Life expectancy in Cystic Fibrosis (CF) has improved dramatically in the last few decades; this is very much due to the emergence of disease-modifying treatments, optimisation of nutritional status and the inception of specialist CF units. However, progressive obstructive lung disease characterised by chronic inflammation, bacterial colonisation and recurrent infections of the lung, resulting in irreversible pulmonary damage, remains the major cause of mortality in individuals with CF. Historically, bacterial infections are the major pathogens accounting for clinical deterioration in CF. More recently, there has been emerging evidence to support respiratory viruses being accountable for the colonisation of bacteria and progression of lung disease in CF. This chapter sought to provide an overview on the impact of respiratory viruses in CF lung disease, the interaction between viruses and bacteria, the preventative and therapeutic measures that are currently available for the management of viral lung disease in CF.
- Cystic fibrosis
- respiratory virus
- Pseudomonas aeruginosa
Cystic Fibrosis (CF) is the most commonly inherited potentially lethal disease amongst the Caucasian ancestry. The prevalence of CF is reported as 0.737 per 10,000 in 27 European Union countries . The United States (US) Cystic Fibrosis Patient Registry reports a similar prevalence of 0.797 CF patients per 10,000 people . It is an autosomal recessive disease and is caused by mutations in the Cystic Fibrosis Transmembrane Conductance Regulator gene (CFTR) . The most common mutation is caused by deletion of phenylalanine at position 508 (Delta F508) of the CFTR on chromosome 7, which accounts for approximately 70% of CF cases. The primary function of CFTR in many tissues is to regulate and participate in the transport of chloride ions across epithelial cell membranes. To date, more than 1,900 mutations have been described in this gene.
CF is a multisystem disease as CFTR is expressed in different organs ; however, the lungs are the predominant organs that bear the brunt of the disease . Recurrent pulmonary infections may start at very early stages in the lives of patients with CF. It has been hypothesised that low airway surface liquid volume and impaired mucociliary clearance are responsible for the pathogenesis of lung infections. These in turn lead to impaired bacterial clearance from respiratory epithelial cells . Pulmonary infections remain the greatest cause of poor life quality, morbidity and mortality in CF that eventually lead to premature death in this condition .
Apart from chronic lung disease with recurrent exacerbations, exocrine pancreatic insufficiency is also a feature that leads to malabsorption and subsequently growth retardation and maturation. Endocrine pancreatic insufficiency is another feature of CF with the manifestation of diabetes. Obstructive azoospermia in male CF patients leads to male infertility.
The median survival from CF has taken great strides over the past 40 years as a consequence of the introduction of specialist centre care, nutritional optimisation, prevention and aggressive management of pulmonary exacerbations . In the United Kingdom (UK) CF population in 2012, the median survival was reported as 43.5 years, compared to 38.8 years for the population in 2008 as per the UK CF Registry . It has been postulated that the continuing improvement in survival of CF patients in successive cohorts means that the previous prediction of patients with CF living beyond a median age of 50 years is not impossible. The recent introduction of Ivacaftor to the management of CF patients with G551D CFTR mutations may further enhance the overall survival .
Historically, bacteria have been the predominant cause for respiratory exacerbations. The presences of some organisms including
In the last 30 years, there have been a number of published studies depicting the impact of respiratory viruses in CF. A number of studies have also demonstrated the relationship between respiratory viruses and bacteria in the pathogenesis of CF exacerbations [15, 32]. The introduction of molecular diagnostic technologies has further enhanced the awareness of respiratory viral aetiology in CF exacerbations as they have much higher detection rates than traditional methods. However, further understanding is required to appreciate their relationship in order to allow the development of potential novel treatment. If indeed respiratory virus does lead to secondary bacterial infection in CF, viral vaccinations and anti-viral therapies would be important therapeutic options for CF. On the other hand, the currently commercially available vaccines and anti-virals for the prevention and treatment of respiratory viral infections are limited; they are primarily for influenza infection. The potential development of new vaccines and anti-virals is an exciting field which may offer alternate therapeutic opportunities for CF exacerbations.
This chapter will focus on the literature regarding respiratory viruses in CF and their clinical implications, the detection techniques for viruses and their differences in sensitivities, the interaction between viruses and bacteria, and the management of viral infections.
2. Viral respiratory infections in CF
Early studies looking at respiratory viruses in CF relied on repeated serological testing, either alone  or in combination with viral cultures for viral detection [21-25]. These methods are relatively insensitive and more recent studies have utilised molecular-based methodologies [18, 26-28, 33-36]. All these studies produced different results in terms of prevalence of respiratory viruses in CF. The differences can be due to different methodologies, different sampling methods; the differences can also be accountable by different populations studied as the prognosis for CF has improved with each successive birth cohort.
Wang et al.  described the relationship between respiratory viral infections and deterioration in clinical status in CF almost 30 years ago. In this 2- year prospective study , viruses were identified through serology and nasal lavage in 49 patients with CF (mean age 13.7 years) on a quarterly basis and at the onset of exacerbations. Although the CF patients had more respiratory illnesses than sibling controls (3.7 versus 1.7/year), there were no differences in virus identification rates (1.7/year). The rate of proven virus infection was significantly correlated with the decline in lung functions, nutritional status, radiology score, and frequency and duration of hospitalisation.
More recent studies suggest no difference in the frequency of either upper respiratory tract illness (URTI) episodes  or proven respiratory viral infections  between children with CF and healthy controls, but children with CF have significantly more episodes of lower airway symptoms than controls [22, 24]. Ramsey et al.  prospectively compared the incidence and effect of viral infections on pulmonary function and clinical scores in 15 school-age patients with CF aged between 5 and 21 years and their healthy siblings. Over a 2-year period, samples were taken at regular two monthly intervals and during acute respiratory illnesses (ARI) for pharyngeal culture and serology for respiratory viruses. There was a total of 68 ARI episodes that occurred in the patients with CF and in 19 episodes there was an associated virus identified. A total of 49 infective agents were identified either during ARIs or at routine testing in the patients with CF; 14 were identified on viral isolation (
Likewise, Hiatt  assessed respiratory viral infections over three winters in 22 infants less than 2 years of age with CF (30 patient seasons), and 27 age-matched controls (28 patient seasons). The average number of acute respiratory illness per winter was the same in the control and CF groups (5.0 vs. 5.0). However, only 4 of the 28 control infants had lower respiratory tract symptoms in association with the respiratory tract illness, compared with 13 out of the 30 infants with CF (Odd ratio – 4.6; 95% confidence interval 1.3 and 16.5; p-value <0.05); 7 of the infants with CF cultured
From previous reports, two viral agents appear to have the greatest effect on respiratory status in CF, namely
In older children and adults with CF,
Over a 1-year period, Smyth et al.  prospectively investigated 108 patients with CF (mean age of 7.9 years) using a combination of viral immunofluorescence, culture and seroconversion to identify respiratory viruses. With the exception of
Collinson et al.  followed 48 children with CF over a 15-month period using viral cultures for viral detection, with the exception of
Punch et al.  used a multiplex reverse transcriptase PCR (RT-PCR) assay combined with an enzyme-linked amplicon hybridization assay (ELAHA) for the identification of seven common respiratory viruses in the sputum of 38 CF patients; 53 sputum samples were collected over 2 seasons and 12 (23%) samples from 12 patients were positive for a respiratory virus (4 for
Olesen and colleagues  obtained sputum and laryngeal aspirates from children with CF over a 12-month period in outpatient clinics. They achieved a viral detection rate of 16%, with
Our group in 2004  utilised ‘real-time’ multiplex Nucleic Acid Sequenced Based Amplification to examine the role of respiratory viruses in CF children. Over an 18-month period, a viral detection rate of 46% was achieved during reported episodes of respiratory illness. The results compared favourably with previous studies and it may be that earlier studies relied heavily on repeated serological testing, either alone  or in combination with viral isolation [21-25]. The viral detection rate was 18.3% from routine nasal samples. However, this was comparable to the seroconversion rate of 12.3% as reported by Wang et al. . Ramsey and colleagues  also achieved a similar seroconversion rate of 16.2% from asymptomatic samples. These results suggest that a laboratory method with a higher sensitivity for viral detection does not increase the detection rate in asymptomatic samples, implying that false positives are not necessarily more common than less sensitive diagnostic methods.
Asner et al.  performed an observational cross-sectional study of CF children from a large paediatric referral centre investigating the association between respiratory viruses and pulmonary exacerbations by taking mid-turbinate swabs, sputum and throat swab samples that were tested by a direct immunofluorescent antibody assay and a multiplex PCR panel. Forty-three patients were recruited into the study. Pulmonary function tests, quality of life and severity scores were recorded. Sputum cell counts, bacterial density and cytokines were measured. Twenty-six (60.5%) subjects were tested positive for at least one respiratory virus by any diagnostic method applied to any sample type. Of the 26 virus positive subjects, 17 (65.4%) were positive for one virus and the remaining 9 (34.6%) were positive for two or more viruses.
A CF centre in Milan (Italy) led by Esposito and colleagues  showed that human
In 2009, a novel swine pandemic
In contrast, the data regarding respiratory viral infection in adults is sparse. An observational study conducted by Hoek et al.  over a 1-year period amongst adult CF patients yielded a viral isolation rate of 33% [8/24] utilising molecular techniques and conventional methods. Etherington and colleagues  from an adult CF centre published a retrospective case control study looking at the prevalence of respiratory viruses during exacerbations. Viral throat swabs were taken from all patients presenting with an acute pulmonary exacerbation requiring intravenous antibiotic treatment over a 12-month period. Viral isolation was performed by PCR. There were 432 pulmonary exacerbations in 180 adults. In total, there was a total positive viral isolation in 42 exacerbations indicating a prevalence of 9.7%.
Flight et al.  followed up 100 adult CF patients prospectively for 12 months. Sputum, nose swabs and throat swabs were collected every 2 months and at the onset of pulmonary exacerbation for virus detection. PCR assays for
Experimental data on the effects of viral infections in CF are limited. Toll-like receptors (TLRs) have recently been identified as key mediators of the innate response and they recognise pathogens through detection of conserved microbial structures that are absent from the host. Kurt-Jones et al.  found that
Some studies have suggested a higher viral replication when there is an impairment of the innate host defence in CF.
Xu et al.  showed that CF cells that were infected with influenza A had less IFN-related antiviral gene induction at 24 h but more significant inflammatory cytokine gene induction at 1 h after infection. Therefore, the lesser antiviral and greater early inflammatory response may explain the severe respiratory illness of CF patients with viral infections. Sutanto and co-workers  showed that CF airway epithelial cells had a marked increase in IL-8 production, a reduction in apoptosis and an increased viral replication compared with airway epithelial cells from healthy children following exposure to human rhinovirus. This is despite the fact that CF and healthy airway epithelial cells have similar basal and stimulated expression of IL-8 in response to pro-inflammatory stimuli. The increment of IL-8, together with a reduction of apoptotic responses by CF cells to human rhinovirus, could contribute to augmented airway inflammation in the setting of recurrent viral infections early in life.
Azithromycin has previously been shown to offer anti-rhinoviral activity in bronchial epithelial cells and, during rhinovirus infection by increasing the production of interferon-stimulated genes . However, the role of anti-viral properties of Azithromycin in CF is not clearly defined. Schögler et al.  showed that primary bronchial epithelial cells from CF children that were pre-treated with Azithromycin had a seven-fold reduction in rhinovirus replication without inducing cell death. Azithromycin also increased RV-induced pattern recognition receptor, IFN and IFN-stimulated gene mRNA levels when measured by real-time quantitative PCR. Therefore, it is likely that Azithromycin pre-treatment reduces RV replication in CF bronchial epithelial cells, possibly through the amplification of the antiviral response mediated by the IFN pathway.
3. Detection of respiratory viruses
The diagnostic accuracy and sensitivity of respiratory viral detection is determined by several factors:
Appropriate respiratory specimen for testing – Nasal swabs, nasopharyngeal aspirates, nasal swabs and mid-turbinate sampling are reasonable sampling methods in young children who may not be able to expectorate. However, in older patients, sputum is easy to obtain, painless and quick. Bronchoalveolar lavage (BAL) is a useful intervention to obtain specimen in the distal airways but it is more invasive. However, it can provide useful information regarding the activities of respiratory viruses and bacteria in the distal airways.
Appropriate specimen transport method – There are recognised procedures for transporting clinical specimens for diagnostic virology testing. These procedures should be adhered to closely to enhance the chances of isolating the viral organism. For instance, nasal swabs should be transported in viral transport medium and all specimens should be refrigerated if there is a delay of more than 2 hours in reaching the laboratory.
Detection methods – Molecular techniques have superseded many conventional methods such as viral culture and serology as they are far more sensitive and specific; in addition, they have a more rapid turn-around, allowing diagnostic technology to have an immediate impact on clinical management. The advantages of such technology will allow the appropriate utilisation of anti-virals, many of which are virus-specific; it may play a role in complying with hospital infection control policy; finally, it can provide useful information to public health authorities such that public health policies can be adjusted accordingly, e.g. the outbreak of SARS and influenza H5N1 virus.
Utilisation of real-time multiplex amplification technique allows multiple viruses being quantified even if the copy number of the viral target is low.
More recently, Virochip has been shown to be a pan-virus microarray platform that is capable of detection of known as well as novel viruses in a single assay simultaneously . Probes chosen for Virochip can identify nodes in the viral taxonomy at the family, genus and species levels. As the Virochip probes are updated regularly, the extent of probes that can be covered are ever increasing, up to 36,000. It has a diagnostic sensitivity comparable to PCR for detecting respiratory genomes at levels as low as 100 genome copies. At the present time, Virochip is very much a research tool, and several issues must be addressed before it can be used as a routine test for virus detection in the clinical setting, including cost, diagnostic accuracy, repeatability, and sensitivity/specificity for virus detection. In addition, the clinical implication of novel viruses in the human respiratory tract is not yet defined. Therefore, the accurate interpretation of Virochip in the clinical setting remains a formidable task. For example, where specimens are polymicrobial or viral material are present at low levels, clinical and epidemiological information might be required to draw clinically meaningful conclusions.
4. Interaction between respiratory viruses and bacteria
In a 25-year retrospective review from the Danish CF clinic, the first isolation of
An increase in immunoglobulin A (IgA) antibodies to the O-antigen of
The first bacterial isolation of a given organism in CF has also been shown to often follow a viral infection. In the 17-month prospective study reported by Collinson et al. , 5 of the 6 first isolations of
Armstrong and colleagues have reported that 50% of CF respiratory exacerbations requiring hospitalisation are associated with isolation of a respiratory virus . In their prospective study of repeated BAL in infants over a 5-year period, a respiratory virus was identified in 52% of infants hospitalised for a respiratory exacerbation, most commonly
Respiratory viruses can disrupt the airway epithelium and precipitate bacterial adherence.
Kim et al.  found that invariant natural killer T cells induce a type of macrophage activation driving the secretion of interleukin-13 leading to the production of globlet cell metaplasia and airway hyperactivity following infection with Sendai virus. The term ‘invariant’ stems from the fact that all invariant natural killer T cells in humans and mice use a unique T cell receptor that is essential for interaction with CD1d. CD1d molecules present lipid antigens to T lymphocytes rather than peptide antigens as in the case of major histocompatibility complex (MHC) class I and II molecules. Historically, MHC class II dependent CD4 and T lymphocytes, through their response to stimulation by environmental allergens, are keys to the pathogenesis of human asthma. The findings by the authors lead to the notion of the use of anti-interleukin-13 therapy as a potential therapy in patients.
Viral infections might predispose to secondary bacterial infections by impairing mucociliary function and triggering host inflammatory receptors [68, 69]. This phenomenon has been demonstrated both in vivo and in vitro [70, 71]. Avadhanula et al.  showed that different respiratory viruses use different mechanisms to enhance the adherence of bacteria to respiratory epithelial cells. In particular,
Mechanisms independent of the expression of conventional receptors for bacteria, such as binding to viral proteins, could be responsible for enhanced adhesion . Immunofluorescence microscopy demonstrates that bacteria binding to
The lower respiratory tract is protected by local mucociliary mechanisms that involve the integration of the ciliated epithelium, periciliary fluid and mucus. Mucus acts as a physical and chemical barrier onto which particles and organisms adhere. Cilia lining the respiratory tract propel the overlying mucus to the oropharynx where it is either swallowed or expectorated.
De Vrankrijker et al.  showed that mice that were co-infected with
Another study also showed that
Stark et al.  showed that mice that were exposed to
More recently, Chattoraj et al.  demonstrated that acute infection of primary CF airway epithelial cells with rhinovirus liberates planktonic bacteria from biofilm. Superinfection with
Contrary to the above reports, Chin et al.  performed a prospective study over a 2-year period on 35 adult CF patients.
Contrary to the above findings, Asner et al.  found the mean total bacterial density in sputum samples in virus-positive patients being two logs lower than that found in virus-negative patients (p=0.299). However, this could be explained by the fact that the median age of the virus-positive group was significantly lower than the virus-negative group. Virus-positive and virus-negative patients had similar IL-8, neutrophil percentage and neutrophil elastase levels.
Similarly, Kieninger et al.  performed a comprehensive investigation of the inflammatory response of CF airway epithelial cells on virus infection. Strong cytokine production was found in all cells studied, with the magnitude and type of inflammation differing depending on cell type and virus used. There was no exaggerated inflammatory response in CF, either during cytokine production or at the transcriptional level. Instead, there was a trend towards lower cytokine production in CF airway epithelial cells after virus infection, which was associated with increased cell death. The lower inflammatory response in CF can also be explained by additional pathophysiological mechanisms, such as interactions between anti-viral and pro-inflammatory pathways, which are likely to be involved . It could also be speculated that because of chronic activation of pro-inflammatory pathways, CF airway epithelial cells are not able to respond sufficiently to further stimuli, such as virus infections. This might, in turn, lead to a lack of recruitment of effector immune cells resulting in longer duration and more severe respiratory symptoms.
TLRs are key mediators of type I interferon (IFN) during viral infections by recognizing various viral components. TLR7 and TLR9 have become apparent as universally important in inducing type I IFN during infection with most viruses, particularly by plasmacytoid dendritic cells . New intracellular viral pattern recognition receptors leading to type I IFN production have been identified. CFTR mutations have been shown to affect the epithelial induction of type I IFN expression by airway cells in response to
Taken together, these findings suggest conflicting data regarding the inflammatory response of the CF airway epithelium on virus infection and to some extent the symbiotic relationship between viruses and bacteria. Nonetheless, respiratory viruses may lead to epithelial disruption, increase neutrophil influx, inhibition of macrophage phagocytosis, destruction of mucociliary escalator, down-regulation of cilia beat, liberation of pro-inflammatory planktonic
5. Prevention and treatment for respiratory viruses
The diversity of viral serotypes in causing infection has made vaccine preparation very difficult. Frequent mutations of viral proteins of RNA viruses (e.g. genetic drift and shift of
In the UK, it has been reported that 2,150 deaths during the 2011/12 season was attributable to influenza , though some of the deaths may be attributed to
Recent vaccines contain antigens of two
Our group  recently showed that
Due to the lack of randomised controlled studies looking at the efficacy of
The development of an
There is currently no licensed
The conventional methods of vaccination are via the intramuscular and subcutaneous routes. Mucosal immunisation has recently been introduced as it represents an attractive manner of delivering vaccines. It is fast, simple, non-invasive and can be carried out by unskilled individuals. The use of mucosal vaccination seems logical in that most of respiratory viral infections initially start at the mucosal sites and therefore induce local immunity. In the autumn/winter of 2014/15 the annual nasal spray flu vaccine (Fluenz Tetra) became available for children aged 2, 3 and 4 year as part of the UK NHS childhood vaccination programme. The nasal spray flu vaccine is also for children aged 2-18 years who are “at risk” from flu, such as children with long-term health conditions.
Amantadine has been the conventional anti-viral against
Ribavarin, a synthetic guanosine nucleoside that has a broad spectrum of anti-viral activity, has been used for treatment of infections related to
Recently, an anti-rhinoviral agent known as Plecoranil, which acts by inhibiting the uncoating of Picornaviruses , the RV 3C protease inhibitor, Ruprintrivir and soluble ICAM-1, Tremacamra have shown promising results in early-stage clinical trials, but each of these medications was derailed by a combination of cost, pharmacokinetics, toxicity, drug interactions, and limited efficacy .
A previous study suggests that the increased morbidity in CF patients after virus infection is not due to an exaggerated inflammatory response of the airway epithelium but rather linked to increased cell death. Thus, they provide a rationale for implementing therapies aimed at controlling viruses and their replication rather than primarily targeting inflammation. In this respect, a promising candidate is the macrolide-antibiotic azithromycin, which is increasingly used in CF patients as a beneficial immunomodulatory agent  and has recently been shown to possess anti-viral properties .
As we become increasingly knowledgeable about the impact of respiratory virus infections in the context of CF exacerbations, screening for respiratory viruses should be part of the routine investigations for any CF patients that present with exacerbation symptoms. Using the appropriate sampling method in conjunction with sensitive and specific diagnostic technology will enable us to make appropriate clinical decisions surrounding the use of anti-virals and antibiotics.
Gaining further understanding in the pathogenesis of virus-induced respiratory exacerbations in CF may allow the development of new therapeutic techniques. If viral infection does predispose to bacterial infection, then influencing the interaction between viruses and bacteria could be a next pathway to diminish respiratory morbidity in patients with CF. The development of novel therapies will be exciting and this may improve their quality of life and prolong the lifespan of patients with CF.
However, there are still a number of research dilemmas that remain unanswered:
What are the standardised definitions of CF pulmonary exacerbation and pulmonary exacerbation severity score?
What is the optimal way for viral sampling?
What is the role of Virochip in routine viral identification?
How do respiratory viruses influence bacterial activities in chronically infected airways?
What influences the rate of respiratory viral clearance in CF respiratory tract?
What are the roles of anti-virals in CF?
What are the anti-viral properties of Azithromycin in CF?
Further understanding in the pathogenesis of viral infection in CF would be beneficial as this may provide insight to the above unresolved mysteries. At the moment,