Initial Positive Result Research Articles

Failure to achieve gene conversion with chimeric circular oligonucleotides: potentially misleading PCR artifacts observed.

Recently, a novel strategy for nucleotide exchange of target DNA using chimeric RNA/DNA oligonucleotides (CO) was reported. The CO can easily be transfected into cells, remain stable within the cells, and migrate to the nucleus. We have in this study used 42 similar constructs for targeting six different human and canine loci. A variety of cationic lipids, electroporation, and microinjection were used for transfection of the CO into lymphoblastoids, Huh7, HT 1080, and Jurkat cell lines, and canine primary fibroblasts and hepatocytes. However, no nucleotide exchange was detected in any of the targeted loci. Using PCR followed by restriction enzyme analysis, nucleotide exchange in approximately 2%-10% of the PCR products was observed during the first 3 days after transfection with CO-vWF-28S2 designed for repairing a mutation in the von Willebrand gene. Surprisingly, the observed exchange reverted after culturing the cells for a longer period of time (14 days). Furthermore, a positive indication of gene conversion (5%) was also obtained using an allele-specific PCR method for analysis of the PAI-1 gene. However, cloning of the PCR products revealed no nucleotide exchange. In our view, the most likely explanation is that the initial false positive result originates from a PCR artifact created by the CO itself. Our results imply that an independent method, that is, Southern blotting, must be used to verify an observed nucleotide exchange.

Discovery of phospholamban. A personal history.

Early efforts to identify mechanisms by which sympathetic stimulation increases myocardial contractility led to studies of effects of beta-adrenergic agonists and cyclic AMP on the cardiac contractile proteins and sarcoplasmic reticulum (SR); initial positive reports, however, could not be confirmed. The discovery that cyclic AMP-dependent protein kinases (PK-A) mediated intracellular actions of cyclic AMP led at least four groups to test the hypothesis that phosphorylation of the cardiac SR played a role in the actions of beta-adrenergic agonists. Three of them (Wollenberger, Wray et al., and LaRaia & Morkin) demonstrated that cardiac SR was a substrate for PK-A phosphorylation; however, the lability of the Ca2+ pump in these membranes made it difficult to demonstrate a functional significance of this finding. Our group, which had extensive experience in measuring SR Ca2+ transport, began by examining the ability of PK-A to activate the SR Ca2+ pump "poised" at half-saturating Ca2+ concentrations. Our initial positive result led to the discovery that a 22,000-dalton protein, named phospholamban by Phyllis B. Katz, mediated effects on Ca2+ transport by the SR that could explain both the inotropic and lusitropic effects of sympathetic stimulation.

A Comparison of Ligase Chain Reaction toPolymerase Chain Reaction in the Detection ofChlamydia trachomatis Endocervical Infections

Objective: To compare the reliability of ligase chain reaction (LCR) to polymerase chain reaction (PCR) in detecting Chlamydia trachomatis endocervical infections.Methods: We conducted a prospective study of 486 patients at risk for chlamydial infection of the endocervix. We obtained two endocervical specimens from each patient and used LCR and PCR to detect C. trachomatis. Discrepant results between the two techniques were resolved by repeat testing and by testing for the major outer membrane protein (MOMP) gene, if necessary. We determined the sensitivity, specificity, positive predictive value, and negative predictive value for each test, using concordant results or MOMP gene results as the “gold standard”.Results: Of the 486 patients, 42 (8.6%) had evidence of C. trachomatis infection after resolution of discrepant results. Of the 42 true positive specimens, 41 were positive by initial LCR and 38 were positive by initial PCR. Of the 444 true negative specimens, none had a positive initial LCR result, while 2 had a positive initial PCR test. Therefore, compared to the gold standard, LCR had a sensitivity of 97.6% and specificity of 100%, while PCR had a sensitivity of 90% and a specificity of 99.5%. The positive and negative predictive values of LCR were 100% and 99.8%, respectively. PCR had a positive predictive value of 95% and a negative predictive value of 99.1%. The difference in sensitivity of LCR versus PCR was not statistically significant (P = .125).Conclusion: LCR and PCR perform equally well in detecting C. trachomatis endocervical infections.

A comparison of ligase chain reaction to polymerase chain reaction in the detection of Chlamydia trachomatis endocervical infections

Objective To compare the reliability of ligase chain reaction (LCR) to polymerase chain reaction (PCR) in detecting Chlamydia trachomatis endocervical infections. Methods We conducted a prospective study of 486 patients at risk for chlamydial infection of the endocervix. We obtained two endocervical specimens from each patient and used LCR and PCR to detect C. trachomatis. Discrepant results between the two techniques were resolved by repeat testing and by testing for the major outer membrane protein (MOMP) gene, if necessary. We determined the sensitivity, specificity, positive predictive value, and negative predictive value for each test, using concordant results or MOMP gene results as the “gold standard.” Results Of the 486 patients, 42 (8.6%) had evidence of C. trachomatis infection after resolution of discrepant results. Of the 42 true positive specimens, 41 were positive by initial LCR and 38 were positive by initial PCR. Of the 444 true negative specimens, none had a positive initial LCR result, while 2 had a positive initial PCR test. Therefore, compared to the gold standard, LCR had a sensitivity of 97.6% and specificity of 100%, while PCR had a sensitivity of 90% and a specificity of 99.5%. The positive and negative predictive values of LCR were 100% and 99.8%, respectively. PCR had a positive predictive value of 95% and a negative predictive value of 99.1%. The difference in sensitivity of LCR versus PCR was not statistically significant (P = .125). Conclusion LCR and PCR perform equally well in detecting C. trachomatis endocervical infections. Infect. Dis. Obstet. Gynecol. 6:57–60, 1998. © 1998 Wiley-Liss, Inc.

Prevalence studies of GB virus-C infection using reverse transcriptase-polymerase chain reaction.

Among the three recently described GB viruses (GBV-A, GBV-B, and GBV-C), only GBV-C has been linked to cryptogenic hepatitis in man. Because of the limited utility of currently available research tests to determine antibody response to GBV-C proteins, the prevalence of GBV-C RNA in human sera was studied using reverse transcription-polymerase chain reaction (RT-PCR). The prevalence of GBV-C is higher among volunteer blood donors with elevated serum alanine aminotransferase (ALT) levels (3.9%) than among volunteer blood donors with normal ALT levels (0.8%). Higher rates were also noted among commercial blood donors (12.9%) and intravenous drug users (16.0%). GBV-C was frequently detected in residents of West Africa, where the prevalence was > 10% in most age groups. Approximately 20% of patients diagnosed with either acute or chronic hepatitis C virus (HCV) were found to be positive for GBV-C RNA. In addition, GBV-C RNA sequences were detected in individuals diagnosed with non-A-E hepatitis, with clinical courses ranging from mild disease to fulminant hepatitis. Fourteen of sixteen subjects with or without clinically apparent hepatitis were positive for GBV-C RNA more than 1 year after the initial positive result.

Immunology: Antiphospholipid antibodies and β2-glycoprotein-I in 500 women with recurrent miscarriage: results of a comprehensive screening approach

Five hundred consecutive women (median age 33 years; range 19-45) with a history of recurrent miscarriage (median 4; range 3-16) were screened for the presence of antiphospholipid antibodies (APA)-lupus anticoagulant (LA) and/or anticardiolipin antibodies (ACA). The prevalence of persistently positive tests for LA was 9.6% and for immunoglobulin G (IgG) and immunoglobulin M (IgM) ACA was 3.3 and 2.2% respectively. Only seven women (1.4%) were LA and ACA positive. Repeat testing, after an interval of at least 8 weeks, demonstrated that only 65.7% of LA positive, 36.6% IgG ACA positive and 36.0% IgM ACA positive women on initial testing had a second positive test result. The dilute Russell's viper venom time detected the LA significantly more often than either the activated partial thromboplastin time or the kaolin clotting time (P < 0.001). There was no difference in the gestation of previous miscarriages between APA positive and APA negative women. There was no difference in the plasma beta 2-glycoprotein-I concentrations between APA positive and APA negative women with miscarriages and normal women. All women with a history of recurrent miscarriage should be tested for the presence of both LA and ACA. A second confirmatory test should be performed in those with an initial positive test result.

Gender Differences in the Noninvasive Evaluation and Management of Patients with Suspected Coronary Artery Disease

To determine if gender-based differences exist in the post-test management and clinical outcome of patients with clinically suspected coronary artery disease who have stress electrocardiographic or myocardial perfusion imaging evaluation. Retrospective cohort study. University medical center. From a cohort of 3975 middle-aged patients referred for outpatient stress testing, 840 (47% women) were evaluated noninvasively for clinically suspected coronary artery disease. The rates of subsequent diagnostic procedures and the incidence of subsequent coronary revascularization, myocardial infarction, or cardiac death were determined for women and men. Pretest cardiac risk profiles were similar, except hypertension and hypercholesterolemia were more frequent in women. Atypical angina was more common in women than in men (57.5% compared with 44.5%, respectively; P < 0.001). Rates of initial test positivity (defined as exercise-induced horizontal or downsloping ST-segment depression > or = 1.0 mm or > or = 1 reversible thallium-201 defect) were similar in women and men. Compared with men, most women with an initial positive test result had no additional coronary artery disease evaluation (62.3% compared with 38.0%; P = 0.002). Coronary revascularization procedures were done more frequently in men (4.9% [22 of 449] compared with 2.0% [8 of 391]; P = 0.03). Cardiac death or myocardial infarction occurred more often in women during 2 years of follow-up (6.9% [27 of 391] compared with 2.4% [11 of 449]; P = 0.002). Women with suspected coronary artery disease have fewer additional diagnostic tests than men after an initial abnormal noninvasive stress test result, even though the incidence of typical angina, cardiac risk factors, and initial diagnostic test positivity rates are similar.

Clinical relevance of testing for antineutrophil cytoplasm antibodies (ANCA) with a standard indirect immunofluorescence ANCA test in patients with upper or lower respiratory tract symptoms.

Reports from specialist nephrological centres have suggested that the antineutrophil cytoplasm antibody (ANCA) test is highly specific and sensitive for patients with Wegener's granulomatosis. To determine the usefulness of the ANCA test in everyday respiratory practice the results of the test were audited in all patients in the south west of England with respiratory symptoms who underwent the test. The results of all 335 patients who had presented with upper or lower respiratory tracts symptoms, or both, and were tested for ANCA by the indirect ANCA test in 1990, as recommended in the broadsheet of the British Association of Clinical Pathologists, were audited. Case notes and necropsy reports were available for review in 231 cases (69%), and in the remainder information was obtained by a standard questionnaire. There were 106 positive results, 45 (44%) from patients with Wegener's granulomatosis. The sensitivity and specificity of a positive ANCA test result in this study were 65% and 77% respectively. For a diagnosis of Wegener's granulomatosis the sensitivity and positive predictive accuracy of a positive cytoplasmic ANCA (c-ANCA) test were greater than of a positive perinuclear ANCA (p-ANCA) test. There were 61 positive tests in 266 patients who did not have Wegener's granulomatosis (23%); of these 27 were from patients with infection, 10 with fibrotic lung disease, nine with underlying connective tissue disease, seven with malignancy, and five following pulmonary emboli. Most of these positive ANCA results were p-ANCA (69%) rather than c-ANCA (31%). Serial ANCA requests were made in 15 cases of patients without Wegener's granulomatosis who had an initial positive ANCA test result. In all cases the ANCA tests subsequently became negative. In this study the sensitivity and specificity of a positive ANCA test result were less than that reported from specialised centres. However, the test was found to be useful in clinical practice, especially c-ANCA, in conjunction with clinical symptoms of respiratory pathology and evidence of renal disease.

Plasmid content and localisation of the STal (STaP) gene in enterotoxigenic Escherichia coli with a non-radioactive polynucleotide gene probe

Enterotoxigenic Escherichia coli (ETEC) strain P2200 of porcine origin possessed eight possibly plasmid-determined characters (K88+ Raf+ Hly+ Col+ Smr Tcr Su(r) STa+) and six plasmid DNA bands of 4.2-93 kb. Analysis of the spontaneous loss of characters and the results of matings with other E. coli strains revealed that the K88, Raf, Hly, Smr, Tcr and Su(r) characters could be transferred, and that the presence of the K88 and Raf characters was associated with an 83-kb plasmid. The presence and location of the STaI gene was investigated in several ETEC strains of bovine or porcine origin. Hybridisation with a non-radioactive polynucleotide probe associated the STaI gene with a plasmid in each strain; these plasmids were of 32-142 kb. In contrast, plasmids from a P2200 STa- variant and plasmids from two STa- variants of the bovine ETEC strain B41* (strain B41 obtained from a different source) did not hybridise with the probe. One of the B41*STa- variants had lost the STa plasmid, whereas the second variant retained a plasmid of the same size which did not hybridise. In contrast, a third B41*STa- variant retained a plasmid of the same size that still hybridised with the STaI probe. Plasmid DNA restriction fragment analysis, followed by hybridisation with the STaI probe, showed that the STaI gene was associated with 8.3-, 6.8- and 3.5-kb plasmid fragments in strain B41, and with 4.9-, 6.8- and 3.5-kb plasmid fragments in strain B41*, following digestion with EcoRI, BamHI, or EcoRI + BamHI, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)

The psychological effects of false-positive results in prenatal screening for fetal abnormality: a prospective study.

The purpose of the study was to describe the impact of false-positive results from initial maternal serum alpha-fetoprotein (MS-AFP) screening. The analyses compared two groups of women, those receiving a negative result (n = 346) and those receiving an initial positive result (n = 26), over four time points--prior to testing, immediately after testing, later in pregnancy, and in the post-partum period. Receiving an abnormal result was associated with high levels of anxiety which were reflected in increased worry about the baby's health and a more negative attitude towards the pregnancy and the baby. Women who had an initial abnormal result were offered a variety of further tests. Those women who went on to have amniocentesis were less worried about their baby's health in the third trimester and also less anxious post-partum than those who did not have amniocentesis. In view of the increasing number of screening tests available, it is necessary to establish whether and how these levels of distress can best be reduced.

Blind comparison of Abbott and Dupont HIV antigen ELISA tests for detecting antigenaemia in asymptomatic human immunodeficiency virus antibody positive homosexual men.

Three hundred and ninety eight serum samples from 270 human immunodeficiency virus (HIV) antibody positive asymptomatic homosexual men were tested in the Abbott and Dupont HIV antigen ELISA tests. In the Abbott test 62 (16%) of the sera were positive, according to the manufacturer's instructions, compared with 55 (14%) in the Dupont test. Twenty six sera were positive with the Abbott test but negative with the Dupont test and 19 sera were positive only by the Dupont test. Only 36 (9%) of the sera were positive in both tests. The Abbott confirmatory neutralisation test gave excellent agreement with the initial Abbott HIV antigen ELISA test; the Dupont confirmatory test was only in agreement with the initial positive Dupont antigen ELISA test in one third of the sera tested. Although the overall sensitivity of each of the two commercial assays tested was similar, the Abbott method may be preferable for clinical purposes if confirmation of an initial ELISA positive test result by neutralisation assay is required.

Pitfalls in Diagnosing Galactosemia

Newborn galactosemia screening programs using the fluorescence spot test to detect red cell galactose‐1‐phosphate uridyltransferase activity are prone to inaccuracy if the screened infants have received blood transfusions. We describe an infant with galactosemia who received packed red cell transfusions in the first few days of life and was misdiagnosed after an initial positive screening test result. Although the patient was thought to have cytomegaloviral hepatitis, a percutaneous liver biopsy helped direct the evaluation toward identifying the galactosemia carrier state in both parents. This case report illustrates the need for careful consideration of the patient's history of transfusion of blood products when evaluating newborn screening results.

Pitfalls in Diagnosing Galactosemia

Newborn galactosemia screening programs using the fluorescence spot test to detect red cell galactose-1-phosphate uridyltransferase activity are prone to inaccuracy if the screened infants have received blood transfusions. We describe an infant with galactosemia who received packed red cell transfusions in the first few days of life and was misdiagnosed after an initial positive screening test result. Although the patient was thought to have cytomegaloviral hepatitis, a percutaneous liver biopsy helped direct the evaluation toward identifying the galactosemia carrier state in both parents. This case report illustrates the need for careful consideration of the patient's history of transfusion of blood products when evaluating newborn screening results.

Validity of Mass Blood Pressure Screening in Children

The validity of a mass blood pressure screening program carried out on Dallas high school students who were followed for at least 3 years is reported. Blood pressure results on initial screening in the tenth grade were related to outcome blood pressure status. The frequency of initial blood pressure elevation (systolic and/or diastolic pressure above the 95th percentile) was 10%, whereas the prevalence of sustained elevation in the tenth grade was less than 2%. The initial screening correctly identified 72% of those who had sustained elevations on all three tenth grade examinations (sensitivity), and correctly identified 91% of those who did not have sustained elevations (specificity). However, the predictive value of an initial positive screening result was low, at only 17% (152/900). The high proportion of false-positive results represents a significant cost to any blood pressure screening program, both in economic terms and in its potential for creating anxiety among families with children having only transiently elevated blood pressure. Because the costs are high and the yield from mass screening of children low, case finding of childhood hypertension should remain the responsibility of the physician who sees children in the clinical setting.

Pneumatic transport of tablets

It is necessary to consider that according to impact theory the maximum stresses arise on collision of bodies not on the surface but at a certain depth. This circumstance may lead to the fact that an initial positive result will be inaccurate; tablets disintegrate some time after the experiment. This was found in our preliminary experiments (cyclone discharges of diameter less than 290 mmwere used). From the collision of tablets moving at significant rates, internal cracks are generated which also lead after some time to disintegration of tablet surfaces. It is particularly necessary to be careful in the final decision as to the suitability of a pneumatic conveyor if it is constructed for tablets with coating, since the strength of the coating is, as a rule, higher than the strength of the tablets and breakdown of the surface may arise after a sufficiently large period of time.

ANTIDEPRESSANT DRUGS AND LIVER DAMAGE.

<h3>Objective</h3> To explore primary care providers’ (PCPs’) role in result notification for newborn screening (NBS) for cystic fibrosis (CF), given that expanded NBS has increased the number of positive screening test results, drawing attention to the role of PCPs in supporting families. <h3>Design</h3> Cross-sectional survey and qualitative interviews. <h3>Setting</h3> Ontario. <h3>Participants</h3> Primary care providers (FPs, pediatricians, and midwives) who received a positive CF NBS result for an infant in their practice in the 6 months before the study. <h3>Main outcome measures</h3> Whether the PCP notified the family of the initial positive CF screening result. <h3>Results</h3> Data from 321 PCP surveys (response rate of 51%) are reported, including 208 FPs, 68 pediatricians, and 45 midwives. Interviews were completed with 34 PCPs. Most (65%) surveyed PCPs reported notifying the infant’s family of the initial positive screening result; 81% agreed that they have an important role to play in NBS; and 88% said it was important for PCPs, rather than the NBS centre, to notify families of initial positive results. With support and information from NBS centres, 68% would be extremely or very confident in doing so; this dropped to 54% when reflecting on their recent reporting experience. More than half (58%) of all PCPs said written point-of-care information from the NBS centre was the most helpful format. Adjusted for relevant factors, written educational information was associated with a lower rate of notifying families than written plus verbal information (risk ratio of 0.79; 95% CI 0.69 to 0.92). In the interviews, PCPs emphasized the challenge of balancing required content knowledge with the desire for the news to come from a familiar provider. <h3>Conclusion</h3> Most PCPs notify families of NBS results and value this role. These data are relevant as NBS programs and other genomic services expand and consider ways of keeping PCPs confident and actively involved.