Vol. 25, No. 10JOURNAL OF CLINICAL MICROBIOLOGY, OCt. 1987, p. 1830-18360095-1137/87/101830-07$02.00/0Copyright © 1987, American Society for Microbiology

Diagnosis of Chronic Pseudomonas aeruginosa Infection in CysticFibrosis by Enzyme-Linked Immunosorbent Assay

SVEND S. PEDERSEN,l2* FRANK ESPERSEN,' AND NIELS H0IBY1

Department of Clinical Microbiology, Statens Seruminstitut,' and Department of Pediatrics, Rigshospitalet,2DK-2100 Copenhagen 0, Denmark

Received 23 March 1987/Accepted 15 June 1987

An easily applicable test for diagnosis of chronic Pseudomonas aeruginosa infection in cystic fibrosis byenzyme-linked immunosorbent assay (ELISA) for determination of serum immunoglobulin G to P. aeruginosawas developed. Soluble antigens obtained by ultrasonication of P. aeruginosa, serotypes 0:1 to 0:17, were usedas antigens immobilized to polystyrene microtiter plates. The intraplate, plate-to-plate, and day-to-dayvariations were 14, 19, and 20%, respectively. Plates coated with the antigens could be stored for at least 64days at +4 and +22°C without any significant change in activity. Normal values were determined in sera from164 controls (100 children and 64 adults). The sensitivity and specificity of the ELISA was determined by usingserum samples from 243 cystic fibrosis patients and were compared to results with crossed im-munoelectrophoresis (CIE). The ELISA could diagnose chronic P. aeruginosa infection with a diagnosticsensitivity of 93% and specificity of 92%. The sensitivity and specificity for the diagnosis of the early stages ofchronic P. aeruginosa infection by a single sample were 90 and 100%, respectively, and by using an increasedantibody response in paired samples, the sensitivity was 93% and specificity was 87%. There was a statisticallysignificant correlation between antibody levels obtained by ELISA and those obtained by CIE. The sensitivityand specificity of the ELISA were equal to those of CIE, and because of its simplicity, the ELISA isrecommended as a routine test in patients with cystic fibrosis.

Chronic Pseudomonas aeruginosa lung infection is theleading cause of death in patients with cystic fibrosis (CF)(24). Sputum may for a time show growth of P. aeruginosawithout any obvious impact on the health of the patient, andthen gradually the clinical condition deteriorates with in-creasing symptoms of respiratory infection. This change isconsidered a transition from harmless colonization of themucosal lining of the respiratory tract to an invasive andtissue-destroying infection (12). Specific antibodies againstP. aeruginosa antigens rise when the infection becomeschronic, and by determination of precipitating antibodiesagainst P. aeruginosa it is possible to diagnose the onset ofinfection (9). Determination of precipitins, however, is time-consuming and requires a laboratory which routinely per-forms crossed immunoelectrophoresis (CIE). An easier, butequally specific and sensitive, method is needed, and wehave therefore developed an enzyme-linked immunosorbentassay (ELISA) for detection of serum immunoglobulin G(IgG) to P. aeruginosa.

MATERIALS AND METHODS

Patients. Diagnosis of CF was established on the basis ofabnormal sweat electrolytes, characteristic lip biopsy, andexocrine pancreatic insufficiency. After diagnosis, the prog-ress of approximately 80% of the Danish CF patients isfollowed with monthly visits to the CF center at Rigshos-pitalet. Clinical data and lower respiratory tract bacteriologyare examined at each visit and have been recorded prospec-tively since 1970.Mucoid and nonmucoid P. aeruginosa strains were iden-

tified in sputum samples by standard methods (16).

* Corresponding author.

The following definitions were used to describe the pa-tients: noncolonized were patients without growth of P.aeruginosa in sputum at regular monthly examinations andwith precipitin bands by CIE between 0 and 1; colonizedwere patients with consistent or intermittent growth of P.aeruginosa in sputum, but with precipitin bands between 0and 1; infection indicated patients with consistent growth ofP. aeruginosa in sputum and with precipitin bands of .2;and early chronic infection indicated patients in whom P.aeruginosa was cultured at monthly intervals for approxi-mately 6 months and in whom precipitin bands remained at-2.

Sera. Serum samples have been obtained from all patientswith cystic fibrosis at 3- to 12-month intervals since 1973 andwere stored at -20°C until analysis. For the purpose of thisstudy serum samples from 164 controls and 243 CF patientswere assayed. The samples were characterized as follows.

(i) Normal. To establish normal values for the ELISA,serum from 164 healthy controls with no history of P.aeruginosa infection were investigated. Median age (range)for 100 children was 6 years (0 to 14 years) and for 64 adultsit was 32 years (20 to 64 years).

(ii) Detection of antibodies in early chronic P. aeruginosa-infected CF patients (CF+P). (a) Single sample. To determinewhether the ELISA could compare with the measurement ofP. aeruginosa precipitins for early serological diagnosis, thefirst serum samples showing precipitin bands of .2 werechosen from 91 patients. The median age (range) was 9.8years (0.9 to 30.8 years). The bacteriological record of eachpatient was reviewed to establish the time of onset forconstant growth of P. aeruginosa in sputum.

(b) Paired samples. To investigate the feasibility of usingan increase in antibodies for early diagnosis of chronic P.aeruginosa infection, paired samples were analyzed. Amongthe patients from the previous group (a, above) serumsamples obtained while the patient was noncolonized were

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DIAGNOSIS OF P. AERUGINOSA IN CF BY ELISA 1831

available for analysis from 64 patients. The earliest availablesample was chosen to diminish the influence of possibleexposure to P. aeruginosa or other bacteria which maycross-react with P. aeruginosa (8). These samples werepaired with the first samples showing precipitin bands of .2as described for the previous group. The median time span(range) between the two samples was 3.4 years (0.1 to 10.8years).

(iii) Detection of antibodies in noncolonized CF patients(CF-P). As controls for the early infected patients, serawere chosen from noncolonized CF patients in the followingway. For a single sample, samples were obtained from 36 CFpatients in whom the CF diagnosis was made during infancyor early childhood at the Danish CF center and who havebeen followed regularly at the center since diagnosis. P.aeruginosa had never been isolated in their sputum. Themedian age (range) at the time of serum sampling was 0.8years (0.1 to 2.0 years). For paired samples, noncolonizedpatients from a nationwide survey (see below) were selected.Two samples, spaced temporally as much as possible, wereavailable for analysis in 77 patients. The median age (range)at the time of the first sample was 1.6 years (0.1 to 16.8years). Nineteen patients from the previous group (singlesample, above) were included in this group. The time spanbetween the two samples was 3.7 years (0.6 to 12.7 years).

(iv) Nationwide survey of CF patients. To investigate thecorrelation between ELISA and CIE, serum samples from243 CF patients were obtained for measurement of ELISAratio and determination of P. aeruginosa precipitins. Thepatients represent >95% of all living Danish CF patients inthe sampling period (1984 to 1985) and include noncolonizedand colonized individuals and those with chronic infection of0.5 to 15 years in duration. Data on sputum bacteriology inthe 6 months immediately preceding the sample were avail-able in 205 patients for analysis of sensitivity and specificityof the CIE and ELISA.

Sequential serum samples from two of the CF patientsassayed above were tested by ELISA and CIE to comparethe two methods in the individual patient. The time intervalanalyzed was up to 10 years and included a period ofnoncolonization and early and late infection.

Preparation of P. aeruginosa antigen I. P. aeruginosastrains representing all 17 serotypes of the InternationalAntigenic Typing Scheme were obtained from Mikkelsen(18) and from the Cross Infection Laboratory, London.From each strain water-soluble antigens were produced asdescribed previously (9). Equal volumes of each antigenwere pooled and designated St-Ag: 1-17. Aliquots of St-Ag: 1-17 were stored at -20°C until use. The protein concentrationwas estimated to 22 g/liter by the method described byLowry et al. (16). In CIE against rabbit antibodies raisedagainst a previously described sonic extract, the St-Ag:1-17contained 64 antigens (8).ELISA. Irradiated 96-well polystyrene microtiter plates

(Immunoplate no. 1; Nunc A/S, Roskilde, Denmark) werecoated with St-Ag: 1-17 (2.2 ,ug of protein per well), diluted inphosphate-buffered saline (pH 6.9), and incubated for 1 h atroom temperature. After two washes with tap water, addi-tional binding sites in the wells were blocked by overnightincubation at 4°C with phosphate-buffered saline containing1% (wt/vol) bovine serum albumin (Sigma Chemical Co., St.Louis, Mo.). Serum samples were diluted 1:4,000 in a bufferof Na2HPO4 (8 mM), KCl (0.2 mM), and NaCi (0.5 M), pH7.2, containing 1% (wt/vol) Triton X-100 (Sigma) and 1%(wt/vol) bovine albumin, and 0.1-ml aliquots were applied toeach well. The samples were allowed to react for 1 h at room

temperature and then were washed three times in tap water.A 0.1-ml portion of horseradish peroxidase-labeled goatanti-human IgG (Tago, Burlingame, Calif.) diluted 1:2,000 inthe above dilution buffer was added to each well andincubated for 1 h at ambient temperature. The plates werethen washed six times in tap water, 0.1 ml of sodium citrate(0.1 M), pH 5.0, containing 1,2-phenylendiamide-dihydro-chloride (2.2 mM) (Sigma) and H202 (6.5 mM) was added perwell, and the plates were incubated in the dark for 60 min.The enzyme reaction was stopped by the addition of 0.1 mlof H2SO4 (1 M). The optical density at 492 nm (OD492) wasread on an automatic plate reader (Titertek Multiskan; FlowLaboratories, Helsinki, Finland). The antigen concentrationand the serum dilution were chosen after preliminary inves-tigation with serial dilutions of sera from five noncolonizedand five chronic P. aeruginosa-infected CF patients in platescoated with various concentrations of St-Ag:1-17. Ail sam-ples were tested in triplicate, and a control sample made bymixing equal volumes of serum from 10 noncolonized CFchildren was tested on each plate. Results were expressed asa ratio by dividing the mean OD value of the test sample bythe mean OD of the pooled control sample. If the test samplegave an OD value of >2, the sample was diluted 1:40,000 andretested and the ratio was defined as the mean OD valuemultiplied by 4.2. This factor was obtained from linearregression analysis of the ratios obtained by serial dilution of10 serum samples.The influence of time and conditions of storage of coated

ELISA plates was examined. Plates were coated andblocked according to the above procedure; each plate wasdried in air, sealed in a polyethylene bag with silica gel, andstored at -20, +4, and +22°C for 1, 2, 4, 8, 16, 32, and 64days. The analysis was then performed with 26 serumsamples as described above.

Analytical variation of ELISA. With 10 serum samples withlow (n = 3), medium (n = 3), and high (n = 4) ELISA OD492,the intraplate, plate-to-plate, and day-to-day varaiationswere determined. The formula used was SD = V(ad212n),where SD is the standard deviation, I.d2 is the sum ofsquared differences of double determinations of the samesample, and n is the number of observations.The intraplate, plate-to-plate, and day-to-day variations

were 14, 19, and 20%, respectively. With 99% confidencelimits the maximal variation of the assay was 20 ± 45%.Thus, a change of >65% in paired samples was consideredsignificant. A single sample was considered positive (i.e.,indicating P. aeruginosa infection) when the ratio was>1.65.

Validation of the ELISA. The specificity and sensitivity ofthe methods were determined by the method of Wullf (25).The diagnostic sensitivity was the predictive value of anegative test (PVneg). The diagnostic specificity was thepredictive value of a positive test (PVP,,). The nosographicsensitivity was the percentage of patients with the diseasewho had a positive test. The nosographic specificity was thepercentage of patients without the disease who had a nega-tive test.

Precipitating antibodies against P. aeruginosa. P. aerugi-nosa precipitins were determined by CIE as previouslydescribed (9). Briefly, 2 ,ul of St-Ag:1-17, containing 22 g ofprotein per liter, was applied to the well and first-dimensionseparation was carried out at 10 V/cm for 1 h. Second-dimension separation was performed for 18 h at 1 to 2 V/cminto patient serum (15 pul/cm2 of gel). Agarose (1%;Indubiose A 37; L'Industrie Biologique Francaise S.A.) andTris-barbital buffer, pH 8.6 (ionic strength; 0.02) were used.

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FIG. 1. Correlation between age and IgG to P. aeruginosa measured by ELISA in control individuals without a history of P. aeruginosainfection (n = 164). Broken lines are median values.

The immunoprecipitates were stained with Coomassie bril-liant blue (Sigma) and counted (= number of precipitinbands). The normal value is O to 1 precipitin band (10).

Statistics. Wilcoxon signed rank statistic for paired andunpaired data, the chi-square test, and the Spearman corre-

lation test were used. The level of significance was 0.05(two-tailed).

RESULTS

Control samples. Of 100 control children, 7% (95% confi-dence limits [95% CL], 3 to 14%) had an ELISA ratio of-1.65, and the median ratio was 0.97 (lOth to 90th percentilerange [10-90 PR], 0.64 to 1.50). Of 64 control adults, 66%(95% CL, 53 to 77%) had a ratio of .1.65, the median ratio(and 10-90 PR) being 1.81 (1.24 to 3.13). There was a

significant correlation between age and ratio (r = 0.68; P <0.001) (Fig. 1).

Sera from 36 noncolonized CF children from sample groupCF-P had a median ELISA ratio of 1.13 (10-90 PR, 0.75 to1.50). None of these patients had a ratio of >1.65. The ratiowas significantly higher for CF-P than for age-matchedcontrol children (Fig. 2).

Diagnosis of early stage of chronic P. aeruginosa infection.(i) Single sample. The median ELISA ratio for 91 CF patientswith early infection (CF+P) was 2.48 (10-90 PR, 1.72 to4.16). Of 91 patients, 96% (95% CL, 89 to 99%) had a ratio of.1.65. The median time from constant presence of P.aeruginosa in sputum to time when the serum sample wastaken was 6 months (range, 2 months to 2.8 years). Thedifference in ratios between noncolonized (CF-P) and in-fected (CF+P) patients was highly significant (P < 0.001;Fig. 2). Furthermore, the ratio of CF+P was significantlyhigher than for adult controls. The sensitivity and specificityof the ELISA as a single sample diagnostic test for chronicP. aeruginosa infection in CF patients were calculated(Table 1): the diagnostic sensitivity and specificity were 90%(95% CL, 76 to 97%) and 100% (95% CL, 96 to 100%),respectively. The nosographic sensitivity and specificitywere 96% (95% CL, 89 to 99%) and 100% (95% CL, 90 to100%). The samples from CF+P patients were chosen when

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FIG. 2. Distribution of lgG to P. aeruginosa measured byELISA in CF patients and controls. CF-P represents CF patientswithout a history of P. aeruginosa coloiiization (n = 36); CF+P isCF patients with chronic P. aeruginosa infection in an early stage (n= 91). The upper limit for normal values in children is 1.65.

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DIAGNOSIS OF P. AERUGINOSA IN CF BY ELISA 1833

TABLE 1. Distribution of CF-P and CF+ P patients accordingto the result of a single serumsample test in the ELISA'

No. of patientsELISA ratio

CF+P CF-P Total

21.65 87 0 87<1.65 4 36 40

Total 91 36a Chi-square test: P < 0.001.

precipitin bands were .2, the median being 3 (10-90 PR, 2 to7).

(il) Paired samples. Changes in ELISA ratio were studiedin paired serum samples obtained from 64 CF+P and 77CF-P patients. The time elapsing between the first andsecond samples was not significantly different between thetwo groups. The median ratios in the CF-P group were 1.21(10-90 PR, 0.61 to 2.54) and 1.09 (10-90 PR, 0.75 to 1.77) forthe first and second samples, which was not significantlydifferent. The median ratio in the CF+P group was 0.95(10-90 PR, 0.53 to 1.67) for the first sample, which issignificantly lower than the first sample in the CF-P group.At the time of chronic P. aeruginosa infection, the ratio hadincreased in all patients with a median of 2.57 (10-90 PR,1.72 to 4.24; P < 0.001). Table 2 shows that 5 (8%) of 64patients (95% CL, 3 to 17%) did not respond to chronic P.aeruginosa infection, with an increase in ratio of .65%. Themean increase among the responders was 230%, whereas itwas 52% among the nonresponders (P < 0.001). On the otherhand, 9 (12%) of 77 patients (95% CL, 5 to 21%) in the CF-Pgroup had a .65% increase in ratio. However, in eight ofthese patients both samples had a ratio lower than 1.65. Thediagnostic sensitivity and specificity of the ELISA for pairedsamples were 93% (95% CL, 85 to 98%) and 87% (95% CL,76 to 94%), respectively. The nosographic sensitivity was

92% (95% CL, 83 to 97%) and the specificity was 88% (95%CL, 79 to 95%).Comparison of antibody levels measured by ELISA and CIE

in chronic P. aeruginosa infection. Data from the nationwidesurvey showed that P. aeruginosa was cultured consistentlyfor 6 months in 108 patients (CF+P), intermittently in 20(CF±P), and not at all in 77 CF patients (CF-P). Table 3shows the distribution of CF+P, CF±P, and CF-P accord-ing to the results of CIE. The diagnostic sensitivity andspecificity for CIE to distinguish between CF+P and CF-Pwere 100% (95% CL, 95 to 100%) and 98% (95% CL, 94 to100%). The nosographic sensitivity and specificity were

100% (95% CL, 97 to 100%) and 97% (95% CL, 91 to 100%).PVneg and PVp., for CF+P versus CF±P were 100% (95%CL, 69 to 100%) and 92% (95% CL, 85 to 96%). The

TABLE 2. Distribution of CF-P and CF+P patients accordingto increase in antibody ratio measured by ELISA

in paired serum samplesa

Increase in No. of patientsELISA ratio (%) CF+P CF-P Total

265 59 9 68<65 5 68 73

Total 64 77

a Chi-square test: P < 0.001.

TABLE 3. Correlation between CIE and sputum bacteriologya

Precipitin No. of patientsbands CF+P CF+P CF-P Total

::-2 108 10 2 120<2 0 10 75 85

Total 108 20 77a CF + P means constant finding of P. aeruginosa, CF-P means complete

absence, and CF±P means intermittent growth of P. aeruginosa in sputumduring 6 months prior to sampling of serum. Chi-square test: CF + P versusCF-P-P < 0.001; CF+P versus CF±P-P < 0.001; CF±P versus CF-P-P < 0.001.

corresponding values for CF±P versus CF-P were 88%(95% CL, 79 to 94%) and 83% (95% CL, 52 to 98%). Table 4shows the results of the ELISA on sera from the same groupof patients as in Table 3. The diagnostic sensitivity was 93%(95% CL, 85 to 98%) and specificity was 92% (95% CL, 85 to96%). The nosographic sensitivity was 95% (95% CL, 89 to96%) and specificity was 88% (95% CL, 79 to 95%). PVnegand PVPOS for the ELISA to diagnose CF+P or CF±P were72% (95% CL, 47 to 90%) and 93% (95% CL, 87 to 98%). ThePVneg and PVp0s of the ELISA to distinguish between CF±Pand CF-P were 84% (95% CL, 74 to 91%) and 44% (95%CL, 20 to 70%).

Figure 3 shows the distribution of ELISA ratios accordingto precipitin bands from 243 CF patients. By Spearmancorrelation analysis, a highly significant correlation wasfound (rho = 0.82; P < 0.001). In Fig. 4 are two typicalexamples of the correlation between CIE and ELISA whenindividual patients are followed longitudinally. Both param-eters increased at the same time as P. aeruginosa wascultured consistently from bronchial secretions, and theytend to follow each other during the ongoing infection.

Storage of precoated ELISA plates. The precoated ELISAplates could be stored under the specified conditions at +4and 22°C without any significant change in ELISA ratio. Themedian ratios on day 1/day 64 at +4 and +22°C were1.80:1.86 and 1.71:1.99, respectively. Storage at -20°C gavesignificantly higher ratios on day 64 than on day 1 (1.93versus 3.49).

DISCUSSION

P. aeruginosa manifests a variety of somatic and extracel-lular antigens, and antibodies against outer membrane pro-teins (5, 7), whole cells (3), exotoxin A (6, 15), phospholipaseC (6), Pseudomonas elastase and alkaline protease (4, 6, 15),lipopolysaccharide (20, 21), and the common antigen of P.aeruginosa (13) have been found in CF patients. The ratio-nale for using a sonicated antigenic mixture containing atleast 64 different antigens is that no single antigen has been

TABLE 4. Correlation between ELISA and sputum bacteriologyNo. of patients

ELISA ratioCF+P CF±P CF-P Total

21.65 103 7 9 119<1.65 5 13 68 86

Total 108 20 77

a CF+P, CF±P. and CF-P denote constant finding, intermittent finding,and complete absence of P. aeruginosa from sputum, respectively, during 6months preceding the serum sample. Chi-square test: CF+ P versus CF-P-P < 0.001; CF+ P versus CF+P-P < 0.001, CF±P versus CF-P-P < 0.05.

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1834 PEDERSEN ET AL.

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found to be able to detect the antibody response in all CFpatients (4, 6, 9, 13).

Adults had higher ratios than children among the controls,and there was a significant correlation between ELISA ratioand age. This has been noted before when antibody titersagainst P. aeruginosa have been measured (6), and it wasfound to be due to normally occurring cross-reacting anti-bodies (8). It is possible that finding the majority of the adultsto have a high ELISA ratio can be explained by the ELISAdemonstrating more of the "normal, acquired immunity" (1)than was revealed by the CIE. As a consequence, in adultpatients a ratio of .1.65 in a single sample cannot beconsidered abnormal and paired samples should be preferredin adults or an upper limit of 3.5 should be defined (Fig. 2).The CIE and ELISA did correlate, but not completely.

This may be due to several factors. (i) The ELISA is moresensitive than CIE. (ii) In the ELISA we measure onlyantibodies of the IgG class. (iii) The ELISA can alsomeasure nonprecipitating antibodies. (iv) The ELISA usesanother principle of quantitating the antigen-antibody reac-tion. (v) Auto-antiimmunoglobulin antibodies, present in90% of CF patients (22), may interfere with the ELISA.The St-Ag:1-17 contains 10 antigens which are cross-

reactive with other gram-negative bacteria including Esche-richia coli and Haemophilus influenza (8), common in theyounger age groups among CF patients (19). This mayexplain why CF-P had a significantly higher ratio thanage-matched normal controls (Fig. 2). Using a whole-cellELISA, Brett et al. found more than one-third of their CF-Pgroup had abnormally high antibody titers (2). Furthermore,increased precipitins against H. influenza may explain twoof the nine false-positive patients from Table 4. An unex-pected finding was that patients who later developed chronicP. aeruginosa infection had a significantly lower ELISAratio before infection than those who did not acquire chronicP. aeruginosa infection. The reason for this remains unex-

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DIAGNOSIS OF P. AERUGINOSA IN CF BY ELISA 1835

plained. The ability to distinguish between colonization andinfection with P. aeruginosa in CF patients is very importantas a poor prognosis is associated with the finding of in-creased antibodies against P. aeruginosa (9). Institution offrequent courses of antipseudomonal chemotherapy as soonas the chronic infection is diagnosed has improved theprognosis significantly (23) and can reduce the rate ofincrease in P. aeruginosa precipitins (S. S. Pedersen, T.Jensen, N. H0iby, C. Koch, and E. W. Flensborg, ActaPaediatr. Scand., in press).Our study confirms the results of earlier studies (9) show-

ing that measurement of precipitating antibodies against P.aeruginosa by CIE is a highly specific and sensitive test fordiagnosis of chronic P. aeruginosa infection in CF patients(Table 3). This study also shows that the measurement ofIgG to a sonic extract of P. aeruginosa by means of anELISA is as sensitive and specific a method as CIE. TheELISA can be used as a single-sample test to distinguishbetween CF patients chronically infected with P. aeruginosaand those not chronically infected. The probability that P.aeruginosa had been cultured consistently from sputum forat least 6 months was 92% if the ELISA ratio was .1.65(Table 4). Some patients may show an increase in P.aeruginosa-specific antibodies very shortly after P. aerugi-nosa organisms have been cultured for the first time, and inother patients colonization with P. aeruginosa can persistfor 1 to 2 years without eliciting a systemic immune re-sponse. The ELISA could clearly distinguish CF patientswho had had growth of P. aeruginosa for an average of 6months from CF patients who had had complete absence ofP. aeruginosa from sputum. The chance of thus beingchronically infected was 100% if the test was positive,whereas the chance of not being infected if the test wasnegative was 90% (Table 1). Our ELISA was also useful formeasuring a significant and abnormal increase in IgG anti-bodies in paired samples from the individual patient. A 65%increase in ELISA was seen in 92% of patients who had beencolonized with P. aeruginosa for approximately 6 monthsand the predictive value of a positive test was 87% (Table 2).The predictive value of a negative ELISA was 72% whenpatients with intermittent P. aeruginosa colonization(CF±P) were compared with patients who were chronicallyinfected (CF+P) (Table 4). This indicates that, in somecases, intermittent colonization may give rise to formation ofspecific antibodies. This is in accordance with the findings ofBrett et al., who noted a considerable overlap betweenantibody titers in a colonized group and an infected group ofpatients (2). As the state of colonization is usually onlytemporary before progression to infection, the finding of P.aeruginosa in sputum should prompt frequent determina-tions of antibody titers.

Antibodies against P. aeruginosa may be harmful in CF asthey may be involved in immune-complex-mediated tissuedamage (11), and hypergammaglobulinemia has been asso-ciated with more severe pulmonary disease (17). It is there-fore important to pinpoint as early as possible when P.aeruginosa colonization leads to a systemic immune re-sponse. The frequency with which patients should be testedfor antipseudomonas antibodies depends mainly on thebacteriological examination of sputum samples, which isperformed monthly in our clinic. We use the followingguidelines: if P. aeruginosa has never been grown, serum istested once a year, but if P. aeruginosa has been culturedfrom sputum once or more or if the clinical conditiondeteriorates without any obvious reason, serum is tested atintervals of 3 months or less. Aggressive chemotherapy is

then initiated as soon as the patient becomes chronicallyinfected.

In conclusion, we find that the ELISA is a diagnostic toolof comparable sensitivity and specificity to CIE. WhereasCIE is a difficult procedure, the ELISA described in thispaper is a simple method, and it is recommended for theearly diagnosis of P. aeruginosa infection in CF patients.

ACKNOWLEDGMENTS

We thank Lena N0rregaard for excellent technical assistance.S.S.P. is recipient of a scholarship from the Danish Medical

Research Council, and this study was supported by grants fromP. Carl Petersens Foundation, the National Association againstRespiratory Diseases, and the Research Foundation at DronningLouises B0rnehospital.

LITERATURE CITED1. Axelsen, N. H. 1976. Analysis of human Candida precipitins by

quantitative immunoelectrophoresis. Scand. J. Immunol. 5:177-189.

2. Brett, M. A., A. T. M. Ghonheim, and J. M. Littlewood. 1986.Serum antibodies to Pseudomonas aeruginosa in cystic fibrosis.Arch. Dis. Child. 61:1114-1120.

3. Brett, M. A., A. T. M. Ghonheim, J. M. Littlewood, and M. S.Losowsky. 1986. Development of enzyme-linked immunosor-bent assay (ELISA) to detect antibodies to Pseudomonas aeru-ginosa cell surface antigens in sera of patients with cysticfibrosis. J. Clin. Pathol. 39:1124-1129.

4. During, G., and N. H0iby. 1983. Longitudinal study of immuneresponse to Pseudomonas aeruginosa antigens in cystic fi-brosis. Infect. Immun. 43:197-201.

5. Fernandes, P. B., C. Kim, K. R. Cundy, and N. N. Huang. 1981.Antibodies to cell envelope proteins of Pseudomonas aerugi-nosa in cystic fibrosis patients. Infect. Immun. 33:527-532.

6. Granstrom, M., A. Ericsson, B. Strandvik, B. Wretlind, O. R.Pavlovskis, R. Berka, and M. L. Vasil. 1984. Relation betweenantibody response to Pseudomonas aeruginosa exoproteins andcolonization/infection in patients with cystic fibrosis. ActaPaediatr. Scand. 73:772-777.

7. Hancock, R. E. W., E. C. A. Mouat, and D. P. Speert. 1984.Quantitation and identification of antibodies to outer membraneproteins of Pseudomonas aeruginosa in sera of patients withcystic fibrosis. J. Infect. Dis. 149:220-226.

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