TB Updates for the Physician Rochester, Minnesota June 19, 2009 Mycobacterial Laboratory Science Update Nancy L. Wengenack, Ph.D. Associate Professor of Laboratory Medicine and Pathology Division of Clinical Microbiology Mayo Clinic Rochester, Minnesota Mycobacterial Laboratory Science Update Nancy L. Wengenack, Ph.D. Associate Professor of Laboratory Medicine and Pathology Division of Clinical Microbiology Mayo Clinic Rochester, Minnesota
Objectives Describe the methods currently in routine laboratory use for detection and identification of M. tuberculosis Provide some examples of the use of these methods in our practice Consider future directions in the molecular detection of M. tuberculosis Why do we need fancy molecular diagnostics for M. tuberculosis? After all, molecular assays generally cost more than non-molecular techniques Ex: MTD for M. tuberculosis - $383 list TB culture w/id $137 list Molecular techniques are generally more complex and require a different skill set for technologists than traditional microbiologic techniques
Advantages of Molecular Diagnostics for Mycobacteria Turn-around-time!!!!! PCR can be completed in only a few hrs after growth of the organism in culture Sequencing identification in 1 day after growth in culture Direct PCR can be performed for some organisms obviating the need for culture Timeline for M. tuberculosis Detection, Identification, and Drug Resistance Testing Day 1 Day 7 Day 14 Day 21 Day 28 1. AFB smear < 24 hours liquid culture; ID by probes or 16S rdna sequencing Drug resistance testing MTD or PCR test for Mtb
Potential Advantages (continued) Improved patient care Appropriate antibiotics can be selected Appropriate care can be started sooner Improved safety for lab workers Reduced manipulation of growing cultures (ex. M. tuberculosis) Traditional methods often lack sufficient discrimination Recognized Species of Mycobacteria (through May, 2008) Classification M. tuberculosis complex Slowly-growing NTMs Rapidly-growing mycobacteria Non-cultivatible mycobacteria Total Number of species and subspecies 8 54 70 1 133
Mycobacteria species grown in culture in 2007 (Mayo Clinic, N=1609) Top 10 species (N) M. avium complex (645) M. gordonae (355) M. tuberculosis (236) M. chelonae/absc. (146) M. fortuitum (40) M. peregrinum/sept. (38) M. kansasii (26) M. arupense (18) M. marinum (13) M. xenopi (13) Ex. of other species: M. asiaticum M. austroafricanum M. heckeshornense M. lentiflavum M. mucogenicum M. obuense M. simiae M. szulgai M. terrae M. wolinskyi Which molecular techniques are commonly used in Mycobacteriology? Nucleic acid hybridization probes Line probe assays PCR or PCR-like assays (TMA) DNA sequencing Sanger dideoxy chain termination Pyrosequencing
Detection from pure culture Nucleic Acid Hybridization Probes (Gen-Probe) Line Probe assays (Innogenetics, Hain Lifesciences) PCR (lab-developed) M. tuberculosis detection M. tuberculosis complex speciation Selected other NTMs Sequencing traditional Sanger dideoxy chain termination (Applied Biosystems and others) Pyrosequencing (Biotage) Direct detection from Specimen For M. tuberculosis identification M. tuberculosis Direct Test (TMA, Gen-Probe) Amplicor (PCR, Roche) Laboratory-developed PCR For M. tuberculosis drug resistance markers and other mycobacteria Line Probe assays (Hain Lifesciences and Innogenetics)
Nucleic Acid Hybridization Probes From culture only no amplification step need lots of target nucleic acid Gen-Probe AccuProbes available for: M. tuberculosis complex M. avium complex M. gordonae M. kansasii Require about 2 hours to complete Nucleic Acid Hybridization Probes Modified from Wolk DM et al., 2001, Infect. Dis. Clin. N. Amer., 15:1157-1204 Target region RNA template obtained from mycobacteria culture Add labeled DNA probe w/ complimentary sequence Target region DNA Probe Probe Label label is hydrolyzed on unbound probe chemiluminescent detection of bound probe
Nucleic Acid Hybridization Probes Mycobacteria M. avium M. intracellulare M. avium complex M. gordonae M. kansasii M. tuberculosis complex Sensitivity 99.3% 100% 99.9% 98.8% 92.8% 99.2% Specificity 100% 100% 100% 99.7% 100% 99.0% http://www.gen-probe.com/prod_serv/mycobac_accuprobe.asp Traditional (Sanger) DNA Sequencing for Mycobacteria Identification Identification from culture Targets 16S rdna rpob ITS of 16S-23S rdna hsp65 others (gyrb, reca...) 400-500bp reads typical
Traditional Sequencing Workflow PCR Product clean-up Target amplification Cycle Sequencing PCR Sephadex clean-up Sequence analysis Cycle sequencing Sequence analysis Compare the isolate sequence to known sequence libraries MicroSeq (AB) Ridom Custom GenBank (NCBI) TAT ~8 hrs with new FAST chemistry
Sequencing of the 16S target does not differentiate all mycobacteria Identical partial 16S sequence; ITS sequence can help differentiate M. abscessus and M. chelonae (hsp65, rpob and ITS targets can help) M. gastri and M. kansasii (M. kansasii is a photochromogen) M. fortuitum subp. acetamidolyticum and M. fortuitum subsp. fortuitum M. peregrinum and M. septicum(rpob and ITS targets can help) M. murale and M. tokaiense M. flavescens sequevar II and M. novocastrense Identical partial 16S sequence and ITS sequence M. tuberculosis complex species (gyrb can speciate except for M. tuberculosis and M. africanum II) M. marinum and M. ulcerans (hsp65 and rpob can provide resolution; photochromogenicity can help) Identical partial 16S sequences; rpob can help differentiate M. houstonense and M. senegalense M. porcinum and M. neworleansense Line Probe Assays Principle DNA extraction PCR Detection Results http://www.hain-lifescience.com
Genotype MTBC Line Probe Assay http://www.hain-lifescience.com Line Probe Assays from Culture Hain Lifesciences GenoType Mycobacterium CM and AS M. tuberculosis complex and 29 nontuberculous mycobacteria on 2 strips GenoType MTBC Differentiation of M. tuberculosis complex GenoType MTBDR plus M. tuberculosis complex plus wt and mutant rpob, katg, inha Innogenetics Inno-LiPA Mycobacteria v2 16 mycobacteria species including M. tuberculosis complex Controls M. avium M. kansasii M. tb complex
Line Probe Assays Directly from Specimen Hain Lifesciences GenoType Mycobacteria Direct M. avium, M. intracellulare, M. kansasii, M. malmoense, M. tuberculosis complex Innogenetics INNO-LiPA Rif.TB detects M. tuberculosis complex and rifampin resistance Detection of Mtb Complex Directly from Respiratory Specimens Mycobacterium tuberculosis Direct Test (MTD, Gen- Probe) TMA method that amplifies M. tuberculosis complex rrna directly from respiratory specimens Limitations Detects only M. tuberculosis complex Negative does not rule out M. tuberculosis infection (still need to do a culture) Cross-reactions can occur w/ M. celatum and possibly a few other rare mycobacteria
Gen-Probe MTD Assay: Transcription Mediated Amplification (TMA) RNA target RNA-DNA duplex Reverse transcriptase (RT) creates a DNA copy RNA degradation by RNase H + RT creates a DNA copy 10 6 increase in RNA target in < 30min RNA (100-1000 copies) transcribed from DNA template by T r RNA polymerase Detection: RNA:labeled DNA hybrid formed; chemiluminescent detection Modified from Wolk et al., (2001) Infect. Dis. Clinics North America 15:1177. Performance of Culture (3 specimens) and MTD test (1st specimen) for diagnosis of pulmonary TB Result Pulmonary TB Status Pos (81) Neg (716) Sensitivity Specificity PPV (%) NPV (%) Mean TAT (days) MTD Positive 73 0 90 100 100 98.9 2 Negative 8 716 Culture (combined) Positive 78 0 96 100 100 99.6 18 Negative 3 716 Moore DF et al., 2005, DMID, 52:247-54
Real-time PCR for M. tuberculosis A Laboratory-Developed Test M. tuberculosis LC PCR assay Specimen Processing 1. 500ul Raw specimen 2. NALC-NaOH treatment 3. 95 C 5 min 4. Bead beat 2 min 5. Extract 6. Assay samples on LightCycler
M. tuberculosis PCR Melt Curves katg S315T T M 59 C M. tuberculosis TM 65 C (Wild-type) Why switch to a laboratory-developed real-time PCR for M. tuberculosis? Sensitivity and specificity equivalent to MTD Expanded specimen types (respiratory, non-respiratory, formalin-fixed paraffinembedded tissue) Lower Cost Closed PCR system less prone to false positives Simplified workflow for the laboratory
Molecular detection of TB resistance markers PYROSEQUENCING SEQUENCING by SYNTHESIS PPi ATP Pyrogram Light E S C A G T C T G C T Nucleotides added individually in a predefined sequential order
Pyrosequencing Work Flow Culture organism PCR amplify target 1.5-3hrs Pyrosequencing (up to 96 samples) Pyrosequencing of Mycobacteria (continued) 96 short-read sequences in about 4 hours user friendly (load and go) equipment cost is lower than traditional sequencing ( $59,000 for 24 sample format) shorter read provide less discrimination than traditional sequencing
Isoniazid Resistance Detection in M. tuberculosis Pyrosequencing Approach wild-type resistant Arnold et al., Clin Microbiol Infect, 2005, 11:122-130. Future Directions? Direct detection of mycobacteria from raw specimens remains challenging PCR assays are limited by the number of different organisms or targets that can be assessed in a single assay
Future Trends Direct Sequencing (non-viable, mixed) web-based algorithm Bacteria 1 Bacteria 2 Kommedal et al., Analysis of mixed sequencing chromatograms and its application in direct 16S rdna sequencing of poly-microbial samples. J. Clin. Microbiol. published ahead of print on 3 September 2008, doi:10.1128/jcm.00213-08. Dual loci sequencing in 1 tube Gene1 (16S) Gene2 (rpob) Sequencing www.ripseq.com www.isentio.com Future? Will low-density, chip-based or liquid bead-based microarrays provide reliable detection of multiple targets or drug resistance markers directly from clinical specimens?
Summary Molecular diagnostics have profoundly reduced the turn-around-time for the detection and identification of M. tuberculosis in the clinical laboratory. Reliable identification of M. tuberculosis directly from clinical specimens or within 24hrs after growth in culture is currently a standard of practice. Multiplex assays using bead or chip microarray technology may offer the potential advances in clinical mycobacteriology including the ability to rapidly and reliably detect drug resistance markers.