Tuberculous meningitis (TBM) is the most devastating form of meningitis and prompt diagnosis holds the key to its management. Conventional microbiology has limited utility and nucleic acidbased methods have not been widely accepted for various reasons. In view of the paucibacillary nature of cerebrospinal fluid (CSF) and the recent demonstration of free Mycobacterium tuberculosis DNA in clinical specimens, the present study was designed to evaluate the utility of CSF 'filtrates' for the diagnosis of TBM using PCR. One hundred and sixty-seven CSF samples were analysed from patients with 'suspected' TBM (n581) and a control group including other cases of meningitis or neurological disorders (n586). CSF 'sediments' and 'filtrates' were analysed individually for M. tuberculosis DNA by quantitative real-time PCR (qRT-PCR) and conventional PCR. Receiver-operating characteristic curves were generated from qRT-PCR data and cut-off values of 84 and 30 were selected for calling a 'filtrate' or 'sediment' sample positive, respectively. Based on these, TBM was diagnosed with 87.6 % and 53.1 % sensitivity (P ,0.001) in 'filtrates' and 'sediments', respectively, and with 92 % specificity each. Conventional devR and IS6110 PCR were also significantly more sensitive in 'filtrates' versus 'sediments' (sensitivity of 87.6 % and 85.2 % vs 31 % and 39.5 %, respectively; P ,0.001). The qRT-PCR test yielded a positive likelihood ratio of 11 and 6.6 by analysing 'filtrate' and 'sediment' fractions, respectively, which establishes the superiority of the 'filtrate'-based assay over the 'sediment' assay. PCR findings were separately verified in 10 confirmed cases of TBM, where M. tuberculosis DNA was detected using devR PCR assays in 'sediment' and 'filtrate' fractions of all samples. From this study, we conclude that (i) CSF 'filtrates' contain a substantial amount of M. tuberculosis DNA and (ii) 'filtrates' and not 'sediments' are likely to reliably provide a PCR-based diagnosis in 'suspected' TBM patients.

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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.

Coronavirus pandemic has caused a vast number of deaths worldwide. Thus creating an urgent need to develop effective counteragents against novel coronavirus disease (COVID-19). Many antiviral drugs have been repurposed for treatment but implicated minimal recovery, which further advanced the need for clearer insights and innovation to derive effective therapeutics. Strategically, Noscapine, an approved antitussive drug with positive effects on lung linings may show favorable outcomes synergistically with antiviral drugs in trials. Hence, we have theoretically examined the combinatorial drug therapy by culminating the existing experimental results with in silico analyses. We employed the antitussive noscapine in conjugation with antiviral drugs (Chloroquine, Umifenovir, Hydroxychloroquine, Favlplravir and Galidesivir). We found that Noscapine-Hydroxychloroquine (Nos-Hcq) conjugate has strong binding affinity for the main protease (Mpro) of SARS-CoV-2, which performs key biological function in virus infection and progression. Nos-Hcq was analyzed through molecular dynamics simulation. The MD simulation for 100 ns affirmed the stable binding of conjugation unprecedentedly through RMSD and radius of gyration plots along with critical reaction coordinate binding free energy profile. Also, dynamical residue cross-correlation map with principal component analysis depicted the stable binding of Nos-Hcq conjugate to Mpro domains with optimal secondary structure statistics of complex dynamics. Also, we reveal the drugs with stable binding to major domains of Mpro can significantly improve the work profile of reaction coordinates, drug accession and inhibitory regulation of Mpro. The designed combinatorial therapy paves way for further prioritized in vitro and in vivo investigations for drug with robust binding against Mpro of SARS-CoV-2.