Antonino Di Caro UOC Microbiologia, Banca Biologica e Cell factory

Transcription

1 Ottimizzazione dei percorsi diagnostici per la diagnosi delle infezioni da micobatteri Antonino Di Caro UOC Microbiologia, Banca Biologica e Cell factory Istituto Nazionale per le Malattie Infettive «L. Spallanzani» IRCCS 20 Marzo 2017, Roma

2 Mycobacterium tuberculosis

3 Diagnosis of tuberculosis Latent Infection Active tuberculosis TST IFN- techniques Molecular methods -Detection -Identification -Detection of AMR Smear examination Solid and liquid culture Identification Susceptibility testing methods NGS Molecular Epidemiology Detection of AMR RFLP MIRU Spoligotyping

4 AFB Acid Fast Bacilli smear microscopy AFB smear microscopy, developed more than 100 years ago, remains the worldwide most common method in use in which bacteria are directly observed in sputum and other respiratory or non-respiratory samples. World health Organization Implementing Tuberculosis Diagnosis recommend that 2 (on induced sputum) to 3 smear tests (sputum) must be performed on suspected Automatic patients. Manual The sensitivity of direct examination after coloration for the diagnosis of pulmonary tuberculosis ranges between 50% and 80% of positive cultures and is limited to samples with more than 10^4 mycobacteria/ml Steingart 2006, Lancet; El Kechine, 2011

5 AFB Acid Fast Bacilli smear microscopy Fast; Cheap; Monitorization of treatment; Low sensitivity Ziehl-Neelsen stain Auramina O stain AFB smear result Colture result for TB Interpretation Positive Positive Presumptive diagnosis for TB Negative Positive Colture is more sensitive than smear so this may occur in people with true disease; may test additional samples using NAAT. Positive Negative Questionable results for TB; an inhibitor (normal flora) may be present in the specimen or the AFB seen on the smear are not M. tuberculosis. A colutre test for the inhibitor may be performed. Negative Negative Symptoms probably not due to active mycobacterial infection.

6 Decontamination Eliminate normal flora from the non-sterile samples (Mycobacteria are acid and alkaline resistant) Homogenization to release the bacteria from the sample and allow access to the nutrient present in the media N-acetyl-cysteine: homogenization NaOH: decontaminant Neutralization by phosphate buffer (PBS) Homogenization Sample mixing Phosphate buffer Centrifugation Pellet

7 Culture in solid and liquid media GOLD STANDARD!!! Decontamination (in non sterile samples) Culture in the adequate media Inoculums!! Growth in liquid media 7-42d Growth in solid media Slow: 15d-2m 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

8 Come ottimizzare l uso dei test molecolari

9 Molecular identification AccuProbe InnoLiPA Mycobacteria GeneXpert GenoType Mycobacterium CM/AS, GenoType MTBC TRCReady-80

10 Molecular identification Available Home made PCR for M. tuberculosis identification Method Material Target Processing Infrastructure* Equipment** Training Sensitivity, Specificity Rererences PCR Decontaminated Sputum, human respiratory samples and extrapulmonary samples IS6110 Manual Biosafety level 2; Biosafety level 3 Low Minimal De Almeida In, 2015 PCR Decontaminated sputum mpt64 Manual Biosafety level 2; Biosafety level 3 Low Minimal Watanabe JM, 2015 PCR Decontaminated sputum sdaa Manual Biosafety level 2; Biosafety level 3 Low Minimal Nimesh M, 2013 PCR Extrapulmonary samples IS1610 Manual Biosafety level 2; Biosafety level 3 Low Minimal Montenegro LM, 2013 PCR Decontaminated IS986 Manual Biosafety level 2; Biosafety level 3 Low Minimal Greco S, 2006 PCR Sputum, human respiratory samples and extrapulmonary samples 16S rrna and 23S rrna Manual Biosafety level 2; Biosafety level 3 Low Minimal Greco S, 2006

11 Molecular identification Commercially available NAAT assays for TB detection in clinical samples using DNA target Assay Manufacturer Method Material Processing Target Infrastructure* Equipment** Training Sensitivity Specificity Rererences COBAS Taq ManW MTB Test Roche Molecular Diagnostics, Pleasanton, CA, USA RT-PCR DNA from decontaminated human respiratory samples Automatic 16S DNA Biosafety level 2 High Moderate 73.6 to Fegou E., 2005 Seegene Anyplex Seegene TECHNOLOGIES Inc.(U.S.A) RT-PCR DNA from decontaminated human respiratory samples; Automatic IS6110 and MPB64 Biosafety level 2 High Minimal Perry D., 2014 BD ProbeTec ET assay Becton Dickinson, Sparks, MD, USA Strand Displacement amplification DNA from decontaminated sputum Automatic IS6110 Biosafety level 2 High Moderate 1.00; ; 95.8 Karadağ A., 2013; Wang JY, 2007) LCx Abbott Laboratories, USA, Abbott Park, IL, USA Ligase chain reaction respiratory samples and CSF Automatic - Biosafety level 2 High Moderate Piersimoni C, 2005; Ribeiro FK, 2004; Chua HC, 2005 Artus W M. tuberculosis PCR Qiagen, Hilden, Germany RT-PCR DNA from decontaminated sputum Automatic 16S DNA Biosafety level 2 High Minimal Hur M, 2011 Loopamp W Tuberculosis Complex Detection Reagent Kit EIKEN Chemical, Tokyo, Japan LAMP Untreated sputum Automatic - Biosafety level 2 High Minimal Mitarai S, 2011 Real Art MTB PCR - qrt-pcr Respiratory samples Automatic 16s rdna Biosafety level 2 High Minimal MTB Q PCR Alert Nanogen Advanced Diagnostic RT-PCR Sputum, Exudates, Urine Automatic IS6110 Biosafety level 2 High Minimal Seagar AL, 2012 Quenching Probe PCR (genecube) Toyobo, Osaka, Japan RT-PCR Respiratory samples Automatic dnaj Biosafety level 2 High Minimal Hida Y, 2012 Real Accurate Mycobacterium tuberculosis PCR kit (PathoFinder) PathoFinder BV, Maastricht, the Netherlands RT-PCR DNA from decontaminated sputum Automatic IS6110 Biosafety level 2 High Moderate - - -

12 Molecular identification Commercially available NAAT assays for Mycobacteria detection in clinical samples using RNA target Assay Manufacturer Method Material Processing Target Infrastructure* Equipment** Training Sensitivity, Specificity Rererences Internal transcribed sequence highresolution melting (ITS- HRM) and Roche HRM master mix (in-house ITS- HRM) Roche diagnostic Nederland BV genusspecific PCR DNA from decontaminate d sputum Automatic 16S rrna of bp 584 Biosafety level 2 High Minimal - - Roth A, 2000 GenoType Mycobacteria direct Hain Lifescience RT-PCR Decontaminate d clinical specimens Automatic 23S rrna Biosafety level 2 High Minimal F. Franco- Álvarez de Luna, 2006 Amplified Mycobacteriu m tuberculosis Direct Test (MTD) Gen-Probe, Inc., San Diego, CA RT-PCR Decontaminate d clinical specimens Automatic 16S rrna Biosafety level 2 High Minimal 0.99; ; 0.97 Kobayashi M, 2014; Chen X, 2012 The Cobas TaqMan MTB Roche Diagnostics (Rotkreuz, Switzerland) RT- PCR Decontaminate d clinical specimens Automatic 16S rrna Biosafety level 2 High Moderate Bloemberg GV, 2013 Transcription- Reverse Transcription Concerned Reaction (TRCR) Tosoh Bioscience, Tokio TRCR Respiratory and non respiratory samples Automatic 16S rrna Biosafety level 2 High Moderate Tanaka H, 2010

13 Molecular identification Main commercial assay for MTB gene resistance Assay Manufacturer Method Material Processing Target Infrastructure * Equipment** Training Sensitivity Specificity Rererences INNO-LiPA Rif.TB Innogenetics, Ghent, Belgium Reverse-Based Hybridisation technique. DNA from decontaminate d sputum Automatic rpob Biosafety level 2 High Minimal - - Bhutia R., 2013 GenoType MTBDR Hain Lifescience, Nehren, Germany RT-PCR DNA from decontaminate d sputum or extrapulmona ry samples Automatic rpob, katg Biosafety level 2 High Minimal Lyu J, 2013 GeneXpert MTB/Rif assay Cepheid, Sunnyvale, USA RT-PCR Sputum or extrapulmona ry Automatic rpob Biosafety level 2 High Minimal Hervé C, 2015

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15 Since 2013, the World Health Organization has recommended the Xpert MTB/RIF as the initial test for tuberculosis microbial diagnosis for patients with suspected pulmonary tuberculosis including new cases; for retreatment cases; for suspected multidrug-resistant (MDR) tuberculosis; and for HIV-infected patients with suspected tuberculosis because of its excellent sensitivity and specificity associated with an extremely short turnaround time.

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17 INMI experience The aim of our work is to evaluate this new molecular assay in comparison with AFB and culture. From October 2015 to May 2016, 978 respiratory samples (sputum, BAL and induced sputum) from 378 patients were collected at L.Spallanzani Hospital in Rome. Samples were analysed by TRCReady-80 MTB, Tosoh Bioscience, Tokyo, Japan, Acid-Fast bacilli (AFB) staining, solid and liquid medium culture were performed on all samples.

18 System Overview Sample, 500 ul Purification Cartridge TRC reagent A TRCReady-80 TRC reagent B Tip set

19 TRCReady MTB vs CULTURE Our study PATIENTS TRCReady MTB TB culture confirmed Total Specificity: 100% Sensibility: 92.0% NVP: PPV: 1.00 Total

20 TRCReady MTB + 2 AFB vs CULTURE PATIENTS TRCReady MTB+ 2 AFB TB culture confirmed Total Specificity: 100% Sensibility: 97.29% NVP: PPV: 1.00 Total

21 TRCReady-80 Result Screen

22 PLOS ONE DOI: /journal.pone August 10, 2016 O. Opota et al. / Clinical Microbiology and Infection 22 (2016) 613e619

23 Diagnosis of tuberculosis Latent Infection Active tuberculosis TST IFN- techniques Molecular methods -Detection -Identification -Detection of AMR Smear examination Solid and liquid culture Identification Susceptibility testing methods NGS Molecular Epidemiology Detection of AMR RFLP MIRU Spoligotyping

24 CHARACTERIZATION OF MTB CLUSTERS BY WGS AT INMI Clusters identified by genotyping and classical epidemiology Additional clusters identified by WGS Naizgy Gebremedhin Brahne Enjay Naizgy Gebremedhin Brahne

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27 CROI 2017

28 Grazie per la Vostra attenzione