The efficacy of the heat killing of Mycobacterium tuberculosis

Abstract

There is concern that current procedures for the heat inactivation of Mycobacterium tuberculosis may not be adequate. This raises serious safety issues for laboratory staff performing molecular investigations such as IS6110 restriction fragment length polymorphism typing. This paper confirms that the protocol of van Embden et al, as performed routinely in this laboratory, is safe and effective for the heat inactivation of M tuberculosis. This procedure involves complete immersion of a tube containing a suspension of one loopfull of growth in a water bath at 80°C for 20 minutes. Seventy four isolates were included in this investigation. Despite prolonged incubation for 20 weeks, none of the heat killed M tuberculosis suspensions produced visible colonies or gave a positive growth signal from liquid culture. This method did not affect the integrity of the DNA for subsequent molecular investigations. I S6110 restriction fragment length polymorphism analysis is considered to be the "gold standard" typing method for DNA fingerprinting of Mycobacterium tuberculosis.1 Before DNA extraction, M tuberculosis must be heat inactivated to render it safe for manipulation outwith a containment level 3 facility. Two reports have raised concerns that some heat killing procedures used for the inactivation of M tuberculosis are not reliably effective. This may put laboratory workers using molecular techniques at risk of laboratory acquired infection (P Bemer-Melchior et al. Transmission of Mycobacterium tuberculosis in a mycobacteriology laboratory. Presented at the 5th International Conference on the Prevention of Infection,1998). Zwadyk and colleagues 2 first suggested that temperatures below 100°C do not consistently kill M tuberculosis. They showed survival of 50% and 25% of the organisms after heat inactivation at 95°C in a dry heat block for 20 and 30 minutes, respectively. These findings were confirmed by Bemer-Melchior and Drugeon, 3 who investigated several different inactivation protocols involving heat killing at either 80°C for 20 minutes or 100°C for five minutes, followed by either lysozyme (0.5 mg/ml) or a combined proteinase K (0.4 mg/ml) and lysozyme (0.5 mg/ml) digestion. They reported the growth of M tuberculosis in 80% of subcultures on Löwenstein-Jensen (L-J) medium after heat inactivation at 80°C for 20 minutes and treatment with lysozyme and in 10% after heat inactivation at 80ºC for 20 minutes and treatment with both lysozyme and proteinase K. In addition, Zwadyk and colleagues 2 showed that increasing exposure time did not always correlate with a decrease in viability. In view of these findings, we investigated the suitability of the heat killing procedure currently used in our laboratory. This assessment involved detailed attention to procedures used during heat inactivation and included extended viability checks before and after heat inactivation."Two reports have raised concerns that some heat killing procedures used for the inactivation of Mycobacterium tuberculosis are not reliably effective" METHODSA...

Extraction of Mycobacterium tuberculosis DNA: a Question of Containment

“…We think it unlikely that our results are due to laboratory contamination, as we manipulated small volumes of cultures, samples were inoculated on five different days, all quality control parameters of all media used were met at the time of these experiments, and BACTEC readings included two uninoculated vials at the end that remained negative throughout. Contrary to previous reports (3,8) these data indicate that neither heating at 80°C nor treatment with lysozyme and proteinase K reliably inactivates M. tuberculosis. In addition, this study confirms previously published data that subsequent to treatment with the chemicals used in this protocol, the growth of M. tuberculosis is better supported by traditional L-J medium than by BACTEC 12B medium (2).…”

“…Heating of the culture is widely used, and at least one report has deemed heating at 80°C as sufficient for inactivation (3). However, other reports have raised concerns as to the efficacy of heating (8) and even of further treatment with a combination of lysozyme and proteinase K (2).…”

Laboratory evaluation of a specimen transport medium for downstream molecular processing of sputum samples to detect Mycobacterium tuberculosis

“…In that regard, safe (inactivated for purpose of infection control) and efficient (preservation and stability of DNA) transportation of sputum specimens is of paramount importance. Several methods have been described for inactivation of viable Mycobacterium tuberculosis from cultures by either heat or a combination of heat and the use of chemicals (Djelouagji and Drancourt, 2006, Doig et al, 2002, Inoue et al, 2014. However, the only relevant published data on the effect of reagents added directly to sputum on the viability of TB bacilli is the sample reagent buffer (SR) of the Xpert assay.…”

Rapid Inactivation of Mycobacterium and Nocardia Species before Identification Using Matrix-Assisted Laser Desorption Ionization–Time of Flight Mass Spectrometry

“…Regardless of which option is used, it is essential that cultures are rendered nonviable prior to processing outside appropriate biocontainment facilities for obvious safety reasons. Previous reports have identified a variety of ways in which mycobacteria, including Mycobacterium tuberculosis can be successfully inactivated prior to manipulation, including heat treatment (9), exposure to lethal irradiation with or without photosensitizing chemicals (10)(11)(12), alcohol exposure (13), or a combination of treatments (14)(15)(16). While most were successful, temperatures and/or exposure times varied in order to achieve uniform cell death.…”