Detection of borreliacidal antibodies in hamsters by using flow cytometry.

Flow cytometry can be used to detect antibody that kills Borrelia burgdorferi. Borreliacidal activity was detected within 3 h of incubating B. burgdorferi with immune serum and complement. Right-angle light scatter and propidium iodide fluorescence were the cytometric parameters which correlated best with in vitro killing of B. burgdorferi. Flow cytometry is a rapid method for determining the presence of borreliacidal activity and may lead to a better serodiagnostic test for the detection of Lyme disease.

Detection of bacteria in blood by centrifugation and filtration.

Culture of blood is the most frequent means of diagnosing bacteremia. However, conventional blood culturing methods are slow in isolating bacteria. We developed a method for isolation of bacteria by centrifugation and filtration. Fresh human whole blood was inoculated with facultatively anaerobic and aerobic microorganisms (3 to 172 microorganisms per 5 ml). Seeded blood was then mixed with Ficoll-Hypaque (density, 1.149 +/- 0.002 g/ml) and centrifuged (386 x g) for 30 min at ambient temperature. The entire gradient (plasma, leukocytes, and Ficoll-Hypaque) was removed and filtered through a 0.22-micron membrane filter (Millipore). The filters were then placed on chocolate agar plates and incubated at 35 degrees C in a humidified atmosphere containing 5% CO2. For each bacterium tested, approximately 35 to 100% of the viable microorganisms were recovered when compared with control cultures (pour plates of seeded blood). All bacteria produced isolated colonies on filters after overnight incubation (18 h). This procedure may prove to be a more rapid method for isolating bacteria from clinical blood samples than the blood culture bottle technique.

Evaluation of two commercial procedures for rapid identification of Neisseria gonorrhoeae using a reference panel of antigenically diverse gonococci.

Two commercial tests for the rapid identification of Neisseria gonorrhoeae were evaluated. Two hundred seventy-nine organisms were tested, including 202 strains of N. gonorrhoeae. The Syva MicroTrak test results were less subjective but required a fluorescence microscope. The Phadebact Monoclonal GC OMNI Test required modification of the manufacturer's interpretive instructions in order to avoid cross-reactions, but it was a practical test. Specificities of both tests were 100%. Sensitivities of the Phadebact Monoclonal GC OMNI and Syva MicroTrak tests were 100% and approximately 100%, respectively.

Rapid detection of bacteria in cerebrospinal fluid by immunofluorescence staining on membrane filters.

A filter-fluorescent-antibody (FFA) staining procedure was developed for detection of bacteria in cerebrospinal fluid (CSF). The sensitivity of this procedure was determined and compared with that of the slide Gram stain of centrifuged samples, latex agglutination, and a previously developed filter Gram stain. Serial 10-fold dilutions of filter-sterilized CSF seeded with logarithmic-phase organisms were examined by each method and cultured to determine colony-forming bacteria. The bacteria included Haemophilus influenzae type b; Neisseria meningitidis group B, C, and W135; Streptococcus pneumoniae types 6A and 23F; and group B Streptococcus species. FFA was found to be equal in sensitivity to the filter Gram stain (P greater than 0.30). Both filter-staining procedures had greater sensitivity than the slide Gram stain of centrifuged sediment (P less than 0.02) and latex agglutination (P less than 0.0001). Addition of human leukocytes at a concentration of 5 to 10 cells per oil immersion field did not decrease sensitivity of the FFA procedure, although background fluorescence increased. FFA is a rapid and dependable procedure for detection of low numbers of bacteria in CSF. Evaluation of FFA with clinical specimens is needed.

Development of a rapid method for determining the susceptibility of Mycobacterium tuberculosis to isoniazid using the Gen-Probe DNA Hybridization System.

A rapid test was developed for determining the susceptibility of Mycobacterium tuberculosis to isoniazid by using nucleic acid hybridization. The method was based on quantification of total mycobacterial rRNA hybridized to a 125I-labeled DNA probe in the absence and presence of various concentrations of isoniazid. The radioactive hybridized complex was isolated by adsorption to hydroxyapatite crystals and measured in a gamma counter. The susceptibilities of four reference strains and 20 clinical isolates were compared by the Gen-Probe DNA Hybridization System and the critical concentration method. Overall agreement between the two methods was excellent. Results were obtained with the DNA probe after 3 to 5 days of incubation instead of the 14 to 21 days required for the critical concentration method. These findings indicate that susceptibility testing of M. tuberculosis by nucleic acid hybridization has merit for the clinical laboratory. Additional studies are needed to determine the efficacy of the DNA probe method for determining the susceptibility of M. tuberculosis to other antimycobacterial agents and its correlation with clinically significant levels of resistance.

Rapid method for the differentiation of gram-positive and gram-negative bacteria on membrane filters.

Microfiltration has become a popular procedure for the concentration and enumeration of bacteria. We developed a rapid and sensitive method for the differentiation of gram-positive and gram-negative bacteria, utilizing a polycarbonate membrane filter, crystal violet, iodine, 95% ethanol, and 6% carbol fuchsin, that can be completed in 60 to 90 s. Gram reactions of 49 species belonging to 30 genera of bacteria were correctly determined by the filter-Gram stain. The sensitivities of the filter-Gram stain and conventional slide-Gram stain were compared by testing dilutions of Escherichia coli, Neisseria meningitidis, Streptococcus pneumoniae, and Haemophilus influenzae suspensions in the presence and absence of whole human blood. The filter-Gram stain was approximately 100-fold more sensitive than the slide-Gram stain. The filter-Gram stain detected 2 to 100 bacteria, whereas the slide-Gram stain failed to detect less than 1,000 bacteria. The sensitivities of the methods were not significantly altered by the addition of whole human blood to the dilutions of bacteria tested. The filter-Gram stain could be a useful tool for the examination of body fluids with very low numbers of bacteria.

Identification of Yersinia spp. with the API 20E system.

The ability of the API 20E system to identify 105 clinical isolates of Yersinia spp. was compared with those of conventional biochemical tests at 28 and 37 degrees C. Elimination of the Voges-Proskauer test (recorded as a negative result) increased the percentage of correct identifications for Yersinia spp. from 66 to 93% when the API 20E strips were incubated at 28 degrees C.

Evaluation of a quantitative fluorescence immunoassay (FIAX) for detection of serum antibody to Borrelia burgdorferi.

A quantitative, indirect, fluorescence immunoassay (FIAX; Whittaker Bioproducts, Inc.) was compared with the conventional indirect fluorescent-antibody test for detection of serum antibody to Borrelia burgdorferi. FIAX correlated well with the indirect fluorescent-antibody test (r = 0.72). FIAX is a convenient and dependable means of measuring serum antibody to B. burgdorferi.

Evaluation of three commercial agglutination tests for the identification of Staphylococcus aureus.

Three commercially available rapid slide agglutination tests for the identification of Staphylococcus aureus were evaluated with 354 recent clinical isolates (165 strains of S. aureus). The test results of two latex agglutination products, SeroSTAT Staph (Scott Laboratories, Inc.) and Staphylatex (American Micro Scan), and one hemagglutination product, Staphyloslide (BBL Microbiology Systems), were compared with the results of the tube coagulase test, which was read at 4 h (4-h tube coagulase test) and, if negative, again after overnight incubation at room temperature (24-h tube coagulase test). Discrepancies between agglutination and tube coagulase identifications were resolved by use of the thermonuclease, mannitol fermentation, and slide coagulase tests. All sensitivities, specificities, predictive values of a positive result, and predictive values of a negative result for the three agglutination tests were at least 98.8% and comparable with the 4-h tube coagulase test. Best results were obtained with the 24-h tube coagulase test, which yielded one false-negative and no false-positive tests. Agglutination identifications may be performed on organisms taken directly from a primary plate when sufficient growth is present. Kit agglutination procedures yield rapid and reliable identifications and are easy to perform. This study also demonstrates the usefulness of the 24-h tube coagulase test.