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Purpose We aimed this study to standardize real time - polymerase chain reaction (RT-PCR) for the detection of Mycobacterium tuberculosis (Mtb) in cerebrospinal fluid (CSF) samples and compare its diagnostic performance with GeneXpert (Xpert), Mycobacteria Growth Indicator Tube (MGIT) and Multiplex PCR (MPCR) for tuberculous meningitis (TBM). Methodology A total of 217 CSF samples were obtained from patients with suspected TBM during the study period between January 2019 and December 2021. The optimal cycle threshold (CT) of RT-PCR was determined by comparing different gene targets of Mtb ( IS6110 , 16SrRNA , HSP65 and Ag85B ). Sensitivity, specificity, positive predictive value (PPV) and negative predictive value (NPV) was determined for RT-PCR, Xpert, MGIT960 and MPCR. Diagnostic accuracy of these assays was compared by using clinical diagnosis as reference standard. Results IS6110 RT-PCR was found to be highly sensitive as compared to other gene targets. Sensitivities of IS6 110 RT-PCR, MPCR, Xpert and MGIT against a reference standard of definite, probable and possible TBM were 36.7%, 21.1%, 16.7% and 6.7%, respectively; specificities were 97.6%, 100%, 100% and 100%, respectively. Xpert, RT-PCR, MPCR and MGIT960 detected 6.91% (n = 15), 5.99% (n = 13), 5.99% (n = 13) and 2.76% (n = 6) of definite TBM, respectively. RT-PCR detected 6.45% (n = 14) and 2.76% (n = 6) of possible TBM and probable TBM, respectively and MPCR detected 1.38% (n = 3) of possible and probable TBM each. Conclusion IS6110 RT-PCR is highly sensitive for primary screening of suspected TB cases, which may help clinicians to start appropriate patient's treatment with clinical suspicion of TBM. Introduction Tuberculous meningitis (TBM), is one of the most devastating manifestations of extra pulmonary tuberculosis (EPTB) caused by Mycobacterium tuberculosis (Mtb) [1]. Although the frequency of TBM accounts for approximately 5–10% of all EPTB and 1% of all TB cases, it causes higher mortality and morbidity rates with disproportionate amount of suffering [2]. The timely diagnosis and immediate treatment for TBM is essential, as the diagnostic delay leads to intracerebral inflammation, neurocognitive disorders, brain damage and death [3]. The diagnosis of TBM is based on clinical findings and detection of Mtb in cerebrospinal fluid (CSF) samples by Ziehl–Neelsen (ZN) smear, solid or liquid culture, nucleic acid amplification tests and CSF adenosine deaminase (ADA) assay. The ZN smear provides low sensitivity ranging from 10 to 60% and MGIT960 liquid culture takes 2–3 weeks to provide results with 40–60% sensitivity or Lowenstein-Jensen (LJ) solid culture can take from 6 to 8 weeks, leading to the diagnostic delay [4,5]. The biochemical possibilities including identification of indirect diagnostic marker like ADA has low specificity due to the increased CSF ADA level in bacterial infections and mixed results in HIV-infected patients [2,6]. The clinical diagnosis of TBM is challenging for clinicians due to paucibacillary nature of Mtb in CSF samples and non-specific clinical presentation of the patients. To overcome these problems, WHO has endorsed GeneXpertMTB/RIF (Xpert) assay as the initial diagnostic test for suspected TBM patients to detect both Mtb and rifampicin resistance simultaneously [[7], [8], [9], [10]]. However, the sensitivity of Xpert was not high enough to rule out TBM from CSF samples, with 45%–67% of sensitivity to detect microbiologically confirmed TBM [7]. A study from Bahur et al. observed that Xpert Ultra (70–95%) was superior to Xpert (43–45%) for TBM diagnosis [9]. In contrast, a recent study reported that Xpert Ultra was not superior to Xpert [10]. Therefore, Xpert negative CSF sample is insufficiently sensitive to exclude TBM cases. In addition, WHO recommended line probe assay (LPA) which detect both Mtb and drug resistance (rifampicin and isoniazid) require sufficient number of target molecules in CSF sample [11]. To overcome these problems, several studies have developed and used in-house nucleic acid amplification tests (NAAT) to detect Mtb in CSF samples [4,12,13]. Hence based on this knowledge, we aimed (i) to standardize a high sensitive real time - polymerase chain reaction (RT-PCR) to detect paucibacillary Mtb in CSF and (ii) to compare the diagnostic performance of RT-PCR with Xpert, MGIT960 and Multiplex PCR (MPCR) by using clinical reference of definite, probable and possible TBM as standard. Section snippets Ethics, study design and sample collection The study was reviewed and approved by the Institute Ethics Committee (IEC) (Human studies) at JIPMER, Pondicherry (Approval No. JIP/IEC/2019/143). In this prospective study, a total of 217 CSF samples (volume, 2 ml) were collected from TBM suspected cases (Male, 55.1% and Female, 44.9%) at south Indian tertiary care hospital in between January 2019 to December 2021 and the medical case records were reviewed. All the study participants were categorized based on definite TBM, probable TBM, LOD analysis of RT-PCR assays The comparative analytical LODs of RT-PCRs targeting IS6110 , 16SrRNA , HSP65 and Ag85B of Mtb were tested by spiking the serial dilutions of quantified bacteria in CSF samples. The Ct value was calculated to optimize the conditions of RT-PCR. IS6 110RT-PCR detected 1.5 × 102 cfu/ml Mtb as positive followed by 16SrRNA (1.5 × 103 cfu/ml), HSP65 (1.5 × 104 cfu/ml) and Ag85B (1.5 × 104 cfu/ml) (Table 2). These results indicate that IS6 110RT-PCR was promising and capable to detect lower Discussion Tuberculous meningitis (TBM) remains a significant health problem worldwide for which earlier and accurate diagnosis is critical and remains challenging to patient management and disease control. To provide accurate results in short period of time, we developed and compared RT-PCR assays targeting four different Mycobacterium tuberculosis (Mtb) genes including IS6110 , 16SrRNA , HSP65 and Ag85B for the purpose of potential substitution for GeneXpert (Xpert), Mycobacteria Growth Indicator Tube Conclusion Results of our study highlighted that IS6 110 RT-PCR is highly sensitive as compared to Xpert, MGIT, MPCR by using clinical reference standards of definite, probable and possible TBM. The IS6 110 RT-PCR assay demonstrated as excellent diagnostic method for the detection of Mtb among CSF samples, shortening turnaround time and reducing economic costs. We recommend that IS6 110 RT-PCR might be used as an initial screening test for tuberculosis. Conflict of interest None. Acknowledgement The authors would like to thank Jawaharlal Institute of Postgraduate Medical Education and Research (JIPMER), Pondicherry, India for providing intramural funding for this study. References (27) N.G. Fomukong et al. Insertion sequence typing of Mycobacterium tuberculosis : characterization of a widespread subtype with a single copy of IS6110 Tuber Lung Dis (1994) K. Sharma et al. Comparative evaluation of Xpert MTB/RIF assay with multiplex polymerase chain reaction for the diagnosis of tuberculous meningitis Tuberculosis (2018) B.W. Lee et al. DNA amplification by the polymerase chain reaction for the rapid diagnosis of tuberculous meningitis. Comparison of protocols involving three mycobacterial DNA sequences, IS6110 , 65 kDa antigen, and MPB64 J Neurol Sci (1994) S.S. Negi et al. Diagnostic potential of IS6110 , 38kDa, 65kDa and 85B sequence-based polymerase chain reaction in the diagnosis of Mycobacterium tuberculosis in clinical samples Indian J Med Microbiol (2007) S. Marais et al. Tuberculous meningitis: a uniform case definition for use in clinical research Lancet Infect Dis (2010) K. Sharma et al. Comparative evaluation of Xpert MTB/RIF assay with multiplex polymerase chain reaction for the diagnosis of tuberculous meningitis Tuberculosis (2018) J. Donovan et al. Xpert MTB/RIF Ultra versus Xpert MTB/RIF for the diagnosis of tuberculous meningitis: a prospective, randomised, diagnostic accuracy study Lancet Infect Dis (2020) N.C. Bahr et al. Diagnostic accuracy of Xpert MTB/RIF Ultra for tuberculous meningitis in HIV-infected adults: a prospective cohort study Lancet Infect Dis (2018) F.V. Cresswell et al. Xpert MTB/RIF Ultra for the diagnosis of HIV-associated tuberculous meningitis: a prospective validation study Lancet Infect Dis (2020) D. Jeon et al. Impact of implementation of an automated liquid culture system on the diagnosis of tuberculous pleurisy Chest (2015) D.T. Nguyen et al. Trends of tuberculosis meningitis and associated mortality in Texas, 2010-2017, a large population-based analysis PLoS One (2019) K. Thakur et al. The global neurological burden of tuberculosis In Semin Neurol (2018) B.D. Daniel et al. Tuberculous meningitis in children: clinical management & outcome Indian J Med Res (2019) Navigate Down View more references Cited by (0) Recommended articles (6) View full text © 2023 Indian Association of Medical Microbiologists. Published by Elsevier B.V. All rights reserved. About ScienceDirect Remote access Shopping cart Advertise Contact and support Terms and conditions Privacy policy We use cookies to help provide and enhance our service and tailor content and ads. By continuing you agree to the use of cookies . Copyright © 2023 Elsevier B.V. or its licensors or contributors. 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