Cloning and secretory expression of VP2 gene of infectious bursal disease virus in eukaryotic cells

VP2 gene coding region of a vaccinal strain [D78] of infectious bursal disease virus [IBDV] was cloned in a eukaryotic expression vector, pSec Tag2A. The gene was placed downstream of Ig kappa chain leader sequence, under the control of human cytomegalovirus [hCMV] immediate early enhancer and promoter. The construct pSec Tag2A-VP2 was transferred in COS-7 cell line and the expression and secretion of VP2 was assessed by dot blotting and antigen capture ELISA. The antibody used in the immunological assays was a neutralizing monoclonal antibody [1A6] against VP2. Positive reaction with the antibody indicated the construct was functional with respect to expression and secretion of a native VP2

Phospholipase C in Beijing strains of mycobacterium tuberculosis

Phospholipase of Mycobacterium tuberculosis plays an important role in pathogenesis through breaking up phospholipids and production of diacylglycerol. In this study, we examined the Beijing strains of Mycobacterium tuberculosis isolated from Iranian patients for the genes encoding this enzyme. DNA extraction was performed using CTAB [cetyltrimethylammonium bromide] from positive culture specimens in tuberculosis patients. PCR was then used to amplify the plcA, plcB, plcC genes of Beijing strain, and non-Beijing strains were identified by spoligotyping. Of 200 specimens, 19 [9.5%] were Beijing strain and 181 [90.5%] were non-Beijing strains. The results of PCR for Beijing strains were as follows: 16 strains [84.2%] were positive for plcA, 17 [89.4%] were positive for plcB and 17 [89.4%] were positive for plcC genes. The standard strain [H37RV] was used as control. The majority of Beijing strains have phospholipase C genes which can contribute to their pathogenesis but we need complementary studies to confirm the role of phospholipase C in pathogenecity of Mycobacterium tuberculosis

[ assessment of hepatitis B and C prevalence in street children of Tehran from Farvardin to Shahrivar 1386]

One of the most important problems for street children is the health issue. Many of these children are suffering from malnutrition, anemia, and respiratory, gastrointestinal and dermatologic disorders and also acquired infections such as hepatitis, AIDS, and tuberculosis. According to the emphasis for performing vaccination of high risk children, recognition of these groups has especial importance. In this descriptive study 203 street children were gathered from different places of Tehran and settled at a welfare center. These children were clinically examined by a pediatrician and requested to answer a questionnaire. Blood samples [3CC] were obtained from each of them in order to determine the existence of Hepatitis B Virus [HBV] and Hepatitis C Virus [HCV] infections by ELISA method. Among 203 street children studied in this research, 196 were boys and 7 were girls. Six cases [3%] were HBsAg positive, 54 cases were HBsAb positive [26.6%] and 16 cases were HBcAb positive [8%]. Seven cases [3.5%] were HCVAb positive. All of the positive cases were boys. According to these positive results for hepatitis B and C, additional laboratory examination for screening of acquired infectious disease such as Hepatitis seems to be necessary

[Detection of human papilloma virus among women with cervical cancer using PCR method]

Cervical cancer is the second leading cancer among women, worldwide. It is also the second malignant cause of death, particularly in women aged 25-65. In order to progress a cancer from dysplasia to invasive carcinoma, a cascade of cellular changes should occur. Since genital HPV carries oncogenes responsible for these essential changes, today HPV is considered as the major risk factor of cervical cancer. It is believed that HPV can increase the rate of cancer progression when associating with other risk factors such as smoking, taking contraceptive drugs, immunosuppression, etc. Paraffin-embedded cervical tissues of 70 patients with cervical cancer were analyzed by PCR method for presence of HPV. In addition, high risk typing of HPV positive samples was performed using HPV high risk typing PCR kit. Among all patients 49% were positive for HPV. HPV16 was the most common type detected in HPV-positive cases. Investigation of age classification showed that a majority of HPV positive cases aged between 35 and 44 years. Considering the prevalence of HPV among young women with cervical cancer and its long premalignant period, we suggest to examine all the women above 20 years of age and also check the suspected cases for HPV

Detection of Chlamydia trachomatis, Mycoplasma hominis and Ureaplasma urealyticum by multiplex PCR in semen sample of infertile men

The aim of this study was to detect Chlamydia trachomatis, Mycoplasma hominis and Ureaplasma urealyticum from semen samples of infertile men by Multiplex PCR and investigation of influence of bacteriospermia on semen parameters. Semen samples of 200 infertile men were evaluated by Multiplex PCR. In addition, analysis of semen parameters was performed according to the WHO guidelines. All the patients were without clinical symptoms of urogenital tract infection. Thirty three percent of cases showed at least one bacterium. We found a noticeable relation between the presence of bacteriospermia and the rate of non motile and morphologically abnormal sperms [P< 0.0001]. In addition, sperm concentration was lower in positive cases [P< 0.04]. There was no relation between leukocytospermia and bacteriospermia [P> 0.05]. Asymptomatic existence of Chlamydia and Mycoplasmas in urogenital tracts might play an important role in sperm impairment due to infertility. Bacteriospermia can influence sperm's motility, morphology and concentration

[Isolation of anaerobic, aerobic bacteria and mycobacterium from foot skin infections of diabetic patients]

Diabetic foot infections are a potentially severe complication of diabetes. Diabetic foot infections can sometimes lead to long-term debilitation and, in the most severe cases, amputation. They are the most common infections in patients with diabetes, whose weakened immune systems put them at an increased risk of acquiring antibiotic resistant infections. For this descriptive study, 120 diabetic patients [30 women and 90 men age ranged between 45-65 years and disease duration of 0.5 to 37 years] were investigated. Immediately after the hospitalization, specimens from infected foot lesions were taken using Thio and BHI as transport medium. Aerobic cultures were carried out in all cases according to conventional methods while anaerobic cultures were performed when appropriate. Finally, susceptibility tests were performed on isolated microorganism. Totally, 75% of cases were polymicrobial infections. We isolated gram positive cocci 95%, gram positive bacilli 35%, gram negative 55% and mycobacterium 10%. Meanwhile, we found that 12.5% of our bacteria were anaerobic and 87.5% were facultative aerobic bacteria. In antimicrobial susceptibility testing, Rifampin was the most effective antibiotic against S.aureus and peptostreptococcus. Surprisingly, E.coli was resistant to all tested antibiotics. Diabetic foot infections have a polymicrobial nature. Antibiotic treatment of infections should be prescribed on the results of microbiological investigation

[Nocardiosis; a case report]

Nocardia could be transmitted to lungs through dust particles; then transmitted to other organs via vascular system. We describe a 11-year old boy presenting with headache and vomiting. CT studies revealed hemorrhage in his right hemisphere as well as cerebral edema. He was hospitalized with primary diagnosis of hydrocephaly and pseudotumor cerebri. Further studies showed nocardia astroides in acid fast and blood agar culture

Genital trct infection in asymptomatic infertile men and its effect on semen quality

Male urogenital tract infection plays an important role in men infertility. Asymptomatic bacteriospermia has been paid attention as a major cause of male infertility. The aim of this study was to microbiological investigation of semen sample of infertile men attending to infertility clinic and evaluation of the effects of bacteriospermia on semen quality. Eighty eight infertile men were evaluated by standard bacterial culture method. Standard semen analysis was performed according to WHO guidelines. Among total cases, 35.22% [31 cases] showed at least one pathogen: 10.22% E.coli, 9.09% Coagulase Negative Staphylococci [Saprophyticcus], 6.81% Group B Streptococci, 5.88% Entrococci, 5.68% Candida sp., 2.27% Gonococci, 2.27% Staphylococcus aureus, 1.13% Klebsiella sp. and 1.13% Providencia sp. There was a significant relation between the bacteriospermia and the rate of no motile and morphologically abnormal sperms [P<0.0001]. The quality of sperm motility was significantly decreased in contaminated semen. The percentage of morphologically normal sperm was lower. E.coli and Entrococci were the most effective agents against sperm parameters. Asymptomatic bacteriospermia has a negative effect on sperm quality. E.coli and Entrococci are the most common bacteria with negative influence on sperm motility and morphology. Moreover, presence of bacteriospermia and leukocytospermia did not correlate with each other [P>0.05]. It seems that leukocytospermia is a poor marker to predict bacteriospermia

Detection of chlamydia trachomatis from urine specimens by pcr inwomen with cervicitis

Chlamydia trachomatis is the most common agent of urogenital infections in both men and women. Diagnosis of chlamy-dial infections is based on isolation of bacteria in tissue culture media that requires at least 48 to 72h. Polymerase chain reaction [PCR] is a sensitive and specific method for detection of small quantity of bacterial DNA in clinical samples. The first goal of this study was to perform a PCR testing for detecting of C. trachomatis from urine samples and after that to identify the frequency of C. trachomatis among cervicitis women and at the end, to identify the potential risk factors for chlamydial genital infection. From August to October 2002, a total of 122 consecutive women with cervicitis who attended Obstetric and Gynecology Clinic of Shoosh, Tehran-Iran were involved into the study. After DNA extraction from urine specimens, PCR tests were performed. C. trachomatis genome was detected in 14 of 94 [14/9%] urine specimens. The highest C. trachomatis cervical infection frequency was found in women with 28 to 38 years old group, elementary education level group, and in users IUD for contraception. The results of this study indicate that PCR technique is a useful method for detecting C. trachomatis in urine

Molecular methods [16s-rRNA] compared to bacteriological diagnosis of meningitis

For treatment of patients with meningitis, rapid diagnosis of the agent is very important. Nowadays all of researches have approved qualification and efficiency of molecular tests. Detection of bacteria from CSF and blood is the major problem as a result of usage of antibiotics by patients. So we researched on CSF samples by PCR test and used DG74 and RDR80 primers for 16s rRNA sequence. Our cases were 51 children with meningitis symptoms that had referred to Mofid Hospital in Tehran. Theses samples were different from culture, cell counter and protein glucose amounts. After researching we reached to these results that 23.5% of cases were positive for bacterial culture and 41.1% of them were positive for PCR test. So sensitivity of PCR was 95.23%, specificity of PCR was 96.66% and efficiency of PCR was 96%. In first group 8 specimen were PCR positive [88.8%]. In second group, all of 12 specimens were PCR positive [100%]. In third, 8 specimens were suspected for viral meningitis, only one case was PCR positive, so it had bacterial agent. In fourth group, all of 22 specimens were PCR negative. Therefore sensitivity and specificity of PCR test with 16s rRNA gene sequence in identification of bacterial agent in CSF was 95.23% and 96.66% respectively

Determination of alpha1-antitrypsin phenotypes and genotypes in Iranian patients

Alpha 1-antitrypsin [AAT] or alpha 1-protease inhibitor [PI] is the principal inhibitor of proteolytic enzyme in serum. Its phenotypic variability has been reported to be associated with liver, lung diseases and rheumatoid arthritis in humans. There is much documentation about high risk phenotypes of PI in some regions of the world, however, there are no reliable reports on these phenotypes and genotypes and their related diseases in Iranian population. The aim of this study was to determine PI phenotypes and genotypes in Iranian patients suffering from PI deficiency. For this purpose, whole blood samples from 307 patients suspected of diseases related to PI deficiency, and 156 healthy persons were examined. PI phenotypes and genotypes were determined by isoelectric focusing [IEF] and polymerase chain reactionrestriction fragment length polymorphism [PCR-RFLP], respectively. Allele frequencies from patients and normal subjects were compared. For reliability, a family study of the patients was also carried out. The PI phenotype frequencies of all six possible combinations of M, S and Z haplotypes in patients were: MM, 77.20%; MS, 6.18%; MZ, 7.17%; SS, 3.91%; ZZ, 4.56%; SZ, 0.98% and in normal subjects were: MM, 78.20%; MS, 5.76%; MZ, 15.38%; SS, 0.64%; 0% for ZZ and SZ. Analysis of data showed that there was a significant difference between patients [with liver, lung diseases and rheumatoid arthritis] and control subjects [p< 0.05]. In Conclusion, the allelic frequencies of S and Z in the patient group were 7.49% and 8.63%, while in the normal subjects were 5.13% and 4.17%, respectively. This is the first report of the prevalence of high risk alleles [Z and S] in patients suspected of PI deficiency and related diseases in Iran

Purification and molecular analysis of BCG antigen 60

Tuberculosis remains as an important socioeconomical and medical problem throughout the world and especially in Iran. Early and timely diagnosis of pulmonary and extrapulmonary tuberculosis is vital to initiate prompt treatment. Current diagnostic methods are either slow or lack enough sensitivity or specificity. Several mycobacterial antigens are involved in the complex interaction with the immune system of the host. Their identification is important for both diagnosis and protection against mycobacteria. Antigen 60 [A60] is a thermostable antigen found in the cytosol of M. bovis and M. tuberculosis. An ELISA test using A60 is designed for diagnosis of tuberculosis with satisfactory results. In previous studies, A60 has also showed a protective effect against experimental infections and useful immunotherapeutic effects in promotion of cancer development. In the present work we tried to purify A60 from the cytoplasm of BCG. A60 was purified by exclusion gel chromatography using sepharose 4B. A60 was recognized by bidimensional immunoelectrophoresis with anti-BCG and anti-A60 antiserum, where it appears as the less mobile component. In agarose electrophoresis, A60 showed only one band but in immunodiffusion it showed two immunoprecipitinogen lines with anti-BCG anti-serum. In analyzing with dot blotting, both cytoplasm and cell wall of BCG showed positive reaction with anti-A60 anti-serum. When A60 was fractionated by SDS-PAGE and analyzed by western blot using anti-A60 antibody, 65,46,40, 38 and 35 KDa protein fractions were identified. It is concluded that A60 is a macromolecular antigen of BCG with a molecular weight of 10[6]-10[7] Da and is a lipoprotein-polysaccharide complex which contains several proteins. A60 is present in both cytoplasm and cell wall of BCG and can easily be purified from BCG vaccine using exclusion chromatography by sepharose 4B, to be used for designing diagnostic tests for TB

comparison between PCR and bacteriology method in diagnosis bacterial meningitis

Meningitis is one of the lethal diseases and it's prompt diagnosis is of utmost importance. Current diagnostic methods [eg. bacterial cultures, cellular cultures, biochemical methods and cell count in CSF] are not sensitive enugh. Therefere dorelopnga new method for quick diagnosis is essential. Material and We extracted DNA from cerebrospinal fluid and PCR was performed by primers which amplified the 16s rRNA sequence. Microbial culture was also performed for comparison. We examined 51 specimens, among which 23.5% were culture positive. By PCR method, we reported 41.1 positive cases. Therefore if PCR is not used, almost half of the positive cases will be missed. We concluded that PCR is the best method of diagnosis, with high accuracy and precision for detecting microorganisms in sterile specimens in 3-4 hours. Since the 16s rRNA sequence has been similar in all prokaryotes, this sequence is considered appropriate for traget for PCR