Validation of Nasal and Bronchial Absorption Sampling Methods for the Assessment of RSV Bronchiolitis in Babies and Children
Phase: NA
Conditions: Bronchiolitis, Bronchiolitis, Viral, RSV Infection, Respiratory Failure
Sponsor: Imperial College Healthcare NHS Trust

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THE RSV-SAM STUDY: Validation of Nasal and 
Bronchial Absorption Sampling Methods for the 
Assessment of Respiratory Syncytial Virus (RSV) 
Bronchiolitis in Babies and Children 
 
 
Imperial College-Pulmocide RSV Collaboration:  
 Imperial College, St Mary’s: Simon Nadel (Principal Investigator), Jethro Herberg, 
Vanessa Sancho Shimizu, Hannah Jarvis, Natasha Gunawardana, Trevor Hansel, 
Peter Openshaw 
 Imperial College, Charing Cross, Virology: David Muir (Consultant Virologist) 
 Pulmocide (IC Spin-Off Company): Garth Rapeport, Lindsey Cass, Kaz  Ito 
 
 
 
Abbreviations 
 
MLF: mucosal lining fluid 
NPA: nasospharyngeal aspirate 
NS: nasosorption with a SAM, consisting of soft absorptive material 
SAM: synthetic absorptive matrix 
RSV: respiratory syncytial virus 
PICU: paediatric intensive care unit 
qPCR: quantitative polymerase chain reaction 
NP: nasopharyngeal 
URTI: upper respiratory tract infections 
NAC: nasal allergen challenge   
RTI: respiratory tract infection 
BS: bronchosorption 
DNA: deoxyribonucleic acid 
mRNA: messenger ribonucleic acid 
 

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INTRODUCTION 
 
Epidemiology of RSV 
Since the discovery of the respiratory syncytial virus (RSV) in 1955, it has been 
recognised as the single most important cause of severe lung infections in infants of less 
than 12 months of age (1). Most children admitted to hospital with RSV are previously 
healthy although prematurity is the main known risk factor for severe disease (2). 
Globally RSV is the main cause of acute lower respiratory tract infections and a 
significant cause of death (3). 
 
RSV at St Mary’s Hospital 
St. Mary’s Hospital is responsible for the clinical care of many babies with RSV infection. 
Of the babies that attend the Accident and Emergency department with upper 
respiratory tract infections (URTI) each winter, over 100 are admitted to the paediatric 
wards with RSV bronchiolitis. Around 40 babies a year require mechanical ventilation 
on the Paediatric Intensive Care Unit (PICU). Although the clinical burden is large, most 
babies respond well to treatment and mortality is relatively low. 
 
Current sampling methods to diagnose RSV 
Studies of the upper and lower airways of human infants with RSV bronchiolitis are 
required to definitively understand pathological mechanisms.  Studies in infants are 
difficult to undertake, especially as the child may be clinically unstable and conventional 
procedures to obtain samples may be too invasive.  Infants who require mechanical 
ventilation for severe RSV bronchiolitis are tracheally intubated, sedated and have 
indwelling vascular catheters. This allows access to the respiratory tract and enables 
blood sampling.   
 
In infants naturally infected with RSV, the presence of virus is generally detected in 
nasopharyngeal aspirate (NPA) samples (5;9). In the UK it is standard clinical practice 
to perform NPA sampling by direct suction of secretions. However, because this 
involves the introduction of a suction catheter into the nostril and then to the back of 
the nose, this procedure may be relatively unpleasant. Sampling by NPA has been found 
superior to nasal swabs manufactured from natural products (10;11).  

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Using nasal swabs in young children is rapid and painless and also accurate for RSV 
diagnosis (12). There is concordance between nasal and nasopharyngeal (NP) swabs for 
viral testing (13). In a comparison with NPA, nose and throat swabs were more 
comfortable and have less risk of harm in a clinical setting, but NPA is still 
recommended in a hospital context (14). Flocked nylon swabs gave higher viral loads 
than cotton swabs when using qPCRs for detection of human rhinovirus (15).  
 
Viral load may be obtained from these samples by quantitative polymerase chain 
reaction (qPCR) of infectious samples. This correlates with disease severity in infected 
infants (4;5).  In adults infected experimentally with RSV, the viral load peaks before 
maximal clinical symptoms (6). It is a major parameter in quantitating the magnitude of 
infection (7). In assessing the effects of novel therapeutics in experimental infection, the 
area under the curve (AUC) of viral load is generally the primary end point (8).  To 
determine this, daily samples over the disease course are required. However, there are 
concerns regarding the accuracy of viral load detection from routine NPA samples as 
this is dependent of the amount of mucous suctioned at the same time, which varies 
enormously from patient to patient. 
 
The current methods of sampling described above have a number of limitations: 
 Samples are often diluted in saline with can lead to poor sensitivity 
 There is a lack of consistency in the location the sample is obtained from which 
can affect both diagnostic specificity and reproducibility 
 Sampling can be uncomfortable and invasive 
 
 

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Proposal for novel absorption sampling methods  
In conjunction with a specialist medical device manufacturing company (Hunt 
Developments (Midhurst, West Sussex) we have produced novel nasosorption and 
bronchosorption kits that have CE marking. Both nasosorption and bronchosorption  
methods use synthetic absorptive matrix (SAM) strips: that look and feel like blotting 
paper, and will be placed onto the mucosal surface. These are comfortable to use and 
can be used at frequent intervals over extended periods of time.  This non-invasive 
technique is ideal for infants and children, and it is possible to obtain neat mucosal 
lining fluid (MLF) even from normal healthy noses. The eluates contain cytokines and 
chemokines at high detectable levels on multiplex immunoassay.  
 
We would like to use these SAMs to take MLF samples from the nasal and bronchial 
mucosal surfaces to see if these novel techniques can overcome the problems with 
current sampling methods. We plan to use these absorption techniques to measure RSV 
viral load. We also aim to look at the immune response in terms of the anti-viral interferon 
response (IFN-γ, IFN-λ, IFN-α2a, IP10, ITAC). In therapeutic studies in the future, it may 
be possible to document levels of drug (pharmacokinetics) in nasal MLF. 
 
Nasosorption (NS) and bronchosorption (BS)  
NS involves using a synthetic absorptive matrix (SAM) to absorb mucosal lining fluid 
(MLF) in a precise manner from the inferior turbinate. At Imperial College we have used 
NS in children with allergic rhinitis (16), infants with a family history of atopy (17), and 
in atopic adults after nasal allergen challenge (NAC)  (18). SAM has also been used after 
experimental human rhinovirus challenge in asthma when both NS and BS were 
performed (19;20). The Stephen Durham group at Imperial have recently compared and 
validated a variety of SAMs (21), and performed a nasosorption study after cat NAC (22)   
 
The swabs are medical devices, have small handles and are enclosed in a sampling tube. 
These devices are CE-marked, clean and sterile, and produced in a specialist medical 
device factory. 
 

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Bronchosorption (BS) involves a similar method to NS with a similar SAM material. The 
SAM is attached to a plastic reinforced catheter that can be introduced through an 
endotracheal tube to access the bronchial mucosa. Professor Onn Min Kon, respiratory 
consultant at St Mary’s Hospital, has successfully carried out over 700 BS sampling 
procedures in adults with a range of respiratory diseases. The technique has been very 
well tolerated, and has permitted cytokines and chemokines to be measured in the 
bronchial mucosal lining fluid (MLF).   
 
Using a similar but smaller device we hope to take samples from infants ventilated due 
to severe RSV bronchiolitis. Babies ventilated with severe RSV infection, have an 
endotracheal tube (ETT) in position that requires suction clearage at regular intervals. 
Hunt Developments have developed a specialised device for introduction down the ETT, 
that can be used for BS in babies ventilated for severe RSV bronchiolitis.  
 
As an initial step we will perform a pilot study on tolerability in ventilated infants, and 
have strict inclusion criteria which will allow inclusion of infants who are stable on the 
ventilator with low-moderate ventilation and oxygen requirements. We intend to 
compare inflammatory mediator levels in mucus that has been directly suctioned from 
the ETT, with levels of mediators in samples obtained using SAM by nasosorption and 
bronchosorption.  
 
Genetics  
Mendelian single gene deficiencies in the form of primary immunodeficiencies have 
demonstrated the important role that host genetics play in infectious disease outcome. 
More recently, single gene disorders predisposing to a narrow spectrum of infections 
have been described. Examples include defects in the IL12-IFNγ pathway predisposing to 
atypical mycobacterial infections, IRAK4-MYD88 defects to invasive pyogenic infections, 
and TLR3-IFN defects to herpes simplex encephalitis. Sequencing of the patient samples 
by whole exome/genome sequencing or linkage analysis will be carried out.  One part of 
this study aims to identify genetic aetiologies of severe RSV infections which will 
provide tools for molecular diagnosis and genetic counselling.  
 
 

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Summary 
In summary this study will compare the novel methods of NS and BS with the standard 
technique of nasophayngeal aspiration (NPA) and routine ETT suction. We shall assess 
the samples for diagnosis of RSV, viral load and immune responses in the airways of 
babies with RSV infection. We shall also assess the genetics of babies included in this 
study, to see if they may be vulnerable to RSV infection. 
 
 
AIMS 
 
The study involves obtaining nasal samples from babies with suspected RSV infection in 
the emergency department (ED), as well as those admitted to the paediatric wards and 
PICU at St. Mary’s Hospital, London. This will help us to understand the epidemiology of 
and the host response to RSV infection in infants both hospitalised and in the 
community. In addition, we shall take nasal samples from infants attending hospital for 
outpatient appointments, routine elective surgery and admitted to PICU for non-
respiratory diagnoses as controls. 
 
The specific aims are: 
 
1. To study different sampling methods; comparing the established method of NPA and 
tracheal suction to nasosorption and bronchosorption. We will assess sample 
volumes, clinical tolerability and virology results: especially viral load.  
 
2. To assess the safety and validity of using bronchosorption to take bronchial MLF 
samples. 
 
3. To study severity of disease and correlate this with quantitative RSV PCR. 
 
4. To study the incidence of RSV A and B infection, different subtypes of RSV infection.  
 
5. To measure the nasal and bronchial mucosal immune response: in terms of 
cytokines and chemokines, especially those relating to interferons. 

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6. To identify if there is a genetic predisposition to severe RSV infection 
 
7. To understand feasibility of these sampling processes in preparation for an 
interventional study potentially in winter 2017-8. 
 
 
 
 

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ENDPOINTS 
 
To compare SAM and nasopharyngeal aspirate (NPA) as sampling methods for 
measuring RSV viral load. 
 
PRIMARY ENDPOINT: RSV viral load 
 
Virology: How do SAM and NPA compare for RSV viral load determination? 
 
We shall also assess: 
 Sensitivity, specificity, quantitation 
 Utility for RSV-A vs RSV-B  
 
SECONDARY ENDPOINTS 
1. Immune Response: Establishing the use of nasal and bronchial sampling to measure 
the host immune response to RSV. These techniques could then be applied to the 
study of drug pharmacokinetics for new therapeutics in future clinical studies.  
 
2. Epidemiology and virology: This study will assess the number of babies with upper 
respiratory tract infections (URTI) presenting to St Mary’s Hospital ED, the number 
of babies with confirmed RSV hospitalised on the Paediatrics Ward and also the 
number admitted to the PICU. The study will assess the relative incidence of RSV A 
and B and allow us to determine whether RSV subtype is associated with disease 
severity.  
 
3. Genetics: To establish whether there is a genetic predisposition to severe RSV 
infection. To carry out genetic analysis by targeted gene sequencing, next generation 
sequencing or whole genome SNP typing in order to evaluate any genetic 
deficiencies. 
 
4. Correlation of qPCR with clinical severity 
 
 

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STUDY DESIGN 
Type of Study: Pilot study 
 
Duration: To take place in RSV infection season between October and March, starting in 
October 2015. 
 
Identification of patients: We shall obtain our study groups from patients attending St. 
Mary’s Hospital, London. 
 
Number and types of Subjects: 
 
A total of 5 participant groups (n = 30 in each group)  
Details of each group and respiratory sampling procedures are given below: 
 Group 1: Babies with suspected respiratory tract infection (RTI) in the ED:  
 
Nasosorption (NS) in each nostril 
NPA  
 Group 2: Babies with diagnosed RSV infection admitted to paediatric wards: 
 
NS  (2 to 3 times daily, 4-6 hours apart) 
NPA daily 
 Group 3: Babies with diagnosed severe RSV infection in PICU requiring mechanical 
ventilation:  
 
 
NS (2 to 3 times daily, 4-6 hours apart) 
NPA daily 
Bronchial aspirate (BA) daily  
 Control Group 1:  Babies without respiratory symptoms, attending routine outpatient 
appointments or undergoing elective surgical procedures:  
 
Nasosorption (NS) only once 
 Control Group 2 : Babies without RSV infection but requiring mechanical ventilation 
in PICU: 
NS (2 to 3 times daily, 4-6 hours apart) 
NPA daily 

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Bronchial aspirate (BA) daily 
PROCEDURES 
Sampling by Absorption:  
 
Nasosorption: SAM strips will be placed inside each of the nostrils for up to 30 seconds. 
We will place the SAM on the inferior aspect of the inferior turbinate and middle 
turbinate. There is minimal discomfort in sampling in these areas. This is a well-
tolerated, non-invasive procedure without the need for local anaesthetic.   
Bronchosorption: Following clearance of excess mucus by suction, we will introduce the 
SAM device that will be passed down the ETT to collect bronchial MLF. The device will 
be passed into the ETT in the same way that a routine suction catheter is introduced. 
The device will be introduced by the consultant paediatric intensivist or lead 
physiotherapist gently to a maximum distance of 10cm beyond the end of the ETT, or 
until it impacts in the bronchial tree. It will then be withdrawn by 5cm and the SAM 
paper will then be extruded. The device will then be kept in this position for 10 seconds 
to allow for maximal absorption. The SAM will then be retracted into the device and the 
device will be withdrawn.  
NB for the 2016-2017 season we will not be carrying out the bronchosorption 
procedure as we have concerns regarding the device and this is being revised 
accordingly. 
 
Bronchial aspirate:  
Routine clearance of excess mucus by suction through the endotracheal tube. We will 
keep this aspirate that is usually discarded. 
 
 
Nasophargeal aspirate:   
NPA will be carried out after the Nasal SAM has been performed. NPAs will be collected 
using a standardized protocol (23-25). The child is supine and a small suction catheter 
is used to take fluid from the back of the nose directly with no saline flush is a routine 
method used to suction babies with a blocked nose in clinical practice. 
 

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In babies who are admitted to the ward, sampling will have to consider the impact of 
nasogastric and other tubes in the airway. If both NS and NPA all samples have to be 
taken from the same nostril there should be a 10 min interval between each sample 
 
Blood sampling: 
If the baby is having a blood test as part of their routine hospital care an additional 1mL 
will be taken and used to measure markers of infection and inflammation, together with 
genetic analysis (subject to consent). If the baby requires further blood tests for clinical 
reasons, we may take further 1ml research samples at up to 2 further time points to see 
changes in the immune response.  
 
A 1ml blood sample will be taken at the first time point for genetic analysis as detailed 
below. 
 
Blood Genetics Methods 
i. 
Sample collection Blood samples from the baby will be collected and 
genetic material extracted  
ii. 
Sample processing  To carry out genetic analysis from DNA/RNA 
isolated from infants by targeted gene sequencing, next generation 
sequencing or whole genome SNP typing in order to evaluate any genetic 
deficiencies. 
iii. 
Data Collection It will be necessary to collect demographic and clinical 
data from the families in order to define clearly clinical phenotypes. This 
will include data on age, sex, ethnic groups and clinical details relating to 
the phenotype. These clinical data will be provided by collaborating 
clinicians from participating hospitals. 
 
 
 
 

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INCLUSION AND EXCLUSION CRITERIA 
 
Group 1 and Group 2 
 
Inclusion Criteria 
 Infants aged 2 weeks-24 months  
 Presentation to the Emergency Department with any upper respiratory tract 
infection (URTI) in the RSV season (Oct-March). 
OR 
 Documented RSV infection, admitted to the paediatric wards at St Mary’s 
Hospital.  
 
Exclusion Criteria  
 Any local or systemic factor that would influence the safety of nasal sampling. 
 Bilateral indwelling nasal catheters or local nasal pathology preventing access 
for nasal sampling. 
 Bleeding disorders. 
 The baby is taking part in another interventional study. 
 The parents or guardians not able to sign the informed consent from due to 
limited English or comprehension despite the use of independent interpreter 
services.  
 Limited life expectancy of the baby, 
 
Group 3 
 
Inclusion criteria  
 Hospitalised Infants admitted to the PICU at St. Mary’s Hospital, aged 2 weeks-24 
months with documented RSV infection (by rapid test and/or PCR). 
 Infants of weight >2kg. 
 On a conventional ventilator with an Endotracheal Tube (ETT) of >2.0mmm diameter 
 

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Exclusion criteria  
 Any local or systemic factor that would influence the safety of nasal sampling. 
 Bilateral nasal catheters or local nasal pathology preventing access for nasal 
sampling. 
 The baby is taking part in another interventional study. 
 Prematurity - corrected gestational age <36 weeks, weight <2kg 
 Significant hypoxia or instability precluding ventilator disconnection 
 Transcutaneous oxygen saturation of <95% on 60% oxygen  
 Risk of bleeding  
 Pneumothorax 
 Infants receiving oral corticosteroid therapy at any time in past month 
 Parents or guardians not able to sign informed consent from due to limited 
English or understanding despite the use of independent interpreter services.  
 Limited life expectancy or a decision to limit management. 
 NB – in the event of an exclusion to carrying out bronchial aspirate, (i.e: Significant 
hypoxia or instability precluding ventilator disconnection 
 Transcutaneous oxygen saturation of <95% on 60% oxygen  
 Risk of bleeding  
 Pneumothorax); 
As long as there is no contra-indication to carrying out NPA or nasal SAM sampling, then 
these children can still be included in the study, but will not require bronchial sampling. 
 
 
 
 
Control Group 1 
Inclusion criteria 

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 
Babies, aged 2 weeks-24 months, attending routine outpatient 
appointments or undergoing elective surgical procedures. 
Exclusion criteria 
 
Any respiratory symptoms 
 
All other exclusion are the same as Groups 1 and 2 
 
 
Control Group 2 
 
Inclusion criteria 
 
 Infants aged 2 weeks-24 months.  
 Infants ventilated on the PICU for any condition  
 Confirmed RSV negative by PCR of respiratory tract samples 
 
Exclusion Criteria 
 All exclusions are the same as Group 3 
 In addition – any concern about raised intracranial pressure 
 
 

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DATA STORAGE 
 
Anonymised data will be stored in Redcap in the Department of Paediatrics at Imperial 
College London. The database will be password protected and will be web-based. Only 
those directly involved in the study will be able to access the data. Individuals will only 
be identified on the database by their unique alphanumeric identifier; data relating to 
the subjects’ names to this identifier will be stored separately. Any data generated may 
be stored indefinitely and shared with other researchers outside of the participating 
sites. This will be done through databases such as ENA and EGA. Any data made 
available on these databases will be strictly confidential.  
 
DATA ANALYSIS 
 
Levels of inflammatory mediators (e.g. cytokines as pg/ml) across the patient groups 
will be presented as: 
 Raw data: linear and log plots as appropriate 
 Box-whisker plots: median/quartile/range 
 Heat maps to show correlation between levels of mediators 
 
Statistical analysis will be performed to demonstrate significance of levels of individual 
biomarkers and molecular signatures. 
Data and all appropriate documentation will be stored for a minimum of 10 years after 
the completion of the study, including the follow-up period.   
 
WITHDRAWAL CRITERIA 
 
Any clinically significant side effects during procedures or withdrawal of consent by the 
parents or guardians of participants.  
 
 
 

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ADVERSE EVENTS 
DEFINITIONS   
 
Adverse Event (AE): any untoward medical occurrence in a patient or clinical study 
subject.   
 
 
 
Serious Adverse Event (SAE): any untoward and unexpected medical occurrence or 
effect that: 
 
Results in death 
 
Is life-threatening – refers to an event in which the subject was at risk of 
death at the time of the event; it does not refer to an event which 
hypothetically might have caused death if it were more severe 
 
 
Requires hospitalisation, or prolongation of existing inpatients’ 
hospitalisation 
 
Results in persistent or significant disability or incapacity 
 
Is a congenital anomaly or birth defect 
 
Medical judgement should be exercised in deciding whether an AE is serious in other 
situations.  Important AEs that are not immediately life-threatening or do not result in 
death or hospitalisation but may jeopardise the subject or may require intervention to 
prevent one of the other outcomes listed in the definition above, should also be 
considered serious. 
REPORTING PROCEDURES 
 
All adverse events should be reported.  Depending on the nature of the event the 
reporting procedures below should be followed.  Any questions concerning adverse 
event reporting should be directed to the Chief Investigator in the first instance.   
Non serious AEs 
All such events, whether expected or not, should be recorded.  
Serious AEs 
An SAE form should be completed and faxed to the Chief Investigator within 24 hours.  
However, relapse and death due to hospitalisations for elective treatment of a pre-
existing condition do not need reporting as SAEs. 
 
All SAEs should be reported to the REC where in the opinion of the Chief Investigator, 
the event was: 
 
‘related’, ie resulted from the administration of any of the research 
procedures; and 
 
‘unexpected’, ie an event that is not listed in the protocol as an expected 
occurrence 
 

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Reports of related and unexpected SAEs should be submitted within 15 days of the Chief 
Investigator becoming aware of the event, using the NRES SAE form for non-IMP 
studies.  The Chief Investigator must also notify the Sponsor of all SAEs. 
 
Local investigators should report any SAEs as required by their Local Research Ethics 
Committee, Sponsor and/or Research & Development Office. 
 
Contact details for reporting SAEs 
Fax: 020 331 25750, attention Dr Simon Nadel  
Please send SAE forms to:  
St, Mary’s Hospital, 
7th Floor, Queen Elizabeth the Queen Mother Building, 
Paddington 
London 
W2 1NY 
Tel: 020 78866077 (Mon to Fri 09.00 – 17.00) 
 
ASSESSMENT AND FOLLOW-UP 
When sampling is well tolerated there will not be follow up of patients, and the patients 
will not generally be informed of the result of analyses.  
 
REGULATORY ISSUES 
 
ETHICS APPROVAL 
 
The Chief Investigator has obtained approval from the West Midlands – The Black 
Country Research Ethics Committee.  The study must be submitted for Site Specific 
Assessment at each participating NHS Trust.  The Chief Investigator will require a copy 
of the Trust R&D approval letter before accepting participants into the study.  The study 
will be conducted in accordance with the recommendations for physicians involved in 
research on human subjects adopted by the 18th World Medical Assembly, Helsinki 
1964 and later revisions. 

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CONSENT  
Consent to enter the study must be sought from each participant’s parents or guardians 
only after a full explanation has been given, an information leaflet offered and time 
allowed for consideration.  Signed consent should be obtained.  The right of the parent 
or guardian to refuse to participate without giving reasons must be respected.  After the 
participant has entered the study the clinician remains free to give alternative 
treatment to that specified in the protocol at any stage if he/she feels it is in the 
participant’s best interest, but the reasons for doing so should be recorded.  In these 
cases the participants remain within the study for the purposes of follow-up and data 
analysis.  All parents and guardians are free to withdraw at any time from the protocol 
treatment without giving reasons and without prejudicing further treatment. 
 
CONFIDENTIALITY 
The Chief Investigator will preserve the confidentiality of participants taking part in the 
study and is registered under the Data Protection Act. 
 
INDEMNITY 
Imperial College Healthcare NHS Trust London holds negligent harm and non-negligent 
harm insurance policies which apply to this study. Imperial College Healthcare NHS 
Trust holds standard NHS Hospital Indemnity and insurance cover with NHS Litigation 
Authority for NHS Trusts in England, which apply to this study. 
 
SPONSOR 
Imperial College Healthcare NHS Trust will act as the main Sponsor for this study.  
Delegated responsibilities will be assigned to the NHS trusts taking part in this study.   
 
PARTICIPANT REIMBURSMENT 
There will be no reimbursement for participating in this study as samples are taken 
opportunistically. 
 FUNDING 
This study is funded by Pulmocide. 
 AUDITS  
The study may be subject to inspection and audit by Imperial College Healthcare NHS 
Trust London under their remit as sponsor and other regulatory bodies to ensure 

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adherence to GCP and the NHS Research Governance Framework for Health and Social 
Care (2nd edition).  
 
 
STUDY MANAGEMENT 
 
The day-to-day management of the study will be co-ordinated through Dr. Simon Nadel, 
consultant paediatrician and intensivist.   
 
PUBLICATION POLICY 
 
Our aim is to ensure the full dissemination of all study results in medical publications of 
the highest impact and citation factor. Individual subjects will be strictly anonymised. 
 
 
 
 
 

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