pathological characteristics of the different stages of acanthamoeba ...

Pathological characteristics of the different stages ofAcanthamoeba keratitis

Yuzhao Sun, Jing Hong,1 Pei Zhang,1 Rongmei Peng1 & Gege Xiao1

Department of Ophthalmology, China Medical University, the First Affiliated Hospital, Shenyang, China, and 1Peking

University Third Hospital, Peking University Eye Centre, Beijing, China

Date of submission 26 April 2013Accepted for publication 21 July 2013Published online Article Accepted 24 July 2013

Sun Y, Hong J, Zhang P, Peng R & Xiao G

(2013) Histopathology 63, 862–868

Pathological characteristics of the different stages of Acanthamoeba keratitis

Aims: To classify the clinical stages of Acanthamoebakeratitis (AK), and clarify the relationship betweenpathological changes and clinical features.Methods and results: Between January 2007 andMay 2012, AK was diagnosed in 11 eyes by patho-logical examination and confocal laser scanningmicroscopy. Pathological investigation of all corneasamples from keratoplasty was done with periodicacid-Schiff and haematoxylin and eosin stains. AKclinical stage, pathological features and postoperativetreatment were studied retrospectively. The 11 caseswere classified into development stage, convalescencestage, or cicatricial stage. In the development stage,marked conjunctival hyperaemia, a corneal epithelialdefect, obvious corneal infiltration and progressiveinflammation were seen; pathological changes com-

prised abundant inflammatory cells and a roundedcyst in the oedematous stroma, as well as a verysmall amount of neovascularization. In the convales-cence cases, moderate conjunctival hyperaemia, cor-neal disciform structures, repair of the cornealepithelial defect and abundant neovascularizationwere seen; pathological changes included significanttissue necrosis and a small, shrunken cyst in thestroma, as well as significant neovascularization. Inthe cicatricial stage, keratoleukoma was seen; patho-logical changes comprised a few inflammatory cellsand shrunken cysts scattered in the stroma. Therewere no cases of recurrence.Conclusions: The pathological features of differentclinical stages confirmed the new clinical classifica-tion of AK.

Keywords: Acanthamoeba keratitis, clinical stage, pathological features

Introduction

In the form of trophozoites and cysts, Acanthamoeba isubiquitously distributed in the environment, and hasbeen isolated from soil, fresh water, sea-water, andeven the air.1–4 Acanthamoeba propagates by formingcysts, and can cause vision-threatening keratitis. Acan-thamoeba keratitis (AK) has distinctive features, such aspseudodendrites of the epithelium, radial keratoneuri-

tis, ring infiltrates, and a disciform structure of the cor-neal stroma. Ishibashi has previously classified these asthe different clinical stages of Acanthamoeba5,6; how-ever, the growth pattern of Acanthamoeba in the corneaand the changes that occur inside the cornea duringAcanthamoeba invasion, as well as the relationshipbetween clinical symptoms and pathological changes,are unknown. Thus, we need a better understanding ofthe pathogenesis of AK for its diagnosis and treatment.Recent studies have shown that the pathophysio-

logy of Acanthamoeba infection involves an intricateseries of sequential events, including the productionof several pathogenic proteases that degrade base-ment membranes, induce cytolysis and apoptosis of

Address for correspondence: J Hong, Peking University Third

Hospital, Peking Univercity Eye Centre, 49 North Garden Road,

Haidian District, Beijing 100191, China.

e-mail: [email protected]

© 2013 John Wiley & Sons Ltd.

Histopathology 2013, 63, 862–868. DOI: 10.1111/his.12237

the cellular elements of the cornea, and culminate inthe dissolution of the collagenous corneal stroma.7,8

However, the relationship between pathologicalchanges and clinical symptoms remains unknown.We present 11 patients with Acanthamoeba keratitis

who underwent keratoplasty. We classified them usingnew methods, according to their different clinicalfeatures. We also examined the pathological features ofthe different clinical stages, and clarified therelationship between pathological changes and clinicalsigns.

Materials and methods

E T H I C S S T A T E M E N T

Our investigation adhered to the tenets of the Decla-ration of Helsinki. This investigation was approved bythe Institutional Review Board of Peking UniversityThird hospital, and, following a full explanation ofthe study, written informed consent was obtainedfrom all patients.

Subjects

Eleven patients with AK who underwent keratoplastyat Peking University Third Hospital, Peking UniversityEye Centre, between January 2007 and May 2012,were studied. These included four males and seven

females, aged 24–68 years. None wore contactlenses, and six gave a history of trauma. The dura-tion of disease ranged from 1 month to 60 years(Table 1).

D I A G N O S I S O F A C A N T H A M O E B A K E R A T I T I S

The 11 patients were diagnosed with AK by patho-logical examination of a corneal scraping stainedwith periodic acid–Schiff (PAS) and haematoxylinand eosin (H&E), and by confocal laser scanningmicroscopy, or by pathological examination of theinfected cornea obtained at keratoplasty.

K E R A T O P L A S T Y

All of the patients underwent a keratoplasty. Thedepth of infiltrates as judged by use of a slit lampmicroscope or confocal laser scanning microscopydetermined whether a deep lamellar keratoplasty(LKP) or penetrating keratoplasty (PKP) was per-formed; if the infiltrates were deeper than three-quar-ters of the corneal stroma, a PKP was performed;otherwise, an LKP was performed.

P O S T O P E R A T I V E T R E A T M E N T A N D F O L L O W - U P

All 11 patients received different anti-amoeba therapypostoperatively, according to their different clinical

Table 1. Characteristics of the eleven patients with Acanthamoeba keratitis

No Age Gender Eye*Traumahistory Course Congestion

Infiltrate size(mm)

Infiltratedepth

Epithelial defect(mm)

1 47 M Os Yes 4 month ++ 8.0 3/4 matrix 8.5

2 53 F Os Yes 1 month +++ 6.75 All cornea 7.5

3 52 M Od Yes 3 month +++ 6.5 All cornea 7

4 56 F Os Yes 3 weeks +++ 7.5 1/2 matrix 7.5

5 24 M Os No 5 month +++ 8.0 All cornea 8

6 43 F Os No 4 month ++ 7.5 All cornea 4

7 55 F Os Yes 4 month +++ 7.25 All cornea 4

8 56 F Os No 4 month +++ 7.0 3/4 matrix 5

9 24 M Os Yes 9 month - 7.5 All cornea –

10 68 F Os No 60 year – 7.25 3/4 matrix –

11 41 F Od No 6 month – 7.5 All cornea –

*“Os” in this line means the left eye, “Od” in this line means the right eye.

© 2013 John Wiley & Sons Ltd, Histopathology, 63, 862–868.

Pathology of Acanthamoeba keratitis 863

stages. All of the patients were followed up for 1–5 years.

Results

Nine of the 11 patients were diagnosed with AK bypathological examination of a corneal scrapingstained with PAS and H&E, and confocal laser scan-ning microscopy. The remaining two patients werediagnosed by pathological examination of the cornealsection post-keratoplasty.

C L I N I C A L S T A G E

According to clinical features, we present a new clas-sification of the clinical stages of AK: the developmentstage, convalescence stage, and cicatricial stage. Thefeatures of the development stage were marked con-junctival hyperaemia, obvious corneal infiltration,progressive inflammation, a corneal epithelial defect,a ring infiltrate corresponding to the epithelial defectarea, and a very small amount of neovascularizationsurrounding the cornea (Figure 1A). The features ofthe convalescence stage were moderate conjunctival

A

C

B

Figure 1. The clinical features and pathomorphological changes observed in the development stage of Acanthamoeba keratitis. A, Preoperative

anterior segment photograph showing clinical features. Obvious conjunctival hyperaemia, a small amount of neovascularization surrounding

the cornea and a ring infiltrate~7 mm in diameter and of the same area as the corneal epithelial defect were seen. B, C, Pathological

changes (PAS stain). The yellow arrows indicate a membrane stained pink with PAS, which was observed in exactly the same area as the

defective epithelium. In C, there were amoeba cysts in the stromal layer where the pink membrane was observed. The stromal layer of the

corneal lesions area was oedematous, and there was infiltration by inflammatory cells, most of which were not intact. There was a small

amount of, or inconspicuous, tissue necrosis. No cysts were observed in the area of tissue necrosis; most of the cysts could be seen in the

shallower or middle stromal layer, and appeared rounded and full. Neovascularization was not obvious. The black arrows indicate abundant

cysts with a full appearance.


864 Y Sun et al.

hyperaemia, corneal disciform structures, repair ofthe corneal epithelial defect corresponding to disci-form structures (areas of corneal epithelial defectsmaller than the areas of the disciform structures),limited lesions, and abundant neovascularization sur-rounding the cornea (Figure 2A). The cicatricial stagewas distinguished by complete repair of the cornealepithelium, degradative inflammation, and kerato-leukoma (Figure 3A).According to this classification, six of the 11 cases

were in the development stage (cases 1–6 in Table 1),

two were in the convalescence stage (cases 7 and 8),and three were in the cicatricial stage (cases 9–11).

P A T H O L O G I C A L F E A T U R E S

Development stageThe corneal epithelium was defective in the lesionarea, and a uniform membrane stained pink withPAS was seen at the site of the defective epithelium.There were amoeba cysts in the stromal layer wherethe pink membrane was seen, whereas no amoeba

A

C

B

Figure 2. The clinical features and pathomorphological changes observed in the convalescence stage of Acanthamoeba keratitis. A, Preopera-

tive anterior segment photograph showing clinical features. Moderate conjunctival hyperaemia, abundant and bulky neovascularization sur-

rounding the cornea, corneal disciform structures ~7 mm in diameter, and a corneal epithelial defect of approximately 5 9 7 mm were

seen. B, Pathological changes (PAS). The corneal epithelium was defective in the lesion area (indicated by the black arrow), but no mem-

brane stained with PAS was present. [Correction added on 25 September 2013, after first online publication: Black arrow inserted to indicate

defective corneal epithelium] C, Pathological changes (PAS). The stromal layer of the corneal lesion area was oedematous, and a large

amount of tissue necrosis and many inflammatory cells were seen. The inflammatory cells were not intact, and the area of oedematous

stroma was larger than that of the corneal epithelial defect. No cysts were observed in the area of tissue necrosis; cysts were occasionally

seen in the stromal layer, and appeared shrunken. Neovascularization was seen in surrounding areas. The black arrows indicate cysts. The

yellow arrows indicate tissue necrosis and inflammatory cells (not intact).



cysts were evident in the stromal layer where thepink membrane was absent. The stromal layer of thecorneal lesion area was oedematous, and inflamma-tory cells had infiltrated, although most of the inflam-matory cells were disrupted. There was little orinconspicuous tissue necrosis. No cysts were seen inthe area of tissue necrosis; most of the cysts wereobserved in the shallower or middle area of the stro-mal layer, and had a rounded and full appearance.Neovascularization was not obvious (Figure 1B,C).

Convalescence stageThe corneal epithelium was defective in the lesionarea, but no PAS-stained membrane was seen at thesite of the defective epithelium. The stromal layer of

the corneal lesion area was oedematous, there was alarge amount of tissue necrosis, and many disruptedinflammatory cells were present. In addition, the areaof oedematous stroma was larger than that of thecorneal epithelial defect. No cysts were seen in thearea of tissue necrosis; cysts were found occasionallyin the stromal layer, and had a shrunken appearance.Neovascularization was seen in the surrounding areas(Figure 2B,C).

Cicatricial stageThe corneal epithelium was complete and thickened,and most of the epithelium was keratinized. Therewere a few inflammatory cells in the shallower layerof the stroma, whereas a large number of eosinophils

A

C

B

Figure 3. The clinical features and pathomorphological changes seen in the cicatricial stage of Acanthamoeba keratitis. A, Preoperative ante-

rior segment photograph showing clinical features. A keratoleukoma of ~7.5 mm was formed, and neovascularization was seen in the kera-

toleukoma. B, Pathological changes of the cicatricial stage (PAS). No PAS-positive membrane was observed. C, Pathological changes of the

cicatricial stage (H&E). The cysts were scattered in the shallower part or middle of the stromal layer, and appeared shrunken, and ‘older’. Eo-

sinophils were seen in the middle and deeper layer of the stroma. The black arrows indicate cysts. The yellow arrows indicate eosinophils.


866 Y Sun et al.

were seen in the middle and deeper layers of thestroma. Cysts were scattered in the shallower or mid-dle part of the stromal layer, and they had a shrun-ken, older appearance (Figure 3B,C).

K E R A T O P L A S T Y

Four of the 11 patients underwent LKP, and theyincluded two development stage cases, one convales-cence stage case, and one cicatricial stage case. Sevenunderwent PKP, and they included four developmentstage cases, one convalescence stage case, and twocicatricial stage cases. The size of the host trephinewas set according to the clinical stage: 1.0 mm largerthan the nidus in development stage cases, 0.5 mmlarger than the nidus in convalescence stage cases,and 0.25 mm larger than the nidus in the cicatricialstage. All of the donor grafts were routinely sized0.25 mm larger than the host.

P O S T O P E R A T I V E T R E A T M E N T A N D F O L L O W - U P

According to the pathology results, we used differenttreatment regimens post-keratoplasty for cases in dif-ferent clinical stages. We continued to use anti-amoeba drugs by intravenous injection (metronida-zole) and eye drops (polyhexamethylene biguanide orchlorhexidine) for the development and convalescencestage cases. When the patient was in the cicatricialstage, the anti-amoeba drug was discontinued.There were no cases of recurrence during the fol-

low-up period of 1–5 years.

Discussion

Acanthamoeba propagates by forming cysts. Cyst adhe-sion to the corneal epithelium is a primary step inAcanthamoeba keratitis.7,8 In this study, pathomorpho-logical observations of the development stage showedthat the corneal epithelia were defective in the lesionarea, and a uniform membrane, stained pink by PAS,was seen at the site of the defective epithelium. Fur-thermore, amoeba cysts were only observed in thestromal layer where the pink membrane was present.We hypothesize that the defective area of the cornealepithelium is the site where the Acanthamoeba cystsinvade, and the pink membrane may representdegraded basement membrane product. We wouldneed to examine the exact composition of the mem-brane to confirm this.Histological examination of cases at the develop-

ment stage showed also that inflammatory cells had

infiltrated, but most of these were not intact, andthere was inconspicuous tissue necrosis. The reasonfor these changes may be that pathogenic Acantha-moeba can degrade chemokines and cytokines, antibod-ies, the complement pathway, and macrophages.9–11

Possibly for the same reason, no tissue necrosisappeared around the cysts, and no cysts wereobserved in the area of tissue necrosis. Meanwhile, theminor inflammatory responses and inconspicuous tis-sue necrosis observed in the development stageensured a relatively intact stroma and rich nutritionfor the cysts, which appeared rounded and full.The minor inflammatory response, inconspicuous

tissue necrosis and abundant cysts observed in thedevelopment stage indicate that the cysts require nutri-ents from the corneal stroma and transfer to anotherposition once the stroma is necrosed and no longerproviding sufficient nutrition. This hypothesis was con-firmed by the pathomorphological changes observed inthe convalescence stage, with a large amount of tissuenecrosis and many inflammatory cells present in thestromal layer of the corneal lesion area. In addition,the cells were obviously no longer intact or whole. Asconspicuous tissue necrosis and degradation of thestromal matrix had destroyed the nutritional environ-ment required by the cysts, the latter were significantlyreduced in number, and appeared shrunken.The changes observed in the convalescence stage

may be attributable to the cytopathic effect (CPE) sub-sequent to adhesion, which involves killing of hostcells, degradation of the epithelial basement mem-brane and underlying stromal matrix, and penetrationinto the deeper layers of the cornea.12–16 The CPEmay also result in degradation of the PAS-stainedmembrane, as this membrane was not seen in the areaof defective epithelium in the convalescence stage. It isknown that the cyst has antigenicity,17,18 and thatthis may cause persistent corneal and scleral inflam-mation in the absence of active amoebic infection.19

This may be another reason for the strong inflamma-tory response seen in the convalescence stage.Escaping from the corneal immune system would

allow cysts to remain in the corneal tissue for anextended period of time. Although they had differingmorphologies, cysts could be seen at all stages,including the cicatricial stage. A histological sectionfrom a cicatricial stage case in our study with a 60-year history also showed cysts. Yang reported thatthe cysts were present for up to 31 months afteranti-amoeba treatment.19 Eosinophils are involved inthe immune response against parasites; thus, theimmune response induced by a long-standing cystand its antigen in the cicatricial stage would recruit a



large number of eosinophils to the middle and deeperlayers of the stroma.Vascularization of the cornea is known to be part of

the process of tissue repair. Our observations showedthat vascularization was not obvious in the develop-ment stage, whereas in the convalescence stage neo-vascularization was observed in surrounding areas.These significant pathomorphological changes were

consistent with our novel clinical classification ofAcanthamoeba keratitis according to clinical features.The histological appearances observed in the develop-ment stage included a defective corneal epithelium inthe lesion area, a uniform PAS-positive membrane,and amoeba cysts in the stromal layer in the area ofthe defective epithelium. The clinical features of thedevelopment stage were a corneal epithelial defectand a ring infiltrate corresponding to the epithelialdefect area, where the amoeba cysts were present.The pathology also showed inconspicuous neovascu-larization, in keeping with the clinical findings of noor minimal neovascularization surrounding the cor-nea. The pathology in the convalescence stageshowed that the corneal epithelium was defective inthe lesional area, and the stromal layer in this areawas oedematous, and showed a large amount of tis-sue necrosis and many inflammatory cells. In addi-tion, the area of oedematous stroma was larger thanthat of the corneal epithelial defect, and abundantand bulky neovascularization was observed in thesurrounding areas. The pathology in the cicatricialstage showed complete and thickened corneal epithe-lium, most of which was keratinized; the stromashowed scattered neovascularization. Clinical featuresof the cicatricial stage included keratoleukomatogether with the neovascularization. The fact thatthe pathological findings were consistent with thechanges in clinical signs confirmed the validity of ournew clinical classification.There were no cases of postoperative recurrence.

The possible reason was that we varied the size of thearea resected according to the clinical stages pro-posed: a larger resection area for the developmentstage, a resection area the same size as the leukomafor the cicatricial stage, and a resection area inbetween the other two for the convalescence stage. Inaddition, we used different treatment regimens post-keratoplasty for cases in different clinical stages: inthe development and convalescence stages the cystswere full, so we continued to use anti-amoeba drugs;in the cicatricial stage the cysts were shrunken, sowe discontinued these drugs.On the basis of the 11 cases presented here, it

appears that the pathological features of the different

clinical stages of AK are different. Our new classifica-tion of the clinical stages is comprehensive, andshould assist in guiding the treatment of Acantha-moeba keratitis, including the pattern of keratoplastyand postoperative treatment.

References

1. Mutoh T, Ishikawa I, Matsumoto Y, Chikuda M. A retrospec-

tive study of nine cases of Acanthamoeba keratitis. Clin. Ophthal-

mol. 2010; 4; 1189–1192.2. Siddiqui R, Khan NA. Biology and pathogenesis of Acantha-

moeba. Parasit. Vectors 2012; 5; 6.

3. Band RN, Mohrlok S. The cell cycle and induced amitosis in

Acanthamoeba. J. Protozool. 1973; 20; 654–657.4. Weisman RA. Differentiation in Acanthamoeba castellanii. Annu.

Rev. Microbiol. 1976; 30; 189–219.5. Ishibashi Y. Acanthamoeba keratitis. Ophthalmologica 1997; 211;

39–44.6. Ishibashi Y, Miyanaga Y. Acanthamoeba keratitis. Nippon No

Ganka 2008; 79; 721–726.7. Clarke DW, Niederkorn JY. The pathophysiology of Acantha-

moeba keratitis. Trends Parasitol. 2006; 22; 175–180.8. Panjwani N. Pathogenesis of Acanthamoeba keratitis. Ocul. Surf.

2010; 8; 70–79.9. Toney DM, Marciano-Cabral F. Resistance of Acanthamoeba spe-

cies to complement lysis. J. Parasitol. 1998; 84; 338–344.10. Na BK, Cho JH, Song CY, Kim TS. Degradation of immunoglob-

ulins, protease inhibitors and interleukin-1 by a secretory pro-

teinase of Acanthamoeba castellanii. Korean J. Parasitol. 2002;

40; 93–99.11. Walochnik J, Obwaller A, Haller-Schober EM, Aspock H. Anti-

Acanthamoeba IgG, IgM, and IgA immunoreactivities in correla-

tion to strain pathogenicity. Parasitol. Res. 2001; 87; 651–658.12. Cao Z, Jefferson DM, Panjwani N. Role of carbohydrate-medi-

ated adherence in cytopathogenic mechanisms of Acantha-

moeba. J. Biol. Chem. 1998; 273; 15838–15845.13. 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. Im-

mun. 2005; 73; 1061–1068.14. Alizadeh H, Neelam S, Niederkorn JY. Effect of immunization

with the mannose-induced Acanthamoeba protein and Acantha-

moeba plasminogen activator in mitigating Acanthamoeba kera-

titis. Invest. Ophthalmol. Vis. Sci. 2007; 48; 5597–5604.15. Hurt M, Niederkorn J, Alizadeh H. Effects of mannose on Acan-

thamoeba castellanii proliferation and cytolytic ability to cor-

neal epithelial cells. Invest. Ophthalmol. Vis. Sci. 2003; 44;

3424–3431.16. Kim WT, Kong HH, Ha YR et al. Comparison of specific activity

and cytopathic effects of purified 33 kDa serine proteinase from

Acanthamoeba strains with different degree of virulence. Korean

J. Parasitol. 2006; 44; 321–330.17. Mathers W, Stevens G, Rodrigues M et al. Immunopathology

and electron microscopy of acanthamoeba keratitis. Am. J. Oph-

thalmol. 1987; 103; 626–635.18. Kremer I, Cohen EJ, Eagle RC Jr, Udell I, Laibson PR. Histo-

pathologic evaluation of stromal inflammation in acanthamoeba

keratitis. CLAO J. 1994; 20; 45–48.19. Yang YF, Matheson M, Dart JK, Cree IA. Persistence of acantha-

moeba antigen following acanthamoeba keratitis. Br. J. Ophthal-

mol. 2001; 85; 277–280.


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