Recovery of Mycobacterium tuberculosis from positive mycobacterium growth indicator tubes stored at room temperature for up to 6 years in low-income and High-Tuberculosis-Burden Country

Abstract

Background: Biobanking of Mycobacterium tuberculosis (Mtb) sputum samples for future research activities recommends the use of -70°C or -80°C freezers. Infrastructure for biobanking is not readily available in the majority of low-income countries. This study aimed to assess the recovery rate of Mtb isolates stored at room temperature for more than 6 years in Zimbabwe.

Methods: Census samples of all confirmed rifampicin-resistant/multidrug-resistant tuberculosis isolates that were stored in mycobacterial growth indicator tubes (MGITs) at room temperature from 2011 to 2016 were identified and retrieved. The samples were subcultured on MGIT and 7H10 solid media for the extraction of genomic deoxyribonucleic acid using the phenol/chloroform method followed by precipitation with isopropanol.

Results: A total of 248/400 (62%) isolates were successfully recovered. Recovery rates increased with declining time since the last culture, with 51% for samples stored for 6 years which increased to 77% for those stored for 1 year. The isolates that grew but were contaminated during the first subculture at the National Microbiology Reference Laboratory in Harare could not be recovered through decontamination because of limited resources. Decontamination was only possible during the second culture at the University of Stellenbosch.

Conclusion: Storage of Mtb isolates at room temperature is a viable option in low-income countries where currently recommended biobanking procedures may not be available. This low-cost biobanking will facilitate research activities years later as new questions arise. Standard infection prevention and control when handling Mtb samples stored under room temperature for long periods is strongly recommended as these bacteria remain viable longer than previously reported.

Keywords: Culture; Mycobacterium tuberculosis; storage.