Journal of

Background and Aim: Yersinia enterocolitica is a Gram-negative bacterium which its strains are involved in human diseases. To differentiate among pathogenic and non-pathogenic types, tests such as Congo Red absorption, Crystal Violet, and Calcium Dependency test are used. These tests are based on existence of 70-75 kb plasmids and sometimes, with respect to plasmids instability, we will face false negative results. Therefore, by setting up a methodology based on stable chromosomal genes of pathogenic agent we can overcome this hurdle. The goal of this survey was comparison among routine and molecular diagnostic approaches in the identification of Y. enterocolitica pathogenic strains.

Materials and Methods: Some Gram-negative bacteria from family Enterobacteriacea and some Y.  enterocolitica strains isolated of human beings and environment were evaluated.

Results: Obtained results showed that 4 Y. enterocolitica strains isolated of human beings were PCR positive while PCR results of environmental strains, one human strain and non-Yersinia strains were negative.

Conclusion: The mentioned approach can be used as a method to differentiate among pathogenic and non-pathogenic strains of Y. enterocolitica.

Background and Aim: Acute promyelocytic leukemia (APL) is associated with the t(1517) ,fusing promyelocytic leukemia (PML) and retinoic acid receptor-a (RARa) genes. This disease is uniquely sensitive to treatment with all-trans retinoic acid (ATRA) and highly responsive to conventional chemotherapy. The t(1117)(q23q21) abnormality associated with a PLZF-RARa rearrangement is the commonest of the alternative translocations accounting for less than 1% APL. Blasts from PLZF-RARa cases have been found to be resistant to the differentiating effects of retinoids. In this study we aimed to determine the PLZF-RARa and fusion genes in patients with APL morphology who referred to Hematology-Oncology and BMT research center, Tehran Shariaty Hospital in 2006.

Materials & Methods: Peripheral blood and/or bone marrow samples were taken from 200 patients with APL morphology and 200 patients with other subtypes of AML. The mono-nuclear cells were enriched by centrifugation over a ficoll-isopaque gradient. RNA extracted by Trizol or TriReagent and then reverse transcribed to cDNA using random primers. PCR performed using specific primers for each fusion. PCR products electerophoresed on a 2% agarose gel containing 0.05% ethidiume bromide.

Results: In 2 (1%) patients with APL-morphology the RT-PCR analysis showed PLZF-RARa fusion transcripts.

Conclusion: It can be concluded that RT-PCR is a rapid and sensitive method for detection of abnormal fusion genes in leukemia and allows the definition of a correct strategy for treatment and subsequent minimal residual disease monitoring

Background and Aim: Chronic Myelogenous Leukemia (CML) is a clonal hematopoietic stem cell disorder characterized by a translocation between chromosome 9 and 22 called Philadelphia Chromosome. Telomerase- essential enzyme that adds telomeric repeats into the telomeres- maintains integrity of  chromosomal ends. Most normal somatic cells do not express hTERT (catalytic subunit of telomerase) but most neoplasia and cancer cells express it. In this study we evaluated the hTERT expression in patients with CML at different phases of the disease.

Materials and Methods: In this cross sectional study, 73 samples of 45 patients with CML were studied. Twenty six of samples were taken from patients in chronic phase before therapy and 26 samples three month after therapy. Nine samples were taken in accelerated phase and 12 in blastic phase. hTERT expression was studied by RT-PCR and the results were analyzed using SPSS 15.

Results: Thirty three (73%) of patients were male and 12 (23%) were female. Patients were divided into three age groups 17-29, 30-40 and 41-75 years. Of 73 samples, 43 samples (58.9%) were positive for hTERT and 30 samples (41.1%) were negative for this gene. In chronic phase (before therapy) 69.2% of the samples were PCR positive, but after therapy only 38.5% of samples were PCR positive. In accelerated and blastic phases, 55.6% and 83.3% of samples were PCR positive respectively. The hTERT positivity was differently significant (p<0.05) among different phases of the disease.

Conclusion: Significant difference between hTERT expression in different phases of CML disease can be used as a useful molecular marker for fallowing up, prognosis and disease progression after treatment

Background & Aim : Chronic myeloid leukemia (CML) is a disorder of pluripotential hematopoietic stem cell that is as a myeloproliferative disease and occurs in about 15 percent of all leukemia. Two cell cycle regulatory proteins that function as tumor suppressor are P16INK4A and P14ARF. The origin of these two proteins is a human INK4A-ARF gene locus that located on chromosome 9p21. P16INK4A control retinoblastoma (Rb) and P14ARF control with p53 thought negative feedback. The purposes of this study, this was that whether these genes are preferable use as a factor in prognosis and progression of disease.

Materials and Methods: This research was a Cross sectional study.  The expression of p16INK4A and p14ARF mRNA in about 73 peripheral bloods (PB) Samples were collected from 45 CML patients at different phases of disease were assayed by reverse transcriptase polymerase chain reaction (RT-PCR). 26 samples were from patients at chronic phase before any treatment, 26 samples 3 month after treatment with imatinib, 9 samples in accelerated phase and 12 samples in Blastic phase.

Results. From 45 patients with CML, 33 patients (73%) were men and 12 patients (27%) were women. About 26 samples (35%) were p16INK4A positive and 55 samples (75%) were p14ARF RT-PCR positive. This expression of the two genes at different phases of disease were not statistically significant (p>0.05).

Conclusion: High percentage of the CML patients expressed P14ARF and P16INK4A genes. The expression of these gene at different phases of disease (diagnosis, accelerate, and Blastic phases) was not statistically significant even though, the expression of these genes was higher after the treatment.  The increased expression of these genes was probably because of the Imatinib treatment.

Background and Aim : Myeloproliferative neoplasms are clonal and heterogeneous disorders of hematopoietic stem cells lead to increase of one or more cell lines in the blood. Recently, the acquired mutation JAK2 V617F has been described in the majority of patients with myeloproliferative neoplasms (MPNs).This mutation is characterized by a G to T transverse at nucleotide 1849 in exon 12 of the JAK2 gene, located on the chromosome 9p, leading to a substitution of valine to phenylalanine at amino acid position 617 in the JAK2 protein. The aim of this study was to assess the prevalence of JAK2 mutation in MPN patients.

Materials and Methods: In this experimental study we evaluated JAK2 mutation in 58 patients with MPNs by simple randomized sampling. The mutation was detected by ARMS-PCR in patients.

Results: The JAK2 V617F mutation was detected in 86.6% (26/30) of patients with polycythemia Vera, 46.6% (7/15) of patients with essential thrombocythemia and 61.5% (8/13) of patients with idiopathic myelofibrosis. Polycythemia Vera patients carrying the mutation displayed a higher levels of WBC (p=0.03) and 61.5% (16/26) of these patients were females. The differences in other groups were not significant. The mutation was confirmed by sequencing.

Conclusions: Our Results show similarity with other studies. Thus, ARMS-PCR can be applied as differential diagnosis test for detection of JAK2 mutation in suspected patients with MPNs.

Background and Aim: Thalassemia is a genetic disease whit relative or complete lack of alpha or beta globin chain. Patients with moderate and severe form need to have multitransfusion in early life. Occurrence of all immunization against blood group antigens in patients with thalassemia is relatively high and may have difficulties in treatment and transfusion. Antibody production against this blood groups cause lots of problems like preparation of compatible blood for transfusion. Correct diagnosis of blood group phenotype due to existence of dual population of donor and patient RBC would be difficult.

Materials and Methods: In this study, randomly from 40 thalassemic patients, before blood transfusion, 4cc of peripheral blood collected in tubes containing EDTA. Also, 10 healthy individuals who had no history of blood transfusion were in the study as control for serological (agglutination) and molecular results . Phenotyping of patients and control group was done by tube agglutination method by commercial antibody. For molecular test, Allele Specific Primer Amplification (ASPA) PCR was performed for each antigens in separate micro tubes.

Results: In this study' we could set up a method that can amlify any of Rh C c E and E gene separately by a pair of specific primer in a same thermal conditions.Comparison of results of two methods showed that in the control group with no transfusion history. Similar results in phenotyping and genotyping was observed. But in patients, results of two methods had lots of differences.

Discussion and Conclusion: The advantage of this method over PCR-RFLP method is that' all four genes can be amplified the in a same concentration and temperature conditions, and ability to determine the individual's antigen, immediately by electrophoresis. Therefore, Since the above method is simple and inexpensive for medical and research centers it is recommended.

Background and Aim: The JAK2 is an acquired mutation that is observed in majority of patients with classical Philadelphia-negative Myeloproliferative neoplasms that include polycythemia vera (PV), essential thrombocythemia (ET), primary myelofibrosis (PMF). This acquired mutation is characterized by a G to T transversion at nucleotide 1849 in exon 12 of the JAK2 gene, leading to a substitution of valine to phenylalanine at amino acid position 617(V617F) of the JAK2 protein, and result in constitutive JAK2 activation that promotes hypersensitivity to growth factors and cytokines.

Materials and Methods: In this study we evaluated RNA from 58 patients with MPNs and statistical analysis was done with mann whitney test. The mutation detected by AS-PCR. In addition, 3 samples were sequenced in Mille gen company.

Results: 46 patients:86.6%(26/30) of those with polycythemia vera, 53.3% (8/15) of those with essential thrombocythemia,61.5% (8/13) of those with idiopathic myelofibrosis polycythemia vera patient carrying the mutation displayed higher levels of WBC (p=0.03). on the other hand,16 out of 26 JAK2V617F positive patients were female there is a demonstrate correlation between the presence of a mutant allele and female gender. The difference in other groups were not significant.

Discussion and Conclusion: The JAK2V617F mutation has been detected in the vast majority of patients with polycythmia vera (65-95%) and in a lower frequency in patients with essential thrombocythemia (23-57%), idiopathic myelofibrosis (23-57%) and chronic myeloid leukemia 19% (3/16 CML Ph-). Detection of the mutation is helpful in differential diagnosis, prognosis, and prediction of therapeutic response.

Background and Aim: Enteroinvasive Escherichia coli (EIEC) strains include a group of diarrheagenic Escherichia coli (DEC) and are known to cause shigellosis-like symptoms in both adults and children. They belong to a limited number of serotypes and their somatic (O) antigens are identical with, or related to, certain Shigella antigens. EIEC strains are confirmed by demonstration of invasiveness by polymerase chain reaction (PCR) for detection of the ipaH (invasive plasmid antigen H)  gene that is specific for these strains among DEC.Since in our country,Iran study for detection of  these strains. hasnot been carried out therefore the aim of this study was detection of EIEC in diarrheal under 5 year old children in Tehran.

Materials and Methods: During the descriptive study,300stool samples were collected from children with diarrhea in Ali Asghar Hospital and children medicinal center of Tehran during 4 months (April-Jul 2008). E.coli species were isolated by standard bacteriological and biochemical tests. Presence of invasive plasmid antigen H (ipaH) gene in confirmed colonies was investigated by PCR technique.

Results: Among 300 stool specimens studied using culture method and biochemical tests,39(13%) E.coli species were isolated. Among these 39 strains,7(2.3%) strains containing ipaH gene (EIEC) were detected by PCR technique.

Conclusions: Enteroinvasive Escherichia coli (EIEC) in our country, Iran, may be as bacterial pathogen causing childhood diarrhea. Therefore we should apply new techniques for investigation of these strains.

Background and Aim: Group B streptococcus(GBS)(Streptococcus agalactiae) is the leading cause of morbidity and mortality of the newborn infant and accounted as a factor leading septicemia after birth in mothers. Infections in infants are usually acquired by contact with the genital tract of the mother during labor and delivery. So a rapid screening test for group B streptococcus that could accurately identify pregnant women who are carrying the bacteria at the time of delivery would obviate the need for prenatal screening.The goal of this study was molecular epidemiology of group B beta Hemolytic Streptococcal(GBS) colonization in the vaginal flora of pregnant women.

Materials and Methods: Samples were taken from mucus of anal and vaginal of 250 pregnant women during 35-37 week's ingestion by swap. Samples were tested by standard culture using Todd Hewitt Broth and Blood Agar and also by PCR using cfb gene.

Results: Culture identified 21(8.4%) women as carriage of GBS from 250 women but PCR assay could identify 24(9/6%) women. In comparison to culture results, sensitivity, NPV Specificity PPV of PCR Were(100%, 100% and 97%, 82%) respectively. The times that used for PCR assay and culture were 2h and 36h respectively.

Conclusion: In conclusion, we found that group B streptococci can be detected rapidly and reliably by a PCR assay of combined vaginal and anal secretions from pregnant women at the time of delivery. Also this study shows that incidence of GBS is at high rate in Iranian pregnant woman, so we recommend screening of pregnant woman for detecting of GBS emphatically.

Background and Aim: Aspergillosis are the most prevalent cause of the respiratory infections. These fungi show invasive aspergillosis(IA) in immunocompromised patients. The number of immunocompromised patients are increasing due to immunodisorder illnesses, grafts and immunosuppressor drugs, so, rapid identification methods are very important. The aim of this study was to detect the Aspergillus spp. In fluid samples by nested PCR, and compare with culture and direct smear.

Materials and Methods: Conventional detection methods such as culture and direct smear are unsensitive and time consuming. Some methods such as immunodetecting methods have high false positive and are unreliable. Nowadays, molecular methos and PCR are very helpful. These methods are both sensitive and reliable and very rapid. In this study, we used Nested PCR, culture and direct smear to detect Aspergillus spp in BAL fluid samples.

Results: This research is a descriptive-comparative study and has been done for rapid identification of fungi related to Aspergillosis such as culture, direct smear and nested- PCR. Findings of this study show that positive results by nested-PCR were more effective and sensitive than culture and direct smear.
Conclusions: We found that positive results by PCR were more effective and sensitive than two other methods.

Background and Aim: Tumor dissemination via blood to distant organs is the main cause of death. Therefore, there is a critical need to set up sensitive methods for the early detection of circulating tumor cells(CTCs) and disseminated tumor cells(DTCs) in peripheral blood (PB) and bone marrow(BM) specimens of breast cancer patients. The aim of this research is to study the detection of micrometastasis using MUC2 in such patients.

Materials and Methods: In this study, PB and BM samples were collected from 50 breast cancer patients after operation and before adjuvant therapy. Mucin 2 (MUC2) was used as a tumor marker and its transcript level in the sample patients was analyzed using gene specific, quantitative real-time PCR reaction with SYBR Green technology. Samples from 20 healthy individuals were used as negative controls. HPRT was used as a reference gene.

Results: MUC2 mRNA was detected in 8 (16%) of PB and BM samples. MUC2 mRNA was not detected in PB samples of healthy individuals. The relapse rate among MUC2-positive patients was higher than MUC2-negative patients and it was statistically significant in BM (P<0.05).

Conclusion: This study shows that MUC2 can be a suitable marker for the detection of micrometastasis in breast cancer patients at early stages of cancer and that it may provide the basis for identifying women at risk of relapse.

Background and Aim: ESR1 gene polymorphism has been found to be associated with breast cancer and clinical features of the disease in Caucasians. Genomic data for ESR1 in either population is therefore of value in the clinical setting for that ethnic group. In this study association of polymorphism in ESR1 gene with breast cancer risk was investigated.

Materials and Methods: A case-control study was conducted to establish a database of ESR1 polymorphisms in Iranian population. The ESR1 gene was scanned in Iranian patients newly diagnosed with invasive breast tumors, (150 patients) and in healthy individuals (147) (healthy control individuals). PCR single-strand conformation polymorphism technology and direct sequencing was performed.

Results: The frequency of heterozygote genotype in exon 8 (ACG → ACA / ) was significantly higher in breast cancer patients (48.0%) than in control individuals (1.4%). We found that mutant allele (ACA) was significantly more common in breast cancer patients with age at menarche

Conclusion: Our data suggest that ESR1 polymorphisms are correlated with various aspects of breast cancer in Iranian ESR1 genotype, as determined during pre-surgical evaluation, might represent a surrogate marker to increase predicting breast cancer in Iranian population.

Background and Aim: Onychomycosis or nail fungus infection has an increasing prevalence with many effects on patients’ social life and mental health dermatophytes, yeasts and non-dermatophyte molds are among the best known agents of fungal infections of nails. The purpose of this study was to determine the prevalence of non-dermatophyte molds using morphological (direct examination and culture) and molecular (PCR) methods in patients referring to Medical Sciences Mycology Laboratory in Tehran, Iran.

Materials and Methods: In this study, samples were taken from 170 patients. For direct microscopic examination (DME), 15% KOH solution was used for the culture of samples, Sabouraud dextrose agar media (S) was applied together with chloramphenicol (SC) and chloramphenicol and cycloheximide (SCC). Meanwhile, differential tests were done for mycological diagnosis (slide culture), and 28SrDNA amplification and sequencing were performed for suspect or unknown samples.

Results: Of the 170 patients, 74 cases (43.5%) had onychomycosis, of which 53 cases (71.62%) were female and 21 cases (28.38%) were male. Also, of the 74 cases of onychomycosis, 40 cases (54.05%) were reported candidiasis, 21 cases (28.37%) non-dermatophyte molds, and 12 cases (16.21 %) dermatophytes.

Conclusion: The prevalence of onychomycosis in this study was 43.5% and the application of polymerase chain reaction (PCR) technique in cases of false positive, false negative and long-term culture was valuable meanwhile, given that all the samples that had positive results in DME with negative cultures were positive in molecular tests, this study reveals the power of molecular techniques compared with culture method.