DOI 10.1378/chest.119.1.243 2001;119;243-249 Chest

 Lurdes Matas and Vicente Ausina José Domínguez, Núria Galí, Silvia Blanco, Pablo Pedroso, Cristina Prat, 

Assay in Urine SamplesImmunochromatographicAntigen by a Rapid

Streptococcus pneumoniaeDetection of

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Detection of Streptococcuspneumoniae Antigen by a RapidImmunochromatographic Assayin Urine Samples*

Jose Domınguez, MSc, PhD; Nuria Galı, MSc; Silvia Blanco, BSc;Pablo Pedroso, BSc; Cristina Prat, MD; Lurdes Matas, MD, PhD; andVicente Ausina, MD, PhD

Study objectives: Evaluation of a newly available rapid (15 min) immunochromatographicmembrane test (ICT) to detect Streptococcus pneumoniae in urine samples, in order to assess itsutility in the diagnosis of bacteremic and nonbacteremic pneumococcal pneumonia.Design: Retrospective study.Setting: We studied urine samples from 51 patients with bacteremic and nonbacteremicpneumonia due to S pneumoniae diagnosed by blood culture and pneumococcal polysaccharidecapsular antigen detection by counterimmunoelectrophoresis in urine samples, 16 patients withprobable pneumococcal pneumonia, 71 patients with nonpneumococcal pneumonia, and 16patients with pneumonia but no pathogen identified. Urine samples were collected and frozenat 2 20°C until used. The ICT test was performed following the instructions of the manu-facturer.Measurements and results: S pneumoniae antigen was detected in 41 of 51 patients withpneumococcal pneumonia (80.4%); results were positive in 23 of 28 bacteremic cases (82.1%) andin 18 of 23 nonbacteremic cases (78.3%). From patients with a diagnosis of presumptivepneumococcal pneumonia, antigen was detected in seven urine samples (43.7%) and also in onecase of the 16 patients with pneumonia but no pathogen identified. The specificity of the ICT testwas 97.2%.Conclusion: The ICT assay is a valuable tool for the diagnosis of pneumococcal pneumonia,especially for the nonbacteremic cases. (CHEST 2001; 119:243–249)

Key words: antigen detection; immunochromatographic test; Streptococcus pneumoniae; urine samples

Abbreviations: CIE 5 counterimmunoelectrophoresis; EIA 5 enzyme immunoassay; ICT 5 immunochromatographictest; PCA 5 polysaccharide capsular antigen; PnC 5 C polysaccharide

Streptococcus pneumoniae prevails as the mostcommon pathogen of community-acquiredpneumonia.1,2 Systemic pneumococcal infec-

tion is a major cause of morbidity and mortality,

especially for young children, people with underlyingdiseases, and the elderly. The increasing number ofstrains with intermediate sensitivity and resistance topenicillin and with resistance to single or multiple

For editorial comment see page 9

broad-spectrum antibiotics3,4 makes the identifica-tion of individuals with pneumococcal infections ofincreased importance.

The diagnosis of pneumococcal infection requiresrecovery of the microorganism from an uncontami-

*From the Servei de Microbiologia, Hospital Universitari Ger-mans Trias i Pujol, Badalona, Facultat de Medicina de laUniversitat Autonoma de Barcelona, Spain.Manuscript received October 18, 1999; revision accepted June27, 2000.Correspondence to: Jose Domınguez, MSc, PhD, Servei de Mi-crobiologia, Hospital Universitari Germans Trias i Pujol, Car-retera del Canyet s/n, 08916 Badalona, Spain; e-mail:jadoming@ns.hugtip.scs.es

laboratory and animal investigations

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nated specimen (blood, pleural fluid, transtrachealaspirate, or transthoracic aspirate). Blood cultureresults are positive in only about one fourth of cases,and prior antibiotic therapy significantly reduces thenumber of positive blood culture results.1 It isestimated that nonbacteremic cases account for 70 to80% of pneumonias caused by S pneumoniae. Inaddition, invasive methods used to obtain uncontam-inated specimens from the infectious focus cannot besystematically and indiscriminately performed be-cause of concern for adverse effects and lack ofpersonnel skilled in the technique.2 Cultures ofexpectorated sputum only provides a probable diag-nosis since pneumococcal organisms are often car-ried in the nasopharynx.5

Polysaccharide capsular antigen (PCA) detectionmethods are an alternative for the diagnosis ofpneumococcal pneumonia. The advantage of agglu-tination tests over counterimmunoelectrophoresis(CIE) for detection of native pneumococcal antigenin respiratory samples has been documented previ-ously, but it is inefficient to detect PCA in urine.6,7

However, CIE is able to detect PCA in urinesamples. Enzyme immunoassay (EIA) has shown thepotential usefulness of PCA detection.8,9 But thesemethods have failed due to a lack of sensitivity. Animportant factor to explain the inefficiency of thesemethods is the necessity of the assay to detect allpneumococcal serotypes, since each PCA varies inimmunogenicity. The number of etiologic diagnosiscould increase, providing therapeutic benefit, if arapid, sensitive, and specific technique for detectingpneumococcal specific antigen was used.

The aim of the present study was to evaluate a newavailable immunochromatographic membrane test(ICT) to detect S pneumoniae in urine samples.

Materials and Methods

Groups of Patients

Urine samples were obtained from four groups of patients.First, we studied 51 urine samples collected at admission from 51patients (34 men and 17 women) with pneumonia due to Spneumoniae. The mean age of this group was 48 years (range, 2to 91 years). In 28 cases, pneumonia was bacteremic; in theremaining 23 cases, pneumonia was nonbacteremic. Of the 28bacteremic cases, 19 were diagnosed by isolating S pneumoniaeby blood culture; in the remaining 9 cases, diagnosis was doneusing blood culture plus PCA detection in urine samples. The 23nonbacteremic cases were diagnosed by PCA detection in urinesamples by CIE. S pneumoniae was isolated by blood cultureBact/Alert (Organon Teknika; Durham, NC), and identificationwas based on the usual criteria.10 CIE was performed withpneumococcal omniserum (Statens Serum Institut; Copenhagen,Denmark), which contains antibodies to each of the 84 sero-types.11 Continuous CIE was performed using a modification ofthe procedures of Ingram et al12 and Rytel13 on 15 3 10-cm glass

plates with 1 mm of 1% agarose solution (Agarose, Type II:Medium EEO; Sigma Chemicals; St. Louis, MO) in sodiumbarbital buffer (pH 8.6) of ionic strength 0.05. Paired wells werepunched 3-mm apart in the prepared plate and filled with 10 mLof the specimen or pneumococcal antiserum. Electrophoresis wasperformed at 20 mA per plate for 60 min. Plates were refriger-ated after electrophoresis in a humid chamber and read imme-diately and after being held overnight. An additional examinationof gel was performed after protein staining of CIE plates withCoomasie brilliant blue. Positive and negative controls wereincluded in each plate.

The second group consisted of 16 urine samples from 16patients (12 men and 4 women) with probable pneumococcalpneumonia. The mean age was 56 years (range, 16 to 84 years).Patients with Gram’s stain and/or sputum culture results positive forS pneumoniae and/or antigen detection positive in respiratorysamples were classified as having probable pneumococcal disease.

Group 3 contained 71 urine samples from 71 patients withnonpneumococcal infection, 28 of whom had pneumonia of otheretiologies (14 Legionella pneumophila, 6 Chlamydia pneumoniae,5 Mycoplasma pneumoniae, 2 Coxiella burnetii, and 1 Pseudo-monas aeruginosa), 12 of whom had bacteremia from a nonres-piratory focus (1 Staphylococcus aureus, 1 Streptococcus mitis, 1Streptococcus agalactiae, 1 Enterococcus faecalis, 1 Lysteriamonocytogenes, 1 Escherichia coli, 1 Klebsiella oxytoca, 1 Paeruginosa, 1 Bacteroides fragilis, 1 Candida albicans, 1 Candidaglabrata, and 1 E coli and Enterobacter aerogenes), and 31 ofwhom had urinary tract infections (5 with E coli, 6 with Proteusmirabilis, 1 with Proteus vulgaris, 1 with Morganella morganii, 4with Klebsiella pneumoniae, 1 with K oxytoca, 1 with E aerogenes,2 with P aeruginosa, 4 with E faecalis, 1 with S agalactiae, 2 withC albicans, 1 Candida spp, and 2 mixed infections: 1 with Paeruginosa and E faecalis, and 1 with E coli and E faecalis). Theurine specimens of the patients with urinary tract infectionsyielded from 50,000 to . 105 bacteria or fungi/mL.

The fourth group comprised 16 urine samples from 16 patientswith pneumonia with no pathogen identified.

Preparation of Clinical Specimens for Antigen Detection

Urine specimens were collected and frozen at 2 20°C until useand thawed immediately before being tested by ICT.

S pneumoniae Urinary Antigen Test

The Binax Now Streptococcus pneumoniae Urinary AntigenTest (Binax; Portland, ME) is a rapid diagnostic technique in aflow membrane format that relies on the detection of C-polysaccharide (PnC) present in urine specimens. PnC wascoupled to spherilose to produce an affinity matrix. A hyperim-mune rabbit serum was used to prepare PnC-specific affinitypurified antibodies. The ICT consists of a hinged test device inwhich rabbit anti-S pneumoniae is adsorbed onto nitrocellulosemembrane (the sample line), and goat anti-rabbit IgG is adsorbedonto the same membrane as a second stripe (the control line). Asecond set of rabbit anti-S pneumoniae antibody are conjugatedto colloidal gold particles dried onto an inert fibrous support (Fig1). The resulting conjugate pad and the striped membrane arecombined to construct the device.

The ICT test was performed according to the instructions ofthe manufacturer, with a swab being dipped into the urinesamples and then inserted into the test device. A citrate bufferwas added to help in the flowing up of the antigen present in thesamples. The conjugate rabbit anti-S pneumoniae antibody bindspneumococcal antigen if present in the urine sample, and theresulting antigen/antibodies-conjugated complexes are captured

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by immobilized rabbit anti-S pneumoniae antibodies, forming thesample line. In addition, immobilized goat anti-rabbit IgG cap-tures excess conjugated antibody, forming the control line. Theresults were read visually in 15 min. The test was interpreted bythe presence or absence of visually detectable pink-to-purplecolored lines. A positive result includes the detection of both thesample and the control line, while a negative assay produces onlythe control line. The reading of the ICT results was blinded andread by two independent persons.

Results

S pneumoniae antigen was detected in 41 of 51patients with pneumococcal pneumonia (80.4%),being positive in 23 of the 28 bacteremic cases(82.1%), and in 18 of the 23 nonbacteremic cases(78.3%). On the other hand, in function of the PCA

detection CIE results, ICT was positive in 27 of 32patients with positive CIE results (87.1%), and in 14of 19 patients (73.7%) with negative CIE results(Table 1). From patients with a diagnosis of pre-sumptive pneumococcal pneumonia, antigen wasdetected in 7 of 16 urine samples (43.7%; Table 2).

Urine samples from patients with no pneumococ-cal infection were negative by ICT, except in onecase of a patient with pneumonia due to L pneumo-phila diagnosed by antigen detection in urine, and inanother case of one patient with a bacteremia due toB fragilis (specificity, 97.1%). In both cases, the ICTwas positive presenting a weak intensity color on thesample line difficult to interpret. No PCA wasdetected in these urine samples by CIE. In thefalse-positive result in L pneumophila pneumonia,

Figure 1. Top view (top, A) and cross section (bottom, B) of the ICT device to detect S pneumoniaein urine samples.

Table 1—ICT Results, by Means of Diagnosis, From Patients With Confirmed Pneumococcal Pneumonia*

ICT Results TotalPositive Blood

Culture Plus CIE

Positive BloodCulture and

Negative CIE

Negative BloodCulture andPositive CIE

Positive 41 9 14 18Negative 10 0 5 5

*Data are expressed as No. of patients.

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we cannot reject a double infection with S pneu-moniae. With regard to the patients with pneumoniawith no pathogen identified, antigen was detected in1 of the 16 urine samples tested, corresponding to apatient whose blood culture was not done, the CIEwas negative, and the sputum was rejected for beinga poor-quality specimen.

The positive results of ICT can be differentiated inthree groups according to the color intensityreached: weak, medium, and very intense. In gen-eral, medium and very intense ICT results correlatedwith positive PCA detection by CIE; in 20 of the 32cases with positive CIE (62.5%), ICT results weremedium or very intense (8 of 9 when blood cultureresults were positive, and 12 of 23 when results werenegative); in contrast, in 12 of the 19 cases withnegative CIE (63.2%), ICT results were weak. Theinterpretation of the presence or absence of visuallydetectable colored lines was easy and unequivocal,but in three patients from group 1 with a weakintense line, and three patients from group 2, also

with weak line results, the interpretation of thesample line was very difficult.

We determined 11 different serotypes from the 24bacteremic pneumococcal pneumonia cases in whichit was possible to serotype the isolated pneumococci.The correlation of the S pneumoniae serotype withour results is difficult due to the wide variety ofserotypes identified. However, no particular sero-type had been identified with higher sensitivity byICT (Table 3).

Discussion

Immunologic tests for detection of PCA in clinicalspecimens have been studied with a variety of tech-niques: CIE,6,14,15 agglutination tests,16–18 andEIA.8,9,19 Although CIE is less sensitive than agglu-tination tests for detection of PCA in sputum sam-ples,6 it has been reported that agglutination tests,unlike CIE, are not good for detecting PCA in

Table 2—Pneumococcal Antigen Detection by ICT in Urine Samples From Presumptive Pneumococcal Pneumonia*

Patient No.

Results

MicroscopySputumCulture

Latex Agglutinationin Sputum Samples

BloodCulture

CIE in UrineSamples

ICT in UrineSamples†

1 2 1 1 2 2 112 1 1 ND 2 2 23 1 1 1 2 2 14 1 1 1 2 2 1115 1 1 1 2 2 26 2 1 2 2 2 27 1 1 2 2 2 18 2 1 ND 2 2 19 1 1 1 2 2 1

10 2 1 1 2 2 211 2 2 1 2 2 212 2 2 1 2 2 213 2 2 1 2 2 114 2 1 1 2 2 215 2 1 1 2 2 216 1 1 1 2 2 2

*1 5 S pneumoniae detected; 2 5 S pneumoniae not detected; ND 5 not done.†2 5 no color (negative); 1 5 weak intense; 11 5 medium intense; 111 5 very intense.

Table 3—S pneumoniae Antigen Detection in Urine Specimens by ICT Test Related to the S pneumoniae Serotype

ICTResult*

Serotypes of S pneumoniae†

1 (5) 3 (2) 4 (1) 5 (4) 7 (1) 9 (3) 12 (1) 13 (1) 14 (3) 19 (2) 23F (1)

2 1 0 0 1 0 1 0 0 0 1 01 2 0 0 1 0 2 0 0 0 0 011 0 0 0 1 1 0 0 0 1 0 0111 2 2 1 1 0 0 1 1 2 1 1

*2 5 no color (negative); 1 5 weak intense; 11 5 medium intense; 111 5 very intense.†The numbers in parentheses are the No. of isolates. Twenty-four of 28 S pneumoniae isolates were typeable.

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urine.18 Several studies have shown the potentialusefulness of antigen detection by EIA.8,9,19,20 How-ever, these assays have not been shown to be uni-formly sensitive or specific. In addition, the detec-tion of PCA in the sputum is obviously not totallyspecific for the presence of pneumococcal pneumo-nia, and detection of PCA in serum and urinesamples have several difficulties. On the one hand, alow antigen concentration, the presence of circulat-ing pneumococcal IgG immune complexes,21 andthe variation in the clearance of the different PCAserotypes22 can explain the low efficiency detectingPCA in serum samples. On the other hand, thereasons for the inefficiency of the agglutination testsand EIA for detecting PCA in urine remains unclear,but the fact that urine PCA has only partial immu-nologic identity with native PCA, and that the uri-nary PCA has fewer binding sites than the nativePCA,23 are probably involved. It has been suggestedthat urine can constitute an unfavorable microenvi-ronment due to the pH, salt content, and proteinconcentration interfering with the interaction ofantigens with antibodies, although it might not inter-fere in the CIE reaction that takes place inside abuffered gel.24

The pneumococci possess, in addition to type-specific PCA, a number of antigens common to thespecies. PnC is a phosphocholine-containing polysac-charide in the pneumococcal cell wall, derived fromteichoic acids, and is common to all pneumococcalserotypes.25 In order to solve the problems to detectPCA, tests have been developed for the detection ofPnC. Although these tests give excellent results insputum,17,26–28 Nielsen and Henrichsen29 obtained alow sensitivity detecting PnC by CIE in urine sam-ples, and Gillespie et al30 detected PnC in five serumsamples of 20 patients with bacteremic S pneu-moniae infections. In contrast, Bromberg et al31

using latex agglutination detected PnC in 23 concen-trated urine samples of 33 patients with pneumococ-cal bacteremia. Until now, the lack of sensitivity ofthe test developed to detect PnC in serum and urinesamples did not allow the consideration of thesetechniques as an alternative to give a definitivediagnosis of pneumococcal pneumonia.

Soluble or particulate microbial antigens are ex-creted in the urine in many systemic infectiousdiseases.23 The superiority of urine as an antigendetection specimen has previously been document-ed.14 The increased detection of antigen in urine vsserum can be attributed to the lack of confoundingantibodies, and the natural concentration of theantigen by the kidneys. The ICT developed to detectS pneumoniae antigen in urine samples, performedcorrectly in our evaluation, showing an excellentspecificity (97.2%) and an excellent overall sensitivity

(80.4%) detecting antigen in pneumococcal pneu-monia cases. The ICT described here is a very usefultest for the detection of S pneumoniae antigen fromurine samples to diagnose pneumococcal pneumo-nia. In addition, the ICT resolves the problem ofnonbacteremic pneumococcal pneumonia diagnosis.

Detection of pneumococcal antigen in urine sam-ples is a useful tool for bacteremic and nonbactere-mic pneumococcal pneumonia. Both CIE and ICTresults may be positive when all other bacteriologicevidence of infection is lacking due to a failure of Spneumoniae isolation, and they do not depend onviable organisms. Nevertheless, the evaluated ICT,as opposed to CIE, is rapid, giving a result in # 15min, depending on the concentration of antigenpresent in the urine specimens. It is also technicallyless complex, and requires less equipment than theCIE. PCA is detected efficiently in urine samples byCIE, but overnight storage at 4°C, Coomasie bril-liant blue staining of plates, and concentration ofurine are important procedures in order to obtainoptimal results,24 with the subsequent delay of 24 to48 h in providing the diagnosis. Moreover, there areimportant variables in PCA detection by CIE, suchas the choice of agar and ionic strength of the bufferused; CIE is not performed in the great majority oflaboratories and requires trained and expert labora-tory personnel. In addition, not all polysaccharidescan be detected by CIE, types 7 and 14 are neutralat pH 8.6 and should be detected using buffer at pH5.0.32 On the contrary, all serotypes determined fromthe bacteremic pneumococcal pneumonia cases eval-uated had been detected by ICT, including serotypes7 and 14 (Table 3).

It is well known that PnC-like antigen is found inthe Streptococcus oralis group of a-hemolitic strep-tococci (comprising S oralis, S mitis, and S pneu-moniae),33 and it has been suggested that the cross-reactions between a-streptococci and pneumococcidepend on the presence of a common antigenicdeterminant.34 This cross reaction cannot be a draw-back for the ICT, since this antigen is released morereadily from S pneumoniae and it is present at higherconcentration in the pneumococci than in the othera-streptococci.33 In addition, S mitis is not associatedwith pneumonia, and is not likely to appear withfrequency in the population tested by the ICT. Thesingle patient with bacteremia due to S mitis wasnegative, according to our results.

In our experience, ICT results are not influencedby oral streptococci and pneumococcal colonizationof the oropharynx. S pneumoniae colonizes the oro-pharynx in 30 to 70% of healthy adults.5 The con-centration of pneumococci in the carrier’s orophar-ynx is below the antigen detection level required forlatex agglutination.35 Taking into consideration the

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high sensitivity of the ICT, detection of antigen inurine of pneumococci present in the oropharynx mayoccur, although it does not seem to be clinicallyrelevant.

In our study, the specificity of the ICT wasexcellent. Bacterial contamination did not affect thedetection of antigen and did not produce false-positive results. We only found two false-positiveresults. It is not known if there are PnC-like struc-tures in L pneumophila or B fragilis that are respon-sible for the false-positive results, but the possibilityof double infection cannot be rejected. In addition,the two false-positive results gave weak-intensity col-ored lines, which were difficult to interpret. In order toavoid these problems, we could consider these resultsas negative, bringing the specificity 100%, and thesensitivity remaining in 74.5%. Because of the highspecificity of the ICT, we can consider the patients witha diagnosis of presumptive pneumococcal pneumoniaand the patient with pneumonia without pathogenidentified with positive ICT result as having certainpneumococcal pneumonia.

The observation that ICT detects antigen in urinesamples with similar sensitivity in both bacteremicand nonbacteremic pneumococcal pneumonia casesare consistent with the idea that antigenuria does notnecessarily signify the presence of demonstratedbacteremia. The polysaccharide in the circulation isnot solely originated from the bloodstream organ-isms.36 If antigens were released readily from thelungs, one might expect a higher incidence of anti-genuria in patients with nonbacteremic pneumococ-cal pneumonia.

In our experience, the intensity of the pink-to-purple color in the sample line increased accordingto the concentration of antigen present in the urinesamples, making reading easier. Reading the coloredlines on the ICT may take some practice to avoidfalse-negative results in samples with low concentra-tion of antigen. In preliminary studies, we found thatconcentration of antigen present in urine samples byselective ultrafiltration improves the diagnosis (datanot shown). Prior administration of antibiotics maynot prevent antigen detection by ICT. We detectantigen in three of five urine samples collected afterthe therapy antibiotic was initiated. Nevertheless,given that PnC is a cell wall component and penicil-lin interferes with the cell wall synthesis, antigenmight be detected more easily in samples collectedin the first days after admission. This fact and thetime to detect pneumococcal antigen in urine sam-ples after the start of antibiotic treatment was notthoroughly evaluated in this study.

In conclusion, the sensitivity and specificity of theICT to detect S pneumoniae antigen in urine sam-ples should prove very useful in the rapid diagnosis

of pneumococcal pneumonia. The ICT is a valuabletool for the detection of pneumococcal antigen,especially for the diagnosis of nonbacteremic pneu-mococcal cases, which are not diagnosed in the greatmajority of the hospitals.

ACKNOWLEDGMENT: We would like to thank Binax, Inc. andC.B.F. LETI, S.A. for providing the test kits.

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DOI 10.1378/chest.119.1.243 2001;119;243-249 Chest

Lurdes Matas and Vicente Ausina José Domínguez, Núria Galí, Silvia Blanco, Pablo Pedroso, Cristina Prat,

Immunochromatographic Assay in Urine Samples Antigen by a RapidStreptococcus pneumoniaeDetection of

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