During 1994–2005, we isolated Mycobacterium microti from 5 animals and 4 humans. Only 1 person was immunocompromised. Spoligotyping showed 3 patterns: vole type, llama type, and a new variant llama type.

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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...

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In recent years, various polymorphic loci and multicopy insertion elements have been discovered in the Mycobacterium tuberculosis genome, such as the direct repeat (DR) locus, the major polymorphic tandem repeats, the polymorphic GC-rich repetitive sequence, IS6110, and IS1081. These, especially IS6110 and the DR locus, have been widely used as genetic markers to differentiate M. tuberculosis isolates and will continue to be so used, due to the conserved nature of the genome ofM. tuberculosis. However, little is known about the processes involved in generating these or of their relative rates of change. Without an understanding of the biological characteristics of these genetic markers, it is difficult to use them to their full extent for understanding the population genetics and epidemiology of M. tuberculosis. To address these points, we identified a cluster of 7 isolates in a collection of 101 clinical isolates and investigated them with various polymorphic genetic markers, which indicated that they were highly related to each other. This cluster provided a model system for the study of IS6110 transposition, evolution at the DR locus, and the effects of these on the determination of evolutionary relationships among M. tuberculosis strains. Our results suggest that IS6110 restriction fragment length polymorphism patterns are useful in grouping closely related isolates together; however, they can be misleading if used for making inferences about the evolutionary relationships between closely related isolates. DNA sequence analysis of the DR loci of these isolates revealed an evolutionary scenario, which, complemented with the information from IS6110, allowed a reconstruction of the evolutionary steps and relationships among these closely related isolates. Loss of the IS6110 copy in the DR locus was noted, and the mechanisms of this loss are discussed.