Deciphering the mechanisms of Isoniazid reisistance to mycobacterium tuberculosis

Nagel, Simone (2018-12)

Thesis (MSc)--Stellenbosch University, 2018.

Thesis

ENGLISH ABSTRACT: Isoniazid (INH) serves as the backbone of combined anti-tuberculosis therapy. However, the effectivity of this drug has been compromised due to increasing resistance to it in Mycobacterium tuberculosis (M. tuberculosis). This resistance arises through spontaneous mutations in certain genes/genomic regions of M. tuberculosis. The current drug resistance testing algorithm for INH consists of a genotypic MTBDRplus line-probe assay (LPA), which reports resistance to INH based on mutations in katG gene and inhA promoter, and a phenotypic drug susceptibility test (DST), used to confirm INH susceptibility in rifampicin resistant cases on LPA. However, the algorithm is problematic as there are discrepancies between mutations causing INH resistance detected on LPA and phenotypic DST. This may result in incorrect diagnoses and the prescription of incorrect drug treatment regimens. The current study investigates 398 clinical isolates obtained from NHLS Port Elizabeth with discrepant status between the LPA and the phenotypic DST. The isolates were investigated by means of a series of standardized methods such as Sanger sequencing, Whole Genome Sequencing and spoligotyping. This was done to determine if the reasons for the discrepancies between the LPA and phenotypic DST were due to novel mechanisms of resistance to INH. Genotyping via spoligotyping indicated a prevalence of the Beijing genotype in this cohort, as well as mixed infections and novel spoligotype patterns that don’t resemble any known lineage or family markers. Sanger sequencing revealed canonical mutations that were missed by the LPA as well as mutations occurring elsewhere in the katG gene, indicating there are blind spots in the LPA that may have detrimental effects on patient response to treatment. Per the whole genome sequencing results, novel mechanisms were associated with spontaneous non-canonical single nucleotide polymorphisms in M. tuberculosis. These polymorphisms occurred in genetic regions outside the LPA region of hybridisation and conferred varying levels of resistance to INH. The clinical isolates in this dataset consisted of 29.2% low-level resisistant (minimum inhibitory concentration between 0.1-1.0 µg/L to INH) and 61% highlevel resistant (minimum inhibitory concentration of > 1µg/ml to INH) isolates.The degree of resistance to INH is relevant, as it informs prescription of treatment regimens. It is recommended that cases of low-level resistance to INH be treated with the short course Bangladesh regimen consisting of high dose INH, whereas high-level resistance to INH requires the removal of this antibiotic from the regimen.Homologous recombineering was also done to functionally confirm the role these novel mechanisms have on INH resistance, and the subsequent level of resistance caused. The results obtained in this study will ultimately be used to improve current diagnostic methods for detection of INH resistance.

AFRIKAANSE OPSOMMING: Isoniazid (INH) dien as die ruggraat van gekombineerde anti-tuberkulose terapie. Tans is die effektiewiteit van hierdie antibioika in gedrang as gevolg van toenemende weerstand daarteen in Mycobacterium tuberculosis (M. tuberculosis). Hierdie weerstand ontstaan deur spontane mutasies in sekere gene/genotipiese streke van M. tuberculosis. Die huidige middel weerstand toets algoritme vir INH bestaan uit 'n genotipiese MTBDRplus (LPA) toets, wat INH weerstandigheid rapporteer gebaseer op mutasies in katG gene en inhA promotor, en 'n fenotipiese middel vatbaarheid toets (DST), gebruik om INH vatbaarheid te bevestig in rifampisien weerstandige gevalle op LPA. Die algoritme is egter problematies omdat daar teenstrydighede is tussen mutasies wat INH weerstand veroorsaak op LPA en fenotipiese DST. Dit kan lei tot verkeerde diagnoses en die voorskrif van verkeerde behandeling vir tuberkulose. Die huidige studie ondersoek 398 kliniese isolate van NHLS Port Elizabeth met teenstrydige weerstandigheids status tussen die LPA en die fenotipiese DST. Die isolate is ondersoek deur middel van gestandardiseerde metodes soos Sanger volgordebepaling, hele genoom volgordebepaling (WGS) en spoligotipering. Dit was gedoen om die redes vir die teenstrydighede tussen die LPA en fenotipiese DST te bepaal. Spoligotipering het aangedui dat die meeste isolate in die studie aan die Beijing genotipe behoort, sowel as gevalle van gemengde infeksies en onbekende patrone. Sanger volgordebepaling het klassieke mutasies wat deur die LPA gemis was asook mutasies wat elders in die katG geen,voorkomgediagnoseer, wat aandui dat daar blinde kolle is in die LPA. Die blinde kolle mag ‘n nadelige effek hê op die behandeling van die pasient. Volgens die hele genoom volgordebepaling resultate, het die unieke meganismes van weerstand gepaardgegaan met punt mutasies in M. tuberculosis. Hierdie mutasies het voorgekom in genetiese streke buite die wat ondersoek word deur die LPA en verleen wisselende vlakke van weerstand teen INH. Die kliniese isolate in hierdie studie bestaan uit 29.2% lae-vlak weerstandige (minimum inhiberende konsentrasie tussen 0.1-1.0 μg/L tot INH) en 61% hoëvlak weerstandige (minimum inhiberende konsentrasie van > 1µg/ml tot INH) isolate. Die vlak van weerstand teen INH is belangrik, aangesien dit die voorskrif van antibiotika vir behandeling beinvloed. Ons beveel aan dat gevalle van lae-vlak weerstand tot INH behandel word met die kort kursus Bangladesh antibiotika kursus wat bestaan uit hoë dosis INH, terwyl hoëvlak weerstand teen INH vereis dat INH uitgesluit word vir behandeling. Homologosiese rekombinasie is gedoen om funksioneel te bevestig dat die rare mutasies ‘n rol het in INH weerstand. Die resultate verkry in hierdie studie sal uiteindelik gebruik word om huidige diagnostiese metodes vir die opsporing van INH weerstand te verbeter.

Please refer to this item in SUNScholar by using the following persistent URL: http://hdl.handle.net/10019.1/105166
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