Anti-Acanthamoeba synergistic effect of chlorhexidine and Garcinia mangostana extract or α-mangostin against Acanthamoeba triangularis trophozoite and cyst forms

Acanthamoeba spp. can cause amoebic keratitis (AK). Chlorhexidine is effective for AK treatment as monotherapy, but with a relative failure on drug bioavailability in the deep corneal stroma. The combination of chlorhexidine and propamidine isethionate is recommended in the current AK treatment. However, the effectiveness of treatment depends on the parasite and virulence strains. This study aims to determine the potential of Garcinia mangostana pericarp extract and α-mangostin against Acanthamoeba triangularis, as well as the combination with chlorhexidine in the treatment of Acanthamoeba infection. The minimal inhibitory concentrations (MICs) of the extract and α-mangostin were assessed in trophozoites with 0.25 and 0.5 mg/mL, for cysts with 4 and 1 mg/mL, respectively. The MIC of the extract and α-mangostin inhibited the growth of A. triangularis trophozoites and cysts for up to 72 h. The extract and α-mangostin combined with chlorhexidine demonstrated good synergism, resulting in a reduction of 1/4–1/16 of the MIC. The SEM results showed that Acanthamoeba cells treated with a single drug and its combination caused damage to the cell membrane and irregular cell shapes. A good combination displayed by the extract or α-mangostin and chlorhexidine, described for the first time. Therefore, this approach is promising as an alternative method for the management of Acanthamoeba infection in the future.

Nanomaterials in the Management of Contact Lens-Associated Ocular Complication

Contact lens is the transparent hemisphere-shaped ophthalmic product worn by a large population all across the globe for improving vision. It is applied to the cornea of the eye not only for improving vision but also for the delivery of drugs. The contact lens has many benefits but it also causes several ophthalmic infections which include amoebic keratitis, fungal, bacterial, and viral keratitis. The development of bacterial biofilm in lens cases exacerbates the ocular infection after the application of contact lenses in the cornea. Contact lens often associated with discomfort and dry eye. Coating these contact lens with different nanomaterials and an antimicrobial agent is a very good approach to improve the quality and biological activity of lenses. Surface functionalization is the most commonly adopted, and versatile method to tailor the optical properties. This review highlights the new technology to achieve the sustained release of drugs from contact lens by controlling their chemical, mechanical, and optical properties..

Diagnosis of Acanthamoeba keratitis in Mashhad, Northeastern Iran: A Gene-Based PCR Assay

Background: The genus Acanthamoeba is a free-living opportunistic protozoan parasite, which widely distributed in soil and fresh water. Acanthamoeba keratitis, which causes a sight-threating infection of the cornea, is going to rise in Iran and worldwide. The aim of this study was to compare direct microscopy, culture and PCR for detection of Acanthamoeba spp. in clinical samples and to determine the genotypes of Acanthamoeba spp. by sequencing 18SrRNA gene. Methods: Among patients clinically suspected to AK referred to a tertiary ophthalmology center at Mashhad, northeastern Iran. During 2017-18, twenty corneal scrapes specimens obtained. The samples were divided into three parts, subjected to direct microscopic examination, culture onto non-nutrient agar and PCR technique. Sensitivity, specificity, accuracy and likelihood ratio were evaluated. Results: Among 20 persons clinically suspected to amoebic keratitis, 13(69.2%) patients definitely diagnosed as Acanthamoeba keratitis. Wearing contact lens, eye trauma due to foreign particle and swimming in fresh water were the main predisposing factors. Most of patients suffered from pain and photophobia. Corneal ring infiltration and epithelial defect were common clinical sings. Direct examination had the lowest sensitivity and sensitivity of both Nelson-PCR and JDP-PCR methods were equal and highest. In addition, the results of sequencing identified that all strains belonged to T4 genotype. Conclusion: Amoebic keratitis is a sporadic parasitic keratitis, which is mainly seen in contact lens user in Mashhad. PCR based on 18S ribosomal DNA with JDP primers is a reliable and highly sensitive method for diagnosis of Acanthamoeba keratitis in clinically suspected cases.

Cytochrome P450 monooxygenase of Acanthamoeba castellanii participates in resistance to polyhexamethylene biguanide treatment

Acanthamoeba spp. are free-living parasites that can cause severe infections such as granulomatous amoebic encephalitis (GAE) and amoebic keratitis (AK). Polyhexamethylene biguanide (PHMB) is a topical application for AK treatment. However, PHMB is not entirely effective against all Acanthamoeba strains or isolates. The mechanisms by which Acanthamoeba protects itself against extreme drug conditions without encystation are still unknown. According to a previous study, cytochrome P450 monooxygenase (CYP450MO) plays an important role in the oxidative biotransformation of numerous drugs related to metabolism. In this study, a CYP450MO fragment was inserted into the pGAPDH-EGFP vector and transfected into Acanthamoeba castellanii. We found that CYP450MO-overexpressing Acanthamoeba had higher survival rates than those of the control cells after PHMB treatment. Moreover, we also found that encystation-related genes such as cellulose synthase I (CSI), encystation-mediating serine proteinase (EMSP), and autophagy-related protein 8 (ATG8) expression levels were not significantly different between Acanthamoeba transfected by pGAPDH-EGFP or pGAPDH-EGFP-CYP450MO. We suggest that Acanthamoeba transfected by pGAPDH-EGFP-CYP450MO may not induce encystation-related genes to resist PHMB treatment. In conclusion, these findings indicate that CYP450MO may be an additional target when PHMB is used for treatment of amoebic keratitis.

Acanthamoeba spp. monoclonal antibody against a CPA2 transporter: a promising molecular tool for acanthamoebiasis diagnosis and encystment study

Free-living amoeba of the genus Acanthamoeba are ubiquitous protozoa involved in opportunistic and non-opportunistic infection in humans, such as granulomatous amoebic encephalitis and amoebic keratitis. Both infections have challenging characteristics such as the formation of the resistant cysts in infected tissues, hampering the treatment and most usual diagnosis depending on time-consuming and/or low sensitivity techniques. The use of monoclonal antibodies presents itself as an opportunity for the development of more effective alternative diagnostic methods, as well as an important and useful tool in the search for new therapeutic targets. This study investigated the possibility of using a previously produced monoclonal antibody (mAb3), as a diagnostic tool for the detection of Acanthamoeba trophozoites by direct and indirect flow cytometry and immunofluorescence. Immunoprecipitation assay and mass spectrometry allowed the isolation of the antibody's target and suggested it is a transporter part of the CPA (cation: proton antiporter) superfamily. In vitro tests indicate an important role of this target in Acanthamoeba's encystment physiology. Our results support the importance of studying the role of CPA2 transporters in the context of acanthamoebiasis, as this may be a way to identify new therapeutic candidates.

Amoebic infections

Two very different groups of amoebic species infect humans. (1) Obligate anaerobic gut parasites, including the major pathogen Entamoeba histolytica, which causes amoebic dysentery and amoebic liver abscess. Infection is especially common in Mexico, South America, Africa and South-east Asia; nearly all cases in temperate countries is acquired elsewhere. Other gut amoebae are Dientamoeba fragilis (which causes relatively mild colonic involvement with diarrhoea), and eight non-pathogenic species including Entamoeba dispar. (2) Aerobic free-living, water and soil amoebae– Naegleria, Acanthamoeba and Balmuthua, which can become facultative tissue parasites in humans after cysts or trophozoites are inhaled, ingested, or enter damaged skin, cornea or mucosae: they may cause primary amoebic meningo-encephalitis, amoebic keratitis or brain abscess.

Isolation and molecular identification of Acanthamoeba spp. from hot springs in Mazandaran province, northern Iran

Acanthamoeba is a free-living protozoan that can be found in natural and artificial environments such as hot tubs, surface water and springs and can cause severe diseases including amoebic keratitis and granulomatous amoebic encephalitis. The present study was conducted owing to the lack of research regarding genotypes of Acanthamoeba in hot springs of Mazandaran province in northern Iran. Twenty-four water samples were collected from all hot springs in Mazandaran province. After filtration through nitrocellulose membrane, samples were cultured on non-nutrient agar medium enriched with TYIS-33. The cultures were microscopically examined for the presence of Acanthamoeba. Positive cultures were analysed by polymerase chain reaction (PCR) and genotypes were determined by targeting the 18 S rRNA gene. The pathogenic potential of all positive isolates was identified using thermotolerance and osmotolerance tests. Eleven (47.8%) samples were positive for Acanthamoeba. Based on sequencing analysis, 100% of isolates belonged to the T4 genotype. Thermo- and osmo-tolerance tests showed that four (36.3%) Acanthamoeba strains were highly pathogenic. According to our research, the occurrence of Acanthamoeba in recreational hot springs could be a hazard for high risk persons. Posting warning signs and regular monitoring of these waters by health planners may therefore be useful for decreasing Acanthamoeba spp. infections.