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Biomedical subjects

M S Ibrahim

Publications and source records attributed to M S Ibrahim.

At least 19 recordsLinked to original sources
PubMed2005

Two-color multiplex assay for the identification of orthopox viruses with real-time LUX- PCR.

The LUX [Light Upon eXtension] is a real-time detection system that can be used for the detection and quantification of pathogens nucleic acids. In this study we used a universal LUX approach, a variation of the LUX detection system, for identifying Orthopoxvirus nucleic acids in real time. This approach enables the design of sequence-specific primer sets in high identity genome sequences. The assay described here is designed to allow simultaneous detection of Variola and other orthopox viruses in a multiplex format, with a limit of detection in the range of 50--100 copies of the Orthopoxvirus genome. Regression analysis showed that the assay was linear over seven orders of magnitude, with 0.97 correlation coefficient. The sensitivity and specificity of the assay, as determined from a panel of 100 samples that contained nucleic acids from a variety of bacteria and viral species, were rated at 98%. Thus, the assay offers a sensitive and specific tool for simultaneous identification and quantification of Variola and other orthopox viruses, and the approach allows more flexible sequence-specific primers design for pox viruses as well as other microbial pathogens.

Animals↗
PubMed2003

Comparison of the voltammetric studies at mercury and glassy carbon electrodes for the interaction of lumichrome with DNA and analytical applications.

The determination of the interaction between lumichrome (LC), one of the products of decomposition of the biologically important flavins, and calf thymus double-stranded DNA was performed by using cyclic voltammetry (CV) and differential pulse stripping voltammetry (DPSV) in connection with a hanging mercury drop electrode (HMDE) or glassy carbon electrode (GCE). The nature of the process taking place at both electrode surfaces was clarified. It was found that the addition of DNA to a buffered LC solution results in the decrease of redox peak currents with changes in the peak potentials at both electrodes. We assume that LC interacting with DNA produces an electrochemically inactive supramolecular complex via intercalation. There was a difference between the electrochemical parameters determined at the HMDE and those at the GCE. The binding constants ( K) of the LC-DNA complex at HMDE and GCE were determined through the changes of peak currents and their values at the 10(5) level and 10(4) level with each nucleotide residue of DNA binding one LC molecule, respectively. Furthermore, the calibration graph for the determination of DNA was obtained by the decrease in the DPSV peak current of LC in the presence of DNA. Different variables, such as the concentration of LC, the accumulation time and solution conditions, were studied and optimised to maximize the sensitivity; in addition, the detection limit and the reproducibility were determined.

Animals↗
PubMed2002

Voltammetric studies of the interaction of lumazine with cyclodextrins and DNA.

The interaction of lumazine, an antibacterial drug, with alpha-, beta-cyclodextrins and DNA in aqueous solution was studied by differential pulse stripping voltammetry and cyclic voltammetry as well as UV-vis spectroscopy. The electrochemical and absorption spectral data indicated a 1:1 complex formation of lumazine with alpha-, beta-cyclodextrins and DNA. The nature of the process, taking place at the hanging mercury drop electrode, was clarified. It was found that the complexation of lumazine molecules enhances the stacking interactions which might facilitate the formation of a perpendicularly stacked layer of lumazine-alpha-cyclodextrin complex on the electrode surface. Based on the variations in the current or absorbance, the formation constants and consequently, the Gibbs energy of these complexes were determined. The small size cavity of alpha-cyclodextrin was found to have a greater affinity for lumazine than the beta-cyclodextrin. Moreover, the interactions of lumazine-alpha-cyclodextrin or lumazine-beta-cyclodextrin inclusion complex with DNA have been investigated by means of voltammetry. The results suggest that lumazine displayed high affinity for DNA and the inclusion complex decomposed when it binds to DNA.

Algorithms↗
PubMed2001

Low or no antibody responses to human immunodeficiency virus type 1 Nef in infected carriers with subtype E, in contrast to subtype B that showed antibodies preferentially recognizing subtype-specific Nef epitopes.

The viral accessory gene product Nef has been shown to play an important role in human immunodeficiency virus type 1 (HIV-1)-induced pathogenesis. Only little information is available regarding the differences in the host immune responses against Nef protein and its function in vivo among different subtypes of HIV-1. In the present study, we showed marked differences in the immune responses to Nef protein between subtypes B and E. The amino acid sequence in subtype E Nef showed 72% homology with that in subtype B. Most murine monoclonal antibodies obtained by immunization with subtype B or E Nef protein showed cross-reactivity with both Nef proteins (80 and 67%, respectively). Next, we focused on the immune responses among infected Japanese and Thai individuals. Subtyping of the individuals into B and E was carried out by enzyme-linked immunosorbent assay (ELISA) using synthetic peptides corresponding to the V3 loop representing the principal neutralizing domain. Most of the sera from these individuals reacted strongly with Gag p24 proteins derived from subtypes B and E at similar levels. However, the immune responses among these individuals to Nef protein were markedly different. Some subtype B-infected Japanese and Thai individuals (40 and 35%, respectively) showed higher levels of anti-Nef antibodies, although these antibodies preferentially recognized epitopes specific to subtype B. On the other hand, most of the subtype E-infected Japanese and Thai individuals showed low or no antibody responses to Nef proteins. Thus, immune responses to Nef were markedly different between subtypes B- and E-infected carriers, suggesting different function(s) for Nef in AIDS pathogenesis. Further, vaccine design must take into account the different subtypes of HIV-1.

Acquired Immunodeficiency Syndrome↗
PubMed2000

Phase-selective a.c. adsorptive stripping voltammetry of lumazine on a hanging mercury drop electrode.

The electrochemical behavior of lumazine (LMZ), an important antibacterial agent, has been studied at the hanging mercury drop electrode (HMDE). The nature of the process taking place at the HMDE was clarified. Its adsorption behavior at HMDE has been studied by using a.c and cyclic voltammetry (CV). Both the molecule and its reduced product appeared to be adsorbed at the surface of the electrode. Controlled adsorptive accumulation of LMZ on the HMDE provides the basis for the direct stripping measurement of that compound in the subnanomolar concentration level. Experimental and instrumental parameters for the quantitative determination were optimized. Phase-selective a.c voltammetry provided the best signal and gave a detection limit of 0.15 microg L(-1) (9.0 x 10(-10) mol/L) LMZ in aqueous solution. Molecules or ions which may interfere were studied.

Adsorption↗
PubMed2000

Detection of Francisella tularensis in infected mammals and vectors using a probe-based polymerase chain reaction.

We investigated the use of a TaqMan 5' nuclease assay (5NA) directed against the Francisella tularensis outer membrane protein (Fop) gene and a polymerase chain reaction-enzyme immunoassay (PCR-EIA) directed against the tul 4 gene for detection of this organism in experimentally infected mice and in field-collected tick vectors. We also evaluated the use of specially formulated filter paper (FTA) for rapid sample preparation. The 5NA had a detection limit of 1 pg of genomic DNA (<100 colony-forming units) and could be completed within several hours. The PCR-EIA could detect 1 pg of genomic DNA and 10 attograms (ag) (22 copies) of cloned insert, but takes longer to perform. Both assays were genus-specific, and successfully detected F. tularensis in mouse tissues (5NA) and in tick extracts (PCR-EIA). The FTA paper provided inexpensive, rapid, template preparation for the tick extracts, mouse tissues, and DNA obtained from clinical specimens. These probe-based assays have the potential to provide rapid, real-time/high-throughput molecular diagnostics in field situations.

Animals↗
PubMed1998

Real-time microchip PCR for detecting single-base differences in viral and human DNA.

This report describes real-time 5' nuclease PCR assays to rapidly distinguish single-base polymorphism using a battery-powered miniature analytical thermal cycling instrument (MATCI). Orthopoxviruses and the human complement component C6 gene served as targets to demonstrate the feasibility of using the MATCI for diagnosis of infectious diseases and genetic disorders. In the Orthopoxvirus assay, consensus Orthopoxvirus PCR primers were designed to amplify 266-281 base-pair (bp) segments of the hemagglutinin (HA) gene in camelpox, cowpox, monkeypox, and vaccinia viruses. A vaccinia virus-specific fluorogenic (TaqMan) probe was designed to detect a single-base (A/G) substitution within the HA gene. In the C6 gene assay, a 73-bp segment of the C6 gene was PCR-amplified from human genomic DNA, and TaqMan probes were used to detect a single-base (A/C) polymorphism in the second position of codon 98. The MATCI correctly identified the nucleotide differences in both viral DNA and human genomic DNA. In addition, using a rapid DNA preparation method, it was possible to achieve sample, preparation of human genomic DNA, DNA amplification, and real-time detection in less than 1 h.

DNA↗
PubMed1998

5' nuclease PCR assay to detect Yersinia pestis.

The 5' nuclease PCR assay uses a fluorescently labeled oligonucleotide probe (TaqMan) to rapidly detect and quantitate DNA templates in clinical samples. We developed a 5' nuclease PCR assay targeting the plasminogen activator gene (pla) of Yersinia pestis. The assay is species specific, with a detection threshold of 2.1 x 10(5) copies of the pla target or 1.6 pg of total cell DNA. The assay detected Y. pestis in experimentally infected Xenopsylla cheopis fleas and in experimentally infected monkey blood and oropharyngeal swabs. The TaqMan assay is simple to perform and rapid and shows promise as a future field-adaptable technique.

Animals↗
PubMed1997

Detection of Rift Valley fever virus in mosquitoes by RT-PCR.

A reverse transcriptase-polymerase chain reaction (RT-PCR) assay to detect Rift Valley fever (RVF) virus RNA in experimentally infected mosquitoes was developed. The specificity of the assay was evaluated with three other phleboviruses; sandfly fever Sicilian (Sabin), sandfly fever Naples (Sabin) and Punta Toro (MSP 3) viruses. The relative sensitivity of the assay, determined by using RVF virus RNA extracted from serial dilutions of virus culture, was approximately 50 plaque forming units. This sensitivity level was 100-fold higher when a nested PCR procedure was used. When the RT-PCR assay was used with coded samples of intrathoracically-infected and uninfected mosquito, the assay detected the virus in all infected mosquitoes. With this assay, it was possible to detect RVF virus RNA in a single infected mosquito in the background of 10, 25 or 50 uninfected mosquitoes.

Animals↗
PubMed1997

The potential of 5' nuclease PCR for detecting a single-base polymorphism in Orthopoxvirus.

A fluorogenic 5' nuclease PCR assay was evaluated for its ability to specifically detect and differentiate DNA of two Orthopoxvirus species. A pair of consensus primers that target a DNA segment of the Orthopoxvirus haemagglutinin gene, and two oligonucleotide probes; each labelled with a different fluorescent reporter dye and the same quencher dye, were used in a single-tube assay. The assay is based on the 5'-->3' nuclease activity of AmpliTaq DNA polymerase that cleaves a fluorescein-labelled hybridized probe. Probe cleavage generates specific fluorescent signals whose intensity can be quantified by fluorometry. After evaluating the effects of various annealing temperatures and probe concentrations and normalizing the emission intensities of the reporter dyes, it was possible to detect and differentiate monkeypox and vaccinia virus DNAs on the basis of a single-base polymorphism. The sensitivity of the 5' nuclease PCR assay is comparable to the sensitivity of ethidium bromide-stained gels, but the assay provides higher specificity and virtually eliminates the need for laborious post-PCR processing.

DNA Primers↗
PubMed1997

A colorimetric PCR-enzyme immunoassay to identify hantaviruses.

BACKGROUND: Hantaviruses cause two serious human diseases: hantavirus pulmonary syndrome and hemorrhagic fever with renal syndrome. At least nine hantaviruses are known to be pathogenic for humans and numerous others, with unknown disease potential, have been detected in rodents. Assays to quickly identify specific hantaviruses would be useful both for clinical diagnosis and in risk assessment studies. OBJECTIVES: The goal of our study was to develop and test a specific and sensitive PCR-based assay for identification and differentiation of hantaviruses. STUDY DESIGN: We developed an assay that combined RNA-PCR amplification and colorimetric enzymatic detection to identify representative European, Asian, and north American hantaviruses. RNAs from 18 hantavirus strains of nine species were amplified in the presence of digoxigenin-dUTP by using a single pair of oligonucleotide primers and polymerase chain reaction (PCR) performed by using rTth DNA polymerase. Digoxigenin-labeled PCR products were hybridized in solution to virus type-specific biotinilated probes, captured onto streptavidin-coated microtiter plates and detected by horseradish peroxidase-labeled anti-digoxigenin antibodies and a chromogenic substrate. RESULTS AND CONCLUSIONS: The assay correctly identified each homologous virus type tested. The detection limit of the assay was approximately 15 PFU or at least 50 copies of the viral genome. The assay is simple and strain-specific and is adaptable for automation, making it more practical than other available techniques for accurate and reliable diagnosis and typing of hantaviruses.

Orthohantavirus↗
PubMed1996

Muscle actin gene from Aedes aegypti (Diptera:Culicidae).

A recombinant phagemid containing a 1,240-bp insert encoding an actin was isolated from a yellow fever mosquito, Aedes aegypti (L.), complementary DNA library. This insert (pBS-Act35) contained an open reading frame of 822 bp whose deduced amino acid sequence exhibited > 95% homology with the carboxyl terminal 274 amino acids of Drosophila melanogaster Meigen and silkworm, Bombyx mori (L.), actin genes. Reverse transcriptase-polymerase chain reaction was used to clone and determine the sequence of the additional 306 nucleotides that comprise the 5' end of the gene. The coding nucleotide sequence of the whole gene (designated Aeact-1) exhibited between 81 and 89% homology with coding sequences of D. melanogaster and B. mori actin genes, and its deduced amino acid sequence exhibited > 95% homology with those genes. The highest similarity of Aeact-1 gene at the amino acid sequence level was with B. mori and D. melanogaster muscle actins. Southern blot analysis indicated that the Aedes genome contains at least 5 actin-related sequences.

Actins↗
PubMed1996

Venezuelan equine encephalitis in BALB/c mice: kinetic analysis of central nervous system infection following aerosol or subcutaneous inoculation.

OBJECTIVE: To investigate the routes of entry of Venezuelan equine encephalitis (VEE) virus into the brain, we infected BALB/c mice with a virulent strain (V3000) by aerosol or subcutaneous inoculation. METHODS: Immunohistochemistry and in situ hybridization methods were used to detect VEE virus in tissues taken at daily intervals postinfection. RESULTS: In both groups, virus in the brain first appeared in olfactory regions. Aerosol exposure caused early massive infection of olfactory epithelium, which developed into bilaterally symmetrical infection of the olfactory nerves, olfactory bulbs, and lateral olfactory tracts by day 2 postinfection. After subcutaneous inoculation, VEE in the brain also appeared first in olfactory regions, but was not detected until day 3 postinfection. By day 4 postinfection, VEE viral infection had spread throughout the brain in both groups. Vascular endothelium and the choroid plexus remained uninfected during the entire study. CONCLUSIONS: Our findings suggest that VEE virus, whether given by aerosol or subcutaneously, first enters the brain through the olfactory tract.

Aerosols↗
PubMed1995

An immunological factor that affects Anopheles gambiae survival.

High titers of antibodies against Anopheles gambiae midguts were produced in New Zealand rabbits to identify midgut targets for an antimosquito vaccine. The serum from one of 8 rabbits (designated R2B6) killed 71.6% (Abbott's adjusted % mortality) of An. gambiae within 7 days. Mosquitoes ingesting R2B6 serum were unable to absorb their blood meal nutrients, resulting in reduced oviposition and egg hatching rates. Anopheles stephensi and Anopheles arabiensis were also killed when ingesting R2B6 serum but Anopheles freeborni, Anopheles albimanus, and Aedes aegypti were not affected. The mosquitocidal factor was a relatively large molecule (> 100,000 MW) maintained at threshold levels in the sera and killing was complement independent. Mortality, however, was not IgG mediated, as determined by protein A-sepharose fractionation. This surprising finding confounds possibilities of using antibodies against whole mosquito midguts as a step in the development of antimosquito vaccines.

Aedes↗
PubMed1993

Delta virus versus HBsAg in chronic active hepatitis and their relation to clinical, laboratory, and morbidity findings in bilharzial and non-bilharzial patients.

This work was done to study the effect of delta virus and HBV infection, as two aetiological factors on clinical presentation, biochemical liver functions and prognosis of chronic active hepatitis (CAH) in schistosomiasis infected and non-infected patients. 66.7% of the patients were carriers for HBsAg, among them 41.7% were infected with delta virus. It was evident that the clinical presentation of the patients with positive serological markers of HBV and HDV demonstrated advancing liver disease than in the other studied groups. Moreover, the biochemical liver profile was significantly affected when the triad of chronic HBV, delta virus and schistosomiasis infection was present. This could be related to the immunosuppression caused by schistosomal infection. Furthermore, mortality rate was significantly higher in schistosomiasis infected individuals.

Adolescent↗
PubMed1992

Surface-associated antigens of Brugia malayi L2 and L3 parasites during vector-stage development.

Surface and metabolic labeling procedures were used to characterize the composition and the time of expression of Brugia malayi L2 and L3 surface-associated molecules as the larvae develop within the mosquito vector. Larvae were harvested from mosquito tissues at 5 (early L2), 8 (late L2) and 11 (L3) days post-infection and labeled with 125I-Iodo-Gen. The results of one-dimensional analysis showed that there is a progressive increase in the complexity of peptides associated with the surface of developing larvae, culminating in the expression of 7 major labeled components on L3s. Both L2 and L3 parasites have surface-associated components of 42, 35, 33, 19 and 17 kDa. Between days 8 and 11 of development in the insect vector, Brugia malayi undergoes the L2 to L3 molt and acquires additional major immunogenic peptides of 40 and 22 kDa. Two-dimensional analyses of extracts from 125I-labeled L2s and L3s revealed that the major 35-, 33-, 19- and 17-kDa molecules are part of a peptide complex that forms a 'ladder' between 17 and 150 kDa. To gain information on the times during which the major surface-associated molecules are produced by the parasite, larvae were labeled with [35S]methionine either in situ as they developed within the mosquito or during culture after exiting the vector. For in situ labeling, [35S]methionine was introduced into the hemolymph of infected mosquitoes by micro-injection at days 2, 5 and 8 post-infection and the larvae were allowed to develop for an additional 3 days. The results of 1- and 2-dimensional analyses of [35S]methionine-labeled extracts from vector-stage or post-vector-stage larvae indicate that the molecules associated with the surface of B. malayi L3s are synthesized between day 5 and day 11 of development in the insect host. Immediately after the larvae exit the vector, the synthesis of the 40 and 22-kDa peptides is drastically reduced or terminated.

Animals↗