Rapid molecular diagnosis of TB and drug-resistant TB
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1 Rapid molecular diagnosis of TB and drug-resistant TB 2 nd European Advanced Course in Clinical Tuberculosis Amsterdam 2014 J. Domínguez Institut d Investigació Germans Trias i Pujol Universitat Autònoma de Barcelona jadomb@gmail.com
2 Utility of the molecular methods in tuberculosis To rapidly diagnose patients with active TB and treat correctly Diagnosis active tuberculosis Smear examination Solid and liquid culture Identification Molecular methods -Detection -Detection of resistance Susceptibility testing methods We are fighting a large & growing epidemic with old tools
3 Detection of M.tuberculosis in clinical samples Conventional PCR Real-time PCR Line probe assays The previous evaluation of the risk of TB is very important for PV
4 Detection of resistance Applied to clinical strain, or directly to clinical sample Sequencing, LPA, GeneXpert and PCR real time. Walter et al, Respirology 2012
5 Pyrosequencing PPi ATP
6 Pyrosequencing Type of sample Drug Value % (no. of positive samples/ total no. of samples) for: Sensitivity Specificity Clinical strains Isoniazid 76.9 (60/78) 100 (11/11) Rifampin 97.2 (34/35) 97.9 (46/47) Overall 83.2 (94/113) 98.3 (57/58) Clinical specimens a Isoniazid 85.7 (6/7) 100 (16/16) Rifampin 85.7 (6/7) 100 (16/16) Overall 85.7 (12/14) 100 (32/32) a A total of 23 patients results were available (one clinical specimen per patient). García-Sierra N. JCM 2011
7 Pyrosequencing Pyrosequencing Agreement between pyrosequencing and BACTEC Drug Sensitivity (%) (95% CI) Specificity (%) (95% CI) Agreement (%) Kappa SE FLQ 20/31 (64.5) ( ) 76/79 (96.2.0) ( ) 96/110 (87.3) KAN/AMK/CM 8/29 (27.6) ( ) 77/81 (96.3) ( ) 85/109 (78.0) EMB 40/60 (66.7) ( ) 43/50 (86.0) ( ) 83/110 (75.5) Molina-Moya B. JI. 2014
8 GenoType MTBDR, Hain Lifescience 1ª línea (INH, RIF) MDR 2ª línea (FLQ, KAN/AMK/CAP, EMB) XDR
9 GenoType MTBDR, Hain Lifescience Lacoma A. JCM 2008 Bactec 460TB system result (no. [%] of strains) MTBDRplus test result Susceptible (n 14) INH Resistant (n 48) Susceptible (n 50) RIF Resistant (n 12) Susceptible 14 (100) 13 (27) 50 (100) 1 (8.3) Resistant 0 35 (73) 0 11 (91.7) Lacoma A. JCM 2012 Drug Clinical strains Sensitivity (%) (95% CI) Specificity (%) (95% CI) FLQ 4/7 (57.1) ( ) 17/22 (77.3) ( ) KM/CM 5/5 (100) ( ) 19/22 (86.4) ( ) EMB 14/23 (60.9) ( ) 9/11 (81.9) ( )
10 Xpert MTB/Rif assay, Cepheid
11 Main results We included 27 unique studies (integrating nine new studies) involving 9557 participants. Sixteen studies (59%) were performed in low- or middle-income countries. For all QUADAS-2 domains, most studies were at low risk of bias and low concern regarding applicability. As an initial test replacing smear microscopy, Xpert MTB/RIF pooled sensitivity was 89% [95% Credible Interval (CrI) 85% to 92%] and pooled specificity 99% (95% CrI 98% to 99%), (22 studies, 8998 participants: 2953 confirmed TB, 6045 non-tb). As an add-on test following a negative smear microscopy result, Xpert MTB/RIF pooled sensitivity was 67% (95% CrI 60% to 74%) and pooled specificity 99% (95% CrI 98% to 99%; 21 studies, 6950 participants). For smear-positive, culture-positive TB, Xpert MTB/RIF pooled sensitivity was 98% (95% CrI 97% to 99%; 21 studies, 1936 participants). For people with HIV infection, Xpert MTB/RIF pooled sensitivity was 79% (95% CrI 70% to 86%; 7 studies, 1789 participants), and for people without HIV infection, it was 86% (95% CrI 76% to 92%; 7 studies, 1470 participants). Among 180 specimens with nontuberculous mycobacteria (NTM), Xpert MTB/RIF was positive in only one specimen that grew NTM (14 studies, 2626 participants). Rifampicin resistance For rifampicin resistance detection, Xpert MTB/RIF pooled sensitivity was 95% (95% CrI 90% to 97%; 17 studies, 555 rifampicin resistance positives) and pooled specificity was 98% (95% CrI 97% to 99%; 24 studies, 2411 rifampicin resistance negatives). Dignosis RIF Resistence Sensitivity Specificity Sensitivity Specificity 51/52 (98.1%) 76/76 (100%) 5/5 (100%) 121/121 (100%)
12 AID TB Resistance A B C
13 AID TB Resistance AID TB Resistance Agreement between AID TB Resistance and BACTEC 460TB Drug Sensitivity (%) (95% CI) Specificity (%) (95% CI) Agreement (%) Kappa SE INH 45/46 (97.8) ( ) 14/14 (100) ( ) 59/60 (98.3) RIF 43/43 (100) ( ) 17/17 (100) ( ) 60/60 (100) FQ 2/6 (33.3) ( ) 52/53 (98.1) ( ) 54/59 (91.5) EMB 21/35 (60.0) ( ) 22/24 (91.7) ( ) 33/59 (72.9) KAN/C M 17/17 (100) ( ) 34/34 (100) ( ) 51/51 (100) STR 22/22 (100) ( ) 28/29 (96.6) ( ) 50/51 (98.0) Molina-Moya B. JI. 2014
14 PCR multiplex (Anyplex II MTB/MDR/XDR)
15 PCR multiplex (Anyplex II MTB/MDR/XDR) Drugs Clinical strains INH RIF FQ KAN Sensitivity (%) (95% CI) 39/51 (76.5) ( ) 35/36 (97.2) ( ) 19/27 (70.4) ( ) 22/27 (81.5) ( ) Specificity (%) (95% CI) 10/10 (100) ( ) 24/25 (96.0) ( ) 29/33 (87.9) ( ) 28/33 (84.8) ( ) AMK c 12/12 (100) ( ) 15/25 (60.0) ( ) CAP 7/7 (100) ( ) 33/53 (62.3) ( ) Molina-Moya B. JI. 2014
16 GenoFlow DR-MTB Array, DiagCor a b c a. INH R, RIF R b. INH S, RIF R c. INH S, RIF R d e f d. INH R, RIF S e. INH S, RIF S f. Invalide Drug Clinical samples Sensitivity (%) Specificity (%) INH 12/13 (92.3) 9/9 (100) RIF 11/11 (100) 11/12 (91.7) Molina-Moya B. 2014
17 Conclusions Some limitations in molecular diagnosis of TB Differences between phenotypic and genotypic methods. Similarities between molecular methods Only the more frequent mutations related to resistances are included in the assays In some cases the mutations identified are silent and are not always related to resistance acquisition. Presence of compensatory mutations. For a correct management of DR-TB patients, the results should be confirmed by a phenotypic method. We could develop guidelines, algorithms, and consensus documents for the optima utilization of the molecular methods.
18 Clinical implications of results from molecular drug resistance testing for M. tuberculosis A TBNET/RESIST-TB consensus statement The document is developed by physicians, microbiologists and molecular biologists to reach a consensus about reporting standards in the clinical use of results of M. tuberculosis molecular DR testing.
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