title: Detection and Quantification of Mycobacterium tuberculosis in the Sputum of Culture-Negative HIV-infected Pulmonary Tuberculosis Suspects: A Proof-of-Concept Study.
abstract: BACKGROUND
  Rapid diagnosis of pulmonary tuberculosis (TB) is critical for timely initiation of treatment and interruption of transmission. Yet, despite recent advances, many patients remain undiagnosed. Culture, usually considered the most sensitive diagnostic method, is sub-optimal for paucibacillary disease.
  
  
  METHODS
  We evaluated the Totally Optimized PCR (TOP) TB assay, a new molecular test that we hypothesize is more sensitive than culture. After pre-clinical studies, we estimated TOP's per-patient sensitivity and specificity in a convenience sample of 261 HIV-infected pulmonary TB suspects enrolled into a TB diagnostic study in Mbarara, Uganda against MGIT culture, Xpert MTB/RIF and a composite reference standard. We validated results with a confirmatory PCR used for sequencing M. tuberculosis.
  
  
  RESULTS
  Using culture as reference, TOP had 100% sensitivity but 35% specificity. Against a composite reference standard, the sensitivity of culture (27%) and Xpert MTB/RIF (27%) was lower than TOP (99%), with similar specificity (100%, 98% and 87%, respectively). In unadjusted analyses, culture-negative/TOP-positive patients were more likely to be older (P<0·001), female (P<0·001), have salivary sputum (P = 0·05), sputum smear-negative (P<0.001) and less advanced disease on chest radiograph (P = 0.05). M. tuberculosis genotypes identified in sputum by DNA sequencing exhibit differential growth in culture.
  
  
  CONCLUSIONS
  These findings suggest that the TOP TB assay is accurately detecting M. tuberculosis DNA in the sputum of culture-negative tuberculosis suspects. Our results require prospective validation with clinical outcomes. If the operating characteristics of the TOP assay are confirmed in future studies, it will be justified as a "TB rule out" test.
authors.affiliation: Section of Infectious Diseases, Department of Medicine, Boston Medical Center and Boston University School of Medicine, Boston, Massachusetts, United States of America.|Thisis Diagnostics Inc., Boston, Massachusetts, United States of America.
authors.forename: Guillermo
authors.initials: G
authors.lastname: Madico
authors.affiliation: Epicentre, Médecins sans Frontières, Mbarara, Uganda.|Mbarara University of Science and Technology, Mbarara, Uganda.
authors.forename: Moses
authors.initials: M
authors.lastname: Mpeirwe
authors.affiliation: Department of Biostatistics, Boston University School of Public Health, Boston, Massachusetts, United States of America.
authors.forename: Laura
authors.initials: L
authors.lastname: White
authors.affiliation: Núcleo de Doenças Infecciosas, Universidade Federal do Espírito Santo, Vitória, Brazil.
authors.forename: Solange
authors.initials: S
authors.lastname: Vinhas
authors.affiliation: Clinical Microbiology Laboratory, Boston Medical Center, Boston, Massachusetts, United States of America.
authors.forename: Beverley
authors.initials: B
authors.lastname: Orr
authors.affiliation: Epicentre, Médecins sans Frontières, Mbarara, Uganda.
authors.forename: Patrick
authors.initials: P
authors.lastname: Orikiriza
authors.affiliation: Clinical Microbiology Laboratory, Boston Medical Center, Boston, Massachusetts, United States of America.|Department of Pathology and Laboratory Medicine, Boston University School of Medicine, Boston, Massachusetts, United States of America.
authors.forename: Nancy S
authors.initials: NS
authors.lastname: Miller
authors.affiliation: Section of Infectious Diseases, Department of Medicine, Boston Medical Center and Boston University School of Medicine, Boston, Massachusetts, United States of America.
authors.forename: Mary
authors.initials: M
authors.lastname: Gaeddert
authors.affiliation: Epicentre, Médecins sans Frontières, Mbarara, Uganda.
authors.forename: Juliet
authors.initials: J
authors.lastname: Mwanga-Amumpaire
authors.affiliation: Núcleo de Doenças Infecciosas, Universidade Federal do Espírito Santo, Vitória, Brazil.
authors.forename: Moises
authors.initials: M
authors.lastname: Palaci
authors.affiliation: Public Health Research Institute (PHRI) - Rutgers University, Newark, New Jersey, United States of America.
authors.forename: Barry
authors.initials: B
authors.lastname: Kreiswirth
authors.affiliation: Thisis Diagnostics Inc., Boston, Massachusetts, United States of America.
authors.forename: Joe
authors.initials: J
authors.lastname: Straight
authors.affiliation: Núcleo de Doenças Infecciosas, Universidade Federal do Espírito Santo, Vitória, Brazil.
authors.forename: Reynaldo
authors.initials: R
authors.lastname: Dietze
authors.affiliation: Epicentre, Médecins sans Frontières, Mbarara, Uganda.
authors.forename: Yap
authors.initials: Y
authors.lastname: Boum
authors.affiliation: Section of Infectious Diseases, Department of Medicine, Boston Medical Center and Boston University School of Medicine, Boston, Massachusetts, United States of America.|Thisis Diagnostics Inc., Boston, Massachusetts, United States of America.
authors.forename: Edward C
authors.initials: EC
authors.lastname: Jones-López
mesh_terms: D000328:Adult; D005260:Female; D015658:HIV Infections / Q000453:epidemiology / Q000382:microbiology*; D059014:High-Throughput Nucleotide Sequencing; D006801:Humans; D008297:Male; D008875:Middle Aged; D009169:Mycobacterium tuberculosis / Q000235:genetics*; D016133:Polymerase Chain Reaction; D013183:Sputum; D014397:Tuberculosis, Pulmonary / Q000175:diagnosis* / Q000235:genetics / Q000382:microbiology; D014454:Uganda