title: A single-nucleotide-polymorphism real-time PCR assay for genotyping of Mycobacterium tuberculosis complex in peri-urban Kampala.
abstract: BACKGROUND
  Accurate and high-throughput genotyping of Mycobacterium tuberculosis complex (MTBC) may be important for understanding the epidemiology and pathogenesis of tuberculosis (TB). In this study, we report the development of a LightCycler® real-time PCR single-nucleotide-polymorphism (LRPS) assay for the rapid determination of MTBC lineages/sublineages in minimally processed sputum samples from TB patients.
  
  
  METHODS
  Genotyping analysis of 70 MTBC strains was performed using the Long Sequence Polymorphism-PCR (LSP-PCR) technique and the LRPS assay in parallel. For targeted sequencing, 9 MTBC isolates (three isolates per MTBC lineage) were analyzed for lineage-specific single nucleotide polymorphisms (SNPs) in the following three genes to verify LRPS results: Rv004c for MTB Uganda family, Rv2962 for MTB lineage 4, and Rv0129c for MTB lineage 3. The MTBC lineages present in 300 smear-positive sputum samples were then determined by the validated LRPS method without prior culturing.
  
  
  RESULTS
  The LSP-PCR and LRPS assays produced consistent genotyping data for all 70 MTBC strains; however, the LSP-PCR assay was 10-fold less sensitive than the LRPS method and required higher DNA concentrations to successfully characterize the MTBC lineage of certain samples. Targeted sequencing of genes containing lineage-specific SNPs was 100 % concordant with the genotyping results and provided further validation of the LRPS assay. Of the 300 sputum samples analyzed, 58 % contained MTBC from the MTBC-Uganda family, 27 % from the MTBC lineage 4 (excluding MTBC Uganda family), 13 % from the MTBC lineage 3, and the remaining 2 % were of indeterminate lineage.
  
  
  CONCLUSIONS
  The LRPS assay is a sensitive, high-throughput technique with potential application to routine genotyping of MTBC in sputum samples from TB patients.
authors.affiliation: Department of Medical Microbiology, College of Health Sciences, School of Biomedical Sciences, Makerere University, P.O BOX 7072, Kampala, Uganda. wamps@covab.mak.ac.ug.|Department of Bio-molecular Resources and Biolab Sciences, College of Veterinary Medicine, Animal Resources and Bio Security, Makerere University, Kampala, Uganda. wamps@covab.mak.ac.ug.
authors.forename: Eddie M
authors.initials: EM
authors.lastname: Wampande
authors.affiliation: Department of Medical Microbiology, College of Health Sciences, School of Biomedical Sciences, Makerere University, P.O BOX 7072, Kampala, Uganda. shatzios@rics.bwh.harvard.edu.|Division of Infectious Diseases, Brigham and Women's Hospital, Boston, Massachusetts, USA Department of Microbiology and Immunobiology, Harvard Medical School, Boston, MA, USA. shatzios@rics.bwh.harvard.edu.
authors.forename: Stavroula K
authors.initials: SK
authors.lastname: Hatzios
authors.affiliation: Department of Medical Microbiology, College of Health Sciences, School of Biomedical Sciences, Makerere University, P.O BOX 7072, Kampala, Uganda. baetriceachan@yahoo.com.
authors.forename: Beatrice
authors.initials: B
authors.lastname: Achan
authors.affiliation: Department of Pediatrics and Child Health College of Health Sciences, Makerere University, Kampala, Uganda. mupez@yahoo.com.
authors.forename: Ezekiel
authors.initials: E
authors.lastname: Mupere
authors.affiliation: Uganda-Case Western Reserve University Research Collaboration, Kampala, Uganda. mnsereko@mucwru.or.ug.
authors.forename: Mary
authors.initials: M
authors.lastname: Nsereko
authors.affiliation: Uganda-Case Western Reserve University Research Collaboration, Kampala, Uganda. hmk@chs.mak.ac.ug.
authors.forename: Harriet K
authors.initials: HK
authors.lastname: Mayanja
authors.affiliation: Department of Pathology, University of Arkansas for Medical Sciences, Little Rock, AR, USA. EisenachKathleenD@uams.edu.
authors.forename: Kathleen
authors.initials: K
authors.lastname: Eisenach
authors.affiliation: Tuberculosis Research Unit, School of Medicine, Case Western Reserve University and University Hospitals of Cleveland, Cleveland, Ohio, USA. whb@case.edu.
authors.forename: W Henry
authors.initials: WH
authors.lastname: Boom
authors.affiliation: Department of Medical Parasitology and Infection Biology, Swiss Tropical and Public Health Institute, Basel, Switzerland. Sebastien.gagneux@unibas.ch.|University of Basel, Basel, Switzerland. Sebastien.gagneux@unibas.ch.
authors.forename: Sebastien
authors.initials: S
authors.lastname: Gagneux
authors.affiliation: Department of Medical Microbiology, College of Health Sciences, School of Biomedical Sciences, Makerere University, P.O BOX 7072, Kampala, Uganda. mlj10@case.edu.|Uganda-Case Western Reserve University Research Collaboration, Kampala, Uganda. mlj10@case.edu.
authors.forename: Moses L
authors.initials: ML
authors.lastname: Joloba
mesh_terms: D001483:Base Sequence; D004269:DNA, Bacterial / Q000032:analysis; D005838:Genotype; D006801:Humans; D008969:Molecular Sequence Data; D009169:Mycobacterium tuberculosis / Q000235:genetics* / Q000302:isolation & purification; D020641:Polymorphism, Single Nucleotide; D060888:Real-Time Polymerase Chain Reaction; D016415:Sequence Alignment; D017422:Sequence Analysis, DNA; D013183:Sputum / Q000382:microbiology; D014376:Tuberculosis / Q000382:microbiology / Q000473:pathology; D014454:Uganda
chemical_list: D004269:DNA, Bacterial