Axenic cultivation and pathogenic assays of acanthamoeba strains using physical parameters

The main goal of the present study was to set up an axenic cultivation of Acanthamoeba and assess the pathogenic ability of T4 genotypes from different clinical and environmental strains of Acanthamoeba using two physical assays. Sixteen Acanthamoeba isolates including 10 environmental and 6 clinical strains were cultured axenically. Axenic cultivation was performed using Proteosepepton, yeast extract and glucose medium and TY-I-S33culture. Pathogenic survey was done using osmotolerance and thermotolerance assay. Briefly, differentosmolarity [0.5 M and 1 M] of non-nutrient agar plates were performed. One hundred fiftymicrol of axenic culture were collected and were inoculated in 1% agar medium. For thermotolerance assay 150 micro L of amoebas from axenic culture were divided into fresh culture mediums. Cultures were incubated at 37degreeC and 42degreeC. All plates were monitored for 24 h, 48 h and 72 h. Overall, 16 strains of Acanthamoeba isolates previously genotyped as T4 were cultivated axenically after several months. Thermotolerance and osmotolerance assay revealed that all of clinical strains, soil and animal feces strains were highly pathogenic isolates. Two dust and water strains did not grow at high temperature [42degreeC] and osmolarity [1.5 M] and thus they were classified as weak pathogens. Most of T4 genotypes are highly pathogenic organisms. This is an important finding since Acanthamoeba belonging to T4 type is the predominate genotype in environmental and clinical samples. The presence of highly pathogenic Acanthamoeba may pose a risk within susceptible people.

Occurrence of thermotolerant hartmannella vermiformis and naegleria spp. in hot springs of Ardebil province, northwest Iran

Geothermal waters could be suitable niches for thermophilic free living amoebae including Naegleria and Hartmannella. Ardebil Province, northwest Iran is popular for having many hot springs for recreational and health purposes activity. The present research is the first molecular based investigation regarding the presence of Naegleria and Hartmannella in the hot springs of Ardebil Province in Iran. Overall, 30 water samples were taken from waters of thermal hot springs in Ardebil Province, Iran during 2010-2011. All collected samples were transferred to Dept. of Parasitology and Mycology, Shahid Beheshti University of Medical Sciences, Tehran, Iran. Cultivation of concentrated water samples was performed using culture-enrichment method. Cloning of the target amoebae was obtained and morphological and molecular analysis was done using page key combined with two sets of primers, respectively. Sequence analysis and homology search was used for strains identification. Of 30 water samples, 8 [26.7%] were positive for thermotolerant Vahlkampfiids and Hartmannella based on morphological characteristics of vegetative form and double walled cysts. Cloning of the target amoebae were done successfully. Sequencing of the positive isolates revealed that the strains belonged to Naegleria [N. carteri and N. spp] and H. vermiformis. The result highlights a need for improved filtration and disinfection and periodic monitoring of recreational thermal waters in order to prevent disease related to free- living amoebae. This is the first comprehensive molecular study of thermophilic Naegleria and Hartmannella in hot springs of Iran

Isolation of free-living amoebae from Sarein hot springs in Ardebil province, Iran

Free-living amoebae [FLA] are a group of ubiquitous protozoan, which are distributed in the natural and artificial environment sources. The main aim of the current study was to identify the presence of FLA in the recreational hot springs of Sarein in Ardebil Province of Iran. Seven recreational hot springs were selected in Sarein City and 28 water samples [four from each hot spring] were collected using 500 ml sterile plastic bottles during three month. Filtration of water samples was performed, and culture was done in non-nutrient agar medium enriched with Escherichia coli. Identification of the FLA was based on morphological criteria of cysts and trophozoites. Genotype identification of Acanthamoeba positive samples were also performed using sequencing based method. Overall, 12 out of 28 [42.9%] samples were positive for FLA which Acanthamoeba and Vahlkampfiid amoebaewere found in one [3.6%] and 11 [39.3%] samples, respectively. Sequence analysis of the single isolate of Acanthamoeba revealed potentially pathogenic T[4] genotype corresponding to A. castellanii. Contamination of hot springs to FLA, such as Acanthamoeba T[4] genotype [A. castellanii] and Vahlkampfiid amoebae, could present a sanitary risk for high risk people, and health authorities must be aware of FLA presence

First report of Vannellidae amoebae [vannella spp.] isolated from biofilm source

Members of the Vannellidae family are free-living amoebae [FLA] distributed mainly in water and soil sources. The present study reports the first isolation of this genus in the biofilm source from hospital environment in Tehran, Iran. Biofilm samples were collected from hospital environment. Cultivation was performed in non-nutrient agar covered with a heat-killed Escherichia coli. Cloning of the suspected amoebae was done. PCR amplification and Homology analysis using the Basic Local Alignment Search Tool [BLASTn] was performed to search for the most similar reference sequences. Microscopic examination showed numerous fan-shaped amoebae and peculiar cysts different to the usual shape of typical FLA. Sequence analysis of the PCR- product revealed that the suspected amoebae are highly homologous with Vannella spp. gene [99% identity and 100% query coverage] available in the gene bank database. Although Vannella spp. is not proved to be pathogenic itself, but they are capable of harboring pathogenic intracellular organisms such as Microsporidian parasites. Thus, identification of such amoebae can be of clinical importance, as they could lead to transmission of other pathogens to human

Enteric protozoan parasites in rural areas of Bandar-Abbas, southern Iran: comparison of past and present situation

The main goal was to address the prevalence of enteric protozoan parasites in rural areas of Bandar-Abbas, southern Iran and to compare the results with the only conducted study in 1978. This descriptive study was performed from 2009 through 2010 on the 565 fecal samples. Formalin-ether concentration technique was performed and the analysis was carried out using Chi-square test in SPSS software version 13.5. Finally, the comparison of our results with the only previous study which was accomplished by Sheiban and Rezaeian in 1978 was done. The overall prevalence of the protozoan parasites was 48.8%. However, the prevalence of pathogen parasites was 23%. Previous research in 1978 showed 80.4% infectivity. The most protozoan parasites were Blastocystis hominis [25.53%], Giardia lamblia [17.2%] and Entamoeba coli [15.95%]. Previous study in 1978 found Entamoeba coli as the most common protozoa. Our finding revealed that the rate of single infectivity was much higher compared to previous research. The most frequency of infection was in children. The remarkable decrease of protozoan parasites is mainly due to progress in health care in the villages; however more effort should be done with the goal of eradicating infectious agents

Molecular identification and sequencing of mannose binding protein [MBP] gene of acanthamoeba palestinensis

Acanthamoeba keratitis develops by pathogenic Acanthamoeba such as A. palestinensis. Indeed this species is one of the known causative agents of amoebic keratitis in Iran. Mannose Binding Protein [MBP] is the main pathogenicity factors for developing this sight threatening disease. We aimed to characterize MBP gene in pathogenic Acanthamoeba isolates such as A. palestinensis. This experimental research was performed in the School of Public Health, Tehran University of Medical Sciences, Tehran, Iran during 2007-2008. A. palestinensis was grown on 2% non-nutrient agar overlaid with Escherichia coli. DNA extraction was performed using phenol-chloroform method. PCR reaction and amplification were done using specific primer pairs of MBP. The amplified fragment were purified and sequenced. Finally, the obtained fragment was deposited in the gene data bank. A 900 bp PCR-product was recovered after PCR reaction. Sequence analysis of the purified PCR product revealed a gene with 943 nucleotides. Homology analysis of the obtained sequence showed 81% similarity with the available MBP gene in the gene data bank. The fragment was deposited in the gene data bank under accession number EU678895 MBP is known as the most important factor in Acanthamoeba pathogenesis cascade. Therefore, characterization of this gene can aid in developing better therapeutic agents and even immunization of high-risk people

Prevalence of Trichomonas vaginalis using parasitological methods in Tehran

Trichomonas vaginalis is a parasitic protozoan with a predilection for human urogenital tract and causative agent for vaginitis, cervicitis and urethritis in females. T. vaginalis is known as a cofactor in transmission of human immunodeficiency virus and may lead to adverse outcomes in pregnant women. The goal of this study was to determine the prevalence of T. vaginalis infection in females attending Mirzakuchak Khan Hospital, Tehran, Iran. During May 2008 to March 2009, 500 vaginal discharges samples were obtained from women attending sexual transmitted disease [STD] clinic of Mirzakuchak Khan Hospital in Tehran, Iran. The samples were examined by Dorsse culture medium and wet-mount methods. The prevalence of T.vaginalis was determined using culture based method and wet-mount examinations. Sixteen positive [3.2%] and 484 negative [96.8%] samples for T. vaginalis were detected by culture based methods. The wet mount examination revealed 13 positive [2.6%] and 487 negative [97.4%] samples. In the above population, prevalence of trichomoniasis was estimated as 3.2% based on culturing method. Due to adverse outcomes of vaginal trichomoniasis and its correlation with HIV transmission, there is a great need for public education regarding implementation of personal hygienic measures and prevention of inappropriate sexual contacts

Comparison of a PCR-based method with culture and direct examination for diagnosis of Acanthamoeba keratitis

The aim was to compare three different methods [direct examination, culture and PCR methods] for the diagnosis of Acanthamoeba keratitis [AK] in corneal scrapes. Twenty eight corneal scrapes and contact lenses were collected from keratitis patients and referred to the Department of Medical Parasitology and Mycology, School of Public Health, Tehran University of Medical Sciences. Corneal scrapes were divided in three parts for direct examination, culture on non-nutrient agar and PCR analysis. PCR analysis was also performed using a 18S rRNA gene primer pair [DF3 region]. DF3 [Diagnostic fragment 3] is a region of the nuclear small subunit ribosomal RNA gene which is specific for detecting Acanthamoeba strains. Acanthamoeba was the causative agent of keratitis in 50% of the patients. Direct smear of all prepared corneal scrapes in AK patients was negative and culture was positive in only 14.3% of the isolates. PCR analysis was positive in 71.4% of AK patients. These three methods were negative in corneal scrapes of non-AK patients. The sensitivity and specificity of PCR technique for the detection of Acanthamoeba sp. were calculated as 71.4% and 100%, respectively. According to high sensitivity and specificity of PCR-based method, this study confirmed that PCR using 18S rRNA gene primers [DF3 region] is more useful for detecting AK cases compare to culture and direct microscopy methods

Isolation of vahlkampfids [willaertia magna] and thecamoeba from soil samples in Iran

Vahlkampfiids contains wide variety of genuses with some known as human pathogens such as Naegleria, Vahlkampfia and non pathogens such as Willaertia. Since there was no evidence of presence of Vahlkampfiids in different sources in Iran, we have analyzed soil samples to clarify the presence of these amebas. Seven soil samples collected in Tehran were analyzed to clarify the presence of Vahlkampfiids in soil sources, using microscopic examination of non nutrient agar cultures and specific Vahlkampfiids primer pair. Vahlkampfiids were detected in 2 out of 7 soil samples by direct examination of cultures. Sequence analysis confirmed that Willaertia magna [W. magna] was present in 2 samples. Additionally, Thecamoeba were detected in all of soil samples. To the best of our knowledge, this is the first report of existing W. magna and Thecamoeba in Iran. Over all, more research should be implicated in Iran for identification of Vahlkampfiids within different environmental sources as well as their pathogenic capability relevant for human beings

survey on intestinal parasitic infections in patients referred to a hospital in Tehran

To determine the prevalence of parasitic infections in people referred to Kashani Hospital Tehran during Summar season in 2005. Fecal samples were examined by direct examination, formalin-ether concentration and staining with Ziehl-Neelsen. To identify Strongyloides stercoralis samples were cultured on the nutrient agar medium. In a total of 205 cases examined 29.75% were found infected with at least one parasite. The rates was as follows: Blastocystis hominis 20.9%, Giardia lamblia 5.36%, lodamoeba butschelii 0.48%, Dientamoeba fragilis 0.48%, Trichomonas hominis 0.48%, Endolimax nana 0.97%, Enterobius vermicularis 0.48% and Taenia 0.48%. Results included the significance of Giordia infection in children along with symptoms. 76.74% of patients with Blastocystis infection have presented with intestinal symptoms. More research is encouraged to identify the relationship of B. hominis and the symptoms

Amoebic keratitis in Iran [1997-2007]

Amoebic keratitis introduced as a painful corneal infection which sometimes lead to poor vision and blindness. The main goal of this study was to report amoebic keratitis during ten years from 1997-2007 in patients who was suspected to have amoebic keratitis and referred to Parasitology laboratory, School of Public Health, Tehran University of Medical Sciences, Iran. Other aim was to assess the major risk factor for developing this sight-threatening disease. Comparison of lens culture and corneal scrapes culture also was performed. During 1997-2007, 142 patients referred to Dept. of Medical Parasitology, School of Public Health, Tehran University of Medical Sciences, Iran. Details of each patient such as age, sex, history of contact lens wear, type of contact lens, clinical symptoms were recorded in questioners. Keratitis was diagnosed on the basis of culture of lenses and/or corneal scrapes on non-nutrient agar overlaid with Escherichia coli and direct microscopy of lenses and/or corneal scrapes. Among 142 patients, 49 [34.5%] had amoebic keratitis. 73.46% of these patients were from Tehran but there were a few cases from other cities. The commonest age was between 15-25 yr [75.5%] and more female [37:12] were identified then male. It is worth to mention that 44 patients [89.79%] were contact lens wearers who among them 41 patients [93.18%] wore soft contact lens and only three patients suffer from amoebic keratitis because of wearing hard contact lens. Other finding of this study demonstrated that the most common sign of the patients was severe pain combined with photophobia. This study indicates that Acanthamoeba keratitis continue to rise in Iran. This is due to increase frequency of lens wearers as well as consideration of ophthalmologist to Acanthamoeba as an agent of keratitis and improvement of laboratory methods. Another finding of this research was the confirmation of soft contact lens as a major risk factor. It is recommended to educate contact lens wearers for regular disinfection. Besides, culture of corneal scrapes was negative in most of cases, so lens culture were performed which had a much better result