Impact of patient characteristics on performance of nucleic acid amplification tests and DNA probe for detection of Chlamydia trachomatis in women with genital infections.
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The performance of nucleic acid amplified tests (NAAT) for Chlamydia trachomatis at the cervix and in urine was examined in 3,551 women, and the impacts of clinical findings (age, endocervical and urethral inflammation, menses, and gonococcal coinfection) were assessed. Ligase chain reaction (LCR) and first-generation uniplex PCR were studied relative to an unamplified DNA probe (PACE2) and to an expanded, independent diagnostic reference standard. Relative to the expanded standard, cervical or urine LCR was generally the most sensitive test in most subgroups. Increased detection by NAAT of cervical C. trachomatis over PACE2 was highest among women without mucopurulent endocervical discharge versus those with (relative increase in positivity with cervical LCR, 46%) and among women > or =20 years old versus younger women (relative increase in positivity with cervical LCR, 45%). The sensitivity of cervical PCR was highest when mucopurulent endocervical discharge was present (84%) and highest for cervical LCR when cervical gonococcal coinfection was detected (91%). Urethral inflammation was associated with higher sensitivities of urine LCR (86 compared to 70% when inflammation was absent) and PCR (82 compared to 62% when inflammation was absent). Menses had no effect on test performance. The effects of patient characteristics on test specificities were less pronounced and were closely related to observed sensitivities. These findings support expanded use of NAAT for screening and diagnosis of C. trachomatis in diverse clinical populations of women. (+info)
Use of ligase chain reaction in early diagnosis of tuberculous meningitis.
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INTRODUCTION: Nucleic acid-based amplification tests are currently licensed only for the detection of Mycobacterium tuberculosis in pulmonary specimens. There are insufficient data for extrapulmonary specimens. The aim of this study is to investigate the diagnostic value of these investigations in tuberculous meningitis. MATERIALS AND METHODS: We performed a prospective study using a commercial ligase chain reaction DNA amplification technique [Ligase chain result (LCx) M. tuberculosis; Abbott Laboratories, Abbott Park, IL, USA] on cerebrospinal fluid (CSF) to diagnose tuberculous meningitis and compared the results with standard microbiological data. Conflicting cases were resolved according to the final clinical diagnosis. A total of 54 CSF specimens from 54 patients were tested. RESULTS: Six (11.1%) specimens were culture-positive for M. tuberculosis; of these, only 1 (1.9%) was smear-positive. The sensitivity, specificity, positive predictive value and negative predictive value of the LCx assay, compared with culture results, were 66.7%, 100%, 100% and 96%, respectively. After resolution according to clinical data, the sensitivity, specificity, positive predictive value and negative predictive value were 33.3%, 100%, 100% and 75%, respectively. All controls had negative LCx results. There were no false positives. CONCLUSION: LCx assay is highly specific and complements conventional laboratory diagnostic methods (CSF smear and culture) in the diagnosis of tuberculous meningitis. In the appropriate clinical context, a positive result is strongly suggestive of tuberculosis and could enable antituberculous treatment to be started immediately. (+info)
Clonal dominance of hematopoietic stem cells triggered by retroviral gene marking.
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Gene marking with replication-defective retroviral vectors has been used for more than 20 years to track the in vivo fate of cell clones. We demonstrate that retroviral integrations themselves may trigger nonmalignant clonal expansion in murine long-term hematopoiesis. All 29 insertions recovered from clones dominating in serially transplanted recipients affected loci with an established or potential role in the self-renewal or survival of hematopoietic stem cells. Transcriptional dysregulation occurred in all 12 insertion sites analyzed. These findings have major implications for diagnostic gene marking and the discovery of genes regulating stem cell turnover. (+info)
Cervical specimen order and performance measures of Chlamydia trachomatis diagnostic testing.
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The orders of three endocervical specimens of 3,561 women for Chlamydia trachomatis testing were randomized to determine whether test performance measures of two nucleic acid amplification tests and a DNA probe were affected by swab order. Specimen collection order did not appear to affect the diagnostic accuracy of these tests. (+info)
Diagnosing infection levels of four human malaria parasite species by a polymerase chain reaction/ligase detection reaction fluorescent microsphere-based assay.
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Improving strategies for diagnosing infection by the four human Plasmodium species parasites is important as field-based epidemiologic and clinical studies focused on malaria become more ambitious. Expectations for malaria diagnostic assays include rapid processing with minimal expertise, very high specificity and sensitivity, and quantitative evaluation of parasitemia to be delivered at a very low cost. Toward fulfilling many of these expectations, we have developed a post-polymerase chain reaction (PCR)/ligase detection reaction-fluorescent microsphere assay (LDR-FMA). This assay, which uses Luminex FlexMAP microspheres, provides simultaneous, semi-quantitative detection of infection by all four human malaria parasite species at a sensitivity and specificity equal to other PCR-based assays. In blinded studies using P. falciparum-infected blood from in vitro cultures, we identified infected and uninfected samples with 100% concordance. Additionally, in analyses of P. falciparum in vitro cultures and P. vivax-infected monkeys, comparisons between parasitemia and LDR-FMA signal intensity showed very strong positive correlations (r > 0.95). Application of this multiplex Plasmodium species LDR-FMA diagnostic assay will increase the speed, accuracy, and reliability of diagnosing human Plasmodium species infections in epidemiologic studies of complex malaria-endemic settings. (+info)
Changing patterns of Plasmodium blood-stage infections in the Wosera region of Papua New Guinea monitored by light microscopy and high throughput PCR diagnosis.
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In Papua New Guinea (PNG), complex patterns of malaria commonly include single and mixed infections of Plasmodium falciparum, P. vivax, P. malariae, and P. ovale. Here, we assess recent epidemiologic characteristics of Plasmodium blood-stage infections in the Wosera region through four cross-sectional surveys (August 2001 to June 2003). Whereas previous studies performed here have relied on blood smear/light microscopy (LM) for diagnosing Plasmodium species infections, we introduce a newly developed, post-polymerase chain reaction (PCR), semi-quantitative, ligase detection reaction-fluorescent microsphere assay (LDR-FMA). A direct comparison of the two methods for > 1,100 samples showed that diagnosis was concordant for > 80% of the analyses performed for P. falciparum (PF), P. vivax (PV), and P. malariae (PM). Greater sensitivity of the LDR-FMA accounted for 75% of the discordance between diagnoses. Based on LM, the prevalence of blood-stage PF, PV, and PM infections was found to be markedly reduced compared with an early 1990s survey. In addition, there were significant shifts in age distribution of infections, with PV becoming the most common parasite in children < 4 years of age. Consistent with previous studies, prevalence of all Plasmodium species infections increased significantly in samples analyzed by the PCR-based LDR-FMA. This increase was most pronounced for PM, PO, and mixed infections and in adolescent (10-19 years) and adult age groups, suggesting that LM may lead to under-reported prevalence of less common Plasmodium species, infection complexity, and a skewed distribution of infections towards younger age groups. This study shows that the application of LDR-FMA diagnosis in large epidemiologic studies or malaria control interventions is feasible and may contribute novel insights regarding the epidemiology of malaria. (+info)
Multiplexed identification of blood-borne bacterial pathogens by use of a novel 16S rRNA gene PCR-ligase detection reaction-capillary electrophoresis assay.
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We have developed a novel high-throughput PCR-ligase detection reaction-capillary electrophoresis (PCR-LDR-CE) assay for the multiplexed identification of 20 blood-borne pathogens (Staphylococcus epidermidis, Staphylococcus aureus, Bacillus cereus, Enterococcus faecalis, Enterococcus faecium, Listeria monocytogenes, Streptococcus pneumoniae, Streptococcus pyogenes, Streptococcus agalactiae, Escherichia coli, Klebsiella pneumoniae, Haemophilus influenzae, Pseudomonas aeruginosa, Acinetobacter baumannii, Neisseria meningitidis, Bacteroides fragilis, Bacillus anthracis, Yersinia pestis, Francisella tularensis, and Brucella abortus), the last four of which are biothreat agents. The method relies on the amplification of two regions within the bacterial 16S rRNA gene, using universal PCR primers and querying the identity of specific single-nucleotide polymorphisms within the amplified regions in a subsequent LDR. The ligation products vary in color and size and are separated by CE. Each organism generates a specific pattern of ligation products, which can be used to distinguish the pathogens using an automated software program we developed for that purpose. The assay has been verified on 315 clinical isolates and demonstrated a detection sensitivity of 98%. Additionally, 484 seeded blood cultures were tested, with a detection sensitivity of 97.7%. The ability to identify geographically variant strains of the organisms was determined by testing 132 isolates obtained from across the United States. In summary, the PCR-LDR-CE assay can successfully identify, in a multiplexed fashion, a panel of 20 blood-borne pathogens with high sensitivity and specificity. (+info)
Increased complexity of wild-type adeno-associated virus-chromosomal junctions as determined by analysis of unselected cellular genomes.
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Adeno-associated virus (AAV) undergoes preferential Rep-mediated integration into the AAVS1 region of human chromosome 19 during latent infection, at least in highly-selected cell cultures. However, integration at the level of the whole eukaryotic genome in unselected cells has not yet been monitored for AAV as it has been for retro- and lentiviruses. Here we have used ligation-mediated PCR (LMPCR) to monitor the formation of AAV-chromosome junctions within unselected genomic DNA after infection. Our analyses show that, in the absence of selection, the complexity of junction formation is much greater than for selected cells. Sequencing of more than 50 authentic LMPCR clones showed that AAV formed junctions with many different chromosomal sites via DNA micro-homologies that frequently involved GGTC motifs located within the AAV p5 element. One site at position 280 was preferred. Even greater complexity was found when unselected junctions identified by LMPCR were analysed by direct PCR amplification and cloning of genomic DNA. No clones containing AAV-AAVS1 chromosome 19 junctions were identified among the LMPCR clones, although they were readily obtained using chromosomal PCR primers, suggesting that junctions with AAVS1 constituted only a small portion of the total. Thus, we have identified an additional means by which AAV sequences may join to human chromosomes, although the detailed molecular mechanisms remain to be elucidated. These data may have implications for the design of new-generation AAV vectors. (+info)