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Identification of 18S Ribosomal DNA Genotype of Acanthamoeba from Patients with Keratitis in North China

From the Beijing Tongren Hospital, Beijing Institute of Ophthalmology, Capital University of Medical Science, Beijing, China.

	Abstract

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PURPOSE. To identify the genotype of 18S ribosomal RNA gene (18S rDNA, Rns) of the Acanthamoeba strains isolated from patients with keratitis in northern China.

METHODS. The genus-specific primers JDP1 and JDP2 were used for the amplification of the amplimer ASA.S1. With DNA PCR and sequencing, Rns genotypes were identified according to the DF3 sequence variances.

RESULTS. Of all 26 DF3 sequences obtained from the 26 Acanthamoeba strains, 18 were unique (69.2%). Of those, 17 were Rns genotype T4 and one was Rns genotype T3.

CONCLUSIONS. Most Acanthamoeba strains isolated from keratitis in northern China were Rns genotype T4. The results are in agreement with recent results in the literature.

The demonstrated clinical relevance of the genus Acanthamoeba and the relationship between the genotype and eye infection suggest that more studies are needed on the identification of the 18S ribosomal DNA (Rns) genotype of Acanthamoeba strains obtained from corneas with keratitis, contact lenses, lens cases, lens case solution, and the home water supply.

At present, sequences of the complete Rns gene appear to provide the most reliable measure of the Rns genotype. Based on the complete Rns sequences of 2300 to 2700 bp, Stothard et al.⁴ and Gast et al.⁵ defined 12 Acanthamoeba Rns sequence types.

The JDP1-JDP2 is a set of genus-specific primers that hybridize exclusively to Acanthamoeba 18S rDNA in regions E23-2' and E-23-6.^{6 7} Sequencing of the amplimer ASA.S1 was determined by sequencing primer 892C. This segment included portions of 18S rDNA conserving stem 29 and all of 29-1. The sequence in region 29-1 provided the genotype discrimination of ASA.S1. Because the strains of Acanthamoeba in our laboratory were fed with Escherichia coli, the JDP1 primer was used as the genotype identification primer.

In this study, 26 strains of Acanthamoeba were collected from 26 eyes of 26 patients between 1994 and 2002. The specimens were cultured at Beijing Institute of Ophthalmology. The purpose of this study is to discriminate the Rns genotype of clinically relevant isolates and to analyze the correlation between our isolates with published strains from other areas.

	Materials and Methods

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Isolation and Culture
Corneal scrapes were obtained from 26 patients with keratitis (13 women; 13 men), for routine Acanthamoeba culture from 1994 to 2002. Patients’ ages ranged from 13 to 72 years (mean, 33 years). Of all 26 patients with AK, 3 (11.5%) wore soft contact lenses, 5 (19.2%) wore orthokeratology contact lenses, and 2 (7.7%) had a history of trauma. The protocol of the study adhered to the provisions of the Declaration of Helsinki for research involving human subjects.

The scrapings from the ulcers of patients with AK were transferred to non-nutrient agar (NNA) plates covered with 100 µL of a 24-hour-old culture of E. coli. The plates were sealed and incubated at 28°C for 14 days and examined for amoeba growth. The specimens that yielded Acanthamoeba were kept at 0°C on NNA overlaid with E. coli. Cultures used for genotype analysis were maintained at 28°C again and then harvested (1 x 10⁶ cells).

DNA Extraction and PCR
Amoeba harvested from the NNA plates were washed three times in phosphate-buffered saline (PH7.4) and resuspended in 500 µL of cell lysis buffer (5 mM EDTA and 50 mM Tris [pH 8.0]). Proteinase K (0.1 mg/mL) and 12 µL sodium dodecyl sulfate (20%) were added. After gentle mixing by inversion, the lysates were incubated at 56°C for 3 hours. The samples were then chilled on ice for 5 minutes and were extracted with equal volumes of phenol-chloroform (1:1). After extraction, the aqueous phase and organic phase were separated by centrifugation at 2000g for 5 minutes at room temperature. Then the supernatant was extracted with an equal volume of chloroform-isoamyl alcohol (24:1). After centrifugation, the upper phase was precipitated by the addition of a 1/30 volume of 5% NaCl and two volumes of cold absolute ethanol and kept at –20°C overnight. The DNA was pelleted by centrifugation, dried at 4°C, dissolved in 300 µL of double-distilled H₂O, and stored at –20°C until use.

PCR was performed with the genus-specific primers JDP1 (5'-GGCCCAGATCGTTTACCGTGAA) and JDP2 (5'-TCTCACAAGCTGCTAGGGGAGTCA). Magnesium concentration was 2 mM in a PCR reaction. After heating for 7 minutes at 95°C, 35 cycles of amplification at 95°C (60 seconds), 62°C (45 seconds), and 72°C (45 seconds) were performed with a thermal cycler (GeneAmp 2400; Perkin-Elmer, Eden Prairie, MN). Amplification products were detected with ethidium bromide staining after electrophoresis on 2% agarose gels. Direct sequencing of the PCR product was performed with an automated fluorescence sequencing system (model 377; Applied Biosystems, Foster City, CA) with the conserved primer 892C(5'-GTCAGAGGTGA AATTCTTGG) to determine the primary DNA sequence of DF3 of Rns. The DF3 sequence nomenclature used in this study was as described previously.⁷ The DF3 sequence alignment for the amoeba specimens from the Beijing Institute of Ophthalmology is provided in Figure 1 . Phylogenetic reconstruction produced a gene tree by using maximum parsimony, neighbor-joining, and minimum evolution methods in the phylogenetic computer program MEGA2 (Molecular Evolution Genetic Analysis software, ver 2.1; http://www.megasofware.net/ developed by Kumar S, et al., Arizona State University, Tempe, AZ). Acanthamoeba sp. strain V006 was at the root of the tree. This strain is the Rns genotype T1 strain that is an outgroup of sequence types T3, T4, and T11. Figure 2 shows a linearized neighbor-joining tree, obtained by using the Kimura two-parameter distance algorithm in MEGA2.

View larger version (85K): [in this window] [in a new window]   FIGURE 1. Primary sequence alignment of a variable region of DF3 (stem 29-1 of 18S rRNA) of Chinese isolates by CLUSTAL X (http://www.ebi.ac.uk/clustalx/ provided in the public domain by the European Bioinformatics Institute, European Molecular Biology Laboratory, Heidelberg, Germany). The alignment shown is a subset of DF3 that contains the highly variable and informative section of this fragment. Sequences are aligned by similarity. *, Variable positions; ----, gaps.

View larger version (34K): [in this window] [in a new window]   FIGURE 2. The 18S rDNA DF3 sequence linearized neighbor-joining gene tree with Chinese isolates. () Sequences from Chinese isolates. The T1, T3, and T11 designations correspond to strains previously determined to be of that particular genotype.

	Results

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The DF3 regions of the Rns genes of the 26 corneal specimens from patients with AK were amplified and sequenced. The DF3 sequence from the 26 isolates sequenced resulted in 26 sequences. Of the 26 sequences obtained, 18 were unique (69.2%). Of the 18 unique sequences, 17 were phylogenetically similar to other genotype T4 Rns isolates previously identified, whereas the remaining one sequence was similar to genotype T3 isolates. No sequence obtained is similar to any Acanthamoeba Rns genotype other than T3 or T4. The Rns genotype classification results demonstrated that nearly all isolates from Acanthamoeba keratitis are genotype Rns T4.

All corneal isolates from patients with AK were Rns genotype T4 and T3. Table 1 summarizes the genotype results. Sequences of the various regions of DF3 in this study are presented in Figure 1 . Of the 26 corneal scrape isolates, 25 were identified as Rns genotype T4, and 1 isolate was identified as Rns T3 (Fig. 2) . Patients A1P1 and A36P35, patients A16P16 and A8P8, patients A5P5 and A29P29, patients A9P9 and A10P10, patients A2P2 and A13P13, patients A22P22 and A31P31, and patients A3P3, A18P18, and A23P23 had identical corneal DF3 sequences, suggesting that the infection may be caused by the same or closely related strains. Different sequences were found in 11 other corneal isolates from the 11 remaining patients.

	Discussion

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The DNA typing method adopted in this study is relatively fast and reliable in identifying individual strains fed with E. coli. However, in an analysis based on complete gene sequences, this genotype forms a clade with T3 and T11. Thus, this clade is named T3-T4-T11.⁶ To obtain more reliable results in which DF3 sequences are identical, cultures should be axenized, and the complete Rns sequence should be determined and compared.

	Footnotes

 
Submitted for publication September 28, 2003; revised November 19 and December 30, 2003; accepted January 12, 2004.

Disclosure: Y. Zhang, None; X. Sun, None; Z. Wang, None; R. Li, None; S. Luo, None; X. Jin, None; S. Deng, None; W. Chen, None

The publication costs of this article were defrayed in part by page charge payment. This article must therefore be marked "advertisement" in accordance with 18 U.S.C. 1734 solely to indicate this fact.

Corresponding author: Xuguang Sun, Beijing Tongren Hospital, Beijing Institute of Ophthalmology, Capital University of Medical Science, Beijing 100005, China; sunxuguang{at}yahoo.com.

	References

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