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The keratinolytic activities of dermatophyte species are accompanied by the secretion of enzymes, such as serine proteases, which are coded by the Subtilisin (SUB) genes. This study aimed to determine the presence of the SUB genes in the clinical and nonclinical samples of Trichophyton verrucosum and Microsporum gypseum. Isolation was carried out by direct and laboratory examination. Following that, for the determination of the presence of the SUB gene, polymerase chain reaction with specific primers was conducted. The frequencies of the SUB gene were observed in almost 66% of the isolates. Statistical analysis showed a significant relationship between the presence of the SUB gene and the samples collected from human, animals, and soil (p ˂0.005). The current investigation has been the first study of the presence/absence of the SUB gene in the clinical and nonclinical isolates of T. verrucosum and M. gypseum in Iran which may be a new step to perform further studies.

Background: The main goal of present study was to detect polymorphism in MSP-1 gene which is a major blood stage candidate for vaccine in Plasmodium vivax by Single Strand Conformational Polymor­phism-Polymerase Chain Reaction (SSCP-PCR).Methods: During 2008 to 2010 fifty samples were collected from Iranian patients with P. vivax in Hormozgan Province, southern Iran. All of the samples were detected by microscopical examination. Amplification of MSP-1 gene was done by PCR after DNA extraction. Single strand DNAs due to using in SSCP, was electrophoresed on polyacrylamid- Bisacrylamid gel then banding patterns were revealed by silver-staining method. Sequencing as a typing method was performed for some isolates.Results: All of the 50 isolates were positive microscopically. Totally 12 (24%) isolates showed 440 bp and 38 (76%) showed 500 bp in PCR assay. SSCP analysis revealed four banding patterns. Pattern I (10/50), Pattern II (12/50), Pattern III (27/50), and Pattern IV (1/50). The results sequencing analy­sis of the MSP-1 gene in 19 isolates revealed diversity in nucleotides and amino acid in Iranian P. vivax isolates.Conclusion: Our study confirms that the SSCP-PCR is a rapid method for detecting polymorphism in MSP-1 gene in P. vivax. The presence of different haplotypes in MSP-1 gene shows that several P. vivax strains exist in malaria endemic areas of Iran.

Background: Infection with Trichomonas vaginalis is one of the most common sexually transmitted diseases (STDs) in humans. The prevalence of infection in Iran has been reported between 2 to 8%, depending on deferent socio-cultural conditions. This study aimed to determine the prevalence of T. vaginalis in women referred to gynecologic clinics in Hamadan city, West of Iran.Methods: This descriptive cross-sectional study was conducted on 750 women who referred to Gyneco­logic clinics in Hamadan from November 2010 to July 2011. Vaginal samples were obtained from them and examined by wet mount and culture methods for the detection of T. vaginalis.Results: Sixteen out of 750 vaginal swab specimens (2.1%) were culture positive for T. vaginalis and 13 of these positive specimens (1.7%) were wet mount positive. Only 12 of 42 patients who were clinically diagnosed as having T. vaginalis infection, confirmed by culture method. Five hundred and fifty of the participants women (73.3%) had at least one of signs and symptoms of trichomoniasis. No statistical correlation was observed between clinical manifestations and parasitological results (p>0.05).Conclusion: This study showed low prevalence of T. vaginalis infection in the study population. Since clinical signs of trichomonal vaginitis are the same of other STDs, a confirmatory laboratory diagnosis is necessary. Wet smear as well as culture are sensitive for detection of T. vaginalis.

Objectives: The present study investigates the state of administration of information technology services in libraries and special information centers in metropolitan Tehran Methodology: A quantitative approach was used using literature survey. 131 libraries and Special Information Center were selected in Tehran Area. Findings: Statistical Analysis confirmed the non-compliance with four out of seven evaluation criteria. These were planning and implementation of service management, service processes, control processes and presentation. No concrete theory was developed regarding the remaining criteria of management system, communication processes, and solution processes

Background: The main goal of the present study was to develop a new sensitive and specific PCR based method for Identification of Cryptosporidium sp. using novel primers from 18S ribosomal RNA. Cryptosporidi­osis in high-risk host groups particularly in neonates and immuno-compromised individuals may result in death. To the best of our knowledge this is the first study regarding develop a new PCR based method to diagnose the cryptosporidiosis in Iran.Methods: A total of 850 human fecal samples from patients clinically suspected to cryptosporidiosis and 100 healthy and diarrheic cattle stool specimens were collected. The simplified formol-ether concentration method was carried out for all samples. They were then examined microscopically by modified Ziehl-Neel­sen staining method. Total DNA was extracted by QIA amp DNA stool mini kit was carried out by using designed prim­ers.Results: Twenty nine cases of cryptosporidiosis infection in human and 30 samples from cattle microscopi­cally were posi­tive. The described primary and nested PCR method could detect all Cryptospori­dium positive samples from human and cattle. Regards to suspected negative samples in pri­mary PCR examination, the Nested PCR could ap­prove two more positive results. Furthermore, Nested PCR analysis was able to detect one more case which was nega­tive in both microscopically examination and primary PCR. Specificity of the test was 100%. Sensitivity of Nested PCR in comparison to our gold standard; microscopy after Ridley concentration modified ziehl-Neelsen, was 100 %.Conclusion: Our developed PCR based method by using new primers devised from 18S ribosomal RNA revealed the ability for identification of the Cryptosporidium species such as C. parvum and C. huminis with high specificity and sensitivity.

"n "nBackground: The main goal of this study was to conduct a comparative population genetic study of Turkish speaking Iranian Azeris as being the biggest ethno-linguistic community, based on the polymorph markers on Y chromosome. "nMethods: One hundred Turkish-speaking Azeri males from north-west Iran (Tabriz, 2008-2009) were selected based on living 3 generations paternally in the same region and not having any relationship with each other. Samples were collected by mouth swabs, DNA extracted and multiplex PCR done, then 12 Single Nucleotide Polymorphisms (SNPs) and 6 Microsatellites (MS) were sequenced. Obtained data were statistically analyzed by Arlequin software."nResults: SNPs and Microsatellites typing were compared with neighboring Turkish-speaking populations (from Turkey and Azerbaijan) and Turkmens representing a possible source group who imposed the Turkish language during 11-15th centuries AD. Azeris demonstrated high level of gene diversity compatible with patterns registered in the neighboring Turkish-speaking populations, whereas the Turkmens displayed significantly lower level of genetic variation. This rate of genetic affiliation depends primarily on the geographic proximity."nConclusion: The imposition of Turkish language to this region was realized predominantly by the process of elite dominance, i.e. by the limited number of invaders who left only weak patrilineal genetic trace in modern populations of the region. "n

"nBackground: We examined a molecular method with a single-PCR for amplification of a part of CP5 gene enabling us to differen­tiate the pathogenic species, Entamoeba histolytica, from the non-pathogenic species, E. dispar. "nMethods: We developed a single PCR method for this purpose. After investigation of GenBank, primer pairs were de­signed from highly conserved regions of cysteine proteinase (CP5) gene. The primers were utilized in PCR using isolated ge­nomic DNA template of E. histolytica and the PCR products were then sequenced. The same primer and method for PCR was used for isolated genomic DNA template of E. dispar. "nResults: A fragment of about 950 bp was isolated in PCR by using DNA from E. histolytica, however, no banding pattern was produced by using the same primers for E. dispar. We characterized CP5 gene at molecular level in E. histolytica iso­lates from 22 positive; including 20 non-dysentery samples isolated from both cities as well as two dysentery samples iso­lated only from Tabriz. Nucleotide sequence comparison in gene data banks (NCBI, NIH) revealed significant homology with CP5 gene in E. histolytica isolates "nConclusion: We developed a PCR method, which could detect simply and rapidly E. histolytica by amplifying a specific PCR fragment.

"nBackground: 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."nMethods: 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."nResults: 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"nConclusion: 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.

"nBackground: 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. "nMethods: 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. "nResults: 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. "nConclusion: 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.

"nBackground: Malassezia are dimorphic, lipid-dependent yeasts, which are responsible for causing several cutaneous and sys­temic conditions. Although cyclophilins (CyPs) are highly conserved cytosolic proteins that catalyze the peptidyl-prolyl cis-trans isomerazation reaction before protein folding process, it has been suggestive of an allergen in a few numbers of fungi such as Aspergillus fumigatus and Malassezia species. Allergenic cyclophilins are IgE-binding components, which have been characterized in other species of Malassezia; and are considered as Mala s 6 in Malassezia sympodialis. In the pre­sent study we tried to identify the molecular characterization of cyclophilin gene in M. furfur."nMethods: Pairs of oligonucleotide primers were designed from highly conserved regions of the gene counterparts in other fungi. The primers were then applied to amplify the primer-specific DNA fragment. Afterward, PCR product fragments were sequenced to be used in further analysis."nResults: About 573 nucleotides, encoding a polypeptide of 190 amino acids, have been sequenced. Sequence comparison was performed in Gene Bank, both for the nucleotides and their deduced amino acid sequence. It revealed a significant homol­ogy with cyclophilin genes and proteins of other eukaryotic cells. The amino acid sequence of the encoded protein was about 86% identical to the sequence of cyclophilin protein from other fungi."nConclusion: The molecular characterization of cyclophilin gene may open the way to disclosure of the functional char­acteris­tics of cyclophilin and is a fundamental step for understanding the molecular basis of its pathogenesis in AEDS dis­ease.

"nBackground: The aim was to compare three different methods (direct examination, culture and PCR meth­ods) for the diagnosis of Acanthamoeba keratitis (AK) in corneal scrapes."nMethods: 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."nResults: 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."nConclusion: 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.

"nBackground: Subtilisin -like proteases are the group of proteases including keratinases found in dermatophytes which de­graded keratin. Determination of the proteases activity of Trichophyton vanbreuseghemii isolates which were obtained from soil and clinical and soil isolates of Microsporum gypseum in Iran and characterization of their genome were aim of present study."nMethods: Ezymatic activity was determined by use of chromogenic substrates. The genes, which coded subtilisin-like pro­teases in above-mentioned dermatophytes, was identified and amplified by using specific primers in PCR."nResults: The highest yield of enzyme production was observed in only one isolate of T. vanbreuseghemii Ir-84 whereas low enzyme activity was observed in M. gypseum isolates. Homology study of obtained nucleotide as well as amino acid sequences indicated different rates of homology with other subtilisin-like proteases genes in other pathogenic dermato­phytes."nConclusion: Intra-strain differences were observed in production of serine proteinases and molecular characterization of genes encoding such enzymes could be of great interest for studies on pathogenicity and other purposes.

"nBackground: Otitis media with effusion (OME) is common in the children. It is proven that pathogenic bacteria as a causative agent of middle ear effusion, however the role of fungal infection in otitis media with effusion remains unclear. Therefore, this study was conducted to assess presence of fungi in OME."nMethods: From January 2005 to September 2006, a number of 62 children with proven otitis media with effusion subjected to the case series study at Amiralmomenin Hospital in Rasht City, Iran. After myringotomy, middle ear effusion was collected. In 48 patients, both ears demonstrated effusion, whereas in 14 patients, only one ear had effusion. Standard mycological culture and polymerase chain reaction (PCR) assay were performed in 110 and 79 samples, respectively."nResults: The growth of fungi was observed in 9 samples (8.8%). The result of our PCR assay showed that 23 samples (29.1%) were positive for fungal DNA."nConclusion: Middle ear effusion from cases with OME contains fungi and it might play a role in the pathogens of OME. PCR assay is a better indicator in detection of fungus in middle ear effusion, compared with fungal culturing method. However, the estimation of its sensitivity and specificity in detection of fungal agents in these patients needs more molecular epidemiological studies.

Background: This study was conducted to determine of molecular epidemiology of the Giardia lamblia by PCR-RFLP method in Tehran, capital of Iran. Methods: Thirty eight stool samples were randomly selected from 125 patients diagnosed with giardiasis using microscopy in Tehran. DNA extraction of some samples were performed by phenol/chloroform/isoamyl alcohol method and to raise the sensitivity of the PCR assay, the genomic DNA of the others were extracted using glass beads and the QIAamp Stool Mini Kit in order to effectively remove the PCR inhibitors. A single step PCR-RFLP assay, targeting the glutamate dehydrogenase (gdh) locus, was used to differentiate within and between assemblages A and B that have been found in humans. Results: Of the 38 isolates, 33 samples (87%) were found as G. lamblia (genotype AII), 3 (7.8%) belonged to assemblage B, genotype BIII, the mixed of genotype AII and B were detected only in two samples (5.2%). Conclusions: PCR-RFLP is a sensitive and powerful analytical tool that allows effective genotype discrimination within and between assemblages at targeting gdh gene, and makes it possible to identify the presence of mixed genotypes. Our data suggest that there is an anthroponotic origin of the infection route, assemblage A group II, in Tehran so it seems that the main reservoir of Giardia infection is humans in the area studies.

Background: Acanthamoeba castellanii is the important cause of amoebic keratitis in Iran. The key molecule in pathogenesis of Acanthamoeba keratitis is Mannose Binding Protein (MBP) led to adhesion of amoeba to corneal epithelium. Subsequent to adhesion other cytopathic effects occur. The goal of this study was to identify the molecular characterization of a gene encoding MBP in an Iranian isolate of A.castellanii in order to pave the way for further investigations such as new therapeutic advances or immunization. Methods: A.castellanii was cultured on non nutrient agar. Extraction of DNA was performed by phenol-chloroform method. After designing a pair of primer for the gene encoding MBP, PCR analysis was performed. Finally, the PCR product has been sequenced and the result submitted to the gene data banks. Results: An MBP gene of 1081 nucleotides was sequenced. This fragment contained three introns and encodes a protein with 194 amino acids. Homology search by Blast program showed a significant homology with the MBP gene in gene data banks (96%). Besides, the identity of amino acids with the other MBPs in gene data banks was about 86%. Conclusion: We isolated and sequenced a gene fragment encoding MBP in an Iranian isolate of A.castellanii. Molecular characterization of this important gene is the first step in pursuing researches such as developing better therapeutic agents, immunization of population at risk or even developing a diagnostic tool by PCR techniques.

Background: To assess the utility of the chromosomal ail virulence gene sequence for detection of pathogenic Yersinia en­terocolitica in raw meet food products (beef, lamb, and chicken). Methods: This study included 39 Yersinia enterocolitica positive cultures from suspicious food samples, in a working pe­riod of six months. These samples were referred to the "Food-Borne Diseases and Chronic Diarrhea Lab at Research Cen­tre for Gastric and Liver Diseases" of the Taleghani Hospital at Shahid Beheshti University of Medical Sciences, Te­hran, Iran. Isolates from 8 cultured Y. intermedia, Y. aldovi, Y. intermedia type O:45, Y. kristensenii, Y. enterocolitica type O:12/26, Y. enterocolitica type1/7/8, Y. frederiksenii type O:39, and Y. enterocolitica type O:8 samples were in­cluded in the study. Four non-Yersinia species Salmonella typhi, Shigella dysenteriae, Shigella flexeneri, and Proteus mirabi­lis were used for specificity testing. An established Yersinia type O:9 was used as positive control and for sensitiv­ity testing. An in-house real-time PCR assay was designed in order to rapidly and specifically identifies the pres­ence of specific Yersinia species. Results: Out of 39 tested Y. enterocolitica samples, 6(2.3%) showed positive results for the ail gene PCR prod­uct, typed as O:8, and O:9, respectively. PCR products were sent for sequencing. Two sequences were registered with the Na­tional Center for Biotechnology Information (NCBI Genbank) as polymorphic ail gene sequences under the acces­sion numbers of DQ157767 and DQ003329. Conclusions: Collectively, this test is well adapted for definite confirmation of pathogenic Y. en­terocolitica in food sam­ples.

Background: Strongyloides stercoralis, the etiological agent of strongyloidiasis, is one of the most common parasitic nema­tode with the unique ability to complete its life cycle and proliferate within the host. Although it is an endemic parasite in Iran, no molecular characterization is available on isolates originated from the country. Therefore, this study was con­ducted for molecular identification of human Strongyloides isolates in the three most prevalent provinces."nMethods: After microscopical examination and agar plate culture of nearly 1500 stool samples, 20 isolates of S. stercoralis filari­form larvae were recovered from (Gilan and Mazandaran in north and Khouzestan in south) of Iran. The genomic DNA was extracted from all these samples and two primer sets were selected for amplification. ITS1 region of the rDNA gene was amplified by a nested Polymerase Chain Reaction (nested-PCR). The PCR prod­ucts were sequenced and analyzed in compari­son with the sequences deposited in GenBank."nResults: DNA sequence analysis of ITS1 region showed that all the 20 isolates were S. stercoralis. There was slight varia­tion in the ITS1 region among the isolates. "nConclusion: ITS1 sequencing seems to be a valid target for molecular identification of S. stercoralis.

Trichophyton rubrum (T. rubrum) is an anthropophilic dermatophyte that is distributed worldwide and causes common cutaneous disease such as mycosis. Although several properties of this fungus have been investigated so far, however a few studies were carried out in the field of molecular biology of this fungus. In the present study we tried to identify its molecular characterization of the goanosin three phosphat (GTP) binding protein gene. Pairs of 21 nt primers were designed from highly conserved regions of the gene in other fungi. The primers were utilized in PCR by using isolated genomic DNA template as well as cytoplasmic RNA of T. rubrum and the PCR and RT-PCR fragments were then sequenced. About 645 nucleotides have been sequenced which encodes a polypeptide with 214 amino acids. Nucleotide sequence comparison in gene data banks (NCBI, NIH) for both the DNA and its deduced amino acid sequence revealed significant homology with GTP binding protein genes and proteins of other eukaryotic cells. The amino acid sequence of the encoded protein was about 64% identical to the sequence of GTP binding protein from other fungi. In summary, we have cloned the first GTP binding protein of dermatophytes and characterized it as a member of this gene family in other eukaryotic cells.

Cryptosporidium is an apicomplexan parasite of humans and a wild range of domestic as well as wild animals. An 833-bp fragment of the 18S-rRNA gene was used to identify Cryptosporidium spp. recovered from children and adult patients, in human immunodeficiency virus (HIV) positive and negative patients in Iran. Initial identification of cryptosporidiosis was carried out by Ziehl-Neelsen acid-fast staining method of stool samples. The samples, then, were identified specifically by nested PCR, targeting the most polymorphic region of the 18S-rRNA gene. The genotype encountered was detected by restriction endonuclease digestion of the nested-PCR product. Among 17 analyzed isolates, two different genotypes of Cryptosporidium were identified; 24% of the isolates belonged to C. parvum human genotype, and 76% to the potentially zoonotic species of C. parvum bovine genotype. The results of the present study showed that in contrast to HIV negative individuals, HIV positive individuals were more likely to be infected with zoonotic genotypes of the parasite; it was also confirmed the fact that zoonotic transmission of the parasite in Iran was as frequent as the transmission of anthroponotic origin. These outcomes are helpful for researchers to establish the corresponding prevention and treatment measures.

Trichophyton rubrum is an anthropophilic fungus causing up to 90% of chronic cases of dermatophytosis. Several properties of this fungus have been investigated so far. However, a few studies were carried out in the field of molecular biology of this fungus. In the present study, we tried to identify the subunit G of its vacuolar ATPase (V-ATPase). Pairs of 21 nt primers were designed from highly conserved regions of the V-ATPase subunit G genes in other fungi. Mentioned primers were utilized in PCR using isolated genomic DNA template as well as cytoplasmic RNA of T.rubrum and the PCR and RT-PCR fragments were then sequenced. About 469 nucleotides were sequenced which encoded a polypeptide with 119 amino acids. Nucleotide sequence comparison in gene data banks (NCBI, NIH) for both the DNA and its deduced amino acid sequence revealed significant homology with V-ATPase subunit G genes and proteins of other eukaryotic cells. The amino acid sequence of the encoded protein was about 84% identical to the sequence of V-ATPase subunit G from other fungi. In summary, we have cloned the first V-ATPase subunit G of dermatophytes and characterized it as a member of this gene family in other eukaryotic cells.

Amebiasis caused by Entamoeba histolytica is still mentioned as one of the major health problems in tropical and subtropical areas. E. histolytica has recently been redescribed as two distinct species; E. histolytica and E. dispar. In the present study, we characterized the 70 kDa Heat Shock Protein (HSP70) of E. dispar at molecular level and compared it with that of E. histolytica. With these findings, we were able to distinguishe E. dispar from the infectious E. histolytica. Pairs of 21 nucleotide primers were designed from highly conserved regions of the same gene in other eukaryotic cells. Mentioned primers were utilized in PCR by using isolated genomic DNA template of E. dispar and the PCR fragments were then sequenced. By the time, 1020 nucleotides have been sequenced and characterized within open reading frame of this new gene which encode a polypeptide with 337 amino acids. Nucleotide sequence comparison in gene data banks (NCBI, NIH) for both the partial DNA and its deduced amino acid sequence revealed significant homology with members of the eukaryotic 70 kDa HSP family. Small parts of the mentioned sequences from E. dispar were about 100% identical to the sequences of 70 kDa HSP from E. histolytica other eukaryotic cells. The new partial gene fragment and its encoded protein have been submitted to the gene data banks (NCBI, NIH) and registered under the accession number of AY763790.

Prostate cancer is the second common form of cancer in men. Detection of circulating Prostate Specific Antigen (PSA) transcripts has effectively been used for early diagnosis of prostate cancer cells. This investigation employed a reverse transcriptase polymerase chain reaction (RT-PCR) technique to distinguish the patients with either localized or metastatic prostate cancer (CaP) vs. Benign Prostate Hyperplasia (BPH) and control subjects, as compared with clinical and pathological records. With reservation of ethical issues, blood samples were collected from 60 cases. Based on pathological and clinical findings, 25 patients (20 with localized cancer, 5 with metastatic), 22 with BPH, and 13 healthy (including 3 females) subjects as negative controls, were selected from Shariati, Mehrad, Sina,, Khatam and Atie Hospitals in Tehran, Iran. RT-PCR for a 260 bp PSA transcript was then performed. Clinical and pathological records were used for the assessment and comparison of PSA RT-PCR results. None of the control subjects and BPH (with 7 exceptions) were found positive by RT-PCR (Relative specificity= 72.7%). In patients with prostate cancer, 21 out of 25 were found PSA positive (Relative sensitivity=83.4%) and the remaining 3 have been shown to be PSA negative (Positive predictive value= 83.4%). All of 5 metastatic patients (100%) revealed PSA positive results. Our data reflects the clinical relevance and significance of RT-PCR results as assessed with clinical and pathological examinations. PSA RT-PCR might be used as a powerful means for diagnosis, even when either pathological or clinical findings are negative, and could be employed for further molecular epidemiology surveys.

Background: The main goal of the present study was to develop a new sensitive and specific PCR based method for Identification of Cryptosporidium sp. using novel primers from 18S ribosomal RNA. Cryptosporidi­osis in high-risk host groups particularly in neonates and immuno-compromised individuals may result in death. To the best of our knowledge this is the first study regarding develop a new PCR based method to diagnose the cryptosporidiosis in Iran. Methods: A total of 850 human fecal samples from patients clinically suspected to cryptosporidiosis and 100 healthy and diarrheic cattle stool specimens were collected. The simplified formol-ether concentration method was carried out for all samples. They were then examined microscopically by modified Ziehl-Neel­sen staining method. Total DNA was extracted by QIA amp DNA stool mini kit was carried out by using designed prim­ers. Results: Twenty nine cases of cryptosporidiosis infection in human and 30 samples from cattle microscopi­cally were posi­tive. The described primary and nested PCR method could detect all Cryptospori­dium positive samples from human and cattle. Regards to suspected negative samples in pri­mary PCR examination, the Nested PCR could ap­prove two more positive results. Furthermore, Nested PCR analysis was able to detect one more case which was nega­tive in both microscopically examination and primary PCR. Specificity of the test was 100%. Sensitivity of Nested PCR in comparison to our gold standard; microscopy after Ridley concentration modified ziehl-Neelsen, was 100 %. Conclusion: Our developed PCR based method by using new primers devised from 18S ribosomal RNA revealed the ability for identification of the Cryptosporidium species such as C. parvum and C. huminis with high specificity and sensitivity.

Background: 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. Methods: 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. Results: 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. Conclusion: 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.

Background: The aim was to compare three different methods (direct examination, culture and PCR meth­ods) for the diagnosis of Acanthamoeba keratitis (AK) in corneal scrapes. Methods: 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. Results: 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. Conclusion: 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.

Background: This study was conducted to determine of molecular epidemiology of the Giardia lamblia by PCR-RFLP method in Tehran, capital of Iran. Methods: Thirty eight stool samples were randomly selected from 125 patients diagnosed with giardiasis using microscopy in Tehran. DNA extraction of some samples were performed by phenol/chloroform/isoamyl alcohol method and to raise the sen­sitivity of the PCR assay, the genomic DNA of the others were extracted using glass beads and the QIAamp Stool Mini Kit in order to effectively remove the PCR inhibitors. A single step PCR-RFLP assay, targeting the glutamate dehydro­genase (gdh) locus, was used to differentiate within and between assemblages A and B that have been found in humans. Results: Of the 38 isolates, 33 samples (87%) were found as G. lamblia (genotype AII), 3 (7.8%) belonged to assemblage B, genotype BIII, the mixed of genotype AII and B were detected only in two samples (5.2%). Conclusions: PCR-RFLP is a sensitive and powerful analytical tool that allows effective genotype discrimination within and between assemblages at targeting gdh gene, and makes it possible to identify the presence of mixed genotypes. Our data sug­gest that there is an anthroponotic origin of the infection route, assemblage A group II, in Tehran so it seems that the main reservoir of Giardia infection is humans in the area studies.

Background: Acanthamoeba castellanii is the important cause of amoebic keratitis in Iran. The key molecule in pathogene­sis of Acanthamoeba keratitis is Mannose Binding Protein (MBP) led to adhesion of amoeba to corneal epithelium. Subse­quent to adhesion other cytopathic effects occur. The goal of this study was to identify the molecular characterization of a gene encoding MBP in an Iranian isolate of A.castellanii in order to pave the way for further investigations such as new therapeu­tic advances or immunization. Methods: A.castellanii was cultured on non nutrient agar. Extraction of DNA was performed by phenol-chloroform method. After designing a pair of primer for the gene encoding MBP, PCR analysis was performed. Finally, the PCR prod­uct has been sequenced and the result submitted to the gene data banks. Results: An MBP gene of 1081 nucleotides was sequenced. This fragment contained three introns and encodes a protein with 194 amino acids. Homology search by Blast program showed a significant homology with the MBP gene in gene data banks (96%). Besides, the identity of amino acids with the other MBPs in gene data banks was about 86%. Conclusion: We isolated and sequenced a gene fragment encoding MBP in an Iranian isolate of A.castellanii. Molecular characteri­zation of this important gene is the first step in pursuing researches such as developing better therapeutic agents, im­mu­nization of population at risk or even developing a diagnostic tool by PCR techniques.

By using two types of different fuels and changing the ratios of these fuels, Reactivity Controlled Compression Ignition Engine (RCCI) is able to provide a more effective control over combustion phase at different loads and speeds. In a typical RCCI engine which could be considered as a type of homogeneous charge compression ignition (HCCI) engine, a low reactive fuel is injected into the intake port and a high reactive fuel is directly injected into the combustion chamber. In this study, a multi-dimensional model coupled with chemical mechanism is developed to simulate an RCCI engine operation fueled with iso-octane as the low reactive fuel and n-heptane as the high reactive one. Initially, the engine map was derived using different quantities of total above-mentioned fuels at different ratios and then engine inappropriate operating points were detected and improved by changing intake air pressure and injection timing strategies. The improved criteria to extend engine map are ringing intensity limit, NOx formation standard and gross indicated efficiency. It was concluded that high ringing intensity and NOx formation can be reduced by increasing intake air pressure; also badfire and misfire points can get improved by retarding the injection timing.