Microbiology
Tahereh Tahmasebi; Rahim Nosrati; Hamed Zare; Horieh Saderi; Reyhaneh Moradi; Parviz Owlia
Volume 11, Issue 4 , October 2016, , Pages 354-362
Abstract
Background: Glutathione (GSH) is a non-protein thiol compound, which plays an important role in the response to oxidative stress and nutritional stress. The aim of this study was to isolate indigenous S. cerevisiae strains capable of effectively produce GSH. Methods: One hundred-twenty sweet fruit samples ...
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Background: Glutathione (GSH) is a non-protein thiol compound, which plays an important role in the response to oxidative stress and nutritional stress. The aim of this study was to isolate indigenous S. cerevisiae strains capable of effectively produce GSH. Methods: One hundred-twenty sweet fruit samples were collected. The strains were isolated on yeast glucose chloramphenicol (YGC) agar medium and identified. The isolates were evaluated for GSH producing on yeast malt (YM) medium. Concentration of glutathione was investigated by recording absorbance of all samples at wavelength 412 nm (Ellman’s method). In addition, optimization of glucose and peptone concentration in culture medium and the effects of various environmental conditions such as temperature (20–35 °C), agitation rate (150–250 rpm), and initial pH (4.0–6.0) were assessed on producing of GSH. Results: From 120 samples, 80 isolates were identified by morphological, biochemical and molecular tests as S. cerevisiae. Five isolates were capable to produce effectively GSH. The optimal culture conditions were agitation rate, 200 rpm; temperature, 30 °C; initial pH, 6; glucose, 30 g/l; and peptone concentration, 5 g/l. In optimal conditions, the amount of derived glutathione was improved compared to YM basal medium and highest GSH concentration (296.8 mg/l) was obtained after cultivation with shaking for 72 h. Conclusion: The possibility of obtaining S. cerevisiae cells with a high GSH intracellular content can be considered an interesting opportunity of furthering the range of application and utilization of this molecule.
Microbiology
Hossein Koupahi; Sahar Honarmand Jahromy; Mohammad Rahbar
Volume 11, Issue 4 , October 2016, , Pages 370-376
Abstract
Background: Methicillin resistant Staphylococcus aureus (MRSA) has been emerged as a nosocomial and community acquired pathogen worldwide. There are many challenges for laboratory detection of MRSA. The aim of this study was to compare different phenotypic methods with PCR based method as a gold standard ...
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Background: Methicillin resistant Staphylococcus aureus (MRSA) has been emerged as a nosocomial and community acquired pathogen worldwide. There are many challenges for laboratory detection of MRSA. The aim of this study was to compare different phenotypic methods with PCR based method as a gold standard for detection of mecA gene. Methods: A total of 220 clinical isolates of S. aureus which were isolated from various clinical specimens from September 2013 until the June of 2014 in Milad Hospital of Tehran, Iran was subject of our study. Methicillin resistance was determined by oxacillin and cefoxitin disks, oxacillin screen agar and CHROMagar™ MRSA medium. The results of these methods were compared with mecA gene based PCR method as a gold standard method. Results: Among 220 isolates from S. aureus, 105 (47.72%) isolates were positive for mecA gene by PCR method. The results of cefoxitin disk diffusion method with 100% sensitivity and specificity was the same as PCR method .CHROMagar™ MRSA medium had 98.13% sensitivity and 100% specificity. Oxacillin disk diffusion and oxacillin screen agar had 95.42% and 97.22% sensitivity respectively. Conclusion: Result of cefoxitin disk diffusion method with 100% sensitivity and specificity was the same as PCR method for detection mecA gene. Cefoxitin disk diffusion method can be used as an alternative method of PCR for detection of MRSA.
Microbiology
Fahimeh Safarnezhad Tameshkel; Mohmmad Hadi Karbalaie Niya; Masuodreza Sohrabi; Mahshid Panahi; Farhad Zamani; Farid Imanzade; Nasser Rakhshani
Volume 11, Issue 3 , July 2016, , Pages 216-221
Abstract
Background: Nowadays, the immune response to hepatitis C (HCV) treatment has become a crucial issue mostly due to the interleukin 28B (IL-28B) polymorphism effects in chronic HCV patients. The aim of this study was to detect the polymorphism of IL-28B gene (rs12979860) in HCV genotype 1 patients treated ...
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Background: Nowadays, the immune response to hepatitis C (HCV) treatment has become a crucial issue mostly due to the interleukin 28B (IL-28B) polymorphism effects in chronic HCV patients. The aim of this study was to detect the polymorphism of IL-28B gene (rs12979860) in HCV genotype 1 patients treated with pegylated Interferon and Ribavirin. Methods: From the 2010 to 2012, a total of 115 peripheral blood mononuclear cells (PBMCs) of HCV patients who presented to Gastrointestinal & Liver Disease Research Center (GILDRC), Firoozgar Hospital, Tehran, Iran were enrolled in this retrospective cross sectional study. Samples were then categorized based on the presence of sustained virologic response (SVR and no-SVR). Variables including age, gender, serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) levels of the two groups were investigated based on different IL-28B genotypes. Results: Analysis by the variables of age and gender showed a mean age ± SD of 42.1±14.0 and gender variability of 44 females (38.2%) and 71 males (61.8%). Adding up these results, the analysis of ALT levels revealed that there was between 293 and 14 mg/ml; AST levels ranged between 217 and 17 mg/ml; the viral load (HCV RNA) ranged between 7,822,000 and 50 IU/ml; the prevalence of CC, CT and TT genotypes were 90.9%, 54% and 25.0%. Conclusion: IL-28B polymorphism has an effective impact on the therapeutic response to ribavirin and peginterferon combination therapy in chronic HCV patients infected by different genotypes. This polymorphism is crucial in natural clearance.
How to cite this article:
Safarnezhad Tameshkel F, Karbalaie Niya MH, Sohrabi M, Panahi M, Zamani F, Imanzade F, et al. Polymorphism of IL-28B Gene (rs12979860) in HCV Genotype 1 Patients Treated by Pegylated Interferon and Ribavirin. Iran J Pathol. 2016; 11(3):216-21.
Microbiology
Roghayeh Teimourpour; Amineh Sadat Tajani; Vahid Reza Askari; Sina Rostami; Zahra Meshkat
Volume 11, Issue 3 , July 2016, , Pages 222-230
Abstract
Background: Hepatitis C virus (HCV) infection is one of the most prevalent infectious diseases responsible for high morbidity and mortality worldwide. Therefore, designing new and effective therapeutics is of great importance. The aim of the current study was to construct a DNA vaccine containing ...
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Background: Hepatitis C virus (HCV) infection is one of the most prevalent infectious diseases responsible for high morbidity and mortality worldwide. Therefore, designing new and effective therapeutics is of great importance. The aim of the current study was to construct a DNA vaccine containing structural proteins of HCV and evaluation of its expression in a eukaryotic system. Methods: Structural proteins of HCV (core, E1, and E2) were isolated and amplified from JFH strain of HCV genotype 2a using PCR method. The PCR products were cloned into pCDNA3.1 (+) vector and finally were confirmed by restriction enzyme analysis and sequencing. The eukaryotic expression of the vector was confirmed by RT-PCR. Results: Recombinant vector containing 2241bp fragment of HCV structural genes was constructed.The desired plasmid was sequenced and corresponded to 100% identity with the submitted sequences in GenBank. RT-PCR results indicated that the recombinant plasmid could be expressed efficiently in the eukaryotic expression system. Conclusion: Successful cloning of structural viral genes in pCDNA3.1 (+) vector and their expression in a eukaryotic expression system facilitates the development of new DNA vaccines against HCV. A DNA vaccine encoding core-E1-E2 antigens was designed. The desired expression vector can be used for further attempts in the development of vaccines.
Microbiology
Faramarz Masjedian Jazi; Gholamreza Irajian; Reza Mirnejad; Vahhab Piranfar; Taghi zahraei salehi; Noor Amir Mozafari; Ehsanollah Ghaznavi-rad; Mahmoud Khormali
Volume 11, Issue 3 , July 2016, , Pages 238-247
Abstract
Background: Brucellosis is an endemic zoonotic disease in the Middle East. This study intended to design a uniplex PCR assay for the detection and differentiation of Brucella at the species level and determining the antibiotic susceptibility pattern of Brucella in Iran. Methods: Sixty-eight Brucella ...
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Background: Brucellosis is an endemic zoonotic disease in the Middle East. This study intended to design a uniplex PCR assay for the detection and differentiation of Brucella at the species level and determining the antibiotic susceptibility pattern of Brucella in Iran. Methods: Sixty-eight Brucella specimens (38 animal and 30 human specimens) were analyzed using PCR (using one pair of primers). Antibiotic susceptibility patterns were evaluated and compared using the E-Test and disk diffusion susceptibility test. Tigecycline susceptibility pattern was compared with other antibiotics. Results: Thirty six isolates of B. melitensis, 2 isolates of B. abortus and 1 isolate of B. suis from the 38 animal specimens, 24 isolates of B. melitensis and 6 isolates of B. abortus from the 30 human specimens were differentiated. The MIC50 values of doxycycline for human and animal specimens were 125 and 10 μg/ml, respectively, tigecycline 0.064 μg/ml for human specimens and 0.125μg/ml for animal specimens, and trimethoprim/ sulfamethoxazole and ciprofloxacin 0.065 and 0.125μg/ml, respectively, for both human and animal specimens. The highest MIC50 value of streptomycin in the human specimens was 0.5μg/ml and 1μg/ml for the animal specimens. The greatest resistance shown was to tetracycline and gentamicin, respectively. Conclusion: Uniplex PCR for the detection and differentiation of Brucella at the strain level is faster and less expensive than multiplex PCR, and the antibiotics doxycycline, rifampin, trimethoprim-sulfamethoxazole, ciprofloxacin, and ofloxacin are the most effective antibiotics for treating brucellosis. Resistance to tigecycline is increasing, and we recommend that it be used in a combination regimen.
Microbiology
Samira Rashidian; Roghayeh Teimourpour; Zahra Meshkat
Volume 11, Issue 2 , April 2016, , Pages 112-119
Abstract
Background: Tuberculosis (TB) remains as a major cause of death around the world. Construction of a new vaccine against tuberculosis is an effective way to control it. Several vaccines against this disease have been developed. The aim of the present study was to cloning of tb10.4 gene in pcDNA3.1+ plasmid ...
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Background: Tuberculosis (TB) remains as a major cause of death around the world. Construction of a new vaccine against tuberculosis is an effective way to control it. Several vaccines against this disease have been developed. The aim of the present study was to cloning of tb10.4 gene in pcDNA3.1+ plasmid and evaluation of its expression in eukaryotic cells. Methods: Firstly, tb10.4 fragment was amplified by PCR and the PCR product was digested with restriction enzymes. Next, it was cloned into pcDNA3.1+ plasmid. Following that, pcDNA3.1+/tb10.4 recombinant plasmid was transfected into eukaryotic cells. Results: 5700 bp band for pcDNA3.1+/tb10.4 recombinant plasmid and 297 bp fragment for tb10.4 were observed. Cloning and transfection were successful and designed recombinant vector was confirmed by sequencing. Conclusion: Successful cloning provides a basis for the development of new DNA vaccines against tuberculosis. In the current study, the aim was cloning of tb10.4 gene in pcDNA3.1+ plasmid and transfection into eukaryotic cells.
Microbiology
Gholamreza Irajian; Mehri Sharifi; Shiva Mirkalantari; Reza Mirnejad; Mohammad reza Jalali Nadoushan
Volume 11, Issue 2 , April 2016, , Pages 138-143
Abstract
Background: In most cases, prostatitis can be caused by a bacterial agent such as Ureaplasma urealyticum. Considering to the cumbersome of the culture method for the detection of Ureaplasma species in clinical samples such as prostate; PCR method that is faster and more appropriate than the cultivation ...
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Background: In most cases, prostatitis can be caused by a bacterial agent such as Ureaplasma urealyticum. Considering to the cumbersome of the culture method for the detection of Ureaplasma species in clinical samples such as prostate; PCR method that is faster and more appropriate than the cultivation methods, can be utilized for the detection of U. urealyticum and U. parvum. PCR-RFLP method can differentiate both biovars and assist in studies of the clinical diagnosis, epidemiology and pathology of this species in human. The aim of this study was to molecular detection of U. urealyticumin in prostate tissue samples based on PCR- RFLP. Methods:Two hundred prostate tissue samples were collected from patient suffering from prostatitis. The PCR assay was used to amplify a 559 bp fragment of 16S-23SRNA interspace region of Ureaplasma. After sequencing, PCR products from positive samples were digested with TaqI restriction enzyme. Results: Seven cases (3.5%) out of 200 prostate tissue samples were positive for U. urealyticum. Results of PCR products sequencing demonstrated that all isolates were U. parvum biovar. PCR-RFLP results shown that there was not any differentiation in pattern of enzymatic digestion, in addition, all isolates were U. parvum, serovar 3. Conclusion: U. urealyticum can be one of the causing agents of prostatitis. Using PCR-RFLP with specific primer and restriction enzyme is a rapid and cost-effect method for detection and differentiation of Ureaplasma from clinical samples.
Microbiology
Sharareh Mohammad Hasani; Reza Mirnejad; Vahhab Piranfar; Jafar Amani; Mohamad javad Vafadar
Volume 11, Issue 2 , April 2016, , Pages 144-150
Abstract
Background: Rapid diagnosis and differentiation of Brucella is of high importance due to the side effects of antibiotics for the treatment of brucellosis. This study aimed to identify and compare PCR-ELISA as a more accurate diagnositc test with other common molecular and serological tests. Methods: ...
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Background: Rapid diagnosis and differentiation of Brucella is of high importance due to the side effects of antibiotics for the treatment of brucellosis. This study aimed to identify and compare PCR-ELISA as a more accurate diagnositc test with other common molecular and serological tests. Methods: In this experimental and sectional study, during March 2014 to Sep 2015, 52 blood samples of suspected patients with clinical symptoms of brucellosis were evaluated in medical centers all over Iran with serum titers higher than 1:80. Using two pairs of specific primers of Brucella abortus, B. melitensis and DIG-dUTP, Fragment IS711 (The common gene fragment in B. melitensis and B. abortus) was amplified. DIG-ELISA was performed using specific probes of these 2 species of Brucella and patterns were subsequently analyzed, then positive responses were compared by detecting gel electrophoresis. Results: PCR-ELISA method detected all 28 samples from 52 positive samples. Its sensitivity was 6.0 pg concentration of genomic DNA of Brucella. In gel electrophoresis method, 22 samples of all positive samples were detected. PCR-ELISA was more efficient than PCR and bacterial culture method at P-value Conclusion: PCR-ELISA molecular method is more sensitive than other molecular methods, lack of mutagenic color and also a semi-quantitative ability. This method is more effective and more accurate compared to PCR, serology and culture of bacteria. PCR-ELISA does not have false responses. The limitation of this method is detection of bacteria in the genus compared to Multiplex PCR and Gel Electrophoresis.
Microbiology
Faezeh Ghasemi; Sina Rostami; Maryam Sadat Nabavinia; Zahra Meshkat
Volume 11, Issue 1 , January 2016, , Pages 41-46
Abstract
Background: Human papillomavirus (HPV) is responsible for the development of cervical neoplasia. Infection with human papillomavirus type 16 (HPV-16) is a major risk factor for the development of cervical cancer. The virus encodes three oncoproteins (E5, E6 and E7), of which, the E7 oncoprotein ...
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Background: Human papillomavirus (HPV) is responsible for the development of cervical neoplasia. Infection with human papillomavirus type 16 (HPV-16) is a major risk factor for the development of cervical cancer. The virus encodes three oncoproteins (E5, E6 and E7), of which, the E7 oncoprotein is the major protein involved in cell immortalization and transformation of the infected cells. The aim of the current study was to develop Michigan Cancer Foundation 7 (MCF7) cells, which could stably express E7 protein of HPV type 16. Methods: E7 gene of HPV type 16 was introduced into MCF7 cells by Lipofectamine 2000 reagent and the transfected cells were treated with G418 antibiotic. After antibiotic selection of the transfected cells, stable expression of E7 gene of HPV16 was confirmed by reverse transcriptase polymerase chain reaction (RT-PCR). Results: Antibiotic selections of transfected cells were performed and transfected cells were alive in cytotoxic concentration of the antibiotic. RNA was extracted from transfected cells and E7 gene of HPV16 was amplified by RT-PCR method and a 350-bp band corresponds to E7 was observed. Conclusion: Results confirmed the stable transfection of cells. The stably transfected cells can be used as a useful tool in future studies on HPV16 and cancers caused by this virus.
Microbiology
Fatemeh Rezaei; Horieh Saderi; Shahram Boroumandi; Soghrat Faghihzadeh
Volume 11, Issue 1 , January 2016, , Pages 47-53
Abstract
Background: In order to select a better antibiotic choice for treatment of Pseudomonas aeruginosa infections, this study was conducted to determine the frequency of resistance to some antipseudomonal β-lactams in P. aeruginosa isolates from patients in Tehran, Iran. In addition, the relation between ...
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Background: In order to select a better antibiotic choice for treatment of Pseudomonas aeruginosa infections, this study was conducted to determine the frequency of resistance to some antipseudomonal β-lactams in P. aeruginosa isolates from patients in Tehran, Iran. In addition, the relation between presence of genes known to be responsible for resistance to β-lactams (ampC, mexC1,2,and mexC3,4 genes) and resistance phenotype among P. aeroginosa isolates was evaluated. Methods: P. aeruginosa strains were isolated and identified by routine methods and PCR for oprL gene. Disk diffusion method was employed to determine the antimicrobial susceptibility pattern according to CLSI recommendations. PCR was used to detect the resistance genes. Results: Among 100 isolates of P. aeruginosa, 82% had ampC, 86% mexC1,2and 89% mexC3,4 genes and combinations of these genes were seen in most of isolates and only 3% of isolates had none of these genes. Resistance to mezlocillin, cefepime, ceftazidime and piperacillin/ tazobactam was seen in 46%, 41%, 36% and 29% of isolates, respectively. Significant relation (P value ≤0.05 by Chi-square or Fisher Exact test) was observed between the presence of ampC gene and resistance to all the studied β-lactams in this study. No relation was observedfor mexC genes,although many ofisolates containing these two genes were phenotypically resistant. Conclusion: This study had shown for the first time, the presence of ampC and mexC genes in significant percent of clinical isolates of P. aeruginosa in Tehran, Iran, and relation between presence of ampC gene and resistance to β-lactams.
Microbiology
Abdolmajid Ghasemian; Shahin Najar Peerayeh; Bita Bakhshi; Mohsen Mirzaee
Abstract
Background:Isolates of Staphylococcus aureus express a myriad of adhesive surface proteins that play important role in colonization of the bacteria on nasal and skin surfaces, beginning the process of pathogenesis. The aim of this study was to screen several of the Microbial Surface Components Recognizing ...
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Background:Isolates of Staphylococcus aureus express a myriad of adhesive surface proteins that play important role in colonization of the bacteria on nasal and skin surfaces, beginning the process of pathogenesis. The aim of this study was to screen several of the Microbial Surface Components Recognizing Adhesive Matrix Molecules (MSCRAMMs) genes among the isolate of S. aureus from hospitalized children.
Methods:A totalof 22 S. aureus isolates were collected from hospitalized children in Tehran from 2012 to 2013. Detection of the mecA and several adhesive surface proteins genes including clfA, B (encoding clumping factors A, B); fnbA, B (encoding finronectin binding proteins A, B); fib (encoding fibrinogen binding protein); eno (encoding laminin binding protein); cna (encoding collagen binding protein); ebps (encoding elastin binding protein) and bbp (encoding bone sialo-protein binding protein), was performed by PCR.
Results: The clfAB genes were detected among all the isolates. The prevalence of fnbA, fnbB, fib, eno, cna, ebps and bbp was 63%, 6%, 50%, 59%, 82%, 63%, 9% and 0%, respectively.
Conclusion: The high prevalence of these genes is important for future plans in vaccine designation. MRSA and MSSA isolates similarly can produce adhesive surface proteins for colonization.
Microbiology
Horieh Saderi; Parviz Owlia
Abstract
Background: This study was done to detect multidrug resistant (MDR) and extremely drug resistant (XDR) of Pseudomonas aeruginosa among strains isolated from patients in Tehran, Iran, due to importance of these phenotypes in treatment of human infections. Methods: Eighty eightP. aeruginosa were isolated ...
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Background: This study was done to detect multidrug resistant (MDR) and extremely drug resistant (XDR) of Pseudomonas aeruginosa among strains isolated from patients in Tehran, Iran, due to importance of these phenotypes in treatment of human infections. Methods: Eighty eightP. aeruginosa were isolated from patients in Tehran, Iran, and identified by routine methods and PCR for oprL gene. Their antimicrobial susceptibility to 16 antimicrobial agents from 7 antimicrobial categories (aminoglycosides, carbapenems, cephalosporins, fluoroquinolones, penicillins/ß-lactamase inhibitors, monobactams, polymyxins) were determined by disk diffusion method, according to recommendation of Clinical and Laboratory Standards Institute. Characterization of P. aeruginosa isolates as MDR and XDR was done according to standardized international terminology presented by European Centre for Disease Prevention and Control as well as the Centers for Disease Control and Prevention in 2011. MDR was defined as acquired non-susceptibility to at least one agent in ≥3 antimicrobial categories and XDR was defined as non-susceptibility to at least one agent in ≥6 antimicrobial categories. Results: The rates of susceptibility to antimicrobials were as follows: gentamicin 27.3%, tobramycin 54.5%, amikacin 56.8%, netilmicin 36.4%, imipenem 55.7%, meropenem 55.7%, doripenem 60.2%, ceftazidime 63.6%, cefepime 56.8%, ciprofloxacin 59.1%, levofloxacin 60.2%, ticarcillin-clavulanic acid 37.5%, piperacillin-tazobactam 63.6%, aztreonam 43.2%, colistin 90.9%, polymyxin 95.5%. Altogether, 48 (54.5%) and 29 (33%) isolates were characterized as MDR and XDR, respectively. Discussion:The high frequency of antibiotic resistance in clinical isolates of P. aeruginosa in Iran makes epidemiological surveillance of susceptibility of this bacterium more essential for the best selection of empirical antibiotics.
Microbiology
Massoud Hajia; Ali Akbar Amirzargar; Mina Nazari; Neda Razavi Davodi; Morteza Karami Zarandi
Abstract
Background: Tuberculous meningitis (TBM) is a severe form of extra pulmonary tuberculosis with high mortality and morbidity rate in all age group patients specific in adults and children. The incidence and prevalence are not exactly known in Iran. In this study, we tried to evaluate the role of rapid ...
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Background: Tuberculous meningitis (TBM) is a severe form of extra pulmonary tuberculosis with high mortality and morbidity rate in all age group patients specific in adults and children. The incidence and prevalence are not exactly known in Iran. In this study, we tried to evaluate the role of rapid diagnosis and to find out the highest risk group patients.
Methods:Totally, 1783-suspected patients with tuberculous meningitis whose CSF specimens were admitted at Noor Pathobiology Laboratory, Tehran, Iran were enrolled in this study from January 2009 until December 2013.All specimens were checked for MTB by direct examination, culture and PCR tests, andfor the adenosine deaminase (ADA).
Results:Confirmed positive cases were aged from 13 to 82 yr old with mean age 46.63 yr (SD±18.84). The number of diagnosed positive MTB was different by the 3 applied protocol, 64 by PCR, 28 by culture and 33 by direct examination. Considering the result of PCR protocol theTBM was approved in 64 patients with rate of 3.59%. Two patients had other infection as well, one 56 years old with VZV and the other patient who was HIV positive was 27 years old. Increased ADA titer higher than cutoff was relevant with other results of positive samples except in two cases.
Conclusion:Analysis of the results proved adults are more at risk for tuberculous meningitis than children in Iran are. It is also confirmed PCR method provide the most efficient, rapid and reliable results for these patients who are at the critical situations.
