The Contribution of Viruses and Bacteria to Childhood Community-acquired Pneumonia: 11-Year Observational Study From Norway.

BACKGROUND: Viruses are associated with pediatric community-acquired pneumonia (CAP) but are also common in the upper airways of healthy children. We have determined the contribution of respiratory viruses and bacteria by comparing children with CAP and hospital controls. METHODS: Children less than 16 years old with radiologically confirmed CAP (n = 715) were enrolled over an 11-year period. Children admitted for elective surgery during the same period served as controls (n = 673). Nasopharyngeal aspirates were tested for 20 respiratory pathogens by semiquantitative polymerase chain reaction tests and cultivated for bacteria and viruses. We used logistic regression to calculate adjusted odds ratios [aOR; 95% confidence intervals (CIs)], and estimated population-attributable fractions (95% CI). RESULTS: At least 1 virus was detected in 85% of cases and 76% of controls, and greater than or equal to 1 bacterium was detected in 70% of cases and controls. The presence of respiratory syncytial virus (RSV) (aOR, 16.6; 95% CI: 9.81-28.2), human metapneumovirus (HMPV) (13.0; 6.17-27.5) and Mycoplasma pneumoniae (27.7; 8.37-91.6) were most strongly associated with CAP. For RSV and HMPV, there were significant trends between lower cycle-threshold values indicating higher viral genomic loads, and higher aORs for CAP. The population-attributable fraction estimates of RSV, HMPV, human parainfluenza virus, influenza virus and M. pneumoniae were 33.3% (32.2-34.5), 11.2% (10.5-11.9), 3.7% (1.0-6.3), 2.3% (1.0-3.6) and 4.2% (4.1-4.4), respectively. CONCLUSIONS: RSV, HMPV and M. pneumoniae were most strongly related to pediatric CAP and accounted for half of all cases. There were positive trends between increasing viral genomic loads of RSV and HMPV, and higher odds for CAP.

Viral and Atypical Bacterial Detection in Young Nepalese Children Hospitalized with Severe Pneumonia.

Respiratory viruses cause a substantial proportion of respiratory tract infections in children but are underrecognized as a cause of severe pneumonia hospitalization in low-income settings. We employed 22 real-time PCR assays and retrospectively reanalyzed 610 nasopharyngeal aspirate specimens from children aged 2 to 35 months with severe pneumonia (WHO definition) admitted to Kanti Childrens' Hospital in Kathmandu, Nepal, from January 2006 through June 2008. Previously, ≥1 of 7 viruses had been detected by multiplex reverse transcription-PCR in 30% (188/627) of cases. Reanalyzing the stored specimens, we detected ≥1 pathogens, including 18 respiratory viruses and 3 atypical bacteria, in 98.7% (602/610) of cases. Rhinovirus (RV) and respiratory syncytial virus (RSV) were the most common, detected in 318 (52.1%) and 299 (49%) cases, respectively, followed by adenovirus (AdV) (10.6%), human metapneumovirus (hMPV) (9.7%), parainfluenza virus type 3 (8.4%), and enterovirus (7.7%). The remaining pathogens were each detected in less than 5%. Mycoplasma pneumoniae was most common among the atypical bacteria (3.7%). Codetections were observed in 53.3% of cases. Single-virus detection was more common for hMPV (46%) and RSV (41%) than for RV (22%) and AdV (6%). The mean cycle threshold value for detection of each pathogen tended to be lower in single-pathogen detections than in codetections. This finding was significant for RSV, RV, and AdV. RSV outbreaks occurred at the end of the monsoon or during winter. An expanded diagnostic PCR panel substantially increased the detection of respiratory viruses in young Nepalese children hospitalized with severe pneumonia. IMPORTANCE Respiratory viruses are an important cause of respiratory tract infections in children but are underrecognized as a cause of pneumonia hospitalization in low-income settings. Previously, we detected at least one of seven respiratory viruses by PCR in 30% of young Nepalese children hospitalized with severe pneumonia over a period of 36 months. Using updated PCR assays detecting 21 different viruses and atypical bacteria, we reanalyzed 610 stored upper-respiratory specimens from these children. Respiratory viruses were detected in nearly all children hospitalized for pneumonia. RSV and rhinovirus were the predominant pathogens detected. Detection of two or more pathogens was observed in more than 50% of the pneumonia cases. Single-virus detection was more common for human metapneumovirus and RSV than for rhinovirus and adenovirus. The concentration of virus was higher (low cycle threshold [CT] value) for single detected pathogens, hinting at a high viral load as a marker of clinical significance.

Parechovirus A in Hospitalized Children With Respiratory Tract Infections: A 10-Year-Long Study From Norway.

BACKGROUND: The role of Parechovirus A (PeV-A) in hospitalized children with respiratory tract infections (RTIs) is unclear. We studied the occurrence and impact of PeV-A over 10 years. METHODS: Children from Sør-Trøndelag County, Norway, hospitalized with RTI and a comparison group of asymptomatic children admitted to elective surgery, were prospectively enrolled from 2006 to 2016. Nasopharyngeal aspirates were cultured and analyzed with polymerase chain reaction tests for PeV-A and 19 other pathogens. The cycle threshold levels of PeV-A were reported as measures of viral genomic loads. Parechovirus A-positive samples were genotyped by amplification and sequencing of the VP3/VP1 junction. RESULTS: Parechovirus A was detected in 8.8% (323/3689) patients with RTI and in 10.1% (45/444) of the children in the comparison group (P = .34). Parechovirus A genotyping (n = 188) revealed PeV-A1 (n = 121), PeV-A3 (n = 15), PeV-A5 (n = 6), and PeV-A6 (n = 46). Viral codetections occurred in 95% of patients and in 84% of the children in the comparison group (P = .016). In multivariable logistic regression analysis, RTI was unrelated to PeV-A genomic loads, adjusted for other viruses and covariates. Similar results were found for PeV-A1 and PeV-A6. CONCLUSIONS: Parechovirus A and viral codetections were common in hospitalized children with RTI and asymptomatic children in a comparison group. Our findings suggest that PeV-A has a limited role in hospitalized children with RTI.

No Correlation Between Nasopharyngeal Human Bocavirus 1 Genome Load and mRNA Detection or Serology in Adeno-/Tonsillectomy Patients.

Human bocavirus 1 (HBoV1) can persist in nasopharynx and tonsils. Using HBoV1 serology, reverse-transcription polymerase chain reaction (PCR) for detecting messenger RNA (mRNA) and quantitative PCR for HBoV1 genome load count, we studied to what extent the HBoV1 DNA loads in nasopharynx correlate with acute infection markers. Tonsillar tissue, nasopharyngeal aspirate, and serum were obtained from 188 elective adeno-/tonsillectomy patients. Relatively high loads of HBoV1 DNA were detected in the nasopharynx of 14 (7%) primarily asymptomatic subjects with negative mRNA and/or serodiagnostic results. Quantitative HBoV1 DNA PCR may have lower specificity than HBoV1 mRNA detection for diagnosing symptomatic infection.

Comparison of procalcitonin, C-reactive protein, white blood cell count and clinical status in diagnosing pneumonia in patients hospitalized with acute exacerbations of COPD: A prospective observational study.

Lower respiratory tract infection is the most common cause of acute exacerbations of chronic obstructive pulmonary disease (AECOPD). The aim of the present study was to compare the accuracy of procalcitonin (PCT), C-reactive protein (CRP) and white blood cell count (WBC) as single diagnostic tests and in combination with clinical signs and symptoms to diagnose pneumonia in patients hospitalized with AECOPD. This was a prospective, single centre observational study. Patients with spirometry-confirmed COPD who were hospitalized due to AECOPD were consecutively recruited at the hospital's Emergency Unit. Pneumonia was defined as a new pulmonary infiltrate on chest X-ray. The values of PCT, CRP and WBC were determined at admission. Receiver operating characteristic (ROC) curve analysis was used to study the accuracy of various diagnostic tests. Of the 113 included patients, 35 (31%) had pneumonia at admission. Area under the ROC curve (AUC) for PCT, CRP and WBC as a single test to distinguish between patients with and without pneumonia was 0.67 (95% CI 0.55-0.79), 0.73 (95% CI 0.63-0.84) and 0.67 (95% CI 0.55-0.79), respectively ( p = 0.42 for the test of difference). The AUC for a model of clinical signs and symptoms was 0.84 (95% CI 0.76-0.92). When biomarkers were added to the clinical model, the AUCs of the combined models were not significantly different from that of the clinical model alone ( p = 0.54). PCT had about the same accuracy as CRP and WBC in predicting pneumonia in patients hospitalized with AECOPD both as a single test and in combination with clinical signs and symptoms.

Human adenovirus in nasopharyngeal and blood samples from children with and without respiratory tract infections.

BACKGROUND: Human adenovirus (HAdV) is a double-stranded DNA virus associated with respiratory tract infections (RTI) in children. Using polymerase chain reaction (PCR) tests, HAdV often is detected together with other virus species, even in healthy controls. OBJECTIVES: The aim of this study was to compare molecular detection of HAdV with culture, and to examine the associations of various methods to RTI. STUDY DESIGN: Nasopharyngeal aspirates (NPA) were collected from 4319 children admitted with RTI and from 361 controls. The NPAs were examined for 23 viral and bacterial pathogens, using inhouse real-time PCR-assays based on TaqMan probes, in addition to bacterial and viral culture. HAdV concentration was evaluated semi-quantitatively from the Ct-value and quantitatively by use of ADENOVIRUS R-gene®. RESULTS: HAdV-DNA was detected in 6.1% patient samples and in 10.5% controls (p< 0.001). Compared to controls, patients had an OR of 3.8 (95% CI 1.4-10.3) for mono-detection of HAdV DNA, and an OR of 5.1 (95% CI 2.0-13.4) for HAdV-positive samples grew adenovirus by culture. HAdV DNA loads from children with RTI consisted of two clusters: one cluster with high viral loads (Ct < 30 and >106 copies/ml) and one cluster with low viral loads, whereas among the controls, nearly all had low viral loads (OR 7.8, 95% CI 2.2-27.1). In 61 available plasma samples, 16.4% were positive for HAdV DNA, all were from patients. CONCLUSION: The detection of HAdV DNA per se by qualitative PCR is not useful as a diagnostic test. Detection of HAdV by use of viral culture and a high viral HAdV DNA load are the two methods most strongly associated with RTI in children.

Direct observation of proton pumping by a eukaryotic P-type ATPase.

In eukaryotes, P-type adenosine triphosphatases (ATPases) generate the plasma membrane potential and drive secondary transport systems; however, despite their importance, their regulation remains poorly understood. We monitored at the single-molecule level the activity of the prototypic proton-pumping P-type ATPase Arabidopsis thaliana isoform 2 (AHA2). Our measurements, combined with a physical nonequilibrium model of vesicle acidification, revealed that pumping is stochastically interrupted by long-lived (~100 seconds) inactive or leaky states. Allosteric regulation by pH gradients modulated the switch between these states but not the pumping or leakage rates. The autoinhibitory regulatory domain of AHA2 reduced the intrinsic pumping rates but increased the dwell time in the active pumping state. We anticipate that similar functional dynamics underlie the operation and regulation of many other active transporters.

Single vesicle biochips for ultra-miniaturized nanoscale fluidics and single molecule bioscience.

One of the major bottlenecks in the development of biochips is maintaining the structure and function of biomolecules when interfacing them with hard matter (glass, plastics, metals, etc.), a challenge that is exacerbated during miniaturization that inevitably increases the interface to volume ratio of these devices. Biochips based on immobilized vesicles circumvent this problem by encapsulating biomolecules in the protective environment of a lipid bilayer, thus minimizing interactions with hard surfaces. Here we review the development of biochips based on arrays of single nanoscale vesicles, their fabrication via controlled self-assembly, and their characterization using fluorescence microscopy. We also highlight their applications in selected fields such as nanofluidics and single molecule bioscience. Despite their great potential for improved biocompatibility, extreme miniaturization and high throughput, single vesicle biochips are still a niche technology that has yet to establish its commercial relevance.

Human bocavirus in children: mono-detection, high viral load and viraemia are associated with respiratory tract infection.

BACKGROUND AND OBJECTIVES: Human bocavirus 1 (HBoV1) has recently been detected in children with respiratory tract infections (RTI). In order to study whether HBoV1 can cause RTI, we investigated its presence in children with upper RTI (URTI), lower RTI (LRTI) and a control group of children without RTI. STUDY DESIGN: Nasopharyngeal aspirates (NPA) and blood samples were collected from children admitted to hospital with RTI from 6 June 2007 to 28 February 2009 (n=1154), and from children admitted for elective surgery who had no RTI (n=162). Using polymerase chain reaction (PCR), the NPAs were examined for 17 infectious agents including HBoV1. Blood samples were tested with HBoV1-PCR only. RESULTS: HBoV1 was detected in NPAs from 10% of patients and 17% of controls. Adjusted for age, gender and the presence of other viruses, HBoV1 was not associated with RTI. In the HBoV1-positive NPAs, at least one other virus was detected in 75% and the virus appeared alone in 25%. Adjusted for age and gender, the detection of HBoV1 as the sole virus was associated with RTI, but not with LRTI. Viraemia was found only in children with RTI. The study showed that it was associated with RTI and LRTI. A high HBoV1-load was associated with LRTI, but not with RTI. No interactions between HBoV1 and other infectious agents were found. CONCLUSIONS: Our data support the hypothesis that HBoV1 causes RTI in children, because detection of HBoV1 alone, viraemia and high viral load are associated with RTI and/or LRTI in this age group. However, HBoV1 is common in healthy children.