ABSTRACT
We experienced significant problems while developing a PyrosequencingTM (Biotage, Uppsala, Sweden) assay to characterize the rifampin resistance-determining region of Mycobacteriumtuberculosis, a target with high guanosine-cytosine content. This paper describes the successful use of a modified pyrosequencing protocol through partial substitution of deoxyguanosine triphosphate with deoxyinosine triphosphate in the polymerase chain reaction.
Subject(s)
DNA, Bacterial/metabolism , Deoxyguanosine/metabolism , Inosine/analogs & derivatives , Pathology, Molecular/methods , Sequence Analysis, DNA/methods , Specimen Handling/methods , Bacterial Proteins/genetics , Base Composition , DNA, Bacterial/chemistry , DNA, Bacterial/genetics , Humans , Inosine/metabolism , Mycobacterium tuberculosis/genetics , SwedenSubject(s)
Cytological Techniques , Equipment Contamination , Plasma/microbiology , Yeasts , Adult , Arthroscopy , Cytological Techniques/methods , Disease Outbreaks , Humans , Knee Joint/microbiology , Knee Joint/surgery , Male , Postoperative Complications/diagnosis , Postoperative Complications/microbiology , Staphylococcal Infections/diagnosis , Staphylococcal Infections/etiology , Staphylococcal Infections/surgeryABSTRACT
After isoniazid and rifampin (rifampicin), the next pivotal drug class in Mycobacterium tuberculosis treatment is the fluoroquinolone class. Mutations in resistance-determining regions (RDR) of the rpoB, katG, and gyrA genes occur with frequencies of 97%, 50%, and 85% among M. tuberculosis isolates resistant to rifampin, isoniazid, and fluoroquinolones, respectively. Sequences are highly conserved, and certain mutations correlate well with phenotypic resistance. We developed a pyrosequencing assay to determine M. tuberculosis genotypic resistance to rifampin, isoniazid, and fluoroquinolones. We characterized 102 M. tuberculosis clinical isolates from the Philippines for susceptibility to rifampin, isoniazid, and ofloxacin by using the conventional submerged-disk proportion method and validated our pyrosequencing assay using these isolates. DNA was extracted and amplified by using PCR primers directed toward the RDR of the rpoB, katG, and gyrA genes, and pyrosequencing was performed on the extracts. The M. tuberculosis H37Rv strain (ATCC 25618) was used as the reference strain. The sensitivities and specificities of pyrosequencing were 96.7% and 97.3%, 63.8% and 100%, and 70.0% and 100% for the detection of resistance to rifampin, isoniazid, and ofloxacin, respectively. Pyrosequencing is thus a rapid and accurate method for detecting M. tuberculosis resistance to these three drugs.
Subject(s)
Antitubercular Agents/pharmacology , Drug Resistance, Bacterial , Isoniazid/pharmacology , Mycobacterium tuberculosis/drug effects , Ofloxacin/pharmacology , Rifampin/pharmacology , Sequence Analysis, DNA/methods , Bacterial Proteins/chemistry , Bacterial Proteins/genetics , Drug Resistance, Bacterial/genetics , Fluoroquinolones/pharmacology , Genotype , Humans , Microbial Sensitivity Tests , Mutation , Mycobacterium tuberculosis/genetics , Phenotype , Time FactorsABSTRACT
The past decade has seen a surge in the development of a variety of molecular diagnostics designed to rapidly identify or characterize medically important microorganisms. We briefly review important advances in molecular microbiology, and then discuss specific assays that have been implemented in clinical microbiology laboratories throughout the country. We also discuss emerging methods and technologies that will soon be more widely used for the prompt and accurate detection of the agents of infectious diseases.
