title: Acanthamoeba DNA can be directly amplified from corneal scrapings.
abstract: This study evaluated the performance of direct amplification of Acanthamoeba-DNA bypassing DNA extraction in the diagnosis of Acanthamoeba keratitis in clinically suspected cases in comparison to direct microscopic examination and in vitro culture. Corneal scrapings were collected from 110 patients who were clinically suspected to have Acanthamoeba keratitis, 63 contact lens wearers (CLW), and 47 non-contact lens wearers (NCLW). Taken samples were subjected to direct microscopic examination, cultivation onto the non-nutrient agar plate surface seeded with Escherichia coli, and PCR amplification. The diagnostic performance of these methods was statistically compared. The results showed that Acanthamoeba infection was detected in 21 (19.1%) of clinically suspected cases (110); 17 (81%) of them were CLW and the remaining 4 (19%) positive cases were NCLW. Regarding the used diagnostic methods, it was found that direct amplification of Acanthamoeba DNA bypassing nucleic acid extraction was superior to microscopy and culture in which 21 cases (19.1%) were positive for Acanthamoeba by PCR compared to 19 positive cases by culture (17.3%) and one case (0.9%) by direct smear. The difference in detection rates between culture and direct smear was highly statistically significant (P = 0.001). On the other hand, there was no significant difference in detection rates between culture and PCR (P = 0.86). On using culture as the gold standard, PCR showed three false-positive samples that were negative by culture and one false-negative sample that was positive by culture. At the same time, direct smear showed 18 false-negative samples. The sensitivity, specificity, positive predictive value, negative predictive value, and diagnostic accuracy of PCR were 94.7, 96.7, 85.7, 98.9, and 96.4, respectively, while those of direct smear were 5.3, 100, 100, 83.5, and 83.6, respectively. In conclusion, direct amplification of Acanthamoeba-DNA bypassing DNA extraction is a reliable, specific, sensitive method in the diagnosis of Acanthamoeba keratitis in clinically suspected cases. It should set up in ophthalmological centers as an easy diagnostic tool.
authors.affiliation: Parasitology Department, Research Institute of Ophthalmology, Giza, Egypt, nag.elsaka@yahoo.com.
authors.forename: Nagwa Mostafa
authors.initials: NM
authors.lastname: El-Sayed
authors.forename: Mohamed Saad
authors.initials: MS
authors.lastname: Younis
authors.forename: Azza Mohamed
authors.initials: AM
authors.lastname: Elhamshary
authors.forename: Amina Ibrahim
authors.initials: AI
authors.lastname: Abd-Elmaboud
authors.forename: Shereen Magdy
authors.initials: SM
authors.lastname: Kishik
mesh_terms: D000048:Acanthamoeba / Q000235:genetics* / Q000302:isolation & purification*; D015823:Acanthamoeba Keratitis / Q000175:diagnosis / Q000469:parasitology; D000328:Adult; D000818:Animals; D016054:DNA, Protozoan / Q000032:analysis* / Q000235:genetics; D005260:Female; D006801:Humans; D008297:Male; D008875:Middle Aged; D021141:Nucleic Acid Amplification Techniques / Q000379:methods*; D012680:Sensitivity and Specificity
chemical_list: D016054:DNA, Protozoan