IN VITRO METHOD FOR DETERMINING THE PROBABILITY OF DEVELOPING RECURRENT WHEEZING AND ASTHMA AFTER BRONCHIOLITIS CAUSED BY RESPIRATORY SYNCYTIAL VIRUS

Abstract

This study relates to the detection of respiratory diseases using molecular markers as a prognostic tool for the evolution of respiratory infections. Specifically, during the differential diagnosis of respiratory infections caused by Respiratory Syncytial Virus and Human Metapneumovirus, the expression pattern of the severity markers IL-3, IL-33, and IL-12p40 will be established. The expression patterns of these molecular markers can be determined in biological samples using ELISA assays, flow cytometry, or real-time PCR. Confirmation of the etiological agent of the infection, in combination with the determination of the IL-3, IL-33, and IL-12p40 molecular marker patterns, will indicate a prognosis for disease severity.

Description

The present invention relates to the use of molecular markers as a tool for detecting the evolution of respiratory infections. Specifically, during the differential diagnosis of respiratory infections caused by Respiratory Syndrome Virus (RSV) and Human Metapneumovirus (hMPV). Specifically, the present invention describes the use of the markers IL-3, IL-12p40 and IL-33, for the prediction of the occurrence of recurrent respiratory conditions as a consequence of bronchiolitis caused by RSV. STATE OF THE ART Respiratory syncytial virus (RSV) is the most prevalent cause of respiratory tract infection and the leading cause of hospitalization for acute lower respiratory tract infection in infants. RSV bronchiolitis has been associated with asthma and recurrent wheezing; however, the mechanisms responsible for this association have not been elucidated. RSV is an enveloped virus belonging to the Paramyxoviridae family and is one of the most prevalent etiological agents of acute respiratory tract infections, causing a large burden on public health systems. RSV causes lower respiratory tract disease in patients of all ages, with the most severe infection occurring in infants under 6 months of age, especially those with underlying chronic conditions. Many infants who experience RSV bronchiolitis during their first months of life develop recurrent asthma and asthma, but the specific mechanisms involved in the immunopathogenesis of RSV bronchiolitis remain poorly defined. Furthermore, current clinical parameters do not allow for predicting the likelihood of developing any of these chronic diseases later in life after an RSV bronchiolitis infection. The inflammation caused by this virus is characterized by massive cellular infiltration, primarily neutrophils, in the lower respiratory tract. These lung alterations have been described as a consequence of RSV infection in airway epithelial cells, alveolar macrophages, and peripheral blood neutrophils, which induce the secretion of specific cytokines and chemokines. These molecules exhibit chemotactic properties on inflammatory cells and other cell types. A Th1 / Th2 cytokine imbalance has also been proposed as a key event in the inflammation with cell recruitment caused by RSV infection. This imbalance could be mediated by eosinophils and other cells associated with allergy induction, such as basophils and mast cells, which may contribute to the development of asthma.Furthermore, some cytokines produced by airway epithelial cells are known to be directly involved in the induction of allergy and asthma, including IL-25, IL-33 and thymic stromal lymphopoietin (TSLP). The state of the art includes some documents that refer to the use of markers as a tool for the differential diagnosis of respiratory diseases, among which are: Patent application WO2012169887A2 relates to a method for predicting the severity of RSV; however, this document works with the markers OLFM4, CD177, MMP8, MMP9, PTX3, IL-8, RANTES, and CD4, and a combination thereof, without mentioning the use of the markers IL-3, IL-12p40, and IL-33. It cannot be inferred from this document that the cytokines IL-33, IL-12p40, or IL-3 would be related to patients who experienced recurrent wheezing or the possibility of developing asthma in the future after RSV infection. Document EP1867734A1 describes the use of molecular markers for the diagnosis and prognosis of diseases. However, the diseases it addresses are cerebrovascular, and it makes no reference to markers for the diagnosis of respiratory diseases. Only a few studies have analyzed the upper and lower respiratory tract regions during RSV bronchiolitis in infants. The objective of the present invention is to disclose the use of the cytokines IL-3, IL-12p40, and IL-33, molecular markers to predict the occurrence of recurrent wheezing in the future (IL-3 and IL-12p40) or possible development of asthma (IL-33), in later stages of life in these infants, due to bronchiolitis caused by RSV infection. DESCRIPTION OF THE FIGURES Figure 1. Figure 1 shows the pattern of cytokine and chemokine production in bronchoalveolar lavage fluid (BALF) from patients infected with RSV compared to controls. Cytokine and chemokine concentrations were determined in BALF from infants with RSV bronchiolitis and from healthy infants as controls, analyzed by Luminex. Cytokine and chemokine concentrations are expressed in pg / ml and grouped according to their median and interquartile range for comparison. The significance of RSV bronchiolitis (N=14) compared to healthy infant controls (N=5) was determined by the non-parametric Mann-Whitney U test (*P-value < 0.05 was considered statistically significant). The production of IL-3, IL-4, IL-10 and IL-13 (a), IL-1 β, IL-6, IL-7 and TNF-β (b), the chemokines CCL2 / MCP-1, CCL3 / MIP 1 a, Eotaxln and IL-8 (c) and IL-12p40, IL-12p70, IFN-y and IL-17 (d) were analyzed in BALF samples. Figure 2. Figure 2 shows the pattern of cytokine and chemokine production in nasopharyngeal aspirate (NPA) samples from RSV-infected patients, compared with The expression of mediators in NPAs from infants with RSV bronchiolitis (N=14) was compared to that of control infants (N=5) by relative quantification (RQ) using TaqMan probes in a quantitative RT-PCR. Specific TaqMan probes were used to amplify the cytokines IL-1α, IL-1β, TNFα, IL-6, and IL-8 (a); and the chemokines CXCL10, CCL2 / MCP-1, CCL3 / MIP-1α, and CCL4 / MIP-1β (b). Relative mediator concentrations are expressed as times the relative induction in controls. Median and interquartile ranges are shown as horizontal bars and rectangles, respectively (a p-value < 0.05 was considered statistically significant). Figure 3. Figure 3 shows the production of cytokines and chemokines in BALF samples from infants with RSV bronchiolitis according to the development of asthma during the follow-up period. The concentrations of cytokines and chemokines: Eotaxin, IL-3, IL-5, TNF-β (a) and IL-12p40, IL-12p70, and IL-7 (b), detected by Luminex in BALF samples from infants with RSV bronchiolitis (N=14), were analyzed according to the development of asthma during the follow-up period. The concentration of mediators is expressed in pg / ml, and the median and interquartile ranges are shown as horizontal bars and rectangles, respectively (an R-value < 0.05 was considered statistically significant). Figure 4. Figure 4 shows the relative quantification of IL-33 messenger RNA induction in nasopharyngeal aspirate from infants with RSV bronchiolitis, analyzed according to family history of atopy. IL-33 gene expression was determined in nasopharyngeal aspirate from infants with RSV bronchiolitis (N=14) with or without a family history of atopy. Results are shown as times the increase in induction relative to controls. Median and interquartile ranges for each group are shown as horizontal bars and rectangles, respectively (a p-value < 0.05 was considered statistically significant). Figure 5. Figure 5 shows the correlation between IL-12p40 and IL-3 in BALF samples from RSV-infected patients with the number of recurrent wheezing episodes. A Pearson correlation was established between IL-12p40 (a) or IL-3 (b) and recurrent wheezing episodes in BALF samples from RSV-infected infants (14). Results are expressed as pg / ml for cytokines and number of episodes during the follow-up period (a p-value < 0.05 was considered statistically significant). DESCRIPTION OF THE INVENTION. The present invention describes the use of the molecular markers IL-3, IL-12p40, and IL-33 as molecular markers for predicting recurrent wheezing (IL-3, IL-12p40) and possible development of asthma (IL-33), in later stages of life in these infants following bronchiolitis caused by RSV, specifically in patients under 12 months of age. The method for predicting the severity of a disease developed by an RSV and hMPV infection consists of: amplify a respiratory secretion sample using a molecular biology procedure such as PCR, using the markers IL-3,1L-12p40 and IL-33; - quantify with some molecular quantification method such as ELISA, the overexpression of the target sequence complementing it with the markers IL-3, IL-33 or IL-12p40; To correlate based on the quantification of the overexpression obtained, the development of the disease caused by RSV: High probability of developing wheezing if a concentration greater than 500pg / ml of IL-12p40 or IL-3 is present in bronchoalveolar lavage fluids; high probability of developing asthma if a concentration greater than 500pg / ml of IL-33 is found in bronchoalveolar lavage fluids. EXAMPLE Previously healthy infants under 12 months of age, hospitalized due to viral bronchiolitis, were enrolled in the study. To qualify for enrollment, patients had to be RSV-positive by immunofluorescence, under 12 months of age, without risk factors for severe illness, and with a progression from mild to moderate illness during their first week, as defined by a clinical score. Infants with chronic medical conditions or who were receiving corticosteroids at the time of enrollment were excluded from the study. During the same period, healthy infants who underwent an invasive procedure for a non-infectious condition (inguinal hernia = 4, and thyroglossal fistula = 1) were also enrolled. Samples from these infants were used as controls. Cytokine and chemokine profiles were evaluated in the upper and lower airways of infants with bronchiolitis due to RSV infection. Clinical outcomes were also assessed over a three-year follow-up period. Nasopharyngeal aspirates and bronchoalveolar lavage fluid (BALF) samples from hospitalized infants with RSV bronchiolitis and healthy controls were collected and analyzed for cytokine and chemokine detection using Luminex and quantitative real-time RT-PCR. Elevated levels of Th2-like cytokines (IL-3, IL-4, IL-10, and IL-13), pro-inflammatory cytokines, and chemokines (IL-1β, IL-6, TNF-β, MCP-1 / CCL2, MIP-1a / CCL3, and IL-8) were observed in BALF samples from infants with RSV bronchiolitis compared to controls. It was identified that those patients who had a family history of atopy produced higher levels of IL-33 than those patients who did not have this family history (Figure 4).Remarkably, a direct correlation was found between IL-3 and IL-12p40 levels in RSV-infected infants and the development of recurrent wheezing later in life. In fact, a significant direct correlation was found between IL-12p40 and IL-3 levels in BALF samples from RSV-infected patients and the number of recurrent wheezing episodes during the follow-up period (Pearson correlation: r = 0.68). ρ=0.0071; r = 0.71; ρ=0.0058, respectively) (Figure 5). These results suggest that IL3 and IL-12p40 are molecular predictors for recurrent wheezing in infants with bronchiolitis caused by RSV. Fourteen patients with RSV bronchiolitis and five controls were enrolled. Infants with RSV bronchiolitis were hospitalized for an average of 4.5 days (range: 1–9 days), with a median severity score of 4. All patients were able to discontinue oxygen therapy within the first two days, prior to the procedure. They experienced no complications from the procedure and were discharged in good condition. All patients were monitored for three years, with no reported hospital readmissions for other reasons. One patient in the control group could not be monitored after 24 months. The clinical variables assessed were physician-reported recurrent wheezing, physician-reported asthma diagnosis, and chronic use of corticosteroid therapy. These characteristics showed no significant differences between the two groups during clinical follow-up.The clinical characteristics are summarized in Table 1. Table 1 Clinical characteristics of registered patients RSV (n=14) Controls (n=5) Age (range) months 2.2 (0-9) 2.8 (1-6) Gender: male / female 06-Aug 03-Feb Parental atopy Jul-14 02-May Clinical score (range) 4 (1-7) - Hospital stay (range) 4.5 (1-9) - Recurrent wheezing Apr-14 02-May Asthma Mar-14 01-May Corticosteroid therapy Jun-14 01-May Cytokine and chemokine production in BALF samples from infants with RSV bronchiolitis Cytokine and chemokine protein concentration levels in BALF samples from infants with RSV bronchiolitis were measured by Luminex compared to those in control infants. Elevated levels of IL-3, IL-4, IL-10, IL-13 (Th2-related cytokines); IL-1β, IL-6, TNF-β (pro-inflammatory cytokines); MCP1 / CCL2, MIP-1a / CCL3, IL-8 (chemokines); IL-12p40 (Th1-related cytokines); and IL-7 (growth factors) were observed in BALF samples from infants with RSV bronchiolitis compared to controls. Significantly reduced levels of Eotaxin (chemokine) and IL-12p70 were also found in BALF samples from infants with RSV bronchiolitis compared to control infants. The production of IFN-γ (Th1-related cytokine) and IL17 (Th17-related cytokine) was not detected in BALF, either from the infected patient or the healthy control (Figure 1). Significantly elevated levels of IL-12p40, IL-12p70 (Th1-related cytokines), IL-3, IL-5 (Th2-related cytokines), TNF-β (a pro-inflammatory cytokine), Eotaxin (a chemokine), and IL-7 (a growth factor) were observed in nasopharyngeal aspirate samples from RSV-infected patients who developed asthma during the follow-up period, compared to patients who did not (Figure 3). Furthermore, a significant increase in IL-33 messenger RNA, normalized to GAPDH, was observed in nasopharyngeal aspirate from RSV-infected patients with a family history of atopy, compared to patients without such a history (Figure 4).

Claims

1. An in vitro method for determining the probability of developing recurrent wheezing and asthma following bronchiolitis caused by Respiratory Syncytial Virus (RSV), characterized in that it consists of: a) obtaining a bronchoalveolar lavage sample previously isolated from a patient; b) amplifying and detecting the markers IL-3, IL-12p40, and IL-33 by PCR and / or ELISA; c) quantifying the overexpression of the markers IL-3, IL-12p40, and IL-33, where: a concentration greater than 500 pg / mL of IL-12p40 and IL-3 indicates a high probability of recurrent wheezing, and a concentration greater than 500 pg / mL of IL-33 indicates a high probability of developing asthma. Sole Claim