T4 Genotype Research Articles

In Vitro Amoebicidal Activity of Titanium Dioxide/UV-A Combination Against Acanthamoeba.

To assess the amoebicidal effect of titanium dioxide (TiO2)/UV-A combination against Acanthamoeba sp trophozoites and cysts. The amoebicidal effect of the TiO2/UV-A combination was tested on trophozoites and cysts of clinical isolates of Acanthamoeba hatchetti and Acanthamoeba sp genotype T4, obtained from two severe cases of ulcerative keratitis. Samples of cultured Acanthamoeba were transferred to a 96-well plate. We tested the effect of sterile water (blank control), TiO2 alone, UV-A alone, TiO2 and additional UV-A exposure, chlorhexidine 0.02% alone, chlorhexidine 0.02% and TiO2, chlorhexidine and UV-A, chlorhexidine 0.02% and TiO2, and additional UV-A exposure. Cell viability assessment was done using the trypan blue dye exclusion method. The combination of TiO2 with UV-A demonstrated antitrophozoite and anticyst activity (P < 0.05). This in vitro study showed a synergistic effect of the association of chlorhexidine with TiO2 and UV-A on cysts (P < 0.001). Given the in vitro synergistic effectiveness of the association of chlorhexidine with TiO2 and UV-A against cysts, the treatment of Acanthamoeba keratitis could be improved by this new therapeutic approach.

Isolation and molecular identification of Acanthamoeba spp. from hot springs in Mazandaran province, northern Iran.

Acanthamoeba is a free-living protozoan that can be found in natural and artificial environments such as hot tubs, surface water and springs and can cause severe diseases including amoebic keratitis and granulomatous amoebic encephalitis. The present study was conducted owing to the lack of research regarding genotypes of Acanthamoeba in hot springs of Mazandaran province in northern Iran. Twenty-four water samples were collected from all hot springs in Mazandaran province. After filtration through nitrocellulose membrane, samples were cultured on non-nutrient agar medium enriched with TYIS-33. The cultures were microscopically examined for the presence of Acanthamoeba. Positive cultures were analysed by polymerase chain reaction (PCR) and genotypes were determined by targeting the 18 S rRNA gene. The pathogenic potential of all positive isolates was identified using thermotolerance and osmotolerance tests. Eleven (47.8%) samples were positive for Acanthamoeba. Based on sequencing analysis, 100% of isolates belonged to the T4 genotype. Thermo- and osmo-tolerance tests showed that four (36.3%) Acanthamoeba strains were highly pathogenic. According to our research, the occurrence of Acanthamoeba in recreational hot springs could be a hazard for high risk persons. Posting warning signs and regular monitoring of these waters by health planners may therefore be useful for decreasing Acanthamoeba spp. infections.

Nuclear Group I introns with homing endonuclease genes in Acanthamoeba genotype T4

Various strains belonging to three Acanthamoeba species, A. griffini (genotype T3), A. lenticulata (T5), and A. jacobsi (T15), have group I introns in their 18S rRNA genes. Group I introns are self-splicing ribozymes that can spread among host lineages either through an intron-encoded endonuclease at the DNA level, or by reverse splicing during the RNA cycle. In Acanthamoeba, introns belong to the subclass IC1, they are located at one out four positions within the rRNA, show low identity values and all lack open reading frames to encode for an endonuclease. Uncharacterized introns from strains of another genotype, T4 (A. castellanii complex), resemble those of genotype T3, and at least one of them contains a non-functional endonuclease gene. Here, we analyzed all available data on Acanthamoeba 18S rDNA sequences to identify the possible presence of open reading frames that could encode endonucleases. We found a total of eight 18S rDNA sequences, all from T4 strains, that have introns containing putative non-functional endonuclease genes. Furthermore, two distinct endonucleases can be identified that are differently inserted in unrelated introns.

Effect of oxidative stress on vital indicators of Acanthamoeba castellanii (T4 genotype).

Acanthamoeba has 22 genotypes with the T4 genotype being the main causative agent of amoebic granulomatous encephalitis and keratitis. Because the molecular mechanisms of the immune defenses of neutrophils and macrophages against histoparasites are based on oxidative stress, parasites may rely on their antioxidant systems to preclude immune defenses. Therefore, understanding of the effect of oxidative stress on vital characteristics of Acanthamoeba castellanii (T4 genotype) and the antioxidant defense responses of Acanthamoeba to oxidative status will cast light on immune cell-parasite interactions. Acanthamoeba T4 cells were cultured in RPMI-1640 medium containing different concentrations of hydrogen peroxide (H2O2). The survival of Acanthamoeba was evaluated by MTT assay and the IC50 concentration was calculated. The total antioxidant capacity (TAC) of the parasite was determined by the cupric reducing antioxidant capacity (CUPRAC) method. Malondialdehyde (MDA) as a marker of lipid peroxidation, protein carbonyl content as a measure of oxidized protein, total thiol (-SH) groups present on proteins as a major source of cellular antioxidants, and total oxidant status (TOS) were evaluated by colorimetric methods. The reactive oxygen species level increased markedly after induction of oxidative stress by the treatment of Acanthamoeba T4 with H2O2. Exposure to H2O2 also significantly increased the MDA and protein carbonyl content. The TOS level and total thiol groups also increased in the treated group compared to those in untreated parasites, although the results were not statistically significant. The TAC level was found to be significantly higher in H2O2-treated parasites, confirming that the parasite fosters its total antioxidant capacity to overcome oxidative conditions. This study showed that under oxidative stress, the defense reactions of the parasite are in part mediated by increasing its antioxidant activity, which is important for the survival of the parasite.

DNA extraction from amoebal isolates and genotype determination of Acanthamoeba from tap water in Latvia.

Acanthamoeba are free-living amoebae found in various ecological environments, and they are major microbial predators. Some Acanthamoeba species can cause keratitis, granulomatous amoebic encephalitis, and infections of the organs. This is the first study in Latvia describing Acanthamoeba detection by molecular techniques and genotyping using tap water from different sources. An 18S rRNA gene investigation of the 20 Acanthamoeba isolates suggested that 19 belonged to the hazardous T4 genotype and one sample was T12. The results indicate that tap water in Latvia may be a source of Acanthamoeba that is potentially pathogenic to humans. All obtained sequences were submitted to the GenBank database under the following accession numbers: MG386295-MG386314.

The cellulose synthase BcsA plays a role in interactions of Salmonella typhimurium with Acanthamoeba castellanii genotype T4.

Pathogenic bacteria share their natural habitat with many other organisms such as animals, plants, insects, parasites and amoeba. Interactions between these organisms influence not only the life style of the host organisms, but also modulate bacterial physiology. Adaptation can include biofilm formation, capsule formation, and production of virulence factors. Although biofilm formation is a dominant mode of bacterial life in environmental settings, its role in host-pathogen interactions is not extensively studied. In this work, we investigated the role of molecular pathways involved in rdar biofilm formation in the interaction of Salmonella typhimurium with the Acanthamoeba castellanii genotype T4. Genes coding for the rdar biofilm activator CsgD, the cellulose synthase BcsA, and curli fimbriae subunits CsgBA were deleted from the genome of S. typhimurium. Assessment of interactions of wild-type and mutant strains of S. typhimurium with A. castellanii revealed that deletion of the cellulose synthase BcsA promoted association and uptake by A. castellanii, whereas the interactions with csgD and csgBA mutants were not changed. Our findings suggest that cellulose synthase BcsA inhibits the capabilities of S. typhimurium to associate with and invade into A. castellanii.

Molecular detection of Toxoplasma gondii in natural surface water bodies in Poland.

The aim of this study was molecular detection of Toxoplasma gondii in 36 natural surface water bodies in Poland, including preliminary genotype identification and determination of co-occurrence of this parasite with other protozoa that have been detected in previous studies. The examined DNA samples were obtained before to detect Cryptosporidium, Giardia and free-living amoebae. Nested polymerase chain reaction (PCR) based on B1 gene and sequencing was performed for both confirmation of parasite presence in water and genotype identification. T. gondii DNA was detected in 19.4% (7/36) water bodies, while in the case of other studies, T. gondii prevalence ranged between 0% and over 56%. These differences may be caused by natural variations in T. gondii occurrence as well as different sample volumes and methods of sample processing or DNA isolation and detection. Two cases of double contamination were reported: T. gondii with Cryptosporidium parvum and T. gondii with potentially pathogenic Acanthamoeba T4 genotype, thus there is a possibility of mixed infection in humans after occasional contact with water. Obtained T. gondii strains were genetically identical or closely similar (99.8%) to RH strain representing genotype I, however, further examinations involving more loci will be conducted to identify the genotype.

Detection of serum antibodies in children and adolescents against Balamuthia mandrillaris, Naegleria fowleri and Acanthamoeba T4

The presence of free-living amoebae of the genera Naegleria, Acanthamoeba and Balamuthia, which contain pathogenic species for humans and animals, has been demonstrated several times and in different natural aquatic environments in the northwest of Mexico. With the aim of continuing the addition of knowledge about immunology of pathogenic free-living amoebae, 118 sera from children and adolescents, living in three villages, were studied. Humoral IgG response against B. mandrillaris, N. fowleri and Acanthamoeba sp. genotype T4, was analyzed in duplicate to titers 1: 100 and 1: 500 by enzyme-linked immunosorbent assay (ELISA). Children and adolescents ages ranged between 5 and 16 years old, with a mean of 9 years old, 55% males. All tested sera were positive for the 1: 100 dilution, and in the results obtained with the 1: 500 dilution, 116 of 118 (98.3%) were seropositive for N. fowleri, 101 of 118 (85.6%) were seropositive for Acanthamoeba sp. genotype T4, and 43 of 118 (36.4%) were seropositive for B. mandrillaris. The statistical analysis showed different distributions among the three communities and for the three species of pathogenic free-living amoebae, including age. Lysed and complete cells used as Balamuthia antigens gave differences in seropositivity.

Development and optimization of new culture media for Acanthamoeba spp. (Protozoa: Amoebozoa)

The isolation and growth in axenic liquid media of Acanthamoeba strains is necessary in order to carry out primary in vitro drug screening. Amoebic isolates which are hard to grow in the current liquid media have been reported. Such circumstances hampers the ability of conducting drug sensitivity tests. Therefore, finding suitable universal growth media for Acanthamoeba species is required. The present study was aimed at the development of liquid medium suitable for growing a fastidious (F) genotype T3 Acanthamoeba isolate, and eventually for other genotypes of this genus as well. Trophozoite growth was indirectly monitored by respiration analysis with oxygen-sensitive microplates (OSM) and further confirmed by manual counting. Media were empirically designed and tested first in a non-fastidious (NF) T3 isolate and then tested with 14 different strains, including the fastidious one. Combinations of nutritive components such as meat/vegetable broth, LB medium, malt and skimmed milk led to the design of new media suitable for culturing all the isolates tested, in conditions similar to those obtained in standard culture media such as PYG or CERVA.

Presence of Acanthamoeba in the ocular surface in a Spanish population of contact lens wearers.

Pathogenic strains of Acanthamoeba are causative agents of a sight-threatening infection of the cornea known as Acanthamoeba keratitis (AK) which mainly affects contact lens wearers and it is commonly related to poor hygiene of contact lenses and their cases. Moreover, treatment of AK is complex due to the existence of a highly resistant cyst stage and if not diagnosed early has poor prognosis, leading to blindness and/or keratoplasty. Even though AK is increasing worldwide as well as awareness among patients and clinicians, it is still a poorly studied pathogen. Additionally, a remaining question to be answered is whether these opportunistic pathogens are present in the ocular surface of healthy contact lens wearers since they are the main group at risk.In order to carry out this study, sterile Schirmer strip tests were collected from a group of individuals all of them contact lens wearers who were attending a local ophthalmology clinic in Tenerife, Canary Islands, Spain. The collected samples (100 eyes of 50 patients) were cultured in 2% Non-Nutrient Agar (NNA) plates and positive plates (16) were then cultured in axenic conditions for further analyses. Molecular analysis classified all isolated strains belonged to Acanthamoeba genotype T4 and osmotolerance and thermotolerance assays revealed that all strains were potentially pathogenic. In conclusion, the ocular surface of contact lens wearers included in this study was colonized by potentially pathogenic strains of Acanthamoeba and should be considered as a risk for AK infection in this region and worldwide.

Acanthamoeba of three morphological groups and distinct genotypes exhibit variable and weakly inter-related physiological properties.

Free-living amoeba of the genus Acanthamoeba can eventually act as parasites, causing infections in humans. Some physiological characteristics of Acanthamoeba have been related to the grade of pathogenicity, allowing inferences about the pathogenic potential. The main goal of this study was to characterize isolates of Acanthamoeba obtained in Brazil and evaluate properties associated with their pathogenicity. A total of 39 isolates obtained from keratitis cases (n = 16) and environmental sources (n = 23) were classified into morphological groups and genotyped by sequencing the 18S rDNA fragments ASA.S1 and GTSA.B1. Samples were also tested regarding their thermo-tolerance, osmo-tolerance, and cytopathogenicity in MDCK cells. Isolates were identified and classified as follows: group I (T17, T18); group II (T1, T3, T4, T11); and group III (T5, T15), with the predominance of genotype T4 (22/39). Clinical isolates were genotyped as T3 (1/16), T4 (14/16) and T5 (1/16). The majority of isolates (38/39) were able to grow at 37°C, but tolerance to 40°C was more frequent among environmental samples. The tolerance to 1M mannitol was infrequent (4/39), with three of these corresponding to clinical samples. The variable ability to cause cytopathic effects was observed among isolates of distinct genotypes and origins. This study identified, for the first time, T1 and T18 in Brazil. It also indicated a weak association between the clinical origin of the isolates and tolerance to high temperatures, high osmolarity, and cytopathogenicity, demonstrating that some in vitro parameters do not necessarily reflect a higher propensity of Acanthamoeba to cause a disease.

Molecular Identification and Genotyping of Acanthamoeba Isolated from Environmental Sources in Kashan, Central Iran

Background: Acanthamoeba is one of the most common opportunistic free-living amoebae, with ubiquitous presence in various en- vironmental sources. Pathogenic strains of Acanthamoeba are the causative agents of amoebic keratitis and granulomatous amoebic encephalitis. Objectives: The aim of the present study was to identify Acanthamoeba genotypes in soil, hospital dust, and stagnant water samples from Kashan, Central Iran. Methods: In this cross sectional study, a total of 122 samples from soil (n, 32), hospital dust (n, 40), and stagnant water (n, 50) were collected and examined for the presence of free-living amoebae and Acanthamoeba species. All the samples were cultured onto non-nutrient agar plates for detection of free-living amoebae. Acanthamoeba species was identified by polymerase chain reaction (PCR) assay, using specific primers. A total of 29 Acanthamoeba isolates were sequenced, and different genotypes were detected via sequence analysis. Results: The results showed that 82.8% (101/122) of samples were positive for free-living amoebae. The PCR assay revealed that 62.5%, 52.5%, and 50% of soil, hospital dust, and stagnant water samples were positive for Acanthamoeba species, respectively. Moreover, T4, T5, T2, T7, and T11 genotypes were identified. The most common genotype was T4 (76%), isolated from stagnant water. Conclusions: Acanthamoeba is a prevalent species in the soil, hospital dust, and stagnant water of Kashan. As this protozoon can cause severe infections, health education and improvement of sanitation services are recommended for prevention of infection. Keywords: Acanthamoeba, Soil, Hospital Dust, Stagnant Water, Genotype, Iran

Genotypic characterization and assessment of infectivity of human waterborne pathogens recovered from oysters and estuarine waters in Brazil

Waterborne, food-borne and sewage-borne pathogens are a major global concern, with the annual recurrence, most notably during the summer, of outbreaks of gastroenteritis of unconfirmed etiology associated with recreational activities in marine environments. The consumption of contaminated water-based foodstuffs is also related to outbreaks of human illness. The main goals of the present study were: i) to identify the genetic assemblages of Giardia duodenalis cysts in growing and depurated oysters destined for human consumption on the southern coast of São Paulo, Brazil; ii) to verify the main circulating G. duodenalis assemblages and their subtypes in different brackish waters used for the production of mollusks and for recreational purposes; iii) to track the contamination of growing and depurated oysters by the human adenovirus and identify the infectivity of adenoviral particles recovered from oysters before and after depuration; iv) to evaluate the occurrence and genotype of the free-living amoebae of the genus Acanthamoeba in brackish water and oysters from all the sites described above. Four sampling sites in the Cananeia estuary were selected to search for pathogenic and amphizoic protozoa (Giardia and Acanthamoeba respectively): site 1: oyster growth, site 2: catchment water (before UV depuration procedure), site 3: filter backwash (filtration stage of water treatment) and site 4: oyster depuration tank. Oysters at sites 1 and 4 were evaluated for the presence of adenovirus (HAdV). Analysis consisted of conventional microbiological as well as molecular methods. Giardia duodenalis were detected in all the water sites analyzed and the molecular analysis revealed that sub-assemblage AII was the most frequently distributed throughout the estuarine environment, although one sample was identified as belonging to the assemblage C. Acanthamoeba were also isolated from different locations of the estuarine area, and were detected at all the analyzed sites. The majority of isolates belonged to the T3 genotype, while the T4 genotype was identified once. The sequencing reaction of Giardia duodenalis revealed the contamination of three batches of depurated oysters by the sub-assemblage AII. With respect to viruses, seven batches of oysters (four growing and three depurated) were found to be harboring infectious HAdV particles when submitted to plaque assay. Overall, the results of the sequencing reactions combined with the plaque assay revealed that the isolates of Giardia duodenalis and the infectious HAdV particles identified in oyster tissues have the potential to infect humans and pose a threat if consumed raw or lightly cooked. This is the first report on the sub-assemblage AII identified in oysters which are submitted to a cleaning and disinfection procedure prior to human consumption in Brazil. Acanthamoeba specific genotypes were also identified for the first time in a recreational estuarine area in Brazil, contributing to knowledge of their molecular and environmental epidemiology, which is considered scarce even in marine and estuarine areas of the world.

Isolation and molecular identification of Acanthamoeba spp from oasis water in Tunisia

In the southern Tunisia Oasis, we conducted 211 water with drawals from various water traffic sites. This water is used for agriculture, swimming or various other human activities. Acanthamoeba genus was detected in 82% of collected samples. Sequencing of the amplification products with primers P892C/P892 has allowed us to detect genotypic variation with predominance of T4 genotype (51%) and presence of the genotypes T14, T5, T3, T16, T15, T10, T11, T9 and T7. They T4, T3, T5, T15, T11 and T10 genotypes have a high potential for pathogenicity and a very high degree of virulence due to their production of serine proteases and extracellular cysteine enzymes involved in tissue degradation of the host. T4 genotype was the most abundant in the environment as well as in infections caused by Acanthamoeba spp. T5 genotype was ranked second and T3 genotype was less abundant in the environment and its pathogenicity is discussed. Acanthamoeba strains with the genotypes T16, T9 and T7 were considered non pathogenic. In fact, they have been isolated only from the environment. However, for these strains, their role as a reservoir can be a real risk to human health.

Detection and molecular characterization of Acanthamoeba spp. in stray cats from Madrid, Spain

Acanthamoeba spp. is a widespread protozoan that has been isolated from air, dust, soil, water and biological samples. An opportunistic pathogen of humans and animals, it may cause ocular keratitis, encephalitis, and even multisystem disease. The frequency of Acanthamoeba in animals is unknown. The aim of present study was determine the presence of Acanthamoeba spp. in immunocompromised stray cats - animals possibly more likely to harbour the infection given their immunocompromised status and frequenting of contaminated environments. Of 307 cats examined, 55 were positive for feline immunodeficiency virus and/or feline leukaemia virus and therefore included in the study. Corneal scrapings were obtained to isolate Acanthamoeba spp. by culture and molecular detection by conventional and real time PCR. None of the samples examined directly by molecular methods were positive for Acanthamoeba spp. However, two (3.6%) cases of the cultured samples provided positive results, which were confirmed by subsequent molecular analysis. Sequencing assigned one isolate to genotype T4 and the other to T2. Since Acanthamoeba spp. may also infect animals and humans, the present findings may raise some public health and veterinary concerns.

Comparison of chlorhexidine disinfectant in vitro effect on environmental and ocular Acanthamoeba strains, the amoebic agents of human keratitis − an emerging sight-threatening corneal disease in Poland

Small amoebae belonging to the Acanthamoeba genus complete their life cycles in different environmental niches as free-living protists however some of them are facultative parasites that can cause severe disease in humans. The sight-threatening Acanthamoeba keratitis develops in immune-competent persons, mainly in contact lens wearers; it is detected with increasing frequency along with the spread of contact lens use. The high abundance of the amoebae in the environment is important for dispersion and transmission of the infections among humans. Emerging threats for the public health generated by these amoebae is the serious medical problem worldwide. Nonspecific symptoms, similar to those occurring in the other eye diseases, diagnostic mistakes, the delay of an appropriate treatment, an exceptional high resistance of the amoebae to chemicals and drugs result in a prolonged course of the disease and often unsuccessful therapeutic management. Thus, different chemicals are still examined for their potential activity in vitro against various species, strains/isolates of Acanthamoeba. As the prolonged therapy often induces encystation subsequently leading to excystment and recurrences of amoebic keratitis, apart from anti-amoebic activity, cysticidal effect of examined agents is desirable. In the present study, results of our comparative investigations showed that cationic antiseptic chlorhexidine digluconate indicated in vitro anti-amoebic effect on environmental Acanthamoeba castellanii Neff strain and pathogenic corneal Acanthamoeba polyphaga T4 genotype. Amoebostatic effect of the disinfectant was expressed in reduced number of surviving amoebae in comparison to the respective control cultures; simultaneously, despite prolonged incubation with the agent no stimulation of encystation was noted. The corneal strain was more resistant to the tested compound than the Neff strain. The cysticidal efficacy of chemicals is very expected, thus further in vitro studies on pathogenic Acanthamoeba strains with different application chemicals pattern are needed.

Can artificial tears prevent Acanthamoeba keratitis? An in vitro approach

BackgroundThe use of contact lenses has increased in recent years as has the incidence of Dry Eye Syndrome, partly due to their use. Artificial tears are the most common treatment option. Since these changes can facilitate Acanthamoeba infection, the present study has been designed to evaluate the effect of three artificial tears treatments in the viability of Acanthamoeba genotype T4 trophozoites. Optava Fusion™, Oculotect®, and Artelac® Splash were selected due to their formulation.MethodsViability was assessed using two staining methods, Trypan Blue stain and CTC stain at different time intervals (2, 4, 6, 8 and 24 h). Trypan Blue viability was obtained by manual count with light microscopy while the CTC stain was determined using flow cytometry.ResultsTrypan Blue staining results demonstrated a decrease in viability for Optava Fusion™ and Artelac® Splash during the first 4 h of incubation. After, this effect seems to lose strength. In the case of Oculotect®, complete cell death was observed after 2 h. Using flow cytometry analysis, Optava Fusion™ and Oculotect® exhibited the same effect observed with Trypan Blue staining. However, Artelac® Splash revealed decreasing cell respiratory activity after four hours, with no damage to the cell membrane.ConclusionsThe present study uses, for the first time, CTC stain analyzed by flow cytometry to establish Acanthamoeba viability demonstrating its usefulness and complementarity with the traditional stain, Trypan Blue. Artelac® Splash, with no preservatives, and Optava Fusion TM, with Purite®, have not shown any useful amoebicidal activity. On the contrary, promising results presented by Ocultect®, with BAK, open up a new possibility for Acanthamoeba keratitis prophylaxis and treatment although in vivo studies should be carried out.

Contamination of swimming pools and hot tubs biofilms with Acanthamoeba.

The members of Acanthamoeba genus are ubiquitous amoeba which could be a pathogenic parasite. The amoeba is resistant to the common chlorine concentration that used for disinfecting the swimming pool water. Therefore, the pools can be suitable environments for the survival and multiplication of the amoeba. In this cross sectional study, 10 indoor recreational water centers from different regions of Tabriz city were selected and sampling was done from fixed and floating biofilms of the swimming pools and hot tubs. The samples were cultured and monitored for the presence of amoeba cyst or trophozoite. For molecular identification of Acanthamoeba, PCR (polymerase chain reaction) and sequencing were conducted based on genus specific fragment of 18S ribosomal DNA (Rns). Acanthamoeba contamination was observed in 6 centers of 10 recreational centers. Based on the amoeba isolation from fixed and floating biofilms, 2 (20%) swimming pools, and 5 (50%) hot tubs were contaminated. Based on the type of the sample, the highest contamination was found in the hot tub water (40%) and the least was found in the swimming pools water (10%) and fixed biofilms of the swimming pools (10%). Out of 8 isolates, 5 (62.5%) were shown expected product in PCR amplification. Sequence analysis showed that Acanthamoeba isolates belonged to the T3 and T4 genotypes. The study revealed a high degree of contamination in the indoor recreational water centers in Tabriz city. So, it is essential to pay closer attention to the hygiene of swimming pools and hot tubs.

Evaluation of the inflammatory response to Kudoa septempunctata genotype ST3 isolated from olive flounder (Paralichthys olivaceus) in Caco-2 cells.

Kudoa septempunctata (Myxosporea, Multivalvulida) is a parasite of the trunk muscle of cultured olive flounder (Paralichthys olivaceus). We investigated whether K. septempunctata genotype ST3 spores induce cell damage and the secretion of inflammatory mediators in Caco-2 cells, which exhibit characteristics similar to human intestinal epithelial cells. Purified K. septempunctata spores were heated at 95 °C for 5 min. Lactate dehydrogenase (LDH) release was measured to determine the efficacy of denaturation. Naïve and heated spores, lipopolysaccharide (positive control) and vehicle (negative control) were added to Caco-2 cells. Cells were subjected to the cytotoxic LDH assay and western blot analysis to examine the expression of inducible nitric oxide synthase (iNOS) and cyclooxygenase (COX)-2. Supernatants were collected to measure nitric oxide (NO) and prostaglandin E2 (PGE2). Most spores were denaturated by heating, and the spore morphology was found to be wrinkled with shell valves and polar capsules. In addition, cytotoxicity and inflammatory mediators, such as NO, PGE2, iNOS, and COX-2, remained unchanged in Caco-2 cells following exposure to naïve and heated spores compared with the positive controls. Collectively, the findings of this study imply that spores of K. septempunctata genotype ST3 do not cause inflammation in Caco-2 cells.

Silver nanoparticle conjugation affects antiacanthamoebic activities of amphotericin B, nystatin, and fluconazole.

Infectious diseases are the leading cause of morbidity and mortality, killing more than 15 million people worldwide. This is despite our advances in antimicrobial chemotherapy and supportive care. Nanoparticles offer a promising technology to enhance drug efficacy and formation of effective vehicles for drug delivery. Here, we conjugated amphotericin B, nystatin (macrocyclic polyenes), and fluconazole (azole) with silver nanoparticles. Silver-conjugated drugs were synthesized successfully and characterized by ultraviolet-visible spectrophotometry, Fourier transform infrared spectroscopy, and atomic force microscopy. Conjugated and unconjugated drugs were tested against Acanthamoeba castellanii belonging to the T4 genotype using amoebicidal assay and host cell cytotoxicity assay. Viability assays revealed that silver nanoparticles conjugated with amphotericin B (Amp-AgNPs) and nystatin (Nys-AgNPs) exhibited significant antiamoebic properties compared with drugs alone or AgNPs alone (P < 0.05) as determined by Trypan blue exclusion assay. In contrast, conjugation of fluconazole with AgNPs had limited effect on its antiamoebic properties. Notably, AgNP-coated drugs inhibited amoebae-mediated host cell cytotoxicity as determined by measuring lactate dehydrogenase release. Overall, here we present the development of a new formulation of more effective antiamoebic agents based on AgNPs coated with drugs that hold promise for future applications.