In the present study we evaluated broad-range 16S rDNA PCR and subsequent sequencing in diagnosis of bacterial meningitis in a clinical setting. Our results show that the molecular findings were in good agreement with those obtained from direct microscopy and bacterial culture. The results were in complete concordance for 206 CSF specimens (91%), including 184 culture-negative and PCR-negative samples and 22 culture-positive and PCR-positive samples.
Our broad-range 16S rDNA PCR proved to be extremely valuable in detecting bacterial meningitis when antibiotics had been started prior to lumbar puncture. For five CSF samples obtained after the start of antimicrobial therapy, a significant bacterial pathogen (S. pneumoniae
[three times], S. agalactiae
[one time], and N. meningitidis
[one time]) was identified by broad-range 16S rDNA PCR, whereas the culture results remained negative. These data confirm the findings of earlier studies (10
Our broad-range 16S rDNA PCR also proved of value in the case of a 2.5-month-old boy with clinical symptoms of sepsis and meningitis. P. bivia
was found in the CSF of this child, while conventional methods remained negative, since anaerobic culture is not part of the standard operation procedure for detecting bacterial meningitis in most microbiological laboratories. The boy was treated empirically with ampicillin and ceftazidime and recovered completely. Recently, isolation of P. bivia
from CSF in a critical ill young patient has been described (8
In two samples, both PCR and culture results were positive, but the microorganism found was different for each method. The first discrepant identification involved a CSF sample from which N. meningitidis
was isolated. The sequence obtained after broad-range bacterial PCR showed clear signs of a mixed sequence in its electropherograms. This sequence was analyzed with the algorithms from the both BLAST and RDP-II databases. The BLAST search resulted in a Lactococcus
species, most closely related to L. lactis
(87.8%), with N. meningitidis
(86.5%) as the next possible match, whereas the RDP-II search resulted in a Neisseria
species (percent similarity values, 0.925 to 0.923). Upon manual comparison, >99% of the sequences from both L. lactis
and N. meningitidis
could be retrieved within the electropherogram. Finally, to confirm the previous findings, the original sequence was also subjected to a third search algorithm, SSEARCH from the Software and Tools for Genome Analysis website (http://www-btls.jst.go.jp/
), resulting in N. meningitidis.
Although lactococci have recently been associated with human disease (1
), we concluded that, based upon the clinical evaluation, the lactococcal DNA was most likely a contaminant introduced at the time of lumbar puncture. The second discrepant result was the detection of F. nucleatum
in the CSF of a 13-year-old girl presenting with mild symptoms of bacterial meningitis. Culture of the CSF yielded N. meningitidis
, and the child was treated accordantly. The detection of F. nucleatum
did not match the clinical presentation of the patient, even though all controls included were valid. Therefore, the original CSF sample, as well as the original DNA isolate, was retested, resulting in the identification of N. meningitidis
for both samples. Resequencing the original PCR product again yielded F. nucleatum
as in the first analysis, confirming that the first PCR had to be contaminated. This case emphasizes that all laboratory findings should always be placed in perspective with the clinical symptoms, even when all controls to prevent false-positive results make a run valid.
One of the shortcomings of our approach seems to be the sensitivity in the lower range of bacterial loads encountered in bacterial meningitis. A total of 13 culture-positive CSF samples remained negative by broad-range 16S rDNA PCR. In four of these samples it was evident that a bacterial pathogen was present, whereas the others were considered to be contaminants of bacterial culture on clinical grounds. All of these samples, which had low numbers of colonies on primary plates, were estimated to have bacterial loads below our limit of detection. However, it should be emphasized that the sensitivity of our assay (100 CFU/ml of CSF) should be sufficient for detecting the majority of bacterial meningitis cases, since La Scolea et al. showed that only 16% of culture-positive CSF samples in bacterial meningitis have bacterial loads of <1,000 CFU/ml (32
The overall performance of our assay, compared to bacterial culture, is in line with other published broad-range PCR methods (30
), with an overall sensitivity of 86%, a specificity of 97%, a PPV of 80%, and an NPV of 98%. It should be emphasized that the relatively low PPV is at least partly explained by the inadequacy of bacterial culture: in six PCR-positive, culture-negative CSF samples a significant bacterial pathogen was identified in a patient with all of the signs of bacterial meningitis (Table ).
The molecular method validated here is not only suitable for the detection of bacteria in CSF but can also be used for the detection of bacteria in many other clinical samples from anatomic sites that are considered to be sterile in healthy subjects. We have demonstrated that we can detect clinically significant bacteria in other clinical samples, such as blood culture fluids, a brain abscess, a liver abscess, pus from a vertebra, synovial fluids, and pericardial fluid, for which conventional methods could not (data not shown).
In conclusion, our broad-range 16S rDNA PCR with subsequent sequencing has proven to be a valuable supplementary test in daily clinical practice. Bacterial culture of CSF remains the cornerstone in the diagnosis of bacterial meningitis. However, especially when antimicrobial therapy has already started at the time of lumbar puncture, conventional methods should always be accompanied by molecular detection, since the sensitivity of direct microscopic examination and bacterial culture drops substantially when therapy has started. We are currently adapting the standard procedures in our laboratory for the detection of bacterial meningitis; broad-range 16S rDNA detection is indicated when antimicrobial therapy has already been started at the time of lumbar puncture or when cultures remain negative, although the suspicion of bacterial meningitis remains.