Research Article: In Vivo Confocal Microscopy Cellular Features of Host and Organism in Bacterial, Fungal, and Acanthamoeba Keratitis

Date Published: June 1, 2018

Publisher: Elsevier Science

Author(s): Jaya D. Chidambaram, Namperumalsamy V. Prajna, Srikanthi Palepu, Shruti Lanjewar, Manisha Shah, Shanmugam Elakkiya, David Macleod, Prajna Lalitha, Matthew J. Burton.


To determine cellular features of fungal (FK), Acanthamoeba (AK), and bacterial keratitis (BK) using HRT3 in vivo confocal microscopy (IVCM).

Prospective observational cross-sectional study.

Eligible participants were adults with microbiologically positive FK, AK, or BK, of size ≥ 3 mm, attending Aravind Eye Hospital from February 2012 to February 2013. Exclusion criteria were descemetocele or perforation. At presentation, IVCM imaging was performed, then corneal scrapes were obtained for culture/light microscopy. An experienced grader (masked to microbiology/clinical features) assessed IVCM images for presence/absence of normal keratocyte-like morphology, stellate interconnected cells with/without visible nuclei, dendritiform cells (DFCs), inflammatory cells in a honeycomb distribution, and organism features. Statistical significance was assessed by logistic regression, adjusted for age, sex, ulcer size, and symptom duration. Main outcome measures were presence/absence of IVCM features in FK, AK, BK.

A total of 183 participants had FK, 18 AK, 17 BK. Acanthamoeba appeared as bright spots (16/18, 89%), double-walled cysts (15/18, 83%), or signet rings (3/18, 17%), and often formed clusters after topical steroid use (univariable odds ratio [OR] 9.98, 95% confidence interval [CI] 1.02-97.96, P = .048). BK was associated with bullae in anterior stroma (OR 9.99, 95% CI: 3.11-32.06, P < .001). Honeycomb distribution of anterior stromal inflammatory cells was associated with FK (univariable OR 2.74, 95% CI: 1.01-7.40, P = .047). Aspergillus ulcers were associated with stromal DFCs (OR 11.05, 95% CI: 1.49-82.13, P = .019) and Fusarium ulcers with stellate appearance of interconnected cell processes with nuclei (OR 0.24, 95% CI: 0.09-0.65, P = .005). Specific cellular and structural features observed using IVCM in microbial keratitis may be associated with organism.

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This study was prospectively approved by the Ethics Committees of the Indian Council for Medical Research, Aravind Eye Hospital, Tamil Nadu, India, and the London School of Hygiene and Tropical Medicine. As previously described, all patients gave written informed consent before enrolment; illiterate participants gave informed consent with a witnessed thumbprint on the study consent form (as approved by the above Ethics Committees).1 The tenets of the Declaration of Helsinki were adhered to during conduct of this study.

Of the 239 participants enrolled in the study, 17 were excluded owing to being microbiologically negative (ie, no organism detected on culture, light microscopy, or IVCM) and 4 were excluded owing to mixed bacterial/fungal infection (culture-positive for bacteria and positive for fungal hyphae on light microscopy and/or IVCM). Of the remaining 218 participants, 183 were diagnosed with fungal keratitis, 18 with Acanthamoeba keratitis, and 17 with bacterial keratitis, as summarized in Table 1. The baseline demographic profile of participants within each group (BK, AK, FK) showed no statistically significant differences in age, sex, presenting visual acuity, or proportion of ulcers with deep involvement of the posterior cornea (Table 2). However, in the AK group the symptom duration (median 30 days, P < .001) and ulcer size (median 6.8 mm diameter, P < .001) were greater than for all other ulcers. A total of 3153 volume scans consisting of 126 120 images were obtained at the enrollment visit in all patients (median 12 volume scans per patient, interquartile range 9-16). We were able to perform IVCM imaging of the posterior half of the cornea in 57 ulcers (Table 3), the majority of which were fungal (81%, 46/57), and culture-positive for Fusarium sp. (n = 22).Table 1Causative Organisms Identified by Culture, Light Microscopy, and In Vivo Confocal MicroscopyN%Fungi (n = 183) Culture-positive (n = 144): Fusarium sp.7333.5% Aspergillus sp.3315.1% Curvularia sp.52.3% Exserohilum sp.41.8% Lasiodiplodia sp.20.9% Cylindrocarpon sp.10.4% Bipolaris sp.10.4% Unidentified hyaline fungi146.4% Unidentified dematiaceous fungi115.0% Culture-negative but light microscopy–positive for fungi3013.8% Culture-negative but IVCM-positive for fungi94.1%Acanthamoeba (n = 18) Culture-positive177.8% Culture-negative but IVCM-positive for Acanthamoeba10.5%Bacteria (n = 17) Culture-positive (n = 17): Streptococcus pneumoniae94.1% Nocardia sp.31.4% Pseudomonas aeruginosa20.9% Aeromonas sp.10.4% Streptococcus viridans10.4% Staphylococcus epidermidis10.4%Total218100%IVCM = in vivo confocal microscopy.Table 2Baseline Characteristics of Study ParticipantsFungal Keratitis (77.4%, N = 183)Acanthamoeba Keratitis (8.3%, N = 18)Bacterial Keratitis (7.8%, N = 17)P ValuebMedian age, years (IQR)50 (36-58)39 (34-55)60 (46-65).104Male sex, n (%)118 (64.8%)11 (61.1%)10 (58.8%).871Symptom duration, median number of days (IQR)7 (4-10)30 (7-60)8 (4-14)<.001Baseline visual acuity, median logMAR (IQR)1.8 (0.6-1.8)1.8 (1.7-1.8)1.7 (1.7-1.8).244Ulcer stromal infiltrate size,a mm (median, IQR)4.4 (3.3-5.5)6.8 (5.3-8.0)3.7 (3.2-5.0)<.001Deep infiltrate involving posterior one-third of cornea, n (%)113 (62.1%)13 (72.2%)12 (70.6%).585aUlcer stromal infiltrate size calculated as geometric mean of longest diameter and perpendicular diameter.bDifferences between all 3 groups assessed for statistical significance using χ2 test for proportions (sex, ulcer depth) and Kruskal-Wallis test for continuous nonparametric variables.Table 3Cellular Features Detected Within In Vivo Confocal Microscopy Images of Bacterial, Fungal, Acanthamoeba, and Microbiologically Negative KeratitisCorneal LocationIVCM FeaturesFungal Keratitis (N = 183)Acanthamoeba Keratitis (N = 18)Bacterial Keratitis (N = 17)P ValueaAnteriorNormal keratocyte-like morphology141 (77%)7 (39%)14 (82%).001Stellate cellular processes with nuclei119 (65%)6 (33%)11 (65%).029Stellate cellular processes no nuclei58 (32%)10 (56%)3 (18%).047Spindles132 (72%)14 (78%)13 (76%).826Granules108 (59%)13 (72%)11 (65%).514Epithelial bullae18 (10%)0 (0%)7 (41%)<.001Stromal bullae19 (10%)1 (6%)8 (47%)<.001Inflammatory cells (honeycomb)90 (49%)1 (6%)6 (35%).001Inflammatory cells (nonspecific)42 (23%)3 (17%)6 (35%).403Basal DFCs97 (53%)7 (39%)14 (82%).027Stromal DFCs19 (10%)5 (28%)4 (23%).043Scar19 (10%)4 (22%)3 (18%).251Fungal spore-like structures6 (3%)0 (0%)0 (0%)-Corneal LocationIVCM FeaturesFungal Keratitis (N = 46)Acanthamoeba Keratitis (N = 7)Bacterial Keratitis (N = 4)P ValueaPosteriorNormal keratocyte-like morphology31 (67%)4 (57%)4 (100%).320Stellate cellular processes with nuclei35 (76%)2 (29%)3 (75%).037Stellate cellular processes no nuclei10 (22%)5 (71%)1 (25%).024Spindles30 (65%)6 (86%)2 (50%).430Granules22 (48%)6 (86%)2 (50%).173Inflammatory cells (honeycomb)11 (24%)1 (14%)0 (0%).476Inflammatory cells (nonspecific)8 (17%)0 (0%)2 (50%).111Stromal DFCs2 (4%)0 (0%)0 (0%).780Scar0 (0%)1 (14%)0 (0%).026Fungal spore-like structures2 (4%)0 (0%)0 (0%)-DFCs = dendritiform cells; IVCM = in vivo confocal microscopy.aStatistical significance of difference between all 3 groups assessed using χ2 test. Here we have described the cellular changes that occur in the cornea in MK as observed with IVCM at first presentation. In FK, which formed the majority of cases in this study of large ulcers, the only IVCM feature weakly associated with this disease was the presence of an anterior stromal honeycomb distribution of inflammatory cells. This specific honeycomb pattern of inflammatory cells is similar to that observed after abrasion injury in real-time in vivo HRT3 IVCM imaging of the mouse cornea; these inflammatory cells were identified as neutrophils using immunohistochemistry in the same tissue ex vivo, and their close interaction with keratocytes was found to be mediated through action of cell adhesion molecules.9 However, to our knowledge, this honeycomb distribution of migrating inflammatory cells has not been formally investigated in FK before. Neutrophils are recruited to the cornea very soon after the onset of infection in MK, even within hours, and this is mediated through release of chemokines in the cornea by host cells (eg, CXCL1, CXCL5, IL8).14, 21   Source:


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