Effect of needle changing and intravenous cannula collection on blood culture contamination rates.

STUDY OBJECTIVES: We tested the hypotheses that blood culture positivity and contamination rates were not increased by not changing needles between venipuncture and inoculation of blood culture bottles or by taking blood for culture by freshly inserted IV cannulae. DESIGN: A prospective study of blood cultures collected by venipuncture or IV cannulae taken from an emergency department population. Venipuncture samples were randomized into needle change (standard method) or no needle change before inoculation into blood culture bottles. PARTICIPANTS: Nine hundred forty patients requiring blood cultures after assessment in the ED. INTERVENTIONS: A standard disinfection procedure using 0.5% chlorhexidine in 70% alcohol was used. Blood was collected by venipuncture and inoculated with or without needle change. Blood collected by IV cannula was inoculated with a fresh needle applied to the collection syringe. MEASUREMENTS AND MAIN RESULTS: There was no statistically significant difference in contamination rates for blood collected by venipuncture with no needle change (6.4%) compared with needle change (4.2%, P > .30). No significant difference in contamination rates was noted for blood taken by freshly inserted IV cannulae (4.3%) compared with venipuncture with needle change after sampling (4.2%, P > .90). Some problems with randomization resulted in unequal numbers in the needle-change (286) versus no-needle-change (141) subgroups, and this may have introduced bias. A higher rate of pathogen growth was observed in blood taken by IV cannula (11.4%) compared with the standard method (6.3%) (P < .025). A significantly greater rate of Gram-negative sepsis was noted in the IV cannula group (6.6%) compared with direct venipuncture with needle change (1.1%) and no needle change (4.2%, P < .01). CONCLUSION: The results of this study do not support the practice of changing needles before inoculating blood samples into blood culture bottles. Collection of blood for culture through freshly inserted IV cannulae is associated with a low contamination rate and is an acceptable alternative to direct venipuncture. Sources of bias in this study suggest that further research is needed to determine the optimal technique for collecting blood cultures.

Partial characterization of a cell-free hemolytic factor produced by Helicobacter pylori.

Maximum cell-free hemolytic activity of helicobacter pylori cultured in broth containing 10% horse serum occurred only after the stationary phase of growth was reached, unlike many hemolysins produced by Gram-negative bacteria which are active during exponential growth. This characteristic of the H. pylori hemolytic factor suggested that it might also possess protease activity. However, because no evidence of albumin degradation was found, the hemolysis by cell-free concentrates of H. pylori appears to be due to a unique factor derived from the organism. Because variable hemolysis results were obtained with culture broths lacking albumin or serum, these proteins may act as carriers or stabilizers of the putative hemolysin.

Haemolytic activity of Campylobacter pylori.

The haemolytic activity of several clinical and reference strains of Campylobacter pylori was determined using cell-free preparations of broth-grown organisms and human, horse, guinea pig, rabbit and sheep erythrocytes. Significant levels of haemolysis were produced only when the cell-free preparations were concentrated tenfold. However, three of 14 strains still gave haemolysis values of less than 50% when tested with guinea pig erythrocytes. Significant haemolytic activity could not be demonstrated with preparations derived from bacteria cultured on solid medium. Irrespective of the strain examined or the type of preparation, strong urease activity was demonstrable. Purified urease had no effect on erythrocytes, and thiourea failed to inhibit the haemolytic activity of Campylobacter pylori cell-free preparations. It was concluded that ureolytic activity was not implicated in the lysis of erythrocytes, either by direct action or via the generation of ammonia. Furthermore, the haemolytic activity produced by Campylobacter pylori was found to be due to a secreted factor, possibly a haemolysin.

Detection of Campylobacter pylori DNA by hybridisation with non-radioactive probes in comparison with a 32P-labelled probe.

A dot-blot hybridisation assay for the detection of Campylobacter pylori was used to compare a 32P-labelled probe with two biotinylated probes and a sulphonated probe. The minimum amount of pure C. pylori DNA that could be detected by the 32P-labelled probe was 100 pg, which corresponded to 5 x 10(4) bacteria. A biotin-labelled DNA (biotin-DNA) probe together with the BluGeneTM detection system produced by Bethesda Research Laboratories (BRL), and a sulphonated probe and ChemiprobeTM detection system (Orgenics) gave similar levels of sensitivity; nylon membranes could be used with both these non-radioactive detection systems. However, a photobiotin-labelled DNA (photobiotin-DNA) probe and detection system produced by Biotechnology Research Enterprises S.A. (BRESA) gave optimum results only with nitrocellulose membranes, and was quantitatively 100 times less sensitive than the other types of probe. The detection systems for the biotin-DNA and photobiotin-DNA probes produced non-specific reactions with crude bacterial blots of heterologous organisms; these non-specific reactions could be removed by treating the dot blots with proteinase K, but not by treatment with RNAase. The sulphonated probe and detection system did not give any reaction with heterologous organism blots.

Cross-reaction between Streptococcus pneumoniae and group C streptococcal latex reagent.

The extent of cross-reactivity between Streptococcus pneumoniae and two commercial streptococcal latex grouping reagents was examined. Cross-reactivity with the group C latex reagent was demonstrated in 91% (20 of 22) of blood cultures tested directly and in 97% (73 of 75) of clinical isolates of various serotypes tested after enzymatic extraction. This is important in the clinical laboratory because it may lead to erroneous identification of organisms present in blood cultures.

In vitro killing of erythromycin-exposed group A streptococci by polymorphonuclear leukocytes.

After exposure to erythromycin, group A streptococci were tested for susceptibility to the antimicrobial activity of human peripheral blood neutrophils in the absence of the antibiotic. Bacterial susceptibility to phagocytic killing increased after prior exposure to supra-inhibitory levels of erythromycin for even as brief as three minutes. Extended exposure and higher concentrations of erythromycin increased phagocytic killing. Although the degree of sensitization varied in different strains of streptococci, all strains tested were significantly more susceptible to phagocytic killing after erythromycin exposure. Killing of erythromycin-treated bacteria that occurred in the absence of antibiotic was dependent upon internalization of the bacteria. Thus, the brief exposure of group A streptococci to inhibitory levels of erythromycin increases their susceptibility to phagocytic killing by peripheral blood neutrophils.

Oxygen-independent killing of Bacteroides fragilis by granule extracts from human polymorphonuclear leukocytes.

Granule proteins from human neutrophils were prepared by extraction with acetate, and their antibacterial activity against Bacteroides fragilis was determined. Activity was highly dependent on pH; greatest killing occurred at the most acid pH tested (pH 5.0). Optimum activity was observed at physiological ionic strength and low bacterial numbers. Killing was inhibited by incubation temperatures of less than 37 degrees C. Eight times more extract was required to kill 50% of stationary-phase bacteria, compared with those growing in logarithmic phase. The antibacterial effect of granule extract was destroyed by boiling, but some activity was retained after heating to 56 degrees C and 80 degrees C. Granule extract activity was tested under conditions in which oxygen-dependent antibacterial systems were inhibited. The rate and extent of killing was not affected by anaerobiosis, sodium azide, or cysteine hydrochloride. These results suggest that the activity of granule extract is independent of oxidative antibacterial systems, and therefore, under conditions that occur in anaerobic infections, potent leukocyte granule-associated mechanisms exist for the destruction of B. fragilis.

Bactericidal activity of a granule extract from human polymorphonuclear leukocytes against Bacteroides species.

The microbicidal activity of an acetate extract of human polymorphonuclear leukocyte granules was tested against Bacteroides fragilis, Bacteroides vulgatus, Bacteroides distasonis and Bacteroides thetaiotaomicron. All strains tested were killed by the extract, and there were no significant differences between the different Bacteroides species.

The effect of exposure of Staphylococcus aureus to penicillin on susceptibility to the bactericidal activity of human leukocytes.

The effect of brief exposure of staphylococci to penicillin upon susceptibility to leukocidal activity was studied. Antibiotic pretreated staphylococci were incubated with normal human leukocytes and viability of the bacteria was determined. Penicillin pretreated staphylococci were more susceptible to killing by leukocytes than untreated control bacteria. The extent of sensitization varied between different strains of staphylococci. When staphylococci were exposed simultaneously to leukocytes and penicillin, the bacteria were protected from the lethal action of penicillin. This study demonstrates that phagocytosed staphylococci are protected from penicillin, but prior exposure to the antibiotic increases their susceptibility to the activity of leukocytes.

Enhanced susceptibility of Escherichia coli to intracellular killing by human polymorphonuclear leukocytes after in vitro incubation with chloramphenicol.

The effect of brief exposure to chloramphenicol of a pathogenic strain of Escherichia coli on susceptibility to normal human leukocytes was examined. Leukocytes killed chloramphenicol-pretreated E. coli more efficiently than they did untreated controls. Phagocytosis of pretreated bacteria, as measured by the uptake of radiolabeled bacteria and by direct visual count of engulfed bacteria, was not significantly increased. The decrease in viability was associated with enhanced intracellular killing of phagocytosed antibiotic-damaged bacteria. Chloramphenicol pretreatment altered the frequency distribution of intracellular bacteria by decreasing the number of leukocytes containing multiple stainable bacteria. Leukocytes failed to kill chloramphenicol-pretreated E. coli in the presence of phenylbutazone, which allowed an accumulation of intracellular bacteria. These results indicate that exposure of E. coli to chloramphenicol renders the bacteria more susceptible to intracellular killing and degradation.

Postantibiotic leukocyte enhancement: increased susceptibility of bacteria pretreated with antibiotics to activity of leukocytes.

Exposure of Escherichia coli and Staphylococcus aureus to high levels of certain antibiotics for brief periods of time increases the susceptibility of the organisms to the antimicrobial action of normal human leukocytes. The degree of sensitization varies with the antibiotic used for the pretreatment of the bacteria and is most pronounced in pretreatment of E. coli with chloramphenicol. Studies on the rates of phagocytosis and intracellular killing of E. coli indicate that the postantibiotic leukocyte enhancement effect operates through increased susceptibility of antibiotic-damaged bacteria to the intracellular killing mechanisms of leukocytes. These observations may explain the efficacy of antimicrobial dosage regimens in which drug levels are below inhibitory concentrations for part of the dosage interval.

Enzyme-linked immunosorbent assay for detection of Haemophilus influenzae type b antigen.

An enzyme-linked immunosorbent assay for the detection of Haemophilus influenzae type b antigen was developed. It was able to detect purified polyribose phosphate at concentrations of greater than or equal to 1 ng/ml in cerebrospinal fluid. This was 50 times more sensitive than counterimmunoelectrophoresis with the same antiserum. The sensitivity for polyribose phosphate in urine was similar, but that in serum was about 10 times less. Nonspecific reactions were observed with blood-stained cerebrospinal fluid and some sera. These were differentiated from true positive reactions by a blocking test with unconjugated immune serum. A wide range of organisms was tested for cross-reactivity in the assay. With the exception of a protein A-rich strain of Staphylococcus aureus, they gave absorbances of < 8% of that of the homologous system. In a series of five cases of proven H. influenzae type b meningitis, the sensitivity of the assay with cerebrospinal fluid was confirmed to be at least 2(5) times greater than that of counterimmunoelectrophoresis. The results indicate that the enzyme-linked immunosorbent assay is highly sensitive and specific in detecting H. influenzae type b antigen. The necessity to perform the blocking assay on all sera limits its usefulness for the examination of these specimens. However, it should prove valuable for the detection of the antigen in cerebrospinal fluid and urine.

Relationship between spontaneous abortion and presence of antibody to Toxoplasma gondii.

Five hundred and twenty-three pregnant women were screened to determine whether a relationship existed between spontaneous abortion, and the presence of antibody to Toxoplasma gondii. The percentage of women with this antibody and a past history of spontaneous miscarriage was not statistically different from the percentage of women without antibody and a past history of spontaneous abortion.