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11. Foulds J, Murray DM, Chai T, Rosner JL: Decreased permeability of cephalosporins through the outer membrane of Escherichia coli grown in salicylates. Antimicrobial Agents and Chemotherapy 1989, 33: 412--417. 12. Domenico P, Hopkins T, Cunha BA: The effect of sodium salicylate on antibiotic susceptibility and synergy in Klebsiella pneumoniae. Journal of Antimicrobial Chemotherapy 1990, 25: 343-351. 13. Sawai T, Hinano S, Yamaguchi A: Repression of porin synthesis by salicylate in Escherichia coil, Klebsiella pneumoniae, and Serratia marcescens. FEMS Microbiology Letters 1987, 40: 233-237. 14. Trias J, Nikaido H: Outer membrane protein D2 catalyzes facilitated diffusion of carbapenems and penems through the outer membrane of Pseudomonas aeruginosa. Antimicrobial Agents and Chemotherapy 1990, 34: 52-57. 15. Fung-TOMC J, Dougherty TJ, DeOrio FJ, SunichJacobson V, Kessler RE: Activity of eefepime and ceftazidime and eefotaxime-resistant gram-negative bacteria and its relationship to [5-1actamase levels. Antimicrobial Agents and Chemotherapy 1989, 33: 498502. 16. Cornick NA, Silva M, Gorbach SL: In vitro antibacterial activity of bismuth subsalieylate. Reviews of Infectious Diseases 1990, 12, Supplement 1: 9-10. 17. Nikaido H, Liu W, Rosenberg EY: OMP and [Mactamase stability of dipolar ionic ccphalosporins containing methoxyimino substituents. Antimicrobial Agents and Chemotherapy 1989, 34: 337-342. 18. Beggs WH, Andrews FS: Role of ionic strength in salt antagonism of aminoglycoside action on Escherichia eoli and Pseudomonas aeruginosa. Journal of Infectious Diseases 1976, 134: 500--504. 19. Trias J, Nikaido H: Protein D2 channel of the Pseudomonas aeruginosa outer membrane has a binding site for basic amino acids and peptides. Journal of Biological Chemistry 1990, 265: 15680--15684. 20. Fukuoka T, Masuda N, Takenouchi T, Sekine N, lijima M, Ohya S: Increase in susceptibility of Pseudomonas aeruginosa to earbapenem antibiotics in low-aminoacid media. Antimicrobial Agents and Chemotherapy 1991, 35: 529-532. 21. Bierer DW: Bismuth subsalicylate: History, chemistry, and safety. Reviews of Infectious Diseases 1990, 12, Supplement 1: 3--8.

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Comparison of a New Commercial Enzyme lmmunoassay for Rapid Detection of Respiratory Syncytial Virus M.T. Garea*, J.M. Lopez, M.L. P e r e z del Molino, A. Coira, E P a r d o

Two rapid methods for detection of respiratory syneytial virus in respiratory specimens were compared: direct immunofluorescence assay (DFA) with monoclonal antibody and an enzyme immunoassay (EIA) (Test-Pack RSV). Ninetyfive nasopharyngeal washings and aspirates from 51 children were examined; the patients were hospitalized during a winter outbreak of RSV infection in the first trimester of 1990. A total of 41.0 % and 56.8 % of these samples were positive by EIA and DFA respectively. Considering only the 51 specimens collected at the onset of illness, EIA detected 72.5 % positive samples and DFA detected 78.4 %. In comparison with DFA, EIA was 92.5 % sensitive and 100 % specific for the acute phase of illness. When all the samples were taken into account, specificity was maintained but sensitivity fell to 72.2 %. The results show that both methods are useful during the acute phase of the illness, when the viral load is important. However, later on in lhe course of the infection DFA appears to be more sensitive than EIA.

Respiratory syncytial virus (RSV) is a major cause of lower respiratory tract infection in infants and young children (1), having a particularly high morbidity and mortality in prematures and children with underlying pulmonary and cardiac disease or immunodeficiency (2, 3). Diagnosis of RSV infection is becoming of increasing importance not only due to its high prevalence but also for the early initiation of antiviral treatment of seriously ill children, and the prevention of nosocomial spread (4, 5). Viral isolation in tissue culture used to be considered the standard diagnostic method. However, due to the slow appearance of cytopathic effects and the great lability of the virus, this Scrvicio de Microbiologfa, Hospital General de Galicia, Galeras s/n, 15705 Santiago de Compostela, Spain.

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method has been largely replaced by newer, more rapid techniques for the detection of viral antigens in clinical specimens (6). The purpose of this study was to compare two of these methods: an enzyme immunoassay (EIA) and a direct immunofluorescence assay (DFA) for the diagnosis of RSV infection in a pediatric unit.

Materials and Methods. A total of 95 nasopharyngeal washes and aspirates were collected from 51 children hospitalized with lower respiratory tract infections suspected of being caused by RSV. Two series of specimens were included in the study: 51 collected at the onset of the symptoms, and the other 44 consecutive specimens with a sampling time ranging from three to seven days after the first sample. The samples were washed with 5 ml of PBS (pH 7), vortexed and centrifuged at 500 g for ten minutes. This procedure was done twice or repeated until the specimens were virtually free of mucous (7). The cell pellet was tested with both EIA and DFA. DFA was performed as follows: the washed cells were resuspended in a few drops of PBS and 30 lal of this suspension were spread on the well of a microscope slide. Specimens with fewer than 50 cells/well were rejected, as described by Miller et al. (8). The slides were air-dried, fixed in cold acetone for ten minutes, and incubated for 15 minutes at room temperature in a moist chamber with 30 ~tl of undiluted fluorescent RSV monoclonal antibody (bioM6rieux, France); then they were washed twice, five minutes each time, in PBS. The specimens were examined under a fluorescence microscope with a halogen lamp. All the clinical samples were read by two observers, considering as positive the specimens with at least one cell showing specific fluorescence (6, 9, 10,

11). EIA was carried out on the cell pellet, using an RSV Test-Pack (Abbott, USA) following the manufacturer's instructions.

Results and Discussion. Table 1 shows sex and age distribution of the patients from whom the 95 nasopharyngeal washings and aspirates studied were taken. Results show a peak of incidence of RSV infection in the group of children younger than six months (70.5 %), 63.9 % of whom were less than three months old, which are similar to those of Freymuth et al. (12). As reported by Denny et al. (13), we did not find any significant

Eur. J. Clin. Microbiol. Infect. Dis.

Table I: Sex and age distributionof patients.

Age (months) 0-3

4--6

7-12

14

6

I

1

22

Male

9

7

7

6

29

Total

23

13

8

7

51

Female

13-20 Total

Table 2: Results of EIA and DFA for RSV in 95 nasopharyngealwashes. DFA Positive

Negative

Total

Positive

39

0

39

Negative

15

41

56

Total

54

41

95

EIA

Table 3: Results of EIA and DFA for RSV in 51

nasopharyngealwashes taken at the acute phase of the illness. DFA

EIA Positive Negative Total

Positive

Negative

Total

37

0

37

3

11

14

40

11

51

difference in sex distribution (Pearson's chi squared with Yates correction). The results obtained by both methods in the 95 samples tested are summarized in Table 2. Three specimens were initially uninterpretable by EIA; this was associated with difficulties in filtration into the test device due to an excess of mucous. The specimens were diluted 1:3 and retested, as described by Warner et al. (7). All the samples positive with EIA were also positive with DFA. Considering only the samples collected at the onset of symptoms (Table 3), EIA detected 37 positive samples (72.5 %), whereas three additional specimens were positive with DFA (78.4 %).

Vol, 11, 1992

In comparison with DFA, EIA was 92.5 % sensitire and 100 % specific for the acute phase of illness. When all the samples were taken into acCOunt, specificity was maintained but sensitivity fell to 72.2 %. These findings suggest that both EIA and DFA are useful in diagnosis during the aCUte phase of illness, when the viral load is imPOrtant. However, EIA offers the advantage of easier manipulation, which might be a decisive factor when a large number of samples are to be Processed; it offers greater objectivity in the interpretation of results, and it does not require either particularly trained technicians or expensive equipment. Nevertheless, the lack of sensitivity of EIA in comparison to DFA during the evolution of the infection supports the use of DFA in the later stages of the illness; these results are in agreement with those reported by Swenson et al. !14), who presented evidence suggesting that test1rig by immunofluorescence assays may result in Positive results in samples taken up to one week after the onset of illness, when specimens have beCOme negative for RSV by cell culture or EIA.

References 1. Mclntosh K, Chanock RM: Respiratory Syncytial Virus. In: Fields BN, Knipe DM, Melnick JL, Chanock RM, Roizman B, Shope RE (ed): Virology. Raven Press, New York, 1985, p. 1285-1304. 2. Hall CB, Powell KR, McDonald NE, Gala CL, Menegus ME, Suffin SC, Cohen H J: RSV infections in children with compromised immune function. New England Journal of Medicine 1986, 315: 77-81.

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8. Miller HR, Phipps PH, Rossier E: Reduction of non

specific fluorescence in respiratory specimens by pretreatment with N-acetyl eysteine. Journal of Clinical Microbiology 1986, 24: 470-471. 9. Mendoza J, Navarre JM, de la Rosa Rojas M: Evaluation of immunofiuorescence, two enzyme immunoassays and the shell-vial assay for detection of respiratory syneytial virus. European Journal of Clinical Microbiology and Infectious Diseases 1991, 10: 40-42, 10. Tong CY, Samuda GM, Chang WK, Yeung CY: Direct and indirect fluorescent-antibody staining techniques using commercial monoelonal antibodies for detection of respiratory syneytial virus. European Journal of Clinical Microbiology and Infectious Diseases 1989, 8: 728~730. 11. Gardner PS, McQuillin J: Rapid virus diagnosis: application of immunofluoreseence. Butterworth, London 1980, p. 94-97.

12. Freymnth F, Pelitjean J, Pothier P, Brouard J, Norrby

E: Prevalence of RSV subgroups A and B in France from 1982 to 1990. Journal of Clinical Microbiology 1991, 29: 653--655. 13. Denny FW, Collier AM, Henderson FW: The epidcmiology of bronehiolitis. Pediatric Research 1977, 11: 234-236. Swenson PD, Kaplan MH: Rapid detection of 14. respiratory syncytial virus in nasopharyngeal aspirates by a commercial enzyme immunoassay. Journal of Clinical Microbiology 1986, 23: 485-488.

Evaluation of the E test in Testing Susceptibility of Pseudomonas aeruginosa to Tobramycin H. Rautelin*, I. R e n k o n e n , O.V. R e n k o n e n

3. McDonald NE, Hall CB, Suffin SC, Alexson C, Harris

4, 5.

6.

7,

PJ, Manning JA: RSV infection in infants with congenital heart disease. New England Journal of Medicine 1982, 307: 397-400. Welliver RC: Detection, pathogenesis and therapy of RSV infections. Clinical Microbiological Reviews 1988, 1: 27-39. Thomas EE, Book LE: Comparison of two rapid methods for detection of respiratory syncytial virus (Test Pack RSV and Ortho RSV Elisa) with direct immunofluorescence and virus isolation for the diagnosis of pediatric RSV infection. Journal of Clinical Microbiology 1991, 29: 632-635. Smilh MC, Creulz C, Huang YT: Detection of respiratory syncytial virus in nasopharyngeal secretions by shell vial technique. Journal of Clinical Microbiology 1991, 29: 463--465. Warner JL, Whilehurs! N J, Todd SJ, Shalaby 1t, Wall LV: Comparison of Directigen RSV with viral isolation and direct immunofluorescence for the identification of respiratory syneytial virus. Journal of Clinical Microbiology 1990, 28: 480--483.

The E test, a new technique for measuring MICs of antimicrobiai agents with the ease of disc diffusion tests, was evaluated in testing the suseeptibility of 94 clinical isolates of Pseudomonas aeruginosa to tobramycin. The use of the E test was found acceptable; 93 % of the MIC results were within one log 2 dilution step and i 0 0 % were within two log 2 dilution steps when the MICs obtained by the E test were compared to those obtained by the conventional agar dilution method. When the E test was compared to the broth microdilution method the corresponding figures were 84 % and 100 %, respectively. Department of Bacteriology and Immunology, University of Hclsinki, Haartmaninkatu 3, 00290 Helsinki, Finland.

Comparison of a new commercial enzyme immunoassay for rapid detection of respiratory syncytial virus.

Two rapid methods for detection of respiratory syncytial virus in respiratory specimens were compared: direct immunofluorescence assay (DFA) with mono...
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