High frequencies of nonviral colds and respiratory bacteria colonization among children in rural Western Uganda.

Introduction: Respiratory illness is the most common childhood disease globally, especially in developing countries. Previous studies have detected viruses in approximately 70-80% of respiratory illnesses. Methods: In a prospective cohort study of 234 young children (ages 3-11 years) and 30 adults (ages 22-51 years) in rural Western Uganda sampled monthly from May 2019 to August 2021, only 24.2% of nasopharyngeal swabs collected during symptomatic disease had viruses detectable by multiplex PCR diagnostics and metagenomic sequencing. In the remaining 75.8% of swabs from symptomatic participants, we measured detection rates of respiratory bacteria Haemophilus influenzae, Moraxella catarrhalis, and Streptococcus pneumoniae by quantitative PCR. Results: 100% of children tested positive for at least one bacterial species. Detection rates were 87.2%, 96.8%, and 77.6% in children and 10.0%, 36.7%, and 13.3% for adults for H. influenzae, M. catarrhalis, and S. pneumoniae, respectively. In children, 20.8% and 70.4% were coinfected with two and three pathogens, respectively, and in adults 6.7% were coinfected with three pathogens but none were coinfected with two. Detection of any of the three pathogens was not associated with season or respiratory symptoms severity, although parsing detection status by symptoms was challenged by children experiencing symptoms in 80.3% of monthly samplings, whereas adults only reported symptoms 26.6% of the time. Pathobiont colonization in children in Western Uganda was significantly more frequent than in children living in high-income countries, including in a study of age-matched US children that utilized identical diagnostic methods. Detection rates were, however, comparable to rates in children living in other Sub-Saharan African countries. Discussion: Overall, our results demonstrate that nonviral colds contribute significantly to respiratory disease burden among children in rural Uganda and that high rates of respiratory pathobiont colonization may play a role. These conclusions have implications for respiratory health interventions in the area, such as increasing childhood immunization rates and decreasing air pollutant exposure.

Common cold viruses circulating in children threaten wild chimpanzees through asymptomatic adult carriers.

Reverse zoonotic respiratory diseases threaten great apes across Sub-Saharan Africa. Studies of wild chimpanzees have identified the causative agents of most respiratory disease outbreaks as "common cold" paediatric human pathogens, but reverse zoonotic transmission pathways have remained unclear. Between May 2019 and August 2021, we conducted a prospective cohort study of 234 children aged 3-11 years in communities bordering Kibale National Park, Uganda, and 30 adults who were forest workers and regularly entered the park. We collected 2047 respiratory symptoms surveys to quantify clinical severity and simultaneously collected 1989 nasopharyngeal swabs approximately monthly for multiplex viral diagnostics. Throughout the course of the study, we also collected 445 faecal samples from 55 wild chimpanzees living nearby in Kibale in social groups that have experienced repeated, and sometimes lethal, epidemics of human-origin respiratory viral disease. We characterized respiratory pathogens in each cohort and examined statistical associations between PCR positivity for detected pathogens and potential risk factors. Children exhibited high incidence rates of respiratory infections, whereas incidence rates in adults were far lower. COVID-19 lockdown in 2020-2021 significantly decreased respiratory disease incidence in both people and chimpanzees. Human respiratory infections peaked in June and September, corresponding to when children returned to school. Rhinovirus, which caused a 2013 outbreak that killed 10% of chimpanzees in a Kibale community, was the most prevalent human pathogen throughout the study and the only pathogen present at each monthly sampling, even during COVID-19 lockdown. Rhinovirus was also most likely to be carried asymptomatically by adults. Although we did not detect human respiratory pathogens in the chimpanzees during the cohort study, we detected human metapneumovirus in two chimpanzees from a February 2023 outbreak that were genetically similar to viruses detected in study participants in 2019. Our data suggest that respiratory pathogens circulate in children and that adults become asymptomatically infected during high-transmission times of year. These asymptomatic adults may then unknowingly carry the pathogens into forest and infect chimpanzees. This conclusion, in turn, implies that intervention strategies based on respiratory symptoms in adults are unlikely to be effective for reducing reverse zoonotic transmission of respiratory viruses to chimpanzees.

Farm animal exposure, respiratory illnesses, and nasal cell gene expression.

BACKGROUND: Farm exposures in early life reduce the risks for childhood allergic diseases and asthma. There is less information about how farm exposures relate to respiratory illnesses and mucosal immune development. OBJECTIVE: We hypothesized that children raised in farm environments have a lower incidence of respiratory illnesses over the first 2 years of life than nonfarm children. We also analyzed whether farm exposures or respiratory illnesses were related to patterns of nasal cell gene expression. METHODS: The Wisconsin Infant Study Cohort included farm (n = 156) and nonfarm (n = 155) families with children followed to age 2 years. Parents reported prenatal farm and other environmental exposures. Illness frequency and severity were assessed using illness diaries and periodic surveys. Nasopharyngeal cell gene expression in a subset of 64 children at age 2 years was compared to farm exposure and respiratory illness history. RESULTS: Farm versus nonfarm children had nominally lower rates of respiratory illnesses (rate ratio 0.82 [95% CI, 0.69, 0.97]) with a stepwise reduction in illness rates in children exposed to 0, 1, or ≥2 animal species, but these trends were nonsignificant in a multivariable model. Farm exposures and preceding respiratory illnesses were positively related to nasal cell gene signatures for mononuclear cells and innate and antimicrobial responses. CONCLUSIONS: Maternal and infant exposure to farms and farm animals was associated with nonsignificant trends for reduced respiratory illnesses. Nasal cell gene expression in a subset of children suggests that farm exposures and respiratory illnesses in early life are associated with distinct patterns of mucosal immune expression.

Rhinoviruses A and C elicit long-lasting antibody responses with limited cross-neutralization.

Rhinoviruses (RVs) can cause severe wheezing illnesses in young children and patients with asthma. Vaccine development has been hampered by the multitude of RV types with little information about cross-neutralization. We previously showed that neutralizing antibody (nAb) responses to RV-C are detected twofold to threefold more often than those to RV-A throughout childhood. Based on those findings, we hypothesized that RV-C infections are more likely to induce either cross-neutralizing or longer-lasting antibody responses compared with RV-A infections. We pooled RV diagnostic data from multiple studies of children with respiratory illnesses and compared the expected versus observed frequencies of sequential infections with RV-A or RV-C types using log-linear regression models. We tested longitudinally collected plasma samples from children to compare the duration of RV-A versus RV-C nAb responses. Our models identified limited reciprocal cross-neutralizing relationships for RV-A (A12-A75, A12-A78, A20-A78, and A75-A78) and only one for RV-C (C2-C40). Serologic analysis using reference mouse sera and banked human plasma samples confirmed that C40 infections induced nAb responses with modest heterotypic activity against RV-C2. Mixed-effects regression modeling of longitudinal human plasma samples collected from ages 2 to 18 years demonstrated that RV-A and RV-C illnesses induced nAb responses of similar duration. These results indicate that both RV-A and RV-C nAb responses have only modest cross-reactivity that is limited to genetically similar types. Contrary to our initial hypothesis, RV-C species may include even fewer cross-neutralizing types than RV-A, whereas the duration of nAb responses during childhood is similar between the two species. The modest heterotypic responses suggest that RV vaccines must have a broad representation of prevalent types.

Viruses associated with ill health in wild chimpanzees.

Viral infection is a major cause of ill health in wild chimpanzees (Pan troglodytes), but most evidence to date has come from conspicuous disease outbreaks with high morbidity and mortality. To examine the relationship between viral infection and ill health during periods not associated with disease outbreaks, we conducted a longitudinal study of wild eastern chimpanzees (P. t. schweinfurthii) in the Kanyawara and Ngogo communities of Kibale National Park, Uganda. We collected standardized, observational health data for 4 years and then used metagenomics to characterize gastrointestinal viromes (i.e., all viruses recovered from fecal samples) in individual chimpanzees before and during episodes of clinical disease. We restricted our analyses to viruses thought to infect mammals or primarily associated with mammals, discarding viruses associated with nonmammalian hosts. We found 18 viruses (nine of which were previously identified in this population) from at least five viral families. Viral richness (number of viruses per sample) did not vary by health status. By contrast, total viral load (normalized proportion of sequences mapping to viruses) was significantly higher in ill individuals compared with healthy individuals. Furthermore, when ill, Kanyawara chimpanzees exhibited higher viral loads than Ngogo chimpanzees, and males, but not females, exhibited higher infection rates with certain viruses and higher total viral loads as they aged. Post-hoc analyses, including the use of a machine-learning classification method, indicated that one virus, salivirus (Picornaviridae), was the main contributor to health-related and community-level variation in viral loads. Another virus, chimpanzee stool-associated virus (chisavirus; unclassified Picornavirales), was associated with ill health at Ngogo but not at Kanyawara. Chisavirus, chimpanzee adenovirus (Adenoviridae), and bufavirus (Parvoviridae) were also associated with increased age in males. Associations with sex and age are consistent with the hypothesis that nonlethal viral infections cumulatively reflect or contribute to senescence in long-lived species such as chimpanzees.

Enhanced Neutralizing Antibody Responses to Rhinovirus C and Age-Dependent Patterns of Infection.

Rationale: Rhinovirus (RV) C can cause asymptomatic infection and respiratory illnesses ranging from the common cold to severe wheezing.Objectives: To identify how age and other individual-level factors are associated with susceptibility to RV-C illnesses.Methods: Longitudinal data from the COAST (Childhood Origins of Asthma) birth cohort study were analyzed to determine relationships between age and RV-C infections. Neutralizing antibodies specific for RV-A and RV-C (three types each) were determined using a novel PCR-based assay. Data were pooled from 14 study cohorts in the United States, Finland, and Australia, and mixed-effects logistic regression was used to identify factors related to the proportion of RV-C versus RV-A detection.Measurements and Main Results: In COAST, RV-A and RV-C infections were similarly common in infancy, whereas RV-C was detected much less often than RV-A during both respiratory illnesses and scheduled surveillance visits (P < 0.001, χ2) in older children. The prevalence of neutralizing antibodies to RV-A or RV-C types was low (5-27%) at the age of 2 years, but by the age of 16 years, RV-C seropositivity was more prevalent (78% vs. 18% for RV-A; P < 0.0001). In the pooled analysis, the RV-C to RV-A detection ratio during illnesses was significantly related to age (P < 0.0001), CDHR3 genotype (P < 0.05), and wheezing illnesses (P < 0.05). Furthermore, certain RV types (e.g., C2, C11, A78, and A12) were consistently more virulent and prevalent over time.Conclusions: Knowledge of prevalent RV types, antibody responses, and populations at risk based on age and genetics may guide the development of vaccines or other novel therapies against this important respiratory pathogen.

Simultaneous outbreaks of respiratory disease in wild chimpanzees caused by distinct viruses of human origin.

Respiratory viruses of human origin infect wild apes across Africa, sometimes lethally. Here we report simultaneous outbreaks of two distinct human respiratory viruses, human metapneumovirus (MPV; Pneumoviridae: Metapneumovirus) and human respirovirus 3 (HRV3; Paramyxoviridae; Respirovirus, formerly known as parainfluenza virus 3), in two chimpanzee (Pan troglodytes schweinfurthii) communities in the same forest in Uganda in December 2016 and January 2017. The viruses were absent before the outbreaks, but each was present in ill chimpanzees from one community during the outbreak period. Clinical signs and gross pathologic changes in affected chimpanzees closely mirrored symptoms and pathology commonly observed in humans for each virus. Epidemiologic modelling showed that MPV and HRV3 were similarly transmissible (R0 of 1.27 and 1.48, respectively), but MPV caused 12.2% mortality mainly in infants and older chimpanzees, whereas HRV3 caused no direct mortality. These results are consistent with the higher virulence of MPV than HRV3 in humans, although both MPV and HRV3 cause a significant global disease burden. Both viruses clustered phylogenetically within groups of known human variants, with MPV closely related to a lethal 2009 variant from mountain gorillas (Gorilla beringei beringei), suggesting two independent and simultaneous reverse zoonotic origins, either directly from humans or via intermediary hosts. These findings expand our knowledge of human origin viruses threatening wild chimpanzees and suggest that such viruses might be differentiated by their comparative epidemiological dynamics and pathogenicity in wild apes. Our results also caution against assuming common causation in coincident outbreaks.

Lethal Respiratory Disease Associated with Human Rhinovirus C in Wild Chimpanzees, Uganda, 2013.

We describe a lethal respiratory outbreak among wild chimpanzees in Uganda in 2013 for which molecular and epidemiologic analyses implicate human rhinovirus C as the cause. Postmortem samples from an infant chimpanzee yielded near-complete genome sequences throughout the respiratory tract; other pathogens were absent. Epidemiologic modeling estimated the basic reproductive number (R0) for the epidemic as 1.83, consistent with the common cold in humans. Genotyping of 41 chimpanzees and examination of 24 published chimpanzee genomes from subspecies across Africa showed universal homozygosity for the cadherin-related family member 3 CDHR3-Y529 allele, which increases risk for rhinovirus C infection and asthma in human children. These results indicate that chimpanzees exhibit a species-wide genetic susceptibility to rhinovirus C and that this virus, heretofore considered a uniquely human pathogen, can cross primate species barriers and threatens wild apes. We advocate engineering interventions and prevention strategies for rhinovirus infections for both humans and wild apes.

Early life rhinovirus wheezing, allergic sensitization, and asthma risk at adolescence.

BACKGROUND: Early life rhinovirus (RV) wheezing illnesses and aeroallergen sensitization increase the risk of asthma at school age. Whether these remain risk factors for the persistence of asthma out to adolescence is not established. OBJECTIVE: We sought to define the relationships among specific viral illnesses and the type and timing of aeroallergen sensitization with the persistence of asthma into adolescence. METHODS: A total of 217 children were followed prospectively from birth to age 13 years. The etiology and timing of viral wheezing illnesses during the first 3 years of life were assessed along with patterns of allergen sensitization. The associations between viral wheezing illnesses, presence and pattern of aeroallergen sensitization, and asthma diagnosis at age 13 years were evaluated. RESULTS: When adjusted for all viral etiologies, wheezing with RV (odds ratio = 3.3; 95% CI, 1.5-7.1), but not respiratory syncytial virus (odds ratio = 1.0; 95% CI, 0.4-2.3), was associated with asthma at age 13 years. Age of aeroallergen sensitization also influenced asthma risk; 65% of children sensitized by age 1 year had asthma at age 13 years, compared with 40% of children not sensitized at age 1 year but sensitized by age 5 years, and 17% of children not sensitized at age 5 years. Early life aeroallergen sensitization and RV wheezing had additive effects on asthma risk at adolescence. CONCLUSIONS: In a high-risk birth cohort, the persistence of asthma at age 13 years was most strongly associated with outpatient wheezing illnesses with RV and aeroallergen sensitization in early life.

Molecular identification and quantification of human rhinoviruses in respiratory samples.

PCR-based molecular assays have become standard diagnostic procedures for the identification and quantification of human rhinoviruses (HRVs) and other respiratory pathogens in most, if not all, clinical microbiology laboratories. Molecular assays are significantly more sensitive than traditional culture-based and serological methods. This advantage has led to the recognition that HRV infections are common causes for not only upper airway symptoms but also more severe lower respiratory illnesses. In addition, molecular assays improve turnaround time, can be performed by technicians with ordinary skills, and can easily be automated. This chapter describes two highly sensitive and specific PCR-based methods for identifying and quantifying HRVs. The first is a two-step PCR method for the detection and typing of HRV. The second is a pan-HRV real-time quantitative (q) PCR method for measuring viral loads in respiratory samples.

Detection of pathogenic bacteria during rhinovirus infection is associated with increased respiratory symptoms and asthma exacerbations.

BACKGROUND: Detection of either viral or bacterial pathogens is associated with wheezing in children; however, the influence of both bacteria and viruses on illness symptoms has not been described. OBJECTIVE: We evaluated bacterial detection during the peak rhinovirus season in children with and without asthma to determine whether an association exists between bacterial infection and the severity of rhinovirus-induced illnesses. METHODS: Three hundred eight children (166 with asthma and 142 without asthma) aged 4 to 12 years provided 5 consecutive weekly nasal samples during September and scored cold and asthma symptoms daily. Viral diagnostics and quantitative PCR for Streptococcus pneumoniae, Haemophilus influenzae, and Moraxella catarrhalis were performed on all nasal samples. RESULTS: Detection rates were 53%, 17%, and 11% for H influenzae, S pneumoniae, and M catarrhalis, respectively, with detection of rhinovirus increasing the risk of detecting bacteria within the same sample (odds ratio [OR], 2.0; 95% CI, 1.4-2.7; P < .0001) or the following week (OR, 1.6; 95% CI, 1.1-2.4; P = .02). In the absence of rhinovirus, S pneumoniae was associated with increased cold symptoms (mean, 2.7 [95% CI, 2.0-3.5] vs 1.8 [95% CI, 1.5-2.2]; P = .006) and moderate asthma exacerbations (18% [95% CI, 12% to 27%] vs 9.2% [95% CI, 6.7% to 12%]; P = .006). In the presence of rhinovirus, S pneumoniae was associated with increased moderate asthma exacerbations (22% [95% CI, 16% to 29%] vs 15% [95% CI, 11% to 20%]; P = .01). Furthermore, M catarrhalis detected alongside rhinovirus increased the likelihood of experiencing cold symptoms, asthma symptoms, or both compared with isolated detection of rhinovirus (OR, 2.0 [95% CI, 1.0-4.1]; P = .04). Regardless of rhinovirus status, H influenzae was not associated with respiratory symptoms. CONCLUSION: Rhinovirus infection enhances detection of specific bacterial pathogens in children with and without asthma. Furthermore, these findings suggest that M catarrhalis and S pneumoniae contribute to the severity of respiratory tract illnesses, including asthma exacerbations.

Genome sequences of rhinovirus a isolates from wisconsin pediatric respiratory studies.

Full-length or nearly full-length RNA genome sequences for 98 rhinovirus (RV) A isolates (from the Enterovirus genus of the Picornaviridae family), representing 43 different genotypes, were resolved as part of ongoing studies to define RV genetic diversity and its potential link to respiratory disease.

Genome sequences of rhinovirus B isolates from wisconsin pediatric respiratory studies.

Nearly full-length RNA genome sequences for 39 rhinovirus B isolates (RV-B), representing 13 different genotypes, were resolved as part of ongoing studies at the University of Wisconsin that attempt to link rhinovirus (RV) diversity and respiratory disease in infants.

Genome sequences of rhinovirus C isolates from wisconsin pediatric respiratory studies.

Human rhinovirus (RV) isolates from the RV-C species are recently discovered infectious agents that are closely linked to asthma and wheezing etiologies in infants. Clinical study samples collected at the University of Wisconsin-Madison describe 41 nearly complete genome sequences representing 21 RV-C genotypes.

Inhaled corticosteroid use is associated with increased circulating T regulatory cells in children with asthma.

BACKGROUND: T regulatory (Treg) cells are important in balancing immune responses and dysregulation of Treg cells has been implicated in the pathogenesis of multiple disease states including asthma. In this study, our primary aim was to determine Treg cell frequency in the peripheral blood of children with and without asthma. The secondary aim was to explore the association between Treg cell frequency with allergen sensitization, disease severity and medication use. METHODS: Peripheral blood mononuclear cells from healthy control subjects (N = 93) and asthmatic children of varying disease severity (N = 66) were characterized by multi-parameter flow cytometry. RESULTS: Our findings demonstrate that children with asthma had a significantly increased frequency of Treg cells compared to children without asthma. Using a multivariate model, increased Treg cell frequency in children with asthma was most directly associated with inhaled corticosteroid use, and not asthma severity, allergic sensitization, or atopic status of the asthma. CONCLUSION: We conclude that low dose, local airway administration of corticosteroids is sufficient to impact the frequency of Treg cells in the peripheral blood. These data highlight the importance of considering medication exposure when studying Treg cells and suggest inhaled corticosteroid use in asthmatics may improve disease control through increased Treg cell frequency.

Comparison of the etiology of viral respiratory illnesses in inner-city and suburban infants.

BACKGROUND: The risk of developing childhood asthma has been linked to the severity and etiology of viral respiratory illnesses in early childhood. Since inner-city infants have unique environmental exposures, we hypothesized that patterns of respiratory viral infections would also be distinct. METHODS: We compared the viral etiology of respiratory illnesses in 2 groups: a cohort of 515 infants from 4 inner-city areas and a cohort of 285 infants from mainly suburban Madison, Wisconsin. Nasal secretions were sampled during periods of respiratory illness and at 1 year of age and were analyzed for viral pathogens by multiplex polymerase chain reaction. RESULTS: Overall, inner-city infants had lower rates of viral detection. Considering specific viruses, sick urban infants had lower rates of detectable rhinovirus or respiratory syncytial virus infection and higher rates of adenovirus infection. Every urban site had a higher proportion of adenovirus-positive samples associated with illnesses (10%-21%), compared with Madison (6%). CONCLUSIONS: These findings provide evidence that inner-city babies have different patterns of viral respiratory illnesses than babies who grow up in a more suburban location. These findings raise important questions about the etiology of virus-negative illnesses in urban infants and the possibility of long-term consequences of early life infections with adenovirus in this population.

Human rhinovirus species and season of infection determine illness severity.

RATIONALE: Human rhinoviruses (HRVs) consist of approximately 160 types that cause a wide range of clinical outcomes, including asymptomatic infections, common colds, and severe lower respiratory illnesses. OBJECTIVES: To identify factors that influence the severity of HRV illnesses. METHODS: HRV species and types were determined in 1,445 nasal lavages that were prospectively collected from 209 infants participating in a birth cohort who had at least one HRV infection. Questionnaires were used during each illness to identify moderate to severe illnesses (MSI). MEASUREMENTS AND MAIN RESULTS: Altogether, 670 HRV infections were identified, and 519 of them were solitary infections (only one HRV type). These 519 viruses belonged to 93 different types of three species: 49 A, 9 B, and 35 C types. HRV-A (odds ratio, 8.2) and HRV-C (odds ratio, 7.6) were more likely to cause MSI compared with HRV-B. In addition, HRV infections were 5- to 10-fold more likely to cause MSI in the winter months (P < 0.0001) compared with summer, in contrast to peak seasonal prevalence in spring and fall. When significant differences in host susceptibility to MSI (P = 0.004) were considered, strain-specific rates of HRV MSI ranged from less than 1% to more than 20%. CONCLUSIONS: Factors related to HRV species and type, season, and host susceptibility determine the risk of more severe HRV illness in infancy. These findings suggest that anti-HRV strategies should focus on HRV-A and -C species and identify the need for additional studies to determine mechanisms for seasonal increases of HRV severity, independent of viral prevalence, in cold weather months.