Skip to main page content

• HOME
• Current Issue
• Archive
• Alerts
• About ASM
• Contact us
• Tech Support
• Journals.ASM.org

This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Reprints and Permissions Copyright Information Books from ASM Press MicrobeWorld Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Scott, J. A. G. Articles by Marsh, K. Search for Related Content PubMed PubMed Citation Articles by Scott, J. A. G. Articles by Marsh, K.

 Previous Article  |  Next Article 

Journal of Clinical Microbiology, June 2003, p. 2554-2559, Vol. 41, No. 6
0095-1137/03/$08.00+0     DOI: 10.1128/JCM.41.6.2554-2559.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.

Diagnosis of Pneumococcal Pneumonia by psaA PCR Analysis of Lung Aspirates from Adult Patients in Kenya

J. Anthony G. Scott,^1,2* Eric L. Marston,³ Andrew J. Hall,⁴ and Kevin Marsh^1,2

Wellcome Trust/Kenya Medical Research Institute, Centre for Geographic Medicine Research—Coast, Kilifi, Kenya,¹ Nuffield Department of Medicine, John Radcliffe Hospital, University of Oxford, Oxford OX3 9DU,² Infectious Disease Epidemiology Unit, London School of Hygiene and Tropical Medicine, London WC1E 7HT, United Kingdom,⁴ Merial Ltd., Atlanta, Georgia 30096³

Received 4 November 2002/ Returned for modification 21 January 2003/ Accepted 12 March 2003

Top Abstract Introduction Materials and Methods Results Discussion References

 
psaA is the gene encoding pneumococcal surface adhesin A (PsaA), a 37-kDa protein expressed on the surface of Streptococcus pneumoniae. PCR primers for psaA have been shown to amplify the target DNA sequence in all 90 serotypes of S. pneumoniae and in none of 67 heterologous pathogens and colonizing bacteria of the upper respiratory tract. Pathogenic bacteria identified in lung aspirate specimens cannot normally be dismissed as contaminants or colonizers, which limit the assay specificity of other respiratory tract specimens. psaA PCR analysis was evaluated in 171 lung aspirates from Kenyan adults with acute pneumonia. The limit of detection was one genome equivalent. Sensitivity, estimated in 35 culture-positive lung aspirates, was 0.83 (95% confidence interval, 0.70 to 0.95). psaA PCR analysis extended the number of identifications of S. pneumoniae in lung aspirates from 35 on culture to 61 by both methods. Of 26 new pneumococcal diagnoses, 19 were corroborated by results of blood culture or urine antigen detection. Sequences of the PCR products from 12 positive samples were identical to the psaA target gene fragment. Using an internal control for the PCR, inhibition of psaA PCR was demonstrated in 17% (8 of 47) of false-negative specimens. The results of a control PCR for the human gene ß-actin suggested that false-negative psaA PCR results are attributable to problems of specimen collection, processing, or DNA extraction in 30% of cases (14 of 47). psaA PCR analysis is a sensitive tool for diagnosis of pneumococcal pneumonia in adults.

Top Abstract Introduction Materials and Methods Results Discussion References

 
Although there is little doubt that pneumococcal pneumonia imposes a considerable burden of morbidity and mortality upon adults in the developing world, the epidemiology of the disease is poorly described (6). The greatest problem in quantifying the burden of disease is diagnosis. Culture of blood is highly specific for pneumococcal pneumonia but has a sensitivity estimated at only 25 to 45% (25). Other techniques, e.g., based on serology or sputum culture, frequently lack specificity (5, 27). PCR is a highly sensitive diagnostic technique for identifying microorganisms in human body specimens. The specificity of the technique depends upon a combination of two factors: the extent to which a gene fragment of similar size can be amplified from an alternative species with the PCR primers and the probability that the target organism could be found in the selected body specimen without necessarily being the cause of the disease.

Pneumococcal surface adhesin A (PsaA) is a 37-kDa lipidated protein expressed on the cell surface of Streptococcus pneumoniae (19), and the gene encoding it, psaA, has been cloned and sequenced (24). In a murine model, PsaA has been shown to stimulate antibodies that are protective against fatal pneumococcal disease (31). Monoclonal antibody studies indicate that PsaA is present at the surface of all 90 serotypes of S. pneumoniae (3) and patterns of restriction enzyme digests of psaA vary little between vaccine serotypes of S. pneumoniae (23). Streptococcus mitis and Streptococcus oralis have been shown to possess a gene with 94 to 95% homology to psaA, but PCR with primers derived from the psaA sequence did not yield an amplified product of the target size in any of 67 heterologous pathogens and colonizing bacteria of the upper respiratory tract but did amplify DNA from all 90 serotypes of S. pneumoniae (10, 15). psaA PCR analysis therefore has the species specificity required for use in diagnosis of pneumococcal pneumonia.

The tissue sample with the greatest specificity, for pneumonia, is lung aspirate fluid obtained by transthoracic percutaneous needle aspiration. Samples from the upper respiratory tract may be contaminated with colonizing strains of S. pneumoniae. Blood fractions also may contain S. pneumoniae caused by intermittent invasion of small inocula from a heavily colonized individual. This is thought to explain the poor specificity of PCR of blood used in young children (4). Although S. pneumoniae may descend the respiratory tract from the nasopharynx to the alveoli directly, finding them there, in the presence of pneumonic consolidation, provides very strong evidence of etiology (26). The purpose of this study was to evaluate the sensitivity of PCR for the diagnosis of pneumococcal pneumonia by using clinical and laboratory techniques likely to maximize specificity.

Top Abstract Introduction Materials and Methods Results Discussion References

 
Study setting and populations. Lung puncture specimens were obtained in a prospective etiological study of acute pneumonia in adults in Kenya (25). Patients who presented to either Coast Province General Hospital, Mombasa, Kenya, or Kilifi District Hospital, Kilifi, Kenya, with radiographic evidence of pneumonia and a history of respiratory illness not exceeding 2 weeks were investigated with the following diagnostic tests: blood cultures, lung aspirate cultures, mycobacterial cultures of sputum and lung aspirate fluid, serotype-specific antigen detection for capsular polysaccharide of S. pneumoniae in urine, and serology for viral pneumonia, Chlamydia pneumoniae, Mycoplasma pneumoniae, and Legionella pneumophila serotype 1 (25). Two hundred fifty-nine patients underwent lung aspiration; from the 89th patient onwards a small aliquot of lung aspirate fluid was set aside for PCR and stored at -70°C. The ethics committees of the London School of Hygiene and Tropical Medicine and of Kenya Medical Research Institute (KEMRI), Nairobi, Kenya, approved the study in accordance with KEMRI guidelines for clinical research.

As it is not possible to obtain control aspirates from healthy humans, lung puncture was performed on 10 C57BL6/J mice that formed part of a separate experiment on nasopharyngeal colonization. Five mice were colonized in the nasopharynx with S. pneumoniae serotype 6B, and five were not colonized.

Clinical methods. Lung puncture was performed on patients with peripheral consolidation by using a 35-mm-long 21-gauge needle attached to a 10-ml syringe. All lung punctures were performed within 24 h, and almost all were performed within 4 h of presentation. The area of diseased lung was localized by clinical signs and by two-plane radiography, and the overlying skin was sterilized with alcohol. During suspended respiration, the needle was passed rapidly into the lung parenchyma through an intercostal space, 0.5 ml of isotonic saline was injected, and the needle was withdrawn over 1 to 2 s under suction. The specimen was distributed to slides for Gram and Ziehl-Neelsen stains and broth cultures. Any residuum in the tip of the needle was expelled into a sterile plastic storage vial and frozen at -70°C for up to 4 years. The specimen volume saved was usually <50 µl (range, 20 to 100 µl). All lung punctures were performed by the same operator. To control for the sterility of the immediate operating environment in Kenya, a similar volume of isotonic saline was expelled from the puncture needle immediately prior to lung aspiration and saved in similar vials. In mice, lung aspirates were performed immediately postmortem with a 25-gauge needle and 100 µl of sterile isotonic saline.

Microbiological methods. Lung aspirates and blood samples were cultured in brain heart infusion broth for 7 days and subcultured on 5% horse blood agar on days 2 and 7 or when indicated by broth turbidity. Colonies of S. pneumoniae were identified by optochin sensitivity and by a positive Quellung reaction with antisera from the Danish checkerboard typing system (30). Pneumococcal pneumonia was also diagnosed from urine specimens by a serotype-specific latex agglutination assay for serotypes 1, 4, 5, 6, 7, 9, 12, 14, 19, and 22; the assay had a sensitivity of 0.46 and a specificity of 0.98 (28).

Urine collected before the administration of treatment in the hospital was also assayed for antimicrobial activity (35). Sterile 6-mm-diameter filter paper disks were placed at the center of a Columbia agar plate evenly inoculated with a fully susceptible Staphylococcus aureus (NCTC 6571). A 20-µl aliquot of urine was dropped onto the disk, and the inhibition zone surrounding the disk was read after overnight incubation.

Control strains. The positive control for psaA PCR was S. pneumoniae serotype 6B (ATCC 51937). Reference strains of Haemophilus influenzae were obtained from J. Elliott (Streptococcal Reference Laboratory, Centers for Disease Control and Prevention [CDC], Atlanta, Ga.) and included 2 isolates of serotype b (one each of biotypes I and II), 1 isolate of serotype f (biotype I), and 5 nontypeable strains (4 of biotype II and 1 of biotype III).

PCR. DNA was extracted from lung aspirates with the QIAamp blood kit protocol (QIAGEN, Chatsworth, Calif.) with modifications that included an overnight digestion with proteinase K (Boehringer-Mannheim, Indianapolis, Ind.) at 42°C and ethanol precipitation of the eluate from the QIAamp spin columns (22). DNA was resuspended in 50 µl of 10 mM Tris-HCl (pH 9.0) (22).

Primers 8229.p (5'-CTTTCTGCAATCATTCTTG-3') and 6496.n (5'-GCCTTCTTTACCTTGTTCTGC-3'), which define an 838-bp fragment of the psaA gene of S. pneumoniae (GenBank accession no. U53509), corresponding to positions 1784 and 2624, respectively, were obtained from J. Sampson (Respiratory Diseases Immunology Laboratory, CDC). PCR was conducted by using the method of Morrison et al. (15); briefly, PCR amplification was performed with 5 µl of extracted DNA in a 50-µl reaction mixture containing 50 mM KCl, 10 mM Tris-HCl (pH 8.3), 1.5 mM MgCl₂, 200 µM concentrations (each) of deoxynucleoside triphosphates (dATP, dCTP, dGTP, and dTTP), 1.5 µM concentrations of each primer, and 1.0 U of AmpliTaq polymerase (Perkin-Elmer, Foster City, Calif.). The thermocycler conditions used were as follows: 95°C for 1 min; followed by 35 amplification cycles of 95°C for 30 s, 52°C for 30 s, and 72°C for 2 min; and held finally at 72°C for 8 min. All PCRs were performed with a Cetus 480 thermocycler (Perkin-Elmer Cetus, Norwalk, Conn.). Ten microliters of each amplification reaction was analyzed by electrophoresis on a 2.0% agarose gel in Tris-borate-EDTA buffer. Gels were stained with ethidium bromide and visualized by UV fluorescence.

Sequencing. PCR products were purified from the post-PCR mix with the QIAquick PCR purification kit (Qiagen) and sequenced in both directions by using the dideoxy-sequencing Prism ready reaction rhodamine dye-terminator kit (Applied Biosystems Incorporated, Foster City, Calif.) with a Cetus 9700 thermocycler (Perkin-Elmer Cetus). The sequencing reactions were resolved with a 4.25% polyacrylamide gel at 51°C with constant voltage (1,500 V) by using an ABI Prism model 373 autosequencer.

Synthetic internal control. An internal control for the psaA primers in this PCR was constructed by inserting the target DNA sequence into a DNA plasmid. The psaA primer sequences were retained in the insert, but the body of the target sequence was replaced with a shorter length of foreign DNA, truncating the resulting amplicon and offering an easy means of identification. The psaA gene was first ligated from positions 1784 to 2624. Using restriction enzymes AciI and HinfI (New England Biolabs, Beverley, Mass.), the sequence was cut at positions 29 and 799, respectively. The fragments were resolved in a 1.5% Tris-acetate-EDTA agarose gel and isolated with GeneClean II (Bio101, La Jolla, Calif.). A portion of the gltA gene from Bartonella henselae was amplified by using primers BhCS781.p (5'-GGGGACCAGCTCATGGTGG-3') and BhCS1137.n (5'-AATGCAAAAAGAACAGTAAAC-3') (16). The gltA amplicons and purified digest construct were blunted with T4 polymerase and ligated by using standard molecular biology techniques (22). The construct was transformed into competent JM109 Escherichia coli (Promega, Madison, Wis.) and selected on ampicillin-X-Gal (5-bromo-4-chloro-3-indolyl-ß-D-galactopyranoside)-IPTG (isopropyl-ß-D-thiogalactopyranoside) plates. Plasmids were harvested from 150-ml overnight cultures by using the QIAGEN plasmid midi kit (Qiagen).

Limit of detection of psaA PCR. First, the concentrations of stock solutions of the internal control were estimated by determining the optical density at 260 nm of serial dilutions in triplicate. Next, the dilutions were amplified in duplicate simultaneous runs with the conditions outlined above for psaA PCR. Standard stoichiometric calculations based upon the optical densities at 260 nm of the dilutions were used to determine the minimum number of plasmids required to produce a positive amplicon.

To determine the limit of detection in terms of viable CFU, psaA PCR analysis was performed on DNA extracts from serial 10-fold dilutions of a 4- to 12-h Todd-Hewitt broth culture and compared with colony counts of a 10-µl aliquot of each dilution cultured overnight on 5% sheep blood agar. Previous investigators have reported a detection limit of <1 CFU (37), but this is most likely to be attributable to DNA from nonviable organisms. To minimize this problem, the experiment was repeated with progressively shorter periods of broth incubation until the detection limit estimate stabilized.

Control of DNA extraction. Human ß-actin was selected as a control gene to monitor specimen processing and DNA extraction. Primers GH20.p (5'-GAAGAGCCAAGGACAGGTAC-3') (GenBank accession no. A26624) and PC04.n (5'-CAACTTCATCCACGTTCACC-3') (GenBank accession no. A26623), which define a 267-bp fragment of the human ß-actin gene (GenBank accession no. L26469), were obtained from the Biotechnology Core Facility (CDC). PCR amplification was performed with 5 µl of extracted DNA in a 50-µl reaction mixture containing 50 mM KCl, 10 mM Tris-HCl (pH 8.3), 1.5 mM MgCl₂, 200 µM concentrations (each) of deoxynucleoside triphosphates (dATP, dCTP, dGTP, and dTTP), 1.5 µM concentrations of each primer, and 0.2 U of thermostable AmpliTaq DNA polymerase (Perkin-Elmer). The thermocycler conditions used were as follows: 95°C for 1 min; followed by 35 amplification cycles of 95°C for 30 s, 58°C for 30 s, and 72°C for 2 min; and held finally at 72°C for 5 min.

Top Abstract Introduction Materials and Methods Results Discussion References

 
Lung aspirate samples were obtained from 171 patients with radiologically confirmed pneumonia. The median age of the patients was 31 years (range, 15 to 76 years), and 107 (63%) patients were male. The median duration of the illness before presentation was 6 days (range, 0 to 14 days). Pneumococcal pneumonia was diagnosed in 95 patients. S. pneumoniae was cultured in the lung aspirates of 35 (20%) patients and in the blood of 40 (23%) patients; there were 19 patients whose cultures of blood and lung aspirate were both positive for S. pneumoniae. The urine antigen test was positive for 65 patients, including 39 whose blood and lung aspirate cultures were both negative.

The limit of detection of the psaA-based PCR, determined by PCR of the synthetic internal control, was one internal control molecule. Because only one copy of the psaA gene has been identified in the S. pneumoniae genome (24), the sensitivity of the PCR under the conditions described here is 1 genomic equivalent. Estimated by assaying fresh broth cultures of S. pneumoniae serotype 6B and comparing them with colony counts, the estimate of the limit of detection was 8 CFU.

The psaA PCR was negative in all 10 aspirate samples from mouse lung and in all 10 sterility controls.

Clinical samples that gave a clear band of the expected molecular size with psaA PCR are shown in Table 1. The sensitivity of psaA PCR in culture-positive lung aspirate specimens was 0.83% (95% confidence interval [CI], 0.70 to 0.95). Among patients with any evidence of pneumococcal pneumonia, psaA PCR was positive in 51% (48 of 95 patients). In total, 55 lung aspirates were positive by psaA PCR, giving a diagnostic yield among all 171 patients of 32%. The proportion of patients with positive results was unaffected by age or sex, nor did it vary significantly with human immunodeficiency virus (HIV) seropositivity or with different radiological patterns of pneumonia (Table 2). Lung aspirate analyses performed on specimens from patients presenting in the first 5 days of the clinical illness were more likely to be positive by psaA PCR than those performed on specimens from patients presenting later (Table 2).

View this table: [in this window] [in a new window]

TABLE 1. Lung aspirate samples positive by psaA PCR analysisa

View this table: [in this window] [in a new window]

TABLE 2. Lung aspirate samples positive by psaA PCR divided according to the presence of antibiotics in the urine of pneumonia patients, the radiological pattern of pneumonia, the HIV status of pneumonia patients, and the duration of illness

Antimicrobial activity was detected in the urine of 81 (54%) of 151 patients; urine from 20 patients was not tested because it was collected after the administration of therapeutic intravenous antibiotics. Blood and lung aspirate cultures were positive in 49% of patients who did not have evidence of antibiotics in urine but in only 17% of patients who did have evidence of antibiotics in urine (Fisher's exact test, P < 0.0005). In contrast, the proportion of positive results on PCR was unaffected by the presence or absence of antibiotics in urine (Table 2). Among patients whose psaA PCR was positive but who also had negative blood and lung aspirate cultures, the prevalence of antibiotic activity in urine was 79% (11 of 14 patients).

In seven cases the positive psaA PCR result was the only evidence suggesting S. pneumoniae as an etiological agent. In this group, five patients had evidence of a second etiological agent (Table 3). For comparison, the proportion of patients with pneumococcal pneumonia diagnosed by culture of blood or lung aspirate who had a second etiological diagnosis was 18% (10 of 56 patients; Fisher's exact test, P = 0.007). Of these seven PCR-positive cases, two had positive lung aspirate cultures for H. influenzae. To test whether the positive results of psaA PCR analysis might be attributable to the presence of H. influenzae, the assay was performed on eight representative clinical isolates of H. influenzae; the psaA PCR was negative in all eight cases.

View this table: [in this window] [in a new window]

TABLE 3. Results of diagnostic tests for 7 patients with pneumonia whose only evidence of a pneumococcal etiology was a positive result by psaA PCR analysis of lung aspirate

Direct dideoxy sequencing was performed on the PCR product of 12 positive samples selected at random. In each case, the result was identical to the sequence of the psaA gene target.

Forty-seven samples that were taken from patients with pneumococcal pneumonia were negative by psaA PCR. These were examined with the synthetic internal control and ß-actin PCR assays. Eight samples were negative on the internal control PCR suggesting the presence of inhibition. After repeat ethanol precipitation, all 8 samples returned positive results on the internal control PCR but remained negative on the psaA PCR. In the ß-actin PCR, 14 samples showed either no band or only a weak band at the expected size of the ß-actin product.

Top Abstract Introduction Materials and Methods Results Discussion References

 
Sensitivity. To derive a sensitivity estimate for psaA PCR analysis, the appropriate specimen group in Table 1 is lung aspirate specimens from which S. pneumoniae has already been obtained by culture. Lung aspirate fluid from patients diagnosed by other techniques may not necessarily contain any pneumococcal DNA. The sensitivity of psaA-based PCR, 0.83, is similar to that of other diagnostic PCR assays which use pneumococcal gene primers (1, 4, 7, 8, 11-13, 17, 18, 21, 32, 33, 37). Table 4 illustrates the small size of the groups in which true sensitivity estimates have so far been obtained; in fact, the study group reported here provides the most precise estimate yet. Particularly striking is the contrast between the small numbers of observations and the relatively large numbers of test variables (target gene, product size, PCR design, specimen examined, and study population). The weighted mean sensitivity of all studies is 0.84, but there is significant heterogeneity among the different estimates (² test, P = 0.001).

View this table: [in this window] [in a new window]

TABLE 4. Summary of sensitivity estimates for PCR directed at pneumococcal genes in the published literature

Given that, under ideal conditions, PCR detects as little as one copy of target DNA, why is sensitivity of pneumococcal PCR so poor in clinical evaluations? There are several potential explanations. Taq polymerase is highly sensitive to porphyrin inhibitors that are generated from the breakdown of hemoglobin (9). Lung aspirate samples may also contain blood either because of traumatic puncture of a small vessel or simply reflecting the natural invasion of a diseased lung by red blood cells during the pathological phase of pneumonia known as red hepatization. Proteolysis, DNA extraction, and ethanol precipitation should eliminate most inhibitors, but when we evaluated inhibition with a synthetic internal control, one in six of the extracted DNA samples would not initially support PCR.

Southern hybridization can extend the limit of detection of PCR (for the pneumolysin gene) 10-fold and may increase sensitivity (32). Theoretically, nested PCR will also increase sensitivity and specificity, though experience with this is variable and it is prone to contamination problems. Salo and colleagues demonstrated a 1,000-fold gain in sensitivity with nested PCR, but Toikka and colleagues, using the same primer pair, found no additional sensitivity advantage to the second (nested) reaction (21, 32). Selecting the optimal specimens for PCR might also maximize sensitivity; however, studies of humans and bacteremic mice have shown little practical difference in the sensitivity of PCR in whole blood, buffy coat samples, or serum (17, 20, 32).

Although the sensitivity of the psaA PCR was 0.83, the assay detected only 51% of all episodes of pneumococcal pneumonia. This is due to the sensitivity limitations of lung aspiration, sample storage and transport, and DNA extraction and purification. The lung aspirate needle may miss the area of consolidation and retrieve only saline and air (26); the size of the specimen is very small compared, for example, to blood aliquots used for culture and may not contain sufficient DNA. In the present study, storage and transportation of the specimen may have led to degeneration of DNA, and finally, in the process of extraction and purification, the small mass of DNA present may be lost. As lung aspirates should also contain human leucocytes and alveolar cells, we made extracted human DNA the control for these factors by using the ß-actin gene as a PCR target; after sampling, storage, transport, processing, and DNA extraction, 30% of the samples taken from patients with negative psaA PCR showed no evidence of human DNA with ß-actin-specific primers.

Specificity. The appropriate control material to evaluate the specificity of PCR in lung aspirates is lung aspirate fluid from sick patients without pneumonia. This is normally only available from healthy controls (14, 34) or, more practically, from patients in whom pneumonia has been diagnosed initially and a different diagnosis arrived at subsequently (36); this is an uncommon eventuality and we were not able to find appropriate specimens in Kenya. psaA PCR analysis was not positive in lung aspirate material from mice, even in those who had experimental nasal colonization with S. pneumoniae. In humans, specificity estimates have also been derived by (i) evaluation of culture-negative lung aspirates (7) or (ii) evaluation of lung aspirates that have yielded nonpneumococcal pathogens on culture (18). However, these strategies are undermined by the fact that culture is insensitive for detecting pneumococci and that isolation of two organisms from one aspirate is not uncommon (2, 7, 26, 29); the absence of pneumococci on culture does not prove that they were absent from the original sample, even if an alternative pathogen was cultured.

Despite this, lung aspirate fluid is likely to have a very high specificity as a specimen because it is uncontaminated by upper respiratory tract secretions. Several findings from this report support this contention. First, 73% (19 of 26) of the patients with positive psaA PCR results but negative lung aspirate cultures had alternative evidence of pneumococcal pneumonia from blood cultures or antigen detection. Second, knowing that culture techniques are sensitive to the presence of antibiotics and that psaA PCR analysis is not, we would predict that among patients whose cultures were negative but whose PCR results were positive there would be a concentration of individuals with evidence of antibiotics. In fact 79% of this group had evidence of antimicrobial activity in urine against a background prevalence of 54%.

The specificity of PCR for pneumococcal disease in previous studies is shown in Table 5 (1, 4, 11-13, 21, 32). Among adults, specificity has been uniformly high with a weighted mean of 0.97 and little heterogeneity between studies (Fisher exact test, P = 0.98). In the study with the lowest estimate, 6 of 100 samples from healthy elderly persons were PCR positive; the investigators speculated that these false-positive results were due to laboratory contamination as the serum vials had been opened for use in previous assays (17). Among children in Israel, 20% of sera were positive by pneumolysin PCR and the distribution of false-positive results with age closely followed the prevalence of nasopharyngeal carriage with age, suggesting that intermittent transient bloodstream invasion occurs in colonized children leading to false-positive PCR results (4). For children, PCR of blood clearly lacks specificity; PCR of lung aspirate fluid may yield more-specific results, though it remains to be tested.

View this table: [in this window] [in a new window]

TABLE 5. Summary of specificity estimates for PCR directed at pneumococcal genes in the published literature

This study has demonstrated that psaA PCR analysis can amplify pneumococcal DNA in lung aspirates from adults with pneumococcal pneumonia in Kenya, that the sequence of this PCR product is identical to that of the psaA gene target, and that the limit of detection is appropriate for the diagnosis of S. pneumoniae in clinical specimens. The sensitivity in culture-positive lung aspirates is 0.83, and the combination of psaA-based PCR analysis and culture of lung aspirates diagnosed nearly twice as many patients as did culture of lung aspirates alone.

 
This paper is published with the permission of the director of KEMRI.

The work was supported by KEMRI and the Wellcome Trust of Great Britain. J.A.G.S. is supported by a Wellcome Trust fellowship (061089).

We are grateful to George Carlone and Jacquelyn Sampson of the Respiratory Diseases Branch, Centers for Disease Control and Prevention (CDC), Atlanta, Ga., for providing primers and advice on the use of psaA PCR analysis; to Richard Facklam and John Elliott, also of CDC, for providing reference strains of H. influenzae; and to Marc Lipsitch of Harvard School of Public Health, Boston, Mass., for assistance with lung aspirates in mice.

 
* Corresponding author. Mailing address: Wellcome Trust/Kenya Medical Research Institute, Centre for Geographic Medicine Research—Coast, P. O. Box 230, Kilifi, Kenya. Phone: 254 415 22063. Fax: 254 125 22390. E-mail: ascott{at}kilifi.mimcom.net.

Top Abstract Introduction Materials and Methods Results Discussion References

 

1
1. Backman, A., P. Lantz, P. Radstrom, and P. Olcen. 1999. Evaluation of an extended diagnostic PCR assay for detection and verification of the common causes of bacterial meningitis in CSF and other biological samples. Mol. Cell. Probes 13:49-60.[CrossRef][Medline]
2
2. Barnes, D. J., S. Narqi, and J. D. Igo. 1988. The role of percutaneous lung aspiration in the bacteriological diagnosis of pneumonia in adults. Aust. N. Z. J. Med. 18:754-757.[Medline]
3
3. Crook, J., J. A. Tharpe, S. E. Johnson, D. B. Williams, A. R. Stinson, R. R. Facklam, E. W. Ades, G. M. Carlone, and J. S. Sampson. 1998. Immunoreactivity of five monoclonal antibodies against the 37-kilodaltan common cell wall protein (PsaA) of Streptococcus pneumoniae. Clin. Diagn. Lab. Immunol. 5:205-210.[Abstract/Free Full Text]
4
4. Dagan, R., O. Shriker, I. Hazan, E. Leibovitz, D. Greenberg, F. Schlaeffer, and R. Levy. 1998. Prospective study to determine clinical relevance of detection of pneumococcal DNA in sera of children by PCR. J. Clin. Microbiol. 36:669-673.[Abstract/Free Full Text]
5
5. Davidson, M., B. Tempest, and D. Palmer. 1976. Bacteriologic diagnosis of acute pneumonia: comparison of sputum, transtracheal aspirates and lung aspirates. JAMA 235:158-163.[Abstract/Free Full Text]
6
6. Fedson, D. S., and J. A. G. Scott. 1999. The burden of pneumococcal disease in developed and developing countries: what is and is not known. Vaccine 17(Suppl. 1):S11-S18.[CrossRef]
7
7. Garcia, A., B. Roson, J. L. Perez, R. Verdaguer, J. Dorca, J. Carratal, A. Casanova, F. Manresa, and F. Gudiol. 1999. Usefulness of PCR and antigen latex agglutination test with samples obtained by transthoracic needle aspiration for diagnosis of pneumococcal pneumonia. J. Clin. Microbiol. 37:709-714.[Abstract/Free Full Text]
8
8. Hassan-King, M., I. Baldeh, O. Secka, A. Falade, and B. Greenwood. 1994. Detection of Streptococcus pneumoniae DNA in blood cultures by PCR. J. Clin. Microbiol. 32:1721-1724.[Abstract/Free Full Text]
9
9. Higuchi, R. 1989. Simple and rapid preparation of samples for PCR, p. 31-38. In H. A. Erlich (ed.), PCR technology: principles and applications for DNA amplification. Stockton Press, New York, N.Y.
10
10. Jado, I., A. Fenoll, J. Casal, and A. Perez. 2001. Identification of the psaA gene, coding for pneumococcal surface adhesin A, in viridans group streptococci other than Streptococcus pneumoniae. Clin. Diagn. Lab. Immunol. 8:895-898.[Abstract/Free Full Text]
11
11. Lorente, M. L., M. Falguera, A. Nogues, A. R. Gonzalez, M. T. Merino, and M. R. Caballero. 2000. Diagnosis of pneumococcal pneumonia by polymerase chain reaction (PCR) in whole blood: a prospective clinical study. Thorax 55:133-137.[Abstract/Free Full Text]
12
12. Menendez, R., J. Cordoba, P. de La Cuadra, M. J. Cremades, J. L. Lopez-Hontagas, M. Salavert, and M. Gobernado. 1999. Value of the polymerase chain reaction assay in noninvasive respiratory samples for diagnosis of community-acquired pneumonia. Am. J. Respir. Crit. Care Med. 159:1868-1873.[Abstract/Free Full Text]
13
13. Michelow, I. C., J. Lozano, K. Olsen, C. Goto, N. K. Rollins, F. Ghaffar, V. Rodriguez-Cerrato, M. Leinonen, and G. H. McCracken, Jr. 2002. Diagnosis of Streptococcus pneumoniae lower respiratory infection in hospitalized children by culture, polymerase chain reaction, serological testing, and urinary antigen detection. Clin. Infect. Dis. 34:E1-E11.[CrossRef][Medline]
14
14. Mimica, I., E. Donoso, J. Howard, and G. W. Ledermann. 1971. Lung puncture in the etiological diagnosis of pneumonia. Am. J. Dis. Child. 122:278-282.[Abstract/Free Full Text]
15
15. Morrison, K. E., D. Lake, J. Crook, G. M. Carlone, E. Ades, R. Facklam, and J. S. Sampson. 2000. Confirmation of psaA in all 90 serotypes of Streptococcus pneumoniae by PCR and potential of this assay for identification and diagnosis. J. Clin. Microbiol. 38:434-437.[Abstract/Free Full Text]
16
16. Norman, A. F., R. Regnery, P. Jameson, C. Greene, and D. C. Krause. 1995. Differentiation of Bartonella-like isolates at the species level by PCR-restriction fragment length polymorphism in the citrate synthase gene. J. Clin. Microbiol. 33:1797-1803.[Abstract]
17
17. Rudolph, K. M., A. J. Parkinson, C. M. Black, and L. W. Mayer. 1993. Evaluation of polymerase chain reaction for diagnosis of pneumococcal pneumonia. J. Clin. Microbiol. 31:2661-2666.[Abstract/Free Full Text]
18
18. Ruiz-Gonzalez, A., A. Nogues, M. Falguera, J. M. Porcel, E. Huelin, and M. Rubio-Caballero. 1997. Rapid detection of pneumococcal antigen in lung aspirates: comparison with culture and PCR technique. Respir. Med. 91:201-206.[CrossRef][Medline]
19
19. Russell, H., J. A. Tharpe, D. E. Wells, E. G. White, and J. E. Johnson. 1990. Monoclonal antibody recognizing a species-specific protein from Streptococcus pneumoniae. J. Clin. Microbiol. 28:2191-2195.[Abstract/Free Full Text]
20
20. Salo, P., K. Laitinen, and M. Leinonen. 1999. Detection of Pneumococcus from whole blood, buffy coat and serum samples by PCR during bacteremia in mice. APMIS 107:601-605.[Medline]
21
21. Salo, P., Å. Örtqvist, and M. Leinonen. 1995. Diagnosis of bacteremic pneumococcal pneumonia by amplification of pneumolysin gene fragment in serum. J. Infect. Dis. 171:479-482.[Medline]
22
22. Sambrook, J., and E. F. Fritsch. 1989. Commonly used techniques in molecular cloning, p. E3-E15. In J. Sambrook, E. F. Fritsch, and T. Maniatis (ed.), Molecular cloning: a laboratory manual, 2nd ed. Cold Spring Harbor Press, Cold Spring Harbor, N.Y.
23
23. Sampson, J. S., Z. Furlow, A. M. Whitney, D. Williams, R. Facklam, and G. M. Carlone. 1997. Limited diversity of Streptococcus pneumoniae psaA among pneumococcal vaccine serotypes. Infect. Immun. 65:1967-1971.[Abstract]
24
24. Sampson, J. S., S. P. O'Connor, A. R. Stinson, J. A. Tharpe, and H. Russell. 1994. Cloning and nucleotide sequence analysis of psaA, the Streptococcus pneumoniae gene encoding a 37-kilodalton protein homologous to previously reported Streptococcus sp. adhesins. Infect. Immun. 62:319-324.[Abstract/Free Full Text]
25
25. Scott, J. A., A. J. Hall, C. Muyodi, B. Lowe, M. Ross, B. Chohan, K. Mandaliya, E. Getambu, F. Gleeson, F. Drobniewski, and K. Marsh. 2000. Aetiology, outcome, and risk factors for mortality among adults with acute pneumonia in Kenya. Lancet 355:1225-1230.[CrossRef][Medline]
26
26. Scott, J. A. G., and A. J. Hall. 1999. The value and complications of percutaneous transthoracic lung aspiration for the etiologic diagnosis of community acquired pneumonia. Chest 116:1716-1732.[Free Full Text]
27
27. Scott, J. A. G., A. J. Hall, and M. Leinonen. 2000. Validation of immune-complex enzyme immunoassays for diagnosis of pneumococcal pneumonia among adults in Kenya. Clin. Diagn. Lab. Immunol. 7:64-67.[Abstract/Free Full Text]
28
28. Scott, J. A. G., A. Hannington, K. Marsh, and A. J. Hall. 1999. Diagnosis of pneumococcal pneumonia in epidemiological studies: evaluation in Kenyan adults of a serotype-specific urine latex agglutination assay. Clin. Infect. Dis. 28:764-769.[Medline]
29
29. Shann, F., M. Gratten, S. Germer, V. Linnemann, D. Hazlett, and R. Payne. 1984. Aetiology of pneumonia in children in Goroka hospital, Papua New Guinea. Lancet ii:537-541.
30
30. Sorensen, U. B. S. 1993. Typing of pneumococci by using 12 pooled antisera. J. Clin. Microbiol. 31:2097-2100.[Abstract/Free Full Text]
31
31. Talkington, D. G., B. G. Brown, J. A. Tharpe, A. Koenig, and H. Russell. 1996. Protection of mice against fatal pneumococcal challenge by immunization with pneumococcal surface adhesin A (PsaA). Microb. Pathog. 21:17-22.[CrossRef][Medline]
32
32. Toikka, P., S. Nikkari, O. Ruuskanen, M. Leinonen, and J. Mertsola. 1999. Pneumolysin PCR-based diagnosis of invasive pneumococcal infection in children. J. Clin. Microbiol. 37:633-637.[Abstract/Free Full Text]
33
33. Vuori-Holopainen, E., E. Salo, H. Saxen, K. Hedman, T. Hyypia, R. Lahdenpera, M. Leinonen, E. Tarkka, M. Vaara, and H. Peltola. 2002. Etiological diagnosis of childhood pneumonia by use of transthoracic needle aspiration and modern microbiological methods. Clin. Infect. Dis. 34:583-590.[CrossRef][Medline]
34
34. Woolf, C. R. 1954. Applications of aspiration lung biopsy with a review of the literature. Dis. Chest 25:286-301.
35
35. World Health Organization. 1986. Acute respiratory infections, p. 91. In Laboratory Manual of Bacteriological Procedures. W. H. O. Regional Office for the Western Pacific, Manila, Philippines.
36
36. Zalacain, R., J. L. Llorente, L. Gaztelurrutia, E. Zenarruzabeitia, F. Uresandi, and V. Sobradillo. 1993. La punción transtorácica aspirativa con aguja ultrafina en las neumonías de alto riesgo adquiridas en la comunidad. Med. Clin. (Barcelona) 100:567-570.
37
37. Zhang, Y., D. J. Isaacman, R. M. Wadowsky, W. J. Rydquist, J. C. Post, and G. D. Ehrlich. 1995. Detection of Streptococcus pneumoniae in whole blood by PCR. J. Clin. Microbiol. 33:596-601.[Abstract]

---

Journal of Clinical Microbiology, June 2003, p. 2554-2559, Vol. 41, No. 6
0095-1137/03/$08.00+0     DOI: 10.1128/JCM.41.6.2554-2559.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.

This article has been cited by other articles:

• Wilson, D., Yen-Lieberman, B., Reischl, U., Warshawsky, I., Procop, G. W. (2004). Comparison of Five Methods for Extraction of Legionella pneumophila from Respiratory Specimens. J. Clin. Microbiol. 42: 5913-5916 [Abstract] [Full Text]  

This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Reprints and Permissions Copyright Information Books from ASM Press MicrobeWorld Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Scott, J. A. G. Articles by Marsh, K. Search for Related Content PubMed PubMed Citation Articles by Scott, J. A. G. Articles by Marsh, K.