Detection of Mycobacterium tuberculosis GlcB or HspX Antigens or devR DNA Impacts the Rapid Diagnosis of Tuberculous Meningitis in Children

Abstract

BackgroundTuberculous meningitis (TBM) is the most common form of neurotuberculosis and the fifth most common form of extrapulmonary TB. Early diagnosis and prompt treatment are the cornerstones of effective disease management. The accurate diagnosis of TBM poses a challenge due to an extensive differential diagnosis, low bacterial load and paucity of cerebrospinal fluid (CSF) especially in children.Methodology/Principal FindingsWe describe the utility of ELISA and qPCR for the detection of Mycobacterium tuberculosis (M. tb) proteins (GlcB, HspX, MPT51, Ag85B and PstS1) and DNA for the rapid diagnosis of TBM. CSF filtrates (n = 532) derived from children were classified as ‘Definite’ TBM (M. tb culture positive, n = 29), ‘Probable and Possible’ TBM (n = 165) and ‘Not-TBM’ including other cases of meningitis or neurological disorders (n = 338). ROC curves were generated from ELISA and qPCR data of ‘Definite’ TBM and Non-Tuberculous infectious meningitis (NTIM) samples and cut-off values were derived to provide ≥95% specificity. devR qPCR, GlcB, HspX and PstS1 ELISAs showed 100% (88;100) sensitivity and 96–97% specificity in ‘Definite’ TBM samples. The application of these cut-offs to ‘Probable and Possible’ TBM groups yielded excellent sensitivity (98%, 94;99) and specificity (98%, 96;99) for qPCR and for GlcB, HspX and MPT51 antigen ELISAs (sensitivity 92–95% and specificity 93–96%). A test combination of qPCR with GlcB and HspX ELISAs accurately detected all TBM samples at a specificity of ∼90%. Logistic regression analysis indicated that these tests significantly added value to the currently used algorithms for TBM diagnosis.ConclusionsThe detection of M. tb GlcB/HspX antigens/devR DNA in CSF is likely to improve the utility of existing algorithms for TBM diagnosis and also hasten the speed of diagnosis.

Diagnosis of pulmonary and extrapulmonary tuberculosis based on detection of mycobacterial antigen 85B by immuno-PCR

“…(Mehta et al, 2012b). Our ELISA results (Table 2b) are almost in agreement with the earlier reports for the detection of Ag85B in serum, cerebrospinal fluid, and ascitic fluid from TB meningitis and abdominal TB patients (Haldar et al, 2012;Kashyap et al, 2007Kashyap et al, , 2010, whereas no report is available for TB pleuritis. While detecting Ag85B from pleural fluids (n = 64) for the diagnosis of TB pleuritis, we demonstrated a sensitivity of 76.5% by I-PCR (Table 2b).…”

“…The number of positive cases recognized from smear-positive and smear-negative PTB cases by I-PCR versus ELISA was 85% versus 77.6% for smear-positive cases and 77% versus 62.5% for smear-negative cases (Table 1). Few positive samples missed by I-PCR may be attributed to either very low undetectable antigen level in the samples or antigen sequestering within antigen-antibody complexes (Haldar et al, 2012). Furthermore, our I-PCR demonstrated 100% sensitivity with smear-positive culturepositive PTB cases.…”

“…The sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV) along with 95% confidence intervals (CIs) were calculated as described by Haldar et al (2012). The comparison of OD values between different groups was made by 2-sample t test with equal variances/Wilcoxon rank sum test.…”

“…The antigen 85 (Ag85) complex is the major culture filtrate protein of M. tuberculosis comprising 3 related proteins, Ag85A, Ag85B, and Ag85C. Among Ag85 complex, Ag85B is the major secreted protein that can be detected in PTB and TB meningitis patients by ELISA and has been suggested as a biomarker for TB diagnosis (Haldar et al, 2012;Kashyap et al, 2007). To improve the sensitivity and shorten the duration, we planned a study to design an I-PCR assay by covalent binding of reporter DNA with the antidetection antibody through succinimidyl 4-[N-maleimidomethyl]-cyclohexane-1-carboxylate (SMCC) Diagnostic Microbiology and Infectious Disease xxx (2015) xxx-xxx for the detection of Ag85B in TB patients, and the results were compared with an analogous ELISA.…”

Laboratory Diagnosis of Central Nervous System Infection

“…Recently several reports revealed potential application of detection of M. tuberculosis −specific antigens in CSF as rapid diagnosis of tuberculosis meningitis 39, 40 . The level of M. tuberculosis Early Secreted Antigenic Target 6 [ESAT-6] was associated with clinical severity, and may be used for the prognosis of tuberculosis meningitis 41, 42 .…”