JOURNAL OF CLINICAL MICROBIOLOGY, July 1996, p. 1649–1653 0095-1137/96/$04.0010 Copyright q 1996, American Society for Microbiology
Vol. 34, No. 7
Rapid Diagnosis of Respiratory Syncytial Virus Infections in Immunocompromised Adults JANET A. ENGLUND,1* PEDRO A. PIEDRA,1 ALAN JEWELL,1 KIRTI PATEL,1 BARBARA B. BAXTER,1 2 AND ESTELLA WHIMBEY Acute Viral Respiratory Diseases Unit, Department of Microbiology and Immunology, Baylor College of Medicine,1 and Division of Infectious Diseases, University of Texas M. D. Anderson Cancer Center,2 Houston, Texas 77030 Received 3 October 1995/Returned for modification 7 December 1995/Accepted 4 April 1996
Although rapid antigen detection methods for the documentation of respiratory syncytial virus (RSV) infections are widely used with pediatric patients, these tests have not been prospectively evaluated in immunocompromised (IC) adults. For bone marrow transplant recipients and adult patients undergoing chemotherapy for leukemia who had recent onset of respiratory symptoms, respiratory samples (combined nasal wash [NW]-throat swab [TS], endotracheal tube [ET] aspirate, or bronchoalveolar lavage [BAL] samples) were collected for simultaneous culture and rapid antigen detection with the Directigen test kit (Becton Dickinson, Cockeysville, Md.). NW specimens from hospitalized pediatric patients with suspected RSV infection were also evaluated. Viral quantitation was performed on aliquots of the original specimens. A total of 539 samples from 372 adult patients were evaluated. RSV was isolated from 56 specimens (40 NW-TS, 7 ET aspirate, and 9 BAL specimens). By using culture as the “gold standard,” rapid antigen detection had a sensitivity of 15% for adult NW-TS specimens, 71.4% for ET aspirate specimens, and 88.9% for BAL specimens; the specificity was >97% for all specimen types. Significantly greater viral quantities were present in pediatric NW specimens than in adult NW specimens. In adults, more virus was present in BAL and ET aspirate specimens than in NW-TS specimens. Rapid detection of antigen respiratory samples obtained from the lower respiratory tracts of IC adults is sensitive and specific, but detection in upper respiratory tract samples is insensitive. The lower sensitivity of antigen detection in NW-TS specimens may be due to decreased viral load. A BAL specimen is more sensitive than an NW-TS specimen for the rapid diagnosis of RSV disease in IC adults.
(BAL) specimens (6, 11, 12), lung biopsy specimens (6, 14), endotracheal tube (ET) aspirate specimens (8, 17), and lung tissue (10, 16). In the present study, we prospectively evaluated the use of different specimen sources and laboratory methods for the diagnosis of RSV in symptomatic immunocompromised adults hospitalized at a large cancer center during two consecutive respiratory virus seasons. (This work was presented in part at the 34th Interscience Conference on Antimicrobial Agents and Chemotherapy, Orlando, Fla., 4 to 7 October 1994.)
Respiratory syncytial virus (RSV) infections in young pediatric patients may be diagnosed by rapid methods of antigen detection (3, 15). Detection of RSV antigen by immunofluorescence or enzyme-linked immunosorbent assay (ELISA) methods is commonly performed in many medical centers. Results of such tests are generally well accepted by clinicians because of their excellent sensitivities and specificities, the rapid availability of results, and the relatively low costs of the tests. The impact of RSV infection on immunocompromised adults has been recognized (6, 11, 12, 17), but the evaluation of laboratory diagnostic methods for these high-risk patients has not been systematically examined. A timely and accurate diagnosis of RSV infection has important implications for patient care in terms of the institution of specific therapy, the potential avoidance of antibiotic therapy, possible postponement of chemotherapy, and the initiation of proper infection control. Although nasal wash (NW) specimens have been established as a good source for detecting RSV by culture or antigen detection pediatric patients (9), the preferred specimen for use in the diagnosis of RSV disease in immunocompromised adults has not been evaluated. Previous studies have used various methods to diagnose RSV infections in adults, including serologic assays (2), sputum culture (6), nasopharyngeal swabs (6, 11), nasopharyngeal washes (10), bronchoalveolar lavage
MATERIALS AND METHODS Patients. All adult patients undergoing bone marrow transplantation or receiving intensive chemotherapy for leukemia at the M. D. Anderson Cancer Center, Houston, Tex., were followed prospectively by a team of infectious disease physicians during the respiratory virus seasons of 2 consecutive years (November 1992 through April 1993 and November 1993 through April 1994). Patients were cultured if new evidence of an acute respiratory illness was noted, with signs and symptoms of acute respiratory illness defined as the onset of rhinorrhea, nasal or sinus congestion, otitis media, pharyngitis, cough, dyspnea, or tachypnea, with or without the onset of fever (16, 17). Specimens from pediatric patients with suspected RSV infection who were hospitalized by their attending physician were cultured. The specimens had been obtained from the pediatric patients at the time of admission to the hospital for determination of potential admission to an antiviral study. Patients were young (median age, 3 months), generally had moderate to severe symptoms associated with RSV disease, and were not immunocompromised (5). Specimens obtained from patients receiving ribavirin were excluded from the study. Specimens. Clinical specimens were collected by designated study physicians. For adult patients who were able to cooperate, a throat swab (TS) and NW specimen were collected and combined prior to transport to the laboratory. TS specimens were collected by swabbing the posterior of the throat bilaterally with a cotton swab and then placing the swab in transport medium. NW specimens
* Corresponding author. Mailing address: Department of Microbiology and Immunology, Baylor College of Medicine, One Baylor Plaza, Houston, TX 77030. Phone: (713) 798-5248. Fax: (713) 798-6802. Electronic mail address:
[email protected]. 1649
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TABLE 1. Titers of RSV types A and B in laboratory specimens quantitated by different methods Titer (log10)a
Dilution and quantitation method
RSV type A
Titer RSV type B
Undiluted Immediate processing Immediate freezing 24 h at 48C and frozen
6.4 6 0.1 5.9 6 0.1 4.3 6 0.5
5.1 6 0.1 4.5 6 0.1 2.3 6 0.2
1:100 Immediate processing Immediate freezing 24 h at 48C and frozen
4.4 6 0.1 3.7 6 0.3 1.7 6 0.5
3.1 6 0.1 2.7 6 0.1 1.1 6 0.2
1:10,000 Immediate processing Immediate freezing 24 h at 48C and frozen
1.8 6 0.5 1.3 6 0.6 Undetectable
Undetectable Undetectable Undetectable
a Results represent quantitation from dilutions of four viral isolates; each specimen was evaluated in duplicate. The lower limit of detection by this assay was 25 PFU/ml (1.4 log10). Data shown are means 6 standard deviations.
were collected by instilling 3 to 6 ml of normal saline with a needleless syringe in the nostril of the patient while the patient was sitting upright with the head tilted back. The patient was instructed to hold his or her breath for 10 s and to then lean the head forward and let the saline drip out from the nostril into a medicine cup. The process was then repeated with the other nostril, with a sample volume of approximately 1 to 2 ml per nostril collected from each patient and placed in viral transport medium (veal infusion broth containing bovine albumin, penicillin, gentamicin, and amphotericin B). When a BAL was performed, an aliquot of lavage fluid was placed in viral transport medium. Respiratory secretions from the ET were collected by instilling approximately 3 to 5 ml of sterile normal saline down the ET and then suctioning the contents into a sterile collection trap. For pediatric subjects, NW specimens were obtained by instilling 2 ml of normal saline in the nares through a plastic tube while the child was recumbent, with the head tipped backward. Fluid was aspirated by suctioning with either a 10-ml disposable syringe or wall suction, and the process was then repeated with the other nostril. A total of approximately 1 to 2 ml of secretory fluid was obtained. All specimens were placed in viral transport medium on ice or were refrigerated immediately following collection and were transported to the laboratory on ice. Culture. Specimens were inoculated in cell culture within 4 h of collection onto four cell culture lines (MDCK, LLC-MK2, HEp-2, and WI-38 cells). Cultures were held for up to 2 weeks. RSV was identified by a characteristic cytopathic effect and was confirmed by staining with a fluorescein-conjugated polyclonal or monoclonal antibody. Antigen detection. Specimens were assayed at the time of inoculation into cell culture for the presence of RSV antigen with a rapid ELISA kit (Directigen RSV test kit; Becton Dickinson Microbiology System, Cockeysville, Md.) according to the manufacturer’s directions. Specimens containing visible blood were excluded from analyses of antigen detection results. Results were obtained within 0.5 h and were reported immediately to the responsible clinician. Viral quantitation. In order to determine the effects of storage and freezing of specimens prior to viral quantitation, concentrations of laboratory isolates of RSV subtype A and B were determined by different processing methods. The samples were processed prior to RSV quantitation in one of three ways: (i) immediate quantitation (within 1 h of processing); (ii) quantitation after the sample was snap frozen in a bath of dry ice plus ethanol within 1 h of processing, stored for 2 weeks at 2708C, and then thawed; or (iii) quantitation after storage at 248C for 24 h and then snap frozen, stored at 2708C for 2 weeks, and then thawed. All specimen handling and dilutions were performed at 48C. A quantitative plaque assay was performed on each dilution of each sample by inoculating them in quadruplicate onto 24-well plates. The lower limit of virus detection was 25 PFU/ml (1.4 log10). Frozen aliquots of clinical specimens from adult patients from whom specimens from which RSV grew in cell culture were obtained were used to quantitate viral load. For pediatric patients, viral quantitation was performed with freshly collected specimens (5). Specimens from patients receiving ribavirin were excluded from the study. Aliquots of the original specimens were inoculated in 10-fold dilutions into 24-well plates of HEp-2 cell monolayers and were allowed to incubate for 1.5 h. The inoculum was then aspirated, the wells were overlaid with methylcellulose and medium, and the plates were incubated for 6 to 8 days in 5% CO2. The minimal level of detection in this assay was 5 PFU/ml; unde-
tectable virus at the undiluted level was considered to be at one-half this level for statistical purposes. Statistical analysis. Fisher’s exact test or the chi-square method was used to determine the associations between variables. Student’s exact t test was used to compare the means of continuous variables.
RESULTS On the basis of previous experience, RSV titers in fresh clinical specimens are not significantly affected when the specimens are maintained at 48C in viral transport medium for up to 4 h. Immediate quantitation of viral titers in specimens obtained from immunocompromised adults was not possible because of practical considerations, such as the fact that ,10% of the cultures ever grew RSV. Therefore, we studied the effect of snap freezing different dilutions of various laboratory isolates of RSV subtypes A and B that were handled in different ways. Analysis of these data by analysis of variance with multiple comparisons revealed that all processing methods resulted in significant differences in their geometric mean titers. However, mean titers of virus fell only ,0.5 log10 (only threefold) when comparing samples processed within 1 h and never frozen and samples that were snap frozen and stored for 2 weeks at 2708C (Table 1). Similar results were seen for both RSV subtypes A and B and for virus at high (undiluted), medium (100-fold dilution), and low (10,000-fold dilution) concentrations. By contrast, the concentration of virus stored for 24 h in the refrigerator prior to freezing fell at least 100fold at all three dilutions of virus. Thus, freezing did have an impact on viral quantitation, but the effect of freezing was not nearly as significant as cold storage at 48C. A total of 539 respiratory specimens from 372 symptomatic immunocompromised adults were evaluated by simultaneous culture and antigen detection over a 2-year period. The most common source was combined NW-TS specimens, with 398 samples collected from 260 patients. Sixty-seven ET aspirate specimens were collected from 41 intubated patients, and 74 specimens were collected from 71 patients who underwent BAL. More than 92% of the specimens were actually inoculated into culture within 4 h of collection; the remainder were tested within 24 h of collection. A total of 56 specimens grew RSV in cell culture (40 from NW-TS, 7 from ET aspirate, and 9 from BAL specimens) (Table 2). Other viruses were isolated from 86 additional specimens, including influenza A or B virus (n 5 38), picornavirus (n 5 20), herpes simplex virus (n 5 9), adenovirus (n 5 7), parainfluenza virus (n 5 7), and cytomegalovirus (n 5 5). Two viruses were isolated from two NW-TS specimens (herpes simplex virus and RSV from one specimen and picornavirus and RSV from the other specimen). For adults, the sensitivity of RSV antigen detection in NW-TS samples was only 15%. The sensitivity of antigen detection was significantly higher for BAL and ET aspirate specimens (P , TABLE 2. Sensitivity, specificity, positive predictive value, and negative predictive value for detection of RSV antigen in different specimen types by rapid antigen detection tests, with culture as the gold standarda Specimen source
No. of samples
NW-TS ET aspirate BAL
398 67 74
No. of samples Sensitivity Specificity PPV NPV Ag1/no. of (%) (%) (%) (%) samples Cx1
6/40 5/7 8/9
15.0 71.4b 88.9b
99.4 98.3 98.5
75.0 91.3 83.3 96.7 88.9 98.5
a Abbreviations: Ag1, antigen positive; Cx1, culture positive; PPV, positive predictive value; NPV, negative predictive value. b Significantly more sensitive than NW-TS (P , 0.05).
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TABLE 3. Viral quantitation in specimens from different sources performed with frozen NW-TS, ET aspirate, and BAL specimens from immunocompromised adults and NW specimens from immunocompetent infants and young childrena Specimen type
No. of specimens
GMT (PFU/ml)
% #5 PFU/ml
No. of specimens Ag1/no. of specimens Cx1
IC adult NW-TS IC adult ET aspirate and BAL Pediatric NW
20 16 15
35 714b 1,450,000c
55 25 0
5/16 13/13b 13/13
a
Abbreviations: GMT, geometric mean titer; IC, immunocompromised; Ag1, antigen positive; Cx1, culture positive. For adult immunocompromised patients, the quantity of virus from ET aspirate or BAL specimens is significantly greater than that from the NW-TS combined specimens (P , 0.05). c The geometric mean titer of virus in NW specimens from pediatric patients is greater than that in either specimen type from adults (P , 0.05). b
0.05; Table 2). The specificity of the test was equally high for all specimen types (Table 2). The positive predictive value and the negative predictive values were highest for BAL specimens. No RSV antigen was detected in clinical specimens which grew other viruses except for the two NW-TS samples which grew RSV and another virus. A discrepancy between antigen detection and culture results was noted for 41 of 539 (8%) specimens. Discordant results were found for 37 NW-TS specimens, with the most frequent finding being a sample which grew RSV in culture but in which RSV was not documented by antigen detection (n 5 34). For three additional NW-TS specimens, antigen was detected but cultures of the specimens were negative. However, two of these three specimens with potential false-positives results by antigen detection on the basis of culture as a “gold standard” were highly likely to represent true RSV antigen detection: one specimen was from a patient who provided a subsequent specimen that grew RSV, and one specimen was from a patient who had previously shed RSV, was treated with ribavirin, and had been discontinued from ribavirin therapy 24 h earlier. No additional specimens for culture or information about the third patient was available. For lower respiratory tract specimens, no pattern in the results of culture versus those of antigen detection methods was seen for the discordant samples: RSV was detected by cell culture but not by antigen detection methods in one BAL and two ET aspirate specimens and by antigen detection but not by cell culture in one additional BAL and two additional ET aspirate specimens. Viral quantitation was assessed in NW-TS, ET aspirate, and BAL specimens from immunocompromised adults and NW specimens from hospitalized young children (Table 3; Fig. 1). On the basis of the geometric mean titer of virus, the viral load in pediatric specimens was significantly greater than that in NW-TS or BAL and ET aspirate specimens from immunocompromised adults (P , 0.05). For specimens known to be positive for RSV by culture, more ET aspirate and BAL specimens from adult patients and NW specimens from pediatric patients than adult NW-TS specimens were RSV antigen positive (P , 0.05). No pediatric specimen collected by nasal washing or via the ET had ,5 PFU of virus per ml, whereas 11 of 20 adult NW-TS specimens had ,5 PFU of virus per ml (P , 0.001). Pediatric NW specimens were therefore significantly different from adult NW-TS specimens in terms of both viral antigen detection and viral load. Paired NW-TS and BAL specimens that were both culture positive and that were collected from the same patients were also evaluated. NW-TS and BAL specimens were obtained either simultaneously or within 4 days (mean, 2.8 days) from six immunocompromised adults. For two patients, NW-TS specimens were obtained first, for three patients, BAL specimens were obtained first, and for one patient, the specimens were obtained simultaneously. Only two of six NW-TS specimens
which grew RSV in culture were positive for RSV by antigen detection methods, but all six BAL specimens demonstrated RSV by both culture and antigen detection methods. The quantity of virus in the various adult specimens varied 10,000fold (range, ,5 to 5 3 104 PFU/ml). The geometric mean titer of RSV was approximately twofold higher in the BAL specimens than in the NW-TS specimens for these paired specimens, a difference which was not statistically significant in this small sample. DISCUSSION The accurate and rapid diagnosis of RSV in immunocompromised adult patients is desirable because of the high rates of morbidity and mortality associated with RSV disease in these patients (6, 10, 11, 17), the potential for antiviral therapy (16), and the serious consequences of nosocomial spread (4). Our data indicate that rapid antigen detection tests are specific but insensitive with a combined NW-TS specimen collected from immunocompromised adult patients. This finding is in contrast to results obtained by the method of specimen collection currently used for pediatric patients (9, 12, 15). In our experience, adults are not nearly as willing to allow nasopharyngeal swabs to be obtained than to allow nasopharyngeal washes to be obtained. Nasopharyngeal swabs or aspirates may be relatively contraindicated for patients with thrombocytope-
FIG. 1. Distribution of RSV titer in cell culture for different specimen types collected from immunocompromised adults. The viral titer obtained by the plaque assay in snap frozen specimens compared with the type of respiratory specimen and results of rapid antigen testing for RSV in that specimen type is depicted. Abbreviations: Ag1, positive result by rapid antigen test detection for RSV; Ag2, negative result by rapid antigen test detection for RSV.
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nia because of the potential for bleeding. In the present study, the TS culture was combined with an NW culture as an economical method of enhancing the yield for respiratory viruses from upper respiratory tract specimens (1). We speculate that similar results would be found if NW specimens without TS specimens from adult patients were studied. The detection of RSV antigen in clinical specimens from adults is significantly affected by the site from which the clinical specimen is obtained: upper respiratory tract specimens were less sensitive than lower respiratory tract specimens (BAL and ET aspirate specimens). When samples were obtained from the same patient at or near the same time, the quantity of virus and the sensitivity of rapid antigen detection for the lower respiratory tract specimen were also found to be higher. Similarly, a significantly higher viral load and better results by antigen detection were found for pediatric patients, indicating that the sensitivity of the rapid antigen test may be a function of viral load. RSV is known to infect respiratory epithelial cells. In clinical specimens from the respiratory tract, individual cells containing many viral particles, antigenic material, and other cellular and inflammatory debris are likely to be present. A direct correlation between the threshold quantity of virus necessary for antigen detection and the quantity of virus present in clinical specimens on the basis of the number of PFU of virus may be somewhat simplistic. Nonetheless, more antigen detection tests were positive when more virus was present in clinical samples, and more virus appeared to be present in lower respiratory tract samples than in upper respiratory tract samples obtained from immunocompromised adults. We speculate that viral loads are likely to be higher in immunologically impaired adult hosts than in immunocompetent adults and that RSV antigen detection may be problematic in NW specimens from adults. For example, infection control measures in previous RSV outbreaks have used rapid antigen detection to assess RSV infections in health care workers (4, 16). During one of those outbreaks (16), culture and antigen detection tests were used to identify potentially infected employees. Antigen detection did not identify the presence of RSV in nasal wash specimens from all three health care workers whose specimens subsequently grew RSV (data not provided). In a study by Falsey et al. (7), who evaluated RSV infection in elderly hospitalized patients with acute cardiopulmonary disease or influenza-like illness, RSV was documented by antigen detection tests in only two of seven nasal swab specimens which grew RSV in culture. Thus, the use of antigen detection alone to detect RSV in upper respiratory tract specimens from adult subjects appears to be insensitive. Our study shows that antigen detection in lower respiratory tract secretions from adults is more sensitive than antigen detection in upper respiratory tract samples. In general, sicker patients are more likely than mildly symptomatic patients to undergo diagnostic bronchoscopy. We attempted to account for this effect by evaluating paired upper and lower respiratory tract specimens. To determine definitively the viral load in various secretions in infected patients, simultaneous sampling of both sites in a larger number of patients would be required. Despite the larger quantity of virus in lower respiratory tract samples in these symptomatic immunocompromised adults, however, the total amount of virus remains at least 1,000-fold lower than that seen in previously healthy, RSV-infected infants. Various factors could have influenced the concentration of RSV recovered from our patients. Viral quantitation of clinical specimens from adults was likely to be somewhat affected by freezing prior to quantation (on the order of a threefold de-
J. CLIN. MICROBIOL.
crease). However, the geometric mean viral load in adults was at least 1,000-fold lower than that in pediatric patients, and therefore, it appears that viral load in immunocompromised adults was significantly lower than that in symptomatic pediatric patients. In addition, various volumes of saline were used to collect different specimen types from patients of different ages. The NW specimens obtained from pediatric patients were collected with a small amount of saline (1 to 2 ml), whereas the NW-TS specimens from adults were collected with approximately twice the volume of saline to obtain a clinical sample. The clinical secretions obtained from adult BAL specimens were even more dilute as a result of the standard lavage procedures used to obtain lavage fluid. Even though BAL specimens were diluted more than NW-TS specimens, the amount of virus per milliliter of specimen was higher in the BAL specimens. The use of antigen detection tests with NW specimens is insensitive but is a noninvasive, inexpensive, and relatively specific screening test which could be considered for use prior to bronchoscopy for symptomatic immunocompromised adult patients. For patients at risk for RSV disease who provide an NW specimen that has a negative RSV antigen result, BAL should be considered as a method of obtaining a clinical specimen which may provide a reliable rapid diagnosis. In our experience, BAL samples are also likely to have fewer problems with high viscosity as a result of mucus than sputum or ET aspirate samples. Highly viscous samples can potentially lead to erroneous results (most commonly, false-positive results) both by immunofluorescent techniques (13) and with antigen detection kits. Tests with BAL specimens appear to have optimal sensitivity for antigen detection, and if they are accompanied by culture and other histopathologic studies, they can provide valuable, rapid information to the clinician. ACKNOWLEDGMENTS We thank Sneha Thakar for assistance with virus isolation and Victor Lewis and the physicians and nurses at the M. D. Anderson Cancer Center for assistance with patient identification and specimen collection. This work was supported in part by Public Health Service contract NO1 AI-15103 from the National Institute of Allergy and Infectious Diseases. REFERENCES 1. Baxter, B. D., R. B. Couch, S. B. Greenberg, and J. A. Kasel. 1977. Maintenance of viability and comparison of identification methods for influenza and other respiratory viruses of humans. J. Clin. Microbiol. 6:19–25. 2. Crane, L. R., J. A. Kish, V. Ratanatharathorn, J. R. Merline, and M. F. T. Raval. 1981. Fatal syncytial virus pneumonia in a laminar airflow room. JAMA 246:366–368. 3. Dominguez, E. A., L. H. Taber, and R. B. Couch. 1993. Comparison of rapid diagnostic techniques for respiratory syncytial and influenza A virus respiratory infections in young children. J. Clin. Microbiol. 31:2286–2290. 4. Englund, J. A., L. J. Anderson, and F. S. Rhame. 1991. Nosocomial transmission of respiratory syncytial virus in immunocompromised adults. J. Clin. Microbiol. 29:115–119. 5. Englund, J. A., P. A. Piedra, Y.-M. Ahn, B. E. Gilbert, and P. Hiatt. 1994. High-dose, short-duration aerosol therapy compared with standard ribavirin therapy in children with suspected respiratory syncytial virus infection. J. Pediatr. 125:635–641. 6. Englund, J. A., C. J. Sullivan, M. C. Jordan, L. P. Dehner, G. M. Vercellotti, and H. H. Balfour, Jr. 1988. Respiratory syncytial virus infection in immunocompromised adults. Ann. Intern. Med. 109:203–208. 7. Falsey, A. R., C. K. Cunningham, W. H. Barker, R. W. Kouides, J. B. Yuen, M. Menegus, L. B. Weiner, C. A. Bonville, and R. F. Betts. 1995. Respiratory syncytial virus and influenza A infections in the hospitalized elderly. J. Infect. Dis. 172:389–394. 8. Guidry, G. G., C. A. Black-Payne, K. Payne, R. M. Jamison, R. B. George, and J. A. Bocchini, Jr. 1991. Respiratory syncytial virus infection among intubated adults in a university medical intensive care unit. Chest 100:1377– 1384.
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