A polymerase chain reaction enzyme immunoassay (PCR-EIA) was developed for the semiquantitative of circulating candidal DNA in disseminated candidiasis due to Candida albicans. Polymerase chain reaction was based on primers from the internal transcribed ribosomal region. Binding of the product to a streptavidin-coated microtitration plate was mediated by a biotinylated capture probe. The product was digoxigenylated during PCR; this was the tag to which antibody was bound in the subsequent EIA. The optical density (OD) endpoint was < 0.1 in 15 sera from patients with no evidence of candidal infection (group 1) and in 13 of the 16 sera from colonized patients (group 2); it was > 0.1 in the other three sera from group 2 blood culture-negative patients who required intravenous amphotericin B for cure. The OD was positive in 28 patients with disseminated candidiasis (group 3), defined as positive blood cultures and successful treatment with amphotericin B (n = 11), positive blood culture confirmed at autopsy (n = 11), or negative blood culture first proven at necropsy (n = 6). In patients from whom multiple samples were available, recovery correlated with an optical density of < 0.1 by day 4 in four patients and by day 13 in the rest. In the five patients with fatal outcome from whom multiple samples were available, the mean OD rose from 0.174 to 0.668. Samples seeded with Candida albicans blastoconidia demonstrated that on OD of 0.220 was equivalent to 10 cfu. Assay of the group 3 sera by a commercial antigen detection test gave a corresponding sensitivity of 60% which rose to 67.9% when an in-house reverse passive latex agglutination test was used.
The two most commonly used targets for diagnosis of pertussis by the polymerase chain reaction have been the pertussis toxin promoter and the repeated insertion sequence IS481. A comparative assessment of these primers was performed on routinely collected nasopharyngeal swabs, stored at -20 C, using novel semiquantitative enzyme immunoassays. Both sets of primers behaved similarly with bacterial suspensions, and the 17 culture-positive nasopharyngeal swabs were also positive with the pertussis toxin promoter primers, with one exception, which had been subject to prolonged storage. Significantly more of the 69 culture-negative swabs were positive with the pertussis toxin promoter primers (n = 36) than with the IS481 primers (n = 18). To determine the effect of inhibitors, a comparative assessment of three primer pairs against human DNA (beta-globin and glyceraldehyde-3-phosphate dehydrogenase) was also performed.
Aim-To develop a polymerase chain reaction enzyme immunoassay (PCR-EIA) to measure levels of circulating aspergillus DNA in invasive aspergillosis caused by Aspergillus fumigatus. Methods-The PCR reaction was based on primers from the 18s rRNA gene.
A new method of extracting bacterial and yeast DNA from blood products dependent on guanidinium thiocyanate acid extraction and proteinase K treatment is described. In spiked samples the sensitivity per 0.1 ml of serum and blood, respectively, was 26 and 150 colony forming units (cfu) for Escherichia coli, 80 and 120 cfu for Staphylococcus aureus and 20 and 26 cfu for Candida albicans. This compared well with existing methodologies, worked on limited clinical samples and was not pathogen specific.
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