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Journal of Cystic Fibrosis 4 (2005) 37 43 www.elsevier.

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Detection of Pseudomonas aeruginosa in patients with cystic fibrosis


G.A. Tramper-Strandersa,*, C.K. van der Enta, T.F.W. Wolfsb
Department of Pediatric Respiratory Medicine, Wilhelmina Childrens Hospital, University Medical Center Utrecht, KH 01.419.0, P.O. Box 85090, 3508 AB, Utrecht, The Netherlands b Department of Infectious Diseases, Wilhelmina Childrens Hospital, University Medical Center Utrecht, Utrecht, The Netherlands Available online 14 June 2005
a

Abstract Chronic pulmonary colonisation with Pseudomonas aeruginosa (PA) in patients with CF is associated with a high morbidity and mortality. Adequate treatment of first acquisition of PA might prevent or postpone chronic colonisation. Early detection of PA is therefore of major importance. Currently, cultures of oropharynx or sputum are most commonly practised. However, oropharyngeal culture has limitations both in the positive and negative predictive value for the presence of PA in the lower respiratory tract. Induction of sputum has little benefit in detection of PA. Serology might have additional value in early detection, when bacterial density is too low to be detected by culture. Molecular techniques are not yet widespread used for detection of PA, but have in general a high sensitivity. In this review, we describe the value of different diagnostic techniques for detecting PA. D 2005 European Cystic Fibrosis Society. Published by Elsevier B.V. All rights reserved.
Keywords: Culture; Serology; Antibodies; PCR; Sensitivity; Infection

1. Introduction Cystic fibrosis is an inherited multi-system disease, caused by a mutation in the CFTR-gene. It is characterised by dysfunction of the exocrine glands and subsequent chronic obstruction in lungs and exocrine pancreatic insufficiency. Chronic obstruction in the lung predisposes for pulmonary infection. Hyperinflammation by persistent infiltration of neutrophils in the airways, even in patients without clinical apparent disease, precedes and accompanies pulmonary infection [1]. In infants, pulmonary infections with Staphylococcus aureus and Haemophilus influenzae are common. Later in childhood, infections with Pseudomonas aeruginosa (PA) become dominant. Twenty percent of 1-year old infants are infected with PA, with an increase in incidence of about 33% in 3-year olds until 80 90% in adulthood [2]. Pseudomonal infection is a multi-stage
Abbreviations: PA, Pseudomonas aeruginosa; CF, cystic fibrosis; BALF, broncho-alveolar lavage fluid; OP, oropharyngeal; ELISA, enzyme-linked immunosorbent assay; CIE, crossed immune electrophoresis; PCR, polymerase chain reaction. * Corresponding author. E-mail address: g.tramper@umcutrecht.nl (G.A. Tramper-Stranders).

process. Initial infection is characterised by adhesion of PA to the epithelial lining of the respiratory tract, and may be transient. If not, aggressive antimicrobial treatment may eradicate PA in this early stage and postpone chronic colonisation [3 8]. Eventually, intermittent colonisation becomes chronic and phenotypes of PA change to alginate producing antibiotic-resistant strains that form biofilms [9]. Chronic PA infection is associated with deterioration of lung function and disease progression, mediated by host inflammatory, mainly neutrophilic, response [10,11]. Standard method for assessing respiratory infections with PA is bacterial culture of sputum, oropharyngeal (cough) swabs or broncho-alveolar lavage fluid (BALF). Culture of BALF can be defined as the reference standard, because it reflects colonisation of the lower respiratory tract. However, BAL is an invasive procedure, and therefore not routinely performed as a diagnostic screening test. Culturing sputum or oropharyngeal swabs is currently the most frequently used method to establish PA carrier status. New identification techniques to trace PA like polymerase chain reaction (PCR) are forthcoming [12 14]. Recently, it was suggested that serologic parameters convert before a culture shows PA [11,15,16].

1569-1993/$ - see front matter D 2005 European Cystic Fibrosis Society. Published by Elsevier B.V. All rights reserved. doi:10.1016/j.jcf.2005.05.009

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G.A. Tramper-Stranders et al. / Journal of Cystic Fibrosis 4 (2005) 37 43

Table 1 Available methods for Pseudomonas aeruginosa screening in cystic fibrosis patients Methods Culture Processing General culture media Gram-negative culture media Selective culture media Site Nasopharynx Oropharynx Bronchi Bronchi/Alveoli Examples

Blood agar McConkey agar Pseudomonas isolation agar, cetrimide agar Nasopharyngeal aspirate Posterior pharynx/tonsils swab Cough swab Sputum spontaneously expectorated Sputum induced by hypertonic saline Broncho-alveolar lavage (BAL)-aspirate

PA is easily cultured on different media. On blood agar, a non-selective medium, PA sometimes is overgrown by commensal throat flora. Gram-negative selective media like McConkey agar makes discrimination of PA from other respiratory pathogens and indigenous flora more convenient. Pseudomonas selective plates like PA isolation agar or cetrimide agar have been developed especially for culture of PA. Within 48 h, PA can be detected on these media [13,17]. The culture site plays an important role in detection of PA. We describe the value of cultures from the upper respiratory tract and cultures of induced sputum in relation to culture of BALF or spontaneously expectorated sputum. 2.1. Cultures from the oropharynx In young children, oropharyngeal (OP) cultures are often used to identify respiratory pathogens. This also counts for older patients who are not able to expectorate sputum. The diagnostic accuracy of OP-culture has important clinical implications, because it is the only non-invasive way of assessing respiratory tract pathogens [17]. Table 2 shows diagnostic and predictive values of OP-cultures compared with BALF-cultures. In young asymptomatic children, PA positive OP-cultures do not reliably predict the presence of bacterial pathogens in the lower airways, however, negative OP-cultures indicate that PA is unlikely to be present in the lower airways [11,17,18]. In older children and symptomatic patients, there is a higher prevalence of PA with a concomitant better positive predictive value [17,19,20]. Combining results of more OP-cultures reduces the number of false negative cultures, and therefore increase negative predictive value [11]. In patients with PA both cultured from BALF and oropharynx, Pulsed Field Gel Electrophoresis (PFGE) showed poor predictive accuracy of OP-isolates for lower airway organisms [18]. This indicated that actual positive predictive value might be different. Fewer data exist on sensitivity of cough swab cultures. Cough swab cultures showed, in a small group of 30 patients, high positive predictive value but low sensitivity in comparison with cultures of spontaneously expectorated sputum [21].

Antibody tests Enzyme-linked immunosorbent assay (ELISA) Western immunoblot Crossed immune electrophoresis (CIE) Diverse Molecular techniques

Non-purified antigens, e.g., whole cell protein Purified antigens, e.g., exoproteins Whole cell protein Precipitins

Polymerase chain reaction (PCR) Fluorescent in situ hybridisation (FISH)

In this review, we evaluate the clinical value of culture, serology and molecular techniques for detection of PA colonisation in patients with CF. Table 1 summarises the techniques that are available.

2. Culture of Pseudomonas aeruginosa from the respiratory tract Bacteria in the respiratory tract are most frequently detected by culture. Conventional qualitative and quantitative culture uses non-selective and selective plate media.

Table 2 Diagnostic values of oropharyngeal cultures for Pseudomonas aeruginosa, with BALF-culture as Freference standard_ Author Armstrong, 1996 Rosenfeld, 1999 No. of BAL 150 141 119 82 108 Age 1 52 months < 60 months Ref. standard BAL PA > 10.5 cfu/ml BAL PA > 10.5 cfu/ml BAL PA any growth < 18 months > 18 months BAL PA any growth 1 OP cult 2 OP cult (t = 3 months, t = 0) BAL PA any growth Sens% 71 82 44 68 Spec% 93 94 95 94 PPV% 57 64 44 76 69 83 83 NPV% 96 97 95 91 85 97 70

Burns, 2001

< 36 months

Ramsey, 1991

43

4 months 25 years

46

93

Ref. standardreference standard; Senssensitivity; Specspecificity; PPVpositive predictive value; NPVnegative predictive value.

G.A. Tramper-Stranders et al. / Journal of Cystic Fibrosis 4 (2005) 37 43

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Its low predictive value for presence of pathogens in the lower respiratory tract, as indicated by BALF-culture as a reference standard, limit oropharyngeal cultures. Noninvasive PA that harbours only temporary in the pharynx possibly explains these low positive predictive values. Errors or differences in sampling technique may also be responsible for low values, although sampling technique was extensively evaluated in cited studies. Sensitivities in Table 2 are to be compared with caution, because different definitions were used for a positive BALF-culture, and so definition of the reference standard. 2.2. Cultures of sputum Simultaneous detection of PA in sputum and BALF samples is not widely described. In 17 sputum-producing adult CF patients, BALF showed seven times an extra PA strain compared with the sputum sample. In two patients, PA infection was newly diagnosed by BAL [22]. In an older study, culture of sputum of CF patients showed high correlation with culture of thoracotomy specimens [23]. Controversy exists whether induction of sputum for detection of PA is of benefit in children with CF. Originally developed for measuring inflammatory parameters in induced sputum in asthmatic subjects, recently, this technique has also been used for detection of lower respiratory tract pathogens in patients with CF [24,25]. Induction of sputum by inhaling nebulized hypertonic saline is a relatively easy and safe procedure [24,26,27]. Volume of induced sputum is significantly larger than spontaneous expectorated sputum, also in previous nonexpectorating children [26,28,29]. It is a reliable procedure as measured by a high reproducibility of inflammatory markers and respiratory pathogens [28]. Nevertheless, it is not frequently used in young children, because it can induce bronchoconstriction. Table 3 shows results of PA status pre- and post-sputum induction. In a young patient group with respiratory symptoms, 32% of 41 patients demonstrated more and/or other specimens of bacteria after sputum induction [30]. In
Table 3 Additional detection of Pseudomonas aeruginosa with sputum induction in patients with CF Author Sample size Age (years) PA preinduction (%)a 7 PA postinduction (%) 7

total, this study group had low number of PA positive patients and only one additional PA was found in induced sputum. In studies with non-symptomatic patients, slight differences were found between pre- and post-induced cultures [24,27,29]. In a small study group, Henig found high agreement between cultures of spontaneous expectorated sputum, induced sputum and BALF regarding mucoid PA, while there was less agreement for non-mucoid PA [26]. Consequently, it seems that induction of sputum for detecting PA has relatively little practical benefit, although existing data regarding PA positive cultures after sputum inductions were derived from small study groups. In addition, most study groups existed of non-symptomatic patients. One reason for low additional detection might be that PA infection by itself gives a higher chance of spontaneous sputum production. Differences in culturing BALF and induced sputum are less clear. However, in comparison with BAL, it is a non-invasive and less intensive procedure.

3. Detection of Pseudomonas aeruginosa with molecular techniques Molecular techniques for detection of PA are still not universally used. PCR has high sensitivity for detection of PA in sputum in culture-positive patients. Karpati et al. demonstrated presence of PA in sputum by PCR-technique and compared them with cultures. A sensitivity of 93% was found [31]. Other studies showed sensitivities between 97 and 100%, depending on primers [12,13,32]. For example, outer membrane protein (oprL) gene locus is more sensitive than Exotoxin A (ETA) gene locus [14]. Molecular detection of PA in sputum might be a useful technique in the early detection of PA. In 10 culture-negative patients with a positive PCR, culture became positive later in 5 patients in a mean time of 4.5 months [13]. In a different study, many PCR-positive culture-negative samples were found. Eighty-three percent of these samples were OPsamples. A sampling error in OP-cultures or misidentification was not ruled out and no follow up was done [12]. FISH is another technique to detect PA. Sensitivity of FISH in comparison with culture is not very high, since the limit for microscopic detection depends on high bacterial density within sputum samples [33]. Advantages of PCR-technique are high sensitivity and possibility of early detection of respiratory pathogens. Another benefit of these molecular techniques is that they can help in establishing cross-infections between patients.

Respiratory symptoms Ho, 2004 41 No symptoms De Boeck, 2000 Sagel, 2001 Suri, 2003 Henig, 2001
a

1.8 12.9

15 19 32 10

4.3 15.2 7 12 7.3 17 19 38 Pa mucoid Pa non-mucoid

33 32 56 60 10

47 21 59 60 30

4. Detection of antibodies against Pseudomonas aeruginosa Infection with PA can be proven both by culturing of the organism itself and by detection of immune response to the micro-organism. Antibody testing with ELISA demonstra-

As determined by OP-, nasopharyngeal or sputum-culture.

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G.A. Tramper-Stranders et al. / Journal of Cystic Fibrosis 4 (2005) 37 43 Table 4 Sensitivity of single sample antibody testing for Pseudomonas aeruginosa Author Sample size Test ELISA Phospholipase C Exotoxin A Alkaline protease Elastase ELISA Phospholipase C Exotoxin A Alkaline protease Elastase ELISA whole cell protein CIE precipitating antibodies Sensitivity (%) 100 68 73 23 100 58 58 15 96 100

ted little or no interference from cross-reacting antibodies directed against other bacteria [15,34]. Chronic infection in general elicits a high antibody response; literature suggests advantages in early infection too [11,16,35]. For the methods, we refer to Table 1. 4.1. Factors that determine extent of antibody response The purified antibodies against PA show different trends in elevation during infection. There is a temporal hierarchy described in development of antibodies: anti-exotoxin A and anti-phospholipase C antibodies seem to develop first, time to titre elevation of anti-elastase and anti-alkaline protease antibodies is prolonged. In addition, frequencies of elevated antibodies seem to vary among different antigens [16,36,37]. There are several factors influencing antibody development. Mucoid PA strains induce a more pronounced antibody response comparing to non-mucoid strains [36,38]. Dual colonisation with Staphylococcus aureus can be accompanied with lower antibody titres of exotoxin A and elastase, but with higher titres of phospholipase C [37,39]. Antibody titres rise during periods of active infection. There is a decrease noted with antimicrobial treatment [37]. Chronically PA infected CF patients who are treated intensively with antibiotics have lower antibody responses [7,37,40,41]. Continuous steroid treatment is accompanied by a decline in antibody titre [15]. Theoretically, other foci of PA infection can induce an increased antibody titre. These foci of PA infection are, e.g., ear or wound infection and cystitis. Not much literature exists on development of anti-PA antibodies in these infections. Few patients with PA cystitis, ear or wound infection showed antibody response, and in general, this response was lower than the response seen in CF patients with pulmonary PA infection [42 44]. Most diagnostic antibody tests are based on IgG antibodies. Other serum immunoglobulins, especially IgA, also show elevation of specific PA antibody titres. Elevation was particularly seen in patients with pulmonary PA infections, other foci elicited fewer specific IgA antibodies [34,45 47]. In addition, titre of anti-PA IgA, like anti-PA IgG, correlates with disease severity and might be a better parameter for follow-up than anti-PA IgG, because it reflects endobronchial infection [45,48]. 4.2. Antibody response in chronic Pseudomonas aeruginosa infection Patients with chronic PA infection show high (more than 2 standard deviations above those of normal control values) IgG titres to proteins of PA [35,49]. Sensitivity and specificity of serology in chronic colonisation differ slightly among distinct methods and antigens. Table 4 shows sensitivities of diverse antibody tests. Antibody levels obtained by whole cell protein ELISA and level of precipitins correlated with

Chronic colonisation Granstrom, 1984 22

Hollsing, 1987

26

Pedersen, 1987

91 108

Intermittent colonisation Pedersen, 1987 20

CIE precipitating antibodies

50

Early colonisation with first positive culture Brett, 1988 33 ELISA whole cell protein Burns, 2001 18 ELISA exotoxin A (BAL+) 11 (OP only+) Immunoblot whole cell protein 18 (BAL+) 11 (OP only+)

88 94 91

94 82

each other and showed both a sensitivity between 96 and 100% for PA colonisation [50]. Instead, ELISA with a single purified antigen has a lower sensitivity for confirming chronic colonisation, and is antigen-dependent [36]. In most alternative studies, no single antigen was able to detect the antibody response in all CF patients [37,51]. 4.3. Antibody response in intermittent Pseudomonas aeruginosa infection From longitudinal studies, it is known that elevated antibody titres are not only observed with chronic infection, but also in an early stage of infection. Intermittent colonisation may give rise to formation of specific antibodies. However, lower titres are found in patients with intermittent colonisation compared with chronic colonisation [35,50]. As antibody titres are also influenced by antimicrobial treatment, titres may vary. 4.4. Antibody response in early Pseudomonas aeruginosa infection Table 5 describes numbers of patients that have evidence of anti-PA antibodies without positive culture. In majority of patients, there is an early antibody response

G.A. Tramper-Stranders et al. / Journal of Cystic Fibrosis 4 (2005) 37 43 Table 5 Prevalence of anti-Pseudomonas aeruginosa antibodies in patients who are culture-negative during study period Author Brett, 1988 Burns, 2001 West, 2002 Sample size 33 11 16 Test ELISA whole cell protein ELISA exotoxin A Immunoblot Whole cell protein ELISA Exotoxin A Cell lysate Elastase Percentage 73 36 91 69 88 44

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on pulmonary infection with PA [15,35]. Serological studies demonstrate a higher incidence of infection than cultures do. In a study performed by Burns et al., time of first detection of antibody for both exotoxin A and whole cell protein immunoblot preceded the detection of PA by culture. By 3 years of age, 97.5% of patients showed evidence of PA infection, measured by a combination of two serologic assays, BALF- and OP-culture. As determined by culture only, 72.5% of patients demonstrated evidence of PA during their first 3 years [11]. Increase of anti-PA antibodies occurred even 6 12 months before PA was isolated from cultures of the respiratory tract in young children. Development of antibodies before isolation of PA occurred in approximately 50 70% of patients [15,16]. Thus, patients with chronic colonisation have in general a firm antibody response to PA. Follow-up of serology gives information about progress of disease and can be useful for monitoring PA infection. Patients with early infection may have elevated antibody titres months before a culture becomes positive. However, not all patients show an early antibody response. Serological testing in addition to culturing can be helpful, particularly for symptomatic patients without positive culture, or patients that have positive OP-culture without symptoms.

detection of PA in comparison with conventional culture of sputum or oropharynx. Early detection of PA is a hallmark of serological and molecular techniques. Antibodies against PA may emerge months before a culture becomes positive, and are a useful parameter for infection monitoring in PA colonised patients as titres may vary with antimicrobial treatment. Polymerase Chain Reaction of sputum or OP-samples demonstrated detection of PA in CF patients in an early stage too, and has a high sensitivity for PA colonisation. Additional benefit of PCR is identifying cross-infections. Serological and molecular techniques are particularly helpful in early or intermittent colonisation, because chronic colonisation is often easily confirmed by culture. Acknowledgements We thank A. Fleer, medical microbiologist, for his comments on this paper. References
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5. Conclusion Early detection of PA in patients with CF is of major importance because aggressive antimicrobial therapy might prevent or postpone chronic PA colonisation and subsequent disease progression. We estimated the value of different detection methods for clinical practise. Conventional cultures have limitations, especially in site of culturing and sampling technique. In young children, PA positive OP-cultures are not good predictors for presence of PA in the lower airways. Also, a negative OP-culture does not definitively exclude the presence of PA in the lower respiratory tract. In older children, positive predictive value is better. Predictive values of OP-cultures can be improved by culturing more regularly. Pathogens in spontaneously expectorated sputum show better concordance with lower respiratory tract pathogens. Sputum induction in children has little additional value for

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