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Laser IVCM appearance of (A) Acanthamoeba cysts demonstrating clustering of cysts, (B) Acanthamoeba cysts demonstrating linear alignment of cysts and (C and D) Acanthamoeba trophozoites.
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Abstract In vivo confocal microscopy (IVCM) is an emerging technology that provides minimally invasive, high resolution, steady-state assessment of the ocular surface at the cellular level. Several challenges still remain but, at present, IVCM may be considered a promising technique for clinical diagnosis and management. This mini-review summarizes...
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... of Acanthamoeba by IVCM was first reported in 1992 12 and was recently supported by The American Academy of Ophthalmology as an adjunctive diagnostic modality. 13 Double-walled cysts appear as hyper-reflective, spherical, occa- sionally ovoid, structures, ranging 15-28 mm in diameter ( Figure 1A and B). The double-wall may not always be apparent by IVCM, making it occasion- ally difficult to differentiate cysts from leukocyte or epithelial nuclei. ...
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... double-wall may not always be apparent by IVCM, making it occasion- ally difficult to differentiate cysts from leukocyte or epithelial nuclei. Clustering ( Figure 1A) and rows ( Figure 1B) of cysts are typically suggestive of active proliferative disease. Trophozoites are 25-40 mm in diameter, 7 and appear as hyper-reflective and ovoid structures on IVCM ( Figure 1C and D) but are difficult to distinguish from leukocytes and keratocyte nuclei. ...
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... double-wall may not always be apparent by IVCM, making it occasion- ally difficult to differentiate cysts from leukocyte or epithelial nuclei. Clustering ( Figure 1A) and rows ( Figure 1B) of cysts are typically suggestive of active proliferative disease. Trophozoites are 25-40 mm in diameter, 7 and appear as hyper-reflective and ovoid structures on IVCM ( Figure 1C and D) but are difficult to distinguish from leukocytes and keratocyte nuclei. ...
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... ( Figure 1A) and rows ( Figure 1B) of cysts are typically suggestive of active proliferative disease. Trophozoites are 25-40 mm in diameter, 7 and appear as hyper-reflective and ovoid structures on IVCM ( Figure 1C and D) but are difficult to distinguish from leukocytes and keratocyte nuclei. ...
Citations
... Clinical anterior segment optical coherence tomography (AS-OCT) [11,12] can provide cross-sectional tomographic imaging of the cornea to determine the depth of disease involvement, but an unmet need for cellular resolution to analyze the pathological changes of cornea and limbus tissues. In vivo confocal microscopy (IVCM) [13] can obtain micrometer-level resolution imaging features of cells and nerves on the ocular surface but works in a contact manner and provides a small field of view (FOV) (below 0.4 mm × 0.4 mm) without the ability of three-dimensional (3D) imaging, which limit its application to some extend in clinics. In recent years, high-resolution OCT technology has significantly improved the quality and efficiency of ophthalmic imaging, especially in the context of the cornea. ...
In this study, a dual-mode full-field optical coherence tomography (FFOCT) was customized for label-free static and dynamic imaging of corneal tissues, including donor grafts and pathological specimens. Static images effectively depict relatively stable structures such as stroma, scar, and nerve fibers, while dynamic images highlight cells with active intracellular metabolism, specifically for corneal epithelial cells. The dual-mode images complementarily demonstrate the 3D microstructural features of the cornea and limbus. Dual-modal imaging reveals morphological and functional changes in corneal epithelial cells without labeling, indicating cellular apoptosis, swelling, deformation, dynamic signal alterations, and distinctive features of inflammatory cells in keratoconus and corneal leukoplakia. These findings propose dual-mode FFOCT as a promising technique for cellular-level cornea and limbus imaging.
... features depend on the stage of the disease. Cysts (dormant form) are easier to detect compared to the trophozoite form: they manifest as highly reflective, round structures in the deep stromal layers usually defined by their dual-layered walls, with diameters between 12 to 25 microns [61,82]. Occasionally, they have been described as contributing to a characteristic "starry sky" pattern [83][84][85]. ...
Acanthamoeba keratitis (AK) is a rare but potentially sight-threatening corneal infection caused by the Acanthamoeba parasite. This microorganism is found ubiquitously in the environment, often in freshwater, soil, and other sources of moisture. Despite its low incidence, AK presents significant challenges due to delayed diagnosis and the complex nature of therapeutic management. Early recognition is crucial to prevent severe ocular complications, including corneal ulceration and vision loss. Diagnostic modalities and treatment strategies may vary greatly depending on the clinical manifestation and the available tools. With the growing reported cases of Acanthamoeba keratitis, it is essential for the ophthalmic community to thoroughly understand this condition for its effective management and improved outcomes. This review provides a comprehensive overview of AK, encompassing its epidemiology, risk factors, pathophysiology, clinical manifestations, diagnosis, and treatment.
... Rarely reported are the much larger hyperreflective trophozoites. [92,93] It has also been reported that the cysts tend to form clusters upon steroid treatment. [94] IVCM has also revealed co-infections, especially with fungi. ...
This is a comprehensive review after a thorough literature search in PubMed-indexed journals, incorporating current information on the pathophysiology, clinical features, diagnosis, medical and surgical therapy, as well as outcomes of Acanthamoeba keratitis (AK). AK is a significant cause of ocular morbidity, and early diagnosis with timely institution of appropriate therapy is the key to obtaining good outcomes. The varied presentations result in frequent misdiagnosis, and co-infections can increase the morbidity of the disease. The first line of therapy continues to be biguanides and diamidines, with surgery as a last resort.
... In vivo confocal microscopy (IVCM) is a non-invasive imaging technique and can generate in vivo images of the cornea with a resolution of 1 µm including images of the epithelium, endothelium, nerves, and cells and able to identify filamentous fungi or Acanthamoeba cysts [84][85][86]. IVCM's accuracy varies from 78 to 91.1%, and its sensitivity ranges from 80 to 94% [84]. It is said to be extremely dependent on the observer's experience and of the varying value in the diagnosis and monitoring of fungal and Acanthamoeba keratitis (AK) [87]. ...
... The "non-invasiveness" of IVCM, realtime and early pathogen identification, monitoring and treatment guidance, and infection depth evaluation are some of its benefits. The incapability to visualize tiny organisms, motion artifacts, and dense corneal infiltrates or scarring can interfere with proper tissue penetration and visualization and are just a few of IVCM's limitations [85,88]. Cases of progressive keratitis or where Acanthamoeba or FK are detected generally require IVCM. ...
Infectious keratitis is the fifth most prevalent cause of blindness worldwide. The primary diagnostic test to identify the pathogenic organism is the culture of corneal scraping. However microbial culture positivity is low and varies widely due to many underlying factors. Therefore, there is a need to understand the prevalence of such cases and what modern tools can be employed to diagnose them.
Contact lens usage, ocular injuries, and ocular surface disease have been reported to be primary risk factors for keratitis with infection of pathogens such as Staphylococcus spp., Pseudomonas aeruginosa, Fusarium spp., Candida spp., and Acanthamoeba spp. Advanced imaging techniques, such as in vivo confocal microscopy and anterior segment optical coherence tomography (OCT), and polymerase chain reaction (PCR) and other molecular techniques have been used to identify the specific causative agents of infectious keratitis more rapidly and accurately than traditional culture methods. However, microbial culture positivity is low and varies widely due to many underlying factors.
In vivo confocal microscopy and polymerase chain reaction (PCR) testing can support the diagnosis of infectious keratitis. Initial treatment for bacterial keratitis (BK) is with antimicrobials primarily fluoroquinolones, while topical natamycin (an antifungal anti-protozoal) is the drug of choice for fungal keratitis. Additionally, PCR and other molecular methods are utilized to corroborate the initial diagnosis or where routine microbial cultures are negative. This review provides current information for diagnosing microbial culture negative keratitis patients. Earlier diagnosis with modern tools could decrease incidence of corneal opacity, vision loss, or even the loss of an eye in keratitis patients.
... Cysts (dormant form) appear as hyper-reflective, spherical and well-defined double-wall structures of ~15-30 µm in diameter in the epithelium or stroma. Trophozoites (active form) appear as hyper-reflective structures of 25-40 µm, which are difficult to discriminate from leukocytes and keratocyte nuclei (Table 2) [4,19,49]. Acanthamoeba spp. can also present as bright spots, signet rings and perineural infiltrates ( Table 2). ...
... Cysts (dormant form) appear as hyper-reflective, spherical and well-defined double-wall structures of~15-30 µm in diameter in the epithelium or stroma. Trophozoites (active form) appear as hyper-reflective structures of 25-40 µm, which are difficult to discriminate from leukocytes and keratocyte nuclei (Table 2) [4,19,49]. Acanthamoeba spp. can also present as bright spots, signet rings and perineural infiltrates ( Table 2). ...
... structures of ~15-30 µm in diameter in the epithelium or stroma. Trophozoites (active form) appear as hyper-reflective structures of 25-40 µm, which are difficult to discriminate from leukocytes and keratocyte nuclei (Table 2) [4,19,49]. Acanthamoeba spp. can also present as bright spots, signet rings and perineural infiltrates ( Table 2). ...
Infectious keratitis (IK) is among the top five leading causes of blindness globally. Early diagnosis is needed to guide appropriate therapy to avoid complications such as vision impairment and blindness. Slit lamp microscopy and culture of corneal scrapes are key to diagnosing IK. Slit lamp photography was transformed when digital cameras and smartphones were invented. The digital camera or smartphone camera sensor’s resolution, the resolution of the slit lamp and the focal length of the smartphone camera system are key to a high-quality slit lamp image. Alternative diagnostic tools include imaging, such as optical coherence tomography (OCT) and in vivo confocal microscopy (IVCM). OCT’s advantage is its ability to accurately determine the depth and extent of the corneal ulceration, infiltrates and haze, therefore characterizing the severity and progression of the infection. However, OCT is not a preferred choice in the diagnostic tool package for infectious keratitis. Rather, IVCM is a great aid in the diagnosis of fungal and Acanthamoeba keratitis with overall sensitivities of 66–74% and 80–100% and specificity of 78–100% and 84–100%, respectively. Recently, deep learning (DL) models have been shown to be promising aids for the diagnosis of IK via image recognition. Most of the studies that have developed DL models to diagnose the different types of IK have utilised slit lamp photographs. Some studies have used extremely efficient single convolutional neural network algorithms to train their models, and others used ensemble approaches with variable results. Limitations of DL models include the need for large image datasets to train the models, the difficulty in finding special features of the different types of IK, the imbalance of training models, the lack of image protocols and misclassification bias, which need to be overcome to apply these models into real-world settings. Newer artificial intelligence technology that generates synthetic data, such as generative adversarial networks, may assist in overcoming some of these limitations of CNN models.
... Moreover, it contains lymphoid tissue, lacrimal accessory glands, and mast cells. The goblet cells secrete MUC5AC [88,89], a mucin that is in the middle of many ocular conditions, and the CALT (lymphoid tissue of conjunctiva) is responsible for the immune response [90]. During ocular surface inflammation present in DED or OA, proinflammatory modulators such as TNF-α, IL-6 intercellular adhesion molecules such as ICAM-1, and various types of immune cells are found in the epithelial conjunctival layer [88]. ...
... The goblet cells secrete MUC5AC [88,89], a mucin that is in the middle of many ocular conditions, and the CALT (lymphoid tissue of conjunctiva) is responsible for the immune response [90]. During ocular surface inflammation present in DED or OA, proinflammatory modulators such as TNF-α, IL-6 intercellular adhesion molecules such as ICAM-1, and various types of immune cells are found in the epithelial conjunctival layer [88]. ...
The most common disorders of the ocular surface are dry eye disease (DED) and ocular allergy (OA). These conditions are frequently coexisting with or without a clinical overlap and can cause a severe impact on the patient’s quality of life. Therefore, it can sometimes be hard to distinguish between DED and OA because similar changes and manifestations may be present. Atopic patients can also develop DED, which can aggravate their manifestations. Moreover, patients with DED can develop ocular allergies, so these two pathological entities of the ocular surface can be considered as mutual conditions that share the same background. Nowadays, by using different techniques to collect tissue from ocular surfaces, the changes in molecular homeostasis can be detected and this can lead to a precise diagnosis. The article provides an up-to-date review of the various ocular surface biomarkers that have been identified in DED, OA, or both conditions.
Abbreviations: DED = dry eye disease, OA = ocular allergy, SS = Sjogren syndrome, TBUT = tear break up time, TFO = tear film osmolarity, AKC = Atopic keratoconjunctivitis, ANXA1 = Annexin 1, ANXA11 = Annexin 11, CALT = Conjunctival associated lymphoid tissue, CCL2/MIP-1 = Chemokine (C-C motif) ligand2/Monocyte chemoattractant protein 1, CCL3/MIP-1α = Chemokine (C-C motif) ligand 3/Macrophage inflammatory protein 1 alpha, CCL4/MIP-1β = Chemokine (C-C motif) ligand 4/Macrophage inflammatory protein 1 beta, CCL5/RANTES = Chemokine (C-C motif) ligand 5 /Regulated on Activation, Normal T cell Expressed and Secreted, CCR2 = Chemokine (C-C motif) receptor 2, CCR5 = Chemokine (C-C motif) receptor 5, CD3+ = Cluster of differentiation 3 positive, CD4+ = Cluster of differentiation 4 positive, CD8+ = Cluster of differentiation 8 positive, CGRP = Calcitonin-gene-related peptide, CX3CL1 C-X3 = C motif -chemokine ligand 1 /Fractalkine, CXCL8 = Chemokine (C-X-C motif) ligand 8, CXCL9 = Chemokine (C-X-C motif) ligand 9, CXCL10 = Chemokine (C-X-C motif) ligand 10, CXCL11 = Chemokine (C-X-C motif) ligand 11, CXCL12 = Chemokine (C-X-C motif) ligand 12, CXCR4 = Chemokine (C-X-C motif) receptor 4, EGF = Epidermal growth factor, HLA-DR = Human leukocyte antigen-D-related, ICAM-1 = Intercellular adhesion molecule 1, IFN-γ = Interferon-gamma, IgG = Immunoglobulin G, IgE = Immunoglobulin E, IL-1 = Interleukin-1, IL-1α = Interleukin-1 alpha, IL-1β = Interleukin-1 beta, CGRP = Calcitonin-Gene-Related Peptide, IL-3 = Interleukin-3, IL-4 = Interleukin-4, IL-6 = Interleukin-6, IL-8 = Interleukin-8, IL-10 = Interleukin-10, IL-17 = Interleukin-17, IL-17A = Interleukin-17A, LPRR3 = Lacrimal proline-rich protein 3, LPRR4 = Lacrimal proline-rich protein 4, MUC5AC = Mucin 5 subtype AC, oligomeric mucus/gel-forming, MUC16 = Mucin 16, OCT = Optical coherence tomography, OGVHD = Ocular graft versus host disease, PAX6 = Paired-box protein 6, VKC = Vernal keratoconjunctivitis, TGF-β = Transforming growth factor β, S100 = proteins Calcium activated signaling proteins, Th1 = T helper 1 cell, Th17 = T helper 17 cell, MGD = Meibomian gland dysfunction, TFOS = Tear film and ocular surface society, SS-KCS = Keratoconjunctivitis Sicca, MMP-9 = Matrix metalloproteinase 9, MMP-1 = Matrix metalloproteinase 1, ZAG = Zinc alpha glycoprotein, CBA = Cytometric bead array, MALDI TOF-MS = matrix assisted laser desorption ionization-time of flight, SELDI TOF-MS = surface-enhanced laser desorption ionization-time of flight, IVCM = in vivo confocal microscopy, AS-OCT = anterior segment optical coherence tomography, iTRAQ = Isobaric tags for relative and absolute quantitation, LC-MS = Liquid chromatography-mass spectrometry, LCN-1 = lipocalin 1, PIP = prolactin induced protein, NGF = Nerve growth factor, PRR4 = proline rich protein 4, VIP = Vasoactive intestinal peptide, ELISA = enzyme linked immunoassay, TNF-α = tumor necrosis factor alpha, PAC = perennial allergic conjunctivitis, SAC = seasonal allergic conjunctivitis, IC = impression cytology, RT-PCR = reverse transcription polymerase chain reaction, PCR = polymerase chain reaction, APCs = antigen-presenting cells, NK cells = natural killer cells, HEL = hexanoyl-lysine, 4-HNE = 4-hydroxy-2-nonenal, MDA = malondialdehyde
... The introduction of in vivo confocal microscopy (IVCM), as a technique for acquiring corneal images, has revolutionized the study and analysis of the structures of this tissue [1,2]. In particular, thanks to this tool, it is possible to quickly and noninvasively acquire images of the sub-basal plexus (specific layer within the epithelium), allowing the visualization of the corneal nerve fibers. ...
... Moreover, several investigators have examined these structures and verified that they provide important clinical B Alessia Colonna alessia.colonna@unipd.it 1 Department of Information Engineering, University of Padova, via Gradenigo, Padova 35131, Italy information related to age, prolonged use of contact lenses, surgery (such as LASIK or PRK), and transplantation [9][10][11][12]. ...
In vivo confocal microscopy is a technique that allows to acquire images of the corneal layers in a rapid and noninvasive way. Analysis of sub-basal nerve allows obtaining important clinical information regarding the eye and the human body’s health. To obtain that information, it is necessary to correctly identify and trace the nerve fibers. Manual analysis is time-consuming and subjective. Numerous automatic algorithms have been proposed to overcome these problems, but none have been included in clinical practice yet. In this work, we take advantage of deep learning techniques. We present an analysis of the performances obtained through UNet (baseline) to which various architectural solutions have been added to boost performance. The variation of the tracing results is also analyzed according to the use of different loss functions, one of which is introduced here: It considers a tolerance margin (Dice with tolerance). The investigated configurations have been shown to be capable of improving the tracing of corneal nerve fibers. The model with attention modules and atrous-spatial pyramid pooling modules showed the greatest improvement compared to the baseline, increasing in the evaluation score from 86.51 to 90.21%. Furthermore, the proposed loss function further increases the results (achieving 92.44%).
... In summary, IVCM is a noninvasive imaging technique that allows for the visualization of living cells and tissues in situ. Although it has limitations, it can provide detailed cellular information that boosts our understanding of ocular surface diseases and improves eye care management for further research and clinical practice [32]. ...
Dry eye disease (DED) is a common ocular disorder affecting millions worldwide. It is characterized by reduced tear production and/or increased tear evaporation, leading to ocular discomfort and impaired vision. Corneal imaging techniques are valuable tools for diagnosing and monitoring DED, as they can provide objective and quantitative information on the structure and function of the ocular surface and the tear film. This chapter will review the principles and applications of various corneal imaging techniques for DED, such as Slit-Lamp Biomicroscopy, Fluorescein CorneoGraphy, In Vivo Confocal Microscopy, Optical Coherence Tomography, Lipid Layer Interferometry, Topography, and Fluorophotometry. The advantages and limitations of each technique are discussed, as well as their potential role in future research and clinical practice, such as monitoring treatment efficacy and guiding personalized treatment approaches.
... Trophozoites (active form) appear as hyper-reflective structures of 25-40 µm which are difficult to discriminate from leukocytes and keratocyte nuclei. [4,11,39]. Acanthamoeba spp. can also present as bright spots, signet rings and perineural infiltrates. ...
Infectious keratitis (IK) is among the top 5 leading causes of blindness globally. Early diagnosis is key to guide an appropriate therapy to avoid complications such as vision impairment and blindness. Culture of corneal scrapes is the initial diagnostic test to grow and identify the causal organism. Alternative diagnostic tools include imaging such as with optical coherence tomography (OCT) and in vivo confocal microscopy (IVCM). OCT’s advantage is its ability to accurately determine the depth and extent of the corneal ulceration, infiltrates and haze; therefore, characterizing the severity and progression of the infection. However, it is not a preferred choice in the diagnostic tool package for infectious keratitis. IVCM is a great aid in the diagnosis of fungal and Acanthamoeba keratitis with overall sensitivities of 66-74% and 80-100%, and specificity of 78-100% and 84-100%, respectively. Recently the use of deep learning (DL) models in IK has been shown to aid diagnosis via image recognition. Most of the studies that have developed DL models to diagnose the different types of IK have utilised photographs from digital camaras, slit-lamp images, or IVCM images. Some studies have used extremely efficient single DL algorithms to train their models and others used ensemble approaches. This technology is likely to assist in the diagnosis of IK in some years. Further work is needed to examine and validate the clinical performance of the DL models in the real-world setting and to evaluate whether this technology improves patient clinical outcomes. The scope of this review was to provide a recent update on the diagnostic imaging tools in IK
... Confocal microscopy can capture images of all layers of the cornea, including sub-basal corneal nerve density, morphology, and the presence of and inflammatory cells, as well as goblet cells and inflammation of the conjunctiva. 116,117 Confocal microscopy requires interpretation by a skilled clinician. ...
Dry eye disease (DED) is a common ocular condition, but the diagnosis relative to other ocular conditions and the evaluation of severity of the condition has often been difficult. This challenge can be due to clinical signs and symptoms not always correlating with each other. An understanding of the various components which create the condition, as well as the diagnostic measures used to evaluate these components, is useful to the clinician working with DED patients. This review paper will discuss traditional diagnostic options, diagnostic imaging, and Advanced Point of Care testing capabilities to determine the severity level of dry eye disease more adequately.
