Clinical features of patients with acute respiratory illness and rhinovirus in their bronchoalveolar lavages

Journal of Clinical Virology 21 (2001) 9 – 16 www.elsevier.com/locate/jcv

Clinical features of patients with acute respiratory illness and rhinovirus in their bronchoalveolar lavages Elsa Malcolm a, Eurico Arruda b, Frederick G. Hayden c, Laurent Kaiser c,* a

Department of Pathology, Uni6ersity of Virginia School of Medicine, Charlottes6ille, VA, USA b Uni6ersity of Sa˜o Paulo School of Medicine, Ribeirao Preto, SP, Brazil c Department of Internal Medicine, Uni6ersity of Virginia School of Medicine, Charlottes6ille, VA, USA Received 28 March 2000; received in revised form 26 July 2000; accepted 17 September 2000

Abstract Background: Several reports in selected populations suggest that human rhinovirus (HRV) may be responsible for lower respiratory tract infections or pneumonia. We describe clinical features of all patients with rhinovirus cultured from their bronchoalveolar lavage (BAL) during a 10-yr period in a tertiary care center. Methods: Results for viral culture of all lower respiratory specimens performed during a 10-year period at the University of Virginia Health Sciences Center were reviewed. A case was defined as any patient with a positive culture for HRV in a BAL specimen. A comprehensive review of the patients’ medical records was performed. In one case, in situ hybridization (ISH) was performed in order to identify whether rhinoviral RNA was present in bronchial biopsy specimens. Results: During the 10-year study period viruses were identified in 431 lower respiratory tract specimens, and were most frequently cytomegalovirus or herpes simplex virus. Twenty patients (ages, 2.5– 86 year) had a bronchoalveolar specimen culture positive for HRV. All had an abnormal chest radiograph, 60% were admitted to the intensive care unit, and 25% expired during their hospitalization. In 18 patients (90%) various severe underlying conditions were identified including solid organ transplants in seven, malignancies in four and AIDS in two. An immunosuppressive disease or condition requiring immunosuppressive therapy was present in all cases. In addition to HRV, one or more potential pathogens were identified in respiratory specimens from 14 patients (70%). Histopathological abnormalities, ranging from fibropurulent debris in alveoli to diffuse alveolar damage, were present in 6 of 13 bronchial biopsies. In two cases without any other significant pathogens than HRV, acute inflammations with fibropurulent debris in alveoli were observed. One lung transplant patient showed intermittent recovery of HRV in her respiratory specimens during a 15-week time period, but ISH did not show HRV RNA in bronchial epithelial cells. Conclusion: Our observations suggest that HRV recovery from BALs or lower respiratory tract samples in highly immunocompromised patients is associated with severe lower respiratory tract illness. Whether HRV directly causes viral pneumonia or predispose to pulmonary injury and/or superinfection remains uncertain. © 2001 Elsevier Science B.V. All rights reserved. Keywords: Human rhinovirus; Bronchoalveolar lavages; Viral pneumonia; Pneumonia

* Corresponding author. Address: Division of Infectious Diseases, University Hospital of Geneva, Rue Micheli-du-Cest 24, 1211 Geneva 14, Switzerland. Tel.: + 41-22-3729802; fax: + 41-22-3729830. 1386-6532/01/$ - see front matter © 2001 Elsevier Science B.V. All rights reserved. PII: S 1 3 8 6 - 6 5 3 2 ( 0 0 ) 0 0 1 8 0 - 3

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1. Introduction Human rhinoviruses (HRV) generally cause self-limited upper respiratory tract illnesses. However, HRV can also be isolated from lower respiratory specimens of patients with severe respiratory tract illnesses (Rabella et al., 1999; Connolly et al., 1994; Kaiser and Hayden, 1999). These observations suggest that HRV may be in some instances responsible for lower respiratory tract infections or pneumonia. Severe HRV lower respiratory illnesses including bronchiolitis and pneumonia have been reported in infants and young children (Kim and Hodinka, 1998; Abzug et al., 1990; Las Heras and Swanson, 1983). In elderly adults HRV infection is associated with lower respiratory symptoms in approximately 60% of cases (Nicholson et al., 1996, 1997; Wald et al., 1995). Although infrequent, HRV infection has also been reported to be associated with pneumonia in immunocompromised hosts (Connolly et al., 1994; Kaiser and Hayden, 1999; Kim and Hodinka, 1998; Abzug et al., 1990; Las Heras and Swanson, 1983; Nicholson et al., 1996, 1997; Wald et al., 1995; Bowden, 1997). A recent study in bone marrow transplant patients suggests that HRV infection may be associated with pneumonia leading to a high mortality rate in this high-risk population (Ghosh et al., 1999). Of note, published reports of patients with a presumed lower respiratory tract HRV infection have focused on selected populations. Thus, little is known about which patients and which conditions might be associated with HRV lower respiratory tract infection. In this retrospective case series, we describe associated conditions, clinical findings, concomitant infections, and outcomes of patients having HRV cultured from bronchoalveolar lavage (BAL) samples during a 10-yr period.

2. Methods

2.1. Patients Results for viral culture of all BALs, bronchial washes, bronchial biopsies or lung biopsies performed between 1988 and 1997 at the University

of Virginia Health Sciences Center were reviewed. In this tertiary care hospital, about one thousand viral cultures from lower respiratory specimens are performed each year. Approximately 40 specimens have positive viral cultures each year and during the examined time period, 1–7 bronchoalveolar specimens were culture positive for HRV per year. A case was defined as any patient with a positive culture for HRV in a BAL specimen. For each case, a comprehensive review of the patients’ medical records was performed. Underlying conditions, clinical features, treatment and antibiotic use, laboratory abnormalities, associated infections and outcome were recorded.

2.2. Rhino6irus isolation BAL fluid was inoculated (0.2 ml) into monolayers of MRC-5 strain of human embryonic lung fibroblasts (Bartels, London, England), Hep-2, A549 and primary monkey kidney cells and incubated in roller drum at 33°C. Monolayers were examined for cythopatic effect every other day during 3 weeks. Primary monkey kidney cell monolayers were hemadsorbed routinely at weeks 1 and 3 with guinea pig red blood cells. Differentiation of rhinoviruses from enteroviruses was done by acid lability testing of picornavirus isolates by standard methods.

2.3. In situ hybridization In situ hybridization (ISH) was performed using DNA probes produced by asymmetric PCR. Total RNA was extracted by affinity chromatography (QIAGEN, Chatsworth, CA) from 100 ml of the original HRV isolate obtained from the BAL of patient 19 in MRC-5 cells. RT-PCR was performed according to published procedures (Pitkaranta et al., 1997). After visualization of a single band of the appropriate size (390 bp) in ethidium bromide stained agarose gel, the PCR product was treated by two consecutive passages through spin column (QIAquick PCR purification kit, QIAGEN, Chatsworth, CA), and each time three washes of the affinity matrix were performed before elution of the PCR product in 50 ml of

E. Malcolm et al. / Journal of Clinical Virology 21 (2001) 9–16

water, in order to minimize contamination of amplicons with non-incorporated primers and nucleotides. Asymmetric PCR was then carried out using as template 5 ml of the product of the first round of amplification in the presence of 0.6 mM 33 P-labeled dCTP and with only the forward or the reverse HRV primer, in order to obtain probes, respectively, for the (+ ) or the (− ) viral RNAs. The PCR buffer was the same as previously published (Pitkaranta et al., 1997) and only three deoxynucleotides (dATP, dGTP and dTTP, 0.6 mM each) were included, in a total reaction volume of 10 ml. A total of 120 cycles of PCR were performed with addition of fresh Taq polymerase after every 30 cycles. Isotope incorporation was calculated by precipitation with 5% trichloroacetic acid and the PCR product was ethanol precipitated twice out of 600 mM ammonium acetate. The ISH assay was performed with this probe as previously published (Arruda et al., 1991) using both cytocentrifuged BAL specimens and bronchial biopsies obtained from patient 19. Positive control slides were prepared with cytocentrifuged HeLa cells infected with the patient’s HRV isolate. Negative controls were non-infected HeLa cells and HRV-infected HeLa cells pretreated with RNase (Arruda et al., 1991).

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were identified throughout the year with no seasonal trends identified. All patients had either symptoms or clinical signs of respiratory tract involvement and all developed an abnormal chest radiograph during hospitalization characterized by an infiltrate, edema-like pattern or opacification. Seven patients also had pleural effusion. Five also underwent thoracic computed tomography, which confirmed the abnormalities observed on the chest radiograph. In seven cases (35%), rhinovirus infection was considered to be nosocomially acquired since respiratory symptoms or signs developed seven days or more after admission. Only two patients had a documented history of upper respiratory tract infection (URI) prior to admission. Nasopharyngeal specimens were obtained during the eight days before or after the BAL in three patients and were positive in only one. Twelve patients (60%) were admitted to the intensive care unit or intubated. Five patients (25%) expired during their hospitalization; all the patients had an active pulmonary process. Four of five patients were treated for a bacterial infection, invasive fungal infection, or metastatic carcinoma.

3.2. Associated conditions 3. Results

3.1. Clinical features and outcomes During the 10-yr study period, 431 lower respiratory specimens had a positive viral culture. The most frequent viruses isolated from these specimens were cytomegalovirus (CMV), herpes simplex viruses, and HRV (Table 1). Twenty cases had a bronchoalveolar specimen culture positive for HRV and consisted of 14 males and 6 females, including three children, with an age range of 2.5 – 86 year. All 20 patients were hospitalized at the time of the bronchoscopic procedure, and eight patients had been hospitalized within the previous six months for a pulmonary illness or were transferred to our hospital for critical pulmonary care. The mean duration of the hospital stay was 36 days (3 – 151). These cases

In 18 of the HRV positive patients (90%) various severe underlying conditions were identified. Seven patients (35%) were solid organ transplant recipients, four of them received grafts during the Table 1 Virus types identified in bronchoscopic lower respiratory tract specimens or biopsies during a 10-yr period at University of Virginia Health Sciences Center Virus identification

Positive culture N= 431

Cytomegalovirus Herpes simplex virus Rhinovirus Adenovirus Influenza/Parainfluenza viruses Echovirus Respiratory syncytial virus

274 (64%) 92 (21%) 28 (7%)a 18 (4%) 13 (3%) 3 (1%) 3 (1%)

a

In 20 patients.

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hospitalization in which HRV was isolated. Bronchoalveolar specimens were obtained an average of 18 days post-transplant (3 – 32 days). Including two lung transplant cases, eight (40%) patients had an underlying pulmonary disease. Four (20%) patients had a malignancy and two had AIDS with B 50 CD4 cell/mm3. During the course of the hospitalization, an immunosuppressive disease or condition requiring immunosuppressive therapy was present in all cases (Table 1). Eighteen patients (90%) were treated with immunosuppressive therapy (cortico-steroids, chemotherapy, or anti-rejection drugs). All of them were receiving prednisone varying in dosages from 5 to 40 mg per day, two patients received chemotherapy and six received cyclosporine, of whom four also received additional immunosuppressive drugs or anti-lymphocyte antibodies. Leukopenia was present in six patients (30%) with a mean white blood cell count of 2400/ml (500 – 3900/ml).

3.3. Associated infections One or more additional pathogens were identified in BAL or respiratory specimens from 14 (70%) of 20 HRV positive patients. Bacteria were detected in 40%, fungi in 45%, other viruses in 25%, and other microorganisms in 10% (Table 2). During the hospitalization bacteremia occurred in three patients, urinary tract infections in two, and abdominal abscess in one. All patients were treated with broad-spectrum antibiotics during their hospitalization.

3.4. Biopsy and autopsy findings Histopathological results were available from 13 patients (65%). Twelve patients had bronchial biopsies and one had an open lung biopsy. In addition, an autopsy was performed on one patient who died during hospitalization. There were no histopathological abnormalities reported in the bronchial biopsies in six of 13 cases. Intranuclear viral inclusions characteristic of CMV were identified in one case, fungal organisms morphologically consistent with Aspergillus sp. in three, acute and chronic inflammation with fibrinopurulent alveolar debris

in two, diffuse alveolar damage with radiation fibrosis in one, and atypical alveolar lining cells in one.

3.5. Case report One patient (Patient 19, Table 2) showed intermittent recovery of rhinovirus in her respiratory specimens during a 15-week time period. This 52-year old female suffered from chronic respiratory failure due to severe emphysema secondary to tobacco. She presented with acute respiratory failure and was placed on cefuroxime and hydrocortisone and intubated and transferred to University of Virginia Medical Center for further management. On admission, she had a normal white blood cells count and right lower lobe lung infiltrate. Five days later, she underwent a right lung transplant. Antirejection drugs included azathioprine, monoclonal anti-lymphocyte globulin, and high doses of prednisone. Post-transplant she required prolonged ventilatory support and serial chest radiographs revealed bilateral pulmonary infiltrates. On posttransplant days three and five, she underwent a BAL/biopsy, both of which were positive for HRV. Subsequent BAL and sputum specimens revealed intermittent presence of HRV during an additional 11-week period (Table 3). Serotyping of these isolates was not performed. Cytomegalovirus, Serratia marcescens and Pseudomonas aeruginosa were also repeatedly isolated. Intravenous ganciclovir was started at the time of transplant and acyclovir, on day seven. Antimicrobial therapy included intravenous gentamicin and ceftazidime. Her discharge diagnoses at the end of her five months hospitalization included pseudomonal pneumonia and cytomegalovirus disease. Six months after discharge, the patient was readmitted for aspiration pneumonia and expired one month later. Autopsy revealed diffuse bronchopneumonia (Pseudomonas aeruginosa) and diffuse alveolar damage in the transplanted lung. Virus cultures were negative. Thirty-six cytocentrifuged preparations from all six BAL samples collected during hospitalization and sections from six bronchial biopsies were tested for HRV by ISH. All assays were negative for HRV RNA, in the presence of properly working positive and negative controls.

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Table 2 Main characteristics of 20 patients with acute respiratory illness and HRV in their BALa Patient

Age (years)

Associated conditions

Associated pathogens in respiratory specimens

Histopathology

1

22

Down’s syndrome, Asthma

–

2

68

Rheumatoid arthritis, COPD

3

26

–

Candida albicans, Cytomegalovirus –

Acute and chronic inflammation, fibropurulent debris in alveoli No abnormalities detected

4

43

5 6 7

2.5 60 52

Neurofibromatosis, Crohn’s disease, Pulmonary hemorrhage Liver transplant COPD, Adenocarcinoma Lung transplant, Neutropenia

8 9 10

3 19 27

Acute myeloid leukemia Liver transplant AIDS

11

7

12

32

Breast cancer with metastasis

Candida albicans, Aspergillus niger

13

59

Chronic lymphoma

14

86

Liver transplant

15

46

Heart transplant

16

60

Lupus erythematosus

17 18 19

57 77 52

Lung trauma Lung trauma Lung transplant

20

45

AIDS

Klebsiella pneumoniae, Enterococcus spp., Candida albicans Staphylococcus aureus, Candida albicans Cytomegalovirus, Herpes simplex virus Aspergillus fumigatus, Cryptococcus neoformans, Nocardia spp. Pseudomonas aeruginosa Acinetobacter spp. Pseudomonas aeruginosa, Serratia marcescens, Cytomegalovirus –

a

Chronic granulomatosis disease

– Klebsiella pneumoniae – Candida albicans, Cytomegalovirus Enterobacter cloacae – Candida albicans, Cytomegalovirus Aspergillus fumigatus

Acute pneumonia, fibropurulent debris in alveoli No abnormalities detected NA NA No abnormalities detected NA No abnormalities detected NA Suppurative granuloma with fungal hyphae Diffuse alveolar damage, radiation fibrosis, invasive aspergillosis No abnormalities detected

NA NA Chronic inflammation, invasive aspergillosis Atypical alveolar lining cells No abnormalities detected Acute and chronic inflammation, viral inclusions NA

NA, not applicable; COPD, chronic obstructive pulmonary disease.

4. Discussion Rhinovirus was the third most common viral respiratory pathogen recovered from lower respiratory specimens at our institution over a 10year period. All patients with HRV in their BAL had an immunosuppressive condition or a pre-existing underlying lung disease. All of these

patients suffered from an acute lower respiratory illness, which suggests that HRV may have played a pathogenic role in their illnesses. These findings are in accordance with several previous reports of HRV lower respiratory tract illness in adults, who invariably presented with an underlying immunosuppressive condition (Rabella et al., 1999; Connolly et al., 1994; Bowden, 1997; Ghosh et al.,

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1999). The frequency of such event has recently been estimated among BMT patients with community respiratory virus infections. In series of 93 patients, 22 had an HRV URTI and seven of them developed pneumonia (Ghosh et al., 1999). HRV infection was documented in approximately 1% of all patients receiving blood or bone marrow transplantation during the five years of this study. Like our study, a high frequency (64%) of nosocomial acquisition was found. Severe respiratory illnesses associated with HRV infection have also been described in young infants, the majority of whom were in the first months of life or suffered from underlying conditions (Kim and Hodinka, 1998; Abzug et al., 1990; Las Heras and Swanson, 1983). A causal link between episodes of lower respiratory tract illnesses and HRV infection can only be established if HRV replication is observed within cells from the lower respiratory tract. We attempted this using ISH in one patient who shed HRV for several weeks and from which bronchial biopsy tissues were available. In this case, we did

not find evidence of HRV RNA in bronchial epithelial cells. However, these results should be interpreted in keeping with the fact that during HRV infection only scarce numbers of HRV infected epithelial cells are found in the upper respiratory tract epithelium amidst non-infected cells (Arruda et al., 1995). Among 78 nasal biopsies performed in 26 subjects within five days after experimental HRV less than half of the samples were ISH positive and only very low numbers of ISH-positive ciliated epithelial cells were found. In addition, in our study, the specimens tested were not handled with special care to minimize RNA degradation, which might also partially explain our negative results. Thus, small size samples, sub-optimal processing and scant numbers of infected cells may have resulted in a false negative result. The ability of HRV to replicate in the lower respiratory epithelium has recently been confirmed in 10 experimentally infected adults. Four days after intranasal and aerosols HRV inoculation the presence of viral RNA has been identified in the bronchial epithelium in 4 of 10

Table 3 Culture results of respiratory specimens in a lung transplant recipients shedding HRV throughout a 15-week time period Date

Specimen

4/28

BAL/Bronchial biopsya BAL/Bronchial biopsy Sputum

4/30 5/2 5/17

8/10

Nasopharyngeal secretions BAL/Bronchial biopsy BAL/Bronchial biopsy BAL/Bronchial biopsy BAL/Bronchial biopsy Sputum

8/22

Sputum

5/25 6/4 6/17 7/27

a

Culture

Histology

Virus Rhinovirus

Other Negative

Non-specific inflammation

Rhinovirus

Negative

Non-specific inflammation

Negative

–

Negative

Serratia marcescens Negative

Rhinovirus and Cytomegalovirus Cytomegalovirus

Serratia marcescens Negative

Non-specific inflammation

Rhinovirus

Negative

Acute cellular rejection, Cytomegalovirus inclusions

Negative

Pseudomonas aeruginosa Pseudomonas aeruginosa Pseudomonas aeruginosa

Non-specific inflammation and atypical respiratory cells

Rhinovirus Negative

–

Cytomegalovirus inclusions

– –

Results of the BAL and the bronchial biopsy performed on the same day were pooled.

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subjects (Papadopoulos et al., 2000). While the results of this report should be confirmed in naturally infected persons, there are strong arguments in favor of the ability of HRV to replicate in the lower respiratory tract. Ruling out contamination from the upper respiratory tract in patients with HRV in their BAL is difficult and in rare instance HRV might be recovered from asymptotic persons (Gwaltney, 1997). In our series, a previous history of URI suggesting HRV infection was documented in a minority of cases, and in two of three nasopharyngeal washes performed at the same period of the BAL, HRV cultures were negative. Of note, the retrospective nature of our study may have been associated with a poor recognition of previous upper respiratory tract events. In one case, BAL specimens were positive for HRV during several weeks, a period during which cultures of nasopharyngeal wash specimens were negative. The presence of HRV in the lower respiratory tract could be explained by dissemination of HRV from the nasopharynx. If favorable conditions for HRV replication exist, HRV may replicate in the tracheobronchial tree. The nasal passage temperature, 33–34°C, is the optimal temperature for HRV replication but data suggest that bronchi temperature is lower than core temperature and approaches the nasal passage temperature (Halperin et al., 1983). In addition, it has been shown recently that some HRV serotypes can replicate effectively in vitro at 37°C (Papadopoulos et al., 1999). Under in vitro conditions HRV grows well on alveolar epithelial cell line (Johnston et al., 1998). These observations suggest that the lower respiratory tract is not likely to be a prohibitive environment for HRV replication. Other indirect evidence comes from the fact that it is possible to recover HRV RNA in BAL specimens several days after experimental HRV inoculation (Halperin et al., 1983; Papadopoulos et al., 1999; Johnston et al., 1998; Gern et al., 1997) and that this RNA is more frequently associated with cells from bronchial specimens compared to BAL fluid. In addition, experimental HRV infection has also been associated with inflammatory changes of the bronchial mucosa in patients with asthma exacerbation (Fraenkel et al., 1995; Folk-

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erts et al., 1998). More recently, the ability of HRV to infect the lower respiratory tract after experimental upper respiratory infection has been conclusively demonstrated showing (using ISH) the presence of replicative strand of HRV RNA in the bronchial epithelium (Papadopoulos et al., 2000). Whether inflammatory changes observed in these experimental studies are produced by direct HRV invasion or indirectly by production of various cytokines and pro-inflammatory mediators is not clear. Interstitial pneumonitis and diffuse alveolar damage have been described in bone marrow transplant patients with presumed rhinoviral pneumonia (Ghosh et al., 1999). In immunocompetent patients experimentally infected by HRV bronchial mucosal lymphocytic and eosinophilic infiltrate have been observed (Fraenkel et al., 1995). In our study, in two cases without any other potential pathogens than HRV, acute inflammation with fibropurulent debris in alveoli were observed. Although no specific findings can be related to HRV, these observations are consistent with other observations showing that HRV can lead to an acute lower respiratory tract inflammatory response and possibly pneumonitis. However, the limited amount of material (transbronchial biopsies in the majority of cases), the high frequency of concomitant infections, and the presence of underlying pulmonary diseases limit the interpretation of these results. In view of numerous associated diseases and concomitant pulmonary infections (70% of cases) observed in our patients, the pathogenic role of each of these conditions is unclear. The concomitant infections can explain several clinical features and histopathological findings but this does not mean that HRV was necessarily an innocent bystander. In these highly immunocompromised patients it is a common finding to identify multiple concomitant opportunistic infections, and even a moderately pathogenic virus could lead to a severe infection. Our observations suggest that in these patients HRV could be responsible for or predispose to lower respiratory tract illness. In the future, prospective therapeutic trials with specific antiviral drugs might help to provide an answer. Although no anti-HRV drugs are commercially

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available at present, new compounds including the capsid inhibitor pleconaril and the 3C protease inhibitor AG7088 are in development. Oral pleconaril appears to reduce the duration and severity of picornavirus upper respiratory illnesses in immunocompetent adults (Hayden et al., 1999) and is active in experimental coxsackie A21 infection (Schiff and Sherwood, 2000). Pleconaril could be considered for use in immunocompromised patients in whom HRV infections are considered to be causing lower respiratory tract disease. Acknowledgements The authors thank Shirley Kyger of the University of Virginia Clinical Laboratory for her assistance in data collection. Laurent Kaiser is a recipient of a grant from the Geneva University Hospital, Geneva, Switzerland. References Abzug MJ, Beam AC, Gyorkyos EA, Levin MJ. Viral pneumonia in the first month of life. Pediatr Infect Dis J 1990;9:881– 5. Arruda E, Mifflin TE, Gwaltney JM, Winther B, Hayden FG. Localization of rhinovirus replication in vitro with in situ hybridization. J Med Virol 1991;34:38–44. Arruda E, Boyle TR, Winther B, Pevear DC, Gwaltney JM, Jr., Hayden FG. Localization of human rhinovirus replication in the upper respiratory tract by in situ hybridization. J Infect Dis 1995;171:1329–33. Bowden RA. Respiratory virus infections after marrow transplant: the Fred Hutchinson Cancer Research Center experience. Am J Med 1997;102(3A):27–30. Connolly MG, Jr., Baugham RP, Dohn MN, Linnermann CC. Recovery of viruses other than cytomegalovirus from bronchoalveolar lavage fluid. Chest 1994;105:1775–81. Folkerts G, Busse WW, Nijkamp FP, Sorkness R, Gern JE. Virus-induced airway hyperresponsiveness and asthma. Am J Respir Crit Care Med 1998;157:1708–20 Review [192 refs]. Fraenkel DJ, Bardin PG, Sanderson G, Lampe F, Johnston SL, Holgate ST. Lower airways inflammation during rhinovirus colds in normal and in asthmatic subjects. Am J Respir Crit Care Med 1995;151:879–86. Gern JE, Galagan DM, Jarjour NN, Dick EC, Busse WW. Detection of rhinovirus RNA in lower airway cells during experimentally induced infection. Am J Respir Crit Care Med 1997;155:1159– 61.

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