TITLE PAGE
Title: Disease course of lower respiratory tract infection with a bacterial etiology in primary care
Authors: Jolien Teepe, MD, MSc1, Berna DL Broekhuizen, MD, PhD1, Katherine Loens, PhD2,
Christine Lammens, MSc2, Margareta Ieven, PhD2, Herman Goossens, MD, PhD2, Paul Little,
MD, PhD3, Chris C. Butler, MD, PhD 4 , Samuel Coenen, MD, PhD2,5, Maciek Godycki-Cwirko,
MD, PhD6, Theo JM Verheij, MD, PhD1, on behalf of the GRACE consortium
Affiliations:
1
Julius Center for Health Sciences and Primary Care, University Medical Center Utrecht,
Utrecht, The Netherlands
2
University of Antwerp, Laboratory of Medical Microbiology, Vaccine & Infectious Diseases
Institute (VAXINFECTIO), Antwerp, Belgium
3
University of Southampton Medical School, Primary Care Medical Group, Southampton,
United Kingdom
4
Nuffield Department of Primary Care Health Sciences Oxford University, New Radcliffe
House, Radcliffe Observatory Quarter, Woodstock Road. Oxford OX2 6NW, and Cwm Taf
University Health Board
5
University of Antwerp, Centre for General Practice, Department of Primary and
Interdisciplinary Care (ELIZA) Antwerp, Belgium
6
Faculty of Health Sciences, Medical University of Lodz, Lodz, Poland
* Corresponding author:
1
J Teepe, University Medical Center Utrecht, Julius Center for Health Sciences and Primary Care,
Stratenum 6.131, PO Box 85500, 3508 GA Utrecht, The Netherlands. E-mail: [email protected]. Tel: +31 88 756 8362, Fax : +31 88 756 8099
Support:
This study was part of the GRACE project (www.grace-lrti.org), funded by the 6th
Framework Program of the European Commission (Reference: LSHM-CT-2005-518226).
The GRACE project has been financially supported through the European Science Foundation
(ESF), in the framework of the Research Networking Programme TRACE
(www.esf.org.trace) and by the Research Foundation Flanders (FWO; Belgium). The current
analysis was supported by the SBOH (www.sboh.nl), employer of Dutch general practitioner
trainees. The funding sources were not involved in the design, conduct, analysis and
interpretation of the data, nor in the writing of the paper.
Word count: 2321
Number of tables: 2
Number of figures: 1
Number of appendices: 4
2
Abstract
Purpose:
Bacterial pathogens are assumed to cause a different illness course than non-bacterial etiologies
of acute cough, but evidence is lacking. We evaluated the disease course of lower respiratory
tract infection (LRTI) with a bacterial etiology in adults presenting with acute cough.
Methods:
Secondary analysis of a multi-centre European trial in which 2061 adults with acute cough (≤ 28
days' duration) were recruited from primary care and randomized to amoxicillin or placebo. For
this analysis only patients in the placebo group (n=1021) were included reflecting natural course
of disease. Standardized microbiological and serological analysis were performed at baseline to
define bacterial etiology. All patients recorded symptoms in a diary for four weeks. Disease
course between those with and without bacterial etiology was compared on symptom severity in
days 2-4, duration of symptoms rated 'moderately bad or worse' and reconsultation.
Results:
Of 1021 eligible patients, 187 were excluded for missing diary results, leaving 834 patients of
whom 162 (19%) had bacterial LRTI. Patients with bacterial LRTI had worse symptoms at day
2-4 after presentation (difference= 0.21, 95% CI 0.04-0.38; p=0.014) and more often reconsulted
27% (44/162) vs. 17% (114/660) than those without bacterial LRTI (OR 1.82, 95% CI 1.21-2.74;
p=0.004). Resolution of symptoms rated ‘moderately bad or worse’ did not differ (HR 0.92, 95%
CI 0.77-1.11; p=0.375).
3
Conclusions:
Patients with acute bacterial LRTI have a slightly worse course of disease when compared to
those without an identified bacterial etiology, but the relevance of this difference is doubtful.
Key words: bacteria, infection, LRTI, cough, prognosis, primary care, randomized clinical trial
4
Abbreviation list
95% CI = 95% confidence interval
Dif = difference
GP = general practitioner
HR = hazard ratio
IQR = interquartile range
LRTI = lower respiratory tract infection
OR = odds ratio
SD = standard deviation
5
Introduction
Lower respiratory tract infections (LRTIs) are one of commonest reasons for consulting in
primary care1 and physicians usually treat LRTI empirically based on clinical assessment without
microbiological testing for the causal pathogen. Bacterial pathogens are often assumed to cause a
different illness course than non-bacterial causes of acute cough, but knowledge of actual
difference is lacking. In the few studies where potential pathogens have been systematically
isolated from primary patients with LRTI, a bacterial pathogen was identified in 19-43% of
patients.1-4 However, difference in illness course was not evaluated in these studies. Knowledge
of actual illness course of bacterial LRTI compared to other LRTI’s may be helpful in several
ways: physicians fear missing bacterial LRTI, because they generally assume that disease course
is more severe and prolonged in these patients. Therefore, patients may be prescribed antibiotics
as a defensive strategy.5 As many of these antibiotics may not benefit patients, cause unwanted
effects, and drive antimicrobial resistance,6-9 insight into the illness course of bacterial LRTI
untreated with antibiotics could help guide empirical antibiotic prescribing, support a strategy of
initial observation (watchful waiting), and help set evidence-based expectations about disease
course for patients.
The aim of the present study was therefore to describe the illness course of patients presenting to
primary care with LRTI in whom a bacterial pathogen was isolated, and to compare this with
LRTI patients with no bacterial pathogen.
6
Methods
Design and study population
This was a secondary analysis of a randomized, placebo controlled trial of amoxicillin for LRTI
in 16 primary care networks in 12 European countries from October 2007 until April 2010. More
details on this GRACE-10 study (Genomics to combat Resistance against Antibiotics in
Community-acquired LRTI in Europe; www.grace-lrti.org) have been reported elsewhere.10
Recruited networks had access to a minimum of 20.000 patients. Given how common LRTIs are,
many more eligible patients presented during the recruitment period than were invited to
participate in this study, and therefore we probably did not achieve the goal of recruiting all
consecutive, eligible patients. Nevertheless, we assume that this selection resulted in limited
selection bias, because participating clinicians reported that the main reason not to include all
eligible patients were time constraints.11 Moreover, we noted no relevant difference in patient
characteristics compared with the observational study of Butler et al.6 done earlier in the same
recruiting network. Eligible patients were aged ≥ 18 years who consulted their general
practitioner (GP) for the first time with an acute cough (duration of ≤ 28 days) as the main
symptom or where cough was not the most prominent symptom, but where the GP considered
acute LRTI was the main diagnosis. Exclusion criteria were clinically suspected pneumonia,12
based on focal chest signs (focal crepitations and bronchial breathing) and systemic features
(high fever, vomiting, severe diarrhea); pregnancy, allergy to penicillin, treatment with
antibiotics in the previous month and immunodeficiency. Additional for this analysis, patients
allocated to amoxicillin and/or patients who did not return their follow up diary were excluded.
The study was approved by ethics committees in all participating countries and all participants
provided written informed consent.
7
Measurements
Symptoms. GPs recorded patients’ clinical signs, and comorbidities on a case report form. They
also registered 14 baseline symptoms (cough, phlegm, shortness of breath, wheeze, runny nose,
fever, chest pain, muscle aching, headache, disturbed sleep, feeling generally unwell,
interference with normal activities/work, confusion/disorientation and diarrhea) on a 4-point
Likert-scale from ‘no problem’ to ‘severe problem’. Baseline symptom severity was calculated
by summing the scores of the symptoms and rescaling them to make them range between 0 and
100. Patients filled in a daily symptom diary during their illness for up to 28 days for the same
symptoms on a 7-point Likert scale from ‘no problem’ to ‘severe problem’. This diary was
previously validated and shown sensitive to change.13
Respiratory sample. A sputum sample, if productive (not available for all), and nasopharyngeal
swab was collected from each patient at day one before any antibiotic therapy was started. Sputa
were sent to the local laboratory and processed immediately. Direct microscopy, gram stain and
culture were performed according to a standardized protocol (appendix 1). Nasopharyngeal
swabs in Universal Transport Medium and in skimmed milk medium were sent to the laboratory
of the University of Antwerp for bacterial and viral polymerase chain reaction (PCR) and
analyzed there.
Chest radiograph. All patients underwent chest radiography within seven days of first
presentation, but preferably within three days. Pneumonia was determined by radiologists, who
8
were blind to all other information when they judged chest radiographs using a uniform standard
operating procedure (appendix 2).11
Definition of bacterial infection. Bacterial infection was defined as the presence of Streptococcus
pneumoniae, Haemophilus influenzae, Mycoplasma pneumoniae, Bordetella pertussis,
Legionella pneumophila in respiratory samples or serological evidence for Mycoplasma
pneumoniae infection. Chlamydia pneumoniae was not included, because its clinical relevance
is unclear.1,14 Streptococcus pneumoniae infection and Haemophilus influenzae infection were
defined present when a predominant microorganism from sputum (ratio of white blood
cells/epithelial cells >=1 as criterion for good quality) was isolated or from nasopharyngeal
swab. Bordetella pertussis infection was determined by PCR (from nasopharyngeal swabs) and
by measurement of IgG antibodies to pertussis toxin in venous blood at day 28. An antibody titre
to pertussis toxin of ≥ 125 IU/ml or positive PCR result in a respiratory sample defined recent
Bordetella pertussis infection. Mycoplasma pneumoniae infection and Legionella pneumophila
infection were determined by PCR from nasopharyngeal swabs. Serological definitive
Mycoplasma pneumoniae infection was defined as the presence of IgM antibodies in the sample
of day one and/or day 28, or a IgG seroconversion or significant increase in IgG between day one
and day 28.
Antibiotic use. During follow-up, patients in the placebo group could be unblinded and switched
to antibiotic treatment according to the responsible treating clinician’s judgments. GPs could
then prescribe any antibiotic they considered appropriate (including amoxicillin, other
penicillins, macrolides, and other). Antibiotic use was defined dichotomously (patient reported
9
intake of antibiotics (one dose or more) for at least 5 days in the first 10 days after the index GP
consultation).
Outcome measures. Outcomes were duration of symptoms rated by patients as ‘moderately bad
or worse’ after initial presentation, symptom severity on days 2-4 after the index consultation,
and worsening of illness, defined as reconsultating the GP with worsening symptoms, new
symptoms, new signs, or illness necessitating admission to hospital within four weeks after the
first consultation.10 In addition, effects on duration of symptoms until complete resolution and
duration of interference with normal activities/work were assessed.
Data analysis
The course of disease of LRTI with a bacterial etiology in adults with acute cough was compared
to those without bacterial LRTI for all four outcomes. Data were analyzed using linear regression
models. Simple linear regression was used for symptom severity, Cox regression for the duration
of symptoms allowing for censoring, and logistic regression for reconsulting for new or
worsened symptoms. In the multivariable analyses we controlled all outcomes for the potentially
confounding factors age, current smoking, co morbidity (pulmonary, cardiac, diabetes mellitus)
and cough duration before consultation. Finally, we repeated the analysis on all outcomes
restricted to the patients who did not use antibiotics to mimic natural course of bacterial LRTI.
Another analysis on all outcomes was done restricted to those without radiologically proven
pneumonia. We expect that Streptococcus pneumonia and Haemophilus influenzae, which are the
most common bacteria, may have driven the course of disease. Therefore we performed two
sensitivity analyses on all outcomes: one restricting to those without Streptococcus pneumoniae
10
infection and one restricting to those without Haemophilus influenzae infection. Data were
analyzed using SPSS (version 20.0) for Windows.
Results
Patients and etiologic diagnosis
Of the 1021 participants randomized to placebo, 834 (82%) returned the diary. The baseline
characteristics of these 834 participants were similar to those who did not return the diary, except
for age (mean age 51 (SD 16) vs. 43 (17)) (appendix 3). Bacterial LRTI was present in 162
(19%) patients, ranging between 14% and 26% in all networks. Streptococcus pneumoniae
(56/834, 7%) and Haemophilus influenzae (56/834, 7%) were the most common bacterial microorganism found. Mycoplasma pneumoniae, Bordetella pertussis and Legionella pneumophila
were found in 40/834 (5%), 31/834 (4%) and 1/834 (0.1%) participants, respectively. Patients
with bacterial LRTI were more frequently current smokers (33% (53/162) vs. 24% (161/672)),
had a longer mean cough duration before index consultation (9 days (SD 7) vs. 8 days (7)),
higher GP rated symptom severity score at day 1 (34 (SD14) vs. 30 (14)) and more often showed
infiltrates on their chest radiograph (6% (9/162) vs. 3% (17/672)) (table 1). Of the 834 eligible
participants, 62 (7%) used antibiotics for at least 5 days in the first 10 days after the index GP
consultation. Patients who used antibiotics had a higher GP rated symptom severity score at day
1 (36 (SD 14) vs. 31 (14)) and were similar to those who did not use antibiotics in terms of mean
age (51 (SD 15) vs. 51 (16)) and comorbidity (31% (19/62) vs. 25% (193/772)) (appendix 4).
Disease course
Patients with bacterial LRTI had higher self-rated mean symptom severity score on days 2-4 than
11
those without bacterial LRTI (difference=0.21, 95% CI (0.04-0.38); p=0.014)) and were more
likely to reconsult for new or worsening symptoms (27% (44/162) vs. 17% (114/660), OR 1.82,
95% CI 1.21-2.74; p=0.004) (table 2)). The vast majority reconsultations considered new or
worsening symptoms and only two patients required hospital admission (one from the bacterial
LRTI group and one from the non-bacterial LRTI group) within four weeks after the first
consultation. No study-related deaths were noted. A Kaplan-Meier curve for the duration of
symptoms rated ‘moderately bad or worse’ (figure 1) showed no difference in median and
interquartile range of symptom duration between patients with and without bacterial LRTI (7
days (IQR 5-15) vs. 7 days (4-13), HR 0.92, 95% CI 0.77-1.11; p=0.375)). The other outcomes
did not differ between those with and without bacterial LRTI.
Repeating the analysis restricted to the patients who used no antibiotics (n=772) showed that
only the outcome measure reconsulting for new or worsening symptoms was different between
patients with and without bacterial LRTI, respectively 23% (33/42) vs. 16% (97/619) (OR 1.67,
95% 1.06-2.63; p=0.029) (table not shown). Furthermore, excluding patients with radiologically
proven pneumonia (n=26) did not result in different findings between those with and without
bacterial LRTI for all outcomes. Repeating the analysis restricted to those patients without
Streptococcus pneumoniae infection (n=778), did not result in different findings. Finally,
repeating the analysis restricted to those patients without Haemophilus influenzae infection
(n=778), showed no differences in course of disease between those with and without bacterial
LRTI for all outcomes.
12
Discussion
Main findings
We found that adult patients who consulted for acute cough in primary care and who had a
potential bacterial pathogen isolated had a slightly worse illness course compared to patients in
whom no bacterial pathogen was isolated. However, the difference is small and clinically not
meaningful.
Strengths and limitations
As far as we are aware, this study is the first to describe illness course of bacterial LRTI in a
large study population in primary care. Moreover, the study population was initially not treated
with antibiotics and only a small number of the patients (7%) were subsequently prescribed
antibiotics for their illness episode. This allowed to describe the illness course of bacterial LRTI
and to assess to what extent it differed from other LRTIs.
A limitation is that more severely ill patients who were immediately referred to a hospital were
not included in this study. The severity of our group of patients with bacterial LRTI might be
milder than in the general population, and therefore the course of disease is probably not
generalizable to sicker patients. However, knowledge on the prognosis of bacterial LRTI among
severely ill patients will not change management, since those with severe bacterial LRTI still
require antibiotic treatment and/or admission to hospital. It should be kept in mind that some
patients with initially mild symptoms subsequently do require hospitalization. However, in our
study, only two patients (one from the bacterial LRTI group and one from the non-bacterial LRTI
group) were admitted to hospital (not immediately).
13
Another possible limitation is misclassification of bacterial LRTI by airway bacterial
colonization. We expect this misclassification is very limited, because estimated colonization of
the lower airways is not likely in symptomatic ambulatory outpatients (5%). Moreover, the
proportion of patients with chronic obstructive pulmonary disease (COPD), in whom airways can
be colonized with Haemophilus influenzae15 merely in severe COPD, in our study was small
(n=45/834, 5%).
Finally, it was not possible to report the full disease course in 7% (59/833) of the patients,
because these patients still had ‘moderately bad or worse’ symptoms at day 28.
Comparison with existing literature
The prevalence of patients with bacterial LRTI and radiographic evidence of pneumonia (1.1%,
9/834) in this study is lower than the 5.9% (14/236) reported in a study from Hopstaken et al.16
The higher proportion of bacterial pneumonia in that study could be explained by difference in
eligibility criteria. Regarding reconsultation, (27% in our study) Macfarlane et al. reported 21%
(28/135) reconsultations for the same illness in LRTI within a month.1
Conclusions and implications for practice
The illness course of bacterial LRTI is generally mild, uncomplicated and similar to nonbacterial LRTI. This suggests that GPs can reassure patients that LRTI, even if bacterial, is a selflimiting condition and that rather than immediate antibiotic prescribing, a strategy of watchful
waiting should be considered.
14
Ethical approval
Ethical approval for the Netherlands was granted by the Medisch Ethische Toetsing
Commissie (METC) of the University Medical Center Utrecht (ref 07-179/O). Competent
authority approval for the Netherlands was granted by De Centrale Commissie Mensgebonden
Onderzoek (CCMO). For the UK ethical approval was granted by Southampton and South
West Hampshire Local Research Ethics Committee (B) (ref 07/H0504/104). Competent
authority approval for the UK was granted by the Medicines and Healthcare Products
Regulatory Agency. Also the other research sites obtained ethical and competent authority
approval from their local organizations. Patients who fulfilled the inclusion criteria were given
written and verbal information on the study and gave informed consent.
Competing interests
All authors declare that we have no relevant conflicts of interest.
15
References
1.
Macfarlane J, Holmes W, Gard P, et al. Prospective study of the incidence, aetiology and
outcome of adult lower respiratory tract illness in the community. Thorax. 2001;56:109-14.
2. Graffelman AW, Knuistingh NA, le CS, et al. A diagnostic rule for the aetiology of lower
respiratory tract infections as guidance for antimicrobial treatment. Br J Gen Pract. 2004;54:204.
3. Hopstaken RM, Stobberingh EE, Knottnerus JA, et al. Clinical items not helpful in
differentiating viral from bacterial lower respiratory tract infections in general practice. J Clin
Epidemiol. 2005;58:175-83.
4. Holm A, Pedersen SS, Nexoe J, et al. Procalcitonin versus C-reactive protein for predicting
pneumonia in adults with lower respiratory tract infection in primary care. Br J Gen Pract.
2007;57:555-60.
5. Wood F, Simpson S, Butler CC. Socially responsible antibiotic choices in primary care: a
qualitative study of GPs' decisions to prescribe broad-spectrum and fluroquinolone antibiotics.
Fam Pract. 2007;24:427-34.
6. Butler C, Hood K, Verheij T, et al. Variation in antibiotic prescribing and its impact on recovery
16
in patients with acute cough in primary care: prospective study in 13 countries. BMJ. 2009;
338:b2242.
7. Akkerman AE, van der Wouden JC, Kuyvenhoven MM, et al. Antibiotic prescribing for
respiratory tract infections in Dutch primary care in relation to patient age and clinical entities. J
Antimicrob Chemother. 2004;54:1116–21.
8. Malhotra-Kumar S, Lammens C, Coenen S, et al. Effect of azithromycin and clarithromycin
therapy on pharyngeal carriage of macrolide-resistant streptococci in healthy volunteers: a
randomised, double-blind, placebo-controlled study. Lancet. 2007;369:482–90.
9. Goossens H, Ferech M,Vander Stichele R, et al. Outpatient antibiotic use in Europe and
association with resistance: a cross-national database study. Lancet. 2005;365:579–87.
10. Little P, Stuart B, Moore M, et al. Amoxicillin for acute lower-respiratory-tract infection in
primary care when pneumonia is not suspected: a 12-country, randomised, placebo-controlled
trial. Lancet Infect Dis. 2013;13:123-129.
11. van Vugt SF, Broekhuizen BD, Lammens C, et al. Use of serum C reactive protein and
procalcitonin concentrations in addition to symptoms and signs to predict pneumonia in patients
presenting to primary care with acute cough: diagnostic study. BMJ. 2013;346:f2450.
12. British Thoracic Society. British Thoracic Society Guidelines for the management of Community
Acquired Pneumonia. Thorax. 2001;56(suppl 4):1-64.
17
13. Watson L, Little P, Moore M, et al. Validation study of a diary for use in acute lower respiratory
tract infection. Fam Pract. 2001;18:553-54.
14. Hyman CL, Roblin PM, Gaydos CA, et al. Prevalence of asymptomatic nasopharyngeal carriage
of Chlamydia pneumoniae in subjectively healthy adults: assessment by polymerase chain
reaction-enzyme immunoassay and culture. Clin Infect Dis. 1995;20:1174-8.
15. Rosell A, Monsó E, Soler N, et al. Microbiologic determinants of exacerbation in chronic
obstructive pulmonary disease. Arch Intern Med. 2005;165:891-7.
16. Hopstaken RM, Coenen S, Butler CC, et al. Prognostic factors and clinical outcome in acute
lower respiratory tract infections: a prospective study in general practice. Fam Pract.
2006;23:512-9.
18
Figure 1. Kaplan–Meier survival curve for the duration of symptoms rated ‘moderately
bad or worse’ in patients with LRTI.
19
Table 1. Baseline characteristics of patients with and without bacterial LRTI
All patients
N= 834(%)
Age, mean (SD)
Male gender
Current smoker
Co morbidity (pulmonary, cardiac, DM)*
Cough duration before index consultation, mean (SD)†
Severity score (all symptoms), mean (SD)‡
Infiltrates on chest radiograph present
Antibiotic use||
51 (16)
345 (41)
214 (26)
212 (26)
9 (7)
31 (14)
26 (3)
62 (7)
Bacterial LRTI
present
N=162(%)
52 (17)
69 (43)
53 (33)
46 (29)
9 (7)
34 (14)
9 (6)
20 (12)
Bacterial LRTI
absent
N=672(%)
51 (16)
276 (41)
161 (24)
166 (25)
8 (7)
30 (14)
17 (3)
42 (6)
P value§
Missing
N (%)
0.450
0.724
0.022
0.311
0.021
0.003
0.046
0.008
0 (0.0)
0 (0.0)
0 (0.0)
1 (0.1)
7 (0.8)
22 (2.6)
29 (3.5)
0 (0.0)
Data are presented as number (%), unless mentioned otherwise. SD = standard deviation.
* Pulmonary co morbidity = history of COPD, asthma or other lung disease. Cardiac co morbidity = history of heart failure, ischemic heart disease or other heart disease. DM =
diabetes mellitus.
† Presented in days.
‡ Score for 14 patients’ GP-recorded symptoms summed and scaled to range between 0 and 100 at day 1.
§ P value compared patients with and without bacterial LRTI.
|| Antibiotic use was defined as patient reported intake of antibiotics (one dose or more) for at least 5 days in the first 10 days after the index GP consultation.
20
Table 2. Prognostic outcomes in patients with and without bacterial LRTI
Bacterial LRTI
present, N=162
7 (5-15)
Bacterial LRTI
absent, N=672
7 (4-13)
Crude analysis
(95% CI)
HR 0.89 (0.74-1.07)
Adjusted analysis
(95% CI)*
HR 0.92 (0.77-1.11)
P value†
Median time to resolution of symptoms rated ‘moderately bad or
0.375
worse’, days (IQR)
Mean symptom severity score on days 2-4 after consultation (SD)‡
1.86 (1.07)
1.67 (1.00)
Dif 0.19 (0.02-0.37)
Dif 0.21 (0.04-0.38)
0.014
Median duration of symptoms until complete resolution, days (IQR)
15 (10-28)
13 (8-27)
HR 0.88 (0.71-1.10)
HR 0.92 (0.74-1.15)
0.471
Worsening of illness (%)§
44/162 (27)
114/660 (17)
OR 1.79 (1.20-2.67)
OR 1.82 (1.21-2.74)
0.004
Median duration of interference with normal activities/work, days (IQR) 6 (1-10)
5 (0-9)
HR 0.91 (0.76-1.09)
HR 0.91 (0.76-1.09)
0.321
CI = confidence interval. IQR = interquartile range. SD = standard deviation. HR = hazard ratio. Dif = difference. OR = odds ratio.
* Adjusted for age (for each year increase), current smoking, co morbidity and cough duration before index consultation.
† For adjusted analysis.
‡ Each symptom was scored by the patient from 0 to 6 (0=no problem, 1=very little problem, 2=slight problem, 3=moderately bad, 4=bad, 5=very bad, 6=as bad as it could be).
§ The vast majority of these represent reconsultation with new or worsening symptoms and only two patients required hospital admission (one from the bacterial LRTI group and
one from the non-bacterial LRTI group) within four weeks after the first consultation. No study-related deaths were noted.
21
Missing
N (%)
1 (0.1)
3 (0.4)
1 (0.1)
12 (1.4)
6 (0.7)
Appendix 2. Standard Operating Procedure (SOP) for the assessment and feedback of
chest radiographs.
As indicated on the GRACE chest radiograph Requisition Form the following questions should be answered regarding each
chest radiograph:
F. Date chest radiography
__ / __
Day
/ ____
Month
G. Chest radiograph of sufficient quality?
H. Consolidation
Year
Yes:
No:
Yes:
No:
Right:
Left:
I. Pleural effusion
Yes:
No:
J. Interstitial pattern/infiltrate
Yes:
No:
If yes:
K. Diagnosis
Normal chest radiograph
Acute bronchitis
Bronchopneumonia
Lobar pneumonia
Other, please
specify:………………………………………………………………………………..
L. Other remarks: ……………………………………………………………………………….
22
As indicated in the GRACE protocol the following definitions should be used when answering these questions:
A. Consolidation: a dense of fluffy opacity that occupies a portion or whole of a lobe or of the entire lung that may or may
not contain air bronchograms
B. Pleural effusion: fluid in the lateral pleural space and not just in the minor oblique fissure
C. Interstitial pattern/infiltrate:
C1: a lacy pattern involving both lungs featuring peribronchial thickening and multiple areas of atelectasis.
C2: minor patchy infiltrates that are not of sufficient magnitude to constitute primary end point consolidation and
small areas of atelectasis that may be difficult to distinguish from consolidation.
The results of chest radiography should be send immediately to the family physician if consolidation (A), pleural effusion
(B) or infiltrate as described under C1 is present. All other categories of results should not be communicated to the FP,
unless the patient or the FP stops participation of the patient in the study. Also if radiographic changes suggestive of a
neoplastic lesion or other major abnormalities are seen on chest radiography the family physician should be informed
immediately.
23
Appendix 3. Baseline characteristics of included (diary returned) and excluded patients (no diary returned)
Age, mean (SD)
Male gender
Current smoker
Co morbidity (pulmonary, cardiac, DM)*
Cough duration before index consultation, mean (SD)†
Severity score (all symptoms), mean (SD)‡
Infiltrates on chest radiograph present
All patients
N=1021 (%)
49 (16)
423 (41)
271 (27)
252 (25)
9 (7)
31 (14)
33 (4)
Included patients
N=834(%)
51 (16)
345 (41)
214 (26)
212 (26)
9 (7)
31 (14)
26 (3)
Excluded patients
N=187 (%)
43 (17)
78 (42)
57 (31)
40 (21)
9 (8)
31 (15)
7 (5)
P value§
<0.001
0.931
0.164
0.245
0.794
0.798
0.307
Missing
N (%)
0 (0.0)
0 (0.0)
1 (0.1)
1 (0.1)
7 (0.7)
25 (2.4)
81 (7.9)
Data are presented as number (%), unless mentioned otherwise. SD = standard deviation.
* Pulmonary co morbidity = history of COPD, asthma or other lung disease. Cardiac co morbidity = history of heart failure, ischemic heart disease or other heart disease. DM =
diabetes mellitus.
† Presented in days.
‡ Score for 14 patients’ GP-recorded symptoms summed and scaled to range between 0 and 100 at day 1.
§ P value compared included versus excluded patients.
24
Appendix 4. Baseline characteristics of patients with and without antibiotic use
Age, mean (SD)
Male gender
Current smoker
Co morbidity (pulmonary, cardiac, DM)*
Cough duration before index consultation, mean (SD)†
Severity score (all symptoms), mean (SD)‡
Infiltrates on chest radiograph present
All patients
N=834 (%)
51 (16)
345 (41)
214 (26)
212 (26)
9 (7)
31 (14)
26 (3)
Antibiotic use
N=62 (%)
51 (15)
23 (37)
13 (21)
19 (31)
7 (7)
36 (14)
8 (13)
No antibiotic use
N=772(%)
51 (16)
322 (42)
201 (26)
193 (25)
9 (7)
31 (14)
18 (2)
P value§
0.910
0.478
0.379
0.329
0.020
0.007
<0.001
Missing
N (%)
0 (0.0)
0 (0.0)
0 (0.0)
1 (0.1)
7 (0.8)
22 (2.6)
29 (3.5)
Antibiotic use was defined as patient reported intake of antibiotics (one dose or more) for at least 5 days in the first 10 days after the index GP consultation.
Data are presented as number (%), unless mentioned otherwise. SD = standard deviation.
* Pulmonary co morbidity = history of COPD, asthma or other lung disease. Cardiac co morbidity = history of heart failure, ischemic heart disease or other heart disease. DM =
diabetes mellitus.
† Presented in days.
‡ Score for 14 patients’ GP-recorded symptoms summed and scaled to range between 0 and 100 at day 1.
§ P value compared included versus excluded patients.
25
Investigator - Laboratory Manual
Contract No: LSHM-CT-2005-518226

Appendix 1. Laboratory manual
Investigator Laboratory
Manual
Project acronym:
GRACE
Project full title:
Genomics to combat Resistance against Antibiotics in Communityacquired LRTI in Europe
Document type:
Investigator - Laboratory Manual
Document status:
Version 3.3
Release date:
31 May 2007

Version 3.3
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Investigator - Laboratory Manual
Contract No: LSHM-CT-2005-518226
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
Version 3.3
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Investigator - Laboratory Manual
Contract No: LSHM-CT-2005-518226
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Table of Contents
1.
INTRODUCTION .................................................................................................................. 29
1.1
Sampling material supplied by the Central Lab (University of Antwerp, Belgium) 29
1.2
Documents ................................................................................................................... 29
2. STUDY PROCEDURES AND OBSERVATIONS ................................................................. 30
2.1
Overview of samples to be taken per visit ................................................................ 30
2.2
Overview of handlings to be performed by the local lab ......................................... 30
2.3
Overview of analysis to be perfomed by the central lab or by the labs from the
other Workpackages ............................................................................................................. 31
3. LABORATORY TEST ASSESSMENTS .............................................................................. 32
3.1
Local Laboratory ......................................................................................................... 32
3.2
Central Laboratory (UA) .............................................................................................. 33
4. SAMPLE HANDLING PROCEDURES ................................................................................ 34
4.1
Investigator Requisition Form .................................................................................... 34
4.2
Material Preparation .................................................................................................... 34
4.3
Overview of Sample Handling per Visit ..................................................................... 35
4.4
Sampling Instructions ................................................................................................. 39
4.4.1
For investigator ...................................................................................................... 39
4.4.2
For Local Laboratory .............................................................................................. 41
5. FROZEN SAMPLE SHIPMENT PROCEDURE.................................................................... 44
6. REPORTING ........................................................................................................................ 45
6.1
Investigator .................................................................................................................. 45
6.2
Local Laboratory ......................................................................................................... 46
6.3
Central Laboratory ...................................................................................................... 48
7. CONTACT PERSONS ......................................................................................................... 50
7.1
Central Lab –University Antwerp – UA ...................................................................... 50
7.2
National Network co-ordinators and Local Labs ...................................................... 50
8. ANNEXES ............................................................................................................................ 52
8.1
Annex 1: Investigator Requisition Form.................................................................... 52
8.2
Annex 2: Local Laboratory Requisition Form ........................................................... 53
8.3
Annex 3: Sputum Result Form ................................................................................... 54

Version 3.3
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Investigator - Laboratory Manual
Contract No: LSHM-CT-2005-518226
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INTRODUCTION
For WP 9, GRACE Contract no. LSHM-CT-2005-518226, the Central Lab has provided you with:
 This Investigator - Laboratory Manual
 An Investigator Requisition Form and a Local Lab Requisition Form
 Visit specific sampling kits for:
Observational study
 Visit 1 (Pre-Treatment/Day 0)
 Visit 2 (4 weeks after visit 1/Day 1)
Control group
 Visit 0
Sampling material supplied by the Central Lab (University of Antwerp, Belgium)






Serum tube and serum transfer tube
EDTA blood sampling tube
Sputum container
Nasopharyngeal swab with Virus Transport Medium
Nasopharyngeal swab with Amies medium
Microbanks with pearl medium (only for use in local labs)
Documents
The material is accompanied by two Requisition Forms: An Investigator Requisition Form (annex 1) and
A Local Lab Requisition Form (annex 2).
For the investigator
 The investigator has to fill in the Investigator Requisition Form on the moment of the sampling.
The Investigator Requisition Form will be kept by the investigator and a fax forwarded to the local
National Network Facilitator (NNF) within 24 h after sampling.
The unfilled Local Lab Requisition Form is accompanied by the samples, the unused sampling
material and the unused labels, at the moment of the transport to the local lab
For the Local Lab
 The Local lab has to fill in the Local Lab Requisition Form on the moment of arrival of the samples
.
The Local Lab Requisition Form will be kept by the local lab and a fax forwarded to the local
National Network Facilitator (NNF) within 24 h after arrival of the samples.
Please make sure that the Forms are filled in completely and legibly. If an error is made, cross out the
mistake, write the correct information and initial and date the change.

Version 3.3
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Investigator - Laboratory Manual
Contract No: LSHM-CT-2005-518226
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STUDY PROCEDURES AND OBSERVATIONS
For all assessments to be performed per visit, please refer to the Sample Handling Procedures in chapter
4.
Overview of samples to be taken per visit
Observational study
Sample
Serum
EDTA blood
Sputum
Nasopharyngeal swab with virus transport
medium
Nasopharyngeal swab with Amies medium
V1 = Day 0 (Pre-treatment)
V2 = Day 1 (4 weeks after visit1)
Control group
V1
V2
S
S
S
S
S
V1
S
S
S
S
S
S
S = Samples to be taken
Overview of handlings to be performed by the local lab
Sample
Handling, Preparation or Test
Serum
Preparation of the serum and storage of the serum tube
preferable at –70°C
EDTA blood
Store tube preferable at –70°C
Sputum
Direct microscopy
Gram Stain
Culture of S. pneumoniae and Haemophilus spp.
Preparation of 2 Microbanks per Streptococcus
pneunomiae and Haemophilus species isolate.
Nasopharyngeal swab with virus Store the swab preferable at –70°C
transport medium
Nasopharyngeal swab with Amies Store the swab preferable at –70°C
medium
V1 = Day 0 (Pre-treatment)
V2 = Day 1 (4 weeks after visit1)

Version 3.3
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Investigator - Laboratory Manual
Contract No: LSHM-CT-2005-518226
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Overview of analysis to be perfomed by the central lab or by the labs from the other
Workpackages
Sample
Serology for M. pneumoniae, C. pneumoniae and L
pneumophila
C-reactive protein
Procalcitonin
Nasopharyngeal swab in
Amies medium
Nasopharyngeal Swab in
Viral transport medium
Sputum
EDTA
blood
Test
Serum
Observational
study
V1
V2
T
T
T
T
T
T
T
Control
group
V1
T
Human Genetics
Susceptibility testing on S. pneumoniae and Haemophilus
spp.
Molecular analysis on S. pneumoniae and Haemophilus spp.
T
T
Viral culture
T
T
Antigen tests
T
T
T
T
T
T
T
T
T
T
T
Virus discovery
PCR for Chlamydophila pneumoniae, Legionella
pneumophila, Mycoplasma pneumoniae, Bordetella sp.,
Streptococcus pneumoniae and respiratory viruses
PCR for atypical bacteria
T
Growth of Streptococcus pneumoniae and Haemophilus spp.
V1 = Day 0 (Pre-treatment)
V2 = Day 1 (4 weeks after visit1)
T = Perform test

Version 3.3
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Investigator - Laboratory Manual
Contract No: LSHM-CT-2005-518226
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LABORATORY TEST ASSESSMENTS
To perform and/or to prepare the specimens for each test, please refer to the detailed description of each
Laboratory Test in chapter 4.4 (Sampling Instructions)
Local Laboratory
 In your Local Laboratory the following tests are to be performed if the sample is present:
 Sputum : Direct microscopy, Gram stain and culture

If sputum cultures are positive for S. pneumoniae and/or Haemophilus spp
Preparation of Microbanks for the Central Lab
 In your Local Laboratory the following specimen have to be prepared for the Central Lab, if the sample
is present:
 A serum sample (in the 10 ml serum transfer tube)
 Microbanks with sputum pathogens S. pneumoniae and/or Haemophilus spp. (see
above)
 Additionally the following samples have to be stored until shipment to the Central Lab, if the sample is
present:





An serum sample (see above)
An EDTA blood sample
Nasopharyngeal swab in virus transport medium
Nasopharyngeal swab in Amies medium
Microbanks (see above)
After preparation, put the samples, together with the unused labels, in a zip-lock bag. Store them in your
freezer, preferably at -70°C until shipment to the Central Lab.
Only in case you do not have the facilities to freeze at -70°C you can store them at -20°C.

Version 3.3
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Investigator - Laboratory Manual
Contract No: LSHM-CT-2005-518226
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Central Laboratory (UA)
The Central Laboratory will perform the following tests on your collected samples:










Serology for M. pneumoniae, C. pneumoniae and L. pneumophila
CRP and procalcitonin in serum
Transfer EDTA blood specimen to Workpackage 4 (WP4) for human genetics.
Susceptibility testing on S. pneumoniae and Haemophilus spp.
Transfer S. pneumoniae and Haemophilus spp. strains to WP6 and WP7, respectively, for
molecular analysis
PCR for C. pneumoniae, L. pneumophila., M. pneumoniae, B. pertussis, S.pneumoniae and
respiratory viruses
Aliquot PCR material and transfer to WP3 and WP5 for virus detection and virus discovery
respectively
Aliquot virus transport medium and transfer to WP3 for viral culture and antigen tests and to WP5
for virus discovery
PCR of atypical bacteria using an nasopharyngeal swab in Amies transport medium
Culturing S. pneumoniae and Haemophilus spp. using a nasopharyngeal swab in Amies transport
medium

Version 3.3
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Investigator - Laboratory Manual
Contract No: LSHM-CT-2005-518226
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SAMPLE HANDLING PROCEDURES
Investigator Requisition Form
First complete the next information on the Investigator Requisition Form (Annex 1):







Patient study number (use same label as in CRF, see 4.2 Material Preparation)
Patient gender (F/M)
Date of Birth (dd/mmm/yyyy)
Collection Date (dd/mmm/yyyy)
Collection Time (24 hours clock)
Visit number (tick the right box)
Specimens collected (tick the right box)
Material Preparation
Observational study
Visit 1 => take the observational study visit 1 sampling kit and open the big zip-lock bag.
Visit 2 => take the observational study visit 2 sampling kit and open the big zip-lock bag.
For Control group
Visit 0 => take the control group visit 1 sampling kit and open the big zip-lock bag.
Each tube has to be identified with a label. Labels are coded with a patient study number and a visit
number.
Labels are to be stuck vertically on the tubes.
The same labels are used for the Patient Registration Form, Case Record Form (CRF), Investigator
Requisition Form , Local Lab Requisition Form, …

Version 3.3
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Investigator- Laboratoy Manual
Contract No: LSHM-CT-2005-518226
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Overview of Sample Handling per Visit
Observational study : Visit 1 (Pre-Treatment / Day 0)
Sample /
Sample Material
Serum sample
EDTA blood sample
Sputum
Nasopharyngeal swab
Nasopharyngeal swab
Preparation at Site
•
•
•
•
Label the serum tube
Draw the blood in the tube
Coagulation (15 mins)
Store the tube until shipment to the local lab
Preparation at Local Lab
• Centrifuge for 15 mins at 4000 rev/min
• Label the serum transfer tube
• Transfer serum into the serum transfer tube
• Label the EDTA- tube
• Draw the blood in the tube
• Store the tube until shipment to the local lab
• Label the sputum container
• Ask the patient for a sputum sample
• Store the container until shipment to the local
lab
Storage (pref. at –70°C) until
shipment to the Central Lab
Store serum frozen until shipment.
Store sample frozen until shipment.
• Perform Direct microscopy, Gram stain and
culture
• If cultures positive for S. pneumoniae and/or
Haemophilus spp, prepare the microbanks
• Label the viral transport tube
• Collect the nasopharyngeal swab and place it in
the viral transport medium tube.
• Store the swab until shipment to the local lab
• Label the Amies tube
• Collect the nasopharyngeal swab and place it in
the Amies medium tube.
• Store the swab until shipment to the local lab

Draft Version 3.0
Page 35 of 56
Store microbanks frozen until shipment.
Store swab frozen until shipment.
Store swab frozen until shipment.
Investigator- Laboratoy Manual
Contract No: LSHM-CT-2005-518226

Observational study: Visit 2 (4 weeks after first visit / Day 1)
Sample /
Sample Material
Serum sample
Nasopharyngeal swab
Nasopharyngeal swab
Preparation at Site
•
•
•
•
Preparation at Local Lab
Label the serum tube
Draw the blood in the tube
Coagulation (15 mins)
Store the tube until shipment to the local lab
Storage (pref. at –70°C) until
shipment to the Central Lab
• Centrifuge for 15 mins at 4000 rev/min
• Label the serum transfer tube
• Transfer serum into the serum transfer tube
• Label the viral transport tube
• Collect the nasopharyngeal swab and place it in
the viral transport medium tube.
• Store the swab until shipment to the local lab
• Label the Amies tube
• Collect the nasopharyngeal l swab and place it
in the Amies medium tube.
• Store the swab until shipment to the local lab

Draft Version 3.0
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Store serum frozen until shipment.
Store swab frozen until shipment.
Store swab frozen until shipment.
Investigator- Laboratoy Manual
Contract No: LSHM-CT-2005-518226

Control group: Visit 0 (day0)
Sample/
Sample Material
EDTA blood sample
Nasopharyngeal swab
Nasopharyngeal swab
Preparation at Site
Preparation at Local Lab
• Label the EDTA- tube
• Draw the blood in the tube
• Store the tube until shipment to the local lab
• Label the viral transport tube
• Collect the nasopharyngeal swab and place it in
the viral transport medium tube.
• Store the swab until shipment to the local lab
• Label the Amies tube
• Collect the nasopharyngeal swab and place it in
the Amies medium tube.
• Store the swab until shipment to the local lab

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Storage (pref. at –70°C) until
shipment to the Central Lab
Store sample frozen until shipment.
Store swab frozen until shipment.
Store swab frozen until shipment.
Investigator- Laboratoy Manual
Contract No: LSHM-CT-2005-518226
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
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
Sampling Instructions
For investigator
1. Serum Sample
a)
b)
c)
d)
Label the required 10 ml serum-tube.
Draw 10 ml of the patient’s blood in the serum-tube.
Leave the sample to coagulate for 15 min’s (without shaking).
Bring the tube in the zip-lock bag, togheter with the other samples from the same
patient.
e) Store the zip-lock bag in your refrigerator until shipment to the Local Lab within 24
hours.
2. EDTA blood sample
a) Label the required EDTA-tube.
b) Draw 10 ml of the patient’s blood in the EDTA-tube.
c) Bring the tube in the zip-lock bag, togheter with the other samples from the same
patient.
d) Store the zip-lock bag in your refrigerator until shipment to the Local Lab within 24
hours.
3. Sputum
a) Label the required sputum container
b) Take a sputum sample.
e) Bring the container in the zip-lock bag, togheter with the other samples from the
same patient.
f) Store the zip-lock bag in your refrigerator until shipment to the Local Lab within 24
hours.
If the patient can not make a sputum at the moment of the sampling an empty sputum
container should be given to the patient for a morning sputum.
4. Nasopharyngeal swab in Viral transport medium and in Amies medium

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
a) Bring the tube with viral transport medium and the tube with Amies medium to room
temperature.
b) Label the tubes.
c) Remove one nasopharyngeal swab out of the package
d) Insert the swab into the nostril parallel to the palate until
resistance is met by contact with the nasopharynx. Leave
the swab in for a few seconds
e) Remove the cap from the transport tube by twisting.
f) Insert the swab into the tube with viral transport medium, cut the swab and close the
vial.
g) Remove the second nasopharyngeal swab out of the package and repeat the
procedure in the other nostril.
h) Bring the swab in the Amies medium tube and snap it off.
i) Bring the tubes in the zip-lock bag, togheter with the other samples from the same
patient.
j) Store the zip-lock bag in your refrigerator until shipment to the Local Lab within 24
hours.

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
For Local Laboratory
1. Arrival of the samples
At the moment of the arrival of the samples in the local lab, the sample kit is containing the
samples, Local Lab Requisition Form, the unused sample material and the unused labels.
The Local lab has to fill in the Local Lab Requisition Form on the moment of arrival of the
samples.
Complete next information on the Local Lab Requisition Form (Annex 2):




Patient study number (use a label)
Arrival Date of the sample kit (dd/mmm/yyyy)
Arrival Time of the sample kit (24 hours clock)
Specimens collected (tick the right box)
o
o
o
o
o
Serum tube
EDTA tube
Sputum
Nasopharyngeal swab with viral transport medium
Nasopharyngeal swab with Amies medium
Check the labels on the specimen with the label used on the Local Lab
Requisition Form. The label information should be identical. If not, please
contact the local National Network Facilitator (NNF).
Please make sure that the Local Lab Requisition Form is filled in completely and legibly. If an
error is made, cross out the mistake, write the correct information and initial and date the
change.
The Local Lab Requisition Form will be kept by the local lab and a fax forwarded to the NNF
within 24 h after arrival of the samples.
Take out from the sample kit if available, the serum tube and the sputum sample for further
sample handling procedures, described below.
Store the rest of the samples in the zip-lock bag in your freezer, preferably at -70°C, until
shipment to the Central Lab.
2. Serum sample
a) Label the required serum transfer tube. Use the unused labels from the sample kit.
b) Centrifuge the sample at 4000 revs/min (1500 g) for 15 min’s in order to separate the
red blood cells (red matrix) from the serum (pale yellow matrix).

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
c) Transfer the serum from the serum tube into the 10 ml serum transfer tube. Be
careful not to pipette any red blood cells.
d) Store the tube, with the other samples from the same patient, in your freezer,
preferably at -70°C, until shipment to the Central Lab.
3. EDTA blood sample
Store the tube in your freezer, preferably at -70°C, until shipment to the Central Lab.
4. Sputum
a) Prepare sputum slides for direct microscopy
b) Prepare sputum slides to be Gram stained
c) Culture the sputum samples according to your local procedures.
Report all findings on the Sputum Result Form
d) If sputum cultures are positive for S. pneumoniae and/or for Haemophilus spp.
Preparation of Microbanks for Central Lab (UA)
1) Label two Microbanks for one positive culture for S. pneumoniae or for Haemophilus spp.
(one microbank for the Central Lab and one microbank as back-up, to be kept at the site)
Use the unused labels from the sample kit and write on the labels “S. pneumoniae” or
“Haemophilus spp.” depending on the organism. The Labels are now coded with a patient
study number, a visit number and organism name.
2) Under aseptic conditions open the screw cap of the Microbank.
3) Inoculate cryopreservative fluid with colonies from a pure culture.
4) Close vial tightly.
5) Invert 4-5 times. Do not vortex or centrifugate!
6) Open the vial again and remove excess fluid.
7) Re-close the vial finger tight.
8) Store one vial, with the other samples from the same patient, preferably at -70°C in your
freezer until shipment to the Central Lab.
9) Keep one back-up in the Local Lab
5. Nasopharyngeal swabs
Store the tubes in your freezer, preferably at -70°C, until shipment to the Central Lab.

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
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Contract No: LSHM-CT-2005-518226
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
FROZEN SAMPLE SHIPMENT PROCEDURE

Every two months (?) shipments on dry ice are foreseen and are to be organised by the
Central Lab. You will not have to call the Central Lab but will be contacted automatically.

One week prior to the next foreseen pick-up, the Central Lab will call you to verify sample
availability and to propose a pick-up date. The agreed pick-up date and time will be
confirmed in writing via e-mail or fax.

At the moment of the shipment one sample kit is containing, all the material from one
patient and the unused labels from the same patient

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Investigator- Laboratoy Manual
Contract No: LSHM-CT-2005-518226
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
REPORTING
Investigator
The investigator will report following data into the databank within 24 hours after collection of the
samples.
 Patient information
o Patient study number
o Gender
o Date of birth
 Sample collection information
o Collection date
o Collection time
 Specimens
 Observational study
 visit 1
o Serum tube
o EDTA tube
o Sputum
o Nasopharyngeal swab with viral transport medium
o Nasopharyngeal swab with Amies medium

visit 2
o Serum transfer tube
o Nasopharyngeal swab with viral transport medium
o Nasopharyngeal swab with Amies medium

Control group
Visit 0
o EDTA tube
o Nasopharyngeal swab with viral transport medium
o Nasopharyngeal swab with Amies medium


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
Local Laboratory
All the information collected by the local laboratory, will be faxed to the NNF. The NNF
will bring all the data into the databank.
Collected data by the local laboratory:
 Local lab Requisition Form (annex 2).
Fax this form within 24 h after receiving of the samples to the NNF

Sputum Result Form (annex 3).
Fax this form (2 pages) within 7 days after receiving of the samples.
The NNF will report the following data, from the Local lab Requisition Form, into the databank:




Patient study number
Arrival Date of the sample kit (dd/mmm/yyyy)
Arrival Time of the sample kit (24 hours clock)
Specimens collected (tick the right box)
 Serum tube
 EDTA tube
 Sputum
 Nasopharyngeal swab with viral transport medium
 Nasopharyngeal swab with Amies medium
The NNF will report the following data, from the Sputum Result Form, into the databank:





Results of Direct microscopy for sputum
Results of Gram stain for sputum
Specimen acceptable for culture
Sputum cultures
Strains frozen for transport

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

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
Central Laboratory
The Central Lab will check the following data into the databank:
 Patient information
 Sample collection information
 Specimens information
The Central Lab will report the following tests into the databank
 Arrival date and time of specimen into the Central Lab
 Results




Susceptibility testing for S. pneumoniae and Haemophilus spp.
CRP
Procalcitonin
Bacterial NAAT-result
o Date NA extraction V1
o Date NA amplification V1
o Remarks
o V1 TS1 M. pneumoniae (pos/neg)
o V1TS1 C. pneumoniae (pos/neg)
o V1 TS1 B. pertussis (pos/neg)
o V1 TS1 L. pneumophila (pos/neg)
o V1 TS1 S. pneumoniae (pos/neg)
o
o
o
o
o
o
o

Date NA extraction V2
Date NA amplification V2
V2 TS1 M. pneumoniae (pos/neg)
V2 TS1 C. pneumoniae (pos/neg)
V2 TS1 B. pertussis (pos/neg)
V2 TS1 L. pneumophila (pos/neg)
V2 TS1 S. pneumoniae (pos/neg)
Viral NAAT-result
o Date NA extraction V1
o Date cDNA production V1
o Date NA amplification V1
o Remarks
o V1 TS1 influenza A (pos/neg)
o V1 TS1 influenza B (pos/neg)
o V1 TS1 parainfluenza 1 (pos/neg)
o V1 TS1 parainfluenza 2 (pos/neg)
o V1 TS1 parainfluenza 3 (pos/neg)


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
o
o
o
o
o
o

V1 TS1 human respiratory syncytial virus (pos/neg)
V1 TS1 human metapneumovirus (pos/neg)
V1 TS1 Bocavirus (pos/neg)
V1 TS1 coronavirus(pos/neg)
V1 TS1 rhinovirus (pos/neg)
V1 TS1 adenovirus (pos/neg)
Serology (values or positive/negative?)
o Date test result
o remarks
o V1: M. pneumoniae IgM
o V1: M. pneumoniae IgA
o V1: M. pneumoniae IgG
o V1: C. pneumoniae IgM
o V1: C. pneumoniae IgA
o V1: C. pneumoniae IgG
o V1: L. pneumophila IgM
o V1: L. pneumophila IgA
o V1: L. pneumophila IgG
o V1: B. pertussis IgM
o V1: B. pertussis IgA
o V1: B. pertussis IgG
o
o
o
o
o
o
o
o
o
o
o
o
V2: M. pneumoniae IgM
V2: M. pneumoniae IgA
V2: M. pneumoniae IgG
V2: C. pneumoniae IgM
V2: C. pneumoniae IgA
V2: C. pneumoniae IgG
V2: L. pneumophila IgM
V2: L. pneumophila IgA
V2: L. pneumophila IgG
V2: B. pertussis IgM
V2: B. pertussis IgA
V2: B. pertussis IgG
Other WP results, to be discussed with other WP leaders.

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
CONTACT PERSONS
Central Lab –University Antwerp – UA
Name:
Function:
Company:
Email address:
Phone number:
Fax number:
Greet Ieven
WP3 leader
UA
[email protected]
+32 3 821 36 44
+32 3 825 42 81
Herman Goossens
GRACE co-ordinator
UA
[email protected]
+32 3 821 37 89
+32 3 825 42 81
Name:
Katherine Loens
Christine Lammens
Function:
Company:
Email address:
Phone number:
GRACE manager
UA
[email protected]
+32 3 820 24 18
Technical co-ordinator
UA
[email protected]
+32 3 820 25 51
Fax number:
+32 3 820 27 52
+32 3 820 26 63
Address:
UA
Laboratory of Medical Microbiology
building S 3rd floor
Universiteitsplein 1
B-2610 Wilrijk, Belgium
National Network co-ordinators and Local Labs
COUNTRY
Name:
Function:
Company:
Email address:
Phone number:
Fax number:
Address:
….

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Investigator- Laboratoy Manual
Contract No: LSHM-CT-2005-518226
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Investigator- Laboratoy Manual
Contract No: LSHM-CT-2005-518226
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
ANNEXES
Annex 1: Investigator Requisition Form
Contract: PL518226
INVESTIGATOR REQUISITION FORM
Site number:
Investigator Name:
Street Address:
Postcode – City:
Country:
Telephone:
UNIQUE NUMBER: (use label)
PATIENT GENDER
F/M
DATE OF BIRTH
d
d
m
m
y
y
COLLECTION DATE
y
y
d
d
m
m
y
y
y
y
COLLECTIONTIME
:
h
h
m
m
Please use one Investigator Requisition Form per visit and tick the revelant box(es)
Yes
No
Observational study
Visit 1
 Serum tube
 EDTA tube
 Sputum
 Nasopharyngeal swab with viral transport medium
 Nasopharyngeal swab with Amies medium
Visit 2
 Serum tube
 Nasopharyngeal swab with viral transport medium
 Nasopharyngeal swab with Amies medium
Control group
Visit 0
 EDTA tube
 Nasopharyngeal swab with viral transport medium
 Nasopharyngeal swab with Amies medium
INSTRUCTIONS:
After collection put the samples, in a zip-lock bag and store them in your refrigerator until shipment to the Local Lab, together with:


the Lab Requisition Form

the unused sample material


the
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3.0unused labels
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The Investigator Requisition Form :

will be faxed to the NNF within 24 h

will be kept by the investigator
Investigator- Laboratoy Manual
Contract No: LSHM-CT-2005-518226


Annex 2: Local Laboratory Requisition Form
Contract: PL518226
LOCAL LABORATORY REQUISITION
FORM
Name Local Laboratory:
Investigator Name:
Street Address:
Postcode – City:
Country:
Telephone:
UNIQUE NUMBER: (use label)
ARRIVAL TIME
ARRIVAL DATE
d
d
m
m
y
y
y
y
:
h
h
m
m
Please use one Local Laboratory Requisition Form per sample kit and tick the relevant box(es)
Yes





No
Serum tube
EDTA tube
Sputum
Nasopharyngeal swab with viral transport medium
Nasopharyngeal swab with Amies medium
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INSTRUCTIONS:
Upon arrival, check all the specimen, and fill in the Local Laboratoy Requisition Form.
Please make sure that the Local Laboratory Requisition Forms is filled in completely and legibly. If an error is made, cross out the
mistake, write the correct information and initial and date the change.
After completing the Form put the samples, which are not be handled in the local laboratory, in a zip-lock bag and store them in your
freezer, preferably at -70°C, until shipment to the Central Lab, together with:

the unused sample material

the unused labels
The Local Laboratoy Requisition Form:

will be faxed to the NNF within 24 h

will be kept by the Local laboratory
Annex 3: Sputum Result Form
Contract: PL518226
SPUTUM RESULT FORM – page 1
Name Local Laboratory:
Investigator Name:
Street Address:
Postcode – City:
Country:
Telephone:
UNIQUE NUMBER: (use label)
Yes
DATE OF TESTING
No
Sputum available
d
d
m
m
y
y
y
y
Please tick the relevant box(es)
Please make sure that the Sputum Result Form is filled in completely and legibly.
If an error is made, cross out the mistake, write the correct information and initial and date the change.
The Sputum Result Form

will be faxed to the NNF within 7 days after receiving the samples
 will be kept by the Local laboratory
Sputum available
Yes
No
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 Microscopic examination
o
o
WBCs
o
o
o
o
o
0-4 per microscopic field
5-10
11-20
21-50
50-100
RBCs
o
o
o
o
o
0-4 per microscopic field
5-10
11-20
21-50
50-100
o
Squamous epithelial cells
o
0-4 per microscopic field
o
5-10
o
11-20
o
21-50
o
50-100
o
Yeasts
o
o
o
1 per microscopic field (few)
1-10 (moderate)
11-20 (many)
Contract: PL518226
SPUTUM RESULT FORM – page 2
UNIQUE NUMBER: (use label)

Gram Stain
o
Gram-positive cocci
o
1 per microscopic field (few)
o
1-10 (moderate)
o
11-20 (many)
o
Gram-negative diplococci
o
1 per microscopic field (few)
o
1-10 (moderate)
o
11-20 (many)
o
Gram negative rods
o
1 per microscopic field (few)
o
1-10 (moderate)
o
11-20 (many)
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
o
Gram positive rods
o
1 per microscopic field (few)
o
1-10 (moderate)
o
11-20 (many)
o
Yeasts
o
o
o
Specimen acceptable for culture:
o
o


1 per microscopic field (few)
1-10 (moderate)
11-20 (many)
Yes
No
Culture:
o
Haemophilus species
o
poor growth (growth in first sector)
o
moderate growth (growth up to second sector)
o
rich growth (growth from third sector)
o
Streptococcus pneumoniae
o
poor growth (growth in first sector)
o
moderate growth (growth up to second sector)
o
rich growth (growth from third sector)
o
Other genera/species:
Please specify: ……………………………………..
o
poor growth (growth in first sector)
o
moderate growth (growth up to second sector)
o
rich growth (growth from third sector)
Strains frozen for transport to Central Lab:
o
o
Haemophilus species
Streptococcus pneumoniae
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