SUMMARY
BACKGROUND: Although phenotypic drug susceptibility testing (DST) is endorsed as the standard for second-line drug testing of Mycobacterium tuberculosis, it is slow and laborious.
METHODS: We evaluated the accuracy of two faster, easier methodologies that provide results for multiple drugs: a genotypic TaqMan® Array Card (TAC) and the Sensititre® MYCOTB™ 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.
RESULTS: 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.
CONCLUSIONS: 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.
Keywords: drug susceptibility testing, MDR-TB, MIC
RESUME
CONTEXTE : Le test de pharmacosensibilité phénotypique est approuvé comme le test standard de deuxième ligne de Mycobacterium tuberculosis, mais il est lent et laborieux.
MÉTHODE : Nous avons évalué l'exactitude de deux méthodes plus rapides et plus faciles qui fournissent des résultats pour de nombreux médicaments : le test génotypique TaqMan® Array Card (TAC) et la plaque Sensititre® MYCOTB™. Les deux méthodes ont été testées dans trois laboratoires centraux au Bangladesh, en Tanzanie et en Thaïlande sur 212 isolats de tuberculose multirésistante et comparées aux méthodes phénotypiques en usage dans ces laboratoires.
RÉSULTATS : L'exactitude d'ensemble pour l'éthambutol, la streptomycine, l'amikacine, la kanamycine, l'ofloxacine et la moxifloxacine contre la méthode phénotypique standard a été de 87% pour le test TAC (fourchette 70–99) et de 88% pour la plaque MYCOTB (fourchette 76–98). Pour régler les discordances, nous avons redéfini le standard comme le consensus des trois méthodes, contre lesquelles le TAC et la plaque MYCOTB aboutissaient à 94–95% d'exactitude tandis que le résultat phénotypique arrivait à 93%. Il y a eu des isolats présentant des mutations génotypiques et une concentration minimale inhibitrice (MIC) élevée qui ont été phénotypiquement sensibles et des isolats sans mutations et avec une MIC faible qui ont été phénotypiquement résistants, ce qui remet en question le standard phénotypique.
CONCLUSIONS : A notre avis, le TAC, la plaque MYCOTB et la méthode phénotypique conventionnelle ont la même performance pour les médicaments de deuxième ligne, mais les méthodes nouvelles offrent la rapidité, plus de capacité de traitement et des informations quantitatives sur la sensibilité.
RESUMEN
MARCO DE REFERENCIA: Las pruebas fenotípicas de sensibilidad se han aceptado como la prueba corriente para los medicamentos de segunda línea contra Mycobacterium tuberculosis, aunque su ejecución es lenta y laboriosa.
MÉTODOS: Se evaluó la precisión de dos métodos más rápidos y sencillos que ofrecen resultados para múltiples medicamentos, a saber: la prueba genotípica TaqMan® con tarjeta de micromatrices (TAC) y la placa Sensititre® MYCOTB™. 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 método fenotípico utilizado en los laboratorios.
RESULTADOS: En comparación con el método fenotípico corriente, la precisión global para etambutol, estreptomicina, amikacina, kanamicina, ofloxacino y moxifloxacino fue 87% con la TAC (entre 70% y 99%) y 88% con la placa MYCOTB (entre 76% y 98%). Con el fin de resolver las discordancias se redefinió la norma de referencia, como el consenso de los tres métodos y en ese caso la precisión de la TAC y la placa MYCOTB fue de 94–95% y el método fenotípico ofreció una precisión de 93%. Se encontraron aislados con mutaciones genotípicas y altas concentraciones mínimas inhibitorias (MIC) que fueron fenotípicamente sensibles y aislados sin mutaciones y baja MIC con fenotipo resistente, lo cual pone en duda la prueba fenotípica como método de referencia.
CONCLUSIÓN: A la luz de estos resultados, se considera que el rendimiento diagnóstico de los métodos con TAC, la placa MYCOTB y el método fenotípico corriente es equivalente para los medicamentos de segunda línea; sin embargo, las dos primeras técnicas ofrecen rapidez, gran productividad e información cuantitativa sobre la sensibilidad a los medicamentos.
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 Xpert® 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® MYCOTB™plate (TREK Diagnostics, Cleveland, OH, USA), which yields a minimum inhibitory concentration (MIC), and a customized TaqMan® Array Card (TAC; Thermo Fisher Scientific, Waltham, MA, USA) for simultaneous detection of mutations in the resistance-determining regions of 10 genes. We evaluated MDR-TB 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.
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 site-specific phenotypic standard (Middlebrook agar proportion, Löwenstein-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 surveil-lance 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 Löwenstein-Jensen proportion method (both read at 21–28 days), and in Tanzania by MGIT™ 960™ (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 μg/ml (n = 40), and then 0.5 μg/ml (n = 58), given updated recommendations).
Table 1.
Critical concentrations, MIC breakpoints, and genotypic resistance-associated mutations probed in this study
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 andanMDR-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 sequence-specific 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 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 GenoType® MTBDRplus and Geno-Type® MTBDRsl version 1.0 (Hain Lifescience, Nehren, Germany), according to the manufacturer's instructions.
Statistical analysis
Means or median MICs were compared using the t-test 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 ± 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 interrogate 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 ± 12% vs. specificity 91 ± 7% for the six drugs EMB, SM, AMK, KM, OFX, MFX; P < 0.05).
Table 2.
Performance of TaqMan®Array Card compared to phenotypic DST
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 μg/ml and 4.0 μg/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 ± 9% vs. 91 ± 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 ± 5%, while categorical agreement was 87 ± 7% for the 10 drugs, P < 0.05 paired t-test).
Table 3.
Performance of Sensititre® MYCOTB™ compared to phenotypic DST
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 ± 10%, 90 ± 5%, and 89 ± 6% for the six drugs EMB, SM, KM, AMK, OFX, MXF (P = NS [not significant]). Specificities were respectively 96 ± 4%, 96 ± 3%, and 92 ± 11% (P = 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%).
Table 4.
Performance of each DST vs. the consensus result
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 with the MIC for various drugs (Figure or Appendix Table A.6).
Figure.
Relationships between genotype and MIC. MDR-TB isolates (n = 212) were tested using the TaqMan® Array Card (genes and mutations shown along x-axis) and the MYCOTB plate (y-axis). The brown horizontal bar indicates the median MIC of each mutation type for the respective drug. MIC = minimum inhibitory concentration; MDR-TB = multidrug-resistant tuberculosis. This image can be viewed online in color at http://www.ingentaconnect.com/content/iuatld/ijtld/2016/00000020/00000008/art00021
As expected, the inhA promoter mutation exhibited resistance to ETH (median MIC 10 μg/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 μg/ml, P = 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 μg/ml, or 2.0 μg/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 = NS). Rifabutin (RBT) MICs were lower than RMP MICs (median MIC 1.0 vs. > 16.0 μg/ml), and this was true across diverse mutations. D516V was common, and had a median MIC of > 16.0 μg/ml for RMP (MYCOTB-resistant), but 0.5 μg/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 = 0.25 vs. 1.25 μg/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 μg/ml for OFX and 1.5–8 μg/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 consensus-resistant 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.
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.
Acknowledgments
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 TaqMan® Array Card testing.
APPENDIX
Table A.1.
Primers and probes used in the TB TaqMan® Array Card
Table A.1.
(continued)
Table A.2.
Performance of TAC compared to Sanger sequencing.
Table A.3.
Performance of TAC compared to the Hain line-probe assay
Table A.4.
Performance of TAC compared to phenotypic DST stratified by agar proportion, L J or MGIT method *
Table A.5.
Performance of Sensititre® MYCOTB compared to phenotypic DST stratified by agar proportion, L J or MGIT methods *
Table A.6.
Relationships between genotypic and MIC *
Table A.6.
(continued)
Footnotes
Footnote: SF, SB and SP contributed equally to this work
* The appendix is available in the online version of this article, at http://www.ingentaconnect.com/content/iuatld/ijtld/2016/00000020/00000008/art00021
Conflicts of interest: none declared.
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