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At least 181 records · Page 10Linked to original sources
PubMed1998

Restriction endonuclease-mediated selective polymerase chain reaction: a novel assay for the detection of K-ras mutations in clinical samples.

The enriched polymerase chain reaction (PCR) assay has been used extensively in the detection of ras gene mutations in many types of human malignancies. Although it is very sensitive, it has a number of features that limit its use in the routine diagnostic laboratory. The aim of this study was to develop a novel enriched PCR strategy, in which the concurrent activity of the restriction enzyme BstNI and Taq polymerase allowed the amplification of mutant K-ras while inhibiting the formation of wild-type product. This restriction endonuclease-mediated selective PCR assay uses three sets of primers, together with BstNI, in the reaction mix, and the amplification products are analyzed by gel electrophoresis. The reliability of the restriction endonuclease-mediated selective PCR assay to detect activated K-ras was determined in a variety of clinical samples, including 139 fresh colorectal carcinomas and 113 paraffin-embedded blocks from 80 separate tumors of the colon and rectum, pancreas, breast, or kidney. Codon 12 mutations of the K-ras oncogene were identified in DNA from both fresh and paraffin-embedded tumors in a rapid, sensitive, and reproducible manner. Mutations were detected in 33 (24%) of the fresh colorectal cancers and 16 (20%) of the paraffin-embedded tumors. These results were 97% concordant in cases in which paraffin blocks and fresh specimens from the same tumor were available for analysis. We conclude that restriction endonuclease-mediated selective PCR is a sensitive, rapid, and robust assay for the detection of point mutations in a variety of clinical samples. Importantly, there is no need for manipulation of the sample once the PCR has been set up, and therefore, the chance of contamination is significantly reduced. In contrast to previous assays, restriction endonuclease-mediated selective PCR is not labor intensive, and its format is suitable for use in routine diagnostic laboratory.

Adenocarcinoma↗
PubMed1994

Comorbidity of empirically based syndromes in matched general population and clinical samples.

Comorbidity of deviance on eight empirically based syndromes was compared in matched general population and clinical samples of 2,705 children aged 4-18, using a bidirectional formula to avoid confounding effects of differential base rates. Syndromes were assessed via parent ratings on the CBCL, teacher ratings on the TRF, and self ratings on the YSR. Significantly higher comorbidity rates were obtained for clinical than general population samples for all 28 pairings of CBCL syndromes, 15 pairings of TRF syndromes, and 22 pairings of YSR syndromes. Bidirectional comorbidity rates for empirically based syndromes were compared to pairings of comparable DSM-III diagnoses.

Adolescent↗
PubMed2025

Amplification of Filovirus Genomes from Clinical Samples for Next Generation Sequencing.

Viral genome sequencing has become a critical tool in outbreak mitigation. Due to their small size relative to the host genome, viral genomes comprise a small fraction of next generation sequencing reads in clinical samples when using unbiased sequencing approaches. Long-range polymerase chain reaction facilitates the amplification of viral genomes from clinical and environmental samples with minimal primer sites, allowing researchers to target regions of the genome that are conserved across available variants. Here, we describe the amplification and sequencing of the Ebola virus genome from tissue samples collected from infected nonhuman primates. This protocol facilitates full viral genome recovery from as low as 103 median tissue culture infectious doses per milliliter.

High-Throughput Nucleotide Sequencing↗
PubMed1997

Representativeness of clinical samples of youths with mental disorders: a preliminary population-based study.

In a household community sample of 1,285, 9-17 years-olds with mental disorders who had received outpatient specialty mental health services in the past year were compared with youths with mental disorders who had not received those services to determine if samples drawn from clinical settings are representative of youths with mental disorders in the general population. Those who had used services were more impaired, less competent, more likely to have comorbid disorders, more likely to belong to non-Hispanic White relative to other ethnic groups, and less likely to be prepubertal girls. Their parents were more educated, but less satisfied with family life, engaged in less monitoring of their children, and more likely to have used mental health services themselves. These findings suggest the hypothesis that samples of youths with mental disorders drawn from outpatient clinical settings are not representative of all youths with mental disorders. If confirmed, this would indicate the importance of population-based samples for the study of psychopathology in youths.

Adolescent↗
PubMed1997

Standardization of a single-cell assay for sensitive detection of multidrug resistance protein expression in normal and malignant cells in archival clinical samples.

Multidrug resistance protein (MRP), like P170, confers multidrug resistance, but its clinical relevance is uncertain, whereas P170 is an accepted cause of chemotherapy failure for which ongoing reversal trials are being conducted. Because such trials have been only modestly successful, we must investigate alternative drug resistance mechanisms such as MRP, which is poorly blocked by P170 inhibitors. The significance of MRP has remained undefined because MRP mRNA is difficult to assay in archival material, does not necessarily reflect MRP levels, and is widely expressed in normal or hematopoietic cells within tumors and bone marrow. Because conventional immunoblot or immunocytochemistry may not be sensitive enough to detect low or heterogeneous MRP expression in clinical samples, we elected to score MRP in single tumor cells by modifying our P170 assays that have proven valuable for correlating P170 expression with the outcome of pediatric cancer chemotherapy. We enhanced the signal-to-noise ratio with several peroxidase-tagged secondary antibody layers and staining refinements, standardizing the assay with MRP-negative and MRP-positive but P170-negative transfected or drug-selected controls in which MRP was quantified by immunoblot. We confirmed sensitivity by staining a very low MRP-expressing revertant line and "mixed" samples containing small numbers of positive cells; we confirmed specificity by applying two antibodies directed against separate MRP epitopes. We examined neuroblastoma, osteosarcoma, rhabdomyosarcoma, and retinoblastoma samples, identifying MRP-positive malignant cells, which were distinguishable from MRP-positive normal cells. This assay may be valuable for early diagnosis of low but potentially important MRP expression, which would allow timely application of alternative therapy, perhaps with MRP-specific blockers.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1995

Comparison of polymerase chain reaction and virus isolation for detection of epizootic hemorrhagic disease virus in clinical samples from naturally infected deer.

We compared our recently reported reverse transcriptase polymerase chain reaction (PCR)-based assay for detection of epizootic hemorrhagic disease virus (EHDV) in clinical samples with different virus isolation (VI) procedures. Thirty-six blood samples and 1 spleen sample from deer were assessed by the EHDV PCR assay and VI in baby hamster kidney (BHK)-21 cells and embryonated chicken eggs (ECE). The EHDV PCR assay detected EHDV RNA from 6 blood samples obtained from deer during 1988-1989 outbreaks of epizootic hemorrhagic disease and from the spleen and blood samples of a deer with clinical hemorrhagic disease in 1992. The 6 blood samples from the 1988-1989 outbreaks and the spleen sample from the 1992 case were VI positive on BHK-21 cell culture. The blood from the same deer with the PCR- and VI-positive spleen was VI negative in BHK-21 cells and ECE. All EHDV isolates were identified as EHDV serotype 2 by a plaque inhibition test. The results of this study indicate that the sensitivity of the previously described EHDV PCR assay is comparable to or greater than that of the VI method in BHK-21 cell culture or ECE. The EHDV PCR assays could provide a superior diagnostic alternative to the current cumbersome and time-consuming VI procedures.

Animals↗
PubMed2006

Assessing the generalization of psychopathy in a clinical sample of domestic violence perpetrators.

This study proposed that domestic violence perpetrators in a clinical sample could be categorized into distinct subgroups and that a particular subgroup of batterers would exhibit sufficient psychopathic characteristics to be clinically meaningful. Participants were interviewed in order to gather a relevant social, familial, educational, criminal, and substance abuse history. They were then administered several psychological measures including the Psychopathy Checklist: Screening Version (PCL:SV). Results lent support to the empirical batterer typology identified by Holtzworth-Munroe, A., Meehan, J. C., Herron, K., Rehman, U., & Stuart, G. L. (2000). However, despite the presence of a more antisocial subgroup, psychopathy did not consistently differentiate among batterers across the measured dependent variables.

Adult↗
PubMedDate not supplied

[Detection of Pneumocystis carinii DNA in clinical samples from children with respiratory diseases].

Method of Pneumocystis carinii DNA detection in clinical samples (sputum) is presented. Primers to one fragment of 16S rRNA gene were used for detection of DNA. Isolation and amplification of DNA were performed in presence of internal DNA control. Analytical sensitivity of method was 200 copies of DNA per 1 ml. Analytical and diagnostic specificities were 100% and 95% accordingly. Sputum from 176 children with frequent respiratory infections were sampled before start of antibacterial therapy and studied simultaneously by the polymerase-chain reaction (PCR) and immunofluorescent assay (IFA). Results of PCR and IFA coincided in 167 (94.89%) children. From them, P.carinii was detected in sputum in 5 (2.85%) children. All children with positive results were treated with antibiotics. Repeated tests of sputum 8 days after start of treatment were all negative. PCR could be recommended as part of complex of clinical diagnostics and control of treatment.

Adolescent↗
PubMed2000

Rapid, sensitive and simple detection of candida deep mycosis by amplification of 18S ribosomal RNA gene; comparison with assay of serum beta-D-glucan level in clinical samples.

We evaluated the clinical usefulness of a polymerase chain reaction (PCR) assay amplifying the 18S ribosomal RNA gene of fungi for the diagnosis of deep candidiasis, compared with that of the beta-glucan test or Cand-Tec test. Thirty critically ill patients who had received prolonged care with intravenous hyperalimentation and endotracheal intubation in the intensive care unit and were suspected of having deep fungal infections were examined. Twenty-one were fungi positive in the PCR assay (70%). Among 24 samples in which the PCR assay, beta-glucan test and Cand-Tec test were performed simultaneously, 75% of the samples (18/24) were fungi positive in the PCR assay, whereas only 54% (13/24) had positive reactions in the beta-glucan test and 21% (5/24) in the Cand-Tec test. The results of the Cand-Tec test showed no relationship with those of the PCR or beta-glucan test. The lower limit of detection in the PCR assay was 4-5 CFU/ml of C. albicans in blood. No fungal organism was amplified from the serum of 20 healthy individuals. The results of the PCR assay and beta-glucan test showed a significant correlation in this study, but the PCR assay proved to be more sensitive than the beta-glucan test (p < 0.05), and to be more useful for the clinical diagnosis and monitoring of deep Candidiasis.

Candida↗
PubMed1996

Multiprimer PCR system for differential identification of mycobacteria in clinical samples.

A novel multiprimer PCR method with the potential to identify mycobacteria in clinical samples is presented. The assay relies on the simultaneous amplification of three bacterial DNA genomic fragments by using different sets of oligonucleotide primers. The first set of primers amplifies a 506-bp fragment from the gene for the 32-kDa antigen of Mycobacterium tuberculosis, which is present in most of the species belonging to the genus Mycobacterium. The second set of primers amplifies a 984-bp fragment from the IS6110 insertion sequence of the bacteria belonging to the M. tuberculosis complex. The third set of primers, derived from an M. tuberculosis species-specific sequence named MTP40, amplifies a 396-bp genomic fragment. Thus, while the multiprimer system would render three amplification fragments from the M. tuberculosis genome and two fragments from the Mycobacterium bovis genome, a unique amplification fragment would be obtained from nontuberculous mycobacteria. The results obtained, using reference mycobacterial strains and typed clinical isolates, show that the multiprimer PCR method may be a rapid, sensitive, and specific tool for the differential identification of various mycobacterial strains in a single-step assay.

Base Sequence↗
PubMedDate not supplied

Headache and sleep: examination of sleep patterns and complaints in a large clinical sample of migraineurs.

OBJECTIVES: This study characterized sleep parameters and complaints in a large clinical sample of migraineurs and examined sleep complaints in relation to headache frequency and severity. BACKGROUND: The relationship between headache and sleep has been documented at least anecdotally in medical literature for well over a century and clinical texts allude to the importance of sleep as a headache precipitant. A small number of empirical studies have emerged, but the precise nature and magnitude of the headache/sleep association and underlying mechanisms remain poorly understood. METHODS: In this investigation, 1283 migraineurs were drawn from 1480 consecutive headache sufferers presenting for evaluation to a tertiary headache clinic. Patients underwent a physical examination and structured interview assessing a variety of sleep, headache, and demographic variables. Migraine was diagnosed according the IHS criteria (1.1 to 1.6 diagnostic codes). Migraineurs were 84% female, with a mean age of 37.4 years. Groups were formed based on patient's average nocturnal sleep patterns, including short, normal, and long sleep groups, and were compared on headache variables. RESULTS: Sleep complaints were common and associated with headache in a sizeable proportion of patients. Over half of migraineurs reported difficulty initiating and maintaining sleep at least occasionally. Many in this sample reported chronically shortened sleep patterns similar to that observed in persons with insomnia, with 38% of patients sleeping on average 6 hours per night. Migraines were triggered by sleep disturbance in 50% of patients. "Awakening headaches" or headaches awakening them from sleep were reported by 71% of patients. Interestingly, sleep was also a common palliative agent for headache; 85% of migraineurs indicated that they chose to sleep or rest because of headache and 75% were forced to sleep or rest because of headache. Patients with chronic migraine reported shorter nightly sleep times than those with episodic migraine, and were more likely to exhibit trouble falling asleep, staying asleep, sleep triggering headache, and choosing to sleep because of headache. Short sleepers (ie, average sleep period 6 hours) exhibited significantly more frequent and more severe headaches than individuals who slept longer and were more likely to exhibit morning headaches on awakening. CONCLUSIONS: These data support earlier research and anecdotal observations of a substantial sleep/migraine relationship, and implicate sleep disturbance in specific headache patterns and severity. The short sleep group, who routinely slept 6 hours per night, exhibited the more severe headache patterns and more sleep-related headache. Sleep complaints occurred with greater frequency among chronic than episodic migraineurs. Future research may identify possible mediating factors such as primary sleep and mood disorders. Prospective studies are needed to determine if normalizing sleep times in the short sleeps would impact headache threshold.

Adolescent↗
PubMed1986

Expectations for reinforcement from alcohol use in a clinical sample.

To examine differences in expectations for reinforcement from alcohol use between a clinical sample of adult alcoholics and general medical controls, subjects were administered the Alcohol Expectancy Questionnaire and were interviewed in a structured format assessing levels of alcohol intake. Alcoholic inpatients were found to expect more global positive changes, social assertiveness, social and physical pleasure and tension reduction from alcohol than did general medical controls. The findings were highly consistent with the results of other recent research examining the relation of alcohol expectancies to drinking patterns in alcoholics.

Adaptation, Psychological↗
PubMed2003

Characterization of blasts in clinical samples containing few blasts.

Characterization (eg, phenotyping) of blasts present at low percentages in clinical samples is often required for decisions regarding the approach to therapy. However, the available methods for cell characterization do not yield reliable data when the target cells are scant, and the existing methods for blast enrichment, such as cell sorting by flow cytometry (FCM), cannot enrich blasts of unknown immunophenotype. Blastretriever is a newly developed density centrifugation reagent for retrieving blasts. We examined the utility of Blastretriever in clinical practice. When normal bone marrow (BM) cells were separated with this reagent, myeloblasts and B-cell precursors were enriched and detected as clusters on the FCM cytogram. Compared with a conventional reagent for mononuclear cell preparation, the Blastretriever reagent markedly enriched leukemic myeloblasts, leukemic lymphoblasts, and blastoid lymphoma cells from 36 test samples (BM cells and peripheral blood). We then applied the Blastretriever reagent to samples from 11 consecutive patients who had been referred to us because they exhibited low percentages of blasts (1 patient had only 0.2% blasts). Characterization was needed but impossible with conventional analyses. Blast enrichment was achieved for all 11 samples, allowing reliable blast characterization by FCM, fluorescence in situ hybridization, and/or G-banding determinations. The revealed blast characteristics were valuable for choosing appropriate therapy for the patients.

Adult↗
PubMed1997

Psychometric properties of the Depression Anxiety Stress Scales (DASS) in clinical samples.

The psychometric properties of the Depression Anxiety Stress Scales (DASS) were evaluated in two studies using large clinical samples (N = 437 and N = 241). In Study 1, the three scales comprising the DASS were shown to have excellent internal consistency and temporal stability. An exploratory factor analysis (principal components extraction with varimax rotation) yielded a solution that was highly consistent with the factor structure previously found in nonclinical samples. Between-groups comparisons indicated that the DASS distinguished various anxiety and mood disorder groups in the predicted direction. In Study 2, the conceptual and empirical latent structure of the DASS was upheld by findings from confirmatory factor analysis. Correlations between the DASS and other questionnaire and clinical rating measures of anxiety, depression, and negative affect demonstrated the convergent and discriminant validity of the scales. In addition to supporting the psychometric properties of the DASS in clinical anxiety and mood disorders samples, the results are discussed in the context of current conceptualizations of the distinctive and overlapping features of anxiety and depression.

Adolescent↗
PubMed2001

Simple form of clinical sample preservation and Leishmania DNA extraction from human lesions for diagnosis of American cutaneous leishmaniasis via polymerase chain reaction.

The diagnosis value of polymerase chain reaction (PCR) was assessed in patients from an area endemic for American cutaneous leishmaniasis (ACL) in Brazil. Different forms of clinical sample preservation and DNA extraction for PCR were tested. The 4 preservation forms of the skin biopsies from patients suspected to have ACL were as follows: imprinted on filter paper (FP); imprinted on nitrocellulose paper (NP); frozen at -20 degrees C (FB); or immersed in 70% ethanol (EB). The DNA was extracted by elution from FP and NP and by enzyme digestion from FB and EB. Clinical examinations and parasitological or immunological tests confirmed the cases of ACL. Of 164 patients suspected to have ACL, 133 patients (81.1%) were confirmed. The PCR was positive in 76.8% of the suspected cases and in 90.2% of the confirmed cases. Polymerase chain reaction alone showed nearly the same positivity of the parasitological and immunological tests together; positivity varied 73.3-82.2%, according to the means by which the samples were preserved or the way the DNA was extracted. This variation was not significantly different (P > 0.05). Therefore, we recommend that clinical samples from patients with ACL should be collected and preserved on FP and the DNA further extracted by elution. The samples can be mailed to reference laboratories for the definitive diagnosis of ACL. This alternative is simple, inexpensive, and adequate for field conditions in developing countries.

Animals↗
PubMed2004

[Investigation of herpes simplex virus DNA by real-time polymerase chain reaction in the clinical samples].

The aim of this study was to investigate the presence of Herpes simplex virus (HSV) type 1 and type 2 DNA from the clinical samples sent to our routine laboratory, by real-time polymerase chain reaction (PCR). A total of 328 samples collected from 306 female and 7 male patients who were admitted to different outpatient clinics were included in the study. The samples included 235 cervical swab samples (of which 150 were from pregnant women), 77 amniotic fluid, 8 blood, 6 cerebrospinal fluid (CSF), one pericardial fluid and one cervical biopsy. DNA extraction were performed with High Pure Viral Nucleic Acid Kit (Roche, Germany) and amplified in Light Cycler (Roche, Germany) with a commercial amplification mix (Metis Biotechnology, Ankara). HSV-DNA positivity were found in 2.1% of the cervical samples (three of 150 pregnant and two of 85 non-pregnant women), two of the blood samples and one of the CSF sample, while there were no positive result for the other clinical specimens. It can be concluded that, real-time PCR would be preferred in conditions requiring rapid diagnosis such as HSV infections of central nervous system and HSV suspected infections of immunosupressed patients, as a rapid and practical method.

Amniotic Fluid↗
PubMed2002

Gender differences in depression risk and coping factors in a clinical sample.

OBJECTIVE: To examine gender differences in depression risk and coping factors in a clinical sample of patients with a diagnosis of DSM-IV major depression. METHOD: Patients were assessed for substance use and abuse, family history of psychiatric disorder, interpersonal depressogenic factors and lifetime history of anxiety disorders. Trait anxiety, coping styles when depressed, parental bonding, marital features and personality style were also measured. Patients were reassessed at 12-month follow-up. RESULTS: There were few gender differences in experience of depression (either in duration, type or severity prior to treatment) in a group with established episodes of major depression but women reported more emotional arousability when depressed. Women reported higher rates of dysfunctional parenting and childhood sexual abuse, and rated their partners as less caring and as more likely to be a depressogenic stressor. Men were more likely to have a generalized anxiety disorder at assessment, to use recreational drugs prior to presentation. Men were rated as having a more rigid personality style and 'Cluster A' personality traits both at assessment and follow-up. CONCLUSION: There were few gender differences in severity or course of established episodes of major depression. Gender differences were related to levels of arousal, anxiety disorders, and repertoires for dealing with depression, rather than depressive symptoms per se.

Adaptation, Psychological↗
PubMed1991

Rapid diagnosis of viral respiratory infections. Comparison between immunofluorescence on clinical samples and immunofluorescence on centrifuged cell cultures.

We compared the technique of immunofluorescence on clinical samples (IFCS) and immunofluorescence on centrifuged cell cultures (IFCC) for the detection of respiratory viruses, using monoclonal antibodies. Four hundred fourty five nasopharyngeal aspirates were tested prospectively for RSV, influenza- (A and B), parainfluenza viruses (1, 2, 3) and adenovirus. IFCS and IFCC detected 94% and 51% of RSV respectively, 78.5% and 82% of influenza viruses, 8.3% and 100% of parainfluenzaviruses and 0 and 100% of adenoviruses. We conclude that for routine rapid virus diagnosis the IFCS procedure is acceptable for RSV, that for influenza virus the IFCC procedure can be omitted for samples positive by IFCS, and that for the detection of parainfluenza and adenoviruses the IFCS procedure is too insensitive.

Cells, Cultured↗