Outcomes of human adenovirus infection and disease in a retrospective cohort of pediatric solid organ transplant recipients.

Information about HAdV infection in SOT recipients is limited. We aimed to describe HAdV infection epidemiology and outcomes in a single-center retrospective cohort during the era of PCR availability. SOT recipients transplanted at the CHOP 2004-2013 were followed up for 180 days post-transplant. HAdV infection was defined as a positive HAdV PCR from a clinical specimen. HAdV disease was defined by organ-specific radiologic and/or laboratory abnormalities. No HAdV surveillance protocols were employed during the study period; testing was solely per clinician discretion. Progression of HAdV infection was defined as HAdV disease or ≥1-log viral load increase since a corresponding site's first positive specimen. Of the assembled 425 SOT recipients, 227 (52.6%) had ≥1 HAdV PCR. Twenty-four (10.6%) had ≥1 HAdV-positive PCR. HAdV-positive subjects were younger than uninfected subjects (2.0 years vs 6.5, P = 0.001). Infection incidence rates were highest in liver recipients (15.3%), followed by heart (8.6%), kidney (8.3%), and lung (4.2%). Four subjects (16.7%) met HAdV disease criteria at virus detection. Five subjects (20.8%) had progression of HAdV infection. All-cause mortality rates in positive and negative subjects were 0% and 3.9%, respectively. HAdV infection was infrequently detected in SOT recipients. Over one-third of HAdV-positive patients met disease criteria at detection or had infection progression, but none died. This low all-cause mortality raises questions about benefits of HAdV surveillance. Larger multicenter studies are needed to assess incidence variance by center and comparative effectiveness of therapeutic interventions.

Immunostimulatory Defective Viral Genomes from Respiratory Syncytial Virus Promote a Strong Innate Antiviral Response during Infection in Mice and Humans.

Human respiratory syncytial virus (RSV) is a major cause of severe respiratory illness in children and susceptible adults. RSV blocks the development of the innate antiviral immune response and can grow to high titers in the respiratory tract. Here we demonstrate that immunostimulatory defective viral genomes (iDVGs) that are naturally generated during RSV replication are strong inducers of the innate antiviral response to RSV in mice and humans. In mice, RSV iDVGs stimulated the expression of antiviral genes, restricted viral replication, and prevented weight loss and lung inflammation. In human cells, the antiviral response to RSV iDVGs was dominated by the expression of IFN-λ1 over IFN-ß and was driven by rapid intranuclear accumulation of the transcription factor IRF1. RSV iDVGs were detected in respiratory secretions of hospitalized patients, and their amount positively correlated with the level of expression of antiviral genes in the samples. Infection of explanted human lung tissue from different donors revealed that most humans can respond to RSV iDVGs and that the rate of accumulation of iDVGs during infection directly correlates with the quality of the antiviral response. Taken together, our data establish iDVGs as primary triggers of robust antiviral responses to RSV and provide the first evidence for an important biological role for naturally occurring iDVGs during a paramyxovirus infection in humans.

Potential antigenic explanation for atypical H1N1 infections among middle-aged adults during the 2013-2014 influenza season.

Influenza viruses typically cause the most severe disease in children and elderly individuals. However, H1N1 viruses disproportionately affected middle-aged adults during the 2013-2014 influenza season. Although H1N1 viruses recently acquired several mutations in the hemagglutinin (HA) glycoprotein, classic serological tests used by surveillance laboratories indicate that these mutations do not change antigenic properties of the virus. Here, we show that one of these mutations is located in a region of HA targeted by antibodies elicited in many middle-aged adults. We find that over 42% of individuals born between 1965 and 1979 possess antibodies that recognize this region of HA. Our findings offer a possible antigenic explanation of why middle-aged adults were highly susceptible to H1N1 viruses during the 2013-2014 influenza season. Our data further suggest that a drifted H1N1 strain should be included in future influenza vaccines to potentially reduce morbidity and mortality in this age group.

Challenges and opportunities in developing respiratory syncytial virus therapeutics.

Two meetings, one sponsored by the Wellcome Trust in 2012 and the other by the Global Virology Foundation in 2013, assembled academic, public health and pharmaceutical industry experts to assess the challenges and opportunities for developing antivirals for the treatment of respiratory syncytial virus (RSV) infections. The practicalities of clinical trials and establishing reliable outcome measures in different target groups were discussed in the context of the regulatory pathways that could accelerate the translation of promising compounds into licensed agents. RSV drug development is hampered by the perceptions of a relatively small and fragmented market that may discourage major pharmaceutical company investment. Conversely, the public health need is far too large for RSV to be designated an orphan or neglected disease. Recent advances in understanding RSV epidemiology, improved point-of-care diagnostics, and identification of candidate antiviral drugs argue that the major obstacles to drug development can and will be overcome. Further progress will depend on studies of disease pathogenesis and knowledge provided from controlled clinical trials of these new therapeutic agents. The use of combinations of inhibitors that have different mechanisms of action may be necessary to increase antiviral potency and reduce the risk of resistance emergence.

Recent H3N2 influenza virus clinical isolates rapidly acquire hemagglutinin or neuraminidase mutations when propagated for antigenic analyses.

Prior to serological testing, influenza viruses are typically propagated in eggs or cell culture. Recent human H3N2 strains bind to cells with low avidity. Here, we isolated nine primary H3N2 viral isolates from respiratory secretions of children. Upon propagation in vitro, five of these isolates acquired hemagglutinin or neuraminidase mutations that increased virus binding to cell surfaces. These mutations can potentially confound serological assays commonly used to identify antigenically novel influenza viruses.

Point: is the era of viral culture over in the clinical microbiology laboratory?

Conventional tube culture systems have long been the mainstay in clinical virology for the growth and identification of viruses from clinical specimens. Innovations such as centrifugation-enhanced shell vial and multiwell plate cultures and the use of genetically engineered and mixed cell lines, coupled with faster detection of viral replication, have allowed for reasonable turnaround times for even some of the most slowly growing clinically important human viruses. However, molecular methods, in particular, the PCR, have usurped the role of viral culture in many laboratories, limiting the use of this traditional method of virus detection or replacing it altogether. Advances and improvements in molecular technology over time have also resulted in newer generations of more rapid and accurate molecular assays for the detection, quantification, and genetic characterization of viruses. For this point-counterpoint, we have asked two individuals, Richard L. Hodinka of the Children's Hospital of Philadelphia, a clinical virologist whose laboratory has completely eliminated viral culture in favor of molecular methods, and Laurent Kaiser, head of the Virology Laboratory at the University of Geneva Hospital, who continues to be a strong advocate of viral culture, to discuss the relevance of viral culture in the molecular age.

Comparison of the GenMark Diagnostics eSensor respiratory viral panel to real-time PCR for detection of respiratory viruses in children.

A novel eSensor respiratory viral panel (eSensor RVP) multiplexed nucleic acid amplification test (GenMark Diagnostics, Inc., Carlsbad, CA) was compared to laboratory-developed real-time PCR assays for the detection of various respiratory viruses. A total of 250 frozen archived pediatric respiratory specimens previously characterized as either negative or positive for one or more viruses by real-time PCR were examined using the eSensor RVP. Overall agreement between the eSensor RVP and corresponding real-time PCR assays for shared analytes was 99.2% (kappa = 0.96 [95% confidence interval {CI}, 0.94 to 0.98]). The combined positive percent agreement was 95.4% (95% CI, 92.5 to 97.3); the negative percent agreement was 99.7% (95% CI, 99.4 to 99.8). The mean real-time PCR threshold cycle (C(T)) value for specimens with discordant results was 39.73 (95% CI, 38.03 to 41.43). Detection of coinfections and correct identification of influenza A virus subtypes were comparable between methods. Of note, the eSensor RVP rhinovirus assay was found to be more sensitive and specific than the corresponding rhinovirus real-time PCR. In contrast, the eSensor RVP adenovirus B, C, and E assays demonstrated some cross-reactivity when tested against known adenovirus serotypes representing groups A through F. The eSensor RVP is robust and relatively easy to perform, it involves a unique biosensor technology for target detection, and its multiplexed design allows for efficient and simultaneous interrogation of a single specimen for multiple viruses. Potential drawbacks include a slower turnaround time and the need to manipulate amplified product during the protocol, increasing the possibility of contamination.

Comparison of the Idaho Technology FilmArray system to real-time PCR for detection of respiratory pathogens in children.

The FilmArray Respiratory Panel (RP) multiplexed nucleic acid amplification test (Idaho Technology, Inc., Salt Lake City, UT) was compared to laboratory-developed real-time PCR assays for the detection of various respiratory viruses and certain bacterial pathogens. A total of 215 frozen archived pediatric respiratory specimens previously characterized as either negative or positive for one or more pathogens by real-time PCR were examined using the FilmArray RP system. Overall agreement between the FilmArray RP and corresponding real-time PCR assays for shared analytes was 98.6% (kappa = 0.92 [95% confidence interval (CI), 0.89 to 0.94]). The combined positive percent agreement was 89.4% (95% CI, 85.4 to 92.6); the negative percent agreement was 99.6% (95% CI, 99.2 to 99.8). The mean real-time PCR threshold cycle (C(T)) value for specimens with discordant results was 36.46 ± 4.54. Detection of coinfections and correct identification of influenza A virus subtypes were comparable to those of real-time PCR when using the FilmArray RP. The greatest comparative difference in sensitivity was observed for adenovirus; only 11 of 24 (45.8%; 95% CI, 27.9 to 64.9) clinical specimens positive for adenovirus by real-time PCR were also positive by the FilmArray RP. In addition, upon testing 20 characterized adenovirus serotypes prepared at high and low viral loads, the FilmArray RP did not detect serotypes 6 and 41 at either level and failed to detect serotypes 2, 20, 35, and 37 when viral loads were low. The FilmArray RP system is rapid and extremely user-friendly, with results available in just over 1 h with almost no labor involved. Its low throughput is a significant drawback for laboratories receiving large numbers of specimens, as only a single sample can be processed at a time with one instrument.

Herpes simplex testing in neonates in the emergency department.

OBJECTIVES: To determine the prevalence of central nervous system (CNS) herpes simplex virus (HSV) infection in neonates evaluated in the emergency department and to identify factors associated with cerebrospinal fluid (CSF) HSV polymerase chain reaction (PCR) testing. An existing testing paradigm was then applied to determine its potential impact on testing frequency. METHODS: This nested case-control study included infants aged 0 to 28 days who had lumbar puncture in the emergency department. Multivariate logistic regression was used to identify factors associated with CSF HSV PCR testing. RESULTS: The CSF HSV PCR testing was performed in 266 (47%) of 570 neonates. The prevalence of CNS HSV infection was 0.5% compared with 1.6% for bacterial meningitis. Performance of CSF HSV PCR testing was not associated with known HSV risk factors. Application of a known HSV testing paradigm would have reduced the proportion of infants tested by 21% without missing any of the cases of CNS HSV infection. CONCLUSIONS: The HSV testing remains common despite the low prevalence of HSV infection. The CSF HSV PCR testing is not well aligned with known risk factors. Future testing strategies should incorporate community HSV prevalence, known neonatal risk factors, and clinical judgment.

Evaluation of the Gen-Probe Aptima HIV-1 RNA qualitative assay as an alternative to Western blot analysis for confirmation of HIV infection.

The Gen-Probe Aptima HIV-1 RNA qualitative assay was evaluated as an alternative to Western blot analysis for the confirmation of HIV infection using serum samples that were repeatedly reactive for HIV antibodies. The Aptima HIV assay readily discriminated between HIV-1-infected and -uninfected individuals and effectively reduced the number of indeterminate results relative to Western blot analysis.

Herpes simplex virus testing and hospital length of stay in neonates and young infants.

OBJECTIVE: To examine whether ordering testing of cerebrospinal fluid (CSF) for herpes simplex virus (HSV) by polymerase chain reaction (PCR) in neonates and young infants is associated with increased hospital length of stay (LOS) or increased hospital charges. STUDY DESIGN: This retrospective cohort study enrolled infants age

Lack of cerebrospinal fluid pleocytosis in young infants with enterovirus infections of the central nervous system.

OBJECTIVES: To identify factors associated with cerebrospinal fluid (CSF) pleocytosis among infants aged 90 days or younger with enterovirus (EV) infections of the central nervous system (CNS). METHODS: This is a retrospective cohort study performed at an urban academic children's hospital. Patients aged 90 days or younger with a positive CSF EV polymerase chain reaction (PCR) test result obtained during the EV seasons from 2000 to 2006 were included. Patients with concomitant serious bacterial illness or herpes simplex virus infection were excluded. Multivariable logistic regression was used to identify factors independently associated with CSF pleocytosis. RESULTS: A total of 159 patients had a positive CSF EV PCR test result during the study period; 5 (3.1%) were excluded for concurrent serious bacterial infection. The median CSF white blood cell (WBC) count was 110/microL (interquartile range, 11-311/microL). Cerebrospinal fluid pleocytosis was present in 109 patients (71%). The proportion of infants with CSF pleocytosis accompanying EV CNS infections increased with age; CSF pleocytosis was present in 59%, 74%, and 90% of infants aged 0 to 28, 29 to 56, and 57 to 90 days, respectively (P = 0.007). Age and peripheral WBC count were independently associated with CSF pleocytosis. CONCLUSIONS: Among infants with EV CNS infections, the absence of CSF pleocytosis is related to younger age and lower peripheral WBC counts, perhaps reflecting the decreased ability of younger infants to mount a robust inflammatory response to EV infection. Thus, CSF EV PCR testing may be warranted in select patients without CSF pleocytosis.

Use of antibiotics in children hospitalized with community-acquired, laboratory-confirmed influenza.

Many children with influenza are treated with antibiotics. In this report, we describe the rate and indications for antibacterial use in children hospitalized with influenza. A total of 333 of 729 (46%) patients received >2 days of treatment with antibacterial medications, of whom 36% did not have an apparent indication for therapy.

Herpes zoster and meningitis resulting from reactivation of varicella vaccine virus in an immunocompetent child.

Herpes zoster complicated by meningitis has been mainly reported in immunocompromised patients after reactivation of wild-type varicella-zoster virus. We present one of the first cases of aseptic meningitis after herpes zoster caused by reactivation of vaccine-type varicella-zoster virus in an immunocompetent child. We also highlight the increasing role of both wild-type and vaccine strains of varicella-zoster virus as a cause of viral meningoencephalitis and the use of appropriate laboratory tools to rapidly and accurately identify the virus in order to provide prompt patient care and management.

Outcomes of Human Adenovirus Infection and Disease in a Retrospective Cohort of Pediatric Hematopoietic Cell Transplant Recipients.

BACKGROUND: Human adenoviruses (HAdVs) are associated with significant morbidity and death after hematopoietic cell transplantation (HCT). In this study, we sought to determine the incidence of HAdV infection among pediatric HCT recipients in the polymerase chain reaction (PCR) testing era, identify risk factors for viremia among patients undergoing HAdV surveillance, and assess the effectiveness of preemptive cidofovir. METHODS: A single-center retrospective cohort of patients who underwent a transplant within a 10-year period was assembled. The incidence of and outcomes of patients with HAdV infection and disease were determined by PCR results and chart review. A Cox regression model was used for surveilled allogeneic HCT recipients to identify factors associated with viremia. We also used a discrete-time failure model with inverse probability treatment weights to assess the effectiveness of preemptive cidofovir for infection. RESULTS: Among 572 HCT recipients, 76 (13.3%) had ≥1 sample that was HAdV PCR positive (3.5% of autologous HCT recipients and 19.7% of allogeneic HCT recipients). Among 191 allogeneic HCT recipients under surveillance, 58 (30.4%) had HAdV detected from any source, and 50 (26.2%) specifically had viremia. The mortality rate was higher in allogeneic HCT recipients with HAdV infection versus those without infection (25.9% vs 11.3%; P = .01). Factors associated with infection included an age of 6 to 12 years, an absolute lymphocyte count of <200 cells/µL, recent prednisone exposure, and recent bacteremia. Preemptive cidofovir was not associated with a reduced risk of infection progression (odds ratio, 0.96 [95% confidence interval, 0.30-3.05]). CONCLUSIONS: HAdV infection is common and associated with an increased rate of death after allogeneic HCT. Using prediction models that incorporate factors associated with HAdV might help target surveillance. Preemptive cidofovir therapy was not protective in a subset of HAdV-positive patients. Larger observational or randomized investigations are necessary, because the utility of surveillance requires effective preemptive therapies.

Detection of echovirus 18 in human breast milk.

We detected enteroviral RNA and cultured infectious virus from a series of banked breast milk samples from the mother of an infant with neonatal sepsis; sequencing of the enterovirus isolate identified it as echovirus type 18. In this case, it is possible that enterovirus transmission occurred through the breast milk.

The clinical and molecular epidemiology of community- and healthcare-acquired rotavirus gastroenteritis.

BACKGROUND: Rotavirus is the most common etiologic agent of healthcare-acquired diarrhea in pediatric patients. There has been little published information on healthcare-acquired rotavirus infection. METHODS: This was a retrospective cohort study of children hospitalized with rotavirus gastroenteritis at our institution between December 1999 and May 2004. Patients with community- and healthcare-acquired rotavirus gastroenteritis were compared with regards to age, time of infection, patient unit, and viral subtype as determined by reverse transcription polymerase chain reaction sequencing. RESULTS: Five hundred seventy-seven children were hospitalized with rotavirus gastroenteritis during the study period. One hundred twenty-one (21%) of these infections were healthcare-acquired. The incidence of healthcare-acquired infection was 4.2 cases per 10,000 patient-days. With the exception of 1 outbreak on an isolated patient unit, community- and healthcare-acquired disease affected similar patient populations, had the same temporal distribution, and were caused by viruses with similar subtypes. However, there was a significant difference between the geographic distribution of community- and healthcare-acquired disease within the hospital (P < 0.001). The majority (83%) of community-acquired cases were admitted to general medicine-surgery units, but only 53% of the healthcare-acquired cases occurred on these units (P = 0.005). The remaining healthcare-acquired infections occurred on units that rarely admitted patients with community-acquired disease. CONCLUSIONS: Healthcare-acquired rotavirus gastroenteritis seems to be caused by repeated introduction of community strains into the hospital setting. Heightened attention to infection control practices and rapid rotavirus identification is necessary on all units, especially those that infrequently admit children with rotavirus gastroenteritis, to prevent the spread of healthcare-acquired disease.

A Multiplex PCR/LDR Assay for Viral Agents of Diarrhea with the Capacity to Genotype Rotavirus.

Rotavirus and noroviruses are major causes of diarrhea. Variable rotavirus vaccination efficacy in Africa and Asia is multifactorial, including the diversity of circulating strains and viral co-infection. We describe a multiplexed assay that detects and genotypes viruses from stool specimens. It includes a one-step reverse transcriptase PCR reaction, a ligase detection reaction (LDR), then hybridization of fluorescent products to micro-beads. In clinical samples it detects rotavirus, caliciviruses (sapovirus and norovirus), mixed infections, and genotypes or genogroups of rotaviruses and noroviruses, respectively. The assay also has the capacity to detect hepatitis A. The assay was validated on reference isolates and 296 stool specimens from the US and Ghana. The assay was 97% sensitive and 100% specific. The genogroup was concordant in 100% of norovirus, and the genotype in 91% and 89% of rotavirus G- and P-types, respectively. Two rare rotavirus strains, G6P[6] and G6P[8], were detected in stool specimens from Ghana. The high-throughput assay is sensitive, specific, and may be of utility in the epidemiological surveillance for rare and emerging viral strains post-rotavirus vaccine implementation.

Neurologic complications in children hospitalized with influenza: characteristics, incidence, and risk factors.

OBJECTIVE: To determine the characteristics, incidence, and risk factors for influenza-related neurologic complications (INC). STUDY DESIGN: A retrospective cohort study of INC in children hospitalized with laboratory-confirmed influenza infection (LCI) from June 2000 to May 2004 was conducted. Systematic chart review was performed to identify clinical characteristics and outcomes. A neighborhood cohort was constructed to estimate the incidence of INC. Logistic regression was used to identify independent risk factors for INC. RESULTS: Of 842 patients with LCI, 72 patients had an INC: influenza-related encephalopathy (8), post-infectious influenza encephalopathy (2), seizures (56), and other (6). Febrile seizures were the most common type of seizures (27). No patient died from an INC. In our neighborhood cohort, the incidence of INC was 4 cases per 100,000 person-years. An age of 6 to 23 months (odds ratio [OR], 4.2; 95% CI, 1.4-12.5) or 2 to 4 years (OR, 6.3; 95% CI, 2.1-19.1) and an underlying neurologic or neuromuscular disease (OR, 5.6; 95% CI, 3.2-9.6) were independent risk factors for the development of INC. CONCLUSION: Seizures are the most common neurologic complication experienced by children hospitalized with influenza. In the United States, encephalopathy is uncommon. Young children and patients with neurologic or neuromuscular disease are at increased risk for INC.