GENOTIPIZZAZIONE MOLECOLARE E ANALISI CLINICA DI PAZIENTI CON TUBERCOLOSI MULTIRESISTENTE NELL¿HINTERLAND MILANESE

Abstract Keywords: tuberculosis, multi-drug-resistance, exogenous reinfection, relapse, fingerprinting, lineage Mycobacterium tuberculosis. Background Tuberculosis (TB) remains a major global health issue. In 2012, an estimated 8.6 million people developed TB and 1.3 million died from the disease (including 320,000 deaths among HIV-positive people). Considering the preventable nature of TB, the number of deaths due to Mycobacterium tuberculosis is unacceptably high. Based on global TB surveillance programs, in 2012 multi-drug resistance was identified among 3.6% of the new TB cases and among 20.0% of previously treated TB cases. The prevalence of MDR TB was particularly worrisome in Eastern Europe and Central Asia: in these areas MDR-TB accounted for more than 20.0% of the new TB cases and more than half of the previously treated TB cases. On the contrary, in Italy in the period 2004-2008 MDR strains represented less than 5% of all TB cases. Objective In this epidemiological retrospective cohort study we aimed to address the phenomenon of MDR-TB in the metropolitan area of Milan in the period 1993-2010. The main objectives of our study were the following: i) to compare epidemiological, microbiological, and clinical features of patients diagnosed with MDR-TB vs. patients diagnosed with non-MDR TB; ii) to evaluate the tendency of M. tuberculosis strains to form clusters and the role played in this setting by MDR-TB; iii) to assess the diffusion of MDR-TB among specific M. tuberculosis lineages; iv) and finally to analyzed the influence of MDR-TB in the recurrence of TB (specifically in the development of relapse vs. exogenous reinfections). Subjects This study was based on the TB dataset maintained at the Infectious Diseases Laboratory at Luigi Sacco Hospital (Milan, Italy), which contains data among TB cases diagnosed in the metropolitan area of Milan. The patients included in the study satisfied the following criteria: i) culture-proven TB; ii) availability of the causative M. tuberculosis strain; iii) presence of antimicrobial in vitro susceptibility data. Material and methods M. tuberculosis DNA was extracted from mycobacterial colonies growing on Lowenstein-Jensen medium according to standard laboratory procedures. Three methods were used to genotype M. tuberculosis strains: the gold standard technique Restriction Fragment Length Polymorphism designed by van Soolingen et al., the Spacer Oligonucleotide Typing (Spoligotyping) as described by Kamerbeek et al., and the Mycobacterial Interspersed Repetitive Unit of Variable Number of Tandem Repeats (MIRU-VNTR) 12-loci genotyping as illustrated by Supply et al. Logistic regression analysis (conducted with SAS Enterprise Guide -Version 5.1, Institute Inc, Cary North Carolina, USA and SPSS Version 20.0, Chicago, Illinois, USA) was used to assess risk factors associated with MDRTB, clustering, TB lineages and recurrent episodes. Results 4,237 TB cases were included in the study. Based on logistic regression analyses, MDR TB cases, were independently associated with presence of comorbidities (aOR 2,5; 95% CI 1,1-5,7), pulmonary TB (aOR 5,2; 95% CI 1,6-16,5), and TB recurrence (aOR 5,2; 95% CI 3,0-9,1). European nationality was associated to a reduced risk of developing MDR-TB (aOR 0,3; 95% CI 0,2-0,6). TB clustered strains were associated to positive microscopic smear exam (aOR 1,9; CI 95% 1,1-3,4). On the contrary there was an inverse correlation between East-European origin of the patient and the tendency of M. tuberculosis strains to clusterize (aOR 0,09; CI 95% 0,01-0,7). Moreover, we showed that East-Asian lineage was independently linked with MDR-TB (aOR 3,4; CI 95% 1,5-7,6). Finally, MDR-TB was more frequently observed in recurrent TB episodes due to relapse; when compared to TB relapse, exogenous TB reinfection were characterized by a reduced risk of MDR (aOR 0,1 CI 95% 0,01-0,9; p 0,04). Conclusions Based on our observations in the metropolitan area of Milan, MDR-TB was associated with foreign origin of the patients, presence of the Beijing lineage of M. tuberculosis and recurrence of TB (specifically, TB relapse). This study highlights the key-role of molecular genotyping in TB epidemiology. Our findings may improve current MDR-TB control strategies. Keywords: tuberculosis, multi-drug-resistance, exogenous reinfection, relapse, fingerprinting, lineage Mycobacterium tuberculosis.