Intrapleural DNase and Tissue Plasminogen Activator in Pediatric Empyema (DTPA Trial)
Phase: PHASE3
Conditions: Pleural Empyema
Sponsor: The Hospital for Sick Children

Study title:  
 
Intrapleural DNase and Tissue Plasminogen Activator in Pediatric Empyema (DTPA Trial) 
 
NCT number: 
NCT01717742 
 
Document date: 
Protocol – June 29, 2017. 
 

Version June 29, 2017. 
 
 
 
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Research Protocol 
 
Intrapleural DNase and Tissue Plasminogen Activator in Pediatric Empyema (DTPA trial) 
 
Principal Investigator: 
Eyal Cohen, Hospital for Sick Children, University of Toronto, Toronto, Ontario 
 
 
Co-Investigators:  
Sanjay Mahant, Hospital for Sick Children, University of Toronto, Toronto, Ontario  
 
 
Felix Ratjen, Hospital for Sick Children, University of Toronto, Toronto, Ontario 
Jeffrey Hoch, St. Michael’s Hospital, University of Toronto, Toronto, Ontario 
 
 
 
Bairbre Connolly, Hospital for Sick Children, University of Toronto, Toronto, Ontario 
Kevin Thorpe, St. Michael’s Hospital, University of Toronto, Toronto, Ontario 
Muhammad Mamdani, St. Michael’s Hospital, University of Toronto, Toronto, Ontario 
 
 
Ian Maclusky, Children’s Hospital of Eastern Ontario, Ottawa, Ontario 
 
Sophie Laberge, CHU Sainte Justine, Montreal, Quebec 
Lucy Giglia, McMaster Children’s Hospital, Hamilton, Ontario 
Mark Walton, McMaster Children’s Hospital, Hamilton, Ontario 
Connie Yang, British Columbia’s Children’s Hospital, Vancouver, British Columbia  
 
 
 
 
 

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SUMMARY 
Pneumonia is one of the most common reasons for hospitalization in children.  In recent years, 
there has been an increase in the incidence of pleural empyema, defined as pneumonia with a 
complicated effusion leading to respiratory compromise and/or extensive loculations.  With modern 
treatments, including antibiotics and intrapleural drainage with instillation of fibrinolytics, long-term 
outcomes are excellent.  However, substantial short-term morbidity persists, including prolonged 
hospital stay with a painful chest tube and ‘treatment failure’ necessitating further procedures. 
DNase (dornase alfa) may work synergistically with fibrinolytic drugs such as tissue 
plasminogen activator (tPA) leading to liquefaction of pleural collections and improved drainage.  
Based on an adult trial, there may be an additional benefit from the addition of intrapleural DNase to 
tPA, but this requires study in a pediatric population as the underlying disease (e.g. microbiology and 
natural history), and other, age-related biological factors may potentially affect treatment response.  
This randomized controlled trial aims to address the efficacy, cost effectiveness and safety of DNase 
combined with tPA compared with tPA alone in the management of childhood pleural empyema.  
 
STUDY AIMS 
Over-arching aim: The overarching aim of this research collaborative is to improve outcomes for 
children with pleural empyema.   
Primary Research Question: In previously well children who present with pleural empyema, does 
intrapleural DNase with tissue plasminogen activator (tPA) via chest drain for three doses over 48 
hours improve the time to hospital discharge compared with three doses over 48 hours of tPA alone?    
Secondary Research Questions: Will there be differences between the groups with respect to other 
outcomes related to efficacy, cost and safety? 
 
TRIAL DESIGN 
Design: The study design will be a blinded, five-centre pragmatic, superiority randomized controlled 
trial in a parallel group, 1:1, two arm design. 
Procedure:  Previously healthy children (6 mo – 18 years) with pleural empyema requiring chest tube 
drainage will be recruited. 
Interventions: At the time of chest tube insertion, subjects will be randomized to intrapleural 
instillation of tPA alone or tPA with DNase for three doses over 48 hours. 
Outcome: Primary outcome is time to hospital discharge.  Secondary outcomes include time to 
meeting discharge criteria, time to drain removal, duration of fever after intervention, need for 
ventilatory support or non-invasive ventilation, serious bleeding, need for further procedural 
interventions, hospital readmission, cost and mortality. 
Sample Size: A sample size of 92 (46 per group) will have the power to detect a 2 day difference in 
length of stay between the groups. While we do not anticipate any post-randomization ineligibility, for 
example from misdiagnosis, we will increase the sample size by 5% to account for this possibility 
(total of 49 patients per group).  It is anticipated that over the study period, 180 children will be 
admitted to the five centres who meet eligibility criteria. 
Analysis: Student’s t-test will assess differences in the primary outcome between groups. 
 
FEASIBILITY 
The investigators are clinical experts with a strong track record in outcomes research in pediatric 
empyema and methodological experts in clinical trials. 
 

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COMPLICATED PNEUMONIA (PLEURAL EMPYEMA) IN CHILDREN 
Pneumonia is one of the most common reasons for hospitalization in childhood, 1 and accounts 
for more inpatient resource utilization (cost) than any other pediatric diagnosis outside of the newborn 
period.2  Up to 50% of children hospitalized with pneumonia have an associated parapneumonic 
effusion.3 Most of these effusions are small and uncomplicated and will resolve with antimicrobial 
treatment of the underlying infection, but in some cases, a complicated effusion can develop 
characterized by enlargement leading to respiratory compromise and/or extensive loculations.  Such 
effusions, commonly referred to as pleural empyema, characteristically lead to substantial morbidity 
including respiratory distress, pain, and prolonged hospitalization leading to child school loss, parental 
work loss and stress on families.  These children generally do not respond to antibiotic therapy alone 
and require management with a drainage procedure. In recent years, there has been a dramatic increase 
noted in the incidence of pediatric pleural empyema in multiple countries. 4-9 A review of Canadian 
data (excluding Quebec and Manitoba) found that there are now over 1,400 new cases each year. 10 
Hospitalization data from the United States seems to suggest that this growth has been most apparent in 
young children, aged 2-4 years,11 with Canadian data documenting a recent increase in incidence rates 
by over 450% in those aged 1-4 years. 10 Although the exact cause of this epidemiologic change is 
somewhat unclear, postulated causes include pneumococcal serotype replacement and increasing 
antibiotic resistance.12  
There are important gaps in the scientific literature regarding the optimal therapy for pleural 
empyema in children that have been highlighted in various recent clinical practice guidelines that note 
that high quality evidence to support therapeutic recommendations is often lacking;13,14 of over 50 
recommendations considered in a recent review, only four (<8%) had high quality evidence.14 This 
problem is pervasive in child health research. High quality clinical evidence is often based on the 
results of randomized controlled trials, and our research group has identified a paucity of randomized 
controlled trials (RCTs) in child health. Over the past 20 years adult RCTs published in leading general 
and subspecialty medical journals have increased substantially, while pediatric RCTs have increased 
only modestly.15,16 This problem is particularly relevant in pharmaceutical (drug) trials, and has 
undoubtedly led to the widespread use of pharmaceutical products in children without sufficient data 
on efficacy or safety;17 Despite some legislative initiatives in the United States and Europe to promote 
drug trials in children, extensive logistical and financial disincentives persist.  Consequently, 79% of 
hospitalized children are treated with drugs for unapproved indications, otherwise known as ‘off-label’ 
use.18  
 
THERAPY FOR PLEURAL EMPYEMA IN CHILDREN 
Antibiotics: In children with pleural empyema, antibiotic coverage for likely causative organisms (S. 
pneumoniae, S.aureus and S. pyogenes) is essential.  Unfortunately, a specific antimicrobial etiology is 
identified in less than 20% of cases, 19 so empiric therapy is commonly used. Although the potential 
choice of agents is wide and guided by local policies and patterns of antibiotic-resistant organisms such 
as penicillin-resistant S. pneumoniae and methicillin-resistant S.aureus (MRSA), recent published 
guidelines in both the United States 13 and Canada 20 suggest that empiric therapy with a third 
generation cephalosporin (ceftriaxone or cefotaxime) with the addition of either clindamycin or 
vancomycin if clinical, laboratory, or imaging characteristics are consistent with infection caused by S. 
aureus. 
 
Drainage procedures: Published clinical practice guidelines support the use of drainage procedures in 
addition to antibiotics in managing pleural empyema in children.13,14,21,22 These guidelines are based on 
aggregate reports suggesting significantly longer hospital stays, duration of antibiotic therapy and 
progression to surgical intervention in children treated with antibiotics alone.23  A variety of different 
drainage procedures have been reported in the literature including chest tube placement with or without 

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fibrinolytics, repeated ultrasound-guided needle thoracentesis, video-assisted thorascopic surgery 
(VATS) and open thoracotomy with decortication.  A systematic review 24 and cost-effectiveness 
analysis 25 of the relative merits of each of these procedures has been conducted by our team.  Based on 
the best-available evidence, either of two approaches is the most effective therapy for treating 
childhood empyema: (a) insertion of a chest drain with regular instillation of fibrinolytics; or (b) 
VATS. Recent Canadian data suggests variability in practice across hospitals, although insertion of a 
chest drain with regular instillation of fibrinolyics may be more cost effective.25 
 
Fibrinolytics: The rationale for utilization of fibrinolytics in parapneumonic effusion is its role in 
dividing pleural septations and loculations that interfere with drainage. A variety of different 
fibrinolytic agents have been described in the literature: streptokinase, urokinase, and tissue 
plasminogen activator (tPA). Streptokinase is rarely used as it is antigenic generates a systemic 
antibody response similar to that found when the drug is given systemically.26 A randomized controlled 
trial in the United Kingdom comparing regular chest tube instillation of urokinase with saline for 3 
days in 60 children with parapneumonic effusions found that length of stay was significantly shorter 
with urokinase (7.4 vs. 9.5 days).27  Another trial from the UK comparing urokinase instillation with 
VATS in 60 children found no difference in the primary outcome of length of stay. 28  Urokinase is not 
available in North America. 
Current fibrinolytic utilization is based on availability in different jurisdictions (e.g. urokinase 
in Europe, tissue plasminogen activator in North America). North American studies of fibrinolyic 
agents have utilized recombinant tPA, and, similar to urokinase, evidence suggests that it substantially 
improves pleural drainage.  Early observational studies suggested that early administration (at the time 
of chest drain insertion) and frequent administration of tPA was associated with shortened hospital 
stays,29 similar efficacy to urokinase,30 and safety.29,30  A randomized controlled trial of 36 children 
comparing once daily tPA x 3 doses (4 mg in 40 mL normal saline at the time of insertion and then at 
24 and 48 hours) with VATS found similar hospital stays (6 vs. 6 days) and lower overall hospital 
charges in the tPA group. 31 
Safety data on fibrinolytics suggests no serious adverse events aside from intrapleural 
bleeding.29,30 Most bleeding is mild and clinically insignificant (i.e. does not lead to a drop in 
hemoglobin).  It is also difficult to differentiate the etiology of cases of clinically significant bleeding 
between the surgical procedure of insertion of the drain and the drug itself.  A recent review of the data 
at the Hospital for Sick Children suggests a prevalence of 2% for clinically significant bleeding 
(leading to a drop in hemoglobin) from chest drain insertion and/or fibrinolytic instillation 
(unpublished data).  Current guidelines support the instillation of tPA as standard of care for pleural 
empyema being treated with a drain.8,10-12 
 
DEOXYRIBONUCLEASE (DNase) 
Despite the utilization of fibrinolytics, pleural drainage can still be challenging leading to 
prolonged hospital stays with a painful chest tube in situ and ‘treatment failure’ whereby pleural 
disease persists that cannot be drained necessitating salvage procedures such as open thoracotomy.  
The need for subsequent procedures is somewhat centre dependent, and, in clinical trials of tPA in 
childhood empyema, ranges from 17%31 to 38%,32 but such procedures are associated with significant 
morbidity including pain and a variety of potential surgical complications.  One potential explanation 
for both the inadequate drainage leading to prolonged hospital stays and the phenomenon of ‘treatment 
failure’ is the presence of extracellular uncoiled DNA liberated from dead leukocytes and other 
bacterial components. Such residual material may increase viscosity, permit biofilm formation, and 
thus interfere with drainage.33-36  Fibrinolytic agents are not thought to reduce pleural viscosity, but 
recombinant human DNase has been shown in vitro to help decrease the viscosity by cleaving free 
DNA and hence liquefying parapneumonic pus.37,38  Subsequent animal studies have demonstrated that 

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the combined effects of tPA and DNAse in a rabbit model of empyema is more effective than either 
agent alone.39 Small human (adult) case series have also described benefit from the addition of DNase 
to the treatment of empyema.40,41 
Safety data on DNase is primarily derived from its currently licensed indication, nebulization at 
a dose of 2.5 to 5 mg once or twice daily for the reduction of sputum viscosity in patients with cystic 
fibrosis. It is well tolerated with rash, voice alteration, chest pain and laryngitis as common side 
effects.42 
DNase RCT in adults: A pivotal randomized controlled trial led by Rahman et al. from the 
UKCRC Oxford Respiratory Trials Unit was recently published in the New England Journal of 
Medicine addressing the effectiveness of DNase in adult patients.42  The authors conducted a blinded 2-
by-2 factorial trial in which 210 adult patients with pleural infections were randomly assigned to four 
study treatments for 3 days: double placebo, intrapleural tPA and DNase, tPA and placebo, or DNase 
and placebo.  The primary outcome was the change in pleural opacity, measured as the percentage of 
the hemithorax occupied by effusion on chest radiography on day 7 compared with day 1.  Secondary 
outcomes included referral for surgery, duration of hospital stay and adverse events.  For the primary 
outcome, the authors found a significantly greater reduction in pleural opacity in the tPA-DNase group 
compared with the placebo group (-29.5±23.3% vs. -17.2±19.6%; mean difference -7.9%; 95% 
confidence interval (CI), -13.4 to  -2.4; p=.005).  The change in pleural opacity observed with tPA 
alone or DNase alone was not statistically different from that observed with placebo.  All secondary 
outcomes also pointed to superiority of tPA-DNase compared with other study arms.  Hospital stay for 
the tPA-DNase group (11.8±9.4 days) was about 50% shorter compared with placebo (24.8±56.1 days), 
whereas in the DNase-only group and the tPA-only group, length of stay was similar to placebo.  The 
frequency of surgical referral at 3 months was lower in the tPA-DNase group compared with placebo 
(4% vs. 16%; odds ratio for surgical referral, 0.17: 95% CI, 0.03 to 0.87, p=.03).  Surgical referrals 
were increased in the DNase-only group (odds ratio, 3.56; 95% CI, 1.30-9.75, p=.01), and were non-
significantly reduced in the tPA-only group (odds ratio, 0.29; 95% CI, 0.07 to 1.29, p=.1). Mortality 
rates were 8% at 3 months and 12% at 12 months, but were similar across all study groups.  
Inflammatory measures (C-reactive protein, systemic white blood cell count and odds of fever) were 
also assessed.  Significant (p<.05) differences were found in mean white count on day 7 and fever on 
day 6 or 7 between tPA-DNase and placebo. The new treatment was not associated with any excess of 
adverse events.  Serious adverse events described included intrapleural hemorrhage (n=2, both in the 
tPA-DNase group), gastrointestinal bleeding (n=2, both in the DNase group), hemoptysis (n=1, in the 
tPA-DNase group, and clinical deterioration (n=1, in the placebo group). 
The findings of this trial can best be explained by the synergistic effects of tPA and DNase.  
The fibrinolytic effects of tPA may disrupt fibrinous septations that would otherwise divide the 
infected collection, allowing DNase to liquefy the thick pus.  Similarly, the thinning of pleural fluid by 
DNase may provide more effective access for tPA to pleural septations. 
In summary, a large well-designed trial in adult patients showed that the combination of tPA and 
DNase therapy improves the drainage of pleural fluid in patients with pleural infection, reduces 
hospital stay and the need for thoracic surgery. Data for children are lacking at the present time. 
     
 
Extrapolating from Adult DNase trial to the management of children: The aforementioned 
review of pediatric pleural empyema suggests that current therapeutic options are still suboptimal. 
Short-term morbidity persists associated with prolonged hospitalization and pain/discomfort from chest 
tubes, as well as the need for secondary salvage surgical procedures with potential complications. 
There may be an additional benefit from the addition of intrapleural DNase to tPA instillation into 
chest drains.  However, extrapolating from adult studies in pleural empyema to children is problematic 
as pediatric pleural empyema is in many ways a different disease for the following reasons: 

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(a) Mortality rates differ substantially.  The mortality rate in adults is thought to be 10-20%,43 44-46 
and comorbidities are common.  Most children who develop pleural empyema are otherwise 
healthy, and mortality is extremely rare in those treated with a drainage procedure. No mortality 
has been described in pediatric empyema RCTs.23,24    
(b) Epidemiologic trends showing a rising incidence in empyema have been primarily 
demonstrated in children.  Although microbiologic confirmation is often elusive, this does 
suggest that the microbial etiology of pediatric empyema (e.g. pneumococcal serotypes not 
covered by conventional vaccines) may differ from adult patients. 
(c) Therapies that have been found to be ineffective in adult patients have been effective in 
children. For example, intrapleural streptokinase was not found to be beneficial in an adult trial 
(MIST1 trial),46 but was found to be useful for some children in a pediatric trial,47 and in a case 
series.48 
Therefore, although there is a biological rationale and clinical efficacy data from adults, currently it is 
not known whether the addition of DNase to tPA will provide improvement in outcomes for pediatric 
pleural empyema. 
 
SUMMARY OF THE EVIDENCE TO DATE FOR PEDIATRIC PLEURAL EMPYEMA 
Pleural empyema is a rising problem in children and leads to significant short-term morbidity. 
Rigorous evidence with pediatric data is essential to inform clinical decision-making.  The proposed 
trial brings clinical experts with a strong track record in outcomes research in pediatric empyema 
together with methodological experts in clinical trials in an effort to improve the outcomes of children 
admitted to hospital with pleural empyema. 
Although there is still some ongoing controversy, the best evidence from randomized controlled 
trials seems to suggest that either VATS or small bore percutaneous chest tube placement with 
instillation of fibrinolytics result in the best outcomes in pediatric pleural empyema as measured by 
hospital length of stay, although chest tube with fibrinolytic therapy may be more cost-effective.  
Fibrinolytics are a safe and effective therapy based on observational and randomized control trial 
evidence.   However, persistent short-term morbidity associated with prolonged hospitalization and 
pain/discomfort from chest tubes, as well as the need for secondary salvage surgical procedures.  Based 
on a well-designed study in adults, there may be an additional benefit from the addition of intrapleural 
DNase to tPA, but this requires study in a pediatric population as the underlying disease (e.g. 
microbiology and natural history), and other, age-related biological factors may potentially affect 
response to treatment.  This randomized controlled trial is designed to address the efficacy, cost 
effectiveness and safety of DNase combined with tPA compared with tPA alone in the management of 
childhood pleural empyema.  
 
STUDY AIMS 
Over-arching aim: The overarching aim of this research collaborative is to improve outcomes for 
children with pleural empyema.   
Primary Research Question: In previously well children who present with pleural empyema, does 
intrapleural DNase with tissue plasminogen activator (tPA) via chest drain for three doses over 48 
hours improve the time to hospital discharge compared with three doses over 48 hours of tPA alone ?    
Primary Hypothesis: Three doses of DNase-tPA, when compared to three doses of tPA via chest drain 
will reduce the time to discharge after chest drain insertion in children hospitalized with pleural 
empyema.  
Secondary Research Questions: Will there be differences between the groups with respect to other 
outcomes related to efficacy, cost and safety? The following outcomes will be assessed: 
a) Time to meeting discharge criteria. 
b) Time to drain removal. 

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c) Duration of fever, defined as the number of days with temperature >38oC. 
d) Need for ventilator support or non-invasive ventilation following intervention. 
e) Risk of serious bleed, defined as intrapleural bleeding resulting in a drop in hemoglobin of greater 
than 20 g/L or needing a transfusion. 
f) 
Need for further interventions, defined as the need for further intervention such as placement of 
another chest drain (by any technique) or surgical intervention such as thoracotomy and 
decortication, video-assisted thorascopic surgery, or pneumonectomy. 
g) Hospital readmission related to pleural empyema or its treatment within three months of discharge. 
h) Cost of the hospitalization. 
i) 
Mortality. 
j) 
Amount of effusion present at the time of drain removal (exploratory outcome). 
Secondary Hypotheses: DNase-tPA, when compared to tPA via chest drain, will: 
a)    Reduce the time of fever, time to meeting discharge criteria, time to drain removal and cost. 
b)    Not result in an increase in need for ventilator support or non-invasive  ventilation following 
intervention, nor an increase in risk of serious bleed, nor any need for further intervention, nor the risk 
of hospital readmission, nor the risk of mortality. 
 
TRIAL DESIGN (see Appendix 1 for trial schematic) 
Design: The study design will be a superiority randomized controlled trial in a parallel group, 1:1, two 
arm design.  
Pragmatic-Explanatory Continuum: The primary purpose of this trial is to inform clinical decision 
making. Therefore, according to the framework described by co-investigator Kevin Thorpe and other 
trial methodologists, this trial has been designed along the pragmatic end of the pragmatic-explanatory 
continuum.49 Specifically, most study domains [eligibility criteria, follow-up intensity, primary 
outcome and primary analysis] will follow pragmatic approaches (“Does this intervention work under 
usual conditions?”); practitioner expertise and adherence and the flexibility of the interventions will 
follow approaches midway along the pragmatic-explanatory continuum (“Can this intervention work 
under ideal conditions?”). This protocol has been designed following the 2010 SPIRIT guidelines 
(Standard Protocol Items for Randomized Trials) 50 and results will be reported according to the 2008 
CONSORT guidelines for pragmatic trials.51 
 
METHODS 
Study Setting: This study will occur at major tertiary care children’s hospitals These sites were 
specifically chosen due to their size (they are among the largest children’s hospitals in Canada), 
relatively close proximity, and interest in collaborative work in improving outcomes in pleural 
empyema.  These centres also preferentially use chest drain with fibrinolytics (as opposed to VATS) as 
their first line treatment of choice for pleural empyema in children.  Furthermore, in allcentres, most 
chest drain insertion is performed using an image guided percutaneous technique performed by 
interventional radiologists and have similar antibiotic prescribing patterns.  Lastly, all centres are also 
implementing clinical pathways to standardize the management of this condition.  
 
Eligibility - Inclusion Criteria: 
1. age 6 months to 18 years  
2. hospitalized with diagnosis of pleural empyema requiring chest tube drainage with fibrinolytics as 
judged by the attending physician with the following criteria: 
(a) pneumonia with pleural effusion as documented on ultrasound of the chest; AND 
(b) need for further intervention in addition to antibiotics based on clinical criteria [(persistent fever 
despite on antibiotics for at least 48 hours OR significant respiratory distress tachypnea, hypoxia) as a 
result of the pleural fluid collection] 

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Eligibility - Exclusion Criteria:  
1. empyema as a result of tuberculosis, fungus or non-infectious causes (e.g. malignancy) 
2. known coagulation impairment  
3. suspected or proven allergy to tPA or DNase 
4. chronic lung disease or other chronic illnesses (e.g. immunodeficiency or neurologic impairment) 
5. child has already undergone a drainage procedure (e.g. chest drain or VATS). 
6. recent administration of an investigational drug (within previous 30 days) 
7. pregnancy 
8. breastfeeding 
 
Interventions: Patients will be randomized to receive either: (a) tPA [alteplase (Cathflo®), Roche] at a 
dose of 4 mg followed by a placebo (saline) administered intrapleural via chest drain once daily for 3 
doses; or, (b) tPA at a dose of 4 mg followed by DNase [dornase alfa (Pulmozyme®), Roche] at a dose 
of 5 mg. Weight-based dosing will not be used for tPA or DNase, as pleural concentrations are 
unpredictable and variable. Pediatric studies of tPA29 31 and guidelines21 have utilized adult dosing. 
DNase will be constituted by the research pharmacies in clear liquids in a polyethylene syringe 42.  
Since the stability of a tPA-DNase admixture is unknown, the drugs will be administered sequentially 
with a 1 hour indwelling time for each drug as described in the aforementioned adult trial.  For tPA, the 
contents of the vial will be diluted to a total volume of 10 ml normal saline (tPA) for children less than 
or equal to 10 kg and 20 ml for children > 10 kg.  A flush of 5 ml of normal saline will be instilled after 
drug administration.  Following the instillation of tPA the drain will be clamped for one hour and then 
will be opened to drain under suction at a pressure of -20 cm H2O for one hour.  Then, either DNase or 
placebo will be instilled as a volume to 10 ml with normal saline for children less than or equal to 10 
kg and 20 ml for children > 10 kg, followed by the same 5 ml normal saline flush, the same one hour 
period of clamping and the same one hour period of drainage at -20 cm H2O.  Thus in total, each cycle 
of tPA-study drug instillation and drainage will take 4 hours to complete. The pharmacies will prepare 
the two arms (DNase or placebo) in a manner such that both are identical (packaging, colour, volume, 
texture, and odour) to ensure blinding.  The contents of the vials (tPA followed by DNase or placebo) 
will be instilled into the chest drain by clinicians caring for the child. The first dose of tPA will be 
given immediately (within 1 hour) after insertion of the chest drain by the interventional radiologist or 
surgeon in the procedure suite or by clinicians on the ward and DNase or placebo will be instilled on 
the ward. On the following day (day 1) and the subsequent day (day 2) a dose of tPA followed by the 
study drug will be administered in the morning, between 9 and 10 am, in a similar fashion.  Thus, each 
patient will receive a total of three doses of either tPA or tPA-DNase over 48 hours.   
 
Clinical Samples at the Hospital for Sick Children: At the SickKids site only, consent will be 
obtained to store samples of chest tube drainage from subjects. These samples will be used for future 
testing to determine the microbiological cause of empyema. 
 
Concomitant Medication: Patients in either arm will not receive any intrapleural drug other than 
directed from this study.   
 
Criteria for discontinuing study interventions: Patients who develop anaphylaxis or serious bleeding 
(requiring a blood transfusion or resulting in a hemoglobin drop of ≥ 20 g/L) while receiving study 
interventions will not receive further study drugs. Patients whose chest drains is displaced will only be 
replaced if clinically indicated for pleural fluid drainage and not for study drug administration alone.  
Outcomes for all patients who do not received the full study interventions will be analyzed in the group 
they were assigned to. 

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Standard care: In keeping with a pragmatic-type trial design, all children will receive standard care 
for this condition including supportive care, laboratory investigations, imaging, antibiotics, chest drain 
care and removal and discharge as outlined in the standard care guidelines.  Although some variability 
is expected in care among treating physicians, rigorous randomization and blinding should ensure that 
confounders are equally distributed between groups.  A detailed care map will be adapted from the 
existing clinical practice guideline co-authored by team investigators (EC and SM) together with the 
Canadian Pediatric Society. Specific elements in the care plan include:  
a) Antimicrobial management - Patients in both treatment arms will receive standard therapy for 
pleural empyema in children as directed by the responsible physician and standardized guidelines. All 
participating hospitals currently recommend a 2nd or 3rd generation cephalosporin (cefuroxime or 
ceftriaxone or cefotaxime) as the empiric antimicrobial therapy with possible addition of clindamycin 
or vancomycin if S. Aureus is a suspected etiology. It is expected that antibiotic regimens will be 
tailored in line with local microbiology advice.  
b) Drain insertion - Chest drains will be placed using an image guided percutaneous technique by 
interventional radiologists or by a surgeon using a standardized technique under anesthesia agreed 
upon by all sites. The recommended size of catheter drain used will be an 8 or 10 French pigtail 
catheter. 
c) Drain management - Chest drain management will be at the discretion of treating physicians.  
Suggested management in the care pathway will include maintaining -20 cm H2O of continuous suction 
and once daily flushes with 10 ml of normal saline (on days where no study drug is administered) to 
maintain patency. 
d) Criteria for drainage removal - Similarly, although recommendations for drainage removal will be 
made in the care map (< 1ml/kg/d), ultimately the decision to remove the chest drain will be made at 
the discretion of the responsible physician likely based on a gestalt of clinical parameters (amount of 
drainage, appearance of the child, fever, etc.). All patients will likely be initially on a similar regime of 
antibiotics and then tailored based on microbiologic results.  
e)  Diagnostic imaging - There is no consensus on the type (ultrasound versus plain radiograph) or 
frequency of diagnostic imaging necessary after drain insertion in hospitalized children.  Therefore, in 
contrast to the aforementioned adult RCT of DNase, follow-up radiographs will only be assessed as an 
exploratory outcome in the study protocol.   
 
Outcomes - Primary outcome: Time to hospital discharge:  This outcome is the most common 
outcome measure in trials of childhood empyema. This is defined as the time, measured in hours, and 
reported in days rounded to a single decimal point, from insertion of the chest drain to discharge from 
hospital.  This will be assessed by a research assistant on a twice daily basis.  
 
Outcomes - Secondary outcomes: Secondary outcomes include measures of effectiveness, harm and 
cost-effectiveness. Formal clinical follow-up and review of other parameters such as radiographic 
resolution and lung function testing will not be performed as preliminary work from this group of 
investigators and others suggests that long-term outcomes in childhood pleural empyema are almost 
universally positive.  Our prospective cohort study has found that by six months, virtually all patients 
are asymptomatic and follow-up radiographs have normalized in 62/65 (95%) and pulmonary function 
tests normalize in 27/28 (96%).52 
a.   Time to meeting discharge criteria: This is defined as the time, measured in hours, and reported in 
days rounded to a single decimal point, from insertion of the chest drain to meeting discharge criteria. 
This will be assessed by a research assistant on a twice daily basis (9 AM and 4 PM) and defined as: no 
fever (temperature less than 38oC) for 24 hours, normal respiratory rate for age [using the World 
Health Organization (WHO) age-specific criteria (< 50 breaths/min for 2-12 months, < 40 breaths/min 

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for 1 to 5 years, and < 20 breaths/min for ≥ 5 years)], no hypoxia (transcutaneous oxygen saturations in 
room air less than 92%), and drinking fluids well.   
b. Time to drain removal: This is defined as the time, measured in hours, and reported in days rounded 
to a single decimal point, from drain insertion to drain removal. 
c.  Duration of fever after intervention:  This is defined as the duration, measured in hours, and 
reported in days rounded to a single decimal point, of fever (defined as temperature >38oC taken by 
any method) from insertion of the chest drain until resolution.  This will be recorded  by a research 
assistant daily. 
d.  Need for ventilatory support or non-invasive ventilation following the intervention. 
e.  Serious bleeding: This is defined as intrapleural bleeding resulting in a drop in hemoglobin of 
greater than 20 g/L or needing a transfusion. 
f.  Need for further interventions: This is defined as the need for further intervention such as placement 
of another chest drain (by any technique) or surgical intervention such as thoracotomy and 
decortication, video-assisted thorascopic surgery, or pneumonectomy. 
g.  Hospital readmission: Any hospital readmission after discharge from hospital for initial treatment 
for pleural empyema within three months related to pleural empyema or its treatment (e.g. harm). All 
hospital readmissions will be categorized as empyema-related or not.  
h.  Cost of the hospitalization: An economic evaluation will compare the relative costs of DNase-tPA 
with tPA alone in previously well children who present with pleural empyema, using patient-level data 
from the trial. We will conduct the analysis from a hospital’s perspective because hospital 
administrators will be making reimbursement decisions for this intervention. Since empyema is an 
acute condition and the long-term consequences are negligible, the time horizon of the analysis will be 
the length of hospital stay. Since the primary effectiveness outcome is length of hospital stay or time to 
hospital discharge, which can be expressed in monetary terms (i.e. hospitalization cost), we will only 
compare the costs of the two strategies to avoid double-counting the effectiveness. The cost of each 
patient includes the cost of intervention (DNase-tPA or tPA alone) and costs incurred during the 
hospital stay (“hospitalization cost”). We will obtain the hospitalization costs of each patient from case 
costing data from hospital financial departments. We will calculate mean cost per patient by 
intervention group, based on initial intervention assignment, and incremental cost using simple linear 
regression: 
Ci = αi + βti +εi 
 
Where Ci is the cost for each patient i, α is the intercept term, t is an intervention dummy term (t=1 if 
the patient received DNase-tPA and t=0 for tPA alone), and ε is the stochastic error term. The 
regression coefficient β estimates the incremental cost of DNase-tPA compared with tPA alone. The 
regression statistics will show mean cost per patient by intervention group and the uncertainty around 
the mean estimates. We will also conduct sensitivity analysis to assess the robustness of the results. 
i.  Mortality: this will include mortality from any cause during the hospitalization for empyema. 
 
Exploratory outcome – chest radiography: In the aforementioned DNase trial in adults, changes in 
pleural opacity, measured as the percent of the hemithorax occupied by effusion on day 7 compared 
with day 1 was a primary outcome.  In pediatric empyema, this outcome is problematic because: (a) 
many children will be discharged prior to day 7; (b) hemithorax size differs substantially across 
different sized children; and, (c) requiring an additional chest radiograph may be a substantial 
disincentive for parental caregiver to provide consent participate particularly given the rising concerns 
of ionizing radiation in developing children. Further, radiographic changes are considered surrogate 
measures of clinical changes as they often lag behind clinical improvement. There is currently no 
standard as to the timing of radiographs in hospital, but it is our experience that virtually all children 
will have a radiograph performed shortly before and/or shortly after chest tube removal.  The 

Version June 29, 2017. 
 
 
 
11 
radiograph closest to the time of drain removal will be reviewed by a blinded study radiologist (BC) to 
determine the percentage of hemithorax occupied using a 5 point ordinal scale utilized in previous 
studies ranging from no fluid present to fluid occupying >75% of the most affected hemithorax.  Given 
that radiographic improvement is time dependent, we do not have any a priori hypotheses about the 
results of this outcome across groups (i.e. those with the worst outcomes (chest drain duration) may 
potentially have better radiographs at the time of drain removal). 
 
Sample size: The primary endpoint is time to hospital discharge after intervention. The mean time to 
hospital discharge after chest tube insertion and fibrinolytics varies. Published studies with treatment 
arms that have included chest drain with fibrinolytics as the primary mode of insertion reported 
hospital days after the intervention from 6 to 15 days.23,24 Hospital length of stay tends to be shorter in 
published RCTs compared with observational studies of empyema, and the most recently published 
RCT using an identical tPA dosing regimen to that which we are proposing described a mean (SD) stay 
of 6.8 (2.9) days. 31 The desired power for the current proposal is 90% to detect a difference of 2 days 
in the mean time to hospital discharge between the treatment arms.  Based on discussions with clinical 
experts in the field, hospital administrators and parents of children with pleural empyema, it is believed 
that a 2 day difference between treatment groups is a minimal clinically meaningful difference, and this 
has been used in previous empyema trials. 27 The adult DNase trial found about a 50% difference in 
length of stay between treatment and placebo groups. 42 
Sample size was thus calculated assuming a type 1 error rate of 0.05 (2 sided), power (1-ß) of 
90%, a standard deviation of 2.9 days for each group to detect a difference of 2 days to be 46 per 
group.  Calculations were performed based on a t test of independent groups. While we do not 
anticipate any post-randomization ineligibility, for example from misdiagnosis, we will increase the 
sample size by 5% to account for this possibility (total of 49 patients per group). There will be no other 
adjustment to the sample size requirements due to loss to follow-up for the primary outcome as the 
primary outcome is assessed in hospital where the research coordinator will be able to ensure complete 
data collection.  
 
Recruitment: All study patients will be initially identified by research assistants who will review both 
(a) all new admissions to the relevant inpatient units in each study centre twice daily; and, (b) all 
referrals for chest drainage insertion at each centre. It is anticipated that most drains at both sites will 
be inserted by interventional radiologists, although a minority may be inserted by general surgeons. 
Physicians and radiologists/surgeons will also be asked to notify the site research assistant and/or site 
investigator of children admitted with pleural empyema who may be eligible for the study.  Patients 
will be approached, eligibility criteria confirmed, consent obtained, and enrolled after the decision to 
refer for chest drainage but before chest drain insertion occurs.  
 
Randomization – Sequence generation, allocation concealment and implementation: After 
informed consent is obtained, patients will be randomized into treatment groups using a random 
allocation sequence facilitated by the Applied Health Research Centre (AHRC), which will be the 
coordination and management centre for the trial and administered by the hospital research pharmacies.  
Randomization will be stratified by centre.  Blocking will be used to ensure that the two comparison 
groups are about the same size throughout the trial for each center and for the trial as a whole (about 49 
per group).  An allocation ratio of 1:1 with random block size will be used within each stratum (centre).  
This will help to ensure that clinicians, investigators, or outcome assessors will not decipher the block 
size. A computer-based pseudo-random number generator will be used to create treatment allocation 
tables for each study center.  After patient eligibility has been confirmed and consent obtained and just 
as the patient leaves the inpatient unit to the radiology suite or operating room for chest drain insertion, 
the site investigator, or his/her delegate, will assign the patient a unique study identification (ID) 

Version June 29, 2017. 
 
 
 
12 
number in sequential order. The study ID will correspond with the randomization table held in the 
research pharmacy for dispensing blinded DNase or placebo.  The biostatistician will maintain a secure 
master list of the randomization codes and the assigned treatments will be checked against the master 
list at the end of the study.    
 
Blinding: Patients, their parents or other caregivers, site investigators, research assistants and 
coordinators, treating physicians (pediatricians, radiologists, and interventional radiologists or 
surgeons), treating nurses, and data managers will be blinded to the treatment allocation.  Group 
allocation will be concealed until the final data analysis is performed.  Intervention drugs will be 
blinded by the research pharmacy. Both arms will be constituted by the research pharmacies in clear 
liquids in a polyethylene container 42 in a manner such that both are identical (packaging, colour, 
volume, texture, and odour) to ensure blinding. After obtaining the treatment number from the central 
randomization centre, the study pharmacist will retrieve the corresponding vial and one of its treatment 
number labels will be attached to the patient’s case report form (CRF).   
 
Unblinding Procedures: We do not anticipate any circumstances that would require unblinding as 
knowledge of study arm is not anticipated to affect any treatments for patients. 
 
Data collection methods: Data collection for outcome measures will be mainly collected in hospital 
by the Research Assistant (RA). At baseline, the following data will be obtained: age, sex, duration of 
antibiotic treatment prior to chest drainage insertion (days), duration of fever prior to chest drain 
insertion (days), hypoxia in room air (oxygen saturations less than 92% in room air prior to 
intervention), microbiologic identification of causative agent (blood culture results, throat swab, pleural 
fluid culture, pleural fluid PCR), ultrasound of chest: pleural effusion size >10 mm <10 mm, stage of 
empyema on ultrasound of chest: Stage 1: anechoic nonseptated fluid; Stage 2: echoic fluid without 
septation; and Stage 3: septated fluid and Stage 4: septations with solid appearing components 
comprising more than one third of the effusion.53  All ultrasounds will be reviewed by a blinded study 
radiologist (BC). In addition, patients will receive a follow-up phone call from the RA at 3 months 
enquiring about any possible readmissions and ongoing symptoms of fever, shortness of breath and/or 
exercise intolerance.   
 
Data management: The Applied Health Research Centre (AHRC) of the Li Ka Shing Knowledge 
Institute of St. Michael’s Hospital will serve as the data management centre under the direction of co-
investigator Muhammad Mamdani. AHRC employs state-of-the-art web based data management 
software, Medidata RAVE™ (5.6.3), a secure encrypted web based clinical trial data management 
system which is fully configurable and incorporates sophisticated data validation rules to ensure high 
quality data capture. RAVE™ allows for remote web-based data entry directly from the hospital sites, 
facilitating real time data access. 
 
Statistical Analysis:  
Baseline characteristics: Patient characteristics and descriptive variables will be presented for each 
treatment arm: age, sex, duration of antibiotic treatment prior to chest drain insertion (days), duration 
of fever prior to chest drain insertion (days), hypoxia, defined as saturations in room air less than 92% 
(yes/no), bacterial identification and subtype, pleural effusion size on ultrasound (>10 mm, <10mm), 
stage of empyema on ultrasound (frequency).  For continuous variables, means and standard deviations 
(SD) or medians (inter-quartile ranges) will be presented.  For categorical variables, proportions will be 
presented.   
 

Version June 29, 2017. 
 
 
 
13 
Primary outcome: Data will be analyzed according to intention to treat principles for the primary 
outcome (e.g. patients who do not receive all three doses of study drug will be analyzed in the group 
they were assigned to). Exceptions to this principle will only include any patient who dies in hospital 
and will be excluded from the analysis of time to hospital discharge. Given that the primary outcome 
and other acute secondary outcomes are obtained during hospitalization, it is anticipated that there will 
be no missing data for these outcomes with the possible, but unlikely exception of in-hospital death, 
which is rare in childhood empyema.  For the follow up outcomes at 3 months, the proportion of 
patients who follow up will be presented for each treatment arm, and patients who are lost to follow up 
will be stated but omitted from the analysis.  The primary outcome, time (in days) to hospital 
discharge, will be described as the difference between the two means with the 95% confidence 
intervals. The student’s t-test (independent two sample test assuming equal group size and variance), 
will be used to detect a difference between the two treatment groups. If the resulting data is 
inappropriate for a t-test (increasing variance with the mean is the biggest concern since the test is quite 
robust to non-normality in the population), a suitable transformation or non-parametric test may be 
used. If feasible, a secondary analysis of the primary outcome will analyze time to discharge, treating 
death as a competing risk, using methods for survival data. Since randomization is stratified between 
five centres, the analysis will adjust for this stratification by including centre as a covariate in a 
multiple linear regression model, to obtain adjusted treatment effects. Additionally, a treatment by 
centre interaction will be tested to see if the treatment effect differs between centres. Subgroup 
analyses will also be conducted to explore any potential differences in outcomes by age or sex. 
 
Secondary outcomes and exploratory outcome: For the secondary and exploratory outcomes that are 
continuous variables (e.g. duration of drain insertion, hospital stay after intervention to meeting 
discharge criteria, duration of fever after the intervention, amount of hemithorax occupied by pleural 
effusion in radiographs) the difference between the two means with the 95% confidence intervals will 
be presented.  The student’s t test will be used to detect a difference between the treatment groups. 
Dichotomous outcomes (serious bleeding, need for further interventions, need for ventilatory support, 
mortality) will be described as the absolute number and proportion.  A Fisher’s exact test will be used 
to detect a significant (p<0.05) difference between the two treatment arms.  The treatment effect will 
also be presented as the relative risk with 95% confidence intervals. These analyses will be viewed as 
hypothesis generating, and therefore, no correction for multiple testing is planned. 
 
Data Monitoring and Safety: Data monitoring will be conducted with a Data Monitoring Committee 
(DMC) composed of a pediatric hospitalist, a respiratory physician and an interventional radiologist.  
The DMC will be completely independent of the investigators, and will be provided with clinical 
information from the trial case report forms for death, surgical events and adverse events, including 
those that were described in the adult DNase trial (hemoptysis, gastrointestinal bleeding, chest pain, 
nausea, transient confusion and rash).  The DMC will be able to request additional information from 
clinicians.   
 
Adverse Event Reporting: All serious unexpected adverse events will be reported to the REB; as per 
SickKids REB guidelines. All serious adverse drug reactions to the study medication will be reported 
to Health Canada within 15 calendar days or for death or life-threatening events, within 7 calendar 
days. In the latter case, a follow-up report will be filed within 8 calendar days. Adverse reactions will 
be managed according to the standard clinical management practices. All adverse events and adverse 
reactions will also be reported to the PI within 24 hours.  
 
Quality Assurance: This project will be monitored by The Applied Health Research Centre (AHRC) 
during the data collection phase of the project. The aim is to ensure that all researchers are maintaining 

Version June 29, 2017. 
 
 
 
14 
the highest ethical, scientific and safety standards for all study participants, and are in compliance with 
all relevant policies, provincial and federal legislation, and international guidelines such as ICH- Good 
Clinical Practice. All studies are categorized according to the Continuing Review Matrix based on the 
type of study and the level of risk (I to IV) to research subjects and the Clinical Research Monitor will 
review at minimum 10% of the research subjects’ records for study eligibility, informed consent, 
adherence to study protocol, reporting of adverse drug reactions and adverse events, and data quality 
including computer database security and storage of records. Findings of the will be presented to the 
Research Ethics Board and lead PI in a written report, and specific recommendations arising from the 
report will be implemented in a timely manner. 
 
ETHICAL CONSIDERATIONS: Informed consent will be obtained from parental caregivers. Given 
the young age of children with empyema (average age of 4 years), it is anticipated that most will not be 
able to consent/assent to participate.  Informed consent/assent will be obtained from all those who are 
able to provide it. Potential known adverse events from study interventions are mainly local as the 
drugs are not systemically absorbed and include intrapleural bleeding, rash, voice alteration, chest pain 
and laryngitis. Aside from receiving the study interventions, participation will require parents to agree 
to be contacted by research personnel for a short (< 5 min) phone call three months post-discharge.   
 
Feasibility and Timelines: Internal data from decision support services at the three participating 
hospitals indicates that in fiscal year 2010-11, there were a total of 92 children with chest drains 
inserted for pleural empyema (25 at Sainte Justine, 33 at CHEO and 34 at SickKids), and, in 2009-10, 
98 children received a chest tube (28 at Sainte Justine, 37 at CHEO and 33 at SickKids).    Based on a 
review of the patients who had chest drains inserted at SickKids from 2008-2011, less than 5% of these 
will be ineligible for this trial (e.g. due to a comorbid condition such as cerebral palsy).  Thus, it is 
anticipated that a total of 180 potentially eligible patients will be available for recruitment, at three of 
the five sites, over two years for this clinical trial.  
Our previous experience with this population indicates a willingness to participate in research.  
In an observational study of long-term outcomes in pediatric empyema requiring participants to 
complete detailed follow-up questionnaires and attend an appointment for physical examination, 
spirometry and radiography for six months, 88/94 (94%) consented to participate, of whom 6 (7%) 
were lost to follow-up at one month and a further 8 (9%) at 6 months.   

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Trial Entry 
Children aged 6 months to 18 years 
Hospitalized with pleural empyema requiring drainage with fibrinolytics as 
defined by: 
a) Pneumonia with pleural effusion as documented on chest ultrasound 
AND 
b) Need for further investigation in addition to antibiotics based on clinical 
criteria, persistent fever despite on antibiotics for at least 48 hours  OR  
Significant respiratory distress (tachypnea, hypoxia) as a result of the 
pleural fluid collection 
Randomize 
tPA arm 
[4 mg tPA in 10 ml (≤10 kg) or 20 ml 
(>10 kg) normal saline]*, then placebo 
(10 ml normal saline (≤10 kg) or 20 ml 
(>10 kg)]* 
tPA-DNase arm 
[4 mg tPA  in 10 ml (≤10 kg) or 20 ml 
(> 10 kg) normal saline]*, then 5 mg 
DNase diluted to 10 ml normal saline 
(≤10 kg) or 20 ml (>10 kg)]* 
Outcomes 
Primary: time to hospital discharge 
 
Secondary: time to meeting discharge criteria, time to drain removal, 
Duration of fever after intervention, need for ventilatory support or 
non-invasive ventilation, serious bleeding, need for further 
interventions, hospital readmissions, cost of the hospitalization, 
mortality, chest radiography (exploratory outcome)