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Microbiological diagnosis of TB

Microbiological diagnosis of TB. José Domínguez 1 and Sabine Rüsch-Gerdes 2 1 Servei de Microbiologia. Fundació Institut en Ciències de la Salut Germans Trias i Pujol. Badalona. Spain 2 Forschungszentrum Borstel, National Referencelaboratory for Mycobacteria. Borstel. Germany.

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Microbiological diagnosis of TB

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  1. Microbiological diagnosis of TB José Domínguez1 and Sabine Rüsch-Gerdes2 1Servei de Microbiologia. Fundació Institut en Ciències de la Salut Germans Trias i Pujol. Badalona. Spain 2 Forschungszentrum Borstel, National Referencelaboratory for Mycobacteria. Borstel. Germany

  2. 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

  3. One of the main objectives for TB control Histology Microbiology Clinical suspicion • 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

  4. Diagnosis of tuberculosis Latent Infection Active tuberculosis Smear examination TST Molecular Epidemiology Solid and liquid culture IFN- techniques Identification RFLP MIRU Susceptibility testing methods • Molecular methods • Detection • Identification • Detection of resistance Spoligotyping

  5. 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

  6. Decontamination Homogenization Phosphate buffer Centrifugation Sample mixing Pellet • Eliminate normal flora from the non-sterile samples • (micobacteria is acid and alkaline resistant) • Homogenizationto 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

  7. Smear microscopy Auramina O stain Ziehl-Neelsen stain Fast; Cheap; Monitorization of treatment; Low sensitivity Hospital Univ. Germans Trias i Pujol 2004 -2007 *Adenopathy, 4/28 (14.2%); Pleural, 1/17 (5.9%)

  8. Culture in solid and liquid media Decontamination (in non sterile samples) Culture in the adequate media Inoculums!! Growth in solid media Slow: 15d-2m Division time 18h Growth in liquid media 7-42d Identification Classical and Molecular methods DST • Sometimes the only place where the mycobacteria can be isolated • Gold standard • Molecular epidemiology • Drug susceptibility testing

  9. Identification molecular methods AccuProbe InnoLiPA Mycobacteria GenoType Mycobacterium CM/AS, GenoType MTBC

  10. Sequencing 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).

  11. PPi ATP Pyrosequencing

  12. M.tuberculosis detection in clinical samples by molecular methods * In smear negative samples the sensitivity is reduced in a 50%

  13. 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.

  14. RFLP (IS6110) Spoligotyping MIRUs Molecular epidemiology

  15. 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. Restriction Fragment Length Polymorphism (RFLP)

  16. Pvu II Electrophoresis Extraction and restriction Radiographic develop Hybridization Transference

  17. 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. Spacer oligonucleotype typing (Spoligotyping)

  18. 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

  19. Micobacterial Interspersed Repetitive Units (MIRU)

  20. MIRU 40 4 3 2 500 pb 1

  21. 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.

  22. Microbiological Diagnosis of TB Drug Susceptibility Testing Borstel 2010 National Reference Laboratory for Mycobacteria Forschungszentrum Borstel Sabine Rüsch-Gerdes

  23. Drug Susceptibility Testing

  24. MDR TB – New Infection WHO: MDR-TB & XDR-TB, The 2008 Report; February 2008

  25. Drug Susceptibility Testing Anteil resistenter Erreger in Deutschland, 2006 For all TB strains isolated, DST has to be performed Quelle: RKI, Bericht zur Epidemiologie der Tuberkulose in Deutschland für 2006; 2008

  26. BiostoffVerordnung (BioStoffV) 1999 Microscopy Culture NAT Microscopy Culture NAT Differentiation DST Level 3 laboratory Level 2 laboratory

  27. 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

  28. Methods for Drug Susceptibility Testing BACTEC 460TB All drugs except cycloserine Results available: 1-2 weeks Radioactive materials, waste

  29. Methods for Drug Susceptibility Testing MGIT 960 For all drugs, except CS Results available: 1-2 weeks

  30. 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

  31. 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

  32. Principle of the Line Probe Assays Chromogen (MBT/BCIP) Colour reaction Alkaline Phosphatase Streptavidin Biotin Biotin-labelled single stranded amplified target DNA-probe Nitrocellulose strip

  33. 1 2 3 4 5 6 7 8 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) - MTBDR – DNA Hybridisation Strip

  34. Genotype® MTBDRplus Hybridization-Strip 1 2 from Culture media 1 RMPr INHr 2 RMPs INHs

  35. 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 + + 3 strains (2,9 %) with a mutation in inhA 1 strain (1%) a mutation outside rpoB cluster I + 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)

  36. Genotype® MTBDRplus Hybridization-Strip 1 2 1 RMPr INHr 2 RMPs INHs 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.

  37. Evaluation of the MTBDRplus Assay on Specimens 72 smear positive sputum specimens: 30 susceptible strains 32 MDR (RMPr/INHr) strains 10 INHr strains Sensitivity RMP detection: 96.8% INH detection: 90.2% Specificity RMP: 95.2% INH: 100%

  38. Line Probe Assays for DST • INNO-LiPA Rif TB • GenoType MTBDRplus • GenoType MTBDRsl Evaluated line probe assays Resistance Sensitivity Specificity RMP 98.1% 98.7% INH 84.3% 99.5% Ling et al., Eur Respir J 2008

  39. Molecular Basis

  40. 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.

  41. GeneXpert Xpert MTB Time-to-result: 1 h 45 min FIND 2009

  42. Evaluation Partner Sites FIND 2009

  43. Xpert MTB High tech for low tech settings: Sensitivity and Specificity seems to be very good for the detection of TB and Rifampicin resistance.

  44. Drug Susceptibility Testing 3 - 4 weeks Solid Media Löwenstein-Jensen (Middlebrook) 7 - 10 days Liquid Media BACTEC 460 TB MGIT Molecular based Methods InnoLipa GenoTypeMTBDR Xpert MTB ‚home made‘- methods Hours – 1day

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