Journal of Ophthalmology & Clinical Research Category: Clinical Type: Review Article
Translational and Reverse Translational Research Supporting Precision Medicine: Acanthamoeba Keratitis as a Model of Linkage between Clinical and Basic Research Focused on Personalized Ophthalmology
- Linda Christian Carrijo-Carvalho1*, Denise De Freitas1, Fábio Ramos De Souza Carvalho1
- 1 Department Of Ophthalmology And Visual Sciences, Paulista School Of Medicine, Federal University Of Sao Paulo, Sao Paulo, Brazil
*Corresponding Author:Linda Christian Carrijo-Carvalho
Department Of Ophthalmology And Visual Sciences, Paulista School Of Medicine, Federal University Of Sao Paulo, Sao Paulo, Brazil
Received Date: May 24, 2014 Accepted Date: Jul 29, 2014 Published Date: Aug 12, 2014
The present picture raises the question of which factor determines the disease severity: the host response or the pathogen virulence. Advanced knowledge on this issue could have saved the patient's eye. The association of the scientific tools and confident findings provided by the research bench, the careful observation and specialized analyses from the clinical practice associated with the application of scientific and practical discoveries to improve patients' health care can potentially benefit the management of AK, and many other ocular diseases. In an ideal scenario, specific profiles of both patient and Acanthamoeba could be recognized, based on previously characterized patterns and prognostic models. The knowledge and technologies translated from research into Precision Medicine (PM) guide the selection of appropriate treatment, based on the patient's predicted response and the pathogen virulence/resistance profile.
Translational Research (TR) aims to apply the knowledge and technologies from basic sciences in the development of studies focused on a medical issue. At the bench, laboratory findings should present a direct means to improve prevention, diagnosis and treatment. Reverse Translational Research (RTR) takes into consideration clinical findings or clinical aspects of a disease observed at the bedside, to direct the design of laboratory assays using appropriate in vitro and in vivo models in the context of TR. Figure 1 represents the association of translational investigations and PM.
Figure 1: The cyclic interaction of translational research, reverse translational research and precision medicine: The black arrows represent the flow of information between laboratory (bench) and clinical findings (bedside). The development of omics technologies, supported by basic and applied sciences, influence translational research and precision medicine.
The following topics describe how the association of TR, RTR and PM is possible, and the current translational findings, taking AK as an example. This article also seeks to open a broad discussion on the contribution of TR and its aspects to the improvement of clinical care and PM. Thus, we present perspectives concerning the current scientific trends which have been applied complementarily to the available technologies for translational studies, with potential diagnostic and therapeutic applications in the field of ophthalmology and visual sciences.
Application of currently available Molecular Biology approaches represents a refinement in the laboratory diagnosis. Identification of Acanthamoeba genotypes through DNA amplification by PCR or real-time PCRoffers rapid results, specificity and sensitivity [13-16]. Real-time PCRcan also give an estimate of parasite load through quantification of DNA copy number, which is associated with poor outcome . Other possible applications of molecular techniques are detection of new species and genotypes of a specific microorganism [18,19] epidemiological survey, establishment of etiological link between source and infection [14,20] recognition of physiological differences (eg growth rate, metabolism) and screening of virulence profiles observed on in traspecific variations of pathogens [21,7]. However, the use of molecular diagnostics alone has not delivered the expected impact in clinical practice and patients' health. In the same way in vitro and in vivo laboratory findings have not been easily translated into clinical application-hence the role of RTR.
REVERSE TRANSLATIONAL RESEARCH
Genetic factors can affect the patient's response to pathogen invasion and represent another issue for translational studies. For example, the activation of inflammatory cascade mediated mainly by toll-like receptor 4 (TRL4) and triggered by virulent strain of Acanthamoeba was recently demonstrated by in vitro and in vivo experimental assays . Single nucleotide polymorphisms of the gene encoding TRL4 have been correlated with susceptibility to infectious and inflammatory diseases in human subjects . Investigation of the genomic profiles among AK patients, by using RTR, could demonstrate the association of TLR4 genetic polymorphisms with increased susceptibility to disease. Further laboratory assays concerning the recognition and polymorphisms of receptors expressed by the cornea tissue in the contact with metabolic products of the protozoa could confirm TLR4 as a potential therapeutic target and a promising prognostic marker in the management of AK.
The omics field in ophthalmology and visual sciences is currently in increasing progress. The advance of genetic therapy focused mainly on earlier detection and treatment of complex pathologies from congenital or acquired primary human source has been motivating an increasing number of ophthalmologists to develop critical analyses about the functional role of basic science applied to the improvement of patients' health at the bedside. Analyses of genomic, transcript omic, proteomic, and metabolomic technologies have demonstrated an integrative function in the investigation and treatment of pathophysiological processes with a higher degree of complexity, related to eye diseases at levels of DNA, RNA, protein and cell/tissue, respectively [32-38]. Furthermore, the use of protein and antibody arrays  and the next-generation DNA sequencing technology [40,41] supports the earlier detection by screening of proteomic, transcript omic and genomic profiles, respectively, in eye diseases and ocular syndromes which are well-defined in clinical practice, but not fully-investigated for gene therapy approaches and treatment outcome. As examples, we have ligneous conjunctivitis, glaucoma, strabismus and retinitis pigmentosa. In this context, the increasing availability of personal genome and validation of biomarkers are promising tools for PM and personalized therapy .
TOWARDS PRECISION MEDICINE
Once AK is a sight-threatening disease, characterized by complex diagnosis and therapy, which requires long-term treatment and long-term follow up, Personalized Medicine, supported by precision diagnosis, is important in the management of the disease. Despite the majority of patients being contact lens wearers [4,44] as the case mentioned at the beginning, some AK cases are not associated with environmental risk factors . Patients usually have a previous clinical diagnosis of bacterial fungal or herpes simplex virus keratitis before a laboratorial diagnosis for AK [3,4,45]. AK may present similar clinical features to herpetic keratitis, contributing to diagnostic delay and late delivery of appropriate medical therapy [4,45,46]. According to the current data early treatment is associated with a better prognosis [4,5].
Prolonged treatment requires discipline of patients with amoebic keratitis in constant application of antimicrobial agents in the first months post-diagnosis and continuous monitoring from the ophthalmologist in order to minimize the cytotoxic effects of chemical drops and avoid possible clinical complications such as iris atrophy, cataracts and glaucoma . In this context, for example, PM could enhance the diagnosis and therapy of AK: early detection of protozoa by conventional culturing method of viable cysts from corneal scrapings, under optimized incubation conditions, associated with estimation of viable parasite load by DNA copy number by molecular techniques have the potential to monitor the length of treatment, dosage adjustments, requirement of combined therapy, and the use of topical steroids. Optimal therapeutic prescription reduces the corneal exposure to the potential toxicity of biocides used for AK treatment [47-49]. Treatment with voriconazole has been proposed for AK unresponsive to conventional therapies with biguanides and/or diamidines [50,51]. Prescription of this triazole derivative chemical requires careful attention to dosage regimen due to drug interactions, genetic variations in drug metabolism and potential hepatic toxicity . Furthermore, Lorenzo-Morales et al. elegantly described in a review article published recently the application of azole compounds other than voriconazolehave been proposed for AK unresponsive to conventional therapies to reduce the infective process in severe cases of the disease for example, the association of miconazole and propamidine or oral it raconazole associated with the topical use of miconazole and ketoconazole . For this reason, serum levels of hepatic enzymes are currently used as standard biomarkers for therapeutic monitoring, mainly of fungal infections. Pharmaco genomic models based on genetic polymorphisms of drug-metabolizing enzymes and drug transporters have the potential to improve medical decisions based on individual genomic profiles .
(Figure 2) presents an outlook of possible benefits of the cyclic interaction of TR, RTR and PM in the context of AK. The proposed scheme can give a general idea of how the flow of information between bench and bedside could benefit an individual case. Table 1 summarizes some examples of potential benefits of translational findings to precision ophthalmology in the management of AK. Discoveries about the pathophysiology of disease, diagnostic and therapy tools currently available and those which should be proven effectual in the near future especially with the increase of omics data are presented in Table 1. As Scientific Investigation is a dynamic and interactive process an ambiguity may exist between the translational findings with current and future applications. The given examples represent a part of the many possibilities to be explored through the association between the three aspects in the study of AK.
Figure 2: Association between translational studies and precision medicine in benefit of Acanthamoeba keratitis management: Blue arrows represent the flow of information from bench to bedside (translational research). Red arrows represent the flow of information from bedside to bench (reverse translational research). Potential applications from translational discoveries are indicated in gray letters.
|Characteristics||Current knowledge||Future perspectives|
|Acanthamoeba||Culture from corneal scraping, followed by amplification and sequencing of 18S ribosomal DNA for accurate diagnosis ||Early diagnosis based on culture independent DNA amplification. Estimate of Acanthamoeba load by real-time PCR , providing data for personalized therapy|
|Acanthamoeba phenotype associated with pathogenic strains: cytopathic effect, higher growth rate and temperature tolerance [21,55,56]||Worse disease prognosis associated with non-T4 genotype [7,15] and genetic profile (eg phylogenetic clustering  and genes coding for differential virulence factors)|
|The mannose induced protein (MIP-133) associated with pathogenesis of AK, inducing epithelial cell death and collagen degradation in the cornea ||MIP-133 as a therapeutic target  or biomarker. Differential expression of serine proteases associated with disease severity [11,12] directing a personalized therapy|
|Cyst resistance to diamidines [1,4] variable drug susceptibility of different Acanthamoeba strains, species or genotypes [6,15,48]||Validation of an in vitro test for identification of resistant strains that correlates with clinical response. New therapeutic agents eg statins  miltefosine  and gene silencing approaches eg siRNA targeting Acanthamoebamyosin  and ergosterol biosynthesis |
|AK patient||Risk associated with contact lens wear [25,44] and with low tear levels of anti-Acanthamoeba IgA antibody ||Improvement of a preventive care for patients with increased risk factors. Development of antibody-based diagnostic test [28,29] and therapeutic approaches|
|Radial keratoneuritis and ring infiltrates as characteristic signs [4,22,24]||Recognition of signs and symptoms for accurate diagnosis. Recognition of signs and symptoms indicative of best/worst prognosis [23,24]|
|Monotherapy with a biguanide or combined therapy using biguanides and diamidines with adjustable therapeutic regimen is effective in several cases, leading to a best outcome [4,25,27]. Ocular toxicity of current anti-Acanthamoeba drugs [4,47,48,49]||Proof of efficacy of off-label drugs [50,51] or novel anti-Acanthamoeba compounds previously approved in pre-clinical trials. Identification of the patient’s drug metabolizing profile through pharmacogenomics tests  to implement personalized therapy|
|Inflammatory response is mediated by TLR4 receptor ||TLR4 as a therapeutic target. Identification of TLR4 polymorphisms  associated with differential prognosis and establishment of personalized therapy|
|Available omics technologies, complex analysis tools and personal genome [39-42]||Development of new biomarkers and genetic tests for prognosis, therapy selection and follow-up |
In conclusion AK is a complicated disease that may be substantially benefit from basic and clinical research. Efforts of TRare necessary for early and accurate diagnosis and the development of new therapies to overcome pathogen resistance and to reduce drug toxicity to the host will support PM. Reverse translational research can lead to advances in disease prognosis in order to distinguish genomic profiles and discovery of new biomarkers, which will direct the future of precision medicine.
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Citation:Carvalho FRS, Carvalho LCC, Freitas DD (2014) Translational and Reverse Translational Research Supporting Precision Medicine: Acanthamoeba Keratitis as a Model of Linkage between Clinical and Basic Research Focused on Personalized Ophthalmology. J Ophthalmic Clin Res, 1: 001.
Copyright: © 2014 Linda Christian Carrijo-Carvalho, et al. 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 author and source are credited.