-
Parasites & Vectors May 2024Proteases produced by Acanthamoeba spp. play an important role in their virulence and may be the key to understanding Acanthamoeba pathogenesis; thus, increasing...
Characterization of novel extracellular proteases produced by Acanthamoeba castellanii after contact with human corneal epithelial cells and their relevance to pathogenesis.
BACKGROUND
Proteases produced by Acanthamoeba spp. play an important role in their virulence and may be the key to understanding Acanthamoeba pathogenesis; thus, increasing attention has been directed towards these proteins. The present study aimed to investigate the lytic factors produced by Acanthamoeba castellanii during the first hours of in vitro co-culture with human corneal epithelial cells (HCECs).
METHODS
We used one old and one recent Acanthamoeba isolate, both from patients with severe keratitis, and subsets of these strains with enhanced pathogenic potential induced by sequential passaging over HCEC monolayers. The proteolytic profiles of all strains and substrains were examined using 1D in-gel zymography.
RESULTS
We observed the activity of additional proteases (ranging from 33 to 50 kDa) during the early interaction phase between amoebae and HCECs, which were only expressed for a short time. Based on their susceptibilities to protease inhibitors, these proteases were characterized as serine proteases. Protease activities showed a sharp decline after 4 h of co-incubation. Interestingly, the expression of Acanthamoeba mannose-binding protein did not differ between amoebae in monoculture and those in co-culture. Moreover, we observed the activation of matrix metalloproteinases in HCECs after contact with Acanthamoeba.
CONCLUSIONS
This study revealed the involvement of two novel serine proteases in Acanthamoeba pathogenesis and suggests a pivotal role of serine proteases during Acanthamoeba-host cell interaction, contributing to cell adhesion and lysis.
Topics: Humans; Acanthamoeba castellanii; Epithelial Cells; Coculture Techniques; Epithelium, Corneal; Peptide Hydrolases; Acanthamoeba Keratitis; Serine Proteases; Protozoan Proteins; Virulence
PubMed: 38812022
DOI: 10.1186/s13071-024-06304-7 -
Veterinary World Apr 2024Keratitis is a serious ocular infection often caused by pathogenic microorganisms such as spp. Among other harmful microbes, keratitis presents a particular challenge...
BACKGROUND AND AIM
Keratitis is a serious ocular infection often caused by pathogenic microorganisms such as spp. Among other harmful microbes, keratitis presents a particular challenge due to its resistance to conventional antimicrobial agents. Linn., commonly known as betel leaf, has been traditionally used for its medicinal properties. This study aimed to assess the potential of the leaf ethanol extract of Linn. in the treatment of in monoculture and co-culture with two prevalent pathogenic bacteria, and , associated with keratitis.
MATERIALS AND METHODS
Minimum inhibitory concentrations (MICs) of , , and extracts in monoculture and coinfected conditions were examined. In addition, this study explored the potential of the extract in preventing adherence in both monoculture and co-culture environments. Scanning electron microscopy (SEM) analysis confirmed the impact of the extract on cell membranes, including acanthopodia. Furthermore, a time-kill kinetic assay was used to validate the amoebicidal activity of the extract against and the tested bacteria.
RESULTS
MICs for trophozoites, cysts, , and in the monoculture were 0.25, 0.25, 0.51, and 0.128 mg/mL, respectively, whereas the MICs for coinfected with bacteria were higher than those in the monoculture. This extract inhibited the growth of trophozoites and cysts for up to 72 h. Moreover, extract effectively prevented the adherence of to contact lenses under monoculture conditions. SEM analysis confirmed that extract affects the cell membrane of , including Acanthopodia. In addition, the time-kill kinetic assay confirmed that the extract contained amoebicidal activity against , including the tested bacteria. Notably, was more susceptible than and to extract treatment. Unexpectedly, our study revealed that negatively affected in the co-culture after 3 days of incubation, whereas facilitated the growth of in the presence of the extract.
CONCLUSION
This study provides compelling evidence of the anti-adhesive and anti- properties of leaf extract against under monoculture and co-culture conditions. The observed impact on cell membranes, coupled with the time-kill kinetic assay results, underscores the potential of leaf extract as a promising agent for combating -related infections in humans and animals.
PubMed: 38798284
DOI: 10.14202/vetworld.2024.848-862 -
Microorganisms Apr 2024Some free-living amoebae can behave as opportunistic parasites, causing rare but dangerous diseases in humans and animals, primarily amoebic keratitis, with loss of...
Some free-living amoebae can behave as opportunistic parasites, causing rare but dangerous diseases in humans and animals, primarily amoebic keratitis, with loss of vision, and encephalitis, which is almost always fatal [...].
PubMed: 38792695
DOI: 10.3390/microorganisms12050865 -
The Science of the Total Environment May 2024Free-living amoebae (FLA) such as Acanthamoeba, Balamuthia mandrillaris, Naegleria fowleri and Sappinia are naturally widespread in freshwater, causing rare but fatal...
Free-living amoebae (FLA) such as Acanthamoeba, Balamuthia mandrillaris, Naegleria fowleri and Sappinia 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 rDNA 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 and N. fowleri using PCR targeting ITS and NFITS (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.
PubMed: 38777057
DOI: 10.1016/j.scitotenv.2024.173318 -
Eye & Contact Lens Jun 2024To report first clinical use of novel medical treatment for Acanthamoeba keratitis. (Observational Study)
Observational Study
PURPOSE
To report first clinical use of novel medical treatment for Acanthamoeba keratitis.
METHODS
Interventional observational case series. Two patients with Acanthamoeba keratitis were unsuccessfully treated with polihexanide (PHMB) 0.02% and propamidine 0.1% for 6 weeks, then all were shifted in a compassionate use of PHMB 0.08% with novel standardized protocol. The postinterventional follow-up of patients was at least 7 months.
RESULTS
PHMB 0.08% eyedrops in a novel standardized protocol improved infection resolution and led to complete healing of the lesion after 4 weeks in the two cases. Corneal opacities and neovascularization decreased slowly, best-corrected visual acuity slightly improved and progressively increased in the further 7 months, and no infection recurrence occurred.
CONCLUSIONS
This preliminary report of two cases shows promising response to polihexanide 0.08% lowering drastically the illness duration, with reduced chance of recurrence, and mostly improving patients' quality of life.
Topics: Acanthamoeba Keratitis; Humans; Biguanides; Adult; Male; Female; Benzamidines; Visual Acuity; Ophthalmic Solutions; Antiprotozoal Agents
PubMed: 38709240
DOI: 10.1097/ICL.0000000000001092 -
Journal of Medicinal Chemistry May 2024are free-living pathogenic protozoa that cause blinding keratitis, disseminated infection, and granulomatous amebic encephalitis, which is generally fatal. The...
are free-living pathogenic protozoa that cause blinding keratitis, disseminated infection, and granulomatous amebic encephalitis, which is generally fatal. The development of efficient and safe drugs is a critical unmet need. sterol 14α-demethylase (CYP51) is an essential enzyme of the sterol biosynthetic pathway. Repurposing antifungal azoles for amoebic infections has been reported, but their inhibitory effects on CYP51 enzymatic activity have not been studied. Here, we report catalytic properties, inhibition, and structural characterization of CYP51 from . The enzyme displays a 100-fold substrate preference for obtusifoliol over lanosterol, supporting the plant-like cycloartenol-based pathway in the pathogen. The strongest inhibition was observed with voriconazole (1 h IC 0.45 μM), VT1598 (0.25 μM), and VT1161 (0.20 μM). The crystal structures of CYP51 with bound VT1161 (2.24 Å) and without an inhibitor (1.95 Å), presented here, can be used in the development of azole-based scaffolds to achieve optimal amoebicidal effectiveness.
Topics: Sterol 14-Demethylase; 14-alpha Demethylase Inhibitors; Structure-Activity Relationship; Acanthamoeba; Acanthamoeba castellanii; Crystallography, X-Ray; Antiprotozoal Agents; Models, Molecular; Molecular Structure
PubMed: 38683753
DOI: 10.1021/acs.jmedchem.4c00303 -
Microorganisms Apr 2024Acanthamoeba keratitis (AK) is a rare but potentially sight-threatening corneal infection caused by the Acanthamoeba parasite. This microorganism is found ubiquitously... (Review)
Review
Acanthamoeba keratitis (AK) is a rare but potentially sight-threatening corneal infection caused by the Acanthamoeba parasite. This microorganism is found ubiquitously in the environment, often in freshwater, soil, and other sources of moisture. Despite its low incidence, AK presents significant challenges due to delayed diagnosis and the complex nature of therapeutic management. Early recognition is crucial to prevent severe ocular complications, including corneal ulceration and vision loss. Diagnostic modalities and treatment strategies may vary greatly depending on the clinical manifestation and the available tools. With the growing reported cases of Acanthamoeba keratitis, it is essential for the ophthalmic community to thoroughly understand this condition for its effective management and improved outcomes. This review provides a comprehensive overview of AK, encompassing its epidemiology, risk factors, pathophysiology, clinical manifestations, diagnosis, and treatment.
PubMed: 38674702
DOI: 10.3390/microorganisms12040758 -
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 -
Digital Journal of Ophthalmology : DJO 2024Laboratory confirmation is crucial for diagnosis and management of herpes simplex virus (HSV) keratitis. However, the sensitivity of polymerase chain reaction (PCR) in...
BACKGROUND
Laboratory confirmation is crucial for diagnosis and management of herpes simplex virus (HSV) keratitis. However, the sensitivity of polymerase chain reaction (PCR) in keratitis is low (25%) compared with that of mucocutaneous disease (75%). We developed an educational intervention aimed at improving the diagnostic yield of PCR.
METHODS
The medical records of keratitis cases seen at the emergency department of a London tertiary ophthalmic referral hospital over two distinct periods, before and after an educational program on swab technique, were reviewed retrospectively.
RESULTS
A total of 252 HSV cases were included. Increases in the laboratory-confirmed diagnosis of HSV-1 were observed, in both first presentations (11.1%-57.7%) and recurrent cases (20%-57.6%). The rate of positive HSV-1 PCR in eyes with an epithelial defect increased from 19% pre-intervention to 62% post intervention. Notably, 3% were positive for varicella zoster virus DNA, and there was a single case of keratitis.
CONCLUSION
Our results suggest that, with proper swabbing technique, PCR may be more sensitive than previously reported.
Topics: Humans; Pilot Projects; Retrospective Studies; DNA, Viral; Keratitis, Herpetic; Herpesvirus 1, Human; Polymerase Chain Reaction; Herpes Simplex
PubMed: 38601900
DOI: 10.5693/djo.01.2024.01.002 -
Microorganisms Mar 2024The genus comprises free-living amoebae distributed in a wide variety of environments. These amoebae are clinically significant, causing opportunistic infections in...
The genus comprises free-living amoebae distributed in a wide variety of environments. These amoebae are clinically significant, causing opportunistic infections in humans and other animals. Despite this, limited data on sequence types and alleles are available in Italy. In the present study, we analyzed all sequences deposited from Italy with new positive clinical samples from symptomatic AK cases, to provide an overview of the genetic variants' spatial patterns from different sources within the Italian context. A total of 137 sequences were obtained. Six sequence types were identified: T2/6, T3, T4, T11, T13, and T15. Only T4 and T15 were found in both sources. The T4 sequence type was found to be the most prevalent in all regions, accounting for 73% (100/137) of the Italian samples analyzed. The T4 sequence type demonstrated significant allelic diversity, with 30 distinct alleles from clinical and/or environmental samples. These outcomes enabled a better understanding of the distribution of isolates throughout Italy, reaffirming its well-recognized ubiquity. isolates analysis from keratitis, together with the environmental strains monitoring, might provide important information on different genotypes spreading. This might be useful to define the transmission pathways of human keratitis across different epidemiological scales.
PubMed: 38543595
DOI: 10.3390/microorganisms12030544