Tuberculosis diagnosis is a challenge in many health centres. The introduction and subsequent endorsement by the World Health Organization of the GenoType MTBDRplus (HAIN Lifesciences, Nehren, Germany),¹ Xpert MTB/RIF and Xpert MTB/RIF Ultra assays’ (Cepheid, Sunnyvale, California, United States),^2,3 and the Truenat MTB, MTB Plus and MTB-RIF Dx assays (Molbio Diagnostics, Goa, India)⁴ highlighted the advantages of using molecular technologies for diagnosis. Although there is a lag in developing new tuberculosis diagnostic assays, in July 2021, another group of technologies received the World Health Organization’s conditional recommendation for use in tuberculosis diagnosis.⁵ These are cobas MTB and cobas MTB-RIF/INH (Roche, Basel, Switzerland),⁶ Fluorotype^® MTBDR (Bruker, Nehren, Germany),⁷ RealTime MTB (Abbott, Illinois, United States),^8,9 and the BD MAX MDR-TB assay (Becton Dickinson, Franklin, New Jersey, United States).¹⁰ The Treatment Action Group report provides information on current and new screening and triage tools.¹¹ The expanding tuberculosis diagnostics pipeline and other non-molecular, culture-based, microscopy and radiology innovations can increase testing and earlier disease detection, even in difficult-to-detect cases, such as in HIV co-infected individuals; expand the spectrum of drug-susceptibility testing; and reduce costs.¹² Once a technology becomes available, it must be validated to ensure conformity to the manufacturer’s claims and/or target product profiles and verified to assess suitability within the intended settings.^{13,14,15,16,17,18} Both validation and verification require suitable reference material that is stable and can be safely and efficiently transported. Mycobacterial species pose a potential biosafety risk, especially for staff responsible for storing and transporting or aliquoting isolates. Culture isolates should also be stored in an appropriate medium and at the correct temperature to maintain viability. These requirements have both cost and space implications.

One of the earliest studies on long-term storage and preservation of mycobacteria was performed in 1972.¹⁹ Mycobacterium tuberculosis complex (MTBC) (cultured on Proskauer and Beck medium) and Mycobacterium bovis (cultured on Sauton’s medium) were stored in nine different diluents. These included sterile skim milk; 1% gelatin buffered at pH 6.8; 1:5 dilution of Sauton’s medium; 0.25% Triton WR 1339 in Sauton’s medium; a solution containing 8.3% dextran, 7.5% glucose, and 0.025% Triton WVR 1339; 15% aqueous solution of lactose at pH 5.0; 5% sodium glutamate; Tween-albumin medium; and distilled water. Archive stocks were stored at two different temperatures (−20 °C and −70 °C) for 3 years. There was a significant reduction in MTBC isolate viability when stored at −20 °C versus −70 °C. In addition, the methods applied had safety concerns, such as the bottling procedures, appropriate container selection, transportation, and the risk of aerosolisation (once the bottle was opened). Although most of these issues were resolved, the storage media and safety considerations were not ideal.¹⁹

In 2005, Huang et al. demonstrated that MTBC isolates from solid media could be stored at −70 °C in 7H9 broth without significant loss in viability (> 90%) for up 7 years, while the viability of strains preserved directly from mycobacteria growth indicator tubes (MGITs) was acceptable for up to 3 years.²⁰ In 2014, the World Health Organization released a laboratory manual recommending MTBC isolate storage in 7H9 broth with glycerol,²¹ media preparation, specific equipment, and sterile work areas. Although the World Health Organization-recommended technique efficiently stores and maintains MTBC strain viability, Metcalfe et al. and Hanekom et al. reported a possible loss of strain fitness due to the subsequent need for sub-culture to maintain stock culture isolates.^22,23 Furthermore, subculturing is prone to contamination, which may further risk the loss of valuable isolates. Kremer et al. reported an alternative option to collect samples from the surface of a frozen medium by scraping the top layer without defrosting the remaining suspension.²⁴ However, the use of a frozen stock may reduce the viability of isolates if the entire tube thaws during the subculturing process. Glass beads have demonstrated medium-term storage for MTBC with strain recovery rates of > 94% within 30 days of incubation by Giampaglia et al.²⁵ However, both the beads and storage vial require pre-sterilisation and preparation of storage medium, which increases the process cost and is labour intensive.

The Microbank^TM platform (www.https://pro-lab.com/products/clinical-microbiology/bacteriology/microbank/) has approximately 25 sterile beads per storage vial and a specifically formulated cryopreservative for low-temperature storage. It is potentially a suitable alternative storage solution for MTBC isolates. The Microbank^TM beads are porous, allowing microorganisms to adhere to the bead surface. A new culture can be grown by inoculating culture media with a single bead from the storage. Thus, the process can be repeated for the 25 beads of each isolate compared to the current method, where the number of possible new cultures depends on how much of the stock is used for each reculture. Some studies^26,27,28 have reported reasonable fungal isolate recovery rates (96% – 100%) from the Microbank^TM beads; however, as far as we know, the Microbank^TM storage solution has not been assessed for MTBC isolate recovery. Nevertheless, the Microbank^TM beads may maintain longer MTBC viability and allow maximum usage with 25 beads, reducing process cost and preserving precious culture isolates.

This study aimed to determine the feasibility of using the Microbank^TM beads to store MTBC isolates from a laboratory in South Africa.

After a storage period of 16 and 21 months, 34 of 40 (85%) archive beads successfully grew MTBC, 5 of 40 (12.5%) did not demonstrate growth after the 42-day incubation, and 1 of 40 (2.5%) demonstrated contamination (Figure 1). The baseline TTD for the positive control culture isolate, ATCC 25177, was 15.9 days (Table 1). The positive control demonstrated a mean TTD of 8.4 days and 20.5 days after 16 months and 21 months. For isolates, where both beads retrieved at the same time point, demonstrated growth of MTBC, 15 of 16 and 13 of 15 after 16 and 21 months showed similar TTDs (Figure 1). Isolates 4 and 17 demonstrated > 8-day difference in TTD between the two beads retrieved simultaneously. The box and whisker plot (Figure 1) displays the distribution of means representing the TTD for the original culture, month 16 and month 21. The one-way analysis of variance indicated a significant difference among the means of the original, month 16 and month 21 TTD (F (2, 57) = 9.17, p < 0.001) (Online Supplementary file Table 1). Post-hoc analysis using Tukey’s Honestly Significant Difference test revealed that the original TTD (mean = 8.06 days) was significantly lower than both month 16 (mean = 19.18 days) and month 21 (mean = 17.10 days) TTD. However, no significant difference was observed between the mean culture isolate TTD at month 16 and month 21.

Regarding usability, laboratory staff reported that the Microbank^TM beads were technically undemanding (requiring only routine laboratory procedures such as centrifugation) and easy to use. Furthermore, no additional preparation of reagents was required.

Storage of MTBC culture isolates on the Microbank^TM beads was performed at −70 °C as recommended^19,21,31 and MTBC isolates were recovered within 39 days of incubation in the BACTEC™ MGIT™ 960 instrument with minimal contamination. For archive cultures where contamination was seen, culture isolates grown from the second bead from the same vial did not show contamination suggesting that contamination occurred during the MGIT reculture process rather than during processing for storage. Since the Microbank^TM bead platform doesn’t address the possible contamination problem due to multiple openings and closing of the vials, sterility during the reculture process is critical.

Although the statistical analysis showed a significant difference in the TTD between the original and post-storage TTD, the TTD between the 16-month and 21-month time points was not statistically significant. This increased TTD could be attributed to decreased metabolic activities of the MTBC bacilli due to their storage at low temperatures.³¹ There was variability in TTD at both time points post storage, even in beads from the same vial, which may suggest that growth depends on the number of bacilli adhering to the bead. Rather than a loss in viability, a decreased number of bacilli on some beads may also be the reason for some culture isolates demonstrating a lack of growth after the 42-day incubation period and highlighting the importance of the uniform distribution of bacteria. It is therefore essential to rigorously invert the Microbank^TM vial at the time of inoculation to assist with the uniform distribution of the bacteria across the beads. The TTD has been described as a time-to-event variable, which represents an indirect measurement of the bacterial load,³² and it is possible to reduce the TTD by increasing the colony-forming units of the initial inoculum. Increasing colony-forming units may be achieved by incubating the culture isolates at 37 °C for a more extended period to allow for increased growth or increasing the starting volume of the inoculum. The susceptibility profile of the culture isolates also did not appear to impact the viability of the culture isolate or the TTD.

Recovery rates of MTBC preserved from culture media (stored at −70 °C for 7 years) can vary from 97.4% to 100%.²⁰ Room temperature storage of culture isolates has also been investigated.³³ Although the bacteria remained viable for up to 6 years, recovery rates were low (54.0% to 66.4% depending on storage conditions, such as the availability of an air conditioner) with a contamination rate of ~7%. Recovery rates of MTBC using the microbead method after a 21-month storage period were demonstrated to be 85%. Although this lower recovery rate appears to be a limitation of this method, further investigation is required using an increased inoculum volume or a higher culture concentration and a longer storage period.

A key advantage of the Microbank™ beads is their safety profile. The minimal liquid volume reduces the risk of leakage and subsequent aerosolisation. The system also does not require the preparation and autoclaving of media. Due to the increased risk of defrosting the entire vial and potential loss of the stock culture isolate, current practice recommends storing two vials of stock material in two separate freezers, which already has cost and space implications.³⁴ With the Microbank™ bead storage, 25 individual isolate recoveries can be made from a single vial, allowing efficient storage, which is ideal for space-constrained environments. With routine subcultures, it has been reported that frequent subcultures may distort drug-susceptibility levels and patterns of strains,³⁵ but with the Microbank™ system, since the primary culture isolate is technically not sub-cultured, this risk is minimal. For ease of identification within biorepository settings, Microbank^TM beads are available in a variety of colours.

Although not investigated in this study, Microbank^TM beads provide an attractive alternative for transporting viable isolates and for applications such as proficiency testing. The current transport of infectious MTBC culture isolates for further confirmatory reflex testing, such as sequencing, requires strict safety adherence criteria and is costly. Typically a secondary watertight container with absorbent material, together with outer packaging,³⁶ is employed for shipping isolates and limitations are placed on the total allowable volume of liquid material in a single shipping container.³⁷ The Microbank^TM bead platform will require the same safety considerations as conventional storage but offers a reduced risk of aerosolisation since the beads are not transported with any liquid.