Pulmonary infection in cystic fibrosis

Journal of Infection (1986) I3, 55-72

REVIEW P u l m o n a r y i n f e c t i o n in c y s t i c f i b r o s i s Peter A. Friend

Clinical Microbiology and Public Health Laboratory, Addenbrooke's Hospital, Cambridge CB2 2QW Accepted for publication II March 1986 Introduction Cystic fibrosis (CF) is the commonest lethal genetic disorder among Caucasians, with an incidence of approximately I in 25oo live births in the U.K. and a carrier frequency estimated as I in 20. T h e search for the basic biochemical defect, t h o u g h t to lie at a single genetic locus, by techniques such as gene probe analysis is proceeding steadily. T h e r e is now a prospect of i m m i n e n t breakthrough following the recent location of a C F gene on chromosome 7 .1-a Identification of the defect may provide the best hope for accurate detection of carriers and treatment of sufferers, t h o u g h similar knowledge has not yet improved therapy for other single gene defects such as sickle-cell anaemia or T a y - S a c h s disease. T h e typical pathological features of C F are apparent in the pancreas as early as 38 weeks of gestation. 4 T h e resultant pancreatic abnormality allows the possibility of screening during the first weeks of life by an immunoreactive trypsin t e c h n i q u e ) For those who survive the neonatal period, progressive lung disease is the most dire complication and the major cause of death. T h e r e are good reasons to expect more effective therapy for pulmonary disease in the future. T h e lungs are histologically normal at birth, 6 with some patients suffering m i n i m u m pulmonary disorder in adult life. 7 Life-expectancy has improved dramatically over the last few decades and, exceptionally in the state of Victoria, Australia, where affected infants have an 8o % chance of survival, to the age of 2o years. 8 Research into m e m b r a n e transport of ions across airway epithelium may lead to a fundamental correction of the abnormal mucous secretions encountered in CF. Very preliminary trials are under way using high doses of inhaled amiloride, a drug blocking sodium ion transport, although researchers recognise the relative youth of their science of airway ion transport. 9 Finally, there is an ever-increasing understanding of the microbial mechanisms which contribute to p u l m o n a r y deterioration. Coupled with the development of new vaccines and o p t i m u m use of conventional and newly developed antimicrobial agents, improved control of lung infections should be feasible.

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© I986 The British Society for the Study of Infection

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P . A . FRIEND Bacteria c o m m o n l y associated with lung disease in cystic fibrosis

T h e bacteria most consistently associated with lung disease in cystic fibrosis are Staphylococcus aureus, Pseudomonas aeruginosa and Haemophilus influenzae. In 1946, di Sant'Agnese and Andersen defined the destructive potential of S. aureus in patients with cystic fibrosis of the pancreas? ° In their report on 14 p o s t - m o r t e m examinations, S. aureus was the main organism cultured from the respiratory tract in I I cases. Haemophilus influenzae and P. aeruginosa were found in only single cases, each in conjunction with a predominant growth of S. aureus. Significantly, all but two of the cases involved patients less than I year of age. These findings set the scene for m u c h of the emphasis on S. aureus in subsequent antimicrobial therapy. Although there were no controlled data confirming the usefulness of such therapy, long-term antistaphylococcal prophylaxis became commonplace, especially in younger children, n-14 In 1979, however, the results of a small well-controlled trial involving 17 adults and children (median age 6"5 years) was published by Mischler and colleagues. 15 Patients served as their own controls by taking oral cephalexin or placebo in alternating 4 - m o n t h periods during a 2-year double-blind crossover study. T h e i r results indicated short-term benefit to patients during periods of cephalexin treatment, particularly in those initially colonised with S. aureus a n d / o r H. influenzae. Unfortunately the acquisition of mucoid strains of P. aeruginosa in six patients with n o n - m u c o i d P. aeruginosa suggest that the long-term effects of such therapy may be less advantageous. At the conclusion of a m u c h larger survey involving 16o patients over 15 years, Kulczycki and colleagues ~6 found a relationship between continuous antibiotic therapy and persistent P. aeruginosa infection, including the appearance of mucoid strains. In those who died, the persistent isolation of P. aeruginosa coincided with the apparent disappearance of S. aureus and other potentially pathogenic flora, as well as with the patient's own eventual clinical deterioration. This change in emphasis from S. aureus was confirmed by p o s t - m o r t e m studies. F r o m almost all C F patients dying from pulmonary complications P. aeruginosa was isolated, 90 % of strains being mucoid. Staphylococcus aureus was rarely recovered. Colonisation of the respiratory tract with P. aeruginosa is now encountered increasingly in many C F clinics t h r o u g h o u t the world. Isolation rates range from 17 % of patients attending E d i n b u r g h clinics (J. R. W. Govan, personal communication), 38% at the Leeds C F centre (M. Miller, personal communication) to 85-90% in parts of the U.S.A. ~7 In the T o r o n t o C F centre, where over 90 % of patients regularly received antibiotics both orally and as an aerosol prophylactically, between 197o and 1982, a rate of 6o-7o % has been reported. 18 Haemophilus influenzae is commonly isolated from sputum, often with other pathogens, during an acute exacerbation of respiratory tract disease. Strains are usually not capsulated and, although the pathogenic mechanisms are ill-understood, 19 a potential for pulmonary destruction is assumed. As in chronic bronchitis and bronchiectasis, bronchial damage and persistence may relate to an ability to destroy mucus and to adhere to epithelial cells or to direct toxic effects on ciliated respiratory tract epithelium. 2° If s u b - o p t i m u m doses

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of antibiotics are used in treating chronic bronchial sepsis, spheroplasts of H. influenzae are a potential reservoir for re-infection. 21 Antimicrobial agents effective against P. aeruginosa infection are often active against H. influenzae also and may reach lethal concentrations against the latter in respiratory secretions. 22 B a c t e r i a less o f t e n a s s o c i a t e d w i t h l u n g disease and the e m e r g e n c e o f

Pseudomonas cepacia Bacteria such as Streptococcus pneumoniae, Klebsiella spp. and other Enterobacteriaciae are not infrequently isolated from the respiratory tract of CF patients.23-26 A range of serological types of S. pneumoniae has been found, with only minor differences in type compared with strains from a control group of children. T h e presence of these strains seems to be of little overall importance in the natural history of lung disorder. Their colonisation of the lower respiratory tract often ceases without specific treatment, thereby suggesting that an effective host immune response accounts for their elimination. 2~ In a study of 72 patients with CF, 49 harboured various species of Enterobacteriaceae in the respiratory tract. 24 Colonisation by these bacteria without P. aeruginosa was seen more often in patients with mild disease. In those with severe disease, Enterobacteriaceae were always accompanied by P. aeruginosa. Their pathogenic potential was less clear, although a significant antibody response against Enterobacteriaceae was found more commonly in the latter group of patients. As well as characteristic mucoid strains of P. aeruginosa, mucoid Enterobacteriaceae have been observed in CF patients. TM 26 T h e physico-chemical nature of the mucoid substances is quite different, however, as referred to later in this review. Reports from the Toronto CF centre 27 point to the emergence of another pseudomonad with sinister potential, Pseudomonas cepacia. In patients with previously mild disease, the acquisition of P. cepacia has been associated with a fulminant and often fatal illness. Patients were typically female, illness being characterised by persistent high fever, leucocytosis and severe progressive respiratory failure, clinically and bacteriologically unresponsive to antibiotics. These features are less commonly seen in the course o f P . aeruginosa infection. T w o other clinical patterns were also observed: (I) chronic asymptomatic carriage of P. cepacia, either alone or in combination with P. aeruginosa; (2) progressive deterioration over many months with recurrent fever, progressive weight loss and repeated admission to hospital. T h e incidence of respiratory tract colonisation by P. cepacia in Toronto clinics rose to 18 % of patients in 198 I. Klinger and colleagues 17have reported a similar incidence of colonisation by P. cepacia in a large population of CF patients treated in Cleveland, Ohio, U.S.A. Pseudomonas aeruginosa continues to be isolated from 85-90 % of their patients. In recent years, however, the isolation rate of other non-fermentative Gram-negative bacilli has risen to 20 ~o. Various species have been encountered, including P. cepacia, Pseudomonas maltophilia, Pseudomonas fluorescens /putida and Achromobacter xylosoxidans. Compared to P. aeruginosa alone, colonisation by non-fermentative Gram-negative bacilli was associated with a poor clinical condition, a correlation that was particularly evident with P. cepacia. These

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organisms were often resistant to a wide range of antimicrobial agents, including the combination of ticarcillin and tobramycin as used in many centres to treat pulmonary exacerbations. Antibiotics with the greatest broadspectrum activity against the isolates were ceftazidime, thienamycin, and azthreonam. T h e new oxyquinolone derivatives were not investigated. In a separate study of a range of antimicrobials, including ceftazidime and thienamycin, ciprofloxacin was the most active in vitro against P. cepacia. 28 Ciprofloxacin also has excellent in vitro activity against other similar non-fermenting organisms. ~9 Epidemiological investigations of infections with P. cepacia have been hampered by the lack of a satisfactory typing system. Recently, however, a technique based on the production of and susceptibility to bacteriocins has been used successfully in epidemiological investigation. 3° It is possible that cross-infection was responsible for the increase in P. cepacia reported by Isles and colleagues, a7 Spirometers and other equipment in the pulmonary function laboratory were found to be contaminated. In the hospital environment there are many other potential sources and vehicles of transmission. T h e entire question of how C F patients become initially colonised and infected with various organisms, especially the potential role of nosocomial, domestic and environmental transmission, would benefit from re-examination. 81, 82 Viral and other non-bacterial infections

T h e precise roles of viral, mycoplasma, coxiella, and chlamydia (so called 'non-bacterial ') infections in the natural history of lung disease have yet to be determined. Of all p u l m o n a r y exacerbations in C F patients, 2o-3o ~o have been found to be related to infections of this nature, 83-36with cultural and serological evidence of infection by all the c o m m o n respiratory viruses. T h e r e is now little doubt that non-bacterial infections may contribute to a worsening of p u l m o n a r y status. Forty-six adult C F patients (age range i 6 - 4 i years) were studied over a period of 4 months by Efthimiou and colleagues. 36 Seroconversions to viruses, Mycoplasma pneumoniae and Coxiella burnettii were found in 29 % of a group of patients with pulmonary deterioration as compared to 4.5°//0 of a stable group. Initial pulmonary function was poorer in the deteriorated group suggesting that patients with more severe lung disease are most susceptible. Wang and colleagues 37prospectively surveyed 49 C F patients over 6 years of age and an age-matched control group of I9 normal siblings. T h e y found a similar incidence of respiratory viral infections in both groups. A very strong correlation emerged over the period of 2 years of the study between the frequency of viral infections and a decline in clinical status and pulmonary function. In contrast to the results of Efthimiou and colleagues, a relationship was not found between initial lung function and the subsequent frequency of these infections. Remarkably, a virus was not isolated from cultures of respiratory secretions; this remains unexplained. Speculation is possible on the pathogenic mechanisms operating in these circumstances. Damage could be due to direct effects of viral infection. Alternatively, some viruses, with their well-known ability to alter p u l m o n a r y bronchociliary defence mechanisms, may alter lower respiratory tract flora and

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intensify the host responses which contribute towards acute and chronic lung damage, as T h e r e may be synergy in viral-bacterial interaction. Petersen and colleagues a5 have found some evidence of this between respiratory syncitial virus and P. aeruginosa. Finally, Strunk and colleagues have found depressed concentrations of complement during respiratory viral infections in C F and suggest that damage may be caused by deposition of i m m u n e complexes in lung tissue29 Despite extensive investigation, some exacerbations of respiratory disease may not be associated with any of the c o m m o n bacteria or viruses. This was noted by Petersen and colleaguesfl 5 who found that one-fifth of exacerbations were unexplained and suggested that factors such as allergy may be operating. Infection by Legionella spp., rhinoviruses, coronaviruses and other viruses would also need to be excluded. Clinical evidence of lung disease during the first few m o n t h s of life in the absence of a bacterial or viral cause has been noted by Kuzemko. 4° He suggested that inflammatory changes in small airways during infancy may be caused by circulating pancreatic proteases and in turn predispose the lung to bacterial colonisation. Longitudinal studies are required to determine immediate and long-term effects of non-bacterial infections in C F and possible interactions with bacterial infection and colonisation. An assessment of these infections during infancy, and any relationship to the persistent bacterial endobronchitis characteristic of the disease, should be made feasible by the early detection of C F by screening procedures. 5 Miscellaneous micro-organisms and lung disease

Legionella pneumophilia T h e r e are three reports on Legionella pneumophilia infection in CF. T h e first, a single epidemiological observation, involved screening Io9 C F patients with stable disease in Philadelphia, U.S.A. 41 By means of a conventional indirect immunofluorescence test ( I F A T ) , 29"4% patients were found to have significantly raised antibody titres to L. pneumophilia (serogroup ~). Although unable to clarify the clinical importance of this finding, the authors c o m m e n t that a high proportion of those with demonstrable antibody had received aerosol therapy or had slept in mist tents. In the second study by Efthimiou and colleagues, a5 formolised yolk-sac antigen in an I F A T was used to detect antibody to L. pneumophilia (serogroups I-6) in 46 adult C F patients. Eight patients (I7"4%) had raised titres of antibody and all serogroups of L. pneumophilia except 3 and 5 were represented. Seven of the eight were in a 'deteriorated' group of patients. Infection by L. pneumophilia was strongly suspected, on both serological and clinical grounds, in three patients. In one case it was accompanied by respiratory illness responding to erythromycin. A relationship was not found between demonstrable antibody and the use of aerosol or corticosteroid therapy. T h e authors discussed alternative explanations for the presence of antibody to L. pneumophilia in patients with CF. These included an anamnestic reaction in the course of acute respiratory illness coupled with over-reactivity of the i m m u n e system as well as serological cross-reactivity between L. pneumophilia and P. aeruginosa. A case report of

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Legionnaires' disease in a patient with C F has been published by Witte and Primavessi. ~ A 5-year-old boy with severe multilobular pneumonia, associated with a significant rise in titre in the I F A T , was treated successfully with erythromycin. Mycobacteria Pulmonary mycobacterial infection is a rare but potentially serious complication of CF.ha, ~4 Smith and colleagues have recently reported on 16 years' experience at the B r o m p t o n Hospital, London. ks D u r i n g the first IO years there were no mycobacterial isolates. In the 6 years following the introduction of routine examination and culture of s p u t u m for acid-fast bacilli in patients with CF, 7 of 223 patients were found to be positive. T h e organisms isolated were Mycobacterium tuberculosis from three patients, unidentified mycobacteria from two patients, and M. cheloni and M. fortuitum in one case each. T h e authors r e c o m m e n d regular s p u t u m examinations for acid-fast bacilli and discuss significant problems in treatment as well as both the clinical and microbiological diagnosis.

Aspergillus fumigatus Serum precipitins and positive skin tests related to Aspergillusfumigatus have been found in up to 30% C F patients. 4~ This finding correlates with the severity of lung disease in adults, although the relationship is lost if the effect of pseudomonas colonisation is removed. T w o explanations for these immunological findings have been advanced. Antigens may gain ready access to p u l m o n a r y i m m u n e mechanisms via bronchial epithelium damaged by Pseudomonas spp. or the viscid secretions may cause 'antigen trapping'. T r u e invasive aspergillus infection, t h o u g h very rare, has been recorded. 47 Allergic bronchopulmonary aspergillosis requiring systemic corticosteroid therapy may also arise? 8 Changing characteristics of Pseudomonas aeruginosa following permanent colonisation M e m b e r s of the genus Pseudomonas are c o m m o n inhabitants of soil and vegetation as well as both fresh and salt water. Because of their metabolic versatility and inherent resistance to many antibiotics and disinfectants, certain species are often found in hospital environments. Pseudomonas aeruginosa is the species usually associated with nosocomial infection and exhibits the typical features of an opportunistic pathogen. As well as innocent colonisation of certain hospital patients, it causes severe life-threatening infection and septicaemia in persons with defective immunity. Isolates of P. aeruginosa from C F patients with chronic lung infection have been found to express unusual properties, absent or infrequent in routine hospital and environmental strains. These changes would appear to be the result of a long-term host-parasite interaction, P. aeruginosa adapting to a new ecological niche in damaged lungs. 'Cystic' strains may, therefore, have one or more of the following characteristics: (I) Production of alginate by mucoid strains. (2) Hypersusceptibility to certain antibiotics.

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(3) Loss of specific O-serogroup reaction. (4) Expression of polyagglutination antigen. (5) Susceptibility to normal h u m a n serum. Mucoid strains o f Pseudomonas aeruginosa and production o f alginate

T h e m u c o i d exopolysaccharide produced by strains of P. aeruginosa isolated from C F patients consists of alginate (a heteropolymer of m a n n u r o n i c and guluronic acids). Alginates are distinct from the exopolysaccharides of Klebsiella spp. and ' m u c o i d ' strains of Escherichia coli, as well as from the characteristic viscid slime produced in broth culture by all strains o f P . aeruginosa particularly when cultured in the presence of gluconate or in 'high carbon media'. 49,5° Alginates are remarkable gelling agents, rapidly forming stable gels in the presence of electrolytes such as ionic calcium. Their potent gelling and colloidal properties have led to commercial exploitation; e.g., in the manufacture of material for taking dental impressions, as an additive to various foodstuffs including table jelly, and for maintaining a good head on beer. T h e commercial source ofalginate is eucaryotic, namely the marine algae. A m o n g the procaryotes, only two bacterial genera, Azotobacter and Pseudomonas, are known to contain species capable of producing alginate. T h e production of copious amounts of alginate is characteristic of most strains of Azotobacter vinelandii isolated from their natural habitat of soil and water. It appears to be related to survival in the environment and to encystment. 5 By contrast, production of alginate by P. aeroginosa is extremely u n c o m m o n among strains from a wide variety of h u m a n , animal and environmental sources. T h e important exception to this rarity is the frequent emergence of alginate-producing P. aeruginosa in the lungs of C F patients. F u r t h e r m o r e , the ionic environment of pulmonary secretions would favour 'gelling' of this alginate into a protective matrix around microcolonies of the organism. 52 Doggett and colleagues 53, ~4 first provided evidence for the emergence of m u c o i d strains of P. aeruginosa in C F patients following initial colonisation by n o n - m u c o i d strains. T h e first report of such emergence in vitro was that of Martin, 55who noticed rings of slimy mucoid growth around areas of phage lysis during the course of routine phage typing. It remains to be determined whether a phage is responsible for selecting mucoid forms in vivo, although phages capable of inducing such a transformation have been isolated from strains of P. aeruginosa present in the lungs of C F patients. 56 A m e t h o d for isolating mucoid variants in vitro depending on selection by antibiotics was developed by Govan and Fyfe. ~v T h e i r m e t h o d was based on the observation that mucoid strains are slightly more resistant to certain antibiotics than isogenic n o n - m u c o i d forms, a difference not apparent on conventional M I C testing that uses doubling dilutions of antibiotics. With this technique, mucoid variants of P. aeruginosa were isolated at a frequency of approximately I in Io 7 with carbenicillin, flucloxacillin or tobramycin as the selective agent. A modified technique has been used by these authors to demonstrate alginate producing variants of P. putida, P. fluorescens and P. mendocina. 58 A similar potential in P. cepacia could not be demonstrated. Fyfe and Govan suggested that all wildtype P. aeruginosa have the necessary genetic information for alginate synthesis. T h e y later located two chromosomal loci involved in repression

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of the alginate biosynthetic pathway. 5° A spontaneous mutation in one of these, or other as yet undiscovered, regulator genes would give rise to the mucoid phenotype. A third possible mechanism by which mucoid strains emerge is related to the biochemical composition of sputum. It is possible that genes governing the synthesis of' slime polysaccharide ' by some Gram-negative bacteria may be regulated by the environment in which they g r o w ) 9 T h e secretions from the respiratory tract in C F are known to have a unique lipid, electrolyte and glycoprotein content. 6°,81 Furthermore, as well as alginate producing strains of P. aeruginosa, mucoid strains of E. coli and other Gram-negative bacilli have been isolated from the respiratory tract of C F patients.85, 86 Hypersusceptibility to antibiotics In 1973, May and Ingold 68 reported on the existence of P. aeruginosa exhibiting increased susceptibility to carbenicillin. In a study involving I I I isolates from the s p u t u m of patients with CF, chronic bronchitis and bronchiectasis, they found that 3 5 % of strains were susceptible to 6 mg/1 carbenicillin (some strains had M I C s as low as 0"7 mg/1). Irvin and colleagues 63 detected strains hypersusceptible to carbenicillin ( M I C < I mg/1) in 55 % of 22 C F patients studied. Increased susceptibility was found also to trimethoprim, tetracycline, and certain penicillins but not to the aminoglycosides. Fyfe and Govan 64 have located two chromosomal genes associated with the hypersusceptibility phenotype. T h e y proposed that strains, generally hypersusceptible to a range ofbeta-lactam antibiotics, could arise after a single-step chromosomal mutation and would then be favoured by the environment of the lung. T h e structural basis of this characteristic is u n k n o w n but several studies suggest that increased permeability of the cell-envelope is responsible. T h e clinical and biological significance of antibiotic hypersusceptibility remains to be elucidated. Superficially there is a paradox; hypersusceptible strains often arise in patients undergoing aggressive chemotherapy with beta-lactam antibiotics. T h e lung, however, is a notoriously difficult site in which to achieve therapeutic concentrations of antipseudomonal antibiotics. Increased permeability, inside a protective alginate matrix or microcolony, may provide organisms with a selective advantage over other strains in the competition for essential nutrients. Other phenotypic changes Loss of the O-serogroup reaction, expression of polyagglutination antigen and susceptibility to normal h u m a n serum are characteristics found in P. aeruginosa isolated from C F patients, but are u n c o m m o n in other hospital strains. These changes relate to alterations in the cell envelope and a deficiency in lipopolysaccharide O-side-chains has been demonstrated. 85 Penketh and colleagues 66 classified IO9 strains o f P . aeruginosa isolated from the s p u t u m of 49 adults with C F as' c o m m o n ' , ' intermediate' o r ' cystic ', the latter possessing all three characteristics, the first none of them. Clinical evaluation of these patients revealed that those colonised with 'intermediate' strains were significantly more severely affected by the disease and spent more time in hospital during the period of the study than those in the other two groups. T h e authors

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suggest that in chronic bronchopulmonary infections in CF caused by P. aeruginosa, environmental adaptation is taking place and is marked by changes in somatic antigens and susceptibility to serum. During this period of adaptation the condition of some patients may greatly deteriorate, although patients surviving the intermediate phase may remain in a relatively stable condition for some time. Antigenic change in the outer membrane of Pseudo° monas may render host defence mechanisms temporarily less effective and therefore, theoretically, treatment should be most vigorous when the organism is changing, allowing the host defence system time to establish a new equilibrium. Pathogenicity o f Pseudornonas aeruginosa in chronic lung infections

Chronic P. aeruginosa infection in CF may become established during infancy or early life. More commonly it is acquired after five years of age, the mean age of onset of infection in the Danish CF centre having been reported as 9 - I 0 years.e vBefore infection becomes chronic, most patients experience intermittent colonisation by non-mucoid strains which subsequently become mucoid and intractable to treatment. 68, 60 A mutational basis for alginate synthesis, leading to the emergence of mucoid forms, has been described. 5°, 51 Initial colonisation by non-mucoid strains may provide an essential microbial reservoir to allow expression of bacterial mutations which contribute to the survival of P. aeruginosa in the lower respiratory tract. T h e precise location of this reservoir is not yet known. T h e necessary population of P. aeruginosa may develop in the lung, oropharynx or maxillary sinuses. TM T h e microflora of the gut of CF patients has not been systematically studied. Decreased resistance to colonisation as a result of prolonged antibiotic therapy could lead to a primary source of P. aeruginosa in the gastro-intestinal tract. 71 Investigations by Woods and colleagues 72-74 seem to indicate that nonmucoid strains of P. aeruginosa may colonise by virtue of their preferential attachment to altered epithelial surfaces in the oropharynx of CF patients. Live buccal mucosal cells were used for in vitro studies of bacterial adherence. Reduced concentrations on the cell surface of fibronectin, a protein involved in maintaining normal bacterial colonisation, were found in CF patients (each of the CF patients studied harboured both mucoid and non-mucoid P. aeruginosa in their respiratory tracts). This was associated with an increase in salivary proteases, an attractive explanation for the decreased concentration of fibronectin. Adherence ofnon-mucoid strains to exposed receptors on epithelial cells may be mediated by their surface pili, a mechanism open to modification by specific immunisation. Similar methods have been employed by Govan and colleagues (personal communication) in a survey of patients attending the Edinburgh CF clinic. Although in vitro results were similar to those of Woods and colleagues, a reservoir o f P . aeruginosa attached to buccal cells in vivo was not observed. Increased adherence of P. aeruginosa to human tracheo-bronchial mucins, which may facilitate colonisation and persistence, has been described by Vishwanath and Ramphal. 75 Pier suggests that the sheer volume of extra mucus produced in CF may explain preferential adherence in this disease. 76 Ramphal

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and Pier 77 have recently shown that mucoid P. aeruginosa binds to acid-injured m u r i n e tracheal cells via alginate exopolysaccharide. Antibody to alginate, produced in significant amounts by C F patients colonised with mucoid P. aeruginosa, may not help to control or clear the infection. 76 Is there a place for an alginate vaccine, given before colonisation by P. aeruginosa? Woods and Bryan 78, using a rat model of chronic lung infection, have found both favourable and unfavourable effects of specific immunisation based on this approach. Pulmonary damage in C F patients infected with P. aeruginosa may result directly from exotoxins and proteases produced by this organism, indirectly from an over-exuberant i m m u n e response to its continued presence, or from a combination of these. 19, 79 It is beyond the scope of this article to discuss in detail the many unresolved questions encountered by those investigating these mechanisms. Interested readers are referred to other sources. 16, 76, 79-82 T h e ability o f P . aeruginosa to evade host defences by phenotypic change has already been mentioned. 66 In patients with chronic infection, a microcolony m o d e of existence may explain both i m m u n e and toxin mediated damage. T h e production of alginate, although unfavourable to P. aeruginosa growing under laboratory conditions, 83 would lead to the formation of a protective matrix in the ionic environment of pulmonary secretions. An i m m u n e response to alginate is n o w known.76 Presented with such a formidable target for phagocytosis a ' frustrated macrophage system' may arise. T h e prospect of damage to the lung mediated by the patients own, highly stimulated, host defences then becomes more plausible. Toxins, produced in reduced amounts in vitro by strains associated with chronic colonisation, 84 would be more easily localised to a target on bronchopulmonary tissues. Finally, bacterial elimination may be hindered by a reduced diffusion rate of antibiotics across an alginate matrix. 85 T h e paradoxical emergence of hypersusceptible strains has already been discussed. S p u t u m bacteriology in cystic fibrosis T h e true relationship between bacteria isolated from s p u t u m samples, and micro-organisms causing infection deep in bronchial or lung tissues, is not known. If pathogenicity is related to the n u m b e r of organisms invading the lower respiratory tract, bacterial counts in s p u t u m may parallel changes in the clinical state. Quantitative s p u t u m culture is a practice in many laboratories. Wong and colleagues s6 have recently described suitable procedures for samples obtained from C F patients. Following liquefaction with dithiothreitol and dilution to allow counting of colonies, five selective media, together with aerobic and anaerobic incubation, were used to facilitate isolation of the bacteria commonly present in the s p u t u m of C F patients (see also methods used by Roberts and Cole 21, 87for the isolation o f H . influenzae). Selective media have also been found to be essential for reliable isolation of t h y m i d i n e - d e p e n d e n t .S. aureus from patients who have received prior treatment with cotrimoxazole, ss Future studies may determine the value of quantitative s p u t u m culture in patients with CF.

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Control o f lung infections

Physical therapy remains a vital adjunct to the m a n a g e m e n t of pulmonary disorder in CF. In order to enhance the clearance of secretions from the lungs by physiotherapy, mucolytic agents such as N-acetylcysteine or mistabron may be administered as an aerosol. 89 Indeed, it is still argued that a prime cause of i m p r o v e m e n t in patients admitted to hospital during pulmonary exacerbations may be the institution of intensive physical treatment regimes. 9° O p t i m u m antimicrobial therapy for C F is not known. In conventional microbiological practice we become accustomed to the concept of eliminating pathogenic bacteria followed by eventual clinical cure. A similar situation does not exist in cystic fibrosis. D u r i n g infancy it may be possible to control p u l m o n a r y infection with one or two courses of intensive chemotherapy, resulting in the complete disappearance of respiratory pathogens for varying periods of time. P u l m o n a r y pathogens, however, reappear and become progressively more difficult to eradicate following further episodes of lung infection. A unique interaction between host and parasite is apparent. Despite heavy colonisation of the b r o n c h o p u l m o n a r y tree with bacteria such as S. aureus, bacteraemia is almost non-existent while p n e u m o n i a and empyema are extraordinarily rare? 1 T h e ultimate frustration is the eventual emergence of P. aeruginosa, a species so manifestly equipped to survive and adapt to adverse conditions. T h e r e are three main approaches to anti-staphylococcal chemotherapy. Staphylococcus aureus may still be involved in early p u l m o n a r y destruction, and despite clinically effective oral antibiotics, complete eradication is often difficult to achieve. 18,14.92T h i s emphasises the problem of antibiotic penetration into diseased lungs. 98, 94 W h e t h e r continuous regimes are more useful than intermittent treatment is not known. T h e study reported by Mischler and colleagues ~5 suggests that there may be some benefit from prophylaxis if courses of antibiotics based on results of s p u t u m culture are used. T h e long-term benefits of such therapy, however, are not yet clear. Encouraging results have been reported by the Danish C F centre in Copenhagen 95 after applying a consistent policy of anti-staphylococcal chemotherapy for I5 years. Whether clinical symptoms of infection were present or not, treatment was instituted following isolation of S. aureus from specimens in which there was microscopical evidence of lower respiratory-tract origin and of inflammation. A high rate of eradication of S. aureus was achieved with an intensive regime of intermittent chemotherapy; problems with resistant strains were not encountered. T h e authors concluded that S. aureus infection could be considered as a minor problem, unrelated to the poor prognosis of C F patients. T h e third approach to therapy is to treat only clinically proven episodes of S. aureus infection, a practice which may be associated with the low isolation rate of P. aeruginosa from patients in Edinburgh. ~2 T h e problems of antibiotic therapy are increased when P. aeruginosa becomes established. Intravenous therapy is usually required for acute pulmonary exacerbations and patients are often admitted to hospital. T o what extent subsequent recovery is due to improved physical treatment in hospital has not been established. T h e only placebo-controlled study examining this 3

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relationship compared intravenous tobramycin with placebo. T h e results obtained favoured the use of tobramycin. 96 Patients with more severe disease, however, were by chance allocated to the placebo group and this may have clouded final interpretation. T h e importance of specific therapy for P. aeruginosa was investigated by Beaudry and colleagues. 97 T w e n t y - t w o patients with advanced lung disease were randomly assigned treatment with either cloxacillin alone or gentamicin plus carbenicillin irrespective of bacteriological findings. N o difference in clinical outcome was found. In contrast to this finding, H o d s o n and colleagues 98 have demonstrated clinical benefit using an aerosol of carbenicillin plus gentamicin in a double-blind randomised cross-over trial in patients with P. aeruginosa infection. Encouraging results have also been obtained at the Danish C F centre with ' m a i n t e n a n c e ' antipseudomonal chemotherapy. 99 T h e i r policy of regular treatment of chronic P. aeruginosa infection, with 2-week courses every 3 months, has resulted in a significant increase in 5-year survival from the time of onset of this infection in their patients. T h e cost implications and side-effects of this approach have been discussed. 79 T h e r e are n u m e r o u s other studies demonstrating the efficacy of various anti-pseudomonal antibiotics, given alone and in combination, but no clearly outstanding regime emerges as the treatment of choice. 9°, 100,101 Can rational guidelines for the treatment of P. aeruginosa be given? T h e study of Beaudry and colleagues 97included patients with advanced pneumonitis. L u n g damage by an overexuberant i m m u n e response as well as other causes of p u l m o n a r y exacerbation such as viral infection m u s t also be considered. Although P. aeruginosa often persists after therapy, and clinical improvement may take place irrespective of in vitro susceptibility, a more subtle antimicrobial effect may operate within the protected environment of the lung. Sub-inhibitory concentrations of tobramycin, azlocillin, ceftazidime and, in particular, ciprofloxacin have been found to suppress release of protease in vitro from certain strains of P. aeruginosa isolated from C F patients. 1°2 These results suggest that other criteria may be used beneficially to select antibiotic therapy in the future. At present, there is evidence to support intensive intravenous treatment of exacerbations associated with P. aeruginosa, particularly during early stages of infection.66.79 Administration of antibiotics by aerosol may be used following these intensive regimes and for home treatment of less severe episodes of respiratory infection in the hope that hospital treatment may be delayed. 89 F u t u r e a p p r o a c h e s to c o n t r o l o f p u l m o n a r y i n f e c t i o n in cystic fibrosis

Knowledge of the underlying biochemical defect is still the best hope for those affected by cystic fibrosis. Should this remain refractory to therapeutic intervention, or only partially correctable, an improved understanding of the pathogenic role of various microbial agents associated with progressive p u l m o n a r y destruction becomes increasingly important. In many centres engaged in the treatment of C F patients, antimicrobial strategies are predominantly anti-staphylococcal. T h e r a p y for pseudomonas infection may be delayed until clinical signs and symptoms are manifest. Host-bacterium interactions remain unclear for organisms that commonly inhabit the lungs. Recent clinical research increases the confusion surrounding the role of

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conventional pathogenic micro-organisms in pulmonary deterioration. Following a limited clinical study involving 45 young CF patients (aged i - I 2 years) in Boston, U.S.A., l°a clinical benefit without significant side-effects was demonstrated in those receiving corticosteroid therapy on alternate days over a period of 4 years. This trial lends support to the hypothesis that an excessive inflammatory response to offending endobronchial micro-organisms is responsible for at least some of the damage. Inhalation of steroids has been tried in patients with pseudomonas lung infection without convincing effect. 8° Clearly, more studies are required to investigate the therapeutic rationale and potential of immunosuppressive agents. Wilmot and colleagues TM have investigated the influence of pseudomonas infection on actuarial survival statistics (percentage survival to age of I6 years) in a group of 234 CF children from the Hospital for Sick Children, London. Those with chronic P. aeruginosa lung infection had significantly worse survival rates than those without. This unfavourable outcome also correlated with the age at which P. aeruginosa was acquired. Respiratory tract colonisation with P. cepacia has also been associated with an adverse clinical outcome. 17, 105 Can the onset of pseudomonas colonisation be delayed? Restricted use of anti-staphylococcal drugs seems to be associated with a low isolation rate of P. aeruginosa from Edinburgh patients. 52T h e polyvalent pseudomonas vaccine (PEV-oI) failed to reduce the rate of colonisation in a small prospective trial in Birmingham TM with evidence of an adverse effect in vaccinated patients who subsequently became colonised. This is perhaps not surprising as PEV-oI is a lipopolysaccharide vaccine developed for protection against septicaemia in burns patients. ~°7 T h e stimulation of specific local immunity to strains of Pseudomonas spp. able to establish significant colonisation in the respiratory tract may be a more hopeful approach. T h e potential of a vaccine blocking initial adherence to respiratory tract mucosa requires further explorationY 3, 76 A third approach is the antibiotic treatment ofP. aeruginosa at its earliest stages of colonisation. Littlewood and colleagues ~°8 at the CF centre in Leeds have reported preliminary findings on the use of nebulised colomycin following isolation of P. aeruginosa from throat swabs or sputum of CF patients in the absence of clinical or laboratory evidence of tissue invasion. A controlled trial of long-term nebulised colomycin is now in progress. It is unlikely that this regime will always be successful in eradicating P. aeruginosa. A study of this kind in a large population of CF patients, however, may allow complete examination of specific host-bacterial interactions associated with the poorer prognosis of those developing chronic pseudomonas lung infection. Knowledge is increasing in many areas of CF research and more effective therapy for various aspects of the disorder seems highly plausible. Certain microbiological aspects remain controversial and require further clarification. It is hoped that further research will resolve many of these difficult problems and contribute towards a brighter future for families affected by cystic fibrosis.

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