The impact of chlorine, ultraviolet-C, and microwave treatment on the survivability of Blastocystis sp. cysts.

Blastocystis sp. is a common widely distributed gut protozoan, with water transmission identified as one of its transmission routes. This study aimed to investigate the effect of chlorine, ultraviolet (UV)-C, and microwave (MW) treatments on the in vitro viability of cysts of Blastocystis sp. Purified Blastocystis sp. cysts were molecularly subtyped. Viable cysts were subjected to different free chlorine concentrations (1, 2, and 4 ppm), different doses of UV-C (5.13, 10.26, 20.52, and 40.47 mJ/cm2), and MW irradiation times (10, 15, 30, and 45 s). Viability reduction percentage, log10 inactivation, and micrometre-based optical microscopy examined cyst number and appearance after each disinfection trial. The three disinfectants' efficacy and application conditions were assessed. The analysed isolates of Blastocystis cysts were subtype 3, possessed varying sizes and shapes, but two identical genomes. The cysts of Blastocystis sp. were resistant to chlorine at all doses and exposure durations tested. UV-C at a dose of 40 mJ/cm2 and MW treatment for 15 s were able to completely disinfect the cysts. The MW was the most effective disinfectant against Blastocystis cysts based on all evaluated factors. MW irradiation is the most efficient water treatment method for eradicating Blastocystis cysts in an easy and safe manner.

Anti-Cryptosporidium parvum activity of Artemisia judaica L. and its fractions: in vitro and in vivo assays.

Background: This study investigates the toxic activity of Artemisia judaica ethanolic extract (ArEx) as well as its phenolic fraction (ArPh), and terpenoid fraction (ArT) against Cryptosporidium parvum (C. parvum) oocysts. Methods: Over a 4 months period, estimation of the total phenolic (TPC), total flavonoids (TFC), and total terpenoids contents (TTC) in ArEx; investigation of the in vitro antioxidant activity of ArEx, ArPh, and ArT; evaluation of ArEx, ArPh, and ArT toxic activity against C. parvum oocysts using MTT assay; parasitological analysis on ArPh-treated C. parvum oocysts and comet assay were performed both in vitro and in vivo (infectivity). Results: The ArEx TPC, TFC, and TTC was 52.6 ± 3.1 mgGAE/g, 64.5 ± 3.1 mg QE/g, and 9.5 ± 1.1 mg Linol/g, respectively. Regarding the phytochemical in vitro antioxidant activity, the ArPh exhibited the highest antioxidant activity compared to the ArEx and ArT. The ArPh showed promising free radical scavenging activity of DPPH and ABTS•+ with IC50 values of 47.27 ± 1.86 µg/mL and 66.89 ± 1.94 µg/mL, respectively. Moreover, the FRAP of ArPh was 2.97 ± 0.65 mMol Fe+2/g while its TAC was 46.23 ± 3.15 mg GAE/g. The ArPh demonstrated toxic activity against C. parvum oocysts with a potent IC50 value of 31.6 µg/mL compared to ArT (promising) and ArEx (non-effective). ArPh parasitological analysis demonstrated MIC90 at 1000 µg/ml and effective oocysts destruction on count and morphology. ArPh fragmented oocysts nuclear DNA in comet assay. Beginning at 200 µg/mL, ArPh-treated oocysts did not infect mice. Conclusion: To combat C. parvum infection, the phenolic fraction of A. judaica L. shows promise as an adjuvant therapy or as a source of potentially useful lead structures for drug discovery.

Identification of a Cyclospora cayetanensis Oocyst Antigens and Their Validity in the Detection of Immunogenic Patterns of Cyclosporiasis Patients.

INTRODUCTION: The diagnosis of cyclosporiasis is currently based on the microscopic detection of oocysts, which may provide invalid results. The availability of simple, objective immunological screening tests would facilitate epidemiological studies of cyclosporiasis. Therefore, the present study aimed to identify the antigens of Cyclospora cayetanensis oocysts and their validity in serodiagnosis. METHODS: According to parasitological and molecular diagnoses, three study groups were specified. Group (G) I included 30 patients with cyclosporiasis, GII included 12 patients with other parasitic infections, and GIII included 16 healthy subjects. SDS-PAGE was used to analyse C. cayetanensis antigens, and the validity of western blotting and enzyme-linked immunosorbent assays (ELISAs) was then assessed amongst the sera of all study groups. RESULTS: The C. cayetanensis antigenic profile showed eight characteristic bands with molecular weights ranging from 14 to 175 kDa. Western blot analysis of sera revealed 93.3% (28/30 of GI) and 92.8% (26/28 of GII and III) sensitivity and specificity, respectively, dividing the patients in GI into four subgroups. The most frequent diagnostic bands (71.4% of GI sera) showed weights of 26-28 kDa, followed by 71 kDa (53.6%). ELISA sensitivity was 90% (27/30), and specificity was 78.6%. Validation showed perfect agreement between the PCR and western blot results, and ELISA presented substantial agreement with both the PCR and western blot results. CONCLUSIONS: Our findings suggest the existence of high immunogenic diversity in C. cayetanensis and indicate that the 26-28 kDa immunogenic groups may potentially be used as a diagnostic marker of cyclosporiasis. Due to the high validity of ELISA, it might be the test of choice for the routine serodiagnosis of cyclosporiasis.

Anti-Blastocystis Activity In Vitro of Egyptian Herbal Extracts (Family: Asteraceae) with Emphasis on Artemisia judaica.

Achillea fragrantissima (Forssk.) Sch. Bip. (known as Qaysoom), Echinops spinosus L. (known as Shoak Elgamal) and Artemisia judaica L.(known Shih Baladi) are members of the Asteraceae family known for their traditional medical use in Egypt. The ethanol extracts of these plants were evaluated for their efficacy against a protozoan parasite (Blastocystis). Two different molecular subtypes of Blastocystis were used (ST1 and ST3). Significant growth inhibition of Blastocystis was observed when exposed to both A. judaica (99.3%) and A. fragrantissima (95.6%) with minimal inhibitory concentration (MIC90) at 2000 µg/mL. Under the effect of the extracts, changes in Blastocystis morphology were noted, with the complete destruction of Blastocystis forms after 72 h with the dose of 4000 µg/mL. Different subtypes displayed different responses to the herbal extracts tested. ST1 exhibited significantly different responses to the herbal extracts compared to ST3. A. judaica was selected as the herb of choice considering all of its variables and because of its effective action against Blastocystis. It was then exposed to further fractionation and observation of its effect on ST1 and ST3. Solvent portioned fractions (dichloromethane (DCM), ethyl acetate (EtOAc) and n-hexane) in A. judaica were found to be the potent active fractions against both of the Blastocystis subtypes used.

Antiprotozoal activity of magnesium oxide (MgO) nanoparticles against Cyclospora cayetanensis oocysts.

Outbreaks of Cyclospora cayetanensis infection have been linked to consumption of food and water contaminated by oocysts that can survive both physical and chemical disinfectants. Magnesium oxide (MgO) nanoparticles (NPs) can be potentially used in food as bactericides. In this study, C. cayetanensis pre- and post-sporulated oocysts were exposed to MgO NPs with different doses ranging from 1.25-25 mg/ml. With comparison to control, the antiprotozoal activity of MgO NPs was evaluated by identifying the median effective concentration dose (EC50), lethal concentration dose (LC90), microscopically changes on treated oocysts and rates of sporulation. Among pre- and post-sporulated oocysts, MgO NPs ≥ EC50 was observed after 24 h at concentrations 10 and 12.5 mg/ml, respectively, while ≥ LC90 was observed after 24 h, 48 h and 72 h at concentrations 15, 12.5 and 10 mg/ml, respectively. MgO NPs treated oocysts showed abnormal morphological changes such as an increase in size, wall injury, deposition of vacuolated homogenous particles in the cytoplasm, evacuation of oocyst's contents, and collapse. Sporocysts of treated oocysts were noticed to be peripherally shifted. Sporulation failure of treated oocysts achieved ≥90% after 24 h and 72 h of incubation with 15 and 12.5 mg/ml, respectively, while it was 10.1% among non-treated. All the differences were statistically significant. Our results demonstrated that MgO NPs has a significant anti-Cyclospora effect on both unsporulated and sporulated oocysts, especially considering that it could be biologically synthesized, that way it can be used safely as a preventive agent in food and water disinfectant treatment.

Nested PCR targeting intergenic spacer (IGS) in genotyping of Giardia duodenalis isolated from symptomatic and asymptomatic infected Egyptian school children.

Distinct sequences of Giardia duodenalis assemblages raised the hypothesis that certain assemblages may contribute to its clinical outcome. However, sequences analysis is time consuming, expensive, and needs many manual operations. Nested PCR targeting intergenic spacer (IGS) region was applied successfully to genotype G. duodenalis. This study aimed to identify the prevalence of G. duodenalis assemblages among giardiasis school children and its relation to the presence of symptoms using nested IGS/PCR. Of 65 microscopically confirmed Giardia-positive samples, 65 samples were genotyped proving high sensitivity (92.3%) of IGS/PCR. Negative IGS/PCR samples were also negative for ß-giardin gene. Subassemblage AI was the commonest with 66.6% (20/30) among asymptomatic children compared to 53.3% (16/30) of symptomatic, while assemblage B was found in 40% (12/30) of symptomatic compared to 20% (6/30) of asymptomatic. The difference was significant. AII was only found in asymptomatic with 13.4% (4/30), while mixed infections (AI&B) were recorded only in 6.6% (2/30) of symptomatic group. A significant relation was found between younger children susceptibility for AI and B infections as presented in 77.7 (12/16) and 83.3% (10/12) of symptomatic, respectively, and 80 (16/80) and 33.4% (2/4) of asymptomatic, respectively. Significant relations were found between AI with intermittent diarrhea and B with chronic. A significant relation was found between assemblage distributions and heavy infection intensity. In conclusion, higher incidence of assemblage B among symptomatic children compared to asymptomatic could denote its possible pathogenic potential.

Molecular characterization of double-stranded RNA virus in Trichomonas vaginalis Egyptian isolates and its association with pathogenicity.

Trichomoniasis is a common human sexually transmitted infection caused by Trichomonas vaginalis. The parasite can be infected with double-stranded RNA viruses (TVV). This viral infection may have important implications on trichomonal virulence and disease pathogenesis. This study aimed to determine the prevalence of T. vaginalis virus among isolates obtained from infected (symptomatic and asymptomatic) women in Ismailia City, Egypt, and to correlate the virus-infected isolates with the clinical manifestations of patients. In addition, the pathogenicity of TVV infected isolates on mice was also evaluated. T. vaginalis isolates were obtained from symptomatic and asymptomatic female patients followed by axenic cultivation in Diamond's TYM medium. The presence of T. vaginalis virus was determined from total extraction of nucleic acids (DNA-RNA) followed by reverse transcriptase-PCR. Representative samples were inoculated intraperitoneally in female albino/BALB mice to assess the pathogenicity of different isolates. A total of 110 women were examined; 40 (36.3 %) samples were positive for T. vaginalis infection. Of these 40 isolates, 8 (20 %) were infected by TVV. Five isolates contained TVV-2 virus species, and the remaining three isolates were infected withTVV-4 variant. A significant association was found between the presence of TVV and particular clinical manifestations of trichomoniasis. Experimental mice infection showed varying degrees of pathogenicity. This is the first report on T. vaginalis infection by TVV in Egypt. The strong association detected between TVV and particular clinical features of trichomoniasis and also the degree of pathogenicity in experimentally infected mice may indicate a possible clinical significance of TVV infection of T. vaginalis isolates.

Study of the pathogenic potential of Dientamoeba fragilis in experimentally infected mice.

Dientamoebafragilis (D. fragilis) is a protozoan parasite whose pathogenic potential is still disputable. The aim of this study was to illustrate the pathogenicity of D. fragilis infection and to determine the infective dose for experimental mice infection. Three groups of mice (8/each) were orally inoculated with in vitro cultured D. fragilis. The infected groups (G1- G3) received 103, 105 and 4 × 106D. fragilis/0.5 ml culture, respectively. A control group (G4) only received parasite-free culture. Two weeks post-inoculation all mice were euthanized for histopathological examination. All mice of G3 (100%) and three mice of G2 (37.5%) were infected, and the results were confirmed by PCR and different staining methods. On the other hand, all mice from group G1 showed a completely negative result. Histopathological examination of the colon and caecum of the highly infected group G3 showed active colitis, with infiltration of mixed inflammatory cells such as eosinophils, neutrophils and lymphocytes within the lamina propria of the intestinal wall. The parasite was not invading the colonic mucosa. This study revealed that infection with D. fragilis is dose-dependent. Moreover, a dose of 105D. fragilis/mouse or higher is necessary to infect mice through the oral route. In addition, this route of infection, although non-invasive, can induce severe inflammatory changes to the colonic and caecal mucosa in experimentally infected mice.

IN VITRO ANTI-PROTOZOAL ACTIVITY OF PROPOLIS EXTRACT AND CYSTEINE PROTEASES INHIBITOR (PHENYL VINYL SULFONE) ON BLASTOCYSTIS SPECIES.

Blastocystis is one of the commonest enteric protozoan parasites worldwide. Despite its controversial clinical significance, frequent association with symptoms has necessitated treatment of cases with persistent symptoms. For long time, metronidzole (MTZ) was considered as a basic drug for blastocystosis treatment, however reports of treatment failure as well as its well-known side effects has promoted the search for more safe and effective alternatives. In vitro antiprotozoal activity of ethanolic extract of Egyptian propolis and a cysteine protease inhibitor, phenyl vinyl sulfone (PVS) on Blastocystis spp. was assessed through challenging with graded concentrations of propolis extract (125, 250, 500 & 1000pg/ml) and PVS (100, 200 and 300 ptg/ml) compared to MTZ (10, 50 and 100 pg/ml) and viable parasites were counted after 24, 48 and 72 hr. of incubation. Molecular subtyling of Blastocystis spp. was done using subtype specific sequence-tagged site (STS) primers. Propolis extract inhibited the growth of Blastocystis spp. in both of the detected subtypes (STI and ST3), which was especially observed in cultures exposed to 500 & 1000 µg/ml through all incuba- tion periods with the later concentration producing comparable results to MTZ. While PVS showed significant parasite count reduction on ST3 isolates, especially with the highest concentration, however the effect on STl isolate was nonsignificant. These findings highlight the potential antiprotozoal activity of propolis extract as a potent natural alternative for MTZ in treatment of blastocystosis.