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Keywords:

  • lytA gene;
  • plasma;
  • ply gene;
  • pneumococcal pneumonia;
  • real-time PCR;
  • Spn9802 fragment;
  • Streptococcus pneumoniae

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Transparency Declaration
  8. References

Clin Microbiol Infect 2010; 16: 1135–1141

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 pneumococcal 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.


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Transparency Declaration
  8. References

Streptococcus pneumoniae is the chief cause of community-acquired pneumonia (CAP) [1]. The diagnosis of pneumococcal pneumonia is hindered by the lack of a highly sensitive and specific ‘gold standard’ method. Culture of sputum and nasopharyngeal secretions is controversial as a result of respiratory tract carriage of pneumococci. Bronchoalveolar lavage or transthoracic needle aspiration are considered to be reliable but they are invasive and cannot be performed routinely. Identification of the bacterium in blood culture provides a definite diagnosis and can serve as an indicator of disease severity. However, the positivity rate of blood cultures rarely exceeds 10% in CAP [2], and can be below 1% if blood samples are obtained during antimicrobial treatment [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 (Roche Diagnostics, Mannheim, Germany) and SepsiTest (Molzym, Bremen, Germany) use conserved PCR targets such as the internal transcribed spacer region and 16S rRNA, for genes that are common to all bacteria. This is a promising strategy for pathogens such as Staphylococcus aureus and Gram-negative enteric bacilli. However, it is difficult for PCR against common bacterial genes to distinguish between S. pneumoniae and alpha-haemolytic streptococci because they are closely related [4,5]. Another target, the DNA fragment Spn9802, has an unknown function, but has been described as a specific target for S. pneumoniae [6]. In the present study, we aimed to evaluate the performance of PCR of three different gene targets of S. pneumoniae (pneumolysin, ply; autolysin, lytA; and the DNA fragment Spn9802), aiming to identify a PCR assay that could be used to detect S. pneumoniae DNA in blood samples from acutely febrile patients.

Materials and Methods

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Transparency Declaration
  8. References

Patients

At the 35-bed Department of Infectious Diseases, Örebro University Hospital, Örebro, Sweden, patients with CAP were enrolled in a prospective study running from November 1999 until April 2002 [7]. The criteria for CAP were: acute illness, radiological signs of pulmonary consolidation, at least two of five signs and symptoms of pneumonia (fever >38°C, dyspnea, cough, pleuritic chest pain, and abnormal lung auscultation), but no hospitalization during the preceding month.

From 235 enrolled patients (median age 71 years) who fulfilled the CAP criteria, an EDTA plasma sample was available in 92 cases where blood culture was also performed. There was no systematic bias selection of patients who provided plasma. These patients were included in the present study.

The 92 patients had a median age of 70 (range 19–93) years; 44 (48%) were females, and 19 (21%) were current smokers. A comorbid illness was noted in 41 patients (45%), including 14 with chronic obstructive pulmonary disease. A Pneumonia Severity Index Risk Class of IV-V was noted in 32 cases (35%), and a CURB-65 score of 3–5 was noted in 17 (18%). Eight patients (9%) were treated in the intensive care unit, and two patients (2%) died within 30 days of hospitalization.

During the study period, adult control patients hospitalized for skin infection, urinary tract infection, arthritis/spondylitis, or planned orthopaedic/urological surgery, but without respiratory symptoms on admission, were enrolled [7].

From 113 enrolled controls, EDTA plasma samples were available from 91 (median age 67 years, range 33–87 years), and these 91 were used in the present study.

Microbiological analysis

Two blood cultures, with two bottles in each, were collected from all patients and controls. Blood culture I was collected at the emergency department and blood culture II was collected on the ward. A Bactec nonradiometric system, 9240 (Becton Dickinson, Sparks, MD, USA), was used for blood culturing. The pneumococcal strains isolated from blood were serotyped by using type-specific pneumococcal rabbit antisera (Statens Serum Institut, Copenhagen, Denmark). Sputum samples were obtained from the CAP patients. Samples with more than five leukocytes per epithelial cell were deemed representative and were subjected to culture. Nasopharyngeal aspirates where obtained from both CAP patients and controls. These aspirates (10 μL) and a diluted aliquot of sputum, giving a detection level of 2 × 105 colony-forming units/mL sputum, were cultured on blood agar with gentian violet. The plates were incubated for 24–48 h at 37°C in 5% carbon dioxide, and bacterial species were identified by standard microbiological methods.

Urinary antigen test

The Binax NOW immunochromatographic test for S. pneumoniae (Binax, Portland, ME, USA) was performed on nonconcentrated urine from the CAP patients and controls.

DNA extraction

Together with blood culture II, an EDTA sample was collected and centrifuged to obtain plasma. After storage at −70°C, the plasma samples (400 μL) were subjected to DNA extraction with the automatic NucliSens easyMAG instrument (bioMérieux, Marcy l’Etoile, France). The sample was eluted in 25 μL of elution buffer. Purified DNA was then run in a duplex real-time PCR for the ply gene and the Spn9802 fragment and a real-time PCR for the lytA gene of S. pneumoniae.

Spn9802 PCR and ply PCR

Spn9802 has been shown as a specific target for S. pneumoniae [6] and we have previously developed a method that also enables detection of S. pseudopneumoniae [8]. This real-time PCR was combined with ply real-time PCR [9] and run as a duplex real-time PCR. The primers and probes are shown in Table 1. This duplex PCR was run under the same thermal conditions as previously described for the ply PCR [10]. As a control for amplification of the specific target and as a standard for quantification, DNA preparations of 500, 2000 and 10 000 bacterial genomes per PCR reaction (reference strain S. pneumoniae CCUG 28588T) were used. A well with a negative control (water) was also used for each reaction mix.

Table 1.   Sequences of oligonucleotide primers and probes for detection of Streptococcus pneumoniae
 Sequences (5′ to 3′)aPositions in target geneb
  1. aPositions with lower case letters indicate a locked nucleic acid [28].

  2. bAccession numbers: Spn9802; AE008434, lytA; NC_003098 and ply; X52474.

Spn9802 F5′-AGTCGTTCCAAGGTAACAAGTCT-3′3370 to 3392
Spn9802 R5′-ACCAACTCGACCACCTCTTT-3′3525 to 3506
Spn9802 FAM5′-FAM-aTcAGaTTgCTgATaAAaCgA-BHQ1-′3 
lytA F5′-CAGCGGTTGAACTGATTGA-′3251 to 269
lytA R5′-TGGTTGGTTATTCGTGCAA-′3423 to 405
lytA P15′-GAAAACGCTTGATACAGGGAGTT-FL-′3 
lytA P25′-LCRed640-AGCTGGAATTAAAACGCACGAG-PH-′3 
ply F5′-TGCAGAGCGTCCTTTGGTCTAT-′3894 to 915
ply R5′-CTCTTACTCGTGGTTTCCAACTTG-′3974 to 950
ply Cy55′-Cy5-TGGCGCCCATAAGCAACACTCGAA-BHQ1-′3 

lytA PCR

In the lytA real-time PCR, the gene was amplified using the LightCycler 2.0 system using LightCycler® Software, version 4.1 (Roche Diagnostics) with probe detection (Table 1). The optimized real-time PCR amplifications were performed in a 20-μL reaction volume, containing 0.7 and 0.5 μM of forward and reverse primers [11] (Scandinavian Gene Synthesis, Köping, Sweden), respectively, 0.3 and 0.2 μM of the probes P1 and P2 (TIB MOLBIOL Syntheselabor, Berlin, Germany), respectively, 3.5 mM MgCl2, and 2 μL of LightCycler Faststart Reaction Mix Hybridization Probes (Roche Diagnostics). The assay used 5 μL of target DNA. The PCR program includes an enzyme activation step at 95°C for 10 min, followed by 45 cycles at 95°C for 5 s, 60°C for 20 s (single measurement of fluorescence) and 72°C for 8 s. As a positive control for the target specific PCRs, DNA preparations of 100–1000 bacterial genomes per PCR (reference strain S. pneumoniae CCUG 36696) were used as well as a negative control for each reaction mix.

Ethics

The study was approved by the Ethics Committee of Örebro County Council. All participants provided their informed consent.

Results

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Transparency Declaration
  8. References

Conventional diagnostic methods

Among the 92 CAP patients, S. pneumoniae was identified by blood culture in ten patients (11%), by urinary antigen test in 24/86 (28%) patients tested, by sputum culture in 13/48 (27%) of patients with representative sputum sample available, and by culturing nasopharyngeal aspirate in 28/84 (33%) patients tested. S. pneumoniae was detected by urinary antigen test and/or sputum culture in 31 cases, including the ten patients with pneumococcal bacteraemia. Thus, there were ten patients with bacteraemic pneumococcal pneumonia and 21 with nonbacteraemic pneumococcal pneumonia. The remaining 61 CAP were deemed to have nonpneumococcal pneumonia.

In the control group, S. pneumoniae was identified by urinary antigen test in two of 89 (2%) of patients tested and by nasopharyngeal aspirate culture in four of 91 (4%) patients tested.

Real-time PCR for ply, Spn9802, and lytA

Table 2 shows the detection rates of PCR for ply, Spn9802, and lytA in the CAP patients and controls. lytA and Spn9802 were detected predominantly in cases with bacteraemic pneumococcal pneumonia, whereas ply was also detected in several patients with nonpneumococcal pneumonia and among the controls.

Table 2.   Detection rates by PCR with three different gene targets of Streptococcus pneumoniae applied to EDTA blood samples from 92 pneumonia patients and 91 adult controls without respiratory symptoms
PCR gene targetPneumonia patientsControls (= 91) Number positive (%)
Bacteraemic pneumococcal pneumonia (= 10) Number positive (%)Nonbacteraemic pneumococcal pneumoniaa (= 21) Number positive (%)Nonpneumococcal pneumoniab (= 61) Number positive (%)
  1. aBlood culture negative but S. pneumoniae detected by culture of representative sputum or by urinary antigen test.

  2. bS. pneumoniae not detected by blood culture, culture of representative sputum, or urinary antigen test.

ply7 (70)6 (29)6 (10)8 (9)
Spn98026 (60)4 (19)1 (2)0
lytA7 (70)1 (5)00

Table 3 shows the results of the individual CAP patients with positive blood culture and/or positive PCR. Among the ten patients with bacteraemic pneumococcal pneumonia, six were positive by all three PCR methods, whereas two were negative with all three PCR methods. No PCR inhibition was seen in these latter two samples when retested with spiked pneumococcal DNA. To study the presence of the three genes in the bacteraemic isolates, we tested the ten S. pneumoniae isolates with the PCR protocols. All ten isolates were positive for ply, Spn9802 and lytA (details not shown).

Table 3.   Community-acquired pneumonia patients with positive blood culture and/or positive PCR for Streptococcus pneumoniae applied to blood samples
Sex, ageBlood culture result and serotypelytA PCR (copies/mL)Spn9802 PCR (copies/mL)ply PCR (copies/mL)S. pneumoniae urinary antigen testSputum culture for S. pneumoniaeCulture of nasopharyngeal aspirate for S. pneumoniae
  1. ND, not done; ‘−’, negative.

F, 83S. pneumoniae 19F9 × 1081 × 10104 × 109+NDND
F, 79S. pneumoniae 9V2 × 1083 × 1091 × 109+NDND
M, 91S. pneumoniae 23F6 × 1022 × 1042 × 104+ND+
F, 89S. pneumoniae 7F2 × 1022 × 1041 × 104+ND+
M, 54S. pneumoniae 33 × 1025 × 1038 × 103+++
F, 74S. pneumoniae 7 F2 × 1021 × 1031 × 103+NDND
F, 84S. pneumoniae 23F8 × 10100+++
F, 57S. pneumoniae 7F003 × 102+ND+
M, 31S. pneumoniae 7F000+ND+
F, 85S. pneumoniae 23F000+ND
M, 231 × 1026 × 1031 × 104++
F, 7802 × 1036 × 101+NDND
M, 5806 × 1022 × 102++
M, 7402 × 1026 × 102+
M, 3104 × 1021 × 102+
M, 87009 × 102+ND+
F, 77003 × 102+++
M, 46006 × 102NDND
F, 69005 × 102ND
M, 51003 × 102
M, 75002 × 102
F, 32002 × 102ND

Among the bacteraemic cases, the DNA copy number per mL was similar for Spn9802 and ply, but was lower for lytA (Table 3). Spn9802 PCR was positive at low copy numbers (14–152 copies/reaction; 200–2000 copies/mL) in five patients with negative blood cultures. In four of these cases, S. pneumoniae was also detected by urinary antigen test or sputum culture. All samples positive by ply PCR alone had low copy numbers (<103 copies/mL).

In the control group, no subject was positive with lytA PCR or Spn9802 PCR, but eight subjects were positive with ply PCR at concentrations of 6 × 101 to 2 × 103 DNA copies/mL. In two of the ply PCR positive controls, other tests were positive for S. pneumoniae (urinary antigen and culture of nasopharyngeal aspirate (n = 1) and culture of nasopharyngeal aspirate (n = 1)). In the controls with all three PCR reactions negative, S. pneumoniae urinary antigen test was positive in one control and culture of nasopharyngeal aspirate was positive in two controls.

The diagnostic performance of the three PCR assays in the CAP cases is shown in Table 4. The sensitivities were low (26–42%) for the diagnosis of pneumococcal pneumonia, although the specificities were high for Spn9802 (98%) and lytA (100%). The sensitivities were higher (60–70%) for the diagnosis of bacteraemic pneumococcal pneumonia, and specificities remained high for Spn9802 (94%) and lytA (99%). Consequently, Spn9802 and lytA had high positive predictive values for pneumococcal pneumonia and high negative predictive values for bacteraemic pneumococcal pneumonia.

Table 4.   Performance of PCR for three different gene targets of Streptococcus pneumoniae applied to EDTA blood samples from 92 pneumonia patients
Reference groupPCR gene targetSensitivityaSpecificitybPositive predictive valuecNegative predictive valued
  1. aReported as percentage (number with positive PCR/number with the defined pneumonia diagnosis).

  2. bReported as percentage (number with negative PCR/number without the defined pneumonia diagnosis).

  3. cReported as percentage (number with the defined pneumonia diagnosis/number with positive PCR).

  4. dReported as percentage (number without the defined pneumonia diagnosis/number with negative PCR).

  5. eS. pneumoniae detected by blood culture, culture of representative sputum, or urinary antigen test.

Pneumococcal pneumoniaeply42 (13/31)90 (55/61)68 (13/19)75 (55/73)
Spn980232 (10/31)98 (60/61)91 (10/11)74 (60/81)
lytA26 (8/31)100 (61/61)100 (8/8)73 (61/84)
Bacteraemic pneumococcal pneumoniaply70 (7/10)85 (70/82)37 (7/19)96 (70/73)
Spn980260 (6/10)94 (77/82)55 (6/11)95 (77/81)
lytA70 (7/10)99 (81/82)88 (7/8)96 (81/84)

Test results in patients with prior antibiotic treatment

Antibiotics were administered prior to blood culturing in 27 cases. In these cases, the positive results for S. pneumoniae noted were: blood culture, urinary antigen test and the three PCR analyses (n = 1), urinary antigen test and the three PCR analyses (n = 1), urinary antigen test, Spn9802 PCR and ply PCR (n = 1), sputum culture alone (n = 1), Spn9802 PCR and ply PCR (n = 1), and ply PCR alone (n = 3). Thus, Spn9802 and/or lytA PCR were positive in three of 26 cases with prior antibiotics and negative blood culture.

Discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Transparency Declaration
  8. References

The present study tested the performance of three real-time PCR assays for the detection of S. pneumoniae in plasma samples from patients with CAP. In general, the sensitivity of the PCR assays was low for the detection of both pneumococcal pneumonia (26–42%) and pneumococcal bacteraemia (60–70%); similar figures have been reported in previous studies [11–13]. This is likely to have been a result of the sample volume used in the DNA extraction. We used 400 μL of plasma, and 5 μL (corresponding to 80 μL of the original plasma) of the purified DNA for PCR. The use of such a small volume limits the detection capacity, especially in cases of low-grade bacteraemia. In comparison, the blood volume in the four blood culture bottles was approximately 40 mL (4 × 10 mL), corresponding to approximately 20–25 mL of plasma. The problem with small sample volumes can be solved by processing a larger volume of blood for PCR, comprising a methodological challenge that has been elaborated in a commercial test system. However, no advantage of PCR detection of bacteria in blood has been reported, even with collection volumes was between 1.5 and 10 mL [14,15]. Methods for selective isolation of bacterial DNA from blood have also been reported [16], but it not yet clear what benefit such techniques provide.

Several studies have used ply PCR to the detect S. pneumoniae DNA in blood samples, with sensitivities in the range 35–100% compared to blood culture [12,17–20]. However, we [10] and others [21] have shown that ply PCR is unspecific for the detection of S. pneumoniae in respiratory secretions. In the present study, the ply gene PCR presumably caused false positivity also in blood, which is a site that is assumed to be ‘sterile’. Healthy adults with false-positive ply PCR with blood samples have been identified previously [17,19,22]. The cause is not known, although laboratory contamination with pneumococcal DNA was suspected [19]. The present study, with discrepant results between those obtained for ply PCR and the other two PCR assays in the control group, demonstrates that laboratory contamination with pneumococcal DNA is not likely to have been a significant cause of false-positive ply PCR results. It has been speculated that such false-positive results can also be caused by alpha-haemolytic streptococci [22]. These streptococci from normal oral flora can enter the bloodstream even after tooth brushing [23,24] and some species, such as Streptococcus mitis and Streptococcus oralis, have been reported to harbour the ply gene [25,26]. In the present study, however, all patients and controls with ply PCR positivity had blood cultures negative for alpha-haemolytic streptococci. Furthermore, in a recent study by our group [10], ply PCR positivity in bronchoalveolar lavage fluid was not correlated with culture positivity for alpha-haemolytic streptococci. Perhaps the detected ply DNA in plasma samples and respiratory secretions from patients with negative cultures originated from degraded bacteria introduced from the oral flora.

In healthy children, nasopharyngeal carriage of S. pneumoniae has been shown to give positive ply PCR results in blood samples [27], as well as a positive S. pneumoniae urinary antigen test [28]. To our knowledge, PCR for lytA and Spn9802 have not been tested on blood samples from paediatric patients and the impact of S. pneumoniae carriage on these tests has only rarely been tested on blood samples from adults. Among four adult controls with S. pneumoniae carriage in the present study, one had a positive urinary antigen test, two were positive with ply PCR and none were positive with lytA PCR or Spn9802 PCR .

As noted, the concentration of ply DNA was low in the patients with no other test positive for S. pneumoniae (Table 3) and in the controls. However, because of its low specificity, we consider that ply should not be used as gene target in PCR assays for S. pneumoniae.

Both lytA and Spn9802 have been found specific for S. pneumoniae [8,11]. The present study clearly shows that lytA PCR and Spn9802 PCR applied to plasma samples are specific for the detection of S. pneumoniae.

The differences in DNA copy numbers between lytA PCR and Spn9802 PCR (Table 3) are most likely to be a result of inter-method variability, showing that quantitative data should be interpreted with caution. However, the high positive predictive values of lytA PCR and Spn9802 PCR (Table 4) make them promising for use in clinical practice, alone or in a multiplex PCR assay for different bacteria. This could facilitate the rapid (<2.5 h) detection of pneumococcal infection in febrile patients. Thus, a patient with positive Spn9802 PCR or lytA PCR could be given suitable treatment at an early stage of the disease.

Detection of pneumococcal antigen in blood samples has been associated with disease severity in CAP [29]. The main reason for this is a high correlation with bacteraemia and the requirement for a large number of S. pneumoniae to give rise to detectable levels of polysaccharides in blood. Bacteraemia has been associated with mortality in CAP [30]. To our knowledge, there have been no studies that correlate disease severity with PCR for S. pneumoniae in blood samples. The present study was not designed to investigate this issue; however, the results obtained suggest that plasma PCR may provide prognostic information. A positive result of lytA PCR or Spn9802 PCR can be used for rapid diagnosis of bacteraemic pneumococcal pneumonia, which is a potentially severe condition [30].

A limitation of the present study is that there were few cases of bacteraemic pneumococcal pneumonia, although sufficient cases to demonstrate a performance of blood PCR similar to that in other studies of this condition [11,13]. There are few reports of the performance of blood-PCR to detect nonbacteraemic pneumococcal pneumonia. The present study demonstrates that blood PCR with small plasma volumes is not useful for the detection of nonbacteraemic pneumococcal pneumonia.

Culture remains the basic method for detection of pneumococci in routine clinical microbiology; it is cheap and enables antibiotic resistance determination. Real-time PCR assays based on specific targets such as lytA and Spn9802 are useful when a rapid analysis is required or when antibiotic treatment has been started prior to sampling.

The use of an unspecific PCR target such as ply should only be considered when there is a research interest with respect to finding strains of S. mitis or S. oralis that are closely related to S. pneumoniae.

We conclude that the detection of Spn9802 and lytA PCRs in plasma is useful for the rapid detection of bacteraemic pneumococcal pneumonia, whereas ply is not specific enough for this organism in blood samples.

Transparency Declaration

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Transparency Declaration
  8. References

The study was supported by funds from the Uppsala-Örebro Regional Research Council. The authors have no conflicting interests to declare.

References

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  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Transparency Declaration
  8. References
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