Direct Detection of Fluoroquinolone Resistance in Sputum Samples from Tuberculosis Patients by High Resolution Melt Curve Analysis.

Multidrug-resistant tuberculosis (MDR-TB) requires treatment with fluoroquinolone (FLQ) drugs, however, the excessive use of FLQ has led to the rise of extensively drug-resistant TB. In 2019, ~ 20% of total MDR-TB cases were estimated to be resistant to FLQ drugs. In the present study, we developed and evaluated the utility of high-resolution melt curve analysis (HRM) for the rapid detection of FLQ-resistant Mycobacterium tuberculosis for the first time directly from sputum samples. A reference plasmid library was generated for the most frequently observed mutations of gyrA gene and was used to discriminate between mutant and wild-type samples in the FLQ-HRM assay. The developed assay was evaluated on n = 25 MDR M. tuberculosis clinical isolates followed by validation on archived sputum DNA (n = 88) using DNA sequencing as a gold standard. The FLQ-HRM assay showed a 100% sensitivity [95% Confidence Interval (CI): 71.5 to 100] and specificity (95% CI: 39.7 to 100) in smear-positive category, and a sensitivity of 88.9% (95% CI: 77.3 to 95.8) with 84.2% (95% CI: 60.4 to 96.6) specificity in smear-negative category. The assay showed a high level of concordance of ~ 90% (κ = 0.74) with DNA sequencing, however, we were limited by the absence of phenotypic drug susceptibility testing data. In conclusion, HRM is a rapid, cost-effective (INR 150/USD 1.83) and closed-tube method for direct detection of FLQ resistance in sputum samples including direct smear-negative samples.

Direct Molecular Detection of Drug-Resistant Tuberculosis from Transported Bio-Safe Dried Sputum on Filter-Paper.

In 2019, amongst half a million new rifampicin-resistant tuberculosis (TB) cases, 78% were multi drug-resistant TB (MDR-TB). Access to rapid and Universal-Drug susceptibility testing (DST) to patients in remote areas is a major challenge to combat drug-resistant TB. To overcome this challenge, we had recently reported the development of 'TB Concentration & Transport kit' for bio-safe ambient temperature transport of dried sputum on filter-paper (Trans-Filter). The present study was conducted to evaluate the utility of DNA extracted from sputum on Trans-Filter in a Multiplex PCR-based sequencing assay (Mol-DSTseq) for diagnosing drug-resistant TB. The developed Mol-DSTseq assays were standardized on Mycobacterium tuberculosis clinical isolates (n = 98) and further validated on DNA extracted from sputum on Trans-Filter (n = 100). Using phenotypic DST as gold standard, the Mol-DSTseq assay showed 100% (95% Confidence Interval [CI] 79.4-100%) and 73.3% (95% CI 54.1-87.7%) sensitivity for detecting rifampicin and isoniazid resistance with a specificity of 85.1% (95% CI 66.2-95.8%) and 100% (95% CI:82.3-100%), respectively. For fluoroquinolones and aminoglycosides, the Mol-DSTseq assay showed a sensitivity of 78.5% (95% CI 49.2-95.3%) and 66.6% (95% CI 9.4-99.1%) with a specificity of 88.2% (95% CI 72.5-96.7%) and 100% (95% CI 93.1-100%), respectively. The Mol-DSTseq assays exhibited a high concordance of ~ 83-96% (κ value: 0.65-0.81) with phenotypic DST for all drugs. In conclusion, the 'TB Concentration and Transport kit' was compatible with Mol-DSTseq assays and has the potential to provide 'Universal-DST' to patients residing in distant areas in high burden countries, like India for early initiation of anti-tubercular treatment.

A novel aptamer-based test for the rapid and accurate diagnosis of pleural tuberculosis.

Pleural tuberculosis (pTB) is diagnosed by using a composite reference standard (CRS) since microbiological methods are grossly inadequate and an accurate diagnostic test remains an unmet need. The present study aimed to evaluate the utility of Mycobacterium tuberculosis (Mtb) antigen and DNA-based tests for pTB diagnosis. Patients were classified as 'Definite TB', 'Probable TB' and 'Non-TB' disease according to the CRS. We assessed the performance of in-house antigen detection assays, namely antibody-based Enzyme-Linked ImmunoSorbent Assay (ELISA) and aptamer-based Aptamer-Linked Immobilized Sorbent Assay (ALISA), targeting Mtb HspX protein and DNA-based tests namely, Xpert MTB/RIF and in-house devR-qPCR. ROC curves were generated for the combined group of 'Definite TB' and 'Probable TB' vs. 'Non-TB' disease group and cut-off values were derived to provide specificity of ≥98%. The sensitivity of ALISA was ∼93% vs. ∼24% of ELISA (p-value ≤0.0001). devR-qPCR exhibited a sensitivity of 50% vs. ∼22% of Xpert (p-value ≤0.01). This novel aptamer-based ALISA test surpasses the sensitivity criterion and matches the specificity requirement spelt out in the 'Target product profile' for extrapulmonary tuberculosis samples by Unitaid (Sensitivity ≥80%, Specificity 98%). The superior performance of the aptamer-based ALISA test indicates its translational potential to bridge the existing gap in pTB diagnosis.

Direct Detection of Rifampin and Isoniazid Resistance in Sputum Samples from Tuberculosis Patients by High-Resolution Melt Curve Analysis.

Drug-resistant tuberculosis (TB) is a major threat to TB control worldwide. Globally, only 40% of the 340,000 notified TB patients estimated to have multidrug-resistant-TB (MDR-TB) were detected in 2015. This study was carried out to evaluate the utility of high-resolution melt curve analysis (HRM) for the rapid and direct detection of MDR-TB in Mycobacterium tuberculosis in sputum samples. A reference plasmid library was first generated of the most frequently observed mutations in the resistance-determining regions of rpoB, katG, and an inhA promoter and used as positive controls in HRM. The assay was first validated in 25 MDR M. tuberculosis clinical isolates. The assay was evaluated on DNA isolated from 99 M. tuberculosis culture-positive sputum samples that included 84 smear-negative sputum samples, using DNA sequencing as gold standard. Mutants were discriminated from the wild type by comparing melting-curve patterns with those of control plasmids using HRM software. Rifampin (RIF) and isoniazid (INH) monoresistance were detected in 11 and 21 specimens, respectively, by HRM. Six samples were classified as MDR-TB by sequencing, one of which was missed by HRM. The HRM-RIF, INH-katG, and INH-inhA assays had 89% (95% confidence interval [CI], 52, 100%), 85% (95% CI, 62, 97%), and 100% (95% CI, 74, 100%) sensitivity, respectively, in smear-negative samples, while all assays had 100% sensitivity in smear-positive samples. All assays had 100% specificity. Concordance of 97% to 100% (κ value, 0.9 to 1) was noted between sequencing and HRM. Heteroresistance was observed in 5 of 99 samples by sequencing. In conclusion, the HRM assay was a cost-effective (Indian rupee [INR]400/US$6), rapid, and closed-tube method for the direct detection of MDR-TB in sputum, especially for direct smear-negative cases.

Operational feasibility and multi-centric evaluation of 'TBDetect sputum microscopy kit' for the direct detection of Mycobacterium tuberculosis in field settings.

BACKGROUND: India relies primarily on direct smear microscopy for tuberculosis (TB) diagnosis. However, the low sensitivity of smear microscopy emphasizes the need to improve its performance. We recently described the development of 'TBDetect' kit which showed improved performance over direct smear microscopy at National Reference Laboratories (NRLs) in India. METHODS: The present study was aimed to assess the operational feasibility of 'TBDetect' microscopy in field settings. This was evaluated by (i) assessing the performance of 'TBDetect' microscopy vs. LED-fluorescence microscopy (LED-FM) on consecutive presumptive pulmonary TB patients (n = 5300) who attended Designated Microscopy Centres (DMCs, n = 13) under 4 NRLs at Bhubaneswar, Bhopal, Chennai, and New Delhi, and (ii) obtaining feedback from Scientists (n = 10) and laboratory technicians (n = 42) using semi-structured questionnaires under the following parameters: feasibility of initiation of 'TBDetect' microscopy in DMCs, sample preparation and testing, training, time-to-result, logistics, and troubleshooting. A scoring questionnaire was also used to assess 'TBDetect' microscopy vs. LED-FM and statistical significance of the scores was calculated using paired t-test. RESULTS: The overall positivity of 'TBDetect' microscopy was 10.32% (547/5300) vs. 8.96% (475/5300) of LED-FM at all sites and the increment in positivity was significant (p = 0.019). In addition, 'TBDetect' microscopy yielded an increment in smear grade status over LED-FM (p = 0.043). The feedback from the study-in-charge and kit users indicated that 'TBDetect' microscopy was easily adapted in point-of-care settings. An analysis of scoring feedback suggested that it was easy to perform and observe in comparison to LED-FM (p < 0.005). CONCLUSIONS: This study established the feasibility of 'TBDetect' microscopy in field settings.

Evaluation of Mycobacterium tuberculosis derived cell-free DNA using pleural fluid and paired plasma samples for the diagnosis of pleural tuberculosis.

Pleural tuberculosis (pTB) is a grave clinical challenge. A novel cell-free M. tuberculosis DNA (cfM.tb-DNA) probe-based-qPCR assay was developed for the diagnosis of pTB. Total cell-free DNA was extracted from pleural fluid (PF) and paired plasma samples and cfM.tb-DNA was quantified by probe-based qPCR targeting devR (109-bp) gene of M. tuberculosis in patients with pleural effusion. Patient categorization was done using 'Composite-Reference-Standard' formulated for the study. Assay cut-offs were determined from samples in the 'Development set' (n = 17; 'Definite & Probable' pTB; n = 9 and 'Non-TB'; n = 8) by ROC-curve analysis and applied to 'Validation set' (n = 112; 'Definite' pTB; n = 8, 'Probable' pTB; n = 34, 'Possible' pTB; n = 28 and 'Non-TB'; n = 42). cfM.tb-DNA qPCR had a sensitivity of 62.5% (95%CI; 24.4,91.4) in 'Definite' pTB category and 59.5% (95%CI; 43.2,74.3) in 'Definite & Probable' pTB category with 95.2% (95%CI; 83.8,99.4) specificity using PF. In plasma (n = 85), the assay had a sub-optimal sensitivity of 7.6% (95%CI; 0.95,25.1) with 88.2% (95%CI; 72.5,96.7) specificity in 'Definite & Probable' pTB group. Xpert MTB/RIF assay detected only six-samples in the 'Validation set'. Logistic regression analysis indicated that PF-cfM.tb-DNA qPCR provided incremental advantage over existing pTB diagnostic algorithms. To the best of our knowledge, this is the first report describing the utility of cfM.tb-DNA for pTB diagnosis in India.

MPT51 and MPT64-based antigen detection assay for the diagnosis of extrapulmonary tuberculosis from urine samples.

In view of WHO's "End-TB" strategy, we developed a non-invasive, urine-based ELISA, targeting 2 Mycobacterium tuberculosis antigens namely MPT51 and MPT64 for extrapulmonary TB (EPTB) diagnosis. Suspected EPTB patients (n = 137) [Pleural TB, Abdominal TB and Tuberculous meningitis] were categorized in "Definite" EPTB (n = 10) [Xpert-MTB/RIF and/or culture-positive], "Probable" EPTB (n = 77) and "Non-EPTB" (n = 50) groups using defined composite reference standards. ROC-curves were generated using ELISA results of "Definite" EPTB and "Non-EPTB" groups for both antigens independently and cut-off values were selected to provide 86.3% (95%CI:73.3-94.2) specificity for MPT51 and 92% (95%CI:80.8-97.8) for MPT64. The sensitivity of MPT51-ELISA and MPT64-ELISA was 70% (95%CI:34.7-93.3) and 90% (95%CI:55.5-99.7) for "Definite" EPTB group and 32.5% (95%CI:22.2-44.1) and 30.8% (95%CI:20.8-42.2) for "Probable" EPTB group, respectively. Combining the results of both ELISAs showed a 100% (95%CI:69.1-100) sensitivity in "Definite" EPTB group and 41.6% (95%CI:30.4-53.4) in "Probable" EPTB group, with an 80% (95%CI:66.3-89.9) specificity. The results demonstrated the potential of urine-based ELISAs as screening tests for EPTB diagnosis.

Assessment of DNA aptamers targeting GlcB and HspX antigens for application in the diagnosis of abdominal tuberculosis.

The diagnosis of abdominal tuberculosis (aTB) is challenging and there is an urgent need for an accurate diagnostic test. We have developed a high affinity DNA aptamer against GlcB antigen of Mycobacterium tuberculosis (Mtb). We further compared the diagnostic utility of in-house-generated high affinity DNA aptamers and polyclonal antibodies against two Mtb antigens, namely GlcB and HspX, in ascitic fluid samples. These diagnostic reagents were assessed in patients (n = 94) who were categorized as 'Definite TB', 'Probable TB', 'Possible TB' (taken together as aTB) and 'Non-TB' disease. Receiver operating characteristic curves were used to derive cut-off values to provide ≥93% specificity. Aptamer Linked Immobilized Sorbent Assay (ALISA) for HspX and GlcB exhibited a sensitivity of ∼84% and 50%, respectively (p-value <0.01). In contrast, antibody-based ELISA exhibited a lower sensitivity of ∼18% and ∼28% for HspX and GlcB, respectively (p-value <0.0001 and p = 0.05 for HspX and GlcB ELISA vs. ALISA, respectively). HspX ALISA detected 32/38 aTB cases, while Xpert detected only 9 samples. In conclusion, HspX aptamer-based test was found to be superior to the other tests for diagnosing aTB and it nearly fulfils the sensitivity criteria of WHO's 'Target Product Profile' for extrapulmonary tuberculosis (sensitivity ≥80%, specificity 98%).

Utility of cell-free transrenal DNA for the diagnosis of Tuberculous Meningitis: A proof-of-concept study.

Tuberculous Meningitis (TBM) diagnosis remains a grave challenge. We evaluated the utility of extracellular vesicles (EVs) as a source of cell-free transrenal-mycobacterial DNA (cf-Tr-MTB DNA) for TBM diagnosis from urine samples. We developed a qPCR-assay targeting a highly repetitive 36-bp sequence specific to Mycobacterium tuberculosis complex. EVs were isolated from urine samples of suspected TBM groups (n = 44) [categorized using composite reference standard as 'Definite' TBM (n = 8), 'Probable' TBM (n = 15), 'Possible' TBM (n = 21)] and 'Non-TBM' group (n = 26). cf-Tr-MTB DNA-based qPCR assay was applied to DNA isolated from EVs (EV-DNA) and EV-free-fraction (EV-free DNA). ROC-curves were generated using qPCR results of 'Definite' TBM and 'Non-TBM' category in both EV-DNA and EV-free DNA samples and cut-off values were selected to provide 100% (95%CI:69.1-100) specificity. The cf-Tr-MTB DNA assay gave a sensitivity of 54.5% (95%CI:38.8-69.6) for EV-DNA and 77.3% (95%CI:62.1-88.5) for EV-free DNA in the TBM group (n = 44). The combination of EV-DNA and EV-free DNA results (corresponding to performance cf-Tr MTB DNA assay in urine), gave an overall sensitivity of 81.8% (95%CI:67.2-91.8) in the TBM group. Our results confirmed EVs as one of the sources of cf-Tr-MTB DNA and we believe the cf-Tr-MTB DNA-based qPCR assay has a potential application for TBM diagnosis.

Compatibility of a novel filter paper-based bio-safe sputum transport kit with line probe assay for diagnosing drug-resistant tuberculosis: a single-site evaluation study.

BACKGROUND: Near-patient access to appropriate tests is a major obstacle for the efficient diagnosis of tuberculosis (TB) and associated drug resistance. METHODS: We recently developed the "TB Concentration & Transport" kit for bio-safe, ambient-temperature transportation of dried sputum on Trans-Filter, and the "TB DNA Extraction" kit for DNA extraction from Trans-Filter for determining drug resistance by DNA sequencing. In the present study, we evaluated the compatibility of Kit-extracted DNA with Hain's line probe assays (LPAs), which are endorsed by National TB programmes for the detection of drug resistance in sputum collected from presumptive multidrug-resistant TB patients (n=207). RESULTS: Trans-Filter-extracted DNA was seamlessly integrated with the LPA protocol (Kit-LPA). The sensitivity of Kit-LPA for determining drug resistance was 83.3% for rifampicin (95% CI 52-98%), 77.7% for isoniazid (95% CI 52-94%), 85.7% for fluoroquinolones (95% CI 42-100%) and 66.6% for aminoglycosides (95% CI 9-99%), with a specificity range of 93.7% (95% CI 87-97) to 99.1% (95% CI 95-100) using phenotypic drug susceptibility testing (DST) as a reference standard. A high degree of concordance was noted between results obtained from Kit-LPA and LPA (99% to 100% (κ value: 0.83-1.0)). CONCLUSIONS: This study demonstrates successful integration of our developed kits with LPA. The adoption of these kits across Designated Microscopy Centres in India can potentially overcome the existing challenge of transporting infectious sputum at controlled temperature to centralised testing laboratories and can provide rapid near-patient cost-effective "Universal DST" services to TB subjects residing in remote areas.

Evaluation of 'TBDetect' sputum microscopy kit for improved detection of Mycobacterium tuberculosis: a multi-centric validation study.

OBJECTIVES: The present study aimed to evaluate the performance of the 'TBDetect' kit-based bio-safe fluorescent microscopy filter (BioFM-Filter) microscopy in comparison with direct smear microscopy and culture for the detection of pulmonary tuberculosis (TB) in a multi-centric setting in India. METHODS: The TBDetect kit enables sputum concentration through filtration using the BioFM-Filter for improved and bio-safe smear microscopy. We evaluated the performance of the TBDetect kit in a six-site multi-centric validation study on sputum collected from 2086 presumptive TB patients. RESULTS: The combined positivity of TBDetect microscopy performed on these sputum samples was 20% (n = 417/2086) vs 16.1% of light-emitting diode fluorescence microscopy (LED-FM, n = 337/2086) and 16% of Ziehl Neelsen (ZN) smear microscopy (n = 333/2086). The increment in positivity of TBDetect over both LED-FM and ZN smears was significant (p < 0.001). The overall sensitivity of TBDetect for six sites was ~55% (202/367, 95% confidence interval (CI): 50, 60%) vs 52% (191/367, 95% CI: 47, 57%) for LED-FM (p 0.14) and 50.9% (187/367, 95% CI: 46, 56%) for ZN smear (p < 0.05), using Mycobacterium Growth Indicator Tube culture (MGIT, n = 1949, culture positive, n = 367) as the reference standard. A bio-safety evaluation at six sites confirmed efficient sputum disinfection by TBDetect; 99.95% samples (1873/1874) were sterile after 42 days of incubation. Scientists and technicians at the study sites indicated the ease of use and convenience of TBDetect microscopy during feedback. CONCLUSIONS: TBDetect added value to the smear microscopy test due to its improved performance, convenience and user safety. These findings indicate that equipment-free TBDetect technology has the potential to improve TB diagnosis in basic laboratory settings by leveraging on the existing nationwide network of designated microscopy centres and primary healthcare centres.

Genotypic characterization of 'inferred' rifampin mutations in GenoType MTBDRplus assay and its association with phenotypic susceptibility testing of Mycobacterium tuberculosis.

In GenoType MTBDRplus assay [line probe assay (LPA)], when Mycobacterium tuberculosis (M. tuberculosis) sample DNA fails to hybridize to at least 1 rpoB wild-type probe and any mutation probe, it is inferred as rifampin (RIF)-resistant. In this study, we sought to identify such 'inferred' mutations in M. tuberculosis isolates (n = 203) by rpoB gene sequencing and determined their association with phenotypic resistance. D516Y, H526N, L511P mutations were associated with both phenotypically sensitive (59%, n = 38/64) and resistant (23.7%, n = 33/139) antimicrobial susceptibility testing (AST) results, whereas S531W mutation was associated with only RIF-resistant isolates (33%, n = 46/139). These results demonstrated that, at standard drug concentrations, some 'inferred' mutations may be missed by RIF-AST (phenotypically sensitive). The use of LPA permits identification of these RIF-resistant isolates, and incorporation of additional mutation probes (e.g., S531W) could further increase LPA specificity. Further studies are needed to establish the significance of the type of 'inferred' mutation with clinical/treatment outcomes.

Utility of circulating cell-free Mycobacterium tuberculosis DNA for the improved diagnosis of abdominal tuberculosis.

Abdominal tuberculosis (ATB) continues to pose a major diagnostic challenge for clinicians due to its nonspecific clinical presentation, variable anatomical location and lack of sensitive diagnostic tools. In spite of the development of several assays till date; no single test has proved to be adequate for ATB diagnosis. In this study, we for the first time report the detection of circulating cell-free Mycobacterium tuberculosis (M. tuberculosis) DNA (cfMTB-DNA) in ascitic fluid (AF) samples and its utility in ATB diagnosis. Sixty-five AF samples were included in the study and processed for liquid culture, cytological, biochemical and molecular assays. A composite reference standard (CRS) was formulated to categorize the patients into 'Definite ATB' (M. tuberculosis culture positive, n = 2), 'Probable ATB' (n = 16), 'Possible ATB' (n = 13) and 'Non-TB' category (n = 34). Two molecular assays were performed, namely, the novel cfMTB-DNA qPCR assay targeting M. tuberculosis devR gene and Xpert MTB/RIF assay (Xpert), and their diagnostic accuracy was assessed using CRS as reference standard. Clinical features such as fever, loss of weight, abdominal distension and positive Mantoux were found to be strongly associated with ATB disease (p<0.05). cfMTB-DNA qPCR had a sensitivity of 66.7% (95% CI:40.9,86.7) with 97.1% specificity (95% CI:84.7,99.9) in 'Definite ATB' and 'Probable ATB' group collectively. The sensitivity increased to 70.9% (95% CI:51.9,85.8) in the combined 'Definite', 'Probable' and 'Possible' ATB group with similar specificity. The cfMTB-DNA qPCR assay performed significantly better than the Xpert assay which demonstrated a poor sensitivity of ≤16.7% with 100% (95% CI:89.7,100) specificity (p<0.001). We conclude that cfMTB-DNA qPCR assay is an accurate molecular test that can provide direct evidence of M. tuberculosis etiology and has promise to pave the way for improving ATB diagnosis.

Efficient diagnosis of tuberculous meningitis by detection of Mycobacterium tuberculosis DNA in cerebrospinal fluid filtrates using PCR.

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 acid-based 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 (n=81) and a control group including other cases of meningitis or neurological disorders (n=86). 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.

Structural switching electrochemical DNA aptasensor for the rapid diagnosis of tuberculous meningitis.

BACKGROUND: Tuberculous meningitis (TBM) is the most devastating manifestation of extra-pulmonary tuberculosis. About 33% of TBM patients die due to very late diagnosis of the disease. Conventional diagnostic methods based on signs and symptoms, cerebrospinal fluid (CSF) smear microscopy or liquid culture suffer from either poor sensitivity or long turnaround time (up to 8 weeks). Therefore, in order to manage the disease efficiently, there is an urgent and unmet need for a rapid and reliable diagnostic test. METHODS: In the current study, to address the diagnostic challenge of TBM, a highly rapid and sensitive structural switching electrochemical aptasensor was developed by combining the electrochemical property of methylene blue (MB) with the molecular recognition ability of a ssDNA aptamer. To demonstrate the clinical diagnostic utility of the developed aptasensor, a blinded study was performed on 81 archived CSF specimens using differential pulse voltammetry. RESULTS: The electrochemical aptasensor developed in the current study can detect as low as 10 pg HspX in CSF background and yields a highly discriminatory response (P<0.0001) for TBM and not-TBM categories with ~95% sensitivity and ~97.5% specificity and has the ability to deliver sample-to-answer in ≤30 minutes. CONCLUSION: In summary, we demonstrate a new aptamer-based electrochemical biosensing strategy by exploiting the target-induced structural switching of H63 SL-2 M6 aptamer and electroactivity of aptamer-tagged MB for the detection of HspX in CSF samples for the diagnosis of TBM. Further, the clinical utility of this sensor could be extended for the diagnosis of other forms of tuberculosis in the near future.

Development and evaluation of novel bio-safe filter paper-based kits for sputum microscopy and transport to directly detect Mycobacterium tuberculosis and associated drug resistance.

India has the highest burden of Tuberculosis (TB) and multidrug-resistant TB (MDR-TB) worldwide. Innovative technology is the need of the hour to identify these cases that remain either undiagnosed or inadequately diagnosed due to the unavailability of appropriate tools at primary healthcare settings. We developed and evaluated 3 kits, namely 'TB Detect' (containing BioFM-Filter device), 'TB Concentration and Transport' (containing Trans-Filter device) and 'TB DNA Extraction' kits. These kits enable bio-safe equipment-free concentration of sputum on filters and improved fluorescence microscopy at primary healthcare centres, ambient temperature transport of dried inactivated sputum filters to central laboratories and molecular detection of drug resistance by PCR and DNA sequencing (Mol-DST). In a 2-site evaluation (n = 1190 sputum specimens) on presumptive TB patients, BioFM-Filter smear exhibited a significant increase in positivity of 7% and 4% over ZN smear and LED-FM smear (p<0.05), respectively and an increment in smear grade status (1+ or 2+ to 3+) of 16% over ZN smear and 20% over LED-FM smear. The sensitivity of Mol-DST in presumptive MDR-TB and XDR-TB cases (n = 148) was 90% for Rifampicin (95% confidence interval [CI], 78-96%), 84% for Isoniazid (95% CI, 72-92%), 83% for Fluoroquinolones (95% CI, 66-93%) and 75% for Aminoglycosides (95% CI, 35-97%), using phenotypic DST as the reference standard. Test specificity was 88-93% and concordance was ~89-92% (κ value 0.8-0.9). The patient-friendly kits described here address several of the existing challenges and are designed to provide 'Universal Access' to rapid TB diagnosis, including drug-resistant disease. Their utility was demonstrated by application to sputum at 2 sites in India. Our findings pave the way for larger studies in different point-of-care settings, including high-density urban areas and remote geographical locations.

Theranostic Application of a Novel G-Quadruplex-Forming DNA Aptamer Targeting Malate Synthase of Mycobacterium tuberculosis.

The successful management of tuberculosis (TB) requires efficient diagnosis and treatment. Further, the increasing prevalence of drug-resistant TB highlights the urgent need to develop novel inhibitors against both drug-susceptible and drug-resistant forms of disease. Malate synthase (MS), an enzyme of the glyoxylate pathway, plays a vital role in mycobacterial persistence, and therefore it is considered as an attractive target for novel anti-TB drug development. Recent studies have also ascribed an adhesin function to MS and established it as a potent diagnostic biomarker. In this study, a panel of Mycobacterium tuberculosis (Mtb) MS-specific single-stranded DNA aptamers was identified by Systematic Evolution of Ligands by EXponential enrichment (SELEX). The best-performing G-quadruplex-forming 44-mer aptamer, MS10, was optimized post-SELEX to generate an 11-mer aptamer, MS10-Trunc. This aptamer was characterized by various biochemical, biophysical, and in silico techniques. Its theranostic activity toward Mtb was established using enzyme inhibition, host cell binding, and invasion assays. MS10-Trunc aptamer exhibited high affinity for MS (equilibrium dissociation constant [KD] ∼19 pM) and displayed robust inhibition of MS enzyme activity with IC50 of 251.1 nM and inhibitor constant (Ki) of 230 nM. This aptamer blocked mycobacterial entry into host cells by binding to surface-associated MS. In addition, we have also demonstrated its application in the detection of tuberculous meningitis (TBM) in patients with sensitivity and specificity each of >97%.

Generation and application of DNA aptamers against HspX for accurate diagnosis of tuberculous meningitis.

Tuberculous meningitis (TBM) is the most severe manifestation of tuberculosis and its diagnosis remains a challenge even today due to the lack of an adequate test. HspX antigen of Mycobacterium tuberculosis was previously established as a reliable diagnostic biomarker for TBM in an ELISA test format using anti-HspX polyclonal antibodies. Towards overcoming the limitations of batch-to-batch variation and challenges of scalability in antibody generation, we utilized Systematic Evolution of Ligands by EXponential enrichment (SELEX) to develop high affinity DNA aptamers against HspX as an alternative diagnostic reagent. Post-SELEX optimization of the best-performing aptamer candidate, H63, established its derivative H63 SL-2 M6 to be superior to its parent. Aptamer H63 SL-2 M6 displayed a specific and high affinity interaction with HspX (Kd ∼9.0 × 10-8 M). In an Aptamer Linked Immobilized Sorbent Assay (ALISA), H63 SL-2 M6 significantly differentiated between cerebrospinal fluid specimens from TBM and non-TBM subjects (n = 87, ***p < 0.0001) with ∼100% sensitivity and ∼91% specificity. Notably, ALISA exhibited comparable performance with previously reported antibody-based ELISA and qPCR. Altogether, our findings establish the utility of HspX aptamer for the reliable diagnosis of TBM and pave the way for developing an aptamer-based point-of-care test for TBM.

Simplified detection of Mycobacterium tuberculosis in sputum using smear microscopy and PCR with molecular beacons.

The prompt diagnosis of smear-negative cases is a prerequisite to controlling tuberculosis (TB). Several new laboratory approaches, including nucleic acid amplification (NAA), are being evaluated in various disease settings to meet this challenge. However, NAA needs simplification before it is widely accepted. Furthermore, a supporting smear result improves confidence in and reliability of PCR. In this context, an asymmetric devR PCR assay using two molecular beacon probes for visual or fluorimetric end-point detection of Mycobacterium tuberculosis was developed. The assays reproducibly detected 25 fg M. tuberculosis DNA versus 100 fg by conventional gel electrophoresis (henceforth referred to as gel assay). The devR and IS6110 PCR assays were blindly evaluated on sputum specimens obtained from a directly observed-treatment short-course centre. Universal sample processing (USP) smear microscopy and culture were used as a supportive test and the 'gold' standard, respectively. Among the 148 specimens analysed, 120 were M. tuberculosis culture-positive. Amongst the 122 direct smear-negative samples, 96 were culture-positive, of which 61 were detected by USP smear microscopy. All 35 USP smear-negative samples were positive by three or more PCR methods. devR PCR had a sensitivity of 92.5 % in the fluorimetric assay versus 86.7 % by visual inspection and 90.8 % by the gel method. IS6110 PCR performed at almost equivalent levels. devR visual and fluorimetric assays considered together yielded an increased sensitivity of 95 % without compromising on a specificity of 92.9 %. The results suggest that the USP smear test is useful for diagnosing direct smear-negative TB and judiciously restricting PCR testing to only smear-negative samples. When used together, these tests can provide rapid diagnosis of smear-negative TB in a cost-effective manner.

Challenges in pleural tuberculosis diagnosis: existing reference standards and nucleic acid tests.

Pleural tuberculosis (pTB) is a grave form of extrapulmonary tuberculosis. Microbiological tests are usually found to be inadequate for pTB diagnosis. The absence of a uniform 'composite reference standard' is challenging; therefore, diagnosis is usually performed using a combination of diversified criteria. Nucleic acid tests vary in diagnostic accuracy and have not yet been integrated into clinical decision making. This review assesses the varied criteria used for pTB classification and the challenges afflicting pleural fluid-based DNA diagnostic tests, namely, PCR and Xpert® MTB/RIF. In the 58 studies (PCR: n = 33; Xpert: n = 25) analyzed, reference standards were heterogeneous and PCR/Xpert pooled sensitivity values (range: 0-100%) were inadequate. However, the consistent high specificity of Xpert (range: 90-100%) indicated its utility as a 'rule-in' test. There is an urgent need to evaluate existing and new molecular tests in well-designed studies  to accurately assess their utility for pTB diagnosis. To conclude, rapid and accurate tests are warranted for pTB diagnosis.