Background: Intranasal inoculation of Streptococcus pneumoniae D39 serotype 2 causes fatal pneumonia in mice. The cytotoxic and inflammatory properties of pneumolysin (PLY) have been implicated in the pathogenesis of pneumococcal pneumonia.
A pneumolysin-specific enzyme-linked immunosorbent assay (PLY-ELISA) for the detection of pneumolysin in urine was developed and evaluated in comparison with the commercially available Binax Now Streptococcus pneumoniae test (Binax, Portland, ME) for the diagnosis of pneumococcal infections. Assay sensitivity was evaluated using urine from 108 patients with culture-confirmed pneumococcal infections. In adults, the sensitivity and specificity of the PLY-ELISA were 56.6% and 92.2%, respectively. In children with nasopharyngeal pneumococcal carriage, PLY-ELISA and Binax Now S. pneumoniae test sensitivities were 62.5% and 87.5%, respectively, while specificities were 94.4% and 27.8%, respectively. In children with nonnasopharyngeal pneumococcal carriage, PLY-ELISA and Binax Now S. pneumoniae test sensitivities were 68.7% and 93.7%, respectively, and test specificities were 94.1% and 41.2%, respectively. The persistence of pneumolysin in urine of pneumococcal pneumonia patients decreased significantly after 4 to 6 days of treatment. Our data suggest that combining the high specificity of the PLY-ELISA with the high sensitivity of the Binax Now S. pneumoniae test would enable pneumococcal infections to be accurately diagnosed in children.
The evolution of imipenem resistance was evaluated in Pseudomonas aeruginosa sequentially isolated from 42 patients with cystic fibrosis. Susceptibility was determined using a commercial microdilution system and imipenem resistance was confirmed by the agar dilution technique. Resistance to imipenem increased during the years from 1988 to 1992. A total of 12 patients (28.5%) carried resistant strains (11.6% of the total P. aeruginosa isolates) but only two of them were treated with the carbapenem. The other patient under imipenem treatment did not harbour resistant isolates. Sixty-four per cent of the imipenem resistant isolates were also meropenem resistant and showed low susceptibility to the other beta-lactams and tobramycin and amikacin. Twenty-one strains were selected for biochemical study. Imipenem susceptible strains showed normal OprD in two strains and diminished OprD in two more. Five strains with MIC of imipenem of 4-8 mg/L lacked OprD while another two had a band with decreased density. All strains with MIC higher than 8 completely lacked this band in western-blot analysis. Imipenem MICs of 0.5-2 mg/L only slightly increased to 1-4 mg/L when a pattern of beta-lactamase derepression was observed. While those with imipenem MICs between 8-16 mg/L increased the imipenem MIC to 16-64 mg/L in the population with a beta-lactamase derepression phenotype.
Recombinant antibodies such as Fab and scFv are monovalent and small in size, although their functional affinity can be improved through tag-specific immobilization. In order to find the optimum candidate for oriented immobilization, we generated Fab and scFv fragments derived from an anti-pneumolysin monoclonal antibody PLY-7, with histidine and cysteine residues added in diverse arrangements. Tagged antibody fragments scFv-Cys7-His6, His6-scFv-Cys7, and Fab-Cys7 lost considerable affinity for the antigen; however, Fab-His6, Fab-Cys1, and scFv-His6-Cys1 were able to detect immobilized antigen, revealing that the position and number of histidine and cysteine residues are involved differently in the reactivity of antibody fragments. Random and orientated immobilizations were carried out using conventional polystyrene and commercial surface-pretreated ELISA plates. The best orientation performance was obtained with Fab-Cys1-biotin on streptavidin-coated plates with increased signal levels of 62%, while oriented immobilization of Fab-His6 and scFv-His6-Cys1 on nickel- and maleimide-coated plates failed to improve the ELISA sensitivity.
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