ORIGINAL ARTICLE BACTERIOLOGY

Usefulness of real-time PCR for lytA, ply, and Spn9802 on plasma
samples for the diagnosis of pneumococcal pneumonia

G. Abdeldaim1, B. Herrmann1, P. Mölling2, H. Holmberg3, J. Blomberg1, P. Olcén2 and K. Strålin3
1) Department of Clinical Microbiology, Uppsala University Hospital, Uppsala, 2) Department of Laboratory Medicine, Clinical Microbiology, Örebro University
Hospital, Örebro and 3) Department of Infectious Diseases, Örebro University Hospital, Örebro, Sweden

Abstract

In the present study, we evaluated rapid real-time PCR assays for ply, Spn9802, and lytA applied to plasma samples for the detection of
Streptococcus pneumoniae in patients with community-acquired pneumonia (CAP). In a prospective study of CAP aetiology, an EDTA
plasma sample was collected together with blood culture in 92 adult CAP patients and 91 adult controls. Among the 92 CAP patients,
lytA PCR was positive in eight (9%), Spn9802 PCR was positive in 11 (12%) and ply PCR was positive in 19 (21%) cases. Of 91 controls,
the ply PCR was positive in eight cases (9%), but no positive cases were noted by Spn9802 or lytA PCRs. Ten CAP patients had pneu-
mococcal bacteraemia. Compared to blood culture, PCR for lytA, Spn9802 and ply had sensitivities of 70% (7/10), 60% (6/10) and 70%
(7/10), and specificities of 96% (79/82), 94% (77/82) and 85% (70/82) respectively. With blood culture and/or culture of representative
sputum, and/or urinary antigen detection, S. pneumoniae was identified in 31 CAP patients. Compared to these tests in combination,
PCR for lytA, Spn9802 and ply showed sensitivities of 26% (8/31), 32% (10/31) and 42% (13/31), and specificities of 100% (61/61), 98%
(60/61) and 90% (55/61) respectively. We conclude that Spn9802 and lytA PCRs may be useful for the rapid detection of bacteraemic
pneumococcal pneumonia, whereas ply PCR is not specific enough for routine use and blood PCR with small plasma volumes is not
useful for the detection of nonbacteraemic pneumococcal pneumonia.

Keywords: lytA gene, plasma, ply gene, pneumococcal pneumonia, real-time PCR, Spn9802 fragment, Streptococcus pneumoniae
Original Submission: 18 June 2009; Revised Submission: 2 September 2009; Accepted: 4 September 2009
Editor: J.-L. Mainardi
Article published online: 14 October 2009
Clin Microbiol Infect 2010; 16: 1135–1141
10.1111/j.1469-0691.2009.03069.x

disease severity. However, the positivity rate of blood cul-
Corresponding author and reprint requests: G. Abdeldaim,
tures rarely exceeds 10% in CAP [2], and can be below 1% if
Department of Clinical Microbiology, Uppsala University Hospital,
SE-751 85 Uppsala, Sweden blood samples are obtained during antimicrobial treatment
E-mail: guma.abdeldaim@medsci.uu.se [3].
The use of PCR protocols to detect multiple bacteria in
blood samples is increasing in clinical practice. Commercial
PCR procedures applied to blood samples, such as SeptiFast
Introduction
(Roche Diagnostics, Mannheim, Germany) and SepsiTest
(Molzym, Bremen, Germany) use conserved PCR targets such
Streptococcus pneumoniae is the chief cause of community- as the internal transcribed spacer region and 16S rRNA, for
acquired pneumonia (CAP) [1]. The diagnosis of pneumococ- genes that are common to all bacteria. This is a promising
cal pneumonia is hindered by the lack of a highly sensitive strategy for pathogens such as Staphylococcus aureus and
and specific ‘gold standard’ method. Culture of sputum and Gram-negative enteric bacilli. However, it is difficult for PCR
nasopharyngeal secretions is controversial as a result of against common bacterial genes to distinguish between
respiratory tract carriage of pneumococci. Bronchoalveolar S. pneumoniae and alpha-haemolytic streptococci because they
lavage or transthoracic needle aspiration are considered to are closely related [4,5]. Another target, the DNA fragment
be reliable but they are invasive and cannot be performed Spn9802, has an unknown function, but has been described as
routinely. Identification of the bacterium in blood culture a specific target for S. pneumoniae [6]. In the present study,
provides a definite diagnosis and can serve as an indicator of we aimed to evaluate the performance of PCR of three differ-

ª2010 The Authors
Journal Compilation ª2010 European Society of Clinical Microbiology and Infectious Diseases

LightCycler 2.0 system using LightCycler Software. and 19 (21%) were current smokers. Portland.1136 Clinical Microbiology and Infection. arthritis/spondylitis. After storage at cultation). Berlin. Volume 16 Number 8. USA) was performed on non- were enrolled in a prospective study running from November concentrated urine from the CAP patients and controls. Faststart Reaction Mix Hybridization Probes (Roche Diagnos- ª2010 The Authors Journal Compilation ª2010 European Society of Clinical Microbiology and Infectious Diseases. including Spn9802 PCR and ply PCR 14 with chronic obstructive pulmonary disease.3 and 0.5 lM of forward were serotyped by using type-specific pneumococcal rabbit and reverse primers [11] (Scandinavian Gene Synthesis. Sweden). were cultured on blood agar with gentian violet.7 and 0. with two bottles in each. pharyngeal aspirates where obtained from both CAP patients lytA. Patients Urinary antigen test At the 35-bed Department of Infectious Diseases. containing 0. Sparks. 3. version lected on the ward. The sample in 92 cases where blood culture was also performed. pneu- University Hospital. ence strain S. patients with CAP moniae (Binax. This real- patients (9%) were treated in the intensive care unit. France). There was eluted in 25 lL of elution buffer. an EDTA sample was col- dyspnea. Microbiological analysis Two blood cultures. 1135–1141 . but no hospitalization during the preceding month. Örebro. S. A well and these 91 were used in the present study.1 (Roche Diagnostics) with probe detection (Table 1). Eight also enables detection of S.2 lM of the probes Sputum samples were obtained from the CAP patients. but without respi. The pneumococcal strains isolated from blood 20-lL reaction volume. )70C. urinary tract infection. the plasma samples (400 lL) were subjected to From 235 enrolled patients (median age 71 years) who ful. autolysin. Spn9802 fragment and a real-time PCR for the lytA gene of The 92 patients had a median age of 70 (range 19–93) years. From 113 enrolled controls. 16. ples with more than five leukocytes per epithelial cell were respectively. Marcy l’Etoile. an EDTA plasma sample was available instrument (bioMérieux. cough. Together with blood culture II. 44 (48%) were females. adult control patients hospitalized shown in Table 1. Sweden. were enrolled [7]. These patients were included in the present study. were collected lytA PCR from all patients and controls. Naso. The criteria for CAP were: acute illness. pneumoniae. niae [6] and we have previously developed a method that and a CURB-65 score of 3–5 was noted in 17 (18%). This duplex PCR was run under the same for skin infection. EDTA plasma samples were 2000 and 10 000 bacterial genomes per PCR reaction (refer- available from 91 (median age 67 years. was used for blood optimized real-time PCR amplifications were performed in a culturing. As a control for amplification of the specific target and ratory symptoms on admission. antisera (Statens Serum Institut. pseudopneumoniae [8]. range 33–87 years). 1999 until April 2002 [7]. Germany). thermal conditions as previously described for the ply PCR or planned orthopaedic/urological surgery. DNA extraction with the automatic NucliSens easyMAG filled the CAP criteria. P1 and P2 (TIB MOLBIOL Syntheselabor. pneumoniae DNA sputum. MD. pneumoniae (pneumolysin. The (Becton Dickinson. Purified DNA was then was no systematic bias selection of patients who provided run in a duplex real-time PCR for the ply gene and the plasma. and bacterial species were identified by stan- Materials and Methods dard microbiological methods. A comorbid illness was noted in 41 patients (45%). A Pneumonia Spn9802 has been shown as a specific target for S. and 2 lL of LightCycler deemed representative and were subjected to culture. The primers and probes are During the study period. giving a detection level of 2 · 105 colony-forming in blood samples from acutely febrile patients. CMI. the gene was amplified using the at the emergency department and blood culture II was col.5 mM MgCl2. at least DNA extraction two of five signs and symptoms of pneumonia (fever >38C. Örebro The Binax NOW immunochromatographic test for S. as a duplex real-time PCR. pneumoniae CCUG 28588T) were used. aiming to identify a and controls. DNA preparations of 500. Blood culture I was collected In the lytA real-time PCR. Copenhagen. Sam. USA). [10]. lected and centrifuged to obtain plasma. Köping. A Bactec nonradiometric system. August 2010 CMI ent gene targets of S. The plates were incubated for 24–48 h at 37C in 5% carbon dioxide. ply. 9240 4. respectively. units/mL sputum. 0. ME. These aspirates (10 lL) and a diluted aliquot of PCR assay that could be used to detect S. pleuritic chest pain. pneumo- Severity Index Risk Class of IV-V was noted in 32 cases (35%). as a standard for quantification. and two time PCR was combined with ply real-time PCR [9] and run patients (2%) died within 30 days of hospitalization. with a negative control (water) was also used for each reac- tion mix. Denmark). and abnormal lung aus. radiological signs of pulmonary consolidation. and the DNA fragment Spn9802).

All participants provided their informed Table 2 shows the detection rates of PCR for ply. lytA and Spn9802 were detected predominantly in cases with bacteraemic pneumococcal pneumonia. including the ten genes in the bacteraemic isolates. there were includes an enzyme activation step at 95C for 10 min. pneumoniae was identified by uri- S. Thus. 16. CMI. culture of representative sputum. NC_003098 and ply. we tested the ten S. No PCR inhibition was able. Conventional diagnostic methods Table 3 shows the results of the individual CAP patients Among the 92 CAP patients. The assay used 5 lL of target DNA. target positive (%) positive (%) positive (%) positive (%) toms ply 7 (70) 6 (29) 6 (10) 8 (9) Spn9802 6 (60) 4 (19) 1 (2) 0 lytA 7 (70) 1 (5) 0 0 a Blood culture negative but S. ª2010 The Authors Journal Compilation ª2010 European Society of Clinical Microbiology and Infectious Diseases. Spn9802. S. pneumoniae detected by culture of representative sputum or by urinary antigen test. pneumococcal DNA. six 24/86 (28%) patients tested. pneumoniae was detected by urinary anti. and lytA County Council. remaining 61 CAP were deemed to have nonpneumococcal tive control for the target specific PCRs. by sputum culture in 13/48 were positive by all three PCR methods. lytA. S. ten patients with bacteraemic pneumococcal pneumonia and followed by 45 cycles at 95C for 5 s. Detection rates by PCR Pneumonia patients with three different gene targets of Streptococcus pneumoniae applied Bacteraemic Nonbacteraemic pneumococcal pneumococcal Nonpneumococcal to EDTA blood samples from 92 pneumonia pneumoniaa pneumoniab Controls (n = 10) (n = 21) (n = 61) (n = 91) pneumonia patients and 91 adult PCR gene Number Number Number Number controls without respiratory symp. Plasma-PCR for S. X52474. Ethics The study was approved by the Ethics Committee of Örebro Real-time PCR for ply. DNA preparations pneumonia. 1135–1141 . by urinary antigen test in ten patients with bacteraemic pneumococcal pneumonia. whereas ply was also detected in Results several patients with nonpneumococcal pneumonia and among the controls. The measurement of fluorescence) and 72C for 8 s. nasopharyngeal aspirate culture in four of 91 (4%) patients tested. pneumoniae was identified by with positive blood culture and/or positive PCR. pneumoniae not detected by blood culture. S. and lytA in the CAP patients and controls.CMI Abdeldaim et al. of 100–1000 bacterial genomes per PCR (reference strain In the control group. 60C for 20 s (single 21 with nonbacteraemic pneumococcal pneumonia. and by culturing nasopharyngeal aspirate in 28/84 (33%) seen in these latter two samples when retested with spiked patients tested. Sequences of oligonu- Positions in cleotide primers and probes for Sequences (5¢ to 3¢)a target geneb detection of Streptococcus pneumo- niae Spn9802 F 5¢-AGTCGTTCCAAGGTAACAAGTCT-3¢ 3370 to 3392 Spn9802 R 5¢-ACCAACTCGACCACCTCTTT-3¢ 3525 to 3506 Spn9802 FAM 5¢-FAM-aTcAGaTTgCTgATaAAaCgA-BHQ1-¢3 lytA F 5¢-CAGCGGTTGAACTGATTGA-¢3 251 to 269 lytA R 5¢-TGGTTGGTTATTCGTGCAA-¢3 423 to 405 lytA P1 5¢-GAAAACGCTTGATACAGGGAGTT-FL-¢3 lytA P2 5¢-LCRed640-AGCTGGAATTAAAACGCACGAG-PH-¢3 ply F 5¢-TGCAGAGCGTCCTTTGGTCTAT-¢3 894 to 915 ply R 5¢-CTCTTACTCGTGGTTTCCAACTTG-¢3 974 to 950 ply Cy5 5¢-Cy5-TGGCGCCCATAAGCAACACTCGAA-BHQ1-¢3 a Positions with lower case letters indicate a locked nucleic acid [28]. tics). pneu- TABLE 2. negative with all three PCR methods. whereas two were (27%) of patients with representative sputum sample avail. consent. Among the blood culture in ten patients (11%). The PCR program patients with pneumococcal bacteraemia. As a posi. pneumoniae CCUG 36696) were used as well as a negative nary antigen test in two of 89 (2%) of patients tested and by control for each reaction mix. b S. b Accession numbers: Spn9802. AE008434. To study the presence of the three gen test and/or sputum culture in 31 cases. Spn9802. or urinary antigen test. pneumonide in CAP 1137 TABLE 1.

b Reported as percentage (number with negative PCR/number without the defined pneumonia diagnosis). The sensitivities were sputum culture. 77 ) 0 0 3 · 102 + + + M. pneumoniae detected by blood culture. 54 S. Volume 16 Number 8. but was lower for lytA reactions negative. age serotype (copies/mL) (copies/mL) (copies/mL) antigen test S. 200–2000 copies/mL) in five rate was positive in two controls. d Reported as percentage (number without the defined pneumonia diagnosis/number with negative PCR). 23 ) 1 · 102 6 · 103 1 · 104 + ) + F. 83 S. 32 ) 0 0 2 · 102 ) ND ) ND. pneumoniae 7F 0 0 0 + ND + F. 78 ) 0 2 · 103 6 · 101 + ND ND M. 31 S. pneumoniae 19F 9 · 108 1 · 1010 4 · 109 + ND ND F. culture of representative sputum. pneumoniae 23F 6 · 102 2 · 104 2 · 104 + ND + F. but eight subjects were positive with diagnosis of bacteraemic pneumococcal pneumonia. Spn9802 and lytA (details not shown). of nasopharyngeal aspirate (n = 1) and culture of nasopha- Among the bacteraemic cases. pneumoniae Sputum nasopharyngeal result and lytA PCR Spn9802 PCR ply PCR urinary culture for aspirate for Sex. S. specificities remained high for Spn9802 (94%) and lytA (99%). pneumoniae 23F 8 · 101 0 0 + + + F. although the specificities were high for Spn9802 (98%) and In the control group. 89 S. 1135–1141 . no subject was positive with lytA lytA (100%). 51 ) 0 0 3 · 102 ) ) ) M. pneumoniae 9V 2 · 108 3 · 109 1 · 109 + ND ND M. Performance of PCR for Positive Negative PCR gene predictive predictive three different gene targets of Reference group target Sensitivitya Specificityb valuec valued Streptococcus pneumoniae applied to EDTA blood samples from 92 Pneumococcal pneumoniae ply 42 (13/31) 90 (55/61) 68 (13/19) 75 (55/73) Spn9802 32 (10/31) 98 (60/61) 91 (10/11) 74 (60/81) pneumonia patients lytA 26 (8/31) 100 (61/61) 100 (8/8) 73 (61/84) Bacteraemic pneumococcal ply 70 (7/10) 85 (70/82) 37 (7/19) 96 (70/73) pneumonia Spn9802 60 (6/10) 94 (77/82) 55 (6/11) 95 (77/81) lytA 70 (7/10) 99 (81/82) 88 (7/8) 96 (81/84) a Reported as percentage (number with positive PCR/number with the defined pneumonia diagnosis). 85 S. moniae isolates with the PCR protocols. The sensitivities were higher (60–70%) for the PCR or Spn9802 PCR. pneumoniae 7F 0 0 3 · 102 + ND + M. In four of these cases. Spn9802 and lytA had high positive predictive TABLE 4. 58 ) 0 6 · 102 2 · 102 + ) + M. 31 ) 0 4 · 102 1 · 102 ) ) + ) M. ‘)’. 16. low copy numbers (<103 copies/mL). pneumoniae 23F 0 0 0 + ND ) M. or urinary antigen test. e S. pneumoniae 7F 2 · 102 1 · 103 1 · 103 + ND ND F. 91 S.1138 Clinical Microbiology and Infection. pneumoniae (urinary antigen and culture positive for ply. 69 ) 0 0 5 · 102 ) ND ) M. other tests Consequently. All samples positive by ply PCR alone had low (26–42%) for the diagnosis of pneumococcal pneumonia. 74 S. ies/mL. patients with negative blood cultures. 84 S. pneumoniae S. 79 S. 57 S. In two of the ply PCR positive controls. In the controls with all three PCR mL was similar for Spn9802 and ply. 87 0 0 9 · 102 + ND + F. pneumoniae 3 3 · 102 5 · 103 8 · 103 + + + F. pneumoniae 7F 2 · 102 2 · 104 1 · 104 + ND + M. Community-acquired pneumonia patients with positive blood culture and/or positive PCR for Streptococcus pneumo- niae applied to blood samples Culture of Blood culture S. pneumoniae was also detected by urinary antigen test or the CAP cases is shown in Table 4. CMI. and ply PCR at concentrations of 6 · 101 to 2 · 103 DNA cop. 75 ) 0 0 2 · 102 ) ) ) F. not done. The diagnostic performance of the three PCR assays in S. the DNA copy number per ryngeal aspirate (n = 1)). ª2010 The Authors Journal Compilation ª2010 European Society of Clinical Microbiology and Infectious Diseases. negative. 74 ) 0 2 · 102 6 · 102 ) + ) M. pneumoniae urinary antigen test was (Table 3). All ten isolates were were positive for S. c Reported as percentage (number with the defined pneumonia diagnosis/number with positive PCR). Spn9802 PCR was positive at low copy numbers positive in one control and culture of nasopharyngeal aspi- (14–152 copies/reaction. 46 ) 0 0 6 · 102 ND ND ) F. August 2010 CMI TABLE 3. pneumoniae F.

fit such techniques provide. although laboratory contamination with pneu. similar figures have been reported in previous studies gen test [28]. have and ply PCR (n = 1). the concentration of ply DNA was low in the plasma. been reported to harbour the ply gene [25. ª2010 The Authors Journal Compilation ª2010 European Society of Clinical Microbiology and Infectious Diseases. In general. two were positive with ply PCR and none were positive in the four blood culture bottles was approximately 40 mL with lytA PCR or Spn9802 PCR . The present study clearly shows that Methods for selective isolation of bacterial DNA from blood lytA PCR and Spn9802 PCR applied to plasma samples are have also been reported [16]. 16. the ply gene PCR presumably caused false a multiplex PCR assay for different bacteria. However.5 and 10 mL [14. as well as a positive S. pneumoniae solved by processing a larger volume of blood for PCR. Perhaps the detected ply DNA in plasma samples and respira- The present study tested the performance of three real-time tory secretions from patients with negative cultures origi- PCR assays for the detection of S. CMI. discrepant results between those obtained for ply PCR and the other two PCR assays in the control group. the sensitivity of the In healthy children. because of its low prising a methodological challenge that has been elaborated specificity. nated from degraded bacteria introduced from the oral flora. The problem with small sample volumes can be patients with no other test positive for S. alone or in the present study. This is likely to have been a result of the sample have not been tested on blood samples from paediatric volume used in the DNA extraction. (Table 3) and in the controls. This could facili- positivity also in blood. pneumoniae urinary anti- 70%). all patients and controls with ply PCR positiv- with prior antibiotics and negative blood culture. 1135–1141 . detection of bacteria in blood has been reported. pneumoniae PCR results. The cause or lytA PCR could be given suitable treatment at an early is not known. Thus. ply PCR positivity in bronchoalveolar lavage fluid was not corre- Discussion lated with culture positivity for alpha-haemolytic streptococci. pneumonide in CAP 1139 values for pneumococcal pneumonia and high negative pre. We used 400 lL of patients and the impact of S. one had a positive urinary antigen of low-grade bacteraemia.26]. Spn9802 PCR cies.15]. with sensitivities in the range 35– inter-method variability. The present study. pneumoniae in plasma sam. a patient with positive Spn9802 PCR samples have been identified previously [17. mococcal DNA was suspected [19]. nasopharyngeal carriage of S. especially in cases riage in the present study. pneumoniae car- small volume limits the detection capacity. urinary antigen test and the three results can also be caused by alpha-haemolytic streptococci PCR analyses (n = 1). Spn9802 PCR and ply bloodstream even after tooth brushing [23. It has been speculated that such false-positive noted were: blood culture. However. urinary antigen test. specific for the detection of S. stage of the disease. no advantage of PCR target in PCR assays for S. even with Both lytA and Spn9802 have been found specific for collection volumes was between 1. showing that quantitative data 100% compared to blood culture [12. Furthermore. however. S. The differences in DNA copy numbers between lytA PCR Several studies have used ply PCR to the detect S. pneumoniae.CMI Abdeldaim et al. ity had blood cultures negative for alpha-haemolytic strepto- cocci. The use of such a adults. such as Streptococcus mitis and Streptococcus oralis. corresponding to approximately 20–25 mL of As noted. but it not yet clear what bene. we consider that ply should not be used as gene in a commercial test system. with dictive values for bacteraemic pneumococcal pneumonia. Plasma-PCR for S. pneumoniae. In comparison. and Spn9802 PCR (Table 3) are most likely to be a result of niae DNA in blood samples. sputum culture alone (n = 1). Thus. demonstrates Test results in patients with prior antibiotic treatment that laboratory contamination with pneumococcal DNA is Antibiotics were administered prior to blood culturing in 27 not likely to have been a significant cause of false-positive ply cases. These streptococci from normal oral flora can enter the analyses (n = 1). and ply PCR alone (n = 3). com. we should be interpreted with caution. In these cases. Among four adult controls with S. PCR for lytA and Spn9802 [11–13].24] and some spe- PCR (n = 1). urinary antigen test and the three PCR [22]. in a recent study by our group [10]. ples from patients with CAP. the high posi- [10] and others [21] have shown that ply PCR is unspecific for tive predictive values of lytA PCR and Spn9802 PCR (Table 4) the detection of S.5 h) detection of pneumococcal infection ‘sterile’. (4 · 10 mL). However.22]. pneumoniae in respiratory secretions. In the present Spn9802 and/or lytA PCR were positive in three of 26 cases study. Healthy adults with false-positive ply PCR with blood in febrile patients.19. pneumoniae carriage on these plasma.17–20]. However. pneumoniae [8. the blood volume test. pneumo. the positive results for S. pneumo- PCR assays was low for the detection of both pneumococcal niae has been shown to give positive ply PCR results in blood pneumonia (26–42%) and pneumococcal bacteraemia (60– samples [27]. and 5 lL (corresponding to 80 lL of the original tests has only rarely been tested on blood samples from plasma) of the purified DNA for PCR. In make them promising for use in clinical practice.11]. which is a site that is assumed to be tate the rapid (<2. To our knowledge.

Olcen P. Emrich T et al. Autolysin- treatment has been started prior to sampling. Blomberg J. Olcen P. Stralin K. 15. 17. Clinical utility of blood cultures in adult patients with community- been associated with disease severity in CAP [29]. the results obtained suggest that plasma PCR may pathogens from whole blood samples. mitis or S. PCR detection of Streptococcus respect to finding strains of S. Evalua- tion of the lightcycler(r) septifast test in the rapid etiologic diagnostic of infectious endocarditis. Holmberg H. J Infect 2009. similar to that in other studies of this condition [11. The main acquired pneumonia without defined underlying risks. Lehmann LE. Smith MD. there have been no studies that correlate disease Streptococcus species by pyrosequencing analysis of the rnpB gene. Herrmann B. for this organism in blood samples. Abdeldaim G. 12. Dis- or Spn9802 PCR can be used for rapid diagnosis of bacterae- crimination of Streptococcus pneumoniae from viridans group strepto- mic pneumococcal pneumonia. Siegel D. Gonzalez AR. meningitis and septicemia using real-time PCR. Lorente ML.1140 Clinical Microbiology and Infection. Ullberg M. Borrow R. Merino MT. The pres. pre-analytic tool molysis. To our 4. Sheppard CL. Rapid detection of pathogens Regional Research Council. Yamashita Y. Dominguez J. oralis that are clo- pneumoniae DNA in serum samples for pneumococcal pneumonia sely related to S. Stralin enables antibiotic resistance determination. culture for hospitalized patients who are receiving antibiotic therapy. Fox AJ. the requirement for a large number of S. 6. Raoult D. Chalasani NP. provide prognostic information. Usefulness of blood rise to detectable levels of polysaccharides in blood. it is cheap and 39: 1553–1558. We conclude that the detection of Spn9802 and lytA PCRs 13. Innings A. etiology of adult community-acquired pneumonia. Kiyoura Y. Habib G. Haemophi- pneumococcal pneumonia. Mayer LW. A positive result of lytA PCR 197: 313–324. Maeno M. Thuny F. Diagn Microbiol Infect Dis 2008. reason for this is a high correlation with bacteraemia and 108: 932–936. Hogan A et al. Korsgaard J. Bactera. Konradsen HB. lus influenzae. assay for rapid detection and differentiation of 25 bacterial and fungal however. with special ref- detect nonbacteraemic pneumococcal pneumonia. CMI. Rudolph KM. 5. 16. J Clin Microbiol 2009. Wellinghausen N. 1135–1141 . Corless CE. Grace CJ. Herrmann B. Eur Respir J Suppl 2002. Morris R. 58: 346–351. Matas L et al. The Clin Microbiol 2005. J Clin Microbiol 2001. Is quantitative PCR for the pneumolysin (ply) gene useful for assays based on specific targets such as lytA and Spn9802 are detection of pneumococcal lower respiratory tract infection? Clin useful when a rapid analysis is required or when antibiotic Microbiol Infect 2009. Guiver M. 18. 43: 5983–5991. Gebert S. Gouriet F. pneumoniae in blood samples. although suf. Harrison TG. pneumoniae. Casalta JP. 9. Gali N. Edwards-Jones V. Jurado RL. Hunfeld KP. Nakano Y. Krabbe M. Falguera M. Olcen P. pneumoniae to give 3. J severity with PCR for S. McGowan JE Jr. Simultaneous detection of Neisseria meningitidis. 32: 1651–1655. Diagnosis of bloodstream infections in immunocompromised patients by real-time PCR. Compar- ison of two urinary antigen tests for establishment of pneumococcal cases of bacteraemic pneumococcal pneumonia. 4528–4534. A multiplex real-time PCR present study was not designed to investigate this issue. Identification of 43 knowledge. George RC. erence to the spn9802 fragment. J Infect 2008. 31: 2661–2666. Paolucci M et al. Hogan A. Evaluation of 1. Thorax 2000. 55: 133–137. Gopal AK. Seki M. A limitation of the present study is that there were few 7. J Clin Microbiol ficient cases to demonstrate a performance of blood PCR 2004. 43: condition [30]. Black CM. August 2010 CMI Detection of pneumococcal antigen in blood samples has 2. Woodhead M. Roux V. Lieberman J. Diagnosis of Streptococcus in plasma is useful for the rapid detection of bacteraemic pneumoniae infections in adults with bacteremia and community- acquired pneumonia: clinical comparison of pneumococcal PCR and pneumococcal pneumonia. 11. The study was supported by funds from the Uppsala-Örebro 16. Volume 16 Number 8. J Med Micro- only be considered when there is a research interest with biol 2004. Eur J Clin Microbiol Infect Dis 2008. Cabal- lero MR. Real-time PCR K. 10. 47: 1046–1049. 53: 189–195. Valdecanas MA. Abdeldaim GM. Kacz- volumes is not useful for the detection of nonbacteraemic marski EB. 28: 569– Transparency Declaration 573. Parkinson AJ. mococci in routine clinical microbiology. 14. Chest 1995. Sheppard CL. Stanzani M. Littenberg B. J Clin Microbiol 2005. and Streptococcus pneumoniae in suspected cases of Culture remains the basic method for detection of pneu. Toward a quantitative DNA-based definition of pneumococcal pneumonia: a There are few reports of the performance of blood-PCR to comparison of Streptococcus pneumoniae target genes. Diagnosis of pneumococcal pneumonia by polymerase chain References reaction (PCR) in whole blood: a prospective clinical study. Varani S. J Clin Microbiol 1993. ª2010 The Authors Journal Compilation ª2010 European Society of Clinical Microbiology and Infectious Diseases. diagnosis. Pierce K. which is a potentially severe cocci by genomic subtractive hybridization. The authors have no conflicting in blood culture bottles by real-time PCR in conjunction with the interests to declare. 7: 164–166. 57: 307–316. Clin Infect Dis 2001. Clin Microbiol Infect 2001. emia has been associated with mortality in CAP [30].13]. 8. pathogens and resistance patterns. 60: ent study demonstrates that blood PCR with small plasma 143–150. Nogues A. Suzuki N. 36: 20s–27s. Kaltoft MS. whereas ply is not specific enough urinary antigen detection. 42: 3620–3625. Community-acquired pneumonia in europe: causative polymerase chain reaction for diagnosis of pneumococcal pneumonia. targeted lightcycler assay including internal process control for detec- The use of an unspecific PCR target such as ply should tion of Streptococcus pneumoniae DNA in clinical samples. Herrmann B. 15: 565–570. Stralin K. Med Microbiol Immunol 2008. Blomberg J.

Griffiths JK. J Clin Microbiol 2003. Beynon KA et al. Pediatr Dent 2002. Efstratiou A. and Streptococcus mitis: characterization of ‘‘atypical’’ pneumo- Commun Dis Public Health 2003. Molecular genetic methods in the diagnosis of lower virulence factor-encoding genes. polysaccharides in urine and serum. Pneumoniae 21. 27. Sempertegui monia. Devine DA. Whatmore AM. 16. respiratory tract infections. J Clin Microbiol 2003. De Baere T et al. pneumonide in CAP 1141 19. 41: 3521–3525. ª2010 The Authors Journal Compilation ª2010 European Society of Clinical Microbiology and Infectious Diseases. Pneumonia severity related to detection of pneumococcal 299. 24: 295– 29. Anderson TP. Salo P. Stralin K. antigen test in children with nasopharyngeal pneumococcal carriage. 20. cocci and organisms allied to s. Estrella B. J Infect Dis 1995. Scand J Infect Dis 2007. 275: 134–141. Smith MA. 39: 645–648. Fine MJ. Mitis harboring s. 28. 1): 42–45. Bhanji S. 68: 1374–1382. Evaluation of a PCR clinical relevance of detection of pneumococcal DNA in sera of chil- assay for detection of Streptococcus pneumoniae in respiratory and dren by PCR. Assessment of the Binax now Streptococcus pneumoniae urinary 23. 36: 669–673. Mancl L. 24. Shriker O. A meta-analysis. Genetic relationships pneumococcal infection by PCR amplification of Streptococcus pneumo. Infect Immun 2000. 112: 713–727. patients with community-acquired pneumonia. Kaijalainen T. Eur Arch Paediatr Dent 2007. Percival RS. Sheppard CL. Hazan I et al. Prognosis and outcomes of bacteraemia associated with tooth brushing using conventional. Verhelst R. Misra S. Dagan R. Comparison of five geno- cal pneumonia by amplification of pneumolysin gene fragment in typic techniques for identification of optochin-resistant pneumococ- serum. Carson CA et al. Diagnosis of invasive 26. CMI. Elwood T. 1135–1141 . brush with a conventional toothbrush. Leinonen M. Clin Infect Dis 2002. oralis. J Clin Microbiol 1998. Ortqvist A. 8 (Suppl 1996. APMIS 2004. Egas J. MacLeod WB. 6: 221–227. Prospective study to determine 22. Hamer DH. elec. Pickerill AP et al. Williams B. Transient bactere.CMI Abdeldaim et al. Murdoch DR. Plasma-PCR for S. Diagnosis of bacteremic pneumococ. cus-like isolates. 41: 63–66. Murdoch DR. between clinical isolates of Streptococcus pneumoniae. mia induced by toothbrushing a comparison of the sonicare tooth. 34: 1025–1028. Sheller B. A pilot study to assess 30. F. nonrespiratory samples from adults with community-acquired pneu. JAMA tric or ultrasonic toothbrushes. Harrison TG. Kearns AM et al. 171: 479–482. Streptococcus niae genomic fragments in blood: a multi-centre comparative study. 25. Duggal MS.