27 results on '"Haldar, Sagarika"'
Search Results
2. Evaluation of Mycobacterium tuberculosis derived cell-free DNA using pleural fluid and paired plasma samples for the diagnosis of pleural tuberculosis
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Sharma, Pratibha, Gupta, Rakesh Kumar, Anthwal, Divya, Dass, Manisha, Yadav, Rakesh, Behera, Ashish, Sethi, Sunil, Singhal, Ritu, Dhooria, Sahajal, Aggarwal, Ashutosh Nath, and Haldar, Sagarika
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- 2023
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3. MPT51 and MPT64-based antigen detection assay for the diagnosis of extrapulmonary tuberculosis from urine samples
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Dass, Manisha, Kaur, Mohinder, Aittan, Simran, Sharma, Pratibha, Punia, Sachin, Muthumohan, Rajagopalan, Anthwal, Divya, Gupta, Rakesh K., Mahajan, Gargi, Kumari, Pooja, Sharma, Neera, Taneja, Rajesh S., Sharma, Lokesh K., Shree, Ritu, Tyagi, Jaya S., Lal, Vivek, and Haldar, Sagarika
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- 2023
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4. Utility of cell-free transrenal DNA for the diagnosis of Tuberculous Meningitis: A proof-of-concept study
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Dass, Manisha, Aittan, Simran, Muthumohan, Rajagopalan, Anthwal, Divya, Gupta, Rakesh Kumar, Mahajan, Gargi, Kumari, Pooja, Sharma, Neera, Taneja, Rajesh S., Sharma, Lokesh Kumar, Shree, Ritu, Lal, Vivek, Tyagi, Jaya Sivaswami, and Haldar, Sagarika
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- 2022
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5. Assessment of DNA aptamers targeting GlcB and HspX antigens for application in the diagnosis of abdominal tuberculosis
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Kumari, Pooja, Dhiman, Abhijeet, Lavania, Surabhi, Sharma, Pratibha, Rath, Deepak, Anthwal, Divya, Gupta, Rakesh Kumar, Kochar, Archit, Sharma, Neera, Gadpayle, A.K., Taneja, R.S., Sharma, Lokesh, Haldar, Sagarika, Sharma, Tarun Kumar, and Tyagi, Jaya Sivaswami
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- 2022
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6. Direct Molecular Detection of Drug-Resistant Tuberculosis from Transported Bio-Safe Dried Sputum on Filter-Paper
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Anthwal, Divya, Jamwal, Shaina, Gupta, Rakesh Kumar, Singhal, Ritu, Verma, Ajoy Kumar, Bhalla, Manpreet, Myneedu, Vithal Prasad, Sarin, Rohit, Choudhary, Sangeeta, Tyagi, Jaya Sivaswami, and Haldar, Sagarika
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- 2022
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7. Genotypic characterization of ‘inferred’ rifampin mutations in GenoType MTBDRplus assay and its association with phenotypic susceptibility testing of Mycobacterium tuberculosis.
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Singhal, Ritu, Anthwal, Divya, Kumar, Gavish, Sah, Grish, Salfinger, Max, Choudhury, Sangeeta, Arora, Jyoti, Bhalla, Manpreet, Myneedu, Vithal P., Sarin, Rohit, and Haldar, Sagarika
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- 2020
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8. Theranostic Application of a Novel G-Quadruplex-Forming DNA Aptamer Targeting Malate Synthase of Mycobacterium tuberculosis
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Dhiman, Abhijeet, Kumar, Chanchal, Mishra, Subodh Kumar, Sikri, Kriti, Datta, Ishara, Sharma, Pradeep, Singh, Tej P., Haldar, Sagarika, Sharma, Neera, Bansal, Anjali, Ahmad, Yusra, Kumar, Amit, Sharma, Tarun Kumar, and Tyagi, Jaya Sivaswami
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- 2019
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9. Generation and application of DNA aptamers against HspX for accurate diagnosis of tuberculous meningitis
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Dhiman, Abhijeet, Haldar, Sagarika, Mishra, Subodh Kumar, Sharma, Neera, Bansal, Anjali, Ahmad, Yusra, Kumar, Amit, Sharma, Tarun Kumar, and Tyagi, Jaya Sivaswami
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- 2018
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10. Evaluation of ‘TBDetect’ sputum microscopy kit for improved detection of Mycobacterium tuberculosis: a multi-centric validation study
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Anthwal, Divya, Gupta, Rakesh Kumar, Gomathi, Narayan Sivaramakrishnan, Tripathy, Srikanth Prasad, Das, Dasarathi, Pati, Sanghamitra, Panwalkar, Nikita, Desikan, Prabha, Bala, Kiran, Singh, Urvashi B., Bhalla, Manpreet, Singhal, Ritu, Verma, Ajoy Kumar, Khayyam, Khalid Umar, Myneedu, Vithal Prasad, Sarin, Rohit, Sharma, Sandeep, Bansal, Avi Kumar, Gupta, Umesh D., Patil, Sripad A., Goyal, Abhinav, Gupta, Ashawant, Singh, Manjula, Gupta, Nalini Kant, Haldar, Sagarika, and Tyagi, Jaya Sivaswami
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- 2021
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11. Efficient diagnosis of tuberculous meningitis by detection of Mycobacterium tuberculosis DNA in cerebrospinal fluid filtrates using PCR
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Haldar, Sagarika, Sharma, Neera, Gupta, V. K., and Tyagi, Jaya Sivaswami
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- 2009
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12. Simplified detection of Mycobacterium tuberculosis in sputum using smear microscopy and PCR with molecular beacons
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Haldar, Sagarika, Chakravorty, Soumitesh, Bhalla, Manpreet, De Majumdar, Shyamasree, and Tyagi, Jaya Sivaswami
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- 2007
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13. Utility of circulating cell-free Mycobacterium tuberculosis DNA for the improved diagnosis of abdominal tuberculosis.
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Sharma, Pratibha, Anthwal, Divya, Kumari, Pooja, Gupta, Rakesh Kumar, Lavania, Surabhi, Sharma, Neera, Sharma, Lokesh Kumar, Rath, Deepak, Soraganvi, Pavan Kumar, Sharma, Ashish, Gadpayle, A. K., Taneja, R. S., Tyagi, Jaya Sivaswami, and Haldar, Sagarika
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CELL-free DNA ,MYCOBACTERIUM tuberculosis ,TUBERCULOSIS ,DNA ,ASCITIC fluids ,ETIOLOGY of diseases - Abstract
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. [ABSTRACT FROM AUTHOR]
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- 2020
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14. 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.
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Anthwal, Divya, Lavania, Surabhi, Gupta, Rakesh Kumar, Verma, Ajoy, Myneedu, Vithal Prasad, Sharma, Prem Prakash, Verma, Hitesh, Malhotra, Viveksheel, Gupta, Ashawant, Gupta, Nalini Kant, Sarin, Rohit, Haldar, Sagarika, and Tyagi, Jaya Sivaswami
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SPUTUM ,DRUG resistance ,MYCOBACTERIUM tuberculosis ,MYCOBACTERIA ,RIFAMPIN ,FLUORESCENCE microscopy ,NUCLEOTIDE sequence ,FILTERS & filtration - Abstract
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. [ABSTRACT FROM AUTHOR]
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- 2019
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15. Structural switching electrochemical DNA aptasensor for the rapid diagnosis of tuberculous meningitis.
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Das, Ritu, Dhiman, Abhijeet, Mishra, Subodh Kumar, Haldar, Sagarika, Sharma, Neera, Bansal, Anjali, Ahmad, Yusra, Kumar, Amit, Tyagi, Jaya Sivaswami, and Sharma, Tarun Kumar
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- 2019
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16. Direct detection of Mycobacterium tuberculosis rifampin resistance in bio-safe stained sputum smears.
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Lavania, Surabhi, Anthwal, Divya, Bhalla, Manpreet, Singh, Nagendra, Haldar, Sagarika, and Tyagi, Jaya Sivaswami
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MYCOBACTERIUM tuberculosis ,RIFAMPIN ,DRUG resistance in bacteria ,SPUTUM examination ,MICROBIAL sensitivity tests ,THERAPEUTICS - Abstract
Direct smear microscopy of sputum forms the mainstay of TB diagnosis in resource-limited settings. Stained sputum smear slides can serve as a ready-made resource to transport sputum for molecular drug susceptibility testing. However, bio-safety is a major concern during transport of sputum/stained slides and for laboratory workers engaged in processing Mycobacterium tuberculosis infected sputum specimens. In this study, a bio-safe USP (Universal Sample Processing) concentration-based sputum processing method (Bio-safe method) was assessed on 87 M. tuberculosis culture positive sputum samples. Samples were processed for Ziehl-Neelsen (ZN) smear, liquid culture and DNA isolation. DNA isolated directly from sputum was subjected to an IS6110 PCR assay. Both sputum DNA and DNA extracted from bio-safe ZN concentrated smear slides were subjected to rpoB PCR and simultaneously assessed by DNA sequencing for determining rifampin (RIF) resistance. All sputum samples were rendered sterile by Bio-safe method. Bio-safe smears exhibited a 5% increment in positivity over direct smear with a 14% increment in smear grade status. All samples were positive for IS6110 and rpoB PCR. Thirty four percent samples were RIF resistant by rpoB PCR product sequencing. A 100% concordance (κ value = 1) was obtained between sequencing results derived from bio-safe smear slides and bio-safe sputum. This study demonstrates that Bio-safe method can address safety issues associated with sputum processing, provide an efficient alternative to sample transport in the form of bio-safe stained concentrated smear slides and can also provide information on drug (RIF) resistance by direct DNA sequencing. [ABSTRACT FROM AUTHOR]
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- 2017
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17. Sub-optimal Specificity of Modified Ziehl-Neelsen Staining for Quick Identification of Tuberculous Meningitis.
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Ting Wang, Guo-Dong Feng, Yu Pang, Yi-Ning Yang, Wen Dai, Lin Zhang, Lin-Fu Zhou, Jia-Lei Yang, Li-Ping Zhan, Marais, Ben J., Yan-Lin Zhao, Gang Zhao, Haldar, Sagarika, and Patel, Vinod Bhagu
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TUBERCULOUS meningitis ,ZIEHL-Neelsen stain ,DIAGNOSIS - Abstract
Background: Microbiological confirmation of tuberculous meningitis (TBM) remains problematic. We assessed the diagnostic performance of a modified Ziehl-Neelsen (MZN) staining method that showed promise in earlier studies. Methods: Patients evaluated for TBM in Shaanxi province, China, were prospectively enrolled from May, 2011 to April, 2013. Cerebrospinal fluid (CSF) specimens were evaluated using the Xpert MTB/RIF® assay, MZN staining, and standard biochemical and microbiological tests, together with detailed clinical and radiological assessment. Results: Among 316 patients included in the study, 38 had definite TBM, 66 probable TBM, 163 possible TBM and 49 "no TBM," using consensus uniform research case definition criteria. Comparing "definite or probable TBM" to "no TBM" MZN staining had higher sensitivity than Xpert MTB/RIF® (88.5 vs. 36.5%), but greatly reduced specificity (71.4 vs. 100.0%); 14/49 (28.6%) cases with "no TBM" tested positive on MZN. Mycobacterium tuberculosis culture was performed in 104/179 (58.1%) of MZN positive samples; 12.5% (13/104) were positive. Using Xpert MTB/RIF® as the reference standard, MZN had a sensitivity of 92.1% (95% CI 79.2-97.3) and specificity of 71.4% (95% CI 57.6-82.2). Conclusion: Xpert MTB/RIF® offered a rapid and specific TBM diagnosis, but sensitivity was poor. MZN was mainly hampered by false positives. Strategies to enhance the sensitivity of Xpert MTB/RIF® or improve the diagnostic accuracy of MZN should be explored. [ABSTRACT FROM AUTHOR]
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- 2016
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18. Detection of Mycobacterium tuberculosis GlcB or HspX Antigens or devRDNA Impacts the Rapid Diagnosis of Tuberculous Meningitis in Children.
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Haldar, Sagarika, Sankhyan, Naveen, Sharma, Neera, Bansal, Anjali, Jain, Vitul, Gupta, V. K., Juneja, Monica, Mishra, Devendra, Kapil, Arti, Singh, Urvashi B., Gulati, Sheffali, Kalra, Veena, and Tyagi, Jaya Sivaswami
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MYCOBACTERIUM tuberculosis , *MENINGITIS diagnosis , *CEREBROSPINAL fluid , *PEDIATRIC diagnosis , *NEISSERIA meningitidis , *LOGISTIC regression analysis - Abstract
Background: Tuberculous 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 Findings: We 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. Conclusions: The 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. [ABSTRACT FROM AUTHOR]
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- 2012
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19. Improved laboratory diagnosis of tuberculosis – The Indian experience.
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Haldar, Sagarika, Bose, Mridula, Chakrabarti, Parul, Daginawala, Hatim F., Harinath, B.C., Kashyap, Rajpal S., Kulkarni, Savita, Majumdar, Anindita, Prasad, H. Krishna, Rodrigues, Camilla, Srivastava, Ranjana, Taori, Girdhar M., Varma-Basil, Mandira, and Tyagi, Jaya S.
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TUBERCULOSIS ,CLINICAL pathology ,MORTALITY ,COMMUNICABLE diseases ,MYCOBACTERIUM tuberculosis ,COST effectiveness ,POLYMERASE chain reaction - Abstract
Summary: Tuberculosis (TB) is the leading cause of death worldwide attributable to a single infectious disease agent. India has more new TB cases annually than any other country. In 2008, India accounted for a fifth of the estimated 9.4 million TB cases globally. There is an overwhelming need for improving TB diagnostics in India through the use of cost effective, patient-friendly methods appropriate to different tiers of the country health system. Substantial progress has been made in India in the field of TB diagnosis and serious efforts have been made to herald the development of diagnostic tests for pulmonary TB, extra pulmonary TB and MDR-TB. Diverse approaches have been attempted towards improving smear microscopy, rapid culture and for differentiation between the Mycobacterium tuberculosis complex and non-tuberculous mycobacteria. Several laboratories have developed in-house PCR assays for diagnosing TB with high accuracy. Approaches for distinguishing M. tuberculosis and/or Mycobacterium bovis infection and disseminated Mycobacterium avium complex infection in HIV-AIDS patients have also been described. Serological tests to detect antigens or antibodies to M. tuberculosis specific components by using cocktails of Excretory/Secretory protein antigens, Ag85 complex antigens, Hsp 65 antigen, RD1 antigens and Rapid Reverse Line Blot Hybridization assays to detect MDR-TB (mutations to rifampicin, isoniazid and streptomycin) have also been developed. Other methods like measurement of adenosine deaminase activity and use of luciferase reporter phages have also been explored for TB diagnosis. These advances in the Indian context are detailed in the present chapter. The validation and application of these methods in laboratory and public health settings is likely to result in improved TB diagnosis and contribute to effective disease management in India. [Copyright &y& Elsevier]
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- 2011
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20. PCR amplification of shorter fragments from the devR ( Rv3133c) gene significantly increases the sensitivity of tuberculosis diagnosis.
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Chakravorty, Soumitesh, Pathak, Divya, Dudeja, Mridu, Haldar, Sagarika, Hanif, M., and Tyagi, Jaya Sivaswami
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TUBERCULOSIS diagnosis ,MYCOBACTERIAL diseases ,TUBERCULIN ,GENETICS ,GENETIC engineering - Abstract
This study was designed to assess the vital issue of gene target length and PCR assay performance in relation to the detection of Mycobacterium tuberculosis in clinical specimens. Two PCR assays that amplify fragments of varying lengths from the devR gene of M. tuberculosis were evaluated. Using M. tuberculosis DNA the ‘short-length’ PCR assay detected 250–500 genome equivalents vs. 500–1000 genome equivalents by the ‘long-length’ assay. In comparison to a highly sensitive smear microscopy test (universal sample processing smear), the sensitivity of the ‘short-length’ assay was 97.8% vs. 69.9% of the ‘long-length’ assay in sputum specimens ( n=506) from patients being evaluated for a possible diagnosis of tuberculosis. The 27.9% absolute increase in sensitivity was statistically significant ( P<0.001). Our results indicate that in a clinical setting when all other conditions are equal, the amplification of a shorter gene fragment of devR increases the sensitivity and efficiency of the PCR assay in spite of using a single copy gene as target. [ABSTRACT FROM AUTHOR]
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- 2006
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21. A novel aptamer-based test for the rapid and accurate diagnosis of pleural tuberculosis.
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Kumari, Pooja, Lavania, Surabhi, Tyagi, Shaifali, Dhiman, Abhijeet, Rath, Deepak, Anthwal, Divya, Gupta, Rakesh Kumar, Sharma, Neera, Gadpayle, A.K., Taneja, R.S., Sharma, Lokesh, Ahmad, Yusra, Sharma, Tarun Kumar, Haldar, Sagarika, and Tyagi, Jaya Sivaswami
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APTAMERS , *MYCOBACTERIUM tuberculosis , *MICROBIOLOGY , *ANTIGENS ,PLEURAL tuberculosis - Abstract
Abstract 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. Highlights • An HspX Aptamer Linked Immobilized Sorbent Assay (ALISA) is developed to detect pleural TB (pTB). • Its performance is compared with qPCR, GeneXpert and ELISA in pleural fluid samples. • HspX ALISA is superior to other tests and detects pTB with ~93% sensitivity and ~98% specificity. • ALISA demonstrates strong potential to bridge the existing gap in pTB diagnosis. [ABSTRACT FROM AUTHOR]
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- 2019
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22. Operational feasibility and multi-centric evaluation of 'TB Detect sputum microscopy kit' for the direct detection of Mycobacterium tuberculosis in field settings.
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Chauhan K, Gupta RK, Anthwal D, Panwalkar N, Desikan P, Bhalla M, Singhal R, Myneedu VP, Umar Khayyam K, Kumar Shanmugam S, Silambu Chelvi K, Radhakrishnan A, Chandrasekaran P, Giri S, Turuk J, Das D, Pati S, Goyal A, Gupta A, Kant Gupta N, Singh M, Sivaswami Tyagi J, and Haldar S
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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 'TB Detect ' 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 'TB Detect ' microscopy in field settings. This was evaluated by (i) assessing the performance of 'TB Detect ' 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 'TB Detect' microscopy in DMCs, sample preparation and testing, training, time-to-result, logistics, and troubleshooting. A scoring questionnaire was also used to assess 'TB Detect ' microscopy vs . LED-FM and statistical significance of the scores was calculated using paired t -test., Results: The overall positivity of 'TB Detect ' 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, 'TB Detect ' 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 'TB Detect ' 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 'TB Detect ' microscopy in field settings.
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- 2024
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23. Direct Detection of Fluoroquinolone Resistance in Sputum Samples from Tuberculosis Patients by High Resolution Melt Curve Analysis.
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Gupta RK, Anthwal D, Bhalla M, Tyagi JS, Choudhary S, and Haldar S
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- Humans, Antitubercular Agents pharmacology, Isoniazid pharmacology, Sputum microbiology, Rifampin pharmacology, Fluoroquinolones pharmacology, Fluoroquinolones therapeutic use, Microbial Sensitivity Tests, Sensitivity and Specificity, Mycobacterium tuberculosis, Tuberculosis, Multidrug-Resistant diagnosis, Tuberculosis, Multidrug-Resistant drug therapy, Tuberculosis, Multidrug-Resistant microbiology
- Abstract
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., (© 2023. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)
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- 2023
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24. 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.
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Anthwal D, Gupta RK, Singhal R, Bhalla M, Verma AK, Khayyam KU, Myneedu VP, Sarin R, Gupta A, Gupta NK, Singh M, Sivaswami Tyagi J, and Haldar S
- Abstract
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., Competing Interests: Conflict of interest: D. Anthwal reports personal fees from the ICMR (fellowship support) during the conduct of the study and a patent, “Apparatus and method for processing a sample for rapid diagnosis of tuberculosis and safe transport of bacteria” (patent application number 201811042155), pending. Conflict of interest: R.K. Gupta has a patent, “Apparatus and method for processing a sample for rapid diagnosis of tuberculosis and safe transport of bacteria” (patent application number 201811042155), pending; and manufactured and provided the TB Concentration & Transport and TB DNA Extraction kits, and participated in training of the laboratory staff who used the kit. Conflict of interest: R. Singhal reports grants from the ICMR during the conduct of the study. Conflict of interest: M. Bhalla reports grants from the ICMR during the conduct of the study. Conflict of interest: A.K. Verma reports grants from the ICMR during the conduct of the study and a patent, “Apparatus and method for processing a sample for rapid diagnosis of tuberculosis and safe transport of bacteria” (patent application number 201811042155), pending. Conflict of interest: K.U. Khayyam reports grants from the ICMR during the conduct of the study. Conflict of interest: C.P. Myneedu reports grants from the ICMR during the conduct of the study and a patent, “Apparatus and method for processing a sample for rapid diagnosis of tuberculosis and safe transport of bacteria” (patent application number 201811042155), pending. Conflict of interest: R. Sarin reports grants from the ICMR during the conduct of the study and a patent, “Apparatus and method for processing a sample for rapid diagnosis of tuberculosis and safe transport of bacteria” (patent application number 201811042155), pending. Conflict of interest: A. Gupta has a patent, “Apparatus and method for processing a sample for rapid diagnosis of tuberculosis and safe transport of bacteria” (patent application number 201811042155), pending; and manufactured and provided the TB Concentration & Transport and TB DNA Extraction kits, and participated in training of the laboratory staff who used the kit. Conflict of interest: N.K. Gupta has a patent, “Apparatus and method for processing a sample for rapid diagnosis of tuberculosis and safe transport of bacteria” (patent application number 201811042155), pending; and manufactured and provided the TB Concentration & Transport and TB DNA Extraction kits, and participated in training of the laboratory staff who used the kit. Conflict of interest: M. Singh has nothing to disclose. Conflict of interest: J. Sivaswami Tyagi reports grants from ICMR during the conduct of the study and a patent, “Apparatus and method for processing a sample for rapid diagnosis of tuberculosis and safe transport of bacteria” (patent application number 201811042155), pending. Conflict of interest: S. Haldar reports grants from the ICMR during the conduct of the study and a patent, “Apparatus and method for processing a sample for rapid diagnosis of tuberculosis and safe transport of bacteria” (patent application number 201811042155), pending., (Copyright ©The authors 2021.)
- Published
- 2021
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25. Challenges in pleural tuberculosis diagnosis: existing reference standards and nucleic acid tests.
- Author
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Tyagi S, Sharma N, Tyagi JS, and Haldar S
- Subjects
- DNA, Bacterial analysis, DNA, Bacterial isolation & purification, Humans, Molecular Diagnostic Techniques methods, Mycobacterium tuberculosis pathogenicity, Polymerase Chain Reaction methods, Sensitivity and Specificity, Tuberculosis, Pleural classification, Tuberculosis, Pleural microbiology, Molecular Diagnostic Techniques standards, Mycobacterium tuberculosis genetics, Nucleic Acids analysis, Tuberculosis, Pleural diagnosis
- Abstract
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.- Published
- 2017
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26. Direct Detection of Rifampin and Isoniazid Resistance in Sputum Samples from Tuberculosis Patients by High-Resolution Melt Curve Analysis.
- Author
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Anthwal D, Gupta RK, Bhalla M, Bhatnagar S, Tyagi JS, and Haldar S
- Subjects
- DNA, Bacterial genetics, Fatty Acid Synthesis Inhibitors, Humans, Isoniazid pharmacology, Mycobacterium tuberculosis isolation & purification, Rifampin pharmacology, Sensitivity and Specificity, Transition Temperature, Antitubercular Agents pharmacology, Drug Resistance, Bacterial, Genotyping Techniques methods, Microbial Sensitivity Tests methods, Mycobacterium tuberculosis drug effects, Sputum microbiology, Tuberculosis, Pulmonary microbiology
- Abstract
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., (Copyright © 2017 American Society for Microbiology.)
- Published
- 2017
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27. Detection of acid-fast bacilli in postlysis debris of clinical specimens improves the reliability of PCR.
- Author
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Haldar S, De Majumdar S, Chakravorty S, Tyagi JS, Bhalla M, and Sen MK
- Subjects
- Bacteriological Techniques, Chelating Agents chemistry, Chelating Agents pharmacology, DNA, Bacterial analysis, Guanidines, Humans, Mycobacterium tuberculosis drug effects, Mycobacterium tuberculosis genetics, Octoxynol pharmacology, Reproducibility of Results, Thiocyanates, Tuberculosis diagnosis, Tuberculosis microbiology, DNA, Bacterial isolation & purification, Mycobacterium tuberculosis isolation & purification, Polymerase Chain Reaction methods, Specimen Handling methods, Sputum microbiology
- Published
- 2005
- Full Text
- View/download PDF
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