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Streptococcus pneumoniae is the chief cause of community-acquired pneumonia (CAP) . 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 , and can be below 1% if blood samples are obtained during antimicrobial treatment .
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 . 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.
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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 , 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  and others  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 . 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 . 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 , 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 , as well as a positive S. pneumoniae urinary antigen test . 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 . 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 . 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 .
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.