Newer nucleic acid techniques, particularly real-time PCR, offer an opportunity to readdress the problem of the diagnosis of S. pneumoniae disease. In the present study we developed three new S. pneumoniae-specific real-time PCRs and demonstrated their abilities to detect S. pneumoniae DNA from cultures and three different types of clinical samples. These new assays were compared with each other and with two previously described real-time PCRs, with promising results.
The LLDs for all five assays were excellent. All demonstrated a linear detection range of 6 orders of magnitude and gave positive amplifications signals with all S. pneumoniae
serotypes and nontypeable S. pneumoniae
strains tested. Specificity evaluations, however, indicated differences among the assays. None of the five assays amplified any of the nonstreptococcal species tested, but in contrast, the specificity was problematic for both ply
real-time PCRs when the amplification of S. pseudopneumoniae
and other isolates of P-LVS was evaluated; these assays lacked sufficient specificity and amplified these S. pneumoniae
-related strains. Several researchers have investigated and reported on atypical alpha-hemolytic streptococci and their likenesses in their phenotypic and genotypic properties to S. pneumoniae
). Arbique et al. (3
) described S. pseudopneumoniae
species and thoroughly discussed the problems posed for S. pneumoniae
disease diagnostics due to both the detection of the ply
and the lytA
genes in these organisms and also the high degree of sequence similarity found in the rRNA gene which is used in the GeneProbe AccuProbe Pneumococcus identification test. The present study added five more isolates of S. pseudopneumoniae
whose identities were confirmed by DNA-DNA reassociation to the original ones described by Arbique et al. (3
), and again the rRNA gene sequence region chosen by the GenProbe investigators for their test failed to discriminate between S. pneumoniae
and S. pseudopneumoniae
. The detection of ply
in several P-LVS isolates and the detection of psaA
in S. pseudopneumoniae
isolates in this study reinforce the call for caution in choosing sequence regions when these genes are used for the development of diagnostic assays. In nature, a taxonomic situation exists in which many mosaic organisms that do not clearly fit any particular species is evolving over time, and this could explain the detection of these genes in pneumococcus-like organisms (3
). The role of P-LVS, if any, in disease has yet to be elucidated. However, a recent publication by Keith et al. (17
) describing a study of patients with chronic obstructive pulmonary disease (COPD) suggests that the isolation of S. pseudopneumoniae
from sputum specimens was associated with both a history of COPD and the exacerbation of COPD (17
). The increasing ability of laboratories to identify this species will help to elucidate its prevalence and clinical relevance. It is difficult to say if the lack of the ability to discriminate between them may have affected previous clinical studies on the detection and identification of S. pneumoniae
, but this cannot be ruled out.
This study described rapid, reliable assays that clearly discriminate S. pseudopneumoniae
and other P-LVS from S. pneumoniae
. The P-LVS isolates in our specificity panel were intentionally selected because of the difficulty in classifying them, and thus, their use provides a strict criterion for assay specificity determinations. Both the lytA
-CDC and the psaA
real-time PCRs were highly specific, showing no amplification with P-LVS isolates. The psaA
real-time PCR was slightly less specific, amplifying two of the S. pseudopneumoniae
isolates. These results correlate with those of an earlier study that used conventional PCR, showing the utility of these genes in discriminating S. pneumoniae
). Undoubtedly, the increasing incidence of isolation of P-LVS is certain to complicate diagnosis and create additional obstacles for pneumococcal assay design; therefore, their inclusion may be required in future specificity evaluations for pneumococcal assay development.
The clinical performance of these pneumococcal real-time PCR assays was assessed with a limited number of samples of three different clinical specimen types. Our studies and those of others indicate that the choice of specimen comes with its own set of problems and that many different factors affect the sensitivities of the assays. Overall, PCR of blood and blood fractions has been reported to be very unpredictable and challenging due to the presence of inhibitors in blood and the low numbers of pneumococci (10
). In previous studies, others have shown that the rate of positivity is greater by PCR than by culture and have suggested that PCR is more sensitive (10
). In our analysis of pneumococcal culture-positive serum specimens, the rates of positivity by the five real-time PCRs correlated well among the various assay but were lower than expected compared with the culture results. There are several possible explanations for this. These include delays in processing and storage of specimens, the drawing of blood at times different from those at which blood for culture was drawn, and the presence of low levels of inhibitors. Additionally, while viable organisms are not required for PCR amplification, the length of time after the initiation of antibiotic treatment and specimen collection may affect the reactivity of the PCR, as reported by Dagan et al. (10
). Other PCR studies that have evaluated blood have cited one or more of these reasons for discordant results (28
). For the specimens evaluated in this study, it is possible that both the storage conditions and the inability to perform PCR at the same time that the blood culture was performed may be factors. Thus, a prospective study with serum specimens collected and stored specifically for PCR would be a better indicator of the true value of these real-time PCRs with serum specimens. Use of MEF and CSF specimens, on the other hand, proved to be 100% sensitive, possibly reflecting the lack of or the presence of lower levels of inhibitors or the ease of removal of inhibitors during extraction and the higher bacterial counts generally seen in MEF specimens than in blood.
Among the culture-negative samples of all three specimen types (serum, MEF, and CSF), some real-time PCR amplification of the samples occurred. Again, this occurred in most cases with identical samples, and there was close to 100% agreement between all five assays, with one or two specimen outliers. In cases in which this occurred, the average of the CT
value was >38, which is very close to the assay CT
limit of 40. The amplification of samples that are culture negative has been reported in previous publications and has been attributed to one or more of the following: (i) the superior sensitivity of PCR methods over that of culture, (ii) amplification of viridans group streptococcus-related sequences, (iii) detection of bacterial DNA from dead organisms, or (iv) contamination (15
). The fact that we detected S. pneumoniae
in MEF specimens that were culture positive only for H. influenzae
is not surprising; other investigators have reported that up to approximately 24% of MEF samples from patients result in mixed S. pneumoniae
culture isolations (4
). Thus, another plausible explanation for these S. pneumoniae
culture-negative, PCR-positive MEF specimens is dual infection not revealed by culture, for even though S. pneumoniae
was undetectable by culture, its presence and/or the presence of its DNA cannot be ruled out. Additionally, S. pneumoniae
has previously been detected in healthy children by PCR (for the ply
gene) of serum samples and was attributed to the carriage of S. pneumoniae
), which might be doubtful, since ply
gene PCRs have been found to be nonspecific (21
). In our study, the high specificities of the lytA
assays make it highly unlikely that atypical alpha-hemolytic streptococci are responsible for the amplification of these culture-negative clinical samples. In addition, our strict laboratory methods regarding PCR procedures and the fact that the same samples that were positive by one assay were positive by the other assays lend support to the true-positive results for these samples and not positive results due to contamination by PCR amplicons. Moreover, with samples from nonsterile sites such as MEF and the difficulty of pneumococcal culture, the real-time PCR positivity of culture negative specimens is not unexpected. Therefore, we suspect that amplification for these culture-negative specimens is most likely indicative of the greater sensitivity of real-time PCR or the amplification of DNA still present in specimens from patients treated with antibiotics.
Despite the limited numbers of specimens tested, there was very good agreement among the results of the assays. The results with clinical samples were good overall and very promising. LLD estimations indicate that the real-time assays are highly sensitive, detecting at least 10 copies for ply and lytA. However, a true clinical assessment must await the execution of much larger, well-defined clinical studies with different types of specimens. Our results clearly demonstrate the enhanced specificities of the lytA-CDC and psaA assays for the detection of true S. pneumoniae strains. Although the two ply assays were shown to be very sensitive, they were much less specific and their use could be problematic, especially if they were applied to the analysis of specimens from nonsterile sites. The true value of real-time PCR for the diagnosis of pneumococcal disease is not yet firmly established, and thus, no one assay is routinely used. The newly developed assays described here present the opportunity to use not only assays with high sensitivities but also assays that have improved specificities compared with those of the assays currently in use. The improvement in specificity allows their use with specimens from nonsterile sites as well as sterile sites, making them suitable for use for diagnosis and in carriage studies.
Currently, the trend is to use multiplex assays for the simultaneous detection of various pathogens, providing a savings in time and money. We have done this for a single pathogen but propose that our lytA-CDC or psaA primer/probe sets would provide high specificity in an array for respiratory pathogen detection multiplexed with primers and probes for the detection of other respiratory pathogens. This potential for multiplexing and the speed of performance make these assays promising tools for molecular detection and epidemiologic carriage studies. This technology should offer an added advantage when it is used in conjunction with other assays for pneumococcal disease diagnosis.