2. 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

3. 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

4. One of the main objectives for TB control “Diagnosis, diagnosis & diagnosis” William Osler Histology Clinical suspicion Microbiology 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

5. Diagnosis of tuberculosis Latent InfectionActive tuberculosis Smear examination Solid and liquid culture Identification Susceptibility testing methods TST IFN-  techniques Molecular Epidemiology RFLP Spoligotyping MIRU Molecular methods -DetectionDetection -IdentificationIdentification -Detection of resistanceDetection of resistance

6. 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

7. 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 Sample mixing Phosphate buffer Centrifugation Pellet

8. Smear microscopy Ziehl-Neelsen stain Auramina O stain Fast; Cheap; Monitorization of treatment; Low sensitivity Hospital Univ. Germans Trias i Pujol 2004 -2007 PulmonaryDisseminatedExtrapulmonary * TOTAL1251860 Positive smear83 (66.4%)11 (61.1%)11 (18.3%) Negative smear42 (33.6%)7 (38.9%)49 (81.7%) *Adenopathy, 4/28 (14.2%); Pleural, 1/17 (5.9%)

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

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

11. Identification molecular methods Sequencing 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).

12. Pyrosequencing PPi ATP

14. M.tuberculosis detection in clinical samples by molecular methods MethodTargetDetection method Sensitivity in respiratory samples (%) Sensitivity in extra respiratory samples (%) Overall specificity (%) AMTD216S rRNAChemiluminometric80-10060-9095-100 LCxb antigenic proteinFluorimetric80-9065-8090-100 AMPLICOR16S rRNAColorimetric75-10045-6090-100 BD ProbeTec IS6110 and 16S rRNA Fluorimetric55-10030-8045-100 INNO-LIPA v2IR16S-23SColorimetric50-9560-8090-100 GenoType Direct 23S rRNAColorimetric60-9560-8095-100 PCR real time16S rRNAFluorimetric70-9065-8585 * In smear negative samples the sensitivity is reduced in a 50%

15. 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.

17. RFLP (IS6110) Spoligotyping MIRUs Molecular epidemiology

18. 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)

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

21. 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)

24. 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)

26. MIRU 40 500 pb 1 4 3 2

27. 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. Conclusions

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

29. Drug Susceptibility Testing

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

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

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

33. 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

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

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

36. Advantages compared to solid media: more rapid high quality of media fully automated system testing of 1 st, 2 nd, and new drugs safety: plastic tubes Disadvantages: expensive higher contamination rate dependency on a company no DST for Cycloserine Liquid media compared to solid media

37. 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

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

39. 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) - 1 2 3 4 5 6 7 8 MTBDR – DNA Hybridisation Strip

40. 1 2 Genotype® MTBDRplus Hybridization- Strip 1 RMP r INH r 2 RMP s INH s from Culture media

41. Results RMP+INH Resistance 100 % concordance between sequencing and MTBDR data 103 MDR strains 91 strains (88.4) with mutations in codon 315 of katG 102 strains (99%) mutations in rpoB cluster I 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) + + +

42. 1 2 Genotype® MTBDRplus Hybridization- Strip 1 RMP r INH r 2 RMP s INH s 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.

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

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

45. Molecular Basis

46. 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 Ofl r strains rrs A1401G AM r /CM r strains embB M306V in EMB r strains The rapid detection of XDR strains is possible with the combined application of Lipas from DNA isolates and directly from sputum specimens. Summary

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

48. Evaluation Partner Sites FIND 2009

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

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

51. Quality Control internal external

52. Internal Quality Control all reagents all techniques all staffs

53. All QC results have to be documented Internal Quality Control

54. 12 3 45 6 78 9 1011 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 Drug Susceptibility testing

55. External Quality Control Participation in international QA programs

56. Reliable results To detect and treat patients properly