Exploring anti-TB leads from natural products library originated from marine microbes and medicinal plants

Multidrug-resistant tuberculosis (MDR-TB) and TB–HIV co-infection have become a great threat to global health. However, the last truly novel drug that was approved for the treatment of TB was discovered 40?years ago. The search for new effective drugs against TB has never been more intensive. Natural products derived from microbes and medicinal plants have been an important source of TB therapeutics. Recent advances have been made to accelerate the discovery rate of novel TB drugs including diversifying strategies for environmental strains, high-throughput screening (HTS) assays, and chemical diversity. This review will discuss the challenges of finding novel natural products with anti-TB activity from marine microbes and plant medicines, including biodiversity- and taxonomy-guided microbial natural products library construction, target- and cell-based HTS, and bioassay-directed isolation of anti-TB substances from traditional medicines.     

Regulatory issues concerning the safety, efficacy and quality of herbal remedies

Herbal remedies and alternative medicines are used throughout the world, and in the past herbs were often the original sources of most drugs. Today we are witnessing an increase in herbal remedy use throughout the Western world raising the question as to how safe are these preparations for the unborn fetus? Many women use herbal products during pregnancy. The dilemma facing most regulatory authorities is that the public considers these products as either traditional medicines or natural food supplements. The user sees no reason for regulation. Most countries have laws concerning foods, drugs, and cosmetics, the details of which seldom clearly define to what section of the law and regulations alternative remedies belong. In most countries alternative remedies are regulated as foods, provided that no medicinal claim is made on the label. The global regulatory sector, however, is changing rapidly. The Therapeutic Goods Administration (TGA) in Australia created a Complimentary Medicines Evaluation Committee in late 1997 to address this issue, and Canada has created a new Natural Health Products Directorate in the realigned Therapeutic Products and Foods Branch in 2000. In parallel, the European Agency for the Evaluation of Medicinal Products has drafted test procedures and acceptance criteria for herbal drug preparations and herbal medicinal products. In the US, the Food and Drug Administration classifies these natural products as dietary supplements. Manufacturers must label a dietary supplement thus: “this statement has not been evaluated by the FDA [, and] this product is not intended to diagnose, treat, cure or prevent any disease.” Whether these products are foods or drugs is undecided. To add complexity to this issue, most of the potential deleterious effects of natural products on the unborn may be related to hormonal effects (e.g., phytoestrogens) and nutriceutical drug interactions (e.g., St. John's Wort and antidepressants), rather than direct embryotoxicity per se. We suggest that ensuring quality of herbal products should receive immediate attention by regulatory authorities, before embarking on the more arduous tasks of safety and efficacy. Birth Defects Res B 68:505–510, 2003. © 2003 Wiley‐Liss, Inc.     

A few specialized issues that should be focused on anti-HIV drug evaluation in vitro

Since the introduction of antiretroviral therapy(ART),the lifespan and quality of life of patients infected with HIV have been significantly improved.But treatment efficacy was compromised eventually by the development of resistance to antiretroviral drugs,and more new anti-HIV drugs with lower toxicity and higher activity were needed.Based on the experience and lessons learned from the treatment in the developed countries,US FDA suggested that more pharmacodynamical researches should be considered ahead of the clinical trials.To facilitate the anti-HIV drug research and development,we reviewed a few specialized issues that should be focused on drug evaluations in vitro,including: 1)Mechanism of action studies,demonstrating the candidate drug's efficacy to specifically inhibit viral replication or a virus-specific function and confirm the drug target.2)Drug resistance studies,selecting the drug-resistant variants in vitro and determining the activities inhibiting HIV isolates resistant to approved antiretroviral drugs of the same class.3)Antiviral activity in vitro in the presence of serum proteins,ascertaining whether an investigational product is significantly bound by serum proteins.4)Combination activity analysis,evaluating in vitro antiviral activity of an investigational product in two-drug combinations with other drugs approved.     

A review on anti‐tuberculosis peptides: Impact of peptide structure on anti‐tuberculosis activity

Antibiotic resistance is a major public health problem globally. Particularly concerning amongst drug‐resistant human pathogens is Mycobacterium tuberculosis that causes the deadly infectious tuberculosis (TB) disease. Significant issues associated with current treatment options for drug‐resistant TB and the high rate of mortality from the disease makes the development of novel treatment options against this pathogen an urgent need. Antimicrobial peptides are part of innate immunity in all forms of life and could provide a potential solution against drug‐resistant TB. This review is a critical analysis of antimicrobial peptides that are reported to be active against the M tuberculosis complex exclusively. However, activity on non‐TB strains such as Mycobacterium avium and Mycobacterium intracellulare, whenever available, have been included at appropriate sections for these anti‐TB peptides. Natural and synthetic antimicrobial peptides of diverse sequences, along with their chemical structures, are presented, discussed, and correlated to their observed antimycobacterial activities. Critical analyses of the structure allied to the anti‐mycobacterial activity have allowed us to draw important conclusions and ideas for research and development on these promising molecules to realise their full potential. Even though the review is focussed on peptides, we have briefly summarised the structures and potency of the various small molecule drugs that are available and under development, for TB treatment.     

Potential of plants to produce recombinant protein products

Plants have great potential as photosynthetic factories to produce pharmaceutically important and commercially valuable biomedicines and industrial proteins at low cost. The U.S. Food and Drug Administration (U.S. FDA) has approved the drug Elelyso (taliglucerase alfa) produced by carrot cells for treatment of type 1 Gaucher’s disease in 2012. The commercial potential of biomedicines produced by molecular farming has dramatically improved due to the success of an experimental drug called ZMapp, which has immunological activity in Ebola patients. A cocktail of three monoclonal antibodies was produced in tobacco (Nicotiana benthamiana) plants (Chen and Davis 2016). At present, very few drugs made by this technology have been approved by worldwide authorities such as the U.S. FDA. However, plants have been proposed as a novel paradigm for commercial production of proteins over the next decade. In recent years, leading researchers on molecular farming have given more priority to the area of animal-free therapeutic proteins such as parenteral and oral vaccines. Although plant-based platforms have considerable advantages over traditional systems such as bacterial and animal systems, there are several obstacles to commercial-scale production, especially with regards to improving the quality and quantity of plant-produced biologics and industrial materials. One of the biggest barriers to commercialization of this technology is the intense scrutiny of these new plant varieties by regulatory agencies and the public as well as the high costs associated with their regulatory approval.     

Bedaquiline: Fallible Hope Against Drug Resistant Tuberculosis

Tuberculosis (TB) is a deadly bacterial infectious disease caused by intra-cellular pathogen Mycobacterium tuberculosis (Mtb). There were an estimated 1.4 million TB deaths in 2015 and an additional 0.4 million deaths resulting from TB among individuals with HIV. Drug-discovery for its cure is very slow in comparison with the causative organism’s fast pace of mutations conferring drug resistance. Moreover, the field of drug-discovery of anti-TB drugs is constantly being challenged by the drug resistant strains of Mtb. Several molecules/inhibitors are being tested across the pharmaceutical industry and research centres for their suitability as drug candidate. It takes immense effort, high costs and a whole lot of screening to bring a single molecule to the clinics for patient cure. In last 60 years, hundreds of molecules have been patented for their probable use to develop drug for treatment of TB. However, only one drug has been successfully approved that is bedaquiline (1-(6-bromo-2 -methoxy-quinolin-3-yl)-4-dimethylamino-2-naphtalen-1-yl-1-phenyl-butan-2-ol). This is a brief review about bedaquiline (BDQ), the only drug in last 45 years approved for curing drug-resistant pulmonary TB, its development, action mechanism and development of resistance against it.     

Raising the Barriers to Access to Medicines in the Developing World – The Relentless Push for Data Exclusivity

Since the adoption of the WTO‐TRIPS Agreement in 1994, there has been significant controversy over the impact of pharmaceutical patent protection on the access to medicines in the developing world. In addition to the market exclusivity provided by patents, the pharmaceutical industry has also sought to further extend their monopolies by advocating the need for additional ‘regulatory’ protection for new medicines, known as data exclusivity. Data exclusivity limits the use of clinical trial data that need to be submitted to the regulatory authorities before a new drug can enter the market. For a specified period, generic competitors cannot apply for regulatory approval for equivalent drugs relying on the originator's data. As a consequence, data exclusivity lengthens the monopoly for the original drug, impairing the availability of generic drugs. This article illustrates how the pharmaceutical industry has convinced the US and the EU to impose data exclusivity on their trade partners, many of them developing countries. The key arguments formulated by the pharmaceutical industry in favor of adopting data exclusivity and their underlying ethical assumptions are described in this article, analyzed, and found to be unconvincing. Contrary to industry's arguments, it is unlikely that data exclusivity will promote innovation, especially in developing countries. Moreover, the industry's appeal to a property rights claim over clinical test data and the idea that data exclusivity can prevent the generic competitors from ‘free‐riding’ encounters some important problems: Neither legitimize excluding all others.     

在美国进行植物药开发的路径、要求及策略探讨

近年来,越来越多药企开始在美国进行植物药的临床试验申请和新药申报,美国 FDA 日益重视植物药的开发,于 2016 年 12 月 发布并实施了修订版的《行业指南:植物药》,为植物药后期开发提供更多专业建议。结合 FDA 已经批准的 2 个植物药案例,详细介绍 植物药 CMC(化学、生产、控制)、临床前研究和过往人用历史方面的基本要求,浅谈 FDA 的申报要求、审评思想和申报策略,以期 为中国中药企业的国际化开发和注册提供参考。     

Use of Fixed Dose Combination (FDC) Drugs in India: Central Regulatory Approval and Sales of FDCs Containing Non-Steroidal Anti-Inflammatory Drugs (NSAIDs), Metformin,or Psychotropic Drugs

Background

In 2012, an Indian parliamentary committee reported that manufacturing licenses for large numbers of fixed dose combination (FDC) drugs had been issued by state authorities without prior approval of the Central Drugs Standard Control Organization (CDSCO) in violation of rules, and considered that some ambiguity until 1 May 2002 about states’ powers might have contributed. To our knowledge, no systematic enquiry has been undertaken to determine if evidence existed to support these findings. We investigated CDSCO approvals for and availability of oral FDC drugs in four therapeutic areas: analgesia (non-steroidal anti-inflammatory drugs [NSAIDs]), diabetes (metformin), depression/anxiety (anti-depressants/benzodiazepines), and psychosis (anti-psychotics).

Methods and Findings

This was an ecologic study with a time-trend analysis of FDC sales volumes (2007–2012) and a cross-sectional examination of 2011–2012 data to establish the numbers of formulations on the market with and without a record of CDSCO approval (“approved” and “unapproved”), their branded products, and sales volumes. Data from the CDSCO on approved FDC formulations were compared with sales data from PharmaTrac, a database of national drug sales. We determined the proportions of FDC sales volumes (2011–2012) arising from centrally approved and unapproved formulations and from formulations including drugs banned/restricted internationally. We also determined the proportions of centrally approved and unapproved formulations marketed before and after 1 May 2002, when amendments were made to the drug rules. FDC approvals in India, the United Kingdom (UK), and United States of America (US) were compared.For NSAID FDCs, 124 formulations were marketed, of which 34 (27%) were centrally approved and 90 (73%) were unapproved; metformin: 25 formulations, 20 (80%) approved, five (20%) unapproved; anti-depressants/benzodiazepines: 16 formulations, three (19%) approved, 13 (81%) unapproved; anti-psychotics: ten formulations, three (30%) approved, seven (70%) unapproved. After 1 May 2002, the proportions of approved FDC formulations increased for NSAIDs (26%/28%) and anti-psychotics (0%/38%) and decreased for metformin (100%/75%) and anti-depressants/benzodiazepines (20%/18%), and the overall proportion approved remained similar before and after that date.FDC formulations gave rise to multiple branded products, ranging from 211 anti-psychotic FDC products from ten formulations to 2,739 NSAID FDC products from 124 formulations. The proportions of FDC sales volumes arising from unapproved formulations were as follows: anti-depressants/benzodiazepines, 69%; anti-psychotics, 43%; NSAIDs, 28%; and metformin, 0.4%. Formulations including drugs banned/restricted internationally comprised over 12% of NSAID FDC sales and 53% of anti-psychotic FDC sales. Across the four therapeutic areas, 14 FDC formulations were approved in the UK and 22 in the US.

Conclusions

There was evidence supporting concerns about FDCs. Metformin excepted, substantial numbers of centrally unapproved formulations for NSAID, anti-depressant/benzodiazepine, and anti-psychotic FDCs were marketed; sales volumes were high. The legal need for central approval of new drugs before manufacture has been in place continuously since 1961, including for FDCs meeting the applicable legal test. Proportions of centrally unapproved formulations after 1 May 2002 did not decrease overall, and no ambiguity was found about states’ licensing powers. Unapproved formulations should be banned immediately, prioritising those withdrawn/banned internationally and undertaking a review of benefits and risks for patients in ceasing or switching to other medicines. Drug laws need to be amended to ensure the safety and effectiveness of medicines marketed in India.     

Pattern of Drug Resistance and Risk Factors Associated with Development of Drug Resistant Mycobacterium tuberculosis in Pakistan

Background

Drug resistant tuberculosis (DR-TB) is a major public health problem in developing countries such as Pakistan.

Objective

The current study was conducted to assess the frequency of drug resistant tuberculosis including multi drug resistance (MDR- TB) as well as risk factors for development of DR-TB, in Punjab, Pakistan.

Methodology

Drug susceptibility testing (DST) was performed, using proportion method, for 2367 culture positive Mycobacterium tuberculosis (MTB) cases that were enrolled from January 2012 to December 2013 in the province of Punjab, Pakistan, against first-line anti-tuberculosis drugs. The data was analyzed using statistical software; SPSS version 18.

Results

Out of 2367 isolates, 273 (11.5%) were resistant to at least one anti-TB drug, while 221 (9.3%) showed MDR- TB. Risk factors for development of MDR-TB were early age (ranges between 10–25 years) and previously treated TB patients.

Conclusion

DR-TB is a considerable problem in Pakistan. Major risk factors are previous history of TB treatment and younger age group. It emphasizes the need for effective TB control Program in the country.     

Spontaneous emergence of multiple drug resistance in tuberculosis before and during therapy

The emergence of drug resistance in M. tuberculosis undermines the efficacy of tuberculosis (TB) treatment in individuals and of TB control programs in populations. Multiple drug resistance is often attributed to sequential functional monotherapy, and standard initial treatment regimens have therefore been designed to include simultaneous use of four different antibiotics. Despite the widespread use of combination therapy, highly resistant M. tb strains have emerged in many settings. Here we use a stochastic birth-death model to estimate the probability of the emergence of multidrug resistance during the growth of a population of initially drug sensitive TB bacilli within an infected host. We find that the probability of the emergence of resistance to the two principal anti-TB drugs before initiation of therapy ranges from 10(-5) to 10(-4); while rare, this is several orders of magnitude higher than previous estimates. This finding suggests that multidrug resistant M. tb may not be an entirely "man-made" phenomenon and may help explain how highly drug resistant forms of TB have independently emerged in many settings.     

Drug resistance mechanisms and novel drug targets for tuberculosis therapy

Drug-resistant tuberculosis (TB) poses a significant challenge to the successful treatment and control of TB worldwide. Resistance to anti-TB drugs has existed since the beginning of the chemotherapy era. New insights into the resistant mechanisms of anti-TB drugs have been provided. Better understanding of drug resistance mechanisms helps in the development of new tools for the rapid diagnosis of drug-resistant TB. There is also a pressing need in the development of new drugs with novel targets to improve the current treatment of TB and to prevent the emergence of drug resistance in Mycobacterium tuberculosis. This review summarizes the anti-TB drug resistance mechanisms, furnishes some possible novel drug targets in the development of new agents for TB therapy and discusses the usefulness using known targets to develop new anti-TB drugs. Whole genome sequencing is currently an advanced technology to uncover drug resistance mechanisms in M. tuberculosis. However, further research is required to unravel the significance of some newly discovered gene mutations in their contribution to drug resistance.     

The potential of plant systems to break the HIV‐TB link

Tuberculosis (TB) and human immunodeficiency virus (HIV) can place a major burden on healthcare systems and constitute the main challenges of diagnostic and therapeutic programmes. Infection with HIV is the most common cause of Mycobacterium tuberculosis (Mtb), which can accelerate the risk of latent TB reactivation by 20‐fold. Similarly, TB is considered the most relevant factor predisposing individuals to HIV infection. Thus, both pathogens can augment one another in a synergetic manner, accelerating the failure of immunological functions and resulting in subsequent death in the absence of treatment. Synergistic approaches involving the treatment of HIV as a tool to combat TB and vice versa are thus required in regions with a high burden of HIV and TB infection. In this context, plant systems are considered a promising approach for combatting HIV and TB in a resource‐limited setting because plant‐made drugs can be produced efficiently and inexpensively in developing countries and could be shared by the available agricultural infrastructure without the expensive requirement needed for cold chain storage and transportation. Moreover, the use of natural products from medicinal plants can eliminate the concerns associated with antiretroviral therapy (ART) and anti‐TB therapy (ATT), including drug interactions, drug‐related toxicity and multidrug resistance. In this review, we highlight the potential of plant system as a promising approach for the production of relevant pharmaceuticals for HIV and TB treatment. However, in the cases of HIV and TB, none of the plant‐made pharmaceuticals have been approved for clinical use. Limitations in reaching these goals are discussed.     

Finding a chink in the armor: Update,limitations, and challenges toward successful antivirals against flaviviruses

Flaviviruses have caused large epidemics and ongoing outbreaks for centuries. They are now distributed in every continent infecting up to millions of people annually and may emerge to cause future epidemics. Some of the viruses from this group cause severe illnesses ranging from hemorrhagic to neurological manifestations. Despite decades of research, there are currently no approved antiviral drugs against flaviviruses, urging for new strategies and antiviral targets. In recent years, integrated omics data-based drug repurposing paired with novel drug validation methodologies and appropriate animal models has substantially aided in the discovery of new antiviral medicines. Here, we aim to review the latest progress in the development of both new and repurposed (i) direct-acting antivirals; (ii) host-targeting antivirals; and (iii) multitarget antivirals against flaviviruses, which have been evaluated both in vitro and in vivo, with an emphasis on their targets and mechanisms. The search yielded 37 compounds that have been evaluated for their efficacy against flaviviruses in animal models; 20 of them are repurposed drugs, and the majority of them exhibit broad-spectrum antiviral activity. The review also highlighted the major limitations and challenges faced in the current in vitro and in vivo evaluations that hamper the development of successful antiviral drugs for flaviviruses. We provided an analysis of what can be learned from some of the approved antiviral drugs as well as drugs that failed clinical trials. Potent in vitro and in vivo antiviral efficacy alone does not warrant successful antiviral drugs; current gaps in studies need to be addressed to improve efficacy and safety in clinical trials.     

2D-QSAR model development and analysis on variant groups of anti-tuberculosis drugs

A quantitative structure activity relationship study was performed on different groups of anti-tuberculosis drug compound for establishing quantitative relationship between biological activity and their physicochemical /structural properties. In recent years, a large number of herbal drugs are promoted in treatment of tuberculosis especially due to the emergence of MDR (multi drug resistance) and XDR (extensive drug resistance) tuberculosis. Multidrug-resistant TB (MDR-TB) is resistant to front-line drugs (isoniazid and rifampicin, the most powerful anti-TB drugs) and extensively drug-resistant TB (XDR-TB) is resistant to front-line and second-line drugs. The possibility of drug resistance TB increases when patient does not take prescribed drugs for defined time period. Natural products (secondary metabolites) isolated from the variety of sources including terrestrial and marine plants and animals, and microorganisms, have been recognized as having antituberculosis action and have recently been tested preclinically for their growth inhibitory activity towards Mycobacterium tuberculosis or related organisms. A quantitative structure activity relationship (QSAR) studies were performed to explore the antituberculosis compound from the derivatives of natural products . Theoretical results are in accord with the in vitro experimental data with reported growth inhibitory activity towards Mycobacterium tuberculosis or related organisms. Antitubercular activity was predicted through QSAR model, developed by forward feed multiple linear regression method with leave-one-out approach. Relationship correlating measure of QSAR model was 74% (R(2) = 0.74) and predictive accuracy was 72% (RCV(2) = 0.72). QSAR studies indicate that dipole energy and heat of formation correlate well with anti-tubercular activity. These results could offer useful references for understanding mechanisms and directing the molecular design of new lead compounds with improved anti-tubercular activity. The generated QSAR model revealed the importance of structural, thermodynamic and electro topological parameters. The quantitative structure activity relationship provides important structural insight in designing of potent antitubercular agent.     

A pivotal registration phase III, multicenter, randomized tuberculosis controlled trial: design issues and lessons learnt from the Gatifloxacin for TB (OFLOTUB) project

ABSTRACT: BACKGROUND: There have been no major advances in tuberculosis (TB) drug development since the first East African/British Medical Research Council short course chemotherapy trial 35 years ago. Since then, the landscape for conducting TB clinical trials has profoundly changed with the emergence of HIV infection, the spread of resistant TB bacilli strains, recent advances in mycobacteriological capacity, and drug discovery. As a consequence questions have arisen on the most appropriate approach to design and conduct current TB trials. To highlight key issues discussed: Is a superiority, equivalence, or non-inferiority design most appropriate? What should be the primary efficacy outcome? How to consider re-infections in the definition of the outcome? What is the optimal length of patient follow-up? Is blinding appropriate when treatment duration in test arm is shorter? What are the appropriate assumptions for sample size calculation? METHODS: Various drugs are currently in the development pipeline. We are presenting in this paper the design of the most recently completed phase III TB trial, the OFLOTUB project, which is the pivotal trial of a registration portfolio for a gatifloxacin-containing TB regimen. It is a randomized, open-label, multicenter, controlled trial aiming to evaluate the efficacy and safety of a gatifloxacin-containing 4-month regimen (trial registration: ClinicalTrial.gov database: NCT00216385). RESULTS: In the light of the recent scientific and regulatory discussions, we discuss some of the design issues in TB clinical trials and more specifically the reasons that guided our choices, in order to best answer the trial objectives, while at the same time satisfying regulatory authority requirements. CONCLUSION: When shortening TB treatment, we are advocating for a non-inferiority, non-blinded design, with a composite unfavorable endpoint assessed 12 months post treatment completion, and added trial procedures specifically aiming to: (1) minimize endpoint unavailability; and (2) distinguish between relapse and re-infection.     

Multidrug resistant to extensively drug resistant tuberculosis: What is next?

Drug resistant tuberculosis is a man made problem. While tuberculosis is hundred percent curable, multidrug resistant tuberculosis (MDR-TB) is difficult to treat. Inadequate and incomplete treatment and poor treatment adherence has led to a newer form of drug resistance known as extensively drug resistant tuberculosis (XDR-TB). XDR-TB is defined as tuberculosis caused by Mycobacterium tuberculosis strain, which is resistant to at least rifampicin and isoniazid among the first line anti tubercular drugs (MDR-TB) in addition to resistance to any fluroquinolones and at least one of three injectable second line anti tubercular drugs i.e. amikacin, kanamycin and/or capreomycin. Mismanagement of tuberculosis paves the way to drug resistant tuberculosis. Emergence of XDR-TB is reported world wide. Reported prevalence rates of XDR-TB of total MDR cases are; 6.6% overall worldwide, 6.5% in industrialized countries, 13.6% in Russia and Eastern Europe, 1.5% in Asia, 0.6% in Africa and Middle East and 15.4% in Republic of Korea. Better management and control of tuberculosis specially drug resistant TB by experienced and qualified doctors, access to standard microbiology laboratory, co-morbitidy of HIV and tuberculosis, new anti-TB drug regimens, better diagnostic tests, international standards for second line drugs (SLD)-susceptibility testing, invention of newer antitubercular molecules and vaccines and knowing the real magnitude of XDR-TB are some of the important issues to be addressed for effective prevention and management of XDR-TB.     

Predicting drug resistance of the HIV-1 protease using molecular interaction energy components

Drug resistance significantly impairs the efficacy of AIDS therapy. Therefore, precise prediction of resistant viral mutants is particularly useful for developing effective drugs and designing therapeutic regimen. In this study, we applied a structure-based computational approach to predict mutants of the HIV-1 protease resistant to the seven FDA approved drugs. We analyzed the energetic pattern of the protease-drug interaction by calculating the molecular interaction energy components (MIECs) between the drug and the protease residues. Support vector machines (SVMs) were trained on MIECs to classify protease mutants into resistant and nonresistant categories. The high prediction accuracies for the test sets of cross-validations suggested that the MIECs successfully characterized the interaction interface between drugs and the HIV-1 protease. We conducted a proof-of-concept study on a newly approved drug, darunavir (TMC114), on which no drug resistance data were available in the public domain. Compared with amprenavir, our analysis suggested that darunavir might be more potent to combat drug resistance. To quantitatively estimate binding affinities of drugs and study the contributions of protease residues to causing resistance, linear regression models were trained on MIECs using partial least squares (PLS). The MIEC-PLS models also achieved satisfactory prediction accuracy. Analysis of the fitting coefficients of MIECs in the regression model revealed the important resistance mutations and shed light into understanding the mechanisms of these mutations to cause resistance. Our study demonstrated the advantages of characterizing the protease-drug interaction using MIECs. We believe that MIEC-SVM and MIEC-PLS can help design new agents or combination of therapeutic regimens to counter HIV-1 protease resistant strains.     

Drug resistance and IS6110‐RFLP patterns of Mycobacterium tuberculosis in patients with recurrent tuberculosis in northern Thailand

The emergence of drug resistant Mycobacterium tuberculosis has become a global threat to tuberculosis (TB) prevention and control efforts. This study aimed to determine the drug resistance profiles and DNA fingerprints of M. tuberculosis strains isolated from patients with relapsed or retreatment pulmonary TB in Chiang Rai province in northern Thailand. Significant differences in multidrug resistance (MDR) (P = 0.025) and resistance to isoniazid (P = 0.025) and rifampin (P = 0.046) between first and second registrations of patients with retreatment TB were found. However, there were no significant differences in resistance to any drugs in patients with relapsed TB. The rate of MDR‐TB strains was 12.2% among new patients at first registration, 22.5% among patients with recurrence who had previously undergone treatment at second registration and 12.5% at third registration. Two retreatment patients whose initial treatment had failed had developed MDR‐TB with resistance to all TB drugs tested, including rifampin, isoniazid, streptomycin and ethambutol. IS6110‐RFLP analysis revealed that 66.7% (10/15 isolates) of MDR‐TB belonged to the Beijing family. In most cases, IS6110‐RFLP patterns of isolates from the same patients were identical in relapse and retreatment groups. However, some pairs of isolates from retreatment patients after treatment failure had non‐identical IS6110‐RFLP patterns. These results suggest that, after failure and default treatment, patients with retreatment tuberculosis have a significantly greater risk of MDR‐TB, isoniazid and rifampin resistance than do other patients.