Evaluation of an in-house-developed PCR for the diagnosis of tuberculous meningitis in Indian children

Abstract

Early and rapid detection of the causative organism is necessary in tuberculosis, particularly tuberculous meningitis, as the disease affects mainly children and if untreated or improperly treated can cause severe central nervous system disorders and can often be fatal. An in-house-developed PCR technique was developed for the detection of Mycobacterium tuberculosis DNA, in which the target for amplification was a 340 bp nucleotide sequence located within the 38 kDa protein gene. The test can detect as small an amount of DNA as 10 fg, which is equivalent to two to three organisms, and is highly specific. Amplified product was detected by ethidium bromide staining after electrophoresis and Southern hybridization. Evaluation of test sensitivity and specificity was carried out using acid-fast bacilli-positive sputum samples from patients with pulmonary tuberculosis and an equal number of non-tuberculosis patient samples as negative controls. In a double-masked study 30 cerebrospinal fluid samples from tuberculous meningitis patients and 30 samples from non-tuberculous meningitis patients were investigated. Out of the 30 samples 22 were positive by ethidium bromide-stained gel electrophoresis and 27 gave positive results by Southern hybridization. All of the 30 control samples showed negative results. The sensitivity of this PCR was 90 % and specificity, 100 %.