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Granulomatous amebic encephalitis, caused by the free-living amebae Balamuthia mandrillaris or Acanthamoeba species, is a rare and deadly infectious syndrome with a current mortality rate of >90%. Much work remains to define the optimal treatment for these infections. Here, we provide a comprehensive overview of the supporting evidence behind antimicrobials currently recommended by the Centers for Disease Control and Prevention (CDC) with updated statistics on survival rates and medication usage from the CDC Free-Living Ameba Database. We also discuss promising treatments, especially the emerging therapeutic agent nitroxoline, and provide recommendations for the next steps in this area.

Free-living amoebae (FLA) are isolated from the hospital environments and known as Trojan horses for medical essential microorganisms. This study aimed to investigate the prevalence and the presence of FLA and two critical agents of nosocomial infections, in the hospital wards. Sixty samples were collected from four communities and cultured onto non-nutrient agar (NNA). After total DNA extraction, FLA were characterized using PCR and sequencing. The presence of Candida albicans and Staphylococcus aureus was evaluated using real-time and conventional PCR, respectively. Acanthamoeba sp. was characterized in 30 (50%) samples. Two (6.6%) and one (3.3%) samples were positive for Vahlkampfiidae and Vermamoeba vermiformis, respectively. S. aureus was detected in 13 (43.3%) of samples, while none of them were positive for methicillin-resistant gene. C. albicans DNA was detected in one (3.3%) FLA-positive sample. The isolation of FLA from hospital suggests an essential role these eukaryotes in the inter-ward circulation of nosocomial infections. [ABSTRACT FROM AUTHOR]

Granulomatous amoebic encephalitis is a rare but likely under‐recognised form of subacute, usually fatal, encephalitis. We present an illustrative case report and literature review of Australian cases, summarising clinical features, diagnostic methods, treatment and outcomes. [ABSTRACT FROM AUTHOR]

In the context of the virosphere, viral particles can compete for host cells. In this scenario, some viruses block the entry of exogenous virions upon infecting a cell, a phenomenon known as superinfection inhibition. The molecular mechanisms associated with superinfection inhibition vary depending on the viral species and the host, but generally, blocking superinfection ensures the genetic supremacy of the virus's progeny that first infects the cell. Giant amoeba-infecting viruses have attracted the scientific community's attention due to the complexity of their particles and genomes. However, there are no studies on the occurrence of superinfection and its inhibition induced by giant viruses. This study shows that mimivirus, moumouvirus, and megavirus, exhibit different strategies related to the infection of Acanthamoeba. For the first time, we have reported that mimivirus and moumouvirus induce superinfection inhibition in amoebas. Interestingly, megaviruses do not exhibit this ability, allowing continuous entry of exogenous virions into infected amoebas. Our investigation into the mechanisms behind superinfection blockage reveals that mimivirus and moumouvirus inhibit amoebic phagocytosis, leading to significant changes in the morphology and activity of the host cells. In contrast, megavirus-infected amoebas continue incorporating newly formed virions, negatively affecting the available viral progeny. This effect, however, is reversible with chemical inhibition of phagocytosis. This work contributes to the understanding of superinfection and its inhibition in mimivirus, moumouvirus, and megavirus, demonstrating that despite their evolutionary relatedness, these viruses exhibit profound differences in their interactions with their hosts. [ABSTRACT FROM AUTHOR]

Free-living amoebae (FLA) are prevalent in diverse environments, representing various genera and species with different pathogenicity. FLA-induced infections, such as the highly fatal amoebic encephalitis, with a mortality rate of 99%, primarily affect immunocompromised individuals while others such as Acanthamoeba keratitis (AK) and cutaneous amebiasis may affect immunocompetent individuals. Despite the prevalence of FLA, there is a lack of standardized guidelines for their detection near human habitats. To date, no studies on the isolation and identification of FLA in environmental soil samples in Warsaw have been published. The aim of this study was to determine the presence of amoebae in soil samples collected from Warsaw parks and squares frequented by humans. The isolated protozoa were genotyped. Additionally, their pathogenic potential was determined through thermophilicity tests. A total of 23 soil samples were seeded on non-nutrient agar plates (NNA) at 26 °C and monitored daily for FLA presence. From the total of 23 samples, 18 were positive for FLA growth in NNA and PCR (78.2%). Acanthamoeba spp. was the most frequently isolated genus, with a total of 13 positive samples (13/18; 72.2%), and the T4 genotype being the most common. Moreover, Platyamoeba placida (3/18; 16.7%), Stenamoeba berchidia (1/18; 5.6%) and Allovahlkampfia sp. (1/18; 5.6%), also potentially pathogenic amoebae, were isolated. To our knowledge, this is the first report of FLA presence and characterization in the Warsaw area. [ABSTRACT FROM AUTHOR]

The molecular classification of Acanthamoeba is currently based on the analysis of 18S rDNA sequences, delimiting around twenty genotypes (T1–T23). In some cases, however, the resolution of 18S is limited, and other genetic markers could be useful for unravelling poorly resolved lineages. In this study, the partial large subunit (LSU) of rDNA and ITS were used to re-examine the Acanthamoeba palestinensis group (T2/T6 lineage), which consists of various poorly defined lineages, including the T2 and T6 genotypes. New sequences overlapping 18S, ITS, and LSU were recovered. The analysis placed previously identified partial ITS-LSU sequences as T2/T6 and further confirmed the separation of the OX1 lineage from T2. In addition, analysis of the second internal transcribed spacer (ITS-2) suggests that multiple species may be present within the T6 and OX1 lineages. The results obtained from the T2/T6 lineage analysis confirm the utility of partial LSU and ITS for the study of Acanthamoeba, suggesting their advantage for disentangling complex lineages. [ABSTRACT FROM AUTHOR]

Acanthamoeba spp. emerged as a clinically important pathogen related to amoebic keratitis. It is among the main causes of corneal transplantation and vision loss in ophthalmology. The treatment protocols have a low cure rate, high toxicity, and need for drug combination. Transition metal compounds have shown promising antiprotozoal effects. This study evaluates the amoebicidal activity of copper(II) coordination compounds in combination with chlorhexidine and the cytotoxicity to topical ocular application. These copper(II) coordination compounds were screened against Acanthamoeba castellanii trophozoites (ATCC 50492). The cytotoxicity on rabbit corneal cell line (ATCC—CCL 60) was performed. The compounds showed high amoebicidal potential, with inhibition of trophozoite viability above 80%. The Cp12 and Cp13 compounds showed Minimal Inhibitory Amoebicidal Concentration (MIAC) at 200 µM and mean inhibitory concentration (IC50) values lower than 10 µM. Against the cysts, Cp12 showed a reduction in viability (48%) in the longest incubation period. A synergistic effect for Cp12 with chlorhexidine was observed. The compounds have a dose-dependent effect against rabbit corneal cells. Compound Cp12 has potential for future application in developing ophthalmic formulations against Acanthamoeba keratitis and its use in multipurpose solutions is highlighted. [ABSTRACT FROM AUTHOR]

Chlorocresol has antibacterial and antifungal properties, yet its effectiveness in eradicating Acanthamoeba spp. remains unexplored. Acanthamoeba species trophozoites are usually sensitive to biocides, whereas cysts tend to be more resistant. This study aimed to evaluate the cysticidal activity of chlorocresol against Acanthamoeba polyphaga. Chlorocresol concentrations of 0.02, 0.04, and 0.08% were prepared and A. polyphaga cysts were incubated at room temperature (28–37 C) for 1, 24, 48, and 72 hr at each concentration. Cyst viability was evaluated using trypan blue staining and the percentage of nonviable cysts was calculated. For qualification assays, treated cysts were cultured on nonnutrient agar medium coated with Escherichia coli, incubated at 30 C, observed under a stereomicroscope for 30 days, and inoculated into peptone–yeast extract–glucose medium at 30 C for 72 hr. The results revealed that the A. polyphaga cysts were susceptible to 0.02, 0.04, and 0.08% chlorocresol. Chlorocresol made a significant difference in viability (P < 0.001) compared with the nontreated control for the same incubation time. This is the first study to examine the efficacy of chlorocresol against A. polyphaga cysts and it was highly effective. Chlorocresol could thus serve as an alternative chemical disinfectant for the eradication of A. polyphaga cysts as well as a prophylactic against transmission of other pathogenic microorganisms for which Acanthamoeba species can act as a carrier. [ABSTRACT FROM AUTHOR]

Purpose: To report real-world data (RWD) on the use of in vivo confocal microscopy (IVCM) in handling cases of suspected Acanthamoeba keratitis (AK) cases at a regional referral center during a 12-year period. Methods: Retrospective study of patients with suspected AK presenting at a regional referral center for IVCM in Sweden from 2010 to 2022. Demographics, symptoms, outcomes, and clinical management were analyzed, and IVCM images were interpreted. Results: Of 74 included patients with suspected AK, 18 (24%) were IVCM-positive, 33 (44%) were IVCM-negative, 15 had inconclusive IVCM results (20.2%), and 8 (11%) were referred for a second opinion based on IVCM, 4 of which were IVCM-positive (5.5%), yielding an overall IVCM-positive rate of 29.5%. Cultures were taken in 38 cases (51%) with only 2 cases (2.7%) culture-positive for AK. Of IVCM-negative cases, cultures were taken in 22 (67%) of cases and 100% of these were AK-negative. IVCM-positive cases had more clinic visits (median 30, P = 0.018) and longer follow-up time (median 890 days, P = 0.009) than IVCM-negative patients, while visual acuity improvement did not differ (P > 0.05). Of IVCM-positive cases, 10 (56%) underwent surgery despite prior anti-amoebic treatment, and 14 (78%) had 3 or more IVCM examinations during follow-up, with cysts (100%), dendritic cells (89%) and inflammatory infiltrate (67%) as the most prevalent features. Longitudinal IVCM indicated improvement in cysts, dendritic cells and subbasal nerves with treatment, while clinical resolution was not always consistent with complete absence of cysts. Conclusions: In a real-world setting, IVCM has a high reliability in classifying AK-negative cases, while IVCM detects AK-positive cases more frequently than the gold-standard culture method, leading to its preferential use over the culture method where time or resources are limited. Despite this, a subset of cases are IVCM-inconclusive, the clinical course of referred patients is long requiring many hospital visits, and visual acuity in most cases does not improve with medical treatment alone. Information sharing across centers and standardization of referral and diagnostic routines is needed to exploit the full potential of IVCM in AK patient management. [ABSTRACT FROM AUTHOR]

Acanthamoeba, are ubiquitous eukaryotic microorganisms, that play a pivotal role in recognizing and engulfing various microbes during predation, offering insights into microbial dynamics and immune responses. An intriguing observation lies in the apparent preference of Acanthamoeba for Gram-negative over Gram-positive bacteria, suggesting potential differences in the recognition and response mechanisms to bacterial prey. Here, we comprehensively review pattern recognition receptors (PRRs) and microbe associated molecular patterns (MAMPs) that influence Acanthamoeba interactions with bacteria. We analyze the molecular mechanisms underlying these interactions, and the key finding of this review is that Acanthamoeba exhibits an affinity for bacterial cell surface appendages that are decorated with carbohydrates. Notably, this parallels warm-blooded immune cells, underscoring a conserved evolutionary strategy in microbial recognition. This review aims to serve as a foundation for exploring PRRs and MAMPs. These insights enhance our understanding of ecological and evolutionary dynamics in microbial interactions and shed light on fundamental principles governing immune responses. Leveraging Acanthamoeba as a model organism, provides a bridge between ecological interactions and immunology, offering valuable perspectives for future research. [ABSTRACT FROM AUTHOR]

Acanthamoebae spp. are considered the most commonly occurring free-living amoebae (FLA) in the environment. Their high resilience enables them to thrive in different types of environments. Using purposive sampling, 80 surface water samples were collected from identified coastal sites in Mariveles, Bataan, and Lingayen Gulf (40 water samples for each). Nineteen (23.75%) of the 80 water samples yielded positive amoebic growth during the 14-day culture and microscopic examination. The polymerase chain reaction confirmed Acanthamoeba spp. DNA in isolates MB1, A3, A4, A7, C5, and D3 using JDP1 and JDP2 primer sets. Further sequencing revealed that the isolates belonged to Acantha- moeba sp., Acanthamoeba culbertsoni, Acanthamoeba castellani, and Acanthamoeba genotype T4. The sequences were deposited in GenBank and registered under accession numbers PP741651, PP767364, PP741728, PP741729, PP767365, and PP767366, respectively. Potential risk factors such as waste disposal, expansion of human settlements to coastal locations, and soil runoffs in these environments should be controlled to mitigate the proliferation of potentially pathogenic strains of FLAs. [ABSTRACT FROM AUTHOR]

Natural hot springs are ideal places and environmental matrices that offer relaxation to people and microorganisms of different types. A total of 40 surface water samples were collected from the five identified collection sites, eight water samples for each site. Collection sites are designated 200 m apart to cover the entire study site. Surface water samples were collected approximately 10–20 cm from the surface. Water samples were filtered, cultured, and microscopically observed for 14 days. After 14 days of cultivation, eight (20%) water samples revealed cystic and trophozoite stages. Polymerase chain reaction using JDP1 and JDP2 specific primers confirmed the presence of Acanthamoeba spp. from two of our isolates in the hot spring, isolates 1.1 and 5.1. Further sequencing revealed that the isolates are Acanthamoeba T20 and Acanthamoeba genotype T7. Sequences were deposited to GenBank and were assigned accession numbers PP741726 and PP741727, respectively. The isolation of Acanthamoeba spp. in hot springs has significant health implications, especially for those who use it for recreational activity. Private resort owners are highly encouraged to regularly monitor and maintain hot spring resorts to avoid future infections. [ABSTRACT FROM AUTHOR]

Acanthamoeba keratitis (AK) is a rare but serious infection of the cornea, typically associated with contact lens wear. Here, we present a case of AK caused by the Acanthamoeba genotype T4D/T4e in a patient without identifiable risk factors: a 34-year-old woman who initially presented with signs and symptoms suggestive of herpetic keratitis, and who did not respond to conventional treatment. Corneal culture and targeted metagenomic analysis (18S rRNA, 16S-like rRNA) revealed the presence of an Acanthamoeba species closely related to the 'Nagington' strain. Despite intensive anti-Acanthamoeba therapy, complications arose necessitating penetrating keratoplasty. In conclusion, this case underscores the importance of considering Acanthamoeba as a causal agent of keratitis in non-contact-lens wearers. The identification of Acanthamoeba genotype T4D/T4e challenges the previous understanding of its pathogenic potential. Furthermore, it emphasizes the need for ongoing research into the pathogenicity of different Acanthamoeba subtypes. Early diagnosis and treatment are essential for preventing vision-threatening complications associated with AK. [ABSTRACT FROM AUTHOR]

Background Acanthamoeba spp. is the causative agent of Acanthamoeba keratitis and granulomatous amoebic encephalitis. Strathclyde minor groove binders (S-MGBs) are a promising new class of anti-infective agent that have been shown to be effective against many infectious organisms. Objectives To synthesize and evaluate the anti- Acanthamoeba activity of a panel of S-MGBs, and therefore determine the potential of this class for further development. Methods A panel of 12 S-MGBs was synthesized and anti- Acanthamoeba activity was determined using an alamarBlue™-based trophocidal assay against Acanthamoeba castellanii. Cross-screening against Trypanosoma brucei brucei , Staphylococcus aureus and Escherichia coli was used to investigate selective potency. Cytotoxicity against HEK293 cells allowed for selective toxicity to be measured. DNA binding studies were carried out using native mass spectrometry and DNA thermal shift assays. Results and discussion S-MGB-241 has an IC50 of 6.6 µM against A. castellanii , comparable to the clinically used miltefosine (5.6 µM) and negligible activity against the other organisms. It was also found to have an IC50 > 100 µM against HEK293 cells, demonstrating low cytotoxicity. S-MGB-241 binds to DNA as a dimer, albeit weakly compared to other S-MGBs previously studied. This was confirmed by DNA thermal shift assay with a Δ T m = 1 ± 0.1°C. Conclusions Together, these data provide confidence that S-MGBs can be further optimized to generate new, potent treatments for Acanthameoba spp. infections. In particular, S-MGB-241, has been identified as a 'hit' compound that is selectively active against A. castellanii , providing a starting point from which to begin optimization of DNA binding and potency. [ABSTRACT FROM AUTHOR]

Acanthamoeba keratitis (AK) is a rare, sight-threating corneal infection. The disease is challenging to diagnose and treat, and the amoeba can rapidly encyst, persisting in the tissue and causing recurrences. Medical therapy is conventionally considered the first line treatment, but advanced cases could require more invasive treatments like a "chaud" corneal transplant. We review the incidence of severe complications in patients affected by AK. Of 439 reports screened, 158 met our inclusion criteria. Incidence of severe complications was low, with 2.21 % patients developing perforation, 1 % requiring evisceration/enucleation and less than 1 % developing endophthalmitis. Corneal transplantation was required in 16.68 % of the cases. According to our results, and considering the reported incidences of these complications in other infectious keratitis, AK patients have an overall low risk of developing perforation, endophthalmitis, and enucleation/evisceration. Nevertheless, data available in the literature remain poor, and further randomized control trials are needed to confirm our findings. [ABSTRACT FROM AUTHOR]

Acanthamoeba, a widely distributed free-living amoeba with 20 genotypes identified through rRNA gene sequencing, exhibits varying degrees of pathogenicity influenced by its genotype. This study focuses on assessing the prevalence of Acanthamoeba species in the surface waters of Ilam, located in western Iran, utilizing morphological analysis and sequencing of the 18S rRNA gene through the PCR method. A total of 50 water samples were collected from various regions within Ilam city, situated in the southwest of Iran. To isolate Acanthamoeba parasites from the samples, a culture method was used, and all utilized culture media were scrutinized through microscopic and molecular techniques. The parasite’s genotype was determined by sequencing a 500-bp fragment of the 18S rRNA gene. Using microscopic and molecular methods, 19 and 16 water samples tested positive, respectively. The 18S rRNA sequences revealed that the isolates belonged to the T4, T2, and T11 genotypes. This study emphasizes the presence and inclination for close contact with highly pathogenic genotypes of Acanthamoeba in the surface waters of Ilam City. [ABSTRACT FROM AUTHOR]

Acanthamoeba is a ubiquitous genus of amoebae that can trigger a severe and progressive ocular disease known as Acanthamoeba Keratitis (AK). Furthermore, current treatment protocols are based on the combination of different compounds that are not fully effective. Therefore, an urgent need to find new compounds to treat Acanthamoeba infections is clear. In the present study, we evaluated staurosporine as a potential treatment for Acanthamoeba keratitis using mouse cornea as an ex vivo model, and a comparative proteomic analysis was conducted to elucidate a mechanism of action. The obtained results indicate that staurosporine altered the conformation of actin and tubulin in treated trophozoites of A. castellanii. In addition, proteomic analysis of treated trophozoites revealed that this molecule induced overexpression and a downregulation of proteins related to key functions for Acanthamoeba infection pathways. Additionally, the ex vivo assay used validated this model for the study of the pathogenesis and therapies of AK. Finally, staurosporine eliminated the entire amoebic population and prevented the adhesion and infection of amoebae to the epithelium of treated mouse corneas. [ABSTRACT FROM AUTHOR]

Acanthamoeba keratitis (AK) is a rare but serious infection of the cornea, typically associated with contact lens wear. Here, we present a case of AK caused by the Acanthamoeba genotype T4D/T4e in a patient without identifiable risk factors: a 34-year-old woman who initially presented with signs and symptoms suggestive of herpetic keratitis, and who did not respond to conventional treatment. Corneal culture and targeted metagenomic analysis (18S rRNA, 16S-like rRNA) revealed the presence of an Acanthamoeba species closely related to the ‘Nagington’ strain. Despite intensive anti-Acanthamoeba therapy, complications arose necessitating penetrating keratoplasty. In conclusion, this case underscores the importance of considering Acanthamoeba as a causal agent of keratitis in non-contact-lens wearers. The identification of Acanthamoeba genotype T4D/T4e challenges the previous understanding of its pathogenic potential. Furthermore, it emphasizes the need for ongoing research into the pathogenicity of different Acanthamoeba subtypes. Early diagnosis and treatment are essential for preventing vision-threatening complications associated with AK.

Abstract Purpose To report real-world data (RWD) on the use of in vivo confocal microscopy (IVCM) in handling cases of suspected Acanthamoeba keratitis (AK) cases at a regional referral center during a 12-year period. Methods Retrospective study of patients with suspected AK presenting at a regional referral center for IVCM in Sweden from 2010 to 2022. Demographics, symptoms, outcomes, and clinical management were analyzed, and IVCM images were interpreted. Results Of 74 included patients with suspected AK, 18 (24%) were IVCM-positive, 33 (44%) were IVCM-negative, 15 had inconclusive IVCM results (20.2%), and 8 (11%) were referred for a second opinion based on IVCM, 4 of which were IVCM-positive (5.5%), yielding an overall IVCM-positive rate of 29.5%. Cultures were taken in 38 cases (51%) with only 2 cases (2.7%) culture-positive for AK. Of IVCM-negative cases, cultures were taken in 22 (67%) of cases and 100% of these were AK-negative. IVCM-positive cases had more clinic visits (median 30, P = 0.018) and longer follow-up time (median 890 days, P = 0.009) than IVCM-negative patients, while visual acuity improvement did not differ (P > 0.05). Of IVCM-positive cases, 10 (56%) underwent surgery despite prior anti-amoebic treatment, and 14 (78%) had 3 or more IVCM examinations during follow-up, with cysts (100%), dendritic cells (89%) and inflammatory infiltrate (67%) as the most prevalent features. Longitudinal IVCM indicated improvement in cysts, dendritic cells and subbasal nerves with treatment, while clinical resolution was not always consistent with complete absence of cysts. Conclusions In a real-world setting, IVCM has a high reliability in classifying AK-negative cases, while IVCM detects AK-positive cases more frequently than the gold-standard culture method, leading to its preferential use over the culture method where time or resources are limited. Despite this, a subset of cases are IVCM-inconclusive, the clinical course of referred patients is long requiring many hospital visits, and visual acuity in most cases does not improve with medical treatment alone. Information sharing across centers and standardization of referral and diagnostic routines is needed to exploit the full potential of IVCM in AK patient management.

Abstract Background This study aimed at measuring the effect of contact lens hygiene risk factors on the prevalence of contact lens-related keratitis and identifying the specific risk factors to both microbial and non-microbial keratitis independently. Methods A cross-sectional study was conducted at Alexandria Ophthalmology Hospital from May to October 2023. All contact lens wearers attending the outpatient clinic had undergone face-to-face interviews using a standardized validated questionnaire which included demographic data and contact lenses (CLs) hygiene risk factors. Participants were classified into two groups; normal group and keratitis group. Keratitis group was further subdivided into non-microbial and microbial group. Results The study included 245 contact lens wearers; 149 normal cases, 50 (20.4%) contact lens-related non-microbial keratitis (CLNK) cases, and 46 (18.8%) contact lens-related microbial keratitis (CLMK) cases. Sharing contact lenses and eye trauma were significant risk factors for both CLNK (p=0.036), (p=0.001) and CLMK (p=0.003), (p=0.017). CLs wear duration for more than 12 hours was associated with an increased risk of CLNK by about 4 times (p=0.030) and overnight wear of contact lenses increased the risk of CLNK by 2.6 times (p=0.030). Showering or swimming in lenses was identified as a significant risk factor for CLMK (p=0.012), moreover washing lenses with tap water increased the risk of CLMK (p=0.030). Conclusions Poor compliance with contact lenses hygiene rules results in a high prevalence of contact lens-related keratitis. Eye trauma and sharing contact lenses were significant hygiene risk factors for both contact lens-related non-microbial keratitis and contact lens-related microbial keratitis.

Background and purpose: Lophomoniasis is a relatively common emerging parasitic disease caused by a pathogenic protozoan called Lophomonas, which mostly affects the lower respiratory tract (lungs and bronchi) of humans. The parasite lives symbiotically in the digestive system of insects such as cockroaches and mites. Acanthamoeba is a free-living amoeba that has two forms of trophozoite and cyst in its life cycle. The respiratory system and airways serve as passage and deployment sites for a wide range of non-pathogenic and pathogenic microorganisms, making it a suitable place for the entry and spread of different species of Acanthamoeba amoeba and the Lophomonas pathogen in the respiratory system, which can cause clinical manifestations similar to tuberculosis. Therefore, the present study aimed to investigate the frequency of these two parasites in sputum clinical samples from suspected tuberculosis patients referred to the tuberculosis laboratory in Babolsar City to rule out the two mentioned infections. Materials and methods: This descriptive-cross-sectional study was conducted on 201 sputum samples of people suspected of tuberculosis who were referred to the tuberculosis laboratory of Babolsar Health Center in Mazandaran province in 202-2023, and all the demographic and epidemiological information of the patients was recorded. In the morphological method, Giemsa staining was used for Lophomonas and Ziehl-Neelsen staining for Acanthamoeba. To identify the presence of Acanthamoeba, sputum samples were cultured in a 1.5% non-nutrient agar culture medium for 72 to 96 hours. Approximately 50 microliters of patients' sputum were added to the NNA medium, along with 20 microliters of trypticase-yeast extract and maltose (TYM) culture medium, and 20 microliters of an autoclaved Escherichia coli bacteria mixture to enrich the culture medium. The cultured plates were then placed in an incubator at 26ºC and checked daily under a light microscope for trophozoite growth. To accurately confirm the presence of parasites, DNA extraction was performed on the patient's sputum samples using the phenol-chloroform-isoamyl alcohol method. Subsequently, conventional polymerase chain reaction (PCR) was conducted using specific primers on the extracted DNA samples. Results: In the study of 201 people suspected of tuberculosis, it was found that 80 individuals (39.8%) lived in urban areas and 121 individuals (60.2%) lived in rural areas. The age range of participants varied from 7 to 88 years old. There were 113 females (56.2%) and 88 males (43.8%) in the study, with 12 individuals from Afghanistan living in urban areas. Out of the 201 sputum samples examined, a total of 23 samples (11.4%) tested positive for the Lophomonas parasite using the PCR method. Among these positive cases, 7 individuals (30.4%) resided in urban areas (Babolsar), while 16 individuals (69.5%) lived in rural areas. However, none of the samples tested positive for Acanthamoeba in this study using staining and PCR methods. Conclusion: The present study showed that due to the similarity of the clinical manifestations of tuberculosis and lophomoniasis, there is a possibility of mistaking these two diseases clinically. Therefore, it is recommended to check fresh sputum samples of people suspected of tuberculosis, especially in endemic areas, in terms of pulmonary lophomoniasis.

Background: This study aimed at measuring the effect of contact lens hygiene risk factors on the prevalence of contact lens-related keratitis and identifying the specific risk factors to both microbial and non-microbial keratitis independently. Methods: A cross-sectional study was conducted at Alexandria Ophthalmology Hospital from May to October 2023. All contact lens wearers attending the outpatient clinic had undergone face-to-face interviews using a standardized validated questionnaire which included demographic data and contact lenses (CLs) hygiene risk factors. Participants were classified into two groups; normal group and keratitis group. Keratitis group was further subdivided into non-microbial and microbial group. Results: The study included 245 contact lens wearers; 149 normal cases, 50 (20.4%) contact lens-related non-microbial keratitis (CLNK) cases, and 46 (18.8%) contact lens-related microbial keratitis (CLMK) cases. Sharing contact lenses and eye trauma were significant risk factors for both CLNK (p=0.036), (p=0.001) and CLMK (p=0.003), (p=0.017). CLs wear duration for more than 12 hours was associated with an increased risk of CLNK by about 4 times (p=0.030) and overnight wear of contact lenses increased the risk of CLNK by 2.6 times (p=0.030). Showering or swimming in lenses was identified as a significant risk factor for CLMK (p=0.012), moreover washing lenses with tap water increased the risk of CLMK (p=0.030). Conclusions: Poor compliance with contact lenses hygiene rules results in a high prevalence of contact lens-related keratitis. Eye trauma and sharing contact lenses were significant hygiene risk factors for both contact lens-related non-microbial keratitis and contact lens-related microbial keratitis. [ABSTRACT FROM AUTHOR]

Acanthamoeba keratitis (AK) is a sight-threatening and difficult-to-treat ocular infection. The significant side effects of current AK treatments highlight the urgent need to develop a safe and effective AK medication. In this study, the amoebicidal activity of Iris setosa Pall. ex Link extract (ISE) against Acanthamoeba was examined and its specific amoebicidal mechanism was explored. ISE induced significant morphological changes in Acanthamoeba trophozoites and exhibited amoebicidal activity against A. castellanii and A. polyphaga. ISE was further fractionated into five subfractions by sequential extraction with n-hexane, chloroform, ethyl acetate, n-butanol, and water, and their amoebicidal activities and underlying amoebicidal mechanisms were investigated. The n-butanol subfraction of ISE (ISE-BuOH) displayed selective amoebicidal activity against the Acanthamoeba species with minimal cytotoxicity in human corneal cells (HCE-2). ISE-BuOH triggered apoptosis-like programmed cell death (PCD) in amoebae, characterized by DNA fragmentation, increased ROS production, and caspase-3 activity elevation. ISE-BuOH also demonstrated a partial cysticidal effect against the amoeba species. ISE-BuOH could be a promising candidate in the development of therapeutic drugs for AK. [ABSTRACT FROM AUTHOR]

Introduction: Often associated with contact lens wear, Acanthamoeba keratitis is an extremely painful blinding infection that has remained significant despite advances in drug discovery and chemotherapies. Current treatment is intricate and demands early diagnosis, aggressive and prolonged application, and may result in infection recurrence. The latter is due to the ability of the parasite to undergo cellular differentiation and transform into a resistant cyst form or infective trophozoite form. Areas covered: Here, we present a review of our current understanding of emerging therapies and priorities tackling Acanthamoeba keratitis. Literature searches were carried out using bibliographic databases and results sifted and reviewed to present recent findings, challenges and novel opportunities in the development of therapeutic interventions and/or preventative measures against Acanthamoeba keratitis. Expert opinion: Nanomedicine-based theranostics to combine diagnosis and treatment of Acanthamoeba keratitis offer remarkable opportunity in the development of clinical applications in the management of Acanthamoeba keratitis. [ABSTRACT FROM AUTHOR]

The Tajogaite Volcano erupted on the western slope of the Cumbre Vieja mountain range on La Palma Island in the Canary Islands, Spain, in 2021. As one of the multiple consequences of this eruption, a layer of tephra was deposited, to a variable extent, over a large part of the island. Tephra deposits affect all aspects of vegetation recovery, the water cycle, and the long-term availability of volcanic nutrients. Protozoa, including free-living amoeba (FLA), are known to be among the first microorganisms capable of colonizing harsh environments. In the present study, the presence of FLA has been evaluated in the Tajogaite Volcano deposits. Samples of the tephra were collected and incubated at 26 °C on 2% non-nutrient agar plates with a layer of heat-killed E. coli. Morphological features, as well as the DF3 region sequence of the 18S rDNA, confirmed the presence of a T4 genotype strain of Acanthamoeba. Thermotolerance and osmotolerance assays were used to evaluate the strain's pathogenic potential. This strain was considered thermotolerant but poorly osmotolerant. To the best of our knowledge, this is the first report of Acanthamoeba being isolated from a recently erupted volcano. [ABSTRACT FROM AUTHOR]

Recent advancements in membrane technologies and disinfection methods have enhanced drinking water quality significantly. However, microorganisms, including free-living amoebae (FLA), persist and pose potential threats to humans. FLA are linked to severe neuro-ophthalmic infections and serve as hosts of pathogenic bacteria. This study examined FLA presence in chlorinated and ultrafiltration drinking water and evaluated chlorine's disinfectant. Of 115 water samples, 21 tested positive for Acanthamoeba sp., Allovahlkampfia sp., and Vermamoeba vermiformis, originating from chlorinated sources. FLA trophozoites withstand temperatures up to 37 °C, while the cysts tolerate heat shocks of 60–70 °C. Trophozoites are susceptible to 5 mg L−1 chlorine, but cysts remain viable at concentrations up to 10 mg L−1. FLAs' survival in chlorinated waters is attributed to high cyst tolerance and lower residual chlorine concentrations. These findings highlight the need for ultrafiltration or enhanced chlorination protocols to ensure safer drinking water. [ABSTRACT FROM AUTHOR]

Introduction: Acanthamoeba infection is a serious public health concern, necessitating the development of effective and safe anti-Acanthamoeba chemotherapies. Poly (ADP-ribose) polymerases (PARPs) govern a colossal amount of biological processes, such as DNA damage repair, protein degradation and apoptosis. Multiple PARP-targeted compounds have been approved for cancer treatment. However, repurposing of PARP inhibitors to treat Acanthamoeba is poorly understood. Methods: In the present study, we attempted to fill these knowledge gaps by performing anti-Acanthamoeba efficacy assays, cell biology experiments, bioinformatics, and transcriptomic analyses. Results: Using a homology model of Acanthamoeba poly (ADP-ribose) polymerases (PARPs), molecular docking of approved drugs revealed three potential inhibitory compounds: olaparib, venadaparib and AZ9482. In particular, venadaparib exhibited superior docking scores (-13.71) and favorable predicted binding free energy (-89.28 kcal/mol), followed by AZ9482, which showed a docking score of -13.20 and a binding free energy of -92.13 kcal/mol. Notably, the positively charged cyclopropylamine in venadaparib established a salt bridge (through E535) and a hydrogen bond (via N531) within the binding pocket. For comparison, AZ9482 was well stacked by the surrounding aromatic residues including H625, Y652, Y659 and Y670. In an assessment of trophozoites viability, AZ9482 exhibited a dose-and time-dependent anti-trophozoite effect by suppressing Acanthamoeba PARP activity, unlike olaparib and venadaparib. An Annexin V-fluorescein isothiocyanate/propidium iodide apoptosis assay revealed AZ9482 induced trophozoite necrotic cell death rather than apoptosis. Transcriptomics analyses conducted on Acanthamoeba trophozoites treated with AZ9482 demonstrated an atlas of differentially regulated proteins and genes, and found that AZ9482 rapidly upregulates a multitude of DNA damage repair pathways in trophozoites, and intriguingly downregulates several virulent genes. Analyzing gene expression related to DNA damage repair pathway and the rate of apurinic/apyrimidinic (AP) sites indicated DNA damage efficacy and repair modulation in Acanthamoeba trophozoites following AZ9482 treatment. Discussion: Collectively, these findings highlight AZ9482, as a structurally unique PARP inhibitor, provides a promising prototype for advancing anti-Acanthamoeba drug research. [ABSTRACT FROM AUTHOR]

Pathogenic and free-living Acanthamoeba are widely distributed in the environment and have been reported to cause keratitis and universally fatal encephalitis. Primary cutaneous acanthamoebiasis caused by Acanthamoeba 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 Acanthamoeba 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 Acanthamoeba so that the cause was identified, and the skin ulcers improved significantly after treatment with multi-drug combination therapy. Acanthamoeba is also a host of pathogenic microorganisms. The presence of endosymbionts enhances the pathogenicity of Acanthamoeba, 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. [ABSTRACT FROM AUTHOR]

Acanthamoeba spp. can cause a sight threatening disease. At present, the current treatments used to treat Acanthamoeba spp. Infections, such as biguanide-based antimicrobials, remain inefficacious, with the appearance of resistant forms and high cytotoxicity to host cells. In this study, an initial screening was conducted against Acanthamoeba castellanii Neff and murine macrophages J774A.1 using alamarBlue™. Among the 160 compounds included in the cited box, 90% exhibited an inhibition of the parasite above 80%, while only 18.75% of the compounds inhibited the parasite with a lethality towards murine macrophage lower than 20%. Based on the amoebicidal activity, the cytotoxicity assay, and availability, Terconazole was chosen for the elucidation of the action mode in two clinical strains, Acanthamoeba culbertsoni and Acanthamoeba castellanii L10. A fluorescence image-based system and proteomic techniques were used to investigate the effect of the present azole on the cytoskeleton network and various programmed cell death features, including chromatin condensation and mitochondria dysfunction. Taking all the results together, we can suggest that Terconazole can induce programmed cell death (PCD) via the inhibition of sterol biosynthesis inhibition. [ABSTRACT FROM AUTHOR]

Background and purpose: Lophomoniasis is a relatively common emerging parasitic disease caused by a pathogenic protozoan called Lophomonas, which mostly affects the lower respiratory tract (lungs and bronchi) of humans. The parasite lives symbiotically in the digestive system of insects such as cockroaches and mites. Acanthamoeba is a free-living amoeba that has two forms of trophozoite and cyst in its life cycle. The respiratory system and airways serve as passage and deployment sites for a wide range of non-pathogenic and pathogenic microorganisms, making it a suitable place for the entry and spread of different species of Acanthamoeba amoeba and the Lophomonas pathogen in the respiratory system, which can cause clinical manifestations similar to tuberculosis. Therefore, the present study aimed to investigate the frequency of these two parasites in sputum clinical samples from suspected tuberculosis patients referred to the tuberculosis laboratory in Babolsar City to rule out the two mentioned infections. Materials and methods: This descriptive-cross-sectional study was conducted on 201 sputum samples of people suspected of tuberculosis who were referred to the tuberculosis laboratory of Babolsar Health Center in Mazandaran province in 202-2023, and all the demographic and epidemiological information of the patients was recorded. In the morphological method, Giemsa staining was used for Lophomonas and Ziehl-Neelsen staining for Acanthamoeba. To identify the presence of Acanthamoeba, sputum samples were cultured in a 1.5% non-nutrient agar culture medium for 72 to 96 hours. Approximately 50 microliters of patients' sputum were added to the NNA medium, along with 20 microliters of trypticase-yeast extract and maltose (TYM) culture medium, and 20 microliters of an autoclaved Escherichia coli bacteria mixture to enrich the culture medium. The cultured plates were then placed in an incubator at 26ºC and checked daily under a light microscope for trophozoite growth. To accurately confirm the presence of parasites, DNA extraction was performed on the patient's sputum samples using the phenol-chloroform-isoamyl alcohol method. Subsequently, conventional polymerase chain reaction (PCR) was conducted using specific primers on the extracted DNA samples. Results: In the study of 201 people suspected of tuberculosis, it was found that 80 individuals (39.8%) lived in urban areas and 121 individuals (60.2%) lived in rural areas. The age range of participants varied from 7 to 88 years old. There were 113 females (56.2%) and 88 males (43.8%) in the study, with 12 individuals from Afghanistan living in urban areas. Out of the 201 sputum samples examined, a total of 23 samples (11.4%) tested positive for the Lophomonas parasite using the PCR method. Among these positive cases, 7 individuals (30.4%) resided in urban areas (Babolsar), while 16 individuals (69.5%) lived in rural areas. However, none of the samples tested positive for Acanthamoeba in this study using staining and PCR methods. Conclusion: The present study showed that due to the similarity of the clinical manifestations of tuberculosis and lophomoniasis, there is a possibility of mistaking these two diseases clinically. Therefore, it is recommended to check fresh sputum samples of people suspected of tuberculosis, especially in endemic areas, in terms of pulmonary lophomoniasis. [ABSTRACT FROM AUTHOR]

Acanthamoeba, a widely distributed free-living amoeba found in various environments, is an opportunistic pathogen responsible for causing Acanthamoeba keratitis, a condition that may lead to blindness. However, identifying the pathogenicity of Acanthamoeba is challenging due to its complex life cycle, ability to adapt to different environments, variable virulence factors, and intricate interactions with the host immune system. Additionally, the development of an effective model for studying Acanthamoeba pathogenicity is limited, hindering a comprehensive understanding of the mechanisms underlying its virulence and host interactions. The aim of this study was to develop an ex vivo model for Acanthamoeba infection using porcine eyeballs and to evaluate the pathogenicity of the Acanthamoeba isolates. Based on slit lamp and biopsy analysis, the developed ex vivo model is capable of successfully infecting Acanthamoeba within 3 days. Histopathological staining revealed that clinical isolates of Acanthamoeba exhibited greater corneal stroma destruction and invasion in this model than environmental isolates. Our results highlight the importance of an ex vivo porcine eye model in elucidating the pathogenesis of Acanthamoeba infection and its potential implications for understanding and managing Acanthamoeba-related ocular diseases. [ABSTRACT FROM AUTHOR]

The impact of different levels of salinity (17.6 ‰, 12.6 ‰, 7.6 ‰ and 2.6 ‰) on growth and development of naked amoebae was studied in beach sands of the Black Sea in the region of Odesa, Ukraine. The decreasing salinity was followed by reduced fecundity and increased generation time of the naked amoeba cells. Vannella devonica, V. aberdonica, V. plurinucleolus, Thecamoeba orbis, T. hilla, Stenamoeba sp. and Acanthamoeba griffini were the most tolerant to gradual salinity decrease, while Saccamoeba marina, Vexillifera armata, Mayorella gemmifera were less tolerant. Cochliopodium gulosum survived at a medium salinity of 17.6 ‰. Under the low medium salinities (7.6 ‰ and 2.6 ‰), most amoebae existed as floating forms. After the saline shock (12.6 ‰, 7.6 ‰ and 2.6 ‰), neither growth nor development were observed in Saccamoeba marina, Сochliopodium gulosum and Mayorella gemmifera and their floating forms remained motionless. [ABSTRACT FROM AUTHOR]

Background The genus Acanthamoeba is reported from various environmental sources and can cause multiple complications, including chronic amoebic aeratitis and amoebic granulomatous encephalitis. This study investigated the presence and genotyping of Acanthamoeba in the soil of parks and patients with malignancies referred to health centers in Zanjan city, Iran. Methods In this cross-sectional study, 200 soil samples were collected from amusement parks in Zanjan city from September 2017 to May 2018. Samples were cultured on 1.5% non-nutrient agar, and the Acanthamoeba genus was identified using the morphological method. PCR was performed on all positive environmental samples, and six microscopically positive clinical samples belonged to our previous study. DNA sequencing of 18S rRNA was performed to analyze the genetic pattern of some PCR-positive isolates. Results Microscopic results showed that 96 (48%) soil samples were positive. PCR confirmed all positive cases of clinical samples and 84 soil samples. Out of the PCR-positive samples, 20 soil samples and five clinical samples were sequenced successfully. All soil isolates belonged to the T4 genotype, and three and two clinical samples belonged to T4 and T5 genotypes, respectively. Conclusion : The presence of Acanthamoeba in both the environment and clinical samples of Zanjan city suggests paying greater attention to the infections caused by it. [ABSTRACT FROM AUTHOR]

Acanthamoeba keratitis (AK) is a severe infection of the cornea. Prevention and treatment are difficult due to the inefficacy of currently available compounds. The impact of many commonly used compounds for routine examinations of Acanthamoeba is unexplored but might offer insight useful in combatting AK. In this study, we demonstrate that sodium metabisulfite, a common preservation constituent of eye care solutions, was found to be active against Acanthamoeba trophozoites at concentrations lower than that commonly found in eye drops (IC50 0.03 mg/mL). We demonstrate that sodium metabisulfite depletes thiamine from growth medium and that Acanthamoeba is a thiamine auxotroph, requiring thiamine salvage for growth. The inhibitory effects of sodium metabisulfite can be overcome by thiamine supplementation. These results are consistent with the lack of key enzymes for thiamine biosynthesis in the genome of Acanthamoeba, an area which might prove exploitable using new or existing compounds. Indeed, this study highlights sodium metabisulfite as a useful inhibitor of Acanthamoeba castellanii trophozoites in vitro and that it acts, at least in part, by limiting available thiamine. [ABSTRACT FROM AUTHOR]

ABSTRACT Acanthamoeba species are among the most common free-living amoeba and ubiquitous protozoa, mainly distributed in water and soil, and cause Acanthamoeba keratitis (AK) and severe visual impairment in patients. Although several studies have reported genomic characteristics of Acanthamoeba, limited sample sizes and sources have resulted in an incomplete understanding of the genetic diversity of Acanthamoeba from different sources. While endosymbionts exert a significant influence on the phenotypes of Acanthamoeba, including pathogenicity, virulence, and drug resistance, the species diversity and functional characterization remain largely unexplored. Herein, our study sequenced and analyzed the whole genomes of 19 Acanthamoeba pathogenic strains that cause AK, and by integrating publicly available genomes, we sampled 29 Acanthamoeba strains from ocular, environmental, and other sources. Combined pan-genomic and comparative functional analyses revealed genetic differences and evolutionary relationships among the different sources of Acanthamoeba, as well as classification into multiple functional groups, with ocular isolates in particular showing significant differences that may account for differences in pathogenicity. Phylogenetic and rhizome gene mosaic analyses of ocular Acanthamoeba strains suggested that genomic exchanges between Acanthamoeba and endosymbionts, particularly potential antimicrobial resistance genes trafficking including the adeF, amrA, and amrB genes exchange events, potentially contribute to Acanthamoeba drug resistance. In conclusion, this study elucidated the adaptation of Acanthamoeba to different ecological niches and the influence of gene exchange on the evolution of ocular Acanthamoeba genome, guiding the clinical diagnosis and treatment of AK and laying a theoretical groundwork for developing novel therapeutic approaches.IMPORTANCEAcanthamoeba causes a serious blinding keratopathy, Acanthamoeba keratitis, which is currently under-recognized by clinicians. In this study, we analyzed 48 strains of Acanthamoeba using a whole-genome approach, revealing differences in pathogenicity and function between strains of different origins. Horizontal transfer events of antimicrobial resistance genes can help provide guidance as potential biomarkers for the treatment of specific Acanthamoeba keratitis cases.

ABSTRACT The cyst wall of the eye pathogen Acanthamoeba castellanii contains cellulose and has ectocyst and endocyst layers connected by conical ostioles. Cyst walls contain families of lectins that localize to the ectocyst layer (Jonah) or the endocyst layer and ostioles (Luke and Leo). How lectins and an abundant laccase bind cellulose and why proteins go to locations in the wall are not known and are the focus of the studies here. Structural predictions identified β-jelly-roll folds (BJRFs) of Luke and sets of four disulfide knots (4DKs) of Leo, each of which contains linear arrays of aromatic amino acids, also present in carbohydrate-binding modules of bacterial and plant endocellulases. Ala mutations showed that these aromatics are necessary for cellulose binding and proper localization of Luke and Leo in the Acanthamoeba cyst wall. BJRFs of Luke, 4DKs of Leo, a single β-helical fold (BHF) of Jonah, and a copper oxidase domain of the laccase each bind to glycopolymers in both layers of deproteinated cyst walls. Promoter swaps showed that ectocyst localization does not just correlate with but is caused by early encystation-specific expression, while localization in the endocyst layer and ostioles is caused by later expression. Evolutionary studies showed distinct modes of assembly of duplicated domains in Luke, Leo, and Jonah lectins and suggested Jonah BHFs originated from bacteria, Luke BJRFs share common ancestry with slime molds, while 4DKs of Leo are unique to Acanthamoeba.IMPORTANCEAcanthamoebae is the only human parasite with cellulose in its cyst wall and conical ostioles that connect its inner and outer layers. Cyst walls are important virulence factors because they make Acanthamoebae resistant to surface disinfectants, hand sanitizers, contact lens sterilizers, and antibiotics applied to the eye. The goal here was to understand better how proteins are targeted to specific locations in the cyst wall. To this end, we identified three new proteins in the outer layer of the cyst wall, which may be targets for diagnostic antibodies in corneal scrapings. We used structural predictions and mutated proteins to show linear arrays of aromatic amino acids of two unrelated wall proteins are necessary for binding cellulose and proper wall localization. We showed early expression during encystation causes proteins to localize to the outer layer, while later expression causes proteins to localize to the inner layer and the ostioles.

Free-living amoebae are rare causes of morbidity and mortality in humans and animals around the globe. Because the route of exposure and clinical progression of disease caused by different species of amoebae may vary in people and animals, determining the species of amoeba present is important. We describe here a fatal infection by the free-living amoeba Balamuthia mandrillaris in a Siberian tiger (Panthera tigris altaica). The 17-y-old patient had a rapid clinical decline after a peracute onset of severe lethargy, dull mentation, and anorexia. Autopsy did not identify a cause of death. Histology revealed inflammation associated with amoebic trophozoites in the brain, lungs, and iris of one eye. These amoebae were confirmed to be B. mandrillaris based on a PCR assay and sequencing. Although there are subtle morphologic differences between cyst stages of Acanthamoeba spp., B. mandrillaris, and Naegleria fowleri when present and identified on routine staining, other modalities, including PCR, immunofluorescence, electron microscopy, and immunohistochemistry, are typically utilized to confirm the pathogen involved in these cases. We review the reports of balamuthosis in animals.

Abstract 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. Graphical Abstract

Objective. To present the results of successful treatment of a patient with corneal lesions caused by Acanthamoeba spp. receiving long-term conservative treatment.Methods. A child with corneal ulcer of mixed etiology with a history of wearing orthokeratology lenses and long-term ineffective treatment in Moscow hospitals was referred to Moscow in S.N. Fyodorov Eye Microsurgery Federal State Institution. The patient underwent a complete ophthalmological examination, including confocal microscopy.Results. The condition of the child’s eye was extremely severe, live and dead cysts of Acantamoeba spp were found in the central zone of the cornea at the depth of 113–149 μm, Along with intensive conservative treatment with catheterisation of the retrobulbar space and introduction of drugs into the irrigation system, UV corneal crosslinking was performed twice.Conclusion. Treatment according to the proposed method allowed to achieve excellent results: there was no corneal syndrome, complete epithelialisation of the corneal defect, disappearance of corneal edema and remission of uveitis manifestations. According to the data of confocal microscopy there is no growth of microflora and acanthamoeba cysts.

Acanthamoeba, a widely distributed free-living amoeba with 20 genotypes identified through rRNA gene sequencing, exhibits varying degrees of pathogenicity influenced by its genotype. This study focuses on assessing the prevalence of Acanthamoeba species in the surface waters of Ilam, located in western Iran, utilizing morphological analysis and sequencing of the 18S rRNA gene through the PCR method. A total of 50 water samples were collected from various regions within Ilam city, situated in the southwest of Iran. To isolate Acanthamoeba parasites from the samples, a culture method was used, and all utilized culture media were scrutinized through microscopic and molecular techniques. The parasite's genotype was determined by sequencing a 500-bp fragment of the 18S rRNA gene. Using microscopic and molecular methods, 19 and 16 water samples tested positive, respectively. The 18S rRNA sequences revealed that the isolates belonged to the T4, T2, and T11 genotypes. This study emphasizes the presence and inclination for close contact with highly pathogenic genotypes of Acanthamoeba in the surface waters of Ilam City. HIGHLIGHTS This study showed the widespread prevalence of Acanthamoeba in water resources.; Using microscopic and molecular methods, 19 and 16 water samples were found positive, respectively.; The 18S rRNA sequences showed that the isolates belong to the T4, T2, and T11 genotypes.; The 18S rRNA gene sequence showed the presence of T4, a highly pathogenic genotype with the highest frequency in the surface water of Ilam.;

Objective: The aim of this study was to evaluate the pathogenicity of Acanthamoeba strains with T4, T5, T11, and T12 genotypes by comparing the osmotolerance and thermotolerance characteristics of Acanthamoeba strains isolated from genotype groups, within species with the same genotype, and from environmental and keratitis cases. Methods: In this study, after axenic cultures of 22 Acanthamoeba strains with T4 (Neff, A, B, D, E), T5, T11, and T12 genotypes isolated from clinical and environmental samples, thermotolerance (37 °C, 39 °C and 41 °C) and osmotolerance (0.5 M, 1 M) tests were performed. Results: All strains showed growth ability at 37 °C and 0.5 M osmolarity. While all five strains isolated from patients with Acanthamoeba keratitis showed growth ability at 37 °C and 0.5 M osmolarity, no growth was detected at 41 °C and 1 M osmolarity. When the tolerance characteristics of the strains with the same genotype were evaluated, the strains with the T5 and T4E genotypes showed the same characteristics. When Acanthamoeba strains with the T4 genotype were evaluated in general, 31.25% of the strains were found to grow at 39 °C and 6.25% at 41 °C. Of the T4Neff strains, only one strain did not show the ability to reproduce at 39 °C and showed a different feature from the other strains. While the strain with the T11 genotype grew at all temperatures, the strain with the T12 genotype did not grow at 41 °C. Conclusion: According to our research results, we believe that tolerance to 39 °C and 1 M mannitol is not an indicator of pathogenicity. More studies with Acanthamoeba strains are required to clarify this issue.

PURPOSE: Outcomes of Acanthamoeba keratitis are often worse in India than in the United States. The goal of this study was to determine whether antiamoebic susceptibility patterns were different when comparing Acanthamoeba isolates from India with those of the United States. METHODS: Acanthamoeba isolates were obtained from corneal scrapings of 43 patients with infectious keratitis seen at the Francis I. Proctor Foundation (N = 23) and Aravind Eye Hospital (N = 20) from 2008 through 2012 and plated on growth media. A previously described minimum cysticidal concentration (MCC) assay was performed by a single laboratory technician to assess susceptibility to 5 antiamoebic agents for all isolates. Testing was conducted in triplicate, with the median MCC chosen for analyses. RESULTS: The MCC (μg/mL) of polyhexamethylene biguanide was 6.25 [IQR 5.47-12.5] for Aravind isolates and 6.25 [IQR 6.25-9.375] for Proctor isolates ( P = 0.75), corresponding values were 6.25 [IQR 3.125-6.25] and 3.125 [IQR 3.125-9.375] for chlorhexidine ( P = 0.81), 2500 [IQR 2500-5000] and 5000 [IQR 1250-20,000] for voriconazole ( P = 0.25), 15.6 [IQR 15.6-39.0625] and 15.6 [IQR 15.6-31.25] for hexamidine ( P = 0.92), and 15.6 [IQR 7.81-15.6] and 15.6 [IQR 7.81-31.25] for propamidine ( P = 0.42). CONCLUSIONS: This study found no statistically significant differences in antiamoebic susceptibility of Indian versus US samples from Acanthamoeba keratitis clinical isolates. These findings suggest that differences in antiamoebic susceptibility are likely not responsible for differential outcomes in Acanthamoeba keratitis between the 2 locations.

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. [ABSTRACT FROM AUTHOR]

Free-living amoebae (FLA) are found in diverse environments, such as soils, rivers, and seas. Hence, they can be used as bioindicators to assess the water quality based solely on their presence. In this study, we determined the presence of FLA in river water by filtering water samples collected from various sites and culturing the resulting filtrates. FLA were detected in all the water samples with varying quality grades (Grades I-V). The significant increase in the size of the amoebae population with the deterioration in the water quality. Monoxenic cultures of the amoebae were performed, and genomic DNAs were isolated, among which 18S rDNAs were sequenced to identify the amoeba species. Of the 12 species identified, 10 belonged to the Acanthamoeba genus; of the remaining 2 species, one was identified as Vannella croatica and the other as a species of Vermamoeba. Acanthamoeba was detected in samples with Grades I to VI quality, whereas the Vermamoeba species was present only in Grade I water. V. croatica was found exclusively in water with Grade II quality. Following morphological observations, genomic DNA was sequenced using 16S rDNA to determine whether the species of Acanthamoeba harbored endosymbionts. Most of the isolated Acanthamoeba contained endosymbionts, among which 4 species of endogenous bacteria were identified and examined using transmission electron microscopy. This study provides evidence that the distribution of amoebae other than Acanthamoeba may be associated with water quality. However, further confirmation will be required based on accurate water quality ratings and assessments using a more diverse range of FLA. [ABSTRACT FROM AUTHOR]

Mycobacterium leprae is an acid-fast bacterium that causes leprosy diseases, which remains a problem worldwide. Even though leprosy prevalence in the world has decreased significantly, many endemic pocket areas continue reporting new cases and harbor M. leprae in the environment, including water and soil. The presence of obligate intracellular bacteria-M. leprae in the environment raises a question on how it survives. Free-living amoeba has been proposed as its reservoir host in the environment. The study was conducted to give evidence that M. leprae can survive inside free-living amoeba isolated from water sources of leprosy endemic areas. M. leprae from leprosy patients was cultured together with Acanthamoeba sp. isolated from the water source of the leprosy endemic area. Viability and duplication of M. leprae inside amoeba then observed at day 14 and 28 using reverse transcriptase PCR and qPCR. The results showed that M. leprae survived inside the amoeba until day 28, but no bacterial replication was observed. The study reveals in vitro evidence of viable M. leprae inside free-living amoeba of leprosy endemic area environment. [ABSTRACT FROM AUTHOR]

Chalcone is a molecule with known biological activities. Based on this, a series of chalcone derivatives bearing methyl, phenyl or furanyl substituents at different positions of A and B rings were synthesised, characterised, and evaluated regarding antiprotozoal activity. Molecules were synthesised via base catalyzed Claisen-Schmidt condensation and characterised by IR and NMR spectral data. Antiprotozoal activity against Phytomonas serpens, Leishmania amazonensis and Acanthamoeba polyphaga was performed. All compounds inhibited more than 50% of the growth of P. serpens while five had this effect on L. amazonensis and all of them no more than 35% of inhibition on A. polyphaga. Remarkably interesting antiprotozoal effects were recorded with compound 5, with IC50 of 1.59 µM for P. serpens and 11.49 µM for L. amazonensis. The addition of a naphthyl group to the B ring can be postulated to be the cause of the 10 times increase observed in its trypanocidal activity. [ABSTRACT FROM AUTHOR]