Marie L. Landry

Human Bocavirus Infection in Young Children in the United States: Molecular Epidemiological Profile and Clinical Characteristics of a Newly Emerging Respiratory Virus

Abstract BackgroundHuman bocavirus (HBoV) is a newly identified human parvovirus that was originally identified in the respiratory secretions of children with respiratory tract disease. To further investigate the epidemiological profile and clinical characteristics of HBoV infection, we screened infants and children <2 years of age (hereafter referred to as “children”) for HBoV MethodsChildren for whom respiratory specimens submitted to a diagnostic laboratory tested negative for respiratory syncytial virus, parainfluenza viruses (types 1–3), influenza A and B viruses, and adenovirus, as well as asymptomatic children, underwent screening for HBoV by use of polymerase chain reaction (PCR). Respiratory specimens were obtained from the children from 1 January 2004 through 31 December 2004 ResultsTwenty-two (5.2%) of the 425 children who had a respiratory specimen submitted to the diagnostic laboratory and 0 of the 96 asymptomatic children were found to be positive for HBoV by PCR (P=.02). Fever, rhinorrhea, cough, and wheezing were observed in ⩾50% of the HBoV-positive children. Of the 17 children who had chest radiography performed, 12 (70.6%) had abnormal findings. HBoV appeared to have a seasonal distribution. Nucleotide polymorphisms were detected in the viral capsid protein (VP) 1/VP2 genes. Two distinct HBoV genotypes circulated during the study period ConclusionsHBoV is circulating in the United States and is associated with both upper and lower respiratory tract disease in infants and young children

Rhinovirus stimulation of interleukin-6 in vivo and in vitro. Evidence for nuclear factor kappa B-dependent transcriptional activation.

To further understand the biology of rhinovirus (RV), we determined whether IL-6 was produced during RV infections and characterized the mechanism by which RV stimulates lung cell IL-6 production. In contrast to normals and minimally symptomatic volunteers, IL-6 was detected in the nasal washings from patients who developed colds after RV challenge. RV14 and RV1A, major and minor receptor group RVs, respectively, were potent stimulators of IL-6 protein production in vitro. These effects were associated with significant increases in IL-6 mRNA accumulation and gene transcription. RV was also a potent stimulator of IL-6 promoter-driven luciferase activity. This stimulation was modestly decreased by mutation of the nuclear factor (NF)-IL-6 site and abrogated by mutation of the NF-kappa B site in this promoter. An NF-kappa B-DNA binding activity, mediated by p65, p50, and p52 NF-kappa B moieties, was rapidly induced in RV-infected cells. Activator protein 1-DNA binding was not similarly altered. These studies demonstrate that IL-6 is produced during symptomatic RV infections, that RVs are potent stimulators of IL-6 elaboration, and that RV stimulation IL-6 production is mediated by an NF-kappa B-dependent transcriptional stimulation pathway. IL-6 may play an important role in the pathogenesis of RV infection, and NF-kappa B activation is likely to be an important event in RV-induced pathologies.

Serum 25-Hydroxyvitamin D and the Incidence of Acute Viral Respiratory Tract Infections in Healthy Adults

Background Declining serum concentrations of 25-hydroxyvitamin D seen in the fall and winter as distance increases from the equator may be a factor in the seasonal increased prevalence of influenza and other viral infections. This study was done to determine if serum 25-hydroxyvitamin D concentrations correlated with the incidence of acute viral respiratory tract infections. Methodology/Findings In this prospective cohort study serial monthly concentrations of 25-hydroxyvitamin D were measured over the fall and winter 2009–2010 in 198 healthy adults, blinded to the nature of the substance being measured. The participants were evaluated for the development of any acute respiratory tract infections by investigators blinded to the 25-hydroxyvitamin D concentrations. The incidence of infection in participants with different concentrations of vitamin D was determined. One hundred ninety-five (98.5%) of the enrolled participants completed the study. Light skin pigmentation, lean body mass, and supplementation with vitamin D were found to correlate with higher concentrations of 25-hydroxyvitamin D. Concentrations of 38 ng/ml or more were associated with a significant (p<0.0001) two-fold reduction in the risk of developing acute respiratory tract infections and with a marked reduction in the percentages of days ill. Conclusions/Significance Maintenance of a 25-hydroxyvitamin D serum concentration of 38 ng/ml or higher should significantly reduce the incidence of acute viral respiratory tract infections and the burden of illness caused thereby, at least during the fall and winter in temperate zones. The findings of the present study provide direction for and call for future interventional studies examining the efficacy of vitamin D supplementation in reducing the incidence and severity of specific viral infections, including influenza, in the general population and in subpopulations with lower 25-hydroxyvitamin D concentrations, such as pregnant women, dark skinned individuals, and the obese.

Interleukin-11: stimulation in vivo and in vitro by respiratory viruses and induction of airways hyperresponsiveness.

To address the role of IL-11 in viral airways dysfunction, we determined whether infectious agents that exacerbate asthma stimulate stromal cell IL-11 production, determined whether IL-11 could be detected at sites of viral infection and evaluated the effects of IL-11 on airway physiology. Respiratory syncytial virus (RSV), parainfluenza virus type 3 (PIV3), and rhinovirus (RV) 14 were potent stimulators while cytomegalovirus and adenovirus only weakly stimulated and herpes simplex virus type 2 and bacteria did not stimulate IL-11 elaboration. IL-11 was not detected or barely detected in nasal aspirates from children without, but was detected in aspirates from children with viral upper respiratory tract infections. The levels of IL-11 were highest in patients with clinically detectable wheezing. IL-11 also caused nonspecific airways hyperresponsiveness in BALB/c mice. These studies demonstrate that three major causes of viral-induced asthma, RSV, RV, and PIV, in contrast to other viruses and bacteria, share the ability to induce stromal cell IL-11 production. They also demonstrate that IL-11 can be detected in vivo during viral respiratory infections, that the presence of IL-11 correlates with clinical bronchospasm and that IL-11 is a potent inducer of airways hyperresponsiveness. IL-11 may be an important mediator in viral airways disorders.

A 1-Year Experience with Human Metapneumovirus in Children Aged <5 Years

Abstract Human metapneumovirus (hMPV) is a recently discovered respiratory pathogen. We tested respiratory specimens for the presence of hMPV by reverse-transcription polymerase chain reaction. These specimens were obtained over a 1-year period from children aged <5 years and had negative results by the direct fluorescent antibody test for respiratory syncytial virus, influenza A and B, parainfluenza viruses 1–3, and adenovirus. Overall, 54 (8.1%) of 668 individuals tested positive for hMPV. During March and April of the study period, hMPV was detected in 17.6% and 25.0% of specimens tested, respectively. At least 2 distinct genotypes of hMPV circulated during the study period. Fever, tachypnea, cough, rhinorrhea, retractions of the chest wall, and wheezing were common findings. Of hMPV-positive children, 60.4% were aged <12 months. hMPV accounted for a small but significant proportion of respiratory-tract disease in infants and children.

Association between a Novel Human Coronavirus and Kawasaki Disease

Abstract Kawasaki disease is a systemic vasculitis of childhood; its etiology is unknown. We identified evidence of a novel human coronavirus, designated “New Haven coronavirus” (HCoVNH), in respiratory secretions from a 6-month-old infant with classic Kawasaki disease. To further investigate the possible association between HCoV-NH infection and Kawasaki disease, we conducted a case-control study. Specimens of respiratory secretions from 8 (72.7%) of 11 children with Kawasaki disease and from 1 (4.5%) of 22 control subjects (children without Kawasaki disease matched by age and the time the specimens were obtained) tested positive for HCoVNH by reverse-transcriptase polymerase chain reaction (Mantel-Haenszel matched odds ratio, 16.0 [95% confidence interval, 3.4–74.4]; P = .0015). These data suggest that HCoV-NH infection is associated with Kawasaki disease.

Evidence of a novel human coronavirus that is associated with respiratory tract disease in infants and young children.

Abstract Background. The etiological agents responsible for a substantial proportion of respiratory tract diseases have not been identified. We sought to determine whether novel human coronaviruses (HCoVs) are circulating in New Haven, Connecticut, and, if so, whether they are associated with respiratory tract disease in infants and young children. Methods. We developed a polymerase chain reaction (PCR)-based approach for screening specimens from the respiratory tracts of symptomatic children. PCR probes that target regions of the replicase 1a gene that are conserved among genetically diverse animal CoVs and HCoVs were designed. Using these probes, we identified genomic sequences of a novel HCoV, designated “New Haven coronavirus” (HCoV-NH). Thereafter, we designed specific probes to screen respiratory specimens from children <5 years old for this novel HCoV. Clinical features associated with HCoV-NH infection were identified. Results. Seventy-nine (8.8%) of 895 children tested positive for HCoV-NH. Cough, rhinorrhea, tachypnea, fever, abnormal breath sounds, and hypoxia were the most common findings associated with HCoV-NH infection. Sequence analysis revealed that HCoV-NH is closely related to a novel HCoV recently reported in The Netherlands. Conclusions. The novel HCoVs identified in New Haven and The Netherlands are similar and likely represent the same species. This newly discovered virus may have worldwide distribution and may account for a significant proportion of respiratory tract disease in infants and young children.

Genotype Prevalence and Risk Factors for Severe Clinical Adenovirus Infection, United States 2004-2006

BACKGROUND Recently, epidemiological and clinical data have revealed important changes with regard to clinical adenovirus infection, including alterations in antigenic presentation, geographical distribution, and virulence of the virus. METHODS In an effort to better understand the epidemiology of clinical adenovirus infection in the United States, we adopted a new molecular adenovirus typing technique to study clinical adenovirus isolates collected from 22 medical facilities over a 25-month period during 2004-2006. A hexon gene sequence typing method was used to characterize 2237 clinical adenovirus-positive specimens, comparing their sequences with those of the 51 currently recognized prototype human adenovirus strains. In a blinded comparison, this method performed well and was much faster than the classic serologic typing method. RESULTS Among civilians, the most prevalent adenovirus types were types 3 (prevalence, 34.6%), 2 (24.3%), 1 (17.7%), and 5 (5.3%). Among military trainees, the most prevalent types were types 4 (prevalence, 92.8%), 3 (2.6%), and 21 (2.4%). CONCLUSIONS For both populations, we observed a statistically significant increasing trend of adenovirus type 21 detection over time. Among adenovirus isolates recovered from specimens from civilians, 50% were associated with hospitalization, 19.6% with a chronic disease condition, 11% with a bone marrow or solid organ transplantation, 7.4% with intensive care unit stay, and 4.2% with a cancer diagnosis. Multivariable risk factor modeling for adenovirus disease severity found that age <7 years (odds ratio [OR], 3.2; 95% confidence interval [CI], 1.4-7.4), chronic disease (OR, 3.6; 95% CI, 2.6-5.1), recent transplantation (OR, 2.7; 95% CI, 1.3-5.2), and adenovirus type 5 (OR, 2.7; 95% CI, 1.5-4.7) or type 21 infection (OR, 7.6; 95% CI, 2.6-22.3) increased the risk of severe disease.

Comparison of BD GeneOhm Cdiff Real-Time PCR Assay with a Two-Step Algorithm and a Toxin A/B Enzyme-Linked Immunosorbent Assay for Diagnosis of Toxigenic Clostridium difficile Infection

ABSTRACT The BD GeneOhm Cdiff assay, a real-time PCR assay for the detection of the Clostridium difficile toxin B (tcdB) gene, was compared with the toxin A/B (Tox A/B) II enzyme-linked immunosorbent assay (ELISA) and a two-step algorithm which includes a C. Diff Chek-60 glutamate dehydrogenase (GDH) antigen assay followed by cytotoxin neutralization. Four hundred liquid or semisolid stool samples submitted for diagnostic C. difficile testing, 200 GDH antigen positive and 200 GDH antigen negative, were selected for analysis. All samples were tested by the C. Diff Chek-60 GDH antigen and cytotoxin neutralization assays, the Tox A/B II ELISA, and the BD GeneOhm Cdiff assay. Specimens with discrepant results were tested by toxigenic culture as an independent “gold standard.” Of 200 GDH-positive samples, 71 were positive by the Tox A/B II ELISA, 88 were positive by the two-step method, 93 were positive by PCR, and 96 were positive by the GDH antigen assay only. Of 200 GDH-negative samples, 3 were positive by PCR only. Toxigenic culture was performed for 41 samples with discrepant results, and 39 were culture positive. Culture resolution of discrepant results showed the Tox A/B II assay to have detected 70 (66.7%), the two-step method to have detected 87 (82.9%), and PCR to have detected 96 (91.4%) of 105 true positives. The BD GeneOhm Cdiff assay was more sensitive in detecting toxigenic C. difficile than the Tox A/B II assay (P < 0.0001); however, the difference between PCR and the two-step method was not significant (P = 0.1237). Enhanced sensitivity and rapid turnaround time make the BD GeneOhm Cdiff assay an important advance in the diagnosis of toxigenic C. difficile infection.

A Standardized Plaque Reduction Assay for Determination of Drug Susceptibilities of Cytomegalovirus Clinical Isolates

ABSTRACT Twelve laboratories collaborated in formulating and testing a standardized plaque reduction assay for cytomegalovirus (CMV) cell-associated clinical isolates. Four characterized and plaque-purified CMV strains, as well as six coded clinical isolates obtained after antiviral therapy, were distributed and tested. Good agreement was obtained for four of the clinical isolates, but a broad distribution of results was obtained for two isolates. Analysis of these results indicates the problems associated with clinical isolates, including the large genetic variability and the highly cell-associated phenotype. This collaborative effort, by addressing these problems, represents a significant step toward the development of a standardized assay.