Skip directly to site content Skip directly to page options Skip directly to A-Z link


Centers for Disease Control and Prevention. CDC twenty four seven. Saving Lives, Protecting People
 Search

		Submit


Tuberculosis (TB)


Section Navigation
			
			CDC Home


Facebook
        Twitter
        LinkedIn
        Syndicate





Report of Expert Consultations on Rapid Molecular Testing to Detect Drug-Resistant Tuberculosis in the United States										Related Pages
										Appendix 1: Molecular Basis of Drug Resistance and Molecular DR tests
Drug resistance in *Mycobacterium tuberculosis* bacteria arises mainly through the acquisition of mutations in the chromosomal sequence that encode changes that 1) block the activity of a drug (mutations in *rpoB* prevent binding of rifampin to RNA polymerase and inhibition of transcription), 2) block activation of a prodrug (e.g., mutations in *katG* lead to loss of the ability of catalase to activate the prodrug isoniazid to its active form), or 3) produce an activity that binds or destroys the drug (e.g., mutations in *inhA* increase the amount of InhA protein which interferes with the activity of isoniazid by binding sufficient isoniazid to reduce its effective concentration in the bacterium to below an inhibitory level) (1,2). The mutations associated with resistance to many of the antituberculosis drugs have been identified, though much work remains to be done to identify the molecular basis of resistance for some of the drugs and to determine the predictive value of finding a particular mutation in a strain of *M. tuberculosis* (1,2). For example, approximately 95% of rifampin-resistant *M. tuberculosis* strains carry mutations within the rifampin-resistance determining region (RRDR), an 81-bp region encoding codons 507 through 533 of the *rpoB* gene.
Molecular genetic tests for detecting drug-resistance are, in general, just a variation of nucleic acid amplification (NAA) tests and can reliably provide information on the presence of mutations associated with drug resistance in 1 to 2 days. Typically, PCR is used to amplify a target sequence followed by a second assay to determine if the sequence contains a mutation associated with resistance. Methods that have been described for the latter include DNA sequencing, pyrosequencing, electrophoretic detection methods (e.g., single strand conformation polymorphism), methods for detecting mismatches in heteroduplexes (e.g., temperature gradient HPLC analysis or branch migration inhibition), and hybridization assays (e.g., molecular beacons, microarrays, membrane hybridization, or line-probe assays). Kits for detecting mutations associated with rifampin resistance that are commercially available in Europe and elsewhere include line-probe assays (INNO-LiPA® Rif.TB, Innogenetics and GenoType® MTBDR(*plus*), Hain LifeScience GmbH) and microarray assays (CombiChip Mycobacteria DR, GENE IN). Some also detect mutations associated with isoniazid resistance. In-house PCR-based tests using molecular beacons have also been used for diagnostic purposes in a few clinical laboratories.
For the hybridization assays, the region of the target gene associated with resistance is PCR amplified, and the labeled PCR products hybridized to oligonucleotide probes immobilized on a nitrocellulose strip or in a microarray. Mutations are detected by lack of binding to wild-type probes and/or by binding to probes specific for commonly occurring mutations. The performance of the line-probe assays relative to culture-based DS tests was evaluated in meta-analyses (3–5). For the INNO-LiPA Rif.TB assay, the pooled sensitivity was 0.97 (95%CI 0.95–0.98) and the pooled specificity was 0.99 (95%CI 0.98–1.00) for detecting rifampin resistance in *M. tuberculosis* isolates. Overall discriminatory ability of the assay was 99% and overall accuracy was 97%, with all studies yielding consistently high performances. In four studies, the INNO-LiPA Rif.TB showed 100% specificity and sensitivity ranging from 80% to 100% for detecting rifampin resistance directly from clinical specimens. For the MTBDR and MTBDR(*plus*) assays, the pooled sensitivity was 0.98 (95%CI 0.96–0.99) and the pooled specificity was 0.99 (95% CI 0.97–0.99) for detecting rifampin resistance in isolates or directly from clinical specimens. Overall discriminatory ability of the assay was 99% and overall accuracy 97%, with all studies yielding consistently high performances.
Molecular beacons are hybridization probes which emit fluorescence only when hybridized to their target. Molecular beacons can discriminate between targets differing by a single nucleotide. Because molecular beacons can use different fluorophores, real-time PCR assays can be designed in which different DNA fragments or mutations can be amplified and detected simultaneously in the same tube. For example, a single-well assay has been developed that uses five molecular beacons to detect mutations associated with rifampin resistance in *M. tuberculosis* bacteria and appears to perform similarly as the line-probe assays. In the California Microbial Diseases Laboratory, molecular beacons were designed to detect mutations in *rpoB, katG*, and *inhA* promoter region genes and directly applied to clinical specimens or to cultures. Comparison of molecular beacons results with results of culture-based drug-susceptibility testing showed 96% to 97% agreement in a series of approximately 1,000 clinical specimens and cultures (6, E. Desmond, personal communication).
Validation studies were conducted at the Wadsworth Center of an approach that combines PCR-amplification of the RRDR with rapid (< 2hrs) DNA sequencing (K. Musser, personal communication). A PyrosequencingTM protocol utilizing two primers was developed to sequence the 81-bp RRDR of the *rpoB* gene and obtain a clear and accurate pyrogram. The detection limit was determined and the pyrosequencing approach was evaluated in primary specimens positive for *M. tuberculosis* complex DNA by real-time PCR. Final results were compared with conventional susceptibility testing results and/or DNA sequencing. This test has a detection limit of <1 colony forming unit, 100% specificity, and 99% agreement in the 188 cultures and specimens tested. (7)
Molecular genetic tests for the other antituberculosis drugs are much less developed and studied than the tests for rifampin resistance. A meta-analysis of the performance of the Hain MTBDR*(plus)* assay for detecting isoniazid revealed a pooled sensitivity of 0.85 (95%CI 0.77– 0.90) which ranged from 57%–100% and a pooled specificity of 0.99 (95%CI 0.98–1.00) which was fairly consistent across studies. Validation studies conducted in the California Microbial Diseases Laboratory that used archived cultures revealed that the molecular beacon test displayed 82.7% sensitivity, 100% specificity, 100% positive predictive value, and 98.1% negative predictive value for detecting isoniazid resistance (6). Tests for the other key resistances, especially the XDR TB defining resistances, are in various stages of development from discovery of the mutations associated with resistance to development of prototype assays and laboratory-based evaluations.
References
Zhang Y and Telenti A. (2000) Genetics of drug resistance in *Mycobacterium tuberculosis*. In Hatfull GF and Jacobs WR Jr (Eds.). Molecular genetics of Mycobacteria (ASM Press, Washington, D.C) pp. 235–54.
Johnson R, Streicher EM, Louw GE, Warren RM, van Helden PD, Victor TC. Drug resistance in *Mycobacterium tuberculosis*. Curr Issues Mol Biol. 2006 Jul;8(2):97-111.
Morgan M, Kalantri S, Flores L, Pai M. A commercial line probe assay for the rapid detection of rifampicin resistance in *Mycobacterium tuberculosis*: a systematic review and meta-analysis. BMC Infect Dis. 2005; 5:62.
Ling DI, Zwerling AA, Pai M. GenoType MTBDR assays for the diagnosis of multidrug-resistant tuberculosis: a meta-analysis. Eur Respir J. 2008;32:1165*–*74.
WHO Expert Group Report. Molecular line probe assays for rapid screening of patients at risk of multi-drug resistant tuberculosis (MDR-TB). Geneva: World Health Organization; 2008.
Lin S-YG, Lin S-Y, Probert W, Lo M , Desmond E. Rapid detection of isoniazid and rifampin resistance mutations in *Mycobacterium tuberculosis* complex from cultures or smear-positive sputa by use of molecular beacons. J Clin Microbiol 2004 42: 4204-08.
Halse TA, Edwards J, Driscoll JR, Escuyer VE, and Musser KA. A pyrosequencing approach to rapidly assess mutations in the *rpoB* gene associated with rifampin resistance in clinical specimens of *Mycobacterium tuberculosis*. 48th Annual ICAAC/IDSA Annual Meeting. Washington, DC. 2008.
							
					
						Page last reviewed:  September 1, 2012					Content source: 	Division of Tuberculosis Elimination, National Center for HIV, Viral Hepatitis, STD, and TB Prevention, Centers for Disease Control and Prevention homeTuberculosisBasic TB Factsplus iconHow TB SpreadsLatent TB Infection and TB DiseaseSigns & SymptomsTB Risk FactorsExposure to TBTB PreventionVaccinesTB & HIV CoinfectionTB and DiabetesTB TermsPersonal StoriesTesting & Diagnosisplus iconWho Should be TestedTesting for TB InfectionTesting in BCG-Vaccinated PersonsTB Screening and Testing of Health Care PersonnelTesting During PregnancyDiagnosing latent TB infection and TB diseaseTreatmentplus iconDeciding When to Treat Latent TB InfectionTreatment Regimens for Latent TB Infection (LTBI)Treatment for TB DiseaseAdverse EventsTB Treatment of Persons Living with HIVTB Treatment and PregnancyTB Treatment for ChildrenFrequently Asked QuestionsDrug-Resistant TBResearchplus iconTB Epidemiologic Studies Consortiumplus iconResearch ProjectsPublicationsTB Trials Consortiumplus iconStudy DescriptionsBackgroundBehavioral & Social Science ResearchHealth Care Settingsplus iconTB Infection Control in Health Care SettingsResources for TB Screening and Testing of Health Care Personnelplus iconFrequently Asked QuestionsTB in Specific Populationsplus iconHealth DisparitiesAsian PersonsBlack or African American PersonsHispanic or Latino PersonsChildrenPregnancyCorrectional FacilitiesPeople Experiencing HomelessnessInternational TravelersLaboratory Informationplus iconDrug Susceptibility TestingFalse-Positive Investigation ToolkitInterim Laboratory Biosafety Guidance for XDR  tuberculosis strainsModel Performance Evaluation Program (MPEP)Rapid Molecular Testing to Detect Drug-Resistant TB in the USplus iconExecutive SummaryIntroductionBackground on Tests for Molecular Detection of DRGeneral Considerations and Principles for a Molecular DR Testing ServicePossible Scenarios and Scope of Testing for a Molecular DR Testing ServiceResearch NeedsGeneral Recommendations of the Expert PanelCommunication Plan for the ReportRecommendationsReferencesPanel Members and CDC ParticipantsAppendix 1Appendix 2Appendix 3The Uses of Nucleic Acid Amplification Tests for the Diagnosis of TBplus iconGeneral ConsiderationsGeneral Recommendations of the Expert PanelRecommendations to ACET, CDC, DTBEData & Statisticsplus iconTB Incidence in the United StatesReported TB in the US, 2022 Surveillance Reportplus iconNational DataReporting Area DataRisk FactorsDemographicsTB Treatment and Case OutcomesDrug-Resistant TBMolecular SurveillanceTuberculosis in the United States, 2022 (Slide Set)Archived Surveillance Reports and Slide Sets2021 State and City ReportArchived State and City ReportsProfessional Resources & Toolsplus iconTools for Healthcare ProvidersResources for TB Programsplus iconStrengthening Civil Surgeons’ Capacity to Improve LTBI Surveillance and Outcomes Among Status AdjustersNotice of Funding Opportunity (NOFO)Program EvaluationTB Centers of ExcellenceTB Elimination AllianceTB Genotypingplus iconConventional TB GenotypingWhole Genome SequencingTB GIMSAnalytic ToolsLatent Tuberculosis Infection ResourcesPublications & Productsplus iconPatient and Public MaterialsFact SheetsGuidelinesplus iconControl & EliminationDrug-Resistant TBInfection Control & PreventionLaboratory & GenotypingTB & HIVTB in Specific PopulationsTesting & DiagnosisTreatmentVaccines & ImmunizationsArchived TB GuidelinesGuides & Toolkitsplus iconAggregate ReportsCDC TB Surveillance TrainingCore CurriculumEffective TB Interviewing for Contact InvestigationEthnographic GuidesFalse-Positive Investigation ToolkitForging Partnerships to Eliminate TuberculosisGuide to Application of GenotypingLTBI: A Guide for Primary Health Care ProvidersMantoux Tuberculin Skin Testing ProductsReport of Verified Case of Tuberculosis (RVCT)Self-Study ModulesTB Contact Investigation Interviewing Skills CourseTB eDOT ToolkitUnderstanding the TB Cohort Review ProcessNewslettersplus iconTB Notes, No. 4, 2023TB Notes, No. 3, 2023TB Notes. No. 2, 2023TB Notes. No. 1, 2023TB Notes, No. 4, 2022TB Notes, No. 3, 2022TB Notes, No. 2, 2022TB Notes, No. 1, 2022TB Notes, No. 4, 2021TB Notes, No. 3, 2021TB Notes, No. 2, 2021TB Notes, No. 1, 2021Pamphlets, Brochures, Bookletsplus iconQuestions and AnswersPatient Education SeriesTuberculosis - Get the FactsTuberculosis - The Connection between TB and HIV12-Dose Regimen for Latent TB Infection-Patient Education BrochurePostersplus iconMantoux Tuberculin Skin Test Wall ChartWorld TB DayThink TBStop TBTake on TBReports & Articlesplus iconDTBE Authored Journal ArticlesTuberculosis Laboratory Aggregate ReportsSlide Setsplus iconIntroduction to TuberculosisEpidemiology of Tuberculosis Among Non-U.S.​–Born Persons in the United States, 1993–2016Self-Study Modules on Tuberculosis, 1-5 Slide SetsThe Tuberculosis (TB) in Correctional Settingsplus iconFacilitator GuideIntroduction to TB GenotypingEpidemiology of Tuberculosis in Correctional Facilities, United States, 1993-2017Prevention and Control of Tuberculosis in Correctional and Detention FacilitiesGuidelines for Preventing the Transmission of M. TB in Health care SettingsInvestigation of Contacts of Persons with Infectious TBEpidemiology of Pediatric Tuberculosis in the United StatesTargeted Tuberculosis Testing and Treatment of Latent Tuberculosis InfectionElectronic Tools & ResourcesWeb-Based Courses & Webinarsplus iconTB 101 for Health Care WorkersContent SyndicationInfographicsplus iconSocial Media GraphicsCustomizable Take on TB Infographic with InstructionsEventsWorld TB Dayplus iconU.S. TB ChampionsHistoryFeaturesplus iconTB Risk FactorsTreating Latent TB Infection with Short-Course RegimensWorld Tuberculosis (TB) Day 2021World Tuberculosis (TB) Day 2020Linksplus iconState TB Control OfficesAbout Usplus iconMission Statement and Activitiesplus iconDTBE Strategic PlanNational TB Program ObjectivesOrganization ChartAdvisory Groupsplus iconFederal TB Task Force To receive email updates about this page, enter your email address: Email Address			What's this?				Submit


About CDC
					Contact Us
					800-232-4636
 Facebook Twitter Instagram LinkedIn Youtube Pinterest Snapchat
 CONTACT CDC
				Contact Us
							Call 800-232-4636
							Email Us
			ABOUT CDC
				About CDC
							Jobs
							Funding
			POLICIES
				Accessibility
							External Links
							Privacy
							Policies
							No Fear Act
							FOIA
							OIG
							Nondiscrimination
							Vulnerability Disclosure Policy
			CONNECT WITH US
				Facebook
								Twitter
								Instagram
								LinkedIn
							Youtube
								Pinterest
								Snapchat
								Email
			LANGUAGES
				Español
							繁體中文
							Tiếng Việt
							한국어
							Tagalog
							Русский
							العربية
							Kreyòl Ayisyen
							Français
							Polski
							Português
							Italiano
							Deutsch
							日本語
							فارسی
							English


U.S. Department of Health & Human Services
			Accessibility
			External Links
			Privacy
			Policies
			No Fear Act
			FOIA
			Nondiscrimination
			OIG
			Vulnerability Disclosure Policy
			USA.gov


Exit Notification / Disclaimer Policy Close
 Links with this icon  indicate that you are leaving the CDC website. The Centers for Disease Control and Prevention (CDC) cannot attest to the accuracy of a non-federal website.
				Linking to a non-federal website does not constitute an endorsement by CDC or any of its employees of the sponsors or the information and products presented on the website.
				You will be subject to the destination website's privacy policy when you follow the link.
				CDC is not responsible for Section 508 compliance (accessibility) on other federal or private website. Website Disclaimers
 Cancel Continue