Resazurin microtitre plate assay and Sensititre® MycoTB for detection of Mycobacterium tuberculosis resistance in a high tuberculosis resistance setting

Background Rapid diagnosis of drug-resistant Mycobacterium tuberculosis is a challenge in low-income countries. Phenotypic drug susceptibility testing using Sensititre® MycoTB assay and the resazurin microtitre plate assay (REMA) are relatively new innovative methods to determine drug susceptibility. Objectives This study aimed to determine the performance of the Sensititre and REMA for M. tuberculosis drug susceptibility testing in a high-volume tuberculosis reference laboratory. Methods A laboratory-based study was performed at the Inkosi Albert Luthuli Central Hospital Tuberculosis Laboratory from January 2014 to June 2015. The Sensititre® MycoTB plate and REMA were compared to the gold standard agar proportion method (APM) using 134 stored isolates. Results Agreement between the Sensititre® MycoTB plate and APM was observed with 98% sensitivity, 82% specificity, 94% positive and 93% negative predictive values of the Sensititre® MycoTB assay for the detection of rifampicin resistance and 97%, 96%, 99% and 88% for isoniazid resistance. Good categorical agreement between the REMA and the APM was observed among isolates with 89% sensitivity, 68% specificity, 89% positive and 68% negative predictive value for the detection of rifampicin resistance and 95%, 96%, 99% and 81% for isoniazid resistance. Results for the second-line drugs showed elevated minimum inhibitory concentrations for multidrug-resistant and extensively drug-resistant tuberculosis isolates. Conclusion The REMA and Sensititre® MycoTB plate are attractive alternatives to the gold standard APM for the phenotypic detection of M. tuberculosis drug resistance.


Introduction
have an extended treatment duration of 18-24 months with harmful second-line anti-tuberculosis drugs. The WHO has recently proposed a 9-12-month shortened regimen duration for MDR tuberculosis, with moxifloxacin replacing gatifloxacin (used in the original Bangladesh regimen). 7,8 The conventional, culture-based phenotypic method is recognized as the gold standard for confirmation of disease and tuberculosis drug susceptibility testing (DST). The agar proportion method (APM) compares growth of Mycobacterium colonies on drug-free and drug-containing mediums, where growth in a particular antibiotic-containing medium determines resistance. It is a low-cost method requiring no special equipment, but with a turnaround time of up to 6 weeks. 9,10 BACTEC Mycobacterium growth indicator tubes (MGIT) 960 liquid culture system (Becton Dickinson) is expensive and prone to contamination but has the advantage of a rapid turnaround time. 2 The WHO has endorsed the GeneXpert MTB/RIF Ultra system, which is a real-time polymerase chain reactionbased assay for tuberculosis diagnosis and detection of rifampicin resistance, yielding results within 2 hours. The Genotype MTBDR Plus identifies common mutations for rifampicin and isoniazid. 1,11 Although molecular methods are rapid, they require costly equipment and staff expertise, and cannot detect resistant strains caused by unidentified mutations. 11 An innovative colorimetric assay has been designed using redox indicators to detect tuberculosis cell viability by a simple colour change. The resazurin microtitre plate assay (REMA) uses resazurin salts in a liquid culture medium. 12,13 This nontoxic compound incorporates into living cells and is reduced to the fluorescent molecule resorufin via a reduction and oxidation reaction. 14 Visible colour change of the reagent from blue to pink demonstrates cell viability and therefore drug resistance. This technique has been applied for high throughput screening and determining minimum inhibitory concentrations (MIC) of antituberculosis drugs. 14,15 Another advance in determining the phenotypic susceptibility of M. tuberculosis is the Sensititre ® MycoTB (MycoTB TREK Diagnostics) plate method. The Sensititre ® MycoTB plate method is a novel 96-well microtitre plate broth microdilution DST method incorporating drug concentrations of 12 anti-tuberculosis drugs for MIC determination. 16 The drugs included consist of both firstline (rifampicin, isoniazid and ethambutol) as well as second-line drugs (moxifloxacin, ofloxacin, paraaminosalicylic acid, rifabutin, streptomycin, amikacin, cycloserine, ethionamide and kanamycin) existing as lyophilised forms in microtitre wells. A 7-21-day period of incubation is needed to observe culture growth noted as turbidity or cellular deposits at the base of a well. 14,16 The aim of this study was to determine the performance of the Sensititre ® MycoTB plate and REMA as potential tools for tuberculosis DST in a high tuberculosis-burden reference laboratory. This entailed comparison of the REMA and Sensititre assay using the APM as a gold standard in order to evaluate turnaround times, sensitivity, specificity, and positive and negative predictive values.

Ethical considerations
The Biomedical Ethics Research Committee (University of KwaZulu-Natal) granted ethical approval for the use of stored study isolates (reference number BE 268/12).

Processing and culture of sputum specimens
Previously stored M. tuberculosis strains isolated from sputum samples received at the Tuberculosis Laboratory based at the Inkosi Albert Luthuli Central Hospital, Durban, South Africa, from January 2014 to June 2015 were used in this study. Briefly, sputum samples were digested and decontaminated using the N-acetyl-L-cysteine-NaOH-sodium citrate (NALC-NaOH, 2% NaOH final concentration) method and cultured in mycobacterium growth indicator tubes (MGIT). Positive cultures were confirmed for the presence of M. tuberculosis using the Ziehl Neelsen or MPT64 antigen assay (SD Bioline, Gyeonggi-do, South Korea). MDR and XDR M. tuberculosis isolates were routinely stored in the laboratory. One hundred and fifty stored M. tuberculosis isolates were subcultured onto Middlebrook 7H11 agar and incubated at 37 °C for 21 days. A total of 134 isolates had confluent growth and were used in the study.

Drug susceptibility testing by the agar proportion method
Drug susceptibility of tuberculosis-positive cultures was determined using the indirect APM on Middlebrook 7H10 agar 2,16 for first-and second-line anti-tuberculosis drugs (rifampicin, isoniazid, kanamycin, moxifloxacin and capreomycin). One hundred microlitres from a positive MGIT was inoculated into each quadrant of the Middlebrook 7H10 DST agar plate and incubated for 21 days. Growth of more than 1% on the antibiotic-containing quadrant when compared to the antibiotic-free growth control was regarded as resistant to the corresponding antibiotic. A susceptible growth control isolate, H37Rv (American Type Culture Collection, 25618), was used in the study.

Resazurin microtitre plate assay
The susceptibility of MDR, XDR and sensitive tuberculosis isolates were evaluated against first-and second-line antituberculosis drugs by the colorimetric REMA method for the 134 isolates. One hundred microlitres of Middlebrook 7H9 (M7H9) broth was aseptically prepared and dispensed carefully into each of the wells of a flat-bottomed, 96-well microtitre plate with lid (Lasec, Midrand, South Africa).
The anti-tuberculosis drugs that were tested using the REMA method included rifampicin, isoniazid, kanamycin, moxifloxacin and capreomycin.
An inoculum turbidity of McFarland standard number 1 was prepared from Middlebrook 7H11 (M7H11) agar, diluted in M7H9 (1:10) broth and thereafter added (100 µL) to each of the drug-free and drug-containing wells. 10,12 A sterile control and a growth control for each isolate were also included. To prevent evaporation during incubation, sterile M7H9 broth was added to all perimeter wells. The plate was incubated at 37 °C after being sealed in a plastic bag. A working solution of resazurin salt (30 µL of 0.02% concentration) was inoculated after 8 days of incubation into each microtitre well. 10 After overnight incubation, plates were then read the next day, a total of 9 days turnaround time for results interpretation. A colour change from blue to pink denoted a positive reaction (reduction of resazurin to resorufin) confirming drug resistance due to M. tuberculosis cell viability. 10

Sensititre® MycoTB assay
Setting up the microtitre plates and their interpretation were performed according to manufacturer's instructions. Colonies from a culture plate (M7H11) were emulsified in a glass tube containing saline, tween and glass beads until a 0.5 McFarland standard was obtained. One hundred microlitres of the suspension was inoculated into each well. The plates were then incubated at 37 °C. Drug concentrations present in each plate were: 4 µg/mL isoniazid, 16 µg/mL rifampicin, 32 µg/mL ethambutol, 40 µg/mL ethionamide, 40 µg/mL kanamycin, 32 µg/mL ofloxacin, 64 µg/mL paraaminosalicyclic acid, 16 µg/mL rifabutin, 32 µg/mL streptomycin, 256 µg/mL cycloserine, 16 µg/mL amikacin and 8.0 µg/mL moxifloixacin. Observations of the plates were made from day 7 to 10 for the presence of turbidity or cellular material confirming growth of M. tuberculosis and therefore resistance. 14,16 Time to results was calculated as the number of days from plate inoculation (day 0) to plate reading with visible growth (day 7-10).

Interpretation of results
Currently, there are no MIC-interpretative breakpoints for the broth microdilution assays when testing M. tuberculosis isolates on Sensititre plates; accordingly, the endpoint was determined as the first well that did not contain any growth. These antimicrobial drug MIC results were recorded on a data sheet per isolate tested. The reproducibility of Sensititre ® MycoTB assay by duplicate testing of the isolates was not performed for conditional (or categorical) agreement between the Sensititre ® MycoTB method and APM on initial testing. In this study, for interpretation of the Sensititre data, true resistance by Sensititre ® MycoTB was established by comparing the Sensititre MIC to the APM critical concentration. This means that an isolate was considered resistant if the Sensititre MIC was greater than the APM critical concentration and therefore truly susceptible if it was less than or equal to the APM critical concentration. For the REMA method, resistance to each drug was determined when there was growth in wells above the APM critical concentrations of 0.25 µg/mL (isoniazid), 1.0 µg/mL (rifampicin), 2.0 µg/mL (moxifloxacin), 4.0 µg/mL (capreomycin) and 5.0 µg/mL (kanamycin). 18 REMA plates were therefore interpreted categorically based on the calorimetric reaction. The REMA and Sensititre plates were interpreted by two readers who were blinded to the APM results. The two readers were in agreement with all plates read and therefore there was no discordance in interpretation.
Of the 100 rifampicin-resistant isolates by APM, 98 were resistant by Sensititre assay. Of these 98 isolates, 9 had an MIC of 1 µg/mL, 7 had an MIC of 2 µg/mL, 16  Of the 111 isoniazid-resistant isolates by APM, 108 were resistant by the Sensititre assay. Of these 108 isolates, 7 had an MIC of 1 µg/mL, 9 had an MIC of 2 µg/mL, 39 had an MIC of 4 µg/mL, 52 had an MIC of greater than 4 µg/mL and 1 had an MIC above 8 µg/mL. The seven isolates that were isoniazid-resistant by the Sensititre ® MycoTB with an MIC of 1 µg/ml (72% MDR and 28% mono-resistant to isoniazid by APM) were also confirmed resistant by the REMA assay. Nine isolates resistant to isoniazid by the Sensititre ® MycoTB with an MIC of 2 µg/mL comprised clinically-derived XDR (n = 6), MDR (n = 2) and pre-XDR (n = 1). Resistance to isoniazid by the REMA method was noted in all 9 isolates. Of the 52 isoniazid-resistant isolates with an MIC of over 4 µg/mL by the Sensititre ® MycoTB (Figure 1), 25 were XDR, 23 MDR and 4 were mono-resistant to isoniazid by the APM. The REMA assay confirmed isoniazid resistance in 51 of the 52 (98%) isolates.     Refer to Figure 1 for isoniazid MIC distributions using the Sensititre ® MycoTB. The Sensititre ® MycoTB assay sensitivity, specificity, positive and negative predictive values for the detection of isoniazid resistance were found to be 97%, 96%, 99% and 88% ( Table 1). The REMA showed sensitivity, specificity, positive and negative predictive values of 95%, 96%, 99% and 81% for the detection of isoniazid resistance (Table 2).
Sensititre ® MycoTB testing for the detection of resistance among the MDR and XDR isolates for rifampicin, isoniazid, ofloxacin and kanamycin correlated well with the APM (Table 1). Discrepancies between APM and REMA were observed with moxifloxacin susceptible isolates (APM) where 12 isolates were falsely designated as resistant by REMA ( Table 2). The overall sensitivity, specificity, positive and negative predictive values of the Sensititre ® MycoTB plate for moxifloxacin-resistance detection were found to be 91%, 98%, 91% and 96% ( Table 1). The REMA assay showed sensitivity, specificity, positive and negative predictive values of 91%, 78%, 64% and 96% for the detection of moxifloxacin resistance ( Table 2).
The overall sensitivity, specificity, positive and negative predictive values of the Sensititre ® MycoTB assay for ofloxacin resistance detection were found to be 100%, 97%, 97% and 100% ( Table 1). The sensitivity of detection for ofloxacin resistance was not assessed by the REMA as the drug could not be procured due to limited funds. The overall sensitivity, specificity, positive and negative predictive values of the Sensititre ® MycoTB plate for kanamycin resistance detection were found to be 92%, 85%, 86% and 92% ( Table 1). The REMA assay showed sensitivity, specificity, positive and negative predictive values of 83%, 94%, 93% and 85% for kanamycin resistance detection ( Table 2). Refer to Figure 3 for kanamycin MIC distributions using the Sensititre ® MycoTB. Good accuracy with regard to resistance to the first-line anti-tuberculosis drugs was observed with the Sensititre method as compared to the REMA (Tables 1 and 2).
The performance of the REMA for the detection of capreomycin resistance was determined. All of the 62 capreomycin-resistant isolates by the REMA were confirmed to be XDR tuberculosis by the APM (Table 2). Capreomycin was not part of the drug panel included in the Sensititre ® MycoTB assay, therefore its performance could not be assessed.
The levels of resistance among the MDR and XDR tuberculosis isolates to the additional first-and second-line antibiotics were further assessed using the Sensititre ® MycoTB assay using established critical concentrations (Figures 4 and 5).
Resistance to streptomycin was observed in 90% (54/60) of XDR isolates with a large proportion of the streptomycin-   http://www.ajlmonline.org Open Access

Discussion
The objective of tuberculosis DST is to determine resistant strains that are prognostic of treatment failure and relapse. The Sensititre ® MycoTB assay provides a faster method of testing first-and second-line tuberculosis drugs and displayed 99.3% concordance with the agar proportion method in prior studies. 19 In our study, the percentage agreements between Sensititre plate and APM for resistant isoniazid (97%), rifampicin (98%), moxifloxacin (91%) and ofloxacin (100%) were found in similar studies; however, kanamycin had the lowest categorical agreement with the APM (92%). 19   analysis would be useful to clarify discordant results, for example, repeat testing of isolates and ruling out technical errors. According to the WHO, current critical concentrations of all anti-tuberculosis drugs will be reviewed. 1 As phenotypic DST is required for the determination of moxifloxacin susceptibility (WHO recommendation due to poor concordance of Genotype MTBDRsl with APM), this might prove problematic if the Sensititre ® MycoTB assay is used. 1 The distribution of MIC with regard to second-line tuberculosis antimicrobials among MDR and XDR isolates was clearly evident as rising MIC values with the increasingly MDR strains. Resistance to capreomycin has been documented in KwaZulu-Natal in approximately 90% of XDR isolates of newly diagnosed individuals. 17,19 Both ethambutol and pyrazinamide resistance has been reported as exceeding 60%, the firstline drugs composing the Bangladesh regimen, as described in previous studies. 8 The Sensititre ® MycoTB plate rapidly produced results in comparison to the APM, however, it produced 44% (15/34) false rifampicin-resistant isolates (sensitive by APM). The treatment implication would include patients who were mismanaged with prolonged, toxic second-line therapy in an MDR facility.
The REMA assay had a relatively short turnaround time of 9 days. 21 Delays in results retrieval and therefore suitable treatment options for resistant tuberculosis may result in the further selection of resistant M. tuberculosis, morbidity and mortality in patients afflicted with the disease. Advantages of the REMA format are that it is faster, low cost, easy to interpret and does not need special equipment. 21 A disadvantage of this method is the potential for aerosolisation since the plates utilise a liquid medium resulting in a biosafety hazard. 15 In our study, REMA proved to be labour-intensive, requiring individual drug and dye preparation, as well as tedious microtitre plate inoculation. The current study showed the sensitivity and specificity of the REMA are comparable to that of the APM for isoniazid, rifampicin, capreomycin, moxifloxacin and kanamycin to be comparable to previous studies. 21 Agreement between REMA and the APM was over 90% for resistance testing among MDR and XDR isolates (isoniazid 95%, capreomycin 93%, moxifloxacin 91%).

Conclusion
The Sensititre ® MycoTB assay is a desirable alternative method compared to the APM for tuberculosis DST.
Simultaneous first-and second-line antimicrobial testing eradicates the need to set up and maintain antimicrobial drug solutions (REMA). The REMA is ideal for use in resource-poor settings due to its low cost and lack of instrumentation. 12 In comparison to the APM, which has a short shelf life, the Sensititre ® MycoTB MIC plate can last for up to 2 years at room temperature. The Sensititre plate may be used together with rapid molecular tests immediately targeting first-and second-line drug testing in scenarios of MDR and XDR tuberculosis. 18 Little information is available to date on a worldwide, regional and local scale on the use of the Sensititre plate. The comparative performance of Sensititre ® MycoTB assay to APM did show much discordance in this study and therefore cannot be recommended as a replacement of the current gold standard. In future, more studies will have to be performed to determine anti-tuberculous drug MIC interpretive breakpoints using wild type and non-wild type isolates, as well as discordant analysis, in order to increase its use in high-burden areas both locally and nationally, especially in the province of KwaZulu-Natal.