Acanthamoeba Keratitis: A Single-Institution Series of Four Cases With Literature Review

Acanthamoeba species are free-living protozoa found pervasively in water and soil, which can cause infections of the central nervous system, skin, and eye. Amoebic keratitis (AK) is a vision-threatening, often chronic infection that is associated with the use of soft contact lenses due to corneal microtrauma and improper cleaning and storage. Although AK infections are rare, they cause significant morbidity including vision loss due to the diagnostic and therapeutic challenges they pose. The clinical course is determined by the organism’s inherent pathogenicity, delay of diagnosis, and the paucity of data on effective therapeutic regimens. The case series and review of literature that follows examine current latest best practices in AK diagnosis including in vivo confocal microscopy (IVCM) and therapeutic interventions including miltefosine.


Introduction
Acanthamoeba species are free-living protozoa found ubiquitously in soil and water and are implicated in serious infections of the central nervous system, skin, and eye [1]. Their life cycle is notable for being biphasic, with a trophozoite form capable of attaching to host epithelium and invading to deeper structures, as well as a cystic form that can endure adverse conditions including treatment attempts [1]. Human studies of serum antibodies to Acanthamoeba show that most individuals have been exposed to the organism; however, clinically significant infections are of relatively low incidence and frequently affect contact lens users and the immunocompromised [2,3].
Amoebic keratitis (AK) is a progressive, vision-threatening infection that is strongly associated with soft contact lenses due to contamination related to improper cleaning and storage with infested tap water. Additionally, corneal microtrauma and epithelial changes are known to occur with chronic contact use, predisposing the user to infection [4]. It is common for these infections to be chronic in nature due to the durability of the cystic form and the possibility for reinfection, which is at least partially attributed to the immunologically privileged nature of this anatomic site. Treatment is challenging due to the diagnostic delay related to initial misdiagnosis, nonspecific presentation of the disease, and the need for specialized diagnostic measures. Additionally, Acanthamoeba infection is oftentimes polymicrobial, including coinfections with fungi and bacteria. Without treatment of coinfections, a symbiotic relationship between the two organisms persists, causing continued damage to the cornea despite treatment of the Acanthamoeba. Untreated and suboptimally treated infections have been observed to spread contiguously into the deeper structures of the eye and central nervous system manifesting as granulomatous amoebic encephalitis [5]. While AK infections are relatively uncommon, they are associated with significant morbidity and vision loss due to the diagnostic and therapeutic challenges they pose.
The series of cases that follow highlight common presenting features, discuss diagnostic assays, and report on experiences of multidrug oral treatment strategies. Rapid referral to an ophthalmologist trained in confocal microscopy can be key to making an early diagnosis of AK. If topical treatments fail, systemic therapies including miltefosine, a pharmacologic agent with recent approval for refractory AK, and voriconazole may be of benefit. With further progression of the disease, she underwent penetrating keratoplasty (PKP). Pathology of Descemet's membrane revealed severe inflammation with numerous cysts and possible trophozoites. Posttransplantation, an aggressive regimen of oral miltefosine, voriconazole, and trimethoprim/sulfamethoxazole was added for six months. The patient continued to improve after surgery, tolerated the medications with monitoring, and remained free of disease at three years follow-up ( Figure 5). Cataract surgery was required to restore best-corrected vision.

Case 2
A 59-year-old female with an unremarkable past medical history presented to the ophthalmology clinic for an evaluation of approximately one month duration of left eye pain and photosensitivity. She had been treated previously for presumptive HSV keratitis and corneal abrasion without improvement. Symptoms progressed to include pain with extraocular movements, reduced visual acuity, and sinus pain despite appropriate topical therapy. Exposure history was significant for use of soft contact lenses, which she changed daily and denied wearing to sleep.
On physical examination, she had a large central epithelial defect, patchy infiltrates, satellite lesions, and a hypopyon. There was initial suspicion for fungal keratitis; therefore, she was started on oral voriconazole. Topical moxifloxacin was added, and oral acyclovir was continued. A confocal examination was performed and demonstrated bright, round double-walled cystic structures, consistent with Acanthamoeba ( Figure 6). Topical chlorhexidine was then added. Corneal scrapings were found to be positive for Acanthamoeba by nucleic acid amplification. She continued to worsen despite topical and systemic therapy. Repeat confocal microscopy demonstrated persistence of cysts, with a deeper spread into the corneal stroma ( Figure 7). The decision was made to proceed with deep anterior lamellar keratoplasty (DALK) with anterior chamber washout. Miltefosine was added to the regimen for approximately one month prior to this procedure. The corneal tissue sent to pathology was negative for Acanthamoeba and fungal elements. She was continued on topical chlorhexidine for an additional two weeks and no longer required oral voriconazole or miltefosine postoperatively. The patient has remained stable for three years after DALK. Cataract surgery was required to restore bestcorrected visual acuity.

Case 3
A 71-year-old female with a past medical history significant for diabetes mellitus type two was referred from an outside ophthalmology clinic due to concern for worsening Acanthamoeba keratitis confirmed four months previously by PCR. She had been treated with propamidine isethionate and ofloxacin. She had sought care initially for an abrupt decline in visual acuity. Notably, she had worn contacts without incident for the preceding 50 years.
Her examination showed central punctate epithelial erosions with stromal edema and haze. She was started on topical chlorhexidine and moxifloxacin. Despite treatment, her vision continued to deteriorate, and she developed eye pain, burning, and tearing. Follow-up confocal microscopic examination demonstrated numerous Langerhans cells (Figure 8), double-walled cysts (Figure 9), and extensive multilevel scarring. She continued to worsen despite treatment with topical chlorhexidine, oral voriconazole, and oral acyclovir. DALK was deemed necessary, and pathology of both anterior and deep corneal stroma demonstrated Acanthamoeba trophozoites. She was continued on topical chlorhexidine and oral acyclovir postoperatively.  The patient's course was complicated by the recurrence of Acanthamoeba keratitis two months after DALK. New ring infiltrate and hypopyon were noted on examination, and corneal scrapings submitted for nucleic acid testing redemonstrated Acanthamoeba cysts. The patient was started on oral trimethoprim/sulfamethoxazole, voriconazole, and miltefosine. She was continued on topical chlorhexidine, and topical polyhexamethylene biguanide (PHMB) was added. Due to rapid progression, urgent penetrating keratoplasty was pursued with persistent Acanthamoeba cysts noted on pathology. The patient had tolerability problems with the miltefosine; therefore, this was discontinued. She was continued on oral voriconazole, trimethoprim/sulfamethoxazole, and topical PHMB until the disease appeared to be in remission. She then underwent cataract surgery to restore vision. Unfortunately, she developed corneal allograft failure necessitating penetrating keratoplasty again. Pathology was negative for protozoa; therefore, PHMB was discontinued. She currently has a guarded long-term prognosis due to chronic Acanthamoeba keratouveitis secondary glaucoma and neurotrophic keratopathy ( Figure 10).

Case 4
A 46-year-old female with a history of eosinophilic esophagitis, positive antinuclear antibody, and trigeminal neuralgia was referred from an outside ophthalmologist due to approximately five months of recurrent right eye burning, tearing, vision changes, photophobia, and unilateral headache. Her symptoms appeared shortly after she began storing her contact lenses in tap water. She was initially treated by an outside optometrist for presumed herpetic keratitis with minimal relief.
Her examination was notable for the presence of a ring infiltrate and geographic ulcer of the right cornea ( Figure 11). A confocal examination demonstrated cystic amoeba forms in the corneal stroma strongly suspicious for Acanthamoeba ( Figure 12); however, the scrapings sent for culture grew only Pseudomonas aeruginosa. She was started on topical PHMB, vancomycin, tobramycin, voriconazole, and oral valacyclovir. Due to incomplete resolution and strong clinical suspicion for Acanthamoeba, oral trimethoprim/sulfamethoxazole, voriconazole, and miltefosine were added to the therapeutic regimen. Repeat confocal microscopy demonstrated attenuated organisms near the surface and deep double-walled cysts ( Figure 13). Penetrating keratoplasty was performed, and pathology demonstrated Acanthamoeba trophozoites, with some "empty" cysts and severe reactive changes of the corneal epithelium. This patient's postoperative course was complicated by disease persistence with progression involving the native sclera and uvea ( Figure 14). Corneal transplantation via sclerokeratoplasty was performed and was again complicated by recurrent infection of the grafted tissue. However, failure of the sclerokeratoplasty was felt to be due to inflammation from Acanthamoeba keratouveitis, a response to dead or attenuated organisms in the corneal stroma. After the inflammation was controlled with oral mycophenolate mofetil, a third penetrating keratoplasty with cataract surgery was done to restore vision. Due to intolerances of trimethoprim/sulfamethoxazole and azole antifungals, she was continued on oral miltefosine with flucytosine for six months after transplantation with no evidence of persistent or recurrent disease ( Figure  15). Her postoperative course was complicated by neurotrophic keratopathy requiring supraorbital nerve transposition to the right cornea and tarsorrhaphy.

Discussion
Amoebic keratitis is an important diagnosis to consider in the presentation of clinical keratitis due to its ability to cause profound loss of vision and potential to cause life-threatening disease through invasion into the central nervous system. Its morbidity may be attributed to a variety of factors, some intrinsic to the pathogen and some ecological. The Acanthamoeba life cycle is inherently virulent due to the ability of the cystic form to survive inhospitable environments, including treatments that would otherwise be efficacious [1]. The trophozoite form thrives using the epithelium of the eye as an ideal portal of entry due to the concentration of mannosylated glycoproteins, which is further upregulated by the microtrauma caused by contact lens use [1,3,4].
Maintaining contact lenses also enhances the probability of direct inoculation through activities such as handling, cleaning, and storing the lenses with contaminated hands or storage products. Estimates suggest that 85% of cases are associated with the use of contact lenses, although studies have been inconclusive as to whether extended wear lenses or disposable daily lenses have higher risk [3]. Infections are not limited to contact lens users, and one study suggests that those infections may behave more aggressively [3]. Activities such as swimming, bathing, and outdoor work can introduce amoeba to the ocular structures in these individuals as well.
Another reason for this pathogen's high morbidity is delay of diagnosis. As demonstrated in the cases above, this may be due to the overlap of symptoms and co-occurrence with other more common etiologies such as trauma, viruses (particularly herpes simplex virus), and bacteria [5]. A further complicating factor is that Acanthamoeba species have a well-documented propensity for symbiosis with fungal and bacterial species; therefore, multi-organismal infections should not be excluded [4,5].
Diagnosis is complicated by various limitations of diagnostic testing. Although culture remains the gold standard (with 50%-75% sensitivity and 100% specificity), multiple recent studies have demonstrated its significantly inferior sensitivity compared to that of in vivo confocal microscopy (>90% sensitivity and specificity) [6,7]. An added benefit of confocal microscopy is that it offers an immediate diagnosis, allowing for rapid initiation of treatment. The caveat is that it requires expensive equipment and a trained specialist and is not readily available in most eye clinics. If a specialist is not readily available, this may result in delay of diagnosis and treatment [5]. Much like culture, PCR relies heavily on specimen collection technique and yield, which requires invasive procurement of specimen, although PCR has improved sensitivity (71.4%-84%) and specificity (100%) [8,9]. Finally, direct cytological evaluation of corneal scrapings has sensitivity and specificity comparable to PCR and culture but still requires an invasive procurement procedure. Even minor delays in diagnosis may have associated morbidity because protozoa can invade deeper into contiguous tissues and transition into more durable and treatment-resistant cystic forms. Therefore, the goal is to initiate treatment as early and aggressively as reasonable.
Optimizing treatment of AK is an active area of investigation. The most widely accepted first-line treatments include a combination of two or three of the following topical therapies aimed at disrupting organism membranes: 0.1% propamidine isethionate, 0.02%-0.04% chlorhexidine, and 0.02% polyhexamethylene biguanide (PHMB) [10]. More concentrated doses of PHMB have demonstrated superior efficacy in locally advanced cases [10]. Greatest efficacy has been shown when doses are "pulsed" in a way that encourages excystment to the more susceptible trophozoite phase [10].
Miltefosine is a recently approved option for treatment-resistant AK with activity against both the trophozoite and cystic forms, which has shown promise in otherwise refractory cases [11]. Notably, it was originally developed in the 1980s as an antineoplastic agent; however, levels required to be sufficiently antineoplastic would be nearly universally lethal [12]. Its mechanism of action is not fully understood; however, it mimics the structure of membrane phospholipids with notable omission of glycerol. This provides the ability to disrupt phospholipid-rich membranes and cytochrome c, inducing apoptosis [12]. This potency was successfully harnessed in early 2017 for leishmaniasis, as the protozoal membranes were found to be even more susceptible than human cells. Because cellular and organelle membranes are a ubiquitous target, this medication is not without risk for significant off-target toxicity, including teratogenicity, nausea, vomiting, fever, headache, and nephrotoxicity. Less frequently, it can have severe outcomes, such as Stevens-Johnson syndrome and profound thrombocytopenia [13]. Laboratory and animal studies have yielded promising results; however, large-scale studies of efficacy in humans are lacking due in part to the rarity of the condition. Numerous case studies have remarked on its anecdotal efficacy by measure of final visual acuity, avoidance of surgery, and decreased time to cure [14,15].
The role of voriconazole also remains uncertain, although anecdotally it has shown compelling benefit. When administered topically, it is of very low risk with the main intolerance being discomfort. The contentious nature of systemic use arises from the relative paucity of in vivo data. One in vitro study suggested that voriconazole could have an antagonistic effect when coadministered with cysticidal chlorhexidine and propamidine [16]. However, another in vitro study speculated that voriconazole should be effective through the inhibition of ergosterol synthesis via 14α-demethylase [17]. Oral voriconazole has also been shown to be curative as monotherapy in two published cases, and other case studies have suggested systemic efficacy in combination with topical agents [18].
The role of steroids in the treatment of AK is often disputed. It increases the pathogenicity of the organism by enhancing excystment whether given intravenously or topically. Although increasing the burden of the active trophozoite form certainly bears the possibility of worsening the infection, it also increases the organisms' overall susceptibility to antiamoebic therapy [3]. Steroids also quell the inflammatory response to the organism, which is responsible for some of the disease morbidity [1].
Because of well-documented coinfections and known synergism between the protozoa and certain bacteria, some therapeutic regimens include an antibacterial agent, such as topical moxifloxacin, ciprofloxacin, trimethoprim-sulfamethoxazole, or erythromycin [6]. These are of unproven efficacy but may be useful empirically or perioperatively and are generally well tolerated.
Medical management is generally attempted prior to surgery unless there is immediate threat to central nervous system penetration. Procedures such as penetrating keratoplasty (PKP) and deep anterior lamellar keratoplasty (DALK), which were originally performed with the intention of debriding diseased tissue and debulking disease, are now generally reserved for either cases refractory to medical management or for restoration of vision due to structural damage [6,19,20].

Conclusions
In conclusion, it is essential to include AK on the differential diagnosis for patients with keratitis, especially, but not exclusively, in those who wear contact lenses. Should a patient on empiric topical/oral therapies develop a further decline in vision, worsened eye pain, photophobia, or increased injection, they should communicate this promptly to their care team and be seen urgently for a repeat examination. Additionally, AK cannot be excluded in patients proven to have a viral, bacterial, or fungal infection, as coinfection is relatively common. Similarly, AK should not be excluded if there is high clinical suspicion, even if culture or PCR is negative due to poor sensitivity.
Rapid referral to an ophthalmologist trained in confocal microscopy can be key to a timely diagnosis and therefore improved outcome. In cases refractory to standard topical treatments, systemic therapies including miltefosine and voriconazole may be of benefit. Surgery should generally be reserved for central nervous system penetration or visual acuity restoration. Table 1 shows the summary of the cases.