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Antioxidants (Basel, Switzerland) Dec 2023is a ubiquitous genus of amoebae that can act as opportunistic parasites in both humans and animals, causing a variety of ocular, nervous and dermal pathologies....
is a ubiquitous genus of amoebae that can act as opportunistic parasites in both humans and animals, causing a variety of ocular, nervous and dermal pathologies. Despite advances in therapy, the management of patients with infections remains a challenge for health services. Therefore, there is a need to search for new active substances against Acanthamoebae. In the present study, we evaluated the amoebicidal activity of nitroxoline against the trophozoite and cyst stages of six different strains of . The strain showed the lowest IC value in the trophozoite stage (0.69 ± 0.01 µM), while the strain L-10 showed the lowest IC value in the cyst stage (0.11 ± 0.03 µM). In addition, nitroxoline induced in treated trophozoites of features compatibles with apoptosis and autophagy pathways, including chromatin condensation, mitochondrial malfunction, oxidative stress, changes in cell permeability and the formation of autophagic vacuoles. Furthermore, proteomic analysis of the effect of nitroxoline on trophozoites revealed that this antibiotic induced the overexpression and the downregulation of proteins involved in the apoptotic process and in metabolic and biosynthesis pathways.
PubMed: 38136200
DOI: 10.3390/antiox12122081 -
Microorganisms Nov 2023Several species of genus are potential pathogens and etiological agents of several diseases. The pathogenic mechanisms carried out by these amoebae in different target...
Several species of genus are potential pathogens and etiological agents of several diseases. The pathogenic mechanisms carried out by these amoebae in different target tissues have been documented, evidencing the relevant role of contact-dependent mechanisms. With the purpose of describing the pathogenic processes carried out by these protozoans more precisely, we considered it important to determine the emission of extracellular vesicles (EVs) as part of the contact-independent pathogenicity mechanisms of , a highly pathogenic strain. Through transmission electronic microscopy (TEM) and nanoparticle tracking analysis (NTA), EVs were characterized. EVs showed lipid membrane and a size between 60 and 855 nm. The secretion of large vesicles was corroborated by confocal and TEM microscopy. The SDS-PAGE of EVs showed proteins of 45 to 200 kDa. Antigenic recognition was determined by Western Blot, and the internalization of EVs by trophozoites was observed through Dil-labeled EVs. In addition, some EVs biological characteristics were determined, such as proteolytic, hemolytic and COX activity. Furthermore, we highlighted the presence of leishmanolysin in trophozites and EVs. These results suggest that EVs are part of a contact-independent mechanism, which, together with contact-dependent ones, allow for a better understanding of the pathogenicity carried out by .
PubMed: 38004773
DOI: 10.3390/microorganisms11112762 -
Pathogens (Basel, Switzerland) Nov 2023Few studies have been conducted in the cooling systems of power plants; they have focused on , leaving a gap in the knowledge of other pathogenic free-living amoebae in...
Few studies have been conducted in the cooling systems of power plants; they have focused on , leaving a gap in the knowledge of other pathogenic free-living amoebae in this environment. The objective of this study was to determine the occurrence of saline-tolerant pathogenic in a geothermal power plant. The identification of isolated amoebae at genus level was carried out, observing their morphological characteristics; the determination of genotype and species of was performed via molecular biology (PCR). Water temperature ranged from 18 to 43 °C and conductivity from 4.0 × 10 to 8.7 × 10 μS/cm; this last value was greater than the seawater value. Only five amoeba genera were found. was in all the sampling sites, showing high saline tolerance. The high temperature, but mainly high conductivity, were the environmental conditions that determined the presence of pathogenic free-living amoebae in the hot water. All the strains of killed the mice, having a mortality of 40 to 100%. genotypes T10 and T5 were identified, T10 is rarely isolated from the environment, while T5 is more frequent. This is the first time that genotypes T5 and T10 have been reported in the environment in Mexico.
PubMed: 38003827
DOI: 10.3390/pathogens12111363 -
Microorganisms Apr 2023Amoebae of the genus cause a sight-threatening infection called keratitis. It is considered a rare disease in humans but poses an increasing threat to public health...
BACKGROUND
Amoebae of the genus cause a sight-threatening infection called keratitis. It is considered a rare disease in humans but poses an increasing threat to public health worldwide, including in Poland. We present successive isolates from serious keratitis preliminary examined in terms of the identification and monitoring of, among others, the in vitro dynamics of the detected strains.
METHODS
Clinical and combined laboratory methods were applied; causative agents of the keratitis were identified at the cellular and molecular levels; isolates were cultivated in an axenic liquid medium and regularly monitored.
RESULTS
In a phase-contrast microscope, sp. cysts and live trophozoites from corneal samples and in vitro cultures were assessed on the cellular level. Some isolates that were tested at the molecular level were found to correspond to , , , genotype T4. There was variability in the amoebic strain dynamics; high viability was expressed as trofozoites' long duration ability to intense multiply.
CONCLUSIONS
Some strains from keratitis under diagnosis verification and dynamics assessment showed enough adaptive capability to grow in an axenic medium, allowing them to exhibit significant thermal tolerance. In vitro monitoring that was suitable for verifying in vivo examinations, in particular, was useful to detect the strong viability and pathogenic potential of successive strains with a long duration of high dynamics.
PubMed: 37317148
DOI: 10.3390/microorganisms11051174 -
BMC Infectious Diseases Sep 2022Acanthamoeba is an emerging pathogen, infamous for its resilience against antiprotozoal compounds, disinfectants and harsh environments. It is known to cause keratitis,...
INTRODUCTION
Acanthamoeba is an emerging pathogen, infamous for its resilience against antiprotozoal compounds, disinfectants and harsh environments. It is known to cause keratitis, a sight-threatening, painful and difficult to treat corneal infection which is often reported among contact lens wearers and patients with ocular trauma. Acanthamoeba comprises over 24 species and currently 23 genotypes (T1-T23) have been identified.
AIMS
This retrospective study was designed to examine the Acanthamoeba species and genotypes recovered from patients with Acanthamoeba keratitis (AK), determine the presence of endosymbionts in ocular isolates of Acanthamoeba and review the clinical presentations.
METHODOLOGY
Thirteen culture-confirmed AK patients treated in a tertiary eye care facility in Hyderabad, India from February to October 2020 were included in this study. The clinical manifestations, medications and visual outcomes of all patients were obtained from medical records. The Acanthamoeba isolates were identified by sequencing the ribosomal nuclear subunit (rns) gene. Acanthamoeba isolates were assessed for the presence of bacterial or fungal endosymbionts using molecular assays, PCR and fluorescence in situ hybridization (FISH).
RESULTS
The mean age of the patients was 33 years (SD ± 17.4; 95% CI 22.5 to 43.5 years). Six (46.2%) cases had AK associated risk factors; four patients had ocular trauma and two were contact lens wearers. A. culbertsoni (6/13, 46.2%) was the most common species, followed by A. polyphaga and A. triangularis. Most of the isolates (12/13) belonged to genotype T4 and one was a T12; three sub-clusters T4A, T4B, and T4F were identified within the T4 genotype. There was no significant association between Acanthamoeba types and clinical outcomes. Eight (61.5%) isolates harboured intracellular bacteria and one contained Malassezia restricta. The presence of intracellular microbes was associated with a higher proportion of stromal infiltrates (88.9%, 8/9), epithelial defect (55.6%, 5/9) and hypopyon (55.6%, 5/9) compared to 50% (2/4), 25% (1/4) and 25% (1/4) AK cases without intracellular microbes, respectively.
CONCLUSIONS
Genotype T4 was the predominant isolate in southern India. This is the second report of T12 genotype identified from AK patient in India, which is rarely reported worldwide. The majority of the Acanthamoeba clinical isolates in this study harboured intracellular microbes, which may impact clinical characteristics of AK.
Topics: Acanthamoeba; Acanthamoeba Keratitis; Disinfectants; Eye Infections; Genotype; Humans; In Situ Hybridization, Fluorescence; Retrospective Studies
PubMed: 36175838
DOI: 10.1186/s12879-022-07741-4 -
PloS One 2022Free-living amoebae (FLA) are gaining attention due to the increasing number of related grave central nervous system (CNS) and sight-threatening eye infections and their...
Isolation and morphological and molecular characterization of waterborne free-living amoebae: Evidence of potentially pathogenic Acanthamoeba and Vahlkampfiidae in Assiut, Upper Egypt.
Free-living amoebae (FLA) are gaining attention due to the increasing number of related grave central nervous system (CNS) and sight-threatening eye infections and their role as Trojan horses for many bacteria and viruses. This study was conducted in Assiut City, Egypt to detect the presence of FLA in different water sources using morphological and molecular approaches and determine their potential pathogenicity. A total of 188 water samples (100 tap, 80 tank, and 8 swimming pool samples) were collected, cultivated on non-nutrient agar seeded with Escherichia coli, and inspected for FLA. Thermo- and osmo-tolerance assays were performed to determine their pathogenicity. Polymerase chain reaction and sequence analysis were performed to confirm the identification and analyze the genotype. Overall, 52 samples (27.7%) were positive for FLA. Of these, 20.7% were identified as Acanthamoeba, 1.6% as Vahlkampfiidae, and 5.3% as mixed Acanthamoeba and Vahlkampfiidae. Seven species of Acanthamoeba were recognized, of which A. triangularis, A. polyphaga, A. lenticulata, and A. culbertsoni are thermo- and osmo-tolerant, and A. astronyxis, A. comandoni, and A. echinulata are non-thermo- and non-osmo-tolerant. The phylogeny analysis revealed T4 and T7 genotypes. Among Vahlkampfiids, 61.5% were identified as thermo- and osmo-tolerant Vahlkampfia, and 30.8% were identified as non-pathogenic Naegleria. One isolate (7.7%) was identified as potentially pathogenic Allovahlkampfia, as confirmed by sequencing. This is the first report documenting the occurrence and phylogeny of waterborne FLA (Acanthamoeba/Vahlkampfiidae) in Assiut, Egypt. The presence of potentially pathogenic FLA highlights the possible health hazards and the need for preventive measures.
Topics: Acanthamoeba; Amoeba; Egypt; Naegleria; Water
PubMed: 35802617
DOI: 10.1371/journal.pone.0267591 -
The Korean Journal of Parasitology Apr 2022Acanthamoeba keratitis (AK) is a rare ocular disease, but it is a painful and sight-threatening infectious disease. Early diagnosis and adequate treatment are necessary...
Acanthamoeba keratitis (AK) is a rare ocular disease, but it is a painful and sight-threatening infectious disease. Early diagnosis and adequate treatment are necessary to prevent serious complications. While AK is frequently diagnosis via several PCR assays or Acanthamoeba-specific antibodies, a more specific and effective diagnostic method is required. This study described the production of a polyclonal peptide antibody against the periplasmic binding protein (PBP) of A. castellanii and investigated its diagnostic potential. Western blot analysis showed that the PBP antibody specifically reacted with the cell lysates of A. castellanii. However, the PBP antibody did not interact with human corneal epithelial (HCE) cells and the other 3 major causative agents of keratitis. Immunocytochemistry (ICC) results revealed the specific detection of A. castellanii trophozoites and cysts by PBP antibodies when A. castellanii were co-cultured with HCE cells. PBP antibody specificity was further confirmed by co-culture of A. castellanii trophozoites with F. solani, S. aureus, and P. aeruginosa via ICC. The PBP antibody specifically reacted with the trophozoites and cysts of A. polyphaga, A. hatchetti, A. culbertsoni, A. royreba, and A. healyi, thus demonstrated its genus-specific nature. These results showed that the PBP polyclonal peptide antibody of A. castellanii could specifically detect several species of Acanthamoeba, contributing to the development of an effective antibody-based AK diagnostics.
Topics: Acanthamoeba; Acanthamoeba Keratitis; Animals; Antibodies; Humans; Peptides; Periplasmic Binding Proteins; Staphylococcus aureus; Trophozoites
PubMed: 35500897
DOI: 10.3347/kjp.2022.60.2.143 -
Pathogens (Basel, Switzerland) Jun 2020Amoebae of the genus are etiological agents of granulomatous amoebic encephalitis (GAE). Recently, through an in vivo GAE model, trophozoites were immunolocalized in...
Amoebae of the genus are etiological agents of granulomatous amoebic encephalitis (GAE). Recently, through an in vivo GAE model, trophozoites were immunolocalized in contact with the peripheral nervous system (PNS) cells-Schwann cells (SC). In this study, we analyzed in greater detail the in vitro early morphological events (1, 2, 3, and 4 h) during the interaction of trophozoites (ATCC 30171) with SC from (ATCC CRL-2941). Samples were processed for scanning and transmission electron microscopy as well as confocal microscopy. After 1 h of interaction, amoebae were observed to be adhered to the SC cultures, emitting sucker-like structures associated with micro-phagocytic channels. In addition, evidence of necrosis was identified since edematous organelles as well as multivesicular and multilamellar bodies characteristics of autophagy were detected. At 2 h, trophozoites migrated beneath the SC culture in which necrosis and autophagy persisted. By 3 and 4 h, extensive lytic zones were observed. SC necrosis was confirmed by confocal microscopy. We reported for the first time the induction of autophagic and necrotic processes in PNS cells, associated in part with the contact-dependent pathogenic mechanisms of trophozoites.
PubMed: 32526974
DOI: 10.3390/pathogens9060458 -
Experimental Parasitology Sep 2018Amoebae from the genus Acanthamoeba are facultative pathogens of humans and other animals. In humans they most frequently infect the eye causing a sight threatening...
Amoebae from the genus Acanthamoeba are facultative pathogens of humans and other animals. In humans they most frequently infect the eye causing a sight threatening infection known as Acanthamoeba keratitis (AK), and also cause an often fatal encephalitis (GAE). A mannose-binding protein (MBP) has been identified as being important for Acanthamoeba infection especially in AK. This lectin has previously been characterized from Acanthamoeba castellanii as consisting of multiple 130 kDa subunits. MBP expression correlates with pathogenic potential and is expressed in a number of Acanthamoeba species. Here we report the purification of a similar lectin from Acanthamoeba culbertsoni and the production of a monoclonal antibody to it. The A. culbertsoni MBP was isolated by affinity chromatography using α-D-mannose agarose and has an apparent molecular weight of 83 kDa. The monoclonal antibody is an IgM that is useful in both western blots and immunofluorescence. We expect that this antibody will be useful in the study of the pathology of A. culbertsoni and in its identification in clinical samples.
Topics: Acanthamoeba; Acanthamoeba Keratitis; Animals; Antibodies, Monoclonal; Antibodies, Protozoan; Antigens, Protozoan; Blotting, Western; Electrophoresis, Polyacrylamide Gel; Enzyme-Linked Immunosorbent Assay; Hybridomas; Immune Sera; Immunoglobulin Isotypes; Immunohistochemistry; Mannose-Binding Lectin; Mice; Mice, Inbred BALB C; Protozoan Proteins
PubMed: 30031120
DOI: 10.1016/j.exppara.2018.07.009 -
Journal of Clinical Microbiology Oct 2015Amoebic keratitis (AK) is a potentially blinding infection, the prompt diagnosis of which is essential for limiting ocular morbidity. We undertook a quality improvement... (Comparative Study)
Comparative Study
Amoebic keratitis (AK) is a potentially blinding infection, the prompt diagnosis of which is essential for limiting ocular morbidity. We undertook a quality improvement initiative with respect to the molecular detection of acanthamoebae in our laboratory because of an unusual case of discordance. Nine ATCC strains of Acanthamoeba and 40 delinked, biobanked, surplus corneal scraping specimens were analyzed for the presence of acanthamoebae with four separate real-time PCR assays. The assay used by the Free-Living and Intestinal Amebas Laboratory of the CDC was considered the reference standard, and the performance characteristics of each individual assay and pairs of assays were calculated. Outcome measures were sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV). Of 49 included specimens, 14 (28.6%) were positive by the gold standard assay, and 35 (71.4%) were negative. The sensitivities of the individual assays ranged from 64.3% to 92.9%, compared to the gold standard, while the specificities ranged from 88.6% to 91.4%. The PPVs and NPVs ranged from 69.2% to 78.6% and from 86.1% to 96.9%, respectively. Combinations of assay pairs led to improved performance, with sensitivities ranging from 92.9% to 100% and specificities ranging from 97.1% to 100%. ATCC and clinical strains of Acanthamoeba that failed to be detected by certain individual assays included Acanthamoeba castellanii, Acanthamoeba culbertsoni, and Acanthamoeba lenticulata. For three clinical specimens, false negativity of the gold standard assay could not be excluded. Molecular diagnostic approaches, especially combinations of highly sensitive and specific assays, offer a reasonably performing, operator-independent, rapid strategy for the detection of acanthamoebae in clinical specimens and are likely to be more practical than either culture or direct microscopic detection.
Topics: Acanthamoeba; Acanthamoeba Keratitis; Algorithms; Humans; Molecular Diagnostic Techniques; Predictive Value of Tests; Real-Time Polymerase Chain Reaction; Sensitivity and Specificity; Time Factors
PubMed: 26202123
DOI: 10.1128/JCM.01607-15