Acanthamoeba keratitis (AK) is an eye disease often occurring in contact lens wearers. AK treatment is prolonged and requires multiple drugs, which can lead to adverse effects. Our study aimed to compare the in vitro activities and safety of Miltefosine with that of conventional antimicrobial agents used to treat AK. Acanthamoeba castellanii genotype T4 was obtained from a patient with keratitis and subjected to in vitro susceptibility testing with various antimicrobial agents, including Chlorhexidine (CHX), Pentamidine isethionate (PI)Polyhexamethylene biguanide (PHMB), and Miltefosine to assess their efficacy against Acanthamoeba trophozoites and cyst. The cytotoxicity of the agents was evaluated in Vero cells, and their selectivity indexes (SI) were calculated. Chlorhexidine exhibited the highest amoebicidal activity with the highest selectivity index against the trophozoite and cyst, ranging from 1.17 to 8.35. The selectivity index of PHMB is slightly comparable to Chlorhexidine, exhibiting significant anti-Acanthamoeba activity. On the other hand, Pentamidine isethionate and Miltefosine displayed low SI among the compounds. Pentamidine isethionate was effective at high concentrations, which was toxic. Miltefosine exhibited the lowest cytotoxicity; nevertheless, due to the lowest anti-Acanthamoeba activity presented a low selectivity against the parasite. Further studies on more clinical samples and prolonged incubation time should be done to investigate the effectiveness and toxicity of drugs in both in vitro and in vivo conditions.

Free-living amoeba of the genus Acanthamoeba are ubiquitous protozoa involved in opportunistic and non-opportunistic infection in humans, such as granulomatous amoebic encephalitis and amoebic keratitis. Both infections have challenging characteristics such as the formation of the resistant cysts in infected tissues, hampering the treatment and most usual diagnosis depending on time-consuming and/or low sensitivity techniques. The use of monoclonal antibodies presents itself as an opportunity for the development of more effective alternative diagnostic methods, as well as an important and useful tool in the search for new therapeutic targets. This study investigated the possibility of using a previously produced monoclonal antibody (mAb3), as a diagnostic tool for the detection of Acanthamoeba trophozoites by direct and indirect flow cytometry and immunofluorescence. Immunoprecipitation assay and mass spectrometry allowed the isolation of the antibody\'s target and suggested it is a transporter part of the CPA (cation: proton antiporter) superfamily. In vitro tests indicate an important role of this target in Acanthamoeba\'s encystment physiology. Our results support the importance of studying the role of CPA2 transporters in the context of acanthamoebiasis, as this may be a way to identify new therapeutic candidates.

Cationic carbosilane dendrimers are branched molecules with antimicrobial properties. Their activity has been tested against Acanthamoeba polyphaga, a causative agent of Acanthamoeba keratitis, a severe ocular disease in humans. A. polyphaga trophozoites and cysts were exposed to different noncytotoxic cationic carbosilane dendrimers with proven antiamoebic activity. The effects of treatment on cell surface and cell ultrastructure were examined by scanning and transmission electron microscopy, respectively. Two of the dendrimers tested induced dramatic alterations of cellular ultrastructure in both trophozoites and cysts, including vacuolization, depletion of cytoplasmic contents, and reduced cell size. Additionally, we observed severe alterations of the plasma membrane with membrane blebbing in trophozoites and disruption in cysts. These alterations were also observed with chlorhexidine, a drug used for treatment of Acanthamoeba keratitis. Our results support that these compounds may target membranes, and their action is critical for parasite integrity.

BACKGROUND: Acanthamoebiasis treatment is a major and challenging problem due to the presence of resistant cyst form. Many herbal extracts and their derivatives have been used against trophozoites and cysts of Acanthamoeba, but no effective therapeutic agent has yet been discovered. Therefore, the present study aimed to evaluate the effect of Rumex obtusifolius (R. obtusifolius) extracts against a clinical strain of Acanthamoeba genotype T4 in vitro.
METHODS: In this experimental study, after genotyping the clinical isolate, the hydroalcohlic extracts of R. obtusifolius seeds and leaves were prepared. Different concentrations (1.25, 2.5, 5 and 10 mg/ml) of extracts were tested in triplicate (24, 48 and 72h) on trophozoites and cysts of Acanthamoeba. The mortality of the parasite was assessed by trypan blue vital staining and flow cytometry analysis.
RESULTS: Results showed that the extract of R. obtusifolius leaves at the concentration of 10 mg/ml killed 100% of trophozoites and cysts after 72 h. However, the seed extract of R. obtusifolius had weak inhibitory effects on trophozoites and cysts of Acanthamoeba. In the presence of 10 mg/ml of hydroalcoholic seed extract of R.obtusifolius in culture medium after 72 h, 28.6% of trophozoites and 0% of cysts of Acanthamoeba were killed. After analysis by flow cytometry, seeds and leaves extract indicated apoptosis effect. Seed and leaf extracts caused 2.6% and 0.4% percent apoptosis.
CONCLUSIONS: These extracts are not promising candidates for further medicine development on acanthamoebiasis. Nonetheless, further research is necessary to clarify the effects of effective fractions of seed and leaf extracts of R. obtusifolius and their mechanisms of action.

BACKGROUND: Giardia duodenalis causes giardiasis in humans, particularly in developing countries. Despite the availability of treatments, resistance to some of the commercial anti-Giardia drugs has been reported in addition to their harmful side effects. Therefore, novel treatments for giardiasis are required. In this study, we aimed to assess the in vitro activity of crude extracts of Ageratum conyzoides against G. duodenalis trophozoites.
METHODS: Plants were classified into three groups based on their flower colors: white (W), purple (P), and white-purple (W-P). Plants were separately cut into leaf (L) and flower (F) parts. Changes in internal organelle morphology of trophozoites following exposure to crude extracts were assessed using transmission electron microscopy (TEM). In subsequent experiments, efficacy of the most active essential oils from crude extracts [half maximal inhibitory concentrations (IC50) ≤ 100 μg/mL] against G. duodenalis trophozoites was tested. In vitro anti-Giardia assays using essential oils were performed in the same way as those performed using crude extracts.
RESULTS: LW-P and FP extracts showed high activity (IC50 ≤ 100 μg/mL) against G. duodenalis trophozoites, with IC50 ± SD values of 45.67 ± 0.51 and 96.00 ± 0.46 μg/mL, respectively. In subsequent experiments, IC50 ± SD values of LW-P and FP essential oils were 35.00 ± 0.50 and 89.33 ± 0.41 μg/mL, respectively. TEM revealed the degeneration of flagella and ventral discs of G. duodenalis trophozoites following exposure to crude extracts.
CONCLUSIONS: Crude LW-P and FP extracts of A. conyzoides showed the highest activity against G. duodenalis. Exposure to crude extract induced changes in the flagella and ventral discs of G. duodenalis trophozoites, which play important roles in attachment to the surface of mucosal cells. Our results suggest that the tested extracts warrant further research in terms of their efficacy and safety as giardiasis treatment.

The aim of this study was to evaluate the inflammation process that resulted from the inoculation of Wistar Rats with Acanthamoeba griffini, a virulent T3 Acanthamoeba genotype that produces keratitis. Haematoxylin and eosin, periodic acid stain, immunohistochemistry and morphometry were used to analyse tissues from rats of an Acanthamoeba keratitis (AK) model. Two weeks after inoculating the rats with A griffini trophozoites, the thickness of the stroma had diminished, followed by an increase in thickness at 4 weeks. At the latter time, an abundance of inflammatory infiltrate cells was observed, some found to express IL-1β, IL-10 and/or caspase 3. Intercellular adhesion molecule-1 was expressed in corneal blood vessels amid the abundant vascularization characteristic of the development of AK. Through an immunohistochemical technique, trophozoites were detected at 2 and 4 weeks post-inoculation. By 8 weeks, there were a low number of trophozoites and cysts and the corneas of infected rats were similar in thickness to those of the controls. Thus, the rats were capable of healing experimental AK in the present rat model. Diverse immunological mechanisms regulated the inflammatory process in acute AK induced by A griffini in a murine model.

We aimed to compare LDH release assay, trypan blue and fluorescent stainings, and non-nutrient Escherichia coli plate assay in determining treatment efficacy of antiamoebic agents against Acanthamoeba castellanii trophozoites/cysts, in vitro. 1BU trophozoites/cysts were challenged with 0.02% polyhexamethylene biguanid (PHMB), 0.1% propamidine isethionate (PD), and 0.0065% miltefosine (MF). Efficacies of the drugs were determined by LDH release and trypan blue assays, by Hoechst 33343, calcein-AM, and ethidium homodimer-1 fluorescent dyes, and by a non-nutrient agar E. coli plate assay. All three antiamoebic agents induced a significant LDH release from trophozoites, compared to controls (p < 0.0001). Fluorescent-dye staining in untreated 1BU trophozoites/cysts was negligible, but using antiamoebic agents, there was 59.3%-100% trypan blue, 100% Hoechst 33342, 0%-75.3% calcein-AM, and 100% ethidium homodimer-1 positivity. On E. coli plates, in controls and MF-treated 1BU trophozoites/cysts, new trophozoites appeared within 24 h, encystment occurred after 5 weeks. In PHMB- and PD-treated 1BU throphozoites/cysts, irregularly shaped, smaller trophozoites appeared after 72 h, which failed to form new cysts within 5 weeks. None of the enzymatic- and dye-based viability assays tested here generated survival rates for trophozoites/cysts that were comparable with those yielded with the non-nutrient agar E. coli plate assay, suggesting that the culture-based assay is the best method to study the treatment efficacy of drugs against Acanthamoeba.

Knowledge of the metabolic profile and exchange processes in the protozoan parasite Giardia lamblia is of importance for a better understanding of the biochemical processes and for the development of drugs to control diseases caused by G. lamblia. In the current paper, 1H High Resolution Magic Angle Spinning (HR-MAS) NMR spectroscopy was directly applied to G. lamblia trophozoite suspensions to analyze the detectable small metabolites with a minimum of intervention. Thirty-one components were identified with main contributions from amino acids such as alanine and ornithine. The reproducibility, variability, and stability of the metabolites were investigated. Citrulline was found to be formed as an intermediate and citrulline levels depended on the stage of cell growth. Glucose-1-phosphate was found to be formed in relatively high amounts after cell harvesting if enzymes were not inactivated. In addition, the metabolic footprint of Giardia trophozoites, i.e. changes in the culture medium induced by G. lamblia, was investigated by liquid state NMR spectroscopy of culture media before and after inoculation. A quantitative comparison of the NMR spectra revealed component changes in the culture media during growth. The results suggested that not glucose but rather arginine serves as main energy supply. Biochemical functions of intracellular components and their metabolic exchange with the culture medium are discussed. The results provide an important basis for the design of HR-MAS NMR based metabolomic studies of G. lamblia in particular and any protozoan parasite samples in general.

Granulomatous amoebic encephalitis (GAE) is a chronic, difficult to resolve infection caused by amphizoic amoebae of the genus Acanthamoeba, which in most cases occurs in immunosuppressed persons or with chronic diseases such as diabetes. In this study, we describe the early events of A. culbertsoni infection of GAE in diabetic mice model. Diabetes was induced in male BALB/c mice, with a dose of streptozotocin (130 mg/kg). Healthy and diabetic mice were inoculated via intranasal with 1 × 106 trophozoites of A. culbertsoni. Then were sacrificed and fixed by perfusion at 24, 48, 72 and 96 h post-inoculation, the brains and nasopharyngeal meatus were processed to immunohistochemical analysis. Invasion of trophozoites in diabetic mice was significantly greater with respect to inoculated healthy mice. Trophozoites and scarce cysts were immunolocalized in respiratory epithelial adjacent bone tissue, olfactory nerve packets, Schwann cells and the epineurium base since early 24 h post-inoculation. After 48 h, trophozoites were observed in the respiratory epithelium, white matter of the brain, subcortical central cortex and nasopharyngeal associated lymphoid tissue (NALT). At 72 h, cysts and trophozoites were immunolocalized in the olfactory bulb with the presence of a low inflammatory infiltrate characterized by polymorphonuclear cells. Scarce amoebae were observed in the granular layer of the cerebellum without evidence of inflammation or tissue damage. No amoebas were observed at 96 h after inoculation, suggesting penetration to other tissues at this time. In line with this, no inflammatory infiltrate was observed in the surrounding tissues where the amoebae were immunolocalized, which could contribute to the rapid spread of infection, particularly in diabetic mice. All data suggest that trophozoites invade the tissues by separating the superficial cells, penetrating between the junctions without causing cytolytic effect in the adjacent cells and subsequently reaching the CNS, importantly, diabetes increases the susceptibility to amoebae infection, which could favor the GAE development.

Plasmodium falciparum parasite life stages respond differently to antimalarial drugs. Sensitive stage-specific molecular assays may help to examine parasite dynamics at microscopically detectable and submicroscopic parasite densities in epidemiological and clinical studies. In this study, we compared the performance of skeleton-binding protein 1 (SBP1), ring-infected erythrocyte surface antigen, Hyp8, ring-exported protein 1 (REX1), and PHISTb mRNA for detecting ring-stage trophozoite-specific transcripts using quantitative reverse transcriptase polymerase chain reaction. Markers were tested on tightly synchronized in vitro parasites and clinical trial samples alongside established markers of parasite density (18S DNA and rRNA) and gametocyte density (Pfs25 mRNA). SBP1 was the most sensitive marker but showed low-level expression in mature gametocytes. Novel markers REX1 and PHISTb showed lower sensitivity but higher specificity for ring-stage trophozoites. Using in vivo clinical trial samples from gametocyte-negative patients, we observed evidence of persisting trophozoite transcripts for at least 14 days postinitiation of treatment. It is currently not clear if these transcripts represent viable parasites that may have implications for clinical treatment outcome or transmission potential.