[Unusual diphtheromorphic gram-positive bacilli in clinical samples: current taxonomic status and clinical significance].
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Corynebacterium diphtheriae↗
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Simultaneous identification of Mycobacterium genus and Mycobacterium tuberculosis complex in clinical samples by 5'-exonuclease fluorogenic PCR.
Early diagnosis of tuberculosis and screening of other mycobacteria is required for the appropriate management of patients. We have therefore developed a 5'-exonuclease fluorogenic PCR assay in a single-tube balanced heminested format that simultaneously detects Mycobacterium tuberculosis complex (MTC) and members of the Mycobacterium genus (MYC) using the 16S ribosomal DNA target directly on clinical samples. One hundred twenty-seven clinical samples (65 smear negative and 62 smear positive) with a positive culture result from 127 patients were tested, including 40 negative control specimens. The finding of both a positive MTC and probe value and a positive MYC probe value confirmed the presence of MTC or mycobacteria with a 100% positive predictive value. However, a negative value for MTC or MYC did not discount the presence of mycobacteria in the specimen. Interestingly, the addition of the MYC probe allowed the diagnosis of an additional 7% of patients with tuberculosis and rapid screening of nontuberculous mycobacteria (NTM). Thus, over 75% of the patients were diagnosed with mycobacterial disease by PCR. The sensitivity was much higher on smear-positive samples (90.3%) than smear-negative samples (49.2%) and was slightly higher for MTC than NTM samples. With regard to the origin of the sample, MTC pulmonary samples gave better results than others. In conclusion, we believe this test may be useful for the rapid detection of mycobacteria in clinical samples and may be a valuable tool when used together with conventional methods and the clinical data available.
DNA, Bacterial↗
A confirmatory factor analysis of the WMS-III in a clinical sample with crossvalidation in the standardization sample.
A maximum likelihood confirmatory factor analysis (CFA) of the Wechsler Memory Scale-III (WMS-III) was performed by applying LISREL 8 to a general clinical sample (n=281). Analyses were designed to determine which of seven hypothesized oblique factor solutions could best explain memory as measured by the WMS-III. Competing latent variable models were identified in previous studies. Results in the clinical sample were crossvalidated by testing all models in the WMS-III standardization samples (combined n=1,250). Findings in both the clinical and standardization samples supported a four-factor model containing auditory memory, visual memory, working memory, and learning factors. Our analysis differed from that presented in the WMS-III manual and by other authors. We tested our models in a clinical sample and included selected word list subtests in order to test the viability of a learning dimension. Consistent with prior research, we were also unable to empirically support the viability of the immediate and delayed memory indices, despite allowing the error terms between the immediate and delayed memory subtests to correlate.
Adult↗
The relationship between agoraphobia symptoms and panic disorder in a non-clinical sample of adolescents.
BACKGROUND: The purpose of this study was to evaluate the clinical correlates of agoraphobic fear and avoidance and panic disorder in a non-clinical sample of adolescents. METHOD: In a sample of 2365 high school students, combined data from a questionnaire and a structured clinical interview were used to classify subjects with agoraphobic fear and avoidance. Panic symptoms, major depression, childhood separation anxiety disorder, anxiety sensitivity and negative affectivity were also assessed. RESULTS: Fifteen subjects met study criteria for agoraphobic fear and avoidance in the past year. Only three (20%) of those with agoraphobia symptoms reported histories of panic attacks and there was no overlap between those with agoraphobic fear and avoidance and the 12 subjects who met DSM-III-R criteria for panic disorder. However, subjects with agoraphobia symptoms and those with panic disorder reported similar levels of anxiety sensitivity and negative affectivity. Childhood separation anxiety disorder was more common among those with agoraphobic fear and avoidance compared to those without. CONCLUSION: Agoraphobic avoidance is rare in non-clinical samples of adolescents and usually not associated with panic attacks. However, adolescents with agoraphobia symptoms and those with panic disorder have similar clinical correlates consistent with a panic/agoraphobia spectrum model.
Adolescent↗
[Detection of Mycobacterium tuberculosis in clinical samples by PCR and DNA probe].
OBJECTIVE: To improve the sensitivity and specificity of polymerase chain reaction (PCR) for detecting M. tuberculosis in uncultural clinical samples. METHODS: PCR amplification products were identified with agarose gel electrophoresis (PCR-electrophoresis), then Southern blotted onto a membrane and hybridized with a digoxigenin-labeled M. tuberculosis DNA probe (PCR-probe). RESULTS: The sensitivity of the purefied DNA for PCR-electrophoresis was 1 picogram, that for PCR-probe was increased to 100 femtogram. In 24 species tested, only M. tuberculosis complex and M. xenopi DNA produced 245-bp amplification bands, but the band from M. bovis did not hybridize with 188-bp digoxigenin-labeled probe. 225 clinical samples were examined by smear, PCR-electrophoresis and PCR-probe, 51 nontuberculous clinical samples were all negative, the positive rates of 173 tuberculous samples were 16.2%, 37.6% and 50.3%, respectively. The smear and culture of 1 sputum sample from a patient infected by nontuberculous mycobacteria were all positive, but its PCR-electrophoresis and PCR-probe were all negative. Identification of species showed that it was a quickly growing mycobacterium. Otherwise, the figures of electrophoresis were classified as four types, and influential factors were also discussed. CONCLUSIONS: The results showed that PCR was a valuable detective tool for early diagnosis and differential diagnosis of tuberculosis, and PCR-probe could increase positive rate of the detection and avoid misjudgements of results.
Adolescent↗
PCR assay based on DNA coding for 16S rRNA for detection and identification of mycobacteria in clinical samples.
A PCR and a reverse cross blot hybridization assay were developed for the detection and identification of mycobacteria in clinical samples. The PCR amplifies a part of the DNA coding for 16S rRNA with a set of primers that is specific for the genus Mycobacterium and that flanks species-specific sequences within the genes coding for 16S rRNA. The PCR product is analyzed in a reverse cross blot hybridization assay with probes specific for M. tuberculosis complex (pTub1), M. avium (pAvi3), M. intracellulare (pInt5 and pInt7), M. kansasii complex-M. scrofulaceum complex (pKan1), M. xenopi (pXen1), M. fortuitum (pFor1), M. smegmatis (pSme1), and Mycobacterium spp. (pMyc5a). The PCR assay can detect 10 fg of DNA, the equivalent of two mycobacteria. The specificities of the probes were tested with 108 mycobacterial strains (33 species) and 31 nonmycobacterial strains (of 17 genera). The probes pAvi3, pInt5, pInt7, pKan1, pXen1, and pMyc5a were specific. With probes pTub1, pFor1, and pSme1, slight cross hybridization occurred. However, the mycobacterial strains from which the cross-hybridizing PCR products were derived belonged to nonpathogenic or nonopportunistic species which do not occur in clinical samples. The test was used on 31 different clinical specimens obtained from patients suspected of having mycobacterial disease, including a patient with a double mycobacterial infection. The samples included sputum, bronchoalveolar lavage, tissue biopsy samples, cerebrospinal fluid, pus, peritoneal fluid, pleural fluid, and blood. The results of the PCR assay agreed with those of conventional identification methods or with clinical data, showing that the test can be used for the direct and rapid detection and identification of mycobacteria in clinical samples.
Bacteriological Techniques↗
Comparative performance of high-density oligonucleotide sequencing and dideoxynucleotide sequencing of HIV type 1 pol from clinical samples.
The performance of the high-density oligonucleotide array methodology (GeneChip) in detecting drug resistance mutations in HIV-1 pol was compared with that of automated dideoxynucleotide sequencing (ABI) of clinical samples, viral stocks, and plasmid-derived NL4-3 clones. Sequences from 29 clinical samples (plasma RNA, n = 17; lymph node RNA, n = 5; lymph node DNA, n = 7) from 12 patients, from 6 viral stock RNA samples, and from 13 NL4-3 clones were generated by both methods. Editing was done independently by a different investigator for each method before comparing the sequences. In addition, NL4-3 wild type (WT) and mutants were mixed in varying concentrations and sequenced by both methods. Overall, a concordance of 99.1% was found for a total of 30,865 bases compared. The comparison of clinical samples (plasma RNA and lymph node RNA and DNA) showed a slightly lower match of base calls, 98.8% for 19,831 nucleotides compared (protease region, 99.5%, n = 8272; RT region, 98.3%, n = 11,316), than for viral stocks and NL4-3 clones (protease region, 99.8%; RT region, 99.5%). Artificial mixing experiments showed a bias toward calling wild-type bases by GeneChip. Discordant base calls are most likely due to differential detection of mixtures. The concordance between GeneChip and ABI was high and appeared dependent on the nature of the templates (directly amplified versus cloned) and the complexity of mixes.
Anti-HIV Agents↗
Factor structure of MicroCog in a clinical sample.
Two factor solutions adequately describe the MicroCog performance data of a clinical sample of substance abusers. Information Processing Speed and Information Processing Accuracy factors seem to explain the Short Form and Standard Form scores of a clinical sample as they do for the normative sample. Clinicians need to be aware that the factor scores do not allow for unambiguous interpretation, but may be more meaningful than independent domain scores.
Cognition Disorders↗
Evaluation of different continuous cell lines in the isolation of mumps virus by the shell vial method from clinical samples.
AIMS: To compare prospectively the efficacy of the Vero, LLC-MK2, MDCK, Hep-2, and MRC-5 cell lines in the isolation of the mumps virus from clinical samples by means of the shell vial method. METHODS: During an epidemic outbreak of parotiditis 48 clinical samples (saliva swabs and CSF) were studied. Two vials of the Vero, LLC-MK2, MDCK, MRC-5, and Hep-2 cell lines were inoculated with 0.2 ml of the samples by the shell vial assay. The vials were incubated at 36 degrees C for two and five days. The vials were then fixed with acetone at -20 degrees C for 10 minutes and stained by a monoclonal antibody against mumps virus by means of an indirect immunofluorescence assay. RESULTS: The mumps virus was isolated from 36 samples. The Vero and LLC-MK2 cell lines showed a 100% isolation capacity, MDCK showed 77.7%, MRC-5 showed 44.4%, and Hep-2 showed 22.2%. The Vero and LLC-MK2 lines were significantly different to the other cell lines (p < 0.001). The sensitivity for the Vero and LLC-MK2 lines at two and five days of incubation was identical (100%). The values obtained in the study of the quantitative isolation capacity (positive isolation with > 5 infectious foci) were 94.4% for Vero, 97.2% for LLC-MK2, 5.5% for MDCK, 5.5% for Hep-2, and 0% for MRC-5. CONCLUSIONS: The Vero and LLC-MK2 cell lines are equally efficient at two and five days incubation for the isolation of the mumps virus from clinical samples, and the use of the shell vial method considerably shortens the time of aetiological diagnosis with higher specificity.
Animals↗
The Stice model of overeating: tests in clinical and non-clinical samples.
The present study tested the dual pathway model of Stice [. A review of the evidence for a sociocultural model of bulimia nervosa and an exploration of the mechanisms of action. Clinical Psychology Review, 14, 633-661 and . A prospective test of the dual-pathway model of bulimic pathology: mediating effects of dieting and negative affect. Journal of Abnormal Psychology, 110, 124-135.] in a non-clinical sample of female adolescents and a clinical sample of female eating disorder patients. The model assumes that negative affect and restrained eating mediates the link between body dissatisfaction and overeating. We also tested an extended version of the model postulating that negative affect and overeating are not directly related, but indirectly through lack of interoceptive awareness and emotional eating. Structural equation modelling was used to test our models. First, in the two samples, body dissatisfaction and drive for thinness were associated with overeating/binge eating. In both clinical and adolescent sample, we found support for the negative affect pathway and not for the restraint pathway. Lack of interoceptive awareness and emotional eating appear to (partly) explain the association between negative affect and overeating. Emotional eating was much more strongly associated with overeating in the clinical than in the adolescent sample. In sum, we found substantial evidence for the negative affect pathway in the dual pathway model. The link between body dissatisfaction and overeating in this respect might be explained by the fact that negative affect, due to body dissatisfaction, is related to a lack of awareness of personal feelings and to eating when dealing with negative emotions, which on its turn is associated with overeating.
Adolescent↗
Optimizing dengue diagnosis by RT-PCR in IgM-positive samples: comparison of whole blood, buffy-coat and serum as clinical samples.
Dengue, a major public health problem in tropical and sub-tropical regions, is the most important arboviral disease of humans. Early diagnosis is very important on follow-up of infected patients, especially those at risk of the severe manifestations of this disease. Aiming at the improvement of the molecular diagnosis of these infections and due to the lack of studies that indicated the best sample for dengue virus detection by RT-PCR, viral detection by RT-PCR in blood, serum and buffy-coat of 75 IgM-positive serum samples for dengue was evaluated. Out of the 75 samples, 17 were positive for dengue using RT-PCR and from these samples, three were positive in the blood, 14 positive in the serum and eight positive in the buffy-coat. These results indicate that serum is the best clinical sample for RT-PCR amplification of dengue genomes.
Animals↗
Rapid and high sensitivity test for direct detection of bovine herpesvirus-1 genome in clinical samples.
A procedure for direct detection of the BHV-1 genome in clinical samples, including semen, was developed. The method is based on the PCR amplification of a highly conserved DNA fragment within the glycoprotein gI sequence of the virus (323 bp between nt 1491 to nt 1814). The method is rapid and highly specific for all 27 subtypes assayed, which are included in the clinical and genetically different groups of BHV-1. The viral origin of the PCR product was assessed by Southern hybridization, with an internal probe. The method for DNA isolation from clinical samples included a fast extraction procedure with Chelex 100 resin allowing the loading of larger amounts of DNA in the PCR and in turn increasing the sensitivity of the method of detection. The level of sensitivity achieved by PCR was in the range of 1 TCID(50). This PCR assay may be an useful tool for BHV-1 monitoring in semen banks at low cost.
Animals↗
A measurement model of the Medical Outcomes Study 36-Item Short-Form Health Survey in a clinical sample of disadvantaged, older, black, and white men and women.
The authors assess the factorial validity of the Medical Outcomes Study 36-Item Short-Form Health Survey (SF-36) for use in a clinical sample of disadvantaged, older adults with significant comorbidities. Confirmatory factor analysis was performed using LISREL VIII on data obtained from baseline face-to-face interviews with a clinical sample of 1,051 patients who were at risk for acute deterioration of their clinical condition due either to their age alone (75 years or older), or to their age (50 to 74 years old) and major comorbid conditions. An acceptable eight-factor measurement model reflecting the original specification (ie, subscales) of the SF-36 was obtained (chi-square to degrees of freedom ratio = 2.14; root mean squared residual = .055; adjusted goodness of fit index = .90). That model, however, required relaxing the assumptions associated with seven correlated error terms. Moreover, an alternative nine-factor model that allowed the ¿getting sick¿ and ¿getting worse¿ items to form their own factor, labeled ¿health optimism,¿ fit the data significantly better (8 degrees of freedom chi-square improvement = 61; P< 0.0001). Although continued use of the SF-36 in older, disadvantaged, clinical samples is appropriate, further assessment of the underlying measurement model in other samples using confirmatory factor analytic techniques is needed to resolve the issue of correlated error structures and the existence of the health optimism factor.
Black or African American↗
Rapid extraction of fungal DNA from clinical samples for PCR amplification.
A hexadecyltrimethylammonium bromide (CTAB) method for isolating fungal DNA from clinical samples, suitable for PCR amplification is described. Yeast and filamentous fungi DNA from clinical samples was amplified with primers complementary to the genes coding for rRNA, amplifying a 105 bp fragment and internal transcribed spacer primers amplifying fragments between 242 and 622 bp. The level of sensitivity was 10 +/- 5 yeast and 28 Aspergillus fumigatus CFU ml-1 of biological fluid.
Blood↗
Sequence variation in the ftsZ gene of Bartonella henselae isolates and clinical samples.
In a search for methods for subtyping of Bartonella henselae in clinical samples, we amplified and sequenced a 701-bp region in the 3' end of the ftsZ gene in 15 B. henselae isolates derived from cats and humans in the United States and Europe. The ftsZ sequence variants that were discovered were designated variants Bh ftsZ 1, 2, and 3 and were compared with 16S rRNA genotypes I and II of the same isolates. There was no ftsZ gene variation in the strains of 16S rRNA type I, all of which were Bh ftsZ 1. The type II strains constituted two groups, with nucleotide sequence variation in the ftsZ gene resulting in amino acid substitutions at three positions, one of which was shared by the two groups. One 16S rRNA type II isolate had an ftsZ gene sequence identical to those of the type I strains. Variants Bh ftsZ 1 and 2 were detected in tissue specimens from seven Swedish patients with diagnoses such as chronic multifocal osteomyelitis, cardiomyopathy, and lymphadenopathy. Patients with similar clinical entities displayed either Bh ftsZ variant. The etiological role of B. henselae in these patients was supported by positive Bartonella antibody titers and/or amplification and sequencing of a part of the B. henselae gltA gene. B. henselae ftsZ gene sequence variation may be useful in providing knowledge about the epidemiology of various B. henselae strains in clinical samples, especially when isolation attempts have failed. This report also describes manifestations of atypical Bartonella infections in Sweden.
Adolescent↗
Using capillary electrophoresis-selective tandem mass spectrometry to identify pathogens in clinical samples.
Analysis of microbial mixtures in complex systems, such as clinical samples, using mass spectrometry can be challenging because the specimens may contain mixtures of several pathogens or both pathogens and nonpathogens. We have successfully applied capillary electrophoresis-selective MS/MS of unique peptide marker ions to the identification of common pathogens in clinical diagnosis. We searched the CE-MS/MS spectra acquired from the proteolytic digests of pure bacterial cell extracts against protein databases. The identified peptides that matched a protein associated with a particular pathogen were selected as marker ions to identify that bacterium in clinical specimens. Thirty-four clinical specimens, obtained from pus, wound, sputum, and urine samples, were analyzed using both biochemical and selective MS/MS methods. The bacteria in these clinical samples were cultivated directly, without prior isolation of a pure colony, before performing the selective MS/MS analyses. The bacteria analyzed included both Gram-positive and -negative strains. The match with respect to the pathogens identified was good between the biochemical and the selective MS/MS methods; the matching rate was 91%. The rate was as high as 97% when not considering two specimens for which the bacteria were not grown successfully. Two of the specimens that we identified using the biochemical method as containing two bacterial species were confirmed also through selective tandem MS analysis.
Electrophoresis, Capillary↗
Detection of Mycobacterium tuberculosis in clinical samples using insertion sequences IS6110 and IS990.
To detect Mycobacterium tuberculosis in clinical samples, we used the M. tuberculosis-complex specific insertion sequence IS990 as the target in a simple DIG-PCR ELISA assay, as this element is present as a single copy in all strains of M. tuberculosis we have examined to date. The IS990 test was compared with a similar PCR that utilizes IS6110 as target. For detection of PCR product, digoxigenin-11-dUTP (DIG-dUTP) was incorporated into the product. After amplification, the PCR product was hybridized with biotinylated capture probe, which was complementary to the inner part of the amplicon. The hybrid was captured onto streptavidin-coated microtiter plate and DIG-labeled PCR product was detected using a peroxidase-conjugated antibody to DIG. We evaluated DIG-PCR ELISA for the detection of M. tuberculosis DNA in 265 respiratory and non-respiratory specimens taken from patients with known and suspected tuberculosis disease or from controls. The sensitivity and specificity of both IS990-based test and IS6110-based test was 96.5% and 95.3% respectively, comparable to the sensitivity and specificity of the IS6110-based test. The results demonstrate that the IS990 PCR ELISA test is a rapid and sensitive tool for the detection and identification of M. tuberculosis in clinical samples, and may have advantages to the more widely used IS6110-based tests, particularly in areas where IS6110-negative strains are found.
DNA Transposable Elements↗
Detection of adenovirus DNA in clinical samples by SYBR Green real-time polymerase chain reaction assay.
BACKGROUND: Adenoviruses are associated with a variety of diseases including upper respiratory tract infections, acute conjunctivitis, cystitis and gastroenteritis. Adenoviruses can also cause fatal disseminated infections in patients undergoing stem cell transplantation. Measurement of adenovirus load in clinical samples from localized adenovirus infections or disseminated adenovirus infections may provide important information for analyzing the pathogenesis of various adenovirus infections. The purpose of the present study was to develop and optimize a highly sensitive real-time polymerase chain reaction (PCR) assay to detect a wide range of adenoviruses and to detect adenovirus DNA in clinical samples from immunocompetent children. METHODS: Clinical samples of throat swabs and blood were collected from 111 patients suspected of having adenovirus infection. The copy number of adenovirus DNA was measured by real-time PCR assay. RESULTS: SYBR Green real-time PCR assay is able to detect 10-10(6) copies of standard adenovirus DNA per run. Adenovirus DNA was detected in all culture-positive samples serotyped as 1, 2, 3, 4, 5, 6, 8 and 11. Viral loads on throat swabs from immunocompetent children with adenovirus infection ranged from 10(5) to 10(11) copies/mL. Adenovirus DNA was detected in 60% of blood samples and copy number ranged from 10(3) to 10(5) copies/mL. CONCLUSION: SYBR Green real-time PCR is a useful quantitative tool for analysis of adenovirus DNA. The present results for immunocompetent children with adenovirus infections provided basic data for comparison with data obtained from immunocompromised patients.
Adenovirus Infections, Human↗