title: A Novel Molecular Strategy for Surveillance of Multidrug Resistant Tuberculosis in High Burden Settings.
abstract: BACKGROUND
  In South Africa and other high prevalence countries, transmission is a significant contributor to rising rates of multidrug resistant tuberculosis (MDR-TB). Thus, there is a need to develop an early detection system for transmission clusters suitable for high burden settings. We have evaluated the discriminatory power and clustering concordance of a novel and simple genotyping approach, combining spoligotyping with pncA sequencing (SpoNC), against two well-established methods: IS6110-RFLP and 24-loci MIRU-VNTR.
  
  
  METHODS
  A total of 216 MDR-TB isolates collected from January to June 2010 from the NHLS Central TB referral laboratory in Braamfontein, Johannesburg, representing a diversity of strains from South Africa, were included. The isolates were submitted for genotyping, pncA sequencing and analysis to the Centre for Tuberculosis in South Africa and the Public Health Research Institute Tuberculosis Center at Rutgers University in the United States. Clustering rates, Hunter-Gaston Discriminatory Indexes (HGI) and Wallace coefficients were compared between the methods.
  
  
  RESULTS
  Overall clustering rates were high by both IS6110-RFLP (52.8%) and MIRU-VNTR (45.8%), indicative of on-going transmission. Both 24-loci MIRU-VNTR and IS6110-RFLP had similar HGI (0.972 and 0.973, respectively), with close numbers of unique profiles (87 vs. 70), clustered isolates (129 vs. 146), and cluster sizes (2 to 26 vs. 2 to 25 isolates). Spoligotyping alone was the least discriminatory (80.1% clustering, HGI 0.903), with 28 unique types. However, the discriminatory power of spoligotyping was improved when combined with pncA sequencing using the SpoNC approach (61.8% clustering, HGI 0.958). A high proportion of MDR-TB isolates had mutations in pncA (68%, n = 145), and pncA mutations were significantly associated with clustering (p = 0.007 and p = 0.0013 by 24-loci MIRU-VNTR and IS6110-RFLP, respectively), suggesting high rates of resistance to pyrazinamide among all MDR-TB cases and particularly among clustered cases.
  
  
  CONCLUSIONS
  We conclude that SpoNC provides good discrimination for MDR-TB surveillance and early identification of outbreaks in South Africa, with 24-loci MIRU-VNTR applied for pncA wild-type strains as needed.
authors.affiliation: Centre for Tuberculosis, National Institute of Communicable Diseases, Sandringham, South Africa.
authors.forename: Halima M
authors.initials: HM
authors.lastname: Said
authors.affiliation: Public Health Research Institute, Rutgers University, Newark, New Jersey, United States of America.
authors.forename: Nicole
authors.initials: N
authors.lastname: Kushner
authors.affiliation: Centre for Tuberculosis, National Institute of Communicable Diseases, Sandringham, South Africa.
authors.forename: Shaheed V
authors.initials: SV
authors.lastname: Omar
authors.affiliation: Centre for Tuberculosis, National Institute of Communicable Diseases, Sandringham, South Africa.
authors.forename: Andries W
authors.initials: AW
authors.lastname: Dreyer
authors.affiliation: Centre for Tuberculosis, National Institute of Communicable Diseases, Sandringham, South Africa.
authors.forename: Hendrik
authors.initials: H
authors.lastname: Koornhof
authors.affiliation: Centre for Tuberculosis, National Institute of Communicable Diseases, Sandringham, South Africa.
authors.forename: Linda
authors.initials: L
authors.lastname: Erasmus
authors.affiliation: Centre for Tuberculosis, National Institute of Communicable Diseases, Sandringham, South Africa.
authors.forename: Yasmin
authors.initials: Y
authors.lastname: Gardee
authors.affiliation: Centre for Tuberculosis, National Institute of Communicable Diseases, Sandringham, South Africa.
authors.forename: Ivy
authors.initials: I
authors.lastname: Rukasha
authors.affiliation: Public Health Research Institute, Rutgers University, Newark, New Jersey, United States of America.
authors.forename: Elena
authors.initials: E
authors.lastname: Shashkina
authors.affiliation: National Health Laboratory Service, Groote Schuur Hospital, Cape Town, South Africa.
authors.forename: Natalie
authors.initials: N
authors.lastname: Beylis
authors.affiliation: The Bill & Melinda Gates Foundation, Seattle, Washington, United States of America.
authors.forename: Gilla
authors.initials: G
authors.lastname: Kaplan
authors.affiliation: Public Health Research Institute, Rutgers University, Newark, New Jersey, United States of America.
authors.forename: Dorothy
authors.initials: D
authors.lastname: Fallows
authors.affiliation: Centre for Tuberculosis, National Institute of Communicable Diseases, Sandringham, South Africa.|Department of Medical Microbiology, Faculty of Health Science, University of Pretoria, Pretoria, South Africa.
authors.forename: Nazir A
authors.initials: NA
authors.lastname: Ismail
mesh_terms: D016000:Cluster Analysis; D017281:Cost of Illness; D060005:Genotyping Techniques; D006801:Humans; D009154:Mutation / Q000235:genetics; D009169:Mycobacterium tuberculosis / Q000145:classification / Q000235:genetics / Q000302:isolation & purification / Q000502:physiology*; D011159:Population Surveillance; D018088:Tuberculosis, Multidrug-Resistant / Q000453:epidemiology* / Q000235:genetics*