Multidrug-Resistant Tuberculosis in Lisbon, Portugal: A Molecular Epidemiological Perspective Joa ˜ o Perdiga ˜o, 1 Rita Macedo, 2 Ine ˆs Joa ˜o, 2 Elisabete Fernandes, 2 Laura Brum, 2 and Isabel Portugal 1,2 Portugal has the fourth highest tuberculosis (TB) incidence rate in the European Union (EU). Thirty-nine percent of all cases originate in Lisbon Health Region. Portugal also presents high levels of multidrug-resistant tuberculosis (MDR-TB) (1.5%, primary rate and 2.4%, in retreatment cases). In the present study we have characterized 58 MDR-TB clinical isolates by: (i) determining the resistance profile to first- and second-line drugs used in the treatment of tuberculosis; (ii) genotyping all isolates by MIRU–VNTR; (iii) analyzing mutations conferring re- sistance to isoniazid, rifampicin, streptomycin, and ethambutol, in katG, mabA-inhA, rpoB, rpsL, rrs, and pncA genes. We have therefore established the prevalence of the most common mutations associated with drug resis- tance in the Lisbon Health Region: C-15T in mabA-inhA for isoniazid; S531L in rpoB for rifampicin; K43R in rpsL for streptomycin; and V125G in pncA for pyrazinamide. By genotyping all isolates and combining with the mutational results, we were able to assess the isolates’ genetic relatedness and determine possible transmission events. Strains belonging to family Lisboa, characterized several years ago, are still responsible for the majority of the MDR-TB. Even more alarming is the high prevalence of extensive drug-resistant tuberculosis (XDR-TB) among the MDR-TB isolates, which was found to be 53%. The TB status in Portugal therefore requires urgent attention to contain the strains continuously responsible for MDR-TB and now, XDR-TB. Introduction P ortugal has the fourth highest tuberculosis (TB) incidence rate in the European Union (EU), having registered in 2004, 33.7 cases per 100,000 habitants nation- wide. Although this rate has diminished in the last 20 years to less than a half, the decline rate has been very slow, having decreased only 2.7 cases per 100,000 people from 2003 to 2004. 12 In the year of 2004, 39% of all new cases reported nation- wide were from Lisbon Health Region. The majority of these cases (72%) were concentrated in Lisbon district, of which 74% were associated with three high-risk groups for TB, namely, human immunodeficiency virus (HIV) infected, drug users, and immigrants. 12 The emergence of multidrug-resistant tuberculosis (MDR- TB), that is, resistance to at least isoniazid and rifampicin, poses a serious threat, not only to national tuberculosis con- trol plans, but also to the implementation of an effective di- rectly observed short-course treatment (DOTS) strategy. Portugal also reports high rates of MDR-TB. In 2004, Portu- guese Health authorities have reported a rate of primary MDR-TB of 1.1%, and 6.7% among retreatment cases. 12 Drug resistance generally occurs as a result of a regimen with the wrong combination of drugs, or due to the patients’ lack of adherence to the regimen. The only known mechanism for resistance development by Mycobacterium tuberculosis, is through mutation acquisition in chromosomal genes associ- ated with drug resistance. Several genes have been associated with drug resistance, namely katG, inhA, kasA, and ahpC, with isoniazid 4,49,58 ; rpoB with rifampicin resistance 20,49,61 ; rpsL and rrs with streptomycin resistance 7,28,29 ; embCAB with eth- ambutol resistance 35,37,42 ; and pncA with pyrazinamide re- sistance. 30,38,46,47 The emergence of MDR-TB strains is due to sequential acquisition of mutations in some of the genes above. Epidemiological surveys concerning MDR-TB strains are necessary to gain insight into the transmission and resistance development mode of TB, and ultimately to manage properly the local TB situation. Strain typing is an invaluable process in epidemiology and, the last year’s epidemiological surveys have benefited from the development of additional molecu- lar epidemiological methods, alternative to the traditional Restriction Fragment Length Polymorphism followed by hy- bridization with Insertion Sequence 6110 (RFLP-IS6110). 2,56 Such an example is the use of Mycobacterial Interspersed Repetitive Units–Variable Number of Tandem Repeats (MIRU–VNTR) for strain typing. MIRU–VNTR typing is based on the determination of the number of repeating units on several chromosomally dispersed loci. 2,33,52 1 Centro de Patoge ´nese Molecular, URIA, Faculdade de Farma ´cia da Universidade de Lisboa, Lisbon, Portugal. 2 Unidade de Micobacte ´rias, Centro de Bacteriologia, Instituto Nacional de Sau ´ de Dr. Ricardo Jorge, Lisbon, Portugal. MICROBIAL DRUG RESISTANCE Volume 14, Number 2, 2008 ª Mary Ann Liebert, Inc. DOI: 10.1089=mdr.2008.0798 133