Limit of molecular detection of strongyloides stercoralis parasite performing two PCR protocols

Low parasite density in chronic infection with S. stercoralis is a challenging diagnostic issue. Generally, molecular techniques don't depend on parasite viability while copro-culture or Baermann concentration method relies on the presence of living larvae in fecal samples. Therefore, evaluation of PCR-based methods is important to increase the detection rates in light or chronic infection. This study was designed to analyze the sensitivity of quantitative PCR [qPCR] and nested PCR [nPCR] regarding the minimum amount of S. stercoralis DNA template that can be reliably detected by each molecular technique. Strongyloides larvae were collected from cultured stool samples from suspected infected Egyptian children. After counting larvae present in a known volume under the microscope, DNA extraction was done and serial dilution of genomic materials was prepared. Then, qPCR and nPCR targeting the small subunit of the rRNA gene were performed. Regarding qPCR, the limit of detection was 0.0005 S.stercoralis larvae/microl, with crossing threshold [Ct] value ranged from 17.8 to 38.7 while, nPCR did not detect from [0.002 to 0.00012 S. stercoralis larvae/microl] with minimum limit 0.004 S. stercoralis larvae/microl. Real-time quantitative PCR is very sensitive technique that can detect very low genomic load up to about 10 [9.765] genome copies/reaction compared to nested PCR which started positivity from 78.125 genome copies/reaction. Therefore, qPCR is recommended to detect chronic strongyloidiasis especially in high-risk groups to prevent lif-etheratening spread of such infection

Genetic polymorphism of Toxoplasma gondii from recently infected aborted Egyptian women

Toxoplasmosis caused by Toxoplasma gondii is one of the most prevalent parasitic diseases in human beings. Human toxoplasmosis can be associated with serious clinical manifestations, particularly in developing fetus. The aim of the current study was to identify the possible lineage type of Toxoplasma gondii, molecularly detected in placental samples of women whose pregnancies were spontaneously terminated in the first trimester. Preliminary detection of Toxoplasma genomic materials was done by a SYBR green qPCR technology. Subsequent identification of Toxoplasma strain was done for the positive samples using PCR- restriction fragment length polymorphism [RFLP] at the SAG2 loci of T. gondii using restriction enzymes HhaI and Sau3AI. Out of 72 tested samples, Toxoplasma B1 gene was detected in 9 cases. Toxoplasma genotypes I and II in addition to unknown type were identified in 4, 3 and 2 cases respectively, while type III was not detected in our samples, hence excluded as a leading cause of abortion in humans in our preliminary study. Nevertheless, it remains uncertain to what extent the genotype of the parasite directly contributes to the clinical severity of human toxoplasmosis. Certainly, advanced molecular techniques targeting different Toxoplasma strains are crucial for better understanding of human toxoplasmosis. For more elucidation, additional studies are recommended intended for genetic characterization of such serious parasitic infection using larger number of samples

Coital retrograde menstruation as a risk factor for pelvic endometriosis

To investigate coitus during menstruation as a possible predisposing factor for endometriosis 78 patients were asked about their coital habitus during menstruation and subsequently were examined by ultrasound transabdominally, transvaginally, or both, before laparoscopy or surgery. MRI was used in six patients only. There was a statistically significant relationship between endometriosis and coital practice during menstruation. The incidence of endometriosis in patients with history of coitus in menses was 66% while the incidence was 34% in patients with no such history. It is concluded that coitus during menses could be a predisposing factor for endometriosis

Role of exercise echocardiography in decision making of balloon valvuloplasty in moderate mitral valve stenosis

Assessment of value of exercise echocardiography using in patients with rheumatic moderate mitral valve stenosis in interventional decision making [percutenous balloon mitral valvuloplasty]. 30 patients with moderate mitral stenosis were participated in the study. All patients were subjected to exercise echocardiography and were divided into two groups. Group I: 20 patients with good exercise tolerance and group II: 10 patients with poor exercise tolerance and mitral valvuloplasty was done, and then reevaluated with exercise echocardiography one month after valvuloplasty. In group [I] mean pressure gradient across mitral valve at peak of exercise was ranged between 12 and 16 mmHg with mean of 134.78 +/- 1.33 mmHg, while in group[II] mean pressure gradient across mitral valve at peak of exercise was ranged between 17 and 20 mmHg with mean of 18.30 +/- 1.25 mmHg. In group [I] pulmonary artery pressure at peak of exercise ranged between 25 and 40 mmHg with mean of 30.00 +/- 5.38 mmHg, while in group [II] it was ranged between 35 and 40 mmHg with mean of 39.00 +/- mmHg at peak of exercise. All members of group II had subvalvular affection [high score] and 50% of them had high score for calcification with poor exercise tolerance while non of group I had subvalvular affection and only 10% of them had law calcific score]. Exercise echocardiography plays an important role in evaluating true symptomatic patients and assesses the hemodynamic severity in patients with moderate mitral stenosis

Evaluation of inhouse HBV DMA PCR, and its comparison with amplicor HBV PCR [Roche] and quantiplex HBV bDNA [Chiron] in detection of hepatitis b virus infection

To evaluate the diagnostic efficacy of inhouse HBV DNA PCR detected either by ELISA or agarose gel electrophoresis techniques this study was designed. 60 patients having either HbsAg antigen or HBc IgG or both were selected from the outpatients medical clinic and inpatient medical section of the National liver institute, Minoufiya University. Inhouse HBV DNA PCR detected either by ELISA or agarose gel electrophoresis techniques were done for those patients, and the results were expressed in comparison with the results of Amplicor HBV PCR [Roche], and quantiplex HBV bDNA [Chiron]. Out of sixty patients who were serologically positive for either HbsAg, anti- HBc IgG or both, the inhouse HBV DNA PCR detected by ELISA was positive in 11 patients [18.33%], while inhouse HBV DNA PCR detected by EB and gel electrophoresis was positive in 9 patients [15%], also, quantiplex HBV bDNA [Chiron] was positive in 8 [17.78%] out of 45 patients only and amplicor HBV DNA PCR [Roche] was positive in 28 [46.67%] out of 60 patients. Also, the study revealed that the sensitivity of inhouse HBV DNA PCR detected by ELISA was 39.28%, with 100% specificity and 71.67% diagnostic accuracy, while inhouse HBV DNA PCR detected by agarose gel electrophoresis after ethidium bromide staining showed 32.14% sensitivity, 100% specificity and 68.33% diagnostic accuracy also, Also, quantiplex HBV bDNA [Chiron] showed 28.57% sensitivity, 91.66% specificity and 62.22% diagnostic accuracy when compared with the positive results obtained with amplicor HBV PCR [Roche]. So, due to its low sensitivity in comparison with amplicor HBV DNA PCR [Roche] we can not rely on inhouse PCR detected either by ELISA or agarose gel electrophoresis after ethidium bromide staining in diagnosis of HBV infection, further optimization of inhouse PCR is recommended for good yield and better diagnosis

Comparative study of different diagnostic modalities of m. tuberculosis and evaluation of rifampicin resistant strains using line probe assay versus conventional proportional method

Tuberculosis remains a major health problem around the world being responsible for 3 million deaths each year. The spread of TB is expected to worsen as infection with multi-drug resistance M. tuberculosis isolates [MDR-M] increases in many countries. Rifampicin is an important component of effective multi-drug therapies of TB. Thus, Rif-resistance means that short course therapy is no longer an option and those second-line drug susceptibilities are required in order to make an informed choice for alternative therapy. Furthermore, resistance to Rif serves as a useful surrogate marker for the detection of MDR isolates. The aim of this study is to shed some light on the line probe assay [LiPA] in comparison to the conventional proportion method for detecting Rif susceptibility genotype and phenotype of M. tuberculosis clinical isolates. This study was conducted on 154 patients with age ranged from 18 to 72 years who were divided into 2 groups. The first group included 130 cases [80 males and 50 females] suspected of having pulmonary TB, and the second group included 24 cases [18 males and 6 females] suspected of having extrapulmonary TB. All cases were subjected to tuberculin skin test. The clinical specimens were collected according to the site of presenting symptoms. At least 3 successive samples of sputum, urine and stool were collected for each case. Contaminated specimens [98 sputum, 13 bronchial lavage, 7 urine and one stool samples] were subjected to a digestion-decontamination procedure. Aseptically collected specimens [13 peritoneal aspirate, 19 pleural aspirate, one fallopian tube and 2 lymph node samples]. All used for smear staining with Kinyoun's stain and culture. Mycobacterial isolates on L-J slants were biochemically typed by niacin accumulation, nitrate reduction and heat stable catalase test. The susceptibility of M. tuberculosis to Rif was performed using both the proportion method on MB7H10 agar and LiPA using LiPA Rif TB strips. The distribution of tuberculous patients according to clinical specimens among all cases showed that 98 sputum samples revealed 26 positive culturers. Two sputum samples were positive by stained smear after processing but negative by either direct smear or culture. The overall positivity of sputum samples were 28 specimens. One positive culture/negative Kinyoun's stain was recovered from fallopian tube specimen. Tuberculin skin test revealed positive induration diameter in 28 [96.6%] out of 29 tuberculous patients, whereas the tuberculin skin test was positive in 78 [62.4%] out of 125 negative smear/culture cases. Direct Kinyoun's stain showed positive smear in 15 out of 154 specimens, which were also positive by L-J culture. Also 12 positive cultures were recovered from the 139 negative direct smears. The sensitivity and specificity of direct Kinyoun's stain versus L-J culture were 55.6% and 100% respectively, and the negative and positive predictive values were 91.4% and 100% respectively. Stained smears after processing revealed positive smear in 22 out of 119 patients, of which 20 were positive by L-J culture. However 6 positive cultures were recovered from 97 negative smears. Its sensitivity and specificity versus L-J culture were 76.9% and 97.8% respectively, and the positive and negative predictive values were 90.9% and 93.8% respectively. There was a statistically significant difference in comparing direct Kinyoun's smear and Kinyoun's smear after processing of the 115 contaminated specimens. The results of mycobacterial biochemical reactions revealed 24 M. tuberculosis and 3 mycobacteria other than tuberculosis [MOTT] isolates. As regard Rif-susceptibility testing of the 24 M. tuberculosis isolates, 8[33.3%] isolates were Rif-resistant and 16 [66.7%] were Rif sensitive by the proportion method, whereas 6[25%] isolates were Rif resistant and 18 [75%] were Rif-sensitive by LiPA. The type of rpoB gene mutations in the 6 Rif-resistant M. tuberculosis isolates by LiPA showed that the most frequently detected mutations were H526Y [57.1%] and S531L [28.6%] and H526D 14.3%. One strain showed double mutations atH526Y and S531L. It is concluded that LiPA is a simple technique which provides clear results requiring only a basic knowledge of molecular technique

Role of Nitric oxide and some cytokines in neonatal septicemia

Bacterial sepsis is a life-threatening event. Some cytokines and recently, nitric oxide [NO] have been incriminated in the pathogenesis of sepsis, especially in the mechanisms by which sepsis progresses to septic shock. The aim of the present work is to determine whether NO level is increased in neonates with bacterial sepsis and if it is related to the levels of the inflammatory mediators namely interleukin-6 [/L-6] and tumor necrosis factor-alpha [TNF-alpha]. The study was conducted on twenty neonates with bacterial sepsis proved by history, clinical examination and laboratory investigations. Ten healthy age and sex matched-neonates were taken as a control. All cases were subjected to blood, cerebrospinal fluid and urine cultures and estimation of serum tumor necrosis factor-alpha [TNF-alpha], interleukin-6 [IL-6] and serum NO [nitrite+nitrate] [the stable and catabolic products of NO]. Neonates with bacterial sepsis had a significantly higher serum TNF-alpha serum /L-6 and serum NO [nitrite + nitrate] than controls. There were significant positive correlations between serum levels of TNF-alpha [ng/ml], 12-6 [pg/ml], NO [nitrite +nitrate] [[micro mol/L] and I/T leukocytic ratios, Complete cure had occurred in 14 cases [70%], while 6 cases had succumbed [30%]. All succumbed cases had low systolic pressure <50 mmHg and oliguria, a picture denoting septic shock. In conclusion, levels of serum IL-6, TNF-alpha and plasma nitric oxide are elevated in neonatal septicemia. However, these mediators may be prognostic for survival rather than diagnostic, since, immature/total leukocytic ratio is still a rapid and easy method for suspicion of the case, together with the clinical picture

Role of different nirogen fertilizer levels on the development and effectiveness of VA mycorrhizal fungi on maize plants

The effects of five rates of ammonium sulfate [0, 22.5, 45, 90 and 180 kg N fed-1] on the development and effectiveness of vesicular- arbuscular [VA] mycorrhizal fungi on maize were studied in a clay loam soil. N fertilization at the rates of 22.5 and 45 kg N fed-1 increased significantly the percentage of mycorrhizal root colonization in both mycorrhizal inoculated and un-inoculated plants. Increasing the rates of nitrogen fertilization above 45 kg N fed-1 had a negative effect on root colonization by VA mycorrhizal fungi. A significant increase in dry matter production, N and P uptakes of maize plants was obtained as the rate of nitrogen fertilization increased. At a low N fertilization rate [below 45 kg N fed-1], inoculation with VA mycorrhizal fungi increased the dry matter production, N and P uptakes of maize plants. However, there was no significant response in dry matter yield, N and P uptake of maize plants inoculated with VA mycorrhizal fungi as nitrogen fertilized increased above 45 Kg N fed-1

Menstrual pattern among adolescent females in Alexandria university Part I: Menstrual disorders

This study was initiated aiming at studying the menstrual pattern among Alexandria University female students.A total sample size of 900 girls were chosen randomly from 6 colleges in the university using multistage stratified random technique. An interviewing questionnaire was constructed covering personal and familial characteristics of the students and detailed menstrual history. An anticipatory guidance for the students suffering from severe premenstrual syndrome [PMS] was performed. The majority of the studied sample had a normal menstrual pattern but also most of them complained from PMS symptoms. No significant difference was found between practical and theoretical colleges as regards oligomenorrhea, poly menorrhea and absenteeism due to dysmenorrhea. Only about 11% of the studied sample suffered from menostaxis. Anticipatory guidance is very important in this period of adolescence to improve the PMS symptoms among the students suffering from it

Menstrual pattern adolescent females in Alexandria university Part II: Some ecological factors

The aim of the present study was to detect the effect of the different ecological factors on the menstrual pattern. The research studied 900 girls at the different levels of education at different colleges in Alexandria University. The work was executed through an interviewing questionnaire covering a variety of items concerning personal and menstrual history of each female student. A battery of psychological tests was executed including the assessment of anxiety, depression, self esteem, life events and stress. Results indicated that medical problems anxiety, self esteem, obesity, the student's negative attitude towards menstruation and not playing sports were the most important ecological factors contributing to irregularities of the cycle length. Anxiety was the only factor affecting the duration of bleeding. Age of menarche, socio-economic standard and student's negative attitude towards menstruation were the main factors affecting the occurrence of dysmenorrhea. On the other hand, depression, life events, not playing sports and anxiety were the most important predictors affecting the premenstrual syndrome

Response of chickling vetch [lathyrus sativus L] and forage peas [pisum spp] to inoculation with rhizobium leguminosarum in north Sinai coastal soils

Field experiments were conducted in sandy soil at Rafah, north sinai, to study the performance of two Rhizobium leguminosarum strains [TAL 634 and F9] with vetch, grasspea [Lathyrus sativus] and forag peas [Pisum spp.]. Inoculation of both crops with these strains, either singly or in combination, induced significant increases in plant dry weight, N2 fixation and seed yield. No significant differences, in plant N content or seed yield, were obtained between the two rhizobial strains and/or their mixtures for both crops. The competitive ability of inoculant strains differed with the host plant. Strain TAL 634 was more competitive than strain F9 with chickling vetch. In double strain inocula, strain TAL 634 occupied 40% of nodules versus 15% for F9. With forage peas, strain F9 was more competitive than strain TAL 634. The strain F9 occupied 45% of nodules versus 30%for strainTAL634

Competition between three bradyrhizobium japonicum strains as affected by strain proportion in inocula

Double strain combinations of Bradyrhizobium japonicum strain USDA 110 with strains USDA 138 or TAL 379 mixed in different ratios [1 : 1. 1 : 5, 1 : 10, 1 : 25. 1 : 50, and 1 : 100] were used as inocula for soybean Crowford. Modulation of soybean plants were not affected by changes in ratios of inoculant strains. Increasing the ratio of inocularil strains 138 or 379 to strain 110 resulted in a gradual decrease in plant dry weight, N content and seed yield. A significant decrease in plant dry weight, N content and seed yield was obtained when the ratio exceeded 25 : 1. Strain USDA 110 was more competitive than strain USDA 138 or TAL 379. Strains, USDA 138 or TAL 379 from nodules, reached the same level of USDA 110 nodule occupancy at ratio 25 : 1 for both strains

Field performance of three rhizobium japonicum strains with two soybean cultivars

A field experiment was conducted in a clay loam soil to study the performance of three R. japonicum strains; USDA 110, USDA 138 AND TAL 379; in relation to their N2 fixing potential and competitive relation on two soybean cultivars [Clark and Calland]. Inoculation of soybean cultivars with these strains either singly or in combination induced significant increase in plant dry weight, N2 fixation and seed yield, strain USDA 110 was the most efficient strain in N2 fixation and in increasing seed yield on both cultivars followed by USDA 138, while TAL 379 was the least effective strain. The two soybean cultivars were similar in their response to inoculation. No significant difference in seed yield were observed between Clark and Calland cultivars. The interaction between inoculant strain and soybean cultivar was not significant

Effect of soil conditioners on some biological properties of sandy soil and the diversity of microbes involved in their biodegradation

The application of bitumen and ureaformaldehyde at the two low application rates stimulated the total bacterial, fungal and Azotobacter counts in sandy soil. The higher rate of conditioners applications, however, delayed the proliferation of soil microorganisms for 60 days. The application of bitumen and ureaformaldehyde to sandy soil planting with peanut inoculated with Rhizobium spp. Induced insignificant increase in nodulation, dry matter yield and N accumulation except the higher application rate which resulted in slight decrease in nodulation. Generally, no marked retardation of nodulation or N2 fixation was noted as a consequence to the conditioner application. The bacterial genera contributing to the biodegradation of bitumen and ureaformaldehyde in the sandy soil are Bacillus, Micrococcus, Pseudomonas, Flavobacterium, and Streptomyces. Bacillus was the most dominant genus for both soil conditioners

Field assessment of the interaction between seven soybean cultivars and three Rhizobium japonicum strains

A field experiment in a clay loam soil was conducted to evaluate the interaction between seven soybean cultivars and three Rhizobium japonicum strains. The associations of soybean cultivars with strain USDA 110 were more efficient compared to those with strain USDA 138 or TAL 379. The more efficient symbiosis was observed between strain USDA 110 with Cumberland and Pella cultivars. However, strain USDA 138 gave the best nodulation and N2 fixation with Williams 79 and Calland cultivars. Whereas strain TAL 379 was better with Corsoy and Sprite than with other cultivars. A positive yield response to inoculation was noted with strain USDA 110, being the superior strain all tested cultivars followed by strain USDA 138. The Calland, Pella and Williams 79 cultivars gave the highest yield production. The competition studies revealed that strain USDA 110 was more competitive than either USDA 138 or TAL 379. Strain USDA 110 showed higher competitive ability with Cumberland, Pella and Calland cultivars and comparatively lower competitive ability with Corsoy and Sprite cultivars. Strain USDA 138 occupied 40 and 45% of nodules formed on Calland and Williams 79 cultivars, respectively versus 20% of nodules formed on Corsoy and Pella cultivars. The total nodule occupancy of strain TAL 379 amounted 40% on Sprite and Corsoy cultivars versus 20% on other soybean cultivars

Symbiotic nitrogen fixation by two lentil cultivars in relation to rhizobium strain, competition and irrigation rates

Two Rhizobium Leguminosarum strains TAL 638 and Nitragin 92 A3 were studied in their relation to fix atmospheric nitrogen with two lentil cultivars. The experiment was conducted in a Delta Nile clay loam soil using two rates of irrigation. regardless the irrigation. regimes, the inoculation of lentil with these strains either singly or in combination induced significant increase in plant dry weight. nitrogen fixation grad seed yield. Giza 370 cultivar in symbioses with any of the strains was superior in nitrogen accumulation and seed yield than Giza 9 cultivar. However. the strain X varicty interaction was not significant. The nitrogen fixation and thee yield of both lentil cultivars were significantly affected by water regime. The frequent irrigation resulted in higher plant dry weight. N accumulation and seed yield with both cultivar The inoculum strain outcompeted the resident rhizobia. Between 72-92% of nodules were occupied by the strains used as single inoculum. In double, strain inocula, R. leguminosarum strains, TAL 638 and Nit. 92 A3 were equailly, competitive on both cultivars under the two irrigation regimes. The incidence of double strain occupancy within the nodules collected from the double strain inocula ranged between 0 to 38%

Histopathological and ultrastructural changes in the cornea in macular corneal dystrophy

Macular corneal dystrophy is a genetically determined disease affecting mainly the keratocytes and corneal endothelium, characterized by the presence of mucopolyaccharides within vacuoles inside comeal keratocytec and endothelium this material is stained positively with PAS. By electron microscopy it is found inside the cisternaeof endoplasmic reticulum. Endothlium also shared with keratocytes in the presence of the same granules with secondary degenerative changes in Descemert's membrane

Biological control of rhizoctonia solam using trichoderma viride and its relation to symbiotic nitrogen fixation by faba bean

A pot experiment was carried out using a clay loamy soil to study the efficacy of Trichoderma viride as a biocontrol for Rhizoctonia solani as well as its effect on biological nitrogen fixation by faba bean. Soil inoculation either with R. solani, Tl viride or in combination had no effect on total microbial counts in the rhizosphere soil of the faba bean. These treatments, however, enhanced the proliferation of fungal population in the rhizosphere. The infection with R. Solani induced retardation of nodulation and symbiotic nitrogen fixation. Control of the disease by T. viride resulted in normal nodulation and nitrogen fixation patterns as produced by the healthy [non infected] plants