Isolation of Acanthamoeba Genotype T4 from a Non-Contact Lens Wearer from the Philippines

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Abstract

We report the case of a 76-year old Filipino male who presented with pain, redness, and blurring of vision of the right eye. Corneal scraping was done and sent to the St. Luke’s Research and Biotechnology Group for detection and identification of the infectious agent. Morphological detection was performed by allowing the organism from the scraping to grow in 1.5% non-nutrient agar plate with heat-killed E. coli. Trophozoites with acanthopodia and double-walled cysts characteristic of Acanthamoeba were observed within the first and second week of observations, respectively. Molecular identification of the amoebae at the genus level based on the presence of Acanthamoeba-specific amplimer S1, ASA.S1 confirmed the morphological identification. Genotyping through sequence revealed that the organism belonged to T4, which is the genotype commonly present in the eye of keratitis patients.

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

Michael J. M. Hay, Susan J. Schroeder, I Niszl … (2001)

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 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.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.

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18S ribosomal DNA typing and tracking of Acanthamoeba species isolates from corneal scrape specimens, contact lenses, lens cases, and home water supplies of Acanthamoeba keratitis patients in Hong Kong.

E Houang, Ruby Lam, Joshua Byers … (2002)

We examined partial 18S ribosomal DNA (Rns) sequences of Acanthamoeba isolates cultured in a study of microbial keratitis in Hong Kong. Sequence differences were sufficient to distinguish closely related strains and were used to examine links between strains obtained from corneal scrape specimens, contact lenses, lens cases, lens case solutions, and home water-supply faucets of patients with Acanthamoeba. We also looked for evidence of mixed infections. Identification of Acanthamoeba Rns genotypes was based on sequences of approximately 113 bp within the genus-specific amplicon ASA.S1. This permitted genotype identification by using nonaxenic cultures. Of 13 specimens obtained from corneal scrapes, contact lenses, lens cases, or lens case solutions, 12 were Rns genotype T4 and the remaining one was Rns genotype T3. The sequences of corneal scrape specimens of two patients also were the same as those obtained from their contact lenses or lens case specimens. A possible triple-strain infection was indicated by three different T4 sequences in cultures from one patient's lenses. Although faucet water used by patients to clean their lenses is a possible source of infections, specimens isolated from the faucets at two Acanthamoeba keratitis patients' homes differed from their corneal scrape or lens specimens. The overall results demonstrate the potential of this Rns region for tracking Acanthamoeba keratitis strains in infections and for distinguishing single-strain and closely related multiple-strain infections even when other microorganisms might be present with the cultured specimens. They also confirm the predominance of Rns genotype T4 strains in Acanthamoeba keratitis infections.

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Molecular identification of t4 and t5 genotypes in isolates from acanthamoeba keratitis patients.

D Ledee, M. Elizabeth Fini, M. A. Diaz … (2009)

Acanthamoeba keratitis (AK) is a rare but sight-threatening ocular infection. Outbreaks have been associated with contaminated water and contact lens wear. The epidemiology and pathology may be associated with unique genotypes. We determined the Rns genotype for 37 clinical isolates from 23 patients presenting at the University of Miami Bascom Palmer Eye Institute with confirmed AK infections in 2006 to 2008. The genus-specific ASA.S1 amplicon allowed for rapid genotyping of the nonaxenic cultures. Of the 37 isolates, 36 were of the T4 genotype. Within this group, 13 unique diagnostic fragment 3 sequences were identified, 3 of which were not in GenBank. The 37th isolate was a T5, the first in the United States and second worldwide to be found in AK. For five patients with isolates from the cornea and contact lens/case, identical sequences within each patient cluster were observed, confirming the link between contact lens contamination and AK infection. Genotyping is an important tool in the epidemiological study of AK. In this study, it allowed for the detection of new strains and provided an etiological link between source and infection. Additionally, it can allow for accurate categorizing of physiological differences, such as strain virulence, between isolates and clades.

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Author and article information

Journal

Journal ID (nlm-ta): Trop Med Health

Journal ID (publisher-id): TMH

Title: Tropical Medicine and Health

Publisher: The Japanese Society of Tropical Medicine

ISSN (Print): 1348-8945

ISSN (Electronic): 1349-4147

Publication date (Print): December 2014

Publication date (Electronic): 23 August 2014

Volume: 42

Issue: 4

Pages: 145-147

Affiliations

[1 ]Department of Virology, Institute of Tropical Medicine, Nagasaki University , Nagasaki, Japan

[2 ]Institute of Biology, University of the Philippines , Diliman, Quezon City 1101, Philippines

[3 ]Research and Biotechnology, St. Luke’s Medical Center , 279 E. Rodriguez Blvd. Quezon City 1112, Philippines

[4 ]Eye Institute, St. Luke’s Medical Center , 279 E. Rodriguez Blvd. Quezon City 1112, Philippines

Author notes

[* ]Corresponding author: Tel: (632) 727-5540 Fax (632) 726-0467 E-mail: ffnatividad@ 123456stluke.com.ph

Article

Publisher ID: JST.JSTAGE/tmh/2014-15 Publisher-manuscript ID: 2014-15

DOI: 10.2149/tmh.2014-15

PMC ID: 4253062

SO-VID: 0b97099e-447d-463e-b98b-8663449b7974

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This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Isolation of Acanthamoeba Genotype T4 from a Non-Contact Lens Wearer from the Philippines

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Abstract

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Sex and gender-sensitive medicine

Most cited references 7

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

18S ribosomal DNA typing and tracking of Acanthamoeba species isolates from corneal scrape specimens, contact lenses, lens cases, and home water supplies of Acanthamoeba keratitis patients in Hong Kong.

Molecular identification of t4 and t5 genotypes in isolates from acanthamoeba keratitis patients.

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