A Study of Synergy of Combination of Eosin B with Chloroquine, Artemisinin, and Sulphadoxine-Pyrimethamine on Plasmodium falciparum In Vitro and Plasmodium berghei In Vivo.

METHODS: Drug assessment was carried out singly or in combination on Plasmodium falciparum in vitro using the candle jar method at three inhibitory concentrations. Percent parasitemia of live cells was obtained by microscopic counting. Peter's suppression test was carried out on mice infected with Plasmodium berghei after 3 administration of the drugs singly and in combination, and parasites were counted by microscopy for 10 days. RESULTS: Synergy was exhibited by isobolograms of eosin B combined with artesunate and sulphadoxine-pyrimethamine with more than 10 fold reduction of all drugs in vitro. A good combination index was obtained with artesunate at 50% inibitory concentration with 3.4 nM eosin B and 1.7 nM artesunate in contrast to 124 nM eosin B and 7.6 nM artesunate singly. In vivo studies also showed a considerable lowering of the effective dose of eosin B 30 mg/kg: artesunate 3 mg/kg with 200 mg/kg eosin B and 60 mg/kg artesunate separately. Sulphadoxine-pyrimethamine seemed to have the greatest synergistic effect with a combination index of 0.007, but this could be due to it consisting of a combination of three drugs. Eosin B's combination index with chloroquine was fair, and in vivo tests too did not show as much competence as the other two drugs. Conclusion and Interpretation. It can be concluded that eosin B can be used in combination with antimalarial drugs with favorable results.

Effect of input voltage frequency on the distribution of electrical stresses on the cell surface based on single-cell dielectrophoresis analysis.

Electroporation is defined as cell membrane permeabilization under the application of electric fields. The mechanism of hydrophilic pore formation is not yet well understood. When cells are exposed to electric fields, electrical stresses act on their surfaces. These electrical stresses play a crucial role in cell membrane structural changes, which lead to cell permeabilization. These electrical stresses depend on the dielectric properties of the cell, buffer solution, and the applied electric field characteristics. In the current study, the effect of electric field frequency on the electrical stresses distribution on the cell surface and cell deformation is numerically and experimentally investigated. As previous studies were mostly focused on the effect of electric fields on a group of cells, the present study focused on the behavior of a single cell exposed to an electric field. To accomplish this, the effect of cells on electrostatic potential distribution and electric field must be considered. To do this, Fast immersed interface method (IIM) was used to discretize the governing quasi-electrostatic equations. Numerical results confirmed the accuracy of fast IIM in satisfying the internal electrical boundary conditions on the cell surface. Finally, experimental results showed the effect of applied electric field on cell deformation at different frequencies.

Expansion of a highly sensitive and specific ELISA test for diagnosis of hydatidosis using recombinant EgB8/2 protein.

OBJECTIVES: Hydatidosis is a zoonotic infection and endemic in Iran. Due to the serological cross-reactivity (of sera) with other parasitic infection, diagnosis of hydatid cyst is considered to be problematic. In this regard, application of recombinant antigens improves serological diagnosis for human hydatidosis. Here, we present an ELISA test based on B8/2 recombinant antigen of Echinococcus granulosus with particular regard to its capability to diagnose human hydatidosis. MATERIALS AND METHODS: The synthesized E. granulosus B8/2 (EgB8/2) gene was sub-cloned into pET28b (+) plasmid. Nde1 and Hind3 restriction enzymes were used to confirm the recombinant plasmid extraction. Cloning was verified by colony PCR, digestion enzymes, and sequence determination methods. To express rtEgB8/2, strains of Escherichia coli BL21 (DE3) pLysS and Rosetta (DE3) were induced with isopropyl ß-D-1-thiogalactopyranoside (IPTG). A Ni-NTA column was used for purification, and the expressed protein was analyzed by SDS-PAGE as well as western blotting. ELISA test was used to identify the antigenicity of produced protein. RESULTS: The presence of EgB8/2 gene fragment in the recombinant plasmid was confirmed. SDS-PAGE showed that the BL21 (DE3) pLysS strain had the highest level of expression and a protein band of 11 kDa was observed in induced bacteria. Western blotting approved the purity of rtEgB8/2 protein, and ELISA test measured sensitivity and specificity as 95% and 97.5%, respectively. CONCLUSION: E. granulosus metacestode contains a high amount of antigen B protein. These results confirm the reproducibility of high-quality rtEgB8/2 recombinant antigen as a reliable candidate in serological test.

Expression and purification of truncated recombinant B8/1 protein of Echinococcus granulosus for diagnosis of hydatid infection in human.

Hydatidosis is one of the most important diseases common between animals and human beings. Caused by Echinococcus granulosus tapeworm, the disease has a global epidemic. The serological diagnostic tests that are now utilized to confirm the imaging approaches have some drawbacks such as low sensitivity and cross-reaction with the serum of the patients infected with other parasites. The application of recombinant and synthetic antigens has proven improvement in the functionality of serological diagnostic tests. The purpose of this study was to demonstrate the expression and purification of truncated recombinant B8/1 (trB8/1) antigen and its application in ELISA for diagnosis of hydatid infection in human. The tEgB8/1 was colonized in the expression vector pET28b (+) and expressed in different strains of E. coli. This protein was purified by Ni2+-NTA chromatography. The antigenicity of the protein was evaluated by Western blotting and ELISA. In the test, 50 positive serum samples from hydatid infected patients, 50 samples from healthy people, and 30 serum samples from patients with other parasitic diseases were used to determine the sensitivity and the specificity of this antigen. The measured sensitivity and specificity of this antigen were identified to be 75.75% and 96.38% respectively. The P value of <0.0001 by using ROC curve, confirmed that this antigen is able to differentiate between healthy and hydatid-infected individuals. Considering the excellent specificity of this antigen and in order to enhance the sensitivity, it is recommended to use a combination of this antigen with other antigens (e.g., EgB8/2-8/5).

Evaluation of a set of refolded recombinant antigens for serodiagnosis of human fascioliasis.

Diagnosis of fascioliasis with high sensitivity and specificity antigens play a vital role in the management of the disease. Majority of commercial serological tests use F. hepatica native antigens and indicate wide diversities in test accuracy. Nowadays, recombinant antigens have been introduced as diagnostic reagents offer better test standardization. A combination of highly pure recombinant antigens associated with correct folding will leads to improve specificity and sensitivity of ELISA for diagnosis of Fascioliasis. In this article, Fasciola hepatica saposin-like protein 2 (SAP-2), ferritin protein (Ftn-1) and leucine aminopeptidase (LAP) recombinant antigens were considered as tools for the detection of F. hepatica immunoglobulin G antibodies in persons with chronic human fasciolasis. The recombinant antigens were obtained as fusion proteins, expressed in Escherichia coli and purified by immobilized metal affinity chromatography (IMAC). The refolding processes of denatured recombinant proteins were performed using dialysis method in the presence of chemical additives, and reduced/oxidized glutathione (in vitro). The immunoreactivity of the recombinant antigens was assessed individually and in a combination compared with excretory/secretory antigen (E/S) in an enzyme-linked immunosorbent assay (ELISA) test. The experiments were optimized using 213 serum samples from humans, including patients with chronic fascioliasis, patients with other parasitic diseases, and healthy subjects. The results indicated 95% sensitivity and 98% specificity for rtFhSAP-2, 96% sensitivity and 91% specificity for E/S, 80% and 83.3% for rtFhFtn-1, 84% and 88% for FhLAP, and also, 96% and 95% for combination of recombinant antigens, respectively. In conclusion, the results of this investigation showed that rtFhSAP-2 with the highest specificity and acceptable sensitivity has a considerable superiority compared to mentioned antigens and even in combination with these antigens in serodiagnosis of human fascioliasis.

Expression, purification and in vitro refolding of the recombinant truncated Saposin-like protein 2 antigen for development of diagnosis of human fascioliasis.

Early diagnosis of fascioliasis is critical in prevention of injury to the liver and bile ducts. Saposin-like protein (FhSAP-2) is probably the most ideal antigen of Fasciola hepatica for development of ELISA kits. SAP-2 has a conserved tertiary structure containing three disulfide bonds and conformational epitopes. Therefore, antigenicity of SAP-2 is greatly depends on disulfide bond formation and proper folding. We produced the recombinant truncated SAP-2 (rtSAP-2) in the SHuffle® T7 and Rosetta strain of Escherichia coli, in soluble and insoluble forms, respectively and purified by immobilized metal affinity chromatography (IMAC). The refolding process of denatured rtSAP-2 was performed using dialysis and dilution methods in the presence of chemical additives, along with reduced/oxidized glutathione (in vitro). Physicochemical studies, including non-reducing gel electrophoresis, Ellman's assay, Western blotting and ELISA showed the most antigenicity and likely correct folding of rtSAP-2, which was obtained by dialysis method. An IgG ELISA test was developed using rtSAP-2 refolded by dialysis and compared with excretory/secretory products of parasite with 52 positive fascioliasis samples, 79 other parasitic samples and 70 negative controls samples. The results exhibited 100% sensitivity and 98% specificity for rtSAP-2, also, 100% and 95.3% for excretory/secretory (E/S) antigen, respectively. In conclusion, it is suggested that rtSAP-2 with the correct folding could be used as a candidate antigen for detection of human fascioliasis.

A Diagnostic and Symptomatological Study on Trichomoniasis in Symptomatic Pregnant Women in Rafsanjan, South Central Iran in 2012-13.

BACKGROUND: Trichomonas vaginalis, the causative agent of trichomoniasis, is responsible for more than half of all sexually transmitted infections (STIs). The present study aimed to determine the frequency of T. vaginalis infection and its clinical manifestations in symptomatic pregnant women in the area based on four different diagnostic methods. METHODS: A total of 162 pregnant women with at least one sign or symptom of vaginosis, referred to two gynecologic and obstetrics clinics in Rafsanjan City, south central Iran, were randomly selected in 2012-13. Through speculum examination of patients by gynecologists, clinical diagnosis determined, vaginal discharge were collected by using two sterile cotton swabs from the posterior fornix and vagina pH was measured. Samples were examined by three diagnostic methods including wet mount, culture in TYI-S-33 medium and polymerase chain reaction (PCR). RESULTS: T. vaginalis was detected in 19.5%, 27.2%, 56.2% and 51.6% of subjects according to diagnostic methods of clinical diagnosis, wet mount, culture and PCR, respectively. There was statistically significant relationship between T. vaginalis infection and patients' age, gestational age, marriage age, residence, educational level, parity. The symptomatological pattern in the 91 women infected with T. vaginalis was as follows: leukorrhea, 96.7%; urine frequency, 65.9%; odorous secretion, 63.3%; urogenital itching and irritation, 53.8%; vaginal inflammation, 47.3%; dyspareunia, 39.6%; and dysuria, 16.5%. CONCLUSION: Our results indicated a high prevalence of T. vaginalis in symptomatic pregnant women, very low sensitivity and relative high specificity of clinical diagnosis and wet mount technique compared to culture and PCR, as well as thatpregnancy increases the susceptibility to the infection in a gestational age-dependent manner.

Reactive nitrogen intermediates in experimental trichomoniasis induced with isolates from symptomatic and asymptomatic women.

Trichomoniasis is the interplay between the infecting parasite, Trichomonas vaginalis, and the host, on which the clinical presentation of the disease depends. Although the clinical spectrum varies from an asymptomatic state to mild, moderate or severe symptoms, the exact virulence markers of T. vaginalis have not been well elucidated. Free radical generation during the disease process and its role in pathogenesis has been reported in various microbial diseases. In the present study, an attempt has been made to study reactive nitrogen intermediate (RNI) concentrations in experimental animals infected with T. vaginalis isolates from symptomatic and asymptomatic women. A significant increase in polymorphs, vaginal epithelial cells and RNI levels was observed in mice infected with isolates from symptomatic and asymptomatic subjects as compared to uninfected controls. The mean concentration of RNI in the vaginal tissue of mice infected with isolates from symptomatic women (75.5+/-7.7) was significantly higher than that of the vaginal tissue of mice infected with isolates from asymptomatic women (47.9+/-7.8), while it was less in the vaginal washes and plasma of mice infected with isolates from symptomatic women (18.7+/-3.6 and 17.1+/-3.3, respectively) compared to those infected with isolates from asymptomatic women (28.9+/-7.3 and 26.7+/-4.4, respectively), which may be due to different macrophage populations with different functional capabilities. Our study indicates that RNI production may play a role in establishing the infection.