Rhinovirus (RV) infections occur frequently in both children and adults and are usually associated with asymptomatic or mild upper respiratory tract infection. RVs can however cause lower respiratory tract disease, in particular acute childhood wheeze.1 Maternal and personal history of atopy, prematurity, presence of underlying disease such as bronchopulmonary dysplasia, asthma or cystic fibrosis, immunosuppression, day care attendance, siblings and infection with other viruses have been identified as risk factors for having more severe RV-associated symptoms in young children.1,2 RV load has also been linked to disease severity, but objective and easily measurable biomarkers to predict RV disease severity are lacking.3
Lactate dehydrogenase (LDH) has been recently identified as a marker predicting disease severity in respiratory syncytial virus (RSV) bronchiolitis.4 LDH is an enzyme implicated in the conversion of lactate to pyruvate in the cells of most body tissues. When found extracellularly or in the bloodstream, it indicates cell damage and inflammation.5 Interestingly, high LDH values in nasal washes were associated with a reduced risk of hospitalization, likely reflecting a robust antiviral and inflammatory response in the upper airways, the primary site of RSV replication.4
We hypothesized that LDH could be associated with disease severity in RV wheezy bronchitis in childhood. We therefore measured LDH levels and potential other biomarkers (RV load, antiviral and proinflammatory cytokines) in nasal washes obtained from a cohort of preschool children hospitalized for RV wheezy bronchitis and related them to disease severity.
We retrospectively analyzed nasal washes obtained from preschool children hospitalized because of a diagnosis of RV wheezy bronchitis between April 2007 and November 2009 at the University Children’s Hospital in Bern. Wheezy bronchitis was defined as auscultatory wheeze finding in presence of an upper respiratory tract infection. Inclusion criteria for the study were (1) age < 6 years and (2) presence of RV in nasopharyngeal secretions. Exclusion criteria were (1) prematurity < 37 weeks of gestation, (2) significant co-morbidity such as congenital heart disease or neuromuscular impairment and (3) presence of other respiratory viruses in nasopharyngeal secretions.
Demographic and clinical data were collected from patients’ medical files and included age, sex, weight and height at admission, personal and family history of asthma and atopy, home tobacco exposure, clinical findings at admission (presence of retractions or nasal flaring, fever, respiratory rate, transcutaneous oxygen saturation) and therapy during hospitalization (oxygen administration, bronchodilatators, topical or systemic steroids, antibiotics, feeding aid). The study protocol was approved by the Ethics Committee of the Canton of Berne.
Nasal Washes and Immune Markers
At admission, nasal washes were obtained in all children by rinsing the nose with 2 mL of saline and immediately tested by Direct Immunofluorescence for following respiratory viruses: Adenovirus, RSV, Influenza virus A and B, Parainfluenzavirus 1–3, Metapneumovirus and Picornavirus.6 Only samples positive for picornavirus and negative for any other virus were considered for analysis.
Presence of RV genome was confirmed in picornavirus-positive samples and quantification of RV load was performed using a one-step real-time reverse transcriptase polymerase chain reaction assay.7 Levels of the cytokines and chemokines IL-4, IL-6, IL-8/CXCL8, IP-10/CXCL10, IL-13, interferon (IFN)-γ and IFN-λ1/3, which have all been shown to be involved in immune responses against RV, were quantified by ELISA according to manufacturer’s instructions (R&D, Minneapolis, MN). Eosinophilic cationic protein was measured by ELISA by following manufacturer’s recommendations (Wuhan EIAab Science, Wuhan, China) and the total release of LDH was quantified by using a LDH detection kit (Roche, Switzerland). IFN-λ1 mRNA levels were measured by quantitative reverse transcriptase polymerase chain reaction. The detection limits of the assays were: RV load 330 copies/mL, IL-4 20 pg/mL, IL-6 5 pg/mL, IL-8/CXCL8 30 pg/mL, IP-10/CXCL10 2 pg/mL, IL-13 40 pg/mL, IFN-γ 10 pg/mL, IFN-λ1/3 25 pg/ml, eosinophilic cationic protein 1.5 ng/mL, LDH 100 U/mL and IFN-λ1 mRNA 250 copies/mL.
Outcome Parameters and Statistical Analysis
Clinical score at admission adapted for age,8 duration of hospitalization, oxygen administration, frequency of bronchodilator inhalation and use of steroids (both topical and systemic) were used as outcome variables assessing disease severity (see Table, Supplemental Digital Content 1, http://links.lww.com/INF/B925, for clinical score at admission). To investigate relationships between nasal wash markers and outcome variables, linear or logistic regression models adjusting for age and atopy were performed. Non-normally distributed predictors were log-transformed. Statistical analysis was computed with STATA 11.2 for Windows (Stata Corp, College Station, TX). Data are presented as median (range) unless otherwise specified.
One-hundred and twenty-six children (64% males) with a median [range] age of 1.65 [0.40–5.81] years were included in the study. Personal and family history or atopy was positive in 33 (26%) and 75 (60%) children, respectively. Median duration of hospitalization was 3 (2–6) days, median clinical score at admission was 4 (0–8). One hundred-three (81.6%) children received oxygen with a median duration of 1.5 days (0–4). All were administered bronchodilators, 41 (34%) were given topical steroids and 73 (60%) systemic steroids. None received antibiotics and 25 (20%) needed feeding aid.
RV was measurable in all 126 samples with a median of 1.60·108 copies/mL (9.71·105 to 1.60·1010). Median levels of IL-6 were 93 pg/mL (5–3414), of IL-8/CXCL8 7918 pg/mL (30–11989), of IP-10/CXCL10 4053 pg/mL (10–25143) and of IFN-λ mRNA 1.06·106 copies/mL (372–4.34·106). The protein levels of IL-4, IL-13, eosinophilic cationic protein, IFN-λ1/3 and IFN-γ were below detection limit.
LDH was measurable in all samples but one with a median of 301.4 U/mL (42.2–2011.4). LDH levels were related to age at admission (r = 0.3, P < 0.001) but not to RV load and to any of the cytokines/chemokines measured. There was a trend for lower LDH values in children who received oxygen therapy compared with those who did not [278.2 U/mL (42.2–2011.4) vs. 410 U/mL (111–1942), p= 0.10]. In a logistic regression model adjusting for age and atopy, LDH levels were inversely related to the need of oxygen therapy (OR 0.52, 95% CI: 0.28–0.96, P = 0.038), but not associated with the other outcome variables (Fig. 1). Neither RV load nor any of the other cytokines/chemokines measured were related to any outcome variable.
In the present study, we identify LDH levels in nasal washes as a biochemical indicator of severity of RV wheezy bronchitis. We observed that nasal wash LDH levels were significantly higher in children not requiring oxygen administration compared with those who needed oxygen, likely reflecting a robust innate immune response towards the virus and therefore a better clinical course.
LDH is an enzyme measured in clinical routine in serum as a marker of cell damage and inflammation. Intuitively, one would expect higher LDH levels in nasal washes in more severe disease, as the enzyme is released extracellularly upon tissue injury. However, and in accordance with recent findings in RSV bronchiolitis showing that higher nasal wash LDH levels were strongly associated with a reduced risk for hospitalization,4 we found that high LDH levels correlated with a better clinical outcome. Although it has long been assumed that exaggerated and aberrant immune responses are the main drivers of disease severity in virus-induced bronchiolitis and wheezy bronchitis, recent evidence suggests that innate immune responses during respiratory virus infections are mainly protective and that the degree of the inflammatory response is inversely related to disease severity.9,10 Thus, children exhibiting an insufficient inflammatory response, for instance because of an immaturity of their immune system or a status of immunodeficiency, are the ones at risk for a more severe course of the disease. Alternatively, LDH levels in nasal washes may reflect the activation of inflammatory cells rather than airway epithelial cell damage. It has indeed been suggested that the main source of LDH in respiratory secretions are polymorphonuclear leukocytes and alveolar macrophages, and LDH levels in respiratory secretions are unrelated to serum levels.4
This study has important limitations that should be considered when interpreting the data. Its design is retrospective and it only includes a small number of patients. Further, only a correlation between LDH values and oxygen requirement, an indirect parameter of disease severity, was found. Therefore, prospective studies in larger populations are required to determine whether nasal wash LDH levels can be used in daily practice for help in clinical decision making.
In summary, our data demonstrate LDH levels in nasal washes indicate disease severity not only in infants with RSV bronchiolitis, but also in children with RV wheezy bronchitis. The measurement of LDH in nasal washes may represent a promising fast and inexpensive test predicting disease severity in respiratory viral disease.
The authors thank all the study participants and their families for their participation. Special thanks to Lara Turin from the Laboratory of Virology, Division of Infectious Diseases, University of Geneva Hospitals and Faculty of Medicine, University of Geneva for her technical and scientific support.
This work was supported by a grant of the Ulrich Müller-Gierok Foundation to N.R.
1. Kieninger E, Fuchs O, Latzin P, et al. Rhinovirus
infections in infancy and early childhood. Eur Respir J. 2013;41:443–452
2. Miller EK, Williams JV, Gebretsadik T, et al. Host and viral factors associated with severity of human rhinovirus
-associated infant respiratory tract illness. J Allergy Clin Immunol. 2011;127:883–891
3. Takeyama A, Hashimoto K, Sato M, et al. Rhinovirus
load and disease severity in children
with lower respiratory tract infections. J Med Virol. 2012;84:1135–1142
4. Laham FR, Trott AA, Bennett BL, et al. LDH concentration in nasal-wash fluid as a biochemical predictor of bronchiolitis severity. Pediatrics. 2010;125:e225–e233
5. Glick JH Jr. Serum lactate dehydrogenase
isoenzyme and total lactate dehydrogenase
values in health and disease, and clinical evaluation of these tests by means of discriminant analysis. Am J Clin Pathol. 1969;52:320–328
6. Sadeghi CD, Aebi C, Gorgievski-Hrisoho M, et al. Twelve years’ detection of respiratory viruses by immunofluorescence in hospitalised children
: impact of the introduction of a new respiratory picornavirus assay. BMC Infect Dis. 2011;11:41
7. Schibler M, Yerly S, Vieille G, et al. Critical analysis of rhinovirus
RNA load quantification by real-time reverse transcription-PCR. J Clin Microbiol. 2012;50:2868–2872
8. Midulla F, Scagnolari C, Bonci E, et al. Respiratory syncytial virus, human bocavirus and rhinovirus
bronchiolitis in infants. Arch Dis Child. 2010;95:35–41
9. Bennett BL, Garofalo RP, Cron SG, et al. Immunopathogenesis of respiratory syncytial virus bronchiolitis. J Infect Dis. 2007;195:1532–1540
10. Welliver TP, Garofalo RP, Hosakote Y, et al. Severe human lower respiratory tract illness caused by respiratory syncytial virus and influenza virus is characterized by the absence of pulmonary cytotoxic lymphocyte responses. J Infect Dis. 2007;195:1126–1136