J. Mahony

A Randomized, Controlled Trial of Doxycycline and Rifampin for Patients with Alzheimer's Disease

Objectives: To assess whether doxycycline and rifampin have a therapeutic role in patients with Alzheimers disease (AD).

Replicate PCR Testing and Probit Analysis for Detection and Quantitation of Chlamydia pneumoniae in Clinical Specimens

ABSTRACT Nucleic acid amplification of clinical specimens with low target concentration has variable sensitivity. We examined whether testing multiple aliquots of extracted DNA increased the sensitivity and reproducibility of Chlamydia pneumoniae detection by PCR. Nested and non-nested C. pneumoniae PCR assays were compared using 10 replicates of 16 serial dilutions of C. pneumoniae ATCC VR-1310. The proportion positive versus theC. pneumoniae concentration was modeled by probit regression analysis. To validate the model, 10 replicates of 26 previously positive patient specimens of peripheral blood mononuclear cells (PBMC), sputum, or nasopharyngeal swabs (NPS) were tested. The proportion of replicates that were positive varied with the concentration of C. pneumoniae in the sample. At concentrations above 5 infection-forming units (IFU)/ml, both nested and non-nested PCR assay sensitivities were 90% or greater. The nested PCR was more sensitive (median detection, 0.35 versus 0.61 IFU/ml; relative median detection, 0.58; 95% confidence interval, 0.31 to 0.99; P = 0.04). In clinical specimens, replicate PCR detected 15 of 26 (nested) versus 1 of 26 (non-nested,P < 0.001). For PBMC specimens, testing 1, 3, or 5 replicates detected 3, 5, or 9 of 10 positive specimens, respectively. Median C. pneumoniae concentrations were estimated at 0.07 IFU/ml for PBMC and at <0.03 IFU/ml for NPS specimens. We conclude that performing 5 or 10 replicates considerably increased the sensitivity and reproducibility of C. pneumoniae PCR and enabled quantitation for clinical specimens. Due to sampling variability, PCR tests done without replication may miss a large proportion of positive specimens, particularly for specimens with small amounts of target C. pneumoniae DNA present.

Evaluation of the NucliSens Basic Kit for detection of Chlamydia trachomatis and Neisseria gonorrhoeae in genital tract specimens using nucleic acid sequence-based amplification of 16S rRNA.

ABSTRACT We evaluated a new RNA amplification and detection kit, the NucliSens Basic Kit (Organon Teknika), for the detection ofChlamydia trachomatis and Neisseria gonorrhoeaein genitourinary specimens. The Basic Kit provides an open platform for RNA amplification and detection and contains isolation reagents for nucleic acid extraction, nucleic acid sequence-based amplification (NASBA) reagents (enzymes and buffers), and a generic ruthenium-labeled probe for electrochemiluminescent (ECL) detection of amplified product. Using freshly purified and titrated stocks of C. trachomatis and N. gonorrhoeae and in vitro-generated RNA transcripts for sensitivity determinations, the Basic Kit detected 1 inclusion-forming unit of C. trachomatis, 1 CFU ofN. gonorrhoeae, and 100 RNA molecules of 16S rRNA for both bacteria. The clinical performance of the Basic Kit was evaluated by testing a total of 250 specimens for N. gonorrhoeae by culture and NASBA and a total of 96 specimens for C. trachomatis by PCR and NASBA. The Basic Kit detected 139 of 142N. gonorrhoeae culture-positive specimens and gave a negative result for 73 of 74 culture-negative specimens, for a sensitivity and specificity of 97.9 and 98.7%, respectively. ForC. trachomatis, the Basic Kit detected 24 of 24 PCR-positive specimens and gave a negative result for 71 of 72 PCR-negative specimens, for a sensitivity and specificity of 100 and 98.6%, respectively. The Basic Kit also detected specimens containing both N. gonorrhoeae and C. trachomatis, using a multiplex NASBA assay using primers for both bacteria. The NucliSens Basic Kit offers a versatile platform for the development of sensitive RNA detection assays for sexually transmitted diseases.

Specimen Processing and Concentration of Chlamydia trachomatis Added Can Influence False-Negative Rates in the LCx Assay but Not in the APTIMA Combo 2 Assay When Testing for Inhibitors

ABSTRACT Inhibitors in clinical specimens can be detected by adding the target of nucleic acid amplification to the sample. Introduction of a Chlamydia trachomatis L2 434 preparation containing 12 elementary bodies (EBs) into first-void urine (FVU) from 225 nonpregnant women and 190 pregnant women before specimen processing by the assays produced false-negative rates of 0.48% (2 of 415 specimens) and 13% (44 of 338 specimens) by the APTIMA Combo 2 and the Chlamydia LCx tests, respectively. Reducing the amount of C. trachomatis added to one EB, a concentration closer to the APTIMA Combo 2 test cutoff, for a subset of 244 FVU specimens increased the number of specimens with false-negative results by the APTIMA Combo 2 assay to 7 (2.9%), suggesting that the strength of the input C. trachomatis per specimen has an influence on the number of specimens with false-negative results. Repeat testing after overnight storage and dilution decreased the APTIMA Combo 2 test false-negative rates to 0% (0 of 415 specimens) with the stronger inoculum and 0.8% (2 of 244 specimens) with the weaker inoculum; the false-negative rate of the LCx assay was reduced to 5.4% (18 of 334 specimens). When an additional 70 FVU specimens from women to which 12 EBs were added before specimen processing were tested by the LCx assay, 34 specimens had false-negative results, whereas 21 specimens had false-negative results when the C. trachomatis EBs were introduced after processing. Nine of the 21 specimens to which EBs were added after processing and all of the 34 urine specimens to which the target was added before processing remained falsely negative on repeat testing at a 1:2 dilution, suggesting that input C. trachomatis DNA was lost during processing by the LCx assay. In contrast, the APTIMA Combo 2 assay appears to have a higher sensitivity and either lost little nucleic acid during processing or demonstrated few problems with inhibitors of transcription-mediated amplification.

Accuracy of Results Obtained by Performing a Second Ligase Chain Reaction Assay and PCR Analysis on Urine Samples with Positive or Near-Cutoff Results in the LCx Test for Chlamydia trachomatis

ABSTRACT Nucleic acid amplification assays such as the ligase chain reaction and PCR have encountered reproducibility problems. The initial extract and a newly extracted aliquot of urine specimens (n = 120) which had signal-to-cutoff (S/CO) ratios above 0.80 by the LCx Chlamydia assay were retested. Nucleic acid was extracted from an additional urine sample for testing by the AMPLICOR PCR Chlamydia assay. Fifteen percent (18 of 120) of the urine specimens were negative by all repeat tests (initial mean S/CO ratio by the LCx Chlamydia assay, 0.93; S/CO ratio range, 0.80 to 3.30). Repeat testing of the 102 specimens with possible positive results by the LCx Chlamydia assay by use of the initially extracted aliquot confirmed the results for 95 (93.1%) of the specimens; repeat testing of a newly extracted aliquot confirmed the results for 87 (85.3%) of the specimens. Twenty specimens had discordant results by the two repeat LCx Chlamydia assays. A total of 78 of 102 (76.5%) of the specimens were positive by the AMPLICOR PCR, and the AMPLICOR PCR confirmed the results for 82.1% (78 of 95) and 89.6% (78 of 87) of the specimens positive by the two repeat LCx Chlamydia assays, respectively. Some of the discrepancies observed by multiple repeat tests may have been due to specimen mislabeling or contamination during performance of the procedure rather than to the LCx Chlamydia assay. Both assays suffered from a lack of reproducibility on repeat testing with a small proportion of specimens, probably due to the presence of low levels of DNA, the presence of variable amounts of amplification inhibitors, and the loss of DNA during extraction.

Circulating Nucleic Acids of Chlamydia pneumoniae and Cytomegalovirus in Patients Undergoing Coronary Angiography

ABSTRACT Peripheral blood mononuclear cells from 208 consecutive patients undergoing elective coronary angiography or angioplasty were collected before, immediately after, and 4 h after the procedure. Nucleic acids of Chlamydia pneumoniae and of cytomegalovirus (CMV) were detected by PCR and confirmed by hybridization. CirculatingC. pneumoniae DNA was identified in 24 patients (11.5%) and was associated with current smoking (odds ratio [OR] = 4.5, 95% confidence interval [CI] = 1.6 to 12.2, P = 0.004) but not with arterial narrowing on coronary angiogram or with serological results positive for C. pneumoniae. Circulating CMV DNA was identified in 36 patients (17.3%) and was associated with anti-CMV immunoglobulin G (OR = 2.7, 95% CI = 1.2 to 6.3, P = 0.02) but not with angiographic arterial narrowing or with the need for revascularization. Neither C. pneumoniae nor CMV DNA detection increased after angioplasty, a procedure in which endothelium is disrupted. Larger prospective studies are needed to determine the prognostic significance of DNA detection.

Cost-effectiveness of screening swab or urine specimens for Chlamydia trachomatis from young Canadian women in Ontario.

Background Undetected and untreated Chlamydia trachomatis infections can result in a significant health burden. Diagnostic testing refers to tests performed on patients with symptoms, whereas screening refers to testing specimens in asymptomatic patients. The goal of diagnostic testing and screening programs are early identification of infections to prevent upper tract infection and transmission to other partners. Goal To compare the costs and outcomes of alternative diagnostic testing and screening programs for women ages 15 to 24 years in the province of Ontario, Canada. Study Design Using outcome probabilities from the literature and a consensus group, together with the costs from insurance billing, a decision analytic model was constructed to determine the baseline risk of C trachomatis and related sequelae. Seven diagnostic testing and screening programs were compared over a 10-year period. The programs compared included the use of nucleic acid amplification assays collected from urine or endocervical swab specimens. Results Largely because of lower sensitivity the urine-based testing or screening programs were dominated by the swab-based programs. The move from swab-based testing to a swab-based screening program for high-risk women costs

Effect of time elapsed since previous voiding on the detection ofChlamydia trachomatis antigens in urine

1873 per case of C trachomatis averted. Expanding the program further to include all women in Ontario between 15 and 24 years of age is considerably more costly at

Lack of association between vascular dementia and Chlamydia pneumoniae infection: a case-control study

5990 per case averted. Conclusions It is more costly and more effective to screen and treat high-risk women ages 15 to 24 years for C trachomatis than to perform only swab-based diagnostic testing on symptomatic women. Expanding the screening program to include all women ages 15 to 24 years is considerably more expensive and only moderately more effective than screening only high-risk women.

Neptune: a bioinformatics tool for rapid discovery of genomic variation in bacterial populations

To determine if the time elapsed since previous voiding affects the sensitivity of an enzyme immunoassay (EIA) to detectChlamydia trachomatis in urine, 882 women and 428 men were tested for chlamydial infection in urethral specimens by isolation in cell culture (women and men) and EIA with blocking confirmation (women only). Of the 36 women (4.1 %) and 38 men (8.9 %) who were positive forChlamydia trachomatis in the urethra, 55.5 % (20/36) and 81.6 % (31/38) respectively were positive in the first void urine (FVU) sediment by confirmed EIA. In women the sensitivity of the EIA performed on FVU was 67.8 % (19/28) if the urine had been in the bladder <3 hours and decreased to 12.5 % (1/8) if longer times had elapsed (odds ratio 13.7; 95 % confidence interval 1.4 to 700.0) with no obvious confounding. In men a weaker association was present (odds ratio 2.7; 95 % confidence interval 0.4 to 22.3). These findings should enable investigators to optimize the analysis of urine to diagnose chlamydial infections.