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PubMedDate not supplied

[Evaluation of severe dysplasia and carcinoma in situ detected by directed biopsy].

Eighty women with Papanicolaou smears groups IIIa and IIIb and an abnormal colposcopic picture, where colposcopic biopsy pathologic results showed severe dysplasia or carcinoma in situ, were analyzed. Severe dysplasia was found in 51 (63.7%) and carcinoma in situ in 29 (36.2%)cases. By using methods of a definitive diagnostic value, in cases of severe dysplasia, the early diagnosis was confirmed in 24 (47.1%) cases, in 14 (27.4%) cases carcinoma in situ was detected, in 3 (5.9%) moderate dysplasia, in 2 (3.9%) mild dysplasia, and in 8 (15.7%) cases benign histologic changes were found. Carcinoma in situ detected by colposcopy-directed biopsies was confirmed, after a definitive diagnostic procedure, in 24 (82.8%) cases, in 3 cases (10.3%) severe dysplasia was found and in 2 (6.9%) cases there were benign histologic changes. The results suggest that carcinoma in situ and severe dysplasia are vulnerable lesions which can be transformed into a lower grade or even completely disappear by directed biopsies. Such an evolution is significantly more to be expected within the population with an abnormal colposcopic picture if lesions cover not more than one fourth of the cervical circumference.

Adolescent↗
PubMed1978

Detection by direct immunofluorescence of antibodies to Treponema pallidum in the cutaneous infiltrates of rabbit syphilomas.

By direct immunofluorescence, with use of fluorescein-labeled sonified Treponema pallidum, specific antibodies were detected in the tissue infiltrates of cutaneous syphilomas in rabbits. Specimens from cutaneous and mucosal inflammatory lesions induced by intradermal injection of a keratinous substance from an epidermal cyst served as controls. Granular fluorescence was detected in the dermis of 11 of the 12 syphiloma specimens and corresponded to areas of heavy plasma cell infiltrates, and some fluorescence was found directly on plasma cells identified by subsequent staining with hematoxylin and eosin. This fluorescence could be blocked by preabsorption. Control slides did not show any fluorescence. Immunopathologic techniques using labeled antigens may be of diagnostic value in syphilis and other infectious disorders which feature specific infiltrates.

Animals↗
PubMed1985

Monoclonal antibody-directed radioimmunoassay detects cytochrome P-450 in human placenta and lymphocytes.

A multiplicity of cytochromes P-450 is responsible for the detoxification and activation of xenobiotics such as drugs and carcinogens. Individual differences in sensitivity to these agents may reside in the cytochrome P-450 phenotype. A monoclonal antibody-directed radioimmunoassay was developed that detects epitope-specific cytochromes P-450 in human placentas and peripheral lymphocytes. Placentas from women who smoked cigarettes contained greater amounts of cytochrome P-450 with the monoclonal antibody-specific epitope than placentas from nonsmokers. The amount of this cytochrome P-450 in human peripheral lymphocytes increased after treatment of the mitogenized lymphocytes with the cytochrome P-450 inducer benz[a]anthracene.

Animals↗
PubMed1983

Glycolipid-lectin interactions: detection by direct binding of 125I-lectins to thin layer chromatograms.

Glycolipids that bind 125I-labeled lectins are detected by autoradiography after thin layer chromatography of glycolipid standards or crude lipid extracts. Soybean agglutinin, Bandeiraea simplicifolia I isolectins A4 and B4, and Helix pomatia lectin are used to detect corresponding cell surface, glycolipid receptors in human and bovine erythrocytes. When lipid extracts from A and AB erythrocyte stroma are analyzed with Helix pomatia lectin, a polymorphic expression of blood group A glycolipid determinants is detected. The Bandeiraea simplicifolia isolectins react weakly with human erythrocyte glycolipids but bind at least 4 glycolipids in bovine stroma extracts. Soybean agglutinin reacts with glycolipids in all erythrocytes analyzed. This technique extends lectin specificity studies from inhibition analyses in aqueous systems using available, known structures to identification of specific, lectin-binding glycolipids in crude lipid extracts of cell membranes.

ABO Blood-Group System↗
PubMed2004

Marking of specific sequences in double-stranded DNA molecules--SNP detection and direct observation.

In this study, we describe a simple method to mark specific sequences in double-stranded DNA molecules. For the marking, we used two specifically designed oligonucleotides, one of which is complementary to the sequence to be marked and the other, serving as a splint, to make the marking stable and detectable by subsequent various analytical means. In the presence of the two deoxyoligonucleotides, whereas RecA protein-mediated reaction converts the sequence to be marked to a regional triple-stranded structure with the complementary (probing) oligonucleotide, DNA ligase transforms it to a stable multi- (possibly quintuple) stranded structure with the splint oligonucleotide. The whole marking process is simple and completed in a single reaction mixture. Because RecA protein makes the marking to proceed with high fidelity, we were able to mark (detect) SNPs in complex genomes like human's. Furthermore, the structure of the marked sequence is stable and quite distinct enough to be readily detectable by biochemical means or direct observation by scanning probe microscopy.

Blotting, Southern↗
PubMed1996

Comparison of a commercial ELISA and an immunoperoxidase monolayer assay to detect antibodies directed against porcine respiratory and reproductive syndrome virus.

A commercially available enzyme-linked immunosorbent assay (ELISA) for the detection of antibodies against porcine respiratory and reproductive syndrome virus (PRRSV) was compared to an immunoperoxidase monolayer assay (IPMA). Serum samples used were collected from pigs experimentally infected with either the American or European antigenic type of PRRSV, and also from piglets born to sows that had been experimentally infected with the European antigenic type of PRRSV. In addition, three sets of European field sera (n = 275, n = 68, n = 349) were tested and evaluated using the IPMA as the gold standard. Results showed that both the IPMA and the ELISA were able to detect antibodies against the two antigenic types of PRRSV. When sera of experimentally infected pigs were tested, the IPMA with homologous antigen detected antibodies 2 to 3 days earlier than the ELISA, and was more sensitive in detecting maternal antibodies. The ELISA was slightly more sensitive for detecting antibodies against the American type than for the European type. When sets of field sera were tested, the relative sensitivity of the ELISA ranged between 0.68 and 0.91, and the relative specificity ranged between 0.75 and 0.97. However, in two of these sets (n = 275, n = 349) we determined that a decrease of the threshold value of ELISA (from 0.4 to 0.3) increased sensitivity without loss of specificity. We concluded that the ELISA is an easy, quick and reliable test to diagnose PRRSV infection in swine herds.

Animals↗
PubMedDate not supplied

Enzyme immunoassays for the detection of infectious antigens in body fluids: current limitations and future prospects.

Enzyme immunoassays are attaining increased usage for the direct detection of microbial antigens in body fluids. Advantages of enzyme immunoassays include a high degree of sensitivity resulting from the inherent magnification of the enzyme-substrate reaction and the use of objective end points without the need for radioactivity. Enzyme immunoassays have been developed for the reliable detection of several important microbial antigens in body fluids, including antigens of rotavirus, hepatitis B virus, and Haemophilus influenzae type b. However, standard enzyme immunoassay techniques are not sufficiently sensitive for the measurement of some antigens from other viruses, bacteria, and parasites in concentrations that commonly occur in body fluids during the course of infectious diseases. This review examines some of the limitations of currently available enzyme immunoassay technology and discusses approaches to increasing the sensitivity and specificity of enzyme immunoassay systems. Methods for improving these assay systems include the use of monoclonal antibodies, improved methods of enzyme-immunoreactant conjugation, more sensitive substrate systems, improved methods of antigen-antibody access, and the direct measurement of microbial enzymes. The use of such techniques should lead to the development of efficient enzyme immunoassay systems for the direct detection of a wide range of bacterial, viral, and parasitic infections.

Absorption↗
PubMed1995

Cytogenetic studies of breast carcinomas: different karyotypic profiles detected by direct harvesting and short-term culture.

Chromosome analysis was performed on samples from 85 consecutive patients with breast cancer by one or more of three different methods: direct harvest, culture after mechanical disaggregation, and culture after collagenase digestion. Metaphases suitable for karyotyping were obtained in 70% of the cases; direct harvest yielded metaphases in 29% and cultures without and with digestion in 40% and 59%, respectively. Chromosomal abnormalities were detected in 37 cases. Cells judged to be phenotypically abnormal in culture were twice as likely to reveal chromosomal aberrations as normal-looking cells. Eight cases showed multiclonal abnormalities. Significant differences were detected in the karyotypic profile depending on the method used. With direct harvest, the yield of complex chromosomal changes was 87%, compared to 44% after culture of digested tissue (P < 0.01), and also polyploidy was more common in direct-harvested samples. Detailed karyotypic analysis was possible in 29 primary tumors. The chromosomes most frequently involved were 1, 3, 7, 11, 16, and 17. Recurrent structural abnormalities were der(1;16)(q10;p10), i(1)(q10), del(6)(q21), and del(1)(p22). Breakpoints clustered to the centromere regions of chromosomes 1, 3, 11, 15, and 16 and to the short arms of chromosomes 7, 17, and 19. Seven of twenty-nine fully analyzed cases had a family history of breast cancer, and changes of chromosomes 1, 3, and 15 seemed to be more common in these cases. There was an association between karyotype and survival: The 3 year survival was 63% in patients with complex karyotypic changes and 92% in those without complex changes.

Breast Neoplasms↗
PubMed2002

Control of propranolol intake by direct chromatographic detection of alpha-naphthoxylactic acid in urine.

A rapid chromatographic procedure with a C18 column, a mobile phase of 0.15 M sodium dodecyl sulfate (SDS)-10% (v/v) 1-propanol at pH 3 (0.01 M phosphate buffer), and fluorimetric detection, is reported for the control of propranolol (PPL) intake in urine samples, which are injected directly without any other treatment than filtration. The peak of PPL was only observed in samples taken a few hours after ingestion of the drug due to its extensive conjugation and metabolisation. The detection of several unconjugated PPL metabolites was therefore considered: desisopropylpropranolol (DIP), propranolol glycol (PPG), alpha-naphthoxylactic acid (NLT) and alpha-naphthoxyacetic acid (NAC). NLT showed the best characteristics: it eluted at a much shorter retention time than PPL, its concentration in urine samples was greater and it did not present any interference from endogeneous compounds in urine, common drugs or drugs administered in combination with PPL. The limit of quantification, measured as the concentration of analyte providing a relative standard deviation of 20%, was 24 ng/ml, and the day-to-day imprecision was below 4% for concentrations above 200 ng/ml. The procedure allows the routine control of PPL at therapeutic urine levels. Urinary excretion studies showed that the detection of NLT is possible at least up to 20-30 h after oral administration.

Adrenergic beta-Antagonists↗
PubMed1999

Visualization of axonally transported horseradish peroxidase using enhanced immunocytochemical detection: a direct comparison with the tetramethylbenzidine method.

Visualization of the neuronal tract tracer horseradish peroxidase (HRP) is commonly achieved through the histochemical detection of its enzymatic activity using 3,3',5,5'-tetramethylbenzidine (TMB) as a chromogen. However, the TMB product is unstable and is incompatible with tissue processing methods that render the enzyme inactive, or when a combination of HRP tract tracing with neuronal phenotype identification is required. In this study we evaluated the applicability of the immunocytochemical detection method for horseradish peroxidase (HRP) visualization using an enhanced detection meth-od based on the Elite ABC peroxidase amplification protocol. The results provide evidence for the immunocytochemical visualization of both anterograde and transganglionic HRP transport in the rat spinal cord. This immunocytochemical method not only showed similar sensitivity to the TMB protocol in detecting HRP-labeled motor neuron perikarya but provided enhanced resolution in the identification of individual neuronal fibers compared to the TMB method. Immunodetection of the HRP tracer also allowed its co-localization with specific neuronal markers using double immunofluorescence techniques. These results offer the first demonstration that sensitive identification of axonally transported HRP can be achieved by immunocytochemistry and provides further support for its use in HRP tract tracing studies.

Animals↗
PubMed2001

Specific detection by PCR of Streptococcus agalactiae in milk.

The aim of this study was to develop a simple and specific method for direct detection of Streptococcus agalactiae from cow's milk. The method was based on polymerase chain reaction (PCR) using species-specific and universal primers derived from the 16S rRNA gene. The amplification product was verified by restriction endonuclease digest and sequencing. Specific identification was proven on a collection of 147 S. agalactiae isolates of bovine and human origin. In addition, 17 strains belonging to different bacterial species that potentially can be found in milk samples also tested negative. The PCR developed was used for direct detection of S. agalactiae in milk, using for the first time with gram-positive bacteria the nucleic acid-binding properties of diatomaceous earth. The test, which has high specificity, high sensitivity (100 cfu/mL), and can be carried out in less than 24 h, represents an innovative diagnostic tool for the detection of S. agalactiae in milk.

Animals↗
PubMedDate not supplied

[Detection of direct markers of cytomegalovirus. Research of the spectrum and avidity of antiviral antibodies in infants and preschool children].

Thirty-three children aged 1 month to 3 years were examined within the case study. spELISA, immunoblot (IB), shell vial method (SVM) and PCR, were used for the detection of anti-CMV IgM and IgG, in the diagnosis of cytomegalovirus (CMV). Clinical signs of CMV infection (CMVI) were registered in 20 children (group 1); no CMVI specific signs were detected in the remaining 13 children (group 2). Class M antibodies were identified in 50% of group-1 sera. Around 80% of children in the group had anti-CMV-IgG. AI < 0.6 was in 3 (20%) of 15 examinees. Direct CMV markers (DNA and infection activity) were detected in 13 (65%) of 20 children. Sera of 13 children with non-specific symptomatology (group 2) had no anti-CNV-IgM, while IgG were found in 54% examinees in the group. The infectious active virus was not detected in a single baby. The used laboratory tools enhance the efficiency of CMVI diagnosis and denote a disease variation.

Antibodies, Viral↗
PubMed2001

[Viral infections and pregnancy: contribution of amniotic fluid and blood samples].

The main viral infections prenatally detected in fetuses are: cytomegalovirus, parvovirus B19, rubella virus and varicellazoster virus infections. Prenatal diagnosis is based on the direct detection of the virus by culture (CMV), of its antigens or of its genome, essentially by PCR. This direct detection can be done either on fetal blood or on amniotic fluid. Prenatal diagnosis can also be performed by detection of specific IgM in fetal blood (rubella). Non specific markers of viral infection can also help in diagnosis. At the present time, prenatal diagnosis is essentially based on the detection of the viral genome in amniotic fluid. In order to better appreciate the severity of fetal infections, some groups have tried to identify prognostic markers of these infections. The viral load as well as the level of specific IgM could play a role in certain infections (CMV).

Amniotic Fluid↗
PubMed1990

Quantification of human cytomegalovirus viremia by using monoclonal antibodies to different viral proteins.

Human cytomegalovirus (HCMV) viremia in peripheral blood polymorphonuclear leukocytes (PMNLs) from 187 immunosuppressed patients (79 heart transplant recipients and 108 patients with acquired immunodeficiency syndrome [AIDS]) was investigated. Five mouse monoclonal antibodies (MAbs), one specifically reactive to HCMV immediate-early antigen (IEA) in PMNLs, two specifically reactive to IEA in infected cell cultures, and two specifically reactive to late antigens, were used in immunofluorescence and/or immunoperoxidase test systems for (i) detection of HCMV IEA in human embryonic lung fibroblast (HELF) cell cultures inoculated with PMNL samples, (ii) direct detection of HCMV IEA in PMNL nuclei of cytospin preparations, and (iii) identification of HCMV isolates from PMNL samples in HELF cells. Quantification of viremia was achieved by counting the number of infected PMNLs per 2 x 10(5) cells examined directly on cytospin preparations as well as by counting the number of IEA-positive HELF cells inoculated with 2 x 10(5) PMNLs. A significant correlation was found between the number of infected PMNLs and the number of infected HELF cells. When the number of infected PMNLs per 2 x 10(5) cells was greater than 10, both methods (PMNL IEA and HELF IEA) gave concordant results; when it was greater than 80/2 x 10(5) cells, clinical symptoms were consistently associated with HCMV viremia. Ten patients with heart transplants and three patients with AIDS who had high or increasing levels of HCMV viremia underwent antiviral treatment with ganciclovir. Treatment was discontinued only after disappearance of IEA-positive PMNLs from blood (the last marker of infection to become negative). On the other hand, in the presence of low levels of viremia (less than 10 infected PMNLs per 2 x 10(5) cells), different methods often provided discordant results and overt clinical symptoms were never observed. IEA-negative results with PMNL samples or HELF cells in the presence of positive virus isolation could never be attributed to the inability of IEA MAbs to recognize individual HCMV strains, since all of the relevant viral isolates were recognized by IEA MAbs. In addition, all five MAbs used in the study were capable of identifying all 135 conventional HCMV isolates obtained from the study population. It was concluded that (i) maximal sensitivity in diagnosing HCMV viremia may be achieved by combining different techniques; (ii) direct detection of HCMV IEA in PMNLs, yielding results on the day of blood collection, is a very rapid and sensitive technique, and thus one can rely on it for clinical management of HCMV infections; and (iii) low levels of viremia are clinically irrelevant, whereas high levels are associated with clinical symptoms.

Acquired Immunodeficiency Syndrome↗
PubMed2002

PCR-based assays for the fish pathogen Aeromonas salmonicida. II. Further evaluation and validation of three PCR primer sets with infected fish.

Two Aeromonas salmonicida-specific polymerase chain reaction (PCR) tests and 1 A. salmonicida subsp. salmonicida-specific PCR test were used to screen salmonid populations that were either overtly or covertly infected with A. salmonicida subsp. salmonicida. It was demonstrated that these PCR assays could be used to replace the biochemical testing currently employed to confirm the identity of A. salmonicida isolates cultured from infected fish. The AP and PAAS PCR assays were also capable of direct detection of A. salmonicida in overtly infected fish, with mucus, gill and kidney samples most likely to yield a positive result. Culture was a more reliable method for the direct detection of A. salmonicida in covertly infected salmonids than was the direct PCR testing of tissue samples, with the AP and PAAS PCRs having a lower detection limit (LDL) of approximately 4 x 10(5) colony-forming units (CFU) g(-1) sample.

Aeromonas↗
PubMed1998

Development and use of nested polymerase chain reaction (PCR) for the detection of adenovirus from conjunctivitis specimens.

BACKGROUND: The standard virus isolation method for detecting adenovirus is time consuming and direct detection of viral antigens in smears has its limitations. Therefore a rapid and a reliable method to identify virus in clinical specimens is desirable. OBJECTIVE: To develop and evaluate nested PCR as a tool for detecting adenovirus from conjunctival swabs of patients with acute conjunctivitis during an epidemic. STUDY DESIGN: A total of 201 patients with acute conjunctivitis were seen between August and November 1996. Conjunctival swabs from the most recently affected eyes were collected from 20 random patients and processed for antigen detection in direct smears, for adenovirus, enterovirus (EV70) and coxsackievirus A24 variant and adenovirus isolation by culture. Nested PCR was performed using oligonucleotides to amplify 1004 basepair (bp) and 956 bp fragments of DNA coding for adenovirus hexon protein. The neutralisation test, to type the adenovirus, was done on four isolates selected at random. RESULTS: The PCR could detect 0.0032 fg of adenovirus DNA (corresponding to 8.3 x 10(-3) adenovirus particles). The EV70 and coxsackievirus A24 antigens were not detected. The specimens were positive for adenovirus by all three techniques in seven patients: (a) by direct smear and PCR in 2; (b) by virus isolation and PCR in 2; and (c) by PCR alone in five patients. In one patient the direct smear alone was positive. The PCR required 3 days to detect the virus, antigen detection provided diagnosis the same day and virus isolation required 8-27 days. A total of four isolates selected at random were identified as serotype 7a. CONCLUSION: The nested PCR is a reliable and rapid technique for detection of adenovirus from conjunctival swabs. The adenovirus serotype 7a was the likely causative agent of this epidemic conjunctivitis.

Acute Disease↗
PubMed2005

Establishment of a practical enzymatic assay method for determination of isovaleryl-CoA dehydrogenase activity using high-performance liquid chromatography.

BACKGROUND: Isovaleric acidemia (IVA) is one of the various target disorders for tandem mass spectrometry (MS/MS) newborn screening. In the diagnosis of IVA, no enzymatic assay method for isovaleryl-CoA dehydrogenase (IVD) activity has been reported whereby the production of enoyl-CoA species was directly detected. We established a direct assay method to detect 3-methylcrotonyl-CoA (MC-CoA) production using high-performance liquid chromatography (HPLC). METHODS: Isovaleryl-CoA dehydrogenase crude enzyme was prepared by sonicating lymphocytes in peripheral blood. Aliquots were incubated with isovaleryl-CoA, flavin adenine dinucleotide, and phenazine methosulfate. 3-Methylcrotonyl-CoA produced in the samples was separated by HPLC and detected using an ultraviolet spectrophotometer. RESULTS: The detection of MC-CoA was reproducible depending upon the concentration of the substrates, the incubation time, and the number of cells contained in the crude enzyme solution. We applied this assay to three patients diagnosed with IVA and showed that neither of them had detectable residual activity. Only a few hours were required from the initial blood sampling to the end of the assay. CONCLUSIONS: These results demonstrate that this method for detecting MC-CoA production, using HPLC, is a practical assay for determining IVD activity. It can be a useful confirmatory test for IVA cases detected through MS/MS screening of newborns.

Chromatography, High Pressure Liquid↗
PubMed1989

[The modern diagnosis of gonorrhea].

Modern identification of Neisseria gonorrhoeae may be either accomplished by direct detection of gonococcal antigen, i.e. without microbiological culturing, or, following culturing, by rapid culture confirmation. The direct detection of gonococcal antigen in an enzyme immuno-assay (Gonozyme) shows reduced specifity especially with mixed microbial flora, and decreased sensitivity with small numbers of organisms. Culture confirmation may be achieved by a rapid sugar degradation test (API----quadFERM+), by specific enzyme detection using chromogenic substrates (Gonochek II), or by demonstration of protein I specific for Neisseria gonorrhoeae by means of monoclonal antibodies (Mikrotrak, Phadebact monoclonal GC, Gonogen II). Detection of protein I is highly sensitive and specific in both the tests of immunofluorescence and coagglutination.

Antigens, Bacterial↗