Detection of Acanthamoeba spp. using carboxylesterase antibody and its usage for diagnosing Acanthamoeba-keratitis

Contact lens usage has contributed to increased incidence rates of Acanthamoeba keratitis (AK), a serious corneal infection that can lead to blindness. Since symptoms associated with AK closely resemble those incurred by bacterial or fungal keratitis, developing a diagnostic method enabling rapid detection with a high degree of Acanthamoeba-specificity would be beneficial. Here, we produced a polyclonal antibody targeting the carboxylesterase (CE) superfamily protein secreted by the pathogenic Acanthamoeba and evaluated its diagnostic potential. Western blot analysis revealed that the CE antibody specifically interacts with the cell lysates and conditioned media of pathogenic Acanthamoeba, which were not observed from the cell lysates and conditioned media of human corneal epithelial (HCE) cells, Fusarium solani, Staphylococcus aureus, and Pseudomonas aeruginosa. High titers of A. castellanii-specific antibody production were confirmed sera of immunized mice via ELISA, and these antibodies were capable of detecting A. castellanii from the cell lysates and their conditioned media. The specificity of the CE antibody was further confirmed on A. castellanii trophozoites and cysts co-cultured with HCE cells, F. solani, S. aureus, and P. aeruginosa using immunocytochemistry. Additionally, the CE antibody produced in this study successfully interacted with 7 different Acanthamoeba species. Our findings demonstrate that the polyclonal CE antibody specifically detects multiple species belong to the genus Acanthamoeba, thus highlighting its potential as AK diagnostic tool.

Acanthoamoeba Keratitis Induced by a Therapeutic, Soft Contact Lens: Diagnosis via Gram Staining

Purpose: We report two cases of Acanthamoeba keratitis diagnosed by Gram staining in patients who had recently worn therapeutic, soft contact lenses and had no history of lens use for visual correction.Case summary: The first patient was initially diagnosed with suspected mixed bacterial or fungal keratitis before a final diagnosis of Acanthamoeba keratitis was confirmed by Gram staining of a corneal smear. The second patient was initially diagnosed with a persistent epithelial defect caused by an earlier lid injury inflicted by a metallic foreign body, and then with a suspected mixed infection combined with herpetic uveitis. The patient was finally diagnosed with Acanthamoeba keratitis by Gram staining of a corneal smear. Both cases were treated with polyhexamethylene biguanide and chlorhexidine.Conclusions: Therapeutic, soft contact lenses are used to enhance corneal, epithelial wound healing in conjunction with antimicrobial prophylaxis. However, application of such a lens to a diseased cornea may predispose to the development of microbial keratitis caused by microorganisms resistant to the usual, prophylactic, antimicrobial eye drops. Therapeutic, soft contact lenses are associated with a risk of Acanthamoeba keratitis; early diagnosis is important. Gram staining of a corneal smear is useful in this context. Acanthamoeba is not eradicated by empirical broad-spectrum antimicrobials.

Evaluation of Amebicidal and Cysticidal Activities of Antifungal Drug Isavuconazonium Sulfate against Acanthamoeba T4 Strains

Acanthamoeba species of amebae are often associated with Acanthamoeba keratitis, a severe corneal infection. Isavuconazonium sulfate is an FDA-approved drug for the treatment of invasive aspergillosis and mucormycosis. This prodrug is metabolized into the active isavuconazole moiety. Isavuconazole was previously identified to have amebicidal and cysticidal activity against Acanthamoeba T4 strains, but the activity of its prodrug, isavuconazonium sulfate, against trophozoites and cysts remains unknown. Since it is not known if isavuconazonium can be metabolized into isavuconazole in the human eye, we evaluated the activities of isavuconazonium sulfate against trophozoites and cysts of three T4 genotype strains of Acanthamoeba. Isavuconazonium displayed amebicidal activity at nanomolar concentrations as low as 1.4 nM and prevented excystation of cysts at concentrations as low as 136 μM. We also investigated the cysticidal activity of isavuconazonium sulfate in combination with a currently used amebicidal drug polyhexamethylene biguanide (PHMB). Although combination of isavuconazonium with PHMB did not elicit an obvious synergistic cysticidal activity, the combination did not cause an antagonistic effect on the cysts of Acanthamoeba T4 strains. Collectively, these findings suggest isavuconazonium retains potency against Acanthamoeba T4 strains and could be adapted for Acanthamoeba keratitis treatment.

Cosmetic contact lenses: beauty can be blinding

Background: Although cosmetic contact lenses are ideally indicated for patients with corneal and iris abnormalities, they are currently fashionable among the younger generation of emmetropes to enhance their physical appearance. Cosmetic contact lens wearers carry a greater risk of microbial keratitis, even more so with counterfeit ones.Case presentation: Here, we report two cases of counterfeit cosmetic contact lens wearers with Acanthamoeba keratitis (AK) who were misdiagnosed as herpes simplex virus (HSV) keratitis.Conclusion: AK is a sight-threatening complication among contact lens wearers. Since clinically AK may masquerade as HSV, early diagnosis of AK is often delayed. As both microorganisms can mimic each other, determining the co-existence of both infections can be challenging. Delay in initiating proper treatment can lead to blinding complications.

In Vitro Activity of Pentamidine Isethionate against Trophozoite and Cyst of Acanthamoeba

Background: Acanthamoebae are a causative agent of Acanthamoeba keratitis (AK) in immunocompetent individuals. Since access to propamidine isethionate (Brolene®) as a first-line treatment has been limited in recent years, in the current study, we examined the effects of pentamidine isethionate against trophozoite and cyst forms of Acanthamoeba. Methods: This experimental study was conducted in the Department of Medical Parasitology and Mycology, School of Public Health, Tehran University of Medical Sciences, Tehran, Iran, during 2019-2020. Pentamidine isethionate at concentrations of 50, 100, 200, 400, 600, 800, and 1000 µM were tested against trophozoites and cyst stages of T4 genotype, at 24- and 48-hour incubation period, and the viability was determined by trypan blue staining. In addition, the cytotoxic effect of the drug was examined in Vero cells using the 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) assay. Results: The 50% inhibitory concentration (IC50) of pentamidine isethionate on trophozoite after 24 and 48h were 97.4 µM and 60.99 µM. These results on cyst after 24 and 48h were 470 µM and 175.5 µM, respectively. In MTT assay, the drug showed an inhibitory effect on Vero cell growth with IC50 values of 115.4 µM and 87.42 µM after 24h and 48h, respectively. Conclusion: Pentamidine isethionate exhibited an inhibitory effect on trophozoite and cyst. Given that the trophozoicidal activity of the drug is in the safe dose, it could be suggested as an alternative in patients with AK; however, further investigation is needed in an animal model to confirm the data.