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      Acanthamoeba-mediated cytopathic effect correlates with MBP and AhLBP mRNA expression

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          Abstract

          Background

          In recent years, the concern of Acanthamoeba keratitis has increased since the infection is often associated with contact lens use. Partial 18S rRNA genotypic identification of Acanthamoeba isolates is important to correlate with pathophysiological properties in order to evaluate the degree of virulence. This is the first report of genotypic identification for clinical isolates of Acanthamoeba from corneal scrapings of keratitis in Malaysia. This study is also the first to correlate the mRNA expression of MBP and AhLBP as virulent markers for axenic strains of Acanthamoeba.

          Results

          In this study, ten clinical isolates were obtained from corneal scrapings. Rns genotype and intra-genotypic variation at the DF3 region of the isolates were identified. Results revealed that all clinical isolates belonged to the T4 genotype, with T4/6 (4 isolates), T4/2 (3 isolates), T4/16 (2 isolates) and one new genotype T4 sequence (T4/36), being determined. The axenic clinical isolates were cytopathogenic to rabbit corneal fibroblasts. MBP and AhLBP mRNA expression are directly correlated to Acanthamoeba cytopathic effect.

          Conclusions

          All ten Malaysian clinical isolates were identified as genotype T4 which is predominantly associated with AK. Measuring the mRNA expression of Acanthamoeba virulent markers could be useful in the understanding of the pathogenesis of Acanthamoeba keratitis.

          Electronic supplementary material

          The online version of this article (doi: 10.1186/s13071-017-2547-0) contains supplementary material, which is available to authorized users.

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          Most cited references54

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          An update on Acanthamoeba keratitis: diagnosis, pathogenesis and treatment

          Free-living amoebae of the genus Acanthamoeba are causal agents of a severe sight-threatening infection of the cornea known as Acanthamoeba keratitis. Moreover, the number of reported cases worldwide is increasing year after year, mostly in contact lens wearers, although cases have also been reported in non-contact lens wearers. Interestingly, Acanthamoeba keratitis has remained significant, despite our advances in antimicrobial chemotherapy and supportive care. In part, this is due to an incomplete understanding of the pathogenesis and pathophysiology of the disease, diagnostic delays and problems associated with chemotherapeutic interventions. In view of the devastating nature of this disease, here we present our current understanding of Acanthamoeba keratitis and molecular mechanisms associated with the disease, as well as virulence traits of Acanthamoeba that may be potential targets for improved diagnosis, therapeutic interventions and/or for the development of preventative measures. Novel molecular approaches such as proteomics, RNAi and a consensus in the diagnostic approaches for a suspected case of Acanthamoeba keratitis are proposed and reviewed based on data which have been compiled after years of working on this amoebic organism using many different techniques and listening to many experts in this field at conferences, workshops and international meetings. Altogether, this review may serve as the milestone for developing an effective solution for the prevention, control and treatment of Acanthamoeba infections.
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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.

            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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              Production of different phenotypes from the same genotype in the same environment by developmental variation.

              The phenotype of an organism is determined by the genes, the environment and stochastic developmental events. Although recognized as a basic biological principle influencing life history, susceptibility to diseases, and probably evolution, developmental variation (DV) has been only poorly investigated due to the lack of a suitable model organism. This obstacle could be overcome by using the recently detected, robust and highly fecund parthenogenetic marbled crayfish as an experimental animal. Batch-mates of this clonal crayfish, which were shown to be isogenic by analysis of nuclear microsatellite loci, exhibited surprisingly broad ranges of variation in coloration, growth, life-span, reproduction, behaviour and number of sense organs, even when reared under identical conditions. Maximal variation was observed for the marmorated coloration, the pattern of which was unique in each of the several hundred individuals examined. Variation among identically raised batch-mates was also found with respect to fluctuating asymmetry, a traditional indicator of the epigenetic part of the phenotype, and global DNA methylation, an overall molecular marker of an animal's epigenetic state. Developmental variation was produced in all life stages, probably by reaction-diffusion-like patterning mechanisms in early development and non-linear, self-reinforcing circuitries involving behaviour and metabolism in later stages. Our data indicate that, despite being raised in the same environment, individual genotypes can map to numerous phenotypes via DV, thus generating variability among clone-mates and individuality in a parthenogenetic species. Our results further show that DV, an apparently ubiquitous phenomenon in animals and plants, can introduce components of randomness into life histories, modifying individual fitness and population dynamics. Possible perspectives of DV for evolutionary biology are discussed.
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                Author and article information

                Contributors
                ngsookluan@gmail.com , ngsl0306@hotmail.com
                anisah.nordin@gmail.com , anisah.nordin@ukm.edu.my
                norzanafi9506@yahoo.com
                yusof88@gmail.com
                noraina79@gmail.com
                ckienhui@gmail.com
                Journal
                Parasit Vectors
                Parasit Vectors
                Parasites & Vectors
                BioMed Central (London )
                1756-3305
                28 December 2017
                28 December 2017
                2017
                : 10
                : 625
                Affiliations
                [1 ]ISNI 0000 0004 1937 1557, GRID grid.412113.4, Department of Physiology, Faculty of Medicine, , Universiti Kebangsaan Malaysia, ; Jalan Yaacob Latif, 56000 Kuala Lumpur, Bandar Tun Razak Malaysia
                [2 ]ISNI 0000 0004 1937 1557, GRID grid.412113.4, Department of Parasitology and Medical Entomology, Faculty of Medicine, , Universiti Kebangsaan Malaysia, ; Jalan Yaacob Latif, 56000 Kuala Lumpur, Bandar Tun Razak Malaysia
                [3 ]ISNI 0000 0004 1937 1557, GRID grid.412113.4, Department of Anatomy, Faculty of Medicine, , Universiti Kebangsaan Malaysia, ; Jalan Yaacob Latif, 56000 Kuala Lumpur, Bandar Tun Razak Malaysia
                Article
                2547
                10.1186/s13071-017-2547-0
                5745754
                29282148
                d1814cb7-fac3-4cd5-862e-2032c3569d04
                © The Author(s). 2017

                Open AccessThis article is distributed under the terms of the Creative Commons Attribution 4.0 International License ( http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver ( http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.

                History
                : 7 June 2017
                : 21 November 2017
                Funding
                Funded by: UKM Fundamental Grant
                Award ID: FF-274-2010
                Award Recipient :
                Categories
                Research
                Custom metadata
                © The Author(s) 2017

                Parasitology
                keratitis,acanthamoeba,genotype,cytopathic,mbp,ahlbp
                Parasitology
                keratitis, acanthamoeba, genotype, cytopathic, mbp, ahlbp

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