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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:

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

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).

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

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

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.

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.

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 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 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 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 micro-organism 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 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.

1

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).

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 literatureThe 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 non-bacterial LRTI. This suggests that GPs can reassure patients that LRTI, even if bacterial, is a self-limiting condition and that rather than immediate antibiotic prescribing, a strategy of watchful waiting should be considered.

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.

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;54:20-4.

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 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.

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.

Figure 1. Kaplan–Meier survival curve for the duration of symptoms rated ‘moderately bad or worse’ in patients with LRTI.

Table 1. Baseline characteristics of patients with and without bacterial LRTI

All patients

N= 834(%)

Bacterial LRTI present

N=162(%)

Bacterial LRTI absent

N=672(%)

P value§

Missing

N (%)

Age, mean (SD)

51 (16)

52 (17)

51 (16)

0.450

0 (0.0)

Male gender

345 (41)

69 (43)

276 (41)

0.724

0 (0.0)

Current smoker

214 (26)

53 (33)

161 (24)

0.022

0 (0.0)

Co morbidity (pulmonary, cardiac, DM)*

212 (26)

46 (29)

166 (25)

0.311

1 (0.1)

Cough duration before index consultation, mean (SD)†

9 (7)

9 (7)

8 (7)

0.021

7 (0.8)

Severity score (all symptoms), mean (SD)‡

31 (14)

34 (14)

30 (14)

0.003

22 (2.6)

Infiltrates on chest radiograph present

26 (3)

9 (6)

17 (3)

0.046

29 (3.5)

Antibiotic use||

62 (7)

20 (12)

42 (6)

0.008

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.

Table 2. Prognostic outcomes in patients with and without bacterial LRTI

Bacterial LRTI present, N=162

Bacterial LRTI absent, N=672

Crude analysis

(95% CI)

Adjusted analysis (95% CI)*

P value†

Missing

N (%)

Median time to resolution of symptoms rated ‘moderately bad or worse’, days (IQR)

7 (5-15)

7 (4-13)

HR 0.89 (0.74-1.07)

HR 0.92 (0.77-1.11)

0.375

1 (0.1)

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

3 (0.4)

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

1 (0.1)

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

12 (1.4)

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

6 (0.7)

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.

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 Year

G. Chest radiograph of sufficient quality? |_| Yes:|_| No:

H. Consolidation |_| Yes:|_| No:

If yes:|_| Right:|_| Left:

I. Pleural effusion|_| Yes:|_| No:

J. Interstitial pattern/infiltrate|_| Yes:|_| No:

K. Diagnosis|_| Normal chest radiograph

|_| Acute bronchitis

|_| Bronchopneumonia

|_| Lobar pneumonia

|_| Other, please specify:………………………………………………………………………………..

L. Other remarks: ……………………………………………………………………………….

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.

Appendix 3. Baseline characteristics of included (diary returned) and excluded patients (no diary returned)

All patientsN=1021 (%)

Included patientsN=834(%)

Excluded patientsN=187 (%)

P value§

Missing N (%)

Age, mean (SD)

49 (16)

51 (16)

43 (17)

<0.001

0 (0.0)

Male gender

423 (41)

345 (41)

78 (42)

0.931

0 (0.0)

Current smoker

271 (27)

214 (26)

57 (31)

0.164

1 (0.1)


252 (25)

212 (26)

40 (21)

0.245

1 (0.1)


9 (7)

9 (7)

9 (8)

0.794

7 (0.7)


31 (14)

31 (14)

31 (15)

0.798

25 (2.4)


33 (4)

26 (3)

7 (5)

0.307

81 (7.9)




‡ 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.

Appendix 4. Baseline characteristics of patients with and without antibiotic use

All patientsN=834 (%)

Antibiotic useN=62 (%)

No antibiotic useN=772(%)

P value§

Missing N (%)

Age, mean (SD)

51 (16)

51 (15)

51 (16)

0.910

0 (0.0)

Male gender

345 (41)

23 (37)

322 (42)

0.478

0 (0.0)

Current smoker

214 (26)

13 (21)

201 (26)

0.379

0 (0.0)


212 (26)

19 (31)

193 (25)

0.329

1 (0.1)


9 (7)

7 (7)

9 (7)

0.020

7 (0.8)


31 (14)

36 (14)

31 (14)

0.007

22 (2.6)


26 (3)

8 (13)

18 (2)

<0.001

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.




‡ 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.

Appendix 1. Laboratory manual

Investigator - Laboratory Manual

Project acronym:GRACE

Project full title:Genomics to combat Resistance against Antibiotics in Community-acquired LRTI in Europe

Document type:Investigator - Laboratory Manual

Document status:Version 3.3

Release date: 31 May 2007

Table of Contents

1.Introduction28

1.1Sampling material supplied by the Central Lab (University of Antwerp, Belgium)28

1.2Documents28

2.Study Procedures and Observations29

2.1Overview of samples to be taken per visit29

2.2Overview of handlings to be performed by the local lab29

2.3Overview of analysis to be perfomed by the central lab or by the labs from the other Workpackages30

3.Laboratory Test Assessments31

3.1Local Laboratory31

3.2Central Laboratory (UA)32

4.SamplE HANDLING Procedures33

4.1Investigator Requisition Form33

4.2Material Preparation33

4.3Overview of Sample Handling per Visit35

4.4Sampling Instructions38

4.4.1For investigator38

4.4.2For Local Laboratory40

5.FROZEN Sample Shipment Procedure42

6.Reporting43

6.1Investigator43

6.2Local Laboratory44

6.3Central Laboratory45

7.Contact persons47

7.1Central Lab –University Antwerp – UA47

7.2National Network co-ordinators and Local Labs47

8.Annexes48

8.1Annex 1: Investigator Requisition Form48

8.2Annex 2: Local Laboratory Requisition Form49

8.3Annex 3: Sputum Result Form50

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.

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

Control group

Sample

V1

V2

V1

Serum

S

S

EDTA blood

S

S

Sputum

S

Nasopharyngeal swab with virus transport medium

S

S

S

Nasopharyngeal swab with Amies medium

S

S

S

V1 = Day 0 (Pre-treatment)

V2 = Day 1 (4 weeks after visit1)

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 transport medium

Store the swab preferable at –70°C

Nasopharyngeal swab with Amies medium

Store the swab preferable at –70°C



Overview of analysis to be perfomed by the central lab or by the labs from the other Workpackages

Sample

Test

Observational study

Control group

V1

V2

V1

Serum

Serology for M. pneumoniae, C. pneumoniae and L pneumophila

T

T

C-reactive protein

T

T

Procalcitonin

T

T

EDTA blood

Human Genetics

T

T

Sputum

Susceptibility testing on S. pneumoniae and Haemophilus spp.

T

Molecular analysis on S. pneumoniae and Haemophilus spp.

T

Nasopharyngeal Swab in Viral transport medium

Viral culture

T

T

Antigen tests

T

T

Virus discovery

T

T

T

PCR for Chlamydophila pneumoniae, Legionella pneumophila, Mycoplasma pneumoniae, Bordetella sp., Streptococcus pneumoniae and respiratory viruses

T

T

T

Nasopharyngeal swab in Amies medium

PCR for atypical bacteria

T

T

T

Growth of Streptococcus pneumoniae and Haemophilus spp.

T



T = Perform test

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.

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

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.

Investigator - Laboratory Manual

Contract No: LSHM-CT-2005-518226

The same labels are used for the Patient Registration Form, Case Record Form (CRF), Investigator Requisition Form , Local Lab Requisition Form, …

Version 3.3Page 33 of 52

Overview of Sample Handling per Visit

Observational study : Visit 1 (Pre-Treatment / Day 0)

Sample /Storage (pref. at –70°C) until

Sample MaterialPreparation at SitePreparation at Local Lab shipment to the Central Lab

Serum sample

· Centrifuge for 15 mins at 4000 rev/min

· Label the serum transfer tube

· Transfer serum into the serum transfer tube

· Label the serum tube

· Draw the blood in the tube

· Coagulation (15 mins)

· Store the tube until shipment to the local lab

Store serum frozen until shipment.

EDTA blood sample

· Label the EDTA- tube



Store sample frozen until shipment.

Store microbanks frozen until shipment.

· Perform Direct microscopy, Gram stain and culture

· If cultures positive for S. pneumoniae and/or Haemophilus spp, prepare the microbanks

Sputum

· Label the sputum container

· Ask the patient for a sputum sample

· Store the container 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

Nasopharyngeal swab

Store swab frozen until shipment.

· Label the Amies tube

· Collect the nasopharyngeal swab and place it in the Amies medium tube.



Nasopharyngeal swab

Observational study: Visit 2 (4 weeks after first visit / Day 1)

Sample /

Sample MaterialPreparation at SitePreparation at Local Lab Storage (pref. at –70°C) until shipment to the Central Lab

Serum sample

Store serum frozen until shipment.

· Centrifuge for 15 mins at 4000 rev/min

· Label the serum transfer tube

· Transfer serum into the serum transfer tube

· Label the serum tube


· Coagulation (15 mins)



Nasopharyngeal swab




Nasopharyngeal swab


· Collect the nasopharyngeal l swab and place it in the Amies medium tube.



Control group: Visit 0 (day0)

Sample/ Storage (pref. at –70°C) until

Sample MaterialPreparation at SitePreparation at Local Lab shipment to the Central Lab

EDTA blood sample

· Label the EDTA- tube



Store sample frozen until shipment.


· Collect the nasopharyngeal swab and place it in the Amies medium tube.







Nasopharyngeal swab

Nasopharyngeal swab

Investigator- Laboratoy Manual

Contract No: LSHM-CT-2005-518226

Draft Version 3.0 Page 52 of 52

Sampling Instructions

For investigator

1. Serum Sample

a) Label the required 10 ml serum-tube.

b) Draw 10 ml of the patient’s blood in the serum-tube.

c) Leave the sample to coagulate for 15 min’s (without shaking).

d) 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

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.

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)


· Serum tube

· EDTA tube

· Sputum

· Nasopharyngeal swab with viral transport 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).

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.

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

Reporting

Investigator

The investigator will report following data into the databank within 24 hours after collection of the samples.

· Patient information

· Patient study number

· Gender

· Date of birth

· Sample collection information

· Collection date

· Collection time

· Specimens

· Observational study

· visit 1

· Serum tube

· EDTA tube

· Sputum



· visit 2

· Serum transfer tube



· Control group

· Visit 0

· EDTA tube



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)


· Serum tube

· EDTA tube

· Sputum



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

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

· Date NA extraction V1

· Date NA amplification V1

· Remarks

· V1 TS1 M. pneumoniae (pos/neg)

· V1TS1 C. pneumoniae (pos/neg)

· V1 TS1 B. pertussis (pos/neg)

· V1 TS1 L. pneumophila (pos/neg)

· V1 TS1 S. pneumoniae (pos/neg)



· 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


· Date cDNA production V1


· Remarks

· V1 TS1 influenza A (pos/neg)

· V1 TS1 influenza B (pos/neg)

· V1 TS1 parainfluenza 1 (pos/neg)



· 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?)

· Date test result

· remarks

· V1: M. pneumoniae IgM

· V1: M. pneumoniae IgA

· V1: M. pneumoniae IgG

· V1: C. pneumoniae IgM

· V1: C. pneumoniae IgA

· V1: C. pneumoniae IgG

· V1: L. pneumophila IgM

· V1: L. pneumophila IgA

· V1: L. pneumophila IgG

· V1: B. pertussis IgM

· V1: B. pertussis IgA

· V1: B. pertussis IgG

· 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.

Contact persons

Central Lab –University Antwerp – UA

Name:Greet IevenHerman Goossens

Function:WP3 leaderGRACE co-ordinator

Company:UAUA

Email address:[email protected] [email protected]

Phone number:+32 3 821 36 44+32 3 821 37 89

Fax number: +32 3 825 42 81+32 3 825 42 81

Name:Katherine LoensChristine Lammens

Function:GRACE managerTechnical co-ordinator

Company:UAUA

Email address:[email protected] [email protected]

Phone number:+32 3 820 24 18+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:

….

Annexes

Contract: PL518226

Annex 1: Investigator Requisition Form

INVESTIGATOR REQUISITION FORM

Site number:

Investigator Name:

Street Address:

Postcode – City:

Country:

Telephone:

UNIQUE NUMBER: (use label)

COLLECTION DATE

d d m m y y y y

PATIENT GENDER

F/M

DATE OF BIRTH

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



Visit 2

· Serum tube



Control group

Visit 0

· EDTA tube



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 unused labels

The Investigator Requisition Form :

· will be faxed to the NNF within 24 h

· will be kept by the investigator

Please make sure that the Investigator 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.

Contract: PL518226

Annex 2: Local Laboratory Requisition Form

LOCAL LABORATORY REQUISITION FORM

Name Local Laboratory:

Investigator Name:

Street Address:

Postcode – City:

Country:

Telephone:


ARRIVAL DATE

d d m m y y y y

ARRIVAL TIME

:

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



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

Contract: PL518226

Annex 3: Sputum Result Form

SPUTUM RESULT FORM – page 1


Name Local Laboratory:

Investigator Name:

Street Address:

Postcode – City:

Country:

Telephone:

YesNo

Sputum available

DATE OF TESTING

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 YesNo

· Microscopic examination

· WBCs

· 0-4 per microscopic field

· 5-10

· 11-20

· 21-50

· 50-100

· RBCs


· 5-10

· 11-20

· 21-50

· 50-100

· Squamous epithelial cells


· 5-10

· 11-20

· 21-50

· 50-100

· Yeasts

· 1 per microscopic field (few)

· 1-10 (moderate)

· 11-20 (many)


SPUTUM RESULT FORM – page 2

Contract: PL518226

· Gram Stain

· Gram-positive cocci


· 1-10 (moderate)

· 11-20 (many)

· Gram-negative diplococci


· 1-10 (moderate)

· 11-20 (many)

· Gram negative rods


· 1-10 (moderate)

· 11-20 (many)

· Gram positive rods


· 1-10 (moderate)

· 11-20 (many)

· Yeasts


· 1-10 (moderate)

· 11-20 (many)

· Specimen acceptable for culture:

· Yes

· No

· Culture:

· Haemophilus species

· poor growth (growth in first sector)

· moderate growth (growth up to second sector)

· rich growth (growth from third sector)

· Streptococcus pneumoniae




· Other genera/species:

Please specify: ……………………………………..




· Strains frozen for transport to Central Lab:

· Haemophilus species

· Streptococcus pneumoniae