Rapid microarray-based assay for detection of pyrazinamide resistant Mycobacterium tuberculosis

Havlicek, Juliane; Dachsel, Beatrice; Slickers, Peter; Andres, Sönke; Beckert, Patrick; Feuerriegel, Silke; Niemann, Stefan; Merker, Matthias; Labugger, Ines

doi:10.1016/j.diagmicrobio.2018.12.011

Cited by 7 publications

(9 citation statements)

References 45 publications

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“…Interestingly, our findings showed that more than 97% of the genetic variants identified in the pncA gene were correlated with phenotypic resistance. Hence, Sanger sequencing could be a reliable and accurate method for the rapid diagnosis of PZA resistant TB [44,45]. Regarding STR, the sensitivity of predicting resistance against this drug was most likely reduced due to the presence of a gidB mutation, which could not be interrogated in this study [46,47].…”

Section: Plos Onementioning

confidence: 90%

Prediction of drug resistance by Sanger sequencing of Mycobacterium tuberculosis complex strains isolated from multidrug resistant tuberculosis suspect patients in Ethiopia

et al. 2022

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Background Ethiopia is one of the high multidrug-resistant tuberculosis (MDR-TB) burden countries. However, phenotypic drug susceptibility testing can take several weeks due to the slow growth of Mycobacterium tuberculosis complex (MTBC) strains. In this study, we assessed the performance of a Sanger sequencing approach to predict resistance against five anti-tuberculosis drugs and the pattern of resistance mediating mutations. Methods We enrolled 226 MTBC culture-positive MDR-TB suspects and collected sputum specimens and socio-demographic and TB related data from each suspect between June 2015 and December 2016 in Addis Ababa, Ethiopia. Phenotypic drug susceptibility testing (pDST) for rifampicin, isoniazid, pyrazinamide, ethambutol, and streptomycin using BACTEC MGIT 960 was compared with the results of a Sanger sequencing analysis of seven resistance determining regions in the genes rpoB, katG, fabG-inhA, pncA, embB, rpsL, and rrs. Result DNA isolation for Sanger sequencing was successfully extracted from 92.5% (209/226) of the MTBC positive cultures, and the remaining 7.5% (17/226) strains were excluded from the final analysis. Based on pDST results, drug resistance proportions were as follows: isoniazid: 109/209 (52.2%), streptomycin: 93/209 (44.5%), rifampicin: 88/209 (42.1%), ethambutol: 74/209 (35.4%), and pyrazinamide: 69/209 (33.0%). Resistance against isoniazid was mainly mediated by the mutation katG S315T (97/209, 46.4%) and resistance against rifampicin by rpoB S531L (58/209, 27.8%). The dominating resistance-conferring mutations for ethambutol, streptomycin, and pyrazinamide affected codon 306 in embB (48/209, 21.1%), codon 88 in rpsL (43/209, 20.6%), and codon 65 in pncA (19/209, 9.1%), respectively. We observed a high agreement between phenotypic and genotypic DST, such as 89.9% (at 95% confidence interval [CI], 84.2%–95.8%) for isoniazid, 95.5% (95% CI, 91.2%–99.8%) for rifampicin, 98.6% (95% CI, 95.9–100%) for ethambutol, 91.3% (95% CI, 84.6–98.1%) for pyrazinamide and 57.0% (95% CI, 46.9%–67.1%) for streptomycin. Conclusion We detected canonical mutations implicated in resistance to rifampicin, isoniazid, pyrazinamide, ethambutol, and streptomycin. High agreement with phenotypic DST results for all drugs renders Sanger sequencing promising to be performed as a complementary measure to routine phenotypic DST in Ethiopia. Sanger sequencing directly from sputum may accelerate accurate clinical decision-making in the future.

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Section: Plos Onementioning

confidence: 90%

Prediction of drug resistance by Sanger sequencing of Mycobacterium tuberculosis complex strains isolated from multidrug resistant tuberculosis suspect patients in Ethiopia

et al. 2022

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“…A DNA microarray for the simultaneous (multiplex asymmetric PCR amplification) detection of ARGs among Staphylococcus clinical isolates based on fluorescently labeled PCR products has been developed [ 61 ]. More recently, Havlicek et al proposed a rapid cartridge based, melting curve assay for the detection of pyrazinamide resistant Mycobacterium tuberculosis [ 62 ]. The assay can be automatically implemented using a closed cartridge coupled with a battery powered Alere™ q analyzer, as a point-of-care test in resource-limited settings [ 62 ].…”

Section: Conventional Amr Diagnostic Methodsmentioning

confidence: 99%

“…More recently, Havlicek et al proposed a rapid cartridge based, melting curve assay for the detection of pyrazinamide resistant Mycobacterium tuberculosis [ 62 ]. The assay can be automatically implemented using a closed cartridge coupled with a battery powered Alere™ q analyzer, as a point-of-care test in resource-limited settings [ 62 ].…”

Section: Conventional Amr Diagnostic Methodsmentioning

confidence: 99%

Rapid Methods for Antimicrobial Resistance Diagnostics

et al. 2021

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Antimicrobial resistance (AMR) is one of the most challenging threats in public health; thus, there is a growing demand for methods and technologies that enable rapid antimicrobial susceptibility testing (AST). The conventional methods and technologies addressing AMR diagnostics and AST employed in clinical microbiology are tedious, with high turnaround times (TAT), and are usually expensive. As a result, empirical antimicrobial therapies are prescribed leading to AMR spread, which in turn causes higher mortality rates and increased healthcare costs. This review describes the developments in current cutting-edge methods and technologies, organized by key enabling research domains, towards fighting the looming AMR menace by employing recent advances in AMR diagnostic tools. First, we summarize the conventional methods addressing AMR detection, surveillance, and AST. Thereafter, we examine more recent non-conventional methods and the advancements in each field, including whole genome sequencing (WGS), matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) spectrometry, Fourier transform infrared (FTIR) spectroscopy, and microfluidics technology. Following, we provide examples of commercially available diagnostic platforms for AST. Finally, perspectives on the implementation of emerging concepts towards developing paradigm-changing technologies and methodologies for AMR diagnostics are discussed.

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“…Using fluorescently labeled PCR sequences (DNA chips), simultaneous detection of ARGs among Staphylococcus clinical isolates was achieved ( Zhu et al, 2007 ). Recently, a rapid cartridge based, melting curve assay to detect pyrazinamide resistant Mycobacterium tuberculosis was proposed ( Havlicek et al, 2019 ).…”

Section: Relevant Diagnostic Tools For Detecting Lateral Gene Transfe...mentioning

confidence: 99%

Antibiotic resistance in plant growth promoting bacteria: A comprehensive review and future perspectives to mitigate potential gene invasion risks

et al. 2022

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Plant growth-promoting bacteria (PGPB) are endowed with several attributes that can be beneficial for host plants. They opened myriad doors toward green technology approach to reduce the use of chemical inputs, improve soil fertility, and promote plants’ health. However, many of these PGPB harbor antibiotic resistance genes (ARGs). Less attention has been given to multi-resistant bacterial bioinoculants which may transfer their ARGs to native soil microbial communities and other environmental reservoirs including animals, waters, and humans. Therefore, large-scale inoculation of crops by ARGs-harboring bacteria could worsen the evolution and dissemination of antibiotic resistance and aggravate the negative impacts on such ecosystem and ultimately public health. Their introduction into the soil could serve as ARGs invasion which may inter into the food chain. In this review, we underscore the antibiotic resistance of plant-associated bacteria, criticize the lack of consideration for this phenomenon in the screening and application processes, and provide some recommendations as well as a regulation framework relating to the development of bacteria-based biofertilizers to aid maximizing their value and applications in crop improvement while reducing the risks of ARGs invasion.

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Rapid microarray-based assay for detection of pyrazinamide resistant Mycobacterium tuberculosis

Cited by 7 publications

References 45 publications

Prediction of drug resistance by Sanger sequencing of Mycobacterium tuberculosis complex strains isolated from multidrug resistant tuberculosis suspect patients in Ethiopia

Prediction of drug resistance by Sanger sequencing of Mycobacterium tuberculosis complex strains isolated from multidrug resistant tuberculosis suspect patients in Ethiopia

Rapid Methods for Antimicrobial Resistance Diagnostics

Antibiotic resistance in plant growth promoting bacteria: A comprehensive review and future perspectives to mitigate potential gene invasion risks

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