Tuberculosis(TB)continues to pose a significant threat to global public health,necessitating rapid and precise diagnostic methods and comprehensive detection of antimicrobial resistance(AMR)to facilitate timely clinic...Tuberculosis(TB)continues to pose a significant threat to global public health,necessitating rapid and precise diagnostic methods and comprehensive detection of antimicrobial resistance(AMR)to facilitate timely clinical management.Traditional diagnostic techniques suffer from extended turnaround times and limited ability to comprehensively profile AMR,often resulting in delayed therapeutic interventions.Highthroughput sequencing(HTS)technologies have revolutionized pathogen research by significantly improving diagnostic speed and accuracy.In the context of TB,diverse sequencing strategies and platforms are being employed to fulfill specific research goals,ranging from elucidating the molecular mechanisms underlying AMR to characterizing the genomic diversity among clinical isolates.This review systematically examines current progress in the application of HTS for rapid pathogen identification,comprehensive AMR profiling,epidemiological studies,advances in novel drugs,and vaccine development.Furthermore,we address existing technological limitations and bioinformatics challenges and explore the future directions necessary for effectively integrating HTS-based methodologies into global TB control efforts.展开更多
Antimicrobial resistance(AMR)represents a substantial threat to global public health,complicating the treatment of common infections and leading to prolonged illness and escalated healthcare expenses.To effectively co...Antimicrobial resistance(AMR)represents a substantial threat to global public health,complicating the treatment of common infections and leading to prolonged illness and escalated healthcare expenses.To effectively combat AMR,timely and accurate detection is crucial for AMR surveillance and individual-based therapy.Phenotypic antibiotic resistance testing(AST)has long been considered the gold standard in clinical applications,serving as the foundation for clinical AMR diagnosis and optimized therapy.It has significantly contributed to ensuring patients′health and the development of novel antimicrobials.Despite advancements in automated culture-based AST technologies,inherent limitations impede the widespread use of phenotypic AST in AMR surveillance.Genotypic AST technologies offer a promising alternative option,exhibiting advantages of rapidity,high sensitivity,and specificity.With the continuous advancement and expanding applications of genotypic AST technologies,such as microfluidics,mass spectrometry,and high-resolution melting curve analysis,new vigor has been injected into the development and clinical implementation of genotypic AST technologies.In this narrative review,we discuss the principles,applications,and advancements of emerging genotypic AST methods in clinical settings.The comprehensive review aims to highlight the significant scientific potential of emerging genotypic AST technologies in clinical AMR diagnosis,providing insights to enhance existing methods and explore novel approaches.展开更多
Gonorrhea is one of the most common sexually transmitted diseases worldwide. To cure infection and prevent transmission,timely and appropriate antimicrobial therapy is necessary. Unfortunately, Neisseria gonorrhoeae, ...Gonorrhea is one of the most common sexually transmitted diseases worldwide. To cure infection and prevent transmission,timely and appropriate antimicrobial therapy is necessary. Unfortunately, Neisseria gonorrhoeae, the etiological agent of gonorrhea, has acquired nearly all known mechanisms of antimicrobial resistance(AMR), thereby compromising the efficacy of antimicrobial therapy. Treatment failure resulting from AMR has become a global public health concern. Whole-genome sequencing is an effective method to determine the AMR characteristics of N. gonorrhoeae. Compared with next-generation sequencing, the MinION sequencer(Oxford Nanopore Technologies(ONT)) has the advantages of long read length and portability. Based on a pilot study using MinION to sequence the genome of N. gonorrhoeae, we optimized the workflow of sequencing and data analysis in the current study. Here we sequenced nine isolates within one flow cell using a multiplexed sequencing strategy. After hybrid assembly with Illumina reads, nine integral circular chromosomes were obtained. By using the online tool Pathogenwatch and a BLAST-based workflow, we acquired complete AMR profiles related to seven classes of antibiotics. We also evaluated the performance of ONT-only assemblies. Most AMR determinants identified by ONT-only assemblies were the same as those identified by hybrid assemblies. Moreover, one of the nine assemblies indicated a potentially novel antimicrobial-related mutation located in mtrR which results in a frame-shift, premature stop codon, and truncated peptide.In addition, this is the first study using the MinION sequencer to obtain complete genome sequences of N. gonorrhoeae strains which are epidemic in China. This study shows that complete genome sequences and antimicrobial characteristics of N.gonorrhoeae can be obtained using the MinION sequencer in a simple and cost-effective manner, with hardly any knowledge of bioinformatics required. More importantly, this strategy provides us with a potential approach to discover new AMR determinants.展开更多
The recent outbreak of the coronavirus disease 2019(COVID-19)pandemic and the continuous evolution of the severe acute respiratory syndrome coronavirus 2(SARS-CoV-2)have highlighted the significance of new detection m...The recent outbreak of the coronavirus disease 2019(COVID-19)pandemic and the continuous evolution of the severe acute respiratory syndrome coronavirus 2(SARS-CoV-2)have highlighted the significance of new detection methods for global monitoring and prevention.Although quantitative reverse transcription PCR(RT-qPCR),the current gold standard for diagnosis,performs excellently in genetic testing,its multiplexing capability is limited because of the signal crosstalk of various fluorophores.Herein,we present a highly effi-cient platform which combines 17-plex assays with matrix-assisted laser desorption/ionization time-of-flight mass spectrometry(MALDI-TOF MS),enabling the targeting of 14 different mutation sites of the spike gene.Diagnosis using a set of 324 nasopharyngeal swabs or sputum clinical samples with SARS-CoV-2 MS method was identical to that with the RT-qPCR.The detection consistency of mutation sites was 97.9%(47/48)com-pared to Sanger sequencing without cross-reaction with other respiratory-related pathogens.Therefore,the MS method is highly potent to track and assess SARS-CoV-2 changes in a timely manner,thereby aiding the continuous response to viral variation and prevention of further transmission.展开更多
Dear Editor,Bacterial antimicrobial resistance(AMR)poses a serious threat to global human health(Antimicrobial Resistance Collaborators,2022).Comprehensive profiling of AMR and accurate molecular typing are important ...Dear Editor,Bacterial antimicrobial resistance(AMR)poses a serious threat to global human health(Antimicrobial Resistance Collaborators,2022).Comprehensive profiling of AMR and accurate molecular typing are important for tracking and controlling the emergence and dissemination of antibiotic-resistant bacteria(Yahara et al.,2021).We previously developed a multiplex amplicon sequencing-based method for directly sequencing AMR-related loci in N.gonorrhoeae from clinical samples(Zhang et al.,2021).展开更多
基金supported by the CAMS Innovation Fund for Medical Sciences(CIFMS)(2021-I2M-1-038 and 2023-I2M-2-001)the Non-profit Central Research Institute Fund of the Chinese Academy of Medical Sciences(2019PT310029 and 2023-PT310-04).
文摘Tuberculosis(TB)continues to pose a significant threat to global public health,necessitating rapid and precise diagnostic methods and comprehensive detection of antimicrobial resistance(AMR)to facilitate timely clinical management.Traditional diagnostic techniques suffer from extended turnaround times and limited ability to comprehensively profile AMR,often resulting in delayed therapeutic interventions.Highthroughput sequencing(HTS)technologies have revolutionized pathogen research by significantly improving diagnostic speed and accuracy.In the context of TB,diverse sequencing strategies and platforms are being employed to fulfill specific research goals,ranging from elucidating the molecular mechanisms underlying AMR to characterizing the genomic diversity among clinical isolates.This review systematically examines current progress in the application of HTS for rapid pathogen identification,comprehensive AMR profiling,epidemiological studies,advances in novel drugs,and vaccine development.Furthermore,we address existing technological limitations and bioinformatics challenges and explore the future directions necessary for effectively integrating HTS-based methodologies into global TB control efforts.
基金supported by the Non-profit Central Research Institute Fund of Chinese Academy of Medical Sciences(2023-PT310-04)。
文摘Antimicrobial resistance(AMR)represents a substantial threat to global public health,complicating the treatment of common infections and leading to prolonged illness and escalated healthcare expenses.To effectively combat AMR,timely and accurate detection is crucial for AMR surveillance and individual-based therapy.Phenotypic antibiotic resistance testing(AST)has long been considered the gold standard in clinical applications,serving as the foundation for clinical AMR diagnosis and optimized therapy.It has significantly contributed to ensuring patients′health and the development of novel antimicrobials.Despite advancements in automated culture-based AST technologies,inherent limitations impede the widespread use of phenotypic AST in AMR surveillance.Genotypic AST technologies offer a promising alternative option,exhibiting advantages of rapidity,high sensitivity,and specificity.With the continuous advancement and expanding applications of genotypic AST technologies,such as microfluidics,mass spectrometry,and high-resolution melting curve analysis,new vigor has been injected into the development and clinical implementation of genotypic AST technologies.In this narrative review,we discuss the principles,applications,and advancements of emerging genotypic AST methods in clinical settings.The comprehensive review aims to highlight the significant scientific potential of emerging genotypic AST technologies in clinical AMR diagnosis,providing insights to enhance existing methods and explore novel approaches.
基金supported by the Chinese Academy of Medical Sciences Innovation Fund for Medical Science (2016-I2M-3-021)。
文摘Gonorrhea is one of the most common sexually transmitted diseases worldwide. To cure infection and prevent transmission,timely and appropriate antimicrobial therapy is necessary. Unfortunately, Neisseria gonorrhoeae, the etiological agent of gonorrhea, has acquired nearly all known mechanisms of antimicrobial resistance(AMR), thereby compromising the efficacy of antimicrobial therapy. Treatment failure resulting from AMR has become a global public health concern. Whole-genome sequencing is an effective method to determine the AMR characteristics of N. gonorrhoeae. Compared with next-generation sequencing, the MinION sequencer(Oxford Nanopore Technologies(ONT)) has the advantages of long read length and portability. Based on a pilot study using MinION to sequence the genome of N. gonorrhoeae, we optimized the workflow of sequencing and data analysis in the current study. Here we sequenced nine isolates within one flow cell using a multiplexed sequencing strategy. After hybrid assembly with Illumina reads, nine integral circular chromosomes were obtained. By using the online tool Pathogenwatch and a BLAST-based workflow, we acquired complete AMR profiles related to seven classes of antibiotics. We also evaluated the performance of ONT-only assemblies. Most AMR determinants identified by ONT-only assemblies were the same as those identified by hybrid assemblies. Moreover, one of the nine assemblies indicated a potentially novel antimicrobial-related mutation located in mtrR which results in a frame-shift, premature stop codon, and truncated peptide.In addition, this is the first study using the MinION sequencer to obtain complete genome sequences of N. gonorrhoeae strains which are epidemic in China. This study shows that complete genome sequences and antimicrobial characteristics of N.gonorrhoeae can be obtained using the MinION sequencer in a simple and cost-effective manner, with hardly any knowledge of bioinformatics required. More importantly, this strategy provides us with a potential approach to discover new AMR determinants.
基金The research project was funded by grants from the CAMS Innovation Fund for Medical Sciences(CIFMS,2021-I2M-1-038)Non-profit Central Research Institute Fund of Chinese Academy of Medical Sciences(2019PT310029)the Fundamental Research Funds for the Central Universities(3332021092).We are grateful to Lili Ren for kindly providing us with clinical isolates of SARS-CoV-2.And we also would like to thank Editage(www.editage.cn)for English language editing.
文摘The recent outbreak of the coronavirus disease 2019(COVID-19)pandemic and the continuous evolution of the severe acute respiratory syndrome coronavirus 2(SARS-CoV-2)have highlighted the significance of new detection methods for global monitoring and prevention.Although quantitative reverse transcription PCR(RT-qPCR),the current gold standard for diagnosis,performs excellently in genetic testing,its multiplexing capability is limited because of the signal crosstalk of various fluorophores.Herein,we present a highly effi-cient platform which combines 17-plex assays with matrix-assisted laser desorption/ionization time-of-flight mass spectrometry(MALDI-TOF MS),enabling the targeting of 14 different mutation sites of the spike gene.Diagnosis using a set of 324 nasopharyngeal swabs or sputum clinical samples with SARS-CoV-2 MS method was identical to that with the RT-qPCR.The detection consistency of mutation sites was 97.9%(47/48)com-pared to Sanger sequencing without cross-reaction with other respiratory-related pathogens.Therefore,the MS method is highly potent to track and assess SARS-CoV-2 changes in a timely manner,thereby aiding the continuous response to viral variation and prevention of further transmission.
基金supported by the CAMS Innovation Fund for Medical Sciences(CIFMS)(2021-I2M-1-037,2021-I2M-1-038)the National Natural Science Foundation of China(82002193)+2 种基金the National Science and Technology Infrastructure of China(National Pathogen Resource Center-NPRC-32)the Non-profit Central Research Institute Fund of Chinese Academy of Medical Sciences(2019-PT310-029,2021-PT310-004)the Fundamental Research Funds for the Central Universities(3332021092)。
文摘Dear Editor,Bacterial antimicrobial resistance(AMR)poses a serious threat to global human health(Antimicrobial Resistance Collaborators,2022).Comprehensive profiling of AMR and accurate molecular typing are important for tracking and controlling the emergence and dissemination of antibiotic-resistant bacteria(Yahara et al.,2021).We previously developed a multiplex amplicon sequencing-based method for directly sequencing AMR-related loci in N.gonorrhoeae from clinical samples(Zhang et al.,2021).
