An In-silico Approach to Design and Validate siRNA against Monkeypox Virus.

INTRODUCTION: The monkeypox virus has emerged as an uncommon zoonotic infection. The recent outbreak of MPXV in Europe and abroad in 2022 presented a major threat to individuals at risk. At present, no specific MPXV vaccinations or medications are available. METHODS: In this study, we predicted the most effective siRNA against the conserved region of the MPXV and validated the activity by performing molecular docking studies. RESULTS: Ultimately, the most efficient siRNA molecule was shortlisted against the envelope protein gene (B6R) based on its toxicity, effectivity, thermodynamic stability, molecular interaction, and molecular dynamics simulations (MD) with the Human Argonaute 2 protein. CONCLUSION: Thus, the strategy may offer a platform for the development of potential antiviral RNA therapeutics that target MPXV at the genomic level.

Antiretroviral action of Rosemary oil-based atazanavir formulation and the role of self-nanoemulsifying drug delivery system in the management of HIV-1 infection.

Atazanavir or ATV is an FDA-approved, HIV-1 protease inhibitor that belongs to the azapeptide group. Over time, it has been observed that ATV can cause multiple adverse side effects in the form of liver diseases including elevations in serum aminotransferase, indirect hyper-bilirubinemia, and idiosyncratic acute liver injury aggravating the underlying chronic viral hepatitis. Hence, there is an incessant need to explore the safe and efficacious method of delivering ATV in a controlled manner that may reduce the proportion of its idiosyncratic reactions in patients who are on antiretroviral therapy for years. In this study, we assessed ATV formulation along with Rosemary oil to enhance the anti-HIV-1 activity and its controlled delivery through self-nanoemulsifying drug delivery system or SNEDDS to enhance its oral bioavailability. While the designing, development, and characterization of ATV-SNEDDS were addressed through various evaluation parameters and pharmacokinetic-based studies, in vitro cell-based experiments assured the safety and efficacy of the designed ATV formulation. The study discovered the potential of ATV-SNEDDS to inhibit HIV-1 infection at a lower concentration as compared to its pure counterpart. Simultaneously, we could also demonstrate the ATV and Rosemary oil providing leads for designing and developing such formulations for the management of HIV-1 infections with the alleviation in the risk of adverse reactions.

A computational approach for designing and validating small interfering RNA against SARS-CoV-2 variants.

AIMS: The aim of this study is to develop a novel antiviral strategy capable of efficiently targeting a broad set of SARS-CoV-2 variants. BACKGROUND: Since the first emergence of SARS-CoV-2, it has rapidly transformed into a global pandemic, posing an unprecedented threat to public health. SARS-CoV-2 is prone to mutation and continues to evolve, leading to the emergence of new variants capable of escaping immune protection achieved due to previous SARS-CoV-2 infections or by vaccination. OBJECTIVE: RNA interference (RNAi) is a remarkable biological mechanism that can induce gene silencing by targeting complementary mRNA and inhibiting its translation. METHOD: In this study, using the computational approach, we predicted the most efficient siRNA capable of inhibiting SARS-CoV-2 variants of concern (VoCs). RESULT: The presented siRNA was characterized and evaluated for its thermodynamic properties, offsite-target hits, and in silico validation by molecular docking and molecular dynamics simulations (MD) with Human AGO2 protein. CONCLUSION: The study contributes to the possibility of designing and developing an effective response strategy against existing variants of concerns and preventing further.

Journey of technological advancements in the detection of antimicrobial resistance.

Increased uses rather an extensive misuse of antibiotics due to easy availability and easy access have resulted in antibiotic resistance as a global crisis. The speed of discovery of new antibiotics has slowed down recently. Therefore, there is a need to reduce the rate of increase in resistance against the presently available antibiotics, or else many infections may be left untreatable or difficult to be treated due to the high prevalence of resistance. The judicious use of broad-spectrum antibiotics can control the increase in resistance profile. Various techniques are presently being used for the detection of antibiotic resistance. Conventional phenotypic methods are preferred that are highly reliable but are much more time-consuming. The patients cannot spare more time as the infection keeps increasing. The results with genotypic methods are obtained within 24 h as compared to phenotypic methods. Hence, recent molecular methods like qPCR can be used for detection. In this review, we present an overview of various methods useful for the detection of antibiotic resistance, with emphasis on their advantages and limitations. The review also emphasizes qPCR to be the most preferred method out of all because of various advantageous factors.

Issues with the current drugs for Mycobacterium tuberculosis cure and potential of cell envelope proteins for new drug discovery.

Mycobacterium tuberculosis has been the smartest pathogen ever and a challenge to drug development. Its replicative machinery is unique, so targeting the same for killing the pathogen remains a challenge. Our body typically throws out the drugs before they see the bacterium multiply. The pathogen has also learned how to remove drugs from its internal chambers and not allow them to reach their targets. Another strategy for Mtb is the mutation of the targets to reject drug binding and bypass the drug's inhibitory actions. In this review, we tried to explore possible targets on the outer side of the bacterial cell. We have also explored if those targets are promising enough and if there are drugs or inhibitors available. We also discuss the essential proteins and why they remain to be a good target. We concluded that the cell envelope has got a few proteins that can be targeted in isolation or maybe along with other machinery while making the outer environment more conducive for penetration of current drugs or newly proposed drugs.

The role of thioredoxin proteins in Mycobacterium tuberculosis probed by proteome-wide target profiling.

Mycobacterium tuberculosis encounters diverse microenvironments, including oxidative assault (ROS and RNS), when it attempts to establish itself within its human host. Therefore, redox sensory and regulation processes are assumed significant importance, as these are essential processes for M. tuberculosis to survive under these hostile conditions. M. tuberculosis contains thioredoxin system to maintain redox homeostasis, which establish a balance between the thiol/dithiol couple. Still very less is known about it. In the present study, we attempted to capture the targets of all the M. tuberculosis thioredoxin proteins (viz., TrxB and TrxC) and a thioredoxin-like protein, NrdH, under aerobic and hypoxic conditions by performing thioredoxin trapping chromatography followed by mass spectrometry. We found that TrxC captured the maximum number of targets in both the physiological conditions and most of the targets of TrxB and NrdH showing overlap with targets of TrxC, indicating that TrxC acts as main thioredoxin. Further the PANTHER classification system provides involvement of targets in various metabolic processes and Gene Ontology analysis suggests that glutamine biosynthetic process and Fe-S cluster biosynthesis are the most enriched processes in the target list of TrxC and TrxB respectively. Also, we suggest that the thioredoxin system might play an important role under hypoxia by targeting those proteins which are responsible to sense and maintain hypoxic conditions. Furthermore, our studies establish a link between TrxB and iron-sulfur cluster biogenesis in M. tuberculosis. Ultimately, these findings open a new direction to target the thioredoxin system for screening new anti-mycobacterial drug targets.

Revisiting the therapeutic potential of homeopathic medicine Rhus Tox for herpes simplex virus and inflammatory conditions.

BACKGROUND: Herpes simplex virus type-1 and type-2 cause a viral disease named Herpes. Genital herpes is mainly caused by HSV-2 with symptoms of painful and itchy blisters on the vagina, cervix, buttocks, anus, penis, or inner thighs with blisters that rupture and convert into sores. The homeopathic remedy Rhus Tox has been widely used to treat herpes and has shown invitro anti-inflammatory effects in previous studies. PURPOSE: The presented review focuses on relapses and harmful effects caused by acyclovir in modern medicine and the probable antiherpetic activity of Rhus Tox on HSV infection based on its pathophysiology, preclinical findings, on primary cultured mouse chondrocytes, mouse cell line MC3T3e1 and a comparative study of Natrum Mur with Rhus Tox on HSV infection. STUDY DESIGN: The design of the study focuses mainly on the descriptive data available in various literature articles. METHOD: Databases such as PubMed, Google Scholar, Medline and ScienceDirect were used to search the articles. Articles are selected from 1994 to 2022 focusing solely on the competence of Rhus Tox against herpes. Keywords used for the study are antiviral, Herpes, Rhus Tox, in vitro and homeopathy. RESULTS: The review includes fifteen articles, including 4 full-text articles on HSV, 6 in vitro studies of homeopathic compounds performed on the herpes virus, and 5 articles based on the pathophysiology and effects of Rhus tox. The review article proposes the anti-inflammatory and antiviral action of the homeopathic remedy Rhus Tox which can be used in crisis conditions when the physician doubts the simillimum, as it prevents further outbreaks of HSV infection. CONCLUSION: The homeopathic medicine Rhus Tox has no cytotoxicity observed under in vitro conditions and can be used to treat herpes infection. Further studies are needed to confirm the results under in vitro and in vivo conditions as well as in clinical trials.

Association of Viral and Host Genetic Architecture with the Status of Neurocognitive Disorder in HIV-Infected Individuals.

The polymorphisms in host genes such as CCR5, CCR2, stromal derived factor (SDF), and MBL (mannose-binding lectin) as well as the viral nef gene have been shown to influence human immunodeficiency virus (HIV) infection, followed by the development of HIV-associated neurocognitive disorder (HAND). In this preliminary study with a limited number of samples, we have tried to associate the genetic polymorphism from the host and viral genetic factors with the neurocognitive status along with immuno-virological parameters. The total RNA was isolated from 10 unlinked plasma samples containing 5 samples from each group with and without HAND based on the International HIV Dementia Scale (IHDS) score <9.5 and >9.5, respectively. The CCR5, CCR2, SDF, MBL, and HIV nef genes were amplified and digested with restriction enzymes, except for the nef gene amplicon. Restrictions fragment length polymorphism (RFLP) was used to determine whether allelic variations were present in the digested host gene products, while sequencing was done for HIV nef amplicons without digestion. CCR5 delta 32 heterozygous variants were present in two samples from the HAND group. Three samples with HAND showed SDF-1 3' heterozygous allelic variant, while the MBL-2 gene presented with a homozygous mutant allele (D/D) in codon 52, heterozygous mutant allele (A/B) in codon 54, and codon 57 (A/C) for all samples except IHDS-2 irrespective of dementia status. Furthermore, amino acid alignment of Nef sequences confirmed the heterogeneity, while prediction of the human leukocyte antigen binding epitopes further explored its effect on functional motifs with variable binding efficiency such as epitopes GAFDLSFFL (aa 83) and LTFGWCFKL (aa 138) binding with HLA molecules at 60% and 80%, respectively. Thus, host genetics evidently influence predisposition to HIV infection and HAND. The genetic variability in the nef gene from both groups resulted in altering the functionality of specific domains and showing its impact on the progression of the disease, which needs to be explored.

Host-microbiota interactions and oncogenesis: Crosstalk and its implications in etiology.

A number of articles have discussed the potential of microbiota in oncogenesis. Several of these have evaluated the modulation of microbiota and its influence on cancer development. Even in recent past, a plethora of studies have gathered in order to understand the difference in microbiota population among different cancer and normal individuals. Although in majority of studies, microbiota mediated oncogenesis has been primarily attributed to the inflammatory mechanisms, there are several other ways through which microbiota can influence oncogenesis. These relatively less discussed aspects including the hormonal modulation through estrobolome and endobolome, production of cyclomodulins, and lateral gene transfer need more attention of scientific community. We prepared this article to discuss the role of microbiota in oncogenesis in order to provide concise information on these relatively less discussed microbiota mediated oncogenesis mechanisms.

Elucidation of Antiviral and Antioxidant Potential of C-Phycocyanin against HIV-1 Infection through In Silico and In Vitro Approaches.

Antiretroviral therapy is the single existing therapy for patients infected with HIV; however, it has drawbacks in terms of toxicity and resistance. Thus, there is a continuous need to explore safe and efficacious anti-retroviral agents. C-Phycocyanin (C-PC) is a phycobiliprotein, which has been known for various biological properties; however, its effect on HIV-1 replication needs revelation. This study aimed to identify the inhibitory effects of C-PC on HIV-1 using in vitro and in silico approaches and to assess its role in the generation of mitochondrial reactive oxygen species (ROS) during HIV-1 infection. In vitro anti-HIV-1 activity of C-PC was assessed on TZM-bl cells through luciferase gene assay against four different clades of HIV-1 strains in a dose-dependent manner. Results were confirmed in PBMCs, using the HIV-1 p24 antigen assay. Strong associations between C-PC and HIV-1 proteins were observed through in silico molecular simulation-based interactions, and the in vitro mechanistic study confirmed its target by inhibition of reverse transcriptase and protease enzymes. Additionally, the generation of mitochondrial ROS was detected by the MitoSOX and DCF-DA probe through confocal microscopy. Furthermore, our results confirmed that C-PC treatment notably subdued the fluorescence in the presence of the virus, thus reduction of ROS and the activation of caspase-3/7 in HIV-1-infected cells. Overall, our study suggests C-PC as a potent and broad in vitro antiviral and antioxidant agent against HIV-1 infection.

The NeuroinflammatoryPotential of HIV-1 NefVariants in Modulating the Gene Expression Profile of Astrocytes.

HIV-1 mediated neurotoxicity is thought to be associated with HIV-1 viral proteins activating astrocytes and microglia by inducing inflammatory cytokines leading to the development of HIV-associated neurocognitive disorder (HAND). In the current study, we observe how HIV-1 Nef upregulates the levels of IL-6, IP-10, and TNF-α around 6.0fold in normal human astrocytes (NHAs) compared to cell and empty vector controls. Moderate downregulation in the expression profile of inflammatory cytokines was observed due to RNA interference. Furthermore, we determine the impact of inflammatory cytokines in the upregulation of kynurenine pathway metabolites, such as indoleamine 2,3-dioxygenase (IDO), and 3-hydroxyanthranilic acid oxygenase (HAAO) in NHA, and found the same to be 3.0- and 3.2-fold, respectively. Additionally, the variation in the level of nitric oxide before and after RNA interference was significant. The upregulated cytokines and pathway-specific metabolites could be linked with the neurotoxic potential of HIV-1 Nef. Thus, the downregulation in cytokines and kynurenine metabolites observed after siRNA-Nef interference indicates the possibility of combining the RNA interference approach with current antiretroviral therapy to prevent neurotoxicity development.

Diagnostic performance of patient self-collected oral swab (tongue and cheek) in comparison with healthcare worker-collected nasopharyngeal swab for severe acute respiratory syndrome coronavirus-2 detection.

The present study was conducted to compare the performance of patient self-collected oral swab (OS) with healthcare worker (HCW)-collected nasopharyngeal swab (NPS) for SARS-CoV-2 detection by reverse transcription polymerase chain reaction (RT-PCR) in real-world setting. Paired OS and NPS were collected from 485 consecutive individuals presenting with symptoms of coronavirus disease-19 (COVID-19) or asymptomatic contacts of COVID-19 cases. Both specimens were processed for RT-PCR and cycle threshold (Ct) value for each test was obtained. Positive percent agreement (PPA), negative percent agreement (NPA), overall percent agreement (OPA) and kappa were calculated for OS RT-PCR compared with NPS RT-PCR as reference. A total of 116/485 (23.9%) participants were positive by NPS RT-PCR. OS had PPA of 71.6%, NPA of 98.8%, OPA of 92.4% and kappa of 0.771. Almost all participants (483/485, 99.6%) reported OS as a convenient and comfortable sample for SARS-CoV-2 testing over NPS. All participants with Ct values <25 and majority (90.8%) with Ct values <30 were detected by OS. To conclude, OS self-sampling was preferred in comparison with NPS due the ease and comfort during collection. The performance of OS RT-PCR for SARS-CoV-2 detection, however, was sub-optimal in comparison with NPS RT-PCR.

The Burden of Respiratory Viruses and Their Prevalence in Different Geographical Regions of India: 1970-2020.

As per the 2019 report of the National Health Portal of India, 41,996,260 cases and 3,740 deaths from respiratory infections were recorded across India in 2018. India contributes to 18% of the global population, with severe acute respiratory infection (SARI) as one of the prominent causes of mortality in children >5 years of age. Measures in terms of the diagnosis and surveillance of respiratory infections are taken up globally to discover their circulating types, detect outbreaks, and estimate the disease burden. Similarly, the purpose of this review was to determine the prevalence of respiratory infections in various regions of India through published reports. Understanding the pattern and prevalence of various viral entities responsible for infections and outbreaks can help in designing better strategies to combat the problem. The associated pathogens comprise respiratory syncytial virus (RSV), rhinovirus, influenza virus, parainfluenza virus, adenovirus, etc. Identification of these respiratory viruses was not given high priority until now, but the pandemic of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has sensitized our system to be alert about the burden of existing infections and to have proper checks for emerging ones. Most of the studies reported to date have worked on the influenza virus as a priority. However, the data describing the prevalence of other respiratory viruses with their seasonal pattern have significant epidemiological value. A comprehensive literature search was done to gather data from all geographical regions of India comprising all states of India from 1970 to 2020. The same has been compared with the global scenario and is being presented here.

HIV-Associated Neurotoxicity: The Interplay of Host and Viral Proteins.

HIV-1 can incite activation of chemokine receptors, inflammatory mediators, and glutamate receptor-mediated excitotoxicity. The mechanisms associated with such immune activation can disrupt neuronal and glial functions. HIV-associated neurocognitive disorder (HAND) is being observed since the beginning of the AIDS epidemic due to a change in the functional integrity of cells from the central nervous system (CNS). Even with the presence of antiretroviral therapy, there is a decline in the functioning of the brain especially movement skills, noticeable swings in mood, and routine performance activities. Under the umbrella of HAND, various symptomatic and asymptomatic conditions are categorized and are on a rise despite the use of newer antiretroviral agents. Due to the use of long-lasting antiretroviral agents, this deadly disease is becoming a manageable chronic condition with the occurrence of asymptomatic neurocognitive impairment (ANI), symptomatic mild neurocognitive disorder, or HIV-associated dementia. In-depth research in the pathogenesis of HIV has focused on various mechanisms involved in neuronal dysfunction and associated toxicities ultimately showcasing the involvement of various pathways. Increasing evidence-based studies have emphasized a need to focus and explore the specific pathways in inflammation-associated neurodegenerative disorders. In the current review, we have highlighted the association of various HIV proteins and neuronal cells with their involvement in various pathways responsible for the development of neurotoxicity.

Modulating catalytic activity of human topoisomerase II α enzyme by fluorescent gold nanoclusters.

Precise monitoring of the enzyme activity by a suitable modulator is one of the very fundamental aspects of drug designing that provides the opportunity to overcome the challenges of several diseases. Herein, inhibition of human Topoisomerase IIα enzyme which serves as a potential target site for several anti-cancer drugs is demonstrated by using ultra-small size gold nanoclusters (Au NCs) with the dimension comparable with size of the active site of the enzyme. Molecular dynamics simulation results demonstrate that the Au NCs strongly interact with the human Topo IIα enzyme at its active site or allosteric site depending on forms of enzyme. Additionally, binding energy and interaction profile provides the molecular basis of understanding of interactions of ultra-small size Au NCs and human Topo IIα enzyme. Enthalpy change (ΔH) and binding constant (K) are measured based on a sequential binding model of the Au NCs with the enzyme as demonstrated by the ITC study. Moreover, the in-vitro inhibition study of the catalytic activity of the enzyme and gel electrophoresis indicates that the ultra-small size Au NCs may be used as a potent inhibitor of human Topo IIα enzyme.

Current status of probiotics for prevention and management of gastrointestinal cancers.

INTRODUCTION: Gastrointestinal cancers contribute to a significant number of cancer- associated mortality. The gastrointestinal tract harbors a multitude of microorganisms, known as the microbiota. Recently, the microbiota is considered to be an accessory organ resulting in several health benefits. The microbiota is involved in almost all aspects of an individual ranging from managing behavior to controlling metabolism, immune status and the response to a disease. Researchers are observing the modulation of microbiota in almost every disease, including cancer. Probiotics are microorganisms that can help to alter the host microbiota toward a healthy state thus providing benefits from many diseases including cancer. AREAS COVERED: We explored the current status of the use of probiotics in cancer patients. Although probiotic bacteria can provide significant benefits to individuals suffering from cancer, the number of cancer-specific clinical products containing probiotics is not comparable to research studies showing their benefits. The lack of available products is due to several factors including a lack of risk assessment data of beneficial probiotics in cancer patients. EXPERT OPINION: Laboratory investigations indicate a huge potential of probiotics for the prevention and management of gastrointestinal cancer, but more clinical studies are required to support their application in clinical settings.

ACE2 and TMPRSS2 polymorphisms in various diseases with special reference to its impact on COVID-19 disease.

BACKGROUND: A carboxypeptidase protein called ACE2 is found in many organs. ACE2 protein can play a pivotal role to regulate the pathological changes of several diseases including COVID-19. TMPRSS2 gene is expressed in many human tissues and plays a critical role in spreading the infection of the viruses including coronavirus and progression of prostate cancer, and hence could be used as a potential drug target. There are limited reports on occurrence of genetic polymorphism of ACE2 and TMPRSS2 in general population, expressions in pathological conditions, and its impact on COVID-19 disease. Hence we comprehended the occurrence of ACE2, TMPRSS2 polymorphism in general population, expression in various diseases and its impact on COVID-19 disease. METHOD: We utilized multiple databases, PubMed (Medline), EMBASE and Google Scholar for literature search. DESCRIPTION: ACE2 polymorphisms have significant linkages with various diseases, including severity of SARS-CoV-2 infection. Genetic variations of these genes contribute to individual's genetic susceptibility to viral infection and its subsequent clearance. The diversity and variations in the population distribution of these genes, might greatly influence and in turn reflect into the observed population and gender differences of the severity and clinical outcomes of SARS-CoV-2 infection. CONCLUSION: There are diversities in distribution of ACE2 and TMPRSS2 polymorphisms among different populations. Analyzing the genetic variants and expression of ACE2 and TMPRSS2 genes, in a population may provide the genetic marker for susceptibility or resistance against the coronavirus infection, which might be useful for identifying the susceptible population groups for targeted interventions and for making relevant public health policy decisions.

Detection of Beijing strains of MDR M. tuberculosis and their association with drug resistance mutations in katG, rpoB, and embB genes.

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.

Genotype analysis of ofloxacin-resistant multidrug-resistant Mycobacterium tuberculosis isolates in a multicentered study from India.

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.