A landscape of genomic alterations at the root of a near-untreatable tuberculosis epidemic

Abstract Background Atypical Beijing genotype Mycobacterium tuberculosis strains are widespread in South Africa and have acquired resistance to up to 13 drugs on multiple occasions. It is puzzling that these strains have retained fitness and transmissibility despite the potential fitness cost associated with drug resistance mutations. Methods We conducted Illumina sequencing of 211 Beijing genotype M. tuberculosis isolates to facilitate the detection of genomic features that may promote acquisition of drug resistance and restore fitness in highly resistant atypical Beijing forms. Phylogenetic and comparative genomic analysis was done to determine changes that are unique to the resistant strains that also transmit well. Minimum inhibitory concentration (MIC) determination for streptomycin and bedaquiline was done for a limited number of isolates to demonstrate a difference in MIC between isolates with and without certain variants. Results Phylogenetic analysis confirmed that two clades of atypical Beijing strains have independently developed resistance to virtually all the potent drugs included in standard (pre-bedaquiline) drug-resistant TB treatment regimens. We show that undetected drug resistance in a progenitor strain was likely instrumental in this resistance acquisition. In this cohort, ethionamide (ethA A381P) resistance would be missed in first-line drug-susceptible isolates, and streptomycin (gidB L79S) resistance may be missed due to an MIC close to the critical concentration. Subsequent inadequate treatment historically led to amplification of resistance and facilitated spread of the strains. Bedaquiline resistance was found in a small number of isolates, despite lack of exposure to the drug. The highly resistant clades also carry inhA promoter mutations, which arose after ethA and katG mutations. In these isolates, inhA promoter mutations do not alter drug resistance, suggesting a possible alternative role. Conclusion The presence of the ethA mutation in otherwise susceptible isolates from ethionamide-naïve patients demonstrates that known exposure is not an adequate indicator of drug susceptibility. Similarly, it is demonstrated that bedaquiline resistance can occur without exposure to the drug. Inappropriate treatment regimens, due to missed resistance, leads to amplification of resistance, and transmission. We put these results into the context of current WHO treatment regimens, underscoring the risks of treatment without knowledge of the full drug resistance profile

Antimycobacterial Activity, Synergism, and Mechanism of Action Evaluation of Novel Polycyclic Amines against Mycobacterium tuberculosis

Mycobacterium tuberculosis has developed extensive resistance to numerous antimycobacterial agents used in the treatment of tuberculosis. Insufficient intracellular accumulation of active moieties allows for selective survival of mycobacteria with drug resistance mutations and accordingly promotes the development of microbial drug resistance. Discovery of compounds with new mechanisms of action and physicochemical properties that promote intracellular accumulation, or compounds that act synergistically with other antimycobacterial drugs, has the potential to reduce and prevent further drug resistance. To this end, antimycobacterial activity, mechanism of action, and synergism in combination therapy were investigated for a series of polycyclic amine derivatives. Compound selection was based on the presence of moieties with possible antimycobacterial activity, the inclusion of bulky lipophilic carriers to promote intracellular accumulation, and previously demonstrated bioactivity that potentially support inhibition of efflux pump activity. The most potent antimycobacterial demonstrated a minimum inhibitory concentration (MIC99) of 9.6 μM against Mycobacterium tuberculosis H37Rv. Genotoxicity and inhibition of the cytochrome bc1 respiratory complex were excluded as mechanisms of action for all compounds. Inhibition of cell wall synthesis was identified as a likely mechanism of action for the two most active compounds (14 and 15). Compounds 5 and 6 demonstrated synergistic activity with the known Rv1258c efflux pump substrate, spectinomycin, pointing to possible efflux pump inhibition. For this series, the nature of the side chain, rather than the type of polycyclic carrier, seems to play a determining role in the antimycobacterial activity and cytotoxicity of the compounds. Contrariwise, the nature of the polycyclic carrier, particularly the azapentacycloundecane cage, appears to promote synergistic activity. Results point to the possibility of combining an azapentacycloundecane carrier with a side chain that promotes antimycobacterial activity to develop dual acting molecules for the treatment of Mycobacterium tuberculosis

Analytical evaluation of thirty-two severe acute respiratory syndrome 2 lateral flow antigen tests demonstrates sensitivity remains with the SARS-CoV-2 Gamma lineage.

Background: The emergence of variants of concern (VOCs) requires an ongoing assessment of the performance of antigen lateral flow tests (Ag-RDTs). The limit of detection (LOD) of 32 Ag-RDTs was evaluated using the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) Gamma variant. Methods: Ag-RDTs were performed according to the manufacturer’s instructions with a clinical isolate of the Gamma variant. Results: Twenty-eight of the 32 Ag-RDTs exceeded the World Health Organization criteria. Conclusions: This comprehensive analytical evaluation of Ag-RDTs demonstrated that the test performance was maintained with Gamma VOC

Diagnostic performance of GENEDIA W and ActiveXpress+ COVID-19 antigens tests among symptomatic individuals in Peru and The United Kingdom

Objectives: In order to generate independent performance data regarding accuracy of COVID-19 antigen-based rapid diagnostic tests (Ag-RDTs), prospective diagnostic evaluation studies across multiple sites are required to evaluate their performance in different clinical settings. This report describes the clinical evaluation the GENEDIA W COVID-19 Ag Device (Green Cross Medical Science Corp., Chungbuk, Korea) and the ActiveXpress+ COVID-19 Complete Testing Kit (Edinburgh Genetics Ltd, UK), in two testing sites Peru and the United Kingdom. Methods: Nasopharyngeal swabs collected from 456 symptomatic patients at primary points of care in Lima, Peru and 610 symptomatic participants at a COVID-19 Drive-Through testing site in Liverpool, England were analyzed by Ag-RDT and compared to RT-PCR. Analytical evaluation of both Ag-RDTs was assessed using serial dilutions of direct culture supernatant of a clinical SARS-CoV-2 isolate from the B.1.1.7 lineage. Results: For GENEDIA brand, the values of overall sensitivity and specificity were 60.4% [95% CI 52.4–67.9%], and 99.2% [95% CI 97.6–99.7%] respectively; and for Active Xpress+ the overall values of sensitivity and specificity were 66.2% [95% CI 54.0–76.5%], and 99.6% [95% CI 97.9–99.9%] respectively. The analytical limit of detection was determined at 5.0 x 102 pfu/ml what equals to approximately 1.0 x 104 gcn/ml for both Ag-RDTs. The UK cohort had lower median Ct values compared to that of Peru during both evaluations. When split by Ct, both Ag-RDTs had optimum sensitivities at Ct<20 (in Peru; 95% [95% CI 76.4–99.1%] and 100.0% [95% CI 74.1–100.0%] and in the UK; 59.2% [95% CI 44.2–73.0%] and 100.0% [95% CI 15.8–100.0%], for the GENDIA and the ActiveXpress+, respectively). Conclusions: Whilst the overall clinical sensitivity of the Genedia did not meet WHO minimum performance requirements for rapid immunoassays in either cohort, the ActiveXpress+ did so for the small UK cohort. This study illustrates comparative performance of Ag-RDTs across two global settings and considers the different approaches in evaluation methods

Multicenter Diagnostic Evaluation of a Novel Coronavirus Antigen Lateral Flow Test among Symptomatic Individuals in Brazil and the United Kingdom

The COVID-19 pandemic has led to the commercialization of many antigen-based rapid diagnostic tests (Ag-RDTs), requiring independent evaluations. This report describes the clinical evaluation of the Novel Coronavirus 2019-nCoV Antigen Test (Colloidal Gold) (Beijing Hotgen Biotech Co., Ltd.), at two sites within Brazil and one in the United Kingdom. The collected samples (446 nasal swabs from Brazil and 246 nasopharyngeal samples from the UK) were analyzed by the Ag-RDT and compared to reverse transcription-quantitative PCR (RT-qPCR). Analytical evaluation of the Ag-RDT was performed using direct culture supernatants of SARS-CoV-2 strains from the wild-type (B.1), Alpha (B.1.1.7), Delta (B.1.617.2), Gamma (P.1), and Omicron (B.1.1.529) lineages. An overall sensitivity and specificity of 88.2% (95% confidence interval [CI], 81.3 to 93.3) and 100.0% (95% CI, 99.1 to 100.0), respectively, were obtained for the Brazilian and UK cohorts. The analytical limit of detection was determined as 1.0 × 103 PFU/mL (Alpha), 2.5 × 102 PFU/mL (Delta), 2.5 × 103 PFU/mL (Gamma), and 1.0 × 103 PFU/mL (Omicron), giving a viral copy equivalent of approximately 2.1 × 104 copies/mL, 9.0 × 105 copies/mL, 1.7 × 106 copies/mL, and 1.8 × 105 copies/mL for the Ag-RDT, respectively. Overall, while a higher sensitivity was claimed by the manufacturers than that found in this study, this evaluation finds that the Ag-RDT meets the WHO minimum performance requirements for sensitivity and specificity of COVID-19 Ag-RDTs. This study illustrates the comparative performance of the Hotgen Ag-RDT across two global settings and considers the different approaches in evaluation methods

Multicenter Diagnostic Evaluation of OnSite COVID-19 Rapid Test (CTK Biotech) among Symptomatic Individuals in Brazil and the United Kingdom

Evaluating rapid diagnostic tests in diverse populations is essential to improving diagnostic responses as it gives an indication of the accuracy in real-world scenarios. In the case of rapid diagnostic testing within this pandemic, lateral flow tests that meet the minimum requirements for sensitivity and specificity can play a key role in increasing testing capacity, allowing timely clinical management of those infected, and protecting health care systems

Identification of mechanisms regulating the intra cellular concentration of rifampicin in Mycobacterium Tuberculosis

Thesis (PhD)--Stellenbosch University, 2013.ENGLISH ABSTRACT: Rifampicin resistance in clinical isolates of Mycobacterium tuberculosis develops through selection of bacterial variants harbouring mutations in the rpoB gene. These mutations infer a fitness-cost in the absence of antibiotic pressure, however, fitness-levels of rifampicin-resistant strains can be restored by compensatory mutations in rpoA and rpoC. This study was the first to investigate the epidemiological relevance of these compensatory mutations in clinical M. tuberculosis isolates collected in South Africa. Through targeted DNA sequencing, we demonstrated a strong association between rpoC mutations and transmission, and the rpoB S531L mutation. Our study emphasises the epidemiological relevance of compensatory evolution in response to the emergence of rifampicin resistance, and illustrates how compensatory mutations may be selected as a function of epistatic interactions. Recently a hypothesis has been developed which suggests that the activation of efflux systems through exposure to rifampicin may explain the observed spectrum of rifampicin resistance phenotypes. To elucidate whether rifampicin dependent activation of efflux systems also increases energy production, the RNA expression profiles of candidate energy metabolism genes were investigated. This study demonstrated that rifampicin exposure induced an overall increase in the expression of energy metabolism genes. Our findings suggest that the response to rifampicin is not universal and may depend on other genomic mutations. From these results we conclude that the stress response induced by exposure to rifampicin increases the energy production which fuels efflux activity thereby enabling the cell to extrude rifampicin in an energy dependent manner. This also provides a platform to explain the mechanism by which the newly developed drug, TMC207, increases the rate of culture conversion when used in combination with second-line anti-TB drugs. We propose that inhibition of ATP synthesis by TMC207 will deprive the efflux pumps and transporter genes of energy, which will result in the accumulation of second-line anti-TB drugs within the bacilli, leading to more efficient binding of the second-line drugs to their targets and ultimately to cell death. To identify the genetic basis governing the level of rifampicin resistance, we sequenced the genomes of MDR clinical isolates and in vitro generated rifampicin resistant mutants. Only minor genetic changes in addition to the rpoB mutation were identified in the genomes of in vitro rifampicin resistant mutants which displayed varying levels of resistance. This suggests that these mutants may either use alternative regulatory mechanisms or have acquired SNPs outside the genetic regions investigated in this study to modulate rifampicin resistance levels. In contrast, the genomes of clinical MDR isolates from the Low Copy Clade showed considerable variability in genes involved in cell wall, cellular processes and lipid metabolism, while the genomes from the Beijing Clade displayed variability in genes known to confer drug resistance and compensatory mechanisms. These results suggest that the structure and processes of the cell wall, as well as lipid metabolism plays a critical role in determining the intra-cellular concentration of rifampicin. Finally, this study illustrated the complexity in the physiology of M. tuberculosis resistant to rifampicin, whereby multiple mechanisms are employed by the bacteria to modulate its resistance levels.AFRIKAANSE OPSOMMING: Rifampisien weerstandigheid in kliniese isolate van Mycobacterium tuberculosis ontwikkel deur die seleksie van bakteriële variante wat mutasies in die rpoB geen het. Alhoewel hierdie mutasies lei tot „n afname in fiksheid van die bakterieë in die teenwoordigheid van antibiotika, kan die fiksheids vlakke van rifampisien weerstandige stamme herstel word deur vergoedende mutasies in rpoA en rpoC. Hierdie is die eerste studie wat die epidemiologiese relevansie van hierdie vergoedende mutasies in kliniese M. tuberculosis isolate wat in Suid-Afrika versamel is, ondersoek. Deur middel van doelgerigte DNA volgordebepaling het ons „n sterk assosiasie tussen rpoC mutasies en transmissie, en die rpoB S31L mutasie getoon. Hierdie studie beklemtoon die epidemiologiese relevansie van regstellende evolusie na aanleiding van die ontwikkeling van rifampisien weerstandigheid en illustreer hoe regstellende mutasies geselekteer mag word as „n funksie van epistatiese interaksies. „n Hipotese is onlangs ontwikkel wat voorstel dat blootstelling aan rifampisien uitvloei sisteme in die bakterium aktiveer, wat moontlik die waargenome spektrum van rifampisien weerstandige fenotipes kan verklaar. Ons het die RNA uitdrukkingsprofiele van kandidaat-energiemetabolisme gene ondersoek om te bepaal of rifampisien afhanklike aktivering van uitvloei sisteme ook energieproduksie verhoog. Hierdie studie demonstreer dat rifampisien-blootstelling „n algehele verhoging in die uitdrukking van energiemetabolisme gene induseer. Ons bevindinge stel voor dat die reaksie van die sel op rifampisien blootstelling nie universeel is nie, en moontlik ook afhanklik is van ander genomiese mutasies. Uit hierdie resultate kan ons aflei dat die stres respons wat geïnduseer word deur rifampisien-blootstelling energieproduksie verhoog, wat weer die uitvloei aktiwiteit aanvuur, en gevolglik die sel in staat stel om rifampisien op „n energie-afhanklike wyse uit te dryf. Dit bied ook „n basis om die meganisme te verklaar waardeur die nuwe middel, TMC207, die tempo van kultuuromskakeling verhoog wanneer dit saam met tweede-linie anti-TB middels gebruik word. Ons stel voor dat die inhibisie van ATP sintese deur TMC207 die uitvloeipompe en transporteerder gene van energie ontneem. Gevolglik veroorsaak dit „n ophoping van tweedelinie anti-TB middels binne-in die bakterium, wat geleentheid bied vir meer effektiewe binding tussen die middels en hulle teikens en uiteindelik seldood veroorsaak. Ons het DNA volgordes bepaal van die genome van MDR kliniese isolate en in vitro selekteerde rifampisienweerstandige mutante om sodoende die genetiese grondslag waarop die vlak van rifampisienweerstandigheid beheer word, te identifiseer. Slegs klein verskille, bo en behalwe die rpoB mutasie, is geïdentifiseer in die genome van in vitro rifampisien weerstandige mutante wat verskillende vlakke van weerstandigheid getoon het. Dit dui aan dat hierdie mutante of ander regulatoriese meganismes gebruik, of hulle het enkelnukleotied polimorfismes buite die genetiese area wat in hierdie studie ondersoek is, waarmee rifampisien weerstandigheid gemoduleer word. In teenstelling hiermee het die genome van kliniese MDR isolate van die “Low Copy Clade” aansienlike variasie getoon in gene wat betrokke is by die selwand, sellulêre prosesse en lipiedmetabolisme. Verder het die genome van die Beijing genotipe variasie in gene getoon wat betrokke is by middelweerstandigheid en regstellende meganismes. Hierdie resultate dui aan dat die struktuur en prosesse van die selwand, asook lipiedmetabolisme, „n kritiese rol speel in die bepaling van die intrasellulêre konsentrasie van rifampisien. Opsommend, hierdie studie toon verskeie meganismes aan wat deur die bakterieë gebruik word om weerstandigheidsvlakke te moduleer en die kompleksiteit van die fisiologie van M. tuberculosis wat weerstandig is teen rifampisien.The National Research Foundation (NRF)South African Medical Research Council (MRC)Harry Crossley Foundatio

Detection of minor variants in Mycobacterium tuberculosis whole genome sequencing data

The study of genetic minority variants is fundamental to the understanding of complex processes such as evolution, fitness, transmission, virulence, heteroresistance and drug tolerance in Mycobacterium tuberculosis (Mtb). We evaluated the performance of the variant calling tool LoFreq to detect de novo as well as drug resistance conferring minor variants in both in silico and clinical Mtb next generation sequencing (NGS) data. The in silico simulations demonstrated that LoFreq is a conservative variant caller with very high precision (≥96.7%) over the entire range of depth of coverage tested (30x to1000x), independent of the type and frequency of the minor variant. Sensitivity increased with increasing depth of coverage and increasing frequency of the variant, and was higher for calling insertion and deletion (indel) variants than for single nucleotide polymorphisms (SNP). The variant frequency limit of detection was 0.5% and 3% for indel and SNP minor variants, respectively. For serial isolates from a patient with DR-TB; LoFreq successfully identified all minor Mtb variants in the Rv0678 gene (allele frequency as low as 3.22% according to targeted deep sequencing) in whole genome sequencing data (median coverage of 62X). In conclusion, LoFreq can successfully detect minor variant populations in Mtb NGS data, thus limiting the need for filtering of possible false positive variants due to sequencing error. The observed performance statistics can be used to determine the limit of detection in existing whole genome sequencing Mtb data and guide the required depth of future studies that aim to investigate the presence of minor variants

Determining cost and placement decisions for moderate complexity NAATs for tuberculosis drug susceptibility testing.

BackgroundAccess to drug resistant testing for tuberculosis (TB) remains a challenge in high burden countries. Recently, the World Health Organization approved the use of several moderate complexity automated nucleic acid amplification tests (MC-NAAT) that have performance profiles suitable for placement in a range of TB laboratory tiers to improve drug susceptibility tests (DST) coverage.MethodsWe conducted cost analysis of two MC-NAATs with different testing throughput: Lower Throughput (LT, ResultsThe base-case per test cost of $18.52 (range:$13.79 - $40.70) for LT test and$15.37 (range: $9.61 -$37.40) for HT test. Per test cost estimates were most sensitive to the number of testing days per week, followed by equipment costs and TB-specific workloads. In general, HT NAATs were cheaper at all testing volume levels, but at lower testing volumes (less than 2,000 per year) LT tests can be cheaper if the durability of the testing system is markedly better and/or procured equipment costs are lower than that of HT NAAT.ConclusionAssuming equivalent performance and infrastructural needs, placement strategies for MC-NAATs need to be prioritized by laboratory system's operational factors, testing demands, and costs