No patient specimen collection was required, and no patient identifiers were recorded on quality assurance tools. Participation in the Xpert MTB/RIF quality assurance and PT programme was voluntary and free of charge. No incentives were provided. This study was approved by the CDC Center for Global Health, Division of Human Research Protection (CGH HSR tracking # 2014-082).

Dried tube specimens for this study were prepared at the United States Centers for Disease Control and Prevention (CDC; Atlanta, Georgia, United States), International Laboratory Branch between 2013 and 2015. The Xpert MTB/RIF cartridge captures and lyses intact M. tuberculosis bacilli.⁵ Dried tube specimens were developed using whole cell inactivation in order to preserve the cell structure of the selected mycobacterial strains. This more closely simulates DNA extraction from a patient specimen. All DTS preparation steps were performed in a Biosafety Level 3 containment laboratory with personal protective equipment, including respiratory protection. Dried tube specimens were prepared using well-characterised rifampicin-resistant and rifampicin-susceptible M. tuberculosis strains and non-tuberculous mycobacteria strains obtained from the American Type Culture Collection, WHO proficiency testing challenges, and the CDC’s collection of laboratory-derived M. tuberculosis strains. M. tuberculosis strains were characterised using phenotypic and genotypic methods, including the Middlebrook 7H10 method of proportion drug susceptibility testing and Sanger sequencing of the rpoB gene.^6,16 Strains were inoculated into PANTA-supplemented BACTEC® MGIT (Mycobacteria Growth Indicator Tube) 7 mL tubes (Modified Middlebrook 7H9 broth base) (Becton, Dickinson and Company; Sparks, Maryland, United States) and incubated in the BACTEC® MGIT 960® instrument (Becton, Dickinson and Company; Sparks, Maryland, United States) until flagged positive for culture growth. Cultures were then incubated an additional 4–6 days at 37 °C in an auxiliary incubator and inoculated onto Middlebrook 7H10 agar and blood agar (Remel, Lenexa, Kansas, United States) for enumeration of mycobacteria and contamination checks.

Mycobacteria were inactivated using a 2:1 ratio of Xpert MTB/RIF Sample Reagent (SR) (Cepheid; Sunnyvale, California, United States) to MGIT culture in sterile 50 mL conical tubes (Figure 1). The suspensions were incubated at 15 °C – 30 °C, and vortexed for 30 seconds every 15 minutes for a total of two hours. MGIT culture/SR suspensions were neutralised with 20 mL – 25 mL of in-house prepared, sterile phosphate buffer pH 6.8 and centrifuged at 3000 × g for 15 min. The pellets were washed with an additional 45 mL phosphate buffer, centrifuged, and the supernatants were discarded. Pellets were then resuspended in 5 mL of phosphate buffer, transferred to sterile glass tubes with 5–10 3 mm glass beads (Thermo Fisher Scientific, Waltham, Massachusetts, United States), vortexed for 5 min, and allowed to settle for 15 min. Supernatants above the beads were transferred to new sterile glass tubes, labelled as stock suspensions, and stored at 2 °C – 8 °C. Inactivation verification was performed by inoculating 0.5 mL of stock suspension into MGIT tubes supplemented with PANTA and incubating for two cycles for a total of 84 days. Only stocks testing negative for growth were considered for panel preparation and distribution.

Xpert MTB/RIF pre-testing of five DTS from each stock was conducted to evaluate DTS accuracy and precision on a limited scale prior to preparing large numbers of PT samples. Dried tube specimens were prepared by diluting an aliquot of each stock suspension 1:10 with in-house prepared, sterile saline, adding food grade dye at a concentration of 0.1% (volume/volume) to each dilution, and transferring 100 µL of each stock dilution into five 4 mL cryovials. Cryovials were left uncapped in a Class II biosafety cabinet (BSC) until all liquid evaporated and aliquots were visibly dry. Dried tube specimen samples were then capped and stored in the dark at room temperature until tested.

Dried tube specimens were rehydrated with 2.5 mL of SR, shaken vigorously 20 times and incubated at room temperature for 15 min with additional shaking repeated after 10 min (Figure 2). The Xpert MTB/RIF cartridges were inoculated with approximately 2.0 mL of rehydrated samples using the transfer pipette provided with the kit, and tested immediately on a GeneXpert IV or GeneXpert VIII using GeneXpert DX software version 4.0 (Cepheid, Sunnyvale, California, United States), according to manufacturer’s instructions.¹⁷ Dried tube specimens prepared in 2013 were tested with Xpert MTB/RIF Assay G4 Research Use Only cartridges. Dried tube specimens prepared in 2014 and 2015 were tested with Food and Drug Administration-cleared Xpert MTB/RIF US In Vitro Diagnostic (IVD) cartridges.

To ensure that DTS yielded accurate Xpert MTB/RIF results, M. tuberculosis detection and rifampicin resistance results from DTS pre-testing were verified against the expected Xpert MTB/RIF results for the parent strain (as determined by prior phenotypic and genotypic testing). Results were considered acceptable if the qualitative M. tuberculosis detection and rifampicin resistance results matched the expected results for the parent strain for all five pre-tested DTS from the stock. Precision was evaluated to verify uniform suspension of the organism in the stock using the cycle threshold (Ct) standard deviation (SD). In line with previous studies, Probe A was selected for most analysis as it was the first probe to reach the detection threshold.¹⁸ The SDs of probe A Ct values were calculated for all DTS and compared between different samples within each panel. Probe C Ct values were used when probe A did not bind. The SD for the cartridge internal specimen processing control (SPC) Ct was also calculated for all samples and panels to investigate whether inherent Xpert MTB/RIF cartridge variability contributed to variation in DTS Ct values. To ensure that adequate amounts of inactivated organism were present in the DTS and that the integrity of DNA was not compromised during inactivation, the average Xpert MTB/RIF semi-quantitative result (i.e. the category assigned to the amount of M. tuberculosis DNA based on the Ct of the first probe detected) was required to fall in the medium (Ct = 16–22) to low (Ct = 22–28) range.

The five most precise and accurate stocks (i.e. the stocks with the lowest Ct SD and lowest mean Ct with DTS all yielding expected test results during pre-testing) were selected to aliquot DTS for use in the PT programme. Each Xpert MTB/RIF PT panel included one DTS per stock, for a total of five samples. Eight unique panels were prepared for use in the PT programme from 2013 (2013-A, 2013-B, 2013-C, and 2013-D) to 2015 (2014-A, 2015-A, 2015-B, and 2015-C) as described above. A strict BSC cleaning protocol (1% sodium hypochlorite followed by 70% ethanol rinse of all inner walls, sash, work surface and under work surface) was employed before aliquoting each panel. In most cases, only one sample was dried at a time. In situations where more than one sample was dried in the same BSC at the same time, all DTS had the same expected result. An aliquoting order was routinely followed: (1) ‘Tuberculosis Not Detected’ (non-tuberculous mycobacteria) samples were first aliquoted, then dried, capped, and removed; (2) ‘Tuberculosis Detected, Rifampicin Resistance Not Detected’ samples followed; and finally (3) ‘Tuberculosis Detected, Rifampicin Resistance Detected’ samples were aliquoted last. No culture manipulation was performed in the BSC while DTS samples were drying. Once prepared, panels were validated by confirming accurate and precise test results for 10% of the DTS produced using the same methodologies outlined above. Ten DTS from each stock underwent validation for panel 2013-A (50 total), 18 for panel 2013-B (90 total), 25 for panels 2013-C, 2013-D, and 2014-A (125 total per panel), 50 for panels 2015-A and 2015-B (250 total per panel), and 55 for panel 2015-C (275 total).

The primary challenge associated with producing DTS was variability between Ct values from separate aliquots of the same dilution. However, the inherent cartridge-to-cartridge variability in Ct values that was observed when comparing the SD of probe A to the SD of the SPC likely plays a role in the variability observed in DTS Ct values. After the Xpert MTB/RIF assay gained Food and Drug Administration clearance in 2013, we transitioned from using Xpert MTB/RIF Assay G4 Research Use Only cartridges in 2013 to Xpert MTB/RIF US IVD cartridges in 2014 and 2015. The range of SDs of probe A Ct values decreased from 1.0–6.2 in 2013 to 0.9–1.9 in 2014–2015. This decrease further suggests that the Ct value variability and high SD values observed were likely due to inconsistencies between cartridges. However, the variability of DTS could also be due to mycobacteria (particularly M. tuberculosis) clumping in MGIT cultures, leading to an uneven DTS stock suspension and difficulty in achieving equal numbers of bacilli in each aliquot. Additional evaluations are underway to improve both the accuracy and precision of DTS.

Another limitation of using the DTS technique for PT is that the DTS sample type (inactivated, dried mycobacteria) does not resemble the most commonly tested clinical sample type, sputum. While the use of DTS for PT does not allow for the complete evaluation of all the procedural steps or potential pitfalls involved in Xpert MTB/RIF testing of sputum, it could be an adequate substitute. For example, creating lyophilised, M. tuberculosis-spiked sputum samples would involve the purchase and maintenance of expensive equipment, such as lyophilisers, and increase biosafety risk for laboratorians. Alternately, the use of liquid samples increases biohazard risk to both shipping and laboratory personnel and increases transportation costs. Therefore, the safety and cost-saving benefits of DTS may outweigh potential disadvantages associated with assessing proficiency of Xpert MTB/RIF testing using dried, inactivated M. tuberculosis in resource-limited settings.

We are continuing to study and refine the technique for producing DTS, including the investigation of heat inactivation of M. tuberculosis for improved biosafety, stability and sample accuracy and precision. Since development of this novel PT technology, a voluntary Xpert MTB/RIF DTS-based PT programme has been rolled out to 26 countries and United States-affiliated territories, as described in ‘A Global Proficiency Testing Program for Xpert MTB/RIF using Dried Tube Specimens’ by Klein et al.²⁴ In addition, in 2016 we began the transfer of DTS technology to countries as part of a PT package that includes feedback and corrective actions to sustainably improve the quality of international diagnostic testing services. In 2019, the scope of the PT programme was increased to include the Xpert MRB/RIF Ultra assay (Cepheid, Sunnyvale, California, United States) where the same panel prepared for Xpert MTB/RIF PT was also successfully validated for use as PT for the Xpert MTB/RIF Ultra assay.

Proficiency testing for the Xpert MTB/RIF assay, as part of a comprehensive QA programme, should be a priority for every national tuberculosis programme. The DTS technique creates accurate, precise, and safe PT panels. Designing this preparation procedure around the availability of equipment and reagents in national tuberculosis reference laboratories in resource-limited countries promotes the transfer of DTS technology to both national and regional programmes and assists countries in implementing consistent, sustainable EQA programmes.