[One novel PMS2 germline mutation in a patient with hereditary non polyposis colorectal cancer]

Hereditary non polyposis colorectal cancer [HNPCC] is an autosomal dominant syndrome. The cancer appears between 40 and 50 years of age. Mutation in mismatch repair genes can lead to this cancer .One of the genes which is involved in this disease is PMS2 gene. Here, we present a case with a novel germline mutation in PMS2 gene. The aim of this report was to examine PMS2 gene and identify novel germline mutations in this gene. A 77-year-old male with diagnosis of colorectal carcinoma was referred for genetic testing. He suffered from a polyp with a diameter of 6.8 cm in hepatic flexure. The patient did not meet Amsterdam Criteria and Bethesda Guidelines, but screening for HNPCC was performed on account of pathological findings. Blood sample was used for identification of mutation and the paraffin embedded block was prepared for MSI analysis. One mutation in PMS2 gene was detected by analysis of the amplicon sequencing. The mutation was a transitional mutation in position 676 which led to transformation of guanine to adenine resulting in substitution of glutamic acid for glycine. Immunohistochemistry confirmed abnormal expression of PMS2 gene and MSI assay showed instability of sequenced amplicons in this gene

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

[Molecular determination of Echinococcus granulosus isolated from hydatid cyst using mitoconderial atp6 gene]

Several strains of the Echinococcus granulosus have been described based on morphological characters, intermediate host specificity and/or genetic analysis of mitochondrial and nuclear DNA. The aim of this study was to characterize different E.granulosus isolates by using sequences of mitochondrial atp6 gene. In this study, Sixty infected liver and lungs of cattle, sheep and goats were collected from the abattoir of Varamin city-Iran during 2008. Protoscoleces were removed from each fertile cyst and DNA extracted. New and specific primers were designed for two existing genotypes [G1 and G6] of E. granulosus known to occur in Iran and applied in PCR reactions. The new primers selectively amplified the G1 and G6 genotypes of E. granulosus with specific bands of 708 and 705 bp respectively. The G1 genotype was identified in all fertile cyst samples. This study showed that the new primer pairs which specifically amplify portions of the mitochondrial atp6 gene of the G1 and G6 strains of Echinococcus granulosus are proper molecular marker for investigating genetic variation in a number of isolates of E. granulosus from a range of hosts [sheep, goats, cattle] in Iran. The result of sequenced samples showed that our sequences were the same as those reported previously for these strains

Molecular epidemiology of cryptosporidiosis in Iranian children, Tehran, Iran

Cryptosporidium is a worldwide protozoan parasite and one of the most common causes of infection and diarrhea in humans and cattle. The aim of the present study was determination of subtypes of Cryptosporidium among children with diarrhea in Tehran by sequence analysis of the highly polymorphic 60-kDa glycoprotein [GP60] gene. Fecal samples were collected from 794 diarrheic children. Initial identification of Cryptosporidium was carried out on stool samples by Ziehl-Neelsen acid-fast staining method. DNA was extracted from positive microscopically samples and Cryptosporidium genotypes and subtypes were determined, accordingly. Out of 794 collected samples, 19 [2.40%] were positive for Cryptosporidium oocysts. Sequences analysis of GP60 gene showed that 17 [89.47%] of the positive isolates were Cryptosporidium parvum and 2 [10.52%] were C. hominis. All subtypes of C. parvum isolates belonged to allele families IIa [6/17] and IId [11/17]. The most common allele in all 17 isolates belonged to IId A20G1a [41.18%]. A22G1 [IF] subtype was detected in two C. hominis isolates of the children. The predominancy of C. parvum species [specially, IId A20G1a subtype] in current study underlines the importance of zoonotic Cryptosporidium transmission in Iran

Prevalence of intestinal parasites in bakery workers in Khorramabad, Lorestan Iran

Food contamination may occur through production, processing, distribution and preparation. In Iran especially in Khorramabad, 33[degree sign] 29' 16" North, 48[degree sign] 21' 21" East, due to kind of nutrition, culture and economic status of people, bread is a part of the main meal and the consumption of bread is high. In this study, the bakery workers were studied for determining of intestinal parasites prevalence. The study was carried out during September to November 2010 in Khorramabad. All the 278 bakeries and the bakery workers including 816 people were studied in a census method and their feces were examined for the presence of parasites by direct wet-mount, Lugol's iodine solution, and formaldehyde- ether sedimentation, trichrome staining, and single round PCR [For discrimination of Entamoeba spp]. Ninety-six [11.9%] stool specimens were positive for different intestinal parasites. Intestinal parasites included Giardia lamblia 3.7%, Entamoeba coli 5.5%, Blastocystis sp. 2.1%, Entamoeba dispar 0.4%, Hymenolepis nana 0.1%, and Blastocystis sp. 0.1%. In order to reduce the contamination in these persons, some cases such as stool exam every three months with concentration methods, supervision and application of accurate health rules by health experts, training in transmission of parasites are recommended

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

Sequence diversity in tRNA gene locus A-L among Iranian isolates of entamoeba dispar

A number of methods for detecting diversity in Entamoeba have been described over the years. In the present study the genetic polymorphism of noncoding locus A-L was analyzed using PCR and sequencing in order to clarify the genotypic differences among E. dispar isolates. A total of 28 E. dispar from patients with gastrointestinal symptoms were determined and the genomic DNA was extracted directly from stool. For genotype analysis; Locus A-L was amplified by PCR and PCR products were sequenced .The sequences obtained were edited manually and aligned using Gene Runner software. With sequencing of PCR products a reliable genetic diversity in size, number and position of the repeat units were observed among the Iranian E. dispar isolates in locus A-L gene. Sequences showed variation in length from 448bp to 507bp and seven distinct types were identified. The genetic diversity of loci like A-L shows them to be suitable for epidemiological studies such as the characterization of the routes of transmission of these parasites in Iran

Fatal sepsis by Bacillus circulam in an immunocompromised patient

An immunosuppressed man was admitted to hospital with diarrhea and a history of urinary tract infection. He was subjected to treatment with antibiotics. The patient died of putative severe sepsis. The etiological agent was a carbapenemase producing isolate of Bacillus circulans with resistance to all prescribed antimicrobial agents

[Cryptosporidium genetic diversity based on analysis of COWP, SSU-rRNA and TRAP-C2 polymorphic genes in children with diarrhea in Tehran and qazvin provinces: a survey from Iran]

Since Cryptosporidium is a worldwide distributed protozoan parasite and is considered as one of the most common causes of infection and diarrhea in humans with autoimmune deficiency, as well as in young live stock, molecular epidemiologic studies of cryptosporidiasis will be helpful for underlying transmission and molecular pathogenesis of Cryptosporidium in humans. The aim of the present study was to determine the species and genotypes of Cryptosporidium among children with diarrhea in Tehran and Qazvin provinces by PCRRFLP using the three polymorphic regions of SSU-rRNA, COWP and TRAP-C2 genes. 1263 stool samples were collected from the children less than 12 years with diarrhea who referred to Pediatrics Medical Centers in Gazvin and Tehran Provinces, Iran, during 2005-2007. After determination of the presence of Cryptosporidium oocytes by ZiehlNeelsen acid, fast staining genomic DNA was extracted. Nested PCR-RFLP was performed by -rRNA, COWP and TRAP-C2 genes. Results of microscopically positive samples showed that the overall prevalence of infection in children was 31 [2.5%]. Results of nested PCR amplification showed that of 31 isolates of children, all of three targeted gene were successfully amplified. Our results indicated that the zoonotic transmission is the main mode of infection in Iran and indicates that direct or indirect contact with animals, especially calf, is possibly the main route of human infection

Detection of acanthamoeba from fresh water using polymerase chain reaction

Acanthamoeba is a genus of amoeba, one of the most common protozoa found worldwide in soil, and also frequently found in fresh water. In healthy individuals, Acanthamoeba spp. can cause ulcerating keratitis which is often associated with the use of improperly sterilized contact lenses. The aim of this study was to detect Acanthamoeba from fresh water collected from some town squares of Tehran by polymerase chain reaction [PCR]. In this study, 22 samples were collected from fresh water. They were cultured on NNA medium after filtration. Culture samples positive for Acanthamoeba were assessed using polymerase chain reaction [PCR]. Thirteen samples [59%] were recognized as Acanthamoeba on culture. Using species-specific primers which amplified a 903 bp fragment of 188 rRNA, 6 [27%] samples from 13 samples which were positive on culture were identified as Acanthamoeba. Acanthamoeba has been recognized as an etiologic agent of Keratitis in people who use contact lenses and also in immunocompromised individuals. So, detection of this organism in water resources and exact assessment of this parasite could have a significant role in prevention of disease

[Differentiation of Entamoeba histolytica and Entamoeba dispar by single polymerase chain reaction]

In most part of the world detection of cysts and trophozoites of Entamoeba is based on morphological structure of this species in stool sample by microscopy. However, microscopic examination is unable to distinguish between similar morphological protozoa such as Entarnoeba histolytica and Entamoeba dispar. A simple and cost-effective method is needed in medical laboratories for detection and differentiation of these two species. Stool samples of patients who were referred from health care centers were examined by direct microscopy and trichrome stain. Polymerase chain reaction [PCR] utilizing pEd30F and pEd21AS primers from Peroxiredoxin gene, was used for differentiation of E. histolytica and E. dispar. Genomic DNA from samples was amplified by these primers. The fragment under 100 bp was related to E. histolytica and in contrast the fragment above the 100 bp was related to E. dispar. In this study from 22 microscopic positive samples, E. histolytica was observed only in one patient and E. dispar was detected in the other 21 samples. The result of this study indicate that the PCR reaction could amplify E. dispar and E. histolytica with just one primer pair and this is a cost-effective method for distinguishing between these two species

Genetic diversity among entamoeba histolytica and entamoeba dispar strains in gastrointestinal disorder patients in Tehran, Iran

Since the first description of Amebiasis, we still do not have a proper answer to the question of why disease and symptoms develop in only 5 to 10% of those infected with E. histolytica. It has been speculated that a spectrum of virulence levels among the E. histolytica strains contribute to the outcome of amebic infection. In this study, beside determination of prevalence rate of E.histolytica and E.dispar in gastrointestinal disorder patients, genetic diversity in non-coding locus 1-2 was investigated to identify genetic differentiation of Entamoeba in positive isolates. A total of 1700 stool samples were checked from patients referred to clinical laboratories affiliated with Shahid Beheshti Medical University; samples were examined by direct and formalin detergent methods. Twenty seven cases of E. histolytica/E. dispar were detected and total genomic DNA was extracted from stool samples. E. histolytica/E. dispar complex were determined by PCR with two sets of species-specific primers from locus 1-2 gene. The purified PCR products were sequenced and the results were compared with known E. histolytica and E. dispar sequenced data. PCR for locus 1-2 gene amplified a fragment of about 430 bp in 21 out of 27 samples and was identified as E. dispar. One isolate showed a band of about 340 bp and was identified as E. histolytica. PCR were negative in five samples which were discarded. With PCR and sequencing of the PCR products a reliable genetic diversity in size, number and position of the repeat units were seen among the Iranian E. dispar isolates in locus 1-2 gene. Eight new E. dispar genotypes were found in this study and submitted to the Gen Bank/EMBL/DDBJ. The only Iranian E. histolytica isolate [NH1 E.h IR] was completely similar with the KU2 [Accession No. AB075706] strain reported from Japan

analysis of hydatid cyst surgeries in patients referred to hospitals in Khorram-Abad, Lorestan during 2002-06

Echinococcosis or hydatid cyst [HC] is considered as one of the major parasitic infections in Iran that causes many health problems and economic losses in communities. The aim of this study was to determine the prevalence of HC in patients referred to surgery wards of three hospitals in Khorram-Abad, the center of Lorestan province in South-West of Iran from 2002 - 2006. Totally, 64513 medical records of patients referred to surgery wards of Shohadaye Ashayer, Tohid and Taamine Ejtemaee hospitals in Khorram-Abad Lorestan were studied. These patients had gone under surgical operations for different reasons. Among these medical records, 43.7% belonged to Shohadaye Ashayer, 8.2% to Tohid and 18.1% to Taamine ejtemaee hospitals. Cysts were found in liver and lung in 61.5% and 20.5% of cases, respectively. In addition, cysts were found in brain, muscle, kidney eye and peritonea in the remaining 18% of cases. A very low level of knowledge about hydatid disease was found in the community. The mean age of the patients was 40.2 years and the highest rate of infection with HC was observed in women. Further studies are required to find the etiologic factors of H.C in Khorram-Abad Lorestan-Iran