title: Comparative Study of a Real-Time PCR Assay Targeting senX3-regX3 versus Other Molecular Strategies Commonly Used in the Diagnosis of Tuberculosis.
abstract: BACKGROUND
  Nucleic acid amplification tests are increasingly used for the rapid diagnosis of tuberculosis. We undertook a comparative study of the efficiency and diagnostic yield of a real-time PCR senX3-regX3 based assay versus the classical IS6110 target and the new commercial methods.
  
  
  METHODS
  This single-blind prospective comparative study included 145 consecutive samples: 76 from patients with culture-confirmed tuberculosis (86.8% pulmonary and 13.2% extrapulmonary tuberculosis: 48.7% smear-positive and 51.3% smear-negative) and 69 control samples (24 from patients diagnosed with non-tuberculous mycobacteria infections and 45 from patients with suspected tuberculosis which was eventually ruled out). All samples were tested by two CE-marked assays (Xpert®MTB/RIF and AnyplexTM plus MTB/NTM) and two in-house assays targeting senX3-regX3 and the IS6110 gene.
  
  
  RESULTS
  The detection limit ranged from 1.00E+01 fg for Anyplex, senX3-regX3 and IS6110 to 1.00E+04 fg for Xpert. All three Xpert, senX3-regX3 and IS6110 assays detected all 37 smear-positive cases. Conversely, Anyplex was positive in 34 (91.9%) smear-positive cases. In patients with smear-negative tuberculosis, differences were observed between the assays; Xpert detected 22 (56.41%) of the 39 smear-negative samples, Anyplex 24 (61.53%), senX3-regX3 28 (71.79%) and IS6110 35 (89.74%). Xpert and senX3-regX3 were negative in all control samples; however, the false positive rate was 8.7% and 13% for Anyplex and IS6110, respectively. The overall sensitivity was 77.6%, 85.7%, 77.3% and 94.7% and the specificity was 100%, 100%, 90.8% and 87.0% for the Xpert, senX3-regX3, Anyplex and IS6110 assays, respectively.
  
  
  CONCLUSIONS
  Real-time PCR assays targeting IS6110 lack the desired specificity. The Xpert MTB/RIF and in-house senX3-regX3 assays are both sensitive and specific for the detection of MTBC in both pulmonary and extrapulmonary samples. Therefore, the real time PCR senX3-regX3 based assay could be a useful and complementary tool in the diagnosis of tuberculosis.
authors.affiliation: Biochemistry, Molecular Biology and Immunology Department, Faculty of Medicine, University of Malaga, Malaga, Spain.
authors.forename: Rocio
authors.initials: R
authors.lastname: Sanjuan-Jimenez
authors.affiliation: Microbiology Service, Regional University Hospital, Malaga, Spain.
authors.forename: Inmaculada
authors.initials: I
authors.lastname: Toro-Peinado
authors.affiliation: Microbiology Service, Regional University Hospital, Malaga, Spain.
authors.forename: Pilar
authors.initials: P
authors.lastname: Bermudez
authors.affiliation: Infectious Diseases Service, Regional University Hospital, Malaga, Spain.
authors.forename: Juan D
authors.initials: JD
authors.lastname: Colmenero
authors.affiliation: Biochemistry, Molecular Biology and Immunology Department, Faculty of Medicine, University of Malaga, Malaga, Spain.
authors.forename: Pilar
authors.initials: P
authors.lastname: Morata
mesh_terms: D000293:Adolescent; D000328:Adult; D000368:Aged; D000369:Aged, 80 and over; D001426:Bacterial Proteins / Q000235:genetics*; D016022:Case-Control Studies; D002648:Child; D002675:Child, Preschool; D005260:Female; D006801:Humans; D008297:Male; D008875:Middle Aged; D025202:Molecular Diagnostic Techniques; D060885:Multiplex Polymerase Chain Reaction; D009169:Mycobacterium tuberculosis / Q000235:genetics*; D010770:Phosphotransferases / Q000235:genetics*; D011446:Prospective Studies; D011933:Reagent Kits, Diagnostic; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods / Q000592:standards; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D016037:Single-Blind Method; D014376:Tuberculosis / Q000175:diagnosis* / Q000382:microbiology*; D055815:Young Adult
chemical_list: D001426:Bacterial Proteins; D011933:Reagent Kits, Diagnostic; C110608:RegX3 protein, Mycobacterium; D010770:Phosphotransferases; C110607:SenX3 protein, Mycobacterium