Tuberculosis (TB) is a leading public health problem across the world. For various reasons, TB and multidrug-resistant tuberculosis (MDR-TB) have increased. Clarification on TB/HIV co-infection and homelessness as risk factors for TB and MDR-TB is required to inform policy interventions to reduce TB-related morbidity, mortality, and healthcare costs. In this quantitative study, data from the Houston Health Department (N = 341) were analyzed to explore the relationship between TB and MDR-TB outcomes and TB/HIV co-infection and type of housing/homelessness. Foreign-born persons are disproportionately affected in the United States. The socio-ecological model provided a theoretical framework for the investigation. Multiple and logistic regression analyses were conducted to investigate the relationships between variables, controlling for age and gender. Results indicate that HIV infected persons were more likely than non-infected persons to contract TB, and homeless persons were more likely than non-homeless persons to contract TB/MDR-TB, suggesting that high TB/HIV co-infection rates increase prevalence of TB and MDR-TB while improvements in housing reduce prevalence of TB and MDR-TB. However, no significant associations between variables were found. The odds ratio, Exp(B) = 0.000, p -?¥ 0.90, 95% Cl [0.000, with no upper bound values] was observed for both independent variables. Regular screening for TB/HIV co-infection among persons with high TB and MDR-TB risk profiles is recommended. Further investigation is required. Inclusion of more covariates could further elucidate more evidence of an association between variables. Study findings may support interventions to reduce TB-related morbidity, leading to positive social change.

Yes / Mutations in the gyrase genes cause fluoroquinolone resistance in Mycobacterium tuberculosis. However, the predictive value of these markers for clinical outcomes in patients with MDR-TB is unknown to date. The objective of this study was to determine molecular markers and breakpoints predicting second-line treatment outcomes in M. tuberculosis patients treated with fourth-generation fluoroquinolones. We analysed treatment outcome data in relation to the gyrA and gyrB sequences and MICs of ofloxacin, gatifloxacin and moxifloxacin for pretreatment M. tuberculosis isolates from 181 MDR-TB patients in Bangladesh whose isolates were susceptible to injectable drugs. The gyrA 90Val, 94Gly and 94Ala mutations were most frequent, with the highest resistance levels for 94Gly mutants. Increased pretreatment resistance levels (>2 mg/L), related to specific mutations, were associated with lower cure percentages, with no cure in patients whose isolates were resistant to gatifloxacin at 4 mg/L. Any gyrA 94 mutation, except 94Ala, predicted a significantly lower proportion of cure compared with all other gyrA mutations taken together (all non-94 mutants + 94Ala) [OR = 4.3 (95% CI 1.4-13.0)]. The difference in treatment outcome was not explained by resistance to the other drugs. Our study suggests that gyrA mutations at position 94, other than Ala, predict high-level resistance to gatifloxacin and moxifloxacin, as well as poor treatment outcome, in MDR-TB patients in whom an injectable agent is still effective.

Thesis (MScMedSc)--Stellenbosch University, 2008. / ENGLISH ABSTRACT: Application of molecular fingerprinting highlights transmission as the driving force behind the drug resistant epidemic in South Africa. Different strains dominate within different geographical regions, which is a reflection of micro-epidemics of drug resistance in the different regions. Cluster analysis shows that strains within the same strain family are different. The Beijing drug resistant strain family is the most dominant strain family (31%) in the Western Cape and of particular concern is the highly transmissible Beijing cluster 220 strain in the Western Cape communities. This cluster is widespread in the region and was previously identified in a MDR outbreak in a high school in Cape Town. Results suggest that the spread of Beijing drug resistant cluster 220 in the community was due to a combination of acquisition of drug resistant markers and transmission. This study also indicate that atypical Beijing can acquire drug resistance and become fit amongst HIV infected individuals. This is contrary to believe that atypical Beijing strains are not frequently associated with drug resistance and are attenuated. This implies that HIV levels the playing field for all drug resistant strains. Mechanisms leading to the evolution of MDR-TB and XDR-TB in a mine setting with a wellfunctioning TB control program which exceeds the target for cure rates set by the WHO were investigated. Despite the excellent control program, an alarming increase in the number of drug resistant cases was observed in 2003 and subsequent years. Phylogenetic analysis shows sequential acquisition of resistance to first and second-line anti-TB drugs leading to the development of MDR and XDR-TB. Contact tracing indicate extensive transmission of drug resistant TB in the shafts, hospital and place of residence. This study shows that despite exceeding the WHO cure rate target, it was not possible to control the spread and amplification of drug resistance. In summary, as a top priority, future TB control plans need to address diagnostic delay more vigorously. / AFRIKAANSE OPSOMMING: Molukulêre tegnieke toon transmissie as die hoofrede vir die toename in die anti-tuberkulose middelweerstandigheid epidemie in Suid-Afrika. Die verskillende Mikobakterium tuberkulose rasse wat domineer in verskillende areas is ‘n refleksie van middelweerstandige mikro-epidemies in verskillende gebiede. Analise van identiese rasgroepe demonstreer dat ras families bestaan uit verskillende rasse. Die Beijing middelweerstandige rasfamilie is die mees dominante familie in die Wes-Kaap (31% van monsters van middelweerstandige families) en van spesifieke belang is die hoogs oordraagbare Beijing 220 groep. Hierdie groep is die mees wydverspreide groep in die studie area en was voorheen geïdentifiseer tydens ‘n meervoudige middelweerstandige uitbreking in ‘n hoërskool in Kaapstad. Die resultate dui aan dat die Beijing middelweerstandige groep 220 in die gemeenskap versprei as gevolg van ‘n kombinasie van middelweerstand verwerwing en transmissie. Hierdie studie dui verder aan dat die atipiese Beijing ook middelweerstandigheid kan verwerf en hoogs geskik is vir infeksie veral in MIV geïnfekteerde individue. Hierdie data is in teenstelling met die algemene denke dat atipiese Beijing nie gereeld geassosieer word met middelweerstandigheid nie en dat dit dikwels geattenueer is. Dit beteken dat MIV die hoof faktor is wat alle middelweerstandige rasse kans gee om te versprei. Hierdie studie het die meganisme wat lei tot die evolusie van middelweerstandigheid en “XDRTB” in die myne ondersoek. Die myn besit ‘n goeie funksioneerde tuberkulose kontrole program wat alreeds die Wêreld Gesondheids Organisasie se mikpunt vir tuberkulose genesing oortref. Ten spyte van ‘n uitstekende tuberkulose kontrole program, is daar ‘n bekommerenswaardige toename in die aantal middelweerstandige tuberkulose gevalle waargeneem in 2003 en in die daaropvolgende jare. Filogenetiese analise wys dat opeenvolgende verwerwing van middelweerstandigheid teen eerste en tweede vlak anti-tuberkulose middels gelei het tot die ontwikkeling van meervoudige middelweerstandigheid en “XDR-TB”. Die opsporing van kontakpersone om transmissie te bewys dui aan dat transmissie van middelweerstandige tuberkulose in die werk plek, hospitaal en woon plek plaasvind. Hierdie studie wys dat ongeag die feit dat die Wêreld Gesondheids Organisasie se genesings verwagtinge oortref is, dit steeds onmoontlik was om die verspreiding en amplifisering van middelweerstandigheid te beheer. ‘n Top prioriteit vir tuberkulose kontrole planne in die toekoms behoort die vertraging van diagnose sterk aan te spreek.

Thesis (MScMedSc (Biomedical Sciences. Molecular Biology and Human Genetics))--University of Stellenbosch, 2009. / ENGLISH ABSTRACT: South Africa is currently facing a major multidrug-resistant tuberculosis (MDR-TB) epidemic and has one of the highest incidences in the world. Aminoglycoside antibiotics are commonly used in this country as a treatment against MDR-TB. A well known side-effect of aminoglycosides is permanent hearing loss and this is thought to have a significant genetic component. To date, at least six mutations in the mitochondrial genome are known to confer susceptibility to aminoglycosideinduced hearing loss. It is imperative that we investigate the frequency of these mutations in our populations and determine whether certain sub-groups are at increased risk. The aim of the present study was therefore to investigate the genetic aetiology of aminoglycoside-induced hearing loss in the South African population. A multiplex method using the ABI Prism® SNaPshotTM Multiplex system was optimised to screen for six mutations in the MT-RNR1: A1555G, C1494T, T1095C, 961delT+C(n), A827G and T1291C. A total of 115 MDR-TB patients from the Brooklyn Chest Hospital in Cape Town who were receiving high doses of either streptomycin, kanamycin or capreomycin were recruited for this study. Furthermore, 439 control samples, comprising of 93 Afrikaner, 104 Caucasian, 112 Black and 130 Mixed Ancestry individuals were recruited and screened for the presence of the six mutations. Identification of novel variants in the MT-RNR1 and the entire mitochondrial genome was performed using High Resolution Melt analysis (HRM) and whole mitochondrial DNA sequencing, respectively. A total of 97 family members from a South African family known to harbour the A1555G mutation were recruited and genotyped using SNaPshot analysis. In addition, mitochondrial functioning in the presence of different streptomycin drug concentrations, in transformed lymphoblasts of an individual harbouring the A1555G, was assessed by means of the MTT colorimetric assay. Detection of heteroplasmic mutations was performed using PCRRestriction Fragment Length Polymorphism (RFLP) analysis and UN-SCAN-IT software. We successfully developed a robust and cost-effective method that detects the presence of all six mutations simultaneously. The method worked equally well on both blood (from adults) and buccal swabs (from children). The C1494T, T1095C and T1291C mutations were not detected in any of the MDR-TB or control groups. Alarmingly, the A1555G mutation was detected in 0.9% of the Black control samples and in 1.1% of the Afrikaner controls (in one sample in the heteroplasmic state 25%). The A827G mutation was present at a frequency of 0.9% in the MDR-TB patients and in 1.1% of the Afrikaner controls. The 961delT + insC(n) mutation was found in relatively high frequencies in both the MDR-TB patients (3.5%) and control groups (1.1% of the Afrikaner, 1.5% of the Mixed Ancestry and 7.1% of the Black samples). Similarly, the T961G mutation was III detected at high frequencies in the Caucasian (2.9%) and Afrikaner (3.2%) controls. Screening for novel variants in MT-RNR1 in MDR-TB patients experiencing ototoxicity revealed two novel variants (G719A and T1040C). However, G719A and T1040C are not likely to be pathogenic since they were detected in ethnic-matched controls: Mixed Ancestry (20.7%) and Black (1.8%) controls. Furthermore, a total of 50 novel variants were identified within the mitochondrial genome of eight MDR-TB patients with ototoxicity. Only five of the 50 variants (one in the MT-TH, ND3, COX3 and two in the CYTB gene) were shown to reside at positions that are evolutionarily conserved across five species from human to frog, and the four variants in the protein coding genes resulted in missense changes. A total of 76 of the 97 family members recruited were found to be A1555Gpositive (on mitochondrial haplogroup L0d) and are therefore at risk of developing irreversible hearing loss. Genes and variants known to act as genetic modifiers: tRNASer(UCN), homozygous A10S in TRMU and 35delG in GJB2 were not present in this family. For the MTT assay, decreased mitochondrial functioning of cells harbouring the A1555G mutation in the presence of streptomycin were (compared to wild type) observed but this was not statistically significant (p-value: 0.615- 0.999). The high frequency of the A1555G mutation (0.9%) in the Black population in South Africa is of concern given the high incidence of MDR-TB in this particular ethnic group. However, future studies with larger numbers of samples are warranted to determine the true frequencies of the aminoglycoside deafness mutations in the general South African population. Our data suggests that the 961delT + insC(n) and T961G variants are common non-pathogenic polymorphisms due to the high frequencies observed in controls (>1%). The identification of the first novel variants within protein coding genes that could possibly be associated with aminoglycoside-induced hearing loss holds great possibilities with regards to the identification of a second gene involved in drug induced hearing loss. Future studies where the possible effect of these variants on the normal functioning of these genes could be assessed would contribute greatly to this field of research. All 76 A1555Gpositive members of the family were given genetic reports and counseled about their risk and that of their children for developing hearing loss due to aminoglycoside use. The development of a rapid and cost-effective genetic method facilitates the identification of individuals at high risk of developing hearing loss prior to the start of aminoglycoside therapy. This is of critical important in a low-resource country like South Africa where, despite their adverse sideeffects, aminoglycosides will be continue to be used routinely and are accompanied with very limited or no audiological monitoring. Future studies and greater public awareness is therefore needed to address this serious problem. / AFRIKAANSE OPSOMMING: Suid Afrika beleef tans „n grootskaalse tuberculose epidemie (veral weerstandige vorme van tuberculose) (MDR-TB), met een van die hoogste voorkomssyfers in die wêreld. Aminoglikosied antibiotikums word baie algemeen gebruik in Suid Afrika vir die behandeling van MDR-TB. ‟n Bekende newe effek van die middels is permanente gehoor verlies en dit is van mening dat dit gekoppel is aan „n genetiese component. Daar is tans ses mutasies in die mitochondriale genoom wat vatbaarheid tot aminoglikosied-geinduseerde gehoor verlies veroorsaak. Daarom is dit van uiterse belang dat die frekwensie van die mutasies in ons populasies bepaal word sodat daar vasgestel kan word watter groepe „n hoë risiko het om gehoor verlies te kan ontwikkel. Die ABI Prism® SNaPshotTM Multipleks sisteem is gebruik en geoptimiseer om te toets vir die ses mutasies in die MT-RNR1: C1494T, T1095C, 961delT+C(n), A827G and T1291C. „n Totaal van 115 MDR-TB pasiente van die Brooklyn Chest Hospital in Kaap Stad is gewerf vir die studie. Hierdie pasiente ontvang daaglikse hoë dosese van een van die volgende aminoglikosiede: streptomycin, kanamycin of capreomycin. Verder is „n totaal van 439 kontrole DNA monsters gewerf vanuit die volgende etniese groepe: 93 Afrikaner, 104 Blank, 112 Swart and 130 Kleurling. Hierdie monsters is ook getoets vir die ses mutatsies. Hoë Resolusie Smelt analise (HRS) is gebruik om nuwe DNS volgorde veranderinge in die MT-RNR geen te identifiseer. Die hele mitochondriale genoom is blootgestel aan DNA volgorde bepaling in „n poging om nuwe DNS volgorde verandering in die genoom te identifiseer wat moontlik betrokke kan wees by aminoglikosied-geinduseerde gehoor verlies. „n Total van 97 lede van „n Suid Afrikaanse familie waar die A1555G mutasie teenwoordig is, is deur middle van die SNaPshot metode gegenotipeer. Verder is die normale funcitoneering van die mitochondrion in getransformeerde witbloed selle, getoets in die teenwoordigheid van verskillende konsentrasies streptomycin met behulp van die MTT kleurmetrie toets. Deteksie van heteroplasmiese mutasies is gedoen deur middle van die PCR-RFLP tegniek en alle analises is gedoen op die UN-SCAN-IT program. Ons was suksesvol in die ontwikkeling van „n vinnige, koste effektiewe en kragtige tegniek wat al ses die mutasies in MT-RNR1 in een reaksie kan optel. Hierdie tegniek het goed gewerk met DNA monsters van bloed en van selle verkry vanuit die wangholte (geneem van kinders jonger as 12 jaar). Die C1494T, T1095C en T1291C mutasies is glad nie waargeneem in enige van ons MDR-TB patiente of kontroles nie. Skrikwekkend is die hoë frekwensie (0.9%) waarby die A1555G mutasie in die Swart kontrole groep waargeneem is. Hierdie mutasie is ook in 1.1% van die Afrikaner kontrole groep opgemerk in heteroplasmie van 25%. Die A827G mutasie was teenwoordig in 0.9% en 1.1% van die MDR-TB patiente en Afrikaner kontrole monsters, onerskeidelik. Die 961delT + insC(n) mutasie is opgemerk in baie hoë frekwensies in beide die MDR-TB (3.5%) en kontrole groepe (1.1% van die Afrikaner, 1.5% van die Kleurling en 7.1% van die Swart monsters). Die T961G mutasie is ook in hoë frekwensies in slegs die Blanke (2.9%) en die Afrikaner (3.2%) kontrole groepe waargeneem. Nuwe DNS volgorde veranderinge in MT-RNR1 is gesoek in „n groep MDR-TB patiente wat gehoor verlies ondervind. Slegs twee nuwe verandering is ontdek (G719A en T1040C). Dit is onwaarskynlik dat hierdie veranderinge patogenies is siende dat hulle teen frekwensies van 20.7% en 1.8% waargeneem is in die Kleurling en Swart kontrole groepe onderskeidelik. Tydens die soeke na nuwe DNS volgorde veranderinge wat moontlik geassosieer is met aminoglikosied-geinduseerde gehoor verlies in die mitochondriale genoom is 50 onbekende veranderinge ontdek (een in die MT-TH, ND3, COX3 en twee in die CYTB gene). Die veranderinge is verder ondersoek vir evolusionêre konservasie op beide die nukliotied en amino suur vlak van mens to padda. Dit is bevind dat 76 uit die 97 familie lede positief is vir die A1555G mutasie en het dus „n hoë risiko om aminoglikosied-geinduseerde gehoor verlies te ontwikkel as hul bloot gestel word aan hierdie antibiotikums. Verder is gevind dat hierdie familie op die L0d mitochondriale haplogroep lê. Geen van die sogenaamde genetiese modifiseerde gene of DNS volgorde veranderinge in hierdie gene (tRNASer(UCN), A10S in TRMU in homosigotiese vorm en die 35delG in GJB2) is gevind in die familie nie. Die MTT toets het „n afname in die mitochondriale funksioneering van selle waar die A1555G mutasie teenwoordig was getoon, alhoewel die verskil tussen selle wat nie die A1555G mutasie het nie, nie statisties betekenisvol was nie (p-waarde: 0.615-0.999). Die hoë frekwensie van die A1555G mutasie (0.9%) in die Swart populasie van Suid Afrika is skrikwekkend siende dat die voorkomssyfer van MDR-TB in hierdie groep baie hoog is. Toekomstige studies met grooter getalle is nodig om die ware frekwensie van die mutasies geassosieer met aminoglikosied-geinduseerde gehoor verlies in die algemende Suid Afrikaanse populasie te bepaal. Ons data dui aan dat die 961delT + insC(n) en die T961G mutasies slegs algemene nie-patogeniese polimorphismis is siende dat dit in sulke hoë frekwensies (>1%) in kontroles opgemerk is. Die identifiseering van die eerste DNS volgorde veranderinge in proteïen kodeerende gene wat moontlik geassosieer is met aminoglikosied-geinduseerde gehoor verlies hou groot en belowende moontlikehede in, interme van die identifiseering van „n tweede geen. Toekomstige studies waarin die effek van hierdie veranderinge op die normale funktioneering van hierdie gene ondersoek word sal „n besondere groot bydrae lewer tot hierdie veld van navorsing. Al 76 van die A1555G positiewe familie lede is voorsien van genetiese verslae en het berading ontvang in verband met hul risiko en die risiko van hul kinders om aminoglikosied-geinduseerde gehoor verlies te ontwikkel. Die ontwikkeling van „n kragtige, vinnige en koste-effektiewe genetiese metode vergemaklik die vinnige identifiseering van hoë risiko individue vir die ontwikkeling van gehoor verlies voordat hulle met hul aminoglikosiede behandeling begin. Dit is veral noodsaaklik in „n derde wêreld land soos Suid Afrika waar, ten spyte van hul gevaarlike newe effekte, aminoglikosied antibiotikums steeds gebruik sal word. Daarom is grooter publieke bewusmaking nodig om hierdie problem te probeer oplos en te verhoed.

Thesis (MScMedSc)--Stellenbosch University, 2013. / ENGLISH ABSTRACT: Multidrug resistant tuberculosis (MDR-TB), defined as having resistance to at least the first-line drugs, isoniazid and rifampicin (RIF), is a global health problem. Mutations in the rpoB gene, encoding the β-subunit of RNA polymerase, are implicated in RIF resistance - with the S531L and H526Y mutations occurring most frequently. The level of RIF resistance varies for strains with identical rpoB mutations, which suggests that other factors play a role in RIF resistance. Efflux has been implicated in determining the intrinsic level of RIF resistance. Increased expression of the multidrug efflux pump, Rv1258c, following RIF exposure was observed in some Mycobacterium tuberculosis MDR clinical isolates and H37Rv RIF mono-resistant mutants, but not others. The factors influencing the induction of Rv1258c are poorly understood. The aim of this study was to investigate the effects of rpoB mutations on expression of Rv1258c and whiB7, a transcriptional regulator of Rv1258c, in M. tuberculosis H37Rv in vitro generated RIF resistant mutants, in the absence and presence of RIF. The promoter region of M. tuberculosis H37Rv Rv1258c was cloned into a position upstream of a lacZ gene (encoding β-galactosidase) in multi-copy episomal and integrating vectors. Vector functioning and the effect of rpoB mutations on Rv1258c promoter activity were initially investigated in the non-pathogenic related species, Mycobacterium smegmatis mc2155 rpoB mutants and subsequently in M. tuberculosis by doing β-galactosidase assays. qRT-PCR was done to investigate the effects of rpoB mutations on native Rv1258c and whiB7 gene expression. Episomal and integrating vectors were functional and the integrating vector system was used for subsequent β-galactosidase assays in M. tuberculosis. Rv1258c promoter activity in the S531L mutant was approximately 1.5 times less and in the H526Y mutant 1.5 times higher than that of the wild-type in M. smegmatis. Similarly, Rv1258c promoter activity in the S531L mutant was approximately half and in the H526Y mutant approximately double that of the wild-type in M. tuberculosis. A similar trend in Rv1258c and whiB7 expression to those observed using β-galactosidase assays were observed when investigating the native Rv1258c and whiB7 gene transcript levels compared to the wild-type using qRT-PCR, although differences were not significant. Exposure of the M. smegmatis and M. tuberculosis rpoB mutants to sub-inhibitory levels of RIF did not affect Rv1258c promoter activity. Mutations in rpoB had a marginal effect on Rv1258c and whiB7 transcript levels, but showed the same trend as that seen for Rv1258c promoter activity. It remains to be determined whether these differences are biologically significant. When considering efflux pumps as new targets for treatment, possible differences in efflux pumps expression due to different rpoB mutations should be considered. / AFRIKAANSE OPSOMMING: Multi-middel weerstandige tuberkulose (MDR-TB) word gedefinieer as weerstandigheid tot ten minste rifampisien (RIF) en isoniasied, wat deel van die eerstelyn anti-tuberkulose behandeling vorm. Mutasies in die rpoB geen, wat die β-subeenheid van die RNA polimerase enkodeer, word geassosieer met RIF weerstandigheid. S531L en H526Y rpoB mutasies kom die algemeenste voor. RIF weerstandigheids vlakke verskil egter tussen isolate met identiese rpoB mutasies, wat impliseer dat ander faktore ook 'n rol in RIF weerstandigheid speel. 'n Toename in transkripsie van die Rv1258c geen, wat 'n multi-middel effluks pomp enkodeer, is waargeneem met blootstelling aan RIF, slegs in sommige M. tuberculosis H37Rv RIF mono-weerstandige mutante and MDR kliniese isolate, maar nie in ander nie. Die faktore wat die induksie van die Rv1258c effluks pomp beïnvloed is nie goed nagevors nie. Die studie ondersoek die effek van die rpoB mutasies op die uitdrukking van die Rv1258c en whiB7,'n transkripsionele regulator van Rv1258c, gene in M. tuberculosis H37Rv in vitro gegenereerde RIF weerstandige mutante, in die teenwoordigheid en afwesigheid van RIF. Die promotor area van die M. tuberculosis H37Rv Rv1258c geen is in 'n posisie stroomop van 'n lacZ geen, wat vir β-galaktosidase enkodeer, in multi-kopie episomale en integreerende vektors ingekloneer. Die funksionaliteit van die vektor en effek van rpoB mutasies op Rv1258c promotor aktiwiteit is ondersoek in die naverwante nie-patogeniese spesies, M. smegmatis en daarna in M. tuberculosis deur β-galaktosidase essais te doen. qRT-PCR is gedoen om die effek van rpoB mutasies op die vlak van transkripsie van die natuurlike Rv1258c geen en die whiB7 geen te bestudeer. Beide die episomale en integreerende vektors was funksioneel en daar is besluit om die integreerende vektor vir daaropeenvolgende β-galaktosidase essais in M. tuberculosis te gebruik. Rv1258c promotor aktiwiteit van die S531L mutant was ongeveer 1.5 keer minder as en die van die H526Y mutant 1.5 keer hoër as die van die ongemuteerde bakterië in M. smegmatis. Soortgelyk was die Rv1258c promoter aktiwiteit van die S531L mutant ongeveer die helfde van en die van H526Y mutant ongeveer dubbel die van die ongemuteerde bakterië in M. tuberculosis 'n Soortgelyke neiging in die vlakke van Rv1258c en whiB7 transkripte van die natuurlike geen is gedurende qRT-PCR waargeneem alhoewel die verskille nie beduidend was nie. Blootstelling aan sub-inhibitoriese konsentrasies van RIF het geen effek op Rv1258c uitdrukking in die M. smegmatis of M. tuberculosis rpoB mutante gehad nie. Die rpoB mutasies het net 'n effense effek op Rv1258c en whiB7 transkrip vlakke in M. tuberculosis rpoB mutante, maar transkrip vlakke het 'n soortgelyke neiging as die Rv1258c promoter aktiwiteit getoon. Of die waargenome verskille biologies betekenisvol is, moet nog bepaal word. Indien effluks pompe as teikens vir bahandeling gebruik sou word, moet in ag geneem word dat effluks pompe moontlik verskillend uitgedruk word in verskillende rpoB mutante. / The DST/NRF Centre of Excellence in Biomedical Tuberculosis Research, Stellenbosch University / DAAD-NRF in Country Scholarship and Ernst and Ethel Eriksen Trust / Harry Crossley Foundation

This quantitative, descriptive study investigated factors that contributed to TB patients registered in four Tembisa clinics in 2001, defaulting treatment. An interview schedule with closed and open-ended questions was used for 30 patients who could be traced who had interrupted treatment. The reasons for treatment interruption were related to socio-economic, TB policy-related and health care worker-related factors. The findings illustrate that TB management requires a multi-sectoral approach and joint efforts to tackle the disease that continues to kill people even though it is curable. / Health Studies / M.A. (Health Studies)

The study investigated the knowledge levels of clients on long-term tuberculosis (TB) treatment at Kwekwe General Hospital, Zimbabwe. A quantitative, descriptive research design was chosen and data was collected using a structured questionnaire with a convenience sample of 60 clients on TB treatment and 10 professional nurses. The major findings of the study were that although clients had some knowledge about their condition, there was a lack of knowledge regarding critical aspects such as information on drug-resistant TB and the Directly Observed Therapy Short Course. The professional nurses experienced constraints such as insufficient time for appropriate health education and home visits. Based on the study findings and conclusions, several recommendations were made. / Health Studies / Thesis(M.A(Health Studies))

Tuberculosis (TB) is the world’s most deadly infectious disease and causes enormous public health problems. The comorbidity with HIV and the rise of multidrug-resistant TB strains impede successful therapy through drug-drug interactions and the lack of efficient second-line treatments. The aim of this thesis was to support the improvement of anti-TB therapy through development of pharmacometric models, specifically focusing on the novel drug bedaquiline, pharmacokinetic interactions and methods for pooled population analyses. A population pharmacokinetic model of bedaquiline and its metabolite M2, linked to semi-mechanistic models of body weight and albumin concentrations, was developed and used for exposure-response analysis. Treatment response was quantified by measurements of mycobacterial load and early bedaquiline exposure was found to significantly impact the half-life of bacterial clearance. The analysis represents the first successful characterization of a concentration-effect relationship for bedaquiline. Single-dose Phase I studies investigating potential interactions between bedaquiline and efavirenz, nevirapine, ritonavir-boosted lopinavir, rifampicin and rifapentine were analyzed with a model-based approach. Substantial effects were detected in several cases and dose-adjustments mitigating the impact were suggested after simulations. The interaction effects of nevirapine and ritonavir-boosted lopinavir were also confirmed in patients with multidrug-resistant TB on long-term treatment combining the antiretrovirals and bedaquiline. Furthermore, the outcomes from model-based analysis were compared to results from conventional non-compartmental analysis in a simulation study. Non-compartmental analysis was found to consistently underpredict the interaction effect when most of the concentration-time profile was not observed, as commonly is the case for compounds with very long terminal half-life such as bedaquiline. To facilitate pooled analyses of individual patient data from multiple sources a structured development procedure was outlined and a fast diagnostic tool for extensions of the stochastic model components was developed. Pooled analyses of nevirapine and rifabutin pharmacokinetics were performed; the latter generating comprehensive dosing recommendations for combined administration of rifabutin and antiretroviral protease inhibitors. The work presented in this thesis demonstrates the usefulness of pharmacometric techniques to improve treatment of TB and especially contributes evidence to inform optimized dosing regimens of new and old anti-TB drugs in various clinical contexts.

KwaZulu-Natal has been ranked as having the fourth highest incidence of transmitted Multiple Drug Resistant-Tuberculosis (MDR-TB) in sub-Saharan Africa. Substantial literature exists indicating the permanent damage that MDR-TB medication has on hearing abilities. The purpose of this study was to describe the hearing function of adults on long term MDR-TB treatment from Murchison Hospital MDR-TB unit in the Ugu District in rural KwaZulu-Natal. The primary aim of the study was to review the possible changes in hearing function in a group of adults on long-term treatment for MDR-TB. Secondly, the study aimed to estimate the number of adults who may present with changes following MDR-TB treatment and establish if relationships exist between the audiological findings and factors such as age and gender. The design of the study was a retrospective comparative data review of 68 patient records, all of which underwent audiological investigations from the start of MDR-TB treatment over a five-month period. The study made use of descriptive and inferential statistics to analyse the data. Specific inferential statistical analysis included analysis of covariance as well as regression analysis. Results from the study showed changes in hearing function in Distortion Product Otoacoustic Emissions (DPOAEs) and Pure Tone Audiometry (PTA) results at all five audiological sessions and across a range of frequencies. 84% of the total sample presented with overall refer readings for DPOAEs and 98.53% of the group of adults presented with criteria indicative of ototoxic hearing loss, specifically a bilateral mild-profound sloping SNHL on clinical PTA results. In the total sample of patient records reviewed in this study, all 68 records showed a change in hearing function, be that changes in DPOAE function and/or changes in PTA thresholds, following long-term treatment for MDR-TB. Variations in the effects of gender and ear difference were minimal and non-significant in all results. Similar presentation, to ototoxic hearing loss, of other degenerative conditions exists; however these conditions were accounted for as exclusion criteria in this study. Therefore the only remaining cause of possible hearing deficit was that of ototoxicity. The study provided valuable data regarding hearing function in a population of adults on long-term MDR-TB treatment in South Africa. Furthermore, the study has highlighted the need for the establishment of standardised audiological monitoring programmes sensitive to ototoxic hearing loss, within the South African context where the incidence of Tuberculosis (TB) and MDR-TB is reportedly high.

A tuberculose (TB) embora passível de tratamento efetivo, ainda é um grave problema de saúde pública em diversos países, inclusive no Brasil. Nas últimas décadas houve progressos consistentes no controle da doença, porém o avanço da resistência bacilar ainda é um desafio a ser superado, já que os mecanismos da resistência são bastante complexos e não totalmente conhecidos, o que dificulta o desenvolvimento de testes de sensibilidade com elevada acurácia. O objetivo deste trabalho foi caracterizar as mutações gênicas de cepas de Mycobacterium tuberculosis de pacientes do Brasil e de Moçambique com doença resistente a drogas através do sequenciamento genômico total, além de descrever padrões de mutações obtidos por testes moleculares comerciais e comparar estes dados com resultados de testes fenotípicos. Estudo descritivo e transversal que incluiu 30 isolados (17 do Brasil e 13 de Moçambique), submetidos aos testes moleculares comerciais Genotype MTBDRplus®, Genotype MTBDRsl®, Xpert MTB/RIF® e teste fenotípico BACTEC MGIT 960 SIRE®. Todos os isolados também foram avaliados pelo sequenciamento genômico realizado pelo Illumina MiSeq Sequencing System® e submetidos a análise de mutações que conferem resistência às drogas contra TB utilizando o TB profiler online tool. A sensibilidade e especificidade do sequenciamento genômico para detecção de resistência a rifampicina foi de 87,5% e 92,3%, respectivamente. Além disso, o sequenciamento detectou a mutação (Val170Phe) no gene rpoB em dois isolados de M. tuberculosis de Moçambique. Esta mutação não é detectada pelos testes genotípicos comerciais. A sensibilidade do sequenciamento para a isoniazida foi de 95,6% e a especificidade de 100%. Para a estreptomicina, a sensibilidade foi de 85,7% e a especificidade de 93,3%. Para o etambutol, observamos sensibilidade de 100% e especificidade de 77,2%. As mutações mais frequentes associadas à resistência à rifampicina foram a Ser450Leu e a His445Tyr no gene rpoB. Em relação à isoniazida, predominou a mutação Ser315Thr no gene katG. O sequenciamento genômico, dado seu alto poder discriminatório, tem grande potencial de fornecer informações mais acuradas sobre mecanismo gênicos da resistência bacilar, possibilitando futuramente o aprimoramento de testes diagnósticos mais precisos. / Although there is an effective treatment for tuberculosis (TB), it is still a serious public health problem in several countries, including Brazil. In the last decades, there has been consistent progress in disease control, but the increasing number of disease caused by resistant strains is still a challenge to be overcome, since the mechanisms of resistance are quite complex and not fully known, which difficult the development of susceptibility tests with high accuracy. The aim of this work was to characterize gene mutations of Mycobacterium tuberculosis strains from Brazilian and Mozambican patients with drug-resistant disease through whole genome sequencing, as well as to describe patterns of mutations obtained by commercial molecular tests and to compare these data with results of phenotypic susceptibility tests. It was a cross-sectional study that included 30 isolates (17 from Brazil and 13 from Mozambique). Commercial molecular tests Genotype MTBDRplus(TM), Genotype MTBDRsl(TM), Xpert MTB / RIF(TM) and BACTEC MGIT 960 SIRE(TM) phenotypic test were performed for all isolates. All of them were also evaluated by whole genome sequencing performed by the Illumina MiSeq Sequencing System(TM) and submitted to analysis of mutations that confer drug resistance against TB using the TB profiler online tool. The sensitivity and specificity of whole genome sequencing for detection rifampicin resistance was 87.5% and 92.3%, respectively. Also, whole genome sequencing detected the mutation (Val170Phe) in the rpoB gene in two isolates of M. tuberculosis from Mozambique. This mutation is not detected by commercial genotypic tests. The sensitivity of the whole genome sequencing for isoniazid was 95.6%, and the specificity was 100%. For streptomycin, the sensitivity was 85.7%, and the specificity was 93.3%. For ethambutol, we observed a sensitivity of 100% and specificity of 77.2%. The most frequent mutations associated with rifampicin resistance were rpoB Ser450Leu and His445Tyr. About isoniazid, the katG Ser315Thr mutation was the most frequent. Whole genome sequencing, given its high discriminatory power, has great potential to provide more accurate information about the gene mechanisms of bacilli resistance, making possible the improvement of more accurate diagnostic tests in the future.