Respiratory syncytial virus genomic load and disease severity among children hospitalized with bronchiolitis: multicenter cohort studies in the United States and Finland.

BACKGROUND: We investigated whether children with a higher respiratory syncytial virus (RSV) genomic load are at a higher risk of more-severe bronchiolitis. METHODS: Two multicenter prospective cohort studies in the United States and Finland used the same protocol to enroll children aged <2 years hospitalized for bronchiolitis and collect nasopharyngeal aspirates. By using real-time polymerase chain reaction analysis, patients were classified into 3 genomic load status groups: low, intermediate, and high. Outcome measures were a length of hospital stay (LOS) of ≥3 days and intensive care use, defined as admission to the intensive care unit or use of mechanical ventilation. RESULTS: Of 2615 enrolled children, 1764 (67%) had RSV bronchiolitis. Children with a low genomic load had a higher unadjusted risk of having a length of stay of ≥3 days (52%), compared with children with intermediate and those with high genomic loads (42% and 51%, respectively). In a multivariable model, the risk of having a length of stay of ≥3 days remained significantly higher in the groups with intermediate (odds ratio [OR], 1.43; 95% confidence interval [CI], 1.20-1.69) and high (OR, 1.58; 95% CI, 1.29-1.94) genomic loads. Similarly, children with a high genomic load had a higher risk of intensive care use (20%, compared with 15% and 16% in the groups with low and intermediate genomic loads, respectively). In a multivariable model, the risk remained significantly higher in the group with a high genomic load (OR, 1.43; 95% CI, 1.03-1.99). CONCLUSION: Children with a higher RSV genomic load had a higher risk for more-severe bronchiolitis.

T lymphocytes contribute to antiviral immunity and pathogenesis in experimental human metapneumovirus infection.

Human metapneumovirus (hMPV), a member of the family Paramyxoviridae, is a leading cause of lower respiratory tract infections in children, the elderly, and immunocompromised patients. Virus- and host-specific mechanisms of pathogenesis and immune protection are not fully understood. By an intranasal inoculation model, we show that hMPV-infected BALB/c mice developed clinical disease, including airway obstruction and hyperresponsiveness (AHR), along with histopathologic evidence of lung inflammation and viral replication. hMPV infection protected mice against subsequent viral challenge, as demonstrated by undetectable viral titers, lack of body weight loss, and a significant reduction in the level of lung inflammation. No cross-protection with other paramyxoviruses, such as respiratory syncytial virus, was observed. T-lymphocyte depletion studies showed that CD4(+) and CD8(+) T cells cooperate synergistically in hMPV eradication during primary infection, but CD4(+) more than CD8(+) T cells also enhanced clinical disease and lung pathology. Concurrent depletion of CD4(+) and CD8(+) T cells completely blocked airway obstruction as well as AHR. Despite impaired generation of neutralizing anti-hMPV antibodies in the absence of CD4(+) T cells, mice had undetectable viral replication after hMPV challenge and were protected from clinical disease, suggesting that protection can be provided by an intact CD8(+) T-cell compartment. Whether these findings have implications for naturally acquired human infections remains to be determined.

The search for adenovirus 14 in children in Houston, Texas.

Adenovirus (Ad)14 has recently emerged in the United States causing outbreaks of severe respiratory disease. To determine if Ad14 circulated in Houston, Texas, during the same time as an outbreak in military recruits in nearby San Antonio, 215 pediatric adenovirus isolates were serotyped using microneutralization. None were Ad14; Ad1, Ad2, and Ad3 were the most common identified serotypes.

Development of a cotton rat-human metapneumovirus (hMPV) model for identifying and evaluating potential hMPV antivirals and vaccines.

Hispid cotton rats were inoculated with two different human metapneumovirus (hMPV) subtype A strains and one subtype B hMPV. Although no overt disease was seen in any virus-inoculated animal, following an eclipse phase, significant pulmonary virus titers were observed in every hMPV-inoculated animal through day 7 post virus inoculation (p.i.) and in most through day 10. Peak virus titers occurred four days p.i., while virus-induced histopathology was most evident in lung sections obtained from animals 7 to 10 days p.i. The latter consisted primarily of desquamating and hypertrophic columnar epithelial cells lining the bronchi and bronchioles and the presence of large numbers of leukocytes in and around the bronchi and bronchioles. In fluorescent antibody studies, virus antigen-specific fluorescence was most evident in the desquamating tall columnar epithelial cells lining bronchi and bronchioles, in pneumocytes lining alveoli and in single or small groups of free cells, most probably leukocytes, present in the lumen of alveoli, bronchi and bronchioles. Virus was generally not detected in inoculated animals >10 days p.i. Although the pattern of virus replication in cotton rats was similar for all the three virus stains, the B subtype consistently grew to lower levels than the two A strains. Regardless, these findings indicate that hMPV replicates in cotton rats and that these animals may be used as a small animal model of hMPV infection and to facilitate the identification and development of vaccines and antivirals for preventing and/or ameliorating infections caused by this virus.

Bordetella pertussis is an uncommon pathogen in children hospitalized with bronchiolitis during the winter season.

BACKGROUND: In the United States (U.S.), Bordetella pertussis incidence has increased. Cough and apnea are common findings in pertussis and also in bronchiolitis, the most common cause of hospitalization in U.S. infants. The objective was to determine the prevalence of B. pertussis infection in children hospitalized with bronchiolitis and to describe its clinical course. METHODS: Children hospitalized with bronchiolitis and age <2 years were eligible for a prospective, multicenter cohort study during 3 consecutive winter seasons (November-March) from 2007 to 2010. Sixteen sites in 12 states participated using a standardized enrollment protocol. Families were asked the 2010 Centers for Disease Control and Prevention pertussis classification questions. Nasopharyngeal aspirates were obtained and tested by real-time polymerase chain reaction for 16 viruses, Mycoplasma pneumoniae and B. pertussis. RESULTS: Two thousand sixty-eight (94%) of 2207 children had 1 or more respiratory pathogens. B. pertussis was identified in 4 children [0.2%; 95% confidence interval (CI): 0.1-0.5%] with 3 having a viral co-infection. All 4 were younger than 4 months; 2 met the Centers for Disease Control and Prevention definition of probable pertussis; and 3 had received at least 1 dose of an acellular pertussis vaccine. During the hospitalization, 2 had paroxysmal cough, 1 required intensive care unit care and the median length of stay was 13 days. CONCLUSIONS: Our data support that B. pertussis is an uncommon pathogen in U.S. children hospitalized with bronchiolitis in the winter. Making a diagnosis of pertussis can be challenging because the disease can be atypical and may not meet the Centers for Disease Control and Prevention definition of probable infection.

Comparison of the inhibition of human metapneumovirus and respiratory syncytial virus by ribavirin and immune serum globulin in vitro.

Human metapneumovirus (hMPV) is a newly recognized pathogen that like its better-known relative, human respiratory syncytial virus (hRSV), appears to be ubiquitous and an important cause of respiratory disease in diverse subpopulations. No antivirals or vaccines are currently approved for the treatment or prevention of hMPV infections. However, ribavirin is licensed to treat serious hRSV-induced infections in children and immune globulin designed for intravenous administration (i.v.IG) and palivizumab (Synagis), a humanized monoclonal antibody preparation, have been utilized as alternatives to vaccines for preventing or reducing the severity of infections caused by this virus. Because both ribavirin and i.v.IG have broad viral specificities, studies were performed to compare the ability of these two agents to inhibit the replication of hRSV and hMPV in tissue culture-based assays. Two experimental chemotherapeutic agents (i.e. VP14637 and JNJ2408068) and different antibody preparations were included in this testing for comparison. Ribavirin and the i.v.IG utilized were found to have equivalent antiviral activity against hMPV and hRSV. In contrast, except for antisera specifically raised against hMPV, all of the other materials tested had marked activity only against hRSV.

Gene sequence variability of the three surface proteins of human respiratory syncytial virus (HRSV) in Texas.

Human respiratory syncytial virus (HRSV) has three surface glycoproteins: small hydrophobic (SH), attachment (G) and fusion (F), encoded by three consecutive genes (SH-G-F). A 270-nt fragment of the G gene is used to genotype HRSV isolates. This study genotyped and investigated the variability of the gene and amino acid sequences of the three surface proteins of HRSV strains collected from 1987 to 2005 from one center. Sixty original clinical isolates and 5 prototype strains were analyzed. Sequences containing SH, F and G genes were generated, and multiple alignments and phylogenetic trees were analyzed. Genetic variability by protein domains comparing virus genotypes was assessed. Complete sequences of the SH-G-F genes were obtained for all 65 samples: HRSV-A = 35; HRSV-B = 30. In group A strains, genotypes GA5 and GA2 were predominant. For HRSV-B strains, the genotype GB4 was predominant from 1992 to 1994 and only genotype BA viruses were detected in 2004-2005. Different genetic variability at nucleotide level was detected between the genes, with G gene being the most variable and the highest variability detected in the 270-nt G fragment that is frequently used to genotype the virus. High variability (>10%) was also detected in the signal peptide and transmembrane domains of the F gene of HRSV A strains. Variability among the HRSV strains resulting in non-synonymous changes was detected in hypervariable domains of G protein, the signal peptide of the F protein, a not previously defined domain in the F protein, and the antigenic site Ø in the pre-fusion F. Divergent trends were observed between HRSV -A and -B groups for some functional domains. A diverse population of HRSV -A and -B genotypes circulated in Houston during an 18 year period. We hypothesize that diverse sequence variation of the surface protein genes provide HRSV strains a survival advantage in a partially immune-protected community.

Lactate dehydrogenase and caspase activity in nasopharyngeal secretions are predictors of bronchiolitis severity.

BACKGROUND: Bronchiolitis is the leading cause of hospitalization in infants. Biomarkers of disease severity might help in clinical management. OBJECTIVE: To determine the clinical predictiveness of NW-LDH, NW-caspase 3/7, and NW-LDH/NW-caspase 3/7 ratio in bronchiolitis. METHODS: Previously healthy children less than 24 months of age with bronchiolitis were recruited from the Texas Children's emergency room and intensive care unit from October 2010 to April 2011. Demographic, clinical information, and NW samples were obtained at enrollment. NW samples were analyzed for respiratory viruses, caspase 3/7, and LDH. RESULTS: A viral pathogen was detected in 91·6% of 131 children, with the most common being respiratory syncytial virus and human rhinovirus. A single infection was found in 61·8% of subjects and co-infection in 29·8%. Children admitted to ICU had significantly higher NW-LDH than children sent home from the ER or admitted to the general floor (P = 0·02). Children infected with RSV had the highest NW-LDH concentration (P = 0·03) compared with other viral infections. NW-LDH and NW-caspase were significantly correlated (r = 0·77, P < 0·0001). The univariate models showed NW-LDH and NW-LDH/NW- caspase 3/7 ratio were directly associated with hospitalization. Mutivariate regression analyses suggested a complex interaction between the biomarkers, demographics, and disposition. CONCLUSIONS: NW-LDH, NW-caspase 3/7 and NW-LDH/NW-caspase 3/7 ratio and their interactions with demographic factors are predictive of bronchiolitis severity and can help distinguish children requiring ICU-level care from those admitted to the general floor, or discharged home from the emergency center.

LDH concentration in nasal-wash fluid as a biochemical predictor of bronchiolitis severity.

OBJECTIVE: Because the decision to hospitalize an infant with bronchiolitis is often supported by subjective criteria and objective indicators of bronchiolitis severity are lacking, we tested the hypothesis that lactate dehydrogenase (LDH), which is released from injured cells, is a useful biochemical indicator of bronchiolitis severity. PATIENTS AND METHODS: We retrospectively analyzed a study of children <24 months old presenting to the emergency department with bronchiolitis. Demographic, clinical information, nasal wash (NW), and serum specimens were obtained. NW samples were analyzed for respiratory viruses, caspase 3/7 activity, and a panel of cytokines and chemokines. Total LDH activity was tested in NW samples and sera. RESULTS: Of 101 enrolled children (median age: 5.6 months), 98 had NW specimens available. A viral etiology was found for 82 patients (83.6%), with respiratory syncytial virus (RSV) (66%) and rhinovirus (19%) being the most common viruses detected. Concentrations of LDH in NW specimens were independent from those in sera and were higher in children with RSV infection or with dual infection. Significant correlations were found between NW LDH and NW cytokines/chemokines. Similarly, NW LDH correlated with NW-caspase 3/7 activity (r = 0.75; P < .001). In a multivariate analysis, NW LDH concentration in the upper quartile was significantly associated with a reduced risk of hospitalization (odds ratio: 0.19 [95% confidence interval: 0.05-0.68]; P = .011). CONCLUSIONS: NW LDH levels in young children with bronchiolitis varied according to viral etiology and disease severity. Values in the upper quartile were associated with approximately 80% risk reduction in hospitalization, likely reflecting a robust antiviral response. NW LDH may be a useful biomarker to assist the clinician in the decision to hospitalize a child with bronchiolitis.

Correlates of immunity to respiratory syncytial virus (RSV) associated-hospitalization: establishment of minimum protective threshold levels of serum neutralizing antibodies.

OBJECTIVE: To determine if respiratory syncytial virus (RSV) specific, serum antibody titers correlate with protection against RSV associated-hospitalization at all ages. DESIGN: Participants who were enrolled in a trial to determine the frequency of specific virus infections associated with hospitalization [J. Am. Med. Assoc. 283 (2000) 499] were included in our analysis if they were enrolled from July 1991 to June 1993, had a culture for virus isolation, and provided blood samples at hospitalization and 14-60 days later. RSV infection was defined by a positive culture and/or serology. Microneutralization, ELISA to the fusion (F) protein and Western blot were the serological assays that were used to determine correlates of immunity. RESULTS: One hundred and seventy-five individuals, 1 month to 89 years old, out of 538 patients hospitalized with an acute respiratory infection met the criteria for analysis. RSV associated-hospitalization occurred in 11 (40.7%) of 27 infants (<1 year), 8 (38.1%) of 21 young children (1 to <5 years), and 15 (11.8%) of 127 children and adults (> or =5 years). At the time of hospitalization, geometric mean neutralizing antibody titers (log(2)) to RSV/A and RSV/B, and geometric mean binding antibody titer (log(2)) to F protein were significantly higher in patients with non-RSV associated-hospitalization compared to those with RSV associated-hospitalization (RSV/A: 7.9 versus 6.1, P<0.001; RSV/B: 9.4 versus 7.3, P<0.001; ELISA-F, 13.9 versus 12.6, P=0.01). For every 1 log(2) increase in titer of neutralizing antibodies to RSV/A and RSV/B, and binding antibody to F protein there was a significant increase in the likelihood of not having an RSV associated-hospitalization by 22.3, 25, and 24.4% respectively. A minimal protective threshold titer of > or =6.0 (odds ratio 3.5; 95% CI 1.4-9.1) and > or =8.0 log(2) (odds ratio 2.9; 95% CI 1.1-7.7) against RSV associated-hospitalization was established for neutralizing antibodies to RSV/A and RSV/B; a threshold titer could not be established for binding antibody to F protein. CONCLUSION: Participants with naturally acquired serum neutralizing antibody levels at least equal to the minimal protective threshold titer were approximately three times more likely not to have an RSV associated-hospitalization. We speculate that achieving a minimal protective threshold antibody titer through active immunization will significantly reduce RSV associated-hospitalization among all ages.

Conserved CTL epitopes on the adenovirus hexon protein expand subgroup cross-reactive and subgroup-specific CD8+ T cells.

Adenoviruses often cause lethal infections in immunocompromised individuals. Adoptive transfer of immune T cells offers a therapeutic option, but this strategy has been hindered by the paucity of information on molecular targets of cellular immunity and by the immunologic heterogeneity of the 51 human adenoviruses, which are grouped from A to F on the basis of genome size, composition, homology, and organization. Clonal analysis of the adenovirus-specific cytotoxic T lymphocyte (CTL) responses of seropositive individuals identified 5 novel CD8(+) T-cell epitopes, all located in conserved regions of the capsid protein hexon. Reactive T cells were cross-reactive between 2 to 4 groups, while no T cells specific for a single subgroup were detected. Thus, by exploiting these peptide targets, it is possible to prepare a T-cell population capable of reacting with most adenoviruses that cause disease in immunocompromised patients.

Fiber-modified adenoviruses generate subgroup cross-reactive, adenovirus-specific cytotoxic T lymphocytes for therapeutic applications.

Adenovirus (Ad) infections are responsible for considerable morbidity and mortality, particularly in pediatric hematopoietic stem cell transplant (HSCT) recipients. To date there is no therapy. The present study was motivated by the potential for using adoptive immunotherapy as either prophylaxis or treatment for Ad infections and associated diseases. The authors have developed a protocol to reactivate Ad-specific memory T cells from peripheral blood mononuclear cells (PBMCs) using a clinical-grade adenoviral vector. Such lines contain a specific CD4 and CD8 T-cell component and are capable of recognizing and lysing target cells infected with wild-type Ad serotypes from different Ad groups. Furthermore, the frequency of Ad-specific precursors can be determined in PBMCs ex vivo and used as a means to assess changes in Ad-specific T-cell memory responses after infusion. This is the first report of a simple and reproducible method to activate and expand Ad-specific cytotoxic T lymphocytes (CTLs), which should be protective against the range of different Ad subtypes that affect transplant recipients.