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At least 127 records · Page 7Linked to original sources
PubMed1995

Routine application of the polymerase chain reaction for detection of Mycobacterium tuberculosis in clinical samples.

AIM: To investigate the use of the polymerase chain reaction (PCR) in the routine laboratory for the detection of Mycobacterium tuberculosis in clinical samples. METHODS: Samples were divided and processed separately for the detection of M tuberculosis by microscopy, culture and PCR. After DNA extraction, PCR was performed with primers specific for the insertion element IS6110 and the product was analysed by agarose gel electrophoresis, Southern blotting or dot blotting and hybridisation with a digoxigenin labelled internal probe. Each sample was tested for inhibitors of Taq polymerase with the aid of an internal control. Multiple negative and positive controls were used to monitor each step of the procedure. RESULTS: The data from two laboratories, using the same operating procedures, were combined. Of 1957 specimens, 79 (4%) were culture and PCR positive, while 1839 (93.9%) were negative in both tests. Thirty specimens (1.5%) were PCR positive only and nine (0.5%) were culture positive but PCR negative. CONCLUSION: Using culture and clinical history as the gold standard, sensitivity and specificity for PCR were 92.1% and 99.8%, respectively. With elaborate precautions, PCR is a suitable and reliable method for the detection of M tuberculosis in clinical samples in a routine microbiology laboratory.

Bacteriological Techniques↗
PubMed2004

Evaluation of real time polymerase chain reaction assays for confirmation of Neisseria gonorrhoeae in clinical samples tested positive in the Roche Cobas Amplicor assay.

OBJECTIVE: Development of a rapid, sensitive, and accurate assay for confirmation of Neisseria gonorrhoeae in clinical samples. METHOD: Two real time polymerase chain reaction (PCR) assays, developed on the LightCycler for amplification of the N gonorrhoeae cppB gene, were utilised for confirmation of this bacterial pathogen in samples positive by the Roche Cobas Amplicor assay. Performance characteristics of the two assays were compared with other commercial nucleic acid amplification assays, including the Abbott LCx and Roche 16S rRNA tests. RESULTS: All related Neisseria as well as other bacterial species tested negative by both cppB gene based assays, whereas 120 N gonorrhoeae clinical isolates from various geographical regions gave in positive results. Both assays had a sensitivity of one copy per reaction. 122 clinical samples positive and another 50 samples negative for N gonorrhoeae by Roche Cobas Amplicor were selected from a specimen pool of more than 3000 women tested previously. Overall, 73 of 122 (59.8%) samples were confirmed as positive. The two real time assays had sensitivities of 99% and 100% and specificities of 98% and 100%, respectively. The 16S and LCx assays produced similar results to the real time assays, indicating a similar sensitivity to and specificity of both real time assays. CONCLUSION: The data from this study highlight the need to confirm N gonorrhoeae positive Cobas Amplicor PCR results as an important part of the testing algorithm of all diagnostic laboratories utilising this assay.

DNA, Bacterial↗
PubMed2001

Long-term stability of the Child Behavior Checklist in a clinical sample of youth with attention deficit hyperactivity disorder.

Evaluated the long-term stability of the Child Behavior Checklist (CBCL) in a longitudinal clinical sample of youth with attention deficit hyperactivity disorder (ADHD), testing the hypothesis that the CBCL scales will show stability over time. Participants were 105 Caucasian, non-Hispanic boys with ADHD between the ages of 6 and 17 assessed at baseline and at a 4-year follow-up. Stability of CBCL scales were computed for dimensional (intraclass correlation coefficients [ICCs], Pearson correlations) and dichotomized scale scores (kappa coefficients and odds ratios [ORs]). Evidence was found for stability of the categorical and dimensional types of scores, as demonstrated by statistically significant stability of the Pearson correlation coefficients, kappas, and ORs. The robust findings obtained from ICCs and kappa coefficients document substantial stability for CBCL scales over time within individuals with ADHD. These results support the informativeness of the CBCL as a useful measure of longitudinal course in clinical samples of youth with ADHD.

Adolescent↗
PubMed2004

Prevalence of Aspergillus species in clinical samples isolated in an Indian tertiary care hospital.

UNLABELLED: CONTEXT (BACKGROUND): In recent times, it has become important to determine the prevalence of different Aspergillus species in clinical samples in view of difference in antifungal susceptibility noted in some species. AIMS: To determine the species prevalence of Aspergillus isolates in various clinical samples received in the Mycology Laboratory at our institute. METHOD: Over a period of 4-years, a total of 18,731 samples were processed, and species identification carried out by standard microbiological methods. RESULTS: Four hundred and fifty six samples (2.43%) were culture positive for Aspergillus species. A.flavus (46.93%) was the most common isolate, followed by A.fumigatus (37.72%) and A.niger (15.35%). It was observed that A.fumigatus was the predominant species isolated from blood and respiratory specimens, A.flavus was predominantly isolated from nasal polyps whereas A.niger predominated in nail specimens. Culture positivity was highest in the age group 12-65 years and in males. Sixty-nine patients (15.13%) were admitted to the intensive care unit. CONCLUSIONS: The study highlights the diverse manifestations caused by Aspergillus species in human beings and also throws light on the different species prevalent locally. The knowledge would prove useful in selecting empirical antifungal therapy and formulating prophylactic and pre-emptive strategies.

Aspergillosis↗
PubMed1975

New technique for quantitative bacteriological sampling of wounds by velvet pads: clinical sampling trial.

A new surface sampling method, the velvet pad rinse technique, was compared with the velvet pad imprint method, in a randomized bacteriological sampling trial, using surgical wounds to evaluate quantitative results. The efficiency of bacterial recovery was increased 20-fold by the new technique, and the improvement was ascribed in particular to the introduction of a procedure of rinsing the velvet pad after sampling.

Bacteria↗
PubMed2006

Infectivity-enhanced adenoviruses deliver efficacy in clinical samples and orthotopic models of disseminated gastric cancer.

PURPOSE: Metastatic gastric cancer remains a common and devastating disease without curative treatment. Recent proof-of-concept clinical trials have validated gene therapy with adenoviruses as an effective and safe modality for the treatment of cancer. However, expression of the primary coxsackie-adenovirus receptor is variable in advanced cancers, and therefore, the use of heterologous receptors could be advantageous. EXPERIMENTAL DESIGN: Here, we used capsid-modified adenoviruses for increasing the transduction and subsequent antitumor efficacy. 5/3 chimeric viruses have a serotype 3 knob which allows binding to a receptor distinct from coxsackie-adenovirus receptor. The fiber of Ad5lucRGD is modified with an integrin-targeted motif. Polylysine motifs, pK7 and pK21, bind to heparan sulfates. Oncolytic adenoviruses replicate in and kill tumor cells selectively. Gastric cancer cell lines and fresh clinical samples from patients were infected with transductionally targeted viruses. Capsid-modified oncolytic adenoviruses were used in cell killing experiments. To test viral transduction and therapeutic efficacy in vivo, we developed orthotopic mouse models featuring i.p. disseminated human gastric cancer, which allowed the evaluation of biodistribution and antitumor efficacy in a system similar to humans. RESULTS: Capsid modifications benefited gene transfer efficiency and cell killing in gastric cancer cell lines and clinical samples in vitro and in vivo. Modified oncolytic adenoviruses significantly increased the survival of mice with orthotopic gastric cancer. CONCLUSIONS: These preclinical data set the stage for the clinical evaluation of safety and efficacy in patients with disease refractory to current modalities.

Adenoviridae↗
PubMed1993

Comparison of three primer sets for the detection of Mycobacterium tuberculosis in clinical samples by polymerase chain reaction.

A number of studies have underlined the interest of the polymerase chain reaction (PCR) in the detection of Mycobacterium tuberculosis in clinical samples. Among the different parameters to be carefully studied the choice of target gene and primers is essential. The amplification of nucleotidic sequences localised on three different target genes (groEL, IS6110, Pab) was examined in 196 clinical samples from patients with suspected tuberculosis or receiving antituberculous therapy. The results obtained after hybridization with non-radioactive labelled probes were compared with the culture data. None of the primer sets studied showed a satisfactory sensitivity (79% to 84%) suitable for it to be used alone. The false-negative specimens with the PCR tests usually corresponded to those that contained few mycobacteria. With the methods described in this study, the use of two or three primer sets located on different target genes allowed to improve the positivity rate compared to the culture and sensitivity of the test (90-98%), particularly for paucibacillary samples. On the other hand, the interpretation was easier when concordant results were obtained.

DNA, Bacterial↗
PubMed2005

Inducible clindamycin resistance in staphylococci isolated from clinical samples.

This study aimed to determine the levels of the macrolides-lincosamides-streptogramins B (MLS(B)) resistance phenotype of Staphylococcus aureus and coagulase-negative staphylococci (CNS) isolates from clinical samples. A total of 521 strains of staphylococci, comprising 230 S. aureus and 291 CNS isolates from various clinical samples, were identified by conventional methods. The double-disc test was applied by placing erythromycin and clindamycin discs on these isolates to investigate the inducible and constitutive MLS(B) resistance phenotypes and MS phenotype. Among the S. aureus strains, 24.3% showed the constitutive and 7.8% the inducible phenotype, while there was no MS phenotype. In the CNS strains, 40.2% showed the constitutive and 14.7% the inducible MLS(B) resistance phenotype, and 18.2% had the MS phenotype. In both S. aureus and CNS strains, the constitutive MLS(B) resistance rate was found to be higher than the rate of inducible resistance. By applying double-disc tests on a routine basis to detect inducible MLS(B) resistance, clindamycin can be effectively used on staphylococcal infections. Additionally, it can be used to survey the MLS(B) resistance of staphylococci strains from specific geographical regions or hospitals.

Anti-Bacterial Agents↗
PubMed1998

Comparison of rhabdomyosarcoma, buffalo green monkey kidney epithelial, A549 (human lung epithelial) cells and human embryonic lung fibroblasts for isolation of enteroviruses from clinical samples.

BACKGROUND: Traditionally, primary monkey kidney (PMK) epithelial cells have been one of the more widely used cell types for the isolation of enteroviruses from clinical samples. For the isolation of coxsackie A viruses, intraperitoneal inoculation of newborn mice has been used in some laboratories. OBJECTIVE: With the discontinued availability of PMK epithelial cells and the reported growth of coxsackie A viruses in rhabdomyosarcoma cells (RD), we compared the use of the latter cell line with our routinely used microwell cell cultures. STUDY DESIGN: Microwell cell cultures of buffalo green monkey epithelial cell line (BGM), human lung carcinoma epithelial cell line (A549) and human embryonic lung (HEL) fibroblasts were compared with RD cell line for the isolation of enteroviruses from clinical samples. RESULTS: A total of 39 enteroviruses was isolated from 3517 specimens. The HEL fibroblasts yielded 28 (72%) enteroviruses, followed by A549 (25 isolates, 64%), BGM (23 isolates, 59%) and RD cells (18 isolates, 46%). CONCLUSIONS: All isolates which grew in RD cells showed specific cytopathic effects in one or more of the other inoculated cell cultures. Quantitative determinations (TCID50) with five different enteroviruses showed that the HEL fibroblasts and RD cell line to be the most sensitive cell types, followed by BGM and A549 cell lines. However, integrity of the inoculated cell monolayers was best with HEL fibroblasts and A549 cells but morphology was not optimal with RD cells during the incubation period of 14 days.

Animals↗
PubMed2003

Evaluation of a rapid bacteriophage-based method for the detection of Mycobacterium tuberculosis in clinical samples.

Rapid, sensitive and low-cost methods are needed urgently for the detection of Mycobacterium tuberculosis in clinical samples, especially in developing countries. To this end, the clinical performance of FASTPlaqueTB(TM) (a bacteriophage-based method) has been studied in parallel with microscopy, standard microbiological culture and in-house IS6110-based PCR methods. A total of 64 samples, including 42 sputum samples and 22 urine samples, were tested in this study. The sensitivity, specificity and overall accuracy values for the FASTPlaqueTB assay relative to that of culture were respectively 76.5, 95 and 90 %. The corresponding values for the in-house IS6110-based PCR assay were 88, 91 and 90 % and, for Ziehl-Neelsen staining, were 59, 95 and 85 %. FASTPlaqueTB gave better clinical performance with urine samples than with sputum samples (sensitivity, specificity and overall accuracy were 100 % with urine samples and 64, 93 and 84 % with sputum samples). The 100 % sensitivity of FASTPlaqueTB was higher than that of the corresponding values for PCR (67 %) with urine samples. In conclusion, FASTPlaqueTB proved to be sensitive, cheap relative to the PCR and rapid. It is able to detect M. tuberculosis in clinical samples within 1 day, reducing the time to diagnosis in comparison with culture.

Bacteriuria↗
PubMed1992

Detection of Mycobacterium tuberculosis in clinical samples by using polymerase chain reaction and a nonradioactive detection system.

A test based on the polymerase chain reaction (PCR) was developed for the detection of the Mycobacterium tuberculosis complex in clinical samples. In this test, a 245-bp sequence of the insertion element IS986 was amplified and detected by agarose gel electrophoresis in the presence of ethidium bromide and by Southern blot and dot blot hybridization by using a 188-bp digoxigenin-labeled probe. We tested clinical specimens from 227 patients suspected of having tuberculosis. These included 102 cerebrospinal fluid, 48 sputum, 18 pleural fluid, 5 bronchoalveolar lavage, 18 blood, 7 pus, 8 bone marrow, and 6 urine samples and 15 tissue biopsy specimens. We also tested sputum samples from 75 patients with diseases other than tuberculosis. Sputum samples were first decontaminated, and all samples were treated with proteinase K-detergent solution to extract the DNA. Part of each sample was spiked with M. tuberculosis to provide a semiquantitative assay and to control for the loss of mycobacteria or interference with the PCR which may cause false-negative results. One femtogram of M. tuberculosis DNA could be detected. PCR was positive for all 32 culture-positive (for M. tuberculosis) and Ziehl-Neelsen staining (ZN)-positive samples, 10 of 12 culture-positive and ZN-negative samples, and all 4 culture-negative and ZN-positive samples. PCR detected M. tuberculosis complex bacteria in 35 of 178 culture- and ZN-negative samples. Clinical data supported the diagnosis of tuberculosis in the majority of the 35 patients from whom those samples were obtained.

Base Sequence↗
PubMed1996

Evaluation and comparison of PCR and hybridization methods for rapid detection of cytomegalovirus in clinical samples.

Rapid diagnosis of cytomegalovirus (CMV) infection may be obtained by molecular techniques, such as the polymerase chain reaction (PCR) and hybridization assays. The optimal technique to detect CMV in clinical samples was assessed. Two different PCR assays were used, targeting either the major immediate early 1 (MIE 1) or the HXLF 4 gene. The PCR products were detected by gel electrophoresis, dot blotting and an easy to use, rapid, solid phase hybridization assay, DNA enzyme immunoassay (DEIA). Standard tissue culture was also used. Cerebrospinal fluids (18), liver biopsies (9) from hepatic transplant recipients, amniotic fluids (7) from mothers with suspected peripartum infection, and samples (6) of miscellaneous origin (brain and fundus biopsy, pericardial and pleural fluid) were tested. Among the 40 samples, CMV was detected in 19 cases. Three were positive by both molecular techniques and tissue culture, 14 by molecular methods and 2 by culture. 16/19 or 9/19 CMV-positive samples were detected by PCR amplification of the HXLF 4 or MIE 1 gene, respectively and 14/16 HXLF 4-positive samples were detected using either dot-blot or DEIA, compared to 9/16 using gel electrophoresis. Thus, the most sensitive assays for the detection of CMV in clinical samples using the methods compared in the current study were PCR amplification of the HXLF 4 gene followed by dot-blot or DEIA hybridization.

Amniotic Fluid↗
PubMed2003

Comparison between indirect immunofluorescence assay and shell vial culture for detection of mumps virus from clinical samples.

We report a prospective comparison of the efficacies of an indirect immunofluorescence assay (IFA) and shell vial culture (SVC) of throat swab and urine samples from patients with mumps. Throat swab samples were used for the IFA; the urine samples and throat swabs were inoculated into vials of Vero cells. We studied 62 patients by using 62 throat swabs and 50 urine samples (50 patients with both samples). Sixty (96.7%) throat samples were positive in the SVC, and 61 (98.3%) were positive in the IFA. For the 50 patients from whom both samples were available, the IFA was positive in 50 (100%) cases, the urine sample was positive in 49 (98%) cases, and the throat swab was positive in 48 (96%) cases (P > 0.05). This comparison of throat swabs and urine samples has shown that the two clinical samples are similar in efficacy.

Fluorescent Antibody Technique, Indirect↗
PubMed2003

The 50 and 100-item short forms of the Paced Auditory Serial Addition Task (PASAT): demographically corrected norms and comparisons with the full PASAT in normal and clinical samples.

While the standard 200-item version of the Paced Auditory Serial Addition Task (PASAT) is a sensitive neuropsychological instrument, it can be quite aversive to some patients due to its length and progressively increasing difficulty. We present demographically-corrected norms for 50 and 100-item short-form versions in a sample of 560 neurologically normal adults. Age, education, and ethnicity (but not gender) were found to be significant predictors of performance. In a clinical sample of 786 HIV-infected adults, diagnostic accuracy of the 50, 100, and 200-item versions was essentially equivalent (using clinical ratings of a comprehensive neuropsychological battery as the gold standard, overall classification rates of the three PASAT versions were 71%, 74%, and 73%, respectively), with better specificity (89-92%) than sensitivity (46-53%). The 50-item version showed moderate ceiling effects, but the 100-item test did not. In a mixed clinical sample of 40 subjects, the 50-item version was administered more than twice as fast as the 200-item version, and was tolerated better (discomfort rating of 4.0 vs. 5.9 on a 10-point scale, p < .05). We conclude that in many cases the PASAT-50 and PASAT-100 provide equivalent diagnostic accuracy with a significant reduction in administration time and patient discomfort.

Adult↗
PubMed2005

Comparison of an internally controlled, large-volume LightCycler assay for detection of Mycobacterium tuberculosis in clinical samples with the COBAS AMPLICOR assay.

We present a sensitive and specific assay for reliable and flexible detection of members of the Mycobacterium tuberculosis complex (MTBC) in clinical samples. This real-time PCR assay, which uses the LightCycler 2.0 instrument and 100-mul glass capillaries, can provide a result within 1 h after DNA extraction. The primers amplify a 206-bp fragment of the MTBC 16S rRNA gene. The sensor hybridization probe targets a region highly specific to members of the MTBC. The assay also includes a novel type of internal control that monitors the function of the reaction components and can detect potential inhibitors. Template DNA was extracted by the same procedure used for the COBAS AMPLICOR M. tuberculosis assay, so the LightCycler assay could be directly compared to the COBAS AMPLICOR assay. The LightCycler assay was evaluated with 146 clinical samples of various types. Very good agreement (100% sensitivity, 98.6% specificity) could be shown between the LightCycler and COBAS AMPLICOR assays. Specificity was checked with a panel of nontuberculous mycobacteria, as well as a large panel of bacterial and fungal organisms.

DNA, Bacterial↗
PubMed2003

Further investigation of the obsessive-compulsive inventory: psychometric analysis in two non-clinical samples.

The obsessive-compulsive inventory [OCI; Psychol. Assessment 10 (1998) 206-214] is a self-report measure of obsessive-compulsive disorder (OCD) intended for use with both clinical and non-clinical samples (). Two reports support its convergence with established measures of OCD and its assessment of specific symptom subtypes (e.g., checking and washing). The current studies investigate the OCI factor structure in two non-clinical samples. Based on correlational analyses and complementary factor analyses, Study 1 results indicate that five subscales, rather than the seven rationally derived ones, best capture the structure of symptoms measured by the OCI. These findings were replicated in Study 2. Based on these analyses, we offer suggestions for revising the original subscales into these five empirically derived subscales that closely resemble structures proposed in the current OCD literature.

Adult↗
PubMed2002

New parachlamydial 16S rDNA phylotypes detected in human clinical samples.

Chlamydiales are important intracellular bacterial pathogens, causing a wide variety of diseases in vertebrates, including humans. Besides the well-known species in the family Chlamydiaceae, new chlamydial organisms have recently been discovered, forming three new families: Parachlamydiaceae, Simkaniaceae and Waddliaceae. Parachlamydia acanthamoebae and Simkania negevensis are currently investigated as emerging human respiratory pathogens. Additional chlamydial lineages have been discovered by 16S rDNA-based molecular studies, and their implication in human infections is poorly known. By using a pan-chlamydia 16S rDNA PCR, we have searched for the presence of chlamydiae in 228 clinical samples that all previously had been shown to be PCR-negative for Chlamydophila pneumoniae: 170 respiratory samples, 45 atheromatic plaques and 13 peripheral blood mononuclear cell samples. Nine respiratory samples tested positive. Sequence analysis has allowed us to assign four sequences to Chlamydophila psittaci, three sequences to Chlamydophila felis, and two sequences to two novel phylotypes belonging to the Parachlamydiaceae. These latter sequences showed similarity values of more than 93% with each other and with the P. acanthamoebae sequence, thus belonging to novel, unrecognized species. In conclusion, this report showed that a variety of non-C. pneumoniae chlamydial respiratory infection is present in humans, and that new parachlamydiae distinct from P. acanthamoebae may be detected in human clinical samples. Future studies will be of interest in order to estimate the diversity of these novel chlamydiae in both clinical and environmental samples, as well as their possible clinical implication in human and animal infections.

Adolescent↗
PubMed1993

Rapid detection of vesicular stomatitis virus New Jersey serotype in clinical samples by using polymerase chain reaction.

Vesicular stomatitis virus of the New Jersey serotype (VSV-NJ) causes vesicular disease in cattle, pigs, and horses throughout the Americas. Vesicular disease is clinically indistinguishable from foot-and-mouth disease (FMD). Therefore, outbreaks of vesicular disease in FMD-free areas must be rapidly diagnosed by laboratory methods and affected farms must be quarantined until laboratory results confirm the absence of FMD. Diagnosis is currently performed in high-containment (biosafety level 3) laboratories by using complement fixation and virus isolation in tissue culture. We describe here an alternative method for the detection of VSV-NJ RNA in clinical samples. This method includes a rapid acid guanidine-phenol RNA extraction procedure coupled with a one-tube polymerase chain reaction (PCR) using reverse transcriptase. By using this test, we were able to detect the largest number of positive samples (53 of 58), followed by complement (48 of 58) and isolation in tissue culture (43 of 58). The primers chosen for this assay amplify a 642-nucleotide region of the phosphoprotein gene of VSV-NJ but not of VSV-IN. Sequencing of the PCR product enables genetic typing of virus isolates and epidemiological studies. Since no infectious materials are necessary to perform this test and any infectious virus in clinical samples is destroyed by acid guanidine-phenol treatment, diagnosis can be safely performed in regular diagnostic laboratories.

Animals↗