The management of tuberculosis: epidemiology, resistance and monitoring.

This PhD thesis is based on 5 studies conducted in the period 2006-2010 during my employment at the International Reference Laboratory of Mycobacteriology, Statens Serum Institut. The overall aim was to assess tuberculosis (TB) treatment in Denmark with specific focus on the risk of relapse of TB disease, and to analyse treatment outcome of patients with multidrug-resistant (MDR) or isoniazid-resistant TB. The project established the need for rapid methods to detect resistance and follow-up of treatment. A rapid method to detect drug resistance was optimised and evaluated for use directly in clinical specimens. The studies were based on data from the Mycobacterial registry in the period 1992-2007, which included the results from microscopy, culture, drug-susceptibility and restriction fragment length polymorphism (RFLP). Information on dates of death/emigration were taken from the CPR-registry and treatment from surveillance data and patient records. The rate of recurrent TB was found to be low in Denmark, during 13.5 years of follow-up. Relapse accounted for 1.3% of the recurrent cases and reinfection was rare, only in 0.5% cases. The relapse hazard increased up to four years after diagnosis. Cavitary disease was associated with relapse as opposed to reinfection and may need prolonged treatment and closer monitoring. The incidence of MDR-TB and isoniazid resistance was confirmed to be low. Successful short- and long-term treatment outcome of MDR-TB and isoniazid-resistant TB was high. High- and low-level isoniazid resistance did not affect treatment outcome. A multiplex PCR hybridization mutation analysis, that simultaneously detects the most frequent rpoB and katG gene mutations conferring rifampin and high-level isoniazid resistance, was optimized for direct use and evaluated in smear-positive specimens as opposed to slow conventional drug-susceptibility testing (DST). The second-generation rifampin and isoniazid resistance mutation assay additionally included detection of mutations within the inhA gene conferring low-level isoniazid resistance. This assay was found to be rapid (< 48 h) and easy to perform in isolates and clinical specimens. A high concordance between mutation and conventional DST results was found for rifampin, while results varied for isoniazid . The mutation analysis identified all MDR-TB cases and the majority of isoniazid-resistant cases in Denmark. Standard 6-month multiple anti-TB drug therapy is necessary to treat drug-susceptible TB. Drug-resistant TB often requires therapy adjustments and extended treatment. MDR-TB particularly poses therapeutic challenges. Rapid detection of resistance mutations directly in smear-positive patient specimens may improve MDR-TB patient treatment, although the impact on isoniazid-resistant TB treatment outcome remains to be determined. The mutation assay is a rapid supplement to the gold standard conventional DST in high-income countries such as Denmark, while in low-income countries it can be used for preliminary DST. The assay may be applied to smear-positive samples from patients suspected of treatment failure, recurrent TB, drug-resistant TB exposure or originating from countries with high levels of DR. The new extended mutation assay has proved to be a useful tool, which has now been included in the World Health Organization's policy to combat and prevent new cases of MDR and extensively drug-resistant TB.