Novel scoring system for differentiating parechovirus-A3 and enterovirus infection in neonates and young infants.

BACKGROUND: Parechovirus-A3 (PeV-A3) and the enteroviruses (EVs) are the most common viral pathogens responsible for sepsis and meningoencephalitis in neonates and young infants; however, differences in the clinical presentations of two infections are not well described. OBJECTIVES: To describe the clinical presentations of PeV-A3- and EVs-related diseases and develop a novel scoring system to differentiate two diseases. STUDY DESIGN: This prospective study used real-time PCR and genetic sequencing to evaluate viral etiologies of febrile neonates and infants <4 months with suspected sepsis or meningoencephalitis in Niigata area, Japan, in 2014-2016. The clinical manifestations of PeV-A3- and EVs-infected patients were compared, and a novel scoring system was developed after identifying the most distinguishable clinical findings, followed by the external cohort validation. RESULTS: In 210 patients evaluated, we identified 56 PeV-A3-infected (27%) and 43 EVs-infected (20%) patients. The following clinical manifestations were significant in PeV-A3-infected patients, as compared with EVs-infected patients; a higher body temperature (38.9°C vs. 38.5°C, P < .01) and heart rate (181/min vs. 168/min, P = .01), cold extremities (72% vs. 34%, P < .01) and skin mottling (65% vs. 23%, P < .01), lower white blood cell count (5,200/µL vs. 8,900/µL, P < .01) and incidence of cerebrospinal fluid (CSF) pleocytosis (2% vs. 63%, P < .01). Using some of these significant findings, the scoring system successfully distinguished the diseases (accuracy: 86% and 83% for the derivative and external validation cohorts, respectively). CONCLUSIONS: We found significant clinical manifestations in PeV-A3-infected patients compared to EVs-infected patients. The scoring system may be helpful to distinguish two infections, especially at onset of outbreak.

Enteric viruses exploit the microbiota to promote infection.

Enteric viruses infect the mammalian gastrointestinal tract which is home to a diverse community of intestinal bacteria. Accumulating evidence suggests that certain enteric viruses utilize these bacteria to promote infection. While this is not surprising considering their proximity, multiple viruses from different viral families have been shown to bind directly to bacteria or bacterial components to aid in viral replication, pathogenesis, and transmission. These data suggest that the concept of a single virus infecting a single cell, independent of the environment, needs to be reevaluated. In this review, I will discuss the current knowledge of enteric virus-bacterial interactions and discuss the implications for viral pathogenesis and transmission.

Bacterial community structure and effects of picornavirus infection on the anterior nares microbiome in early childhood.

BACKGROUND: Little is known regarding the nasal microbiome in early childhood and the impact of respiratory infection on the infants' nasal microbial composition. Here we investigated the temporal dynamics and diversity of the bacterial composition in the anterior nares in children attending daycare centers. RESULTS: For our investigation, we considered 76 parental-taken nasal swabs of 26 children (aged 13 to 36 months) collected over a study period of 3 months. Overall, there was no significant age-specific effect or seasonal shift in the nasal bacterial community structure. In a sub-sample of 14 healthy children the relative abundance of individual taxa as well as the overall diversity did not reveal relevant changes, indicating a stable community structure over the entire study period. Moreover, the nasal bacterial profiles clustered subject-specific with Bray-Curtis similarities being elevated in intra-subject calculations compared to between-subject calculations. The remaining subset of 12 children provided samples taken during picornavirus infection (PVI) and either before or after a PVI. We detected an association between the relative abundance of members of the genus Streptococcus and PV when comparing both (i) samples taken during PVI with samples out of 14 healthy children and (ii) samples taken during PVI with samples taken after PVI within the same individual. In addition, the diversity was higher during PVI than after infection. CONCLUSIONS: Our findings suggest that a personalized structure of the nasal bacterial community is established already in early childhood and could be detected over a timeframe of 3 months. Studies following infants over a longer time with frequent swab sampling would allow investigating whether certain parameter of the bacterial community, such as the temporal variability, could be related to viral infection.

Nasal microbiota clusters associate with inflammatory response, viral load, and symptom severity in experimental rhinovirus challenge.

The role of nasal and fecal microbiota in viral respiratory infections has not been established. We collected nasal swabs and washes, and fecal samples in a clinical study assessing the effect of probiotic Bifidobacterium animalis subsp. lactis Bl-04 on experimental rhinovirus infection. The nasal and fecal microbiota were characterized by 16S rRNA gene sequencing. The resulting data were compared with nasal inflammatory marker concentrations, viral load, and clinical symptoms. By using unsupervised clustering, the nasal microbiota divided into six clusters. The clusters predominant of Staphylococcus, Corynebacterium/Alloiococcus, Moraxella, and Pseudomonadaceae/Mixed had characteristic inflammatory marker and viral load profiles in nasal washes. The nasal microbiota clusters of subjects before the infection associated with the severity of clinical cold symptoms during rhinovirus infection. Rhinovirus infection and probiotic intervention did not significantly alter the composition of nasal or fecal microbiota. Our results suggest that nasal microbiota may influence the virus load, host innate immune response, and clinical symptoms during rhinovirus infection, however, further studies are needed.

Role of airway glucose in bacterial infections in patients with chronic obstructive pulmonary disease.

BACKGROUND: Patients with chronic obstructive pulmonary disease (COPD) have increased susceptibility to respiratory tract infection, which contributes to disease progression and mortality, but mechanisms of increased susceptibility to infection remain unclear. OBJECTIVES: The aim of this study was to determine whether glucose concentrations were increased in airway samples (nasal lavage fluid, sputum, and bronchoalveolar lavage fluid) from patients with stable COPD and to determine the effects of viral infection on sputum glucose concentrations and how airway glucose concentrations relate to bacterial infection. METHODS: We measured glucose concentrations in airway samples collected from patients with stable COPD and smokers and nonsmokers with normal lung function. Glucose concentrations were measured in patients with experimentally induced COPD exacerbations, and these results were validated in patients with naturally acquired COPD exacerbations. Relationships between sputum glucose concentrations, inflammatory markers, and bacterial load were examined. RESULTS: Sputum glucose concentrations were significantly higher in patients with stable COPD compared with those in control subjects without COPD. In both experimental virus-induced and naturally acquired COPD exacerbations, sputum and nasal lavage fluid glucose concentrations were increased over baseline values. There were significant correlations between sputum glucose concentrations and sputum inflammatory markers, viral load, and bacterial load. Airway samples with higher glucose concentrations supported more Pseudomonas aeruginosa growth in vitro. CONCLUSIONS: Airway glucose concentrations are increased in patients with stable COPD and further increased during COPD exacerbations. Increased airway glucose concentrations might contribute to bacterial infections in both patients with stable and those with exacerbated COPD. This has important implications for the development of nonantibiotic therapeutic strategies for the prevention or treatment of bacterial infection in patients with COPD.

Nasopharyngeal microbiome in premature infants and stability during rhinovirus infection.

RATIONALE: The nasopharyngeal (NP) microbiota of newborns and infants plays a key role in modulating airway inflammation and respiratory symptoms during viral infections. Premature (PM) birth modifies the early NP environment and is a major risk factor for severe viral respiratory infections. However, it is currently unknown if the NP microbiota of PM infants is altered relative to full-term (FT) individuals. OBJECTIVES: To characterize the NP microbiota differences in preterm and FT infants during rhinovirus (RV) infection. METHODS: We determined the NP microbiota of infants 6 months to ≤2 years of age born FT (n=6) or severely PM<32 weeks gestation (n=7). We compared microbiota composition in healthy NP samples and performed a longitudinal analysis during naturally occurring RV infections to contrast the microbiota dynamics in PM versus FT infants. RESULTS: We observed significant differences in the NP bacterial community of PM versus FT. NP from PM infants had higher within-group dissimilarity (heterogeneity) relative to FT infants. Bacterial composition of NP samples from PM infants showed increased Proteobacteria and decreased in Firmicutes. There were also differences in the major taxonomic groups identified, including Streptococcus, Moraxella, and Haemophilus. Longitudinal data showed that these prematurity-related microbiota features persisted during RV infection. CONCLUSIONS: PM is associated with NP microbiota changes beyond the neonatal stage. PM infants have an NP microbiota with high heterogeneity relative to FT infants. These prematurity-related microbiota features persisted during RV infection, suggesting that the NP microbiota of PM may play an important role in modulating airway inflammatory and immune responses in this vulnerable group.

Prospective etiological investigation of community-acquired pulmonary infections in hospitalized people living with HIV.

The study of the etiological agents of community-acquired pulmonary infections is important to guide empirical therapy, requires constant updating, and has a substantial impact on the prognosis of patients. The objective of this study is to determine prospectively the etiology of community-acquired pulmonary infections in hospitalized adults living with HIV. Patients were submitted to an extended microbiological investigation that included molecular methods. The microbiological findings were evaluated according to severity of the disease and pneumococcal vaccine status. Two hundred twenty-four patients underwent the extended microbiological investigation of whom 143 (64%) had an etiology determined. Among the 143 patients with a determined etiology, Pneumocystis jirovecii was the main agent, detected in 52 (36%) cases and followed by Mycobacterium tuberculosis accounting for 28 (20%) cases. Streptococcus pneumoniae and Rhinovirus were diagnosed in 22 (15%) cases each and influenza in 15 (10%) cases. Among atypical bacteria, Mycoplasma pneumoniae was responsible for 12 (8%) and Chlamydophila pneumoniae for 7 (5%) cases. Mixed infections occurred in 48 cases (34%). S pneumoniae was associated with higher severity scores and not associated with vaccine status. By using extended diagnostics, a microbiological agent could be determined in the majority of patients living with HIV affected by community-acquired pulmonary infections. Our findings can guide clinicians in the choice of empirical therapy for hospitalized pulmonary disease.

Changes in microbiota during experimental human Rhinovirus infection.

BACKGROUND: Human Rhinovirus (HRV) is responsible for the majority of common colds and is frequently accompanied by secondary bacterial infections through poorly understood mechanisms. We investigated the effects of experimental human HRV serotype 16 infection on the upper respiratory tract microbiota. METHODS: Six healthy volunteers were infected with HRV16. We performed 16S ribosomal RNA-targeted pyrosequencing on throat swabs taken prior, during and after infection. We compared overall community diversity, phylogenetic structure of the ecosystem and relative abundances of the different bacteria between time points. RESULTS: During acute infection strong trends towards increases in the relative abundances of Haemophilus parainfluenzae and Neisseria subflava were observed, as well as a weaker trend towards increases of Staphylococcus aureus. No major differences were observed between day-1 and day 60, whereas differences between subjects were very high. CONCLUSIONS: HRV16 infection is associated with the increase of three genera known to be associated with secondary infections following HRV infections. The observed changes of upper respiratory tract microbiota could help explain why HRV infection predisposes to bacterial otitis media, sinusitis and pneumonia.

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.

Outgrowth of the bacterial airway microbiome after rhinovirus exacerbation of chronic obstructive pulmonary disease.

RATIONALE: Rhinovirus infection is followed by significantly increased frequencies of positive, potentially pathogenic sputum cultures in chronic obstructive pulmonary disease (COPD). However, it remains unclear whether these represent de novo infections or an increased load of organisms from the complex microbial communities (microbiome) in the lower airways. OBJECTIVES: To investigate the effect of rhinovirus infection on the airway bacterial microbiome. METHODS: Subjects with COPD (n = 14) and healthy control subjects with normal lung function (n = 17) were infected with rhinovirus. Induced sputum was collected at baseline before rhinovirus inoculation and again on Days 5, 15, and 42 after rhinovirus infection and DNA was extracted. The V3-V5 region of the bacterial 16S ribosomal RNA gene was amplified and pyrosequenced, resulting in 370,849 high-quality reads from 112 of the possible 124 time points. MEASUREMENTS AND MAIN RESULTS: At 15 days after rhinovirus infection, there was a sixfold increase in 16S copy number (P = 0.007) and a 16% rise in numbers of proteobacterial sequences, most notably in potentially pathogenic Haemophilus influenzae (P = 2.7 × 10(-20)), from a preexisting community. These changes occurred only in the sputum microbiome of subjects with COPD and were still evident 42 days after infection. This was in contrast to the temporal stability demonstrated in the microbiome of healthy smokers and nonsmokers. CONCLUSIONS: After rhinovirus infection, there is a rise in bacterial burden and a significant outgrowth of Haemophilus influenzae from the existing microbiota of subjects with COPD. This is not observed in healthy individuals. Our findings suggest that rhinovirus infection in COPD alters the respiratory microbiome and may precipitate secondary bacterial infections.

Pneumococcal bacterial load colonization as a marker of mixed infection in children with alveolar community-acquired pneumonia and respiratory syncytial virus or rhinovirus infection.

BACKGROUND: The main aim of this study was to evaluate whether nasopharyngeal Streptococcus pneumoniae colonization in children with alveolar community-acquired pneumonia (CAP) and respiratory syncytial virus (RSV) or rhinovirus (RV) infection indicates a mixed lung infection. METHODS: The nasopharyngeal secretions of 530 children with radiographically confirmed CAP were tested using the Luminex x TAG respiratory virus panel fast assay. Real-time polymerase chain reaction for the autolysin-A (LytA) and wzg (cpsA) genes of S. pneumoniae was performed on the RSV- and RV-positive samples. RESULTS: Sixty-five of the 126 RSV-positive children (51.6%) were colonized with S. pneumoniae. Mean bacterial load was significantly higher in the patients with alveolar involvement (4.54±1.47 log10 DNA copies/mL vs. 3.75±1.62 log10 DNA copies/mL; P=0.04). Serotypes 5 and 19A were almost exclusively identified in the children with RSV and alveolar CAP, although the difference was statistically significant only for serotype 19A (P=0.03). Eighty-three of the 134 RV-positive children (61.9%) were colonized with S. pneumoniae and again mean bacterial load was significantly higher in the patients with alveolar involvement (4.21±1.37 log10 DNA copies/mL vs. 3.41±1.47 log10 DNA copies/mL; P=0.03). Serotypes 1, 5 and 19A were more frequently identified in the children with RV and alveolar CAP, although the difference was statistically significant only for serotype 5 (P=0.04). CONCLUSIONS: In children with alveolar CAP and RSV or RV infection, the determination of nasopharyngeal pneumococcal bacterial load and identification of the serotypes can contribute to the diagnosis of mixed lung infection.

Asthma exacerbations. 2: aetiology.

The natural history of asthma involves relatively stable periods that are often punctuated by significant exacerbations of symptoms. There are many aetiologies that may lead to an increase in asthma severity including respiratory infection (bacterial/viral), allergens, irritants, and occupational exposures. Each trigger probably acts through different mechanisms, but a final common pathway of multicellular inflammation, enhanced bronchial responsiveness, and airflow obstruction is a likely consequence. This review discusses the most common causes of asthma exacerbations with a focus on their microbiology and immunopathogenesis. Through an understanding of underlying causes of asthma exacerbations, treatments with increased effectiveness may be developed, and it is these future developments that may directly influence the morbidity and mortality of the disease.

Prevalence of neutralizing antibodies to Equine rhinitis A and B virus in horses and man.

Equine rhinitis viruses (ERVs) are the causative agents of mild to severe upper respiratory infections in horses worldwide. Immunologically, four serotypes of ERVs have been identified. Equine rhinitis A virus (ERAV) and Equine rhinitis B virus 1 (ERBV1) are the most frequent serotypes in Europe. Both viruses have a broad host range in cultured cells with ERAV being able to infect humans. Since there is neither information on the seroprevalence of ERAV and ERBV1 in Austria nor on the zoonotic potential of ERBV1, we investigated 200 horse and 137 veterinary sera for the presence of neutralizing antibodies relating to ERAV and ERBV1. One hundred and eighty (90%) and 173 (86%) horse sera neutralized ERAV and ERBV1, respectively. In contrast, only four (2.7%) and five (3.6%) human sera showed weak neutralizing activity to ERAV and ERBV1, respectively. These results indicate that ERAV and ERBV1 are widespread in the Austrian horse population; however, the risk of acquiring zoonotic infection among veterinarians appears low.

Relationships among specific viral pathogens, virus-induced interleukin-8, and respiratory symptoms in infancy.

Both virus-mediated damage to airway tissues and induction of pro-inflammatory cytokines such as interleukin-8 (IL-8) could contribute to symptom severity during viral respiratory infections in children. To test the hypothesis that IL-8 contributes to the pathogenesis of respiratory symptoms during naturally acquired respiratory viral infections in children, nasal wash samples collected from infants with acute viral infections (n = 198) or from healthy uninfected infants (n = 31) were analysed for IL-8. Nasal wash IL-8 was positively related to age in uninfected children (rs = 0.36, p < 0.05). Respiratory syncytial virus (RSV) infection caused more severe respiratory symptoms compared to infections with influenza A, parainfluenza viruses, or rhinoviruses. In addition, RSV, parainfluenza and rhinovirus infections increased levels of IL-8 in nasal lavage fluid, and there were some differences in the ability of the viruses to induce IL-8 production (RSV>influenza, p < 0.05). Finally, there were significant correlations between nasal wash IL-8 levels and symptom scores during infections with rhinovirus (rs = 0.56, p < 0.001) or influenza A (rs = 0.45, p < 0.05), but not with parainfluenza virus or RSV. These findings provide evidence of a close relationship between the generation of IL-8 and symptoms during acute community-acquired infections with rhinovirus or influenza A. In contrast, for RSV and parainfluenza infections, factors in addition to IL-8 production appear to contribute to the generation of clinical symptoms.

Nested PCR for specific detection and rapid identification of human picornaviruses.

A nested PCR for the detection and rapid identification of human picornaviruses is described. Enteroviruses and rhinoviruses were amplified with the same set of four primers from the 5'-noncoding region. The nested primers allowed the detection of far less than 1 PFU in diluted virus stocks without Southern blot hybridization. In patients with neurological disorders (mainly aseptic meningitis), 43% of 37 specimens (11 of 21 cerebrospinal fluid specimens, 2 of 10 serum specimens, and 3 of 6 stool specimens) were positive by PCR. A total of 21% (10 of 47 specimens) of heart biopsy specimens from patients with dilative cardiomyopathy were PCR positive, whereas 3% (2 of 70 specimens) of control biopsy specimens from patients with coronary artery disease were PCR positive. PCR-amplified fragments from 27 of 29 clinical isolates and 14 of 28 patient samples were successfully serotyped by restriction enzyme digestion. Two specimens were further investigated by direct sequencing of PCR products, leading to the identification of a poliovirus type 3 isolate with a sequence that was highly divergent from previously published sequences.