Molecular Diagnosis and Vaccines - latest Issue
Volume 4, Issue 1, 2006
Insulin receptor substrate-1 and transforming growth factor beta-1 gene polymorphisms in the metabolic syndromeSource: Molecular Diagnosis and Vaccines 4, pp 1 –12 (2006)More Less
The metabolic syndrome (MS) encompasses a cluster of abnormalities including dysregulation of glucose and lipid metabolism, abdominal obesity and hypertension. This study aimed to investigate the association between insulin receptor substrate-1 (IRS-1) Glycine972Arginine (Gly972Arg) and transforming growth factor ?1 (TGF?1) Leucine10Proline (Leu10Pro) gene polymorphisms with MS and its different components. Fifty adult males with features of MS according to criteria set by the Adult Treatment Panel III (ATPIII) constituted the group of patients, 50 healthy males of comparable age served as controls. All participants were subjected to history taking, blood pressure, waist and hip circumference measurements. Laboratory investigations included fasting serum glucose, HbA1C, lipid profile, serum insulin and serum TGF?1. Insulin resistance was determined by the computer version of Homeostasis Model Assessment (HOMA2-IR). IRS-1 Gly972Arg polymorphism was determined by PCR / restriction digestion and TGF?1 Leu10Pro by the 5' nuclease assay. Compared to controls, MS patients were more obese, more insulin resistant and had an atherogenic lipid profile. Genotype distributions of both polymorphisms were not different between patients and controls. In MS patients, carriers of the IRS-1 mutant Arg972 allele were significantly more obese than non-carriers (p = 0.023), no other differences were detected. On the other hand, obese carriers of the Arg allele had significantly greater waist circumference, higher serum glucose, insulin, triglycerides and HOMA2-IR than non-carriers (p = 0.019, 0.03, 0.014, 0.02 and 0.01 respectively). Homozygous and heterozygous carriers of the mutant TGF?1 Pro 10 allele were more obese than Leu homozygotes (p = 0.012). Homozygous carriers of Pro10 had higher serum insulin, TGF?1 and HOMA2-IR than the other 2 genotypes (p = 0.0026, 0.0002 and 0.012 respectively). Waist circumference was an independent predictor of HOMA2-IR in patients. TGF?1 Pro 10 allele was more frequent in patients with than those without CHD (p = 0.023). HOMA2-IR was an independent predictor of CHD in MS patients. Our results indicate that, in the setting of MS, IRS-1 Arg972 and TGF?1 Pro10 variants are associated with obesity and insulin resistance. The TGF?1 Pro10 variant increases the risk of CHD. Understanding molecular mechanisms underlying such associations may help to plan treatment strategies to reduce health hazards related to MS.
Free fetal DNA in maternal blood: new horizons in non-invasive prenatal diagnosis; prediction of fetal RhD status in RhD negative mothersSource: Molecular Diagnosis and Vaccines 4, pp 13 –22 (2006)More Less
RhD incompatibility between the pregnant mother and her fetus is a significant problem due to the possibility of haemolytic disease of the fetus and newborn (HDF/N). In paternal RhD heterozygosity antenatal diagnosis of fetal RhD status for all RhD negative pregnant women is highly desirable for both financial and ethical reasons. Free fetal DNA in maternal plasma has been proved to be a good source of genetic prenatal non invasive diagnosis. Different sensitivities have been quoted on its use to detect RhD status in RhD negative pregnant women. In this study we aimed to assess whether fetal DNA in maternal plasma can be used to determine the fetal RhD status. The reliability of the PCR technique at different gestational ages was evaluated. Plasma and serum samples were collected from 40 RhD negative women at different stages of pregnancy. Samples were genotyped for RhD using polymerase chain reaction. Cord blood RhD serologic examination was done for all delivered babies as a gold standard. Samples from ten non-pregnant women (5 RhD+ and 5 RhD-) were included as controls for genotyping. Twenty seven babies were serologically proven to be RhD positive and thirteen were RhD negative. Starting from the second trimester, the PCR based assay accurately (100%) predicted the fetal RhD status and showed 100%specificity and sensitivity. No false positive results were reported.There was one false negative case in the first trimester. Maternal plasma samples were better than maternal serum (sensitivity96% vs 88%). It is concluded that maternal free fetal DNA is a promising non-invasive prenatal diagnosis. It can accurately be used starting from the second trimester to predict RhD positive cases where further measures to protect the fetus would be used.
Diagnosis of bacterial endophthalmitis by polymerase chain reaction using universal primers for eubacterial genomeSource: Molecular Diagnosis and Vaccines 4, pp 23 –30 (2006)More Less
Endophthalmitis is a devastating ocular disease, the incidence of which has been reported as 0.1-0.3% after intraocular surgery and 2.0-3.0% after penetrating ocular trauma. Confirmation of the diagnosis of bacterial endophthalmitis is dependent onmicrobiological isolation of organisms. We evaluated polymerase chain reaction (PCR) in the diagnosis of bacterial endophthalmitis using vitreous fluid (VF) and aqueous humour (AH) specimens. Intraocular specimens from 50 (20 VF and 30 AH) cases with endophthalmitis and 20 (6 VF and 14AH) cases with non-infective disorders, were processed for microbiological investigations. Nested PCR directed at the 16S rDNA using universal primers for eubacterial genome was done. Of the 20 controls 2 (10%) were positive using eubacterial primer. On the other hand PCR for eubacterial genome showed 100% correlation with the 22 (44%) bacteriologically positive specimens. Eubacterial genome, was detected in 9 (18%) of 28 bacteriologically negative specimens. Among the 19 eubacterial PCR negative specimens, 8 were fungus positive Candida (5) and Aspergillus (3). By inclusion of PCR, microbiologically positive specimens increased from 46.5% to 75.8%. Application of PCR on AH was as sensitive as that on VF for the detection of eubacterial genome. It is concluded that PCR is a reliable tool for the diagnosis of bacterial endophthalmitis.
Source: Molecular Diagnosis and Vaccines 4, pp 31 –38 (2006)More Less
High-quality coast-effective virus-free blood transfusion is the major goal in blood banks - here, a comparative evaluation of Nucleic acid testing (NAT) versus the currently implemented enzyme immunoassays (EIA) was carried out utilizing three thousands donors' samples for detection of HBV and HCV. Study design was based on obtaining two samples from each donor, a serum sample for EIA screening of HbsAg, Hbs Ab, HBcAb and HCV Ab, and plasma for NAT. Individual plasma specimens were pooled to make 125 pools (24 specimen/pool) for HBV-DNA and HCV-RNA testing. According to the results, donors pools were categorized into seven groups: Group 1; included 97 seronegative HCV Ab, among them one pool (1.03%) was found HCV-NAT positive, Group 2; included 28 seropositive HCV Ab, among them 26 (92.9%) were HCV-NAT positive, Group 3; demonstrated that the detection limit of a single HCV-PCR -positive sample per pool (> 103 IU/ml), Group 4; included 85 seronegative HBV pools, among them 5 (5.9%) were HBV-NAT positive, Group 5; included 40 pools positive only for HBc IgG and 29 (72.5%) HBV-NAT positive, Group 6; included 16 pools seropositive for HBc Ab and HBs Ab - which are allowed for transfusion an allarming result of 12.5% (2 pools) were HBV-NAT positive. Finally, group 7; demonstrated that the detection limit of one single HBV-PCR positive sample per pool was 103 copy/ml. It is concluded that, implementation of NAT screening for HBV and HCV targets in blood banks would provide more conclusive data and consequently reduce the risk of post-transfusion infections. Optimization of pooling size versus detection limit is needed specially in endemic area.
Th1 and Th2 cytokine production at the level of transcriptional regulation in the peripheral blood from rheumatoid arthritis patientsSource: Molecular Diagnosis and Vaccines 4, pp 39 –51 (2006)More Less
T-box expressed in T cells (T-bet) and GATA-binding protein 3 (GATA-3) are transcriptional factors that play a crucial role in Th1 and Th2 development. We investigated the immunomodulatory roles of T-bet and GATA-3 and Th1/Th2 related cytokines IFN-? and IL-4 in the pathogenesis of rheumatoid arthritis (RA) and their association with disease activity. The mRNA markers level was measured in the peripheral blood of RA patients and controls by real-time PCR. Total RNA was isolated directly from peripheral blood of 21 RA patients and 10 healthy controls. The mRNA levels of T-bet, IFN-? and IFN-?/IL-4 ratio were significantly lower in RA patients than controls (p= 0.001, p= 0.000, p=0.014 respectively). In all RA patients, there were significant positive correlations in mRNA expression of T-bet with IFN-? (r =0.895, p= 0.000), and of GATA-3 with IL-4 (r =0.089, P =0.000). RA patients were divided into two groups according to C reactive protein (CRP) levels. In comparison to RA patients with a low positive CRP (CRP < 12 mg/L), patients with a high positive CRP (CRP ? 12 mg/L) had lower T-bet, IFN-? expression as well as IFN-?/IL-4 and T-bet/GATA-3 expression ratios. Conversely, the mRNA expression of GATA-3 and IL-4 increased in the low positive CRP group compared to the high positive CRP group but it did not reach a significant value (p =0.204, p =0.259 respectively). In conclusion, this study demonstrates that peripheral blood from RA patients showed a decreased Th1 response as indicated by low T-bet and IFN- ? mRNA expression, which was found mainly in patients with the most active disease. The suppressed function of Th1, associated with the suppression of both T-bet and IFN-? gene expression, may be one of the immunological characteristics of rheumatoid arthritis.
Association between bacterial contamination of the uterine cervix at embryo transfer and success rate in in-vitro fertilization/intracytoplasmic sperm injectionSource: Molecular Diagnosis and Vaccines 4, pp 53 –60 (2006)More Less
In the last two decades, in-vitro fertilization and embryo transfer (IVF-ET) have been successful in the alleviation of long-standing infertility due to female or male factors. It is well documented that the results of IVF in the presence of male factor are not as good as those in patients with normal semen parameters. Intracytoplasmic sperm injection (ICSI) is now one of the most successful and viable techniques in assisted fertilization. It leads to an increase in the fertilization rates but without corresponding increase in implantation rates. Therefore, efforts were directed to create a perfect relationship between good quality embryos and receptive endometrium. The aim of this study was to evaluate the effect of Chlamydia trachomatis infection and microbial flora of the cervix at the time of embryo transfer on fertilization and implantation in women undergoing ICSI procedure. From June 2003 to November 2003, thirty participants with a male cause of infertility were enrolled in the study in Ain Shams University Maternity hospital. All were recruited from the outpatient clinic of Assisted Reproduction unit. After embryo transfer (ET), cervical scrapings were taken and tested for Chlamydia trachomatis DNA by PCR technique. Also the distal tip of the embryo transfer catheter was cut off and tested for bacterial growth by conventional culture techniques. Pregnancy tests were done 2 weeks later, and participants were accordingly divided into pregnant and nonpregnant groups. Chlamydia was detected in 30% of the non-pregnant group versus 10% of the pregnant one but with no statistical significance. Non significant difference was found between both groups regarding cervical bacterial growth. E. coli had border line significance in the non pregnant group. In conclusion, the present study supports the hypothesis that microbial flora of the cervix detected during ET have no role in the implantation process, and does not affect pregnancy rates in women undergoing ICSI procedure for infertility. However further larger scale studies are recommended to assess the possible role of Chlamydia trachomatis and E. coli.
Molecular characterization of Vancomycin-resistant enterococci isolates from hematology-oncology patientsSource: Molecular Diagnosis and Vaccines 4, pp 61 –73 (2006)More Less
Vancomycin-resistant enterococci (VRE) are important nosocomial pathogens in many countries with the genotype vanA and vanB being the most important is hospital environment. The objectives of this study is the Molecular characterization of VRE isolated from hematology-oncology patients. Fecal/rectal samples from 50 randomly selected patients together with blood samples from the 11 patients who developed bacteremia. Enterococcal isolates were identified and subjected to antimicrobial susceptibility testing to vancomycin by agar screen method. Vancomycin resistance was confirmed by determining its minimum inhibitory concentration by broth dilution method. Susceptibility of the VRE isolates to different antimicrobials was also determined using the disk diffusion method. Multiplex PCR was used to detect vanA and vanB genes among the isolated VRE strains. Fifty enterococcal strains were isolated from the fecal-rectal samples, of which six (12 %) were VRE (3 E. faecium, 2 E. faecalis and one E. gallinarum). On the other hand, blood cultures from patients with bacteremia were all negative for enterococci. The most significant risk factor for colonization with VRE was previous hospitalization. Other factors included prolonged hospitalization, previous ICU admission, febrile neutropenia, current and previous vancomycin administration. 83.3% of the VRE strains were sensitive to nitrofurontoin, while 83.3% were resistant to ampicillin and erythromycin. vanA gene was detected in two isolates while van B gene was detected in another three isolates. One isolate was found to be devoid of both vanA and vanB. It is concluded that Vancomycin resistance genes are present among the enterococci colonizing the gastrointestinal tract of hematology-oncology patients. This represents a critical risk factor for hospital aquired infection by these pathogens as well as a threat of spread to other pathogens. Surveillance for VRE in high-risk patients together with effective infection control measures and adapted antibiotic policies are important to control transmission of VRE among patients.
Source: Molecular Diagnosis and Vaccines 4, pp 75 –84 (2006)More Less
Survivin, and Aven are members of the inhibitor of apoptosis (IAP) proteins family. They are reported to be over-expressed in many cancers including leukemia. Previous studies suggested that Survivin is implicated in both control of apoptosis and regulation of cell division. The aim of this study was to examine the expression of the two apoptotic inhibitors - Survivin and Aven-at the messenger (m)RNA level in acute myeloid leukemias (AML) in relation to the clinical and hematological findings. Thirty adult patients with acute myeloid leukemia (AML) and 10 healthy subjects were studied. Patients were subjected to complete blood picture, bone marrow examination and immunophenotyping. Based on the French American British classification, they were subdivided to M1, M2, M3, M4 and M5. A reverse transcriptase-polymerase chain reaction (RTPCR) was used to investigate the expression of Survivin, and Aven mRNA. The AML patients showed expression of Survivin (206 bp) but not Aven mRNA. In contrast, neither Survivin nor Aven were expressed in the control group. Out of the 30 patients, 23 patients (76.6 %) showed detectable levels of Survivin expression (p<0.001). Quantitative analysis revealed differences in expression levels of Survivin mRNA between different FAB subtypes. Three subtypes (M1, M4 and M5) showed a slightly higher expression level of Survivin mRNA (mean values of 0.56, 0.57 and 0.81 respectively) than the other two subgroups (M2 and M3) (mean range 0.04 and 0.1 respectively). A significant correlation was found between Survivin expression level and peripheral blood (PB) blast % in M5 subtype (p=0.02). It is concluded that, Survivin is an important antiapoptotic signal in acute myeloid leukemia. Up-regulation of Survivin expression in AML may be involved in its pathogenesis and may provide a useful tool for prognosis.
Source: Molecular Diagnosis and Vaccines 4, pp 85 –97 (2006)More Less
The aim of the present study was to investigate the frequency of FLT3 activating mutations; Internal tandem duplication (ITD)& the point mutation in exon 20 at aspartate (Asp) residue 835 within the tyrosine kinase domain (D835) in acute myloid leukemia (AML) and their correlation to prognostic factors and clinical outcomes. The study was conducted on 50 (23 males and 27 females) adult patients with newly diagnosed AML. Twenty apparently healthy and haematologically normal individuals of matched age and sex were also studied to verify the possibility of the occurrence of FLT3 mutations in normal subjects. We evaluated patients after induction chemotherapy which consisted of the standard 3+7 induction regimen. The evaluation was done in accordance with standard criteria for complete remission (CR), remission failure (RF) or remission death (RD) after 28 days from induction chemotherapy. Detection of FLT3 mutation was done by polymerase chain reaction (PCR) for the (ITD) mutation and by restriction fragment length polymorphism mediated polymerase chain reaction (RFLPPCR) for the (D835) mutation. We analyzed FLT3 mutations according to age, sex, WBC, FAB subtypes, percentage (%) of blast cells in peripheral blood (PB) and bone marrow (BM) and response to induction therapy. Tandem duplication (ITD) was found in (18/50) 36.0%. There were (9/18) 50.0% males and (9/18) 50.0% females, with no significant correlation between age or sex and FLT3-ITD. On the other hand only (3/50) cases were positive for FLT3-D835 6.0%. The three positive cases were males. A statistically significant relationship was found between D835 and male sex while no significant relation was found with age. The total white blood cell count (WBCs) was the only parameter that showed a significant increase in positive FLT3-ITD and FLT3-D835. Neither hemoglobin levels nor Blast % in PB or BM had significant difference between positive and negative cases of FLT3-ITD and FLT3-D835. On the other hand a significant relationship between FLT3-ITD and FAB subtype was found, while all positive FLT3-D835 cases were of M3 subtype. Among the 16 positive cases with ITD that could be followed after induction, only 3 achieved induction remission. In contrast, none of the 3 cases with FLT3-D835 mutation achieved induction remission. A statistically significant relationship was found between FLT3/ITD, FLT3-D835 mutation and failure to achieve induction remission. It is concluded that the prevelance of FLT3/ITD in our study population is a little higher than that reported else where, while that of FLT3-D835 is almost similar. FLT3 mutations are identified as poor prognostic factors in Adult AML as regards remission rate. Further study on a larger scale is recommended.
Source: Molecular Diagnosis and Vaccines 4, pp 99 –105 (2006)More Less
Leprosy is a chronic infectious disease caused by Mycobacterium leprae (M. leprae), which is one of the few known pathogenic bacteria that can not be cultivated in vitro. In the present study, real time PCR was introduced as a diagnostic tool to monitor individuals eight to ten years after completion of antileprosy therapy and to compare it with conventional method of acid fast bacillus (AFB) detection. The study was carried out on twenty four individuals who had leprosy eight to ten years ago and completed a course of Dapsone monotherapy. Slit skin smears were taken from all individuals. Three out of twenty four individuals (12.5%) had positive AFB while eight out of twenty four individuals were positive by real time PCR (33.3%). All individuals who had positive AFB were positive by real time PCR with a sensitivity of 67.19% and specificity of 100%. Nineteen out of twenty four individuals had residual lesion of leprosy but this had no significant association with detection of Mycobacterium leprae by AFB or real time PCR. So it is concluded that real time PCR may be a useful tool to monitor previously treated leprotical individuals for the purpose of infection control.