Browsing by Author "Kayigire, Xavier A"
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- ItemSputum derived biomarkers of anti-tuberculosis drug activity in early bactericidal activity (EBA) studies.(Stellenbosch : Stellenbosch University, 2017-03) Kayigire, Xavier A; Diacon, Andreas H; Stellenbosch University. Faculty of Medicine and Health Sciences. Dept. of Biomedical Sciences: Molecular Biology and Human Genetics.ENGLISH ABSTRACT: Sputum sample is a crucial material to diagnose tuberculosis (TB), the resistance to different drugs and the assessment of new drug effectiveness in clinical trials. The first step to evaluate the efficacy of a new anti-TB drug is the determination of its early bactericidal activity (EBA) which is the decline of bacterial load per milliliter of sputum per day on solid agar plates during the first two weeks of treatment. The count of colony forming units (CFU) of Mycobacterium tuberculosis (Mtb) on agar plates and the time to positivity (TTP) in liquid media are two parameters currently used in EBA studies. These two methods are time consuming, require highly skilled staff, an expensive infrastructure and are prone to contamination. Therefore, new methods which are more sensitive, specific, fast and automated are urgently needed. In this study, we compared the EBA determined by CFU and TTP to the EBA determined by Xpert MTB/RIF assay (Xpert). Culture methods proved to be superior to Xpert to assess the two weeks EBA of different compounds tested. Sputum samples collected from TB patients on treatment contain a mixture of dead, injured and viable cells of Mtb. We hypothesized that the poor performance of Xpert in the determination of the EBA was due to the presence of DNA from bacteria unable to grow which was amplified together with DNA from viable ones. To overcome this problem, before performing Xpert, we have pre-treated pan-susceptible and extensively drug resistance isolates subjected to prior standard drug susceptibility testing with propidium monoazide (PMA), a reagent that penetrates only non viable cell, binds to its DNA and prevents its amplification. Then, we applied PMA pre-treatment protocol to clinical isolates from TB patients under standard TB treatment. The combination of Xpert and PMA improved the specificity to detect viable Mtb compared to Xpert alone. This improvement was statistically significant in pan-susceptible isolates incubated with isoniazid (INH) and ethambutol (EMB) and extensively drug resistant isolates incubated with EMB. Unfortunately, the effect of PMA was not statistically significant in clinical isolates from TB patients on standard treatment. Due to these conflicting results, we could not recommend the use of Xpert-PMA combination to quantify viable Mtb in preference of culture media. Dormant mycobacteria are believed to be the result of internal and external stress including the effects of anti-TB drugs on viable mycobacteria and they are assumed to be the reason for the prolongation of TB treatment up to 6 months. In this work, the activity of SQ109, an investigational drug, Rifampicin (RMP) an already established anti-TB drug and their combination (SQ109/RMP) was assessed on both, replicating and non-replicating forms of Mtb, by using a combination of Auramine O/Nile Red staining and confocal microscopy. We found that SQ109 and RMP monotherapy increase the number of non-replicating Mtb while SQ109/RMP combined prevents this increase. These findings show that the pressure of SQ109 alone causes Mtb to switch to dormancy and once combined with RMP, SQ109 enhances the sterilizing activity of RMP. Monotherapy is the underlying cause of the emergence of drug resistance. We evaluated the change in proportion of RMP mutants in patients under RMP monotherapy for two weeks from baseline to day 14. We have applied statistical modelling to estimate when a patient kept on RMP monotherapy beyond two weeks would become clinically resistant. We found that RMP monotherapy beyond two weeks will induce clinical relevant resistance only after 30 days of treatment. This indicates that during TB treatment RMP resistance develops gradually due to pharmacodynamic and pharmacokinetic factors as it was previously reported. In this work, we showed that Xpert, the combination of Xpert with PMA and staining of sputum smears with Nile Red/Auramine O are promising biomarker candidates to determine the EBA of novel anti-TB drugs. Furthermore, we demonstrated that if RMP was the only drug used for TB treatment, a resistance against RMP would become clinically relevant after 30 days.