Rapid molecular diagnosis of TB and drug-resistant TB

J. Domínguez Institut d’Investigació Germans Trias i Pujol

Universitat Autònoma de Barcelona jadomb@gmail.com

2nd European Advanced Course in Clinical Tuberculosis Amsterdam 2014

Utility of the molecular methods in tuberculosis

Diagnosis active tuberculosis

Smear examination

Solid and liquid culture

Identification

Susceptibility testing methods Molecular methods - Detection - Detection of resistance

To rapidly diagnose patients with active TB and treat correctly

We are fighting a large & growing epidemic with old tools

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

Detection of resistance ¡  Applied to clinical strain, or directly to clinical sample ¡  Sequencing, LPA, GeneXpert and PCR real time.

Walter et al, Respirology 2012

Pyrosequencing

PPi

ATP

Pyrosequencing sensitivity and specificity values are shown inTable 2.

Regarding INH resistance detection, several authors havepreviously reported good sensitivity values when analyzing clin-ical strains. Bravo et al. (5) and Marttila et al. (17) foundsensitivity values of 63.8% and 66.7%, respectively. Our sensi-tivity result for INH resistance (76.9%) is slightly higher be-cause we also assessed mutations in the inhA gene, which werenot included in the study by Marttila et al. Regarding RIF,values similar to our results were obtained by Bravo et al.(97.2%) (5) and Marttila et al. (97.4%) (17).

Interestingly, we obtained a higher value of pyrosequencingsensitivity for the INH resistance mutation in clinical samples(93.1%) than in clinical strains (76.9%). This might be due tothe bias introduced by analyzing more than one sample perpatient. In fact, all M. tuberculosis isolates from the clinicalspecimens carried the mutation AGC3ACC in katG (data notshown), which is easily detected by pyrosequencing. Whentaking into account one sample per patient, the sensitivity fordetecting INH resistance falls to 85.7%.

Taking into account only strains in which the katG gene hada wild-type sequence, mutations in the inhA promoter regionwere responsible for INH resistance in 25.3% of the isolatesbut in none of the direct specimens. Thus, we were able todetect INH-resistant strains by pyrosequencing the inhA pro-moter region that would not have been identified as resistantjust by analyzing katG mutations.

Eighty percent of the smear-positive samples according toZiehl-Neelsen staining had a valid pyrosequencing result,whereas for smear-negative samples, the percentage of valid

results was 33.4%. Despite the low percentage of valid resultsfor smear-negative samples, the test enables the early detec-tion of resistance in some samples with a bacterial load. Boe-hme et al. (4), in a multicenter evaluation of the Xpert MTB/RIF, found that the test obtained an indeterminate result in192 of 5,190 (3.7%) tests performed and that 1% of sampleswith a positive result for M. tuberculosis detection had an in-determinate result for rifampin resistance.

Among the 41 rpoB codons analyzed for mutations by pyro-sequencing, the pyrosequencing identified an rpoB 516 muta-tion in a RIFs strain only in one case (2.4%) that was con-firmed by GenoType (in this case, no sequencing result wasavailable). This mutation usually confers low-level RIF resis-tance, and the lack of phenotypic detection might be due to alow RIF critical concentration (24). The level of sensitivityreached for the detection of INH resistance is not very high,and therefore, it is not clear whether it could be useful forclinical use. The low sensitivity in comparison with the sensi-tivity of detection of RIF resistance is due to the complexmolecular basis of INH resistance and the existence of still-unidentified genes involved in the INH resistance mechanism(18, 21). As has been previously described (10, 16, 25), themost common mutation involved in INH resistance is in katG,which has been related to high levels of INH resistance. Incontrast, mutations causing low levels of INH resistance arenot as clearly elucidated, as they are much more complex andinvolve different genes; however, a firm relationship has beenfound between mutations in the inhA regulatory region andlow or intermediate levels of resistance (6, 10). Therefore, thecombination of a susceptible result by the pyrosequencing as-say but a pattern of resistance with the Bactec 460TB systemfor INH could reflect an infrequent mutation and could berelated to low-level resistance. This misclassification probablywould not affect a patient’s treatment, especially if treatmentwith the four first-line drugs is prescribed. However, it could beprudent to have the phenotypic result available before switch-ing to the dual therapy. In addition, by considering RIF resis-tance alone as an accurate marker for multidrug resistance(MDR) (21), the sensitivity of the test for the detection ofMDR increases, even though the INH pattern can be misclas-sified as susceptible. Therefore, genotyping methods, includingpyrosequencing, give rapid detection of strains resistant to themain first-line tuberculosis drugs, allowing better clinical man-agement until the results for phenotypic pattern are obtained.

In conclusion, our observations show that pyrosequencing isa valuable tool in the clinical setting, as it allows the detectionof INH- and RIF-resistant M. tuberculosis strains both from

TABLE 1. Pyrosequencing results compared to those of Bactec 460TB for isolated clinical strains and directly from clinical samplesa

Pyrosequencingresult

Bactec 460TB result !no. (%)" for:

Clinical strains Clinical samples

Isoniazid Rifampin Isoniazid Rifampin

Susceptible(n # 11)

Resistant(n # 78)

Susceptible(n # 47)

Resistant(n # 35)

Susceptible(n # 16)

Resistant(n # 29)

Susceptible(n # 16)

Resistant(n # 29)

Susceptible 11 (100) 18 (23.1) 46 (97.9) 1 (2.9) 16 (100) 2 (6.9) 16 (100) 2 (6.9)Resistant 0 60 (76.9) 1 (2.1) 34 (97.1) 0 27 (93.1) 0 27 (93.1)a Clinical samples included 42 sputum specimens, 2 bronchoalveolar lavage specimens, 3 bronchial aspirate specimens, and 1 pleural effusion sample.

TABLE 2. Sensitivity and specificity values of pyrosequencing fordetecting overall, isoniazid, and rifampin resistance in clinical

strains and clinical specimens in comparison to the resultsusing the Bactec 460TB method

Type of sample DrugValue !% (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 specimensa 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 perpatient).

VOL. 49, 2011 NOTES 3685

García-Sierra N. JCM 2011

Pyrosequencing

Pyrosequencing

29

Table 4. Pyrosequencing sensitivity and specificity for detecting FQ, 549

KAN/AMK/CM and EMB resistance and agreement values between 550

pyrosequencing and BACTEC (460TB or MGIT). 551

Pyrosequencing Agreement between

pyrosequencing and BACTEC

Drug

Sensitivity (%) (95% CI)

Specificity (%) (95% CI)

Agreement (%) Kappa SE

FLQ 20/31 (64.5)

(45.4-80.2) 76/79 (96.2.0)

(88.5-99.0) 96/110 (87.3) 0.659 0.083

KAN/AMK/CM 8/29 (27.6)

(13.4-47.5) 77/81 (96.3)

(88.7-99.0) 85/109 (78.0) 0.297 0.100

EMB 40/60 (66.7)

(53.2-77.9) 43/50 (86.0)

(72.6-93.7) 83/110 (75.5) 0.515 0.079

552

553 Molina-Moya B. JI. 2014

GenoType MTBDR, Hain Lifescience

1ª línea (INH, RIF) → MDR 2ª línea (FLQ, KAN/AMK/CAP, EMB) → XDR

detect a C3T inhA mutation at position !15, whereas it wasidentified by sequencing. For the 17 correctly identified INH-resistant strains, the mutations detected in inhA were as fol-lows: 14 had a C3T mutation at position !15 and 2 had aT3C mutation at position !8, while the remaining strain hada S315T mutation in katG (Table 1).

The concordance between the results obtained with theBactec 460TB system and by the MTBDRplus assay for thedetection of INH resistance in the group of 21 strains withhigh-level resistance was 85.71% (18/21). In two strains, se-quencing detected mutations in katG different from the onescovered by the assay: one strain had two deletions in codons234 and 155 to 158, and the other one had a stop mutation incodon 204. The remaining strain had two mutations in katG: aS315T mutation that was detected by sequencing but not by theMTBDRplus test and a G3T mutation at codon 463. For the18 strains identified to be INHr, 17 had an S315T mutation inkatG, while the remaining one had a C3T mutation at position!15 in inhA.

Overall, by consideration of the group of 48 INHr strains, in14 cases the pattern of susceptibility revealed by the MTBDR-plus assay did not match the one obtained with the Bactec460TB system. In 3 cases, the responsible mutations shouldhave been identified by the assay, whereas the 11 remainingdisagreements could be explained: in 6 cases DNA sequencing

indicated wild-type sequences, and in the other 5 cases, themutations identified were outside the regions covered by theassay.

MTBDRplus assay results for the clinical specimens.Among the 65 clinical specimens tested directly, 31 strainswere fully susceptible and 34 were resistant (29 were RIFr andINHr and 5 were RIFs and INHr). Among the 26 smear-negative samples, the strains isolated were fully susceptible in19 cases, had the INHr amd RIFs pattern in 4 cases, and hadthe INHr and RIFr pattern in 3 cases. In the 39 smear-positivesamples, 12 strains were fully susceptible, 1 had the INHr andRIFs pattern, and 26 had the INHr and RIFr pattern. Figure 3shows the distribution of the MTBDRplus assay results accord-ing to the results obtained with Bactec 460TB system for the 65samples. We obtained valid test results for 51/65 (78.4%) sam-ples (Table 4). For the 14 remaining samples, all of which weresmear negative, the results were invalid. However, the M. tu-berculosis complex-specific (TUB) control band was reportedin nine of these samples. The overall rate of concordancebetween the results of the MTBDRplus assay and those of theBactec 460TB system for the assessment of RIF resistance was98% (50/51) (" # 0.960; 95% CI # 0.859). The level of agree-ment for RIF resistance for the smear-positive samples was 100%(39/39; " # 1; 95% CI # 0), and that for smear-negative spec-imens was 91.6% (25/26; " # 0.8; 95% CI # 0.625). Thediscordant sample was RIF susceptible according to the Bactec460TB system, but the pattern obtained by the MTBDRplusassay indicated that the sample tested was resistant because theband for the WT8 rpoB probe was missing. The overall rate ofconcordance between both tests for the assessment of INHsusceptibility was 96.2% (49/51; " # 0.920; 95% CI # 0.797).The levels of agreement for INH susceptibility were 94.8%(37/39; " # 0.885; 95% CI # 0.740) for the smear-positivesamples and 100% (26/26; " # 1; 95% CI # 0) for the smear-negative samples. The two discordant samples were INH re-sistant according to the Bactec 460TB system but INH suscep-tible according to the MTBDRplus assay. Both samples werefrom the same patient.

Given that more than one specimen was collected fromsome patients, the rate of concordance was also calculatedby taking into account only one sample per patient (n # 28).The overall rate of concordance between the MTBDRplusassay and the Bactec 460TB system was 92.85% (26/28).Therefore, the rate of concordance between the MTBDR-plus assay and the Bactec 460TB system for the assessmentof RIF resistance was 96.42% (27/28; " # 0.909; 95% CI #0.089). The levels of agreement for RIF resistance were100% (20/20; " # 1; 95% CI # 0) for smear-positive samples

TABLE 2. MTBDRplus assay results according to Bactec 460TBsystem results for the 62 clinical strains

MTBDRplustest result

Bactec 460TB system result (no. [%] of strains)

INH RIF

Susceptible(n # 14)

Resistant(n # 48)

Susceptible(n # 50)

Resistant(n # 12)

Susceptible 14 (100) 13 (27) 50 (100) 1 (8.3)Resistant 0 35 (73) 0 11 (91.7)

FIG. 1. Representative DNA patterns obtained by the MTBDR-plus assay. Examples of invalid results are also included. Lane 1,example of a pattern of RIFs and INHs; lane 2, example of a patternof RIFr and INHr; lane 3, example of a pattern of RIFs and INHr; lanes4 to 9, examples of invalid results. The positions of the oligonucleotidesand the control probes are given on the left. The targeted genes andthe specific probes lines are shown from top to bottom, as follows:conjugate control (CC); amplification control (AC); M. tuberculosiscomplex-specific control (TUB); rpoB amplification control; rpoB wild-type probes WT1 to WT8 (505 to 533); four rpoB mutant probes(probes MUT1, MUT2A, MUT2B, and MUT3) in codons D516V,D526Y, H526D, and S531L, respectively; katG amplification control;katG codon 315 wild-type probe; two katG codon 315 mutant probes(probes MUT1 and MUT2) with AGC-ACC (S315T1) and AGC-ACA(S315T2) mutations, respectively; inhA amplification control; inhAwild-type probes WT1 and WT2 covering positions !15 and !16 ofthe gene regulatory region; four inhA mutant probes (probes MUT1,MUT2, MUT3A, and MUT3B) with mutations C3T at position !15,A3G at position !16, T3C at position !8, and T3A at position !8,respectively. M, colored marker.

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of eight cases (three strains and five clinical samples) had a gyrAwild-type pattern according to GenoType MTBDRsl and pyrose-quencing, while Bactec 460TB indicated that these cases were re-sistant. Given that phenotypical methods are not standardized forsecond-line drugs, we cannot exclude the possibility that Bactec460TB might have misclassified some results. Different mutationsat gyrB have also been related to FLQ resistance (5, 32, 36). Giventhat mutation probes for gyrB are not included in the assay, it ispossible that some isolates might have had mutations in thesepositions, which the assay could not detect. In seven cases (fivestrains and two clinical samples), we detected a mixture of WT andMUT bands for the same position, indicating the presence of het-eroresistance. For two strains, the GenoType MTBDRsl profilewas WT1, WT2, WT3, and MUT2; for another two strains, theprofile was WT1, WT3, and MUT 1; and for the last strain, theprofile was WT1, WT2, and MUT 1. Regarding clinical samples(from two different patients), profiles detected were WT1, WT2,WT3, and MUT3B and WT1, WT2, WT3, and MUT3C. The pres-ence of a mixed wild-type and resistant population with regard tothis drug has also been reported in previous studies (7, 13). Spec-ificity values were around 80% and similar to values in the litera-ture (7, 8, 13, 14, 18, 20). A natural polymorphism at gyrA codon95 (ACC) unrelated to FLQ resistance has been described (31),and by pyrosequencing, we found it in 9 clinical samples (1 perpatient) and 28 clinical strains.

The sensitivity of confirmation of KM/CM resistance by tar-geting rrs in clinical strains and samples was 100%, indicating thatthe test performs well in detecting the presence of mutations at rrspositions 1401, 1402, and 1482 (25, 26). Values from other studiesare also in an acceptable range, from 75% to 80% (7, 13, 18). Thespecificity of detecting KM/CM resistance in clinical strains was86.4%, which is slightly lower than values reported in the litera-ture (7, 13, 14, 18). For two clinical strains, we reported the pres-ence of WT1, WT2, and MUT1 bands, indicating a mixture ofresistant and susceptible bacilli. The other example found corre-sponded to a clinical sample which also had the pattern WT1,WT2, and MUT1.

The low detection rate of EMB resistance is consistent withprevious reports (7, 18, 40). It has been reported that 30 to 70% ofEMB-resistant strains have a mutation in embB (3, 16, 40). Indeed,mutations at codon 306 have shown a strong correlation withEMB resistance. However, the low sensitivity implies that there isa need to identify other mutations conferring resistance to thisdrug. Huang et al. identified several mutations in embB other thanthat at codon 306 (14). Interestingly, we found that two EMB-susceptible strains harbored a mutation at codon 306. Brossier etal. also reported this fact, highlighting the need for a better under-standing of the molecular basis of EMB resistance (7). Two strains

resistant to EMB according to Bactec 460TB had an MTBDRslprofile indicating the absence of WT1, MUT1A, and MUT1Bbands. By pyrosequencing, these strains showed an ATG ¡ ATCmutation. One clinical sample also harbored a mixture of WT1and MUT1A bands.

The agreement between the phenotypic reference method(Bactec 460TB) and GenoType MTBDRsl varies according to thedrug considered. In our experience, the concordance for clinicalstrains was 88.8% and 72.4% for KM/CM and FLQ, respectively,whereas for EMB, the value was 67.6%. For clinical samples,agreement values were higher for FLQ and KM/CM and slightlylower for EMB. Values reported in the literature, obtained only forclinical strains, are around 90% for FLQ and KM/CM and 70% forEMB (14, 18).

It is important to remember that the main limitation of themolecular tests is that they are unable to target all possible muta-tions involved in resistance, and as a consequence, some resistantstrains will not be detected. This is one of the explanations for oursensitivity values. Since only the more frequent mutations relatedto resistance are detected by the assay, results must be confirmedby phenotypic methods. Although common mutations predictiveof resistance are well known for some drugs, in some cases themutations identified are silent and are not always related to theacquisition of resistance. It is important to bear in mind that mu-tation prevalences differ by geographical region and setting. Inaddition, the exact ratio of resistant to susceptible bacilli that re-sults in phenotypic resistance is unclear. In general, if the propor-tion of resistant cells in an isolate is less than 10% of mutant DNA,it will be difficult to detect by molecular methods, while the phe-notypical methods might give a more sensitive test result in thesecases (29). This means that in practice, as we have noticed, theGenoType MTBDRsl result can differ from that obtained by aproportion-based method, such as the Bactec 460TB system.Overall, we found 55 discordant drug results between these twoassays (22 for strains and 33 for clinical samples). Although nosystematic confirmation of Bactec 460TB was performed, one ofthese discordant strains was studied with the Bactec MGIT 960system (Becton Dickinson Microbiology System, Sparks, MD),confirming the FQL, KM/CM, and EMB resistance result obtainedwith the Bactec 460TB. For clinical strains, pyrosequencing con-firmed GenoType MTBDRsl results in 90% of the cases, while forclinical specimens, pyrosequencing confirmed 81% of the cases.These results confirm that GenoType MTBDRsl is an accuratemethod for the detection of mutations involved in M. tuberculosisresistance. However, the presence of discordant results, especiallywhen GenoType MTBDRsl detected mutations but the Bactec460TB obtained a susceptible pattern, raises some concerns aboutits utility in low-prevalence areas.

TABLE 6 Sensitivity and specificity of GenoType MTBDRsl for detecting resistance to FLQ, KM/CM, and EMB in clinical strains andclinical samplesa

Drug

Clinical strains Clinical samples

Sensitivity (%) (95% CI) Specificity (%) (95% CI) Sensitivity (%) (95% CI) Specificity (%) (95% CI)

FLQ 4/7 (57.1) (20.2–88.1) 17/22 (77.3) (54.1–91.3) 1/3 (33.3) (1.7–87.4) 11/13 (84.6) (53.6–97.2)KM/CM 5/5 (100) (46.2–100) 19/22 (86.4) (64–96.4) 4/4 (100) (39.5–100) 7/12 (58.3) (28.5–83.5)EMB 14/23 (60.9) (38.7–79.5) 9/11 (81.9) (47.7–96.7) 3/9 (33.3) (9–69) 5/7 (71.4) (30.2–94.8)a A total of 16 patients were included (one sample per patient). Sensitivity values are expressed as numbers of true detections of resistance by GenoType MTBDRsl/total number ofresistant cases by Bactec 460TB. Specificity values are expressed as numbers of true detections of susceptibility by GenoType MTBDRsl/total number of susceptible cases by Bactec460TB. CI, confidence interval.

Lacoma et al.

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Lacoma A. JCM 2008

Lacoma A. JCM 2012

Xpert MTB/Rif assay, Cepheid

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%)

C B A

AID TB Resistance

1

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) (87.0-99.9)

14/14 (100) (73.2-100)

59/60 (98.3) 0.955 0.045

RIF 43/43 (100) (89.8-100)

17/17 (100) (77.1-100)

60/60 (100) 1.000 0.000

FQ 2/6 (33.3) (6.0-75.9)

52/53 (98.1) (88.6-99.9)

54/59 (91.5) 0.473 0.216

EMB 21/35 (60.0) (42.2-75.6)

22/24 (91.7) (71.5-98.5)

33/59 (72.9) 0.479 0.103

KAN/CM

17/17 (100) (77.1-100)

34/34 (100) (87.4-100)

51/51 (100) 1.000 0.000

STR 22/22 (100) (81.5-100)

28/29 (96.6) (80.4-99.8)

50/51 (98.0) 0.963 0.036

AID TB Resistance

Molina-Moya B. JI. 2014

PCR multiplex (Anyplex II MTB/MDR/XDR)

33

Table 5. Sensitivity and specificity of Anyplex II MTB/MDR/XDR for detecting drug 1

resistance considering one strain and sample per patient. 2

3

a Twenty-nine samples were included for INH and RIF sensitivity and specificity calculations. 4 b Twenty-three samples were included for FLQ and KAN/CAP calculations. 5 c AMK phenotypic DST for the clinical samples was not performed. 6

CI, confidence interval. 7

8

9

10

Drugs Clinical strains Clinical samples

Sensitivity (%)

(95% CI)

Specificity (%)

(95% CI)

Sensitivity (%)

(95% CI)

Specificity (%)

(95% CI)

INH 39/51 (76.5) (62.2-86.8)

10/10 (100) (65.5-100)

14/15 (93.3) (66.0-99.7)a

14/14 (100) (73.2-100)a

RIF 35/36 (97.2) (83.8-99.9)

24/25 (96.0) (77.7-99.8)

14/14 (100) (73.2-100)a

15/15 (100) (74.7-100)a

FQ 19/27 (70.4) (49.7-85.5)

29/33 (87.9) (70.9-96.0)

2/4 (50.0) (0.09-90.8)b

19/19 (100) (79.1-100)b

KAN 22/27 (81.5) (61.3-93.0)

28/33 (84.8) (67.3-94.3)

5/5 (100) (46.3-100)b

17/18 (94.4) (70.6-99.7)b

AMKc 12/12 (100) (69.9-100)

15/25 (60.0) (38.9-78.2) - -

CAP 7/7 (100) (56.1-100)

33/53 (62.3) (47.9-74.9)

5/5 (100) (46.3-100)b

17/18 (94.4) (70.6-99.7)b

PCR multiplex (Anyplex II MTB/MDR/XDR)

Molina-Moya B. JI. 2014

GenoFlow DR-MTB Array, DiagCor

a. INHR, RIFR

b. INHS, RIFR c. INHS, RIFR

Drug Clinical samples

Sensitivity (%) Specificity (%)

INH 12/13 (92.3) 9/9 (100)

RIF 11/11 (100) 11/12 (91.7)

d. INHR, RIFS

e. INHS, RIFS

f. Invalide

a b

d

c

f e

Molina-Moya B. 2014

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.

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.