Deoxyribonucleic Acid Polymerase Chain Reaction Research Articles

Assessing Responses to Treatment of Opportunistic Mycoses and Salvage Strategies

Invasive fungal disease (IFD) in immunocompromised patients remains a major cause of morbidity and mortality and there is a pressing need for studies of novel antifungal agents and strategies to improve outcomes. Trial design details often determine not only the appropriate interpretation of the results, but also their translation into clinical practice. However, the conduct of IFD clinical trials remains challenging due to the rarity of IFD, heterogeneity of underlying diseases, and the lack of clear and standardized response criteria. Response assessments are influenced by host, underlying disease and treatment factors as well as eligibility criteria. In addition, the criteria used to assess response, when response is assessed and the type of antifungal therapy under study can impact response evaluations. This article will discuss recent trials of primary, salvage, empiric, and prophylactic antifungal therapy with specific attention to the design of these antifungal therapy trials and how their designs influence their interpretation. The potential role of surrogate markers, such as the galactomannan index, fungal deoxyribonucleic acid polymerase chain reaction assay, and (18F) fluorodeoxyglucose positron emission tomography scans in establishing the early diagnosis of IFD, as well as enhancing the ability to assess outcomes to antifungal therapy, and thereby the optimal duration of antifungal therapy, will be discussed.

Mother-To-Child Transmission Of Hiv At The University Of Benin Teaching Hospital, Benin City, Nigeria

Introduction: A variety of drugs including single dose (SD) nevirapine, zidovudine and its combination, given to mother in pregnancy and baby postnatally have been used to reduce mother-to-child HIV transmission rate. The experience at the University of Benin Teaching Hospital (UBTH), using highly active antiretroviral therapy (HAART) is reported. Methods: A total of 317 Mothers/ babies pairs recruited at the Prevention of Mother to Child Transmission (PMTCT) programme of UBTH who had HAART, were recruited for the study. HIV status of the infants was determined by deoxyribonucleic acid polymerase chain reaction (DNA PCR). Results: Of the 285 mothers/ infants pairs who adhered to our protocol, the transmission rate was 2.46 %. The babies who had mixed feeding had significantly higher rates than those who were exclusively breast-fed or had replacement feeding. P =

The Impact of Prevention of Mother to Child Transmission of HIV Programme In the Federal Capital Territory, Abuja

Human Immunodeficiency virus (HIV)/ acquired immune deficiency syndrome (AIDS) is spreading rapidly among the world's children especially in Sub-Saharan Africa. Mother-to-child transmission (MTCT) is the major route of acquired this disease in children. Prevention of mother-to-child transmission (PMTCT) of HIV programme is aimed at reducing HIV/AIDS in children to its barest minimum. The aim of the present study is to determine the impact of PMTCT programme on HIV exposed infants in the Federal Capital Territory (FCT), Abuja. A six month prospective study of 160 HIV exposed infants attending Paediatric Outpatient Special Treatment Clinic (POSTC) of the University of Abuja Teaching Hospital (UATH) was carried out. Exposed infants were categorized based on their participation in the PMTCT programme. Deoxyribonucleic acid (DNA) Polymerase Chain Reaction (PCR) test was used for early diagnosis of HIV infection in the study infants. Overall transmission rate of HIV infection among the study subjects was 33.7%. Transmission was found to occur in 6.7% of infants who participated in PMTCT programme and in 68.6% of those not involved in the programme, P < 0.001. For infants in the full programme, transmission occurred in 2.7% of cases and in 25.0% among those involved partially, P < 0.05. The study shows that transmission of HIV infection to exposed infants is high in FCT, Abuja. Transmission was however found to be significantly lower in infants who participated in PMTCT programme and even much lower in those involved in the full programme. It is therefore recommended that there is an urgent need to establish full PMTCT programme in many health care facilities across the nation as a major way of reducing paediatric HIV/AIDS in the country.

Molecular and cytogenetic evidence of three sibling species of the Anopheles barbirostris Form A (Diptera:Culicidae) in Thailand

Nine isoline colonies of Anopheles barbirostris Form A, derived from individual isofemale lines from Chiang Mai, Phetchaburi, and Kanchanaburi, were established in our insectary at Chiang Mai University. All isolines shared the same mitotic karyotype (X(1), X(2), Y(1)). Molecular analysis of deoxyribonucleic acid (DNA) sequences and polymerase chain reaction (PCR) products of ITS2, COI, and COII regions revealed three distinct groups: A1 (Chiang Mai), A2 (Phetchaburi), and A3 (Kanchanaburi). Crossing experiments among the three groups exhibited strong reproductive isolation, producing low and/or non-hatched eggs, and inviable and/or abnormal development of the reproductive system of F(1)-progenies. Asynaptic regions along the five polytene chromosome arms of F(1)-hybrid larvae clearly supported the existence of three sibling species within A. barbirostris Form A, provisionally named species A1, A2, and A3.

The role of biotechnology in the socio-economic advancement and national development: An Overview

Biotechnology is any technique which involves the application of biological organisms or their components, systems or processes to manufacturing and service industries to make or modify products, to improve plants or animals or to develop micro-organisms for special uses. Since 1953, when James Watson and Francis Crick identified the structure of deoxyribonucleic acid (DNA) as the genetic basis of all living organisms, the scientific understanding of biological and genetic processes has dramatically accelerated. The present molecular techniques such as cloning, genetic engineering, recombinant DNA technique and polymerase chain reaction (PCR) involve genetic manipulations using microorganisms such as bacteriophages and bacterial plasmids as vectors and bacterial cells as hosts. Biotechnology revolution has spawned new industries focused on manipulating human, animal, plant and microbial agents to create heretofore unattainable products and services. Biotechnology occupies a very strategic position in the socio-economic advancement and development of the nation in particular and the world at large.

The relationship between ‘wild’ and ‘building’ isolates of the dry rot fungus Serpula lacrymans

Molecular and morphological parameters of Serpula lacrymans isolates from various sites in the built environment in Europe and Australia were compared to similar parameters of ‘wild’ isolates from India, the Sumava Mountains (Czech Republic) and Mount Shasta (USA). The Indian, Czech Republic and all of the building isolates bar one showed identity in both molecular and morphological features. The Australian and the USA isolates (BF-050 and USA’94 respectively) showed specific morphological differences and could be separated on the basis of randomly amplified polymorphic deoxyribonucleic acid polymerase chain reaction (RAPD PCR) with the USA isolate being least closely related to the S. lacrymans type strain of FPRL12C. ITS sequence data revealed two base differences between FPRL12C and BF-050 in the 673 sequenced, nine differences between FPRL12C and USA’94 and 16 differences between USA’94 and the closely related organism Serpula himantioides. The possible evolutionary relationships between the various isolates are discussed along with suggestions for the origin of S. lacrymans as a scourge of the built environment in many temperate areas of the world.

Systemically administered mesenchymal stromal cells transduced with insulin-like growth factor-I localize to a fracture site and potentiate healing.

To determine to the ability of systemically administered pluripotential mesenchymal stromal cells to localize to a fracture site and whether transduction with a therapeutic gene, insulin-like growth factor-I (D1-IGF-I), could potentiate healing. Murine model, basic science study. Laboratory. Closed, transverse, mid-shaft femur fractures were produced in 108 Balb/c mice after intramedullary stabilization. A cloned, pluripotential, mesenchymal cell line, termed D1, was stably transfected with either the gene beta-galactosidase (D1-BAG) as a histologic marker or with the gene IGF-I (D1-IGF-I) growth factor. Mice received systemic injections of either D1-BAG cells for in vivo localization or D1-IGF-I for therapeutic intervention. A third group received lactated Ringer's solution and served as control. Sections obtained from the fracture site and contralateral femurs were examined histologically and by deoxyribonucleic acid-polymerase chain reaction (DNA-PCR) to detect the presence of transplanted cells at 2, 4, and 6 weeks after fracture. Matrix mineralization and callus maturation were evaluated by histology. At all time points, using histologic staining with X-gal and deoxyribonucleic acid-polymerase chain reaction for marker genes, there was a statistically greater number of transplanted cells ( p< 0.001) and significantly higher DNA-PCR for marker genes ( p< 0.001) in the fractured femurs than in the nonfractured femurs. Mice receiving D1-IGF-I cells demonstrated a greater percent of mineralized callus than controls at two weeks (p < 0.05). At 4 and 6 weeks, treated mice demonstrated on average greater mineralized matrix and accelerated progression to an osseous callus as compared with the control group. Cell-based gene therapy has the potential to deliver higher therapeutic levels of growth factors specifically at the site of cell localization while minimizing wider systemic side effects. This study demonstrates that systemically injected IGF-I transduced, mesenchymal cells are able to return to and repopulate the bone marrow. More importantly, these cells localize preferentially to a fracture site and accelerated fracture healing.

Detection of Neisseria gonorrhoeae and Chlamydia trachomatis colonization of the gravid cervix

Objectives: The aims of the study were to determine whether a Gram stain of cervical mucus can accurately rule out infection with Neisseria gonorrhoeae or Chlamydia trachomatis and to compare a diagnostic test that is based on the polymerase chain reaction with a deoxyribonucleic acid probe in the detection of these organisms. Study Design: Gravid patients were screened for N gonorrhoeae and C trachomatis with a deoxyribonucleic acid probe, Gram stain, and analysis with the polymerase chain reaction. A normal, noninfected sample was defined by <10 polymorphonuclear leukocytes per high-power field on the Gram stain. Standard statistical methods were used to compare results of the Gram stain and the deoxyribonucleic acid probe, as well as to compare results of deoxyribonucleic acid probe hybridization and polymerase chain reaction analysis. A P value of <.05 was considered statistically significant. Results: Patient enrollment totaled 519. The prevalence of infection as determined by deoxyribonucleic acid probe hybridization was 1.4% for N gonorrhoeae (7/518) and 6.8% for C trachomatis (35/518). The cervical Gram stain predicted the absence of infection in 17% (90/518) of patients, with a negative predictive value of 99% for N gonorrhoeae and 97% for C trachomatis. African American race, age <20 years, and unmarried status were all predictors of the presence of C trachomatis or N gonorrhoeae cervicitis. For the patients who lacked these risk factors (n = 74), the Gram stain had 100% negative predictive value. Analysis with the polymerase chain reaction detected 8 additional patients with C trachomatis and 105 additional patients with N gonorrhoeae, in comparison with deoxyribonucleic acid probe hybridization. Conclusion: The cervical Gram stain can accurately predict the absence of N gonorrhoeae and C trachomatis in gravid women. Analysis with the polymerase chain reaction indicates that N gonorrhoeae and C trachomatis are significantly more prevalent in this population than previously reported. (Am J Obstet Gynecol 2000;183:340-6.)

Molecular Papanicolaou tests in the twenty-first century: Molecular analyses with fluid-based Papanicolaou technology

Objective: This study was undertaken to demonstrate the feasibility of performing molecular analyses at the deoxyribonucleic acid, ribonucleic acid and protein levels of cervical cytologic examination with a methanol fluid-based Papanicolaou specimen collection system. Study Design: Genomic deoxyribonucleic acid and total ribonucleic acid were extracted from cell pellets obtained from the residual fluid-based Papanicolaou specimen collection buffer after clinical processing. Genomic and human papillomavirus deoxyribonucleic acid polymerase chain reaction and reverse transcriptase–polymerase chain reaction were performed. Messenger ribonucleic acid transcript analysis and human papillomavirus 16 E6 mutational analysis were also performed. Methylation-specific polymerase chain reaction was used to evaluate hypermethylation status of the p16 gene and the gene for E-cadherin. Immunohistochemical staining for protein expression was performed on the processed monolayer slides. Results: Cell pellets from the residual fluid-based cytologic specimen yielded good quality deoxyribonucleic acid and ribonucleic acid. Molecular analyses of genomic deoxyribonucleic acid were successful for the identification of human papillomavirus E6 and p53 polymorphism status by means of restriction enzyme digestion and direct sequencing. Methylation status of the promotor regions of the p16 tumor suppressor gene and the gene for E-cadherin were also successfully identified. Ribonucleic acid was used as the template for transcript analysis and mutational analysis of the corresponding complementary deoxyribonucleic acid of the p53 gene. Protein expression analysis was demonstrated by immunohistochemical staining for carcinoembryonic antigen. Conclusion: It is feasible to conduct multiple molecular analyses at the deoxyribonucleic acid, ribonucleic acid, and protein levels of the cervicovaginal cell pellets from the residual fluid-based Papanicolaou cytologic specimen. This relatively simple and widely used collection system will allow significant advances in molecular epidemiology and eventual development of a molecular Papanicolaou test. (Am J Obstet Gynecol 2000;183:39-45.)

The cytochrome P450 2D6 (CYP2D6) enzyme polymorphism: screening costs and influence on clinical outcomes in psychiatry.

This study examined factors that affect cost, reliability, and the value of determining the cytochrome P450 2D6 (CYP2D6) polymorphism in clinical practice. The method of deoxyribonucleic acid isolation, sample preparation, oligonucleotide primers, and polymerase chain reaction procedures were scrutinized for their effect on CYP2D6 genotyping efforts. The determination of the CYP2D6 A, B, D, E, and T alleles was used to identify the deficiency in CYP2D6 expression in 161 individuals phenotyped for CYP2D6 activity with dextromethorphan. The CYP2D6 genotype was assessed in 74 outpatients who had received diagnoses of depression. Eighteen of these patients were screened because of an adverse response to a tricyclic or antidepressant known or suspected to be a CYP2D6 substrate. The CYP2D6 A, B, C, D, E, and T alleles could be detected in 13 hours at a cost of $84 per sample by judicious selection of conditions and procedures. The genotype provided an accurate predictor of CYP2D6 expression in all 134 subjects who expressed the enzyme and in all 27 unrelated individuals phenotyped as deficient in CYP2D6 activity. In the patient group that experienced adverse effects, 44% of all CYP2D6 gene copies contained the A, B, D, E, or T allele(s) associated with inactive CYP2D6 expression. This was more than twice the rate for the occurrence of mutant alleles in the other 56 psychiatric patients (21%) and in 80 random subjects from the general population (20%; p < 0.05). Screening psychiatric patients for CYP2D6 expression may distinguish metabolic-based therapeutic problems from drug sensitivity caused by other mechanisms.

Testing for in utero human immunodeficiency virus infection with fetal blood sampling

OBJECTIVE: Our purpose was to evaluate the potential of midtrimester fetal blood sampling to detect in utero human immunodeficiency virus type 1 infection. STUDY DESIGN: Ultrasonographically guided fetal blood sampling was performed before pregnancy termination in 28 women infected with human immunodeficiency virus type 1. Mean gestational age was 22 weeks (range 15 to 29 weeks). Samples were tested for human immunodeficiency virus with undissociated p24 antigen and deoxyribonucleic acid polymerase chain reaction or viral isolation by cell culture. After terminations fetal thymuses were also tested for human immunodeficiency virus infection by polymerase chain reaction. Polymerase chain reaction was also performed on maternal cells to confirm that the primer pairs used were able to detect human immunodeficiency virus type 1 strains present. RESULTS: All fetal blood samples had negative results by p24 antigen and polymerase chain reaction or virus culture and all fetal thymuses were negative by polymerase chain reaction. CONCLUSION: Early in utero human immunodeficiency virus infection appears infrequent, supporting the hypothesis that mother-to-child transmission predominantly occurs late in pregnancy. Therefore midtrimester prenatal diagnosis is not currently of use to women in making reproductive decisions. (Am J Obstet Gynecol 1996;175:489-93.)

Prenatal diagnosis of Kell blood group genotypes: KEL1 and KEL2

OBJECTIVE: Our purpose was to devise diagnostic test(s) that determine fetal KEL1 and KEL2 genotypes. STUDY DESIGN: KEL1 and KEL2 polymorphisms are due to a single C to T base substitution at nucleotide 698 of exon 6 of KEL. This allowed us to develop two polymerase chain reaction tests that distinguish KEL1/1 and KEL2/2 homozygotes and KEL1/2 heterozygotes. The first test uses a Bsm I restriction fragment length polymorphism in a genomic deoxyribonucleic acid polymerase chain reaction product containing the single base polymorphism, and the second test uses allele-specific primers to distinguish KEL1 and KEL2 genotypes. These tests were applied in a blind study to 15 amniotic fluid deoxyribonucleic acid samples. The corresponding KEL1 and KEL2 fetal red blood cell phenotypes were determined serologically. The tests were also applied to two families in which the mothers had antibodies to KEL1. RESULTS: In all cases results of analysis of Kell genotypes from the amniotic fluid deoxyribonucleic acid samples agreed with the fetal red blood cell Kell phenotypes. The tests were also successfully used to determine fetal Kell genotype by use of peripheral blood deoxyribonucleic acid. CONCLUSION: Two polymerase chain reaction - based tests can be used for prenatal diagnosis of KEL1 and KEL2 genotypes; these procedures should prove useful in the proper management of Kell-sensitized pregnancies. (Am J Obstet Gynecol 1996;175:455-9.)

Relation between the presence of human papillomavirus type 16 deoxyribonucleic acid in cervicovaginal cells and general health condition

Cervicovaginal cell samples were analyzed for the presence of human papillomavirus type 16 deoxyribonucleic acid. All 99 women included in the study had normal Papanicolaou smear results, normal findings on wet smear, and no clinical signs of genital papillomavirus or any other genital infection. Deoxyribonucleic acid polymerase chain reaction was performed with three different human papillomavirus type 16-specific primer pairs from the early 6, early 7, and upstream regulatory regions. Human papillomavirus type 16 was detected in 21% of the 99 women. Seven of 69 women (10%) who were not taking any medication except for cyclic estrogen or progestin replacement therapy were carriers of human papillomavirus type 16. Seven of 19 women (37%) who used hormonal contraceptives or who sought early pregnancy termination were carriers of human papillomavirus type 16. All 7 women with diseases that required frequent hospital care were carriers of human papillomavirus type 16. The 4 women who had never had sexual intercourse were not carriers of human papillomavirus type 16. Our results indicate that the human papillomavirus type 16 prevalence in women may reflect to some extent the general health conditions of the patients.