Gwendolyn Sanderson

Community study of role of viral infections in exacerbations of asthma in 9-11 year old children.

Abstract Objective: To study the association between upper and lower respiratory viral infections and acute exacerbations of asthma in schoolchildren in the community. Design: Community based 13 month longitudinal study using diary card respiratory symptom and peak expiratory flow monitoring to allow early sampling for viruses. Subjects: 108 Children aged 9-11 years who had reported wheeze or cough, or both, in a questionnaire. Setting: Southampton and surrounding community. Main outcome measures: Upper and lower respiratory viral infections detected by polymerase chain reaction or conventional methods, reported exacerbations of asthma, computer identified episodes of respiratory tract symptoms or peak flow reductions. Results: Viruses were detected in 80% of reported episodes of reduced peak expiratory flow, 80% of reported episodes of wheeze, and in 85% of reported episodes of upper respiratory symptoms, cough, wheeze, and a fall in peak expiratory flow. The median duration of reported falls in peak expiratory flow was 14 days, and the median maximum fall in peak expiratory flow was 81 1/min. The most commonly identified virus type was rhinovirus. Conclusions: This study supports the hypothesis that upper respiratory viral infections are associated with 80-85% of asthma exacerbations in school age children. Key messages Key messages In this study common cold viruses were found in 80-85% of reported exacerbations of asthma in children Rhinoviruses, which cause most common colds, accounted for two thirds of viruses detected Analysis of diary cards also showed large numbers of similar but less severe episodes that may also be viral in origin

Rhinoviruses Infect the Lower Airways

Rhinoviruses are the major cause of the common cold and a trigger of acute asthma exacerbations. Whether these exacerbations result from direct infection of the lower airway or from indirect mechanisms consequent on infection of the upper airway alone is currently unknown. Lower respiratory infection was investigated in vitro by exposing primary human bronchial epithelial cells to rhinoviruses and in vivo after experimental upper respiratory infection of human volunteers. Bronchial infection was confirmed by both approaches. Furthermore, rhinoviruses induced production of interleukin-6, -8, and -16 and RANTES and were cytotoxic to cultured respiratory epithelium. This evidence strongly supports a direct lower respiratory epithelial reaction as the initial event in the induction of rhinovirus-mediated asthma exacerbations. The frequency of infection and the nature of the inflammatory response observed are similar to those of the upper respiratory tract, suggesting that rhinovirus infections may be one of the most important causes of lower in addition to upper respiratory disease.

Frequency, severity, and duration of rhinovirus infections in asthmatic and non-asthmatic individuals: a longitudinal cohort study.

BACKGROUND Rhinovirus infections cause exacerbations of asthma. We postulated that people with asthma are more susceptible to rhinovirus infection than people without the disease and compared the susceptibility of these groups. METHODS We recruited 76 cohabiting couples. One person in every couple had atopic asthma and one was healthy. Participants completed daily diary cards of upper-respiratory-tract (URT) and lower-respiratory-tract (LRT) symptoms and measured peak expiratory flow twice daily. Every 2 weeks nasal aspirates were taken and examined for rhinovirus. Mixed models were used to compare risks of infection between groups. We also compared the severity and duration of infections. FINDINGS We analysed 753 samples. Rhinovirus was detected in 10.1% (38/378) of samples from participants with asthma and 8.5% (32/375) of samples from healthy participants. After adjustment for confounding factors, asthma did not significantly increase risk of infection (odds ratio 1.15, 95% CI 0.71-1.87). Groups did not differ in frequency, severity, or duration of URT infections or symptoms associated with rhinovirus infection. First rhinovirus infection was associated more frequently with LRT infection in participants with asthma than in healthy individuals (12 of 28 infections vs four of 23, respectively, p=0.051). Symptoms of LRT associated with rhinovirus infection were significantly more severe (p=0.001) and longer-lasting in participants with asthma than in healthy participants (p=0.005). INTERPRETATION People with atopic asthma are not at greater risk of rhinovirus infection than healthy individuals but suffer from more frequent LRT infections and have more severe and longer-lasting LRT symptoms.

Rhinoviruses replicate effectively at lower airway temperatures

Rhinoviruses are epidemiologically connected to the majority of acute asthma exacerbations; however, their ability to infect and replicate in the lower airways is disputed. A frequent argument against this possibility involves the temperature preference for rhinovirus replication, generally accepted to be 33°C, the temperature of the nasal passages. However, this argument is based on studies with a single rhinovirus serotype. In this study, differences in temperature preferences were evaluated between several serotypes and relative titers were determined than can be achieved at upper and lower airway temperatures. Rhinovirus serotypes 1b, 2, 7, 9, 14, 16, 41, and 70 were titrated in Ohio‐HeLa cell cultures at either 33°C or 37°C. Possible selection by culture temperature was examined by continuous culture at 33°C and 37°C for 2–4 passages and subsequent titration at both temperatures. Finally, nasal aspirate samples derived from patients with wild‐type rhinoviral common colds were cultured at 33°C and 37°C and RT‐PCR was used to assess rhinovirus replication at each temperature. The majority of the serotypes and wild‐type viruses replicated slightly better at 33°C than at 37°C. However, titers achieved after one or more replicative cycles at 37°C were still high enough to initiate infection. Furthermore, in some instances equal or even better replication was observed at 37°C. It is concluded that temperature preferences may vary between rhinoviruses and are not likely to be a prohibitive factor for infection of the lower airways. J. Med. Virol. 58:100–104, 1999.

Amplified rhinovirus colds in atopic subjects

Abstract. Evidence suggests that atopic individuals may be predisposed to more severe rhinoviral colds coupled to a worsening of existing airway disease than those with asthma. The role of atopy and IgE levels, as well as their relationship to clinical disease expression have not been defined. We hypothesized that an allergic diathesis modulates rhinoviral colds and have initiated studies of normal, atopic and asthmatic subjects employing experimental rhinoviral infection, with measurements of symptom scores, viral shedding and cultures, albumin in nasal washes and serological responses. Twenty‐two subjects (11 normal, 5 atopic, 6 atopic and asthmatic) participated and were inoculated with human rhinovirus serotype 16 (HRV 16). Measurements of neutralizing antibody and viral culture were performed at screening, pre‐inoculation, during the cold and at 8–10 weeks convalescence. Daily symptoms were noted, nasal washes done, IgE measured and atopy was diagnosed by skin tests. Seventeen volunteers developed clinical colds as assessed by symptom scores, virus shedding was demonstrated (with positive culture) in all subjects and a fourfold or higher seroconversion occurred in 11/22. Neutralizing HRV antibody developed unexpectedly in 10 subjects between screening and inoculation and the presence or absence of this pre‐inoculation antibody determined subsequent severity of colds in normal but not in atopic subjects. Atopic antibody positive individuals developed severe clinical colds that were independent of preinoculation antibody in contrast to normal subjects who developed mild colds in the presence of a neutralizing antibody (.P= 0.01). Both atopic and normal antibody negative subjects developed severe colds. This differential response was matched by nasal wash albumin levels which were significantly increased (P= 0–01) during the cold in atopic (but not in normal) volunteers with pre‐inoculation antibody. Levels of IgE were not correlated with severity of clinical disease or viral shedding. Our studies of HRV disease in atopic subjects suggest heightened susceptibility to the detrimental effects of colds; additional studies are needed to clarify the relevant mechanisms.

Rhinovirus infection increases 5-lipoxygenase and cyclooxygenase-2 in bronchial biopsy specimens from nonatopic subjects.

Rhinovirus infections cause wheeze, cough, and bronchial hyperresponsiveness. To investigate the involvement of cysteinyl-leukotrienes and prostanoids in these symptoms, bronchial biopsy specimens from 9 normal subjects (nonatopic and with no history of chronic lung disease) were immunostained for 5-lipoxygenase (5-LO) and cyclooxygenase (COX) pathway enzymes 2 weeks before and 4 days after experimental infection with human rhinovirus serotype 16. 5-LO-positive cell counts increased 9-fold (from 0.48 to 4.4 cells/mm(2); P <.05), and 5-LO-activating protein (FLAP)-positive cell counts increased 3.6-fold (from 1.8 to 6.5 cells/mm(2); P =.09). Levels of leukotriene A(4) hydrolase and leukotriene C(4) synthase were unchanged. COX-2--positive cell counts increased from 0 to 2.6 cells/mm(2) (P =.009), with no change in COX-1 levels. Increases of 3-4-fold were seen in levels of macrophages (P =.02) and mast cells (P =.07) but not of eosinophils (P >.4), and bronchoalveolar lavage fluid cysteinyl-leukotriene levels doubled (from 11.2 to 20.4 pg/mL; P =.13). Cold symptom scores correlated with bronchial immunostaining for FLAP (rho = 0.93; P =.001). In normal subjects, rhinovirus colds induce bronchial inflammation with markedly enhanced expression of 5-LO pathway proteins and COX-2.

Rhinovirus identification by BglI digestion of picornavirus RT-PCR amplicons

Abstract Rhinoviruses are the main cause of the common cold and precipitate the majority of asthma exacerbations. RT-PCR followed by internal probe hybridisation or Southern blotting, or nested PCRs are currently the most sensitive methods for their identification. However, none of the published techniques can differentiate satisfactorily rhinoviruses from other picornaviruses. Examination of the restriction maps of sequenced rhinoviruses, revealed a highly conserved BglI restriction site (GCCnnnnnGGC), located exactly in the middle of the 380-bp amplicon generated with the OL26–OL27 primer pair, which has been used extensively in the past to identify picornaviruses. Such a site was either not present, or positioned differently in other picornaviruses of known sequence. It was, therefore, considered that digestion of rhinovirus amplicons with this enzyme would result in two equal length fragments, generating a single 190-bp band in gel electrophoresis. In contrast, either one undigested 380-bp band or a double-band pattern would appear in amplicons from other picornaviruses. To test this hypothesis, Bgl digestions of OL26–OL27 amplicons from cultured and wild-type rhinoviruses, whose identity was confirmed by acid lability, as well as from echo, polio and coxsackie viruses were carried out. All rhinovirus samples were digested successfully generating single bands. Among the other picornaviruses, only 6.6% presented a single band pattern, while the rest were as predicted from the model. With a sensitivity of 100% and a specificity over 90%, the method described, which is rapid and remarkably easy to perform, can be used to distinguish rhinoviruses from other picornaviruses to a considerable extent.

Experimental rhinovirus infection in volunteers

Experimental viral disease studies in volunteers have clarified many aspects of the pathogenesis of human viral disease. Recently, interest has focused on rhinovirus-associated asthma exacerbations, and new volunteer studies have suggested that airway responsiveness (AR) is enhanced during a cold. For scientific, ethical and safety reasons, it is important to use validated methods for the preparation of a virus inoculum and that the particular virological characteristics and host responses should not be altered. We have prepared a new human rhinovirus (HRV) inoculum using recent guidelines and assessed whether disease characteristics (for example, severity of colds or changes in AR) were retained. Studies were conducted in 25 clinically healthy volunteers using a validated HRV inoculum in the first 17 and a new inoculum in the subsequent eight subjects. Severity of cold symptoms, nasal wash albumin levels and airway responsiveness were measured, and the new inoculum was prepared from nasal washes obtained during the cold. The new inoculum was tested using standard virological and serological techniques, as well as a polymerase chain reaction for Mycoplasma pneumoniae. No contaminating viruses or organisms were detected and the methods suggested were workable. Good clinical colds developed in 20 of the 25 subjects and median symptom scores were similar in the validated and new inoculum groups (18 and 17.5, respectively; p=0.19). All subjects shed virus, and there were no differences noted in viral culture scores, nasal wash albumin and rates of seroconversion in the two groups. Although airway responsiveness increased in both groups (p=0.02 and p=0.05), the degree of change was similar. We have performed experimental rhinovirus infection studies and demonstrated similar clinical disease in two inoculum groups. Amplified airway responsiveness was induced; continuing studies will define the mechanisms and suggest modes of treatment.

Peak expiratory flow changes during experimental rhinovirus infection

Rhinovirus (RV) colds are associated with asthma exacerbations and experimental infections are commonly used to investigate the mechanisms involved. However, a temporal association between experimental RV infections and falls in peak expiratory flow (PEF) have not been demonstrated. PEF was measured in 22 volunteers (11 normal, five atopic, six atopic asthmatic) who developed RV serotype 16 colds after inoculation. PEF was measured twice daily for 2 weeks prior and 6 weeks after RV infection and episodes of respiratory morbidity based on changes in PEF were defined using validated criteria. Six significant reductions in PEF were temporally related to the RV infections (in two (18%) normal, one (20%) atopic, three (50%) atopic asthmatic subjects, p=0.1) and occurred 4-9 days (median 6) after inoculation. Mean+/-SEM PEF at day 6 was 87.8+/-1.8% of the predicted value in the six subjects with reductions versus 99.4+/-1.4% pred in those without (p=0.01). Symptom scores were significantly different at day 6 in the two groups (10.6+/-1.9 versus 6.8+/-1.0, p=0.03), but no differences were noted in the viral culture scores and changes in nasal albumin. In subjects with significant PEF reduction, the decrease in the provocative concentration causing a 20% fall in the forced expiratory volume in one second (FEV1) (PC20) was 1.7+/-1.3 mg x mL(-1) versus 1.2+/-1.1 mg x mL(-1) in the negative group (p=0.06). The degree of seroconversion to RV was significantly higher in the group with reduced PEF (median change dilutions 8 versus 4, p=0.02). The results of the present study suggest that rhinovirus-associated, respiratory morbidity occurs during experimental infection in some but not all normal and asthmatic subjects and also that experimental colds are a valid model for the study of rhinovirus-associated airway symptoms and asthma exacerbations.

A multiplex RT-PCR for the detection of parainfluenza viruses 1-3 in clinical samples

Parainfluenza viruses (PIV) are an important cause of respiratory morbidity. Conventional diagnostic methods for detection of PIV are time consuming or lack sensitivity. A multiplex PCR that detects PIV 1-3 was developed using novel primers for PIV viruses 1 and 2 and primers for PIV 3 described previously. Following RNA extraction a single multiplex reverse transcription was undertaken using antisense primers specific for each virus type. This was followed by a 40-cycle multiplex PCR using primers directed towards the haemagglutinin-neuraminidase coding region of each virus type. Products were probed with type-specific fluorescein labelled internal probes and detected by chemiluminescence. Cultured PIV viruses were detectable to a sensitivity of 1 TCID50. The technique was applied to 57 nasal aspirates taken from children presenting with various acute respiratory conditions and analysed previously by culture, immunofluorescence and/or serology. It was possible to detect PIV 1, 2 or 3 in 13/13 samples found previously positive for PIV by tissue culture, 13/15 found previously positive by immunofluorescence and 6/10 that coincided with positive serology. None of the samples found previously positive for other viruses (26) or negative to virus detection (6) were found positive by RT-PCR. It is concluded that this method is as sensitive as combined immunofluorescence and tissue culture for the detection of the PIV viruses 1-3 and should be useful for rapid diagnosis of PIV 1-3 infections.