Rapid detection of simulated bacteremia by centrifugation and filtration.

A centrifugation-filtration procedure was developed to expedite the recovery of microorganisms from blood. Fresh whole human blood was inoculated with various aerobic and facultatively anaerobic microorganisms (3 to 18 per ml). The seeded blood was carefully overlaid on a Ficoll-Hypaque gradient (density, 1.114 g/ml) and centrifuged (400 x g) for 45 min at ambient temperature. The entire gradient (plasma, leukocytes, and Ficoll-Hypaque) was removed and filtered through a 0.22-micrometer membrane filter. The filters were then placed on chocolate agar and incubated at 35 degrees C in humidified air containing 5% CO2. No statistically significant differences were detected between the numbers of microorganisms recovered by filtration and by direct culture of the original inoculum. Most microorganisms were detected within 18 h after filtration. This system has excellent sensitivity and negligible toxicity.     

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.     

Detection and quantitation by lysis-filtration of bacteremia after different oral surgical procedures.

Patients with bacteremia after dental extraction, third-molar surgery, dental scaling, endodontic treatment, and bilateral tonsillectomy were studied by means of lysis-filtration of blood samples with subsequent aerobic and anaerobic incubation. Samples were obtained before, during, and 10 min after treatment. Bacteremia was observed in 100% of patients after dental extraction, 55% of patients after third-molar surgery, 70% of patients after dental scaling, 20% of patients after endodontic treatment, and 55% of patients after bilateral tonsillectomy. Anaerobic microorganisms were isolated more frequently than aerobic microorganisms were, and viridans group streptococci were the most commonly isolated bacteria. Ten minutes after treatment, the frequency as well as the magnitude of bacteremia showed pronounced reduction.     

Detection of anaerobic bacteria in blood cultures by lysis filtration

An anaerobic adaptation of the lysis-filtration system for detection of anaerobic microorganisms in blood is described. The method was compared with a conventional broth bottle system in detection of anaerobic bacteremia after oral surgery. Of 43 blood samples obtained during and after surgery, 31 were positive with the lysis-filtration system and 17 were positive with the broth bottle system. Sixteen aerobic and 62 anaerobic strains were isolated with the lysis-filtration system versus 9 aerobic and 22 anaerobic strains with the broth bottles. The lysis-filtration technique was thus superior to the conventional broth bottle method in detecting anaerobic bacteria.     

Presumptive diagnosis of anaerobic bacteremia by gas-liquid chromatography of blood cultures.

By quantitative gas-liquid chromatography of glucose-containing blood cultures at the moment of first signs of growth, a presumptive diagnosis of anaerobic bacteremia could be made in 24 out of 26 cultures yielding obligate anaerobes upon subsequent culture. With Bacteroides sp. (20 strains isolated), elevated levels of isovaleric acid (greater than or equal to 0.1 mumol/ml) and/or succinic acid (greater than or equal to 5 mumol/ml) were detected in the medium used. An exception to this were two strains of Bacteroides thetaiotaomicron that did not produce sufficient quantities of these acids. In the case of butyrate-producing gram-positive cocci (four strains), butyric acid in amounts of greater than or equal to 0.8 mumol/ml was detected. Propionibacteria (five strains) produced propionic acid in amounts of greater than or equal to 3.9 mumol/ml. No false positive results were found in 103 blood cultures with growth of aerobic or facultatively anaerobic bacteria only.     

Non-selective lymphocyte isolation from human blood by nylon wool filtration and density centrifugation.

A new method was employed to isolate lymphocytes from human peripheral blood. Continuous flow filtration through a nylon wool filter, at a flow rate of 1.4 ml/min, produced a lymphocyte yield of 90.5% and a purity of 96% without any shift in the B-T cell ratio. Ficoll-Isopaque with a specific gravity of 1.085 g/ml instead of 1.077 g/ml could be used to remove the erythrocytes. An overall recovery, including defibrination, filtration, Ficoll-Isopaque centrifugation and washing step, of 74.5% was achieved.     

Detection of bacteremia by Difco ESP blood culture system.

In a multicenter study, the Difco ESP blood culture system (Difco Laboratories, Detroit, Mich.) was compared with the BACTEC NR660 system (Becton Dickinson Diagnostic Instrument Systems, Sparks, Md.). The ESP system monitors each blood culture bottle every 12 to 24 min to detect changes in oxygen consumption and gas production by microbes. Equal volumes of blood were inoculated into aerobic ESP-80A and BACTEC 6A, 16A, or PEDS Plus broths and anaerobic ESP-80N and BACTEC 7A or 17A broths and were incubated for up to 7 days. ESP bottles contain supplemented tryptic soy broth without antimicrobial agent-adsorbing resins. From 7,532 aerobic compliant sets, the ESP system detected 356 clinically significant positive cultures and the BACTEC NR660 system detected 329. From 6,007 anaerobic cultures, the ESP system detected 234 clinically significant positive cultures and the BACTEC NR660 system detected 198. In aerobic broths, 292 organisms were isolated from both systems and 78 organisms were isolated from the ESP system alone, whereas 54 organisms were isolated from the BACTEC NR660 system alone (P < 0.05). Among individual organisms, pneumococci were isolated significantly more often in ESP aerobic broths. In anaerobic broths, 180 organisms were isolated from both systems and 68 organisms were isolated from the ESP system alone, whereas 35 organisms were isolated from the BACTEC NR660 system alone (P < 0.05). Aerobic gram-positive organisms as a group and Candida spp. were isolated significantly more often in ESP anaerobic broths. Both systems detected 207 clinically significant bacteremic episodes and the ESP system alone detected 63, whereas the BACTEC NR660 system alone detected 32 (P < 0.05). Significantly more episodes of bacteremia caused by Staphylococcus epidermidis and anaerobes were detected by the ESP system. The differences in the numbers of organisms detected >6h earlier in ESP broths compared with BACTNEC NR660 broths were significant, as were earlier times to detection. Although the total number of organisms detected was not significantly different, the ESP system alone detected more organisms in a shorter time than did the BACTEC NR660 system alone. The continuous monitoring capability of the ESP system makes it an attractive alternative to the BACTEC NR660 system.     

Detection and quantitation of antibodies against Rhizopus by enzyme-linked immunosorbent assay.

In sawmills high concentrations of spores from the mould Rhizopus microsporus may occur, causing allergic alveolitis in exposed workers. Both symptomatic and asymptomatic exposed workers may develop antibodies. An enzyme-linked immunosorbent assay (ELISA) for the detection of antibodies to Rhizopus has been developed in order to study the relationship between antibody levels and exposure levels. The precision of the measurements of Rhizopus antibodies by ELISA carried out on the same microtiter plate was estimated to be 11%. It is therefore possible to detect changes in antibody levels of approximately 25% or more. Antibodies were studied longitudinally by ELISA in 60 wood trimmers. The observed changes in antibody levels exceeded the precision of the ELISA method substantially, indicating significant variability in antibody levels in wood trimmers. The ELISA test was compared with the double immunodiffusion test (DID). Sera from 67 wood trimmers were analyzed by both methods. Antibodies were detected by ELISA in 70% and by DID in 28% of the workers in this group, clearly demonstrating that the ELISA test is the most sensitive method for the detection of antibodies to Rhizopus.     

Detection and quantitation of hepatitis-B surface antigen immune complexes (HBsAg-ICs) by an antigen-specific method. I. Detection and quantitation of in vitro prepared HBsAg-ICs

An antigen-specific method has been developed for direct detection and quantitation of HBsAg-ICs. The method involves (1) precipitation of HBsAg-ICs with 3.5% PEG; (2) dissociation of the PEG precipitated ICs by treatment with NaSCN, NaI, KBr, low or high pH buffers, trypsin or papain; and (3) detection and titration of HBsAg and/or anti-HBs liberated from ICs. Treatment with 2 M and 3 M NaSCN, papain or trypsin liberates HBsAg, while following treatment with 3 M and NaI free anti-HBs is detectable. Trypsin digestion (2 mg/ml, 30 min at 37 degrees C) proved to be most effective for disrupting HBsAg-ICs formed at equivalence as well as in excess of antigen or antibody. After trypsin digestion of the sample RPHA, RIA and ELISA may be used as a third step. Th     

Detection of experimental bacteremia and fungemia by examination of buffy coat prepared by a micromethod.

Rabbits received intravenous injections of bacteria or fungi, and a comparison was made of the abilities of broth cultures, plating after dilution either in saline solution or in distilled water containing Triton X-100, and buffy coat examinations to detect the organisms in heart blood. The most sensitive method was broth culture. By microscopy or subculture of buffy coat cells prepared by centrifugation of blood in microhematocrit tubes, organisms were rapidly and regularly detected when their viable counts increased to 300--1,000/ml as detected by plating. By micromodification, buffy coat examination is technically easy to perform, and the method is only slightly less sensitive than when a larger amount of blood is used. Thus, it would be ideal for rapid provisional diagnosis of sepsis in patients, e.g., neonates, when the use of only a small blood sample is preferred.     

Detection ofChlamydia trachomatis by direct immunofluorescence improved by centrifugation of specimens

During a study of women with laparoscopically investigated pelvic pain, genital tract specimens were examined forChlamydia trachomatis using a direct fluorescent antibody (DFA) technique (MicroTrak, Syva) and culture. Some smears, particularly those from the upper genital tract, contained an inadequate number of cells when examined by the DFA technique and many cell monolayers were destroyed by the specimens. To obtain results for such samples, or to confirm the original DFA result, additional specimens which had been frozen at –70 °C or in liquid nitrogen were centrifuged at high speed and the resulting deposit examined by the DFA technique. By this means, 32 negative results were confirmed for specimens from 10 chlamydia-negative patients with pelvic inflammatory disease or with high chlamydial antibody titres, and 26 negative results were confirmed for 19 patients who were positive at other sites. In addition, three chlamydia-positive and six chlamydia-negative results were obtained for sites where the original smear for DFA testing had been inadequate (few epithelial cells) and six specimens that were negative originally were found to be positive. Thus, of 73 specimens that were either inadequate or negative by DFA testing originally, 9 (12 %) were positive by DFA testing after centrifugation.     

Sperm selection by PD-10 sephadex columns: comparison with SpermPrep filtration and Percoll centrifugation

The efficacy of a disposable, prepacked column (PD-10) containingSephadex G-25, to select motile spermatozoa, was compared withanother column for sperm filtration (SpermPrep) and centrifugationthrough Percoll gradients. Aliquots of washed sperm suspensionswere processed by the three techniques. The number of motilecells and the proportion of total spermatozoa selected was similarfor all methods. Recovery of spermatozoa showing optimal movementwas 145.9 ± 30% (mean ± SEM) with PD-10 columnsand 131.9 ± 32% with Percoll, both significantly higherthan SpermPrep (71.9 ± 11%; P < 0.05). The straightline velocity of motile cells was lower in samples processedby SpermPrep (29.3 ± 2 µm/s) compared to both PD-10(34.7 ± 1 µm/s) and Percoll (34.9 ± 2 µm/s;P = 0.07). When whole semen was processed, total sperm recoverywith PD-10 was 61.7 ± 8% versus 47.7 ± 7% withPercoll (P < 0.001). Percoll centrifugation improved thepercentage of morphologically normal spermatozoa more than PD-10.Similar proportions of motile spermatozoa and cells with optimalmotility were obtained by both methods. We conclude that PD-10filtration columns can be used to prepare semen in the laboratoryas a practical alternative to other methods.     

Detection and quantitation of tetanus antitoxin in blood donations.

Passive haemagglutination and IEOP have been used both to detect and to measure tetanus antitoxin in human donor sera. Forty percent of blood donors had detectable antitoxin but only 9% had levels suitable for production of human antitetanus immuoglobulin (larger than or equal to 2 IU/ml). The incidence of high titre antitoxin was significantly greater in men and was unrelated to the ABO blood group system. The prevalence of antitoxin in selected donor groups and immunized staff is shown.     

Detection of fungemia obscured by concomitant bacteremia: in vitro and in vivo studies.

Our recent clinical experience suggested that bacteremia may interfere with the detection of concomitant fungemia when standard blood culture methods are used. To determine the extent to which bacteria may interfere with fungal isolation from blood cultures, an in vitro model simulating blood cultures taken during concomitant fungemia and bacteremia was created. Each of six bacteria (Staphylococcus epidermidis, Staphylococcus aureus, Streptococcus faecalis, Escherichia coli, Klebsiella pneumoniae, and Pseudomonas aeruginosa) was combined with each of three pathogenic yeasts (Candida albicans, Candida tropicalis, and Torulopsis glabrata) in vented blood culture bottles containing enriched brain heart infusion broth and fresh normal human blood. Blood culture bottles were analyzed at 1, 2, and 7 days of incubation. Gram strains and subcultures onto chocolate and MacConkey agars failed to detect fungi in 37.0, 66.7, and 100% of samples, respectively. However, subcultures onto Sabouraud dextrose agar failed in only 13% of the samples (occurring only with P. aeruginosa). In a rabbit model of concomitant fungemia with C. albicans and bacteremia with P. aeruginosa, no yeasts were recovered from blood cultures despite 100% detection of P. aeruginosa. Therefore, the usual microbiological techniques may be inadequate to detect fungemia when concomitant bacteremia is present.     

Density gradient centrifugation and glass wool filtration of semen remove spermatozoa with damaged chromatin structure.

The ability of double-layered density gradient centrifugation (DGC) or glass wool filtration (GWF) of semen to remove spermatozoa with damaged chromatin structure was assessed by the flow cytometric sperm chromatin structure assay (SCSA), which measures the susceptibility to sperm nuclear denaturation in situ. Ejaculates from 26 men attending a university-affiliated assisted reproduction laboratory were processed by DGC and GWF. Unprocessed, DGC- and GWF-processed specimens were assessed by the SCSA and by conventional semen parameters. Changes in chromatin structure were compared with conventional semen parameters. Both sperm preparation techniques yielded sperm suspensions with improved sperm chromatin structure as well as motility (%), forward progression (1-4) and viability (%). DGC was superior to GWF in the efficiency of recovering motile, morphologically normal, mature sperm suspensions. However, GWF produced improved chromatin integrity (SDalpha(t)) and viability. Moderate correlations between SCSA and conventional sperm parameters were observed. Nevertheless, the SCSA provides additional information about the biochemical integrity of sperm DNA and may be used in future studies to provide insight into assisted reproduction technology outcomes not explained by conventional sperm parameters.     

Controlled evaluation of BacT/alert standard anaerobic and FAN anaerobic blood culture bottles for the detection of bacteremia and fungemia.

FAN medium was formulated to improve microbial recovery, particularly for fastidious microorganisms and for microorganisms causing sepsis in patients receiving antimicrobial therapy. In a controlled clinical evaluation performed at four university-affiliated hospitals, FAN anaerobic bottles were compared with standard anaerobic bottles for yield, speed of detection of microbial growth, and detection of septic episodes. A total of 10,431 blood culture sets were received; both anaerobic bottles of 7,694 blood culture sets were adequately filled with blood. Altogether, 925 isolates were recovered: 557 that were the cause of sepsis, 99 that were indeterminate as the cause of sepsis, and 269 contaminants. More Staphylococcus aureus (P < 0.001), coagulase-negative staphylococci (P < 0.001), Escherichia coli (P < 0.02), and all microorganisms combined (P < 0.005) were recovered from FAN bottles; more nonfermentative gram-negative bacilli (P < 0.05), Torulopsis glabrata (P < 0.001), and other yeasts (P < 0.01) were recovered from standard bottles. Growth of S. aureus (P < 0.001), coagulase-negative staphylococci (P < 0.001), Enterococcus faecalis (P < 0.025), streptococci other than Streptococcus pneumoniae (P < 0.01), and all microorganisms combined (P < 0.001) was detected earlier in standard bottles; growth of more isolates of E. coli (P < 0.05) and anaerobic bacteria (P < 0.01) was detected earlier in FAN bottles. The mean times to detection were 14.2 and 16.1 h for standard and FAN bottles, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)     

Lysis centrifugation and slide blood culture systems for diagnosis of bacteremia in immunocompromised patients

Compared with a slide blood culture system, a lysis centrifugation system detected significantly more positive blood cultures and more episodes of bacteremia in immunocompromised patients (even when on antibiotics), while giving results more rapidly and even at significantly lower CFUs. However, it had a high contamination rate and did not detect Pseudomonas aeruginosa as often. Use of the two systems together is recommended for diagnosing bacteremia early in immunocompromised patients.     

Detection of simulated candidemia by the BACTEC 9240 system with plus aerobic/F and anaerobic/F blood culture bottles

We studied the ability of the BACTEC 9240 automated blood culture system to detect simulated candidemia, including both Candida albicans and non-albicans Candida species. Simulated blood cultures were produced using 50 Candida isolates and BACTEC Plus Aerobic/F and Anaerobic/F blood culture bottles. Ten milliliters of blood and a suspension of each isolate containing 1,000 CFU were introduced into each bottle and then incubated at 35 degrees C in the BACTEC 9240 system. The system detected growth in 56 of 100 bottles. Four isolates did not have growth detected in either bottle after 21 days of incubation, resulting in four missed episodes of candidemia. If the blood culture bottles had been incubated for 5 days, an additional episode of candidemia would have remained undetected. If the bottles had been incubated for only 3 days, another episode would have been missed, resulting in up to six missed episodes of candidemia (four Candida glabrata isolates, one C. albicans isolate, and one Candida rugosa isolate). Terminal subculture of bottles without detected growth recovered yeast in 93% (41 of 44) of the bottles, representing 41 false negatives. In bottles where growth was detected, the time to detection was approximately 24 h. However, the mean time to growth detection for C. glabrata isolates in anaerobic medium was 22.14 +/- 2.47 h, but it was 120.89 +/- 35.33 h in aerobic medium (P < 0.001). The BACTEC 9240 system detected growth of most Candida isolates; however, the delayed time to detection of C. glabrata is clinically significant. Given the high rate of false negatives, terminal subcultures may be helpful in certain situations.