Isolation of eastern equine encephalitis virus in A549 and MRC-5 cell cultures.

Eastern equine encephalitis (EEE) has been diagnosed either serologically or by virus isolation. Until now, the recovery of EEE virus has been delegated to reference laboratories with the expertise and resources needed to amplify the virus in a susceptible vertebrate host and/or to isolate and identify the virus in cell culture. We report a case in which EEE virus was recovered directly from a patient's cerebrospinal fluid in A549 and MRC-5 cell cultures. Many clinical virology laboratories routinely use these cells to recover adenovirus, herpes simplex virus, and enterovirus. To the best of our knowledge, this is the first report of isolation of EEE virus in A549 cell culture. This report demonstrates the possibility of recovery of EEE virus in cell culture without the necessity of bioamplification or maintaining unusual cell lines.

Hemorrhage decreases macrophage inflammatory protein 2 and interleukin-6 release: a possible mechanism for increased wound infection.

OBJECTIVE: To determine whether alteration in wound exudate cell immune function occurs after trauma-hemorrhage. BACKGROUND: Although clinical and experimental studies indicate that the rate of wound infection is increased after trauma and hemorrhagic shock, the underlying mechanism for this increased susceptibility remains unknown. METHODS: Male C3H/HeN mice were subjected to a midline laparotomy and polyvinyl alcohol sponges were implanted subcutaneously in the abdominal wound before hemorrhage (35+/-5 mm Hg for 90 minutes and resuscitation) or sham operation. The wound exudate cells from the sponges were harvested on the first, third, and fifth postoperative day and cultured for 24 hours in the presence of lipopolysaccharide (10 microg/ml) or heat-killed Staphylococcus aureus. Interleukin (IL)-1beta, IL-6, monocyte chemotactic protein 1, macrophage inflammatory protein 2, and nitrite levels were determined in the supernatants. The distribution of macrophages and polymorphonuclear leukocytes was assessed in the sponge with and without in vivo injection of S. aureus. The phagocytic activity of isolated wound exudate cells was determined using fluorescent S. aureus. RESULTS: The composition of exudate cells was unaltered by hemorrhagic shock; however, in vivo injection of S. aureus significantly decreased the percentage of macrophages under such conditions. Wound exudate cell phagocytic activity and the release of IL-1beta, IL-6, monocyte chemotactic protein 1, and macrophage inflammatory protein 2 was decreased on the first postoperative day. The release of IL-1beta and IL-6 was also decreased on the third postoperative day in hemorrhaged mice. On the fifth postoperative day, wound exudate cell cytokine production was comparable to that in shams. CONCLUSIONS: Because most wound infections occur early after severe trauma, these results suggest that the dysfunction of wound exudate cells after hemorrhage might contribute to the increased incidence of wound infections. Therefore, attempts to enhance or restore wound cell immune function might be helpful for decreasing the incidence of wound infections in trauma victims.

Outbreak of Shigella sonnei in a clinical microbiology laboratory.

Laboratory technologists (22%) developed infections with Shigella sonnei. The isolates had the same antibiogram and pulse-field gel electrophoresis pattern as an unknown isolate handled by a laboratory student. Covering faucet handles with paper towels during hand washing in the laboratory was protective. No further cases occurred after the laboratory was cleaned with a phenolic agent and a handle-free faucet was installed.

Association of Legionnaires' disease with construction: contamination of potable water?

OBJECTIVE: To describe two cases of nosocomial legionellosis and discuss the epidemiology of this infection. DESIGN: Potable water was collected from multiple sites. Patient and environmental isolates were characterized by the Legionella slide agglutination test and monoclonal antibody subtyping. Concordance among isolates was confirmed by pulsed-field gel electrophoresis (PFGE). SETTING: A 713-bed university-affiliated hospital. RESULTS: There was widespread contamination of potable water with Legionella pneumophila during a period of major construction; cooling towers were without growth of Legionella. One patient's isolate was the same by PFGE as the environmental isolate collected from the water faucet in his room. Control measures included superheating water used in all patient care areas to 75 degrees C for 72 hours and flushing superheated water through faucets and showers; cleaning shower heads with a sonicator washer; and raising the hot water storage tank temperature from 43 degrees C to 52 degrees C. After these interventions, repeat environmental cultures over the next 6 months were without growth of Legionella, and no further cases of nosocomial legionnaires' disease were documented. An association between legionnaires' disease and construction is postulated. Heightened surveillance and preventive measures may be warranted during periods of excavation on hospital grounds or when potable water supplies are otherwise shut down and later repressurized.

Use of urinary gram stain for detection of urinary tract infection in infants.

STUDY OBJECTIVE: To determine whether Gram stain of urine is more sensitive than urinalysis in detecting urinary tract infection in infants. DESIGN: Prospective series. SETTING: Urban teaching hospital emergency department. PARTICIPANTS: Two hundred seven infants 6 months old or less, from whom a catheterized or suprapubically aspirated urine specimen was obtained for culture. INTERVENTIONS: Urinary Gram stain, culture, and urinalysis were performed. With culture results as the validating standard, the Gram stain sensitivity, specificity, and predictive values were compared with urinalysis, including leukocyte esterase, nitrite, pyuria, and bacteriuria. RESULTS: The prevalence of positive cultures was 8.7% (18 of 207). Gram stain had higher sensitivity than overall urinalysis (94% versus 67%, P < .05), higher specificity (92% versus 79%, P < .05), and higher positive predictive value (53% versus 23%, P < .05). CONCLUSION: Urinary Gram stain appears to be more reliable than urinalysis in detecting urinary tract infection in young infants.

A pseudo-epidemic involving bone allografts.

Preimplantation cultures of four sterile bone allograft specimens grew Comomonas acidovorans and Pseudomonas species. An epidemiological investigation, including molecular subtyping methods, revealed that the allograft specimens were contaminated in a microbiology laboratory sonicator water bath.

Polymerase chain reaction amplification of herpes simplex virus DNA from clinical samples.

The polymerase chain reaction was used to amplify herpes simplex virus (HSV) DNA from 79 clinical specimens from the female genital tract, and the results were compared with cell culture. Combining the polymerase chain reaction with visualization of amplified products using a direct gel analysis, HSV DNA was detected in 38 specimens, six of which were negative for virus by cell culture. Hybridization of the amplified products detected HSV in three other specimens. One specimen was positive for HSV by cell culture but negative for viral DNA by polymerase chain reaction. Specimen purification before the polymerase chain reaction improved the detection of viral DNA. Restriction endonuclease cleavage of amplified DNA seen by direct gel analysis, used to differentiate HSV-1 from HSV-2, was correct in each case. The analysis time using the polymerase chain reaction was 8-10 hours, making this technique potentially valuable in the clinical setting of parturition.

Detection of herpes simplex virus using the polymerase chain reaction followed by endonuclease cleavage.

The polymerase chain reaction (PCR) was used to amplify herpes simplex virus DNA using a single set of primers that amplify both herpes simplex virus I (HSVI) and II (HSVII). The viruses can be differentiated by a single restriction enzyme cleavage. Virus from dilutions of HSV-infected A549 cell suspensions were amplified and the infectivity endpoints of cell culture were compared with the PCR, and with another direct detection method, the enzyme-linked immunosorbent assay (ELISA). The PCR was capable of detecting virus at a 10(-4) dilution for both HSVI and HSVII, when the corresponding TCID50 endpoints were 10(-5.9) and 10(-5.7), respectively. The ELISA detected virus only down to the 10(-1) dilution. The amplification procedure showed the greatest sensitivity when an initial protease digestion was followed by filtration. The PCR may have use in detection of HSV in clinical situations in which a rapid result is desirable.

Investigation of atypical western blot (immunoblot) reactivity involving core proteins of human immunodeficiency virus type.

Serum specimens which originally exhibited a narrow (indeterminate) 24-kilodalton core protein (p24) or p24/p55 pattern of reactivity with human immunodeficiency virus (HIV) in the Western blot (immunoblot) test were studied to gather information on antibody specificity. A total of 12 specimens were initially reevaluated with an indirect immunofluorescence assay (IFA), three enzyme-linked immunosorbent assays (ELISAs), and Western blot analyses. Five of the specimens were IFA positive and contained anti-gp160/gp120 antibodies which were observed only when an HIV Western blot antigen rich in gp160 and gp120 was used. The remaining seven serum specimens were nonreactive by IFA and showed variable reactivity in HIV antibody ELISAs. The specimens did not cross-react with core antigens for human T-cell leukemia virus types 1 and 2 or contain detectable levels of HIV p24 antigen. The p24/p55 reactivity of six of the seven indeterminate specimens could be reduced or eliminated by preincubating the specimens with disrupted, HIV-infected H9 cells but not with uninfected H9 cells. The six specimens also exhibited discernible reactivity with recombinant HIV p24 antigen. When an additional 23 indeterminate specimens were assayed, all of the serum specimens were nonreactive by IFA while 65% (15 of 23) showed various degrees of reactivity with the recombinant p24 protein. There was no indication that any of the HIV core antibody reactivity was caused by HIV infection. Indeterminate results for five patients with specific p24 reactivity, who were retested after a period of weeks or months, remained indeterminate for HIV antibody with no significant change in ELISA or Western blot reactivity.

Purification of lipoteichoic acid by chromatography in water-organic solvent systems.

Lipoteichoic acid (LTA), extracted from Streptococcus mutans 10449 by hot aqueous phenol, was partially purified by Sepharose 6B column chromatography in 0.01 M sodium acetate, pH 6.0, containing 0.25 M sodium chloride and 0.001 M EDTA. Nucleic acid and polysaccharide were precipitated from the LTA-containing column peak by the addition of 2 volumes of chloroform-methanol (1:5). The resulting single-phase chloroform-methanol-water (1:5:3) supernatant contained LTA and small amounts of several contaminating substances as indicated by reverse-phase high-pressure liquid chromatography and chemical analyses. LTA was purified further by DEAE-cellulose chromatography, using a concentration gradient of sodium chloride in chloroform-methanol-water (1:5:3). Two column peaks of LTA were found to contain phosphate, glycerol, glucose, and fatty acids at molar ratios of 1:1:0.11:0.10 and 1:1:0.09:0.04, respectively. The LTA polymers contained 18 and 22 repeating units of unsubstituted glycerophosphate and two glucose residues. The LTA in one column peak had two fatty acids per molecule, whereas that in the second peak contained only one. The yield of LTA was 1.68 mg per g of cell dry weight or 65 mg per g of phenol-water-extracted material. The specific activity of the LTA preparation was increased 128-fold by the purification scheme as determined by a erythrocyte-binding assay. Reverse-phase high-pressure liquid chromatography may be used for rapid separation of LTA molecules containing different numbers of acyl groups.

Concanavalin A-induced agglutination of Naegleria.

Concanavalin A (Con A) agglutinated all Naegleria gruberi strains tested but did not agglutinate any N. fowleri strains tested. Agglutination was time and temperature dependent and Con A concentration and ameba concentration dependent over certain ranges. Agglutination increased to maximum up to 1 h incubation with Con A. At least 1 X 10(6) amebae/ml were needed for maximum agglutination, and Con A concentrations higher than 100 microgram/ml did not appreciably increase agglutination. Incubation of 4 degrees C or with 10 mM alpha-methyl-D-mannoside inhibited agglutination of N. gruberi. These data indicate a difference in polysaccharide structure of cell membranes of N. fowleri and N. gruberi.