Use of Subgenic 18S Ribosomal DNA PCR and Sequencing for Genus and Genotype Identification of Acanthamoebae from Humans with Keratitis and from Sewage Sludge

Abstract

This study identified subgenic PCR amplimers from 18S rDNA that were (i) highly specific for the genus Acanthamoeba, (ii) obtainable from all known genotypes, and (iii) useful for identification of individual genotypes. A 423-to 551-bp Acanthamoeba-specific amplimer ASA.S1 obtained with primers JDP1 and JDP2 was the most reliable for purposes i and ii. A variable region within this amplimer also identified genotype clusters, but purpose iii was best achieved with sequencing of the genotype-specific amplimer GTSA.B1. Because this amplimer could be obtained from any eukaryote, axenic Acanthamoeba cultures were required for its study. GTSA.B1, produced with primers CRN5 and 1137, extended between reference bp 1 and 1475. Genotypic identification 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 with culture results for 11 of 15 culture-positive specimens and detected Acanthamoeba in one of nine culturenegative 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.B1 sequences identified nine South African AK-associated isolates as genotype T4 and three isolates from sewage sludge as genotype T5. Our results demonstrate the usefulness of 18S ribosomal DNA PCR amplimers ASA.S1 and GTSA.B1 for Acanthamoeba-specific detection and reliable genotyping, respectively, and provide further evidence that T4 is the predominant genotype in AK.The demonstrated pathogenicity for humans and animals of organisms belonging to the genus Acanthamoeba (17, 26), coupled with the difficulty of using morphological criteria for subgeneric identification of isolates (30,38), has stimulated a number of laboratories to pursue molecular methods for detection and identification. The objective is to develop methods that are suitable for both clinical and environmental applications. The identification of amoebic isolates should be very reliable and, at least for clinical use, the detection system should be very sensitive. Several research groups, including our own, have demonstrated the usefulness of PCR methods for detection of acanthamoebae (10,15,21,25,27,40). As few as 1 to 10 trophozoites can be detected. It also is possible to enhance detection of individual amoeba in very dilute liquid clinical samples with fluorescent in situ hybridization (FISH) (36). Several molecular approaches increase the reliability of specimen identification, but the use of DNA sequence variation appears to be the most promising. The variation is observed in restriction fragment length polymorphisms of complete or partial nuclear 18S rRNA genes (8,20,21,22), of complete mitochondrial 16S rRNA genes (7, 46), and of the complete mitochondrial genome (3,7,13,18,22,45). It also is observed in the DNA seq...

Rapid detection and simultaneous molecular profile characterization of Acanthamoeba infections

“…HRM may therefore provide relevant additional information for the comprehensive classification of protozoa (Ledee et al, 2003;Marciano Cabral, 2003) and confirm hypothesis suggesting that the named taxa based on 18S rDNA Rns phylogenetic relationship genotyping are not monophyletic entities. In previous 18S rDNA-based reports, the genotype T4 was predominant (~85%) (Boggild et al, 2009;Booton et al, 2009;Ledee et al, 2003;Lorenzo-Morales et al, 2010;Schroeder et al, 2001;Zhao et al, 2010). However, in Greece, the genotypes T2, T3, T5, T6, and T11 have been identified (Spanakos et al, 2006) and in Slovakia and the Czech Republic the genotypes T15, T4, and T3 were detected in AK (Nagyová et al, 2010).…”

“…Great improvement was obtained using TaqMan rtPCR tests but they are unable to detect strains from genotype T5 (Qvarnstrom et al, 2006) and A. astronyxis (Da Rocha-Azevedo et al, 2009;Qvarnstrom et al, 2006). Moreover, the cost for the numerous TaqMan probes and for the postamplification procedures required for characterizing Acanthamoeba is high (Gatti et al, 2010;Goldschmidt et al, 2009b;Khairnar et al, 2011;Rivera and Adao, 2009;Schroeder et al, 2001;Yera et al, 2007).…”

GENOTYPING OF BALAMUTHIA MANDRILLARIS BASED ON NUCLEAR 18S AND MITOCHONDRIAL 16S rRNA GENES

“…25 Extensive studies in the laboratory at Ohio State University and elsewhere have revealed many distinct Acanthamoeba genotypes of nuclear 18S rDNA. 24,[26][27][28] They also have shown, however, that most Acanthamoeba keratitis cases are associated with a single sequence type. 24,26 It is not known whether there is any correlation between sequence types and the pathogenic potential of Balamuthia.…”

Morphological, physiological, molecular and phylogenetic characterization of new environmental isolates of Acanthamoeba spp. from the region of Bratislava, Slovakia

“…The analysis based on these partial sequences has been proven equally as effective as that of the entire gene in obtaining the information required for the detection of Acanthamoeba as well as for phylogenetic analysis (Schroeder et al 2001). Schroeder et al (2001) also used the PCR product ASA.S1, which was acquired with primers JDP1 and JDP2, specifically for the detection of Acanthamoeba spp. The product is highly genus-selective and can be acquired from all known 18S rDNA genotypes.…”