title: Real-time PCR using the 529 bp repeat element for the diagnosis of atypical ocular toxoplasmosis.
abstract: BACKGROUND
  Ocular toxoplasmosis may present in atypical fashion, particularly in immunosuppressed patients, and PCR is an important diagnostic tool especially when differentiating from other infectious causes.
  
  
  METHODS
  A descriptive case-series demonstrating the use of a novel real-time PCR protocol targeting 529 bp repeat element, a multicopy and highly conserved fragment, in Toxoplasma gondii genome. This was designed and established by our microbiology service following independent, external validation.
  
  
  RESULTS
  Three immunosuppressed patients presenting to a tertiary uveitis referral centre with unilateral, severe, sight-threatening uveitis are described. One patient presented with a large focus of sight-threatening retinitis and occlusive vasculitis while on systemic immunosuppression with azathioprine and adalimumab for Crohn's disease. One patient with chronic lymphocytic leukaemia presented with severe posterior uveitis and total retinal detachment. Finally, the third patient presented with severe retinitis adjacent to the optic nerve and vitritis causing acute vision loss. HIV infection was subsequently identified. In all three cases, the cause of inflammation was not clear from clinical examination alone and prompt treatment was required to prevent permanent vision loss. Intraocular sampling and PCR testing was performed including testing for toxoplasmosis, herpesviruses and syphilis.
  
  
  CONCLUSIONS
  The novel real-time PCR assay described is more sensitive than those targeting the Toxoplasma B1 gene owing to the higher number of repeats and highly conserved sequence level. This technique can be applied in clinical practice and provides a valuable tool for the rapid diagnosis of ocular toxoplasmosis.
authors.affiliation: Manchester Royal Eye Hospital, Manchester, UK.
authors.forename: Laura R
authors.initials: LR
authors.lastname: Steeples
authors.affiliation: Public Health England, Public Health Laboratory, Manchester Medical Microbiology Partnership, Manchester Royal Infirmary, Manchester, UK.
authors.forename: Malcolm
authors.initials: M
authors.lastname: Guiver
authors.affiliation: Manchester Royal Eye Hospital, Manchester, UK.
authors.forename: Nicholas P
authors.initials: NP
authors.lastname: Jones
mesh_terms: D000368:Aged; D017963:Azithromycin / Q000627:therapeutic use; D020029:Base Pairing; D017931:DNA Primers / Q000737:chemistry; D015342:DNA Probes / Q000737:chemistry; D016054:DNA, Protozoan / Q000235:genetics*; D004338:Drug Combinations; D015822:Eye Infections, Parasitic / Q000175:diagnosis* / Q000188:drug therapy / Q000469:parasitology; D005260:Female; D006801:Humans; D007166:Immunosuppressive Agents / Q000627:therapeutic use; D008297:Male; D008875:Middle Aged; D011239:Prednisolone / Q000627:therapeutic use; D060888:Real-Time Polymerase Chain Reaction; D012091:Repetitive Sequences, Nucleic Acid / Q000235:genetics*; D014122:Toxoplasma / Q000235:genetics / Q000302:isolation & purification*; D014126:Toxoplasmosis, Ocular / Q000175:diagnosis* / Q000188:drug therapy / Q000469:parasitology; D015662:Trimethoprim, Sulfamethoxazole Drug Combination / Q000627:therapeutic use; D014605:Uveitis / Q000175:diagnosis* / Q000188:drug therapy / Q000469:parasitology; D014822:Vitreous Body / Q000469:parasitology
chemical_list: D017931:DNA Primers; D015342:DNA Probes; D016054:DNA, Protozoan; D004338:Drug Combinations; D007166:Immunosuppressive Agents; D015662:Trimethoprim, Sulfamethoxazole Drug Combination; D017963:Azithromycin; D011239:Prednisolone
keywords: Diagnostic tests/Investigation; Infection; Inflammation; Microbiology