Download
slide1 n.
Skip this Video
Loading SlideShow in 5 Seconds..
Thank you for viewing this presentation. PowerPoint Presentation
Download Presentation
Thank you for viewing this presentation.

Thank you for viewing this presentation.

99 Vues Download Presentation
Télécharger la présentation

Thank you for viewing this presentation.

- - - - - - - - - - - - - - - - - - - - - - - - - - - E N D - - - - - - - - - - - - - - - - - - - - - - - - - - -
Presentation Transcript

  1. Thank you for viewing this presentation. • We would like to remind you that this material is the property of the author.It is provided to you by the ERS for your personal use only, as submitted by the author. • 2012 by the author

  2. The new horizons of molecular diagnosis: do we still need conventional microbiology? D. Cirillo Emerging Bacterial Pathogens Unit WHO CC for integrated laboratory strengthening on Tb and other emerging infections S.Raffaele Scientific Institute ,Milan

  3. Outline • Introduction: role of the laboratory in TB diagnosis • Development and spreading of MDR-TB • Challenges of conventional laboratory diagnosis of tuberculosis • Molecular tests for 1st line drugs resistance detection: • LPAs; Xpert TB Rif • Interpretation of the results • Performance on EP samples • WHO recommendations and diagnostic algorithms • Conclusions • Molecular epidemiology tools

  4. Introduction • Care of TB patients starts with a QA diagnosis • A robust network of Tb laboratories is required: • Adaquate biosafety • Modern diagnostics • SOPs • QAs • Integrated laboratory network

  5. Laboratory biosafety • Mtb is a class 3 risk pathogen • All biosafety strategies (minimum requirements) should be based on procedures risk assessment • Based on: • Bacillary load of samples and workload • Viability of bacilli • Aerosol generation • TB local epidemiology • Fitness of the staff

  6. Technologies and laboratory appropriateness Introducing new technology requires addressing of core elements: • Infrastructure, biosafety measures and maintenance • Equipment validation and maintenance • Specimen transport and referral mechanisms • Management of laboratory commodities and supplies • Laboratory information data and management system • Laboratory quality management • Strategies for HR development and retention GLI road map at :http://www.who.int/tb/dots/laboratory/policy/en

  7. Role of molecular biology • Fast detection and confirmation of pTB cases • Detection of MDR-TB • Detection of EP-TB • Performances and cost implications: • LAMP assay has not been endorsed for incomplete evidences • Other PCR/molecular tests never examined by WHO for absence of large scale evidences

  8. Samples appropriateness and patients selection • Quality and amount of samples are crucial for molecular tests as well • Pretest probability highly influences the parameters of molecular tests

  9. Role of the clinical suspicion level inthe evaluation of the molecular methods Catanzaro A. et al JAMA 2000 Gold standard: microbiology or clinical?

  10. Development and spreading of DR-TB MDR-TB in Europe XDR-TB in the world WHO, Global Report 2011 Devaux I et al 2009. Emerg Infect Dis 15(7):1052-60 18 clusters ,7 belonging to Beijing lineage 2007: 41 Countries 2010: 58 Countries • Causes: • patient’s related • physician’s related • Drug’s/ program’s related • Crucial health problem: • long/expensive treatment • second line drug •  decreased cure rate, increased side effects

  11. Conventional DST: technical challenges • Adequate infrastructures and biosefety levels • MGIT DST: thegold standard • MDRTB : 3-6 weeks; XDRTB : 6-9 weeks • Reproducibility and accuracy of results are drugs dependent: • Rifampicin, isoniazid : good results • Second-line: non raccommended in the absence of CQ (200 samplea ad high risk/year) Standardization and correlation with the clinical outcome is difficult to be achieved Van Deun A. et al 2011. IJTLD 15(1):116-124

  12. MOLECULAR DST ON M. tuberculosis • Based on single nucleotide mutations detection • Data on DST from specimens not suitable for culture • Cross-resistance prediction • Large number of specimens analysed at the same time • Standardization (automated systems) and TAT • Cost-effectiveness • Only available for selected drugs • Only available for selected specimens • Low sensitivity in AFB-negative and non-respiratory samples • Genetic diversity may influence statistical parameters of molecular tests

  13. Genes involved in drug-resistance for major anti-tubercular drugs First-line drugs Second-line drugs Zhang Y et al 2009. IJTLD 13(11):1320–1330

  14. Commercial tests for MDR-TB Diagnosis WHO Global plan (2006-2015):development and roll out of new technologies to be adopted in resources-limited settings • GenoType MTBDRplus, InnoLiPA Rif.TB • Reverse hybridization, colorimetric reaction • Results in 6-7 h • some flexibility (n° probes/strip: 30-40) • Technical expertise: some • Xpert MTB/RIF • Integrated/automated qPCR • Results in 2h • Closed system (limited number of probes: <10) • Technical expertise: none

  15. LiPA Tests: work flow

  16. LPAs performance Isoniazid Rifampicine Inno-LiPA Rif.TB GenoType MTBDRplus GenoType MTBDRplus Ling DI et al 2008. Eur Respir J 32:1165-1174 Morgan M et al 2005. BMC Infect Dis 5:62 Ling DI et al 2008. Eur Respir J 32:1165-1174 cod. 315 gene katG nt -8,-15,-16 gene inhA Hot-spot gene rpoB Sensitivity 95-98% Specificity 98-100% Sensitivity 95-99% Specificity 97-100% Clinical samples Sensitivity 95-99% Specificity 97-99% Sensitivity 82-93% Specificity 95-100% Clinical samples Sensitivity 72-92% Specificity 96-99%

  17. LPAs Performance Few data available on the LPAs performance on smear negative samples • MTBDR vs MTBDRplus • Improvement of performance of 20-25% • Decreasing of indeterminates of about 10-15% • LPAs are approved for AFB positive respiratory samples Miotto P et al 2008. J Clin Microbiol 46(1):393-4 TaT for LPA: 1-2 days • 3° generation (GenoType MTBDRplus v. 2) commercially available since January • AFB-negative and «scanty» • Master mix ready and stabilized • Easily interpretable hybridization «pattern»

  18. Possible automation on LiPA LiPAs require: Level II biosafety areas Skilled laboratory staff Amplicon Contamination control

  19. Xpert MTB/RIF: work flow Boehme CC et al 2010. N Engl J Med 363(11):1005-15

  20. Xpert MTB/RIF: performance Hot-spot gene rpoB Indeterminate: <2.5% (culture contamination: 4.7%) Tb cases identification Specificity 98-99% Sensitivity 97-100%, AFB-pos. 75-84%, AFB-neg. Xpert MTB/RIF, microscopy 0-1 d Liquid Culture 13-21 d Solid Culture 23-43 d Rifampicin-Res Identification Specificity 97-99% Sensitivity 91-97% Co-infection TB-HIV Sensitivity 86% (HIV-neg: 92%) No differences in performances in AFB-neg. (microscopy: 47% in HIV-positive vs 65% in HIV-negative) Boehme CC et al 2011. Lancet 377(9776):1495-505 Theron G et al 2011. Am J Respir Crit Care Med 184:132-140

  21. LiPA e Xpert MTB/RIF Rifampicin resistance identification Time to report to treatment center Boehme CC et al 2011. Lancet 377(9776):1495-505 Xpert MTB/RIF: 0-1 d LPA: 10-26 d* DST in culture: 30-124 d** Xpert MTB/RIF: 0-1 d (Microscopy: 1-2 d) LPA: 27-53 d* DST in culture: 38-102 d** (Culture: 42-62 d) Some results not reported/lost * Test on direct AFB pos sample + test on strain for culture pos smear neg samples ** DST on MGIT + DST on LJ

  22. Xpert MTB/RIF WHO/HTM/TB/2011.2

  23. Potential limits of Xpert MTB/RIF technology • Unknown performance at a district level • Unknown performance in children • If RFP resistance is diagnosed in a low level MDR-TB prevalence setting , the assay needs to be confirmed • Testing for Rif-R only • Need to perform a culture for DST to evaluate other drug resistance • Need to perform a culture for monitoring issue (culture conversion) • It requires uninterrupted and stable electronic power supplies and yearly calibration • Storage of reagents

  24. Cost consideration • Cost–effectiveness modeling: • Increase of 30% of case finding if replacement or add-on to microscopy • Cost-comparison • Current cost (16.86$) per test higher than microscopy, lower than culture/DST on solid and liquid media may drop to 10$ in the future • Initial capital cost: higher that microscope lower than a biosafe culture laboratory

  25. Cost effectiveness Courtesy of C Boheme

  26. WHO recommended policy • Approved for smear-negative cases • Biosafety at microscopy level • No technical skill required • Annual module’s calibration • Distrect peripheral labs • Appropriate transport and storage of reagents • High NPV (99%) • Rif-RES cases to be reconfermed by LiA /colture if prevalence of RIF-R è <10% • Strains or AFB positive respiratory samples • Adequate infrastructures (biosafety, molecolar biology) • Tecnical capacities (supervision, QC) • Appropriate transport and storage of reagents • Central or Regional level • INH drug-sensitive cases needs to be confermed by culture • Test to be adopted in settings with adequate capacity and resources in agreement with local NTP and WHO reccomandations • Reference test for MDR suspects ,TB/HIV

  27. Performance of Xpert on extra-pulmonary specimens (adults and pediatrics)* * Tortoli et. Al ERJ 2012

  28. Comparison GenoType® MTBDRplus and XpertTB/MDR Intermediate Reference labs Patients testing from Sm/ C+ (Rif) Fast tool Surveillance purposes Potential to District level as fast patients diagnostic tool, needs evaluation at district level

  29. Heteroresistance: mixed population of sensitive and resistant bacteria “…Our data show that cultures are not necessarily representative of what is in the clinical specimen. Out of 54 PCR products amplified from DNA isolated from sputum samples of 48 patients in whom drug resistances were known or suspected, a large proportion (17%) showed more than one genotype by RFLP analysis…” [Rinder H et al 2001. IJTLD 5:339-345] Mixed bacterial population cannot be identified by sequencing but can be risolved by LPAs.

  30. Molecolar diagnosis of resistance to drugs other than R and H: the GenoType MTBDRsl test (Hain Lifescience) Analyzed codons: gyrA : 85-97 rrs:1401, 1402,1484 embB: 306

  31. GenoType MTBDRsl: performance • High PPV and specificity  rapid identification of resistant cases • Low sensitivity and NPV  need to confirm SENSITIVE cases by conventional DST • Can be used for screening MDR-TB cases at high risk to develop XDR-TB • For ETB sensitivity is increased (15-20%) when using the presence of mutations as marker for resistance • Overall diagnosis of XDR-TB: 44.4%  additional studies and markers are needed Miotto P et al. ERJ 2012

  32. Ethambutol resistance: is molecular detection of resistance better than MGIT DST? • Ethambutol in MGIT: • Decreaesed sensitivity and specificity • Reduced riproducibility • False-sensitive using 5 μg/mL as break point Scarparo C et al 2008. J Clin Microbiol 42(3):1109-1114 Van Deun A et al 2011. IJTLD 15(1):116-124 70-80% of ETB-resistant isolates are mutated in the codon 306 of embB Gene 84 clinical isolates ETB-R (MGIT, 5 μg/mL) M306V 42,9% Other mutations cod. 306 13,1% Non mutated 44,0% 91 clinical isolatesi ETB-S (MGIT, 5 μg/mL) M306V 18,6% Other mutations cod. 306 24,3% Non mutated 57,1% >95% results ETB-resistant if MIC is determined Miotto P et al. Manuscript submitted 2011

  33. Genetic diversity in M. tuberculosis Mutation rate is different in different geografic areas: Phiilogeographic distribution in M. tuberculosis: Gagneux S et al 2006. PNAS 103:2869-2873

  34. Genetic diversity in M. tuberculosis and implications on genotypic detection of Drug Resistance Experimental evidences show the association of specific mutations responsable for MDR phenotype to selected genotypes (eg. Beijing) Little is known for second line drugs? Miotto P et al ERJ 2012

  35. Common problems in interpreting molecular results • Discrepancies genotype/phenotype: • False negative due to duplication • Double pattern

  36. Common problems in interpreting molecular results: INH, E • Is therapy modified based on resistance data? • inhA - 15 alone: increased mic, needs to follow closely over time • Resistance to Ethionamide • Eth 306: main mechanism fot ETH resistance

  37. A common problem: the “double pattern” • Hetero-resistance = equal representation of susceptible and resistant mutants of the same strain • Mixed pattern = mutual presence of a resistant strain and a second, susceptible strain • Not pure culture • Carry-over contamination Further research is needed to clarify the clinical role of selected mutations or mixed infections

  38. PropidiumMonoazideTM (PMA) • PMA is a membrane impermeant intercalating into free extracellular DNA and DNA from nonviable bacteria • PMA is excluded from viable bacteria. • Exposure to a light source makes the PMA-DNA complex not amplifiable. Codons analysed:

  39. PMA pretreatment of clinical samples allows selective amplification of the DNA derived from live bacteria Comparison between DNA amplified from PMA treated (- -) and untreated () sputum samples collected at diagnosis (t0) and at 14 days from beginning of antitubercular therapy Not treated PMA treated Miotto P et al. ERJ 2012

  40. Non-tuberculousmycobacteria (NTM) • Most mycobacteria are saprophytic bacteria but some NTM are occasionally pathogenic to both humans and animals, causing pulmonary, skin diseases, lymphadenitis and disseminated infections. • Increase in infections caused by NTM • Diseases caused by NTM are often associated with various forms of immunosuppression, particularly HIV infection • Mycobacterium avium complex (MAC) • M. avium (subspparatuberculosis, lepraemurium, and silvaticum), M. intracellulare • Mycobacterium ulcerans(skin infections) • Mycobacterium marinum • Mycobacterium xenopi, Mycobacterium malmoense(lung disease) • Mycobacterium kansasii

  41. Molecular DST in TB: clinical impact • The use of MTBDRplus test, even in the absence of culture and • DST, allowed readjustment of patients’ treatment in Burkina Faso: • - Patients who were classified and treated as MDR cases harbouring RIF- and INH-S strains (n 26); • - Patients negative for MTB complex DNA (n 18); • - Patients with a non-tuberculous mycobacteria (NTM) infection (n 14). • Conclusions • Smear microscopy is not sufficient for management of chronic patients (NTM infections) • Molecular assays can identify MDR-TB cases in the absence of culture facilities. • Molecular assay for second-line drug resistance identification allowed identification of XDR cases if mutations are present but can’t exclude resistance Miotto P et al. BMC Infect. Dis. 2009; 9:142 Badoum et al. ERJ 2011 41

  42. Drug resistance: what’s new? • March 2012, WHO: RIF-R is not equivalent to MDR for surveillance purposes • There is an increasing need for DST testing on anti-TB drugs: Moving forward to personalized therapeutic regimens Characterization of novelmutationsinvolved in drug-resistantphenotype and virulencemarkers • AMK/KAN/CAP: Rv3919c (gidB), Rv2416 (eis) • Characterization of mutations occurring in genes encoding putative targets for new drugs (nitroimidazopyran, linezolid) • Compensatory mutations in MDR-TB strains (Comas et al. 2011, Nat Genetics ) • Better understanding of genetic diversity and drug resistance relationships

  43. Molecular DST in TB: wrap up 2/2 • Clinically relevant when/where culture facilities are not easily available • Possibility/need to perform molecular DST on an increasing number of drugs • Increasing possibility to use molecular approaches not only in case detection step, but also in further steps (e.g. treatment monitoring) • Increasing need to better understand infection aethiology and/or mixed infection (e.g. NTM) • However… • Sensitivity affected by MTB epidemiology • Limited number of targets that are detectable per assay (thus limiting molecular approach potential usefulness) • cannot exclude phenotypic resistance (wild-type result)

  44. Molecular DST in TB: wrap up 2/2 • Clinically relevant when/where culture facilities are not easily available • Possibility/need to perform molecular DST on an increasing number of drugs • Increasing possibility to use molecular approaches not only in case detection step, but also in further steps (e.g. treatment monitoring) • Increasing need to better understand infection aethiology and/or mixed infection (e.g. NTM) • However… • Sensitivity affected by MTB epidemiology • Limited number of targets that are detectable per assay (thus limiting molecular approach potential usefulness) • cannot exclude phenotypic resistance (wild-type result)

  45. Lab-on-Chip (LoC) platforms: a possible answer? Tuberculosis Malaria • Rapid identification of MTB complex • Rapid diagnosis of MDR cases • Rapid genotyping of MTBC • Rapid identification of clinically relevant NTMs Rapid identification of Plasmodium pathogenic species Rapid diagnosis of malaria drug resistant cases The integrated PCR and Microarray lab-on-chip tool should represent a clear innovation over the conventional molecular diagnostics for its robustness, simplicity of use and low-cost.

  46. Lab-on-Chip for molecular diagnostics PCR: • Ultra-Fast PCR • Asymmetric Cy-5 PCR strategy Microarray: • Orientation probes • Hybridization Control probes • Hybridization Negative Controls probes Lab-on-chip architecture 2 PCR reactors of 12.5 uL volume each (Total 25 ul) 1 Hybridization chamber of 30 uL A 126 spots DNA microarray 2 in-let ports compatible with standard micro-pipettor tips Integrated Heaters and Sensors All the reaction modules are fluidically integrated

  47. Drug resistance Species identification Statisticalanalysis on clinicalisolates • Detection limit: • rpoB target: 104/mL (AFB: 1+ / scanty) • Allother targets: < 102/mL (AFB: scanty) • Speciesidentified: • M. tuberculosiscomplex • M. avium • M. intracellulare • M. simiae, M. kansasii, M. scrofulaceum • M. abscessus, M. chelonae • M. xenopi • M. fortuitum

  48. E@syCheck software Chip information Summary of results Automatic generation of final report

  49. TB LoC: adds on • Genotyping by spoligotyping • Directly used for specimens • “Real-time” typing (nosocomial transmission, prisons, community etc…) • Laboratory cross-contaminations • M. tuberculosis complex genotype identification (family) • TaT: 6-7 h, up to 43 samples per run nnnnnnnooooonnnnnn…. (binary-code translation) Automatic report

  50. Geographical distribution: the advantages of multi-purposes platform TB TB/HIV Malaria Neglected tropical diseases Dengue Buruli ulcer treponematoses Leprosy cysticercosis dracunculiasis lymphatic filariasis human rabies Trachoma …