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RATIONALE: Early diagnosis and treatment of tuberculous meningitis saves lives, but current laboratory diagnostic tests lack sensitivity. OBJECTIVES: We investigated whether the detection of intracellular bacteria by a modified Ziehl-Neelsen stain and early secretory antigen target (ESAT)-6 in cerebrospinal fluid leukocytes improves tuberculous meningitis diagnosis. METHODS: Cerebrospinal fluid specimens from patients with suspected tuberculous meningitis were stained by conventional Ziehl-Neelsen stain, a modified Ziehl-Neelsen stain involving cytospin slides with Triton processing, and an ESAT-6 immunocytochemical stain. Acid-fast bacteria and ESAT-6-expressing leukocytes were detected by microscopy. All tests were performed prospectively in a central laboratory by experienced technicians masked to the patients' final diagnosis. MEASUREMENTS AND MAIN RESULTS: Two hundred and eighty patients with suspected tuberculous meningitis were enrolled. Thirty-seven had Mycobacterium tuberculosis cultured from cerebrospinal fluid; 40 had a microbiologically confirmed alternative diagnosis; the rest had probable or possible tuberculous meningitis according to published criteria. Against a clinical diagnostic gold standard the sensitivity of conventional Ziehl-Neelsen stain was 3.3% (95% confidence interval, 1.6-6.7%), compared with 82.9% (95% confidence interval, 77.4-87.3%) for modified Ziehl-Neelsen stain and 75.1% (95% confidence interval, 68.8-80.6%) for ESAT-6 immunostain. Intracellular bacteria were seen in 87.8% of the slides positive by the modified Ziehl-Neelsen stain. The specificity of modified Ziehl-Neelsen and ESAT-6 stain was 85.0% (95% confidence interval, 69.4-93.8%) and 90.0% (95% confidence interval, 75.4-96.7%), respectively. CONCLUSIONS: Enhanced bacterial detection by simple modification of the Ziehl-Neelsen stain and an ESAT-6 intracellular stain improve the laboratory diagnosis of tuberculous meningitis.

Original publication




Journal article


Am J Respir Crit Care Med

Publication Date





475 - 481


Adolescent, Adult, Aged, Aged, 80 and over, Antigens, Bacterial, Bacterial Proteins, Biomarkers, Child, Child, Preschool, Female, Humans, Immunohistochemistry, Leukocytes, Male, Middle Aged, Mycobacterium tuberculosis, Prospective Studies, Sensitivity and Specificity, Staining and Labeling, Tuberculosis, Meningeal, Young Adult