Dissertations / Theses on the topic 'Resistance to anti-malaria drugs'

Malaria is a major health problem, mainly in developing countries, and causes an estimated 1 million deaths per year. Plasmodium falciparum is the major type of human malaria parasite, and causes the most infections and deaths. Malaria drugs, like any other drugs, suffer from possible side effects and the potential for emergence of resistance. Chloroquine, which was a very effective drug, has been used since about 1945, but its use is severely limited by resistance, even though it has mild side effects, and is otherwise very efficacious. Research has shown that there are chloroquine reversal agents, molecules that can reinstate antimalarial activity of chloroquine and chloroquine-like drugs; many such reversal agents are composed of two aromatic groups linked to a hydrogen bond acceptor several bonds away. By linking a chloroquine-like molecule to a reversal agent-like molecule, it was hoped that a hybrid molecule could be made with both antimalarial and reversal agent properties. In the Peyton Lab, such hybrid "Reversed Chloroquine" molecules have been synthesized and shown to have better antimalarial activity than chloroquine against the P. falciparum chloroquine-sensitive strain D6, as well as the P. falciparum chloroquine-resistant strains Dd2 and 7G8. The work reported in this manuscript involves simplifying the reversal agent head group of the Reversed Chloroquine molecules, to a single aromatic ring instead of the two rings groups described by others; this modification retained, or even enhanced, the antimalarial activity of the parent Reversed Chloroquine molecules. Of note was compound PL154, which had IC50 values of 0.3 nM and 0.5 nM against chloroquine-sensitive D6 and chloroquine-resistant Dd2. Compound PL106 was made to increase water solubility (a requirement for bioavailability) of the simplified Reversed Chloroquine molecules. Molecular modifications inherent to PL106 were not very detrimental to the antimalarial activity, and PL106 was found to be orally available in mice infected with P. yoelli, with an ED50 value of about 5.5 mg/kg/d. Varying the linker length between the quinoline ring and the protonatable nitrogen, or between the head group and the protonatable nitrogen, did not have adverse effects on the antimalarial activities of the simplified Reversed Chloroquine molecules, in accord with the trends observed for the original design of Reversed Chloroquine molecules as found from previous studies in the Peyton Lab. The simplified Reversed Chloroquine molecules even tolerated aliphatic head groups (rather than the original design which specified aromatic rings), showing that major modifications could be made on the Reversed Chloroquine molecules without major loss in activity. A bisquinoline compound, PL192, was made that contained secondary nitrogens at position 4 of the quinoline ring (PL192 is a modification of piperaquine, a known antimalarial drug that contains tertiary nitrogens at position 4 of the quinoline ring); this compound was more potent than piperaquine which had an IC50 value of 0.7 nM against CQS D6 and an IC50 of 1.5 nM against CQR Dd2, PL192 had IC50 values of 0.63 nM against chloroquine sensitive D6 and 0.02 nM against chloroquine resistant Dd2. Finally, the mechanism of action of these simplified "Reversed Chloroquines" was evaluated; it was found that the simplified "Reversed Chloroquines" behaved like chloroquine in inhibiting β-hematin formation and in heme binding. However, the simplified "Reversed Chloroquines" were found to inhibit chloroquine transport for chloroquine resistant P. falciparum chloroquine resistance transporter expressed in Xenopus oocytes to a lesser extant than the classical reversal agent verapamil. From these studies it was noted that the simplified "Reversed Chloroquines" may not behave as well as classical reversal agents would in restoring chloroquine efficacy, but they are very potent, and so could be a major step in developing drug candidates against malaria.

Drug resistance is one of the major obstacles facing malaria control. Resistance to the combination sulfadoxine/pyrimethamine (S/P) (Fansidar) is now widespread, although the mechanism by which this arises is still not fully understood. Therefore, the molecular basis of S/P resistance was studied in the rodent malaria parasite, Plasmodium chadaubi. S/P resistant mutants were selected from a clone already resistant to the pyrimethamine, AS (PYR), caused by the presence of an asparagine at position 106 in its dihydrofolate reductase (DHFR). Two S/P resistant clones, AS (50S/P) and AS (75S/P), were selected and chosen for further analysis. AS (PYR) parasites were eliminated by S/P treatment, whereas AS (50S/P) and AS (75S/P) recrudesced following S/P pressure. However, each mutant possessed a different drug resistant phenotype. The AS (75S/P) clone always recrudesced before the AS (50S/P) clone following treatment with S/P, while AS (50S/P) always appeared before AS (75S/P) when treated with either sulfadoxine or pyrimethamine alone. Mutations in the genes encoding the targets of sulfadoxine and pyrimethamine, dihydropteroate synthase (DHPS) and DHFR, have been implicated in the mechanism of S/P resistance in P. falciparum. The P. chabaudi dhps gene was cloned by homology and sequenced. The sequence analysis of the both dhfr and dhps genes of AS (75S/P) and AS(50S/P) did not reveal any polymorphisms when compared to the sequences of the AS (PYR) genes. The mechanism of resistance of S/P in these drug-selected lines is not therefore conferred by additional mutations in these genes. To determine the genetic basis of the S/P resistance, AS (50S/P) was crossed with a drug sensitive clone, AJ. Sixteen independent recombinant progeny clones were phenotyped for their susceptibility to S/P and pyrimethamine and genotyped for the influence of 30 chromosome-specific markers. Linkage analysis shows that mutant dhfr is a major determinant of S/P resistance in P. chabaudi. Quantitative trait analysis suggested that other loci, which may be involved in S/P resistance, are present on chromosomes 4, 5, and 9.

Thesis (MScMedSc)--Stellenbosch University, 2015.
ENGLISH ABSTRACT: Drug resistance in Mycobacterium tuberculosis is an increasing global problem. Drug resistance is mostly caused by single nucleotide polymorphisms (SNPs) within the bacterial genome. This observed increase in global incidence of drug resistant tuberculosis (TB) has sparked the search for new anti-TB drugs and the repurposing of drugs that are currently used against other organisms or species of mycobacteria. One such repurposed drug, clofazimine (CFZ), is currently used for the treatment of leprosy, caused by Mycobacterium leprae. The mechanism of action of CFZ is not clear, but it is hypothesized that CFZ is reduced by a mycobacterial type II NADH oxidoreductase (NDH-2). The reduction of CFZ drives the production of reactive oxygen species (ROS) which is toxic to the pathogen. The aim of this study was to elucidate the mechanism of CFZ resistance. Towards this aim, spontaneous in vitro CFZ resistant mutants were selected, characterized and whole genome was used identify SNPs which may cause CFZ resistance. Mutations were identified in a transcriptional regulator encoded by Rv0678, fatty-acid-AMP ligase, or FadD28 (Rv2941) and glycerol kinase or GlpK (Rv3696c). Mutations in Rv0678 have previously been shown to play a role in both CFZ resistance and bedaquiline (BDQ) cross-resistance, while no link has been found between CFZ resistance and mutations in fadD28 and glpK. The novel, non-synonymous SNP identified in Rv0678 resulted in the replacement of an alanine residue with threonine at codon 84, which is located in the DNA binding domain. Virtual modelling of the mutated Rv0678 protein showed that the A84T mutation may influence DNA binding, possibly due to its proximity to the DNA binding domain. This mutation caused a change in hydrophobicity, which may influence binding to DNA. Previous studies showed that mutations in Rv0678 resulted in the upregulation of mmpL5, a putative efflux pump. However, the mechanism whereby CFZ resistance occurs via increased abundance of this efflux pump in the cell wall is not clear and needs further investigation. The cross-resistance between CFZ and BDQ, caused by mutations in Rv0678, is of concern and may influence the planning of anti-TB drug regimens for the future. The roles of the other two mutations identified in this study in CFZ resistance is also not clear and requires further investigation. Finally, the findings of this study support the role of Rv0678 in CFZ resistance thereby suggesting that this gene could be useful as a diagnostic marker to test for CFZ resistance in clinical isolates.
AFRIKAANSE OPSOMMING: Middelweerstandigheid in Mycobacterium tuberculosis is 'n wêreldwye toenemende probleem. Middelweerstandigheid word meestal veroorsaak deur enkel nukleotied polimorfismes (SNPs) in die bakteriële genoom. Hierdie toename in middelweerstandige tuberkulose (TB) het gelei tot die soektog na nuwe anti-TB-middels en die alternatiewe aanwending van middels wat tans teen ander organismes of spesies van mikobakterieë gebruik word. Een so 'n alternatiewe middel, clofazimine (CFZ), word tans gebruik vir die behandeling van melaatsheid wat veroorsaak word deur Mycobacterium leprae. CFZ se meganisme van werking is nie duidelik nie, maar dit word vermoed dat CFZ gereduseer word deur 'n mikobakteriële tipe II NADH oksidoreduktase (NDH-2). Die reduksie van CFZ dryf die produksie van reaktiewe suurstof spesies wat giftig is vir die patogeen. Die doel van hierdie studie was om die meganisme van CFZ weerstandigheid te ondersoek. Om hierdie doel te bereik was spontane in vitro CFZ weerstandige mutante gekies, gekarakteriseer en heel genoom volgorde bepaling is gebruik om SNPs te identifiseer wat CFZ weerstandigheid veroorsaak. Mutasies in Rv0678, 'n transkripsie reguleerder, vetsuur-AMP ligase, of FadD28 (Rv2941) en gliserol kinase of GlpK (Rv3696c) geïdentifiseer. Dit is al voorheen gevind dat mutasies in Rv0678 ‘n rol speel in beide CFZ weerstandigheid en bedaquiline (BDQ) kruis-weerstandigheid, terwyl geen verband gevind is tussen CFZ weerstandigheid en mutasies in fadD28 en glpK nie. Die nuwe, nie-sinonieme SNP, geïdentifiseer in Rv0678 het gelei to die vervanging van 'n alanien aminosuur met treonien by kodon 84, wat geleë is in die DNS bindings domein. Virtuele modellering van die gemuteerde Rv0678 proteïen het getoon dat die A84T mutasie DNS binding moontlik kan beïnvloed, as gevolg van sy nabyheid aan die DNS bindings domein. Hierdie mutasie veroorsaak 'n verandering in die hidrofobiese natuur, wat DNS binding kan beïnvloed. Vorige studies het getoon dat mutasies in Rv0678 lei tot die opregulering van mmpL5, 'n waarskynlike uitvloei pomp. Die meganisme waardeur CFZ weerstandigheid veroorsaak, deur ‘n groot aantal van hierdie uitvloei pompe in die selwand, is nie duidelik nie en moet verder ondersoek word. Die kruis-weerstandigheid tussen CFZ en BDQ, wat veroorsaak word deur mutasies in Rv0678, is van belang en kan die beplanning van anti-TB middel behandeling vir die toekoms beïnvloed. Die rolle van die ander twee mutasies, wat in hierdie studie geïdentifiseer is, in CFZ weerstandigheid is ook nie duidelik nie en vereis verdere ondersoek. Ten slotte, die bevindinge van hierdie studie steun die rol van Rv0678 in CFZ weerstandigheid en dit dui daarop dat hierdie geen gebruik kan word as 'n diagnostiese merker om vir CFZ weerstandigheid te toets in kliniese isolate.

Despite the array of medical advances of our modern day society, infectious diseases still plague millions of people worldwide. Malaria, in particular, causes substantial suffering and death throughout both developed and developing countries. Aside from the socioeconomic challenges presented by the disease's prevalence in impoverished nations, one of the major difficulties scientists have encountered while attempting to eradicate the disease is the parasite's ability to become resistant to new drugs and methods of treatment. In an effort to better understand the dynamics of malaria, we analyze a mathematical model that accounts for both the treatment aspect as well as the drug resistance that accompanies it. Simulations demonstrating the effects of treatment rates and the level of resistance are studied and discussed in hopes of shedding additional light on the characteristics of this devastating epidemic.

The abundant nuclear enzyme poly (ADP-ribose)polymerase (P ARP) catalyses the formation of long homopolymeric chains of ADP-ribose, utilising NAD+ as a substrate, as the immediate cellular response to DNA damage. PARP recognises a damaged section of DNA and initiates polymer synthesis, which is believed to act as a signal to effect the repair of the lesion. A selective, potent PARP inhibitor could block the recognition, and hence repair, of DNA damage induced by cancer chemotherapy. Since increased DNA repair is regarded as a mechanism whereby tumour cells can become resistant to treatment, PARP inhibitors have therapeutic potential as resistance modifying agents. From a study of PARP inhibitors such as 3-hydroxybenzarnide (A), benzimidazole derivatives (B) were proposed as inhibitors of the enzyme, and the synthesis and biological evaluation of a series of such molecules has been achieved. Substituted 2-aryl benzirnidazoles have proved to be highly potent PARP inhibitors (B;R= 4'NO2Ph, IC5o= 59 nM), under a permeabilised cell assay the nitro phenyl derivative (B; R= 4'N02Ph) is the most potent compound reported to date (IC50= 19 nM). 2-Methyl benzirnidazole-4-carboxamide (B; R= Me) has been shown to potentiate the in vitro cytotoxicity of the antitumour agent temozolomide in L1210 cells, and the synthesis of benzimidazole inhibitors suitable for pre-clinical in vivo eluation has also been investigated, This thesis demonstrates that benzimidazole PARP inhibitors have promising potential for clinical development as resistance modifying agents.

Tuberculosis (TB) is the leading cause of death due to a single infectious disease and remains a major threat to global public health. The increasing emergence of multi-drug resistance to current anti-TB drugs, exacerbated by the long treatment duration, highlights the need for new effective treatments or strategies to shorten the treatment duration, improve patient adherence and curb the alarming rates of resistance. A key challenge to current strategies employed in the development of anti-TB drugs is the complexity in TB disease pathology which presents as a wide spectrum of lesions in patients presenting with the disease. These lesions occur at different anatomical loci in the same individual and at different stages as the disease progresses. In addition, the interaction between the causative agent, Mycobacterium tuberculosis, and its obligate human host induces physiologic and metabolic changes in the infecting bacillus that are specific to each lesion compartment, and dynamic. This is likely to influence M. tuberculosis susceptibility to antibiotic treatment and, consequently, affect treatment duration and possibly the development of drug resistance. A major limitation in current strategies to address this problem is translation of in vitro compound potency to in vivo efficacy. To reach the target site, a drug must first distribute and accumulate in the lesion microenvironments where bacteria reside: the macrophage host cell and the caseum. This thesis focused on the development of an in vitro infection model that could be used to predict drug penetration into M. tuberculosis-infected macrophages. Of particular interest was the extent to which host intracellular drug concentrations translate into effective antimycobacterial activity. To this end, the thesis comprised three key aspects: (i) characterization of physicochemical properties, antimycobacterial activities and M. tuberculosis-mediated metabolism of selected antiTB compounds; (ii) determination of intracellular drug permeation in resting, activated and foamy macrophages; and (iii) determination of the correlation (or not) between intracellular drug concentration and effective M. tuberculosis growth inhibition. The highly lipophilic natural product, fusidic acid (FA), its known human metabolite, 3-ketofusidic (3-ketoFA or GKFA37), and two C-3 alkyl esters (GKFA16 and GKFA17) as FA prodrugs were utilized in the study. In addition, another chemical class, the less lipophilic benzoxazole-based oxime derivatives were also investigated. Moxifloxacin (MXF), levofloxacin (LVF), bedaquiline (BDQ), rifampicin (RIF) and clofazimine (CFZ) were included for reference as known anti-TB drugs with varying lipophilicities. In chapter 2, FA and derivatives showed potent antimycobacterial activity (~1 µM) with selectivity indices (SI) >20 against the THP-1 macrophage cell line. Predicted artificial membrane permeability assay (PAMPA) results suggested that FA and derivatives would readily permeate the cell membrane. M. tuberculosis metabolized the C-3 alkyl-ester prodrug GKFA17 to form both FA and 3-ketoFA, with complete hydrolysis of the prodrug. FA was metabolized to 3-ketoFA, but the low levels of the metabolite suggested that another unidentified metabolite, presumed to be 3-epifusidic acid (3-epiFA), was formed. In vitro assays revealed that the potent benzoxazole-based oxime carbamates (PMN1-201, PMN1-136 and PMN2-09) were rapidly hydrolyzed by M. tuberculosis and were also susceptible to spontaneous degradation in media, forming the poorly active corresponding free oximes (PMN1-199, PMN1-135 and PMN1-157). In chapter 3, the in vitro macrophage drug uptake assay showed that FA C-3 alkyl prodrugs, GKFA16 and GKFA17, accumulated in significantly higher amounts in resting macrophages in comparison to FA and GKFA37. Accumulation of MXF was comparable to the least accumulated FA derivative, GKFA37, and showed steady state intracellular concentrations over a 6-day period. While GKFA16 and GKFA17 showed continued increasing accumulation, intracellular concentrations of FA and GKFA37 decreased after 48 hours, suggesting a likely susceptibility to macrophage efflux. In infected macrophages, the presence of intracellular bacteria or increasing bacterial burden did not affect the host cell ability to accumulate the drugs. FA and derivatives exhibited bacteriostatic inhibition of intracellular mycobacterial growth. MXF showed a potent bactericidal effect, reducing intracellular bacterial counts significantly at 10x MIC, with complete sterilization at 50x MIC even though MXF accumulation was significantly less than that of FA alkyl esters. These results suggested that both the inherent activity of a compound and ability to accumulate within host cells drive cellular efficacy. Given that the C-3 alkyl ester prodrugs accumulated at significantly higher concentrations than FA and GKFA37, this demonstrates the limitations of this assay in ascertaining the impact of intracellular concentration on drug efficacy for bacteriostatic drugs while highlighting its ability to correlate drug penetration and intracellular activity for cidal drugs. The prodrug GKFA17 was shown to undergo metabolism in resting host cells and during infection to form FA and then 3-ketoFA. Therefore, the prodrug strategy could be used to increase intracellular exposure of FA as GKFA17 showed superior macrophage accumulation. Benzoxazole-based oxime carbamates and their corresponding free oximes failed to accumulate in host macrophages and this was corroborated by their failure to control host cell bacterial growth despite the potent in vitro activity against M. tuberculosis of the carbamates, suggesting that they are poorly permeable. Chapter 4 investigated drug permeation in different macrophage phenotypes known to exist in the granuloma during TB disease, including foamy and activated macrophages. The activation state of the host cell did not affect the ability to accumulate anti-TB drugs such as RIF and BDQ. However, FA and its prodrug GKFA17 were significantly reduced in M1 activated macrophages. Despite the significantly reduced intracellular concentration, activated macrophages treated with FA and derivatives showed superior intracellular M. tuberculosis growth inhibition, suggesting that macrophage activation potentiates the activity of these compounds. In order to assess the effect of foamy macrophage lipid bodies (LBs) on drug uptake and intracellular localization, oleic acid-induced foamy macrophages were treated with selected antiTB drugs and experimental compounds. FA and derivatives showed early increased accumulation in foamy cells compared to resting macrophages, while MXF, BDQ and RIF levels were not significantly changed. Intracellular:extracellular (I/E) ratios increased with increase in lipophilicity, with FA C-3 alkyl prodrugs exhibiting the highest I/E ratios of >100. Despite exhibiting increased foamy macrophage concentrations, FA and derivatives exhibited a similar reduction (bacteriostatic) in bacterial counts in both resting and foamy macrophages. The intracellular activity of RIF was also not affected by presence of LBsin foamy macrophages. BDQ, LVF and MXF, however, showed reduced intracellular efficacy against M. tuberculosis in foamy macrophages compared to resting macrophages, suggesting a role for LBs to impact intracellular drug distribution. In conclusion, this thesis demonstrates the potential utility in combining advanced analytical methods and an in vitro infection model to determine cellular drug permeation profiles that might be applied to prioritize compounds and combinations optimized for distribution to target bacterial populations. This will facilitate well-informed decision-making processes in progression of lead compounds in pre-clinical development and, therefore, may offer the potential to reduce high rates of attrition of compounds which enter clinical phase of development.

Thesis (MScMedSc)--Stellenbosch University, 2014.
ENGLISH ABSTRACT: Central dogma suggests that mutations in target genes is the primary cause of resistance to first and second-line anti-TB drugs in Mycobacterium tuberculosis. However, it was previously reported that approximately 5% of Rifampicin mono-resistant clinical M. tuberculosis did not harbor mutations in the rpoB gene. The present study hypothesized that active efflux plays a contributory role in the level of intrinsic resistance to different anti-TB drugs (Isoniazid, Ethionamide, Pyrazinamide, Ethambutol, Ofloxacin, Moxifloxacin, Ciprofloxacin, Streptomycin, Amikacin and Capreomycin in RIF mono-resistant clinical M. tuberculosis isolates with a rpoB531 (Ser-Leu) mutation. This study aimed to define the role of Efflux pump inhibitors (verapamil, carbonylcyanide m-chlorophenylhydrazone and reserpine) in enhancing the susceptibility to different anti-TB drugs in the RIF mono-resistant clinical isolates. The isolates were characterized by determining the level of intrinsic resistance to structurally related/unrelated anti-TB drugs; determining the effect of EPIs on the level of intrinsic resistance in the isolates and comparing the synergistic properties of the combination of EPIs and anti-TB drugs. To achieve this, genetic characterization was done by PCR and DNA sequencing. Phenotyping was done by the MGIT 960 system EpiCenter software to determine the MICs of the different anti-TB drugs and the effect of verapamil and carbonylcyanide m-chlorophenylhydrazone on determined MICs. Due to inability to test reserpine in a MGIT, a different technique (broth microdilution) was used for the reserpine experiment. Additionally; fractional inhibitory concentrations (FIC) indices were calculated for each of these drugs. The FIC assess the anti-TB drugs/inhibitor interactions. STATISTICA Software: version 11 was used for statistical analysis. Results revealed that the RIF mono-resistant isolates were sensitive at the critical concentrations of all 10 drugs tested, with the exception of Pyrazinamide. This could be explained by the technical challenges of phenotypic Pyrazinamide testing. A significant growth inhibitory effect was observed between the combination of EPI and anti-TB drug exposure in vitro. This suggests that verapamil, carbonylcyanide m-chlorophenylhydrazone and reserpine play a significant role in restoring the susceptibility (decrease in intrinsic resistance level) of the RIF mono-resistant isolates to all anti-TB drugs under investigation. Additionally, a synergistic effect was observed by the combination treatment of the anti-TB drugs with the different EPIs. Based on these findings, we proposed a model suggesting that efflux pumps are activated by the presence of anti-TB drugs. The activated pumps extrude multiple or specific anti-TB drugs out of the cell, this in turn decrease the intracellular drug concentration, thereby causing resistance to various anti-TB drugs. In contrast, the addition of EPIs inhibits efflux pump activity, leading to an increase in the intracellular drug concentration and ultimate cell death. This is the first study to investigate the effect of different efflux pumps inhibitors on the level of intrinsic resistance to a broad spectrum of anti-TB drugs in drug resistant M. tuberculosis clinical isolates from different genetic backgrounds. The findings are of clinical significance as the combination of treatment with EPI and anti-TB drugs or use of EPIs as adjunctives could improve MDR-TB therapy outcome.
AFRIKAANSE OPSOMMING: Sentrale dogma beweer dat mutasies in teiken gene die primêre oorsaak van die weerstandheid teen anti-TB-middels in Mycobacterium tuberculosis is. Vorige studies het getoon dat ongeveer 5% van Rifampisien enkelweerstandige kliniese M. tuberculosis isolate nie ‘n mutasie in die rpoB geen het nie. Die hipotese van die huidige studie was dat aktiewe pompe 'n bydraende rol speel in die vlak van intrinsieke weerstandheid teen 10 verskillende anti-TB-middels (Isoniasied, Ethionamied, Pyrazinamied, Ethambutol, Ofloxacin, Moxifloxacin, Siprofloksasien, Streptomisien, Amikasien and Capreomycin) in RIF enkelweerstandige kliniese M . tuberculosis isolate met 'n rpoB531 (Ser-Leu) mutasie. Die doel van hierdie studie was om die rol van uitpomp inhibeerders (verapamil, carbonylcyanide m-chlorophenylhydrazone en reserpien) te definieer in die verbetering van die werking vir verskillende anti-TB-middels in die RIF enkelweerstandige kliniese isolate. Die doelstellings van die studie was om die vlak van intrinsieke weerstandigheid teen struktureel verwante/onverwante anti-tuberkulose middels asook die effek van die EPIs op die vlak van intrinsieke weerstand in die isolate is bepaal. Verder is sinergistiese eienskappe van die kombinasie van EPIs en anti-TB-middels ondersoek. Hierdie doelstellings is bereik deur genetiese karakterisering deur PKR en DNS volgorde bepaling. Fenotipering is gedoen deur gebruik te maak van MGIT 960 EpiCenter sagteware om die Minimum Inhibisie Konsentrasie (MIC) van die verskillende anti-TB-middels en die effek van verapamil en carbonylcyanide m-chlorophenylhydrazone op die MIC te bepaal. Reserpien kan nie in die MGIT sisteem getoets word nie, and daarom is 'n ander tegniek (mikro-verdunning) is gebruik om die effek van reserpien te toets. Fraksionele inhiberende konsentrasies (FIC) is bereken vir elk van hierdie middels die anti-TB-middels / inhibeerder interaksies te bepaal. STATISTICA v11 sagteware is gebruik vir alle statistiese analises. Resultate van hierdie studie toon dat die RIF enkelweerstandige isolate sensitief is teen kritieke konsentrasies van al die middels, met die uitsondering van Pyrazinamied. Weerstandigheid van Pyrazinamied kan wees as gevolg van welbekende tegniese probleme met die standaard fenotipiese pyrazinamied toets. ‘n Beduidende groei inhiberende effek is waargeneem tussen die kombinasie van EPI en anti-TB middel blootstelling in vitro. Dit dui daarop dat verapamil, CCCP en reserpine 'n belangrike rol speel in die herstel van die sensitiwiteit (afname in intrinsieke weerstand vlak) van die RIF enkelweerstandige isolate aan alle anti-TB-middels wat ondersoek is. Daarbenewens is 'n sinergistiese effek waargeneem deur die kombinasie van die verskillende anti-TB-middels en die verskillende EPIs. Op grond van hierdie bevindinge het ons ‘n model voorgestel wat toon dat uitvloei pompe geaktiveer word deur die teenwoordigheid van anti-TB-middels en die geaktiveerde pompe dan verskeie of spesifieke anti-TB-middels uit die sel pomp. Dus verminder die intrasellulêre konsentrasie van die middel en veroorsaak daardeur weerstandigheid teen verskeie anti-TB-middels. Die byvoeging van EPIs inhibeer uitvloei pompe se werking en lei tot 'n toename in die intrasellulêre konsentrasie van die middels en uiteindelik die dood van die selle. Hierdie is die eerste studie wat die effek van verskillende uitvloei pompe inhibeerders op die vlak van intrinsieke weerstand teen 'n breë spektrum van anti-TB-middels in die middel-weerstandige kliniese isolate ondersoek. Die bevindinge kan van belangrike kliniese belang wees aangesien die kombinasie van behandeling met EPI en anti-TB-middels die uitkoms MDR-TB terapie kan verbeter.

Ce manuscrit décrit la distibution géographique des génotypes de Mycobacterium tuberculosis dans une large partie de la Chine, et l’étude d’une éventuelle corrélation avec la vaccination BCG et la résistance aux antibiotiques. La prévalence de la résistance a été analysée dans 10 provinces, et les mutations responsables de la résistance à la rifampicin et à l’INH ont été recherchées. La distribution des différents génotypes a été analysée par spoligotypage, identification de délétions génomiques et par MLVA. Le génotype Beijing représente 55 à 93% des souches dans les provinces étudiées, avec un gradient du Sud vers le Nord de la Chine. Plusieurs autres familles sont identifiées toutes appartenant à la clade dite « Moderne ». Les familles Chine 2 et Chine 3 semblent être spécifiques de la Chine. On identifie également quelques souches de la famille CAS (Central Asia) dans une province et un ancêtre possible de la lignée Beijing dans une autre. La prévalence de la résistance aux antibiotiques a été mesurée parmis plus de 2000 isolats de 10 provinces. Le pourcentage de souches résistantes à au moins une drogue est de 45% et celui des multirésistantes est environ 29%. En Chine on observe aucune corrélation entre le génotype Beijing et la vacination BCG ce qui n’est pas le cas dans d’autres pays. La distribution des mutations du gène rpoB a été déterminée ainsi que les mutations responsables de la résistance à l’INH. Aucune mutation n’est observée dans les gènes oxyR et ahpC. Cette étude est la première étude de grande envergure effectuée en Chine sur la diversité génétique de M. Tuberculosis et sur les mécanismes de résistance aux antibiotiques
This thesis describes the geographical distribution of Mycobacterium tuberculosis genotypes in a major part of China and a study on the possible correlation with BCG vaccination and drug-resistance. The prevalence of drug resistance was investigated in 10 provinces, and drug resistance mutations in M. Tuberculosis related to rifampicin and INH resistance were determined. The distribution of different genotypes of M. Tuberculosis was investigated by using spoligotyping, large sequence polymorphism, and MLVA typing. The Beijing genotype represented 55 to 93% of the strains in the different provinces examined, with a gradient from South to North of China. Several other genotype families of M. Tuberculosis were identified all belonging to the so-called “Modern” clade. Two of them, the China 2 and China 3 families, appear to be specific for China. Some members of the Central Asian family were found in one province whereas a possibly more ancestral strain was traced in another. The prevalence of drug resistance was determined among more than 2000 isolates from 10 provinces. Resistance against at least one drug was found in 45% of the isolates and MDR in about 29%. In China, no correlation was observed between the Beijing genotype and resistance/BCG vaccination, which is different from results in other countries. The distribution of the mutations in the rpoB gene of rifampicin resistant strains was determined. In addition, in INH resistant strains, the distribution of mutation in several genes was analysed. No mutation was detected in the oxyR and ahpC genes. This is the first time that such a large scale study is performed on the genetic diversity of M. Tuberculosis in China

Drug resistance is a significant challenge in the fight against malaria. Importantly, reduced efficacy has been reported against artemether (ATM)/Lumefantrine (LM) (LM-ATM), amodiaquine (AQ)/artesunate (AS) (AQ-AS), two important combination treatment regimens in Africa, and against piperaquine (PQ), a drug which has been evaluated as a potential alternative in Africa, in combination with dihydroarteminisin (DHA). Chloroquine (CQ) resistance in P.falciparum is associated with two main transporters PfCRT and PfMDR1. I investigated the mechanisms of resistance to PQ, LM and AQ, with the overall goal of identifying molecular markers that can be used to track resistance. I used CQ as a reference. The key antimalarial drugs were highly active against clinical isolates from Kilifi, Kenya with median inhibitory concentrations (IC₅₀s) of <5nM for DHA and <55 nM for CQ, AQ, PQ, LM and DEAQ (desethylamodiaquine, the active metabolite of AQ). pfcrt-76 and pfmdr1-86 mutations were associated with AQ, DEAQ and LM but not DHA or PQ activity. Interestingly, > 20% of analysed isolates had decreased susceptibility to LM (IC₅₀ >100nM); these isolates were the most susceptible to CQ and carried wild type genotypes at pfcrt-76 and pfmdr1-86. I observed that CQ resistance had been declining in Kilifi since 1993 (prior to CQ withdrawal) to 2006 (7 years after its withdrawal), similar to observations in Malawi. My results support the hypothesis that susceptibility to antimalarial drugs returns when drug pressure is removed, and suggest that the use of LM-ATM may hasten the return of CQ susceptibility. Continued monitoring of drug susceptibility is crucial. pfcrt-76 and pfmdr1-86 may be useful molecular markers of LM-ATM efficacy in Kilifi and other African sites. Using a microarray approach, I identified additional genes (including various transporters) that may contribute to LM resistance. I recommend further studies to clarify the exact roles of the identified genes.

Orientador: Eduardo Mello de Capitani
Tese (doutorado) - Universidade Estadual de Campinas, Faculdade de Ciencias Medicas
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Resumo: A tuberculose, a resistência aos medicamentos antituberculose e o HIV são hoje em dia três grandes endemias, com tendências similares e com prejuízos incalculáveis para a humanidade, em particular nos países de baixos recursos. Obiectivos do trabalho: Determinar o padrão de resistência aos medicamentos antituberculose, em pacientes HIV positivos, portadores de doença pulmonar por micobactéria tuberculosa e micobactéria não tuberculosa na região de Maputo e a prevalência de MOTT nesta população. População e métodos: estudo elaborado em dois hospitais da cidade de Maputo, Moçambique. Foram estudados 503 doentes com tuberculose pulmonar e HIV+. Obteve-se 282 amostras da expectoração e ou lavagem brônquica nas quais foi solicitado; baciloscopia, cultura de BK, cultura de micobactérias nào tuberculosas e teste de sensibilidade para os antibacilares. Foram também avaliadas as características clínicas, radiográficas, a contagem de CD4 e o perfil hematológico. Resultados: Em 229 (98,7%) dos isolados, o M tuberculosis foi a principal micobactéria identificada. As micobactérias não tuberculosas, surgiram em apenas 3 (1,3%) casos, identificadas na expectoração e com clínica compatível. Dos 282 doentes, 232 (82%) apresentaram sensibilidade aos medicamentos antituberculose (MAT) e 50 (17%) resistência a qualquer MAT. Quanto ao padrão de resistência 27 (13,6%) eram casos novos e 21 (26,6%) casos previamente tratados. Resistências mais observadas a qualquer MAT, foram de 13,6% nos CN e 26,6% nos PT. As resistências gerais aos diferentes MAT foram: 10 H- 14,9%, 20 S- 7,8%, 30 R - 6,4%. A resistência à R foi aumentada tanto nos CN como nos PT. A tuberculose multiresistente combinada, foi de 5,7%, sendo nos CN, 3% e PT, 11,4%. Factores de risco de resistência e de TB-MR, foram identificados: tratamento anterior de TB e CD4<200. Estes doentes apresentaram mediana de CD4 de 151 cels/mm3, mediana de Hgb de 7,8g1dl e de CTL 1140. Do ponto de vista radiológico o padrão atípico foi o mais frequente e cavidades foram observadas num grupo reduzido, predominando nos PT e nos casos que apresentaram poliresistência aos MAT.O sarcoma de kaposi pulmonar surgiu em 4,8% dos doentes e as infecções fiíngicas/bacterianas em 27,9% Conclusões: A tuberculose multiresistente (TB-:MR)apresentou níveis elevados pelo que se deve reduzir o risco de transmissão da TB com medidas de controle da transmissão nosocomial e na comunidade e ampliar a DOTS estratégia a um maior número de população. Face à resistência elevada à H, aconselhamos a introdução de um 30MAT na fase de manutenção no regime de tratamento dos CN, teste de sensibilidade aos MAT no início dos retratamentos. A profilaxia com H em HIV+ na prevenção de TB e tratamento de infecção latente deverá ser analisada com cuidado devido á elevada resistência à H. Uso de cotrimoxazol para redução das causas de morte associadas ao HIV!TB. Tratamento antiretroviral e aconselhamento para teste voluntário de HIV em todos os doentes TB
Abstract: Tuberculosis, multidrug-resistant tuberculosis and mv are, today, the three greatest endemics with similar trends and immeasurable impairment to humanity,particularin low resources countries. Obiectives: Determine the resistance pattern to anti-tuberculosis drugs (ATD) in mv positive patients with pulmonary disease by mycobacteria tuberculosis and mycobacteria non tuberculosis in the Maputo region and the prevalence of MOTT in this population. Population and methods: the study was conducted in 503 patients with pulmonary tuberculosis and mv positive in two hospitaIs of Maputo City, Mozambique. Two hundred and eighty two (282) sputum samples and/or bronchial wash were submitted for testing of baciloscopy, BK culture, MOTT and Drug Susceptibility Testing (DST) for ATD. Clinical and radiographic characteristics, CD4 counting and hematological profile were also evaluated. Results: M tuberculosis was the main organism identified in 229 (98,7%) of the isolated samples. Non-tuberculosis mycobacteria were identified in sputum of only 3 (1,3%) of the cases with compatible clinic. Drug sensitivity was observed in 232 (82%) patients and resistance in 50 (17%) of the 282 sampled cases. In relation to the drug resistance pattems, 27 (13,6%) were in New Cases (NC) and 21 (26,6%) in Previously Treated cases (PT). More observed resistance to ATD was recorded in 13,6% in NC and 26,6% in PT. General resistant to different ATD was: 1° H- 14,9%, 2° S- 7,8%, 3° R- 6,4%. The resistance to R increased both in the NC as in PT. Overall, the MDR-TB was 5,7%, being 3% in NC and 11,4% in PT. The risk factors identified for resistance and MDR-TB were: previous treatment to TB and CD4 <200. These patients presented a median CD4 of 151 cells/mm3, a median of Hgb of 7,8 g/dl and of CTL 1140. From a radiological point ofview, the atipic pattem was the most frequent and cavities were observed in a small group predominant1yin PT and in-the cases that presented poliresistance to ATD. The pulmonary Kaposi sarcoma was observed in 4,8% ofthe patients and the fungall bacterial infections in 27,9%. Conclusions: The high levels of MDR-TB recorded in this study suggests that the risk of TB transmission should be reduced through control measures of nosocomial transmission and in the community and amplify the DOTS strategy to a greater population number. As a result of the high resistance to H, it is advised to introduce one of the 3o ATDin the maintenance phase of the treatment regime of NC, and sensitivity tests to ATD in the beginning of retreatment. Prophylaxis with H in HIV positive for TB prevention and latent infection treatment should be analyzed carefully, due to the high resistance to H. The use of cotrimoxazol to reduce the death causes associated to lllV/TB. Anti-Retroviral Treatment (ATRV) is important and also counceling for HIV voluntary testing in all TB patients
Doutorado
Clinica Medica
Doutor em Clínica Médica

O aumento da resistência do parasita da malária a maioria da drogas antimaláricas disponíveis, tornandose necessário pesquisar novos compostos com potencial atividade antimalárica. O objetivo desta tese foi inicialmente caracterizar a atividade do risedronato contra as formas intraeritrocitárias do parasita in vivo, além de identificar seu possível mecanismo de ação. A IC50 do risedronato foi de 20 mM em culturas de Plasmodium falciparum. Risedronato reduziu a biossíntese de FOH e GGOH e a isoprenilação de proteínas, inibindo a transferência do grupo FPP para as proteínas farnesiladas, entretanto, a transferência do GGPP para as proteínas geranilgeraniladas não foi inibida, isto também ocorreu quando proteínas ras e rab foram analisadas, sugerindo que a droga está inibindo a enzima FPPS. A enzima FPPS de P. falciparum foi expressa e obtivemos uma proteína recombinante fusionada a GST (rPfFPPS). Os substratos IPP, DMAPP, GPP e FPP foram utilizados para determinação da atividade catalítica da enzima, demonstrando FPP e GGPP como principais produtos. Os valores de Km para os diferentes substratos foi determinado. Demonstramos também que rPfFPPS é inibida por risedronato, podendo ser explorado como potencial alvo antimalárico.
The increased resistance of the malaria parasite most of antimalarial drugs are available, making it necessary to search for new compounds with potential antimalarial activity. The aim of this thesis was initially characterize the activity of risedronate against intraerythrocytic forms of the parasite in vivo, and identify its possible mechanism of action. The IC50 of risedronate was 20 mM in cultures of Plasmodium falciparum. Risedronate reduced biosynthesis and FOH, GGOH and protein isoprenylation, inhibiting the transfer of FPP group for farnesylated proteins, however, the transfer of GGPP to geranygeranylated proteins was not inhibited, this also occurred when ras and rab proteins were analyzed, suggesting that the drug is inhibiting the enzyme FPPS. The FPPS enzyme from P. falciparum was expressed and obtained a recombinant protein fused to GST (rPfFPPS). The substrates IPP, DMAPP, GPP and FPP were used to determine the catalytic activity of the enzyme, demonstrating FPP and GGPP as main products. The Km values for the various substrates were determined. We also demonstrate that rPfFPPS is inhibited by risedronate, which can be exploited as potential antimalarial target.