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...

Background In a 12 month period, three Irish-born adult cases with pulmonary TB were initially diagnosed by Xpert® MTB/RIF Ultra assay, which detected a rifampicin resistance-conferring mutation prompting treatment as potential MDR cases. Methods Further laboratory investigations on the cultured isolates included GenoType MTBDRplus assay, phenotypic drug susceptibility tests using the BD BACTEC MGIT culture system and MIC broth microdilution tests. Sequencing of the rpoB gene was performed using Sanger sequencing and WGS. Results Phenotypic drug susceptibility tests determined the isolates to be rifampicin susceptible. Molecular investigations identified an A451V (codon 532) mutation in the Mycobacterium tuberculosis rpoB gene that has not previously been found to cause rifampicin resistance. Genome sequencing revealed that the three isolates’ genomes differed by ≤5 SNPs, indicating a high likelihood of recent transmission events. Furthermore, a cluster of six related M. tuberculosis isolates from our in-house typing database showed four were highly related; all were rifampicin susceptible and lacked this mutation. Conclusions False detection of rifampicin resistance, albeit rare, should be considered possible with Xpert® MTB/RIF Ultra assay, particularly in low TB incidence settings. Confirmatory sequencing methods should be performed to prevent the unnecessary use of second-line anti-tuberculous drugs.