M. tuberculosis Clinical isolates This laboratory 3, 5, 10 M. tuberculosis Clinical isolates (Ghana) T. van der Werft 92, 93, 95-101 M. tuberculosis Clinical isolates (outbreak in The Netherlands) This laboratory 107-128, 130-133 M. tuberculosis Clinical isolates (Tilburg, The Netherlands) P. L. van Puttenb 38 M. africanum Clinical isolate This laboratory 40, 41 M. bovis Clinical isolates This laboratory 43, 45, 105, 106 M. bovis BCG Clinical isolates This laboratory 102 M. bovis BCG Vaccine strain Organon Teknikac 103 M. bovis BCG Vaccine strain

The gene encoding a 23 kilodalton protein antigen has been cloned from Mycobacterium tuberculosis by screening of a recombinant DNA library with monoclonal antibodies. The product of the gene has been identified as the superoxide dismutase (SOD) of M. tuberculosis on the basis of sequence comparison and by expression of the recombinant protein in a functionally active form. The derived amino acid sequence of M. tuberculosis SOD reveals a close similarity to manganese-containing SODs from other organisms, in spite of the fact that previous studies using the purified enzyme have identified iron as the preferred metal ion ligand. SOD is present in the extracellular fluid of logarithmic-phase cultures of M. tuberculosis, but the structural gene is not preceded by a signal peptide sequence. Insertion of the M. tuberculosis SOD gene into a novel shuttle vector demonstrated the mycobacteria but is ineffective in Escherichia coli.

PCR is, in principle, a simple and rapid test for use in the detection ofMycobacterium tuberculosis. However, virtually no data are available on the reliability and reproducibility of the method. In order to assess the validity of PCR for the detection of mycobacteria in clinical samples, seven laboratories participated in a blinded study of 200 sputum, saliva, and water samples containing either known numbers ofMycobacterium bovis BCG cells or no added organisms. Each laboratory used its own protocol for pretreatment, DNA extraction, and detection of the amplification product. Insertion sequence IS6110 was the target for DNA amplification. Several participating laboratories reported high levels of false-positive PCR results, with rates ranging from 3 to 20%o and with one extreme value of 77%. The levels of sensitivity also ranged widely among the different participants. A positive PCR result was reported for 2 to 90% of the samples with 103 mycobacteria. Although most participants did include control tests to check the sensitivity and specificity of the PCR, the sequence of operations from sample pretreatment to purification of DNA from bacteria was not always monitored adequately. During these procedures cross-contaminating DNA was introduced and/or bacterial DNA was lost. The results of the study show that the implementation of an effective system for monitoring sensitivity and specificity is required before the PCR can be used reliably in the diagnosis of tuberculosis.

A repetitive element (IS986), previously isolated from Mycobacterium tuberculosis and shown to detect multiple restriction fragment-length polymorphisms (RFLPs), has been sequenced. It consists of a potential insertion sequence of 1358bp, with 30-bp inverted repeat ends. IS986 has four potentially significant open reading frames (ORFs): ORFa1, ORFa2 and ORFb on one strand and ORFc on the complementary strand. The sequences of the potential translated products identify IS986 as a member of the IS3 family, with an apparent frameshift between ORFa1 and ORFa2. IS986 has potential as a highly specific probe for detection and typing of M. tuberculosis, as well as for transposon mutagenesis of mycobacteria. The sequence of IS986 is virtually identical to that of another recently described element, IS6110 (Thierry et al., 1990).

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