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PubMed1989

Direct visual detection of aflatoxin synthesis by minicolonies of Aspergillus species.

Single-spore colonies of Aspergillus flavus and Aspergillus parasiticus, grown for 4 to 5 days at 25 degrees C on a coconut extract agar containing sodium desoxycholate as a growth inhibitor, produced aflatoxin, readily detectable as blue fluorescent zones under long-wave (365 nm) UV light. Over 100 colonies per standard petri dish were scored for aflatoxin production by this procedure. Progeny from some strains remained consistently stable for toxin production after repeated subculture, whereas instability for toxin synthesis was revealed among progeny from other strains. Spore color markers were used to rule out cross-contamination in monitoring strains. A yellow-spored and nontoxigenic strain of A. flavus, reported previously to produce aflatoxin in response to cycloheximide treatment, proved to be toxin negative even after repeated exposure to cycloheximide. Extended series of progeny from another strain of A. flavus and from a strain of A. parasiticus were each compared by this plating procedure and by fluorometric analysis for aflatoxin when grown in a coconut extract broth. Both of these strains showed variation for toxin synthesis among their respective progeny, and specific progeny showed a good correlation for aflatoxin synthesis when examined by the two procedures.

Aflatoxins↗
PubMed1980

Direct immunofluorescent detection of Legionella pneumophila in respiratory specimens.

Respiratory secretions from patients with clinically suspected Legionnaires pneumonia were examined by direc immunofluorescent tests at the Medical Center Hospital of Vermont and at the Center for Disease Control. No fluorescent bacteria were found by either laboratory in eight specimens from eight patients who were seronegative. Twenty specimens were obtained from seven patients who had serologically confirmed Legionnaires disease. Four of the seven cases were identified at the Medical Center Hospital of Vermont, and six of the seven were identified at the Center for Disease Control. Of 20 specimens, 8 were positive at the Center for Disease Control (six or more bacilli per slide), and 7 specimens were suspicious (one to five bacilli per slide); at the Medical Center Hospital of Vermont, 4 of 20 specimens were positive, and 2 were suspicious. The inclusion of a rhodamine-conjugated counterstain at the Center for Disease Control facilitated the examination and may have improved the sensitivity. Smears from transtracheal aspirates, bronchoscopic aspirates, transcutaneous lung aspirates, pleural fluids, and tracheal aspirate-expectorated sputum produced positive results. Several specimens contained fluorescing bacilli when stained for serogroup 2 as well as serogroup 1, perhaps reflecting the presence of cross-reacting antigens in vivo.

Bacteria↗
PubMed2001

Detection of direct binding of human herpesvirus 8-encoded interleukin-6 (vIL-6) to both gp130 and IL-6 receptor (IL-6R) and identification of amino acid residues of vIL-6 important for IL-6R-dependent and -independent signaling.

Human herpesvirus 8 (HHV-8) is associated with Kaposi's sarcoma, primary effusion lymphoma, and multicentric Castleman's disease; in all of these diseases, interleukin-6 (IL-6) has been implicated as a likely mitogenic and/or angiogenic factor. HHV-8 encodes a homologue of IL-6 (viral IL-6 [vIL-6]) that has been shown to be biologically active in several assays and whose activities mirror those of its mammalian counterparts. Like these proteins, vIL-6 mediates its effects through the gp130 signal transducer, but signaling is not dependent on the structurally related IL-6 receptor (IL-6R; gp80) subunit of the receptor-signal transducer complex. However, as we have shown previously, IL-6R can enhance vIL-6 signal transduction and can enable signaling through a gp130 variant (gp130.PM5) that is itself unable to support vIL-6 activity, indicating that IL-6R can form part of the signaling complex. Also, our analysis of a panel of vIL-6 mutants in transfection experiments in Hep3B cells (that express IL-6R and gp130) showed that most were able to function normally in this system. Here, we have used in vitro vIL-6-receptor binding assays to demonstrate direct binding of vIL-6 to both gp130 and IL-6R and vIL-6-induced gp130-IL-6R complex formation, and we have extended our functional analyses of the vIL-6 variants to identify residues important for IL-6R-independent and IL-6R-dependent signaling through native gp130 and gp130.PM5, respectively. These studies have identified residues in vIL-6 that are important for IL-6R-independent and IL-6R-mediated functional complex formation between vIL-6 and gp130 and that may be involved directly in binding to gp130 and IL-6R.

Antigens, CD↗
PubMed1987

Pseudarthrosis following lumbar fusion: detection by direct coronal CT scanning.

Twenty patients with fusions of the lumbar spine (seven with pseudarthrosis, 11 with solid bony fusions, and two with fusions that appeared solid but assessment was complicated by the presence of surgical hardware) underwent computed tomographic (CT) scanning in the supine position in the axial plane and, employing a specially designed seat, in the coronal plane. Three-millimeter contiguous sections were acquired. The direct coronal images were compared with those reformated from the axial images. The higher-resolution direct coronal images facilitated the diagnosis of pseudarthrosis and increased confidence in interpretation of normal studies. Direct coronal imaging is easy to perform and in many cases requires fewer scans and less radiation than reformations. Because it provides more useful diagnostic information, direct coronal imaging should replace current methods for evaluating fusions of the lumbar spine.

Adult↗
PubMed1996

Localization of apoptotic cells by direct immunogold detection of digoxigenin-labeled genomic DNA in semithin sections.

In this study, which correlates apoptosis with avian ovarian physiology, we modified an in situ DNA nick end-labeling method using immunogold reagents to detect apoptotic cells in semithin sections of quail ovaries embedded in glycol methacrylate resin. Special attention was paid to the prevention of background staining. The results are comparable with those of the ApopTag peroxidase kit in paraffin-embedded ovaries.

Animals↗
PubMed2005

SeqDoC: rapid SNP and mutation detection by direct comparison of DNA sequence chromatograms.

BACKGROUND: This paper describes SeqDoC, a simple, web-based tool to carry out direct comparison of ABI sequence chromatograms. This allows the rapid identification of single nucleotide polymorphisms (SNPs) and point mutations without the need to install or learn more complicated analysis software. RESULTS: SeqDoC produces a subtracted trace showing differences between a reference and test chromatogram, and is optimised to emphasise those characteristic of single base changes. It automatically aligns sequences, and produces straightforward graphical output. The use of direct comparison of the sequence chromatograms means that artefacts introduced by automatic base-calling software are avoided. Homozygous and heterozygous substitutions and insertion/deletion events are all readily identified. SeqDoC successfully highlights nucleotide changes missed by the Staden package 'tracediff' program. CONCLUSION: SeqDoC is ideal for small-scale SNP identification, for identification of changes in random mutagenesis screens, and for verification of PCR amplification fidelity. Differences are highlighted, not interpreted, allowing the investigator to make the ultimate decision on the nature of the change.

Base Sequence↗
PubMed2006

Thin-film technology for direct visual detection of nucleic acid sequences: applications in clinical research.

Certain optical conditions permit the unaided eye to detect thickness changes on surfaces on the order of 20 A, which are of similar dimensions to monomolecular interactions between proteins or hybridization of complementary nucleic acid sequences. Such detection exploits specific interference of reflected white light, wherein thickness changes are perceived as surface color changes. This technology, termed thin-film detection, allows for the visualization of subattomole amounts of nucleic acid targets, even in complex clinical samples. Thin-film technology has been applied to a broad range of clinically relevant indications, including the detection of pathogenic bacterial and viral nucleic acid sequences and the discrimination of sequence variations in human genes causally related to susceptibility or severity of disease.

Base Sequence↗
PubMed1996

Co-infection with two JC virus genotypes in brain, cerebrospinal fluid or urinary tract detected by direct cycle sequencing of PCR products.

The human polyomavirus JC (JCV), which exists in different geographically based genotypes, causes the central demyelinating disease known as progressive multifocal leukoencephalopathy (PML). A coding region recombinant JCV Type 1/Type 3 (Type 4) is excreted in the urine of some 16% of individuals in the USA. In addition, occasional 'crossovers' in viral DNA sequence at type-specific sites in the coding region occur between JCV genotypes amplified from PML brain. For recombination to occur requires the existence of two different genotypes in the same host. Here we provide evidence from direct cycle sequencing of PCR products that different genotypes of JCV can be found in a single tissue sample. After non-type-specific PCR amplification, cycle sequencing produced 'split bands' at type determining sites which were resolved into type or subtype-specific sequences by subcloning of the PCR products. PCR products with split bands at typing sites were found in two brain samples and in one cerebrospinal fluid (CSF) from AIDS patients with PML and in the urine of four immunocompetent individuals. This indicates that co-infection with two viral types does not depend on severe immunocompromise. Combinations of genotypes found were Types 1A & 1B, 1A & 2, 1B & 2 and 2 & 3. In one doubly infected patient the major JCV type excreted in the urine changed within 1 week.

Adult↗
PubMed1994

Molecular biology techniques in the diagnosis of monogenic diseases.

Monogenic diseases are defined by their patterns of inheritance: autosomal dominant, autosomal recessive, or X-linked. This article examines the molecular biology techniques available for their study and detection. These techniques comprise direct detection of mutations (including trinucleotide repeats) and linkage analysis. The usefulness of these techniques is compared to conventional biochemical tests for the diagnosis of various inherited diseases. The authors stress the need for caution in the interpretation of test results and the importance of genetic counseling.

Forecasting↗
PubMed2000

[Incidence of genitourinary infection caused by Chlamydia trachomatis in a STD center calculated by direct antigen detection].

OBJECTIVE: Chlamydia trachomatis is one of the most common sexually transmitted agents which causes a wide spectrum of diseases including urethritis in men and endocervicitis in women. We analyzed patients with genitourinary C. trachomatis infections evaluating risk factors and the association with other sexually transmitted infections. MATERIAL AND METHODS: We processed 1,180 specimens from 913 patients (772 women and 141 men), attended at a Sexually Transmitted Diseases (STD) Center. The diagnostic of C. trachomatis infection was made by an enzyme-linked fluorescent immunoassay, Vidas Chlamydia test (bioMérieux). RESULTS: The incidence of C. trachomatis infection was 4.8% (57 cases) and was higher in women (70.1%) than in men (29.8%). The risk groups observed were: 26 prostitute, 7 contact with prostitute or risk partner, 5 homosexual, 5 promiscuous heterosexual and 14 without risk groups. Associated with this infection we observed other: 10 bacterial vaginosis, 8 Papillomavirus infection, 3 Trichomonas vaginalis infections, 2 Neisseria gonorrhoeae infections and 2 Candidiasis. The 53.4% of these patients didn't have any symptomatology at the consult moment. CONCLUSIONS: The control of patients with risk factors is important for the diagnostic of C. trachomatis and other sexually transmitted infections, because most of them were prostitutes and asymptomatic. Within men, homosexuality, contact with prostitute or risk partner were the practices with higher risk.

Antigens, Bacterial↗
PubMed2002

Optimal tuberculosis case detection by direct sputum smear microscopy: how much better is more?

SETTING: A tuberculosis control project in Bangladesh. OBJECTIVE: To define the efficiency of numbers of microscopic fields screened and the sputum collection scheme used for diagnostic smear examination. DESIGN: Quality controllers noted cumulative numbers of acid-fast bacilli per 100 fields screened. The incremental diagnostic yield of different sputum sampling strategies was determined. Doubtful series were re-checked and/or further samples examined. RESULTS: Acid-fast bacilli were found in 99.6% of 1412 positive and in 79.3% of 576 scanty slides in the first 100 fields. Examination of a third specimen yielded a maximum of 2.7% positives incrementally. The most efficient strategy, using three morning specimens, yielded 94.2% positives on the first and 1.0% on the third sputum; although 10% of suspects did not return, only 1.5% of the positives were among them and more cases were confirmed and treated. The positive predictive value of a single positive or scanty smear was very high (99.2%). CONCLUSIONS: Reading more than 100 fields per smear or examining a third sputum has insufficient marginal returns to justify the workload. Examining morning samples only is more efficient, and their collection does not necessarily inconvenience patients. Treatment can be started on the basis of one positive smear. Provided that a well functioning system of smear-microscopy quality control is in place, we propose a strategy based on examination of two morning sputum samples for negative suspects, with the diagnosis based on a single positive result.

False Negative Reactions↗