Minimum number of bacteria needed for antigen detection by counterimmunoelectrophoresis: in vivo and in vitro studies

Abstract

Threshold concentrations of Streptococcus pneumoniae type 3, Haemophilus influenza type b, and Streptococcus sp. group B type Ib required for positive counterimmunoelectrophoresis reactions were determined in vivo and in vitro. Animals were infected intraperitoneally with various concentrations of microorganisms: adult mice with S. pneumoniae, suckling rats with H. influenza, and 3week-old mice with Streptococcus sp. group B. At 24 h after infection a minimum blood concentration of 103 colony-forming units (CFU)/ml was needed for S. pneumoniae or H. influenza before antigen was detected in the serum. A minimum concentration of 106 CFU/ml was needed for Streptococcus sp. group B at 10 h after infection. Larger threshold concentrations (104 CFU/ml for S. pneumoniae, 105 CFU/ml for H. influenza, and 107 CFU/ml for Streptococcus) were required in broth-grown cultures before cell-free antigens could be demonstrated by counterimmunoelectrophoresis in the medium. Marked levels of antigen release by group B streptococci were observed as the cultures entered early stationary phase. This study provides evidence of a long-accepted, though poorly substantiated, hypothesis that a threshold concentration of microorganism is necessary before counterimmunoelectrophoresis reactions become positive. Counterimmunoelectrophoresis results for clinical specimens should be interpreted cautiously in light of this evidence.

Diagnosis of rotavirus infection with cloned cDNA copies of viral genome segments

“…12 and 19), the PAGE-SS assay suffered from a high level of background staining, whereas in the case of sample 20, the apparent concentration of rotavirus genome RNA, as indicated by the dot hybridization assay, was below that detectable by the PAGE-SS assay (<20 ng). The counterimmunoelectrophoresis assay (5) showed good agreement with the combined results of the PAGE-SS assay and the dot hybridization assay, except for a false-negative result in sample 7. On the other hand, the enzyme-linked immunosorbent assay (21) suffered from a high level of apparent false-positive results and low-positive values that were difficult to interpret.…”

Differential Complement Resistance Mediates Virulence of Haemophilus influenzae Type b

“…For these reasons, the effect of complement on the six H. influenzae types was studied. An inoculum of about 1,000 to 2,000 CFU/ml was chosen based on data showing that this was the usual concentration of organisms in the blood in patients with meningitis (12,15,22,33). Furthermore, Moxon and Murphy (22) proposed that the initial penetration of type b organisms from the nasopharynx involved only few (possibly one) organisms.…”

Laboratory diagnosis of bacterial meningitis.

“…Microscopy Samples of all CSF specimens should be stained with the Gram stain (or other comparable stain) and examined microscopically. Because the diagnostic usefulness of staining procedures depends on the concentration of bacteria in the CSF of patients with bacterial meningitis (10 to 109 CFU/ml), all CSF specimens of sufficient quantity should be processed to concentrate pathogens prior to microscopic examination and culture (42,45,60,62,94,120,154). A small degree of additional concentration for Gram stain purposes can be achieved by sequentially layering small amounts of previously concentrated CSF onto the same area of a microscope slide and allowing each amount to dry thoroughly (9).…”