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 Table of Contents  
ORIGINAL ARTICLE
Year : 2019  |  Volume : 8  |  Issue : 2  |  Page : 185-189

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


1 Division of Molecular Biology and Human Genetics, NRF/DST Centre of Excellence for Biomedical Tuberculosis Research, South African Medical Research Council Centre for Tuberculosis Research, Faculty of Medicine and Health Sciences, Stellenbosch University, Cape Town, South Africa; College of Health Sciences, University of Zimbabwe, Harare, Zimbabwe
2 College of Health Sciences, University of Zimbabwe, Harare, Zimbabwe
3 Division of Molecular Biology and Human Genetics, NRF/DST Centre of Excellence for Biomedical Tuberculosis Research, South African Medical Research Council Centre for Tuberculosis Research, Faculty of Medicine and Health Sciences, Stellenbosch University, Cape Town, South Africa
4 Ministry of Health and Child Care, National Microbiology Reference Laboratory, Harare Central Hospital, Harare, Zimbabwe

Date of Web Publication14-Jun-2019

Correspondence Address:
Joconiah Chirenda
Division of Molecular Biology and Human Genetics, NRF/DST Centre of Excellence for Biomedical Tuberculosis Research, South African Medical Research Council Centre for Tuberculosis Research, Faculty of Medicine and Health Sciences, Stellenbosch University, Cape Town

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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/ijmy.ijmy_46_19

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  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


How to cite this article:
Chirenda J, Chipinduro M, de Kock M, Spies C, Sakubani C T, Warren RM, Sampson SL, Streicher EM. 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. Int J Mycobacteriol 2019;8:185-9

How to cite this URL:
Chirenda J, Chipinduro M, de Kock M, Spies C, Sakubani C T, Warren RM, Sampson SL, Streicher EM. 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. Int J Mycobacteriol [serial online] 2019 [cited 2019 Jul 15];8:185-9. Available from: http://www.ijmyco.org/text.asp?2019/8/2/185/260380




  Introduction Top


Tuberculosis (TB) remains a major cause of morbidity and mortality with a reported incidence of 10.4 million cases and 1.674 million deaths worldwide in 2016.[1] The global prevalence of rifampicin-resistant (RR) TB was 600,000, with 490,000 of these being multidrug-resistant TB (MDR-TB), that is, TB resistant to at least rifampicin and isoniazid. The transmission of RR and MDR-TB remains a threat to the achievement of the global target of reducing TB deaths by 90% by 2030.[2] Effective control and management of TB is anchored on rapid, accurate, and definitive diagnosis with prompt initiation of anti-TB therapy. Prompt TB treatment increases the likelihood of positive treatment outcomes, reduces transmission risks, and reduces the risk of the emergence of drug-resistant Mycobacterium tuberculosis (Mtb). The current recommended drug-resistant TB treatment duration of 12 months or more is expensive to both patient and health-care system and is associated with serious adverse drug reactions.

Development of better diagnostics and treatment modalities requires an understanding of the natural history of evolution of the bacilli, its structures and metabolism, and mechanisms of drug resistance and persistence. Such knowledge can be studied prospectively under programmatic conditions from clinical Mtb isolates or retrospectively using stored Mtb isolates. With the limited resources in low-income countries, room-temperature storage of Mtb isolates remains the feasible option to facilitate future research.

The diagnostic algorithm for TB in Zimbabwe requires that all persons with clinical suspicion of TB will be tested using GeneXpert MTB/RIF and/or sputum smear microscopy as first-line diagnostic tools. GeneXpert MTB/RIF-positive samples are submitted for culture using mycobacterial growth indicator tube (MGIT) and Lowenstein–Jensen (LJ). Mtb culture and drug-sensitivity testing (DST) services were available from two tertiary public health laboratories: the National Microbiology Reference Laboratory (NMRL) in Harare and the National Tuberculosis Reference Laboratory (NTRL) in Bulawayo. The NMRL provided DST services for districts in the Northern part of the country and the NTRL provided services to Southern region districts. The NTRL was the official TB reference laboratory since the time Zimbabwe introduced the National TB Program (NTP) in the 1970s, whereas the NMRL started providing TB culture and DST services in 2010. The laboratories process other routine clinical and research samples besides TB diagnostics. As a resource-limited and high-TB-burden country, storage of mycobacteria culture isolates at subzero temperatures has been a challenge due to unavailability of − 80°C freezers, space, limited constant power supply, limited skilled staff, and inconsistent supply of cryovials. As a result, routine clinical mycobacteria-positive cultures in MGIT tubes are kept at room temperature.

The storage of mycobacteria for long periods at −70°C in enriched liquid broth without loss of metabolic activity, viability, or virulence is the recommended standard protocol since its first description in 1972.[3],[4],[5],[6] Suboptimal freezing of mycobacteria cells has been shown to result in intracellular injury, leading to loss of viability despite the preservation of the cell envelope.[4],[7] Information on the viability of mycobacteria following long-term storage at room temperature is scarce. Available studies either stored sputum samples with cetylpyridinium chloride–sodium chloride or in OMNIgene preservatives for short periods of time, maximum 3 weeks.[8],[9] Studies on viability of Mtb in stored samples at room temperature only assessed storage for 4 weeks with mixed results. In Malawi, authors compared samples stored at room temperature and refrigeration at 4°C and at 4 weeks of follow up: only 37% versus 67% room temperature to refrigerated were recovered.[10] In this study, the authors did not indicate which medium was used to store the samples. Other studies showed mixed results when the samples were stored for a maximum period of 4 weeks.[11],[12] No studies have described the utility of storing Mycobacterium- positive cultures under room temperature for future studies. This study sought to determine the recovery rate of Mtb from MGIT-positive tubes that had been stored at room temperature from 2011, 2012, and 2015 at the NTRL and from 2015 to 2016 at the NMRL.


  Methods Top


Positive GeneXpert RR samples were routinely cultured in BACTEC Mycobacterium growth indicator tube (MGIT 960; BD) and LJ for DST. Positive MGIT tubes were then kept at room temperature. As part of a larger drug-resistant TB epidemiological survey, drug-resistant Mtb isolates and RR and MDR-TB isolates stored in MGIT tubes were retrieved using the laboratory number and year of treatment.

Setting

A descriptive cross-sectional study was conducted on Mtb isolates stored at the NMRL in Harare and the NTRL in Bulawayo for 2011, 2012, 2015, and 2016. The isolates from NMRL were available for 2015 and 2016 and the isolates from the NTRL were available for 2011, 2012, and 2015. There were no samples for 2016 from the NTRL because all 2016 samples were used for the National Drug resistance Survey and therefore not available.

Physical storage

All samples from the two laboratories were stored in cupboards or on the floor within the TB laboratory, at room temperature. At the NTRL, the samples were stored in a dedicated locked room, and at the NMRL, the samples were in the TB culture room which had access control. There was no order in terms of year of sample processing or by drug-resistant status. Retrieving the samples took 5 full days, 2 days at NMRL and 3 days at NTRL. At NMRL, there was no electronic database of all TB culture-positive samples, and the laboratory paper register was used to identify RR/MDR-TB samples. A health and safety officer at the NTRL ensured that face masks were used during sample identification. There was no health and safety officer at the NMRL.

Laboratory procedures

All available RR/MDR-TB isolates were subcultured using the BACTEC MGIT 960 system's standard procedure. Inoculation was done by transferring 0.5 ml of well-mixed stored sample into a new labeled MGIT tube, cap tightly closed, and tube mixed by inverting several times. The tubes were incubated at 37°C until the instrument flagged them as positive or up to a total of 42 days after which they were flagged as culture negative. The cultures were monitored daily for positivity. All positive cultures were confirmed as Mtb using the SD Bioline TB Ag MPT64 Antigen Rapid Test (Standard Diagnostics Inc., Kyonggi, South Korea).

Before aliquoting and shipping to the University of Stellenbosch (SU), bacterial contamination was checked by inoculating positive MGIT culture onto blood agar plates using the isolation technique. Approximately 0.8 ml of MGIT culture was aseptically added to 0.8 ml of 25% glycerol in a cryovial. Prepared aliquots were kept at −20°C for <24 h before shipment. At SU, a volume of 0.8 ml of PANTA antibiotic mixture (Becton Dickinson, USA) was added to the MGIT tube before inoculation to decontaminate the Mtb isolates. The cultures were kept at room temperature for a maximum of 3 days before being cultured on MGIT and 7H10 solid media under biosafety level-3 conditions utilizing aseptic technique. A positive 7H10 slant culture was defined as a growth of at least one colony of Mtb confirmed by MPT64 antigen rapid test. Extraction of deoxyribonucleic acid (DNA) for future molecular work was done using the phenol/chloroform method followed by precipitation with isopropanol.[11],[12] Purified DNA was re-dissolved in TE pH 8.0 buffer and stored at −20°C. Recovery and contamination rates for the first and second subculture were calculated as percentages.

Data management and analysis

Data were entered into Microsoft Excel, which was password protected. Frequencies of number of isolates that successfully grew, number contaminated, and number that failed to grow were reported. Chi-square test was used to compare proportions using Stata version 12.0 (StataCorp. 2011).

Study approval was obtained from the Institutional Review Boards for the University of Zimbabwe, College of Health Sciences and SU, the Medical Research Council of Zimbabwe, and the Research Council of Zimbabwe in Harare.


  Results Top


Recovery rate

A total of 400 stored MGIT cultures identified as having resistant Mtb strains were successfully retrieved and subcultured. Stored MGIT cultures from NTRL were from 2011 (n = 47), 2012 (n = 91), and 2015 (n = 121), whereas those obtained from NMRL were from 2015 (n = 59) and 2016 (n = 82). Of the 400 subcultures, 248 (62%) successfully grew Mtb and were all shipped to SU, South Africa. Overall recovery rate was lower, 54.0% for samples from NMRL, and 66.4% for samples from NTRL. Recovery rate from the second subculture was higher and showed no difference with year of diagnosis [Figure 1]. In 2015, the only year samples were available from both laboratories, recovery rates ranged from 47.5% for samples from NMRL to 77.7% for samples from NTRL. Recovery rate for 2016 was 58.6%, but the samples were available from the NMRL only. More than 50% of subcultured samples were recovered in both 2011 and 2012 [Figure 2].
Figure 1: Subculture recovery rates, National Microbiology Reference Laboratory and National Tuberculosis Reference Laboratory, Zimbabwe

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Figure 2: Percentage Mycobacterium tuberculosis growth recovery rates, by year, laboratory source (National Microbiology Reference Laboratory and National Tuberculosis Reference Laboratory), Harare, Zimbabwe

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Contamination

Contamination rates during the initial subculture in Harare were high (64; 16%), and there was no capacity to decontaminate. These were excluded from the samples shipped to the SU. The contamination rates for isolates retrieved from NTRL were significantly lower than isolates retrieved from NMRL (9/172 [0.052] vs. 16/76 [0.211]; P = 0.0001). Of the 248 MGIT re-subcultured at the SU, 218 (87.9%) were positive for Mtb, 5 were culture negative (2%), and 25 (10.1%) were contaminated. Among the contaminated cultures, 19 (76.0%) contained acid-fast bacilli positive, of these 13 (68.4%) were successfully decontaminated and 6 (24%) could not be successfully decontaminated. The remaining six cultures did not contain acid-fast bacilli and were not processed further. The total samples successfully recovered including decontaminated samples were 231 (93.1%) of 248 re-subcultures.


  Discussion Top


Utility of storing Mycobacterium tuberculosis samples at room temperature

This study demonstrates that, where resources are limited, Mtb samples can be stored at room temperature in MGIT tubes with good recovery after 6 years. Despite the current recommendation of Mtb storage at −70°C, based on 1972 studies,[4],[5],[13] our findings demonstrated that Mtb isolates can remain viable at room temperature in MGIT tubes for a period longer than currently recommended. Without any additional conditions besides the air-conditioned room, Mtb isolates in low-income countries can be stored at room temperature for the purposes of future studies.

Recovery rate

Our findings showed that more than 50% recovery was possible after 6 years of storage at room temperature. Other studies showed recovery rates of above 80% using different storage methods raging from filter paper to subzero freezers.[14] However, the storage periods were for periods of at most 8 weeks, compared to 6 years demonstrated by our study. Studies recommending subzero temperatures have argued that high temperatures and storage media used affect the viability of Mtb strains.[6] In our study, the isolates were stored in the original MGIT tubes used for culture confirmation which contains Middlebrook 7H9 Broth supplemented with OADC, which may indicate that the MGIT media allow Mtb isolates to preserve viability over long periods. Thirty-eight percent of the primary isolates that failed to grow from the initial subcultured may be attributed to loss of viability due to storage at room temperature or contamination. Although storage at room temperature may not be optimal, this approach is more feasible in settings where subzero storage capacity is limited.

While there may be a need to validate our findings under experimental conditions, these findings have the important implications for future biobanking of Mtb in low-income and high-TB-burden countries. The findings provide hope toward sustaining basic science research in resource-limited settings by making storage of Mtb isolates a more practical and feasible procedure.

Standard precautions for infection prevention and control

Our findings confirmed that Mtb strains retain their viability and possibly virulence even when stored at room temperature for about 6 years. The samples were not stored in an orderly fashion or hampering retrieval. Both drug-sensitive and RR- and MDR-TB-confirmed samples were mixed in the same MGIT boxes. At the NMRL, there was no health and safety officer to ensure that retrieval of Mtb isolates was done with minimal risk to infection from stored samples. The safety officer from NTRL provided and supervised the use of face masks to the researcher during retrieval of Mtb samples. The findings have important public health implications. First, there was a potential risk of TB infection among laboratory workers, which has been reported to be three times more than in the general population.[15] Other health-care workers who may be involved in handling of the stored MGIT tubes during disposal of medical waste may have an increased risk of TB infection.[16],[17],[18] Health-care workers, particularly laboratory workers, are recommended to handle TB samples stored in MGIT according to the standard TB infection prevention and control measures to avoid contracting hospital-acquired TB. The NTP must provide adequate resources for TB infection prevention and control including creating awareness among health-care workers and providing biosafe conditions for storing the samples. Some studies have shown that health-care workers have low-risk perception to TB transmission.[15],[19]


  Conclusions and Recommendations Top


Biobanks have become an important means of storing specimens for future use in public health, especially with the emergence of new drug-resistant pathogens.[20] The growing epidemic of drug-resistant pathogens requires biobanking of samples to facilitate epidemiological surveillance and research on the development of new drugs and new diagnostic technologies. Future research on stored Mtb isolates will facilitate understanding of mutations associated with drug resistance and therefore diagnostic tools and new drugs to treat the emerging infections.

The authors propose the revision of the current Mtb storage protocol to include storage in MGIT tubes at room temperature in settings where facilities for −80° are not available. New storage guidelines should include specifications on clear labeling of stored samples and in a well-contained area to minimize breakages. The storage area must be locked at all times for the safety of laboratory workers and safe keeping of samples. MGIT tubes are relatively strong and should not pose any risk to breakage, especially if the room is secure and always locked up with controlled entry.

During retrieval of the samples, numbers in laboratory registers were used and compared with numbers on the MGIT tube. We propose that the labeling on the MGIT tubes should be clear and should indicate resistance pattern of the Mtb isolates. This will improve the ease of identifying the samples in the future and reduce research time. In addition, all Mtb isolates with RR-TB, MDR-TB, poly-resistant TB, and any form of drug resistant must be stored separately in a cool dry place. The use of ordinary refrigerators where air-conditioning facilities are not available could improve recovery rates.

The need for infection control procedures such as daily bench surface swabbing/decontamination, orderly packing, and storage documentation will need to be emphasized to minimize the risk of infection. The successful decontamination at the SU laboratory demonstrates the importance of good laboratory infrastructure for improved recovery rates in stored samples. We, therefore, recommended that the NTRL should build capacity to decontaminate samples for routine diagnostic services to improve case detection and early treatment initiation.

Limitations

Samples were stored in a haphazard manner without storage documentation and proper filing of resistance patterns. As a result, all stored RR/MDR-TB samples could not be retrieved, and the time taken to retrieve the identified samples was long. This may have affected the ability to sample all drug-resistant TB cases ever reported by the two laboratories.

The two reference laboratories provided samples for different years. The NTRL provided samples for 2011, 2012, and 2015, and the NMRL provided samples for 2015 and 2016. This affected an adequate comparison between the two laboratories. Despite the lack of data for some years at the different reference laboratories, the study did show a trend in Mtb growth recovery rate over time.

Because we did not have capacity to decontaminate, resulting in us missing potential positives from the 38% contaminated samples, it was not possible to compare recovery rates across laboratories, given the higher contamination rates at NMRL. Samples from NMRL presented with higher contamination rates than those from NTR. It is possible that differences in efficient laboratory skills, as well as good laboratory practices, may have impacted on the statistically significant contamination rates between the two source laboratories. Therefore, it was not possible to estimate the optimal recovery rates of Mtb under the storage conditions. This, therefore, could have introduced bias in either direction. Optimal recovery rates for MGIT have been varied, ranging from as low as 63% to high rates of more than 90%.[17],[18]

Financial support and sponsorship

This work was funded by Letten Foundation, Wellcome Trust, and University of Stellenbosch. Dr. Elizabeth M. Streicher was supported by the National Research Foundation (NRF) Research Career Advancement Award. Professor Samantha L. Sampson is funded by the South African Research Chairs Initiative of the Department of Science and Technology and National Research Foundation (NRF) of South Africa, award number UID 86539. Professor Rob Warren is funded by the DST-NRF Centre of Excellence for Biomedical Tuberculosis Research; South African Medical Research Council, Centre for Tuberculosis Research. The content is solely the responsibility of the authors and does not necessarily represent the official views of the sponsors.

Conflicts of interest

There are no conflicts of interest.



 
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