Efficacy of Off-Label Anti-Amoebic Agents to Suppress Trophozoite Formation of Acanthamoeba spp. on Non-Nutrient Agar Escherichia Coli Plates.

Acanthamoeba keratitis (AK) is a dangerous infectious disease, which is associated with a high risk of blindness for the infected patient, and for which no standard therapy exists thus far. Patients suffering from AK are thus treated, out of necessity, with an off-label therapy, using drugs designed and indicated for other diseases/purposes. Here, we tested the capability of the off-label anti-amoebic drugs chlorhexidine (CH; 0.1%), dibromopropamidine diisethionate (DD; 0.1%), hexamidine diisethionate (HD; 0.1%), miltefosine (MF; 0.0065%), natamycin (NM; 5%), polyhexamethylene biguanide (PHMB; 0.02%), povidone iodine (PVPI; 1%), and propamidine isethionate (PD; 0.1%) to suppress trophozoite formation of Acantamoeba castellanii and Acanthamoeba hatchetti cysts on non-nutrient agar Escherichia coli plates. Of the eight off-label anti-amoebic drugs tested, only PVPI allowed for a complete suppression of trophozoite formation by drug-challenged cysts for all four Acanthamoeba isolates in all five biological replicates. Drugs such as NM, PD, and PHMB repeatedly suppressed trophozoite formation with some, but not all, tested Acanthamoeba isolates, while other drugs such as CH, DD, and MF failed to exert a relevant effect on the excystation capacities of the tested Acanthamoeba isolates in most, if not all, of our repetitions. Our findings suggest that pre-testing of the AK isolate with the non-nutrient agar E. coli plate assay against the anti-amoebic drug intended for treatment should be performed to confirm that the selected drug is cysticidal for the Acanthamoeba isolate.

The Effect of Anti-Amoebic Agents and Ce6-PDT on Acanthamoeba castellanii Trophozoites and Cysts, In Vitro.

Purpose: The purpose of this study was to analyze the concentration-dependent effects of biguanides (polyhexamethylene biguanide [PHMB], chlorhexidine [CH]); diamidines (hexamidine-diisethionate [HD], propamidine-isethionate [PD], dibromopropamidine-diisethionate [DD]); natamycin (NM); miltefosine (MF); povidone iodine (PVPI), and chlorin e6 PDT on Acanthamoeba trophozoites and cysts, in vitro. Methods: Strain 1BU was cultured in peptone-yeast extract-glucose medium. Trophozoites or cysts were cultured in PYG medium containing each agent at 100%, 50%, and 25% of maximum concentration for 2 hours. The percentage of dead trophozoites was determined using a non-radioactive cytotoxicity assay and trypan blue staining. Treated cysts were also maintained on non-nutrient agar Escherichia coli (E.coli) plates and observed for 3 weeks. Results: All tested drugs displayed significant cytotoxic effects on 1BU cells based on the biochemical and staining-based viability assays tested. On non-nutrient agar E. coli plates, neither trophozoites nor freshly formed cysts were observed after PHMB, PD, NM, and PVPI treatment, respectively, within 3 weeks. However, CH-, HD-, DD-, and MF-treated cysts could excyst, multiply, and encyst again. Conclusions: The off-label drugs PHMB, PD, NM, and PVPI are under in vitro conditions more effective against strain 1BU than CH, HD, DD, and MF. Our findings also suggest that the non-nutrient agar E.coli plate assay should be considered as method of choice for the in vitro analysis of the treatment efficacy of anti-amoebic agents. Translational Relevance: Ophthalmologists may optimize the treatment regime against Acanthamoeba keratitis by pre-testing the in vitro susceptibilities of the Acanthamoeba strain against drugs of interest with the non-nutrient E.coli agar plate assay.

Comparison of in vitro assays to study the effectiveness of antiparasitics against Acanthamoeba castellani trophozoites and cysts.

We aimed to compare LDH release assay, trypan blue and fluorescent stainings, and non-nutrient Escherichia coli plate assay in determining treatment efficacy of antiamoebic agents against Acanthamoeba castellanii trophozoites/cysts, in vitro. 1BU trophozoites/cysts were challenged with 0.02% polyhexamethylene biguanid (PHMB), 0.1% propamidine isethionate (PD), and 0.0065% miltefosine (MF). Efficacies of the drugs were determined by LDH release and trypan blue assays, by Hoechst 33343, calcein-AM, and ethidium homodimer-1 fluorescent dyes, and by a non-nutrient agar E. coli plate assay. All three antiamoebic agents induced a significant LDH release from trophozoites, compared to controls (p < 0.0001). Fluorescent-dye staining in untreated 1BU trophozoites/cysts was negligible, but using antiamoebic agents, there was 59.3%-100% trypan blue, 100% Hoechst 33342, 0%-75.3% calcein-AM, and 100% ethidium homodimer-1 positivity. On E. coli plates, in controls and MF-treated 1BU trophozoites/cysts, new trophozoites appeared within 24 h, encystment occurred after 5 weeks. In PHMB- and PD-treated 1BU throphozoites/cysts, irregularly shaped, smaller trophozoites appeared after 72 h, which failed to form new cysts within 5 weeks. None of the enzymatic- and dye-based viability assays tested here generated survival rates for trophozoites/cysts that were comparable with those yielded with the non-nutrient agar E. coli plate assay, suggesting that the culture-based assay is the best method to study the treatment efficacy of drugs against Acanthamoeba.

Genotyping of Acantamoeba spp. from rhisophere in Hungary.

The protista Acanthamoeba is a free-living amoeba existing in various environments. A number of species among protista are recognized as human pathogens, potentially causing Acanthamoeba keratitis (AK), granulomatous amoebic encephalitis (GAE), and chronic granulomatous lesions. In this study, 10 rhizosphere samples were collected from maize and alfalfa plants in experimental station at Institute of Genetics, Microbiology and Biotechnology, Szent István University. We detected Acanthamoeba based on the quantitative real-time PCR assay and sequence analysis of the 18S rRNA gene. All studied molecular biological methods are suitable for the detection of Acanthamoeba infection in humans. The quantitative real-time PCR-based methods are more sensitive, simple, and easy to perform; moreover, these are opening avenue to detect the effect of number of parasites on human disease. Acanthamoeba species were detected in five (5/10; 50%) samples. All Acanthamoeba strains belonged to T4 genotype, the main AK-related genotype worldwide. Our result confirmed Acanthamoeba strains in rhizosphere that should be considered as a potential health risk associated with human activities in the environment.

Clinical course of Acanthamoeba keratitis by genotypes T4 and T8 in Hungary.

Genus Acanthamoeba is an opportunistic protozoan that is widely distributed in the environment. Within this genus, numerous species are recognized as human pathogens, potentially causing Acanthamoeba keratitis (AK). AK is a corneal disease, associated predominantly with contact lens (CL) wear; its epidemiology is related to the specific Acanthamoeba genotypes. This study reports seven CL wearer, Acanthamoeba PCR-positive patients with AK, diagnosed between January 2015 and 2018. Patients had the diagnosis of AK 1.36 months after first symptoms. Genotyping allowed the identification of six isolates of the T4 and one of the T8 genotypes. At first presentation, pseudendritiformic epithelopathy/dirty epithelium (four eyes, 57.1%), multifocal stromal infiltrates (five eyes, 71.4%), ring infiltrate (three eyes, 42.8%), and perineuritis (one eye, 14.3%) were observed. AK was healed without later recurrence in two eyes (28.5%) using triple-topical therapy, in three eyes (42.8%) following additional penetrating keratoplasty. In one patient (14.3%), AK recurred following successful application of triple-therapy and was treated successfully with repeated triple-topical therapy and in one patient (14.3%), no follow-up data were available after diagnosis. We could not observe correlation of genotype and clinical course or the necessity of corneal transplantation in our case series.

First report of Acanthamoeba genotype T8 human keratitis.

Acanthamoeba has a worldwide distribution in the environment and it is capable of causing a painful sight-threatening disease of the cornea designated as Acanthamoeba keratitis (AK). Nowadays, the cases of AK have surged all over the world along with its disease burden due to increasing use of contact lenses used not only for optical correction but also for cosmetic purposes. In our present work, epithelial abrasion of a 27-year-old female soft contact lens wearer with keratitis was examined. Genotype identification was carried out with a real-time fluorescence resonance energy transfer polymerase chain reaction (PCR) assay based on sequence analysis of the 18S rRNA gene. Genotyping allowed the identification of a T8 group isolate. The analysis confirmed the importance of a complete diagnostic protocol, including a PCR assay, for the clinical diagnosis of AK from human samples. Acanthamoeba T8 should be considered as potential causative organism in keratitis in human.

Laboratory diagnosis of Acanthamoeba keratitis in Hungary.

Acanthamoeba species are free-living amebae that can be found in almost every range of environments. Within this genus, numerous species are recognized as human pathogens, potentially causing Acanthamoeba keratitis (AK). AK is a corneal disease that is predominantly associated with contact lens use, the epidemiology of which is related to the specific genotype of Acanthamoeba. This study reports seven (7/16; 43.75%) positive cases. Detection of Acanthamoeba in corneal scrapings is based on cultivation and polymerase chain reaction (PCR) combined with the molecular taxonomic identification method. By PCR, seven samples were positive; cultivation was successful for five samples, probably because of the low quantity of samples. Genotype identification was carried out with a real-time fluorescence resonance energy transfer PCR assay based on sequence analysis of the 18S rRNA gene, and sensitivity and specificity were evaluated in comparison with traditional parasitological techniques. All seven detected Acanthamoeba strains belonged to the T4 genotype, the main AK-related genotype worldwide. These results confirmed the importance of a complete diagnostic protocol, including a PCR assay, for the clinical diagnosis of AK from human samples. Genotyping allowed the identification of all isolates in the T4 group, thus demonstrating the prevalence of this genotype in Hungary.

Isolation of Acanthamoeba from the rhizosphere of maize and lucerne plants.

Acanthamoeba species are free-living amoebae that can be found in almost every range of environments. Within this genus, a number of species are recognized as human pathogens, potentially causing Acanthamoeba keratitis, granulomatous amoebic encephalitis, and chronic granulomatous lesions. Soil and water samples were taken from experimental station at Julianna Major of Plant Protection Institute of Centre for Agricultural Research, Hungarian Academy of Sciences (CAR HAS). We detected living Acanthamoeba spp. based on culture-confirmed detection combined with the molecular taxonomic identification method. Living Acanthamoeba spp. were detected in thirteen (65%) samples. The presence of Acanthamoeba spp. in the samples depends significantly on the rhizosphere plants. The most frequently identified living Acanthamoeba genotype was T4 followed by T11, T2/T6 and T17. Genotypes T4 and T11 of Acanthamoeba, are responsible for Acanthamoeba keratitis as well as granulomatous amoebic encephalitis, and should therefore be considered as a potential health risk associated with human activities in the environment.

Real-time PCR assay for rapid qualitative and quantitative detection of Entamoeba histolytica.

Simple real-time PCR assay with one set of primer and probe for rapid, sensitive qualitative and quantitative detection of Entamoeba histolytica has been used. Consensus sequences were used to amplify a species-specific region of the 16S rRNA gene, and fluorescence resonance energy transfer hybridization probes were used for detection in a LightCycler platform (Roche). The anchor probe sequence was designed to be a perfect match for the 16S rRNA gene of Entamoeba species, while the acceptor probe sequence was designed for Entamoeba histolytica, which allowed differentiation. The performed characteristics of the real-time PCR assay were compared with ELISA antigen and microscopical detection from 77 samples of individuals with suspected clinical diagnosis of imported E. histolytica infection. Stool and liver abscess pus samples were examined with analytical sensitivity of 5 parasites per PCR reaction. The melting curve means Tms (standard deviation) in clinical isolates were 54°C. The real-time assay was 100% sensitive and specific for differentiation of Entamoeba histolytica, compared with conventional ELISA or microscopy. This real-time PCR assay with melting curve analysis is rapid, and specific for the detection and differentiation of Entamoeba histolytica. The suitability for routine use of this assay in clinical diagnostic laboratories is discussed.