title: Evaluation of immunoperoxidase staining technique in the diagnosis of Acanthamoeba keratitis.
abstract: OBJECTIVE
  We describe a simple procedure of Immunoperoxidase (IP) technique, using indigenously raised antibody, to screen corneal scrapings for Acanthamoeba cysts and trophozoites. This study sought to determine the utility of this test in the diagnosis of Acanthamoeba keratitis.
  
  
  METHODS
  A high titre polyclonal antibody against a local clinical isolate (axenic) of Acanthamoeba species (trophozoite lysate antigen) was raised in rabbits and used for standardization of IP technique for corneal scrapings. Twenty two smears of corneal scrapings, collected from patients showing Acanthamoeba cysts in corneal scrapings stained with calcofluorwhite (pool-1) and patients showing no cysts in similar scrapings (pool-2), were coded and stained by IP technique by a masked technician. All 22 patients had also been tested for bacteria, fungus, and Acanthamoeba in their corneal scrapings by smears and cultures. IP stained smears were examined for organisms including cysts and trophozoites of Acanthamoeba and background staining by two observers masked to the results of other smears and cultures. The validity of the IP test in detection of Acanthamoeba cysts and trophozoites was measured by sensitivity, specificity, positive predictive value and negative predictive value in comparison (McNemar test for paired comparison) with calcofluor white staining and culture.
  
  
  RESULTS
  Based on the readings of observer 1 and compared to calcofluor white staining, the IP test had a sensitivity of 100%, a specificity of 94%, positive predictive value of 80% and negative predictive value of 100%. When compared to culture, the values were 83%, 100%, 100% and 94% respectively. Trophozoites missed in calcofluor white stained smears, were detected in 2 out of 6 cases of culture-positive Acanthamoeba keratitis. The Kappa coefficient of interobserver agreement was determined as fair (30.4%).
  
  
  CONCLUSIONS
  The immunoperoxidase technique is a simple and useful test in the diagnosis of Acanthamoeba keratitis. This can supplement the culture results.
authors.affiliation: Jhaveri Microbiology Centre, L.V. Prasad Eye Institute, Hyderabad, India. savitri@lvpeye.stph.net
authors.forename: S
authors.initials: S
authors.lastname: Sharma
authors.forename: S
authors.initials: S
authors.lastname: Athmanathan
authors.forename: M
authors.initials: M
authors.lastname: Ata-Ur-Rasheed
authors.forename: P
authors.initials: P
authors.lastname: Garg
authors.forename: G N
authors.initials: GN
authors.lastname: Rao
mesh_terms: D000048:Acanthamoeba / Q000276:immunology / Q000302:isolation & purification*; D015823:Acanthamoeba Keratitis / Q000175:diagnosis* / Q000469:parasitology; D000818:Animals; D000913:Antibodies, Protozoan; D003315:Cornea / Q000469:parasitology / Q000473:pathology*; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods / Q000706:statistics & numerical data; D006801:Humans; D015588:Observer Variation; D011237:Predictive Value of Tests; D011817:Rabbits; D015203:Reproducibility of Results
chemical_list: D000913:Antibodies, Protozoan