USA.
Acknowledgements
This work has been accepted as a poster presentation at the European
Congress of Clinical Microbiology and Infectious Diseases, London, 2012.
We thank all GPs who participated in the survey as well as members of
the supervisory committee of the French Regional Panel of General
Practices (DRESS; ORS and URML of Provence–Alpes– Côte d’Azur) and
supportive Region Council.
Funding
The French Regional Panel of General Practices received funding from:
Direction de la Recherche, des Etudes, de l’Evaluation et des Statistiques
(DREES)—Ministère de la Santé et des Solidarités in the frame of the
2006– 2008 convention with Fédération Nationale des Observatoires
Régionaux de Santé (FNORS); Direction Générale de la Santé; Institut de
Recherche en Santé Publique (IReSP) in the frame of the 2008 call for
proposals
IReSP-Assurance
Maladie-INSERM-HAS-DREESRSI-INPES
‘Research on health services’; Groupement Régional de Santé Publique
Rapid antigen diagnostic tests in acute pharyngitis
JAC
Provence– Alpes–Côte d’Azur (GRSP PACA); Haute Autorité de Santé (HAS);
and Union Régionale des Caisses d’Assurance Maladie (URCAM PACA) in
the frame of the Fonds d’Aide à la Qualité des Soins de Ville (FAQSV)
and Fonds d’Intervention pour la Qualité et la Coordination des Soins
de la région Provence– Alpes–Côte d’Azur (FIQCS)/Union Régionale des
Médecins Libéraux Provence– Alpes–Côte d’Azur (URML PACA). The work
of C. P. was supported by the ‘Infectiopole Sud’ foundation (a
non-profit-making foundation, http://www.infectiopolesud.com/), which
paid for her accommodation and travel expenses when coming to
Marseille. None of the above bodies had any role in: study design; the
collection, analysis and interpretation of data; the writing of the paper;
or the decision to submit this paper for publication.
10 Humair JP, Revaz SA, Bovier P et al. Management of acute pharyngitis
in adults: reliability of rapid streptococcal tests and clinical findings. Arch
Intern Med 2006; 166: 640– 4.
Transparency declarations
14 Linder JA, Bates DW, Lee GM et al. Antibiotic treatment of children
with sore throat. JAMA 2005; 294: 2315– 22.
None to declare.
Author contributions
C. P. designed the study and wrote the protocol and the article. L. P.
performed the statistical analysis and reviewed the article. A. P.,
P. V. and B. V. reviewed the study protocol and the article.
Supplementary data
Supplementary data are available at JAC Online (http://jac.oxfordjournals.
org/).
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