Background: Although cerebrospinal fluid (CSF) culture is the diagnostic reference standard for bacterial\r\nmeningitis, its sensitivity is limited, particularly when antibiotics were previously administered. CSF Gram staining\r\nand real-time PCR are theoretically less affected by antibiotics; however, it is difficult to evaluate these tests with an\r\nimperfect reference standard.\r\nMethods and findings: CSF from patients with suspected meningitis from Salvador, Brazil were tested with culture,\r\nGram stain, and real-time PCR using S. pneumoniae, N. meningitidis, and H. influenzae specific primers and probes.\r\nAn antibiotic detection disk bioassay was used to test for the presence of antibiotic activity in CSF. The diagnostic\r\naccuracy of tests were evaluated using multiple methods, including direct evaluation of Gram stain and real-time\r\nPCR against CSF culture, evaluation of real-time PCR against a composite reference standard, and latent class\r\nanalysis modeling to evaluate all three tests simultaneously.\r\nResults: Among 451 CSF specimens, 80 (17.7%) had culture isolation of one of the three pathogens (40 S. pneumoniae,\r\n36 N. meningitidis, and 4 H. influenzae), and 113 (25.1%) were real-time PCR positive (51 S. pneumoniae, 57 N.\r\nmeningitidis, and 5 H. influenzae). Compared to culture, real-time PCR sensitivity and specificity were 95.0% and 90.0%,\r\nrespectively. In a latent class analysis model, the sensitivity and specificity estimates were: culture, 81.3% and 99.7%;\r\nGram stain, 98.2% and 98.7%; and real-time PCR, 95.7% and 94.3%, respectively. Gram stain and real-time PCR sensitivity\r\ndid not change significantly when there was antibiotic activity in the CSF.\r\nConclusion: Real-time PCR and Gram stain were highly accurate in diagnosing meningitis caused by S. pneumoniae,\r\nN. meningitidis, and H. influenzae, though there were few cases of H. influenzae. Furthermore, real-time PCR and Gram\r\nstaining were less affected by antibiotic presence and might be useful when antibiotics were previously administered.\r\nGram staining, which is inexpensive and commonly available, should be encouraged in all clinical settings.
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