Bacterial meningitis is a life-threatening disease which requires rapid diagnosis and antimicrobial therapy. Novel molecular methods offer the opportunity to establish a quick and reliable diagnosis. We evaluated real-time PCR and FISH for their applicability to the diagnosis of bacterial meningitis.
For PCR analysis, a real-time LightCycler approach was chosen to maximize the speed of diagnostics. A eubacterial broad-range PCR assay, based on a previously published primer pair (5
), was adapted to a real-time LightCycler PCR protocol using SYBR green as fluorescent dye. In comparison to specific hybridization probes, SYBR green offered the advantage that its fluorescence interferes to a lesser extent with sequencing reactions and thus allowed direct sequencing of the LightCycler PCR products. The eubacterial SYBR green PCR proved highly sensitive for the diagnosis of bacterial DNA in CSF samples. Due to this high analytical sensitivity of the eubacterial PCR and the ubiquity of bacterial DNA in reagents and plastic materials, etc., a background level of DNA was detected in most clinical samples. Therefore, we performed a ROC analysis of the crossing points of the measurements and determined a cutoff level for interpretation of samples. By applying this cutoff value, we even detected meningeal pathogens in three microscopy- and culture-negative clinical samples. Considering the clinical symptoms of the patients and the finding of granulocytes in the CSF, these results can be regarded as true positives. False-positive results were obtained in only 2/113 (1.8%) samples. Previously published hybridization probe LightCycler PCR assays (30
) were, in addition, evaluated in terms of their utility to detect bacterial pathogens in CSF samples for the first time (Tables and ). The analytical sensitivity of the specific PCR assays in CSF was comparable to that previously published for DNA preparations in physiological saline (30
). Among all assays, the Staphylococcus
spp.-specific PCR and the S aureus
-specific PCR had the lowest analytical sensitivities. This may be a disadvantage of the assays, but, on the other hand, it lowers the risk of false-positive results due to traces of contaminating staphylococcal DNA. However, due to the low sensitivity of the S. aureus
-specific PCR, three CSF samples with negative microscopy but cultural detection of S. aureus
had false-negative results with the S. aureus
-specific PCR assay (Table ). Nevertheless, in microscopy-positive samples, all specific PCR assays showed a diagnostic sensitivity of 100%, detecting all respective pathogens.
FISH has already been successfully implemented for the rapid detection and differentiation of various pathogens (7
). It had, however, not been evaluated on CSF samples before, probably because no probes for N. meningitidis
had been available. In this study, a probe for N. meningitidis
was newly introduced. Because the previously published probes for E. coli
) showed rather weak fluorescence in preliminary experiments (data not shown), a new probe with strong binding activity was designed by consulting a publication on the accessibility of the ribosome (4
). The newly designed probe showed bright fluorescence and only cross-reacted with Shigella
species, which is inevitable, since Shigella
and E. coli
have identical 16S rRNA sequences. Due to the extreme rareness of Shigella
as a cause of meningitis and the similar therapeutic options, this cross-reactivity is not regarded as relevant for the investigation of CSF. Also, with regard to sensitivity, we decided to implement PNA probes for the detection of staphylococci. FISH of staphylococci with DNA probes requires extensive permeabilization treatments and only stained about 50% of the bacterial cells (data not shown). PNA probes penetrate the cell walls more easily, since they are electrically neutral and shorter. In addition to a previously published probe for S. aureus
, we designed a new genus-specific probe for staphylococci. This probe has only one mismatch to some Bacillus
spp. and therefore cross-reacts with these bacteria. This cross-reactivity was regarded as not relevant, since staphylococci and Bacillus
clearly differ in their Gram stain appearance.
The analytical sensitivity of the FISH method was comparable to that of the Gram stain, which had been expected, since both are slide-based techniques.
A eubacterial probe on a PNA base (Bac-Uni1) was chosen for the investigation of microscopy- and culture-negative samples to cover all bacteria, including those with a thick cell wall, like staphylococci. As expected from the experiments on the analytical sensitivity, the method did not allow the recognition of otherwise undetected pathogens. However, it did not produce any false-positive results, stressing the specificity of the FISH method.
When evaluating microscopy- and culture-positive CSF samples by FISH, 13 of 18 samples were judged positive (Table ; Fig. ). In the remaining samples, no bacteria were detected, neither with DAPI nor FISH staining. It is possible that the missed bacteria had a low metabolic activity and therefore a low ribosome content, leading to only faint FISH signals. The negative DNA stain, however, argues for the hypothesis that too-low numbers of bacteria were present on these slides. Thus, the main reason for the relatively high number of FISH-negative samples is probably the uneven distribution of bacteria on the different slides. This is further supported by the fact that all positive samples missed by FISH showed only a low concentration of bacteria on the Gram stain. In addition, FISH was positive in three of four samples that were negative by Gram stain but positive by culture (Table ).
As a conclusion from this work, we propose an approach for the rapid diagnosis of meningitis that combines FISH and PCR based on the Gram stain. FISH is the method of choice for samples in which multiple bacteria are detected by Gram staining. In these cases, FISH allows the identification of the pathogen within 2 h. Advantages of the FISH technique are the low price and the little demands concerning technical equipment compared to PCR, which requires costly reagents and instruments, such as the LightCycler. The low price also makes FISH interesting for small laboratories, even in less industrialized countries. Due to its relatively low diagnostic sensitivity, the FISH method cannot be recommended for the work-up of samples with a low bacterial load that show no or very few bacteria in the Gram stain. Such cases can rapidly be solved by real-time PCR with very high sensitivity and specificity. For cases with low numbers of bacteria on the Gram stain, we propose implementing specific PCR assays according to the result of the Gram stain according to the algorithm used in this study (Table ). In cases of negative microscopy, the eubacterial PCR may be combined with the specific PCR for N. meningitis
to allow a quick decision on therapy and on prophylaxis of contact persons. Thus, two cases of meningococcal meningitis but negative Gram stain results (Table ) could be detected in this study. The eubacterial PCR allows further identification of bacterial DNA due to sequencing of the PCR product, as shown previously (28
) and in this study. Additionally, the eubacterial PCR can be used for the identification of bacteria that are not covered by the specific primers and probes. Taken together, PCR and FISH have the potential to considerably accelerate the diagnosis of bacterial meningitis. To benefit from the different advantages of PCR and FISH, both methods should be combined in a reasonable way.