Browsing by Author "Pallavi Sinha"

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Association of risk factors and drug resistance pattern in tuberculosis patients in North India
(Medknow Publications, 2017) Pallavi Sinha; G.N. Srivastava; Anamika Gupta; Shampa Anupurba
Context: India is one of the high tuberculosis (TB) burden countries in the world. Improper implementation in the guidelines for the management of TB and high rate of defaults on the part of the patients are most important risk factors for the development of multi-drug resistant TB. Aims: This study examines the drug resistance profile and the effect of demographic, clinical and behavioral risk factors on the prevalence of TB and multidrug resistance (MDR) in north India. Settings and Design: This was a prospective, observational study carried out from May 2012 to February 2014 in tertiary care hospital of Varanasi. Subjects and Methods: The study was performed on 721 pulmonary and extrapulmonary specimens of suspected TB patients based on history, was subjected for the Ziehl-Neelsen staining and culture on Lowenstein-Jensen (LJ) media. Statistical Analysis: The features of groups were compared by Chi-square (χ2) and odds ratio. Results: Out of 721 clinically suspected pulmonary and extrapulmonary TB patients, 222 (30.8%) patients were smear positive for acid-fast bacilli and 244 (38.3%) were positive for Mycobacterium species cultured on LJ medium. The prevalence of resistance to at least one anti-TB drug was 71.1% and MDR was 53.5%. Age, gender, HIV status, nature of TB, smoking, and alcohol consumption risk factors were significantly associated with TB prevalence; while prior history of TB infection, pervious household exposure, smoking, and alcohol consumption were significantly associated with MDR. Conclusion: This study showed a high prevalence of drug resistance TB in this region. It also provides evidence in our circumstance, of the role of prior history of TB infection, alcohol and smoking in increasing the risk of developing TB and MDR-TB. Therefore, it is necessary for the public health community to incorporate and strengthen alcohol and smoking nonparticipation interference in TB control program. © 2017 Journal of Global Infectious Diseases | Published by Wolters Kluwer-Medknow.
Detection of Beijing strains of MDR M. tuberculosis and their association with drug resistance mutations in katG, rpoB, and embB genes
(BioMed Central Ltd, 2020) Anamika Gupta; Pallavi Sinha; Vijay Nema; Pramod K. Gupta; Pampi Chakraborty; Savita Kulkarni; Nalin Rastogi; Shampa Anupurba
Background: Molecular epidemiological studies of Mycobacterium tuberculosis (MTB) are the core of current research to find out the association of the M. tuberculosis genotypes with its outbreak and transmission. The high prevalence of the Beijing genotype strain among multidrug resistance (MDR) TB has already been reported in various studies around India. The overall objective of this study was to detect the prevalence of Beijing genotype strains of MDR M. tuberculosis and their association with the clinical characteristics of TB patients. Methods: In this study 381 M. tuberculosis clinical isolates were obtained from sputum samples from 2008 to 2014. The multiplex-PCR and Spoligotyping (n = 131) methods were used to investigate the prevalence of the Beijing genotype strain by targeting the Rv2820 gene and their association with drug resistance and clinical characteristics of TB patients. The drug susceptibility testing of first-line anti-TB drugs was performed by using the proportion method and MGIT960. A collection of isolates having Beijing and non-Beijing strains were also characterized to see if Beijing genotype strains had a higher rate of mutations at codons 516, 526 and 531 of the 81-bp region of the rpoB gene, codon 315 of the katG gene, and codon 306 of the embB gene. Results: The sensitivities and specificities of multiplex-PCR assay compared to that of standard Spoligotyping was detected to be 100%. Further, we observe that the multi drug-resistance was significantly associated with Beijing genotype strains (p = 0.03) and a strong correlation between Beijing genotype strains and specific resistance mutations at the katG315, rpoB531, and embB306 codons (p = < 0.0001, < 0.0001 & 0.0014 respectively) was also found. Conclusions: This rapid, simple, and cost-effective multiplex PCR assay can effectively be used for monitoring the prevalence of Beijing genotype strains in low resource settings. Findings of this study may provide a scientific basis for the development of new diagnostic tools for detection and effective management of DR-TB in countries with a higher incidence rate of Beijing genotype strains. © 2020, The Author(s).
Detection of mutations in the rpoB gene of rifampicin-resistant Mycobacterium tuberculosis strains inhibiting wild type probe hybridization in the MTBDR plus assay by DNA sequencing directly from clinical specimens
(BioMed Central Ltd, 2020) Pallavi Sinha; G.N. Srivastava; Rajneesh Tripathi; Mukti Nath Mishra; Shampa Anupurba
Background: The potential of genetic testing for rapid and accurate diagnosis of drug-resistant Mycobacterium tuberculosis strains is vital for efficient treatment and reduction in dissemination. MTBDR plus assays rapidly detect mutations related to drug resistance and wild type sequences allied with susceptibility. Although these methods are promising, the examination of molecular level performance is essential for improved assay result interpretation and continued diagnostic development. Therefore this study aimed to determine novel mutations that were inhibiting wild type probe hybridization in the Line probe assay by DNA sequencing. Using data collected from Line Probe assay (GenoType MTBDRplus assay) the contribution of absent wild type probe hybridization to the detection of rifampicin resistance was assessed via comparison to a reference standard method i.e. DNA sequencing. Results: Sequence analysis of the rpoB gene of 47 MTB resistant strains from clinical specimens showed that 37 had a single mutation, 9 had double mutations and one had triple mutations in the ropB gene. Conclusions: The absence of wild type probe hybridization without mutation probe hybridization was mainly the result of the failure of mutation probe hybridization and the result of the novel or rare mutations. Additional probes are necessary to be included in the Line probe assay to improve the detection of rifampicin-resistant Mycobacterium tuberculosis strains. © 2020 The Author(s).
Differentiation of Mycobacterium tuberculosis complex from non-tubercular mycobacteria by nested multiplex PCR targeting IS6110, MTP40 and 32kD alpha antigen encoding gene fragments
(BioMed Central Ltd., 2016) Pallavi Sinha; Anamika Gupta; Pradyot Prakash; Shampa Anupurba; Rajneesh Tripathi; G.N. Srivastava
Background: Control of the global burden of tuberculosis is obstructed due to lack of simple, rapid and cost effective diagnostic techniques that can be used in resource poor-settings. To facilitate the early diagnosis of TB directly from clinical specimens, we have standardized and validated the use of nested multiplex PCR, targeting gene fragments IS6110, MTP40 and 32kD α-antigen encoding genes specific for Mycobacterium tuberculosis complex and non-tubercular mycobacteria (NTM), in comparison to smear microscopy, solid culture and single step multiplex PCR. The results were evaluated in comparison to a composite reference standard (CRS) comprising of microbiological results (smear and culture), clinical, radiological and cytopathological findings, clinical treatment and response to anti-tubercular therapy. Methods: The nested multiplex PCR (nMPCR) assay was evaluated to test its utility in 600 (535 pulmonary and 65 extra-pulmonary specimens) clinically suspected TB cases. All specimens were processed for smear, culture, single step multiplex PCR and nested multiplex PCR testing. Results: Out of 535 screened pulmonary and 65 extra-pulmonary specimens, 329 (61.5 %) and 19 (29.2 %) cases were culture positive for M. tuberculosis. Based on CRS, 450 patients had "clinical TB" (definitive-TB, probable-TB and possible-TB). Remaining 150 were confirmed "non-TB" cases. For culture, the sensitivity was low, 79.3 % for pulmonary and 54.3 % for extra-pulmonary cases. The sensitivity and specificity results for nMPCR test were evaluated taken composite reference standard as a gold standard. The sensitivity of the nMPCR assay was 97.1 % for pulmonary and 91.4 % for extra-pulmonary TB cases with specificity of 100 % and 93.3 % respectively. Conclusion: Nested multiplex PCR using three gene primers is a rapid, reliable and highly sensitive and specific diagnostic technique for the detection and differentiation of M. tuberculosis complex from NTM genome and will be useful in diagnosing paucibacillary samples. Nested multiplex PCR assay was found to be better than single step multiplex PCR for assessing the diagnosis of TB. © 2016 Sinha et al.
Genotype analysis of ofloxacin-resistant multidrug-resistant mycobacterium tuberculosis isolates in a multicentered study from india
(Indian Council of Medical Research, 2020) Anamika Gupta; Pallavi Sinha; Sunita Rathod; Siva Kumar Shanmugam; K.R. Uma Devi; Shampa Anupurba; Vijay Nema
Background & objectives: Drug resistance surveillance offers useful information on trends of drug resistance and the efficacy of control measures. Studies and reports of drug-resistant mutations and phenotypic assays thus become important. This study was conducted to investigate the molecular characteristics of ofloxacin (OFX)-resistant, multidrug-resistant tuberculosis (MDR-TB) isolates from different geographical regions of India and their association with strains of different genotypes. Further, the nitrate reductase assay (NRA) was tested against Mycobacteria Growth Indicator Tube (MGIT) for the determination of OFX resistance as an alternative and cost-effective method. Methods: A total of 116 Mycobacterium tuberculosis isolates were used to assess the mutations in the gyrA, gyrB genes and resistance levels to OFX. Mutational analysis in gyrA and gyrB genes and genotype analysis of M. tuberculosis isolates was done by gene-specific polymerase chain reaction (PCR) followed by DNA sequencing and spoligotyping, respectively. Results: Three (6.25%), 12 (44.44%) and 12 (29.27%) MDR-TB isolates from western, northern and southern India, respectively, were found to be OFX-resistant MDR-TB isolates. OFX resistance was observed to be significantly higher in MDR-TB cases for all study regions. Beijing genotypes from northern India were observed to be associated with OFX-resistant MDR-TB cases (P<0.05). Among 35 (30.15%) phenotypically OFX-resistant isolates, 22 (62.86%) had mutations in the gyrA gene and two (5.71%) isolates had mutations in the gyrB gene. Interpretation & conclusions: These results caution against the PCR-based prediction of OFX resistance patterns and highlight the need for searching other genetic loci for the detection of mutations conferring resistance to OFX in M. tuberculosis. Our study also showed the usefulness of NRA as an alternative method to detect OFX resistance. © 2020 Indian Journal of Medical Research, published by Wolters Kluwer-Medknow for Director-General, Indian Council of Medical Research.
Genotype analysis of ofloxacin-resistant multidrug-resistant Mycobacterium tuberculosis isolates in a multicentered study from India
(Wolters Kluwer Medknow Publications, 2020) Anamika Gupta; Pallavi Sinha; Sunita Rathod; Siva Shanmugam; K. Uma Devi; Shampa Anupurba; Vijay Nema
Background & objectives: Drug resistance surveillance offers useful information on trends of drug resistance and the efficacy of control measures. Studies and reports of drug-resistant mutations and phenotypic assays thus become important. This study was conducted to investigate the molecular characteristics of ofloxacin (OFX)-resistant, multidrug-resistant tuberculosis (MDR-TB) isolates from different geographical regions of India and their association with strains of different genotypes. Further, the nitrate reductase assay (NRA) was tested against Mycobacteria Growth Indicator Tube (MGIT) for the determination of OFX resistance as an alternative and cost-effective method. Methods: A total of 116 Mycobacterium tuberculosis isolates were used to assess the mutations in the gyrA, gyrB genes and resistance levels to OFX. Mutational analysis in gyrA and gyrB genes and genotype analysis of M. tuberculosis isolates was done by gene-specific polymerase chain reaction (PCR) followed by DNA sequencing and spoligotyping, respectively. Results: Three (6.25%), 12 (44.44%) and 12 (29.27%) MDR-TB isolates from western, northern and southern India, respectively, were found to be OFX-resistant MDR-TB isolates. OFX resistance was observed to be significantly higher in MDR-TB cases for all study regions. Beijing genotypes from northern India were observed to be associated with OFX-resistant MDR-TB cases (P <0.05). Among 35 (30.15%) phenotypically OFX-resistant isolates, 22 (62.86%) had mutations in the gyrA gene and two (5.71%) isolates had mutations in the gyrB gene. Interpretation & conclusions: These results caution against the PCR-based prediction of OFX resistance patterns and highlight the need for searching other genetic loci for the detection of mutations conferring resistance to OFX in M. tuberculosis. Our study also showed the usefulness of NRA as an alternative method to detect OFX resistance. © 2020 Indian Council of Medical Research. All rights reserved.
Performance of nested multiplex PCR assay targeting MTP40 and IS6110 gene sequences for the diagnosis of tubercular lymphadenitis
(Microbiological Society of Korea, 2017) Pallavi Sinha; Pradyot Prakash; Shashikant C. U. Patne; Shampa Anupurba; Sweety Gupta; G.N. Srivastava
The conventional methods for diagnosis of tubercular lymphadenitis (TBLN) such as - fine needle aspiration cytology, Ziehl-Neelsen staining and culture have limitations of low sensitivity and/or specificity. So, it becomes essential to develop a rapid, sensitive, and specific method for an early diagnosis of TBLN. Therefore, the present study was conducted to evaluate nested multiplex polymerase chain reaction (nMPCR) targeting MTP40 and IS6110 gene sequences of Mycobacterium tuberculosis and Mycobacterium tuberculosis complex, respectively in 48 successive patients of TBLN and 20 random patients with non-tubercular lymph node lesions. Out of the 48 cases of TBLN, 14 (29.2%) were found to be positive by Ziehl-Neelsen staining, 15 (31.2%) were positive by culture and 43 (89.6%) cases were positive after first round of PCR while 48 (100%) cases were positive by nMPCR assay. The sensitivity and specificity of nMPCR was found to be 100% for the diagnosis of TBLN. The results thus obtained indicate that nMPCR assay is a highly sensitive and specific tool for the diagnosis of TBLN. © 2017, The Microbiological Society of Korea and Springer-Verlag Berlin Heidelberg.
PknB remains an essential and a conserved target for drug development in susceptible and MDR strains of M. Tuberculosis
(BioMed Central Ltd., 2017) Anamika Gupta; Sudhir K. Pal; Divya Pandey; Najneen A. Fakir; Sunita Rathod; Dhiraj Sinha; S. SivaKumar; Pallavi Sinha; Mycal Periera; Shilpa Balgam; Gomathi Sekar; K.R. UmaDevi; Shampa Anupurba; Vijay Nema
Background: The Mycobacterium tuberculosis (M.tb) protein kinase B (PknB) which is now proved to be essential for the growth and survival of M.tb, is a transmembrane protein with a potential to be a good drug target. However it is not known if this target remains conserved in otherwise resistant isolates from clinical origin. The present study describes the conservation analysis of sequences covering the inhibitor binding domain of PknB to assess if it remains conserved in susceptible and resistant clinical strains of mycobacteria picked from three different geographical areas of India. Methods: A total of 116 isolates from North, South and West India were used in the study with a variable profile of their susceptibilities towards streptomycin, isoniazid, rifampicin, ethambutol and ofloxacin. Isolates were also spoligotyped in order to find if the conservation pattern of pknB gene remain consistent or differ with different spoligotypes. The impact of variation as found in the study was analyzed using Molecular dynamics simulations. Results: The sequencing results with 115/116 isolates revealed the conserved nature of pknB sequences irrespective of their susceptibility status and spoligotypes. The only variation found was in one strains wherein pnkB sequence had G to A mutation at 664 position translating into a change of amino acid, Valine to Isoleucine. After analyzing the impact of this sequence variation using Molecular dynamics simulations, it was observed that the variation is causing no significant change in protein structure or the inhibitor binding. Conclusions: Hence, the study endorses that PknB is an ideal target for drug development and there is no pre-existing or induced resistance with respect to the sequences involved in inhibitor binding. Also if the mutation that we are reporting for the first time is found again in subsequent work, it should be checked with phenotypic profile before drawing the conclusion that it would affect the activity in any way. Bioinformatics analysis in our study says that it has no significant effect on the binding and hence the activity of the protein. © 2017 The Author(s).
Rapid detection of drug-resistant Mycobacterium tuberculosis directly from clinical specimens using allele-specific polymerase chain reaction assay
(Wolters Kluwer Medknow Publications, 2019) Pallavi Sinha; Tuhina Banerjee; G. Srivastava; Shampa Anupurba
Background & objectives: Rapid detection of drug resistance in Mycobacterium tuberculosis (MTB) is essential for the efficient control of tuberculosis. Hence, in this study a nested-Allele-specific (NAS) PCR, nested multiple allele-specific PCR (NMAS-PCR) and multiple allele-specific (MAS) PCR assays were evaluated that enabled detection of the most common mutations responsible for isoniazid (INH) and rifampicin (RIF) resistance in MTB isolates directly from clinical specimens. © 2019 Indian Journal of Medical Research.
Rapid screening of MDR-TB in cases of extra pulmonary tuberculosis using geno type MTBDRplus
(Public Library of Science, 2016) Richa Kumari; Rajneesh Tripathi; Alok Prakash Pandey; Tuhina Banerjee; Pallavi Sinha; Shampa Anupurba
Background: Drug resistance in tuberculosis is a major public health challenge in developing countries. The limited data available on drug resistance in extra pulmonary tuberculosis stimulated us to design our study on anti-tuberculosis drug resistance pattern in cases of extra pulmonary tuberculosis in a tertiary referral hospital of North India. We performed Geno Type MTBDRplus assay in comparison with conventional drug susceptibility testing by proportion method to study the mutation patterns in rpoB, katG and inhA genes. Methods: A total of 510 extra pulmonary samples were included in this study. After the smear microscopy, all the specimens were subjected for culture on Lowenstein Jensen (LJ) media. Phenotypic drug susceptibility testing (DST) was performed on LJ media for all the MTB isolates and compared with the results of Geno Type MTBDRplus assay which was performed with the DNA isolated from the culture by conventional method. Results: Of 510 specimens cultured, the total culture positivity obtained was 11.8% (60) encompassing 54 (10.6%) Mycobacterium tuberculosis and 6 (1.2%) non-tubercular mycobacteria (NTM). DST results by Geno Type MTBDRplus assay and solid culture methods were compared in 51 MTB isolates excluding the two Rif indeterminate and one invalid test. Geno Type MTBDRplus accurately identified 13 of 14 rifampicin-resistant strains, 14 of 15 isoniazid-resistant strains and 13 of 14 as multi drug resistant tuberculosis (MDR-TB) in comparison with conventional method. Sensitivity and specificity were 92.86% and 97.30% respectively for detection of RIF resistance, 93.33% and 94.44% respectively for detection of INH resistance, 92.86% and 97.30% respectively for detection of MDR-TB, while the overall concordance of Geno Type MTBDRplus assay with conventional DST was 94.11%. The turn-around time for performing Geno Type MTBDRplus assay test was 48 hours. Conclusion: The problem of MDR in extra pulmonary tuberculosis (EPTB) cannot be overlooked and due attention on patients should be given. Routine use of Geno Type MTBDRplus assay for the diagnosis of MDR-EPTB can substantially reduce the time between diagnosis and drug therapy. Culture along with Geno Type MTBDRplus assay could be a solution for rapid and accurate diagnosis of MDR-TB in low bacillary non sputum specimens. © 2016 Kumari et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Speed breeding 3.0: mainstreaming light-driven plant breeding for sustainable genetic gains
(Elsevier Ltd, 2025) Pramod Gorakhanath Kabade; S. Veeresh J. Kumar; Ajay Kohli; Uma Maheshwar Singh; Pallavi Sinha; Vikas Kumar Singh
Advances in photobiological tools are revolutionizing plant breeding by enabling precise control of light parameters, addressing yield stagnation, and mitigating climate challenges. Full-spectrum light-emitting diodes (LEDs) and optimized light protocols have significantly reduced breeding cycles. This review highlights light-driven strategies that are accessible and practical for plant breeders worldwide including the role of light spectrum, intensity, and photoperiod in acceleration of plant growth in both short- and long-day crops. Speed breeding 3.0, with tailored rapid generation advancement (RGA) protocols designed for diverse crop populations, has the potential to significantly sustain genetic gains in a more efficient and targeted manner. These innovative approaches hold the potential to transform global agriculture and secure food systems in the face of rising populations and environmental uncertainties. © 2025 Elsevier Ltd
SpeedFlower: a comprehensive speed breeding protocol for indica and japonica rice
(John Wiley and Sons Inc, 2024) Pramod Gorakhanath Kabade; Shilpi Dixit; Uma Maheshwar Singh; Shamshad Alam; Sankalp Bhosale; Sanjay Kumar; Shravan Kumar Singh; Jyothi Badri; Nadimpalli Rama Gopala Varma; Sanjay Chetia; Rakesh Singh; Sharat Kumar Pradhan; Shubha Banerjee; Rupesh Deshmukh; Suresh Prasad Singh; Sanjay Kalia; Tilak Raj Sharma; Sudhanshu Singh; Hans Bhardwaj; Ajay Kohli; Arvind Kumar; Pallavi Sinha; Vikas Kumar Singh
To increase rice yields and feed billions of people, it is essential to enhance genetic gains. However, the development of new varieties is hindered by longer generation times and seasonal constraints. To address these limitations, a speed breeding facility has been established and a robust speed breeding protocol, SpeedFlower is developed that allows growing 4–5 generations of indica and/or japonica rice in a year. Our findings reveal that a high red-to-blue (2R > 1B) spectrum ratio, followed by green, yellow and far-red (FR) light, along with a 24-h long day (LD) photoperiod for the initial 15 days of the vegetative phase, facilitated early flowering. This is further enhanced by 10-h short day (SD) photoperiod in the later stage and day and night temperatures of 32/30 °C, along with 65% humidity facilitated early flowering ranging from 52 to 60 days at high light intensity (800 μmol m−2 s−1). Additionally, the use of prematurely harvested seeds and gibberellic acid treatment reduced the maturity duration by 50%. Further, SpeedFlower was validated on a diverse subset of 198 rice accessions from 3K RGP panel encompassing all 12 distinct groups of Oryza sativa L. classes. Our results confirmed that using SpeedFlower one generation can be achieved within 58–71 days resulting in 5.1–6.3 generations per year across the 12 sub-groups. This breakthrough enables us to enhance genetic gain, which could feed half of the world's population dependent on rice. © 2023 The Authors. Plant Biotechnology Journal published by Society for Experimental Biology and The Association of Applied Biologists and John Wiley & Sons Ltd.