Vol 7 No 3 (2012)
Production and Evaluation of Toxoplasma gondii Recombinant Surface Antigen 1 (SAG1) for Serodiagnosis of Acute and ChronicToxoplasma Infection in Human Sera
Background: The assays currently available for the detection of specific anti-Toxoplasma antibodies may vary in their abilities to detect serum immunoglobulins, due to the Lack of a purified standardized antigen. The aim of this study was evaluation the recombinant Toxoplasma gondii SAG1 antigen for the serodiagnosis of acute and chronic toxoplasmosis.
Methods: This study describes an ELISA using recombinant SAG1 for detection of IgM and IgG antibodies against Toxoplasma gondii in human sera. Genomic DNA of T. gondii (RH Strain) was isolated and PCR reaction was performed. Recovered DNA was cloned into PTZ57R cloning vector. The recombinant plasmid was detected by restriction analysis. The SAG1 gene was subcloned in the pET- 28a expression vector. Protein production was then induced with 1 mM isopropyl-D - thiogalactopyranoside (IPTG). A total of 204 sera were tested using a commercial IgG and IgM ELISA kit (Trinity, USA) as gold standard prior to testing them with the recombinant antigen.
Results: Tested sera were divided into the following groups:(a) The 74 T. gondii IgG positive (b) 70 T.gondii IgM positive (c) 60 sera who had no serological evidence of toxoplasmosis as negative sera.To determine the specificity of the test, we used other parasitic diseases including echinococusis (N=5), malaria (N=14), leishmaniasis (N=7),fasciolasis (N=4 ), sterengyloidiasis (N=1 ). Sensitivity and specificity of the generated recombinant IgG ELISA in comparison with commercial ELISA (Com ELISA) were 93% and 95%, and the sensitivity and specificity of the generated recombinant IgM ELISA were 87% and 95% respectively.
Conclusion: The results acquired here show that this antigen is useful for diagnostic purposes and could be replaced by lysed, whole cell antigens for diagnosis of chronic toxoplasmosis.
Background: The objective of this study was to determine the prevalence of cystic echinococcosis (CE) in Qom Province, central Iran using ELISA test.
Methods: Overall, 1564 serum samples (800 males and 764 females) were collected from selected subjects by randomized cluster sampling in 2011-2012. Sera were analyzed by ELISA test using AgB. Before sampling, a questionnaire was filled out for each case. Data were analyzed using Chi-square test and multivariate logistic regression for risk factors analysis.
Results: Seropositivity was 1.6% (25 cases). Males (2.2%) showed significantly more positivity than females (0.9%) (P= 0.03). There was no significant association between CE seropositivity and age group, occupation, and region. Age group of 30-60 years encompassed the highest rate of positivity. The seropositivity of CE was 2.1% and 1.2% for urban and rural cases respectively. Binary logistic regression showed that males were 2.5 times at higher risk for infection than females.
Conclusion: Although seroprevalence of CE is relatively low in Qom Province, yet due to the importance of the disease, all preventive measures should be taken into consideration.
Interleukin-10 and Transforming Growth Factor-β in Early and Late Lesions of Patients with Leishmania major Induced Cutaneous Leishmaniasis
Background: Cutaneous leishmaniasis is a neglected parasitic disease, which imposes massive human distress and financial costs to the endemic countries. Better understanding of host immune response to the parasite leads to helpful strategies for disease control. Interleukin (IL)-10 and transforming growth factor (TGF)-β are important immune regulatory cytokines, which appear to develop non-healing forms of leishmaniasis. However, there is little information about the function of IL-10 and TGF-β in old world cutaneous leismaniasis. The aim of this study was to analyze the role of IL-10 and TGF-β in human cutaneous leishmaniasis due to Leishmania major infection.
Methods: Biopsies were obtained from lesions of twenty proven cases of L. major induced cutaneous leishmaniasis. IL-10 and TGF-β positive cells were detected by immunofluorescence staining of frozen sections and compared between two groups of patients with early and late lesions.
Results: The mean percentage of IL-10 positive cells were significantly (P= 0.035) higher in late lesions (0.51±0.24) than early ones (0.15±0.07). Similar results were obtained for TGF-β with mean percentages of 0.16±0.05 and 0.53±0.28 in early and late lesions respectively (P= 0.008).
Conclusion: IL-10 and TGF-β are present in lesions of L. major induced cutaneous leishmaniasis and contribute to the pathogenesis of long lasting disease forms.
Comparison of Resistant and Susceptible Strains of Trichomons vaginalis to Metronidazole Using PCR Method
Background: Metronidazole is drug of choice recommended by WHO for treatment of trichomoniasis, however, some reports claims drug resistance in Trichomonas vaginalis isolates recently. The objective of this study was to determine the minimum lethal concentration (MLC) of metronidazole in resistant and sensitive strains, as well as genetic patterns of these stains by PCR method.
Methods: From February 2006 to March 2007, in a cross sectional study, clinical and wet mount examination of vaginal smear along with culture were performed on 683 women attending to public and private outpatient clinics in Hamadan. Trichomoniasis marked based on major clinical symptoms. Diagnosis confirmed using wet mount microscopically and culture in Diamond medium. A serial concentration of metronidazole was provided and all isolated Trichomonas strains (resistant and sensitive) tested by standard method. Finally, all sensitive and resistant strains examined by PCR technique.
Results: Only 15/683, (2.2%) of patients clinically diagnosed trichomonal vaginitis were positive for T. vaginalis by wet smear and culture. The minimum lethal concentration (MLC) for clinically sensitive isolates was 25 µg/ml; however, this concentration for resistant isolates was 200 µg/ml after 24 h and 100 µg/ml after 50 h. The results of PCR examination of DNA from sensitive and resistant isolates had same pattern. The lanes appeared by two primers were 98 bp and 261 bp for both clinically sensitive and resistant strains.
Conclusion: Resistance to metronidazole in T. vaginalis has not relation to genetic variations and might be related to some physiologic pathways of organism.
Background: This study aimed at determining the prevalence of malaria and anemia among children in rural community of Okada, Edo State Nigeria, as well as to assess the level of use of Insecticide treated bed nets and its impact on prevalence of malaria and anemia among study population.
Methods: Thick blood films from 226 children with signs and symptoms of malaria in Okada community were stained and examined for presence of malaria parasites. Hemoglobin concentration of all children was also determined using standard method.
Result: A total of 185 (81.9%) children were infected with malaria parasite. Malaria parasitaemia was significantly affected by age (P =0.003). A significantly higher number of positive cases of malaria and anemia was observed in rainy season as compared to dry season (P<0.05). The prevalence of anemia in children was 47.3%. Malaria was a risk factor for development of anemia in children (OR=2.551; 95% CI=1.227, 5.305; P=0.015). Use of insecticide treated bed nets was recorded in 11(4.9%) of children studied, and did not significantly reduce the prevalence of malaria and anemia. However among malaria parasite infected children, its use significantly reduced the prevalence of anemia (OR=0.126; 95%CI = 0.015, 1.047; P= 0.031).
Conclusion: Malaria and anemia among children was high malaria intervention progammes by relevant agencies is strongly advocated.
Background: The objective of the present research was to determine the frequency of Toxocara spp. eggs in soil samples of public parks, in the city of Tehran, Iran.
Methods: A total of 600 soil samples were taken from 120 parks between Aprils to November, 2008. Soil samples were collected from 5 distinct sites in the parks. The samples were washed with saline solution and the collected sediment from each park were equally divided and examined by floatation and Petri dish methods for Toxocara eggs.
Result: Ten percent were contaminated with Toxocara spp. eggs. The number of observed Toxocara eggs in each microscopic field was varied from 1-3. No significant differences were observed between floatation and Petri dish methods.
Conclusion: Our public parks showed a high risk of toxocariasis and the need for preventive studies.
Comparison of Agar Dilution, Broth Dilution, Cylinder Plate and Disk Diffusion Methods for Evaluation of Anti-leishmanial Drugs on Leishmania promastigotes
Background: Leishmaniases are a group of diseases caused by Leishmania parasites. Growing of drug unresponsiveness in leishmaniasis patients necessitates the development of new drugs and accordingly a suitable assay is needed for evaluation of any modalities. The aim of this study was to compare four drug assays methods, agar dilution, broth dilution, cylinder plate and disk diffusion, for evaluation of anti-leishmanial drugs on Leishmania promastigotes, using glucantime as a currently available drug for treatment of leishmaniasis.
Methods: For broth dilution method, different concentration of glucantime was added to the parasite culture (promastigotes of Leishmania), while in cylinder plate method wells were punched in agar gel and filled with different concentration of drug and zone of inhibition was measured in each well. In disk diffusion method, the parasites were cultivated on the surface of agar; filter paper disks were enriched with various concentration of glucantime and were placed on the surface of agar. In agar dilution method, various concentrations of drug were incorporated onto blood agar and the parasites were cultivated on the surface of the agar.
Results: A direct correlation was found between the drug concentration and size of inhibitory zones in cylinder plate and disk diffusion methods. These two drug assays methods provided much better performance in comparison with broth and agar dilution methods.
Conclusion: Cylinder plate and disk diffusion methods seem to be acceptable methods for susceptibility testing of anti-leishmanial compounds on Leishmania promastigotes.
Background: Toxoplasma gondii is an obligate intracellular protozoan parasite, capable of infecting all species of mammals including man. Congenital toxoplasmosis is more important during pregnancy for the first time. In this study we expressed and purified P43 Toxoplasma gondii tachyzoite and bradyzoite specific surface antigen.
Methods: The recombinant pGEMEX-1 contained Toxoplasma P43 coding sequence was transformed into E. coli and mass cultured in LB medium contained 100 μg/ml ampicillin at 37°C over night .The T7 promoter was induced by 1mM isopropyl-1-thio-ß-D-galactopyranoside (IPTG. Recombinant protein was purified by affinity chromatography and confirmed by gel diffusion dot blot and western blot,-using specific anti Toxoplasma antibodies.
Results: Recombinant plasmid was induced by IPTG and analyzed by SDS-PAGE. Recombinant protein was confirmed by Western-blot and dot blot using anti human Toxoplasma antibody.
Conclusion: Recombinant Toxoplasma P43 was produced successfully.
Background: There are only four drugs for treating African trypanosomiasis, a devastating disease in sub-Saharan Africa. With slow discovery of better drugs, vaccination is viewed as the best method of control. We previously showed that antibodies to native Trypanosoma brucei brucei tubulin inhibit the growth of trypanosomes in culture. Here, we aimed to determine the effect of antibodies to bacterially expressed trypanosome tubulin on T. brucei brucei growth.
Methods: T. brucei brucei alpha and beta tubulin genes were individually expressed in Escherichia coli under the tryptophan promoter. Monoclonal tubulin antibodies reacted specifically with the expressed tubulins with no cross-reaction with the opposite tubulin. Rabbits were immunized with 450μg each of the concentrated recombinant tubulin, and production of antibodies assessed by ELISA and Western blotting. The effect of polyclonal antibodies on trypanosome growth was determined by culturing bloodstream T. brucei brucei in up to 25% of antisera.
Results: Low antisera dilutions (25%) from the immunized rabbits inhibited trypanosome growth. The most cytotoxic antisera were from one rabbit immunized with a mixture of both alpha and beta tubulins. However, the result was not reproduced in other rabbits and there was no apparent effect on growth at higher antisera dilutions.
Conclusion: Antibodies to bacterially expressed trypanosome tubulin are not effective at killing cultured bloodstream trypanosomes.
Detection and Identification of Toxoplasma gondii Type One Infection in Sheep Aborted Fetuses in Qazvin Province of Iran
Background: The aim of this study was to apply the nested-PCR and bioassay methods in detection and genotyping of Toxoplasma gondii infection in provided sheep aborted fetus samples from Qazvin Province of Iran.
Methods: Eighteen sheep aborted fetal samples were studied by nested-PCR-RFLP, histopathological observation and microbiological assay. Bioassay in mice was carried out by inoculating the brain samples intraperitoneally.
Results: The results demonstrated the frequency of 66% infected sheep aborted fetal samples with T. gondii type one. Although we could not isolate any parasite from inoculated mice even after three passages, but it was confirmed histopathologically formation of cyst like bodies in prepared mice brain sections.
Conclusion: The results of the performed nested-PCR and formation of brain cyst in inoculated mice exhibited that T. gondii type one infection might be considered as one of the major causative agents for abortion in ewes.
Background: Gnathostoma spinigerum causes larva migran in human which is endemic in Southeast Asia. Information regarding larva migration is limited. In this study, we investigated the parasite migration by recovery of worms from the whole body of mouse after oral infection with advanced third stage larvae (AL3). The percentage of blood eosinophils was examined in parallel.
Methods: Mice were orally infected with AL3 and histological study of organs was investigated in order to study the migration of AL3, along with blood eosinophilia.
Results: At 1 hr post infection (PI), the larvae remained in the stomach, thereafter at 3, 5, 7, 10 and 24 hr PI; they were recovered from various organs including liver, mesentery, esophagus, diaphragm, lung, heart and dorsal fat. At day 15 PI, they were mostly found in muscles (76.47%). The average worm recovery (5 months) was 78.03%. The worms were found in the liver at every time point. Larva encystment was detected. There was a significant difference in blood eosinophils between the 8 larvae- (average 9.33% + 6.25%) and the 15 larvae-infected groups (average 22.66% + 11.03%). Surprisingly, the blood eosinophils (average 19.00% + 2.92%) were not higher in the higher infective dose- group (25 larvae).
Conclusion: Liver was involved by G. spinigerum throughout the study. We detected larva encystment which had never been reported in human gnathostomiasis. The highest percentage of eosinophil occurred during the invasive stage.
Background: Malaria is one of the most important parasitic diseases in tropical and temperate regions. The aim of this study was to determine the trend of malaria in Mazandaran Province, northern Iran during 1997-2012.
Methods: This retrospective study was conducted from 1997 to 2012. The population's study was individuals who registered at health centers of Mazandaran Province. Peripheral blood smear were prepared for each case, stained with Giemsa and examined by light microscope. In addition to demographic data, other parameters including Slide Positive Rate (SPR), Annual Parasite Incidence (API) and Annual Blood Examination Rate (ABER) were analyzed.
Results: In total, 844 cases of malaria were reported. Plasmodium vivax was predominant species with 821 cases (97.4%). The number of malaria cases increased from 1997 to 2005 and then decreased to 3 cases in 2011. Some cities had not reported any cases during last three years. The highest infection rate, 163(20.07%), was seen in 2001-02. The SPR had the highest value (0.54%) in 2004-05. The maximum API and ABER were observed in 2001-02 and 1997-98. 641(75.9%) of cases were imported from hyperendemic areas such as Afghanistan and South-eastern Iran and 94 (11.1%) malaria patients were recorded as introduced cases. The highest infection rate of malaria (21.3%) was seen in Babolsar.
Conclusion: Extensive malaria control should be continued to Mazandaran to become malaria-free region and in prevention of re-introduction stage.
Comparison of a Commercial ELISA with the Modified Agglutination Test for Detection of Toxoplasma gondii Antibodies in Sera of Naturally Infected Dogs and Cats
Background: Toxoplasma gondii can infect all warm-blooded animals. Modified agglutination test (MAT) and ELISA are widely used for the detection of T. gondii antibodies. However, there is little information on their acceptability for detecting antibodies in companion animals.
Methods: This study compared ELISA and MAT for their ability to detect T. gondii infection in naturally infected dogs and cats. Blood samples were collected from dogs and cats in different areas of Beijing, China and analyzed by ELISA and MAT. The χ2 test and k analysis were used to evaluate their efficiency and agreement.
Results: For dogs, the seroprevalence of T. gondii antibodies detected by ELISA was 34.7%, which was significantly higher than that detected by MAT (P<0.05). There was no significant difference between ELISA and MAT for detecting T. gondii antibodies in cats. Good agreements between MAT and ELISA were seen in both dogs and cats; however, inconsistent results were demonstrated by k analysis and in MAT titer assay.
Conclusion: Serum-based ELISA may be more satisfactory for screening test of T. gondii infection in dogs, whereas both methods could be acceptable in cats.
Urinary Schistosomiasis with Simultaneous Bladder Squamous Cell Carcinoma and Transitional Cell Carcinoma
This is a case report concerning a 60 years old man who lived for a short period in an endemic area of Khuzestan Province (neighboring province of Persian Gulf in Iran) for approximately 20 years ago. Recently he referred to the Urology Department of Kerman University of Medical Sciences with hematuria and dysuria. In the sonography a polypoid mass on the bladder floor was observed. In the cystoscopy and biopsy a bladder tumor (Simultaneous squamous cell carcinoma and Transitional Bladder Cell Carcinoma) and schistosomiasis (Schistosoma haematobium) was diagnosed.
Malaria is a major problem in tropical and sub-tropical countries, with high morbidity and mortality. Splenectomy makes patients more susceptible to serious bacterial and parasitic infections. We report for the first time in Iran a fatal case of Plasmodium vivax malaria, confirmed by microscopic and molecular (Semi-nested multiplex PCR) tests in a patient who had undergone splenectomy due to hemolytic anemia.
Cases of Sparganum mansoni, caused by the plerocercoid larva of the tapeworm S. mansoni, occur throughout the world, particularly in Asian, Middle Eastern, and European countries. However, cases of infection with this parasite are rarely seen in Japan. Here, we present a case of a 61-year-old woman with a solitary subcutaneous nodule in left inner aspect of the thigh, from which a long, slender, whitish worm was surgically removed. The parasite was histopathologically identified as S. mansoni. Serological testing confirmed cure of the infection after surgical removal of the parasite. The authors advocate immunoserological examination in case of S. mansoni.