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Microbiological diagnosis of TB José Domínguez1 and Sabine Rüsch-Gerdes2 1Servei 2 de Microbiologia. Fundació Institut en Ciències de la Salut Germans Trias i Pujol. Badalona. Spain Forschungszentrum Borstel, National Referencelaboratory for Mycobacteria. Borstel. Germany Microbiological diagnosis of TB: Detection, identification and molecular epidemiology José Domínguez Servei de Microbiologia. Fundació Institut en Ciències de la Salut Germans Trias i Pujol One of the main objectives for TB control Objectives for TB control •To rapidly diagnose patients with active TB and treat them correctly. •To have rapid diagnostic methods, with high sensitivity and specificity to diagnose diseased patients at the beginning of the symptoms for an adequate treatment prescription “Diagnosis, diagnosis & diagnosis” William Osler Clinical suspicion Histology Microbiology Diagnosis of tuberculosis Latent Infection Active tuberculosis Smear examination TST Solid and liquid culture Molecular Epidemiology IFN- techniques Identification RFLP MIRU Susceptibility testing methods Molecular methods -Detection -Identification -Detection of resistance Spoligotyping Clinical samples Samples!! Respiratory and extra-respiratory Quantity: high inoculums means fast growth Quality, including sputum, high yield Localization, biopsies when possible Rapid shipment Previous to starting treatment Think of histology The most important: Clinical-Microbiologist-Pathologist Communication Decontamination • Eliminate normal flora from the non-sterile samples (micobacteria is acid and alkaline resistant) • Homogenization to release the bacteria from the sample and allow access to the nutrient present in the media i.e. Kubica N-acetyl-cysteine: homogenization NaOH: decontaminant Neutralization by phosphate buffer Homogenization Phosphate buffer Centrifugation Sample mixing Pellet Smear microscopy Fast; Cheap; Monitorization of treatment; Low sensitivity Ziehl-Neelsen stain Auramina O stain Hospital Univ. Germans Trias i Pujol 2004 -2007 Pulmonary Disseminated Extrapulmonary* TOTAL 125 18 60 Positive smear 83 (66.4%) 11 (61.1%) 11 (18.3%) Negative smear 42 (33.6%) 7 (38.9%) 49 (81.7%) *Adenopathy, 4/28 (14.2%); Pleural, 1/17 (5.9%) Culture in solid and liquid media Decontamination (in non sterile samples) Culture in the adequate media Inoculums!! Growth in solid media Slow: 15d-2m Growth in liquid media 7-42d Division time 18h Identification Classical and Molecular methods DST • Sometimes the only place where the mycobacteria can be isolated • Gold standard • Molecular epidemiology • Drug susceptibility testing Identification molecular methods AccuProbe InnoLiPA Mycobacteria GenoType Mycobacterium CM/AS, GenoType MTBC Identification molecular methods PRA (Polymorphism Restriction Amplification) Amplification, by PCR of a fragment of the hsp65 gen, followed by a restriction with 2 restriction enzymes (BstEII y HaeIII). Sequencing Pyrosequencing PPi ATP M.tuberculosis detection in clinical samples by molecular methods Sensitivity in respiratory samples (%) Sensitivity in extra respiratory samples (%) Overall specificity (%) Method Target Detection method AMTD2 16S rRNA Chemiluminometric 80-100 60-90 95-100 b antigenic protein Fluorimetric 80-90 65-80 90-100 AMPLICOR 16S rRNA Colorimetric 75-100 45-60 90-100 BD ProbeTec IS6110 and 16S rRNA Fluorimetric 55-100 30-80 45-100 INNO-LIPA v2 IR16S-23S Colorimetric 50-95 60-80 90-100 GenoType Direct 23S rRNA Colorimetric 60-95 60-80 95-100 PCR real time 16S rRNA Fluorimetric 70-90 65-85 85 LCx * In smear negative samples the sensitivity is reduced in a 50% Role of the clinical suspicion level in the evaluation of the molecular methods Catanzaro A. et al JAMA 2000 Problem: there are positive results in negative smear and culture samples. Molecular epidemiology • RFLP (IS6110) • Spoligotyping • MIRUs Restriction Fragment Length Polymorphism (RFLP) • Insertion sequence present exclusively in the M.tuberculosis complex: IS6110 • High polymorphism between no related strains regarding the number of copies and their localization in the chromosome. • Advantages: High discriminative power. • Disadvantages: Slow, laborious and with certain complexity. Pvu II Electrophoresis Extraction and restriction Radiographic develop Hybridization Transference Spacer oligonucleotype typing (Spoligotyping) • The DR sequences (direct repeat) are repeated sequences of 36 bp in only one locus of the M.tuberculosis chromosome, separated by sequences of 34 to 41 bp. • The technique is based on a PCR of the locus where the DR sequences are located. The amplification product is hybridized with oligos synthesized from the inter-DR spaces. • The presence or absence of different DR allows a specific pattern for each strain. • Advantages: Few DNA is required, easy interpretation • Disadvantages: Lesser discriminative power than the RFLP. Micobacterial Interspersed Repetitive Units (MIRU) • Determine the number of repetitive units in 12 (15 or 20) different locus of one genetic sequence called “mycobacterial interspersed repetitive units (MIRUs)”. The number of repetitions is detected by PCR. • The number of repetitive units in each locus is calculated by the size of the fragment amplified with the specifics primers. • MIRU-VNTR is more discriminative than the spoligotyping and similar to the RFLP-IS6110. • Advantages: rapid, simple and automatic. • Disadvantages: In study Micobacterial Interspersed Repetitive Units (MIRU) MIRU 40 500 pb 43 1 2 Conclusions • The microbiological diagnosis of TB will be rapid and accurate if adequate samples are collected and adequate inoculums are used. Don’t forget histology. • The future of TB diagnosis remains in the application of new molecular techniques but, at the moment a cautious interpretation of the results is required. • The sensitivity of the molecular tests vary, and is affected by the amount of bacteria present in the samples, and also by the clinical suspicion level. Low sensitivity is present in samples with low bacterial load, especially in extra respiratory samples. • At the moment, new molecular methods can not substitute the conventional ones. The gold-standard is the culture, and the other methods have to be considered and interpreted as complementary diagnostic methods. • Communication between clinicians and microbiologists is imperative. Microbiological Diagnosis of TB Drug Susceptibility Testing Borstel 2010 National Reference Laboratory for Mycobacteria Forschungszentrum Borstel Sabine Rüsch-Gerdes Drug Susceptibility Testing MDR TB – New Infection WHO: MDR-TB & XDR-TB, The 2008 Report; February 2008 Drug Susceptibility Testing Anteil resistenter Erreger in Deutschland, 2006 For all TB strains isolated, DST has to be Quelle: RKI, Bericht zur Epidemiologie der Tuberkulose in Deutschland für 2006; 2008 performed BiostoffVerordnung (BioStoffV) 1999 Microscopy Culture NAT Level 2 laboratory Microscopy Culture NAT Differentiation DST Level 3 laboratory Methods for Drug Susceptibility Testing Proportion method on Löwenstein-Jensen medium H, R, E, S, PTH, CM, OFL, CS, NSA (instead of P) Results available: 4-6 weeks No critical concentrations for new substances Methods for Drug Susceptibility Testing BACTEC 460TB All drugs except cycloserine Results available: 1-2 weeks Radioactive materials, waste Methods for Drug Susceptibility Testing MGIT 960 For all drugs, except CS Results available: 1-2 weeks Liquid media compared to solid media Advantages compared to solid media: • more rapid • high quality of media • fully automated system • testing of 1st, 2nd, and new drugs • safety: plastic tubes Disadvantages: • expensive • higher contamination rate • dependency on a company • no DST for Cycloserine Infection control strategies Proposed measures: • • • • • • Improved ventilation system Reduced hospitalsation Mask use Isolation of patients HIV-testing and therapy Rapid drug-susceptibility testing Time for the detection has a potential to reduce the extend of spread of resistant strains Principle of the Line Probe Assays Chromogen (MBT/BCIP) Alkaline Phosphatase Streptavidin Biotin DNA-probe Colour reaction Biotin-labelled single stranded amplified target Nitrocellulose strip MTBDR – DNA Hybridisation Strip 1 Control of the conjugate Amplification control Amplification control MTBC Control rpoB rpoB Wild type 1 rpoB Wild type 2 rpoB Wild type 3 rpoB Wild type 4 rpoB Wild type 5 rpoB Mut D516V rpoB Mut H526Y rpoB Mut H526D rpoB Mut S531L Control katG katG wild type katG S315T1 (ACC) katG S315T2 (ACA) - 2 3 4 5 6 7 8 Genotype® MTBDRplus HybridizationStrip 1 from Culture media 2 1 RMPr INHr 2 RMPs INHs Results RMP+INH Resistance 100 % concordance between sequencing and MTBDR data 103 MDR strains 102 strains (99%) mutations in rpoB cluster I 91 strains (88.4) with mutations in codon 315 of katG + + 1 strain (1%) a mutation outside rpoB cluster I 3 strains (2,9 %) with a mutation in inhA + 2 strains (1,9 %) with a mutation in ahpC + + 7 strains (6,8 %) with no mutation in katG, inhA and ahpC 1 strain (1%) not detected as MDR (rpoB outside cluster I, ahpC) Genotype® MTBDRplus HybridizationStrip 1 from specimens Hillemann D, Rüsch-Gerdes S, Richter E. Application of the Genotype MTBDR assay directly on sputum specimens. Int J Tuberc Lung Dis 2006. 10:1057-1059. 2 1 RMPr INHr 2 RMPs INHs Evaluation of the MTBDRplus Assay on Specimens 72 smear positive sputum specimens: 30 susceptible strains 32 MDR (RMPr/INHr) strains 10 INHr strains Sensitivity Specificity RMP detection: 96.8% INH detection: 90.2% RMP: 95.2% INH: 100% Line Probe Assays for DST Evaluated line probe assays INNO-LiPA Rif TB GenoType MTBDRplus Resistance Sensitivity Specificity RMP 98.1% 98.7% INH 84.3% 99.5% Ling et al., Eur Respir J 2008 GenoType MTBDRsl Molecular Basis Drug Gene Gene function Locus Percent of Resistance Fluorochinolones gyrA DNA-Gyrase A appr.80-90% Amikacin, Capreomycin, Kanamycin rrs 16S rRNA appr. 80 % tlyA Methylase Ethambutol embB appr. 30-60% Summary • Overall sensitivity for OFL, AM, CM and EMB was 90.2 %, 83.3 %, 86.8 % and 59.0 %, respectively. • Specificity was 100 % for FLQ, AM, and EMB, and 99.1 % for CM. • Most prevalent mutations were: gyrA D94G in Oflr strains rrs A1401G AMr/CMr strains embB M306V in EMBr strains • The rapid detection of XDR strains is possible with the combined application of Lipas from DNA isolates and directly from sputum specimens. Xpert MTB GeneXpert Time-to-result: 1 h 45 min FIND 2009 Evaluation Partner Sites FIND 2009 Xpert MTB High tech for low tech settings: Sensitivity and Specificity seems to be very good for the detection of TB and Rifampicin resistance. Drug Susceptibility Testing Solid Media 3 - 4 weeks Liquid Media 7 - 10 days Molecular based Methods Hours – 1day Löwenstein-Jensen (Middlebrook) BACTEC 460 TB MGIT InnoLipa GenoTypeMTBDR Xpert MTB ‚home made‘- methods Quality Control internal external Internal Quality Control all reagents all techniques all staffs Internal Quality Control All QC results have to be documented Drug Susceptibility testing 1 2 3 4 5 7 6 embB MUT1B embB MUT1A embB WT1 embB rrs MUT2 rrs MUT1 rrs WT2 rrs WT1 rrs gyrA MUT3D gyrA MUT3C gyrA MUT3B gyrA MUT3A gyrA MUT2 gyrA MUT1 gyrA WT3 gyrA WT2 gyrA WT1 gyrA TUB AC CC 8 9 10 11 External Quality Control Participation in international QA programs Reliable results To detect and treat patients properly