Assessment of diagnosis and DNA damage in microfilarenzic and amicrofilaremic patients in Menoufiya governorate

Lymphatic filariasis is considered an endemic parasitic disease in some localities in Menoufiya Governorate. There is much controversy regarding diagnosis of amicrofilaremic 'endemic normal' population. To evaluate PCR-based assay, indirect immunofluorescent antibody test [IFAT] in microfilaremic and amicrofilaremic patients with lymphatic filariasis in an endemic area. A second objective was to detect individual DNA damage, its relation to microfilaremia and correlation to infection in different age groups. Three villages [Fesha El-Kobra, Serowheit and Ezbet El-Eslah] were selected to perform this study. The study included 128 individuals representing both sex [59 males and 69 females] of different age groups [5-60 years old]. One hour after receiving diethylcarbamazine citrate tablets [DEC], they were subjected to daytime blood film by fresh finger-prick [stained by Giemsa and Wrights stains], and venous blood samples were taken for indirect immunofluorescent antibody test [IFAT, using non-human species of Setaria equina microfilaria], polymerase chain reaction [PCR] based method for detection of filarial parasite DNA and electrophoresis to detect associated DNA damage. Microfilaremic patients diagnosed by examination of blood films were 24/128 [18.75], by IFAT 33/128 [25.78%] and PCR reacted positively in 42/128 [32.8%] of cases. From the latter IFAT missed detection of 8 cases and blood films missed 18 cases. Analysis of study results revealed that PCR was the most sensitive test [95.5%] than IFAT [75%] and lastly the blood film [54.5%]. Significant variable degrees of DNA damage were associated with positive cases than negative ones. Moreover, there was a significant association between DNA damage and microfilaremia. Severe DNA damage was statistically significant in 71.4% of patients within the age group of 20-40 years indicating specific correlation of DNA damage and bancroftian filariasis. Detection of W bancrofti DNA by PCR improved diagnostic sensitivity of bancroftian filariasis because of its high capacity. The method can also be used as a rapid and reliable epidemiological tool for screening villages to locate endemic areas. DNA damage showed considerable relation to microfilaremia and to the patient's age as severe DNA damage was statistically significant in the infected age group of 20-40 years [71.4%]

Haemochromatosis gene mutation H63D is a risk factor for iron overload in Egyptian beta- thalassemic children

Iron overload is the main cause of morbidity and mortality in patients with beta-thalassemia. The aim of this study was to evaluate the prevalence of genetic markers [HFE mutations C282Y and H63D] among Egyptian beta-thalassemic Children and its effect on their iron status. 59 beta-thalassemic children attending the pediatric hematology clinic in Menoufiya University Hospital [23 thalassemia major, 23 thalassemia intermedia and13 thalassemia trait] with 50 apparently healthy, Egyptian children [control group] were screened for the prevalence of these two mutations by digestion of PCR products [RFLP]. Serum ferritin level was measured by ELISA. Neither carrier status for the C282Y allele nor homozygous status for the H63D allele were detected in any of the thalassemic children or the 50 controls. The H63D heterozygous state was detected in 15 [25.4%] thalassemic patients with an allele frequency of 12.71% and in 11 [22%] controls with an allele frequency of 11%. with no significant difference between the thalassemic groups and the controls. The prevalence of carriers for the H63D mutation was 26.1% with an allele frequency of 13.04% in patients with either beta- thalassemia major or intermedia, while in beta- thalassemia trait the prevalence of this mutation was 23.1% with an allele frequency of 11.54%. There were significant higher levels of the mean yearly serum ferritin in both beta-thalassemia major and intermedia patients who are heterozygotes for the H63D mutation compared to those without this mutation. The mean serum ferritin levels were positively correlated with the age of the patients. On the other hand, the prevalence of iron -induced complications was not statistically different between patients carrying or not carrying this mutation [among TM and TI]. There is no difference in the prevalence of H63D mutation between beta-thalassemic patients and the normal children and the presence of a heterozygous H63D status and older age are two risk factors for iron overload in Egyptian beta-thalassemic children. RFLP= Restriction Fragment Length Polymorphism, HCV=Hepatitis C Virus, ALT = Alanine aminotransferase, AST =Aspartate aminotransferase

Estimation of time passed since death by ultrastructure, histopathology and DNA degradation detection by gel electrophoresis in albino rats

The estimation of postmortem interval [PMI] is of great importance in both criminal and civil cases. However this aim remains critical, difficult if not impossible but an imprecise task in forensic investigation. While the traditional thanatochronological changes upon the dead had been used for estimation of PMI inaccurately, advances in molecular biology, have propelled the analysis of DNA to criminal laboratories. Upon the death of an individual, internal nucleases contained within the cell should cause chromosomal DNA to degrade into increasingly smaller fragments over time that can be used as a predictor of PMI. Liver cells for being rich in nuclear DNA and mitochondria were proved to be the best material for the purpose. The aim of the work was to determine the time passed since death by measuring the rate of DNA degradation in samples of livers of albino rats at different post mortem intervals by using gel electrophoresis method, and to asses the changes of liver cells by histopatological examination at the same periods, with exploring the nature of the post mortem liver cell changes by using the Transmission Electron Microscope [TEM]. Passive engorgement of the sinusoids and extravasations of blood elements appeared early after death in histopathology then cell necrosis, shrunken cells, and apoptotic bodies were increasing by time. TEM examinations revealed the presence of chromatin condensations and fragmentation of the nuclear material. Finally cell lysis and apoptotic bodies appeared while cell necrosis was prominent. Electron microscope could add much to understanding postmortem cellular changes, also assessment of the relative existence of necrosis stages in histopathology might help in identifying PMI. The Gel Electrophorsis detection, measurement and analysis by computer pro analyzer revealed the presence of sequential relationship between DNA degradation level and time passed since death at the DNA visualizing positions [200 bp, 400 bp, 600 bp] up to 24 hours. Statistical evaluation showed a highly significant dependence of DNA degradation on time with P < 0.001. By using linear regression analysis an equation to define the time passed since death could be conducted where: Time after death=[DNA degradation - constant] / B. So it is recommended to apply the method of the gel electrophoresis to detect time passed since death by measuring DNA fragmentation from liver cells. These mathematical relations offer a simple and valuable means of estimating the PMI, and its validity should be tested for human being

Assessment of genome damage in a population of Egyptian workers occupationally exposed to cotton dusts

Earlier reports described byssinosis syndrome among workers in cotton industry, while recent studies have shown that workers occupationally exposed to cotton dusts have an increased risk of development of many types of cancer. Hence this study was conducted to assess genotoxic effects [as a measure of carcinogenic risk] of chronic cotton dust inhalation in workers with byssinosis and to combine clinical and occupational data with the results of genotoxicity assays in order to reach quantitativness in risk assessment. Clinically, byssinosis was diagnosed mainly in workers employed at early production areas of yarn preparation: opening, blowing and carding [80%] and those working as machine operators [8.5%]. There was significant correlation between the duration of exposure to cotton dusts and the clinical severity of the disease. Study of cytogenetic markers in exposed workers showed significant increase in the percent of total chromosomal breaks and aberrations as well as the mean value of sister chromatid exchanges accompanied with significant decrease in mitotic index value as compared to controls. Assessment of total genomic damage of DNA by visual comparing of the density of released [migrated, damaged] DNA bands and by measuring the optical density of damaged DNA bands using Gel-Pro computer program revealed 20% increase in DNA damage in blood lymphocytes of workers chronically exposed to cotton dusts when compared to non-exposed controls. Also, there was 50% increase in the optical density of the released RNA in blood lymphocytes of exposed workers than controls which might be used as an index of stress of pollution applied on cotton industry workers. Comet assay endpoints revealed more than twice times higher number of migrated DNA spots [damaged, strongly damaged] in blood lymphocytes from cotton industry workers compared to non-exposed subjects. The genotoxicity burden measured as% of total chromosomal breaks, and aberrations, mean values of SCEs/metaphase and DNA damage endpoints [the number of damaged DNA spots and the optical density of damaged DNA] was correlated significantly with the duration of exposure to cotton dusts. Therefore the fact that workers occupationally exposed to cotton dusts have distinctly more chromosomal mutations and DNA damage may be an important indicator in the chronic effect of cotton dust-associated carcinogenesis. Combining the clinical and occupational data with the results of genotoxicity assays showed that the severity of byssinosis syndrome was associated with the degree of genome damage