Evaluation of the Sensititre MycoTB Plate for Susceptibility Testing of the Mycobacterium tuberculosis Complex against First- and Second-Line Agents

Abstract

The Sensititre MycoTB plate (TREK Diagnostic Systems, Cleveland, OH) uses a microtiter plate MIC format for susceptibility testing of Mycobacterium tuberculosis complex isolates against first- and second-line antituberculosis agents. Categorical agreement versus the agar proportion method for 122 M. tuberculosis complex isolates was 94% to 100%.

TEXT

The gold standard method for Mycobacterium tuberculosis complex susceptibility testing is the 1% indirect agar proportion method (APM), which is laborious and requires 2 to 3 weeks for results (4, 7). This two-site study evaluated a new system, the Sensititre MycoTB plate (MycoTB), for susceptibility testing of M. tuberculosis complex. The plate uses a 96-well microtiter broth format and contains 12 lyophilized first- and second-line antimycobacterial drugs. In contrast to other M. tuberculosis complex susceptibility methods, which test one or two critical concentrations of a drug, the MycoTB plate examines a range of drug concentrations and produces an MIC result. Recently, Abuali et al. evaluated the MycoTB plate using 37 M. tuberculosis complex isolates (1). Our study confirms and extends these results by testing a larger number of isolates, including singly and multiply resistant isolates, and provides precision data for the MycoTB plate. We also compared a manual, mirror plate-reading method with a commercially available, manual plate reader with data management software (Vizion System, TREK Diagnostics).

(This study was presented in part at the 50th Annual Interscience Conference on Antimicrobial Agents and Chemotherapy Boston, MA, 12 to 15 September 2010.)

One hundred twenty-two M. tuberculosis complex isolates (59 from site 1 and 63 from site 2) were tested using the APM and the MycoTB plate. An additional 15 M. tuberculosis complex “challenge” strains with known susceptibility patterns were provided by the Centers for Disease Control and Prevention (CDC) and tested using the MycoTB plate. Inoculum preparation and plating were performed using a biological safety cabinet (BSC) inside a biosafety level 3 (BSL3) laboratory as previously described (5). Several colonies were selected from Middlebrook 7H10 medium using a sterile loop and inoculated into a test tube containing saline-Tween and glass beads (TREK Diagnostics). After being vortexed for 30 to 60 s, the inoculum was allowed to settle for 15 min and adjusted to a 0.5 McFarland standard equivalent using a nephelometer. The numbers of CFU were determined for each isolate tested to verify that the inoculum was within a specific targeted amount (∼10⁵ CFU/ml). One hundred microliters of the inoculum was transferred to 11 ml of Middlebrook 7H9 broth containing oleic acid-albumin-dextrose-catalase (TREK Diagnostics) and vortexed for 20 s. One hundred microliters was transferred to the MycoTB plate wells containing the lyophilized antibiotics. MycoTB plates were covered with plastic seals provided by the manufacturer, and the entire outer surface of the plate was disinfected with a tuberculocidal agent. Plates were incubated at 37°C in 5 to 10% CO₂ and examined on the benchtop without opening at 7, 10, 14, and 21 days using both a manual read aided by a mirrored viewer and the Vizion system. The first well with no visible growth was determined to be the endpoint. The APM plates containing Middlebrook 7H10 medium were examined at 10, 14, and 21 days inside a BSC using a manual read assisted by a backlight.

The MycoTB plate contained a well with a drug concentration either equivalent to the APM critical concentration or within 1 doubling dilution of the critical concentration (Table 1). The MycoTB plate result was considered susceptible when the MIC breakpoint listed was equivalent to or lower than the APM critical concentration and resistant when the MIC breakpoint was higher than the APM critical concentration. The overall categorical agreement between the MycoTB plate MIC results and the APM critical concentration results for the 122 isolates ranged from 96 to 100% for the three first-line agents and 94 to 100% for 9 second-line agents (Table 2). The MycoTB plate sensitivity versus the APM ranged from 93.2 to 100%. The MycoTB plate specificity ranged from 90 to 100% for the six agents that had >10 APM-resistant isolates tested. The manual mirror read and the Vizion plate reader produced highly consistent results with an agreement within 1% for all drugs (data not shown). The results of the MycoTB plate were also excellent for the 15 challenge strain isolates, with each site correctly reporting susceptible or resistant results for all isolates.

Table 1.

Comparison of the APM critical concentrations and MycoTB plate ranges

Agent	APM critical concn(s) tested (μg/ml)	MycoTB plate range (μg/ml)	MycoTB plate concn(s) nearest to the APM critical concn(s)a (μg/ml)
First-line agents
Ethambutol	5.0, 10.0	0.5–32	4.0, 8.0
Isoniazid	0.2, 1.0	0.03–4	0.25, 1.0
Rifampin	1.0	0.12–16	1.0
Second-line agents
Amikacin	5.0	0.12–16	4.0
Cycloserine	25.0	2.0–256	32.0
Ethionamide	5.0	0.3–40	5.0
Kanamycin	5.0	0.6–40	5.0
Moxifloxacin	2.0	0.06–8.0	2.0
Ofloxacin	2.0	0.25–32	2.0
p-Aminosalicylic acid	2.0	0.5–64	2.0
Rifabutin	0.5	0.12–16	0.5
Streptomycin	2.0, 10.0	0.25–32	2.0, 8.0

^aConsidered equivalent to the APM critical concentration for the purposes of categorical (susceptible or resistant) assignment. For agents with two concentrations tested, the lower concentration was used for method comparison purposes.

Table 2.

MycoTB plate MIC results compared to APM critical concentration results using a Vizion read

Agent	APM concn (μg/ml)	MycoTB plate result	No. of isolates with each APM resulta		Sensitivity (%)	Specificity (%)b	% categorical agreement
			Susceptible	Resistant
First-line agents
Ethambutol	5	Susceptible	109	0	98.2	100	98.4
		Resistant	2	11
	10	Susceptible	120	0	100	100	100
		Resistant	0	2
Isoniazid	0.2	Susceptible	69	0	93.2	100	95.9
		Resistant	5	48
	1	Susceptible	78	0	97.5	100	98.4
		Resistant	2	42
Rifampin	1	Susceptible	90	0	98.9	100	99.2
		Resistant	1	31
Second-line agents
Amikacin	5	Susceptible	113	0	99.1	ND	99.1
		Resistant	1	8
Cycloserine	25	Susceptible	122	0	100	ND	100
		Resistant	0	0
Ethionamide	5	Susceptible	98	1	96.1	95.0	95.9
		Resistant	4	19
Kanamycin	5	Susceptible	113	2	100	ND	98.4
		Resistant	0	7
Moxifloxacin	2	Susceptible	119	1	98.4	ND	97.5
		Resistant	2	0
Ofloxacin	2	Susceptible	113	0	95.8	ND	95.9
		Resistant	5	4
Para-aminosalicylic acid	2	Susceptible	114	0	94.2	ND	94.3
		Resistant	7	1
Rifabutin	0.5	Susceptible	93	3	100	90.0	97.5
		Resistant	0	26
Streptomycin	2	Susceptible	93	0	94.9	100	95.9
		Resistant	5	24
	10	Susceptible	105	0	98.1	100	98.4
		Resistant	2	15

^aOut of 122 total isolates.

^bND, not determined. Specificity was not calculated for agents with <10 resistant isolates as determined by APM.

The between-run precision of the MycoTB plate was determined by testing M. tuberculosis strain 27294 (ATCC, Manassas, VA) at site 1 weekly for 23 weeks using 3 operators for both the manual mirror read and the Vizion reader. For all agents, with the exception of ethionamide, which was within 1 doubling dilution of the mode MIC for >91% of isolates, the MIC range was within 1 doubling dilution of the mode MIC for 100% of isolates using both a mirror read and the Vizion read.

The TREK MycoTB plate represents a unique susceptibility test format for the M. tuberculosis complex. The plate contains lyophilized drugs with the concentrations prepared and quality controlled by the manufacturer, and the plate is stable at room temperature for up to 2 years. The additional equipment required is a BSC in a BSL3 lab for inoculum preparation, a simple incubator, and a mirror box, and this can reduce testing costs since specialized incubation instrumentation is not necessary.

The ability to easily and reliably obtain MIC results for M. tuberculosis complex susceptibility testing using the MycoTB plate is a potentially paradigm-changing advance. Testing against a critical concentration of the drug is sufficient to provide the information needed by clinicians for some M. tuberculosis complex isolates. However, there are isolates for which reproducible susceptibility results are difficult to obtain using the critical concentration method (2, 3, 6, 8). Repeated testing of these problematic isolates produces a mixture of susceptible and resistant results even when tested using the same inoculum on the same day with the same technologist performing the test. The MycoTB plate ranges will allow laboratorians to confirm what they have long suspected in these instances, that the isolate MIC is teetering on the breakpoint between susceptible and resistant. The availability of an MIC alerts the clinician to the fact that although the isolate may be technically susceptible, it is sitting at the junction between susceptible and resistant and the patient should be carefully monitored for the emergence of drug resistance.

In addition to producing an MIC result, the ability to prepare a single seed bottle for both rapid broth testing (e.g., Bactec or VersaTREK) and the MycoTB plate allows for use of a consistent inoculum and permits nearly simultaneous confirmation of resistance in a single laboratory if desired. At this time, there are only a few highly specialized laboratories that provide susceptibility testing of second-line drugs for M. tuberculosis complex due to the labor associated with preparation and quality control of agar plates containing precise quantities of numerous drugs. The MycoTB plate is the first commercially available product for second-line drug testing and should decrease result variation and increase the number of laboratories able to provide reliable results for these agents.

A limitation of this study is the small number of resistant M. tuberculosis complex isolates that were available for selected second-line agents. In addition, it would be ideal if pyrazinamide were included in this plate so that all M. tuberculosis complex susceptibility testing was able to be performed using a single method, but that is not feasible at this time due to the need for acidic test conditions with this agent.

In conclusion, the MycoTB plate provides reliable results compared to those of the APM reference method. Results were obtained approximately 1 week earlier with MycoTB (10 to 14 days) than with the APM method (21 days). Although more-rapid molecular methods exist for selected agents (e.g., rifampin), they are not available for all agents or in all areas of the world, and they do not detect all instances of resistance. Therefore, phenotypic methods, such as the MycoTB plate, remain important. The MycoTB plate also allows for MIC determination, testing of second-line agents, and the opportunity for digital plate reading and data storage using the Vizion plate reader. The MIC data represent new information for which additional studies must be done to determine the clinical impact. The MycoTB plate now provides laboratories with a means to collect those data.