Echocardiographic assessment of myocardial infarction: comparison of a rat model in two strains

The purpose of this study was to induce myocardial infarction [MI] and compare the echocardiographic parameters and mortality ratio of Lewis inbred and Wistar outbred strain before and after the procedure to help choose the best one for MI studies. In this study MI was induced in 46 Lewis and 34 Wistar by occlusion of left anterior descending artery [LAD]. Doppler, two-dimensional [2-D] and 2-D guided M-mode images were recorded from parasternal long-axis and parasternal short-axis and apical four-chamber views. The following parameters were acquired. Interventricular septum diastolic and systolic dimension [IVSd, s], diastolic and systolic left ventricular internal diameter [LVIDd, s], diastolic and systolic left ventricular posterior wall dimension [LVPWd, s], ejection fraction [EF], and fractional shortening [FS]. The significant changes were observed in systolic IVS, LVID and EF and FS before and after MI and no significant difference was detected between Lewis and Wistar. The high mortality rate of 51% was seen in the procedure, including anesthesia in Lewis compared to 34% in Wistar. As a conclusion the echocardiographic parameters of these two strains were similar, but according to mortality rate and more cardiac anatomic variation in Lewis rats, Wistar is better for MI studies

Detection of human Papillomavirus 18 in cervical cancer samples using PCR-ELISA [DIAPOPS]

Background and Objectives: Human Papillomavirus [HPV] infection is a major risk factor for adenocarcinoma of the cervix. The high-risk types of the virus such as HPV16 and HPV18, which possess the E6 and E7 oncogenes, are responsible for approximately 50% of all cervical cancers. A rapid, sensitive and specific test has been proposed for detection of HPV to improve cervical cancer screening programs. The aim of this study was to develop a fast PCR-ELISA assay designated as DIAPOPS [Detection of Immobilized Amplified Products in a One Phase System] for detection of HPV16 and HPV18 types in SCC samples and Pap smears. The type specific primers and probes were designed for PCR and PCR-ELISA. The amplified products were hybridized with a specific biotin-labeled probe for HPV18 inner amplicons. The hybrids were detected with peroxidase conjugated avidin. The test was performed on the paraffin block and Pap smear samples from the cervical cancer patients, and the results of DIAPOPS were compared with conventional PCR assay. The 70 samples [SCC and Pap smear samples] were collected from Imam Khomeini and Mirzakoochak Khan Hospitals in Tehran. The PCR-based method detected six HPV16 positive, three HPV18 positive and Two HPV33 positive samples. DIAPOPS results were compared with the conventional PCR results and they showed an increase in sensitivity of the DIAPOPS test. Not only all of them were confirmed by PCR-ELISA but also three samples that conventional PCR showed negative for HPV18, were demonstrated positive by the PCR-ELISA method. The results of the study show that modified PCR-ELISA assay is more sensitive to detect HPV types and can be used for diagnostic purposes

Quantitative comparison of NASBA-ELISA and RT-PCR-ELISA sensitivities for measurement of the BCR-ABL genes fusion transcript in CML patients

Quantitative comparison of NASBA-ELISA and RT-PCR-ELISA sensitivities for measurement of the BCR-ABL genes fusion transcript in CML patients Nazemi A1, Sadeghizadeh M2, Forouzandeh Moghaddam M3, Javadi Gh4, Hashemi M5 1 Student of PhD of Molecular Genetics, Department of Biology, Islamic Azad University, Science and Research Campus, Tehran, Iran. 2Associate Professor, Department of Genetics, Tarbiat Modarres University, Tehran, Iran. 3 Associate Professor, Department of Medical Biotechnology, Tarbiat Modarres University, Tehran, Iran. 4 Associate Professor, Department of Biology, Islamic Azad University, Science and Research Campus, Tehran, Iran. 5 Assistant Professor,Department of Molecular Genetics, Islamic Azad University, Tehran Medical Branch, Tehran, Iran. Chronic myeloid leukemia [CML] is characterized by neoplastic overproduction of myelocytes and neutrophils. Affected patients have a Philadelphia chromosome which arises following a translocation between long arms of chromosome 9 and 22 [q34; q11]. This results in abelson murine/breakpoint cluster region [BCR/ABL] fusion. Detection of cells carrying BCR/ABL fusion is extremely important in monitoring response to treatment, remission and relapse in CML patients. In this study, we compared RT-PCR and NASBA techniques to determine quantitatively the number of bcr/abl transcripts. Fusion transcripts were synthesized and RNA was extracted from K562 leukemic cell line. A serial dilution of both fusion transcript and RNA was prepared; then sensitivities of both techniques were determined. RT-PCR and NASBA reaction products were labeled using equal ratios of DIG-11-dUTP and DIG-11-UTP respectively. Following denaturation, hybridization reactions were carried out with specific probes. The products were incubated in streptavidin coated microplates. Then, the plates were washed, anti-DIG conjugated with peroxidase added and using ATBS as substrate, enzymatic activity was determined by absorption at 405 nm. The results showed that specificity of two techniques was equal but RT-PCR-ELISA sensitivity was about 100-fold more than NASBA-ELISA as it could detect 100 pg RNA less than NASBA-ELISA [0.006 versus 0.06 pg RNA]. Furthermore, leukemia cell detection precision by RT-PCR-ELISA and NASBA-ELISA was 4 and 400 cells, respectively. While NASBA technique does not need thermal cycler PCR but has less sensitivity than RT-PCR and is not suitable for quantitative assessment

Resistance to vancomycin in enterococcus faecium and faecalis clinical isolates

The aim of this study was to investigate antibacterial resistance among enterococci species isolated in Tehran Baghyatallah Hospital. It consisted of 126 isolates of E. faecalis [86%], E. faecium [9%] and other Enterococus Spp. [5%] isolated from urine [34.92%], blood [27.77%], wound swabs [19.84], stool [5%] endotracheal secretions [3.37%], abscess [3.4%], dialysis fluids [1.7%] and catheter [4%]. Twelve [9.5%] isolates were resistant to vancomycin. The VRE isolates were resistant to ampicillin [75%], erythromycin [50%], tetracycline [58%], ciprofloxacin [41.6%], chloramphenicol [33.3%] and gentamicin [41.6%]. Two [16.66%] of VRE isolates were multidrug resistant. Eight [66.6%] of the vancomycin-resistant strains and all of the MDR strains carried the vanA phenotype and genotype. The MIC of VRE isolates were between 32-512 micro g/ml. Our results show that most glycopeptide resistant E. faecalis and E. Faecium carried vanA. It is also possible that frequency of infections caused by glycopeptide-resistant enterococci will increase in our geographical area

Detection of icaAD gene and biofilm formation in staphylococcus aureus isolates from wound infections

Wound infections are a common cause of staphylococcal infections. An ability of S.aureus is to adhere and form biofilm on host surfaces. Biofilm is an exopolysaccharide, a slime matrix around multiple layers of cells and is mediated by expression of the icaADBC operon. The present study evaluated the biofilm forming capacity and the presence of icaAD gene among S.aureus isolated from wound infections. Slime production assay was performed by cultivation on Congo Red Agar plate. In addition, Quantitative biofilm formation determined by microtiter plate assay PCR method used for detection of icaAD gene. Fifty strains were identified, 54% of the isolates produced black colonies on CRA plate, 52% were positive biofilm forming, and all strains carried the icaAD gene. Regarding the ability of S.aureus to form biofilms helps the bacterium to survive hostile environments within the host, suggests that biofilm production is a risk factor for infection. It is important in rapid diagnosis and treatment biofilm forming strains, because biofilm formation may lead to increased antimicrobial resistance and create a significant impediment to wound healing

Quantitative analysis of Epstein - Barr virus DNA load in bone marrow transplant recipients by using real-time PCR

Quantification of Epstein - Barr virus [EBV] in peripheral blood mononuclear cells [PBMNC] of allogenic bone marrow transplant [BMT] recipients is important because EBV-associated posttransplant Lymphoproliferative disease [PTLD] can occur after transplantation due to immunosup-pression therapy. To this end we chose Real-Time PCR using TaqMan probe. For the standard curve, we cloned BALF5 gene of EBV into a plasmid vector. After purification of the EBV-clone and calculation of plasmid copy number, the standard curve was constructed by using serial dilution of the plasmid clone. We were able to detect from 2 to 107 copies per 2x105 PBMNC with wide linear range. The mean EBV DNA copy number was 103.7 copies per 2x105 PBMNC. In this study, No patient of 35 BMT recipients [275 PBMNC samples] developed PTLD during five months follow up post transplant. EBV copy numbers in 22 samples [3 patients] out of 35 BMT recipients were higher than cut off value with symptoms like fever and pulomonary noddes [9%]. The virus load in one patient in the last sample obtained was 72400 copies. We detected low levels of EBV DNA in 20 BMT patients [57/1%]. Real-Time PCR is useful to measure virus load in PBMNC. Detection of EBV in PBMNC samples may be valuable predictive marker to prognosis PTLD. Further studies need to determine ac-curate viral cut off value for treatment patients at risk for PTLD