الفهرس 
-Trichomonas vaginalisOnly 14 pages are availabe for public view
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Abstract
T. vaginalis is a flagellated protozoan parasite that inhabits the human vagina and urethra. It is the most common curable STI, with an estimated incidence of more than 248 million new cases affected annually worldwide.
T. vaginalis cytopathogenicityis a multi- factorial process that can be classified into; contact dependent mechanism presented in the cytoadherence process of the parasite and contact independent mechanisms due to secretion of soluble substances causing the cytotoxic and haemolytic effects.
The nitroimidazoles involve the only class of drugs suitable for treatment of trichomoniasis. It includes metronidazole and tinidazole that are available for trichomoniasis treatment. Resistance of T. vaginalis to metronidazole has been reported to reach up to 9.6% of clinical T. vaginalis isolates. To overcome this resistance, increasing doses of metronidazole were tried. However, this can lead to many adverse effects. So much efforts have been made to find another drug with different mechanism of action to overcome T. vaginalis resistant pathway. In the past years, these efforts were directed to use DNA binder drugs.
Netropsin is a non-covalent DNA minor groove binding ligand with antibiotic and antiviral properties. It exhibits a preference for binding with at least four consecutive AT base pairs sequences in the binding site. It was reported that when netropsin binds to double stranded DNA at specific base sequences, it has the ability to block topoisomerase,
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DNA helicase as well as endonuclease and to interfere with transcription factors binding in these regions.
On cell level, DNA minor groove binders were thought to stop the cell cycle in its G2-M phase. Netropsin causes protracted cell growth consisting of a prolongation of the G1 phase of the cell cycle along with arrest in the G2 compartment causing mitotic arrest.
So, the aim of the present study was to evaluate the effect of a DNA minor groove binder drug ’’Netropsin dihydrochloride’ ’in-vitro
Diagnosis of T. vaginalis infection in referred freshly female-derived biological samples was based on microscopic detection of the organism in wet mounts and/or in in-vitro culture of immediately prepared samples. Out of positive in-vitro cultures, two flourishing isolates from vaginal washouts and a urinary sample were maintained for study and were designated as; the G and U isolates respectively. The study included the metronidazole-resistant (ATCC 50138) strain and was named the R isolate.
The in-vitro growth pattern of T. vaginalis isolates was done using two inoculum size (1x104/ml and 20x104/ml) and followed up by trypan blue exclusion dye test. Concentration 20x104 was chosen for the subculture and for susceptibility tests.
Metronidazole sensitivity assay was done for local isolates, followed by protein profile analysis of isolates using 10% SDS-PAGE.
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Netropsin susceptibility assay was done using different concentrations (3.5-200 μg/ml). Comparative assessment of pathogenic activity before and after co-culture with netropsin was evaluated as regard the hemolytic activity; through monitoring of the interaction with human erythrocytes by inverted microscopy, SEM and measurement of haemoglobin % .
The cytotoxic activity was done through monitoring the interaction of T. vaginalis trophozoites with ’’MDCK’’ cell line by inverted microscopy and measurement of cytotoxicity through MTT cytotoxicity assay.
Both local isolates (G and U isolates) were found to be metronidazole sensitive isolates with lag period of 6-9 hours according to drug concentrations. Both isolates have MIC of 4 μg/ml (0.0237µM/ml = 23.7µM/L) after 48 hours.
SDS-PAGE electrophoresis analysis of the cellular proteins using 20 and 40 μg/lane concentrations was done. The 40μg/lane protein concentration analysis demonstrated a total of 26 protein bands. The bands ranged in molecular weight from 12 to 187 kDa with difference between the isolates, indicating that they were of different phenotypes.
Netropsin effect showed to have a lag period of 9-12 hours according to drug concentration. The MIC was 12.5μg/ml (0.0248 µM/ml = 24.8 µM/L) over 48 hours for G and U isolates and 25μg/ml (0.0496 µM/ml = 49.6 µM/L) over 54 hours for the R isolate. Adding booster doses of R isolates, gave the same results obtained with MIC of R isolate ’’25
μg/ml’’.
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The estimated IC50 for the G and U isolates was 5.21μg/ml (10.34 µM) and 6.4μg/ml (12.7 µM) at 24 hours, respectively. For the metronidazole resistant (R) isolate, the estimated IC50 was 20.43μg/ml (40.58 µM) at 24 hours exposure.
T. vaginalis was found to induce haemolysis with variable degrees, on using different human blood groups, with the higher haemolytic response with blood group O. G isolate exhibited the higher haemolytic activity of 80% haemolysis. U and R isolates exhibited 60% and 40% haemolysis, respectively with blood group O.
Morphological changes of trophozoites were visible upon attachment with RBCs. G isolate exhibited amoeboid transformation, while U isolate showed some abnormal forms with stumps and knobs formation. These changes disappeared upon exposure to netropsin drug (12.5 μg/ml) for 24 hours, indicating that these changes are a part of T. vaginalis virulent mechanisms. Also the appearance of multiple lobose pseudopodia and fine filopodia in all isolates upon attachment with RBCs indicated a role of contact dependent mechanism.
Netropsin-treated for 24 hours and 42 hours showed zero% haemolysis, and no RBCs affection. Trophozoites showed extensive membrane blebbing, intense vacuolation, membrane disruption and pellicular vacuoles as signs of necrosis with affection of the cytoskeletal structures of trophozoites.
There was variability in the effect of different isolates on cell line, the highest cytotoxicity was found in (8:1) trophozoites to
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MDCK cells ratio for all isolates ranging from 66.33% for G isolate, 72.67% for U isolate and 79.47% for R isolate.
The higher the ratio of drug-free trophozoites, the higher the percentage of cell death. It was found that the un-treated trophozoites caused more cytotoxicity than treated trophozoites with netropsin. Also there was a decrease in cytotoxicity value with increasing the duration of inoculation with netropsin. For R isolate, there was non-significant cytotoxicity difference between treated trophozoites with netropsin concentrations 12.5 μg/ml and 25 μg/ml for 24 and 42 hours in all three trophozoites to MDCK cells ratio.
Netropsin was found to have a promising effects on T. vaginalis trophozoites, affecting its replication, virulence and viability, with a different mechanism of action that can overcome the mechanism of T. vaginalis resistance and can substitute metronidazole in patients suffering from allergy to 5-nitroimidazole compounds.