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PubMed1981

[Methods of direct detection of sarcocysts and their diagnostic reliability].

The methods of the direct determination of sarcocysts in bovine muscle were studied. The results obtained with the compression, histological, homogenization and digestion methods were compared and evaluated. The criteria included capture rate, labor requirement, economic requirements, expediency and diagnostic reliability. The compression method was found to be the cheapest but, at the same time, the least reliable. The highest capture rate and diagnostic reliability were found in the digestion method with trypsin used as the diagnostic agent.

Animals↗
PubMed1994

Evaluation of a commercial rRNA amplification assay for direct detection of Mycobacterium tuberculosis in processed sputum.

A commercial assay (Amplified Mycobacterium tuberculosis Direct Test, Gen Probe) which combines transcription-mediated amplification of target rRNA with amplicon detection by a chemiluminescent DNA probe for the rapid detection of Mycobacterium tuberculosis in sputum was evaluated. The test was applied to consecutively collected, NALC/NaOH processed sputum sediments from two laboratories (H and L), each serving a different population of patients with pulmonary tuberculosis. Results were compared to those of fluorochrome staining and culture. A total of 760 specimens obtained from 246 patients were used for the study. The test was positive in 141 of 144 (98%) specimens that were fluorochrome-positive and culture-positive for Mycobacterium tuberculosis. Fifteen of 31 specimens that were fluorochrome-negative, culture-positive were also assay-positive. A total of 312 specimens (100 patients) from laboratory H (prevalence = 10%) and 448 specimens (146 patients) from laboratory L (prevalence = 34%) were analyzed. Compared to culture, test sensitivity, specificity, positive predictive and negative predictive values were 65%, 99%, 94% and 97%, respectively, for laboratory H and 93%, 99%, 99% and 97%, respectively, for laboratory L. If the results were analyzed on the basis of at least one concordant result between the amplification assay and culture in three sputum samples per patient, then the sensitivity, specificity, positive and negative predictive values for identifying infected patients was 70%, 99%, 87% and 97%, respectively, for laboratory H, and 100%, 98%, 96% and 100%, respectively, for laboratory L.

Bacteriological Techniques↗
PubMed1999

Direct detection of Sabin poliovirus vaccine strains in stool specimens of first-dose vaccinees by a sensitive reverse transcription-PCR method.

A multiplex reverse transcription-PCR method was optimized to monitor the duration of excretion of Sabin poliovirus strains in stools of vaccinees following administration of the first dose of the trivalent oral vaccine. The assay detected approximately 1 50% tissue culture infective dose of each poliovirus serotype spiked into cell culture media. Although PCR inhibitors were frequently encountered in the stool specimens, a 1:20 dilution of the extracted RNA was sufficient to obtain a positive PCR result. Analysis of 195 stool specimens collected from 26 vaccinees showed that poliovirus types 1, 2, and 3 were identified more frequently by PCR than by tissue culture isolation. The percentages of specimens positive by PCR for poliovirus types 1, 2, and 3 were 67.2, 82.6, and 53.8, respectively. In contrast, the culture method identified types 1, 2, and 3 virus in 55.4, 64.1, and 27.7% of the samples, respectively. Poliovirus type 2 excretion was detected by PCR in practically all of the oral poliovirus vaccine recipients for 4 to 8 weeks following vaccination. In contrast, excretion of type 1 and 3 viruses was more variable, with a range of 1 to 8 weeks. Shedding of type 3 virus ceased in approximately 70% of vaccinees within a week after immunization. In addition to an enhanced sensitivity for the detection of poliovirus, this PCR method permits the direct characterization of virus in stool specimens without further passage in culture, which may select for genetic variants that may not accurately reflect the virus composition in the original specimen.

Animals↗
PubMed2005

Direct detection of human Staphylococcus aureus carriage in the nose using the Lightcycler Staphylococcus kit.

The Lightcycler Staphylococcus kit is a diagnostic tool for direct real-time detection of Staphylococcus aureus in clinical materials. We show here that detection of S. aureus nasal carriage using this test is hampered by competition of DNA from coagulase-negative staphylococci. However the test is well suited for species identification after culture and the identification of high-load S. aureus carriers.

Carrier State↗
PubMed1986

Double-enzyme conjugates, producing an intermediate color, for simultaneous and direct detection of three different intracellular immunoglobulin determinants with only two enzymes.

A new double-enzyme conjugate was synthesized by coupling alkaline phosphatase (AP) to horseradish peroxidase (HRP). After AP (blue) and subsequent HRP (red) cytochemistry, this new conjugate produced a stable intermediate-colored (violet) product. By coupling this double-enzyme conjugate to an antigen (trinitrophenyl, TNP) or an antibody (anti-mouse immunoglobulin G2a), anti-TNP or -IgG2a-producing cells could be demonstrated as violet cells in spleen sections. This led to the development of a rapid one-step incubation--two-step cytochemical procedure for simultaneous detection of three different determinants in a single tissue section. To demonstrate this novel triple staining method, we coupled three different antigens to, respectively, AP, HRP, and AP-HRP. When spleen sections of immunized animals were incubated with a mixture of these three antigen-enzyme conjugates, we could distinguish antibody-forming cells against each of these three antigens simultaneously as red (HRP), blue (AP), and violet (AP-HRP) cells. The simultaneous detection of three different classes of intracellular antibodies in a single section also proved to be possible with this method. With this study we provide a new direct method for detection of three different intracellular immunoglobulins after a one-step incubation and a two-step standard cytochemical procedure.

Alkaline Phosphatase↗
PubMed1996

[Direct detection of human cytomegalovirus in urine specimens from bone marrow transplant patients by polymerase chain reaction].

Human cytomegalovirus (HCMV) infection is a major causative life-threatening agent that results in opportunistic infections in bone marrow transplant patients. Since antiviral therapy is available for severe HCMV infections, methods to rapidly identify infected patients are needed so that therapy can be promptly instituted. In this study, we used direct method to detect HCMV in urine specimens by nested polymerase chain reaction (nested-PCR) to monitor urinary excretion of HCMV in patients undergoing bone marrow transplantation. The viral DNA was amplified directly from preheated urine without further treatment prior to amplification. Five microliter of urine proved to give the most efficient amplification. The detection limit of the PCR assay for detection of HCMV was 10 copies/microl. The positive rate of the PCR assay and the tissue culture method for detection of HCMV were 15 of 61 (24.6%) and 4 of 61 (6.6%), respectively. Sensitivity and specificity of the PCR assay were 100% and 81% respectively. Based on these data, urinary excretion of HCMV in 21 patients undergoing bone marrow transplantation were monitored weekly by the PCR assay. As a result, in addition to a higher sensitivity, the PCR assay allowed to identify HCMV infection 3.1 (1 approximately 5) weeks earlier than culture method or antibody elevation. Repeated monitoring of virus excretion by this rapid and simple method was useful to promptly detect HCMV infection, allowing proper institution of antiviral drug therapy in patients undergoing bone marrow transplantation.

Adolescent↗
PubMed1990

Herpes simplex virus antigen direct detection in standard virus transport medium by Du Pont Herpchek enzyme-linked immunosorbent assay.

A commercial 5-h direct herpes simplex virus (HSV) antigen detection enzyme immunoassay kit (Du Pont Herpchek) was compared with a cell culture isolation system by using primary rabbit kidney and MRC-5 cells with 779 clinical specimens received in virus transport medium and with stock tissue culture preparations of HSV types 1 and 2. In the first study of 422 specimens from symptomatic patients, Herpchek detected 110 of 111 HSV-positive specimens (26.3% of all specimens), with a sensitivity of 99% and a specificity of 100%. In the second study of 357 specimens primarily from asymptomatic pregnant women, however, Herpchek detected 70 of 119 HSV-positive specimens (33% of all specimens), with a sensitivity of 58.8% and a specificity of 99.5%. Stock virus dilution experiments showed that Herpchek was 10 to 100 times less sensitive than culture. Herpchek was found to be an acceptable test for symptomatic patients, but for asymptomatic patients shedding a low titer of HSV it was not as sensitive and cell culture of Herpchek-negative specimens is recommended for such cases.

Animals↗
PubMed1999

[Evaluations of MTD and Amplicor Mycobacterium for direct detection of Mycobacteria from clinical specimens].

MTD (GEN-PROBE AMPLIFIED MYCOBACTERIUM TUBERCULOSIS DIRECT TEST) for Mycobacterium tuberculosis, and Amplicor Mycobacterium for Mycobacteria (AMP-M. tb for M. tuberculosis, AMP-M. av for M. avium and AMP-M. in for M. intracellulare) were used for the detection of relevant Mycobacterium. Their sensitivity and specificity were evaluated. Total 244 clinical specimens including 164 sputa were examined by the above two tests. The results were compared with those obtained by the conventional methods. Of 244 samples, number of the M. tuberculosis positive samples by microscopy, cultural test, MTD and AMP-M. tb were 32, 33, 38 and 35, respectively. Among 33 culture positive samples, 25 were MTD positive and 26 were AMP-M. tb positive. Therefore, sensitivity of MTD and AMP-M. tb were 75.8% and 78.8%, and their specificity were 93.8% and 95.7%, respectively. When only sputa were used for the tests as the clinical specimens, both sensitivity of MTD and AMP-M. tb were increased to 94.4%. For MAC, positive samples of M. avium complex by culture, M. avium by AMP-M. av and M. intracellulare by AMP-M. in were 13, 16, and 8, respectively. Sensitivity and specificity of AMP-M. av/M. in were 100% and 95.2%, respectively. Clinical findings of the patients whose MTD tests were positive but negative by culture were reexamined. Three of 9 specimens were also positive in AMP-M. tb. From the records of the isolations of tubercle bacilli or other important pathogens from the other kind of clinical specimens, smear tests and patients' response to tuberculosis chemotherapy, four of 9 specimens were confirmed as true positive, three were suspected as positive, and two other specimens were false positive which might be caused by contamination. From these observations, it could be concluded that MTD and AMP-M. tb are more sensitive than conventional culture method, and MTD is more sensitive than AMP-M. tb but needs more careful treatment to avoid the contamination.

Bacteriological Techniques↗
PubMed1999

Direct detection of antipyrine metabolites in rat urine by (13)C labeling and NMR spectroscopy.

Antipyrine is a useful probe to evaluate variation of in vivo activities of oxidative hepatic drug-metabolizing enzymes. Here we describe a new approach using (13)C labeling and NMR spectroscopy for the direct and simultaneous detection of all phase I and phase II metabolites of antipyrine in rat urine. [C-methyl-(13)C]Antipyrine was synthesized and administered orally to rats (100 mg/kg), and the 0- to 24-h postdose urine was analyzed by 100-MHz (13)C NMR spectroscopy under the conditions of distortionless enhancement by polarization transfer without any pretreatments such as deconjugation, chromatographic separation, and solvent extraction. Consequently, all the major metabolites in urine were successfully detected with favorable signal-to-noise ratios in the limited acquisition time (30 min). The assignments of the resonances were performed by enzymic modification and spiking authentic samples. The reproducibility of the NMR detection was sufficient for the quantitative evaluation of the metabolic profile. Effects of 3-methylcholanthrene on antipyrine metabolism were examined by this approach to evaluate variation of in vivo phase I and phase II metabolism of antipyrine in rats. The present approach is useful and practical to evaluate variation of in vivo activities of conjugation enzymes as well as oxidation enzymes responsible for the formation of antipyrine metabolites in rats. This direct approach would enhance the value of the antipyrine test because of the simplicity and convenience.

Animals↗
PubMed1994

Evaluation of Gen-Probe Amplified Mycobacterium Tuberculosis Direct Test and PCR for direct detection of Mycobacterium tuberculosis in clinical specimens.

The Gen-Probe Amplified Mycobacterium Tuberculosis Direct Test (AMTD) is a direct specimen assay for the identification of Mycobacterium tuberculosis from respiratory samples. rRNA is amplified, and the product is detected with a specific chemiluminescent probe. We performed a retrospective evaluation of three separate respiratory specimens from each of 250 patients by using the AMTD and compared the results with those of microscopy, culturing, and a patient chart review. From the latter results, 198 patients (594 specimens) were found negative for M. tuberculosis by culturing and clinical criteria. The overall specificity of the AMTD after discrepancy resolution was 98.5% (585 of 594). There were 52 patients with culture-proven and/or clinically diagnosed tuberculosis. Of these 156 specimens, the organism was cultured from 142 (91%), and acid-fast microscopy was positive for 105 (67.3%). The AMTD was positive for 142 (91%) specimens from these patients. Tuberculosis patient samples were tested by a PCR assay which uses primers for amplification of the IS6110 insertion sequence of the M. tuberculosis complex. The PCR assay detected 144 of the 156 (92.3%) specimens. Overall, when three specimens per patient were examined, the AMTD found all 52 patients positive for tuberculosis, while the PCR assay found 51 patients positive by agarose gel analysis and all 52 patients positive by Southern blot hybridization.

Bacteriological Techniques↗
PubMed2002

Direct detection of hydrogen bonds in monomeric superoxide dismutase: biological implications.

Hydrogen bonds were directly determined via NMR with different experimental approaches at 600 and 800 MHz for reduced monomeric superoxide dismutase (Q133M2SOD, 16 kDa). This protein contains a copper and a zinc ion and shows the classical superoxide dismutase (SOD) eight-stranded beta-barrel fold. The best results for this intermediate molecular mass protein were obtained using a TROSY version of the long-range HNCO experiment at high magnetic field (800 MHz) or with a cryoprobe at 600 MHz. The backbone hydrogen bond network that defines the secondary structure of the protein was detected. Thirty-five backbone hydrogen bonds were identified. The lower limit for their detection, their relation to the TROSY R(2) rates, and the correlation between hydrogen bond detectability and signal line width are discussed. Experiments were also optimized to detect hydrogen bonds involving key side chains, which lead to the observation of five hydrogen bonds. In particular, the hydrogen bonds involving the side chain of Asp 124 were observed, which show significant differences with respect to the bonds expected on the basis of the crystal structure. The relevance of this finding relies also on the fact that Asp 124 is a key residue in determining the affinity of the protein for zinc. It has now been determined that the gain of the toxic function of peroxynitrite formation in SOD mutants related to amyotrophic lateral sclerosis (ALS) is due to SOD species lacking the zinc ion, as a consequence of a reduced affinity for zinc. Therefore, this study provides structural hints for understanding the origin of the enzymatic behavior of the Zn-deficient SOD.

Catalysis↗
PubMed1986

A new method to detect directly in culture cell surface membrane immunoglobulins.

An ELISA assay is described for the measurement of the smIgG. The method is based on the detection of cell-smIgG directly on the same microplate used for the culture. The cells, preincubated at 37 degrees C for one hour, were cultured in the presence of S-ConA and serum-free medium for two days. Using this strategy, the background noise due to non specific adsorption of IgG to plastic wells and cytophilic antibodies was eliminated. The cells in the presence of S-ConA and serum-free medium adhered to the plastic wells, and the cell-smIgG were detected using an anti-human IgG covalently linked to alkaline phosphatase or its F(ab')2 fragment. The possibility of measuring the modulation of the expression of the cell-smIgG without any additional manipulation is stressed.

Adult↗
PubMed1987

Direct detection of rhinoviruses by an enzyme-linked immunosorbent assay.

This paper describes the first enzyme-linked immunosorbent assay for the detection of rhinovirus antigens in clinical specimens (nasal washings), either directly or following overnight cell culture amplification. The assay takes approximately 48 hours to perform and utilizes the same rabbit antirhinovirus hyperimmune serum as both the capture and detecting antibody. The latter has been biotin-labelled and is detected via a streptavidin beta-galactosidase preformed complex. This new assay has been found to be very sensitive, detecting human rhinovirus (HRV)-EL and HRV-2 at titres as low as 10(1.8) TCID50 100 microliter-1 and less than 10(1)TCID50 100 microliter -1, respectively. Furthermore, when 57 different human rhinovirus serotypes were tested in both the HRV-EL and HRV-2 ELISA systems a total of 49 (86%) were found to be cross-reactive. Of 36 clinical specimens tested by virus isolation, cell-culture-amplified (CCA) ELISA, and direct ELISA, 15 were positive by isolation, 11 by CCA-ELISA, and 11 by direct ELISA. The overall correlation of the CCA and direct ELISA techniques with virus isolation was found to be 88.9% and 66.7%, respectively. The present study demonstrates that the ELISA system developed is a sensitive technique for the diagnosis of rhinovirus infections.

Antigens, Viral↗
PubMed1986

Rapid coagglutination test for the direct detection of group A streptococci from throat swabs.

A one-minute antigen detection test was compared with a conventional culture method for detecting group A beta-hemolytic streptococci. The test detects coagglutination between protein A and streptococcal antigen extracted directly from throat swabs. Of the 307 specimens tested, 66 (21.5%) were positive for group A streptococci by culture and 16 specimens (5.2%) were positive for other beta-hemolytic streptococci. The direct test agreed with the culture in 274 of 307 specimens (accuracy 89.3%). The sensitivity of the test was 86.4% (57/66), the specificity 90% (217/241), the positive predictive value 70.4% and the negative predictive value 96%. If only throat cultures with more than 100 colonies of group A streptococci per plate were considered, the sensitivity of the direct test rose to 96%. If only a strong agglutination was considered positive, the specificity of the direct test rose to 98%. Further studies are needed to determine whether this test could be used alone or in addition to culture.

Agglutination Tests↗
PubMed2006

Direct detection of Mycobacterium tuberculosis complex DNA and rifampin resistance in clinical specimens from tuberculosis patients by line probe assay.

The INNO-LiPA.Rif TB test (LiPA) has only been applied to a limited number of clinical specimens. To assess the utility of this test for detecting Mycobacterium tuberculosis complex DNA and rifampin (RMP) resistance, 420 sputum samples comprising specimens from untreated (n=160) and previously treated (n=260) patients from 11 countries in Asia, Africa, Europe, and Latin America were tested. DNA was extracted from sputum samples by using a modification of the Boom's method, while the rpoB core region was amplified by nested PCR. The results were analyzed in conjunction with those obtained by Ziehl-Neelsen (ZN) microscopy and by culture on solid media. The LiPA test was positive for M. tuberculosis complex DNA in 389 (92.9%) specimens, including 92.0% (286 of 311) ZN-positive and 94.5% (103 of 109) ZN-negative specimens. Of these, 30.6% were RMP resistant. In contrast, 74.3% of the specimens were positive for M. tuberculosis by culture, and 30.8% of them were RMP resistant. LiPA detected M. tuberculosis complex DNA in 92.4% (110 of 119) of the culture-positive and 100.0% (41 of 41) of the culture-negative specimens from untreated patients. There was a 99.6% concordance between the RMP resistance as determined by culture and by the LiPA test. With an optimal DNA extraction method, LiPA allows rapid detection of M. tuberculosis complex DNA and RMP resistance directly from sputum specimens. LiPA can still provide useful information when culture fails for various reasons. The rapid availability of this information is necessary to adjust patient treatment and avoid the risk of amplification of drug resistance.

Antitubercular Agents↗
PubMed1990

Direct detection and automated sequencing of individual alleles after electrophoretic strand separation: identification of a common nonsense mutation in exon 9 of the human lipoprotein lipase gene.

Large-scale screening by direct sequencing of DNA to detect molecular variants remains a laborious endeavor whose difficulty is compounded by heterozygosity. We show that mobility shifts of single-stranded DNA electrophoresed under nondenaturing conditions can be used not only to detect variants (Orita,M. et al., 1989, Genomics, 5, 874-879), but also to separate and sequence directly individual alleles. In this manner, we have identified a common variant of human lipoprotein lipase resulting from a nonsense mutation in exon 9 of the gene. Whether this variant is of functional significance remains to be determined.

Alleles↗
PubMed1993

Direct detection and genotyping of Chlamydia trachomatis in cervical scrapes by using polymerase chain reaction and restriction fragment length polymorphism analysis.

Detection and genotyping of Chlamydia trachomatis were optimized by using a polymerase chain reaction (PCR) and restriction fragment length polymorphism (RFLP) analysis performed directly with crude cells of cervical scrapes. Different PCR pretreatment methods were evaluated on samples which were positive for C. trachomatis by cell culture. In comparison with DNA extraction and different proteolytic digestion methods, a simple pretreatment of 10 min of boiling appeared to be optimal for PCR amplification. Crude samples (n = 209) were first screened for C. trachomatis by both cell culture and plasmid PCR. Subsequently, positive samples found by plasmid PCR were subjected to a direct omp1 PCR-based RFLP analysis to differentiate C. trachomatis serovars A to K, Ba, Da, and L1 to L3 and serovariant D-. All cervical scrapes that were found positive for C. trachomatis by cell culture (n = 30) were also positive by plasmid PCR and omp1 PCR and could be easily genotyped. In addition, of the culture-negative group, eight samples were found positive by plasmid PCR. Five of these eight samples were also positive by omp1 PCR; of these five, two were positive by a nested omp1 PCR. Genotyping by RFLP analysis of the 35 omp1 PCR-positive samples showed that serovars D, E, and F are the most prevalent types found in cervical scrapes, while serovariant D- was also detected. This study shows that direct PCR and PCR-based RFLP analysis are feasible for detection and genotyping of C. trachomatis in cervical scrapes and are more sensitive than culture-based serotyping.

Bacterial Typing Techniques↗
PubMed2003

Direct detection of multidrug-resistant Mycobacterium tuberculosis in clinical specimens in low- and high-incidence countries by line probe assay.

The INNO-LiPA Rif.TB assay is designed for the detection of rpoB gene mutations causing rifampin resistance in isolates. We applied the method directly to 60 Lithuanian and Danish clinical specimens to detect rifampin resistance rapidly. Results were obtained in 78.3% of clinical specimens, and all were concordant with those obtained by BACTEC 460. The assay could have major impact on the management of multidrug-resistant tuberculosis.

DNA-Directed RNA Polymerases↗