Isolation and genotyping of Acanthamoeba strains from corneal infections in Italy.
Acanthamoeba keratitis (AK) is a corneal disease caused by members of a genus of free-living amoebae and is associated predominantly with contact lens (CL) use. This study reports 16 cases of culture-proven AK diagnosed in northern Italy. Genotype identification was carried out with a PCR assay based on sequence analysis of the 18S rRNA gene, and sensitivity and specificity were evaluated in comparison with traditional parasitological techniques. A 405&emsp14;bp region of the 18S rRNA gene (ASA.S1) including diagnostic fragment 3 (DF3) was amplified using the genus-specific primers JDP1 and JDP2. Genotype assignment was based on phenetic analysis of the ASA.S1 subset of the nuclear small-subunit rRNA gene sequence excluding the highly variable DF3 region. Phylogenetic analysis was also performed on the sequences obtained. All patients complained of monolateral infection; 11 (68.75%) admitted improper CL disinfection. In 14/16 (87.5 %) subjects, corneal scrapings were stained with calcofluor white and haematoxylin and eosin and, in ten cases (62.5 %), microscopy was positive for Acanthamoeba cysts. In vitro culture on 3 % non-nutrient agar plates was obtained in all cases (100 %), whereas cloning and axenic growth were positive for 14 amoebic stocks (87.5 %). PCR analysis had 100 % sensitivity and specificity compared with in vitro axenic culture, showing positive amplification from 15 isolates. All Acanthamoeba strains belonged to the T4 genotype, the main AK-related genotype worldwide. These results confirmed the importance of a complete diagnostic protocol, including a PCR assay, for the clinical diagnosis of AK on biological samples. Genotyping allowed inclusion of all isolates in the T4 group, thus demonstrating the prevalence of this genotype in northern Italy.
Gatti
Simonetta
S
Parasitology Laboratory, Virology and Microbiology Service, Foundation IRCCS Policlinico San Matteo, 27100 Pavia, Italy.
Rama
Paolo
P
Ophthalmology Unit, San Raffaele Scientific Institute, 20132 Milano, Italy.
Matuska
Stanislav
S
Ophthalmology Unit, San Raffaele Scientific Institute, 20132 Milano, Italy.
Berrilli
Federica
F
Department of Public Health and Cell Biology, University of Rome 'Tor Vergata', 00133 Roma, Italy.
Cavallero
Annalisa
A
Laboratory Medicine Unit, IRCCS Centro Cardiologico Monzino, 20162 Milano, Italy.
Carletti
Silvia
S
Microbiology and Virology Laboratory, San Raffaele Scientific Institute, 20132 Milano, Italy.
Bruno
Antonella
A
Infectious Diseases Research Laboratories, Foundation IRCCS Policlinico San Matteo, University of Pavia, 27100 Pavia, Italy.
Maserati
Roberta
R
Infectious Diseases Research Laboratories, Foundation IRCCS Policlinico San Matteo, University of Pavia, 27100 Pavia, Italy.
Di Cave
David
D
Department of Public Health and Cell Biology, University of Rome 'Tor Vergata', 00133 Roma, Italy.
D000048:Acanthamoeba / Q000145:classification* / Q000235:genetics / Q000254:growth & development / Q000302:isolation & purification*; D015823:Acanthamoeba Keratitis / Q000453:epidemiology* / Q000469:parasitology*; D000328:Adult; D016054:DNA, Protozoan / Q000235:genetics*; D004275:DNA, Ribosomal / Q000235:genetics; D005260:Female; D005838:Genotype; D006801:Humans; D007558:Italy / Q000453:epidemiology; D008297:Male; D008853:Microscopy; D008875:Middle Aged; D010274:Parasitology / Q000379:methods*; D016133:Polymerase Chain Reaction / Q000379:methods*; D016053:RNA, Protozoan / Q000235:genetics; D012337:RNA, Ribosomal, 18S / Q000235:genetics; D012680:Sensitivity and Specificity; D013194:Staining and Labeling / Q000379:methods
D016054:DNA, Protozoan; D004275:DNA, Ribosomal; D016053:RNA, Protozoan; D012337:RNA, Ribosomal, 18S