Improved diagnosis of central nervous system tuberculosis by MPB64-Target PCR

Abstract

Central nervous system (CNS) tuberculosis is a serious clinical problem, the treatment of which is sometimes hampered by delayed diagnosis. Clearly, prompt laboratory diagnosis is of vital importance as the spectrum of disease is wide and abnormalities of the cerebrospinal fluid (CSF) are incredibly variable. Since delayed hypersensitivity is the underlying immune response, bacterial load is very low. The conventional bacteriological methods rarely detect Mycobacterium tuberculosis in CSF and are of limited use in diagnosis of tuberculous meningitis (TBM). This double blind study was, therefore, directed to the molecular analysis of CNS tuberculosis by an in-house-developed PCR targeted for amplification of a 240bp nucleotide sequence coding for MPB64 protein specific for Mycobacterium tuberculosis. Based on the clinical criteria, 47 patients with CNS tuberculosis and a control group of 10 patients having non-tubercular lesions of the CNS were included in the study. Analyses were done in three groups; one group consisting of 27 patients of TBM, a second group of 20 patients with intracranial tuberculomas and a third group of 10 patients having nontubercular lesions of the CNS acted as control. There were no false positive results by PCR and the specificity worked out to be 100%. In the three study groups, routine CSF analysis (cells and chemistry), CSF for AFB smear and culture were negative in all cases. PCR was positive for 21/27 patients (77.7% sensitivity) of the first group of TBM patients, 6/20 patients (30% sensitivity) of the second group with intracranial tuberculomas were positive by PCR and none was PCR-positive (100% specificity) in the third group. Thus, PCR was found to be more sensitive than any other conventional method in the diagnosis of clinically suspected tubercular meningitis.

Comparison of DNA Extraction Protocols and Molecular Targets to Diagnose Tuberculous Meningitis

“…The rapid identification of TBM through molecular analysis of CSF is an important factor for proper and early institution of antimicrobial treatment [7–9]. …”

Rapid detection of Mycobacterium tuberculosis complex by real-time PCR in sputum samples and its use in the routine diagnosis in a reference laboratory

“…Many regions of the mycobacterial genome have been used in molecular diagnosis of TB, such as IS6110, 16S rRNA, hsp65, rpoB, sdaA, devR and mpt64 (Manjunath et al, 1991;Lachnik et al, 2002;Kim et al, 2004;Chakravorty et al, 2005;Negi et al, 2007;Hwang et al, 2013;Nimesh et al, 2013). mpt64 is found as a single copy in the genome of MTBC species (Lee et al, 1994), and has been used for both pulmonary and extrapulmonary TB diagnosis (Tan et al, 1995;Dar et al, 1998;Martins et al, 2000;Bhanu et al, 2005;Therese et al, 2005;Takahashi & Nakayama, 2006;Dil-Afroze et al, 2008;Kusum et al, 2012;Sethi et al, 2012).…”

Detection of Mycobacterium Tuberculosis among Infertile Patients Suspected with Female Genital Tuberculosis

“…Primer selection of present study was done based on local characteristics of strains and their importance in the diagnosis of the disease (tabulated in Table 4). The nucleotide sequences of primers used for the detection of Mycobacterium in this study were described and validated as diagnostic markers in the past [17,18,29,[37][38][39][40][41][42][43][44][45][46]. This antigen would provide a target, which is universally present.…”

“…All samples collected were examined by H & E staining [30,31,32], Z-N staining for AFB [32,33,34], cultured on L-J egg media [34,35,36] and MTB specific multi-gene PCR method [17,18,29,[37][38][39][40][41][42][43][44][45][46] by which FGTB was confirmed and correlated with laparoscopic findings. The diagnosis was done based on morphological [19] and molecular investigations.…”