Culture of normally sterile body fluids is generally considered to be very specific but can suffer from lack of sensitivity, particularly in the presence of antibiotics. This phenomenon can make the diagnostic test appear to be less specific than it truly is when culture negative specimens are included. We attempted to circumvent this problem by measuring specificity first by using only specimens that were CP-358774 culture positive for another pathogen and second after correcting for what appeared to be false positive RT-PCR results by testing these false positives using a second gene target. In this study, the calculated specificities were very high using both approaches, which makes us confident that we have accurately assessed the true specificity of the assay. The results of this study suggest that surveillance programs that use RT-PCR for diagnosis of bacterial meningitis can establish WBC count cut-offs that optimize both detection of cases and utilization of laboratory resources. For example,RAD001 a large number of samples could be excluded without missing a substantial proportion of bacterial meningitis cases using WBC count cut-offs of, for example, 500 or 1000. Programs with patient characteristics that differ from those in our study should consider similar analyses to determine the optimal cut-offs for their setting. We were able to determine the meningococcal group among culture-negative cases using a PCR-based assay; a similar approach is available for H. influenzae. We are also exploring non-culture approaches for determining pneumococcal serotype. These serotype and serogroup data are important for monitoring the impact of the conjugate vaccines that are currently being used in Brazil. We found that the most important risk factor for being culture negative/RT-PCR positive was presence of antibiotic in CSF, which has been previously described. This finding is not surprising because antibiotics are widely available over-thecounter in Brazil. In addition, it is likely that some patients were given antibiotics by healthcare workers before CSF and/or blood were obtained for culture. We suspect that the finding that being from one of three hospitals was a risk factor for being culture negative, RT-PCR positive was due to residual confounding because these hospitals had the highest rates of CSF specimens with the presence of antibiotic.