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1.
Methods Mol Biol ; 2833: 195-210, 2024.
Artigo em Inglês | MEDLINE | ID: mdl-38949712

RESUMO

Whole genome sequencing of Mycobacterium tuberculosis complex (MTBC) isolates has been shown to provide accurate predictions for resistance and susceptibility for many first- and second-line anti-tuberculosis drugs. However, bioinformatic pipelines and mutation catalogs to predict antimicrobial resistances in MTBC isolates are often customized and detailed protocols are difficult to access. Here, we provide a step-by-step workflow for the processing and interpretation of short-read sequencing data and give an overview of available analysis pipelines.


Assuntos
Antituberculosos , Biologia Computacional , Testes de Sensibilidade Microbiana , Mycobacterium tuberculosis , Sequenciamento Completo do Genoma , Mycobacterium tuberculosis/genética , Mycobacterium tuberculosis/efeitos dos fármacos , Sequenciamento Completo do Genoma/métodos , Testes de Sensibilidade Microbiana/métodos , Humanos , Antituberculosos/farmacologia , Biologia Computacional/métodos , Genoma Bacteriano , Farmacorresistência Bacteriana/genética , Mutação , Tuberculose/microbiologia , Tuberculose/tratamento farmacológico
2.
Methods Mol Biol ; 2833: 185-193, 2024.
Artigo em Inglês | MEDLINE | ID: mdl-38949711

RESUMO

Whole genome sequencing (WGS) is becoming an important diagnostic tool for antimicrobial susceptibility testing of Mycobacterium tuberculosis complex (MTBC) isolates in many countries. WGS protocols usually start with the preparation of a DNA-library: the critical first step in the process. A DNA-library represents the genomic content of a DNA sample and consists of unique short DNA fragments. Although available DNA-library protocols come with manufacturer instructions, details of the entire process, including quality controls, instrument parameters, and run evaluations, often need to be developed and customized by each laboratory to implement WGS technology effectively. Here, we provide a detailed workflow for a DNA-library preparation based on an adapted Illumina protocol optimized for the reduction of reagent costs.


Assuntos
Genoma Bacteriano , Testes de Sensibilidade Microbiana , Mycobacterium tuberculosis , Sequenciamento Completo do Genoma , Mycobacterium tuberculosis/genética , Mycobacterium tuberculosis/efeitos dos fármacos , Sequenciamento Completo do Genoma/métodos , Testes de Sensibilidade Microbiana/métodos , Humanos , Antituberculosos/farmacologia , Biblioteca Gênica , DNA Bacteriano/genética , Tuberculose/microbiologia , Tuberculose/diagnóstico , Sequenciamento de Nucleotídeos em Larga Escala/métodos
3.
Lab Chip ; 24(1): 74-84, 2023 12 20.
Artigo em Inglês | MEDLINE | ID: mdl-37999937

RESUMO

Globally, tuberculosis (TB) remains the deadliest bacterial infectious disease, and spreading antibiotic resistances is the biggest challenge for combatting the disease. Rapid and comprehensive diagnostics including drug susceptibility testing (DST) would assure early treatment, reduction of morbidity and the interruption of transmission chains. To date, rapid genetic resistance testing addresses only one to four drug groups while complete DST is done phenotypically and takes several weeks. To overcome these limitations, we developed a two-stage workflow for rapid TB diagnostics including DST from a single sputum sample that can be completed within three days. The first stage is qPCR detection of M. tuberculosis complex (MTBC) including antibiotic resistance testing against the first-line antibiotics, isoniazid (Inh) and rifampicin (Rif). The test is automated by centrifugal microfluidics and designed for point of care (PoC). Furthermore, enriched MTBC DNA is provided in a detachable sample tube to enable the second stage: if the PCR detects MTBC and resistance to either Inh or Rif, the MTBC DNA is shipped to specialized facilities and analyzed by targeted next generation sequencing (tNGS) to assess the complete resistance profile. Proof-of-concept testing of the PoC test revealed an analytical sensitivity of 44.2 CFU ml-1, a diagnostic sensitivity of 96%, and a diagnostic specificity of 100% for MTBC detection. Coupled tNGS successfully provided resistance profiles, demonstrated for samples from 17 patients. To the best of our knowledge, the presented combination of PoC qPCR with tNGS allows for the fastest comprehensive TB diagnostics comprising decentralized pathogen detection with subsequent resistance profiling in a facility specialized in tNGS.


Assuntos
Mycobacterium tuberculosis , Tuberculose Resistente a Múltiplos Medicamentos , Tuberculose , Humanos , Rifampina/farmacologia , Antituberculosos/farmacologia , Antituberculosos/uso terapêutico , Tuberculose Resistente a Múltiplos Medicamentos/diagnóstico , Tuberculose Resistente a Múltiplos Medicamentos/tratamento farmacológico , Tuberculose Resistente a Múltiplos Medicamentos/microbiologia , Mycobacterium tuberculosis/genética , Testes de Sensibilidade Microbiana , Sistemas Automatizados de Assistência Junto ao Leito , Microfluídica , Sensibilidade e Especificidade , Tuberculose/diagnóstico , Tuberculose/tratamento farmacológico , Tuberculose/microbiologia , Isoniazida/farmacologia , Isoniazida/uso terapêutico , Resistência Microbiana a Medicamentos , DNA
4.
Sci Rep ; 11(1): 15333, 2021 07 28.
Artigo em Inglês | MEDLINE | ID: mdl-34321545

RESUMO

Whole genome sequencing (WGS) is revolutionary for diagnostics of TB and its mutations associated with drug-resistances, but its uptake in low- and middle-income countries is hindered by concerns of implementation feasibility. Here, we provide a proof of concept for its successful implementation in such a setting. WGS was implemented in the Kyrgyz Republic. We estimated needs of up to 55 TB-WGS per week and chose the MiSeq platform (Illumina, USA) because of its capacity of up to 60 TB-WGS per week. The project's timeline was completed in 93-weeks. Costs of large equipment and accompanying costs were 222,065 USD and 8462 USD, respectively. The first 174 WGS costed 277 USD per sequence, but this was skewed by training inefficiencies. Based on real prices and presuming optimal utilization of WGS capacities, WGS costs could drop to 167 and 141 USD per WGS using MiSeq Reagent Kits v2 (500-cycles) and v3 (600-cycles), respectively. Five trainings were required to prepare the staff for autonomous WGS which cost 48,250 USD. External assessment confirmed excellent performance of WGS by the Kyrgyz laboratory in an interlaboratory comparison of 30 M. tuberculosis genomes showing complete agreeance of results.


Assuntos
DNA Bacteriano/genética , Sequenciamento de Nucleotídeos em Larga Escala/economia , Mycobacterium tuberculosis/genética , Tuberculose Resistente a Múltiplos Medicamentos/diagnóstico , Tuberculose Resistente a Múltiplos Medicamentos/economia , Sequenciamento Completo do Genoma/economia , Antituberculosos/farmacologia , Farmacorresistência Bacteriana Múltipla/genética , Sequenciamento de Nucleotídeos em Larga Escala/instrumentação , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Humanos , Quirguistão/epidemiologia , Mutação , Mycobacterium tuberculosis/classificação , Mycobacterium tuberculosis/efeitos dos fármacos , Filogenia , Tuberculose Resistente a Múltiplos Medicamentos/epidemiologia , Tuberculose Resistente a Múltiplos Medicamentos/microbiologia , Sequenciamento Completo do Genoma/instrumentação , Sequenciamento Completo do Genoma/métodos
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