A practical approach to render tuberculosis samples safe for application of tuberculosis Molecular Bacterial Load Assay in clinical settings without a biosafety level 3 laboratory

Bariki Anyamkisye Mtafya; Paschal Qwaray; Joseph John; Emanuel Sichone; Alice Shoo; Stephen Henry Gillespie; Nyanda Elias Ntinginya; Wilber Sabiiti

doi:10.1016/j.tube.2022.102275

A practical approach to render tuberculosis samples safe for application of tuberculosis Molecular Bacterial Load Assay in clinical settings without a biosafety level 3 laboratory

Bariki Anyamkisye Mtafya^*, Paschal Qwaray, Joseph John, Emanuel Sichone, Alice Shoo, Stephen Henry Gillespie, Nyanda Elias Ntinginya, Wilber Sabiiti

^*Corresponding author for this work

Research output: Contribution to journal › Article › peer-review

Abstract

Background
Mycobacterium tuberculosis is a category B pathogen requiring level-3-containment laboratories for handling. We assessed the efficacy of heat and Guanidine thiocyanate (GTC) to inactivate M. tuberculosis prior to performance of tuberculosis Molecular Bactrial Load Assay (TB-MBLA).

Method
We performed in vitro experiments using H37Rv reference strain and replicated in sputum specimens. A 0.5 MacFarland standard of M. tuberculosis was serially diluted to 1x10 CFU/mL and pooled sputum was homogenised prior to serial dilutions and Xpert MTB/RIF Ultra. Three replicates for each containing 1 mL for M. tuberculosis and sputum were inactivated at 80 °C for 20 minutes and with GTC for 15 minutes. Inactivated samples were processed for culture and TB-MBLA.

Results
No M. tuberculosis growth was observed in MGIT for GTC or heat treated H37Rv cultures. All untreated H37Rv dilutions were MGIT positive except the most diluted specimens. Heat and GTC treatment of H37Rv reduced TB-MBLA load by 2.1log10 (P = 0.7) and 1.8log10 (P = 0.7) respectively, compared to controls. In contrast, heat treated sputum had TB-MBLA bacterial load of 3.47 ± 3.53 log10 compared to 5.4 ± 3.1 log10 eCFU/mL for GTC (p = 0.57). All heat and GTC treated sputum were culture negative.

Conclusion
Heat or GTC renders M. tuberculosis non-viable and eliminates the need for BSL3 laboratory for performing TB-MBLA in routine healthcare settings.

Original language	English
Article number	102275
Number of pages	5
Journal	Tuberculosis
Volume	138
Early online date	24 Nov 2022
DOIs	https://doi.org/10.1016/j.tube.2022.102275
Publication status	Published - Jan 2023

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Copyright © 2022 The Authors. Published by Elsevier Ltd. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).
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Mtafya, B. A., Qwaray, P., John, J., Sichone, E., Shoo, A., Gillespie, S. H., Ntinginya, N. E., & Sabiiti, W. (2023). A practical approach to render tuberculosis samples safe for application of tuberculosis Molecular Bacterial Load Assay in clinical settings without a biosafety level 3 laboratory. Tuberculosis, 138, Article 102275. https://doi.org/10.1016/j.tube.2022.102275

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title = "A practical approach to render tuberculosis samples safe for application of tuberculosis Molecular Bacterial Load Assay in clinical settings without a biosafety level 3 laboratory",

abstract = "BackgroundMycobacterium tuberculosis is a category B pathogen requiring level-3-containment laboratories for handling. We assessed the efficacy of heat and Guanidine thiocyanate (GTC) to inactivate M. tuberculosis prior to performance of tuberculosis Molecular Bactrial Load Assay (TB-MBLA).MethodWe performed in vitro experiments using H37Rv reference strain and replicated in sputum specimens. A 0.5 MacFarland standard of M. tuberculosis was serially diluted to 1x10 CFU/mL and pooled sputum was homogenised prior to serial dilutions and Xpert MTB/RIF Ultra. Three replicates for each containing 1 mL for M. tuberculosis and sputum were inactivated at 80 °C for 20 minutes and with GTC for 15 minutes. Inactivated samples were processed for culture and TB-MBLA.ResultsNo M. tuberculosis growth was observed in MGIT for GTC or heat treated H37Rv cultures. All untreated H37Rv dilutions were MGIT positive except the most diluted specimens. Heat and GTC treatment of H37Rv reduced TB-MBLA load by 2.1log10 (P = 0.7) and 1.8log10 (P = 0.7) respectively, compared to controls. In contrast, heat treated sputum had TB-MBLA bacterial load of 3.47 ± 3.53 log10 compared to 5.4 ± 3.1 log10 eCFU/mL for GTC (p = 0.57). All heat and GTC treated sputum were culture negative.ConclusionHeat or GTC renders M. tuberculosis non-viable and eliminates the need for BSL3 laboratory for performing TB-MBLA in routine healthcare settings.",

author = "Mtafya, {Bariki Anyamkisye} and Paschal Qwaray and Joseph John and Emanuel Sichone and Alice Shoo and Gillespie, {Stephen Henry} and Ntinginya, {Nyanda Elias} and Wilber Sabiiti",

note = "Funding: This work was supported by the United Kingdom Research and Innovation (UKRI) for the TB-MBLA translation study in routine healthcare settings through the Food Security and Health for East Africa Project (Grant number# EP/T01525X/1) as part of postdoctoral work for Dr Bariki Mtafya at the National Institute for Medical Research Centre-Mbeya Medical Research Centre, Mbeya, Tanzania with the University of St Andrews, UK.",

year = "2023",

month = jan,

doi = "10.1016/j.tube.2022.102275",

language = "English",

volume = "138",

journal = "Tuberculosis",

issn = "1472-9792",

publisher = "Elsevier Inc.",

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TY - JOUR

T1 - A practical approach to render tuberculosis samples safe for application of tuberculosis Molecular Bacterial Load Assay in clinical settings without a biosafety level 3 laboratory

AU - Mtafya, Bariki Anyamkisye

AU - Qwaray, Paschal

AU - John, Joseph

AU - Sichone, Emanuel

AU - Shoo, Alice

AU - Gillespie, Stephen Henry

AU - Ntinginya, Nyanda Elias

AU - Sabiiti, Wilber

N1 - Funding: This work was supported by the United Kingdom Research and Innovation (UKRI) for the TB-MBLA translation study in routine healthcare settings through the Food Security and Health for East Africa Project (Grant number# EP/T01525X/1) as part of postdoctoral work for Dr Bariki Mtafya at the National Institute for Medical Research Centre-Mbeya Medical Research Centre, Mbeya, Tanzania with the University of St Andrews, UK.

PY - 2023/1

Y1 - 2023/1

N2 - BackgroundMycobacterium tuberculosis is a category B pathogen requiring level-3-containment laboratories for handling. We assessed the efficacy of heat and Guanidine thiocyanate (GTC) to inactivate M. tuberculosis prior to performance of tuberculosis Molecular Bactrial Load Assay (TB-MBLA).MethodWe performed in vitro experiments using H37Rv reference strain and replicated in sputum specimens. A 0.5 MacFarland standard of M. tuberculosis was serially diluted to 1x10 CFU/mL and pooled sputum was homogenised prior to serial dilutions and Xpert MTB/RIF Ultra. Three replicates for each containing 1 mL for M. tuberculosis and sputum were inactivated at 80 °C for 20 minutes and with GTC for 15 minutes. Inactivated samples were processed for culture and TB-MBLA.ResultsNo M. tuberculosis growth was observed in MGIT for GTC or heat treated H37Rv cultures. All untreated H37Rv dilutions were MGIT positive except the most diluted specimens. Heat and GTC treatment of H37Rv reduced TB-MBLA load by 2.1log10 (P = 0.7) and 1.8log10 (P = 0.7) respectively, compared to controls. In contrast, heat treated sputum had TB-MBLA bacterial load of 3.47 ± 3.53 log10 compared to 5.4 ± 3.1 log10 eCFU/mL for GTC (p = 0.57). All heat and GTC treated sputum were culture negative.ConclusionHeat or GTC renders M. tuberculosis non-viable and eliminates the need for BSL3 laboratory for performing TB-MBLA in routine healthcare settings.

AB - BackgroundMycobacterium tuberculosis is a category B pathogen requiring level-3-containment laboratories for handling. We assessed the efficacy of heat and Guanidine thiocyanate (GTC) to inactivate M. tuberculosis prior to performance of tuberculosis Molecular Bactrial Load Assay (TB-MBLA).MethodWe performed in vitro experiments using H37Rv reference strain and replicated in sputum specimens. A 0.5 MacFarland standard of M. tuberculosis was serially diluted to 1x10 CFU/mL and pooled sputum was homogenised prior to serial dilutions and Xpert MTB/RIF Ultra. Three replicates for each containing 1 mL for M. tuberculosis and sputum were inactivated at 80 °C for 20 minutes and with GTC for 15 minutes. Inactivated samples were processed for culture and TB-MBLA.ResultsNo M. tuberculosis growth was observed in MGIT for GTC or heat treated H37Rv cultures. All untreated H37Rv dilutions were MGIT positive except the most diluted specimens. Heat and GTC treatment of H37Rv reduced TB-MBLA load by 2.1log10 (P = 0.7) and 1.8log10 (P = 0.7) respectively, compared to controls. In contrast, heat treated sputum had TB-MBLA bacterial load of 3.47 ± 3.53 log10 compared to 5.4 ± 3.1 log10 eCFU/mL for GTC (p = 0.57). All heat and GTC treated sputum were culture negative.ConclusionHeat or GTC renders M. tuberculosis non-viable and eliminates the need for BSL3 laboratory for performing TB-MBLA in routine healthcare settings.

U2 - 10.1016/j.tube.2022.102275

DO - 10.1016/j.tube.2022.102275

M3 - Article

SN - 1472-9792

VL - 138

JO - Tuberculosis

JF - Tuberculosis

M1 - 102275

ER -

A practical approach to render tuberculosis samples safe for application of tuberculosis Molecular Bacterial Load Assay in clinical settings without a biosafety level 3 laboratory

Abstract

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