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Molecular analysis of drug resistant TB
  1. M Melzer1,
  2. T J Brown1,
  3. G L French1,
  4. A Dickens2,
  5. T D McHugh2,
  6. L R Bagg3,
  7. R A Storring3,
  8. S Lacey3
  1. 1Department of Infection, St Thomas' Hospital, London SE1 7EH, UK
  2. 2Department of Medical Microbiology, Royal Free and University College Medical School, London NW3 2PF, UK
  3. 3King George Hospital, Goodmayes, Essex IG3 8YB, UK
  1. Correspondence to:
    Dr M Melzer, Department of Infection, St Thomas' Hospital, London SE1 7EH, UK;
    markmelzer{at}hotmail.com

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Since the mid 1980s the number of notified cases of TB in the UK has continued to rise with the largest increases noted in London and inner city areas.1 King George Hospital in Goodmayes, Essex provides clinical services to a population of approximately 230 000; 17% are non-white subjects including immigrants from countries with high rates of M tuberculosis infection and drug resistance. From September 1996 to July 1997 47 adult cases of culture proven TB were identified including seven with drug resistant isolates. None was identified by contact tracing. A previous TB audit of African born patients revealed a high rate of drug resistance (6/24 (25%)) and delays in obtaining drug sensitivities which could have been detrimental to patient management.2

Under these circumstances the rapid identification of drug resistance in M tuberculosis isolates would have been helpful. The aim of this study was to determine retrospectively the usefulness of PCR-reverse hybridisation methods for screening for mutations within or adjacent to M tuberculosis genes associated with rifampicin (rpoB) and isoniazid (inhA, katG, and ahpC) resistance. We also determined whether resistance genotyping combined with IS6110 typing could help to identify clusters of drug resistant cases not previously identified by contact tracing.

Seven consecutive drug resistant M tuberculosis culture isolates were analysed for rifampicin and isoniazid resistance and the results were compared with conventional susceptibility testing. The commercially available assay INNO-LiPA Rif.TB3 was used to detect rpoB mutations and an in-house PCR-reverse hybridisation line probe was used to detect mutations in or adjacent to the katG, inhA, and ahpC genes.4 The isolates were also IS6110 typed.5

The single rifampicin and isoniazid resistant isolate had an rpoB gene mutation associated with rifampicin resistance (table 1). Four of the five isoniazid resistant isolates had the same single point mutation upstream of the inhA gene and the other a single katG point mutation. Isolates 3 and 5 had indistinguishable IS6110 types that could represent isolates where recent transmission had occurred. No mutations were detected in the 40 fully susceptible isolates.

PCR-reverse hybridisation methods were highly sensitive and specific at detecting mutations that predict for isoniazid and rifampicin resistance. We also demonstrated that different point mutations can be used to discriminate between isoniazid resistant isolates. We believe that with automation and the addition of oligonucleotide probes designed to detect mutations associated with pyrazinamide (pncA)6 and ethambutol (embB)7 resistance, a system capable of detecting resistance to four front line antituberculous drugs will soon be commercially available. Rapid resistance detection by PCR-reverse hybridisation is likely to have a major impact on patient management and our understanding of the epidemiology of drug resistant TB.

Table 1

Demographic data, site, phenotypic and genotypic resistance of the seven resistant study isolates

Acknowledgments

We would like to thank the Steering Group Members of the “Molecular Epidemiology of Tuberculosis in London” for allowing us access to their M tuberculosis IS6110 type database and to the Mycobacterial Reference Laboratory (Dulwich) for conventional susceptibility testing.

References

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