<p dir="ltr"><i>Candida glabrata</i> is a member of the gut microbiota that can become an opportunistic pathogen under certain conditions. It is known for its inherent resistance to azole antifungal drugs and its ability to rapidly develop resistance during treatment. However, the regulatory mechanisms that enable this commensal organism to survive in low-oxygen environments, such as the gut, and to develop antifungal resistance when it becomes pathogenic, are not fully understood. In this study, we demonstrate for the first time the roles of two zinc cluster transcription factors in <i>C. glabrata</i>, Hap1A and Hap1B, in contributing to azole drug resistance in both laboratory strains and drug-resistant clinical isolates, adaptation to hypoxia, and resistance to other antifungal drugs like polyenes and echinocandins under specific conditions.</p><p dir="ltr">Azole drugs, which target the Erg11 protein, are widely used to treat <i>Candida</i> infections. The regulation of azole-induced <i>ERG</i> gene expression and activation of drug efflux pumps in <i>C. glabrata</i> has primarily been linked to the zinc cluster transcription factors Upc2A and Pdr1. Here, we investigated the roles of <i>S. cerevisiae</i> Hap1 orthologs, Hap1A and Hap1B, in <i>C. glabrata</i> as direct regulators of <i>ERG</i> genes upon azole exposure.</p><p dir="ltr">Our research shows that deleting <i>HAP1</i> in the yeast model <i>S. cerevisiae</i> increases sensitivity to fluconazole due to the failure to induce <i>ERG11 </i>expression in the <i>hap1Δ</i> mutant compared to the wild-type strain. Although <i>C. glabrata</i> is closely related to <i>S. cerevisiae</i>, a whole genome duplication (WGD) event allowed <i>C. glabrata</i> to retain two HAP1 ohnologs, while <i>S. cerevisiae</i> lost one copy. Through phylogenetic and syntenic analyses, we identified Hap1A and Hap1B in <i>C. glabrata</i> as ohnologs of Hap1 in <i>S. cerevisiae</i>, which is known to regulate <i>ERG</i> gene expression under both aerobic and hypoxic conditions. Interestingly, deleting <i>HAP1B</i> in <i>C. glabrata</i> increased sensitivity to both triazole and imidazole drugs, similar to Hap1 in <i>S. cerevisiae</i>, while deleting <i>HAP1A </i>did not affect azole sensitivity.</p><p dir="ltr">Gene expression analysis revealed that the increased azole sensitivity in the <i>hap1BΔ </i>strain was due to reduced azole-induced <i>ERG</i> gene expression, leading to lower total endogenous ergosterol levels. Additionally, the loss of <i>HAP1B</i> in <i>C. glabrata</i> clinical isolates like SM1 and BG2, as well as in drug-resistant strains like SM3, also led to increased azole hypersusceptibility. While it was already known that losing <i>UPC2A</i> in <i>C. glabrata</i> increases azole sensitivity, our study is the first to demonstrate that the combined loss of both <i>HAP1B </i>and <i>UPC2A</i> makes <i>C. glabrata</i> strains even more sensitive to azoles than losing either gene alone. Additionally, we show that the loss of both <i>HAP1B </i>and the H3K4 histone methyltransferase <i>SET1</i> increases azole hypersensitivity more than the loss of either gene alone.</p><p dir="ltr">Interestingly, the Hap1A protein is barely detectable under aerobic conditions but is specifically induced under hypoxia, where it plays a crucial role in repressing <i>ERG</i> genes. In the absence of Hap1A, Hap1B compensates by acting as a transcriptional repressor. Our RNA sequencing analysis further showed that losing both <i>HAP1A</i> and <i>HAP1B</i> not only affects genes in the ergosterol biosynthesis pathway but also upregulates iron transport-related genes <i>FET3 </i>and <i>FTR1</i>. Moreover, we found that the hypoxic growth defect caused by the loss of both <i>HAP1A</i> and <i>HAP1B</i> is exacerbated when treated with the echinocandin caspofungin and the cell wall-damaging agent calcofluor white, indicating that these Hap1 ohnologs contribute to maintaining cell wall integrity under hypoxic conditions. Since <i>HAP1A</i> transcript levels remain stable under aerobic conditions, we suspect that Hap1A expression is regulated post-transcriptionally.</p><p dir="ltr">Furthermore, we discovered that the simultaneous loss of both HAP1A and HAP1B leads to increased hypersensitivity to the polyene antifungal drug amphotericin B, though the exact mechanism behind this phenotype remains unclear. Altogether, our study is the first to show that Hap1A and Hap1B have evolved distinct roles, enabling <i>C. glabrata</i> to adapt to specific host and environmental conditions.</p>

Malariakontrolle ist von Resistenzen gegen Malariamedikamente wie Chloroquin (CQ) und Artemisininderivaten (ART) bedroht. Hier untersuchten wir das Ausmaß dieser Resistenzen in Fünf Feldstudien in Nigeria, Ruanda und Südwestindien unter Beurteilung der Prävalenzen Arzneimittelresistenz-assoziierter Mutation der Plasmodium-Parasiten (P. falciparum: K13, dhps, dhfr, mdr1 und P. vivax: mdr1) z.T. in Korrelation mit klinischen Patientendaten und ex-vivo Überlebensraten (ÜLR) unter Zugabe von ART. K13 wurde in 360 zwischen 2010-2018 gesammelte ruandischen P. falciparum Isolaten genotypisiert. Erstmals fanden wir dort niedrige Frequenzen der mit ART-Resistenz assoziierten K13-Mutation. Jedoch lassen Mutation mit niedrigen ÜLR, sowie ein Isolat mit hohen ÜLR aber ohne K13-Mutation eines Patienten der die Infektion unter Therapie nicht eliminieren konnte, Fragen offen. Ca.100 indische P. falciparum und P. vivax Isolaten aus 2015 wurden auf Mutationen in P. falciparum Markern für die Resistenz gegen Sulfadoxin-Pyrimethamin (SP) (d.h. pfdhps/pfdhfr), Artesunat (AS) (d.h. K13) und Lumefantrin (d.h. pfmdr1) sowie P. vivax Marker für CQ-Resistenz (pvmdr1) untersucht. Der Großteil der Isolate zeigt Mutationen die SP-Resistenz hervorrufen, daher könnte die Effizienz der AS+SP-Therapie begrenzen sein. Außerdem eignet sich Lumefantrin nicht als alternatives Medikament auf Grund der beobachteten Dominanz des pfmdr1-Haplotyps „NFD“. Die Abwesenheit der pvmdr1-Mutation Y976F und erfolgreiche Behandlungen zeigen, die Wirksamkeit von CQ gegen vivax Malaria im Studiengebiet. Auch Isolate von nigerianischen Schwangeren mit asymptomatischer P. falciparum Infektion zeigten hohe Prävalenzen von pfdhfr/pfdhps Vier- und Fünffachmutanten darum ist die Wirksamkeit der präventiver Therapie Schwangerer mit SP in Nigeria ernsthaft gefährdet. Die Daten spiegeln die Häufigkeit der Resistenzen gegen Malariamittel in diesen Gebieten wieder mit großen Unterschieden zwischen Regionen und Medikamenten. / The spread of resistance to antimalarial drugs such as chloroquine (CQ) and artemisinins (ART) is a great threat to malaria control. Here, we investigated the extent of such resistance in Nigeria, Rwanda and south-western India. We assessed the prevalence of mutations in few Plasmodium parasites’ markers of resistance, namely P. falciparum genes K13 (ART), pfdhps/pfdhfr (sulfadoxine-pyrimethamine, SP) and pfmdr1 (lumefantrine) as well as P. vivax gene pvmdr1 (CQ) in 5 field studies conducted in 2010-2018, and partially correlated the results to patients’ clinical outcome. Few isolates from Rwanda, were also evaluated for their parasite ex vivo survival rates (SR) upon exposure to ART. We tracked ART resistance in Rwanda by genotyping K13 in 360 P. falciparum isolates from 2010-2018. We showed for the first time that K13 mutations associated with ART resistance are present here, thus in Africa, at a low frequency. However, mutations occurred in patients who recovered and/or had low SR. Of note, one patient with high SR but no K13 mutation was still parasitemic after ART treatment. Moreover, we assessed the presence of mutations in K13, pfdhps/pfdhfr, pfmdr1 and pvmdr1 in ca 100 P. falciparum and 100 P. vivax isolates from south-western India. Most of P. falciparum isolates carried pfdhfr/pfdhps mutations conferring SP resistance, menacing the efficacy of SP-ART treatment. Also, the high prevalence of pfmdr1 haplotype “NFD” advised against the introduction of lumefantrine. The low rates of P. vivax pvmdr1 Y976F and patients’ successful parasite clearance, indicated that CQ remains effective in the area. Finally, a high rate of pfdhfr/pfdhps quadruple and quintuple mutant was observed in Nigerian pregnant women with asymptomatic P. falciparum infection, hence the effectiveness of preventive treatment with SP in pregnancy might be threatened. The data reflected the abundance of antimalarials resistance in these areas with important differences between regions and drugs.

La leishmaniose est une maladie zoonotique affectant à la fois les chiens et les humains, transmise par la piqûre d’un phlébotome. Elle est causée par un parasite protozoaire du genre Leishmania et se manifeste sous trois formes principales, dont la plus grave est souvent mortelle chez l’humain. Or, la pharmacopée utilisée pour combattre cette maladie est très limitée, avec des médicaments qui sont non-spécifiques au parasite en plus d’être les mêmes chez les chiens et les humains. Cela contribue à l'aggravation de la résistance aux antiparasitaires, en particulier à l'antimoine, l'un des médicaments de première ligne contre cette maladie. Une découverte récente de notre laboratoire a mis en cause les vésicules extracellulaires (EVs) dans le développement de résistance chez Leishmania, en raison de leur capacité à transmettre horizontalement des gènes au sein d’une population de parasites. Dans ce projet de maîtrise, nous avons cherché à exploiter cette découverte et à créer des EVs qui faciliteraient le transfert de gènes permettant la resensibilisation des parasites résistants. Pour ce faire, nous avons créé des parasites surexprimant le gène AQP1, qui code pour une protéine de transport de l’antimoine. Nous avons ensuite produit des EVs à partir de ce parasite modifié, et nous en avons caractérisé la taille, la morphologie et le contenu protéique. Enfin, nous avons tenté la resensibilisation des parasites résistants, en utilisant ces EVs surexprimant AQP1. L’impact sur les parasites résistants a ensuite été évalué en termes de leur sensibilité à l’antimoine. Nous avons pu déterminer que certaines des méthodes utilisées avaient un effet significatif de resensibilisation à l’antimoine. Ces découvertes mettent de l’avant une approche innovante pour lutter contre la résistance aux antimicrobiens, et explorent une application potentielle des EVs dans la médecine moderne. / Leishmaniasis is a zoonotic disease affecting both dogs and humans, transmitted by the bite of a sandfly. It is caused by a protozoan parasite of the genus Leishmania and manifests in three main forms, the most severe of which is often fatal in humans. However, the pharmacopeia used to combat this disease is very limited, with drugs that are non-specific to the parasite and are the same for both dogs and humans. This contributes to the worsening of resistance to antiparasitic drugs, particularly to antimony, one of the first-line drugs against this disease. A recent discovery from our team implicated extracellular vesicles (EVs) in the development of resistance in Leishmania, due to their ability to horizontally transmit genes within a parasite population. In this master's project, we sought to exploit this discovery and create EVs that would facilitate the transfer of genes enabling the resensitization of resistant parasites. To do this, we created parasites overexpressing the AQP1 gene, which encodes an antimony transport protein. We then produced EVs from this new strain and characterized their size, morphology, and protein content. Finally, we attempted to resensitize resistant parasites using these AQP1-overexpressing EVs. The impact on resistant parasites was then assessed in terms of their sensitivity to antimony. We determined that some of the methods we used had a significant resensitization effect to antimony. These discoveries highlight an innovative approach to combat drug resistance and explore a potential application of EVs in modern medicine.

Little is still known about what allows mature Arabidopsis thaliana (Arabidopsis) plants to respond with an Age-Related Resistance response (ARR). To better understand how mature plants initiate and establish ARR, gene expression in the leaves of young and mature plants responding to Pseudomonas syringae pv. tomato (Pst) was investigated using RNA-sequencing analysis. Genes involved in the biosynthesis of N-hydroxypipecolic acid (NHP) were upregulated in ARR-responding leaves leading to the idea that NHP, a signaling molecule in Systemic AcquiredResistance (SAR) may also be required for ARR. The ARR response was examined in NHP biosynthesis mutants and revealed that NHP biosynthesis is required for ARR. During ARR, NHP biosynthesis mutants were also shown to accumulate less salicylic acid (SA) compared to wild-type leaves in response to Pst. Healthy untreated leaves had modest accumulation of NHP and modest expression of several cell-surface receptors was observed compared to the healthy untreated leaves of young plants, suggesting that ARR competence in mature untreated plants involves a primed/immune ready state similar to what is observed in systemic leaves of plants induced for SAR in a local leaf. The ARR response also requires the accumulation of intercellular SA which is involved in inhibiting biofilm-like aggregate formation of Pst in mature plant leaves. To understand how SA is transported from the cytosol to the intercellular space during ARR, the ARR response of the PDR-type transporter mutants pdr8-4 and pdr12-3 was examined. The pdr8-4 pdr12-3 double mutant was partially ARR-defective and SA accumulation in leaf intercellular spaces was reduced by ~50% compared to wild-type mature plants during ARR, demonstrating that PDR8 and PDR12 are required for the intercellular localisation of SA during ARR. To obtain evidence that PDR8 and PDR12 act as transporters of SA, SA transporter assays were performed with yeast expressing PDR8 and PDR12. PDR8- and PDR12-expressing yeast accumulated less intracellular SA than empty vector containing yeast cells, suggesting that PDR8 and PDR12 act as SA transporters. Together, this work found ARR shared signaling components with SAR and found additional support for SA as an antimicrobial and signaling molecule during ARR. / Thesis / Doctor of Science (PhD)

Apoptoseresistenzmechanismen spielen bei der Pathogenese maligner Lymphome eine zentrale Rolle. So konnte bei Hodgkin/Reed-Sternbergzellen eine Deregulation des Transkriptionsfaktors NF-_B beobachtet werden, die zu verstärkter Apotoseresistenz führt und so zum malignen Wachstum dieser Zellen wahrscheinlich entscheidend beiträgt. Es konnte gezeigt werden, dass die selektive Blockade von NF-_B sowohl zu erhöhter Apoptosesensitivität als auch zur Inhibition der Zellzyklusprogression in kultivierten Hodgkinzellen führt. Der genaue molekulare Mechanismus der NF-_B-Deregulation in Hodgkinzellen ist jedoch noch unklar. Apoptoseresistenzmechanismen sind nicht nur bei der Pathogenese maligner Lymphome, sondern auch bei der Entstehung von Therapieresistenz von Bedeutung. So konnte gezeigt werden, dass die Überexpression proapoptotischer Gene der bcl-2 Familie in resistenten malignen Zellen sowohl die Empfindlichkeit gegenüber Zytostatika als auch gegenüber Antikörperbehandlung wiederherstellen kann. Neben der bcl-2 Familie spielt wahrscheinlich auch das Apo-I/Fas-System eine wichtige Rolle bei der Entstehung von Zytostatikaresistenz und immunologischer Resistenz. Somit stellt neben der Überexpression des P-Glykoproteins (MDR1), das als transmembranes "Pumpenprotein" Zytostatika aus der Tumorzelle heraustransportieren kann, die Deregulation Apoptose-steuernder Gene einen weiteren wichtigen Therapie-Resistenzmechanismus dar. Eine Möglichkeit, intrazelluläre Resistenzmechanismen zu umgehen, stellt die indirekte Induktion von Zelltod mit Hilfe bispezifische Antikörper dar. Durch diese Moleküle kann eine T-Zell-vermittelte Zellyse von Lymphomzellen erreicht werden. / Resistance towards apoptosis plays an important role in the pathogenesis of malignant lymphomas. It could be demonstrated that deregulation of the transcription factor NF-kB is a common molecular defect of Hodgkin/Reed-Sternberg cells that leads to enhanced resistance towards apoptosis and therefore probabaly contributes to the malignant growth of these cells. It couldbe shown that blocking of NF-kB leads to increased sensitivity towards apoptosis and decreased cell cycle progression. The precise molecular mechanism that leads deregulation of NF-kB is still unknown. Besides its role in the pathogenesis of malignant lymphoma resistance towards apoptosis plays an important role in the development of drug resistance. It could be shown that overexpression of pro-apoptotic members of the bcl-2 family in resistant tumor cells can restore sensitivity towards both cytotoxic drugs as well as antibody treatment. In addition to the bcl-2 family the Apo-I/Fas system is also involved in the development of drug resistance. Thus, besides overexpression of p-glycoprotein (MDR-1) that might pump cytotoxic drugs out of a malignant cell deregulation of apoptosis regulating genes is another important mechanisms of drug resistance development. One possibility to overcome drug resistance is the induction of cell death via bispecific antibodies. These molecules can induce T cell mediated lysis of lymohoma cells.

Thesis (Ph. D.)--University of Virginia, 2008. / Includes bibliographical references. Also available online through Digital Dissertations.

The aim of this thesis was to provide a picture of the trypanosomosis and drug resistance prevalence in Eastern Province of Zambia, to understand the underlying factors of drug resistance (drug use habits), to improve the diagnosis of trypanosomosis in livestock and finally, to improve the diagnosis of isometamidium resistance in T.congolense. After an introductory part where available trypanosomosis and trypanocide resistance diagnostic methods are described and discussed, the body of the thesis is divided in two main sections. In the first section are presented the results of a cross-sectional and a longitudinal epidemiological survey describing the geographical distribution of trypanosomosis cases, of resistant isolates and of cattle treated with isometamidium chloride. The results of the monitoring of unsupervised treatments of cattle with isometamidium by farmers and veterinary assistants with the Isometamidium-ELISA technique are also presented. The second section describes the development of two new diagnostic methods, the first one allowing the diagnosis of trypanosome infections with high sensitivity and specificity through semi-nested polymerase chain reaction and restriction fragment length polymorphism. This is the first report of a pan-trypanosome PCR test (a single PCR test for the diagnosis of all important pathogenic trypanosomes of cattle). The second new method that was developed allows the diagnosis of isometamidium resistant T.congolense strains by PCR-RFLP. This is the first report of a PCR based diagnostic test of trypanocide resistance in T. congolense.<p> / Doctorat en sciences, Spécialisation biologie moléculaire / info:eu-repo/semantics/nonPublished

MSc (Microbiology) / Department of Microbiology

Thesis (PhD )--University of Stellenbosch, 2010. / ENGLISH ABSTRACT: Diagnostic delay is regarded as a major contributor to the continuous rise in tuberculosis (TB) cases and the emergence and transmission of multidrug-resistant tuberculosis (MDR-TB) and extensively drug resistant tuberculosis (XDR-TB). It is therefore essential that more rapid diagnostic methods are developed. Molecular-based assays have the potential for the rapid species-specific diagnosis of TB and associated drug-resistances directly from clinical specimens. We investigated whether high resolution melting analysis (HRM) could enable the rapid diagnosis of TB and associated drug resistance, since the HRM apparatus and reagents are relatively inexpensive and the methodology can easily be implemented in high incidence, low income regions. Application of this methodology allowed for the rapid identification of mycobacterial lymphadenitis from fine-needle aspiration biopsy (FNAB) samples in 2 studies. This was done by targeting the region of deletion 9 (RD9), present in M. tuberculosis and M. canettii, but absent from all other members of the complex. However, the sensitivity of the method was low (51.9% and 46.3%, respectively) when compared to the reference standard (positive cytology and/or positive culture). Despite this limitation our method was able to provide a rapid diagnosis in more than half of the infected patients with a relatively high specificity (94.0% and 83.3%, respectively). We therefore proposed a diagnostic algorithm allowing the early treatment of patients with both HRM and cytology results indicative of mycobacterial disease. We developed the Fluorometric Assay for Susceptibility Testing of Rifampicin (FAST-Rif) which allowed the rapid diagnosis of MDR-TB by detecting rifampicin (RIF) resistance mutations in the rpoB gene with a sensitivity and specificity of 98% and 100%, respectively. The FAST-Rif method was easily adapted to detect ethambutol (EMB) resistance due to mutations in the embB gene with a sensitivity and specificity of 94.4% and 98.4% respectively, as compared to DNA sequencing. The FAST-EMB method was a significant improvement over the inaccurate culture based method. We identified a strong association between EMB resistance (and pyrazinamide resistance) and MDR-TB and subsequently advised modifications to the current (2008) South African National TB Control Programme draft policy guidelines. Due to the potential for amplicon release, we adapted the FAST-Rif and FAST-EMB methods to a closed-tube one-step method using the detection of inhA promoter mutations conferring isoniazid (INH) resistance as a model. The method (FASTest-inhA) was able to identify inhA promoter mutations with a sensitivity and specificity of 100% and 83.3%. These mutations are of particular interest as they confer low level INH resistance and cross-resistance to ethionamide (Eto). Since inhA promoter mutations are strongly associated with XDR-TB in the Western and Eastern Cape Provinces of South Africa, data generated by the recently implemented GenoType® MDRTBPlus assay may allow individualised treatment regimens to be designed for a patient depending on their INH mutation profile. Our proposed treatment algorithm may be particularly useful in XDR-TB cases, for which only few active drugs remain available. Since current diagnostic methods all carry advantages and disadvantages, a combination of phenotypic and genotypic-based methodologies may be the best scenario while awaiting superior methods. / AFRIKAANSE OPSOMMING: Die onvermoë om tuberkulose (TB), multi-weerstandige tuberkulose (MDR-TB) en uiters weerstandige tuberkulose (XDR-TB) vinnig te diagnoseer, is ‘n belangrike oorsaak vir die volgehoue toename en verspreiding daarvan. Dit is noodsaaklik dat diagnostiese toetse wat vinniger resultate oplewer, ontwikkel word. Molukulêre toetsing het die potensiaal om vinnig spesie-spesifieke diagnoses van TB en die weerstandigheid teen TB-medikasie te lewer. Hierdie studie wil vasstel of hoë-resolusie smeltingsanalise (HRS) ‘n vinnige diagnose van TB en die weerstandigheid teen TB-medikasie kan oplewer aangesien die relatiewe lae koste van reagense en apparaat, asook die minimale infrastruktuur en vaardighede wat vir dié toets benodig word, dit uiters geskik maak vir pasiënte in gebiede met ‘n hoë TB-insidensie en lae inkomste. Die toepassing van die HRS-metode op fynnaald-aspiraatbiopsies in twee afsonderlike studies, het gelei tot die vinnige identifisering van mikrobakteriële-limfadenitis. Dit is bemiddel deur die gebied van delesie 9 (RD9) teenwoordig in Mycobacterium tuberculosis en M. canettii, maar afwesig in al die ander lede van die kompleks, te teiken. Die sensitiwiteit van die metode was (51.9% en 46.3%, vir die twee studies onderskeidelik) in vergelyking met die verwysingstandaard (positiewe sitologie en/of positiewe kultuur). Ten spyte van dié beperking was ‘n vinnige diagnose in meer as die helfte van geïnfekteerde pasiënte met ‘n redelike hoë spesifisiteit (94.0% en 83.3%, onderskeidelik) moontlik. ‘n Diagnostiese algoritme wat gebaseer is op die resultate van die HRS en sitologie-toetse, is voorgestel om pasiënte vroeër te behandel. ‘n Fluorometriese toets (FAST-Rif) is ontwikkel vir die vinnige diagnose van MDR-TB deur mutasies in die rpoB-geen op te spoor met ‘n hoë sensitiwiteit en spesifisiteit (98% en 100%, onderskeidelik). Hierdie mutasies is verantwoordelik vir weerstandigheid teen die antibiotikum rifampicin (FAST-Rif) en word beskou as ‘n vinnige diagnose vir MDR-TB. Die FAST-Rif metode kon maklik aangepas word om mutasies in die embB-gene, verantwoordelik vir weerstandigheid teen die antibiotikum ethambutol (EMB), op te spoor. Die FAST-EMB-metode het ‘n sensitiwiteit en spesifisiteit van 94.4% en 98.4% onderskeidelik getoon in vergelyking met DNS volgordebepaling. Die FAST-EMB-metode was ‘n betekenisvolle verbetering op die onakkurate kultuurgebaseerde metodes. ‘n Sterk korrelasie tussen EMB-weerstandigheid (en weerstandigheid teen pyrazinamide) en MDR-TB is geïdentifiseer. Vervolgens is veranderinge aan die Suid-Afrikaanse Nasionale TB-beheerprogram se Konsepbeleidsgids (2008) voorgestel. Om die potensiële vrylating van amplikone te verhoed, is die FAST-Rif en FAST-EMB aangepas tot ‘n enkelstap geslote buissisteem deur gebruik te maak van die opsporing van inhA promotormutasies wat weerstandigheid teen isoniazid (INH) veroorsaak. Die metode het ‘n sensitiwiteit en spesifisiteit van 100% en 83.3% onderskeidelik, getoon. Hierdie mutasies veroorsaak laevlak weerstandigheid teen INH, maar ook kruisweerstandigheid teen ethionamide (Eto). Aangesien daar ‘n sterk verbintenis tussen inhA-promotormutasies en XDR-TB in die Oos en Wes-Kaapprovinsies van Suid-Afrika is, kan data van die GenoType® MDRTBPlus-toets moontlik gebruik word om ‘n meer geïndividualiseerde behandeling te ontwerp afhangende van die pasiënt se INH-mutasieprofiel. Ons behandelingsalgoritme is veral geskik vir XDR-TB pasiënte vir wie daar weinig aktiewe antibiotika beskikbaar is. Huidige diagnostiese metodes het almal voor- en nadele, dus bied ‘n kombinasie van fenotipiese en genotipiese metodes moontlik die beste oplossing totdat beter metodes ontwikkel word.

Thesis(PhD)--Stellenbosch University, 2013. / ENGLISH ABSTRACT: The epidemic levels of drug-resistant tuberculosis (DR-TB) in high-burden countries such as South Africa, which is currently ranked as third highest in the world, is the result of a synergistic relationship between the increased transmission of DR strains, poor patient adherence as well as Human-Immunodeficiency Virus (HIV)-coinfection. The impact of these combined factors on the rise of DR-TB led to an urgent need for the development of new diagnostic tools to rapidly detect TB and its associated drug susceptibility profile. The Foundation for Innovative New Diagnostics (FIND) has taken the onus upon them to ensure that laboratory strengthening becomes a reality by having developed, and still developing, new diagnostic assays in order to improve the laboratory turn-around time (TAT), whereby the transmission of DR-TB strains can be stopped. Laboratory strengthening does not solely rely on new diagnostic assays alone, and thus a Quality Management System, discussed in the dissertation, must be in place to ensure that the rapid result is accurate and reliable. The series of studies encompassed in this dissertation includes methodological validations (both technical and operational) of rapid TB diagnostic assays in order to rapidly and accurately diagnose the disease, and thus reducing the diagnostic delay associated with conventional diagnostic platforms. The studies were conducted “in-house” at the National Health Laboratory Service (NHLS) Reference TB laboratory in Green Point, Cape Town, which is a high-volume public health laboratory. The need to rapidly detect resistance to the first line anti-tubercular drugs Isoniazid and Rifampicin was a priority and thus the performance of a commercial line probe assay (LPA), the GenoType®MTBDRplus Ver1.0 LPA, was assessed for use on smear positive direct patient material. The performance characteristics was superior to that of conventional drug susceptibility testing, where the sensitivity and specificity for the detection of multi-drug resistant TB (MDR-TB) was 98.8 and 100%, respectively, with results in 1-2 days. Based on this study, the World Health Organization (WHO) endorsed the use of molecular LPA for the rapid detection of DR-TB. Furthermore, the need for quality assurance associated with the GenoType®MTBDRplus LPA in the diagnostic laboratory is essential and thus a user manual for the molecular detection of Drug Resistant Tuberculosis in resource-limited settings has also been developed (http://www.finddiagnostics.org/export/sites/default/resource-center/reports brochures/docs/LPA LaboratoryManual22Mar2012.pdf) for which Global Laboratory Initiative (GLI) status is pending. With the outbreak of extensively drug resistant TB (XDR-TB) in Tugela Ferry area in KwaZulu-Natal and the rest of the world, the need to rapidly detect resistance to the second line drugs arose, and thus the performance characteristics of the GenoType®MTBDRsl LPA was assessed for use on smear positive direct patient material. The performance characteristics proved to be excellent once again, with a 93.3% reduction in TAT. The data was scrutinized by the WHO, where it may be used as a triage test to guide treatment, but to date, no final policies on the use thereof has been finalized. The need for rapid point-of-care (POC) testing led to the implementation of the Xpert®MTB/RIF assay in the referral laboratories, for use on both smear positive and smear negative direct patient material. In order to accommodate for laboratories where the LPA has been implemented already, the GenoType®MTBDRplus Ver2.0 LPA was developed, which is aimed for use on all smear types as well. A head-to-head assessment was done between these assays to determine their performance characteristics and it was shown to be equally good. In this study we have shown the utility of molecular diagnostic assays to rapidly diagnose TB and its associated drug susceptibility patterns. This will have a significant impact on diagnostic delay and clinical decision making as well as patient outcome. / AFRIKAANSE OPSOMMING: Die epidemiese vlakke van middel-weerstandige tuberkulose (MW-TB) in hoë-lading lande, soos Suid Afrika wat tans derde hoogste op die wêreld ranglys is, is die nagevolge van 'n sinergistiese verband tussen die verhoogde voorkoms van transmissie van MW stamme, swak pasiënt deelname aan die voorgeskrewe behandelings programme, asook Menslike Immuniteitsgebreksvirus (MIV) ko-infeksie. Die impak van hierdie drie faktore saam, gee aanleiding tot 'n verhoging in MW-TB en dus was daar 'n daadwerklike behoefte vir die ontwikkeling van nuwe diagnostiese toetse wat nie net TB kan identifiseer nie, maar wat ook die gepaardgaande middel-weerstandigheids profiel aandui. Die “Foundation for Innovative New Diagnostics” (FIND) het die onus van laboratorium versterking op hulself geneem, deur te verseker dat die nuut ontwikkelde diagnostiese toetse, asooks steeds ontwikkelende diagnostiese toetse, gebruik kan word om die konsep van laboratorium versterking 'n realiteit te maak. Die doel is dus om sodoende die tyd-tot-resultaat tussen geneesheer en laboratorium te verbeter, terwyl die transmissie van MW-TB ook die hok geslaan kan word. Nietemin, laboratorium versterking berus nie net op nuwe diagnostiese toetse nie, en dus is dit noodsaaklik dat 'n Kwaliteitbestuursisteem, soos bespreek in hierdie verhandeling, in plek is om te verseker dat die resultaat spoedig, akkuraat en betroubaar is. Die samevattende reeks studies in hierdie verhandeling behels metodologiese validasies (beide tegnies en operasioneel van aard) van spoedige TB diagnostiese toetse met die doel om die siekte so vinnig en akkuraat as moontlik te diagnoseer en dus die diagnostiese vertraging, wat histories met konvensionele metodes geassosiëerd is, te verminder. Al die studies is uitgevoer in die “National Health Laboratory Service (NHLS)” TB verwysingslaboratorium in Groenpunt, Kaapstad, wat 'n hoë-volume publieke gesondheidslaboratorium is. Die noodsaaklikheid om weerstandigheid teenoor die eerste-linie antituberkulose middels isoniasied en rifampisien so spoedig moontlik te diagnoseer het 'n groot bekommernis geword, en dus is die laboratorium daartoe genoop om die prestasie eienskappe van 'n kommersiëel beskikbare “line probe assay” (LPA), die “Genotype®MTBDRplus Ver1.0 LPA”, te asseseer vir die gebruik daarvan op direkte pasientmateriaal wat smeer positief is. Die prestasie eienskappe was beter as die van konvensionele middelvatbaarheidstoetse, waar die sensitiwiteit en spesifisiteit vir die diagnosering van MW-TB 98.8 en 100%, respektiewelik, was. Verder was die resultate ook binne 1-2 dae beskikbaar. Op grond van dié bevindinge het die Wêreldgesondheidsorganisasie (WGO) die gebruik van hierdie molekulêre “LPA” vir die spoedige diagnose van MW-TB onderskryf. Nietemin, die belangrikheid van gehalteversekering wat met die “GenoType®MTBDRplus LPA” in die diagnostiese laboratorium geassosieerd is, is essentiëel en dus is 'n gebruikershandleiding vir die molekulêre diagnose van MW-TB in beperkte hulpbron-instellings ontwikkel (http://www.finddiagnostics.org/export/sites/default/resource-center/reports brochures/docs/LPA LaboratoryManual22Mar2012.pdf) waarvoor daar op„n “Global Laboratory Initiative (GLI)” status in afwagting is. Met die uitbraak van ekstensiewemiddelweerstandige TB (EMW-TB) in die Tugela Ferry distrik in KwaZulu-Natal asook in die res van die wêreld, het die noodsaaklikheid onstaan om weerstandigheid teenoor die tweede-linie middels ook so spoedig moontlik te diagnoseer, en die laboratorium is dus weereens daartoe genoop om die prestasie eienskappe van die “GenoType®MTBDRsl LPA” (ook vir die gebruik op direkte pasient materiaal wat smeer positief is) te asseseer. Die prestasie eienskappe was weereens verbysterend, en het „n 93.3% afname in tyd-tot-resultaat getoon. Die data is deur die WGO aangevra, en daar is besluit dat die toets gebruik kan word om behandeling in werking te stel, maar geen finale onderskrywings is tot op hede nog gemaak nie. Die behoefte aan 'n punt-van-sorg toets het gelei tot die implementering van die “Xpert®MTB/RIF” toets in die verwysingslaboratorium, en is geoogmerk vir die gebruik op beide smeer positiewe en -negatiewe direkte pasient materiaal. Omrede die “LPA” al in verskeie laborotoriums geimplementeer was, is die “GenoType®MTBDRplus Ver2.0 LPA” ontwikkel, waarvan die gebruik onafhanklik is van die smeerresultaat. 'n Direkte assesering tussen die twee toetse was gedoen en daar is bevind dat beide se prestasie eienskappe vergelykend was. In hierdie studies het ons bewys dat die gebruik van molekulêre diagnostiese toetse in staat is om TB en die gepaargaande middel-weerstandigheids profiel spoedig te diagnoseer. Hierdie bevindinge sal 'n groot impak hê op die vetraging van tyd-tot-resultaat, op die mediese besluitneming asook op die uitkoms van die pasiënt. / FIND (Foundation for Innovative New Diagnostics) / Hain Lifescience / National Health Laboratory Service (NHLS)