Normalised quantitative polymerase chain reaction for diagnosis of tuberculosis-associated uveitis

Tuberculosis (Edinb). 2018 May;110:30-35. doi: 10.1016/j.tube.2018.03.005. Epub 2018 Mar 23.

Abstract

Polymerase chain reaction (PCR)-based diagnosis of tuberculosis-associated uveitis (TBU) in TB-endemic countries is challenging due to likelihood of latent mycobacterial infection in both immune and non-immune cells. In this study, we investigated normalised quantitative PCR (nqPCR) in ocular fluids (aqueous/vitreous) for diagnosis of TBU in a TB-endemic population. Mycobacterial copy numbers (mpb64 gene) were normalised to host genome copy numbers (RNAse P RNA component H1 [RPPH1] gene) in TBU (n = 16) and control (n = 13) samples (discovery cohort). The mpb64:RPPH1 ratios (normalised value) from each TBU and control sample were tested against the current reference standard i.e. clinically-diagnosed TBU, to generate Receiver Operating Characteristic (ROC) curves. The optimum cut-off value of mpb64:RPPH1 ratio (0.011) for diagnosing TBU was identified from the highest Youden index. This cut-off value was then tested in a different cohort of TBU and controls (validation cohort, 20 cases and 18 controls), where it yielded specificity, sensitivity and diagnostic accuracy of 94.4%, 85.0%, and 89.4% respectively. The above values for conventional quantitative PCR (≥1 copy of mpb64 per reaction) were 61.1%, 90.0%, and 74.3% respectively. Normalisation markedly improved the specificity and diagnostic accuracy of quantitative PCR for diagnosis of TBU.

Keywords: Normalised; PCR; Quantitative; Tuberculosis; Uveitis; Youden index.

Publication types

  • Research Support, Non-U.S. Gov't
  • Validation Study

MeSH terms

  • Adolescent
  • Adult
  • Antigens, Bacterial / genetics
  • Aqueous Humor / microbiology
  • Bacterial Proteins / genetics
  • Case-Control Studies
  • Child
  • Child, Preschool
  • Female
  • Humans
  • Male
  • Middle Aged
  • Mycobacterium tuberculosis / genetics
  • Mycobacterium tuberculosis / isolation & purification
  • Polymerase Chain Reaction / methods
  • Prospective Studies
  • ROC Curve
  • Reproducibility of Results
  • Sensitivity and Specificity
  • Tuberculosis, Ocular / diagnosis*
  • Uveitis / diagnosis*
  • Uveitis / microbiology
  • Vitreous Body / microbiology
  • Young Adult

Substances

  • Antigens, Bacterial
  • Bacterial Proteins
  • MPB64 protein, Mycobacterium