Abstract

Background and Objective: Staphylococcus aureus is an important opportunistic pathogen causing a wide range of infections in human .Most clinical isolates of S.aureus are resistant to a number of antibiotics. For appropriate antimicrobial therapy, this study was conducted to determine antibacterial drug resistance patterns of S.aureus isolates obtained from different clinical samples in Rasht.

Material and Methods:  the clinical isolates of S.aureus were collected from different clinical laboratories in Rasht. Thirty coagulase positive S.aureus strains were identified using biochemical tests and amplification of 23SrRNA and coa genes by polymerase chain reaction.  Finally, the resistance pattern of the isolates to 16 selected antimicrobial agents was evaluated by disk diffusion method.

Results:  the S.aureus isolates (75%) were resistant to methicillin and all of them were multidrug resistance. The isolates were high resistance to ampicillin (73%), amoxicillin (60%), cloxacillin (53%) and low resistance to vancomycin (7%) and gentamicin (10%).

Conclusion: given the high prevalence of methicillin resistant, multi drug resistant and presence of vancomycin resistant S.aureus isolates in Rasht, continuously monitoring of drug resistance pattern of S.aureus isolates is recommended for having appropriate therapeutic regime.

Keywords: Staphylococcus Aureus, Coagulase, Drug Resistance, PCR

Abstract

Background and Objective: Influenza is an acute respiratory infection caused by Influenza virus with three kinds of A, B and C . This virus  spreads througout  the world   and produce some epidemics with different intensities . This study aimed to determine the prevalence of influenza B in patients reffering to health centers.

Material and Methods: this study was conducted on 878 samples in 2011-2013.  Using PureLink^TM Viral RNA/DNA Kit,  Influenza-RNA was extracted. Then Influenza B was distinguished by using  SuperScript III Platinum, Quantitive Real Time PCR System from Invitrogen^TM , specific primers and probs.

Results: the rate of Influenza B positive was %5.58 of the patients that %55.10 of them were female and %44.89 male. The highest rate was related to 31-40 and 51-60 year old patients.

Conclusion:

given the prevalence of  influenza B virus and lack of genetic changes , it is recommended that a proper vaccine for improving immunty and effective drugs for treatmet be used.

Keywords: Influenza B Virus; Respiratory Tract Infections; Common Cold; RT-PCR

Abstract

Background and Objective: Ophthalmic pterygium is a potentially vision-threatening lesion of unknown etiology that often extends on the corneal surface and has a worldwide distribution. Despite various studies, the pathogenesis of pterygium remains unclear and the involvement of human papillomavirus is controversial. We aimed to investigate the involvement of papillomavirus in pterygium formation.

Material and Methods: This case-control study was conducted on 50 tissue specimens of pterygium from the patients who had pterygium surgery as the case group and 10 conjunctival biopsy specimens of individuals without pterygium including the patients with  cataract surgery, as controls. The evidence of papillomavirus infection was tested by polymerase chain reaction (PCR).

Results: All samples, case and control, were not positive for papillomavirus. Both groups were positive for beta-globulin gene used to check the quality of extracted DNA.

Conclusion: In this study, due to the absence of papillomavirus in the context of Pterygium it seems that other factors are involved in causing the disease.

Keywords: Pterygium; Human Papilloma Virus; PCR.

Abstract

      Background and objectives: More Candida albicans strains are reported resistant to fluconazole in patients with AIDS, cancer and organ recipients. Fluconazole resistance can be attributed to changes in pathways of sterol biosynthesis, mutation in or overexpression of ERG11 and the expression of CDR1, CDR2, and MDR1. This study aimed to compare the expression of CDR1, CDR2, and MDR1 in C. albicans resistant and susceptible to fluconazole.

       Methods: MIC testing for fluconazole was performed on C. albicans isolates isolated from patients with oral and vaginal candidiasis to determine resistant and susceptible strains. Then real time PCR was performed on the resistant and susceptible isolates and the expression of CDR1, CDR2, and MDR1 was compared in C. albicans.

     Results: Of 46 Candida albicans isolates, 20 susceptible isolates, 12 semi-susceptible isolates and 14 resistant isolates were identified by MIC. After real time PCR was performed, Candida albicans isolates susceptible to fluconazole showed moderate expression of CDR1, CDR2, and MDR1 genes, while resistant isolates showed slight or no expression.

      Conclusion: Increased expression of CDR1, CDR2, and MDR1 had less and insignificant role in resistance to fluconazole.

      Keywords: Candida Albicans, Gene Expression, Real time PCR method

ABSTRACT

      Background and Objective: Chlamydia trachomatis is one of the most common causes of genital infection in men and women. Genital chlamydial infections in women are clinically asymptomatic in 70-80% of the cases; therefore, the lack of timely diagnosis and treatment leads to complications such as infertility and ectopic pregnancy. The aim of this study was to evaluate the frequency of chlamydial infection in symptomatic and asymptomatic women in the Golestan province.

      Methods: This cross-sectional study was conducted on 150 cervical swab samples obtained from 150 women referred to the clinic, after obtaining written consent and completion of questionnaires. The swab samples were transferred to laboratory in phosphate-buffered saline solution and DNA extraction was carried out using phenol-chloroform and boiling methods. The frequency of chlamydial infection was evaluated by PCR.

       Results: None of the tested samples were found as Chlamydia-positive.

      Conclusion: The findings require that some more extensive research with larger sample sizes and dispersed population be performed to determine the true prevalence.  Considering the serious complications of chlamydial infections and its asymptomatic nature, a highly sensitive and specific method such as PCR should be used to detect Chlamydia. It is suggested that this method be used along with a complementary test to obtain the results that are more accurate. Furthermore, conducting simultaneous studies on other populations at risk will be very helpful in obtaining representable national data.

 

ABSTRACT

       Background and Objective: Herpes simplex encephalitis is a life-threatening consequence of the central nervous system (CNS) infection with Herpes simplex virus (HSV). Although it is a rare disease, mortality rates reach 70% in the absence of therapy and only a minority of individuals can return to normal function. The aim of this study was to determine possible correlation between HSV infection and the incidence of encephalitis in patients with neurological signs.

        Methods: Overall, 152 CSF samples were tested from patients with neurological signs referred to Mahdieh Clinical Laboratory in Isfahan from 2010 to 2013. After cerebrospinal fluid (CSF) collection, DNA was extracted and real-time polymerase chain reaction (PCR) was performed for HSV detection.

          Results: Of 152 patients tested, 50 were diagnosed with encephalitis. HSV DNA was present in the CSF of 13 patients with encephalitis. HSV was significantly higher (p< 0.05) in patients with encephalitis, which shows the significance of infection as an etiological factor of this disease. About 60% of the encephalitis cases were in age range of 1-24 months.

         Conclusion: According to the findings of the present study, Cesarean section is recommended for HSV-positive mothers. A routine real-time PCR test is suggested for HSV detection in patients with encephalitis to avoid unnecessary antiviral treatments.

       

ABSTRACT

        Background and Objective: Antibiotic-resistant Staphylococcus aureus strains have become a problem in treatment of infections caused by S. aureus. This study aimed to evaluate antibiotic resistance in S. aureus isolates from raw milk and detect femA gene in these isolates, as a confirmatory test for identification of S. aureus species.

        Methods: This cross-sectional study was performed on 110 raw milk samples. After culture in Cooked Meat broth, presence of S. aureus in grown colonies was confirmed in accordance with Iranian National Standard, No. 1194. Antibiotic resistance was then evaluated according to guidelines recommenced by the Clinical Laboratory Standards Institute. FemA-specific polymerase chain reaction was performed on antibiotic-resistant strains using specific primers and standard strains to differentiate S. aureus from other species.

         Results: S. aureus were found in 43 (39.09%) of the 110 collected samples. Among these isolates, 79.07% and 76.75% were phenotypically resistant to penicillin and ceftazidime, respectively. In addition, the femA gene was detected in all isolates.

          Conclusion: The results of this study show a high prevalence of resistance to penicillin and ceftazidime among S. aureus strains isolated from raw milk.

        Keywords: Staphylococcus aureus, Antibiotic Resistance, PCR.

ABSTRACT
            Background and Objectives: Diagnosis of hepatitis E virus (HEV) infection could be missed in some cases if serological tests are used solely. Molecular characterization of HEV is essential for diagnosis of acute and chronic HEV infections, and evaluating the chronic HEV infection status in immunocompromised patients. The aim of this study was to prepare a suitable HEV positive control, determine the limit of detection (LOD) of HEV RNA for a specific molecular test, and evaluate the efficiency and precision of the test.
           Methods: Genomic region of HEV NCBI reference sequence was constructed. LOD, intra-assay precision, and inter-assay precision were calculated to evaluate the efficiency and precision of the test. Then, tenfold serial dilutions of the HEV positive control were prepared. Real time PCR was performed three times for each dilution. Mean, standard deviation, and coefficient of variation of cycle thresholds obtained in three independent and simultaneous tests were calculated, and the results were analyzed.
          Results: The LOD of this test was determined as 1.4×104 copy/ml or 42 copy/reaction or 14 copy/µl. Intra-assay precision and inter-assay precision for all assays were lower than 2.5% and 10%, respectively.
          Conclusion: We propose that the real time PCR assay targeting the ORF2/3 overlapping conserved region is suitable for detection of a wide range of different HEV genotypes found in acute and chronic HEV infections. However, the precision of the test should be improved for detecting HEV RNA lower than 103 copy/ml.
          Keywords: Hepatitis E virus, Limit of Detection, Real Time PCR.

ABSTRACT
        Background and Objectives: Biofilm is a population of bacteria growing on a surface and enclosed in an exopolysaccharides matrix, which increases resistance to antimicrobial agents and immune response. Uropathogenic Escherichia coli (UPEC) are biofilm-forming bacteria and the most common cause of urinary tract infections (UTIs). This study evaluated the effect of different concentrations of glucose, NaCl, blood, serum and urine on biofilm formation and antigen 43 (Ag43) gene expression, as a main gene involved in biofilm formation.
        Methods: Among E. coli isolates from patients with UTI, four extended-spectrum beta-lactamase (ESBL) and non-ESBL strains, and a standard UPEC strain were selected. Biofilm formation of the strains in brain heart infusion (BHI) broth with different concentrations of glucose, NaCl, sheep blood, serum and human urine was evaluated using microplate method and crystal violet staining. Ag43 gene expression was investigated using Real-Time polymerase chain reaction, SYBR Green dye, and specific primers.
           Results: Presence of glucose at all concentrations reduced biofilm formation. Presence of 1% NaCl, 1% sheep blood, 10% bovine serum, and 5% urine significantly increased biofilm formation. Expression of Ag43 by the strains grown under 1% glucose, 1% NaCl, 1% sheep blood, 10% bovine serum and 5% urine decreased.
         Conclusion: All environmental factors other than glucose may increase biofilm formation of E. coli at different concentrations. This is not affected by factors such as isolation from inpatient or outpatients and type of strains (ESBL or non-ESBL). Contrary to our expectations, Ag43 expression is independent of environmental factors and decreases even under the most suitable concentrations.
          Keywords: Biofilms, Uropathogenic Escherichia coli, UTI, Antigen 43, Real-Time PCR.

ABSTRACT
        Background and Objective: Campylobacters are infectious zoonotic agents, and among the main bacterial causes of gastroenteritis in humans. Studies have shown that Campylobacter jejuni is of the main causes of infection among humans. Detection of these infectious agents in water resources is of great importance for maintaining the health of humans. Therefore, the aim of this study was molecular detection of C. jejuni strains in surface water samples collected from Rasht, Iran.
       Methods: This cross-sectional descriptive study was performed on 45 surface water samples collected from the city of Rasht. After culture and isolation of bacteria, the molecular detection of C. jejuni was carried out using hipO-specific primers. Presence of cytolethal distending toxin (cdt) gene in positive samples was evaluated by polymerase chain reaction using cdtC-specific primers.
        Results: Of 45 samples, seven (15.5%) were positive for C. jejuni contamination, five of which (71.4%) had the cdtC gene.
Conclusion: The prevalence of toxin-producing C. jejuni in surface waters of Rasht is notable. Therefore, it is recommended to take necessary measures for controlling the spread of this microorganism.
       Keywords: Campylobacter jejuni, Surface water, cdt gene, PCR.

ABSTRACT
        Background and Objectives: Human cytomegalovirus (HCMV) is the most common viral cause of morbidity and mortality in immunocompromised patients. The aim of this study was to evaluate the frequency of active CMV infection in hemodialysis patients in Gorgan, Iran.
        Methods: Plasma samples were obtained from 149 hemodialysis patients at Hemodialysis Unit of Panje-Azar Medical Centre in Gorgan, Iran. Presence of CMV-DNA in plasma samples was evaluated by polymerase chain reaction (PCR) using specific primers for highly conserved regions of major capsid protein gene of HCMV. In addition, level of CMV-IgM antibody was measured by serological testing. Demographic information and past medical history of patients were also recorded. Data was analyzed by SPSS software (version 18).
       Results: Total prevalence of CMV infection was 6.7% (10/149) among the patients receiving hemodialysis. CMV-DNA and anti-CMV IgM antibody were detected in 2.68% and 4.69%, of the samples, respectively. One case was found positive for both CMV-DNA and anti-CMV IgM antibody. CMV infection did not have any correlation with gender, age, ethnicity, duration of hemodialysis, and history of blood transfusion.
        Conclusion: A notable proportion of hemodialysis patients in Gorgan have active CMV infection. Accurate detection of these individuals is important for preventing infection spread, especially in immunocompromised individuals. Simultaneous diagnosis of CMV infection using serological testing and PCR assay could help reduce the risk of infection spread.
          Keywords: HCMV, Hemodialysis, PCR, Iran.

ABSTRACT
          Background and Objectives: Determining the genetic relationship between S. aureus isolates is important for epidemiological surveillance and control of infections caused by this bacterium. The present study was conducted to determine polymorphisms of coagulase gene (coa) among S. aureus isolates from pastry and cheese samples using restriction fragment length polymorphism (RFLP) analysis.
         Methods: Overall, 65 S. aureus isolated from pastry (n=45) and cheese (n=20) samples were examined for the coa gene by polymerase chain reaction (PCR). PCR products were digested with AluI enzyme and the products were assessed using gel electrophoresis.
          Results: Except for two isolates, all isolates were positive in coa-PCR and produced four different PCR products, with molecular sizes ranging from 570 to 970 bp. Overall; five distinct RFLP patterns were detected (I-V). Although pattern types I and III were present in isolates from both samples, types I and IV were mainly present in isolates from cheese and pastry samples, respectively.
        Conclusion: PCR-RFLP analysis of the coa gene indicates that S. aureus isolates from pastry and cheese samples may be originated from different sources. However, as one pattern type was predominant in each group, it can be concluded that majority of the isolates may have the same origin.
          Keywords: Staphylococcus aureus, PCR-RFLP, Coagulase, Pastry, Cheese.

ABSTRACT
        Background and Objectives: Previous studies have demonstrated the relationship between viral infections and risk of developing type 1 diabetes. The aim of this study was to investigate the frequency of Herpes simplex virus (HSV) in patients with type 2 diabetes and healthy control individuals using PCR and ELISA.
          Methods: Blood samples were taken from 180 diabetic patients and 187 healthy controls referred to the Pasteur medical laboratory in Tonekabon, in 2016. Human beta-globin gene was used as internal control to ensure extraction accuracy. Specific primers were used for amplification of the UL30 gene. In addition, level of anti-HSV IgG antibody was measured using a commercial ELISA kit (Euroimmun, Germany).
         Results: DNA of HSV was found in the samples of 11 patients (6.1%) and five healthy controls (2.7%). In addition, anti-HSV IgG was found in the samples of 117 patients (65%) and 108 healthy controls (57.75%). There was a statistically significant relationship between frequency of anti-HSV IgG and diabetes.
          Conclusion: Similar to previous studies, the present study demonstrated a relationship between frequency of HSV infection and type 2 diabetes. However, further studies should be performed to eliminate the effect of other risk factors to help clarify the exact role of viral infections in increasing the risk of diabetes.
            Keywords: Diabetes, Herpes Simplex Virus, ELISA, PCR.
 

 
 

ABSTRACT
            Background and Objectives: Leptospirosis is a widespread zoonotic disease that is transmitted directly or indirectly from animals to humans. Humans mainly acquire pathogenic leptospires through mucosal or percutaneous exposure to environment contaminated with urine from an infected animal. We aimed to identify pathogenic leptospiral serovars by detection of the ompL37 gene using polymerase chain reaction (PCR).
            Methods: Sixteen pathogenic leptospiral serovars and a saprophytic serovar, L. biflexa were cultured in modified semisolid Ellinghausen-McCullough-Johnson-Harris medium containing 5% rabbit serum. Genomic DNA extraction was done using the phenol-chlorophorm method. The ompL37 gene was amplified using specific primers. PCR products were analyzed by agarose gel electrophoresis.
            Results: The ompL37 gene was amplified only in the pathogenic leptospiral serovars. We detected no amplified fragment for the saprophytic serovar.
Conclusion: Leptospirosis may be confused with other infectious diseases, and therefore, its early and accurate diagnosis is crucial. We showed that molecular detection of pathogenic leptospires based on the ompL37 gene could be used for laboratory diagnosis of leptospirosis.
            Keywords: Leptospirosis, PCR, ompl37 Gene, Pathogenic Leptospires.

ABSTRACT
            Background and objectives: Pterygium is a non-cancerous growth of conjunctival tissue that can extend onto the corneal surface. The presence of some oncogenic viruses in pterygium and the neoplastic nature of these lesions led us to the postulated involvement of the viruses in the etiology of pterygium. Given the association of human herpesvirus 6 (HHV-6) with ocular diseases, we aimed to investigate presence of this virus in pterygium.
            Methods: Fifty tissue specimens were collected from patients with pterygium who underwent pterygium surgery between February 2013 and May 2015. The specimens were tested by real-time PCR using Maxima SYBR Green/ROX qPCR Master Mix (2X) kit. Demographic and clinical data were collected and analyzed using SPSS software (version 18).
            Results: Six (12 %) specimens were positive for HHV-6 DNA. There was no statistically significant correlation between pterygium and presence of HHV-6.
            Conclusion: Based on the results, a direct association between HHV-6 and development of pterygium seems less probable, which suggests that other etiologic agents must be involved in the multistep process of the disease.
            Keywords: Human Herpesvirus 6; pterygium; Real-time PCR.

ABSTRACT
           Background and Objective: Bilophlia spp. are gram-negative, pleomorphic rod, obligate anaerobe, oxidase-negative, catalase-positive and non-motile bacteria. B. wadsworthia is type species of genus Bilophila with the additional characteristic of urea hydrolysis. B. wadsworthia can be found in a variety of anaerobe infections, particularly appendicitis and intra-abdominal infection that are considered as important opportunistic pathogens.
           Methods: This study was designed to identify Bilophila spp. in clinical specimens by culture and PCR. We examined 91 DNA samples extracted from infected appendix tissues with specific primers.
           Results: Data showed that Bilophila spp. DNA existence in 53.85% (n=49) provided appendiceal tissue.
           Conclusion: The pathological and molecular examination of infected appendiceal tissues revealed that B. wadsworthia is able to act as the primary cause of significant lesions in the appendicle tissues.
           Key words: Bilophila spp., Appendectomy, Appendicle specimens, PCR, Nucleotide sequencing