Loop-mediated isothermal amplification assay for identification of clinical Enterococcus species

Background: Enterococci are recognized as a cause of nosocomial infections and a major public health problem. The reliable identification to the species level of enterococci should be considered. Objectives: The study aimed to develop a LAMP assay for the rapid and accurate detection of Enterococcus faecalis and E. faecium. Methods: In total, 57 enterococcal isolates from UTI patients were identified using conventional microbiological methods. Two sets of specific primers were designed for E. faecalis and E. faecium targeting the mtlf and efmC genes, respectively. The LAMP assays were conducted using specific primers, dNTPs, MgSO4, Bst DNA polymerase, and templates. Results: The results of phenotypic testing indicated that of the 57 enterococcal isolates, 49 (85.9) were identified as E. faecalis and eight (14.1) as E. faecium. The optimal reaction temperatures in the LAMP assays were 60 and 61°C for the detection of E. faecalis and E. faecium, respectively. All the 57 enterococcal isolates were identified as E. faecalis by the LAMP assay. Conclusions: The present study highlights the importance of the LAMP assay as a rapid and confirmatory tool for the identification of clinical Enterococcus spp. © 2019, Archives of Clinical Infectious Diseases

Optimization and efficient purification in production of Brucella melitensis recombinant HSP and TF proteins with low endotoxin contents

Background: The development of an effective subunit vaccine against brucellosis is a research area of intense. But optimization of recombinant proteins production in Escherichia coli and content of endotoxins associated with final recombinant proteins are very important. Objectives: In the present study, expression and purification of Brucella melitensis rHSP and rTF were optimized to reduce endotoxin contaminants. Materials and Methods: pDEST-tf and pDEST-hsp were transformed into E. coli BL21 (DE3), and then B. melitensis recombinant HSPA and TF proteins were overexpressed. Purification of these proteins was optimized to remove most of endotoxin contaminants from the end product using 0.1 Triton X-114 in washing buffers. Results: An endotoxin reduction of less than 0.05 EUmg/1 was achieved with protein recovery close to an 80 yield. Conclusions: As this new protocol requires only one step to simultaneously purify tagged proteins and eliminate endotoxins, it represents a substantial advantage in time, effort, and expense. © 2013, Ahvaz Jundishapur University of Medical Sciences; Licensee Kowsar Ltd

Carbapenem-resistant Acinetobacter baumannii recovered from burn patients

Purpose. Emergence of carbapenem-resistant Acinetobacter baumannii (CRAB) and their prolonged presence in burn units increases the risk of acquisition of CRAB. Methods. From November 2012 to September 2013, 1474 burn patients were screened for CRAB isolates through testing susceptibility to imipenem and its comparators meropenem, and doripenem. Furthermore, the in vitro activity of other antibiotics against CRAB was investigated. Results. Three patients were infected with carbapenem-susceptible A. baumannii (CSAB) and 168 were infected with CRAB. Approximately one-fifth (n=32) of CRAB isolates were obtained from patients hospitalized in Burn Intensive Care Unit (BICU). Most of CRABs were isolated from wound. The mean length of stay (LOS) in hospital prior to A. baumannii isolation was significantly higher for CRAB compared to CSAB cases (P=0.04). Amongst the independent variables, percentage of total burn surface area (TBSA) significantly increased the mortality rate using multivariate logistic regression (P=0.001, OR= 16.5; 95 CI: 4.72-57.7). The majority of tested isolates were resistant to imipenem (94.8), and to its comparators, doripenem (97.7), and meropenem (97.7). The susceptibility of CRAB isolates was less than 10 to all tested antibiotics except for colistin (100), doxycycline (61.9), gentamicin (18.5), and tigecycline (11.9). Conclusion. Resistance to carbapenem reduces the number of effective antibiotics. The coordinated and intensive efforts of healthcare personnel are required to meet the challenge of dissemination of CRA

Vaccine potential of lena and lcpa proteins of leptospira interrogans in combination with escherichia coli heat-labile enterotoxin, b subunit (LTB)

Background and Objectives: Leptospirosis is a zooanthroponosis caused by the genus of Leptospira. It is an emerging public health problem due to its increasing incidence. The achievement to a vaccine that prevent from entrance of Leptospira interrogans to the deeper tissues of the host is needed. This study aimed to investigate the immunogenicity of LcpA (rLcpA) and LenA (rLenA) recombinant proteins in combination with LTB (rLTB) recombinant protein as an adjuvant against leptospiral infection in hamsters. Materials and Methods: The genes encoding these proteins were cloned into pGH cloning vector and then lenA, lcpA and ltb genes subcloned into pET-15b and pET-28a expression vectors, respectively. The hamsters were immunized with the purified recombinant proteins and challenged with Leptospira interrogans for evaluation of their survival. The antibody responses to the recombinant proteins were determined by ELISA. Then, data entered into SPSS software. Statistical Kruskal-Wallis test was used to compare the significant differences among different groups. The groups with significant differences were further analyzed by post hoc tests. The p value < 0.05 statistically was considered significant. Results: Immunized hamsters with rLenA-plus-rLTB, rLcpA-plus-rLTB and rLenA-plus-rLcpA-plus-rLTB proteins showed 60, 74, and 80 survival rates, respectively. A significant amount of interleukin-17 (IL-17), interleukin-4 (IL-4) and gamma interferon (IFNγ) cytokines were produced in immunized hamsters. Conclusion: Based on our findings, rLcpA and rLenA proteins in combination with rLTB can protect the hamsters against L. interrogans and effectively induce a protective antibody response. Thus, these proteins can be used as an additional prophylactic tool against leptospira. © 2018, Tehran University of Medical Science. All rights reserved

Molecular detection of prostate specific antigen in patients with prostate cancer or benign prostate hyperplasia the first investigation from Iran

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

Preliminary report of a nationwide case-control study for identifying risk factors of tuberculosis following renal transplantation

Background. Tuberculosis (TB) is an important infection encountered posttransplantation, especially among patients in developing countries, where there are high incidences of morbidity and mortality. Materials and Methods. One hundred and twenty subjects (1) from 15 major kidney transplantation centers in Iran from 1984 to 2003 were compared with 440 controls who were matched for operative time, treatment center, and surgical team. Results. Mean ages of research subjects and controls were 38.6 and 36.6 years (P = .04), respectively. The mean duration of pretransplantation hemodialysis was 29 months (range, 2 to 192 months) in research subjects and 20 months (range, 1 to 180 months) in controls (P = .003). Positive past history of tuberculosis was detected in 4 (3.3) research subjects and in 7 (1.5) controls (P = .2). Fifty-two research subjects (43.3) and 241 controls (54.8) had pretransplantation purified protein derivative of tuberculin less than 5 mm (P = .02). Mean dosages of initial and maintenance immunosuppressive drugs in research subjects and in controls were not significantly different. Sixty research subjects (50) and 152 controls (34.5) had rejection prior to diagnosis of TB (P = .03). Conclusion. To our knowledge, this is the first study that demonstrates an increased risk of posttransplant TB by prolonged duration of pretransplant hemodialysis and number of posttransplant rejection episodes. Further study is needed to clarify these findings specifically with respect to various immunosuppressive regimens. © 2005 by Elsevier Inc. All rights reserved

Factors Associated with Revision Surgery after Internal Fixation of Hip Fractures

Background: Femoral neck fractures are associated with high rates of revision surgery after management with internal fixation. Using data from the Fixation using Alternative Implants for the Treatment of Hip fractures (FAITH) trial evaluating methods of internal fixation in patients with femoral neck fractures, we investigated associations between baseline and surgical factors and the need for revision surgery to promote healing, relieve pain, treat infection or improve function over 24 months postsurgery. Additionally, we investigated factors associated with (1) hardware removal and (2) implant exchange from cancellous screws (CS) or sliding hip screw (SHS) to total hip arthroplasty, hemiarthroplasty, or another internal fixation device. Methods: We identified 15 potential factors a priori that may be associated with revision surgery, 7 with hardware removal, and 14 with implant exchange. We used multivariable Cox proportional hazards analyses in our investigation. Results: Factors associated with increased risk of revision surgery included: female sex, [hazard ratio (HR) 1.79, 95% confidence interval (CI) 1.25-2.50; P = 0.001], higher body mass index (fo

Identification of tigecycline- and vancomycin-resistant Staphylococcus aureus strains among patients with urinary tract infection in Iran

Methicillin-resistant Staphylococcus aureus (MRSA) is one of the major causes of hospital- and community-acquired infections worldwide. Although S. aureus rarely accounts for urinary tract infections (UTI), untreated UTI can lead to several complications. For decades vancomycin has been used for the treatment of MRSA infections. This study was performed to assess the in vitro activity of vancomycin, tigecycline, linezolid and quinupristin/dalfopristin against MRSA isolates from UTI patients. Thirty MRSA strains from 54 S. aureus isolates were isolated from patients with UTI. The antimicrobial susceptibility patterns of the strains were determined by the Kirby-Bauer disk diffusion and broth microdilution methods. PCR assays were used to detect the vanA gene. The MRSA isolates resistant to vancomycin were confirmed using the broth microdilution method. The results revealed that the MRSA isolates were 100% susceptible to linezolid and quinupristin/dalfopristin but 93.3% susceptible to vancomycin and tigecycline respectively. The broth microdilution method confirmed two MRSA strains (6.6%) to be resistant to vancomycin and tigecycline. The study identified vancomycin resistance among the MRSA isolates from UTI patients. This vancomycin resistance in MRSA isolates poses a challenge in managing S. aureus infections. Our study's results highlight the need to correctly identify patients in whom last-resort therapy such as linezolid and quinupristin/dalfopristin should be administered

Methicillin-resistant Staphylococcus aureus tracking spread among health-care workers and hospitalized patients in critical wards at a university hospital, Tehran, Iran

Health-care workers may serve as a reservoir for dissemination of methicillin-resistant Staphylococcus aureus (MRSA) to patients in hospital settings. The present study aimed to screen MRSA in nasal swabs of health-care workers and clinical specimens from patients and investigate the possible relationship between these isolates at a university hospital in Tehran, Iran. Additionally, we aimed to identify potential risk factors for MRSA colonization in health-care workers. Staphylococcus aureus strains were isolated from health-care workers and inpatients who completed a questionnaire on risk factors. Cefoxitin disc diffusion test was also used for detection of MRSA. Moreover, all of the MRSA isolates were subjected to pulsed-field gel electrophoresis (PFGE). Colonization rate of MRSA among health-care workers was 22.5%. Furthermore, out of 24 S. aureus isolates obtained from patients, nine (37.5%) were MRSA. Regarding risk factors, the prevalence of nasal MRSA carriage among hospital personnel who used masks was significantly lower than in those without masks (p 0.007). Using PFGE, 10 clusters and 14 singletons were identified among the MRSA isolates. In this regard, most of the MRSA isolates recovered from health-care carriers and patients in intensive care wards, especially general intensive care units, were grouped in certain clusters, indicating intra-ward transmission of the mentioned isolates in these restricted areas. We concluded that screening and decolonization of carriers, contact precautions, prudent use of antibiotics and implementation of active surveillance are recommended strategies for the prevention and control of MRSA transmission in hospital settings. Keywords: Health-care workers, methicillin-resistant Staphylococcus aureus, patients, pulsed-field gel electrophoresis, risk factor

An investigation of extended-spectrum β-lactamases (ESBLs) in Klebsiella isolated from foodborne outbreaks in Iran

Aims and backgrounds: Antibiotic resistance of Enterobacteriaceae such as Klebsiella which usually caused by Extended-Spectrum β-Lactamases (ESBLs) has been an issue for public health. The aim of this study was to evaluate the presence of ESBLs in Klebsiella isolates obtained from foodborne outbreaks diarrheal samples by phenotypic and genotypic methods. Materials and methods: In this study, 416 diarrheal samples were collected from 120 foodborne outbreaks from March 2017 to March 2018 throughout Iran. Isolation and identification of Klebsiella isolates were performed by phenotypic methods, and antibiotic susceptibility testing was accomplished by disk diffusion method. Production of ESBLs was performed using combined disc method. The presence of blaSHV, blaTEM, and the blaCTX-M genes was investigated by PCR, and the data was analyzed using SPSS version 18. Results: Of 416 diarrheal samples, 32 isolates (7.69) were found positive for Klebsiella, of them 24 isolates (75) were identified as Klebsiella pneumoniae, and 8 isolates (25) as Klebsiella oxytoca. In Klebsiella pneumoniae isolates, the highest susceptibility was seen to imipenem and piperacillin (91.7), while the highest resistance was observed to amoxicillin and penicillin (100). Klebsiella oxytoca isolates were also completely (100) resistant to amoxicillin and penicillin and completely (100) susceptible to other antibiotics. Five isolates were identified as ESBLs positive, phenotypically. Of the 32 isolates, 22 isolates (68.7) were positive for the presence of the blaSHV, 5 isolates (15.6) for blaCTX-M, and all 32 isolates (100) for blaTEM genes. Conclusion: Although Klebsiella isolates are not very common in foodborne outbreaks, but they are important due to the presence of ESBLs introducing high resistance to the common antibiotics. © 202