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PLoS Neglected Tropical Diseases Mar 2024Acanthamoeba keratitis (AK) is a corneal sight-threatening infection caused by the free-living amoebae of the genus Acanthamoeba. Early and appropriate treatment...
BACKGROUND
Acanthamoeba keratitis (AK) is a corneal sight-threatening infection caused by the free-living amoebae of the genus Acanthamoeba. Early and appropriate treatment significantly impacts visual outcomes. Mucoadhesive polymers such as chitosan are a potential strategy to prolong the residence time and bioavailability of the encapsulated drugs in the cornea. Regarding the recent administration of miltefosine (MF) for treating resistant AK, in the present study, we synthesized miltefosine-loaded chitosan nanoparticles (MF-CS-NPs) and evaluated them against Acanthamoeba.
METHODOLOGY/PRINCIPAL FINDINGS
Chitosan nanoparticles (CNPs) were prepared using the ionic gelation method with negatively charged tripolyphosphate (TPP). The zeta-potential (ZP) and the particle size of MF-CS-NPs were 21.8±3.2 mV and 46.61±18.16 nm, respectively. The release profile of MF-CS-NPs indicated linearity with sustained drug release. The cytotoxicity of MF-CS-NPs on the Vero cell line was 2.67 and 1.64 times lower than free MF at 24 and 48 hours. This formulation exhibited no hemolytic activity in vitro and ocular irritation in rabbit eyes. The IC50 of MF-CS-NPs showed a significant reduction by 2.06 and 1.69-fold in trophozoites at 24 and 48 hours compared to free MF. Also, the MF-CS-NPs IC50 in the cysts form was slightly decreased by 1.26 and 1.21-fold at 24 and 48 hours compared to free MF.
CONCLUSIONS
The MF-CS-NPs were more effective against the trophozoites and cysts than free MF. The nano-chitosan formulation was more effective on trophozoites than the cysts form. MF-CS-NPs reduced toxicity and improved the amoebicidal effect of MF. Nano-chitosan could be an ideal carrier that decreases the cytotoxicity of miltefosine. Further analysis in animal settings is needed to evaluate this nano-formulation for clinical ocular drug delivery.
Topics: Animals; Rabbits; Drug Carriers; Chitosan; Acanthamoeba; Nanoparticles; Phosphorylcholine
PubMed: 38527059
DOI: 10.1371/journal.pntd.0011976 -
BMJ Open Ophthalmology Apr 2024Microbial keratitis (MK) is a significant cause of blindness in sub-Saharan Africa. We investigated the feasibility of using a novel corneal impression membrane (CIM)...
OBJECTIVE
Microbial keratitis (MK) is a significant cause of blindness in sub-Saharan Africa. We investigated the feasibility of using a novel corneal impression membrane (CIM) for obtaining and processing samples by culture, PCR and whole-genome sequencing (WGS) in patients presenting with suspected MK in Malawi.
METHODS AND ANALYSIS
Samples were collected from patients presenting with suspected MK using a 12 mm diameter polytetrafluoroethylene CIM disc. Samples were processed using culture and PCR for , herpes simplex virus type 1 (HSV-1) and the bacterial 16S rRNA gene. Minimum inhibitory concentrations of isolates to eight antimicrobials were measured using susceptibility strips. WGS was used to characterise isolates.
RESULTS
71 eyes of 71 patients were included. The overall CIM isolation rate was 81.7% (58 positive samples from 71 participants). 69 (81.2%) of isolates were Gram-positive cocci. Coagulase-negative 31.8% and species 14.1% were the most isolated bacteria. Seven (9.9%) participants were positive for HSV-1. Fungi and were not detected. Moxifloxacin and chloramphenicol offered the best coverage for both Gram-positive and Gram-negative isolates when susceptibility was determined using known antimicrobial first quartile concentrations and European Committee on Antimicrobial Susceptibility Testing breakpoints, respectively. WGS identified known virulence genes associated with keratitis.
CONCLUSIONS
In a resource-poor setting, a CIM can be used to safely sample the cornea in patients presenting with suspected MK, enabling identification of causative microorganisms by culture and PCR. Although the microbiological spectrum found was limited to the dry season, these preliminary results could be used to guide empirical treatment.
Topics: Humans; Pilot Projects; Malawi; Male; Female; Adult; Middle Aged; Eye Infections, Bacterial; Young Adult; Bacteria; Microbial Sensitivity Tests; Cornea; Keratitis; Anti-Bacterial Agents; Aged; Polymerase Chain Reaction; Adolescent; Acanthamoeba; RNA, Ribosomal, 16S
PubMed: 38653537
DOI: 10.1136/bmjophth-2024-001682 -
BMC Infectious Diseases Mar 2024To compare the outcomes of big-bubble deep anterior lamellar keratoplasty (BB-DALK) and penetrating keratoplasty (PKP) in the management of medically unresponsive...
PURPOSE
To compare the outcomes of big-bubble deep anterior lamellar keratoplasty (BB-DALK) and penetrating keratoplasty (PKP) in the management of medically unresponsive Acanthamoeba keratitis (AK).
METHODS
This retrospective study included 27 eyes of BB-DALK and 24 eyes of PKP from a tertiary ophthalmology care centre. Glucocorticoid eye drops were subsequently added to the treatment plan 2 months postoperatively based on the evaluation using confocal laser scanning microscopy. The clinical presentations, best-corrected visual acuity (BCVA), postoperative refractive outcomes, graft survival, and Acanthamoeba recurrence were analyzed.
RESULTS
The AK patients included in the study were in stage 2 or stage 3, and the percentage of patients in stage 3 was higher in the PKP group (P = 0.003). Clinical presentations were mainly corneal ulcers and ring infiltrates, and endothelial plaques, hypopyon, uveitis and glaucoma were more common in the PKP group (P = 0.007). The BCVA and the graft survival rate showed no statistically significant differences between the two groups at 1 year after surgery. However, 3 years postoperatively, the BCVA of 0.71 ± 0.64 logMAR, the graft survival rate of 89.5%, and the endothelial cell density of 1899 ± 125 cells per square millimeter in the BB-DALK group were significantly better than those of the PKP group (P = 0.010, 0.046, and 0.032, respectively). 3 eyes (11.1%) in the BB-DALK group and 2 eyes (8.3%) in the PKP group experienced Acanthamoeba recurrence, but the rates showed no statistically significant difference between the two groups (P = 1.000). In the PKP group, immune rejection and elevated intraocular pressure were observed in 5 and 6 eyes, respectively.
CONCLUSION
Corneal transplantation is recommended for AK patients unresponsive to antiamoebic agents. The visual acuity and graft survival can be maintained after BB-DALK surgery. Acanthamoeba recurrence is not related to the surgical approach performed, whereas complete dissection of the infected corneal stroma and delayed prescribing of glucocorticoid eye drops were important to prevent recurrence.
Topics: Humans; Acanthamoeba Keratitis; Keratoplasty, Penetrating; Glucocorticoids; Retrospective Studies; Corneal Transplantation; Glaucoma; Ophthalmic Solutions
PubMed: 38438857
DOI: 10.1186/s12879-024-09147-w -
Scientific Reports Jun 2024Natural products play a significant role in providing the current demand as antiparasitic agents, which offer an attractive approach for the discovery of novel drugs....
Natural products play a significant role in providing the current demand as antiparasitic agents, which offer an attractive approach for the discovery of novel drugs. The present study aimed to evaluate in vitro the potential impact of seaweed Padina pavonica (P. pavonica) extract in combating Acanthamoeba castellanii (A. castellanii). The phytochemical constituents of the extract were characterized by Gas chromatography-mass spectrometry. Six concentrations of the algal extract were used to evaluate its antiprotozoal activity at various incubation periods. Our results showed that the extract has significant inhibition against trophozoites and cysts viability, with complete inhibition at the high concentrations. The IC of P. pavonica extract was 4.56 and 4.89 µg/mL for trophozoites and cysts, respectively, at 24 h. Morphological alterations of A. castellanii trophozoites/cysts treated with the extract were assessed using inverted and scanning electron microscopes and showed severe damage features upon treatment with the extract at different concentrations. Molecular Docking of extracted compounds against Acanthamoeba cytochrome P450 monooxygenase (AcCYP51) was performed using Autodock vina1.5.6. A pharmacokinetic study using SwissADME was also conducted to investigate the potentiality of the identified bioactive compounds from Padina extract to be orally active drug candidates. In conclusion, this study highlights the in vitro amoebicidal activity of P. pavonica extract against A. castellanii adults and cysts and suggests potential AcCYP51 inhibition.
Topics: Acanthamoeba castellanii; Molecular Docking Simulation; Acanthamoeba Keratitis; Plant Extracts; Antiprotozoal Agents; Trophozoites; Animals; Humans
PubMed: 38871751
DOI: 10.1038/s41598-024-63691-8 -
Case Reports in Ophthalmology 2023The aim of this study was to report a case with the use of amniotic membrane transplant and deep anterior keratoplasty in a patient with bilateral Acanthamoeba...
The aim of this study was to report a case with the use of amniotic membrane transplant and deep anterior keratoplasty in a patient with bilateral Acanthamoeba infectious keratitis as a treatment. A 20-year-old male presented with bilateral Acanthamoeba keratitis (AK) who was initially diagnosed with herpetic keratitis receiving full antiviral and corticosteroid topical treatment without any improvement. Corneal biopsy was performed to confirm the suspected diagnosis, and Acanthamoeba stromal cysts were identified in the sample. Treatment was initiated with 0.02% chlorhexidine, 0.1% propamidine isethionate, neomycin, and tropicamide/phenylephrine. Symptoms and clinical improvement were achieved between the 8th and 10th weeks, so corticosteroids were initiated. Treatment was continued until we observed a poor response in the left eye; therefore, an epithelial scraping and amniotic membrane placement were performed. Lately, the right eye underwent a deep anterior lamellar keratoplasty. A challenging case of bilateral AK managed with topical medications, amniotic membrane, and corneal keratoplasty. The earlier the disease is diagnosed, the better the outcome. If the diagnosis is delayed, the amoebas have penetrated deep into the corneal stroma, and successful therapy becomes difficult. A surgical option can be an early solution with a good prognosis for these cases.
PubMed: 37901622
DOI: 10.1159/000533988 -
ISME Communications Jan 2024, a free-living amoeba in water and soil, is an emerging pathogen causing severe eye infection known as keratitis. In its natural environment, performs a dual function...
, a free-living amoeba in water and soil, is an emerging pathogen causing severe eye infection known as keratitis. In its natural environment, performs a dual function as an environmental heterotrophic predator and host for a range of microorganisms that resist digestion. Our objective was to characterize the intracellular microorganisms of phylogenetically distinct spp. isolated in Australia and India through directly sequencing 16S rRNA amplicons from the amoebae. The presence of intracellular bacteria was further confirmed by hybridization and electron microscopy. Among the 51 isolates assessed, 41% harboured intracellular bacteria which were clustered into four major phyla: Pseudomonadota (previously known as Proteobacteria), Bacteroidota (previously known as Bacteroidetes), Actinomycetota (previously known as Actinobacteria), and Bacillota (previously known as Firmicutes). The linear discriminate analysis effect size analysis identified distinct microbial abundance patterns among the sample types; species was abundant in Australian corneal isolates ( < 0.007), Enterobacteriales showed higher abundance in Indian corneal isolates ( < 0.017), and Bacteroidota was abundant in Australian water isolates ( < 0.019). The bacterial beta diversity of isolates from keratitis patients in India and Australia significantly differed ( < 0.05), while alpha diversity did not vary based on the country of origin or source of isolation ( > 0.05). More diverse intracellular bacteria were identified in water isolates as compared with clinical isolates. Confocal and electron microscopy confirmed the bacterial cells undergoing binary fission within the amoebal host, indicating the presence of viable bacteria. This study sheds light on the possibility of a sympatric lifestyle within , thereby emphasizing its crucial role as a bunker and carrier of potential human pathogens.
PubMed: 38500701
DOI: 10.1093/ismeco/ycae016 -
Turkiye Parazitolojii Dergisi Sep 2023, one of the free-living amoeba, has been detected in many environmental samples, mainly in water, soil and air. keratitis and granulomatous amoebic encephalitis are...
OBJECTIVE
, one of the free-living amoeba, has been detected in many environmental samples, mainly in water, soil and air. keratitis and granulomatous amoebic encephalitis are among the most important clinical manifestations caused by . In this study, it was aimed to determine the sensitivity of the rapid loop mediated isothermal amplification (LAMP) test designed with primers specific to 18S rRNA gene to detect more rapidly the presence of in clinical and environmental samples.
METHODS
strain grown in culture was diluted in 200 μL as 1x106 trophozoites and DNA was isolated, and the amount of DNA was determined by Nano-Drop Spectrophotometer. The purified DNAs were diluted from 1000 pg to 0.001 pg and used in colorimetric and fluorescence-based LAMP reactions. The LAMP reaction mixture was incubated for 60 minutes at 63 °C in a total volume of 25 μL.
RESULTS
To determine the sensitivity of the test, positivity of genomic DNA was observed at 1, 10, 100 and 1000 pg/reaction in both colorimetric and fluorescence-based LAMP tests. The lowest analytical sensitivity of both calorimetric and fluorescent LAMP assay was determined as 1 pg/reaction. In addition, as a result of LAMP reaction applied with other parasite DNAs to evaluate the specificity of the test, no LAMP product was detected in calorimetric and 1% agarose gel electrophoresis, except for positive control, and the specificity of the test was determined as 100%.
CONCLUSION
It has been demonstrated that the LAMP assay designed by targeting 18S rRNA gene of has a detection limit of 1 pg of genomic DNA. It is promising that LAMP test is more sensitive and faster than culture method, as well as simple, inexpensive and highly sensitive. For this reason, it is thought that developed test can be applied in the diagnosis of in environmental and clinical samples.
Topics: Acanthamoeba; Genes, rRNA; Biological Assay; Coloring Agents
PubMed: 37724360
DOI: 10.4274/tpd.galenos.2023.46362 -
Cureus Nov 2023keratitis is a potentially sight-threatening infection of the cornea that can lead to blindness. Over the past few decades, there has been a rise in the reported cases...
keratitis is a potentially sight-threatening infection of the cornea that can lead to blindness. Over the past few decades, there has been a rise in the reported cases worldwide, which is in line with the increase of contact lens wearers. Here, we present three cases of keratitis with different outcomes.
PubMed: 38046722
DOI: 10.7759/cureus.48129 -
The Science of the Total Environment Sep 2024Free-living amoebae (FLA) such as Acanthamoeba, Balamuthia mandrillaris, Naegleria fowleri and Sappinia pedata are naturally widespread in freshwater, causing rare but...
Free-living amoebae (FLA) such as Acanthamoeba, Balamuthia mandrillaris, Naegleria fowleri and Sappinia pedata are naturally widespread in freshwater, causing rare but fatal and debilitating infections in humans. Although recent studies have shown an increase in infection rates, there is a paucity of epidemiological studies regarding the presence of these emerging pathogens in water. Herein, we studied the diversity and relative abundance of thermophilic FLA in different recreational baths in a tropical climate for 5 years. From 2018 to 2022, a total of 96 water samples were collected from 7 recreational baths (natural, tiled, regularly cleaned or not, and with temperatures ranging from 27 to 40 °C). DNA was extracted from FLA cultivated at 37 °C to detect thermophilic culturable FLA. Metabarcoding studies were conducted through FLA 18S rRNA gene amplicons sequencing; amplicon sequence variants (ASV) were extracted from each sample and taxonomy assigned against PR2 database using dada2 and phyloseq tools. We also searched for Naegleria sp. and N. fowleri using PCR targeting ITS and NFITS genes (respectively) and we quantified them using an optimized most probable number (MPN) method for FLA. Our results showed that differences in FLA diversity and abundance were observed amongst the 7 baths, but without a clear seasonal distribution. Naegleria, Vermamoeba and Stenamoeba were the most represented genera, while the genera Acanthamoeba and Vahlkampfia were mainly found in 2 baths. The MPN values for Naegleria sp. (NT/l) increased between 2018 and 2022, but the MPN values for N. fowleri (NF/l) seemed to decrease. Globally, our results showed that since we cannot establish a seasonal distribution of FLA, the regular presence of FLA (namely Naegleria and Acanthamoeba) in recreational waters can pose a potential threat in terms of neuroinfections as well as Acanthamoeba keratitis. It is thus imperious to perform the regular control of these baths as a preventive health measure.
Topics: Amoeba; Guadeloupe; Environmental Monitoring; Fresh Water; Bathing Beaches
PubMed: 38777057
DOI: 10.1016/j.scitotenv.2024.173318 -
Frontiers in Cellular and Infection... 2024Pathogenic and free-living are widely distributed in the environment and have been reported to cause keratitis and universally fatal encephalitis. Primary cutaneous...
Pathogenic and free-living are widely distributed in the environment and have been reported to cause keratitis and universally fatal encephalitis. Primary cutaneous acanthamoebiasis caused by is exceedingly rare and presents as isolated necrotic cutaneous lesions without involvement of the cornea or central nervous system. Cutaneous acanthamoebiasis often occurs in immunocompromised patients and is likely overlooked or even misdiagnosed only by cutaneous biopsy tissue histopathological analysis. Here, we report a HIV-infected 63-year-old female with oral leukoplakia for 4 months and scattered large skin ulcers all over the body for 2 months. The cause of the cutaneous lesions was unclear through cutaneous specimens histopathological analysis, and subsequently were detected by metagenomic next-generation sequencing (mNGS), which may be the cause of cutaneous lesions. Based on the mNGS results, a pathologist subsequently reviewed the previous pathological slides and found trophozoites of so that the cause was identified, and the skin ulcers improved significantly after treatment with multi-drug combination therapy. is also a host of pathogenic microorganisms. The presence of endosymbionts enhances the pathogenicity of , and no other pathogens were reported in this case. mNGS is helpful for rapidly diagnosing the etiology of rare skin diseases and can indicate the presence or absence of commensal microorganisms.
Topics: Humans; Female; High-Throughput Nucleotide Sequencing; Amebiasis; Metagenomics; Middle Aged; Acanthamoeba; HIV Infections; Skin; Treatment Outcome
PubMed: 38863830
DOI: 10.3389/fcimb.2024.1356095