Table 1 - available via license: Creative Commons Zero 1.0
Content may be subject to copyright.
Source publication
Drug resistance in presents an enormous public health threat. It is typically defined as >1% of drug resistant colonies using the agar proportion method. Detecting small numbers of drug resistant Tb in a population, also known as heteroresistance, is challenging with current methodologies. Here we have utilized digital PCR to detect heteroresistanc...
Citations
... Therefore, our assay was determined to achieve a LOD of 5000 copies/ml, equivalent to 125 copies/reaction, but it must be clarified that these outcomes pertain solely to the input DNA samples. Given the increasing prevalence of infection with mixed susceptibility and resistance to a drug in the MDR population as evidenced by recent studies [45,46], an alternative LOD validation experiment was performed to investigate the extent to which our assay could unequivocally identify hetero-resistant mutants mixed with WT drug-sensitive strains. Our data corroborated the assay's analytical rigor, with its capacity to distinguish as little as 40% of the desired hetero-resistant mutants mixed with WT being particularly noteworthy. ...
Background
Escalating cases of multidrug-resistant tuberculosis (MDR-TB) pose a major challenge to global TB control efforts, necessitating innovative diagnostics to empower decentralized detection of gene mutations associated with resistance to rifampicin (RIF) and isoniazid (INH) in Mycobacterium tuberculosis (M. tuberculosis) in resource-constrained settings.
Methods
Combining multiplex fluorescent PCR and Multiple Probes Melting Analysis, we identified mutations in the rpoB, katG, ahpC and inhA genes from sputum specimens. We first constructed a reference plasmid library comprising 40 prevalent mutations in the target genes’ resistance determining regions and promoters, serving as positive controls. Our assay utilizes a four-tube asymmetric PCR method with specifically designed molecular beacon probes, enabling simultaneous detection of all 40 mutations. We evaluated the assay’s effectiveness using DNA isolated from 50 clinically confirmed M. tuberculosis sputum specimens, comparing our results with those obtained from Sanger sequencing and retrospective validation involving bacteriological culture and phenotypic drug susceptibility testing (pDST). We also included the commercial Xpert MTB/RIF assay for accuracy comparison.
Results
Our data demonstrated remarkable sensitivity in detecting resistance to RIF and INH, achieving values of 93.33% and 95.24%, respectively, with a specificity of 100%. The concordance between our assay and pDST was 98.00%. Furthermore, the accuracy of our assay was comparable to both Sanger sequencing and the Xpert assay. Importantly, our assay boasts a 4.2-h turnaround time and costs only $10 per test, making it an optimal choice for peripheral healthcare settings.
Conclusion
These findings highlight our assay’s potential as a promising tool for rapidly, accurately, and affordably detecting MDR-TB.
... 73 So, digital PCR offers significant advantages in detecting and quantifying heterologous drug resistance in MTB populations. 74,75 Next-Generation Sequencing Next-generation sequencing (NGS), also termed high-throughput or massively parallel sequencing, is used for pathogen detection and monitoring the hospital microbiome and its drug resistance. It is a technology genre that allows thousands to billions of DNA/RNA fragments to be tested and independently sequenced. ...
Multidrug-resistant tuberculosis (MDR-TB) is an essential cause of tuberculosis treatment failure and death of tuberculosis patients. The rapid and reliable profiling of Mycobacterium tuberculosis (MTB) drug resistance in the early stage is a critical research area for public health. Then, most traditional approaches for detecting MTB are time-consuming and costly, leading to the inappropriate therapeutic schedule resting on the ambiguous information of MTB drug resistance, increasing patient economic burden, morbidity, and mortality. Therefore, novel diagnosis methods are frequently required to meet the emerging challenges of MTB drug resistance distinguish. Considering the difficulty in treating MDR-TB, it is urgently required for the development of rapid and accurate methods in the identification of drug resistance profiles of MTB in clinical diagnosis. This review discussed recent advances in MTB drug resistance detection, focusing on developing emerging approaches and their applications in tangled clinical situations. In particular, a brief overview of antibiotic resistance to MTB was present, referred to as intrinsic bacterial resistance, consisting of cell wall barriers and efflux pumping action and acquired resistance caused by genetic mutations. Then, different drug susceptibility test (DST) methods were described, including phenotype DST, genotype DST and novel DST methods. The phenotype DST includes nitrate reductase assay, RocheTM solid ratio method, and liquid culture method and genotype DST includes fluorescent PCR, GeneXpert, PCR reverse dot hybridization, ddPCR, next-generation sequencing and gene chips. Then, novel DST methods were described, including metabolism testing, cell-free DNA probe, CRISPR assay, and spectral analysis technique. The limitations, challenges, and perspectives of different techniques for drug resistance are also discussed. These methods significantly improve the detection sensitivity and accuracy of multidrug-resistant tuberculosis (MRT) and can effectively curb the incidence of drug-resistant tuberculosis and accelerate the process of tuberculosis eradication.
... This reproducible method detects bacilli at a count as low as 1000 CFU/ml. With these benefits, DDPCR enables the early detection of emerging mutations that evolve in treatment and may necessitate a medication adjustment.185 ...
Tuberculosis (TB) is a significant public health challenge, especially in developing nations. Developing a TB eradication strategy is hampered by the global health concern of drug-resistant (DR) TB. Effective patient treatment, preventing TB transfer and avoiding the upsurge of DR strains depend primarily on the timely and accurate identification of DR TB. Due to inadequate sensitivity, the necessity of trained laboratory personnel, the sluggish growth pattern of Mycobacterium bacilli in culture, and the small number of bacilli that are usually found in extrapulmonary TB samples, TB diagnosis is still tricky in clinical practice. Although mycobacterial culture is the gold standard to identify TB and determine drug resistance, it takes 2 to 8 weeks to develop. Despite their high cost, nucleic acid amplification tests (NAATs) and whole-genome sequencing (WGS) are the commonly employed molecular-based methods for diagnosing and identifying TB. The WHO suggested the GeneXpert MTB/RIF to identify TB and detect resistance to rifampicin. In comparison, numerous molecular techniques were developed, including allele-specific PCR (MAS-PCR), solid-phase hybridization, real-time PCR (RT-PCR) and droplet digital PCR-based technique (DDPCR). This manuscript is intended to overview the current approaches for the phenotypic and genotypic diagnosis of TB disease and identifying resistance to antitubercular drugs depending on recently published articles, WHO and CDC reports, and commercially available diagnostic tools.
... DdPCR has previously proven its efficiency in detecting and quantifying pathogenic microorganisms including bacteria, fungi, viruses, and parasites (King et al., 2016;Koepfli et al., 2016;Baltrušis et al., 2018;Li et al., 2018). Furthermore, it has also shown its ability to detect rare allele changes in mixed populations of Mycobacterium tuberculosis (Pholwat et al., 2013). Here, the potential of ddPCR based assays for rapid and cost-effective detection of resistance in D. immitis isolates, based on their genotypic signatures, was investigated. ...
... Compared to conventional mutation screening assay, the ddPCR mutation detection assay showed higher sensitivity and specificity to detect mutations in patients with acute leukemia and myelodysplastic syndrome (Findlay et al., 2016;Christenson et al., 2017;Rowlands et al., 2019). Importantly, ddPCR identified mutant sequence in mixtures of Mycobacterium tuberculosis populations and detected resistance-predicting alleles in Haemonchus contortus strains with similar results as next-generation sequencing (Pholwat et al., 2013;Baltrušis et al., 2018). In our study, we employed the ddPCR technology to rapidly and precisely genotype D. immitis isolates as being resistant or susceptible to ML based on the presence or absence of mutant SNP alleles (SNP1, SNP2, SNP3 and SNP7) which have been previously correlated with ML-resistance status (Bourguinat et al., 2017b;Ballesteros et al., 2018). ...
Prevention of canine heartworm disease, caused by Dirofilaria immitis, relies on macrocyclic lactones for which drug resistance is now a concern. Although genetic polymorphisms have been associated with resistance in D. immitis populations, the mechanism is still not well understood. The lack of reliable in vitro assays to detect resistance is a limitation for confirming resistance. Ten single nucleotide polymorphisms (SNPs) were previously clinically validated in D. immitis resistant isolates, using the MiSeq platform. This technique although useful for research studies is expensive and does not facilitate rapid detection of these markers in small numbers of clinical samples. We developed a droplet digital PCR protocol for detecting SNPs correlating with ML resistance. Specific primers and hydrolysis probes encompassing the wildtype and mutant alleles were designed to amplify the SNP targets from genomic DNA of different D. immitis isolates. Allele frequencies were determined and the suitability of the ddPCR assay was assessed and compared with MiSeq data. The ddPCR assay accurately detected and quantified alternate nucleotides in two isolates of reference, the ML-susceptible Missouri (MO) and ML-resistant JYD-34, at the previously identified SNP positions. The presence of the SNPs was also determined in additional isolates with known or putative susceptible or resistant phenotypes. We observed SNP1 and SNP2 are more predictive markers and appear suitable for rapid detection and monitoring of drug resistance. Our results suggested that ddPCR could be employed to distinguish infection due to actual genetic resistance from infection with susceptible parasites and also for rapid detection of isolates not only with ML susceptible and resistant genotypes but also mixed genotypes that correspond to heterogeneous isolates containing a mixed population of ML susceptible and resistant parasites. DdPCR may be a useful tool for conducting surveys, or assessments of individual isolates, for genetic evidence of resistance or developing resistance.
... Heteroresistance refers to a bacterial phenotype where subpopulations of cells show significant differences in antibiotic susceptibility, with the term used in the context of MTB to indicate the presence of varying susceptibility within the same clone (monoclonal heteroresistance) or several coexisting clones (polyclonal heteroresistance) [13]. Digital PCRs have been used to detect and quantify the heteroresistance of DR-TB in MTB H37Rv and XDR-TB mixed samples [14]. Droplet Digital PCR (ddPCR) is a multiplex PCR system that can simultaneously detect copies of mutant and wild-type alleles of target genes, including genes conferring drug-resistance in MTB. ...
Heteroresistance in MTB refers to the presence of distinct subpopulations of bacteria with varying levels of antibiotic susceptibility within a population. Multidrug-resistant and rifampicin-resistant TB are serious global health concerns. In this study, we aimed to determine the prevalence of heteroresistance in MTB from sputum samples of new TB cases using Droplet Digital PCR mutation detection assays for katG and rpoB genes, which are commonly associated with resistance to isoniazid and rifampicin, respectively. We found that out of 79 samples, 9 (11.4%) exhibited mutations in katG and rpoB genes. INH mono-resistant TB, RIF mono-resistant TB, and MDR-TB samples constituted 1.3%, 6.3%, and 3.8% of new TB cases, respectively. Heteroresistance in katG, rpoB, and both genes were found in 2.5%, 5%, and 2.5% of total cases, respectively. Our results suggest that these mutations may have arisen spontaneously, as the patients had not yet received anti-TB drugs. ddPCR is a valuable tool for the early detection and management of DR-TB, as it can detect both mutant and wild-type strains in a population, enabling the detection of heteroresistance and MDR-TB. Overall, our findings highlight the importance of early detection and management of DR-TB for effective TB control (in katG, rpoB, and katG/rpoB).
... The third-generation digital PCR (dPCR) can detect trace drug resistance mutations of pathogenic microorganisms and achieve "accurate attack" in the selection of effective anti-infective drugs [65]. In view of the H275Y point mutation of neuraminidase H275Y in 2009 H1N1 influenza virus resistant to oseltamivir, Taylor and co-workers compared the ability of the second-generation fluorescent quantitative PCR (qPCR) and dPCR to detect H275Y under the background of high-abundance wild-type H1N1. ...
Kary B. Mullis developed a revolutionary method name polymerase chain reaction (PCR) in 1983, which can synthesize new strand of DNA complementary to the template strand of DNA and produce billions of copies of a DNA fragment only in few hours. Denaturation, annealing, and extension are the three primary steps involved in the PCR process, which generally requires thermocyclers, DNA template, a pair of primers, Taq polymerase, nucleotides, buffers, etc. With the development of PCR, from traditional PCR, quantitative PCR, to next digital PCR, PCR has become a powerful tool in life sciences and medicine. Applications of PCR techniques for infectious diseases include specific or broad-spectrum pathogen detection, assessment and surveillance of emerging infections, early detection of biological threat agents, and antimicrobial resistance analysis. Applications of PCR techniques for genetic diseases include prenatal diagnosis and screening of neonatal genetic diseases. Applications of PCR techniques for cancer research include tumor-related gene detection. This chapter aimed to discuss about the different types of PCR techniques, including traditional PCR, quantitative PCR, digital PCR, etc., and their applications for rapid detection, mutation screen or diagnosis in infectious diseases, inherited diseases, cancer, and other diseases.
... With a sufficiently large M. tuberculosis population in infected tissue (greater than 10 9 organisms) [7], spontaneous mutants with resistance to a single drug are likely to be present, which could then be selected by inadequate drug pressure [3]. Heteroresistant TB is defined as a disease in which drug-resistant and drugsusceptible M. tuberculosis strains co-exist in the same patient [8][9][10]. In the conventional proportional drug sensitivity tests (pDST), heteroresistance is defined as 1-99% bacterial colony growth on drug-containing media [11]. ...
... Critically, the major limitation of traditional polymerase chain reaction (PCR) methodology is the inability to detect rare mutant sequences in a background of abundant WT DNA. With recent advances, such as digital PCR [10] and next-generation sequencing [15], the limit of detection of rare mutants may approach the 0.1% threshold (corresponding to the ratio of mutant DNA/WT DNA), which remains far above the level required to detect the baseline presence of heteroresistant strains in clinical samples or dynamic changes in the emergence of drug-resistant M. tuberculosis [7]. Even with the limitations of current tools, the clinical impact of heteroresistant TB is increasingly recognized [8,11,14,16]. ...
... We report the successful use of SuperSelective PCR primers for detecting RIF and INH heteroresistance in TB with a detection limit of 0.01% for the mutant DNA/WT DNA ratio. Our method shows a 10-fold improvement in detecting mutant gene copies in the presence of abundant WT genes of M. tuberculosis genome over the most sensitive methods reported to date, such as digital PCR [10]. ...
The emergence of drug-resistant tuberculosis is a significant global health issue. The presence of heteroresistant Mycobacterium tuberculosis is critical to developing fully drug-resistant tuberculosis cases. The currently available molecular techniques may detect one copy of mutant bacterial genomic DNA in the presence of about 1–1000 copies of wild-type M. tuberculosis DNA. To improve the limit of heteroresistance detection, we developed SuperSelective primer-based real-time PCR assays, which, by their unique assay design, enable selective and exponential amplification of selected point mutations in the presence of abundant wild-type DNA. We designed SuperSelective primers to detect genetic mutations associated with M. tuberculosis resistance to the anti-tuberculosis drugs isoniazid and rifampin. We evaluated the efficiency of our assay in detecting heteroresistant M. tuberculosis strains using genomic DNA isolated from laboratory strains and clinical isolates from the sputum of tuberculosis patients. Results show that our assays detected heteroresistant mutations with a specificity of 100% in a background of up to 104 copies of wild-type M. tuberculosis genomic DNA, corresponding to a detection limit of 0.01%. Therefore, the SuperSelective primer-based RT-PCR assay is an ultrasensitive tool that can efficiently diagnose heteroresistant tuberculosis in clinical specimens and contributes to understanding the drug resistance mechanisms. This approach can improve the management of antimicrobial resistance in tuberculosis and other infectious diseases.
... Digital PCR has been used with M. tuberculosis by combining wild-type DNA with DNA carrying resistance alleles in gyrA, katG, rpoB, and rrs. This assay can reveal heteroresistance of 1 mutant to 1,000 wild-type cells (Pholwat et al., 2013). For such sensitivity with sputum, the samples must have more than 1,000 bacilli per ml (M. ...
With tuberculosis, the emergence of fluoroquinolone resistance erodes the ability of treatment to interrupt the progression of MDR-TB to XDR-TB. One way to reduce the emergence of resistance is to identify heteroresistant infections in which subpopulations of resistant mutants are likely to expand and make the infections fully resistant: treatment modification can be instituted to suppress mutant enrichment. Rapid DNA-based detection methods exploit the finding that fluoroquinolone-resistant substitutions occur largely in a few codons of DNA gyrase. A second approach for restricting the emergence of resistance involves understanding fluoroquinolone lethality through studies of antimicrobial tolerance, a condition in which bacteria fail to be killed even though their growth is blocked by lethal agents. Studies with Escherichia coli guide work with Mycobacterium tuberculosis. Lethal action, which is mechanistically distinct from blocking growth, is associated with a surge in respiration and reactive oxygen species (ROS). Mutations in carbohydrate metabolism that attenuate ROS accumulation create pan-tolerance to antimicrobials, disinfectants, and environmental stressors. These observations indicate the existence of a general death pathway with respect to stressors. M. tuberculosis displays a variation on the death pathway idea, as stress-induced ROS is generated by NADH-mediated reductive stress rather than by respiration. A third approach, which emerges from lethality studies, uses a small molecule, N-acetyl cysteine, to artificially increase respiration and additional ROS accumulation. That enhances moxifloxacin lethality with M. tuberculosis in culture, during infection of cultured macrophages, and with infection of mice. Addition of ROS stimulators to fluoroquinolone treatment of tuberculosis constitutes a new direction for suppressing the transition of MDR-TB to XDR-TB.
... Therefore, our ddPCR gyrA assay offers a significant advantage for the detection of low-abundance FQ-resistant C. jejuni mutants, especially in mixed infections of FQ-resistant and susceptible strains. Recently, ddPCR was also used for detecting resistance mutations in Mycobacterium tuberculosis and Legionella pneumophila, reaching 1% to 0.1% distinguishability (32,33). A maximum distinguishability of ddPCR of 0.001% could be reached using an improved method to obtain a higher number of individual droplets (24). ...
Fluoroquinolone (FQ)-resistant Campylobacter jejuni is a serious problem worldwide that limits effective treatment of infections. The traditional detection method depends on bacterial isolation and MIC testing, or traditional PCR, which is time-consuming and hard to identify the FQ-resistant C. jejuni in a high abundance wild-type background. This study aimed to develop a rapid and accurate ddPCR assay to detect FQ-resistant C. jejuni mutants based on the crucial resistance mutation C257T (Thr-86-Ile) in gyrA. Our ddPCR gyrA assay showed high specificity and accuracy. Sanger sequencing and the qPCR assay could only recognize gyrA mutant sequences when the ratios of wild-type/mutant were 1:1 or 10:1, respectively. Our ddPCR gyrA assay was able to detect gyrA mutant sequences in the mixtures with up to at least 1000:1 wild-type/mutant ratios, which suggested a significant advantage to distinguish the low mutant signal from the wild-type background. We further monitored the occurrence of gyrA mutations under ciprofloxacin pressure using our ddPCR gyrA assay, and clearly showed that the transition of a dominant C. jejuni subpopulation from wild-type to gyrA C257T mutant, resulting in FQ-resistance. We tested 52 samples from live chickens and retail chicken meat and showed that four samples contained wild-type/mutant mixtures comprising 1.7%, 28.6%, 53.3%, and 87.0% gyrA C257T mutants, respectively. These results demonstrated that the ddPCR gyrA assay was a highly sensitive alternative method to distinguish and quantify FQ-resistant C. jejuni infections that could help guide the appropriate use of FQs in clinical practice.
... Historically, mixed infection was detected using traditional genotyping methods, such as spoligotyping, IS6110 restriction fragment length polymorphism (RFLP), and Mycobacterial Interspersed Repetitive-Unit variable-number tandem repeat (MIRU-VNTR) (Mathema et al., 2006;Pholwat et al., 2013;Naidoo and Dookie, 2018). However, given the limited sensitivity of these methods, as an abundance of at least 10% of the minor strain is required for detection, the estimated rate based on mathematical modeling is much higher (Cohen et al., 2012). ...
The World Health Organization’s End TB Strategy prioritizes universal access to an early diagnosis and comprehensive drug susceptibility testing (DST) for all individuals with tuberculosis (TB) as a key component of integrated, patient-centered TB care. Next generation whole genome sequencing (WGS) and its associated technology has demonstrated exceptional potential for reliable and comprehensive resistance prediction for Mycobacterium tuberculosis isolates, allowing for accurate clinical decisions. This review presents a descriptive analysis of research describing the potential of WGS to accelerate delivery of individualized care, recent advances in sputum-based WGS technology and the role of targeted sequencing for resistance detection. We provide an update on recent research describing the mechanisms of resistance to new and repurposed drugs and the dynamics of mixed infections and its potential implication on TB diagnosis and treatment. Whilst the studies reviewed here have greatly improved our understanding of recent advances in this arena, it highlights significant challenges that remain. The wide-spread introduction of new drugs in the absence of standardized DST has led to rapid emergence of drug resistance. This review highlights apparent gaps in our knowledge of the mechanisms contributing to resistance for these new drugs and challenges that limit the clinical utility of next generation sequencing techniques. It is recommended that a combination of genotypic and phenotypic techniques is warranted to monitor treatment response, curb emerging resistance and further dissemination of drug resistance.
