title: Genetic diversity and molecular epidemiology of multidrug-resistant Mycobacterium tuberculosis in Minas Gerais State, Brazil.
abstract: BACKGROUND
  We aimed to characterize the genetic diversity of drug-resistant Mycobacterium tuberculosis (MTb) clinical isolates and investigate the molecular epidemiology of multidrug-resistant (MDR) tuberculosis from Minas Gerais State, Brazil.
  
  
  METHODS
  One hundred and four MTb clinical isolates were assessed by IS6110-RFLP, 24-locus mycobacterial interspersed repetitive units variable-number tandem repeats (MIRU-VNTR), TB-SPRINT (simultaneous spoligotyping and rifampicin-isoniazid drug-resistance mutation analysis) and 3R-SNP-typing (analysis of single-nucleotide polymorphisms in the genes involved in replication, recombination and repair functions).
  
  
  RESULTS
  Fifty-seven different IS6110-RFLP patterns were found, among which 50 had unique patterns and 17 were grouped into seven clusters. The discriminatory index (Hunter and Gaston, HGDI) for RFLP was 0.9937. Ninety-nine different MIRU-VNTR patterns were found, 95 of which had unique patterns and nine isolates were grouped into four clusters. The major allelic diversity index in the MIRU-VNTR loci ranged from 0.6568 to 0.7789. The global HGDI for MIRU-VNTR was 0.9991. Thirty-two different spoligotyping profiles were found: 16 unique patterns (n = 16) and 16 clustered profiles (n = 88). The HGDI for spoligotyping was 0.9009. The spoligotyped clinical isolates were phylogenetically classified into Latin-American Mediterranean (66.34 %), T (14.42 %), Haarlem (5.76 %), X (1.92 %), S (1.92 %) and U (unknown profile; 8.65 %). Among the U isolates, 77.8 % were classified further by 3R-SNP-typing as 44.5 % Haarlem and 33.3 % LAM, while the 22.2 % remaining were not classified. Among the 104 clinical isolates, 86 were identified by TB-SPRINT as MDR, 12 were resistant to rifampicin only, one was resistant to isoniazid only, three were susceptible to both drugs, and two were not successfully amplified by PCR. A total of 42, 28 and eight isolates had mutations in rpoB positions 531, 526 and 516, respectively. Correlating the cluster analysis with the patient data did not suggest recent transmission of MDR-TB.
  
  
  CONCLUSIONS
  Although our results do not suggest strong transmission of MDR-TB in Minas Gerais (using a classical 100 % MDR-TB identical isolates cluster definition), use of a smoother cluster definition (>85 % similarity) does not allow us to fully eliminate this possibility; hence, around 20-30 % of the isolates we analyzed might be MDR-TB transmission cases.
authors.affiliation: Post-Graduate Program in Infectious Diseases and Tropical Medicine, Department of Internal medicine, Faculty of Medicine, Federal University of Minas Gerais, Belo Horizonte, Brazil. ngtdantas@gmail.com.
authors.forename: Nayanne Gama Teixeira
authors.initials: NG
authors.lastname: Dantas
authors.affiliation: Laboratory of Molecular Biology Applied to Mycobacteria, Oswaldo Cruz Institute, FIOCRUZ, Rio de Janeiro, Brazil. psuffys@gmail.com.
authors.forename: Phillip Noel
authors.initials: PN
authors.lastname: Suffys
authors.affiliation: Laboratory of Molecular Biology and Public Health, Department of Social Pharmacy, Faculty of Pharmacy, Federal University of Minas Gerais, Belo Horizonte, Brazil. waniasilvacarvalho@gmail.com.
authors.forename: Wânia da Silva
authors.initials: Wda S
authors.lastname: Carvalho
authors.affiliation: Laboratory of Molecular Biology Applied to Mycobacteria, Oswaldo Cruz Institute, FIOCRUZ, Rio de Janeiro, Brazil. magdinier@gmail.com.
authors.forename: Harrison Magdinier
authors.initials: HM
authors.lastname: Gomes
authors.affiliation: Post-Graduate Program in Infectious Diseases and Tropical Medicine, Department of Internal medicine, Faculty of Medicine, Federal University of Minas Gerais, Belo Horizonte, Brazil. isabelanalmeida@gmail.com.
authors.forename: Isabela Neves
authors.initials: IN
authors.lastname: de Almeida
authors.affiliation: Laboratory of Molecular Biology and Public Health, Department of Social Pharmacy, Faculty of Pharmacy, Federal University of Minas Gerais, Belo Horizonte, Brazil. lidajouca@hotmail.com.
authors.forename: Lida Jouca
authors.initials: LJ
authors.lastname: de Assis
authors.affiliation: Ezequiel Dias Foundation, Belo Horizonte, Brazil. augustocj@gmail.com.
authors.forename: Claudio José
authors.initials: CJ
authors.lastname: Augusto
authors.affiliation: Institut for Integrative Cell Biology, I2BC, UMR9198 CEA-CNRS-UPSaclay, Orsay, France. gomikir@yahoo.fr.|Centre Muraz, Bobo-Dioulasso, Burkina Faso. gomikir@yahoo.fr.
authors.forename: Michel Kireopori
authors.initials: MK
authors.lastname: Gomgnimbou
authors.affiliation: Institut for Integrative Cell Biology, I2BC, UMR9198 CEA-CNRS-UPSaclay, Orsay, France. guislaine.refregier@i2bc.paris-saclay.fr.
authors.forename: Guislaine
authors.initials: G
authors.lastname: Refregier
authors.affiliation: Institut for Integrative Cell Biology, I2BC, UMR9198 CEA-CNRS-UPSaclay, Orsay, France. christophe.sola@i2bc.paris-saclay.fr.
authors.forename: Christophe
authors.initials: C
authors.lastname: Sola
authors.affiliation: Post-Graduate Program in Infectious Diseases and Tropical Medicine, Department of Internal medicine, Faculty of Medicine, Federal University of Minas Gerais, Belo Horizonte, Brazil. silvanaspindola@gmail.com.
authors.forename: Silvana Spíndola
authors.initials: SS
authors.lastname: de Miranda
mesh_terms: D000483:Alleles; D000995:Antitubercular Agents / Q000627:therapeutic use; D001426:Bacterial Proteins / Q000235:genetics; D001938:Brazil / Q000453:epidemiology; D016000:Cluster Analysis; D004269:DNA, Bacterial / Q000032:analysis; D012321:DNA-Directed RNA Polymerases; D014644:Genetic Variation; D005838:Genotype; D006801:Humans; D007538:Isoniazid / Q000627:therapeutic use; D018598:Minisatellite Repeats / Q000235:genetics; D009169:Mycobacterium tuberculosis / Q000145:classification / Q000235:genetics* / Q000302:isolation & purification; D010802:Phylogeny; D016133:Polymerase Chain Reaction; D011110:Polymorphism, Genetic; D012293:Rifampin / Q000627:therapeutic use; D018088:Tuberculosis, Multidrug-Resistant / Q000188:drug therapy / Q000453:epidemiology* / Q000382:microbiology
chemical_list: D000995:Antitubercular Agents; D001426:Bacterial Proteins; D004269:DNA, Bacterial; C505805:rpoB protein, Mycobacterium tuberculosis; D012321:DNA-Directed RNA Polymerases; D007538:Isoniazid; D012293:Rifampin