Evaluation of the Efficiency of the Sample Inactivation Reagent in the Abbott RealTime MTB Assay for Inactivation of Mycobacterium tuberculosis

Abstract

The Abbott RealTime MTB assay is a nucleic acid amplification test (NAAT) for the detection of Mycobacterium tuberculosis complex DNA. The sample inactivation procedure used in the assay, consisting of one part sample treated with 3 parts inactivation reagent for 60 min, effectively reduced viscosity and inactivated M. tuberculosis in clinical specimens.

TEXT

The Abbott RealTime MTB assay is a nucleic acid amplification test (NAAT) for the detection of Mycobacterium tuberculosis complex DNA in respiratory specimens (sputum and bronchoalveolar lavage [BAL] fluid and N-acetyl-l-cysteine [NALC] sediments of sputum and bronchoalveolar lavage fluid. A sample inactivation reagent and procedure were developed by Abbott Molecular to liquefy viscous samples and reduce M. tuberculosis viability to allow for safe testing of samples outside a biosafety cabinet. The study was performed to assess the effectiveness of the sample inactivation procedure and to determine the stability of the inactivation reagent (IR).

For the viscosity reduction study, 150 sputum samples were mixed with IR (0.6% sodium hydroxide [wt/vol], 60% isopropanol [vol/vol], and 1.8% Tween 20 [vol/vol]) at a ratio of 1:2 or 1:3. The mixtures were vortexed vigorously and incubated at room temperature. The mixture was vortexed again after 20 to 30 min of incubation. Reduction in viscosity was assessed by visual examination after 30 min, 60 min, and 24 h of incubation.

For the inactivation study, two M. tuberculosis clinical isolates and the M. tuberculosis ATCC 27294 isolate were used to prepare mock M. tuberculosis-positive respiratory samples by mixing 1 ml of M. tuberculosis cell suspension at concentrations of 1 × 10⁶, 1 × 10⁷, or 1 × 10⁸ CFU/ml with 4 ml of pooled M. tuberculosis-negative NALC-treated respiratory sample. Each mock M. tuberculosis NALC sample was then mixed with IR at ratios of 1:2 or 1:3. A mock sample treated with sterile phosphate-buffered saline (PBS) buffer at a sample-to-PBS ratio of 1:2 was used as the positive control. Negative controls were prepared by adding sterile PBS to the pooled M. tuberculosis-negative NALC sample at a ratio of PBS to NALC of 1:2. All samples/controls were vortexed vigorously and incubated for 60 min at room temperature. Vortexing was repeated 30 min into the incubation. At the end of the incubation, the IR-treated samples were transferred to new 50-ml tubes, vortexed, and centrifuged at 3,000 × g for 15 min. The sediment was resuspended in 10 ml of sterile PBS and centrifuged at 3,000 × g for an additional 15 min. The pellets were each resuspended in 10 ml of sterile PBS. One milliliter of the suspension was used to inoculate a mycobacterial growth indicator tube (MGIT). The final M. tuberculosis concentration added to each MGIT culture ranged from 1 × 10⁴ to 2 × 10⁴ up to 1 × 10⁶ to 2 × 10⁶ CFU. In addition, a total of 51 M. tuberculosis-positive clinical NALC sediments of sputum, 20 from Northwestern Memorial Hospital and 31 from Lancet Laboratories, were tested for growth after the IR treatment at a sample-to-IR ratio of 1:3, using the same procedure. Ten of the 20 samples from Northwestern Memorial Hospital were treated at a sample-to-IR ratio of 1:2. The remaining 41 samples were treated with a sample-to-IR ratio of 1:3. Culture was performed with the Bactec MGIT 960 system (Becton Dickinson, Sparks, MD) for 42 days. Positive growth was identified using the Gen-Probe AccuProbe system (Gen-Probe, Inc., San Diego, CA). Initial studies to demonstrate the inactivation efficiency of direct respiratory samples (M. tuberculosis smear- and culture-positive sputum samples) were also performed in combination with an IR stability study, as described below.

To determine the optimal storage conditions for IR, three aliquots of IR were stored for 39 days under storage conditions of 15 to 30°C and 33 to 37°C in glass or polypropylene bottles. Each aliquot of IR under each storage condition was examined for changes in the appearance and volume and tested for M. tuberculosis inactivation efficacy after 39 days of storage with 12 M. tuberculosis smear- and culture-positive sputum samples obtained from Sage Bionetworks (Dhaka, Bangladesh) and the Foundation for Innovative New Diagnostics (FIND) M. tuberculosis specimen bank using a sample-to-IR ratio of 1:3. An M. tuberculosis strain H37Rv cell panel obtained from ZeptoMetrix Corporation (Buffalo, NY) was used as the positive control.

The viscosity reduction study showed that 60 min of incubation was sufficient to reduce the viscosity of the samples. For the inactivation study, none of the mock M. tuberculosis samples prepared with the three M. tuberculosis isolates at 1 × 10⁸, 1 × 10⁷, and 1 × 10⁶ CFU/ml showed M. tuberculosis growth after being treated with IR at a sample-to-IR ratio of 1:3. One IR-treated sample prepared with 1 × 10⁷ CFU/ml M. tuberculosis and treated with IR at a sample-to-IR ratio of 1:2 showed growth after 27 days of incubation, although two repeats at the same bacterial concentration tested negative for growth. None of the 20 M. tuberculosis-positive NALC sputum sediments showed M. tuberculosis growth following treatment with IR at a sample-to-IR ratio of 1:2 or 1:3. Further, 31 clinical NALC sputum sediments that previously tested positive for M. tuberculosis by culture tested negative for M. tuberculosis growth after receiving IR treatment at a sample-to-IR ratio of 1:3.

Changes in appearance after storage were not observed after 39 days. A volume loss of 0 to 6% was observed after 39 days of storage. The highest volume loss of 6% was observed when IR was stored in polypropylene containers at 33 to 37°C. However, the efficiency of the IR solution to inactivate M. tuberculosis was not affected after storage. The 12 M. tuberculosis-positive sputum samples showed no growth after being treated with the IR stored under the various conditions described above.

It was noted that some M. tuberculosis in clinical samples survived the recommended Cepheid GeneXpert MTB/RIF sample inactivation process (15-min incubation period and a sample-to-reagent ratio of 1:2) (1). The authors suggested that complete M. tuberculosis inactivation may require a longer incubation time. Experimental data generated by our study demonstrated that sample inactivation performed for 60 min using the recommended vortexing steps was sufficient for complete M. tuberculosis inactivation.

When a 1:2 sample-to-IR ratio was used, one replicate of a 1 × 10⁷ M. tuberculosis CFU/ml culture (2 × 10⁵ CFU/ml in the MGIT culture) showed growth after 27 days of incubation of the MGIT culture. A previous study showed that MGIT cultures containing 10 CFU/ml M. tuberculosis became positive after 16 days of incubation; the result suggested that a very low number M. tuberculosis survived the inactivation process when a sample-to-IR ratio of 1:2 was used (2, 3). To achieve the optimal inactivation efficiency, the sample-to-IR ratio of 1:3 was used for the rest of the inactivation experiments.

In conclusion, the IR evaluated in this study was able to liquefy sputum samples and to achieve effective inactivation of M. tuberculosis in clinical specimens when treated with IR at a sample-to-IR ratio of 1:3 for 60 min. This inactivation procedure enables these samples to be safely handled outside a biosafety cabinet after the proper inactivation procedure.