Sputum derived biomarkers of anti-tuberculosis drug activity in early bactericidal activity (EBA) studies.

Thesis (PhD)--Stellenbosch University, 2017.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.AFRIKAANSE OPSOMMING: Sputum monsters is belangrike materiaal vir die diagnose van TB, om die weerstandigheid van sekere anti-TB middels te bepaal en om die effektiwiteit van nuwe middels te bepaal in geneesmiddel evaluering. Die eerste stap in die evalueringsproses van ‘n nuwe anti-TB middel is om die vroeë bakerisidiese aktiwiteit (EBA) te bepaal. Dit is ‘n bepaling van die bakteriële lading per milliliter sputum per dag op soliede agar plate gedurende die eerste twee weke van TB behandeling. Die bepaling van kolonie-vormende eenhede (CFU) van M.tb op agar plate en die tyd-tot-positiwiteit (TTP) in vloeibare groeimedia is twee metodes wat tans wyd in gebruik is by EBA studies. Hierdie twee metodes is tydrowend, vereis ‘n groot mate van tegniese vernuf, benodig duur infrastruktuur en is geneig tot kontaminering. Daarom word nuwe metodes wat sensitief, spesifiek, vinnig en geoutomatiseerd is benodig. In hierdie studie vergelyk ons die EBA bepalings deur middel van CFU en TTP met EBA bepalings deur middel van Xpert MTB/RIF ontledings. Metodes deur kultuur tegnieke word bewys as superieur teenoor Xpert vir die bepaling van die twee-week EBA vir verskillende middels. Sputum monsters wat versamel is vanaf TB pasiënte wat op behandeling is, bevat ‘n mengsel van dooie, beskadigde en lewensvatbare M.tb selle. Ons hipotiseer dat die swak resultate van Xpert gedurende EBA bepalings as gevolg van die teenwoordigheid van DNA vanaf bakterië is wat nie lewensvatbaar is nie en wat dan saam met lewensvatbare bakterië geamplifiseer word. Om hierdie probleem te oorkom, en voordat die Xpert tegniek gebruik word, het ons pan-vatbare en uiters middelweerstandige M.tb isolate vooraf behandel met propidium monoazied (PMA) wat nie-lewensvatbare selle binnedring en aan hul DNA bind om sodoende amplifikasie te verhoed. Ons het dan die PMA voorafbehandelingsprotokol toegepas op kliniese isolate van TB pasiënte wat op standaard anti-TB behandeling is. Die kombinasie van Xpert en PMA het die spesifisiteit verhoog om lewensvatbare bakterieë waar te neem teenoor die Xpert metode alleen. Hierdie verbetering was statisties betekenisvol in pan-vatbare isolate wat behandel is met isoniazied (INH) en ethambutol (EMB) en uiters middelweerstandige isolate wat behandel is met EMB. Ogelukkig was die effek van PMA nie statisties betekenisvol vir kliniese isolate vanaf TB pasiënte op standaard behandeling nie. As gevolg van hierdie teenstrydige resultate kon ons nie die gebruik van die Xpert-PMA kombinasie vir die kwantifisering van lewensvatbare M.tb bo die gebruik van kultuurmedia bepalings aanbeveel nie. Daar word aanvaar dat dormante mycobacteria die gevolg is van interne en eksterne stres, insluitend die effek van anti-TB middels, op lewensvatbare mycobacteria is en dat hierdie effekte verantwoordelik is vir die verlengde behandelings periode van 6 maande vir TB infeksie. In hierdie navovorsingswerk word die aktiwiteit van SQ109, ‘n middel nog onder evaluering Rifampisien (RMP), ‘n gevestigde anti-TB middel, en hul kombinasie (SQ109/RMP) geëvalueer teen repliserende en nie-repliserende vorme van M.tb deur gebruik te maak van ‘n kombinasie van Auramine O/Nylrooi kleuring en konfokale mikroskopie. Ons het gevind dat SQ109 en RMP monoterapie verhoog die aantal nie-repliserende M.tb terwyl SQ109/RMP kombinasie hierdie toename verhoed. Hierdie bevindings toon dat SQ109 stres alleen lei daartoe dat M.tb oorskakel na ‘n sluimertoestand en, gekombineer met RMP, verhoog dit die steriliserende effek van RMP. Monoterapie is die onderliggende oorsaak van middelweerstandigheid. Ons het die verandering in die verhouding van RMP mutante in pasiënte op RMP terapie vir twee weke geëvalueer. Ons het ‘n statistieke model gebruik om te bepaal of ‘n pasiënt wat vir twee weke op RMP monoterapie gehou word, kliniese weerstandigheid ontwikkel. Ons het gevind dat RMP monoterapie vir meer as twee weke lei tot klinies relevante weerstandigheid eers na 30 dae behandeling. Dit dui daarop dat gedurende TB behandeling met RMP weerstandigheid stelselmatig ontwikkel as gevolg van farmadinamiese en farmakinetiese faktore soos voorheen aangedui. Hierdie navorsingk bewys dat Xpert, die kombinasie van Xpert en PMA en die kleuring van sputumsmere met Nylrooi/Auramine O belowende biomerker kandidate is om die EBA van nuwe anti-TB middels te bepaal. Ons wys ook dat indien RMP die enigste anti-TB middel is wat gebruik is vir behandeling, weerstandigheid teen RMP klinies relevant word na 30 dae behandeling

Suitability of Xpert MTB/RIF and Genotype MTBDRplus for Patient Selection for a Tuberculosis Clinical Trial ▿

Participation criteria for clinical trials in pulmonary tuberculosis commonly include confirmation of sputum positive for mycobacteria and an indication of drug susceptibility before treatment is initiated. We investigated the suitability of two novel sputum-based nucleic acid amplification methods for patient selection in a recent early bactericidal activity study. Spontaneously expectorated sputum samples of 140 consecutive pulmonary tuberculosis patients were examined with direct fluorescence microscopy, Genotype MTBDRplus assay (MTBDR), Xpert MTB/RIF assay (Xpert), and liquid mycobacterial culture. The methods detected mycobacteria or mycobacterial DNA in 96.8%, 90.5%, 92.9%, and 92.1% of samples, respectively. MTBDR, Xpert, and liquid culture were 100% concordant for detection of resistance to rifampin. Sensitivity and specificity of MTBDR for detection of isoniazid resistance were 83.3% and 100%, respectively. For quantification of mycobacterial sputum load, we found a correlation between Xpert DNA amplification cycle thresholds, time to positivity, and microscopy smear grade. The best correlation was found between Xpert and time to positivity (r = 0.54), which were both correlated with smear microscopy with r values equal to −0.40 and −0.48, respectively. We conclude that MTBDR and Xpert are suitable screening tools for determining rifampin resistance in sputum microscopy smear-positive patients before participation in tuberculosis trials. Xpert should be further explored as a surrogate measurement for sputum mycobacterial load

Direct comparison of Xpert MTB/RIF assay with liquid and solid mycobacterial culture for quantification of early bactericidal activity

The early bactericidal activity of antituberculosis agents is usually determined by measuring the reduction of the sputum mycobacterial load over time on solid agar medium or in liquid culture. This study investigated the value of a quantitative PCR assay for early bactericidal activity determination. Groups of 15 patients were treated with 6 different antituberculosis agents or regimens. Patients collected sputum for 16 h overnight at baseline and at days 7 and 14 after treatment initiation. We determined the sputum bacterial load by CFU counting (log CFU/ml sputum, reported as mean ± standard deviation [SD]), time to culture positivity (TTP, in hours [mean ± SD]) in liquid culture, and Xpert MTB/RIF cycle thresholds (TC, n [mean ± SD]). The ability to discriminate treatment effects between groups was analyzed with one-way analysis of variance (ANOVA). All measurements showed a decrease in bacterial load from mean baseline (log CFU, 5.72 ± 1.00; TTP, 116.0 ± 47.6; TC, 19.3 ± 3.88) to day 7 (log CFU, −0.26 ± 1.23, P = 0.2112; TTP, 35.5 ± 59.3, P = 0.0002; TC, 0.55 ± 3.07, P = 0.6030) and day 14 (log CFU, −0.55 ± 1.24, P = 0.0006; TTP, 54.8 ± 86.8, P &lt; 0.0001; TC, 2.06 ± 4.37, P = 0.0020). The best discrimination between group effects was found with TTP at day 7 and day 14 (F = 9.012, P &lt; 0.0001, and F = 11.580, P &lt; 0.0001), followed by log CFU (F = 4.135, P = 0.0024, and F = 7.277, P &lt; 0.0001). TC was not significantly discriminative (F = 1.995, P = 0.091, and F = 1.203, P = 0.316, respectively). Culture-based methods are superior to PCR for the quantification of early antituberculosis treatment effects in sputum. </p

Does discovery of differentially culturable M tuberculosis really demand a new treatment paradigm? Longitudinal analysis of DNA clearance from sputum.

BackgroundAccording to the traditional tuberculosis (TB) treatment paradigm, the initial doses of treatment rapidly kill most Mycobacterium tuberculosis (Mtb) bacilli in sputum, yet many more months of daily treatment are required to eliminate a small, residual subpopulation of drug-tolerant bacilli. This paradigm has recently been challenged following the discovery that up to 90% of Mtb bacilli in sputum are culturable only with growth-factor supplementation. These "differentially culturable" bacilli are hypothesized to be more drug-tolerant than routinely culturable bacilli. This hypothesis implies an alternative paradigm in which TB treatment does not rapidly reduce the total Mtb population but only the small, routinely culturable subpopulation. To evaluate these competing paradigms, we developed a culture-independent method for quantifying the viable fraction of Mtb bacilli in sputum during treatment.MethodsWe used GeneXpert MTB/RIF to quantify Mtb DNA in sputa collected longitudinally from Ugandan adults taking standard 4-drug treatment for drug-susceptible pulmonary TB. We modeled GeneXpert cycle thresholds over time using nonlinear mixed-effects regression. We adjusted these models for clearance of DNA from killed-but-not-yet-degraded bacilli, assuming clearance half-lives ranging from 0 to 1.25&nbsp;days. We used a convolution integral to quantify DNA from viable bacilli only, and converted cycle thresholds to Mtb genomic equivalents. We replicated our results in a South African cohort.ResultsWe enrolled 41&nbsp;TB patients in Uganda. Assuming a DNA-clearance half-life of 0&nbsp;days, genomic equivalents of viable sputum bacilli decreased by 0.22 log/day until 8.8&nbsp;days, then by 0.07 log/day afterwards. Assuming a DNA-clearance half-life of 1.25&nbsp;days, genomic equivalents of viable bacilli decreased by 0.36 log/day until 5.0&nbsp;days, then by 0.06 log/day afterwards. By day 7, viable Mtb had decreased by 97.2-98.8%. We found similar results for 19&nbsp;TB patients in South Africa.DiscussionUsing a culture-independent method, we found that TB treatment rapidly eliminates most viable Mtb in sputum. These findings are incompatible with the hypothesis that differentially culturable bacilli are drug-tolerant.ConclusionsA culture-independent method for measuring viable Mtb in sputum during treatment corroborates the traditional TB treatment paradigm in which a rapid bactericidal phase precedes slow, elimination of a small, residual bacillary subpopulation

Does discovery of differentially culturable M tuberculosis really demand a new treatment paradigm? Longitudinal analysis of DNA clearance from sputum

Abstract Background According to the traditional tuberculosis (TB) treatment paradigm, the initial doses of treatment rapidly kill most Mycobacterium tuberculosis (Mtb) bacilli in sputum, yet many more months of daily treatment are required to eliminate a small, residual subpopulation of drug-tolerant bacilli. This paradigm has recently been challenged following the discovery that up to 90% of Mtb bacilli in sputum are culturable only with growth-factor supplementation. These “differentially culturable” bacilli are hypothesized to be more drug-tolerant than routinely culturable bacilli. This hypothesis implies an alternative paradigm in which TB treatment does not rapidly reduce the total Mtb population but only the small, routinely culturable subpopulation. To evaluate these competing paradigms, we developed a culture-independent method for quantifying the viable fraction of Mtb bacilli in sputum during treatment. Methods We used GeneXpert MTB/RIF to quantify Mtb DNA in sputa collected longitudinally from Ugandan adults taking standard 4-drug treatment for drug-susceptible pulmonary TB. We modeled GeneXpert cycle thresholds over time using nonlinear mixed-effects regression. We adjusted these models for clearance of DNA from killed-but-not-yet-degraded bacilli, assuming clearance half-lives ranging from 0 to 1.25 days. We used a convolution integral to quantify DNA from viable bacilli only, and converted cycle thresholds to Mtb genomic equivalents. We replicated our results in a South African cohort. Results We enrolled 41 TB patients in Uganda. Assuming a DNA-clearance half-life of 0 days, genomic equivalents of viable sputum bacilli decreased by 0.22 log/day until 8.8 days, then by 0.07 log/day afterwards. Assuming a DNA-clearance half-life of 1.25 days, genomic equivalents of viable bacilli decreased by 0.36 log/day until 5.0 days, then by 0.06 log/day afterwards. By day 7, viable Mtb had decreased by 97.2–98.8%. We found similar results for 19 TB patients in South Africa. Discussion Using a culture-independent method, we found that TB treatment rapidly eliminates most viable Mtb in sputum. These findings are incompatible with the hypothesis that differentially culturable bacilli are drug-tolerant. Conclusions A culture-independent method for measuring viable Mtb in sputum during treatment corroborates the traditional TB treatment paradigm in which a rapid bactericidal phase precedes slow, elimination of a small, residual bacillary subpopulation