This review summarises recent advances made in microscopic techniques (fluorescence and peptide nucleic acids) and culture techniques (solid, liquid, radiometric, and non-radiometric systems) and in the development of rapid methods for the identification of mycobacterial cultures (high performance liquid chromatography, thin layer chromatography, RNA sequencing, and polymerase chain reaction restriction enzyme assays). The role of molecular amplification systems in identifying Mycobacterium tuberculosis is described. Most methods record high specificity and sensitivity for smear positive sputum but have variable sensitivity for sputum smear negative and extrapulmonary specimens. Specimen quality will aVect the performance of these assays and organisational delays, such as the batching of specimens, can reduce the time saved. In house assays can be as eVective as commercial systems as long as appropriate controls are used. (J Clin Pathol 2000;53:727-732)

A clinical, microbiological and economic study of a national rapid molecular service for the identification of Mycobacterium tuberculosis and the determination of rifampicin resistance in smear-positive sputum samples (and other primary specimens) was performed. Ninety-one primary specimens, of which 55 were smear-positive sputum, were examined by molecular and conventional assays. Concordance of molecular results from smear-positive sputum specimens with tuberculosis diagnosis and rifampicin resistance by conventional analysis was 52 (94.5%) of 55 and 44 (91.7%) of 48, respectively. Concordance of molecular analysis on all primary specimens was 81 (89.0%) of 91 (diagnosis) and 55 (90.2%) of 61 (rifampicin resistance). Approximately 28 days were saved in the time to diagnosis by using the molecular assay. Hospitals can reduce the cost of inappropriate isolation of patients with risk factors for multiple drugresistant tuberculosis (MDRTB) who subsequently are shown to have drug-sensitive tuberculosis. At one hospital potential annual savings were between £50 000 and £150 000. Of the nine MDRTB cases identified, all had a previous diagnosis of tuberculosis, 78% were born overseas, 44% were known to be non-compliant with therapy, but only one case (12.5%) was HIV positive. HIV status was not significantly different between MDRTB and drug-sensitive tuberculosis cases. Over 75% of specimens were taken while the patient was on therapy. Isolates from >50% of the MDRTB cases were resistant to three or more drugs and one was resistant to seven drugs. All patients were placed on additional therapy once the molecular result was known; this was subsequently modified based on the results of in-vitro drug susceptibility testing. All survived at least 6 months of follow-up. There was no difference in the proportion of successful cultures from smear-positive samples from patients with drug-sensitive tuberculosis or MDRTB who were on therapy. Molecular rifampicin resistance assays are reliable for diagnosis in cases with smear-positive disease.

Multidrug-resistant Mycobacterium tuberculosis (MDR-TB) is an emerging problem of great importance to public health, with higher mortality rates than drug-sensitive TB, particularly in immunocompromised patients. MDR-TB patients require treatment with more-toxic second-line drugs and remain infectious for longer than patients infected with drug-sensitive strains, incurring higher costs due to prolonged hospitalization. It is estimated that 90% of United Kingdom rifampin-resistant isolates are also resistant to isoniazid, making rifampin resistance a useful surrogate marker for multidrug resistance and indicating that second- and third-line drugs to which these isolates are susceptible are urgently required. Resistance in approximately 95% of rifampin-resistant isolates is due to mutations in a 69-bp region of the rpoB gene, making this a good target for molecular genotypic diagnostic methods. Two molecular assays, INNO-LiPA Rif.TB (Innogenetics, Zwijndrecht, Belgium) and MisMatch Detect II (Ambion, Austin, Tex.), were performed on primary specimens and cultures to predict rifampin resistance, and these methods were compared with the resistance ratio method. A third method, the phenotypic PhaB assay, was also evaluated in comparison to cultures in parallel with the genotypic assays. In an initial evaluation 16 of 16, 15 of 16, and 16 of 16 rifampin-resistant cultures (100, 93.8, and 100%, respectively), were correctly identified by line probe assay (LiPA), mismatch assay, and PhaB assay, respectively. Subsequently 38 sputa and bronchealveolar lavage specimens and 21 isolates were received from clinicians for molecular analysis. For the 38 primary specimens the LiPA and mismatch assay correlated with culture and subsequent identification and susceptibility tests in 36 and 38 specimens (94.7 and 100%), respectively. For the 21 isolates submitted by clinicians, both assays correlated 100% with routine testing.

The performance of a commercial line probe assay (LiPA) (Inno-LiPA Mycobacteria; Innogenetics, Belgium) for the detection and identification of Mycobacterium species from liquid and solid culture was evaluated at five routine clinical laboratories. The LiPA method is based on the reverse hybridization principle, in which the mycobacterial 16S-23S ribosomal RNA (rRNA) spacer region is amplified by polymerase chain reaction (PCR). Amplicons are subsequently hybridized with oligonucleotide probes arranged on a membrane strip and detected by a colorimetric system. The test detects the presence of Mycobacterium species and specifically identifies Mycobacterium tuberculosis complex, Mycobacterium kansasii, Mycobacterium xenopi, Mycobacterium gordonae, Mycobacterium avium complex, Mycobacterium avium, Mycobacterium intracellulare, Mycobacterium scrofulaceum, and Mycobacterium chelonae - Mycobacterium abscessus complex. The results of LiPA were compared with the results obtained using traditional biochemical and molecular tests (DNA probe-based techniques, PCR restriction enzyme analysis of the 65 kDa heat-shock protein gene, and sequencing of the 16S rDNA). A total of 669 isolates, 642 of which were identified as Mycobacterium species and 27 as non- Mycobacterium species, were tested by LiPA. After analysis of 14 initially discordant results and exclusion of one isolate, concordant results were obtained for 636 of 641 Mycobacterium isolates (99.2% accuracy). All Mycobacterium species reacted with the MYC ( Mycobacterium species) probe (100% sensitivity), and all non- Mycobacterium species were identified as such (100% specificity).