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

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)

No description available.

Tuberculosis remains a major public health issue globally, with an estimated 9.2 million new cases in 2006. A new threat to TB control is the emergence of drug resistant strains. These strains are harder to cure as standard anti-tuberculosis first line treatments are ineffective. Multi Drug Resistant Tuberculosis (MDR-TB) is defined as Mycobacterium tuberculosis that has developed resistance to at least rifampicin and isoniazid, and these strains now account for greater than 5% of worldwide cases. Mutations within the Rifampicin Resistance Determining Region (RRDR) of the rpoB gene are present in greater than 95% of strains that show rifampicin resistance by conventional drug susceptibility testing. As rifampicin mono resistance is extremely rare, and rifampicin resistance is usually associated with isoniaizd resistance, the RRDR region of the rpoB gene is a very useful surrogate marker for MDR-TB. Many molecular assays have been attempted based on this theory and have had varied levels of success. The three methods evaluated in this study are DNA sequencing of the rpoB, katG and inhA genes, the Genotype MTBDRplus line probe assay (Hain Lifesciences) and a novel method incorporating Real-Time PCR with High Resolution Melt analysis targeted at the RRDR using the Rotorgene 6000 (Corbett Lifesciences). The sensitivity for the detection of rifampicin resistance was far better using DNA sequencing or the commercially available line probe assay than detection by the Real-Time PCR method developed in this study.

The thesis entitled “Syntheses and Characterization of Novel Materials for Efficacious Anticancer Drug Delivery and Selective Sensing of Bioanalytes” encompasses the syntheses and characterization of various novel materials those are primarily used for efficacious pH-targeted chemotherapy, selective sensing and quantification of ATP inside a single living cell and also for specific sensing of female sex pheromone of certain agriculturally important pests. In recent era of cancer research, pH guided anticancer drug delivery is an emerging field by which not only the drug-sensitive, but also the drug-resistant cancer cell lines can be targeted efficiently. Scientists have paid lot of attentions to this area of research to design biocompatible, pH-responsive drug delivery vehicles, where most of the literatures are end up with complex, elaborated synthetic procedures and use of expensive chemicals. There are only a few reports in the literature on small molecule based drug delivery vehicles, which is not well explored. Herein some of the biocompatible, pH-sensitive lipid and short peptide sequences are synthesized in easy and short synthetic procedures and successfully tested for their efficacious anticancer drug delivery properties by various biophysical and biological techniques. A pH and reduction dual bio-responsive short peptide sequences are also generated in simple steps for the same cause. The formation of different nanostructures from the self-assembly of these short peptides is probed from high level of theoretical calculations and ultimately a well known chemotherapeutic drug, doxorubicin, has been delivered efficiently both to the drug-sensitive and drug-resistant cancer cell lines. In a particular case, in vivo study has also been performed to establish the drug delivery efficacy of those serum-stable vehicles that led to proficient reduction of tumour volume as compared to the free drug. On the other hand, a few of the molecules are synthesized and characterized by various analytical means for the selective sensing and quantification of adenosine 5’-triphosphate (ATP) inside a single living cell. Unique surface functionalized templates are also fabricated over MEMS devices for specific sensing of female sex pheromone of Helicoverpa armigera and Bactocera oleae pest in an agricultural field to detect the early pest infestation. Toward this end, an extensive study on the design, syntheses and characterization of different novel materials is presented below.

Magister Pharmaceuticae - MPharm / Many studies have investigated the pharmacokinetics of anti-tuberculosis drugs in patients infected with tuberculosis. However, little is known about the pharmacokinetics of the drugs that are used in the treatment of multi-drug resistant tuberculosis (MDRTB).Therefore, the objective of the present study was to investigate the steady state concentrations and the pharmacokinetics of ofloxacin, one of the drugs used in the treatment of MDR-TB in patients infected with MDR-TB and patients with MDR-TB co-infected with HIV Plasma samples were drawn at different times over 24 hours after ofloxacin oral administration. For the determination of ofloxacin plasma concentrations, the liquid chromatography coupled with mass spectrometry analysis method was used.The method was validated over a concentration range of 0.1-10 μg/ml. The lower limit of ofloxacin detection was 0.05μg/ml, while the lower limit of quantification was 0.1μg/ml. The response was linear over the range used with a mean recovery of 97.6%. Ofloxacin peak was well separated at a retention time of 9.6 minutes.The pharmacokinetic parameters obtained were presented as mean ± standard deviation(SD). The peak concentration of ofloxacin (Cmax) was 4.71± 2.27 μg/ml occurred at Tmax 3±1.29 hours after ofloxacin oral administration. The mean (±SD) for the area under the concentration-time curve (AUC0-24) and the area under the concentration-time curve(AUC0-∞) were 68.8±42.61 μg/ml.hr and 91.93±76.86 μg/ml.hr, respectively. Ofloxacin distributed widely with a mean (±SD) volume of distribution (Vd) 2.77±1.16 L/kg and it was eliminated with a mean (±SD) total clearance rate of 0.27±0.25 L/hr/kg. Ofloxacin mean (±SD) half-life was 9.55± 4.69 hours and mean (±SD) of the mean residence time (MRT) was 1512± 6.59 hours.In summary, compared with the previous findings in the literature, ofloxacin pharmacokinetic was altered in MDR-TB patients with or without HIV co-infection.The AUC and Cmax were reduced, while the half-life and the time to reach the peak concentration were prolonged. This suggests that, both the rate and the extent of ofloxacin absorption were decreased. Furthermore, ofloxacin was highly eliminated in patients, which may be related to the altered liver function in this group of patients.Further studies investigating the effect of HIV, liver and kidney dysfunctions on ofloxacin pharmacokinetics are recommended in large number of patients infected with MDR-TB.in addition to the therapeutic drug monitoring to maintain the desired concentration of ofloxacin in the patients.

No description available.

The purpose of the study was to develop best practice guidelines to prevent permanent hearing loss associated with the management of multi-drug resistant tuberculosis (MDR-TB) through raised awareness and monitoring. The Human Immunodeficiency Virus (HIV) and MDR-TB are global public health problems requiring urgent scale-up of treatment services. Irreversible sensorineural hearing loss (SNHL) is one of the adverse drug reactions of the current World Health Organization (WHO) recommended MDR-TB chemotherapy fuelling another public health problem, that disabling hearing loss, which is the second highest contributor of Years Lived with Disability (YLD) according to the World Health Report (2003). Expansion of MDR-TB treatment threatens to increase incidence of SNHL unless there is urgent implementation of intervention towards preservation of hearing for patients on treatment. This empirical study determined and documented the incidence of SNHL in HIV positive and HIV negative patients on MDR-TB treatment, the risk factors for SNHL, from the time treatment initiation to SNHL. Based on the findings, developed and improved the understanding of best practice guidelines for monitoring and prevention of MDR-TB treatment-related SNHL. The empirical study recruited a cohort of 173 patients with normal hearing status, after diagnosis with MDR-TB and enrolled on MDR-TB therapy over thirteen month period. Patients in the cohort received monthly hearing sensitivity testing during the intensive MDR-TB therapy when injectable aminoglycoside antibiotics are part of the treatment regimen. The three study endpoints included completion of the eight-month intensive treatment phase without developing hearing loss, development incident hearing loss or loss to follow up. Data was analysed using STATA statistical software and summarised using frequencies, means, proportions, and rates. The study documented incidence of SNHL, time to hearing loss and risk factors for hearing loss. Recommendations to prevent and monitor hearing loss are made based on the the study findings. / Health Studies / D. Litt. et Phil. (Health Studies)

Philosophiae Doctor - PhD / Tuberculosis is a bacterial infection which is a major cause of death worldwide. TB is a curable disease, however the bacterium can become resistant to the first line treatment against the disease. This leads to a disease called drug resistant TB that is difficult and expensive to treat. It is well-known that TB disease thrives in communities in overcrowded environments with poor ventilation, weak nutrition, inadequate or inaccessible medical care, etc, such as in some prisons or some refugee camps. In particular, the World Health Organization discovered that a number of prisoners come from socio-economic disadvantaged population where the burden of TB disease may be already high and access to medical care may be limited. In this dissertation we propose compartmental models of systems of differential equations to describe the population dynamics of TB disease under conditions of crowding. Such models can be used to make quantitative projections of TB prevalence and to measure the effect of interventions. Indeed we apply these models to specific regions and for specific purposes. The models are more widely applicable, however in this dissertation we calibrate and apply the models to prison populations.