Rapid detection of bacterial contamination of platelet-rich plasma-derived platelet concentrates using flow cytometry

Flow cytometry can be used to detect bacterial contamination of platelet products. In this study, we investigated whether the incubation of a minimal volume of platelet-rich plasma (PRP)-derived platelet concentrates (PCs) with growth medium improved the analytical sensitivity of flow cytometry. Five bacterial strains (Staphylococcus aureus, Staphylococcus epidermidis, Bacillus cereus, Klebsiella pneumoniae, and Escherichia coli) were used. Platelets were inoculated with 10, 10(2), and 10(3) CFUs per mL; 0.5 mL, 1.0 mL, and 2.0 mL aliquots of spiked platelets were incubated with growth medium at 37°C for 24 hours. During the incubation period, the numbers of events were analyzed every 4 hours by flow cytometry. We could detect a low concentration (10 CFUs per mL) of bacteria in a small volume (minimum 0.5 mL) of PCs. Irrespective of spiking concentrations and incubation volumes, the detection times of S. aureus and S. epidermidis were 24 hours or less, while those of B. cereus, K. pneumoniae, and E. coli were 16 hours or less. A higher spiking concentration made it possible to shorten the detection time. The numbers of detected bacteria increased during the incubation. However, the graphs corresponding to K. pneumoniae and E. coli showed peak levels and decreasing patterns during the incubation period. The incubation of small volumes of PC with growth medium increased the analytical sensitivity of flow cytometry for bacterial detection. Therefore, flow cytometry can serve as a useful method for sterility testing using PRP-derived PCs with only low levels of consequent platelet loss.     

Rapid method for detection of adherent bacteria on Foley urinary catheters.

An accurate, rapid, and inexpensive method was developed for detecting and enumerating bacteria adherent to Foley urinary catheters based on malachite green staining of acridine orange-prestained specimens. This method has proven to be quick and reliable and will find application in quantitative studies of biomaterial-related sepsis.     

Rapid bioluminescence method for bacteriuria screening.

A study was performed to evaluate the UTIscreen (Los Alamos Diagnostics, Los Alamos, N. Mex.), a rapid bioluminescence bacteriuria screen. The UTIscreen was compared with three other rapid bacteriuria screens: the Bac-T-Screen (Vitek Systems, Hazelwood, Mo.), an automated filtration device; the Chemstrip LN (Boehringer Mannheim Diagnostics, BioDynamics, Indianapolis, Ind.), an enzyme dipstick; and the Gram stain. A semiquantitative plate culture was used as the reference method. Of the 1,000 specimens tested, 276 had colony counts of greater than 10(5) CFU/ml by the culture method. Of these, the UTIscreen detected 96% (265 of 276) using greater than or equal to 5% of the integrated light output of the standard reading as a positive interpretive breakpoint, the Bac-T-Screen detected 96% (266 of 276), the Chemstrip LN detected 90% (249 of 276), and the Gram stain detected 96% (264 of 276). Of the 214 probable pathogens isolated at greater than 10(5) CFU/ml, the UTIscreen detected 95% (204 of 214), the Bac-T-Screen detected 98% (210 of 214), the Chemstrip LN detected 92% (198 of 214), and the Gram stain detected 98% (209 of 214). The predictive values of negative test results at greater than 10(5) CFU/ml for the UTIscreen, the Bac-T-Screen, the Chemstrip LN, and the Gram stain were 98, 97, 93, and 98%, respectively. The overall specificities at greater than 10(5) CFU/ml for the UTIscreen, the Bac-T-Screen, the Chemstrip LN, and the Gram stain were 70, 48, 51, and 69%, respectively. There were 532 specimens with colony counts of >10(3) CFU/ml, and of these, the UTIscreen, the Bac-T-Screen, the Chemstrip LN, and the Gram stain detected 72, 81, 76, and 73%, respectively. Of the 249 probable pathogens isolated at >10(3) CFU/ml, the UTIscreen, the Bac-T-Screen, the Chemstrip LN, and the Gram stain detected 91, 95, 89, and 93%, respectively. The overall specificities at > 10(3) CFU/ml for these methods were 79, 55, 57, and 78%, respectively. The cost per test for detection was approximately $0.50 for the Chemstrip LN. Overall, the UTIscreen is rapid and easy to perform; its sensitivity compared favorably with those of the other screening methods; it had higher specificity than the Bac-T-Screen and Chemstrip LN; and it allowed for bathing of specimen.     

Rapid method for the detection and identification of mycolic acids in aerobic actinomycetes and related bacteria.

A rapid method for the identification of lipids characteristic of the genera Corynebacterium, Mycobacterium, Nocardia, and the "rhodochrous group" has been developed. Modifications of previously described methods make this procedure suitable for use in the clinical laboratory. Thin-layer chromatography is used to demonstrate the presence of the lipid characteristic of Nocardia spp. (type A) in some corynebacteria, nocardias, and members of the "rhodochrous group." Precipitation in ether and ethanol is used to demonstrate the presence of mycobacterial mycolic acids. Since this procedure can be carried out in less than 2 days and the lipids are extracted from the same batch of cells grown for diaminopimelic acid and whole-cell sugar analyses, it can readily be added to the battery of tests performed in reference laboratories that deal with aerobic actinomycetes and related bacteria.     

Rapid screening method for enterotoxigenic Escherichia coli.

A rapid screening method for detection of Escherichia coli producing heat-labile enterotoxin is described. Single colonies are transferred directly from primary culture plates into 96-well microculture plates containing 0.3 ml of brain heart infusion broth in each well. After 24 h at 37 degrees C, each brain heart infusion broth culture is assayed by the miniculture method in the corresponding well of a microculture plate in which Y-1 mouse adrenal tumor cells have been grown. All enterotoxigenic isolates detected by this method were confirmed in the assay but with culture supernatants.     

Rapid methods of detection of bacteria in the bile

Rapid methods are described for assessing the bacterial contamination, using direct phase-contrast microscopy, triphenyltetrazolium chloride (TTC) test, and a standard loop 3 mm in diameter, or by the detection of the biliary 'active' leukocytes and the microflora sensitivity to antibacterial drugs with the use of the TTC test and turbidimetric technique. These methods permit getting an answer in 4-24 hrs, are simple, and do not require special bacteriologic equipment, this recommending them for routine clinical studies.     

Detection of bacteria in red blood cell concentrates by the Scansystem method

Bacterial contamination remains one of the major risks associated with blood product transfusion. The kinetics of bacterial growth in red blood cell concentrates (RBCC) is different than otherwise due to storage at 4 degrees C, conditions in which most bacteria do not survive. Psychrophilic bacteria such as Yersinia enterocolitica, however, can proliferate from a very low level of contamination to clinically significant levels at 4 degrees C and are known to cause severe transfusion-related infections. A screening method allowing the early detection of very low levels of bacteria in RBCC would improve transfusion safety. The Scansystem method has been previously described for detection of bacteria in platelet concentrates. We present here a modification of the system for detection of low levels of bacteria in RBCC. The Scansystem RBC kit protocol requires three steps, i.e., the agglutination and selective removal of RBCs, a labeling stage during which bacteria are labeled with a DNA-specific fluorophore, and finally recovery of bacteria on the surface of a black membrane for analysis using the Scansystem. The entire procedure from sampling to result can be completed in 90 min. Both gram-negative and gram-positive bacteria in RBCC are detected with a higher sensitivity than with currently available culture-based methods. The Scansystem RBC kit is shown to be sensitive enough to identify low-level bacterial contamination in a single unit tested in a pool of up to 20 RBCC samples (detection limit of between 1 and 10 CFU/ml depending on the bacterial strain). The method therefore lends itself to incorporation into high-sample-throughput screening programs.     

Rapid screening method for monoclonal antibodies against cytoskeletal proteins

A horseradish peroxidase/anti-horseradish peroxidase monoclonal mouse antibody complex was prepared and used for the detection of mouse monoclonal antibodies against proteins of the whole cytoskeleton. A rapid qualitative assay of large number of antibody samples on the whole cytoskeleton and an early determination of their specificity by the immunoblot technique is described.     

Rapid method for detection of gram-positive and -negative bacteria in milk from cows with moderate or severe clinical mastitis

A rapid method for demonstration of gram-positive and gram-negative bacteria in milk is described. The technique is based on dilution of the sample in a medium, followed by filtration through a porous polysulfone membrane with a pore size retaining and concentrating bacteria from the sample. The bacteria concentrated on the surface of the membrane are stained with a cationic dye (toluidine blue) that can be visualized by the naked eye. After staining, the membrane is treated with ethanol-acetic acid (pH 2.8 to 3.0), which causes decolorization of gram-negative bacteria, whereas gram-positive bacteria retain the stain. The method does not require heat fixation, electrical power, microscopic examination, or specially trained personnel. The time needed to perform the test is approximately 5 min. The technique was applied to artificially infected milk and milk from cows with moderate or severe clinical mastitis for detection and differentiation of bacteria. The sensitivity of the filtration method was 92 and 100% for gram-positive and gram-negative bacteria, respectively, compared with traditional bacteriological culture of milk samples. The detection limit was 5 x 10(6) CFU/ml for Staphylococcus aureus and 1 x 10(6) CFU/ml for Escherichia coli in spiked milk samples. The overall specificity of the method was 86%. This diagnostic method can provide on-site guidance to the veterinarian to optimize use of antibiotics in mastitis therapy.     

Rapid method for detecting beta-lactamase producing bacteria in clinical specimens.

The use of liquid media to detect the production of beta-lactamase by beta-lactamase producing organisms has been compared with the conventional method of inoculation on to agar media. Pharyngeal cultures were obtained from 162 children treated with penicillin for acute tonsillitis. beta-lactamase producing organisms were detected within 72 h in 80 (49%) of the specimens inoculated on to agar media, while beta-lactamase production was found in 76 (47%) of the specimens after their incubation in liquid media for 24 h. Twenty one of the cultures were positive only after anaerobic incubation while in liquid media while nine were positive only after aerobic incubation. Incubation in liquid media enabled detection of beta-lactamase activity in 53 of the 76 (70%) specimens within 12 h.     

Optimization of real-time PCR assay for rapid and sensitive detection of eubacterial 16S ribosomal DNA in platelet concentrates

A real-time PCR assay was developed for rapid detection of eubacterial 16S ribosomal DNA in platelet concentrates. The sensitivity of this assay can be hampered by contaminating DNA in the PCR reagents. Digestion of the PCR reagents with Sau3AI prior to PCR amplification was effective in eliminating this contaminating DNA without affecting the sensitivity of the assay.     

Slide method for detection of antibody-coated bacteria in urine sediments.

An immunofluorescent slide method incorporating 0.1% (wt/vol) Evans blue as a counterstain was developed and compared with a recently described tube method. Seventy-one urine specimens were tested concurrently by both methods. Results of the two methods agreed in 70 specimens and disagreed in only 1. We found the slide method to be less costly and time consuming than the tube method.     

Rapid assays for detection of anti-islet autoantibodies: implications for organ donor screening

The purpose of the current study was to develop and evaluate rapid assays for autoantibodies to GAD65 (GAA), ICA512bdc/IA-2 (ICA512AA), and insulin (microIAA, mIAA) as a potential tool for identification of cadaveric pancreas donors who were at high risk for developing diabetes. The study included 154 new onset diabetic, prediabetic, and healthy control subjects. Subjects were evaluated for all three autoantibodies in three separate assays: (1) standard (std) assay with a 24-h or 72-h incubation at 4 degrees C (combined GAA/ICA512AA or mIAA, respectively), (2) rapid assay with 1-h room temperature (RT) incubation, and (3) rapid assay with 2-h RT incubation. The serum samples from 777 organ donors were also evaluated for all three autoantibodies and all the positive samples from standard assay evaluated with the 1-h incubation assay. Simple linear regression analyses revealed excellent correlation between the standard assay and the rapid assays for all three autoantibodies, as follows: (1) GAA: std vs. 1 h (R2=0.85) and std vs. 2 h (R2=0.83), (2) ICA512AA: std vs. 1 h (R2=0.85) and std vs. 2 h (R2=0.84), and (3) mIAA: std vs. 1 h (R2=0.70) and std vs. 2 h (R2=0.64). Comparison of assay correlation rates between subject cohorts revealed no significant differences. Compared to their respective standard assays, the 1-h RT GAA assay missed 3.2% and identified an additional 1.3% of samples, the 1-h RT ICA512AA assay had no discordant samples, and the 1-h RT mIAA assay missed 7.1% and identified an additional 5.8% of samples. We analysed a series of 777 stored serum samples from cadaveric donors. Two of 777 (0.25%) were positive for two autoantibodies (both GAA and ICA512AA) and 23 of 777 (3.0%) one autoantibody (11 IAA; 12 GAA). The rapid analysis for all three autoantibodies could be completed in less than 3 h with comparable concordance rates to the more time-consuming standard assays, making these assays an attractive option for organ donor screening to identify potential pancreata for immunopathogenetic research.     

Rapid screening of urine for bacteria and cells by using a catalase reagent.

Five hundred urine samples were tested for cells and bacteria by using a commercial dipstick, a catalase screening device, standard culturing, and chamber counts. The sensitivities of the catalase test were 83% for all samples and 97.6% for specimens containing significant numbers of leukocytes and bacteria. The catalase screening test is simple to perform and should prove useful for the detection of urinary tract infections.