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      Fluorescence amplified fragment length polymorphism for subtyping of genotypes of Acanthamoeba isolated from patients with keratitis

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          Abstract

          Background & objectives:

          Acanthamoeba keratitis (AK) is a painful and vision-threatening ocular infection. The differentiation of Acanthamoeba at the species and subspecies level is complicated. Nearly all the AK isolates have been shown to belong to T4 genotype when analysed by ribosomal RNA gene sequences and there is no universally acceptable method for differentiation of different subtypes of T4. The purpose of this study was to attempt further discrimination of T4 genotypes.

          Methods:

          In the present investigation, 15 Acanthamoeba isolates obtained from cornea of keratitis patients were subjected to fluorescence amplified fragment length polymorphism (FAFLP) genotyping to differentiate T4 subtypes.

          Results:

          FAFLP profiles showed five distinct clusters (I to V) within T4 clonal complex which clearly depicted genetic differences among the isolates of T4 sequence type of Acanthamoeba.

          Interpretation & conclusions:

          Our study demonstrated the usefulness of FAFLP for reliable differentiation of T4 clonal complex of Acanthamoeba.

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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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            Acanthamoeba keratitis update-incidence, molecular epidemiology and new drugs for treatment.

            Katherine A. D. Seal (2003)
            A reliable figure for the expected incidence of Acanthamoeba keratitis of one per 30000 contact lens wearers per year has now been obtained from a combination of three cohort and three Questionnaire Reporting Surveys; 88% of cases wore hydrogel lenses and 12% wore rigid lenses. This figure now provides a basis for the expected number of cases against which to judge either epidemic outbreaks or effects of prevention with disinfecting solutions, better hygiene, or the use of disposable lenses. Molecular biology of Acanthamoeba has advanced considerably in the last 10 years with new automated sequencing technology. This has allowed the construction of a genotype identification scheme with 13 different genotypes against which to compare clinical isolates for epidemiological investigations or pathogenicity markers. So far, only four genotypes have been associated with keratitis of which the majority have been T4 but T3, T6, and T11 have each caused individual cases. Each genotype is heterogenous and can be further subdivided by comparison of sequences of diagnostic fragments of 18S rDNA, riboprinting by PCR-RFLP of 18S rDNA, or by mitochondrial DNA RFLP. Drug therapy has been revolutionised with the introduction of the biguanides-chlorhexidine or polyhexamethylene biguanide-with most but not all infections quickly resolving. Failure can still occur occasionally and further research is needed on more effective combination chemotherapy. A number of guanidines have been identified in this paper that could be usefully pursued as part of combination chemotherapy along with the alkylphosphocholines.
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              Patient characteristics, diagnosis, and treatment of non-contact lens related Acanthamoeba keratitis.

              S. Sharma, Bhuwan P. Garg, Gautam Rao (2000)
              To review the clinical characteristics, diagnosis, and visual outcome in patients with non-contact lens related Acanthamoeba keratitis and compare the findings with reported series of contact lens associated Acanthamoeba keratitis. Medical and microbiology records of 39 consecutive patients with a diagnosis of Acanthamoeba keratitis, at a tertiary eyecare centre in India between January 1996 and June 1998, were analysed retrospectively. A majority of the patients presented with poor visual acuity and large corneal stromal infiltrates (mean size 38.20 (SD 26. 18) mm). A predisposing factor was elicited in 19/39 (48.7%) patients (trauma 15, dirty water splash three, leaf juice one). None of the patients had worn contact lenses. Most patients (26/39 (66. 6%)) came from a low socioeconomic background. Complaint of severe pain was not a significant feature and radial keratoneuritis was seen in 1/39 (2.5%) patients. A ring infiltrate was present in 41.1% of cases. A clinical diagnosis of fungal keratitis was made in 45% of the patients before they were seen by us. However, all patients were diagnosed microbiologically at our institute based on demonstration of Acanthamoeba cysts in corneal scrapings (34/39) and/or culture of Acanthamoeba (34/39). Treatment with biguanides (PHMB, 15/38 (39.4%), PHMB with CHx, 23/38 (60.5%), one patient did not return for treatment) resulted in healing with scar formation in 27 out of 31(87.0%) followed up patients (mean time to healing 106.9 days). Overall visual outcome was poor with no statistical difference between cases diagnosed within 30 days (early) or 30 days after (late) start of symptoms. The visual outcome in cases requiring tissue adhesive (five) and keratoplasty (three) was also poor. This is thought to be the largest series of cases of Acanthamoeba keratitis in non-contact lens wearers. In such cases, the disease is advanced at presentation in most patients, pathognomonic clinical features are often not seen, disease progression is rapid, and visual outcome is usually poor. Possible existence of Acanthamoeba pathotypes specifically associated with non-contact lens keratitis and unique to certain geographical areas is suggested.
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                Author and article information

                Journal
                Journal ID (nlm-ta): Indian J Med Res
                Journal ID (publisher-id): IJMR
                Title: The Indian Journal of Medical Research
                Publisher: Medknow Publications (India )
                ISSN (Print): 0971-5916
                Publication date (Print): January 2011
                Volume: 133
                Issue: 1
                Pages: 83-87
                Affiliations
                [* ] Department of Biotechnology, School of Life Sciences, Pondicherry University, Pondicherry, India
                [** ] Centre for DNA Fingerprinting & Diagnostics, Hyderabad, India
                [+ ] L V Prasad Eye Institute, Hyderabad Eye Research Foundation, Hyderabad, India
                [# ] Ocular Microbiology Service, L V Prasad Eye Institute, Bhubaneswar, India
                Author notes
                Reprint requests: Dr Prashanth K., Assistant Professor in Biotechnology, Department of Biotechnology, School of Life Sciences, Pondicherry University, R. Venkataraman Nagar, Kalapet, Pondicherry 605 014, India e-mail: prashi2k@ 123456gmail.com
                Article
                Publisher ID: IJMR-133-83
                PMC ID: 3100151
                PubMed ID: 21321424
                SO-VID: 48aca0ab-cc26-487b-b46f-e5f1803065cc
                Copyright © © The Indian Journal of Medical Research
                License:

                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.

                History
                Date received : 20 July 2009
                Categories
                Subject: Original Article

                ScienceOpen disciplines: Medicine
                Keywords: t4 genotype differentiation,acanthamoeba keratitis
                Data availability:
                ScienceOpen disciplines: Medicine
                Keywords: t4 genotype differentiation, acanthamoeba keratitis

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