- Email: [email protected]
Tandem Scanning Confocal Corneal Microscopy in the Diagnosis of Suspected Acanthamoeba Keratitis
Tandem Scanning Confocal Corneal Microscopy in the Diagnosis of Suspected Acanthamoeba Keratitis Dipak N. Parmar, BSc(Hons), FRCOphth, Shady T. Awwad, MD, W. Matthew Petroll, PhD, R. Wayne Bowman, MD, James P. McCulley, MD, FRCOphth, H. Dwight Cavanagh, MD, PhD Objective: To evaluate the role of in vivo corneal tandem scanning confocal microscopy (TSCM) in the definitive diagnosis of suspected Acanthamoeba keratitis (AK). Design: Noncomparative interventional single-institution case series. Method: A retrospective case review of patients consecutively referred with suspected AK and undergoing corneal TSCM was performed. Results: A total of 63 cases that met the inclusion criteria for the study were referred for diagnostic evaluation. Tandem scanning confocal microscopy demonstrated Acanthamoeba cysts/trophozoites in 54 cases and fungal hyphae in 2, whereas 1 case was positive for both Acanthamoeba and fungus. Culture of the cornea or contact lenses was carried out in 35 cases, 9 of which were positive for Acanthamoeba. Six of the TSCM-positive cases also underwent corneal biopsy, being positive for Acanthamoeba in only 2. Six patients were negative for Acanthamoeba on TSCM, the etiology being fungal in 1 case, as shown by subsequent culture. One patient was positive on culture for Acanthamoeba but falsely negative by TSCM, which was limited by poor cooperation during the examination. Two cases initially masqueraded as Acanthamoeba keratitis but showed fungus on TSCM. Mean follow-up was 14 months. Conclusion: In vivo corneal TSCM can establish the diagnosis of Acanthamoeba keratitis rapidly and noninvasively, particularly when conventional microbiology is inconclusive. Ophthalmology 2006;113:538 –547 © 2006 by the American Academy of Ophthalmology.
Acanthamoeba keratitis (AK) is a rare condition, with a recent study estimating a severe infection rate of 1.2 per million adults and 0.2 per 10 000 contact lenses wearers per year.1,2 However, the protozoan is difficult to culture, and the true incidence could be almost 10 times higher than previously suggested. During the 1980s, a dramatic rise in the incidence of AK was seen in conjunction with the growing clinical use of soft contact lenses, largely attributed to the use of nonsterile lens solutions, swimming while wearing lenses, and inadequate disinfection practices.3,4 To date, contact lens wear remains the single most prevalent risk factor, particularly when associated with environmental Originally received: June 5, 2005. Accepted: December 14, 2005. Manuscript no. 2005-489. From the Division of Cornea, External Disease and Refractive Surgery, Department of Ophthalmology, University of Texas Southwestern Medical Center, Dallas, Texas. Presented at: American Academy of Ophthalmology meeting, October, 2004; New Orleans, Louisiana. Supported in part by National Eye Institute, Bethesda, Maryland (grant nos.: EY10738 [HDC], EY016664); The Pearle Vision Foundation, Dallas, Texas; and an unrestricted grant from Research to Prevent Blindness, Inc., New York, New York. Correspondence and reprint requests to H. Dwight Cavanagh, MD, PhD, Department of Ophthalmology, University of Texas Southwestern Medical Center at Dallas, 5323 Harry Hines Boulevard, Dallas, TX 75390-9057. E-mail: [email protected].
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water exposure, although Acanthamoeba species are also increasingly recognized as an important cause of keratitis in non– contact lens wearers.5,6 Early diagnosis is invaluable and is associated with a relatively satisfactory outcome, whereas the consequences of late diagnosis can be devastating.7,8 The variability in presentation of patients with AK often makes clinical diagnosis difficult, as the early signs can be nonspecific.1,7 Current laboratory diagnostic techniques relying on culture are limited in yield, with positive rates quoted from 0% to 68% in cases negative for contact lens case culture, whereas in some series Acanthamoeba has been grown from contact lenses or storage cases where corneal epithelial samples were culture negative.5,7–9 The advent of direct testing using the polymerase chain reaction (PCR) is encouraging, although this is not yet a firmly established diagnostic modality and requires particular laboratory expertise.6,10 However, the lack of specific multispecies diagnostic epitopes necessitates the use of multiple low-yield single probes. This increases the expense involved and makes the widespread clinical use of PCR in AK unlikely. The principal disadvantage of laboratory diagnostic techniques lies in the reliance on blind invasive sampling of the cornea, either by scraping or biopsy, and subsequent delay in culture results. Tandem scanning confocal microscopy (TSCM) is a noninvasive diagnostic modality allowing both a qualitative and a quantitative analysis of the entire cornea ISSN 0161-6420/06/$–see front matter doi:10.1016/j.ophtha.2005.12.022
Parmar et al 䡠 Confocal Microscopy in Suspected Acanthamoeba Keratitis to be performed rapidly, delivering immediate diagnosis in vivo.11–13 It is particularly useful when relatively large infecting organisms (ⱖ15 m) are present, as are seen in Acanthamoeba, filamentous fungal, microsporidial, and, possibly, Lyme Borrelia keratitis.14 –19 Confocal microscopy can clearly demonstrate both the cyst and often the trophozoite forms of Acanthamoeba in suspected keratitis. It also shows the enlarged corneal nerves accompanying radial neurokeratitis and the characteristic honeycomb-pattern intrastromal microcavities seen during the late stages of the disease. The rapidity of diagnosis, relative ease of use, and noninvasive nature of confocal microscopy are clearly advantageous when compared to traditional laboratory techniques in suspected AK.14 We report 63 patients with suspected AK who were referred consecutively for establishment of diagnosis by TSCM. This represents the largest such series to date with complete follow-up, as confirmed by an extensive literature search through the Medline and Ovid biomedical databases. We discuss the merits, potential uses, and possible disadvantages of TSCM in this setting, as well as the implications of early diagnosis by this modality.
Materials and Methods Institutional review board approval was obtained before commencement of this study. A detailed retrospective case analysis was performed of patients with suspected AK consecutively referred to the University of Texas Southwestern Medical Center, Dallas, Texas, from 1994 to 2004. All patients had undergone TSCM (Tandem Scanning Corp., Reston, VA [currently Advanced Scanning Corp., New Orleans, LA]) for diagnostic evaluation. Of these patients, only those with complete follow-up data qualified for inclusion in the study; of a total of 90 referred cases, 63 met these inclusion criteria. A video camera connected to both an S-VHS video recorder and a black-and-white monitor was used to record images, which were archived accurately for later reference on S-VHS videotape. Confocal microscopy through focusing was performed, which allowed a 3-dimensional reconstruction of the cornea to be created using established personal computer– based analysis software.20 Features unique to Acanthamoeba or fungi were sought for specifically in the captured images. Data acquisition identified predisposing risk factors for AK, method of treatment, and initial and final diagnoses, the latter confirmed by clinical response to treatment. In those patients who had undergone culture of the cornea and/or contact lens case, standard microbiological cultures had been carried out for bacteria, fungi, and Acanthamoeba, the latter specifically involving growth on Escherichia coli–seeded nonnutrient agar plates.21 To assess the effects of initial vision on final visual outcome, the patients were divided arbitrarily into 3 groups of similar logarithm of the minimum angle of resolution (logMAR) vision ranges according to presenting visual acuities (VAs) of (1) 20/15 to 20/60, (2) 20/70 to 20/200, and (3) 20/400 or worse. The length of time to diagnosis of suspected AK was also recorded and divided into 3 groups of (1) ⬍7 days, (2) 7 to 28 days, and (3) ⬎28 days. The relationship between initial VA and final visual outcome was examined. The effects of the following parameters on the mean change in VA after treatment were also analyzed: (1) initial VA, (2) time to suspected Acanthamoeba diagnosis, and (3) prior topical corticosteroid use. Statistical analysis employing nonpaired t tests was performed with SigmaStat software (Systat Software, Inc., Point Richmond, CA).
Results Demographics A total of 90 cases of suspected AK were referred to us for TSCM. Many of these patients were sent from other centers, so that only 63 had complete follow-up data to meet the study inclusion criteria. Nineteen patients were male and 44 female, with a median patient age of 34 years (range, 13–72) and a mean follow-up of 14 months.
Predisposing Factors Contact lens wear was the most significant risk factor, prevalent in 54 patients with a mean length of prior contact lens wear of 111 months (range, 1– 470). The remaining identified risk factors are summarized in Table 1. The majority of these were soft contact lens wearers, although one case was documented in a patient undergoing the fourth year of orthokeratology treatment for myopia with rigid gas-permeable lenses.
Treatment A total of 56 cases were treated for AK after diagnosis by confocal microscopy, culture, or biopsy. Thirty-four of these cases were treated with ⱖ3 topical acanthamoebicidal agents, 20 with 2 drugs, and 2 cases with only propamidine isethionate (Brolene, Aventis Ltd., West Malling, United Kingdom). Other antiamoebic drugs used include topical polyhexamethylbiguanide, chlorhexidine, neomycin, and clotrimazole. The mean duration of treatment for anti-Acanthamoeba treatment was 8.4 months. Table 1. Predisposing Factors in Patients with Suspected Acanthamoeba Keratitis n CTL wear Soft, 40 RGP, 12 Toric, 1 Orthokeratology, 1 Sleeping with CTL Recent new CTL fit Corneal ectasia Keratoconus, 2 Post-LASIK/RK ectasia, 1 Previous corneal surgery RK, 1 LASIK, 1 RK and LASIK, 1 PK, 1 Cleaning with tap water; swimming in freshwater lakes, rivers, or swimming pools, or hot tub use; exposure to bath/shower water with CTL in place Inappropriate cleaning solution Tap water, 15 Well water, 2 Previous trauma Dry eye Immunocompromised Systemic steroid, 4 Systemic steroid ⫾ methotrexate ⫾ infliximab, 1 Topical cyclosporine 0.05%, 1
54
15 4 3 4
25 17 11 2 6
CTL ⫽ contact lens; HIV ⫽ human immunodeficiency virus; PK ⫽ penetrating keratoplasty; RGP ⫽ rigid gas permeable; RK ⫽ radial keratotomy.
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Figure 1. Chart showing relationship between mean final logarithm of the minimum angle of resolution (LogMAR) visual acuity (VA) and initial VA. Snellen equivalent mean final VAs are also displayed. The differences were statistically significant between group 1 (20/15–20/60) and group 2 (20/70 –20/200) and between group 1 and group 3 (20/400 or worse), but not between groups 2 and 3. Using the nonpaired t test (2 tailed), Pⱕ0.002 (1 vs. 2), Pⱕ0.385 (2 vs. 3), and Pⱕ0.001 (1 vs. 3).
Visual Outcome Figure 1 shows the mean final logMAR VA for all 3 groups. The best outcome was seen in group 1 (20/47), whereas those in group 2 (20/510) and group 3 (20/1100) fared much worse. The differences were statistically significant between group 1 (20/15–20/60) and group 2 (20/70 –20/200) and between group 1 and group 3 (20/400 or worse), but not between group 2 and group 3. Using the nonpaired t test (2 tailed), Pⱕ0.002 (1 vs. 2), Pⱕ0.385 (2 vs. 3), and Pⱕ0.001 (1 vs. 3). Importantly, although the final visual outcome in group 3 was poor (20/1100), this represented a mean posttreatment VA improvement of 0.38 logMAR for this group (Fig 2). Figure 2 shows the change in mean logMAR VA (final minus initial) after treatment for patients with initial VAs of 20/15 to 20/60 (group 1), 20/70 to 20/200 (group 2), or 20/400 or worse (group 3). There was a slight worsening in VA for group 1, and this was more marked in group 2, although an improvement was seen in group 3. The differences between all groups were statistically significant: Pⱕ0.045 (1 vs. 2), Pⱕ0.006 (2 vs. 3), and Pⱕ0.041 (1 vs. 3), using the nonpaired t test (1 tailed).
Figure 2. Chart showing relationship between change in mean logarithm of the minimum angle of resolution (LogMAR) visual acuity (VA) (final minus initial) versus initial VA. The differences between group 1 (20/15– 20/60), group 2 (20/70 –20/200), and group 3 (20/400 or worse) were all statistically significant, so that Pⱕ0.045 (1 vs. 2), Pⱕ0.006 (2 vs. 3), and Pⱕ0.041 (1 vs. 3), using the nonpaired t test (1 tailed).
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Figure 3. Chart showing relationship between change in mean logarithm of the minimum angle of resolution (LogMAR) visual acuity (VA) (final minus initial) and time to suspected diagnosis of Acanthamoeba. Differences between groups did not reach statistical significance (Pⱕ0.24, nonpaired t test).
Six patients had a final visual outcome of light perception (LP) or worse, 1 of these with no LP and another 3 who underwent enucleation. In these cases, the visual outcome at 6 months was taken as the final VA for Figures 1 to 4. Mean length of time from initial symptoms to the suspected diagnosis of Acanthamoeba was 52 days (range, 1–500), most of these patients having been referred from other centers for diagnosis. However, final diagnosis with confocal microscopy was immediate in all but 2 cases, one of which later cultured positive for fungal keratitis, whereas another was highly uncooperative during the confocal examination but later cultured positive for Acanthamoeba. Figure 3 shows the relationship between change in mean logMAR VA (final minus initial) and time from initial symptoms to the suspected diagnosis of Acanthamoeba. The mean VA remained virtually unchanged in those patients with a time to suspected diagnosis of ⬍7 days or ⬎28 days, whereas those between 7 and 28 days showed a relative worsening in vision; however, these differences did not reach statistical significance (Pⱕ0.24). Figure 4 shows the relationship between change in mean logMAR VA (final minus initial) and topical corticosteroid use before final diagnosis. A history of topical steroid use before presentation was present in 30 cases and was associated with a trend toward greater worsening in vision as compared with those without prior steroid use; however, these differences did not reach statistical significance (Pⱕ0.32).
Figure 4. Chart showing relationship between change in mean logarithm of the minimum angle of resolution (LogMAR) visual acuity (VA) (final minus initial) and topical corticosteroid use before final diagnosis. Differences between groups did not reach statistical significance (Pⱕ0.32, nonpaired t test).
Parmar et al 䡠 Confocal Microscopy in Suspected Acanthamoeba Keratitis Table 2. Initial Misdiagnosis of Patients Later Found to Have Acanthamoeba, Some Patients Having Conditions That Masqueraded with Multiple Diagnoses Herpes simplex keratitis Bacterial keratitis Fungal keratitis Varicella zoster keratitis Ebstein–Barr viral keratitis Anesthetic abuse Keratoconjunctivitis sicca
26 30 3 1 1 1 3
Initial Misdiagnosis (Masquerade) Only 11 cases of 63 were suspected as AK from the outset. The remaining 52 initially masqueraded with a variety of misdiagnoses (Table 2).
Clinicopathological Correlation with Confocal Microscopy All patients underwent in vivo diagnostic TSCM, the results of which are shown in Tables 3 and 4. Cultures of cornea and contact lenses, cases, or solutions were performed in 35 patients, whereas corneal biopsy was only done in 6. In those 28 cases that did not undergo culture, confocal microscopy results were predominantly unequivocal, and the suspect contact lens and case were often unavailable. Culture would have yielded little additional diagnostic information in these patients, and thus, unnecessary corneal trauma was prevented. Cases that were equivocal for either Acanthamoeba or fungi on confocal microscopy were reported as negative. Figures 5 and 6 are clinical slit-lamp photographs demonstrating the diverse spectrum of early and late clinical presentation in AK. Indeed, the correlation between clinical (Figs 5, 6) and histopathological findings in AK has been well established in the Yucatan micropig model, after infection by Acanthamoebaladen soft contact lenses.22 Figure 5 thus shows epithelial and subepithelial disease, whereas increasingly advanced stages are shown in Figure 6, with segmental disease (Fig 6A), a ring infiltrate (Fig 6B), and classic radial keratoneuritis (Fig 6C) all clearly seen. Figure 6D shows keratitis secondary to combined infection with Acanthamoeba and filamentous fungi; the corresponding confocal micrographs are seen in Figure 8A–C. Figures 7 and 8 are tandem scanning confocal micrographs demonstrating the characteristic features of AK on TSCM. Figure 7 shows the ovoid Acanthamoeba cyst, which is characteristically surrounded by an optically lucent halo representing a microcavity. The double-walled nature is not always apparent, depending on the obliqueness of the optical section from the TSCM. The thinner linear nature of the trophozoite also is demonstrated (Fig 7B), together with its proximity to subepithelial corneal nerves that are
presumably being ingested. It is important to note, however, that with TSCM it is much easier to see cysts than trophozoites. Figure 8 shows tandem scanning confocal micrographs of keratitis caused by simultaneous infection with Acanthamoeba and filamentous fungi (Fig 8A–C), corresponding to the clinical slitlamp photograph shown in Figure 6D. Multiple filamentous fungi and Acanthamoeba cysts are clearly apparent (Fig 8A), whereas the presence of deep lacunae represents focal tissue loss associated with filamentous fungal hyphal elements (Fig 8B). Longstanding tissue destruction by Acanthamoeba characteristically leaves a honeycomb pattern of optically lucent microcavities in the stroma, which is clearly demonstrated here (Fig 8C). Fungal hyphal elements can be differentiated clearly from microcrystalline deposits, as seen in Figure 8D, which shows a tandem scanning confocal micrograph of gatifloxacin crystals in the anterior stroma of a donor cornea after penetrating keratoplasty. Note the regular stellate pattern of these crystals and also the much thicker nature of the crystal deposits compared with the filamentous branching nature of hyphal elements seen in Figure 8A–C, which are distributed in a much more random, irregular pattern. It is clear from Figures 5 to 8 that both cystic and trophozoite forms of Acanthamoeba can be recognized by TSCM and distinguished from infection with filamentous fungi, infectious microcrystalline changes, or crystalline drug deposits.
Discussion In this retrospective single institutional case series, in vivo corneal TSCM rapidly and noninvasively established the underlying diagnosis in 63 patients referred with suspected AK. Because the clinical appearance of AK can vary, the early signs are often nonspecific, masquerading as other infections.7,8,23 The most common initial misdiagnosis is herpes simplex keratitis, and this was the case in 26 (41%) of our patients. Indeed, the number of patients suspected as having AK from the outset was only 11, leading to a significant delay in diagnosis in the other cases. Two patients whose conditions had clinically first masqueraded as AK were later found to have a fungal etiology based on fungal-positive TSCM. Contact lens wear is well established as a major risk factor for AK, and this is clearly confirmed in our series, where it was documented in 54 patients (86%).2– 4,24 Indeed, the higher prevalence of contact lens wear in females may explain their higher incidence of AK relative to males in this series. The greater incidence in soft lens wearers compared with patients wearing rigid gas-permeable lenses also agrees with previous studies.4,24 One patient developed AK in the fourth year of orthokeratology treatment for myopia. This is
Table 3. Diagnosis by Confocal Microscopy and Culture
Cultured Culture-positive Acanthamoeba Culture-positive fungus Culture-positive Pseudomonas Culture-negative Not cultured Total confocal
Confocal, Acanthamoeba Positive
Confocal, Fungus Positive
Confocal, Positive for Fungus and Acanthamoeba
Confocal, Negative
9 1 0 19 25 54
0 0 0 1 1 2
0 0 0 1 0 1
1 0 1 2 2 6
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Biopsied Biopsy-positive Acanthamoeba Biopsy-positive fungus Biopsy-negative Not biopsied Total confocal
Confocal, Acanthamoeba Positive
Confocal, Fungus Positive
Confocal, Positive for Fungus and Acanthamoeba
Confocal, Negative
2 0 2 50 54
0 0 1 1 2
0 0 0 1 1
0 0 1 5 6
a rare complication of orthokeratology, previously reported in 12 patients in the literature.25–27 The incidence of contact lens–related AK is still uncertain but has been estimated as 1.36 per million in the United States.4 However, the actual rate may be 10 times higher when confocal microscopy is used diagnostically, as compared with culture.10 At these infection rates, 1 in 300 to 1500 contact lens wearers may develop some form of AK over a 30-year period of wearing contacts.1 In view of the ubiquitous presence of Acanthamoeba in the environment, the actual rate of infection would be expected to be much higher, but local immunity and host defense may serve to limit the incidence of corneal involvement.28,29 A significant risk factor for AK is the exposure to environmental sources of water, as was seen in 25 cases of our study.2– 4 This included the use of tap water to clean lenses; swimming in freshwater lakes, rivers, or swimming pools; and the use of hot tubs (Table 1). The use of well water does not seem to have been documented clearly in previous reports, but it was a definite risk factor in 2 of our patients. The presence of minor preceding trauma has been reported to be a risk factor, as was seen in 11 of our patients.2 Similarly, a compromised ocular surface is a known risk factor for AK, as was described in 2 of our patients with keratoconjunctivitis sicca; this also may have been a predisposing factor in 4 of our patients with a history of corneal surgery, 3 of whom had had previous keratorefractive procedures. Acanthamoeba is known to be an opportunistic pathogen, such that skin, sinus, cerebral, and disseminated involvement all have been well documented in AIDS, together with a few cases of systemic immunosuppression in the setting of chronic debilitating illness.30 –32 There is a previous report of AK in a non– contact lens wearer with AIDS, but the exact role of this disease in contributing to corneal involvement remains unclear.33 The presence of 6 immunocompromised patients in our series might suggest immunosuppres-
sion to be a risk factor, with 4 having been on a systemic steroid, 1 on systemic steroid/methotrexate/infliximab, and 1 on topical cyclosporine 0.05%. However, systemic immunosuppression also may limit the degree of pathological tissue destruction, as it has been successfully used to control pain and inflammation after eradication of the organism in Acanthamoeba sclerokeratitis.34 Conventional light microscopy is hindered by reflection and diffraction of light from structures surrounding the point of observation, blurring the resulting image.7,11 These effects limit the ideal resolution of slit-lamp biomicroscopy to only 20 m, but practical observation of the cornea clinically is further hindered by any decreased transparency of this tissue in the presence of edema or scarring. To overcome these problems, in 1957 Minsky proposed the concept of confocal microscopy, in which the illumination (condenser) and observation (objective) systems share the same focal plane and are thus confocal.35 The elimination of nonfocused information dramatically improved the lateral resolution to an order of 1 to 2 m and the axial resolution to 5 to 10 m.7,11,36 The limited field of view provided by confocal systems makes it necessary to scan the focal point across the sample rapidly and to reconstruct the image to allow a real-time on-screen view.11 There are currently only 3 such designs approved by the U.S. Food and Drug Administration: (1) the tandem scanning confocal microscope, (2) slit-scanning confocal microscope, and (3) Heidelberg II retina– cornea tomographic unit (with a special anterior segment attachment) (Heidelberg Engineering, Inc., Vista, CA).11,37 The tandem scanning confocal microscope (Tandem Scanning) used in our study utilizes a Nipkow disc, which is a metal plate with 20- to 30-m-diameter pinholes arranged in an archimedean spiral. All of these pinholes provide multiple single-spot illuminations, so that rapid rotation of this disc allows scanning of the entire specimen. This instrument has been used to examine the cornea qual-
™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™3 Figure 5. Clinical photographs showing early stages of Acanthamoeba keratitis. A–C, Patient at initial presentation showing diffuse epithelial and subepithelial involvement. D, Another patient showing subepithelial disease.
™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™3 Figure 6. Clinical photographs showing the diverse spectrum of Acanthamoeba keratitis, increasingly advanced compared with Figure 5. A, Confocal microscopy photograph of segmental subepithelial involvement. B, C, Confocal microscopy photographs of a different patient’s classic ring infiltrate (B) and radial keratoneuritis (C) after orthokeratology treatment for myopia with overnight rigid gas-permeable contact lens wear. D, Another patient’s keratitis secondary to combined infection with Acanthamoeba and filamentous fungi.
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Figure 7. Tandem scanning confocal micrographs of Acanthamoeba keratitis (width of each micrograph represents 400 m). A, Typical cyst (15–30 m) is seen as a bright oval particle, the double-walled nature partially apparent (to the left of the main cyst) through the oblique optical section taken here. B, Typical rounded cyst (arrowhead) and linearly elongated trophozoite adjacent to and presumably ingesting a subepithelial corneal nerve (arrow). C, Two cysts (arrows) surrounded by a nonreflecting circumferential cavity. The stroma has several areas in the background that are optically lucent distributed in a honeycomb pattern, reflecting microcavities of focal stromal tissue destruction.
itatively and to estimate corneal thickness, haze, nerve fiber density, and cell density of the epithelium, stroma, and endothelium.11,38 The technology of this instrument was recently acquired by Advanced Scanning. Our 63 patients undergoing tandem scanning confocal corneal microscopy for suspected AK would seem to represent the largest clinical experience in the current literature. Mathers et al reported 51 patients who underwent TSCM for suspected AK, 36 of them clearly positive on confocal examination.39 The remaining 15 cases were equivocal on confocal scanning, 7 of them positive for Acanthamoeba on biopsy. Garg et al recently compared the Confoscan 3.0 scanningslit confocal microscope (Nidek Technologies, Greensboro, NC) with conventional microbiology for the diagnosis of
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infectious keratitis in a prospective, double masked, nonrandomized clinical trial (Garg P, Krishma PV, Sharma S, et al. Role of confocal microscopy in the diagnosis of microbial keratitis. Paper presented at: Cornea Society and Eye Bank Association of America at the American Academy of Ophthalmology Annual Meeting, October 2004; New Orleans, LA). Confocal microscopy correctly diagnosed 83 of 93 fungal cases and 8 of 10 AK cases, whereas it excluded fungus or Acanthamoeba in 41 of 45 cases that were negative on microbiological testing. Another series also has been reported in 15 eyes using the Nidek Confoscan 2.0 scanning-slit confocal microscope to confirm the diagnosis of AK in biopsy- or culture-proven disease.15 Tandem scanning confocal microscopy successfully diagnosed 61 of 63 cases in our series, with 1 false-positive
Parmar et al 䡠 Confocal Microscopy in Suspected Acanthamoeba Keratitis
Figure 8. Tandem scanning confocal micrographs of keratitis caused by simultaneous infection with Acanthamoeba and filamentous fungi (A–C), corresponding to a clinical photograph (D) (width of each micrograph represents 400 m. A, Multiple filamentous fungi (arrow) and an adjacent Acanthamoeba cyst (arrowhead) are clearly apparent. B, Deep lacunae (arrows) are seen secondary to focal tissue loss by filamentous fungal hyphal elements (arrowhead). C, Tissue destruction by Acanthamoeba has left a honeycomb pattern of optically lucent microcavities in the stroma (arrowheads), as seen in Fig 7C. The filamentous fungi are also apparent (arrow). D, For comparison, a tandem scanning confocal micrograph of gatifloxacin crystals in the anterior stroma of a donor cornea after penetrating keratoplasty (reprinted with permission from Awwad ST, Haddad W, Wang MX, et al. Corneal intrastromal gatifloxacin crystal deposits after penetrating keratoplasty. Eye Contact Lens 2004;30:169 –72). Note the regular pattern of these crystals and also the much thicker nature of the crystal deposits compared with the filamentous-branching nature of hyphal elements seen in A–C, which are distributed in a much more random, irregular pattern.
and 1 false-negative based on clinical data. One case was equivocally (false) positive on confocal for Acanthamoeba but clearly positive on culture for fungus (Fusarium species), responding favorably to appropriate antifungal treatment. Another case was falsely negative on confocal examination but clearly positive on culture for Acanthamoeba, responding favorably to anti-acanthamoebal treatment. Both of these patients were in great pain and intensely photophobic, with uncontrolled eye movements making an adequate examination difficult to obtain on TSCM. The majority of cases (54/63) were positive for AK on TSCM, and all of them showed a favorable response to treatment. However, the yield of positive cultures in this group was relatively low (9/29 [31%]), whereas the yield of positive corneal biopsy was only 50% (2/4). Only 6 cases were negative for AK on TSCM, of which 4 underwent corneal culture, 1 was culture
positive for Acanthamoeba, and 1 was culture positive for Pseudomonas. Two cases initially masqueraded clinically as Acanthamoeba but were subsequently positive for fungus on TSCM. They responded well to standard antifungal treatment for keratitis, despite negative corneal biopsy and culture results. One patient was positive for both fungal hyphae and Acanthamoeba on TSCM, but negative on both corneal culture and biopsy. She responded favorably to combined antifungal and anti-acanthamoebal treatment. The mean final VA (20/47) was better in those patients who started with the best initial VA of 20/15 to 20/60 (Fig 1). Those with an initial VA of 20/70 to 20/200 or 20/400 and worse had mean final VAs of 20/510 or 20/1100, respectively. This is not altogether surprising, as patients with a poor initial VA are more likely to have had worse scarring and pathological corneal damage from the outset. Most
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Ophthalmology Volume 113, Number 4, April 2006 interestingly, those patients with the worst initial VA (20/ 400 or worse) had the greatest scope for visual improvement (0.38 logMAR), confirming that treatment was most helpful in those patients with the greatest initial visual disability (Figs 1, 2). The early diagnosis of AK has been shown to be associated with a more favorable outcome.7,8,40,41 Our results suggest that a slight improvement in vision is possible if the delay in diagnosis is ⬍7 days, although this was not statistically significant (Fig 3). Worsening of vision was seen with a diagnostic delay between 7 to 28 days, but above 28 days this was not as marked. The earlier diagnosis certainly would be expected to contribute to a better visual outcome, in line with previous studies. However, a few patients in our series who were diagnosed early had poor outcomes, whereas some diagnosed later had excellent visual results. This may be related to the variable virulence of the many strains of pathogenic Acanthamoeba as well as differential susceptibility to current therapeutic agents.24 This may explain why patients with the greatest delay in diagnosis had worsening of vision lesser than that of those with a diagnostic delay between 7 and 28 days (Fig 3). In addition, many of our patients were referred from other centers, where the diagnosis was uncertain and drug treatment for Acanthamoeba had already been initiated, together with topical steroids, thus modifying the healing response. There seemed to be a trend toward greater worsening of VA (Pⱕ0.07) in those patients with a history of topical corticosteroid use compared with those without (Pⱕ0.01), although this difference was not statistically significant. It seems logical that previous corticosteroid use would decrease the local immunity to Acanthamoeba, which is well documented in the tear film, potentially worsening the pathological damage caused by the organism and delaying the clearing response.29 However, the exact degree of topical steroid use before presentation in most cases was difficult to quantify, limiting the statistical significance of such a comparison. Moreover, a previous retrospective study of 38 patients showed that topical corticosteroid use was not associated with a higher rate of medical treatment failure in AK, so the issue remains equivocal.41 The majority of cases were treated successfully with 2, 3, or more anti-Acanthamoeba drugs; however, 3 patients underwent enucleation for persistent pain and blindness. Although drug treatment has been revolutionized by the use of biguanides such as chlorhexidine and polyhexamethylbiguanide, a few cases may still fail to respond.24,40 The greatest challenge remains the effective eradication of Acanthamoeba cysts, which may be resistant even to multiple intensive drug treatment in individual cases. Further research has focused on several drugs of the guanidine family along with the alkylphosphocholines and, possibly, topical metronidazole as potential treatments for the future.1,24,42 The yield of positive cultures in confocal positive patients was relatively low (9/29 [31%]), as was the yield of positive corneal biopsy (2/4 [50%]). The results in the literature show variability in these diagnostic methods. In Mathers et al’s series, 13 patients who were positive for Acanthamoeba by either TSCM or epithelial biopsy underwent culture of corneal scrapings plated on E. coli–laden
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nonnutrient agar, but these were all negative.39 Acanthamoebic culture of the contact lens case in one of these cases was also negative. Bacon et al’s series quotes positive corneal cultures in 10 of 14 (71%) intermediate and 17 of 23 (74%) late cases, although culture was not done in patients presenting before 28 days of initial symptoms.8 This variability in yield may be laboratory or experience specific. A further disadvantage with reliance on culture yield is the delay in diagnosis, which may take days to weeks; certainly this can be obviated by the immediate diagnosis offered through TSCM. Many cases that were diagnosed as positive for AK were negative on corneal culture, but theoretically, many of these cases may have been positive at a later date with repeat biopsy. In summary, TSCM is a noninvasive diagnostic method that can provide immediate diagnosis in suspected AK with a high degree of confidence. This is particularly useful when microbiology or histopathology is inconclusive or not readily available to the initial clinician. The rapidity of diagnosis, relative ease of use, and noninvasive nature of in vivo confocal microscopy are clearly advantageous when compared with traditional laboratory techniques, which all rely on blind invasive sampling of the cornea and are associated with a subsequent delay in the culture result. Importantly, in this consecutive case series spanning 10 years there were only one false-positive and one false-negative TSCM result, all encountered in uncooperative patients. Immediate diagnosis by TSCM from the outset avoids diagnostic delay and would certainly be expected to lead to an improved visual outcome in AK. In cases in which a satisfactory TSCM examination cannot be acquired, however, traditional diagnostic methods such as corneal culture and biopsy must be utilized.
References 1. Mathers WD. Acanthamoeba: a difficult pathogen to evaluate and treat. Cornea 2004;23:325. 2. Radford CF, Minassian DC, Dart JK. Acanthamoeba keratitis in England and Wales: incidence, outcome, and risk factors. Br J Ophthalmol 2002;86:536 – 42. 3. Radford CF, Lehmann OJ, Dart JK. Acanthamoeba keratitis: multicentre survey in England 1992– 6. National Acanthamoeba Keratitis Study Group. Br J Ophthalmol 1998;82:1387–92. 4. Stehr-Green JK, Bailey TM, Visvesvara GS. The epidemiology of Acanthamoeba keratitis in the United States. Am J Ophthalmol 1989;107:331– 6. 5. Illingworth CD, Cook SD, Karabatsas CH, Easty DL. Acanthamoeba keratitis: risk factors and outcome. Br J Ophthalmol 1995;79:1078 – 82. 6. Sharma S, Pasricha G, Das D, Aggarwal RK. Acanthamoeba keratitis in non-contact lens wearers in India: DNA typingbased validation and a simple detection assay. Arch Ophthalmol 2004;122:1430 – 4. 7. Claerhout I, Goegebuer A, Van Den Broecke C, Kestelyn P. Delay in diagnosis and outcome of Acanthamoeba keratitis. Graefes Arch Clin Exp Ophthalmol 2004;242:648 –53. 8. Bacon AS, Dart JK, Ficker LA, et al. Acanthamoeba keratitis. The value of early diagnosis. Ophthalmology 1993; 100:1238 – 43. 9. Moore MB, McCulley JP. Acanthamoeba keratitis associated
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11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25. 26.
with contact lenses: six consecutive cases of successful management. Br J Ophthalmol 1989;73:271–5. Mathers WD, Nelson SE, Lane JL, et al. Confirmation of confocal microscopy diagnosis of Acanthamoeba keratitis using polymerase chain reaction analysis. Arch Ophthalmol 2000; 118:178 – 83. Kaufman SC, Musch DC, Belin MW, et al. Confocal microscopy: a report by the American Academy of Ophthal mology. Ophthalmology 2004;111:396 – 406. Petroll WM, Cavanagh HD, Jester JV. Clinical confocal microscopy. Curr Opin Ophthalmol 1998;9:59 – 65. Jalbert I, Stapleton F, Papas E, et al. In vivo confocal microscopy of the human cornea. Br J Ophthalmol 2003;87:225–36. Pfister DR, Cameron JD, Krachmer JH, Holland EJ. Confocal microscopy findings of Acanthamoeba keratitis. Am J Ophthalmol 1996;121:119 –28. Nakano E, Oliveira M, Portellinha W, et al. Confocal microscopy in early diagnosis of Acanthamoeba keratitis. J Refract Surg 2004;20(5 suppl):S737– 40. Avunduk AM, Beuerman RW, Varnell ED, Kaufman HE. Confocal microscopy of Aspergillus fumigatus keratitis. Br J Ophthalmol 2003;87:409 –10. Muallem MS, Alfonso EC, Romano AC, et al. Bilateral Candida parapsilosis interface keratitis after laser in situ keratomileusis. J Cataract Refract Surg 2003;29:2022–5. Linna T, Mikkila H, Karma A, et al. In vivo confocal microscopy: a new possibility to confirm the diagnosis of Borrelia keratitis? Cornea 1996;15:639 – 40. Shah GK, Pfister D, Probst LE, et al. Diagnosis of microsporidial keratitis by confocal microscopy and the chromatrope stain. Am J Ophthalmol 1996;121:89 –91. Li J, Jester JV, Cavanagh HD, et al. On-line 3-dimensional confocal imaging in vivo. Invest Ophthalmol Vis Sci 2000; 41:2945–53. D’Aversa G, Stern GA, Driebe WT Jr. Diagnosis and successful medical treatment of Acanthamoeba keratitis. Arch Ophthalmol 1995;113:1120 –3. He YG, McCulley JP, Alizadeh H, et al. A pig model of Acanthamoeba keratitis: transmission via contaminated contact lenses. Invest Ophthalmol Vis Sci 1992;33:126 –33. Bernauer W, Duguid GI, Dart JK. Early clinical diagnosis of acanthamoeba keratitis. A study of 70 eyes [in German]. Klin Monatsbl Augenheilkd 1996;208:282– 4. Seal DV. Acanthamoeba keratitis update—incidence, molecular epidemiology and new drugs for treatment. Eye 2003;17: 893–905. Lu L, Zou L, Wang R. Orthokeratology induced infective corneal ulcer [in Chinese]. Zhonghua Yan Ke Za Zhi 2001; 37:443– 6. Xuguang S, Lin C, Yan Z, et al. Acanthamoeba keratitis as a
27. 28.
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38. 39. 40. 41. 42.
complication of orthokeratology. Am J Ophthalmol 2003;136:1159 – 61. Young AL, Leung AT, Cheng LL, et al. Orthokeratology lens-related corneal ulcers in children. A case series. Ophthalmology 2004;111:590 –5. Alizadeh H, Neelam S, Hurt M, Niederkorn JY. Role of contact lens wear, bacterial flora, and mannose-induced pathogenic protease in the pathogenesis of amoebic keratitis. Infect Immun 2005;73:1061– 8. Alizadeh H, Apte S, El-Agha MS, et al. Tear IgA and serum IgG antibodies against Acanthamoeba in patients with Acanthamoeba keratitis. Cornea 2001;20:622–7. Rosenberg AS, Morgan MB. Disseminated acanthamoebiasis presenting as lobular panniculitis with necrotizing vasculitis in a patient with AIDS. J Cutan Pathol 2001;28:307–13. Martinez AJ, Janitschke K. Acanthamoeba, an opportunistic microorganism: a review. Infection 1985;13:251– 6. Martinez AJ. Acanthamoebiasis and immunosuppression. Case report. J Neuropathol Exp Neurol 1982;41:548 –57. Hansen B, Kronborg G. Acanthamoeba keratitis in a non-contact lens wearer with human immunodeficiency virus. Scand J Infect Dis 2003;35:207–9. Lee GA, Gray TB, Dart JK, et al. Acanthamoeba sclerokeratitis: treatment with systemic immunosuppression. Ophthalmology 2002;109:1178 – 82. Minsky M. Memoir on inventing the confocal scanning microscope. Scanning 1988;10:128 –38. Petroll WM, Cavanagh HD, Jester JV. Three-dimensional imaging of corneal cells using in vivo confocal microscopy. J Microsc 1993;170:213–9. Stave J, Zinser G, Grummer G, Guthoff R. Modified Heidelberg Retinal Tomograph HRT. Initial results of in vivo presentation of corneal structures [in German]. Ophthalmologe 2002;99:276 – 80. McLaren JW, Nau CB, Kitzmann AS, Bourne WM. Keratocyte density: comparison of two confocal microscopes. Eye Contact Lens 2005;31:28 –33. Mathers WD, Sutphin JE, Folberg R, et al. Outbreak of keratitis presumed to be caused by Acanthamoeba. Am J Ophthalmol 1996;121:129 – 42. Duguid IG, Dart JK, Morlet N, et al. Outcome of acanthamoeba keratitis treated with polyhexamethyl biguanide and propamidine. Ophthalmology 1997;104:1587–92. Park DH, Palay DA, Daya SM, et al. The role of topical corticosteroids in the management of Acanthamoeba keratitis. Cornea 1997;16:277– 83. van der Bijl P, van Eyk AD, Seifart HI, Meyer D. Diffusion of metronidazole released from aqueous solution and a gel through human and rabbit corneas. J Ocul Pharmacol Ther 2004;20:421–9.
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Acanthamoeba keratitis (AK) is a rare condition, with a recent study estimating a severe infection rate of 1.2 per million adults and 0.2 per 10 000 contact lenses wearers per year.1,2 However, the protozoan is difficult to culture, and the true incidence could be almost 10 times higher than previously suggested. During the 1980s, a dramatic rise in the incidence of AK was seen in conjunction with the growing clinical use of soft contact lenses, largely attributed to the use of nonsterile lens solutions, swimming while wearing lenses, and inadequate disinfection practices.3,4 To date, contact lens wear remains the single most prevalent risk factor, particularly when associated with environmental Originally received: June 5, 2005. Accepted: December 14, 2005. Manuscript no. 2005-489. From the Division of Cornea, External Disease and Refractive Surgery, Department of Ophthalmology, University of Texas Southwestern Medical Center, Dallas, Texas. Presented at: American Academy of Ophthalmology meeting, October, 2004; New Orleans, Louisiana. Supported in part by National Eye Institute, Bethesda, Maryland (grant nos.: EY10738 [HDC], EY016664); The Pearle Vision Foundation, Dallas, Texas; and an unrestricted grant from Research to Prevent Blindness, Inc., New York, New York. Correspondence and reprint requests to H. Dwight Cavanagh, MD, PhD, Department of Ophthalmology, University of Texas Southwestern Medical Center at Dallas, 5323 Harry Hines Boulevard, Dallas, TX 75390-9057. E-mail: [email protected].
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water exposure, although Acanthamoeba species are also increasingly recognized as an important cause of keratitis in non– contact lens wearers.5,6 Early diagnosis is invaluable and is associated with a relatively satisfactory outcome, whereas the consequences of late diagnosis can be devastating.7,8 The variability in presentation of patients with AK often makes clinical diagnosis difficult, as the early signs can be nonspecific.1,7 Current laboratory diagnostic techniques relying on culture are limited in yield, with positive rates quoted from 0% to 68% in cases negative for contact lens case culture, whereas in some series Acanthamoeba has been grown from contact lenses or storage cases where corneal epithelial samples were culture negative.5,7–9 The advent of direct testing using the polymerase chain reaction (PCR) is encouraging, although this is not yet a firmly established diagnostic modality and requires particular laboratory expertise.6,10 However, the lack of specific multispecies diagnostic epitopes necessitates the use of multiple low-yield single probes. This increases the expense involved and makes the widespread clinical use of PCR in AK unlikely. The principal disadvantage of laboratory diagnostic techniques lies in the reliance on blind invasive sampling of the cornea, either by scraping or biopsy, and subsequent delay in culture results. Tandem scanning confocal microscopy (TSCM) is a noninvasive diagnostic modality allowing both a qualitative and a quantitative analysis of the entire cornea ISSN 0161-6420/06/$–see front matter doi:10.1016/j.ophtha.2005.12.022
Parmar et al 䡠 Confocal Microscopy in Suspected Acanthamoeba Keratitis to be performed rapidly, delivering immediate diagnosis in vivo.11–13 It is particularly useful when relatively large infecting organisms (ⱖ15 m) are present, as are seen in Acanthamoeba, filamentous fungal, microsporidial, and, possibly, Lyme Borrelia keratitis.14 –19 Confocal microscopy can clearly demonstrate both the cyst and often the trophozoite forms of Acanthamoeba in suspected keratitis. It also shows the enlarged corneal nerves accompanying radial neurokeratitis and the characteristic honeycomb-pattern intrastromal microcavities seen during the late stages of the disease. The rapidity of diagnosis, relative ease of use, and noninvasive nature of confocal microscopy are clearly advantageous when compared to traditional laboratory techniques in suspected AK.14 We report 63 patients with suspected AK who were referred consecutively for establishment of diagnosis by TSCM. This represents the largest such series to date with complete follow-up, as confirmed by an extensive literature search through the Medline and Ovid biomedical databases. We discuss the merits, potential uses, and possible disadvantages of TSCM in this setting, as well as the implications of early diagnosis by this modality.
Materials and Methods Institutional review board approval was obtained before commencement of this study. A detailed retrospective case analysis was performed of patients with suspected AK consecutively referred to the University of Texas Southwestern Medical Center, Dallas, Texas, from 1994 to 2004. All patients had undergone TSCM (Tandem Scanning Corp., Reston, VA [currently Advanced Scanning Corp., New Orleans, LA]) for diagnostic evaluation. Of these patients, only those with complete follow-up data qualified for inclusion in the study; of a total of 90 referred cases, 63 met these inclusion criteria. A video camera connected to both an S-VHS video recorder and a black-and-white monitor was used to record images, which were archived accurately for later reference on S-VHS videotape. Confocal microscopy through focusing was performed, which allowed a 3-dimensional reconstruction of the cornea to be created using established personal computer– based analysis software.20 Features unique to Acanthamoeba or fungi were sought for specifically in the captured images. Data acquisition identified predisposing risk factors for AK, method of treatment, and initial and final diagnoses, the latter confirmed by clinical response to treatment. In those patients who had undergone culture of the cornea and/or contact lens case, standard microbiological cultures had been carried out for bacteria, fungi, and Acanthamoeba, the latter specifically involving growth on Escherichia coli–seeded nonnutrient agar plates.21 To assess the effects of initial vision on final visual outcome, the patients were divided arbitrarily into 3 groups of similar logarithm of the minimum angle of resolution (logMAR) vision ranges according to presenting visual acuities (VAs) of (1) 20/15 to 20/60, (2) 20/70 to 20/200, and (3) 20/400 or worse. The length of time to diagnosis of suspected AK was also recorded and divided into 3 groups of (1) ⬍7 days, (2) 7 to 28 days, and (3) ⬎28 days. The relationship between initial VA and final visual outcome was examined. The effects of the following parameters on the mean change in VA after treatment were also analyzed: (1) initial VA, (2) time to suspected Acanthamoeba diagnosis, and (3) prior topical corticosteroid use. Statistical analysis employing nonpaired t tests was performed with SigmaStat software (Systat Software, Inc., Point Richmond, CA).
Results Demographics A total of 90 cases of suspected AK were referred to us for TSCM. Many of these patients were sent from other centers, so that only 63 had complete follow-up data to meet the study inclusion criteria. Nineteen patients were male and 44 female, with a median patient age of 34 years (range, 13–72) and a mean follow-up of 14 months.
Predisposing Factors Contact lens wear was the most significant risk factor, prevalent in 54 patients with a mean length of prior contact lens wear of 111 months (range, 1– 470). The remaining identified risk factors are summarized in Table 1. The majority of these were soft contact lens wearers, although one case was documented in a patient undergoing the fourth year of orthokeratology treatment for myopia with rigid gas-permeable lenses.
Treatment A total of 56 cases were treated for AK after diagnosis by confocal microscopy, culture, or biopsy. Thirty-four of these cases were treated with ⱖ3 topical acanthamoebicidal agents, 20 with 2 drugs, and 2 cases with only propamidine isethionate (Brolene, Aventis Ltd., West Malling, United Kingdom). Other antiamoebic drugs used include topical polyhexamethylbiguanide, chlorhexidine, neomycin, and clotrimazole. The mean duration of treatment for anti-Acanthamoeba treatment was 8.4 months. Table 1. Predisposing Factors in Patients with Suspected Acanthamoeba Keratitis n CTL wear Soft, 40 RGP, 12 Toric, 1 Orthokeratology, 1 Sleeping with CTL Recent new CTL fit Corneal ectasia Keratoconus, 2 Post-LASIK/RK ectasia, 1 Previous corneal surgery RK, 1 LASIK, 1 RK and LASIK, 1 PK, 1 Cleaning with tap water; swimming in freshwater lakes, rivers, or swimming pools, or hot tub use; exposure to bath/shower water with CTL in place Inappropriate cleaning solution Tap water, 15 Well water, 2 Previous trauma Dry eye Immunocompromised Systemic steroid, 4 Systemic steroid ⫾ methotrexate ⫾ infliximab, 1 Topical cyclosporine 0.05%, 1
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15 4 3 4
25 17 11 2 6
CTL ⫽ contact lens; HIV ⫽ human immunodeficiency virus; PK ⫽ penetrating keratoplasty; RGP ⫽ rigid gas permeable; RK ⫽ radial keratotomy.
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Figure 1. Chart showing relationship between mean final logarithm of the minimum angle of resolution (LogMAR) visual acuity (VA) and initial VA. Snellen equivalent mean final VAs are also displayed. The differences were statistically significant between group 1 (20/15–20/60) and group 2 (20/70 –20/200) and between group 1 and group 3 (20/400 or worse), but not between groups 2 and 3. Using the nonpaired t test (2 tailed), Pⱕ0.002 (1 vs. 2), Pⱕ0.385 (2 vs. 3), and Pⱕ0.001 (1 vs. 3).
Visual Outcome Figure 1 shows the mean final logMAR VA for all 3 groups. The best outcome was seen in group 1 (20/47), whereas those in group 2 (20/510) and group 3 (20/1100) fared much worse. The differences were statistically significant between group 1 (20/15–20/60) and group 2 (20/70 –20/200) and between group 1 and group 3 (20/400 or worse), but not between group 2 and group 3. Using the nonpaired t test (2 tailed), Pⱕ0.002 (1 vs. 2), Pⱕ0.385 (2 vs. 3), and Pⱕ0.001 (1 vs. 3). Importantly, although the final visual outcome in group 3 was poor (20/1100), this represented a mean posttreatment VA improvement of 0.38 logMAR for this group (Fig 2). Figure 2 shows the change in mean logMAR VA (final minus initial) after treatment for patients with initial VAs of 20/15 to 20/60 (group 1), 20/70 to 20/200 (group 2), or 20/400 or worse (group 3). There was a slight worsening in VA for group 1, and this was more marked in group 2, although an improvement was seen in group 3. The differences between all groups were statistically significant: Pⱕ0.045 (1 vs. 2), Pⱕ0.006 (2 vs. 3), and Pⱕ0.041 (1 vs. 3), using the nonpaired t test (1 tailed).
Figure 2. Chart showing relationship between change in mean logarithm of the minimum angle of resolution (LogMAR) visual acuity (VA) (final minus initial) versus initial VA. The differences between group 1 (20/15– 20/60), group 2 (20/70 –20/200), and group 3 (20/400 or worse) were all statistically significant, so that Pⱕ0.045 (1 vs. 2), Pⱕ0.006 (2 vs. 3), and Pⱕ0.041 (1 vs. 3), using the nonpaired t test (1 tailed).
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Figure 3. Chart showing relationship between change in mean logarithm of the minimum angle of resolution (LogMAR) visual acuity (VA) (final minus initial) and time to suspected diagnosis of Acanthamoeba. Differences between groups did not reach statistical significance (Pⱕ0.24, nonpaired t test).
Six patients had a final visual outcome of light perception (LP) or worse, 1 of these with no LP and another 3 who underwent enucleation. In these cases, the visual outcome at 6 months was taken as the final VA for Figures 1 to 4. Mean length of time from initial symptoms to the suspected diagnosis of Acanthamoeba was 52 days (range, 1–500), most of these patients having been referred from other centers for diagnosis. However, final diagnosis with confocal microscopy was immediate in all but 2 cases, one of which later cultured positive for fungal keratitis, whereas another was highly uncooperative during the confocal examination but later cultured positive for Acanthamoeba. Figure 3 shows the relationship between change in mean logMAR VA (final minus initial) and time from initial symptoms to the suspected diagnosis of Acanthamoeba. The mean VA remained virtually unchanged in those patients with a time to suspected diagnosis of ⬍7 days or ⬎28 days, whereas those between 7 and 28 days showed a relative worsening in vision; however, these differences did not reach statistical significance (Pⱕ0.24). Figure 4 shows the relationship between change in mean logMAR VA (final minus initial) and topical corticosteroid use before final diagnosis. A history of topical steroid use before presentation was present in 30 cases and was associated with a trend toward greater worsening in vision as compared with those without prior steroid use; however, these differences did not reach statistical significance (Pⱕ0.32).
Figure 4. Chart showing relationship between change in mean logarithm of the minimum angle of resolution (LogMAR) visual acuity (VA) (final minus initial) and topical corticosteroid use before final diagnosis. Differences between groups did not reach statistical significance (Pⱕ0.32, nonpaired t test).
Parmar et al 䡠 Confocal Microscopy in Suspected Acanthamoeba Keratitis Table 2. Initial Misdiagnosis of Patients Later Found to Have Acanthamoeba, Some Patients Having Conditions That Masqueraded with Multiple Diagnoses Herpes simplex keratitis Bacterial keratitis Fungal keratitis Varicella zoster keratitis Ebstein–Barr viral keratitis Anesthetic abuse Keratoconjunctivitis sicca
26 30 3 1 1 1 3
Initial Misdiagnosis (Masquerade) Only 11 cases of 63 were suspected as AK from the outset. The remaining 52 initially masqueraded with a variety of misdiagnoses (Table 2).
Clinicopathological Correlation with Confocal Microscopy All patients underwent in vivo diagnostic TSCM, the results of which are shown in Tables 3 and 4. Cultures of cornea and contact lenses, cases, or solutions were performed in 35 patients, whereas corneal biopsy was only done in 6. In those 28 cases that did not undergo culture, confocal microscopy results were predominantly unequivocal, and the suspect contact lens and case were often unavailable. Culture would have yielded little additional diagnostic information in these patients, and thus, unnecessary corneal trauma was prevented. Cases that were equivocal for either Acanthamoeba or fungi on confocal microscopy were reported as negative. Figures 5 and 6 are clinical slit-lamp photographs demonstrating the diverse spectrum of early and late clinical presentation in AK. Indeed, the correlation between clinical (Figs 5, 6) and histopathological findings in AK has been well established in the Yucatan micropig model, after infection by Acanthamoebaladen soft contact lenses.22 Figure 5 thus shows epithelial and subepithelial disease, whereas increasingly advanced stages are shown in Figure 6, with segmental disease (Fig 6A), a ring infiltrate (Fig 6B), and classic radial keratoneuritis (Fig 6C) all clearly seen. Figure 6D shows keratitis secondary to combined infection with Acanthamoeba and filamentous fungi; the corresponding confocal micrographs are seen in Figure 8A–C. Figures 7 and 8 are tandem scanning confocal micrographs demonstrating the characteristic features of AK on TSCM. Figure 7 shows the ovoid Acanthamoeba cyst, which is characteristically surrounded by an optically lucent halo representing a microcavity. The double-walled nature is not always apparent, depending on the obliqueness of the optical section from the TSCM. The thinner linear nature of the trophozoite also is demonstrated (Fig 7B), together with its proximity to subepithelial corneal nerves that are
presumably being ingested. It is important to note, however, that with TSCM it is much easier to see cysts than trophozoites. Figure 8 shows tandem scanning confocal micrographs of keratitis caused by simultaneous infection with Acanthamoeba and filamentous fungi (Fig 8A–C), corresponding to the clinical slitlamp photograph shown in Figure 6D. Multiple filamentous fungi and Acanthamoeba cysts are clearly apparent (Fig 8A), whereas the presence of deep lacunae represents focal tissue loss associated with filamentous fungal hyphal elements (Fig 8B). Longstanding tissue destruction by Acanthamoeba characteristically leaves a honeycomb pattern of optically lucent microcavities in the stroma, which is clearly demonstrated here (Fig 8C). Fungal hyphal elements can be differentiated clearly from microcrystalline deposits, as seen in Figure 8D, which shows a tandem scanning confocal micrograph of gatifloxacin crystals in the anterior stroma of a donor cornea after penetrating keratoplasty. Note the regular stellate pattern of these crystals and also the much thicker nature of the crystal deposits compared with the filamentous branching nature of hyphal elements seen in Figure 8A–C, which are distributed in a much more random, irregular pattern. It is clear from Figures 5 to 8 that both cystic and trophozoite forms of Acanthamoeba can be recognized by TSCM and distinguished from infection with filamentous fungi, infectious microcrystalline changes, or crystalline drug deposits.
Discussion In this retrospective single institutional case series, in vivo corneal TSCM rapidly and noninvasively established the underlying diagnosis in 63 patients referred with suspected AK. Because the clinical appearance of AK can vary, the early signs are often nonspecific, masquerading as other infections.7,8,23 The most common initial misdiagnosis is herpes simplex keratitis, and this was the case in 26 (41%) of our patients. Indeed, the number of patients suspected as having AK from the outset was only 11, leading to a significant delay in diagnosis in the other cases. Two patients whose conditions had clinically first masqueraded as AK were later found to have a fungal etiology based on fungal-positive TSCM. Contact lens wear is well established as a major risk factor for AK, and this is clearly confirmed in our series, where it was documented in 54 patients (86%).2– 4,24 Indeed, the higher prevalence of contact lens wear in females may explain their higher incidence of AK relative to males in this series. The greater incidence in soft lens wearers compared with patients wearing rigid gas-permeable lenses also agrees with previous studies.4,24 One patient developed AK in the fourth year of orthokeratology treatment for myopia. This is
Table 3. Diagnosis by Confocal Microscopy and Culture
Cultured Culture-positive Acanthamoeba Culture-positive fungus Culture-positive Pseudomonas Culture-negative Not cultured Total confocal
Confocal, Acanthamoeba Positive
Confocal, Fungus Positive
Confocal, Positive for Fungus and Acanthamoeba
Confocal, Negative
9 1 0 19 25 54
0 0 0 1 1 2
0 0 0 1 0 1
1 0 1 2 2 6
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Biopsied Biopsy-positive Acanthamoeba Biopsy-positive fungus Biopsy-negative Not biopsied Total confocal
Confocal, Acanthamoeba Positive
Confocal, Fungus Positive
Confocal, Positive for Fungus and Acanthamoeba
Confocal, Negative
2 0 2 50 54
0 0 1 1 2
0 0 0 1 1
0 0 1 5 6
a rare complication of orthokeratology, previously reported in 12 patients in the literature.25–27 The incidence of contact lens–related AK is still uncertain but has been estimated as 1.36 per million in the United States.4 However, the actual rate may be 10 times higher when confocal microscopy is used diagnostically, as compared with culture.10 At these infection rates, 1 in 300 to 1500 contact lens wearers may develop some form of AK over a 30-year period of wearing contacts.1 In view of the ubiquitous presence of Acanthamoeba in the environment, the actual rate of infection would be expected to be much higher, but local immunity and host defense may serve to limit the incidence of corneal involvement.28,29 A significant risk factor for AK is the exposure to environmental sources of water, as was seen in 25 cases of our study.2– 4 This included the use of tap water to clean lenses; swimming in freshwater lakes, rivers, or swimming pools; and the use of hot tubs (Table 1). The use of well water does not seem to have been documented clearly in previous reports, but it was a definite risk factor in 2 of our patients. The presence of minor preceding trauma has been reported to be a risk factor, as was seen in 11 of our patients.2 Similarly, a compromised ocular surface is a known risk factor for AK, as was described in 2 of our patients with keratoconjunctivitis sicca; this also may have been a predisposing factor in 4 of our patients with a history of corneal surgery, 3 of whom had had previous keratorefractive procedures. Acanthamoeba is known to be an opportunistic pathogen, such that skin, sinus, cerebral, and disseminated involvement all have been well documented in AIDS, together with a few cases of systemic immunosuppression in the setting of chronic debilitating illness.30 –32 There is a previous report of AK in a non– contact lens wearer with AIDS, but the exact role of this disease in contributing to corneal involvement remains unclear.33 The presence of 6 immunocompromised patients in our series might suggest immunosuppres-
sion to be a risk factor, with 4 having been on a systemic steroid, 1 on systemic steroid/methotrexate/infliximab, and 1 on topical cyclosporine 0.05%. However, systemic immunosuppression also may limit the degree of pathological tissue destruction, as it has been successfully used to control pain and inflammation after eradication of the organism in Acanthamoeba sclerokeratitis.34 Conventional light microscopy is hindered by reflection and diffraction of light from structures surrounding the point of observation, blurring the resulting image.7,11 These effects limit the ideal resolution of slit-lamp biomicroscopy to only 20 m, but practical observation of the cornea clinically is further hindered by any decreased transparency of this tissue in the presence of edema or scarring. To overcome these problems, in 1957 Minsky proposed the concept of confocal microscopy, in which the illumination (condenser) and observation (objective) systems share the same focal plane and are thus confocal.35 The elimination of nonfocused information dramatically improved the lateral resolution to an order of 1 to 2 m and the axial resolution to 5 to 10 m.7,11,36 The limited field of view provided by confocal systems makes it necessary to scan the focal point across the sample rapidly and to reconstruct the image to allow a real-time on-screen view.11 There are currently only 3 such designs approved by the U.S. Food and Drug Administration: (1) the tandem scanning confocal microscope, (2) slit-scanning confocal microscope, and (3) Heidelberg II retina– cornea tomographic unit (with a special anterior segment attachment) (Heidelberg Engineering, Inc., Vista, CA).11,37 The tandem scanning confocal microscope (Tandem Scanning) used in our study utilizes a Nipkow disc, which is a metal plate with 20- to 30-m-diameter pinholes arranged in an archimedean spiral. All of these pinholes provide multiple single-spot illuminations, so that rapid rotation of this disc allows scanning of the entire specimen. This instrument has been used to examine the cornea qual-
™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™3 Figure 5. Clinical photographs showing early stages of Acanthamoeba keratitis. A–C, Patient at initial presentation showing diffuse epithelial and subepithelial involvement. D, Another patient showing subepithelial disease.
™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™3 Figure 6. Clinical photographs showing the diverse spectrum of Acanthamoeba keratitis, increasingly advanced compared with Figure 5. A, Confocal microscopy photograph of segmental subepithelial involvement. B, C, Confocal microscopy photographs of a different patient’s classic ring infiltrate (B) and radial keratoneuritis (C) after orthokeratology treatment for myopia with overnight rigid gas-permeable contact lens wear. D, Another patient’s keratitis secondary to combined infection with Acanthamoeba and filamentous fungi.
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Figure 7. Tandem scanning confocal micrographs of Acanthamoeba keratitis (width of each micrograph represents 400 m). A, Typical cyst (15–30 m) is seen as a bright oval particle, the double-walled nature partially apparent (to the left of the main cyst) through the oblique optical section taken here. B, Typical rounded cyst (arrowhead) and linearly elongated trophozoite adjacent to and presumably ingesting a subepithelial corneal nerve (arrow). C, Two cysts (arrows) surrounded by a nonreflecting circumferential cavity. The stroma has several areas in the background that are optically lucent distributed in a honeycomb pattern, reflecting microcavities of focal stromal tissue destruction.
itatively and to estimate corneal thickness, haze, nerve fiber density, and cell density of the epithelium, stroma, and endothelium.11,38 The technology of this instrument was recently acquired by Advanced Scanning. Our 63 patients undergoing tandem scanning confocal corneal microscopy for suspected AK would seem to represent the largest clinical experience in the current literature. Mathers et al reported 51 patients who underwent TSCM for suspected AK, 36 of them clearly positive on confocal examination.39 The remaining 15 cases were equivocal on confocal scanning, 7 of them positive for Acanthamoeba on biopsy. Garg et al recently compared the Confoscan 3.0 scanningslit confocal microscope (Nidek Technologies, Greensboro, NC) with conventional microbiology for the diagnosis of
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infectious keratitis in a prospective, double masked, nonrandomized clinical trial (Garg P, Krishma PV, Sharma S, et al. Role of confocal microscopy in the diagnosis of microbial keratitis. Paper presented at: Cornea Society and Eye Bank Association of America at the American Academy of Ophthalmology Annual Meeting, October 2004; New Orleans, LA). Confocal microscopy correctly diagnosed 83 of 93 fungal cases and 8 of 10 AK cases, whereas it excluded fungus or Acanthamoeba in 41 of 45 cases that were negative on microbiological testing. Another series also has been reported in 15 eyes using the Nidek Confoscan 2.0 scanning-slit confocal microscope to confirm the diagnosis of AK in biopsy- or culture-proven disease.15 Tandem scanning confocal microscopy successfully diagnosed 61 of 63 cases in our series, with 1 false-positive
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Figure 8. Tandem scanning confocal micrographs of keratitis caused by simultaneous infection with Acanthamoeba and filamentous fungi (A–C), corresponding to a clinical photograph (D) (width of each micrograph represents 400 m. A, Multiple filamentous fungi (arrow) and an adjacent Acanthamoeba cyst (arrowhead) are clearly apparent. B, Deep lacunae (arrows) are seen secondary to focal tissue loss by filamentous fungal hyphal elements (arrowhead). C, Tissue destruction by Acanthamoeba has left a honeycomb pattern of optically lucent microcavities in the stroma (arrowheads), as seen in Fig 7C. The filamentous fungi are also apparent (arrow). D, For comparison, a tandem scanning confocal micrograph of gatifloxacin crystals in the anterior stroma of a donor cornea after penetrating keratoplasty (reprinted with permission from Awwad ST, Haddad W, Wang MX, et al. Corneal intrastromal gatifloxacin crystal deposits after penetrating keratoplasty. Eye Contact Lens 2004;30:169 –72). Note the regular pattern of these crystals and also the much thicker nature of the crystal deposits compared with the filamentous-branching nature of hyphal elements seen in A–C, which are distributed in a much more random, irregular pattern.
and 1 false-negative based on clinical data. One case was equivocally (false) positive on confocal for Acanthamoeba but clearly positive on culture for fungus (Fusarium species), responding favorably to appropriate antifungal treatment. Another case was falsely negative on confocal examination but clearly positive on culture for Acanthamoeba, responding favorably to anti-acanthamoebal treatment. Both of these patients were in great pain and intensely photophobic, with uncontrolled eye movements making an adequate examination difficult to obtain on TSCM. The majority of cases (54/63) were positive for AK on TSCM, and all of them showed a favorable response to treatment. However, the yield of positive cultures in this group was relatively low (9/29 [31%]), whereas the yield of positive corneal biopsy was only 50% (2/4). Only 6 cases were negative for AK on TSCM, of which 4 underwent corneal culture, 1 was culture
positive for Acanthamoeba, and 1 was culture positive for Pseudomonas. Two cases initially masqueraded clinically as Acanthamoeba but were subsequently positive for fungus on TSCM. They responded well to standard antifungal treatment for keratitis, despite negative corneal biopsy and culture results. One patient was positive for both fungal hyphae and Acanthamoeba on TSCM, but negative on both corneal culture and biopsy. She responded favorably to combined antifungal and anti-acanthamoebal treatment. The mean final VA (20/47) was better in those patients who started with the best initial VA of 20/15 to 20/60 (Fig 1). Those with an initial VA of 20/70 to 20/200 or 20/400 and worse had mean final VAs of 20/510 or 20/1100, respectively. This is not altogether surprising, as patients with a poor initial VA are more likely to have had worse scarring and pathological corneal damage from the outset. Most
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Ophthalmology Volume 113, Number 4, April 2006 interestingly, those patients with the worst initial VA (20/ 400 or worse) had the greatest scope for visual improvement (0.38 logMAR), confirming that treatment was most helpful in those patients with the greatest initial visual disability (Figs 1, 2). The early diagnosis of AK has been shown to be associated with a more favorable outcome.7,8,40,41 Our results suggest that a slight improvement in vision is possible if the delay in diagnosis is ⬍7 days, although this was not statistically significant (Fig 3). Worsening of vision was seen with a diagnostic delay between 7 to 28 days, but above 28 days this was not as marked. The earlier diagnosis certainly would be expected to contribute to a better visual outcome, in line with previous studies. However, a few patients in our series who were diagnosed early had poor outcomes, whereas some diagnosed later had excellent visual results. This may be related to the variable virulence of the many strains of pathogenic Acanthamoeba as well as differential susceptibility to current therapeutic agents.24 This may explain why patients with the greatest delay in diagnosis had worsening of vision lesser than that of those with a diagnostic delay between 7 and 28 days (Fig 3). In addition, many of our patients were referred from other centers, where the diagnosis was uncertain and drug treatment for Acanthamoeba had already been initiated, together with topical steroids, thus modifying the healing response. There seemed to be a trend toward greater worsening of VA (Pⱕ0.07) in those patients with a history of topical corticosteroid use compared with those without (Pⱕ0.01), although this difference was not statistically significant. It seems logical that previous corticosteroid use would decrease the local immunity to Acanthamoeba, which is well documented in the tear film, potentially worsening the pathological damage caused by the organism and delaying the clearing response.29 However, the exact degree of topical steroid use before presentation in most cases was difficult to quantify, limiting the statistical significance of such a comparison. Moreover, a previous retrospective study of 38 patients showed that topical corticosteroid use was not associated with a higher rate of medical treatment failure in AK, so the issue remains equivocal.41 The majority of cases were treated successfully with 2, 3, or more anti-Acanthamoeba drugs; however, 3 patients underwent enucleation for persistent pain and blindness. Although drug treatment has been revolutionized by the use of biguanides such as chlorhexidine and polyhexamethylbiguanide, a few cases may still fail to respond.24,40 The greatest challenge remains the effective eradication of Acanthamoeba cysts, which may be resistant even to multiple intensive drug treatment in individual cases. Further research has focused on several drugs of the guanidine family along with the alkylphosphocholines and, possibly, topical metronidazole as potential treatments for the future.1,24,42 The yield of positive cultures in confocal positive patients was relatively low (9/29 [31%]), as was the yield of positive corneal biopsy (2/4 [50%]). The results in the literature show variability in these diagnostic methods. In Mathers et al’s series, 13 patients who were positive for Acanthamoeba by either TSCM or epithelial biopsy underwent culture of corneal scrapings plated on E. coli–laden
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nonnutrient agar, but these were all negative.39 Acanthamoebic culture of the contact lens case in one of these cases was also negative. Bacon et al’s series quotes positive corneal cultures in 10 of 14 (71%) intermediate and 17 of 23 (74%) late cases, although culture was not done in patients presenting before 28 days of initial symptoms.8 This variability in yield may be laboratory or experience specific. A further disadvantage with reliance on culture yield is the delay in diagnosis, which may take days to weeks; certainly this can be obviated by the immediate diagnosis offered through TSCM. Many cases that were diagnosed as positive for AK were negative on corneal culture, but theoretically, many of these cases may have been positive at a later date with repeat biopsy. In summary, TSCM is a noninvasive diagnostic method that can provide immediate diagnosis in suspected AK with a high degree of confidence. This is particularly useful when microbiology or histopathology is inconclusive or not readily available to the initial clinician. The rapidity of diagnosis, relative ease of use, and noninvasive nature of in vivo confocal microscopy are clearly advantageous when compared with traditional laboratory techniques, which all rely on blind invasive sampling of the cornea and are associated with a subsequent delay in the culture result. Importantly, in this consecutive case series spanning 10 years there were only one false-positive and one false-negative TSCM result, all encountered in uncooperative patients. Immediate diagnosis by TSCM from the outset avoids diagnostic delay and would certainly be expected to lead to an improved visual outcome in AK. In cases in which a satisfactory TSCM examination cannot be acquired, however, traditional diagnostic methods such as corneal culture and biopsy must be utilized.
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