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PubMedDate not supplied

Current advances in the diagnosis and treatment of AIDS: an introduction.

Significant progress has been made in the diagnosis and treatment of AIDS. Laboratory tests available for assessment of human immunodeficiency virus (HIV) infection include the detection of antibodies to HIV type 1, the direct detection of the virus, the identification of surrogate markers, and the phenotypic analysis of peripheral blood mononuclear cells. Clinicians have made great strides in the treatment of tumors, opportunistic infections, and complications associated with AIDS as well as in the treatment of the infection itself. In selected patients, treatment with interferon-alpha has been successful against AIDS-related Kaposi's sarcoma. Attempts to treat the leukopenia and anemia of patients with AIDS by the administration of hematopoietic growth factors have resulted in increased white blood cell counts and a decrease in erythrocyte transfusion requirements. In addition to zidovudine, several antiretroviral agents are undergoing testing, including the nucleoside analogues dideoxycytidine and dideoxyinosine, soluble CD4, and the glycosidase inhibitor N-butyldeoxynojirimycin.

Acquired Immunodeficiency Syndrome↗
PubMed2000

Monitoring bioluminescent Staphylococcus aureus infections in living mice using a novel luxABCDE construct.

Strains of Staphylococcus aureus were transformed with plasmid DNA containing a Photorhabdus luminescens lux operon (luxABCDE) that was genetically modified to be functional in both gram-positive and gram-negative bacteria. S. aureus cells containing this novel lux construct, downstream of an appropriate promoter sequence, are highly bioluminescent, allowing the detection of fewer than 100 CFU in vitro (direct detection of exponentially dividing cells in liquid culture). Furthermore, these bacteria produce light stably at 37 degrees C and do not require exogenous aldehyde substrate, thus allowing S. aureus infections in living animals to be monitored by bioluminescence. Two strains of S. aureus 8325-4 that produce high levels of constitutive bioluminescence were injected into the thigh muscles of mice, and the animals were then either treated with the antibiotic amoxicillin or left untreated. Bioluminescence from bacteria present in the thighs of the mice was monitored in vivo over a period of 24 h. The effectiveness of the antibiotic in the treated animals could be measured by a decrease in the light signal. At 8 h, the infection in both groups of treated animals had begun to clear, as judged by a decrease in bioluminescence, and by 24 h no light signal could be detected. In contrast, both groups of untreated mice had strong bioluminescent signals at 24 h. Quantification of CFU from bacteria extracted from the thigh muscles of the mice correlated well with the bioluminescence data. This paper shows for the first time that bioluminescence offers a method for monitoring S. aureus infections in vivo that is sensitive and noninvasive and requires fewer animals than conventional methodologies.

Amoxicillin↗
PubMed1991

Detection and identification of Helicobacter pylori by the polymerase chain reaction.

A polymerase chain reaction for the specific detection of Helicobacter pylori was developed using a primer pair derived from the nucleotide sequence of the urease A gene of H pylori. Specific amplification of a 411 base pair DNA fragment from all strains of H pylori tested was achieved. Ten organisms were detected using the PCR and the technique permitted direct detection of H pylori in clinical biopsy samples. PCR will be useful for both prospective and retrospective investigation of the aetiology and epidemiology of H pylori associated disease.

Base Sequence↗
PubMed2004

Mass spectrometric detection of protein, lipid and heme components of cytochrome c oxidase from R. sphaeroides and the stabilization of non-covalent complexes from the enzyme.

The cytochrome c oxidase enzyme from the Rhodobacter sphaeroides bacteria exists as a complex of four peptide subunits, two hemes, and a variety of lipids and metal ions held together by non-covalent forces. While the native enzyme functions as an associated unit, this complex usually dissociates during MALDI- TOF analysis. Through the use of matrix additives such as sucrose, the complete complex and partial complexes can be stabilized in the MALDI-TOF experiment. The dissociation of the complex allows for the detection of the components of the enzyme. The direct detection of associated lipids from an aqueous solution of the intact enzyme may eliminate the need for enzyme disruption and lipid extraction. The partial dissociation of multisubunit enzymes in such experiments may allow for the determination of subunit-subunit and subunit-lipid interactions

Amino Acid Sequence↗
PubMed1995

Vestibular function in bilateral progressive sensorineural hearing loss.

To investigate the vestibular function in patients with bilateral progressive sensorineural hearing loss, we examined 5 cases using electronystagmography. Cases 1, 2 and 3 were adult type, and Cases 4 and 5 juvenile type. All patients had dizzy spells in the early stage of the disease, and showed spontaneous nystagmus. Bilateral reduction of caloric response and very low vestibulo-ocular reflex (VOR) gain on rotation testing were observed in Cases 1, 2 and 3. Case 4 showed right canal paresis upon the caloric test and left directional preponderance upon the rotation test. Case 5 showed good responses to both tests. Optokinetic afternystagmus (OKAN) in Cases 2, 3 and 5 was not brisk, and Cases 1 and 2 showed directional preponderance of OKAN. OKAN was useful for detecting directional preponderance. We believe there are two types of this disease which correlate with vestibular function. One type is associated with high grade vestibular dysfunction while, in the other, vestibular function is reasonably good.

Adult↗
PubMed2005

Changes in the EU legislation on Trichinella inspection--new challenges in the epidemiology.

The European Union (EU) countries are searching for new ways to certify meat free of Trichinella; however, with the expansion of the EU, the acceptance of a unilateral method is complicated by the variability of pig and human trichinellosis among EU countries, where significantly higher prevalence rates have been observed in the newly added eastern countries. Several attempts have been made to define Trichinella-free areas, but certification of Trichinella-free pig production farms appears to be the only feasible approach. The increasing prevalence of the non-encapsulating species, Trichinella pseudospiralis, in game, domestic pigs and humans has eliminated the compression technique from the new EU legislation to be enacted in 2006. Also, the observation that several species of Trichinella tolerate freezing in horse meat for up to 4 weeks has forced a change in legislation as well where freezing is no longer an option for certifying horse meat. Because current serological detection methods are not suited for meat inspection, classical direct detection methods and inactivation by freezing remain the methods of choice for pork. It has been proposed, therefore, to automate direct inspection methods as a cost effective alternative to certify pig farms free of Trichinella.

Animal Husbandry↗
PubMed1990

Use of nucleic acid probes in genetic tests.

Advances in molecular genetics have led to the development of clinical assays for several genetic diseases. Two general testing approaches are available: direct detection of the genetic mutation or indirect detection using DNA markers close to, or within, the defective gene. Direct testing at the nucleic acid level is available for diseases in which the basic defect is well characterized, such as sickle cell anemia. Several methods are available for detection of the point mutation that causes sickle cell anemia including: routine Southern blot analysis, allele specific oligonucleotides, and polymerase chain reaction gene amplification. For diseases such as cystic fibrosis, in which the basic genetic defect has not yet been characterized, an indirect approach is used. This approach relies on linkage analysis using DNA markers close to the genetic defect. Inheritance of the DNA marker is followed through the family. Unlike direct testing, the DNA marker does not detect the actual genetic defect. Therefore, a prediction of inheritance of the linked disease is given based on the risk of recombination between the disease locus and the DNA marker. An appreciation of the differences between the direct and indirect approaches is necessary to understand their attributes and their limitations.

Base Sequence↗
PubMed2005

A method for profiling gangliosides in animal tissues using electrospray ionization-tandem mass spectrometry.

Gangliosides are critical in many functions of mammalian cells but present as a minor lipid component with many molecular species of subtle differences. Conventional strategies for profiling gangliosides suffer from poor reproducibility, low sensitivity, and low-throughput capacity. Prior separation of gangliosides by thin-layer chromatography and/or high-performance liquid chromatography not only was laborious and tedious but also could introduce uneven losses of molecular species. We developed a new strategy of using electrospray ionization-tandem mass spectrometry (ESI-MS/MS) to profile gangliosides with high-throughput potential. This strategy involves three new findings: (i) collision-induced fragmentation of gangliosides gave rise to a common ion of m/z 290, a derivative of N-acetylneuraminic acid; (ii) phospholipids exert a profound suppression of ganglioside detection in ESI-MS/MS to prevent a direct detection in total cellular lipid extracts; and (iii) enrichment of gangliosides in the aqueous phase from total cellular lipid extracts eliminates the damping effect of phospholipids and permits direct precursor scan.

Animals↗
PubMed2005

Label-free impedance detection of oligonucleotide hybridisation on interdigitated ultramicroelectrodes using electrochemical redox probes.

The direct detection of oligodeoxynucleotide (ODN) hybridisation using electrochemical impedance spectroscopy was made on interdigitated array (IDA) gold (Au) ultramicroelectrodes manufactured by silicon technology. The immobilisation of single stranded ODNs (ssODNs) was accomplished by self-assembling of thiol-modified ODNs onto an Au-electrode surface. Faradaic impedance was measured in the presence of K(3)[Fe(CN)(6)]. Double strand formation was identified by a decrease of approximately 50% in impedance in the low frequency region in the presence of K(3)[Fe(CN)(6)], compared to the spectrum of single stranded ODN. The frequency dependent diffusion of Fe(CN)(6)(3-) ions through defects in the ODN monolayer determines the impedance of Au-ssODN surface. The influence of DNA intercalator methylene blue on the impedance of both, single and double strands, was examined along with K(3)[Fe(CN)(6)] and confirmed by cyclic voltammetry. The layer densities and the hybridisation have been further corroborated by chronoamperometric redox recycling of para-aminophenol (p-AP) in ELISA like experiments. It can be concluded, that a performed impedance spectroscopy did not change the layer density. The impedance spectroscopy at ultramicroelectrodes combined with faradaic redox reactions enhances the impedimetric detection of DNA hybridisation on IDA platforms.

DNA↗
PubMed1997

A closed tube format for amplification and detection of DNA based on energy transfer.

A new method for the direct detection of PCR-amplified DNA in a closed system is described. The method is based on the incorporation of energy transfer-labeled primers into the amplification product. The PCR primers contain hairpin structures on their 5'ends with donor and acceptor moieties located in close proximity on the hairpin stem. The primers are designed in such a way that a fluorescent signal is generated only when the primers are incorporated into an amplification product. A signal to background ratio of 35:1 was obtained using the hairpin primers labeled with fluorescein as a donor and 4-(4'-dimethylaminophenylazo) benzoic acid (DABCYL) as a quencher. The modified hairpin-primers do not interfere with the activity of DNA polymerase, and both thermostable Pfu and Taq polymerase can be used. This method was applied to the detection of cDNA for prostate specific antigen. The results demonstrate that the fluorescent intensity of the amplified product correlates with the amount of incorporated primers, and as few as 10 molecules of the initial template can be detected. This technology eliminates the risk of carry-over contamination, simplifies the amplification assay and opens up new possibilities for the real-time quantification of the amplified DNA over an extremely wide dynamic range.

Base Sequence↗
PubMed2000

A new enzyme immunoassay for the detection of enteroviruses in faecal specimens.

A new enzyme immunoassay (EIA) for direct detection of enteroviruses based on a group-specific monoclonal antibody was evaluated using stool samples from patients with suspected enteroviral infection. The EIA was compared with polymerase chain reaction (PCR) and virus isolation in cell culture. Of 204 samples tested, 20 were positive by EIA, 34 by PCR, and 18 by cell culture. Compared with PCR, the most sensitive method, the sensitivity of EIA was 58% (20/34); the sensitivity of cell culture isolation was 52% (18/34). The results of both assays correlated in only 60% of cases. The combination of EIA and cell culture isolation detected 76% of PCR-positive stool samples. Enterovirus EIA provides results within 3-4 hr and requires only standard EIA equipment. It represents a rapid, reliable, and cost-effective diagnostic tool for enterovirus diagnosis from faecal samples. Negative results must be confirmed by other techniques, such as PCR or virus isolation in cell culture.

Adult↗
PubMed1997

[Monitoring of inhibitors of the polymerase chain reaction for the detection of hepatitis C virus using the positive internal control].

In the direct detection of pathogens by polymerase chain reaction (PCR) from clinical samples, false negative results due to the presence of inhibitor are problematic. In order to monitor such an inhibitor, we evaluated the detection of the positive internal control in PCR assay for HCV-RNA in serum samples positive for anti-HCV antibodies, of which 41 samples were positive for HCV-RNA by competitive RT-PCR assay. The positive internal control was coamplified with HCV-RNA and hybridized to the specific probe on magnetic beads and then hybrids were detected by colorimetric measurement using automatic PCR machine (COBAS AMPLICOR). Detection of the positive internal control was negatively correlated with viral copy number in sera assayed by competitive RT-PCR. Five of 52 samples (9.6%) with high HCV-RNA copy number (10(7) or 10(8) copies/ml) showed negative results for the internal control. The negative results for the internal control turned out to be positive when the sera were diluted and re-assayed, suggesting competitively inhibitory effects of high viral copy number on amplification of the internal control. Addition of heparin in the serum sample showed an inhibitory effect with a dose dependent manner on the detection of both HCV-RNA and the internal control, with a more effect on the lower copy number of HCV. On the other hand, addition of hemoglobin in the sample with concentration of up to 450mg/dl had no inhibitory effect on the detection of either HCV-RNA or the internal control. Coamplification and detection of the positive internal control was demonstrated to be useful to estimate effects of inhibitors, which may be present in clinical samples, in the detection of HCV-RNA by PCR.

False Negative Reactions↗
PubMed2004

Evaluation of a modified gen-probe amplified direct test for detection of Mycobacterium tuberculosis complex organisms in cerebrospinal fluid.

Laboratory evidence for tuberculous meningitis is difficult to acquire due to the low numbers of organisms present in cerebrospinal fluid (CSF) and the presence of nucleic acid amplification inhibitors. The Amplified Mycobacterium tuberculosis Direct Test (MTD) is sensitive and specific for the direct detection of M. tuberculosis complex in respiratory samples but has not been approved for CSF. We evaluated a modified version of the current MTD, optimized for use with CSF samples. Samples were prepared by spiking CSF with various numbers of M. tuberculosis complex organisms. The modified MTD performance was compared with results obtained using a purified RNA sample extracted using the Qiagen RNeasy Protect Bacteria Mini Kit. By use of CSF artificially spiked with M. tuberculosis complex, the sensitivity of the modified MTD was 100% (six of six) for CSF samples containing approximately 600 CFU/ml, 78% (seven of nine) for approximately 60 CFU/ml, 50% (three of six) for 6 CFU/ml, and 17% (one of six) for samples with <1 CFU/ml. The specificity of the modified MTD method was 100% (22 of 22). The sensitivity of the Qiagen MTD method was 100% for CSF samples containing approximately 600 CFU/ml (six of six) and approximately 60 CFU/ml (nine of nine), 50% for samples with approximately 6 CFU/ml (three of six), and 50% for samples with <1 CFU/ml (three of six). The specificity of the Qiagen MTD method was 86% (19 of 22). With the Qiagen MTD method, however, initial results were equivocal for 14 of the 27 (52%) positive samples, requiring repeat analysis, whereas with the modified MTD, only 1 of 27 (4%) was equivocal. The modified MTD for CSF samples was less time-consuming and less expensive and resulted in considerably fewer equivocal results than the Qiagen MTD method did.

Cerebrospinal Fluid↗
PubMed1997

Dot immunoperoxidase assay for detection of parvovirus B19 antigens in serum samples.

We describe a simple and rapid dot immunoperoxidase assay for the direct detection of parvovirus B19 capsid antigens in human sera. The assay was performed with serum specimens dotted onto nylon membranes. VP1 and VP2 B19 antigens, which represent 4 and 96% of the capsid, respectively, were detected with a pool of monoclonal antibodies directed against the two proteins, and the complex was visualized by immunoperoxidase staining. The assay could be performed in about 4 h, and positive results were revealed at the end of the reaction as dark blue spots on the nylon membrane at the site of positive specimens. A total of 541 serum samples from different subjects and with different laboratory evaluations with regard to B19 infection were analyzed. The results obtained by the dot immunoperoxidase assay were compared with the results obtained for the presence of B19 DNA by dot blot hybridization and nested PCR. With optimized working conditions, the dot immunoperoxidase assay was able to detect the presence of B19 with a sensitivity comparable or slightly higher than that achieved by dot blot hybridization but less than that achieved by nested PCR. Since the level of sensitivity of the dot immunoperoxidase assay proved to be appropriate for the detection of acute B19 infection, and since the cost, time to a result, and versatility of the assay are important issues, from our evaluation, the dot immunoperoxidase assay described may be particularly suitable for large-scale screening of samples and a good alternative to DNA detection methods in the routine laboratory evaluation of B19 infection.

Antibodies, Monoclonal↗
PubMed2003

A surface acoustic wave biosensor concept with low flow cell volumes for label-free detection.

Special surface acoustic wave (SAW) devices using horizontally polarized surface shear waves can be operated in water. They allow an easy detection of molecules with biological relevance (e.g., proteins) via direct detection of the adsorbed mass. The transducer structures of conventional SAW devices are usually connected to the electronics by bond wires. In consequence, flow cell volumes can hardly be designed smaller than 50 microL. A new type of SAW device that is coupled capacitively with the electronics enables the reduction of flow cell volumes down to 60 nL, which decreases sample consumption and reduces the length of the measurement cycles down to a few minutes. To create an immunosensor, the SAW devices first are coated with a thin parylene layer for creating a sensor surface that is chemically homogeneous. Then OptoDex, a dextran containing both photoactive and functional groups is immobilized photochemically. Finally, antibodies are coupled via conventional EDC/NHS chemistry. The technique has been used to monitor urease binding at anti-urease-coated SAW devices in real time and with good resolution. Because of the simple sensor handling and the economical sample use, the new SAW device is particularly suitable for the design of an array.

Journal Article↗
PubMed1980

An analysis of perceptual and cognitive factors in radiographic interpretation.

Detection of small tumors in chest x-ray films was studied under tachistoscopic viewing conditions designed to stimulate single fixations varying in dwell time, found in free search. Two questions were asked: How much dwell time is required to detect a nodule (experiment 1)? How effective are peripheral inputs in the detection of a nodule (experiment 2)? Our findings indicate that a dwell time of 300 ms was sufficient to detect 85% of the nodules when they were viewed directly. Detection accuracy was reduced by one-half when the tumor was located 5 degrees from the axis of gaze. Taken together with data from eye movement experiments, these results provide useful estimates of perceptual and cognitive parameters of visual search.

Cognition↗
PubMed1990

Ultrastructural immunocytochemistry of the intact tissue cyst of Toxoplasma in the brains of mice with congenital toxoplasmosis.

Electron immunocytochemistry, using rabbit polyclonal anti-Toxoplasma IgG as the primary layer in immunogold staining, was used to assess the distribution of Toxoplasma antigen within, and in relation to, intact tissue cysts in brain tissue from mice with congenital toxoplasmosis. Toxoplasma antigen was widely distributed in the matrix of the cyst, being most concentrated in the proximity of the inner surface of the parasitophorous vacuole membrane (PVM). Relatively little Toxoplasma antigen was detected directly associated with cystozoites. Small amounts of antigen were detected within the host cell components exterior to the PVM and in the host neuropil immediately adjacent to the tissue cyst.

Animals↗
PubMed2004

Detection of lipid radicals using EPR.

Cells oxidize molecules to generate energy and to make the materials to build and support the structures and functions needed for life. However, unwanted oxidations can damage these same structures and impair function. Lipids (the lipids in membranes and lipoproteins) are targets of unwanted oxidations. The primary mechanism of these oxidations is free radical-mediated chain reactions. Here we provide an overview of how electron paramagnetic resonance (EPR) can be used to detect the free radicals formed during lipid peroxidation. Although direct detection of lipid-derived radicals has been accomplished, the approach is not feasible for detecting these radicals in cells. Spin trapping with alpha-(4-pyridyl-1-oxide)-N-tert-butylnitrone and 5,5-dimethyl-pyrroline-1-oxide has provided the most information on cellular lipid peroxidation. We present some considerations for successful detection of lipid radicals by EPR.

Cell Line, Tumor↗