Multidrug-resistant Acanthamoeba keratitis caused by the T4 Genotype: A case report

Abstract

This report describes the case of a 28-year-old woman, who presented with a two-week history of severe ocular pain and photophobia in her right eye. She was a soft contact lens wearer with no history of ocular trauma or systemic illness. Ophthalmic examination revealed conjunctival hyperemia, epithelial irregularities, and a developing ring-shaped stromal infiltrate. In vivo confocal microscopy demonstrated double-walled hyperreflective cysts, and culture followed by PCR and sequencing confirmed the presence of Acanthamoeba T4 genotype. Despite intensive treatment with topical polyhexamethylene biguanide, chlorhexidine, propamidine, and voriconazole, as well as oral miltefosine, the infection progressed, leading to stromal necrosis. The patient underwent two therapeutic penetrating keratoplasties; however, the disease continued to advance, eventually resulting in phthisis bulbi and permanent vision loss. This case highlights the potential for aggressive, clinically refractory Acanthamoeba keratitis caused by the T4 genotype, emphasizing the importance of early diagnosis, vigilant monitoring, and timely surgical intervention in resistant cases.

Introduction

Acanthamoeba keratitis (AK) is a sight-threatening corneal infection primarily associated with contact lens wear, particularly under poor hygiene conditions or when lenses are exposed to water ¹. Early signs of infection include epitheliopathy, which may progress to deeper stromal involvement and formation of a ring-shaped infiltrate (Raghavan and Rammohan 2024; Lorenzo-Morales et al. 2015). Traditionally, Acanthamoeba species were classified based on morphological characteristics such as size and shape; however, modern classification relies on molecular biology techniques, particularly polymerase chain reaction (PCR) (Carnt et al. 2016 May; Booton et al. 2009). The genus Acanthamoeba is divided into 23 genotypes (T1–T23), with the T4 genotype being the most prevalent in both clinical and environmental samples (Xu et al. 2025).

Topical biguanides and diamidines are commonly used in combination for treatment (Raghavan and Rammohan 2024; Lorenzo-Morales et al. 2015). Despite an appropriate therapeutic approach, infections may persist or worsen, sometimes leading to enucleation in severe cases (Ficker et al. 1990). Approximately a decade ago, in vitro resistance to polyhexamethylene biguanide (PHMB) was reported, and miltefosine began to be prescribed as a salvage therapy for refractory cases (Hirabayashi et al. 2019). Miltefosine, an alkylphosphocholine, has demonstrated efficacy against several protozoal infections, including visceral leishmaniasis, Trichomonas vaginalis, Trypanosoma cruzi, and Entamoeba histolytica, and has recently been administered for the treatment of refractory AK (Hirabayashi et al. 2019; Thulasi et al. 2021).

Here, we report a clinically refractory case of AK caused by a T4 Acanthamoeba isolate, which ultimately resulted in phthisis bulbi of the affected eye.

Case report

A 28-year-old woman, a soft contact lens wearer, presented with a two-week history of severe ocular pain and photophobia in her right eye. Past medical and ocular histories were unremarkable. Best-corrected visual acuity (BCVA) was 0.2 Log MAR. Slit-lamp examination revealed conjunctival hyperemia, epithelial irregularity, and mild subepithelial edema. (Fig. 1a). In vivo confocal microscopy (IVCM) demonstrated double-walled hyperreflective cysts typical of Acanthamoeba (Fig. 2). Corneal scrapings were cultured on non-nutrient agar with Escherichia coli overlay and subjected to PCR testing. After five days, cultures showed heavy growth of Acanthamoeba spp. PCR sequencing confirmed the T4 genotype. Treatment with hourly PHMB 0.02% and chlorhexidine 0.02% was initiated. Propamidine 0.1% was unavailable. Despite this, after 10 days, a 5 × 4 mm central ring-shaped stromal infiltrate developed (Fig. 1b). Voriconazole 1% drops were added hourly, followed by oral voriconazole 200 mg twice daily, and betamethasone eye drop four times a day. After three weeks, her condition did not improve, and the size and depths of ring shape stromal infiltration progressed so, propamidine 0.1% was provided and prescribed hourly. (Fig. 1c).

Fig. 1.

Sequential slit-lamp photographs showing the clinical progression of Acanthamoeba keratitis in the right eye. (a) Central epitheliopathy with epithelial irregularities. (b) A 5 × 4 mm central, ring-shaped stromal infiltrate with mild corneal edema. (c) Enlargement of the infiltrate to approximately 7 × 7 mm, extending into the mid-stroma with diffuse edema. (d) Further progression toward the limbus with increased stromal depth and an overlying epithelial defect. (e) Diffuse corneal thinning, most prominent inferonasally, accompanied by superior and inferior limbitis and hypopyon. (f) Failed graft and Phthisis bulbi eye

Fig. 2.

In vivo confocal microscopy image shows hyperreflective chain-like structures, (indicated by arrow), and multiple hyperreflective bright spot structures measuring 10–25 µm (indicated by asterik), characteristic of double-walled cysts Acanthamoeba cysts

Over the next two weeks the patient continued to experience ocular pain, and the corneal lesion enlarged and deepened, extending toward the mid-stroma and near the limbus. A corneal debridement and biopsy were performed to rule out bacterial or fungal infection and to confirm ongoing Acanthamoeba infection. PHMB was increased to 0.08%. When no improvement was observed after three additional weeks (Fig. 1d), oral miltefosine (50 mg twice daily for three weeks) was introduced alongside ongoing topical therapy. However, corneal thinning, necrosis, hypopyon, and limbitis ensued (Fig. 1e), prompting a large limbus-to-limbus therapeutic penetrating keratoplasty (T-PKP). Postoperatively, PHMB 0.08% and betamethasone eye drop was continued. Two months later, the patient revealed decreased vision and ocular pain. Hypotony developed, and ultrasound biomicroscopy (UBM) revealed atrophy of one quadrant of the ciliary body with membrane formation. Pars plana vitrectomy, accompanied by the injection of silicone oil and extraction of the lens, was conducted. Three months later, graft melt and perforation occurred, necessitating repeat T-PKP. Despite these interventions, the eye progressed to phthisis bulbi with no light perception.

Molecular analysis

DNA was extracted from corneal tissue cultures containing trophozoites and cysts using a commercial kit (Qiagen, Germany). (Fig. 3). PCR amplification using primers JDP1/JDP2 targeted the 18S rRNA gene. Electrophoresis confirmed a positive band, which was sequenced and analyzed by BLAST (NCBI). (Fig. 4). The sequence showed 100% homology with Acanthamoeba T4 genotype and was deposited in GenBank (Accession No. PX068354).

Fig. 3.

Light micrograph showing Acanthamoeba cysts cultured on non-nutrient agar overlaid with Escherichia coli, observed at × 100 magnification (specimen isolated from corneal scraping)

Fig. 4.

Gel electrophoresis results:(A) 100 bp DNA ladder; (B) corneal scraping sample (490 bp); (C) positive control (490 bp); (d) negative control

Discussion

Early diagnosis and appropriate treatment are essential for improving the visual prognosis in Acanthamoeba keratitis (AK) (Lorenzo-Morales et al. 2015). A high index of suspicion is required in contact lens wearers presenting with epithelial irregularities or pseudo-dendritic lesions (Raghavan and Rammohan 2024). Nearly half of AK cases are initially misdiagnosed as Herpes simplex keratitis (Sanchez et al. 2022). The advent of polymerase chain reaction (PCR) and in vivo confocal microscopy (IVCM) has markedly enhanced diagnostic accuracy (Raghavan and Rammohan 2024; Lorenzo-Morales et al. 2015; Carnt et al. 2016 May). Nevertheless, some cases remain clinically refractory and progress to advanced disease despite early diagnosis and aggressive therapy (Ficker et al. 1990; Hirabayashi et al. 2019; Thulasi et al. 2021; Sanchez et al. 2022; Bang et al. 2010; Iovieno et al. 2010). The genus Acanthamoeba comprises 23 genotypes (T1–T23), with T4 being the most prevalent in both environmental and clinical isolates (Xu et al. 2025). Although T4 is typically the dominant genotype, several studies have suggested that certain isolates can display severe, treatment-resistant behavior. (Soleimani et al. 2021) Iovieno et al. reported a drug-resistant AK case associated with the rare T5 genotype, suggesting that atypical genotypes may be linked to poor outcomes (Iovieno et al. 2010). Our case demonstrates that even the common T4 genotype can present as clinically refractory and lead to devastating visual loss.

Standard therapy generally combines a diamidine (propamidine 0.1% or hexamidine 0.1%) with a biguanide (PHMB 0.02% or chlorhexidine 0.02%). High-dose PHMB (0.08%) monotherapy has also shown outcomes comparable to combination therapy (Dart et al. 2024). Voriconazole, both topical and systemic, has been used as an adjunctive agent in resistant infections [(Raghavan and Rammohan 2024; Lorenzo-Morales et al. 2015; Carnt et al. 2016; Bang et al. 2010). Despite escalating therapy from PHMB 0.02% to 0.08% and adding voriconazole, our patient showed no improvement. Although in vitro drug resistance is uncommon, reports of PHMB tolerance have emerged over the past decade (Iovieno et al. 2010 May; Hasby Saad and Khalil 2018). However, in vitro susceptibility does not always correlate with clinical response. In our case, susceptibility testing revealed no resistance, yet the infection progressed clinically. This highlights the gap between laboratory findings and therapeutic outcomes.

Miltefosine, an alkylphosphocholine approved for leishmaniasis, has shown variable success in refractory AK. Some studies have reported rapid improvement within weeks of treatment, while others achieved only partial or no recovery (Hirabayashi et al. 2019; Thulasi et al. 2021). Recently, miltefosine has received orphan drug designation from the United States Food and Drug Administration (FDA). However, there exists limited data regarding the use of oral miltefosine for the treatment of AK in human subjects (Wouters et al. 2022; Brondfield et al. 2017). Hirabayashi et al. described a resistant AK case where oral miltefosine led to clinical improvement within five weeks (Hirabayashi et al. 2019). In our study, tablet miltefosine 50 mg (was administered twice daily alongside anti ameboma agents. In contrast to previous studies, after five weeks, the infiltration progressed, leading to corneal thinning and the detection of impending perforation.

In a study of 15 patients with AK unresponsive to standard therapy, adjuvant systemic miltefosine was administered and the use of steroids was emphasized due to inflammatory responses during treatment with this drug ⁸. Although steroid use prior to diagnosis is associated with poor outcomes, adjunctive administration with anti-amoebic and miltefosine therapy may be beneficial.

Raghavan and Rammohan 2024; Lorenzo-Morales et al. 2015; Carnt et al. 2016). The use of corticosteroids in the management of AK remains controversial. Some authors emphasize the beneficial effects of topical steroids on severe corneal inflammation, intense pain, and the limitation of neovascularization (Carnt et al. 2016; Dart et al. 2024). However, others caution that corticosteroids may increase the pathogenicity of amoebic infection, potentially leading to poorer outcomes, particularly if these agents are used before anti-amoebic therapy is initiated (Wouters et al. 2022). A study by Carnt et al. provided reassurance for clinicians and patients, indicating that the potential benefits of topical corticosteroid therapy in alleviating pain and discomfort do not lead to worse visual outcomes when initiated after starting modern anti-amoebic therapy (Carnt et al. 2016). In our study, we began topical betamethasone 10 days after the start of anti-amoebic therapy when the infiltration progressed and formed a ring shape.

Unfortunately, the size of the ring-shaped infiltration extended closer to the limbus, and large size T-PKP was considered.

PKP remains the only solution in cases of severe infection and has the potential to eradicate the infection. However, in our patient, clear margins free of Acanthamoeba would not have been achievable due to the involvement of the near to limbus. Kitzmann et al. analyzed 31 eyes with advanced AK and found that those who underwent T-PKP had a significantly higher rate of repeat keratoplasty, recurrent infiltration, glaucoma, and phthisis bulbi (Kitzmann et al. 2009). Similarly, our patient required two T-PKPs in an attempt to salvage the globe, but ultimately developed ischemic complications that culminated in phthisis bulbi.

On the other hand, it has recently been recommended that in cases exhibiting resistance to treatment, early keratoplasty following intensive anti-amoebic therapy tends to yield a more favorable overall prognosis. This approach allows for earlier visual recovery and rehabilitation compared to situations where keratoplasty is delayed for therapeutic reasons or globe salvage (Abu Dail et al. 2025; Zazzo et al. 2022).

Additionally, there is a condition called reactive ischemic posterior segment inflammation (RIPSI), a rare complication that can arise from prolonged and severe acute keratitis AK. It is characterized by sudden vision loss and a rapid decrease in ocular pain (Palme et al. 2017). This condition is commonly observed in patients with underlying hypercoagulation or immune disorders. Histopathological findings confirm the diagnosis, revealing chronic chorioretinal inflammation characterized by perivascular lymphocytic infiltration and retinal vascular thrombosis (Palme et al. 2017; Awwad et al. 2007). Intraocular surgeries, even those that appear uncomplicated, can significantly elevate both intraocular and systemic levels of pro-inflammatory cells, contributing to the development of this complication. Palme et al. reported a case of AK-related RIPSI following intraocular surgery in a patient with primary Sjögren’s syndrome. They concluded that autoimmune diseases may increase the risk of RIPSI triggered by such procedures 20. We observed ischemic conditions characterized by the ciliary body atrophy, significant decreased ocular pain, and phthisis bulbi following prolonged prolong f multi-drug-resistant AK after an uncomplicated T-PKP. These findings align with previous reports in the literature.

Conclusion

We present a case of multidrug-resistant AK. Despite the expected therapeutic benefits of oral miltefosine, this particular case did not respond adequately to the treatment. Although in vivo studies have not demonstrated definitive drug resistance in Acanthamoeba, clinicians should nonetheless remain cautious the possibility of clinical resistance to treatment, particularly given the high prevalence of the T4 genotype and also should remain consider for the reactive ischemic posterior segment inflammation, particularly when there is a rapid decline in vision despite improvement in the corneal infection.

Author Contribution

Mehrnaz Atighehchian.Alireza Latifi and Elham Kazemirad. Maryam Kasiri wrote the main manuscript text and Mehran Zarei-Ghanavati prepared Fig. 1-3. All authors reviewed the manuscript.

Funding

Not applicable

Data availability

The datasets used and/or analyzed during the current study available from the corresponding author on reasonable request.

Declarations

Ethics approval and consent to participate

Not applicable (case report).

Consent for publication

Written informed consent was obtained from the patient for publication of this case report and any accompanying images.

Clinical trial number

Not applicable.

Conflicts of interest

The authors declare no competing interests.