Using urine metabolomics to understand the pathogenesis of infant respiratory syncytial virus (RSV) infection and its role in childhood wheezing.

BACKGROUND: Respiratory syncytial virus (RSV) infection in infants causes significant morbidity and is the strongest risk factor associated with asthma. Metabolites, which reflect the interactions between host cell and virus, provide an opportunity to identify the pathways that underlie severe infections and asthma development. OBJECTIVE: To study metabolic profile differences between infants with RSV infection, and human rhinovirus (HRV) infection, and healthy infants. To compare infant metabolic differences between children who do and do not wheeze. METHODS: In a term birth cohort, urine was collected while healthy and during acute viral respiratory infection with RSV and HRV. We used 1H-NMR to identify urinary metabolites. Multivariate and univariate statistics were used to discriminate metabolic profiles of infants with either RSV ARI, or HRV ARI, and healthy infants. Multivariable logistic regression was used to assess the association of urine metabolites with 1st-, 2nd-, and 3rd-year recurrent wheezing. RESULTS: Several metabolites in nicotinate and nicotinamide metabolism pathways were down-regulated in infants with RSV infection compared to healthy controls. There were no significant differences in metabolite profiles between infants with RSV infection and infants with HRV Infection. Alanine was strongly associated with reduced risk of 1st-year wheezing (OR 0.18[0.0, 0.46]) and 2nd-year wheezing (OR 0.31[0.13, 0.73]), while 2-hydroxyisobutyric acid was associated with increased 3rd-year wheezing (OR 5.02[1.49, 16.93]) only among the RSV infected subset. CONCLUSION: The metabolites associated with infant RSV infection and recurrent-wheezing are indicative of viral takeover of the cellular machinery and resources to enhance virulence, replication, and subversion of the host immune-response, highlighting metabolic pathways important in the pathogenesis of RSV infection and wheeze development.

MicroRNA Profiling from RSV-Infected Biofluids, Whole Blood, and Tissue Samples.

Several studies have shown that respiratory syncytial virus (RSV) can modulate the host innate immune response by dysregulation of host microRNAs (miRNAs) related to the antiviral response, a feature that also affects the memory immune response to RSV (Thornburg et al. MBio 3(6), 2012). miRNAs are small, endogenous, noncoding RNAs that function in posttranscriptional gene regulation. Here, we explain a compilation of methods for the purification, quantification, and characterization of miRNA expression profiles in biofluids, whole blood samples, and tissue samples obtained from in vivo studies. In addition, this chapter describes methods for the isolation of exosomal miRNA populations. Understanding alterations in miRNA expression profiles and identifying miRNA targets genes, and their contribution to the pathogenesis of RSV, may help elucidate novel mechanism of host-virus interaction (Rossi et al., Pediatr Pulmonol, 2015).

Leukotriene synthesis during respiratory syncytial virus bronchiolitis: influence of age and atopy.

Respiratory syncytial virus (RSV) infection is the most common cause of bronchiolitis in infants and an important risk factor for the development of recurrent wheezing and asthma. Cysteinyl leukotrienes were implicated in the pathophysiology of these diseases, and are being targeted for their diagnosis and therapy. We measured urinary leukotriene E4 (LTE4) in infants with RSV bronchiolitis in comparison with controls without respiratory infection, and investigated whether medical and family history, age, and passive exposure to tobacco smoke are related to urinary leukotriene excretion. We studied 33 infants with bronchiolitis and 25 controls, 1-12 months of age. Demographic and historical data were obtained from informed-consent forms and questionnaires completed by the parents. RSV was detected in nasal secretions by enzyme-linked immunoassay. Urine samples were collected on day of admission and were analyzed for LTE4 with an enzyme-linked immunoassay. Urinary LTE4 was 8-fold higher in infants with bronchiolitis than in controls. Leukotriene excretion was significantly higher in infected infants <6 months of age with a medical history of eczema or dry cough and/or family history of asthma. Multivariate analysis revealed that eczema and dry cough are independently associated with high LTE4 excretion during bronchiolitis. Exposure to tobacco smoke did not affect urinary LTE4. Our study shows that leukotriene synthesis during bronchiolitis is particularly elevated in younger infants with an atopic/asthmatic background. Urinary LTE4 may become a valuable, noninvasive marker for the identification of patients who will benefit most from therapy with leukotriene modifiers for management of bronchiolitis.

[Clinical evaluation of urinary leukotriene e4 levels in children with respiratory syncytial virus infection].

The levels of leukotriene E4 (LTE4) of the urine were determined in 24 pediatric patients with infectious diseases due to respiratory syncytial virus (RSV), i.e., bronchitis, pneumonia, and bronchiolitis, and compared with those in controls without allergic disease. The level for LTE4 of the acute-phase urine was 620+/-562 pg/mg. cr in the pediatric patients infected with RSV, being significantly higher than 190+/-67 pg/mg. cr in controls (P<0.005). The levels for LTE4 of the urine in the recovery phase showed a tendency toward decrease, as compared to those in the acute phase. However, there was no significant difference in the level for LTE4 of the acute-phase urine between the presence and the absence of each of the following conditions: expiratory wheezing; the association of pneumonia; family history of allergic diseases; the association of atopic dermatitis; and a past history of expiratory wheezing. An allergological study also revealed that there was no significant difference in LTE4 level between the presence and the absence of peripheral eosinophilia or between the presence and the absence of the high total level for IgE of the serum or positivity for the specific IgE level in the serum. These results suggest that LT is involved with the pathological conditions of RSV infection, but there are no direct relation between atopic diathesis or expiratory wheezing and the amounts of LT production.

[Evaluation of a new ELISA (Bartels) for detection of Legionella pneumophila antigen in urine]. / Evaluación de un nuevo ensayo de ELISA (Bartels) para la detección de antígeno de Legionella pneumophila en orina.

BACKGROUND: Detection of Legionella pneumophila soluble antigens allows rapid diagnosis of pneumonia caused by these bacteria. A new ELISA (Bartels) for antigenuria detection has recently been commercialized. We compared the new ELISA with another well-established ELISA (Binax). METHODS: To evaluate ELISA-Bartels (Legionella Urinary Antigen, Intracel, Issaquah, Washington, United States), urine samples previously characterized by ELISA Binax (Legionella Urinary Antigen Enzyme Immunoassay Kit, Binax, Portland, Maine, United States) were used. Samples came from Legionella outbreaks (n = 48), from sporadic legionellosis (n = 38), and from children with viral pneumonia (n = 21). Samples from the External Quality Control of Legionella of the European Working Group on Legionella Infections (n = 102) were also tested. Of the samples analyzed, 109 were positive in ELISA-Binax, 2 were equivocal and 98 were negative. Samples showing equivocal results were excluded from the analysis. RESULTS: The sensitivity of ELISA-Bartels in comparison with that of ELISA-Binax was 98.2% (107/109) and specificity was 82.7% (81/98). In the 17 samples that were positive in ELISA-Bartels and negative in ELISA-Binax, 10 were positive in ELISA-Binax after concentration by selective ultrafiltration and 6 further cases showed serology indicating or compatible with recent Legionella infection and were thus classified as true positives. CONCLUSIONS: ELISA-Bartels showed good sensitivity and specificity. Sensitivity was even higher than that of ELISA-Binax. Thus, we consider it to be an appropriate method for diagnosis of Legionella pneumonia.

Diagnosis of respiratory virus infections using GACELISA of urinary antibodies.

Sensitive and specific methods are needed to diagnose respiratory virus infections using body fluids such as urine that, unlike blood samples, are readily obtained by non-invasive means. Immunoglobulin G antibody capture enzyme-linked immunosorbent assays were developed for detection of antibody rises to respiratory syncytial virus and influenza A/Taiwan (H1N1) after initial quantification and adjustment of urinary IgG concentration. Of 24 elderly subjects whose sera were assayed by the complement fixation test for antibody to RSV, seven had convalescent titres > or = 32, and five had > or = 4-fold rises in titre. Acute and convalescent urines for six of these seven subjects were tested for virus-specific urinary IgG by GACELISA. Four of four persons with > or = 4-fold rises in CFT had urine ELISA convalescent to acute ratios of > or = 1.8 whereas two subjects with convalescent CF titres > 16, but no increase in serum antibody titre, had urine convalescent/acute ratios of 1.0. Ten subjects with > or = 4-fold rises in CFT or HI antibodies to influenza A/Taiwan had urine ELISA ratios of > or = 1.4 when samples taken on the day of influenza vaccination and 16 days later were compared. These preliminary observations demonstrate clinically significant rises in respiratory pathogen antibody levels between acute and convalescent urine samples, provided that total urinary IgG concentrations are quantified and then standardised.