HPMA-PLGA Based Nanoparticles for Effective In Vitro Delivery of Rifampicin.

PURPOSE: Tuberculosis (TB) chemotherapy witnesses some major challenges such as poor water-solubility and bioavailability of drugs that frequently delay the treatment. In the present study, an attempt to enhance the aqueous solubility of rifampicin (RMP) was made via co-polymeric nanoparticles approach. HPMA (N-2-hydroxypropylmethacrylamide)-PLGA based polymeric nanoparticulate system were prepared and evaluated against Mycobacterium tuberculosis (MTB) for sustained release and bioavailability of RMP to achieve better delivery. METHODOLOGY: HPMA-PLGA nanoparticles (HP-NPs) were prepared by modified nanoprecipitation technique, RMP was loaded in the prepared NPs. Characterization for particle size, zeta potential, and drug-loading capacity was performed. Release was studied using membrane dialysis method. RESULTS: The average particles size, zeta potential, polydispersity index of RMP loaded HPMA-PLGA-NPs (HPR-NPs) were 260.3 ± 2.21 nm, -6.63 ± 1.28 mV, and 0.303 ± 0.22, respectively. TEM images showed spherical shaped NPs with uniform distribution without any cluster formation. Entrapment efficiency and drug loading efficiency of HPR-NPs were found to be 76.25 ± 1.28%, and 26.19 ± 2.24%, respectively. Kinetic models of drug release including Higuchi and Korsmeyer-peppas demonstrated sustained release pattern. Interaction studies with human RBCs confirmed that RMP loaded HP-NPs are less toxic in this model than pure RMP with (p < 0.05). CONCLUSIONS: The pathogen inhibition studies revealed that developed HPR-NPs were approximately four times more effective with (p < 0.05) than pure drug against sensitive Mycobacterium tuberculosis (MTB) stain. It may be concluded that HPR-NPs holds promising potential for increasing solubility and bioavailability of RMP.

Smartly Engineered PEGylated Di-Block Nanopolymeric Micelles: Duo Delivery of Isoniazid and Rifampicin Against Mycobacterium tuberculosis.

In an attempt to deliver multiple drugs through a nanoparticulate platform, the present study was designed to deliver isoniazid (INH) and rifampicin (RMP) together through conjugation/encapsulation approaches using PEG-PLA (polyethylene glycol-poly-L-lactic acid) polymeric micelles. The objective of this study is to identify the preparation and evaluation of PEGylated polymeric micelles with dual drug delivery of INH and RMP for the effective treatment of tuberculosis (TB). Synthesized PEG-PLA di-block-copolymer was further conjugated to INH-forming PEG-PLA-INH (PPI) conjugate. Separately, these conjugates were loaded with RMP building the rifampicin-loaded PEG-PLA-INH polymeric micelles (PMC). The critical micelle concentration (CMC) for the PEG-PLA copolymer was found to be 8.9 ± 0.96 mg/L, and the size and zeta potential were observed to be 187.9 ± 2.68 nm and - 8.15 ± 1.24 mV (0.251 ± 0.042 pdi), respectively. Percent drug loading of PMC was 16.66 ± 1.52 and 23.07 ± 1.05 with entrapment efficiency of 72.30 ± 3.49 and 78.60 ± 2.67% for RMP and INH, respectively. RBC hemolysis capacity of PMC was significantly less than pure RMP and INH. Microplate Alamar blue assay (MABA) along with microscopy showed that the nanoconstructed PMC were more effective than the drugs, and approximately 8-fold reduction in overall minimum inhibitory concentration (MIC) was observed. The prepared duo drug-loaded nano-engineered polymeric micelles were highly effective against sensitive Mycobacterium tuberculosis strains and found to be less hemolytic in nature. The micelles could be further explored (in the future) for in vivo anti-TB studies to establish further to achieve better treatment for TB.

Conjugated and Entrapped HPMA-PLA Nano-Polymeric Micelles Based Dual Delivery of First Line Anti TB Drugs: Improved and Safe Drug Delivery against Sensitive and Resistant Mycobacterium Tuberculosis.

PURPOSE: First line antiTB drugs have several physical and toxic manifestations which limit their applications. RIF is a hydrophobic drug and has low water solubility and INH is hepatotoxic. The main objective of the study was to synthesize, characterize HPMA-PLA co-polymeric micelles for the effective dual delivery of INH and RIF. METHODS: HPMA-PLA co-polymer and HPMA-PLA-INH (HPI) conjugates were synthesized and characterized by FT-IR and 1H-NMR spectroscopy. Later on RIF loaded HPMA-PLA-INH co-polymeric micelles (PMRI) were formulated and characterized for size, zeta potential and surface morphology (SEM, TEM) as well as critical micellar concentration. The safety was assessed through RBC's interaction study. The prepared PMRI were evaluated through MABA assay against sensitive and resistant strains of M. Tuberculosis. RESULTS: Size, zeta and entrapment efficiency for RIF loaded HPMA-PLA-INH polymeric micelles (PMRI) was 87.64 ± 1.98 nm, -19 ± 1.93 mV and 97.2 ± 1.56%, respectively. In vitro release followed controlled and sustained delivery pattern. Sustained release was also supported by release kinetics. Haemolytic toxicity of HPI and PMRI was 8.57 and 7.05% (p < 0.01, INH Vs PMRI; p < 0.0001, RIF Vs PMRI), respectively. MABA assay (cytotoxicity) based MIC values of PMRI formulation was observed as ≥0.0625 and ≥0.50 µg/mL (for sensitive and resistant strain). The microscopic analysis further confirmed that the delivery approach was effective than pure drugs. CONCLUSIONS: RIF loaded and INH conjugated HPMA-PLA polymeric micelles (PMRI) were more effective against sensitive and resistant M tuberculosis. The developed approach can lead to improved patient compliance and reduced dosing in future, offering improved treatment of tuberculosis.

Establishment of Biosafety Level-3 (BSL-3) laboratory: important criteria to consider while designing, constructing, commissioning & operating the facility in Indian setting.

Since the enactment of Environmental Protection Act in 1989 and Department of Biotechnology (DBT) guidelines to deal with genetically modified organisms, India has embarked on establishing various levels of biosafety laboratories to deal with highly infectious and pathogenic organisms. Occurrence of outbreaks due to rapidly spreading respiratory and haemorrhagic fever causing viruses has caused an urgency to create a safe laboratory environment. This has thus become a mandate, not only to protect laboratory workers, but also to protect the environment and community. In India, technology and science are progressing rapidly. Several BSL-3 [=high containment] laboratories are in the planning or execution phase, to tackle biosafety issues involved in handling highly infectious disease agents required for basic research and diagnosis. In most of the developing countries, the awareness about biocontainment has increased but planning, designing, constructing and operating BSL-3 laboratories need regular updates about the design and construction of facilities and clear definition of risk groups and their handling which should be in harmony with the latest international practices. This article describes the major steps involved in the process of construction of a BSL-3 laboratory in Indian settings, from freezing the concept of proposal to operationalization phase. The key to success of this kind of project is strong institutional commitment to biosafety norms, adequate fund availability, careful planning and designing, hiring good construction agency, monitoring by experienced consultancy agency and involvement of scientific and engineering personnel with biocontainment experience in the process.

microRNAs associated with the pathogenesis and their role in regulating various signaling pathways during Mycobacterium tuberculosis infection.

Despite more than a decade of active study, tuberculosis (TB) remains a serious health concern across the world, and it is still the biggest cause of mortality in the human population. Pathogenic bacteria recognize host-induced responses and adapt to those hostile circumstances. This high level of adaptability necessitates a strong regulation of bacterial metabolic characteristics. Furthermore, the immune reponse of the host virulence factors such as host invasion, colonization, and survival must be properly coordinated by the pathogen. This can only be accomplished by close synchronization of gene expression. Understanding the molecular characteristics of mycobacterial pathogenesis in order to discover therapies that prevent or resolve illness relies on the bacterial capacity to adjust its metabolism and replication in response to various environmental cues as necessary. An extensive literature details the transcriptional alterations of host in response to in vitro environmental stressors, macrophage infection, and human illness. Various studies have recently revealed the finding of several microRNAs (miRNAs) that are believed to play an important role in the regulatory networks responsible for adaptability and virulence in Mycobacterium tuberculosis. We highlighted the growing data on the existence and quantity of several forms of miRNAs in the pathogenesis of M. tuberculosis, considered their possible relevance to disease etiology, and discussed how the miRNA-based signaling pathways regulate bacterial virulence factors.

Identification of Mycobacterium tuberculosis genes preferentially expressed during human infection.

The identification of Mycobacterium tuberculosis genes, specifically expressed during infection is a key step in understanding molecular mechanism of mycobacterial pathogenesis. Such genes likely encode proteins required for mycobacterium's survival and progressive infection within the host. In this study, we applied in-vivo-induced antigen technology (IVIAT) to M. tuberculosis and identified 11 putative in-vivo induced genes encoding for immunogenic proteins of diverse functions; these included transcriptional regulators (Rv1460 and Rv2565), biosynthesis and macromolecule metabolism (leuD, guaB1, plcC, hupB and glyS), polyketide synthases (pks6 and pks9), cell processes (ctpA) and one with unknown function (Rv3701c). Quantitative real time-PCR analysis of these genes in the specimens obtained from TB patients demonstrated induced expression of eight genes as compared with bacteria grown in-vitro. In addition, distribution of these genes in different strains of M. tuberculosis was analyzed using PCR and their nucleotide sequence alignments and they were found to be widely distributed among M. tuberculosis isolates including multiple-drug resistant (MDR) and extensively-drug resistant (XDR). This study identified several antigenic determinants of M. tuberculosis expressed during infection, which might help pathogens adapt to or counter hostile environments and suggesting their role during disease process.

Rise of Clinical Microbial Proteogenomics: A Multiomics Approach to Nontuberculous Mycobacterium-The Case of Mycobacterium abscessus UC22.

Nontuberculous mycobacterial (NTM) species present a major challenge for global health with serious clinical manifestations ranging from pulmonary to skin infections. Multiomics research and its applications toward clinical microbial proteogenomics offer veritable potentials in this context. For example, the Mycobacterium abscessus, a highly pathogenic NTM, causes bronchopulmonary infection and chronic pulmonary disease. The rough variant of the M. abscessus UC22 strain is extremely virulent and causes lung upper lobe fibrocavitary disease. Although several whole-genome next-generation sequencing studies have characterized the genes in the smooth variant of M. abscessus, a reference genome sequence for the rough variant was generated only recently and calls for further clinical applications. We carried out whole-genome sequencing and proteomic analysis for a clinical isolate of M. abscessus UC22 strain obtained from a pulmonary tuberculosis patient. We identified 5506 single-nucleotide variations (SNVs), 63 insertions, and 76 deletions compared with the reference genome. Using a high-resolution LC-MS/MS-based approach (liquid chromatography tandem mass spectrometry), we obtained protein coding evidence for 3601 proteins, representing 71% of the total predicted genes in this genome. Application of proteogenomic approach further revealed seven novel protein-coding genes and enabled refinement of six computationally derived gene models. We also identified 30 variant peptides corresponding to 16 SNVs known to be associated with drug resistance. These new observations offer promise for clinical applications of microbial proteogenomics and next-generation sequencing, and provide a resource for future global health applications for NTM species.

Whole Genome Sequencing of Mycobacterium tuberculosis Clinical Isolates From India Reveals Genetic Heterogeneity and Region-Specific Variations That Might Affect Drug Susceptibility.

Whole genome sequencing (WGS) of Mycobacterium tuberculosis has been constructive in understanding its evolution, genetic diversity and the mechanisms involved in drug resistance. A large number of sequencing efforts from across the globe have revealed genetic diversity among clinical isolates and the genetic determinants for their resistance to anti-tubercular drugs. Considering the high TB burden in India, the availability of WGS studies is limited. Here we present, WGS results of 200 clinical isolates of M. tuberculosis from North India which are categorized as sensitive to first-line drugs, mono-resistant, multi-drug resistant and pre-extensively drug resistant isolates. WGS revealed that 20% of the isolates were co-infected with M. tuberculosis and non-tuberculous mycobacteria species. We identified 12,802 novel genetic variations in M. tuberculosis isolates including 343 novel SNVs in 38 genes which are known to be associated with drug resistance and are not currently used in the diagnostic kits for detection of drug resistant TB. We also identified M. tuberculosis lineage 3 to be predominant in the northern region of India. Additionally, several novel SNVs, which may potentially confer drug resistance were found to be enriched in the drug resistant isolates sampled. This study highlights the significance of employing WGS in diagnosis and for monitoring further development of MDR-TB strains.

The role of T regulatory cell-associated markers in monitoring tuberculosis treatment completion and failure.

Monitoring tuberculosis (TB) treatment success is crucial for clinical decision-making. The only available tool in this regard is sputum microscopy, but it has demerits. Moreover, in case of smear negatives and extrapulmonary TB, an efficient tool is still sought for. Therefore, we evaluated T regulatory cell (Treg)-associated markers (CD25, CD39, and FoxP3) and cellular subsets in monitoring treatment success in treatment-completed groups. Expression profile of various markers and subsets were compared real time among treatment-naive pulmonary TB patients (TN-PTB), followed-up treatment-completed (TC-fu) cohort, and a not followed-up (TC-nfu) cohort. Peripheral blood mononuclear cells from various groups were incubated overnight and were stained with antibodies for specific markers and studied by flow cytometry. In both the treatment-completed groups, a decline in frequencies of CD25+ marker and CD4+CD25+, CD4+CD25+FoxP3, CD4+CD25+CD39+ Treg was observed with clearance of infection, indicating their potential in monitoring treatment success. However, in the case of treatment failure patient (Tfp), a drastic increase in frequency of CD4+CD25+FoxP3+ Treg subset was found, indicating its usefulness in predicting treatment failure. Although the investigation unveils markers useful in predicting treatment success or failure, the findings from this study needs to be validated in a larger cohort.

Spoligotype defined lineages of Mycobacterium tuberculosis and drug resistance: Merely a casual correlation?

Drug-resistant tuberculosis (TB) is a major challenge to TB control strategy worldwide. Analysis of genetic polymorphism among drug resistant Mycobacterium tuberculosis (MTB) strains may help provide some insight into the transmission dynamics of these strains. Spoligotyping is a widely used technique to identify genetic polymorphism, based on 43 known spacers interspersed between direct repeat regions. Considerable work has been done in various parts of the world using this technique to identify and analyse the polymorphic nature of MTB. Many studies have been carried out to determine the association of drug resistance with spoligotype defined lineages, and much data has been produced over the years. New information continues to be generated. This review aims to put together the findings of relevant studies in an attempt to understand the correlation of drug resistance with spoligotype defined lineages of MTB. This would help provide a perspective of the available data that can be used as a starting point to understand the molecular epidemiology of drug resistant TB.

Trends in spoligotype patterns of Mycobacterium tuberculosis strains in central India.

This study aims to understand trends in spoligotype patterns of Mycobacterium tuberculosis (MTB) in Central India. Elucidation of these trends may provide baseline information to understand the transmission dynamics of strains of MTB in the region. Spoligotyping was carried out on 340 MTB strains isolated from clinical samples received from 2007 to 2011. The prevalence of ST26/CAS1_Del, ST11/EAI3_IND, ST288/CAS2, ST25/CAS1_Del and Beijing lineages showed waxing and waning trends. ST26/CAS1_Del and ST11/EAI3_IND lineages were consistently present and were predominant. Well-established lineages showed a consistent presence in the community. New orphan lineages appeared to be less capable of establishing themselves.

PCR targeting IS6110 in diagnosing tuberculosis in children in comparison to MGIT culture.

BACKGROUND: Diagnosis of tuberculosis (TB) in children is difficult in children especially in extrapulmonary tuberculosis (EPTB). This study was conducted to evaluate the use of polymerase chain reaction (PCR) targeting IS6110 in the diagnosis of TB in children with pulmonary TB and EPTB and also to compare its performance with MGIT 960 culture and conventional microscopy. METHODS: A total of 142 cases (50 pulmonary, 92 extrapulmonary) of suspected TB patients <15 years of age were included in the study. The clinical specimens obtained from these cases were subjected to Ziehl-Neelsen staining (ZN), MGIT 960 TB culture and PCR targeting insertion sequence IS6110. Sensitivity and specificity of PCR were calculated in pulmonary and extrapulmonary specimens. The results were compared to MGIT culture. RESULTS: PCR targeting IS6110 sequence had sensitivity of 69.01% in various clinical specimens which was significantly more than MGIT culture showing a sensitivity of 47.41% (p<0.05). Sensitivity of PCR IS6110 in extrapulmonary specimens was 65.21% which was lower than sensitivity in pulmonary specimens (76%) but was not statistically significant (p>0.05). CONCLUSIONS: Diagnostic efficacy of PCR IS6110 in pulmonary and extrapulmonary TB cases was similar. PCR using IS6110 primer had significantly better efficiency than MGIT culture in diagnosing TB in children.

Status of multidrug resistant tuberculosis (MDR-TB) among the Sahariya tribe of North Central India.

BACKGROUND: The incidence/prevalence of tuberculosis (TB) is reported to be high in the Sahariya tribe of North Central India. The outbreaks of different drug-resistant isolates of Mycobacterium tuberculosis emphasized the need for continuous monitoring of resistance to anti-tuberculosis drugs. This study aimed to assess the profile of multidrug resistant TB among the Sahariya tribe and their non-tribal neighbors for first line drugs through field-based investigations. METHODOLOGY: A total of 274 sputum positive pulmonary TB individuals were enrolled and studied for their drug susceptibility profile by the proportion method. RESULTS: A total of 21 cases from Sahariya and 6 from non-tribes were identified with MDR-TB. Thus Sahariya tribe showed a 1.95-fold increased risk of developing drug resistance than non-tribes. Significant differences were observed for developing drug sensitivity between Sahariya males and females when analyzed for resistance developed to any drug and overall drug resistance vs. sensitive isolates, respectively. A 4.46-fold risk was found for MDR-TB among the smokers of Sahariya tribe, whereas, the non-tribes did not show any significant association. CONCLUSION: The drug susceptibility profile developed in the present study indicates that drug-resistant tuberculosis is emerging as a serious public health concern in Sahariya tribe. Urgent and effective control measures and better management policies are needed for the prevention of MDR-TB in the tribe.

Assessment of the N-PCR assay in diagnosis of pleural tuberculosis: detection of M. tuberculosis in pleural fluid and sputum collected in tandem.

BACKGROUND: The nonspecific clinical presentation and paucibacillary nature of tuberculous pleuritis remains a challenge for diagnosis. Diagnosis of tuberculous pleural effusion depends on the demonstration of the presence of tubercle bacilli in the sputum, pleural fluid, or pleural biopsy specimen, or demonstration of granuloma in pleura by histological examination. We examined the clinical utility of the diagnosis of pleural tuberculosis using the in house N-PCR assay, AFB smear microscopy and culture. Besides pleural fluid the inclusion of sputum in the efficacy of diagnosis of pleural tuberculosis was scrutinized. METHODOLOGY/PRINCIPAL FINDINGS: Pleural fluid and sputum samples of 58 tuberculous and 42 non-tuberculous pleural effusion patients were processed for AFB smear microscopy, culture and the N-PCR assay. Mycobacteria were detected exclusively in tuberculous pleural effusion samples. None of the non-tuberculous pleural effusion samples were positive for mycobacteria. Comparative analysis showed that the N-PCR assay had the highest sensitivity. Inclusion of sputum along with pleural fluid increased N-PCR sensitivity from 51.7 to 70.6% (p<0.0001).This improved sensitivity was reflected in AFB smear microscopy and isolation by culture. The sensitivity enhanced on inclusion of sputum from 3.4 (p = 0.50) to 10.3% (p = 0.038) for AFB smear microscopy and for isolation of mycobacteria from 10.3(p = 0.03) to 22.4% (p = 0.0005). Thirteen isolates were obtained from 58 pleural tuberculosis patients. Eleven mycobacterial isolates were identified as M. tuberculosis and two as M. fortuitum and M. chelonae. Complete concordance was seen between the biochemical identification of isolates and the N-PCR identification of mycobacterial species prior to isolation. CONCLUSIONS/SIGNIFICANCE: To the best of our knowledge this is the first PCR based report on utility of sputum for diagnosis of pleural tuberculosis. The present study demonstrates that a combination of pleural fluid with sputum sample and N-PCR improved the diagnosis of pleural tuberculosis.

Predominance of ancestral lineages of Mycobacterium tuberculosis in India.

Although India has the highest prevalence of tuberculosis (TB) worldwide, the genetic diversity of Mycobacterium tuberculosis in India is largely unknown. A collection of 91 isolates originating from 12 different regions spread across the country were analyzed by genotyping using 21 loci with variable-number tandem repeats (VNTRs), by spoligotyping, by principal genetic grouping (PGG), and by deletion analysis of M. tuberculosis-specific deletion region 1. The isolates showed highly diverse VNTR genotypes. Nevertheless, highly congruent groupings identified by using the 4 independent sets of markers permitted a clear definition of 3 prevalent PGG1 lineages, which corresponded to the "ancestral" East African-Indian, the Delhi, and the Beijing/W genogroups. A few isolates from PGG2 lineages and a single representative of the presumably most recent PGG3 were identified. These observations suggest a predominance of ancestral M. tuberculosis genotypes in the Indian subcontinent, which supports the hypothesis that India is an ancient endemic focus of TB.

In situ hybridization in the histological diagnosis of early and clinically suspect leprosy.

The present study tests the utility of the in situ hybridization procedure for M. leprae rRNA in the histological diagnosis of early leprosy and clinically suspect leprosy, both diagnostically demanding situations. The histological confirmation obtained with routine histopathology (Haematoxylin-Eosin staining for studying morphologic alterations and Fite-Faraco staining for demonstration of acid-fast bacilli) were 32% for early leprosy and 25% for clinically suspect leprosy. With performance of the in situ hybridization on the histologically unconfirmed cases, the positivity rates obtained were 58.8% and 55%, respectively. The results of the study confirm the utility of the procedure in the diagnostically difficult situations of early and suspect leprosy, and it is proposed that the procedure be employed in situations of clinical doubt.