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INT J TUBERC LUNG DIS 20(8):1105–1112 Q 2016 The Union http://dx.doi.org/10.5588/ijtld.15.0896

Comparison of TaqManW Array Card and MYCOTBTM with conventional phenotypic susceptibility testing in MDR-TB S. Foongladda,* S. Banu,† S. Pholwat,‡ J. Gratz,‡ S. O-Thong,* N. Nakkerd,* R. Chinli,* S. S. Ferdous,† S. M. M. Rahman,† A. Rahman,† S. Ahmed,† S. Heysell,‡ M. Sariko,§ G. Kibiki,§ E. Houpt‡ *Department of Microbiology, Faculty of Medicine Siriraj Hospital, Mahidol University, Bangkok, Thailand; † International Center for Diarrheal Diseases and Research, Dhaka, Bangladesh; ‡Division of Infectious Diseases and International Health, Department of Medicine, University of Virginia, Charlottesville, Virginia, USA; §Kilimanjaro Clinical Research Institute, Moshi, Tanzania SUMMARY B A C K G R O U N D : Although phenotypic drug susceptibility testing (DST) is endorsed as the standard for secondline drug testing of Mycobacterium tuberculosis, it is slow and laborious. M E T H O D S : We evaluated the accuracy of two faster, easier methodologies that provide results for multiple drugs: a genotypic TaqManw Array Card (TAC) and the Sensititrew MYCOTBTM plate. Both methods were tested at three central laboratories in Bangladesh, Tanzania, and Thailand with 212 multidrug-resistant tuberculosis (MDR-TB) isolates and compared with the laboratories’ phenotypic method in use. R E S U LT S : The overall accuracy for ethambutol, streptomycin, amikacin, kanamycin, ofloxacin, and moxifloxacin vs. the phenotypic standard was 87% for TAC (range 70–99) and 88% for the MYCOTB plate (range

76–98). To adjudicate discordances, we re-defined the standard as the consensus of the three methods, against which the TAC and MYCOTB plate yielded 94–95% accuracy, while the phenotypic result yielded 93%. Some isolates with genotypic mutations and high minimum inhibitory concentration (MIC) were phenotypically susceptible, and some isolates without mutations and low MIC were phenotypically resistant, questioning the phenotypic standard. C O N C L U S I O N S : In our view, the TAC, the MYCOTB plate, and the conventional phenotypic method have similar performance for second-line drugs; however, the former methods offer speed, throughput, and quantitative DST information. K E Y W O R D S : drug susceptibility testing; MDR-TB; MIC

MULTIDRUG-RESISTANT TUBERCULOSIS (MDR-TB), defined as resistance to at least isoniazid (INH) and rifampin (RMP), is extremely difficult to treat.1 Better outcomes are obtained with treatment regimens that use active drugs based on the results of second-line drug susceptibility testing (DST).2 In the era of the Xpertw MTB/RIF assay (Cepheid, Sunnyvale, CA, USA), a broader role for DST is recommended not only to confirm MDR-TB but also to define extensively drug-resistant TB (XDR-TB).3,4 However, second-line DST is rare in many parts of the world, as the endorsed phenotypic culture-based methods are complicated,5 requiring multiple manual tests and changes in critical concentration, in addition to concerns about cost, slow turnaround time and reproducibility.

In the present study, we evaluated two second-line DST methodologies that are feasible for central laboratories, the Sensititre w MYCOTBe plate (TREK Diagnostics, Cleveland, OH, USA), which yields a minimum inhibitory concentration (MIC), and a customized TaqManw Array Card (TAC; Thermo Fisher Scientific, Waltham, MA, USA) for simultaneous detection of mutations in the resistancedetermining regions of 10 genes. We evaluated MDRTB isolates in three laboratories serving TB-endemic settings in Bangladesh, Tanzania, and Thailand. The MYCOTB plate has already been described in previous studies.6–8 TAC, which has not been rigorously examined in the field, also offers a DST result for pyrazinamide (PZA). Furthermore, the mutational information from TAC allows specific mutations vs. the quantitative degree of resistance (MIC) to be examined.

Footnote: SF, SB and SP contributed equally to this work

Correspondence to: Eric Houpt, Division of Infectious Diseases and International Health, Department of Medicine, University of Virginia, Charlottesville, VA 22908, USA. Fax: (þ1) 434 924 0075. e-mail: [email protected] Article submitted 31 October 2015. Final version accepted 27 March 2016.

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The International Journal of Tuberculosis and Lung Disease

Table 1

Critical concentrations, MIC breakpoints, and genotypic resistance-associated mutations probed in this study Critical concentration

Drug

Agar proportion method lg/ml

LJ lg/ml

MGIT lg/ml

MYCOTBEMIC breakpoint lg/ml

INH RMP

0.2 1

0.2 40

0.1 1

0.25 1

EMB

5

2

5

4

SM AMK KM OFX

2 4 5 2

4 Not done 30 2

1 Not done 2.5 2

2 4 5 2

MFX

0.5 2.0 5 2 NA

ETH PAS PZA*

NA 40 NA NA

0.25 5 2 100

0.5 2.0 5 2 NA

Resistance-associated mutations inhA (8)C, inhA (15)T, katG 315T rpoB511P, rpoB513L, rpoB513E, rpoB516V, rpoB526Y, rpoB526D, rpoB526L, rpoB531L, rpoB531W, rpoB533P embB306V, embB306Iatc, embB306Iata , embB306L, embB328Y, embB328G, embB406A rpsL43R, rpsL88R, rpsL88M, rrs(514)C, rrs(517)T, rrs(906)G rrs(1401)G, rrs(1484)T, eis(10)A, eis(14)T rrs(1401)G, rrs(1484)T, eis(10)A, eis(14)T gyrA90V, gyrA94G, gyrA94Y, gyrA94A, gyrB447F, gyrB461H, gyrB499D, gyrB501D gyrA90V, gyrA94G, gyrA94Y, gyrA94A, gyrB447F, gyrB461H, gyrB499D, gyrB501D Not done Not done pncA

* pncA mutations were detected using high-resolution melt analysis. ¨ MIC ¼ minimum inhibitory concentration; LJ ¼ Lowenstein-Jensen proportion method; MGIT ¼ Mycobacterium Growth Indicator Tube; INH ¼ isoniazid; RMP ¼ rifampin; EMB ¼ ethambutol; SM ¼ streptomycin; AMK ¼ amikacin; KM ¼ kanamycin; OFX ¼ ofloxacin; MFX ¼ moxifloxacin; NA ¼ not applicable (no endorsed critical concentration); ETH ¼ ethionamide; PAS ¼ para-aminosalicylic acid; PZA ¼ pyrazinamide.

MATERIALS AND METHODS Mycobacterial strains and culture conditions Mycobacterial strains included Mycobacterium tuberculosis H37Rv (American Type Culture Collection 27294) and 212 clinical TB isolates from unique patients from 2013 to 2015 determined to have resistance to INH and RMP using the sitespecific phenotypic standard (Middlebrook agar proportion, Lowenstein-Jensen or MGIT [BD, ¨ Sparks, MD, USA]). These included 98 isolates from a large clinical microbiology laboratory in Thailand (Department of Microbiology, Faculty of Medicine, Siriraj Hospital, Mahidol University, Bangkok), 83 isolates from MDR/XDR-TB surveillance projects among high-risk patients in Bangladesh (International Centre for Diarrheal Diseases and Research [ICDDR], Dhaka, Bangladesh), and 31 isolates from the Kibong’oto National Tuberculosis Hospital in Tanzania (Kilimanjaro Clinical Research Institute, Moshi, Tanzania). All work was reviewed and approved by the Institutional Biosafety and Human Investigation Committees at the University of Virginia (Charlottesville, VA, USA), the Tanzania National Institute for Medical Research (Dar es Salaam, Tanzania), the ICDDR (Dhaka, Bangladesh), and Mahidol University (Bangkok, Thailand). Phenotypic drug susceptibility testing Isolates underwent DST according to local laboratory practices: in Thailand using the agar proportion method on Middlebrook 7H10 media, in Bangladesh using the Lowenstein-Jensen propor¨ tion method (both read at 21–28 days), and in

Tanzania by MGITe 960e (Mycobacterium Growth Indicator Tube, BD, Sparks, MD), read at 7–14 days. The MYCOTB plate was used on all 212 isolates according to the manufacturer’s instructions and read at 10 days; it was reread at 21 days if no growth was observed in the control well. The critical concentrations of the drugs and the MIC breakpoint for MYCOTB used are shown in Table 1. Phenotypic testing, MYCOTB, and TAC (below) were all performed simultaneously, except in Thailand, where agar proportion was tested first before storing the isolates (initially using a moxifloxacin [MFX] critical concentration of 2.0 lg/ml (n ¼ 40), and then 0.5 lg/ml (n ¼ 58), given updated recommendations). The laboratorians (JG, SO, NN, RC, SSF, SMMR, AR, MS) performed all tests, and the results were not blinded. All laboratories participate in biannual species identification and DST proficiency testing through the College of American Pathologists, Northfield, IL, USA. Quality control was performed on each lot of DST media and was tested with the laboratory H37Rv strain and an MDR-TB strain. Genotypic testing All isolates were characterized genotypically using a custom-developed TAC,9 which utilizes real-time polymerase chain reaction (RT-PCR) and sequencespecific probes for the main resistance-associated mutations of the inhA, katG, rpoB, embB, rpsL, rrs, eis, gyrA and gyrB genes and high-resolution melt analysis of the pncA gene (the mutations detected are shown in Table 1 and the primer and probe sequences

TaqMan Array Card and MYCOTB for MDR-TB

Table 2

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Performance of TaqManW Array Card compared to phenotypic DST Phenotypic DST (n ¼ 212)

Drug

TAC result

Resistant

Susceptible

INH

Mutant No mutant Mutant No mutant Mutant No mutant Mutant No mutant Mutant No mutant Mutant No mutant Mutant No mutant Mutant No mutant Variant Wild-type

193 19 194 18 68 47 102 36 6 1 9 1 26 9 9 5 66 24

0 0 0 0 17 80 13 60 1 90 1 191 13 152 9 96 33 84

RMP EMB SM* AMK* KM* OFX* MFX* PZA*

Sensitivity % (95%CI)

Specificity % (95%CI)

Accuracy %

91 (86–94)

NA

NA

92 (87–95)

NA

NA

59 (50–68)

83 (73–89)

70

74 (66–81)

82 (71–90)

77

86 (42–99)

99 (94–100)

98

90 (55–99)

99 (97–100)

99

74 (57–88)

92 (87–96)

89

64 (35–87)

91 (84–96)

88

73 (63–82)

72 (63–80)

73

* As not all results are available for all methodologies or all drugs, not all comparisons sum to n ¼ 212. DST ¼ drug susceptibility testing; CI ¼ confidence interval; INH ¼ isoniazid; NA ¼ not applicable; RMP ¼ rifampin; EMB ¼ ethambutol; SM ¼ streptomycin; AMK ¼ amikacin; KM ¼ kanamycin; OFX ¼ ofloxacin; MFX ¼ moxifloxacin; PZA ¼ pyrazinamide.

in Appendix Table A.1).* Each isolate underwent DNA extraction (DNeasy Blood and Tissue kit, Qiagen Inc, Valencia, CA, USA) and run on a Viia7 RT-PCR cycler (Life Technologies Corp, Carlsbad, CA, USA) for 2 h. The positive control and H37Rv were included on each card along with six isolates. For quality assurance, the respective areas of these genes from 98 of the Thai isolates were sequenced using Sanger sequencing (1st BASE, Seri Kembangan, Selangor, Malaysia), as described previously;9 DNA from 86 of the Bangladesh and Tanzania isolates was tested using GenoTypew MTBDRplus and GenoTypew MTBDRsl version 1.0 (Hain Lifescience, Nehren, Germany), according to the manufacturer’s instructions. Statistical analysis Means or median MICs were compared using the ttest or Mann-Whitney test, respectively. Receiver operating characteristic (ROC) analysis was performed using Predictive Analytics SoftWare (Statistical Package for the Social Sciences, IBM Corp, Armonk, NY, USA) to define an optimal MIC breakpoint. Data are shown as mean 6 standard deviation, unless otherwise stated. All P values were two-tailed.

RESULTS Field evaluation of TaqMan Array Card In a previous study, we developed a TAC that included 27 primer pairs and 40 probes to interro* The appendix is available in the online version of this article, at http://www.ingentaconnect.com/content/iuatld/ijtld/2016/ 00000020/00000008/art00021

gate critical regions of the inhA, katG, rpoB, embB, rpsL, rrs, eis, gyrA, gyrB, and pncA genes.9 We shipped the TACs and protocols to each central laboratory to perform the test. Mutations were observed for each gene using the specific probes; the high-resolution melt analysis was used only for pncA. We previously reported 96% accuracy between TAC and Sanger sequencing, and a similar 94% fidelity was seen on a subset of 98 samples from Thailand (Appendix Table A.2) as well as in comparison to the Hain line-probe assay (Appendix Table A.3). Most of the Sanger-TAC discrepancies were instances where Sanger detected less common mutations, some of uncertain significance, for which we did not design TAC probes (Appendix Table A.2). TAC was therefore used as the common genotypic method for all isolates and was compared to the phenotypic standard (Table 2). TAC detected respectively 91% and 92% of phenotypic resistance to INH and RMP. Overall accuracies were 70% for ethambutol (EMB), 77% for streptomycin (SM), 98% for amikacin (AMK), 99% for kanamycin (KM), 89% for ofloxacin (OFX), 88% for moxifloxacin (MFX), and 73% for PZA. There was no evident difference in the performance of the TAC, regardless of the method used as standard (agar proportion, LJ, or MGIT) (stratified data shown in Appendix Table A.4). As with our earlier findings,9 inaccuracies with the TAC were contributed more by low sensitivity for phenotypic resistance (no mutations found despite phenotypic resistance) than low specificity for susceptibility (e.g., overall sensitivity 75 6 12% vs. specificity 91 6 7% for the six drugs EMB, SM, AMK, KM, OFX, MFX; P , 0.05).

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Table 3 Performance of SensititreW MYCOTBEcompared to phenotypic DST

MYCOTB MIC result lg/ml

Drug INH RMP EMB SM* AMK* KM* OFX* †

MFX* ETH* PAS*

R . 0.25 S 6 0.25 R.1 S61 R.4 S64 R.2 S62 R.4 S64 R.5 S65 R.2 S62 R . 0.5/2.0 S 6 0.5/2.0 R.5 S65 R.2 S62

Phenotypic DST (n ¼ 212) R

S

185 27 197 15 80 35 104 34 5 2 8 2 32 3 9 5 29 35 5 7

0 0 0 0 15 82 12 61 1 90 3 189 19 146 7 98 5 133 9 98



Accuracy %

Accuracy if using ROC-optimized MIC breakpoint lg/ml (%)

87 (82–91)

NA

NA

93 (0.125)

93 (88–96)

NA

NA

93 (0.5)

70 (60–77)

85 (76–91)

76

78 (2.0)

75 (67–82)

84 (73–91)

78

No change

71 (29–96)

99 (90–100)

97

97 (8)

80 (44–97)

98 (95–99)

98

99 (20)

91 (77–98)

88 (83–93)

89

No change

64 (35–87)

93 (87–97)

90

87 (1)

45 (33–58)

96 (92–99)

80

81 (2.5)

42 (15–72)

92 (85–96)

87

89 (4)

* As not all results are available for all methodologies or all drugs, not all comparisons sum to n ¼ 212. † MFX was tested using agar proportion at 0.5 lg/ml on some isolates and at 2.0 lg/ml on others and the corresponding MYCOTB breakpoint was used. DST ¼ drug susceptibility testing; MIC ¼ minimum inhibitory concentration; R ¼ resistant; S ¼ susceptible; CI ¼ confidence interval; ROC ¼ receiver operating characteristic; INH ¼ isoniazid; NA ¼ not applicable; RMP ¼ rifampin; EMB ¼ ethambutol; SM ¼ streptomycin; AMK ¼ amikacin; KM ¼ kanamycin; OFX ¼ ofloxacin; MFX ¼ moxifloxacin; ETH ¼ ethionamide; PAS ¼ para-aminosalicylic acid.

Field evaluation of the MYCOTB Plate We also compared the MYCOTB results against the phenotypic standard. As with previous studies,6–8 the critical concentration on agar proportion was used as the breakpoint to interpret the MIC value, whereby isolates were considered susceptible by the MYCOTB plate if the MIC was less than or equal to the critical concentration, and resistant if greater than the critical concentration (due to the plate layout, for INH and EMB, MIC breakpoints of 0.25 lg/ml and 4.0 lg/ml were used). The MIC plate detected respectively 87% and 93% of phenotypic resistance to INH and RMP. Overall accuracies were 76% for EMB, 78% for SM, 97% for AMK, 98% for KM, 89% for OFX, 90% for MFX, 80% for ethionamide (ETH), and 87% for para-aminosalicylic acid (PAS) (Table 3). There was no significant difference in the performance of MYCOTB, regardless of the method used as standard (agar proportion, LJ, or MGIT) (Appendix Table A.5). As with TAC, MYCOTB inaccuracies were also more due to low sensitivity for resistance than low specificity for susceptibility (75 6 9% vs. 91 6 6% for the six drugs EMB, SM, AMK, KM, OFX, MFX; P , 0.05). Optimization of the MIC breakpoints using ROC analysis did not appreciably improve accuracy (Table 3). As expected, agreement improved if the MIC result was relaxed to allow a one-dilution margin of error (e.g., conditional agreement was 92 6 5%, while categorical agreement was 87 6 7% for the 10 drugs, P , 0.05 paired t-test).

Performance of each DST compared to consensus results As we had three methods to adjudicate each isolate, we redefined the gold standard as the consensus result. Against this consensus standard, the performance of the TAC and MYCOTB improved and became similar to that of the phenotypic method (Table 4). The average sensitivities of the TAC, MYCOTB, and conventional phenotypic DST method were respectively 90 6 10%, 90 6 5%, and 89 6 6% for the six drugs EMB, SM, KM, AMK, OFX, MXF (P ¼ NS [not significant]). Specificities were respectively 96 6 4%, 96 6 3%, and 92 6 11% (P ¼ NS). It is to be noted that the TAC method was particularly insensitive for EMB resistance (74%), the phenotypic method was particularly non-specific for SM susceptibility (74%, largely contributed by the LJ method), all methods were excellent for AMK and KM, and MYCOTB was highly sensitive for detecting OFX resistance (98%). Relationships between genotype and MIC Both the TAC and the MYCOTB plate provide additional information beyond a DST result, namely, the identity of specific mutations and quantitative susceptibility, respectively. There is emerging information that specific mutations correlate with the degree of resistance, with some mutations conferring high-level and others low-level resistance.10 We thus examined the extent to which mutations correlated

TAC

10 100 5 83 0 91 0 192 4 154 8 100 0 6 0 6

S

74 (64–82) 89 (83–94) 100 (59–100) 100 (69–100) 83 (69–93) 91 (59–100) 94 (89–97) 94 (90–97) 90 6 10

91 (84–96) 94 (87–98) 100 (96–100) 100 (98–100) 98 (94–99) 93 (86–97) 100 (54–100) 100 (54–100) 96 6 4 94 6 6

94

94

92

95

100

100

92

83 .4 64 .2 62 .4 64 .5 65 .2 62 .0.5/2.0 60.5/2.0 .0.25 60.25 .1 61

Sensitivity Specificity Accuracy MYCOTB % (95%CI) % (95%CI) % MIC lg/ml 87 15 112 11 6 1 9 1 41 1 10 1 185 21 197 9

R 8 102 5 83 0 91 2 190 10 148 6 102 0 6 0 6

S

Consensus

85 (77–93) 91 (84–95) 86 (42–99) 90 (55–99) 98 (87–100) 91 (59–100) 90 (85–94) 96 (92–98) 90 6 5

93 (86–97) 94 (87–98) 100 (96–100) 99 (96–100) 94 (89–97) 94 (88–98) 100 (54–100) 100 (54–100) 96 6 3 95 6 4

96

90

94

95

99

99

92

89

R S R S R S R S R S R S R S R S

Sensitivity Specificity Accuracy Phenotypic % (95%CI) % (95%CI) % 95 7 115 8 6 1 9 1 33 9 10 1 206 0 206 0

R 20 90 23 65 1 90 1 191 2 156 4 104 6 0 6 0

S

Consensus

89 6 6

*

93 (85–97) 94 (87–97) 86 (42–99) 90 (47–99) 79 (63–90) 91 (59–100) *

92 6 11

*

82 (74–89) 74 (63–83) 99 (94–100) 99 (97–100) 99 (95–100) 96 (91–99) *

93 6 6

*

*

96

95

99

98

85

87

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

* As all isolates in this study were INH- and RMP-resistant based on phenotypic testing, these samples cannot inform on the sensitivity or specificity of the phenotypic method of INH or RMP susceptibility/resistance. † MFX was tested using agar proportion at 0.5 lg/ml on some isolates and at 2.0 lg/ml on others, and the corresponding MYCOTB breakpoint was used. ‡ Average 6 SD sensitivity and specificities are for EMB, SM, AMK, KM, OFX, MFX to compare across methodologies. DST ¼ drug susceptibility testing; TAC ¼ TaqManW Array Card; R ¼ resistant; S ¼ susceptible; CI ¼ confidence interval; MIC ¼ minimum inhibitory concentration; EMB ¼ ethambutol; SM ¼ streptomycin; AMK ¼ amikacin; KM ¼ kanamycin; OFX ¼ ofloxacin; MFX ¼ moxifloxacin; INH ¼ isoniazid; RMP ¼ rifampin; SD ¼ standard deviation.

75 27 110 13 7 0 10 0 35 7 10 1 193 13 194 12

R

Consensus

Performance of each DST vs. the consensus result

Mutant No mutant SM Mutant No mutant AMK Mutant No mutant KM Mutant No mutant OFX Mutant No mutant MFX† Mutant No mutant INH Mutant No mutant RMP Mutant No mutant Average‡

EMB

Drug

Table 4


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with the MIC for various drugs (Figure or Appendix Table A.6). As expected, the inhA promoter mutation exhibited resistance to ETH (median MIC 10 lg/ml). We observed ‘low-level’ resistance to INH with the inhA C(15)T mutation compared to katG S315T mutations (median MIC 1.0 vs. 2.0 lg/ml, P ¼ 0.05). There was a higher INH MIC in isolates with both inhA C(15)T and katG S315T mutations (median MIC . 4.0 vs. 1.0 lg/ml, or 2.0 lg/ml with only one mutation, P , 0.05). TAC included the most common high-level rpoB mutations (S531L, H526Y, H526D, H526L, D516V) as well as some low-level mutations (L511P, Q513L, L533P); however, we observed no RMP MIC differences between these mutations in this repository (P ¼ NS). Rifabutin (RBT) MICs were lower than RMP MICs (median MIC 1.0 vs. .16.0 lg/ml), and this was true across diverse mutations. D516V was common, and had a median MIC of .16.0 lg/ml for RMP (MYCOTB-resistant), but 0.5 lg/ml for RBT (MYCOTB-susceptible). The various embB mutations tested were associated with MICs that hovered around the critical concentration. For SM, the rpsL K43R mutation was by far the most common mutation observed (82/115, 71%) and correlated tightly with very high MICs. We observed 100% sensitivity/specificity for AMK and KM by including both the rrs A1401G and G1484T mutations, as there were no eis mutations in this repository. MICs were lower for AMK than KM (e.g., in all strains median MIC ¼ 0.25 vs. 1.25 lg/ml; P , 0.05). For fluoroquinolones (FQs), the TAC card included probes for the well-described mutations gyrA D94G, D94Y, and D94A, as well as A90V.11–13 Mutations of gyrA at codon 94 were most prevalent (27/212, 13%), followed by 4.2% (9/212) for A90V. MFX yielded lower MICs than OFX (median MIC of gyrA mutants was 6–16 lg/ml for OFX and 1.5–8 lg/ ml for MXF; Appendix Table A.6).

DISCUSSION M. tuberculosis DST for second-line drugs is important for optimal clinical care2 and surveillance. The challenges of the conventional phenotypic DST methods are well described.10,14 Most diagnostic evaluations examine a new method against the phenotypic result, which is held as the standard. In this evaluation of the genotypic TAC and the MYCOTB MIC plate, performance against this phenotypic standard was modest (average accuracy 87–88% for EMB, SM, AMK, KM, OFX and MFX), lower than that needed to convincingly replace conventional phenotypic DST.5 However, because we evaluated three methodologies, we were able to adjudicate the results, and using a consensus standard we found much better performance with the genotypic TAC or the MYCOTB plate, equal to that of the

conventional phenotypic DST, each with an average accuracy of 93–95%. Consensus standards are widely utilized if no one test is perfect,15 are logical to apply to this setting, and are clinically commonplace—physicians may encounter MDR-TB strains that reveal discrepancies between multiple DST methods and conclude that the majority result is likely. Using this standard we encountered a number of OFX-susceptible strains that were resistant according to both MYCOTB (high MIC) and genotypic methods (gyrA mutant). In other words, exclusive use of OFX phenotypic testing could mistriage a number of patients for enhanced XDR-TB treatment; for example, 21% of OFX consensusresistant strains in this study would have been missed. A worrying number of phenotypically OFX-susceptible isolates with elevated MYCOTB MIC and gyrA mutations have also been seen in other studies.7 The phenotypic results for MFX had better correlation with the consensus; however, the number of resistant isolates was small, and results are complicated by the fact that the recommended critical concentrations of MFX vacillate. A laboratory must choose a method based on its capabilities and resistance patterns. For second-line DST in our settings, given that the overall accuracy was similar to that of phenotypic DST, we prefer either the MYCOTB or the TAC (particularly a future version that includes gyrA mutations S91P, D94H, D94N, G88C, D89N, and D89G, which would have increased the sensitivity for OFX/MFX resistance). TAC requires a sophisticated RT-PCR cycler, but yields a result within 2 h and has greater biosafety due to minimal manipulation of the isolate. It is also cheaper than generating and sequencing multiple amplicons using Sanger. The MYCOTB plate has been reviewed in other studies and has generally yielded similar results.8 We think that the MIC result is appealing for individualized patient care, assuming all tested drugs are locally available, as it gives a quantitative result that may inform potential dose increases or within-class changes,16 and is much less onerous than performing phenotypic testing, particularly at multiple MFX concentrations. The MYCOTB method was the most sensitive for detecting fluoroquinolone resistance (91–98%); however, there may be a specificity cost that needs further evaluation. DST of EMB is known to be problematic,17 and our results do not give rise to any enthusiasm about any method, particularly genotypic. DST of SM worked well with TAC or MYCOTB, but less well for the conventional phenotypic methods, particularly due to false resistance with the LJ method.17 As 87 isolates in our MDR-TB repository (42%) were likely susceptible with two methods, SM could still be listed among possible drugs for the treatment of MDR-TB in patients who had not previously received it; however, the LJ method should be used with caution.


Figure Relationships between genotype and MIC. MDR-TB isolates (n ¼ 212) were tested using the TaqManW Array Card (genes and mutations shown along x-axis) and the MYCOTB plate (yaxis). The brown horizontal bar indicates the median MIC of each mutation type for the respective drug. MIC ¼ minimum inhibitory concentration; MDR-TB ¼ multidrug-resistant tuberculosis. This image can be viewed online in color at http://www.ingentaconnect.com/content/iuatld/ijtld/2016/ 00000020/00000008/art00021

1111

1112


The role of individual mutations in the degree of resistance is emerging, particularly for RMP and rpoB,12,18,19 and FQs and gyrA.11,13,20 Clearly rpoB S531L and probably gyrA D94G are high-level resistance mutations. Obtaining robust MIC90 data will be critical to informing the interpretation of these genotypic methods. The rpoB D516V mutation, which exhibited high-level RMP resistance but RBT susceptibility, was quite common in our repository (23/212, 11%).19 Investigation of RBT efficacy in these patients should be evaluated, as this would offer a highly potent drug. There were limitations to this study. As each laboratory performed only one phenotypic method, discrepancies between the agar proportion vs. LJ vs. MGIT methods could not be described. The number of strains with resistance to FQs or injectable agents was relatively low. What is urgently needed are better human outcome data to understand the impact of quantitative MICs, and prospective studies that compare clinical outcomes when second-line DST is performed using different methods. In the case of RMP, there are case reports of poor outcome with phenotypically susceptible strains that are genotypically resistant,10,21 but these data are scarce. This would give decision makers the most relevant information to decide whether the conventional phenotypic standards are truly gold standard or to scale up faster, easier methods for second-line DST. Acknowledgements This work was supported by National Institutes of Health (NIH; Bethesda, MD, USA) grants R01 AI093358 and K24 AI102972 (to EH). SH was also supported by NIH grant K23 AI099019. The authors thank the laboratory staff at the Armed Forces Research Institute of Medical Sciences, Bangkok, Thailand, for the use of their real-time polymerase chain reaction machine for TaqManw Array Card testing. Conflicts of interest: none declared.

References 1 World Health Organization. Guidelines for the programmatic management of drug-resistant tuberculosis: 2011 update. WHO/HTM/TB/2011.6. Geneva, Switzerland: WHO, 2011. 2 Bastos M L, Hussain H, Weyer K, et al. Treatment outcomes of patients with multidrug-resistant and extensively drug-resistant tuberculosis according to drug susceptibility testing to first- and second-line drugs: an individual patient data meta-analysis. Clin Infect Dis 2014; 59: 1364–1374. 3 World Health Organization. Companion handbook to the WHO guidelines for the programmatic management of drugresistant tuberculosis. WHO/HTM/TB/2014.11Geneva, Switzerland: WHO, 2014. 4 World Health Organization. Xpert MTB/RIF implementation manual: technical and operational ’how-to’; practical considerations. Geneva, Switzerland: WHO, 2014. 5 World Health Organization. The use of molecular line probe assay for the detection of resistance to second-line antituberculosis drugs. WHO/HTM/TB/2013.01. Geneva, Switzerland: WHO, 2013.

6 Abuali M M, Katariwala R, LaBombardi V J. A comparison of the Sensititrew MYCOTB panel and the agar proportion method for the susceptibility testing of Mycobacterium tuberculosis. Eur J Clin Microbiol Infect Dis 2012; 31: 835– 839. 7 Hall L, Jude K P, Clark S L, et al. Evaluation of the Sensititre MYCOTB plate for susceptibility testing of the Mycobacterium tuberculosis complex against first- and second-line agents. J Clin Microbiol 2012; 50: 3732–3734. 8 Lee J, Armstrong D T, Ssengooba W, et al. Sensititre MYCOTB MIC plate for testing Mycobacterium tuberculosis susceptibility to first- and second-line drugs. Antimicrob Agents Chemother 2014; 58: 11–18. 9 Pholwat S, Liu J, Stroup S, et al. Integrated microfluidic card with TaqMan probes and high-resolution melt analysis to detect tuberculosis drug resistance mutations across 10 genes. mBio 2015; 6: e02273. 10 Van Deun A, Aung K J, Bola V, et al. Rifampin drug resistance tests for tuberculosis: challenging the gold standard. J Clin Microbiol 2013; 51: 2633–2640. 11 Kambli P, Ajbani K, Sadani M, et al. Correlating minimum inhibitory concentrations of ofloxacin and moxifloxacin with gyrA mutations using the GenoType MTBDRsl assay. Tuberculosis (Edinb) 2015; 95: 137–141. 12 Kambli P, Ajbani K, Sadani M, et al. Defining multidrugresistant tuberculosis: correlating GenoType MTBDRplus assay results with minimum inhibitory concentrations. Diagn Microbiol Infect Dis 2015; 82: 49–53. 13 Willby M, Sikes R D, Malik S, Metchock B, Posey J E. Correlation between gyrA substitutions and ofloxacin, levofloxacin, and moxifloxacin cross-resistance in Mycobacterium tuberculosis. Antimicrob Agents Chemother 2015; 59: 5427–5434. 14 Madison B, Robinson-Dunn B, George I, et al. Multicenter evaluation of ethambutol susceptibility testing of Mycobacterium tuberculosis by agar proportion and radiometric methods. J Clin Microbiol 2002; 40: 3976–3979. 15 Miller W C. Can we do better than discrepant analysis for new diagnostic test evaluation? Clin Infect Dis 1998; 27: 1186– 1193. 16 Heysell S, Pholwat S, Mpagama S, et al. Sensititre MYCOTB MIC plate compared to Bactec MGIT 960 for first and secondline anti-tuberculosis drug susceptibility testing in Tanzania: a call to operationalize minimum inhibitory concentrations. Antimicrob Agents Chemother 2015; 59: 7104–7108. 17 Banu S, Rahman S M, Khan M S, et al. Discordance across several methods for drug susceptibility testing of drug-resistant Mycobacterium tuberculosis isolates in a single laboratory. J Clin Microbiol 2014; 52: 156–163. 18 Zaczek A, Brzostek A, Augustynowicz-Kopec E, Zwolska Z, Dziadek J. Genetic evaluation of relationship between mutations in rpoB and resistance of Mycobacterium tuberculosis to rifampin. BMC Microbiol 2009; 9: 10. 19 Jamieson F B, Guthrie J L, Neemuchwala A, Lastovetska O, Melano R G, Mehaffy C. Profiling of rpoB mutations and MICs for rifampin and rifabutin in Mycobacterium tuberculosis. J Clin Microbiol 2014; 52: 2157–2162. 20 Sirgel F A, Warren R M, Streicher E M, Victor T C, van Helden P D, Bottger E C. gyrA mutations and phenotypic susceptibility levels to ofloxacin and moxifloxacin in clinical isolates of Mycobacterium tuberculosis. J Antimicrob Chemother 2012; 67: 1088–1093. 21 Williamson D A, Roberts S A, Bower J E, et al. Clinical failures associated with rpoB mutations in phenotypically occult multidrug-resistant Mycobacterium tuberculosis. Int J Tuberc Lung Dis 2012; 16: 216–220.


i

APPENDIX

Table A.1 Primers and probes used in the TB TaqManW Array Card Gene

Primers/probes

inhA

rpoB Amplicon 2

embB Amplicon 1

embB Amplicon 2

embB Amplicon 3

rpsL Amplicon 1

rpsL Amplicon 2

rrs Amplicon 1

rrs Amplicon 2

rrs Amplicon 3

Assay region

Product size bp

Forward Reverse T(8)C C(15)T

5 0 -GCTCGTGGACATACCGATTT-3 0 5 0 -TCCGGTAACCAGGACTGAAC-3 0 VIC-5 0 -CGAGACGATAGGCTG-3 0 -MGB VIC-5 0 -ACCTATCATCTCGCC-3 0 -MGB

(54) – codon 22

120

Forward Reverse 315 Thr*

5 0 -CTCGTATGGCACCGGAAC-3 0 5 0 -CCGTACAGGATCTCGAGGAA-3 0 HEX-5 0 -ATCAþCCAþCCGþGCAþTCG-3 0

Codon 303–338

108

Forward Reverse 511Pro 513Leu 513Glu 516Val*

5 0 -GCCGCGATCAAGGAGTTCT-3 0 5 0 -CACGCTCACGTGACAGACC-3 0 VIC-5 0 -AGCCAGCCGAGCC-3 0 -MGB VIC-5 0 - CCAGCTGAGCCTAT-3 0 -MGB VIC-5 0 -CTGAGCGAATTCA-3 0 -MGB HEX-5 0 -AATTCAþTGGþTCCþAGAþACAA-3 0

Codon 500–543

130

Forward Reverse 526Tyr 526Asp 526Leu* 531Leu 531Trp 533Pro

5 0 -AGCCAGCTGAGCCAATTCAT-3 0 5 0 -CACGCTCACGTGACAGACC-3 0 VIC-5 0 -CGGGGTTGACCTACA-3 0 -MGB VIC-5 0 -GTTGACCGACAAGC-3 0 -MGB HEX-5 0 -GTþTGAþCCCþTCAþAGC-3 0 VIC-5 0 - CCGACTGTTGGCGC-3 0 -MGB NED-5 0 -CCGACTGTGGGCG-3 0 -MGB VIC-5 0 - ACTGTCGGCGCCGG-3 0 -MGB

Codon 509–543

103

Forward Reverse 306Val 306Ile 306Ile2 306Leu

5 0 -TGATATTCGGCTTCCTGCTC-3 0 5 0 -GAACCAGCGGAAATAGTTGG-3 0 VIC-5 0 -CTACATCCTGGGCGTG-3 0 -MGB VIC-5 0 -ATCCTGGGCATCGC-3 0 -MGB VIC-5 0 -ATCCTGGGCATAGC-3 0 -MGB VIC-5 0 -TACATCCTGGGCCTG-3 0 -MGB

Codon 283–323

122

Forward Reverse 328Tyr 328Gly

5 0 -GCTACATCCTGGGCATGG-3 0 5 0 -AGCGCCAGCAGGTTGTAATA-3 0 VIC-5 0 -CCGGAGTATCCCTT-3 0 -MGB VIC-5 0 -CCGGAGGGTCCCT-3 0 -MGB

Codon 301–339

115

Forward Reverse 406Ala

5 0 -GGCCATGGTCTTGCTGAC-3 0 5 0 -ACCGCTCGATCAGCACATAG-3 0 VIC-5 0 -CGGAGGCCATCAT-3 0 -MGB

Codon 388–422

104

Forward Reverse 43Arg

5 0 -GCAGCGTCGTGGTGTATG-3 0 5 0 -CCTCGACCTGACTCGTCAAC-3 0 VIC-5 0 -CCACCACTCCGAGGA-3 0 -MGB

Codon 29–62

104

Forward Reverse 88Arg* 88Met*

5 0 -CACAACCTGCAGGAGCACT-3 0 5 0 -TCTTGACACCCTGCGTATCC-3 0 HEX-5 0 - GGþTGAþGGGþACCT-3 0 HEX-5 0 - CGGGþTGAþTGGþACCT-3 0

Codon 72–108

112

Forward Reverse A(514)C C(517)T

5 0 -TCTCTCGGATTGACGGTAGG-3 0 5 0 -CGAGCTCTTTACGCCCAGTA-3 0 VIC-5 0 -TACGTGCCAGCCGC-3 0 -MGB VIC-5 0 -CAGCAGCTGCGGTA-3 0 -MGB

nt 460–571

112

Forward Reverse A(906)G

5 0 -GATCCGTGCCGTAGCTAACG-3 0 5 0 -GTTGCATCGAATTAATCCACATG-3 0 VIC-5 0 -CTAAAACTCGAAGGAATTG-3 0 -MGB

nt 840–962

123

Forward Reverse A(1401)G

5 0 -AAGTCGGAGTCGCTAGTAATCG-3 0 5 0 -TTCGGGTGTTACCGACTTTC-3 0 VIC-5 0 -CCCGTCGCGTCAT-3 0 -MGB

nt 1320–1427

104

katG

rpoB Amplicon 1

Sequences

ii


Table A.1 (continued) Gene rrs Amplicon 4

Primers/probes

gyrB Amplicon 3

pncA Amplicon 1 pncA Amplicon 2 pncA Amplicon 3

pncA Amplicon 4

pncA Amplicon 5 pncA Amplicon 6 pncA Amplicon 7 pncA Amplicon 8 pncA Amplicon 9

Product size bp

5 0 -AAAGTCGGTAACACCCGAAG-3 0 5 0 -CCGGTACGGCTACCTTGTTA-3 0 VIC-5 0 -CGATTGGGACTAAGTC-3 0 -MGB

nt 1409–1510

102

Forward Reverse C(14)T G(10)A

5 0 -TGATCCTTTGCCAGACACTG-3 0 5 0 -CTCGGTCGGGCTACACAG-3 0 VIC-5 0 - CGGCATATGCTACAGT-3 0 -MGB VIC-5 0 -TATGCCACAATCGGATT-3 0 -MGB

(73) – codon 10

103

Forward Reverse 90Val 94Gly† 94Tyr† 94Ala†

5 0 -CCGGTCGGTTGCCGAGACC-3 0 5 0 -CCAGCGGGTAGCGCAGCGACCAG-3 0 VIC-5 0 -AGATCGACACGTCGCC-3 0 -MGB VIC-5 0 -CCAGGSTGCCGTAGATC-3 0 –MGB VIC-5 0 -TCGATCTACTACASCCT-3 0 -MGB VIC-5 0 -ATCTACGCCASCCTG-3 0 -MGB

Codon 75–109

107

Forward Reverse 447Phe

5 0 -CGTAAGGCACGAGAGTTGGT-3 0 5 0 -GCCGAGTCACCTTCTACGAC-3 0 VIC-5 0 -ATTGCCGTTTCACG-3 0 -MGB

Codon 421–463

128

Forward Reverse 461His

5 0 -CGCAAGTCCGAACTGTATGT-3 0 5 0 -CGCTTTCTCCACATTGATGA-3 0 VIC-5 0 - CCGAGTGACCTTC-3 0 -MGB

Codon 451–491

123

Forward Reverse 499Asp 501Asp

5 0 -GGCAAGATCATCAATGTGGA-3 0 5 0 -GATATCGAACTCGTCGTGGA-3 0 VIC-5 0 -TGCTAAAGGACACCGAA-3 0 -MGB VIC-5 0 -AACACCGACGTTCAG-3 0 -MGB

Codon 483–519

111

Forward Reverse

5 0 -GCGTCGGTAGGCAAACTG-3 0 5 0 -GAGCCACCCTCGCAGAAGT-3 0

(50) – codon 18

103

Forward Reverse

5 0 -ATCATCGTCGACGTGCAGA-3 0 5 0 -CCACGACGTGATGGTAGTCC-3 0

Codon 5–45

124

Forward Reverse 57Asp

5 0 -CGCCATCAGCGACTACCT-3 0 5 0 -ACGAGGAATAGTCCGGTGTG-3 0 VIC-5 0 -CCGGGTGACGACT-3 0 -MGB

Codon 30–66

113

Forward Reverse 65Ser

5 0 -ACCCGGGTGACCACTTCT-3 0 5 0 -TGTCCAGACTGGGATGGAA-3 0 VIC-5 0 -CGGACTATTCTTCGTCGT-3 0 -MGB

Codon 53–86

102

Forward Reverse

5 0 -CATTGCGTCAGCGGTACTC-3 0 5 0 -CCGTTCTCGTCGACTCCTT-3 0

Codon 71–113

128

Forward Reverse

5 0 -AATCGAGGCGGTGTTCTACA-3 0 5 0 -ATACCGACCACATCGACCTC-3 0

Codon 90–133

132

Forward Reverse

5 0 -CACGCCACTGCTGAATTG-3 0 5 0 -ATTGCGTACCGCGTCCTC-3 0

Codon 114–149

109

Forward Reverse

5 0 -AGGTCGATGTGGTCGGTATT-3 0 5 0 -ACCCGCTGTCAGGTCCAC-3 0

Codon 128–162

107

Forward Reverse

5 0 -GAGGACGCGGTACGCAAT-3 0 5 0 -ATCAGGAGCTGCAAACCAAC-3 0

Codon 144–187

133

Forward Reverse M. tuberculosis

5 0 -GGATAAGCCTGGGAAACTGG-3 0 5 0 -ACCCCACCAACAAGCTGATA-3 0 VIC-5 0 -CCACGGGATGCAT-3 0 -MGB

nt 142–259

118

gyrA

gyrB Amplicon 2

Assay region

Forward Reverse G(1484)T

eis

gyrB Amplicon 1

Sequences

16S

* Locked nucleic acid bases are indicated by ‘þ’. † A polymorphism at codon 95 Ser/Thr was included by using ‘S’ G/C. TB ¼ tuberculosis; bp ¼ base pair; nt ¼ nucleotide.

iii


Table A.2 Performance of TAC compared to Sanger sequencing. Sequencing (Thailand; n ¼ 98) Drug

TAC

Mutation

Wild-type

INH

Mutant No mutant Mutant No mutant Mutant No mutant Mutant No mutant Mutant No mutant Mutant No mutant Mutant No mutant Mutant No mutant Variant Wild-type

87 2* 88 3† 33 4‡ 62 3§ 6 1¶ 6 1¶ 16 5# 16 5# 37 9

3 6 2 5 0 61 2 31 1 90 1 90 1 76 1 76 5 47

RMP EMB SM AMK KM OFX MFX PZA



Accuracy %

98 (92–100)

67 (30–93)

95

97 (91–99)

71 (29–96)

95

89 (75–97)

100 (94–100)

96

95 (87–99)

94 (80–99)

95

86 (42–100)

99 (94–100)

98

86 (42–100)

99 (94–100)

98

76 (53–92)

99 (93–100)

94

76 (53–92)

99 (93–100)

94

80 (66–91)

90 (79–97)

86

* 1 S315T (ACA) and 1 W328Q. † 1 D516Y, 1 H526R, 1 S531Y. ‡ 1 M306L*, 1 M306V*, 2 Y319S. § 1 K43R*, 1 R86P, 1 R86Q. ¶ eis C(12)T. # 3 S91P, 1 D94H, 1 D94G*. Note: all of these excess Sanger-detected mutations were less common mutations, some of uncertain significance, for which we did not design probes, with the exception of * above (where Sanger detected mutations while the probe did not). TAC ¼ TaqMan Array Card; CI ¼ confidence interval; INH ¼ isoniazid; NA ¼ not applicable; RMP ¼ rifampin; EMB ¼ ethambutol; SM ¼ streptomycin; AMK ¼ amikacin; KM ¼ kanamycin; OFX ¼ ofloxacin; MFX ¼ moxifloxacin; PZA ¼ pyrazinamide.

Table A.3 Performance of TAC compared to the Hain line-probe assay Hain line-probe assay (Bangladesh and Tanzania; n ¼ 86) Drug

TAC

Mutation

Wild-type

INH

Mutant No mutant Mutant No mutant Mutant No mutant Mutant No mutant Mutant No mutant Mutant No mutant Mutant No mutant

77 1 73 4 30 3 3 0 3 0 17 2 17 2

2 6 6 2 5 26 0 60 0 60 0 45 0 45

RMP EMB* AMK* KM* OFX* MFX*

Accuracy % 97 88 88 100 100 97 97

* As not all results are available for all methodologies or all drugs, not all comparisons sum to n ¼ 86. TAC ¼ TaqManW Array Card; INH ¼ isoniazid; RMP ¼ rifampin; EMB ¼ ethambutol; AMK ¼ amikacin; KM ¼ kanamycin; OFX ¼ ofloxacin; MFX ¼ moxifloxacin.

‡

6 2 8 3 1 1

90 8 90 8 22 17 52 7 6 1 6 1

R

12 76 0 47 9 29

0 0 0 0 10 49 12 27 1 90 1 90

S

75 (35–97) 73 (38–94) 50 (1–99) NA

92 (84–96) 92 (84–96) 56 (40–72) 88 (77–95) 86 (42–100) 86 (42–100)


86 (77–93) 100 (92–100) 76 (60–88) NA

83 (71–91) 69 (52–83) 99 (94–100) 99 (94–100)

NA

NA


84 6 11

NA

75

95

85

98

98

81

72

NA

NA

Accuracy %

20 7

3 0

75 8 76 7 43 25 36 27 ND

R

1 55

0 80

0 0 0 0 1 14 0 19

S

LJ (n ¼ 83)

NA

NA

74 (54–89) NA

100 (29–100)

90 (82–96) 92 (83–96) 63 (51–75) 57 (44–69) ND


NA

NA

98 (90–100) NA

100 (95–100)

93 (68–100) 100 (82–100) ND

NA

NA


82 6 16

NA

NA

NA

90

100

ND

67

69

NA

NA

Accuracy %

66 24

0 0 0 1

0 0

28 3 28 3 3 5 14 2 ND

R

33 84

0 21 0 20

0 21

0 0 0 0 6 17 1 14

S

MGIT (n ¼ 31)

73 (63–82)

0 (0–97) NA

NA

NA

90 (74–98) 90 (74–98) 38 (9–75) 88 (62–98) ND


72 (63–80)

100 (84–100) 100 (83–100) NA

100 (84–100)

74 (52–90) 93 (68–100) ND

NA

NA


89 6 17

73

NA

95

100

100

ND

90

65

NA

NA

Accuracy %

* Each isolate (n ¼ 212) was tested with only one phenotypic method. † As not all results are available for all methodologies or all drugs, not all comparisons sum to n ¼ 212. ‡ Average 6 SD accuracies are for EMB, SM, KM, OFX to allow comparison across phenotypic DST methodologies (P ¼ NS) ¨ proportion method; MGIT ¼ Mycobacterium Growth Indicator Tube; R ¼ resistant; S ¼ susceptible; CI ¼ confidence interval; INH ¼ isoniazid; NA ¼ not TAC ¼ TaqManW Array Card; DST ¼ drug susceptibility testing; L J ¼ Lowenstein-Jensen applicable; RMP ¼ rifampin; EMB ¼ ethambutol; SM ¼ streptomycin; ND ¼ not done; AMK ¼ amikacin; KM ¼ kanamycin; OFX ¼ ofloxacin; MFX ¼ moxifloxacin; PZA ¼ pyrazinamide; SD ¼ standard deviation; NS ¼ not significant.

Average

PZA†

MFX 2.0

†

MFX 0.5/0.25†

OFX†

KM†

AMK†

SM†

EMB

Mutant No mutant Mutant No mutant Mutant No mutant Variant Wild-type

Mutant No mutant Mutant No mutant Mutant No mutant Mutant No mutant Mutant No mutant Mutant No mutant

INH

RMP

TAC

Drug

Agar proportion method (n ¼ 98)

Table A.4 Performance of TAC compared to phenotypic DST stratified by agar proportion, L J or MGIT method*

iv The International Journal of Tuberculosis and Lung Disease

R . 0.25 S 6 0.25 R.1 S61 R.4 S64 R.2 S62 R.4 S64 R.5 S65 R.2 S62 R . 0.5 S 6 0.5 R.2 S62 R.5 S65 R.2 S62

78 20 86 12 20 19 51 8 5 2 5 2 8 0 8 3 1 1 4 11 5 7

R

0 0 0 0 7 52 7 32 1 90 3 88 9 79 3 44 2 36 3 80 7 79

S 80 (70–87) 88 (80–93) 51 (35–68) 86 (75–94) 71 (29–96) 71 (29–96) 100 (63–100) 73 (39–94) 50 (1– 99) 27 (8–55) 42 (15–72)


88 (77–95) 82 (66–92) 99 (94–100) 97 (91–99) 90 (81–95) 94 (82–99) 95 (82–99) 96 (90–99) 92 (84–97)

NA

NA


86 6 9

86

86

93

90

91

95

97

85

73

NA

NA

Accuracy %

21 22

3 0 24 3

78 5 82 1 53 15 40 23

R

1 39

0 80 8 48

0 0 0 0 1 14 1 18

S

LJ (n ¼ 83)

49 (33–64) NA

NA

100 (29–100) 89 (71–97) NA

94 (86–98) 99 (93–100) 78 (66–87) 63 (50–75) ND


98 (87–100) NA

NA

100 (95–100) 86 (74–94) NA

93 (68–100) 95 (74–100) ND

NA

NA


85 6 12

NA

72

NA

NA

87

100

ND

71

81

NA

NA

Accuracy %

4 2 0 0

0 0 0 0 0 1

29 2 29 2 7 1 13 3

R

1 14 2 19

0 21 2 19 2 18

0 0 0 0 7 16 4 11

S

MGIT (n ¼ 31)

67 (22–96) NA

0 (0–97) NA

NA

NA

94 (78–99) 94 (78–99) 88 (47–99) 81 (54–96) ND


93 (68–100) 90 (70–99)

100 (84–100) 90 (69–99) 90 (68–99) NA

70 (47–87) 73 (45–92) ND

NA

NA


86 6 12

90

86

NA

86

90

100

ND

77

74

NA

NA

Accuracy %

* Each isolate (n ¼ 212) was tested with only one phenotypic method. † As not all results are available for all methodologies or all drugs, not all comparisons sum to n ¼ 212. ‡ Average 6 SD accuracies are for EMB, SM, KM, OFX to allow comparison across phenotypic DST methodologies (P ¼ NS) ¨ DST ¼ drug susceptibility testing; L J ¼ Lowenstein-Jensen proportion method; MGIT ¼ Mycobacterium Growth Indicator Tube; MIC ¼ minimum inhibitory concentration; R ¼ resistant; S ¼ susceptible; CI ¼ confidence interval; INH ¼ isoniazid; NA ¼ not applicable; RMP ¼ rifampin; EMB ¼ ethambutol; SM ¼ streptomycin; ND ¼ not done; AMK ¼ amikacin; KM ¼ kanamycin; OFX ¼ ofloxacin; MFX ¼ moxifloxacin; ETH ¼ ethionamide; PAS ¼ para-aminosalicylic acid; SD ¼ standard deviation; NS ¼ not significant.

Average‡

PAS†

ETH†

MFX 2.0†

MFX 0.5†

OFX†

KM†

AMK†

SM†

EMB

RMP

INH

Drug

MYCOTBMIC result lg/ml

Agar proportion method (n ¼ 98)

Table A.5 Performance of SensititreW MYCOTB compared to phenotypic DST stratified by agar proportion, L J or MGIT methods*


v

vi


Table A.6 Relationships between genotypic and MIC* Drug

Gene mutation

Median MIC lg/ml

MIC range lg/ml

inhA(8)C (n ¼ 0) inhA(15)T (n ¼ 17) katG315T (n ¼ 169) inhA(15)TþkatG315T (n ¼ 7) Negative (n ¼ 19)

NA 1 2 .4 4

NA 0.06–.4 ,0.03–.4 2–.4 0.03–.4

rpoB531L (n ¼ 135) rpoB531W (n ¼ 0) rpoB526Y (n ¼ 13) rpoB526D (n ¼ 9) rpoB526L (n ¼ 1) rpoB516V (n ¼ 23) rpoB513L (n ¼ 2) rpoB513E (n ¼ 7) rpoB511P (n ¼ 1) rpoB533P (n ¼ 3) Negative (n ¼ 18)

.16 NA .16 .16 8 .16 .16 .16 .16 .16 .16

,0.125–.16 NA 0.25–.16 .16 8 ,0.125–.16 .16 2–.16 .16 0.5–.16 ,0.125–.16

rpoB531L (n ¼ 135) rpoB531W (n ¼ 0) rpoB526Y (n ¼ 13) rpoB526D (n ¼ 9) rpoB526L (n ¼ 1) rpoB516V (n ¼ 23) rpoB513L (n ¼ 2) rpoB513E (n ¼ 7) rpoB511P (n ¼ 1) rpoB533P (n ¼ 3) Negative (n ¼ 18)

2 NA 2 2 0.25 0.5 24 2 .16 0.5 0.5

,0.125–16 NA ,0.125–4 1–.16 0.25 ,0.125–16 8–.16 0.25–.16 .16 ,0.125–1 ,0.125–.16

INH

RMP

RBT

EMB embB306Iatc (n ¼ 11) embB306Iata (n ¼ 21) embB306L (n ¼ 10) embB306V (n ¼ 40) embB328Y (n ¼ 3) embB328G (n ¼ 0) embB406A (n ¼ 5) Negative (n ¼ 122)

8 8 8 8 4 N/A 4 2

2–8 1–16 4–32 2–32 4–8 N/A 4–8 ,0.5–32

rpsL43R (n ¼ 82) rpsL88R (n ¼ 6) rpsL88M (n ¼ 8) rrs(514)C (n ¼ 9) rrs(517)T (n ¼ 7) rrs(906)G (n ¼ 3) Negative (n ¼ 96)

.32 .32 24 8 1 1 1

0.5–.32 16–.32 1–.32 2–.32 0.5–16 ,0.25–2 ,0.25–.32

rrs(1401)G (n ¼ 9) rrs(1484)T (n ¼ 1) eis(10)A (n ¼ 0) eis(14)T (n ¼ 0) Negative (n ¼ 202)

.16 .16 NA NA 0.25

0.25–.16 .16 NA NA 0.125–4

rrs(1401)G (n ¼ 9) rrs(1484)T (n ¼ 1) eis(10)A (n ¼ 0) eis(14)T (n ¼ 0) Negative (n ¼ 202)

.40 .40 NA NA 1.25

,0.625–.40 .40 NA NA 0.625–20

gyrA94G (n ¼ 15) gyrA94Y (n ¼ 8) gyrA94A (n ¼ 4) gyrA90V (n ¼ 9) gyrB501D (n ¼ 3) gyrB447F (n ¼ 0) gyrB461H (n ¼ 0) gyrB499D (n ¼ 0) Negative (n ¼ 173)

16 16 6 8 8 NA NA NA 1

1–32 4–32 2–8 4–16 0.5–32 NA NA NA ,0.25–32

SM

AMK

KM

OFX


vii

Table A.6 (continued) Drug

Median MIC lg/ml

MIC range lg/ml

gyrA94G (n ¼ 15) gyrA94Y (n ¼ 8) gyrA94A (n ¼ 4) gyrA90V (n ¼ 9) gyrB501D (n ¼ 3) gyrB447F (n ¼ 0) gyrB461H (n ¼ 0) gyrB499D (n ¼ 0) Negative (n ¼ 173)

3 8 1.5 2 .8 NA NA NA 0.25

0.125–8 0.5–8 0.5–4 0.125–8 0.5–.8 NA NA NA ,0.0625 –.8

inhA(8)C (n ¼ 0) inhA(15)T (n ¼ 17)

NA 10

Gene mutation

MFX

ETH NA ,0.03–.40

* Negative ¼ no mutation with any probe. MIC ¼ minimum inhibitory concentration; INH ¼ isoniazid; NA ¼ not applicable; RMP ¼ rifampin; RBT ¼rifabutin; EMB ¼ ethambutol; SM ¼ streptomycin; AMK ¼ amikacin; KM ¼ kanamycin; OFX ¼ ofloxacin; MFX ¼ moxifloxacin; ETH ¼ ethionamide.

viii


R E S U M E´ C O N T E X T E : Le test de pharmacosensibilit e´ phénotypique est approuvé comme le test standard de deuxième ligne de Mycobacterium tuberculosis, mais il est lent et laborieux. M E´ T H O D E : Nous avons e´ valué l’exactitude de deux méthodes plus rapides et plus faciles qui fournissent des résultats pour de nombreux médicaments : le test génotypique TaqManw Array Card (TAC) et la plaque Sensititrew MYCOTBTM. Les deux méthodes ont e´ té testées dans trois laboratoires centraux au Bangladesh, en Tanzanie et en Tha¨ılande sur 212 isolats de tuberculose multirésistante et comparées aux méthodes phénotypiques en usage dans ces laboratoires. R E´ S U L T A T S : L’exactitude d’ensemble pour l’ e´thambutol, la streptomycine, l’amikacine, la kanamycine, l’ofloxacine et la moxifloxacine contre la méthode phénotypique standard a e´ té de 87% pour le test TAC (fourchette 70–99) et de 88% pour la plaque

MYCOTB (fourchette 76–98). Pour r e´ gler les discordances, nous avons redéfini le standard comme le consensus des trois méthodes, contre lesquelles le TAC et la plaque MYCOTB aboutissaient a` 94–95% d’exactitude tandis que le re´ sultat ph e´ notypique arrivait a` 93%. Il y a eu des isolats présentant des mutations génotypiques et une concentration minimale inhibitrice (MIC) e´ levée qui ont e´ té phénotypiquement sensibles et des isolats sans mutations et avec une MIC faible qui ont e´ té phénotypiquement résistants, ce qui remet en question le standard phénotypique. C O N C L U S I O N S : A notre avis, le TAC, la plaque MYCOTB et la m e´ thode phe´ notypique conventionnelle ont la même performance pour les médicaments de deuxième ligne, mais les méthodes nouvelles offrent la rapidite´ , plus de capacite´ de traitement et des informations quantitatives sur la sensibilité. RESUMEN

Las pruebas fenot´ıpicas de sensibilidad se han aceptado como la prueba corriente para los medicamentos de segunda l´ınea contra Mycobacterium tuberculosis, aunque su ejecucion ´ es lenta y laboriosa. M E´ T O D O S: Se evaluo ´ la precision ´ de dos métodos ma´s ra´pidos y sencillos que ofrecen resultados para multiples ´ medicamentos, a saber: la prueba genot´ıpica TaqManw con tarjeta de micromatrices (TAC) y la placa Sensititrew MYCOTBTM. Ambos métodos se ensayaron en tres laboratorios centrales en Bangladesh, Tanzania y Tailandia con 212 aislados de casos de tuberculosis multirresistente y se compararon con el m e´ todo fenot´ıpico utilizado en los laboratorios. R E S U L T A D O S: En comparaci on ´ con el m e´ todo fenot´ıpico corriente, la precisi on ´ global para etambutol, estreptomicina, amikacina, kanamicina, ofloxacino y moxifloxacino fue 87% con la TAC M A R C O D E R E F E R E N C I A:

(entre 70% y 99%) y 88% con la placa MYCOTB (entre 76% y 98%). Con el fin de resolver las discordancias se redefinio´ la norma de referencia, como el consenso de los tres métodos y en ese caso la precision ´ de la TAC y la placa MYCOTB fue de 94–95% y el método fenot´ıpico ofrecio´ una precision ´ de 93%. Se encontraron aislados con mutaciones genot´ıpicas y altas concentraciones m´ınimas inhibitorias (MIC) que fueron fenot´ıpicamente sensibles y aislados sin mutaciones y baja MIC con fenotipo resistente, lo cual pone en duda la prueba fenot´ıpica como método de referencia. ´ N: A la luz de estos resultados, se considera CONCLUSIO que el rendimiento diagnostico ´ de los métodos con TAC, la placa MYCOTB y el método fenot´ıpico corriente es equivalente para los medicamentos de segunda l´ınea; sin embargo, las dos primeras técnicas ofrecen rapidez, gran productividad e informacion ´ cuantitativa sobre la sensibilidad a los medicamentos.