Use of subgenic 18S ribosomal DNA PCR and sequencing for genus and genotype identification of acanthamoebae from humans with keratitis and from sewage sludge

This study identified subgenic PCR amplimers from 18S rDNA that were (i) hi ghly specific for the genus Acanthamoeba, (ii) obtainable from all known ge notypes, and (iii) useful for identification of individual genotypes. A 423 - to 551-bp Acanthamoeba-specific amplimer ASA.S1 obtained with primers JDP 1 and JDP2 was the most reliable for purposes i and ii. A variable region w ithin this amplimer also identified genotype clusters, but purpose iii was best achieved with sequencing of the genotype-specific amplimer GTSA.B1. Be cause this amplimer could be obtained from any eukaryote, axenic Acanthamoe ba cultures were required for its study. GTSA.B1, produced with primers CRN 5 and 1137, extended between reference bp 1 and 1475. Genotypic identificat ion relied on three segments: bp 178 to 355, 705 to 926, and 1175 to 1379. ASA.S1 was obtained from single amoeba, from cultures of all known 18S rDNA genotypes, and from corneal scrapings of Scottish patients with suspected Acanthamoeba keratitis (AK). The AK PCR findings were consistent,vith cultu re results for 11 of 15 culture-positive specimens and detected Acanthamoeb a in one of nine culture-negative specimens. ASA.S1 sequences were examined for 6 of the 11 culture-positive isolates and were most closely associated with genotypic cluster T3-T4-T11, A similar distance analysis using GTSA.B 1 sequences identified nine South African AK-associated isolates as genotyp e T4 and three isolates from sewage sludge as genotype T5. Our results demo nstrate the usefulness of 18S ribosomal DNA PCR amplimers ASA.S1 and GTSA.B 1 for Acanthamoeba-specific detection and reliable genotyping, respectively , and provide further evidence that T4 is the predominant genotype in AK.