Evaluating the ALERT algorithm for local outbreak onset detection in seasonal infectious disease surveillance data.

Estimation of epidemic onset timing is an important component of controlling the spread of seasonal infectious diseases within community healthcare sites. The Above Local Elevated Respiratory Illness Threshold (ALERT) algorithm uses a threshold-based approach to suggest incidence levels that historically have indicated the transition from endemic to epidemic activity. In this paper, we present the first detailed overview of the computational approach underlying the algorithm. In the motivating example section, we evaluate the performance of ALERT in determining the onset of increased respiratory virus incidence using laboratory testing data from the Children's Hospital of Colorado. At a threshold of 10 cases per week, ALERT-selected intervention periods performed better than the observed hospital site periods (2004/2005-2012/2013) and a CUSUM method. Additional simulation studies show how data properties may effect ALERT performance on novel data. We found that the conditions under which ALERT showed ideal performance generally included high seasonality and low off-season incidence.

Identification of Enteropathogens by Multiplex PCR among Rural and Urban Guatemalan Children with Acute Diarrhea.

Multiplex polymerase chain reaction (PCR) platforms have enhanced understanding of intestinal pathogens in low- and middle-income countries (LMICs). However, few such studies have been performed in Latin America, where poverty, poor sanitation, and undernutrition persist. Multiplex PCR (BioFire, Salt Lake City, UT) was used to identify viral, bacterial, and parasitic pathogens in stool collected on day 1 and 31 from children aged 6 to 35 months with acute, non-bloody diarrhea in two locations (rural and urban) in Guatemala. We analyzed correlation between pathogens and clinical, demographic, and socioeconomic variables; described patterns of pathogen acquisition, persistence, and clearance over the 30-day period; and calculated population attributable fractions (PAFs) for diarrheal causation for individual pathogens. We analyzed 316 subjects (144 urban; 172 rural) enrolled between March 2015 and January 2016. Rural subjects had significantly more malnutrition, animal exposure, and unimproved water/sanitation infrastructure. The majority of subjects had multiple pathogens/sample (4.8 rural and 2.7 urban). Few meaningful correlates were identified between individual pathogens and clinical, demographic, or environmental variables. Escherichia coli pathotypes, Shigella, Campylobacter, and Giardia had high rates of persistence between initial and 30-day follow-up. Statistically significant adjusted PAFs were identified for Campylobacter (14.9%, 95% CI: 3.2-23.1), norovirus (10.2%, 95% CI: 0.4-17.1), sapovirus (7.6%, 95% CI: 2.3-10.9), and adenovirus 40/41 (5.6%, 95% CI: 0.3-8.7). These observations further characterize the diversity and complexity of enteric pathogens in children in LMICs. Patterns of chronic symptomatic and asymptomatic infection among Latin American children are similar to those observed in other LMIC regions. Findings have direct implications for practitioners treating individuals with acute infectious diarrhea and should inform regional public health strategies.

Validation of a novel molecular diagnostic panel for pediatric musculoskeletal infections: Integration of the Cepheid Xpert MRSA/SA SSTI and laboratory-developed real-time PCR assays for clindamycin resistance genes and Kingella kingae detection.

BACKGROUND: Pathogen detection in pediatric patients with musculoskeletal infections relies on conventional bacterial culture, which is slow and can delay antimicrobial optimization. The ability to rapidly identify causative agents and antimicrobial resistance genes in these infections may improve clinical care. METHODS: Convenience specimens from bone and joint samples submitted for culture to Children's Hospital Colorado (CHCO) from June 2012 to October 2016 were evaluated using a "Musculoskeletal Diagnostic Panel" (MDP) consisting of the Xpert MRSA/SA SSTI real-time PCR (qPCR, Cepheid) and laboratory-developed qPCRs for Kingella kingae detection and erm genes A, B, and C which confer clindamycin resistance. Results from the MDP were compared to culture and antimicrobial susceptibility testing (AST) results. RESULTS: A total of 184 source specimens from 125 patients were tested. The sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV) of the Xpert MRSA/SA SSTI compared to culture and AST results were 85%, 98%, 93%, and 95% respectively for MSSA and 82%, 100%, 100%, and 99% for MRSA. Compared to phenotypic clindamycin resistance in S. aureus isolates, the erm A, B, and C gene PCRs collectively demonstrated a sensitivity, specificity, PPV, and NPV of 80%, 96%, 67%, and 98%. In comparison to clinical truth, Kingella PCR had a sensitivity, specificity, PPV, and NPV of 100%, 99.5%, 100%, and 100%. CONCLUSIONS: This novel MDP offers a rapid, sensitive, and specific option for pathogen detection in pediatric patients with musculoskeletal infections.

Letter to the editor: economic analysis lacks external validity to support universal syndromic testing for suspected meningitis/encephalitis.

In Response to: Duff S, et al. "Economic analysis of rapid multiplex polymerase chain reaction testing for meningitis/encephalitis in pediatric patients" Future Microbiology (2018) (Epub ahead of print).

Automated Real-Time Collection of Pathogen-Specific Diagnostic Data: Syndromic Infectious Disease Epidemiology.

BACKGROUND: Health care and public health professionals rely on accurate, real-time monitoring of infectious diseases for outbreak preparedness and response. Early detection of outbreaks is improved by systems that are comprehensive and specific with respect to the pathogen but are rapid in reporting the data. It has proven difficult to implement these requirements on a large scale while maintaining patient privacy. OBJECTIVE: The aim of this study was to demonstrate the automated export, aggregation, and analysis of infectious disease diagnostic test results from clinical laboratories across the United States in a manner that protects patient confidentiality. We hypothesized that such a system could aid in monitoring the seasonal occurrence of respiratory pathogens and may have advantages with regard to scope and ease of reporting compared with existing surveillance systems. METHODS: We describe a system, BioFire Syndromic Trends, for rapid disease reporting that is syndrome-based but pathogen-specific. Deidentified patient test results from the BioFire FilmArray multiplex molecular diagnostic system are sent directly to a cloud database. Summaries of these data are displayed in near real time on the Syndromic Trends public website. We studied this dataset for the prevalence, seasonality, and coinfections of the 20 respiratory pathogens detected in over 362,000 patient samples acquired as a standard-of-care testing over the last 4 years from 20 clinical laboratories in the United States. RESULTS: The majority of pathogens show influenza-like seasonality, rhinovirus has fall and spring peaks, and adenovirus and the bacterial pathogens show constant detection over the year. The dataset can also be considered in an ecological framework; the viruses and bacteria detected by this test are parasites of a host (the human patient). Interestingly, the rate of pathogen codetections, on average 7.94% (28,741/362,101), matches predictions based on the relative abundance of organisms present. CONCLUSIONS: Syndromic Trends preserves patient privacy by removing or obfuscating patient identifiers while still collecting much useful information about the bacterial and viral pathogens that they harbor. Test results are uploaded to the database within a few hours of completion compared with delays of up to 10 days for other diagnostic-based reporting systems. This work shows that the barriers to establishing epidemiology systems are no longer scientific and technical but rather administrative, involving questions of patient privacy and data ownership. We have demonstrated here that these barriers can be overcome. This first look at the resulting data stream suggests that Syndromic Trends will be able to provide high-resolution analysis of circulating respiratory pathogens and may aid in the detection of new outbreaks.

Comparison of Herpes Simplex Virus PCR with Culture for Virus Detection in Multisource Surface Swab Specimens from Neonates.

The American Academy of Pediatrics currently recommends herpes simplex virus (HSV) culture or PCR for testing of swabs of the conjunctivae, mouth, nasopharynx, and rectum (surface swabs) from neonates. The objectives of this study were to compare the performance and time to results of HSV PCR with those of HSV culture with surface swabs from neonates. Banked multisource surface swab samples that were collected from infants less than or equal to 30 days old from January 2017 to December 2017 and that had previously been cultured for HSV were identified and tested retrospectively by HSV PCR. Surface swab samples from 97 patients were included in the study. Of these 97 patients, 7 (7%) had clinical HSV disease. Of the 7 neonates with HSV disease, 3 (42.9%) had surface swabs positive by culture and 6 (85.7%) had swabs positive by PCR. Limiting the analysis to specimens that were positive only by culture or only by PCR, the specificity for both methods was 100%, but the sensitivity of PCR was 100%, whereas it was 50% for culture. During the study period, 341 HSV cultures and 426 HSV PCRs were performed. The median time from swab collection to reporting of results was 7.6 days (interquartile range [IQR], 7.1 to 7.9 days) for culture and 0.8 days (IQR, 0.6 to 1.0 days) for PCR. HSV PCR of surface swabs from neonates was considerably more rapid and sensitive than HSV culture without yielding false-positive results. Although larger studies are needed to support our findings, strong consideration should be given to utilize PCR instead of culture for the detection of HSV in surface swabs from neonates.

Efficacy of a bovine colostrum and egg-based intervention in acute childhood diarrhoea in Guatemala: a randomised, double-blind, placebo-controlled trial.

BACKGROUND: Treatments for paediatric diarrhoeal disease are limited. We assessed the impact of a bovine colostrum and egg-based treatment designed to reduce diarrhoea duration through non-specific and pathogen-directed mechanisms in children. METHODS: Randomised, double-blind, placebo-controlled trial of PTM202, derived from bovine colostrum and hyperimmune hen's egg on the duration of acute diarrhoeal disease in Guatemalan children. PTM202 contains specific immunoglobulins that target rotavirus, enterotoxigenic Escherichia coli, Shiga toxin-producing E. coli and Salmonella. Children aged 6-35 months presenting to three sites (one rural and two urban) with acute non-bloody diarrhoea were computer randomised to receive three daily doses of PTM202 or placebo. The primary outcome was the post-treatment duration of diarrhoea assessed in the per protocol population. Diarrhoeal pathogens were identified in stool by multiplex PCR (FilmArray Gastrointestinal-Panel, BioFire, Salt Lake City, Utah, USA). Key secondary outcomes included postdiarrhoeal weight gain and impact on diarrhoeal duration stratified by study site and presence of PTM202-targeted organisms in stool at enrolment. Safety was assessed in all participants. RESULTS: From 9 March 2015 to 25 January 2016, 325 children were enrolled, and 301 (154 intervention and 147 placebo) were analysed for the primary outcome. No difference in diarrhoea duration was observed between intervention and placebo in the total population, but a significant reduction was observed in the treatment group among children with at least one targeted pathogen in stool (HR=1.46, P=0.02), an effect most pronounced in urban subjects (HR 2.20, P=0.007) who had fewer stool pathogens and better nutritional status. No impact on 2-week or 4-week weight gain was noted. No adverse events attributed to PTM202 occurred. CONCLUSION: Results demonstrate the potential to target specific pathogens occurring in children with acute non-bloody diarrhoea and shorten illness duration using a novel, safe, nutrition-based intervention. PTM202 may represent a new tool to ameliorate the effects of acute diarrhoeal disease in low/middle-income populations. TRIAL REGISTRATION NUMBER: NCT02385773; Results.

Surveillance for enterovirus D68 in colorado children reveals continued circulation.

BACKGROUND: The largest, most widespread outbreak of enterovirus D68 respiratory disease occurred from August to December of 2014 in the United States with 1153 confirmed infections in 49 states. The epidemiology of enterovirus D68 following the 2014 outbreak is unknown. OBJECTIVES: This study seeks to describe the epidemiology of enterovirus D68 circulation amongst Colorado children from 2014 to 2016. STUDY DESIGN: This is a prospective observational surveillance study of enterovirus D68 infection amongst children tested for respiratory pathogens from July-October 2014-2016 at Children's Hospital Colorado (CHCO), a quaternary care children's hospital in Aurora, CO. RESULTS: Amongst rhinovirus/enterovirus positive respiratory specimens from intensive care unit patients, ninety-eight of 314 (31.2%) in 2014, none of 307 (0%) specimens in 2015, and 19 of 240 (7.9%) specimens in 2016 were identified as enterovirus D68. Amongst respiratory specimens from all patients during the prospective active surveillance period, none of 1469 (0%) in 2015 and 46 of 1403 (3.3%) were positive for enterovirus D68. CONCLUSIONS: Surveillance for enterovirus D68 amongst respiratory specimens at a quaternary care children's hospital revealed a seasonal pattern of circulation in the late summer to early fall of 2014 and 2016. Continued surveillance of respiratory specimens is necessary to define the circulation pattern and understand the epidemiology of this emerging pathogen.

Epidemiology and Molecular Characteristics of Mycoplasma pneumoniae During an Outbreak of M. pneumoniae-associated Stevens-Johnson Syndrome.

BACKGROUND: An increase in Mycoplasma pneumoniae-associated Stevens-Johnson syndrome (SJS) cases at a Colorado pediatric hospital led to an outbreak investigation. We describe the epidemiologic and molecular characteristics of M. pneumoniae among SJS case-patients and surrounding community members during the outbreak. METHODS: M. pneumoniae polymerase chain reaction-positive respiratory specimens from 5 Colorado hospitals and 4 referral laboratories underwent confirmatory polymerase chain reaction testing; positive specimens then underwent multilocus variable-number tandem-repeat analysis (MLVA) and macrolide resistance testing. Three SJS-M. pneumoniae case-patient households were surveyed using a standardized questionnaire, and nasopharyngeal/oropharyngeal swabs were obtained from all consenting/assenting household contacts. International Classification of Diseases, 9th revision codes were used to identify pneumonia cases among Colorado patients 5-21 years of age from January 2009 to March 2014. RESULTS: Three different M. pneumoniae MLVA types were identified among the 5 SJS case-patients with confirmed infection; MLVA type 3-X-6-2 was seen more commonly in SJS case-patients (60%) than in 69 non-SJS community specimens (29%). Macrolide resistance was identified in 7% of community specimens but not among SJS case-patients. Of 15 household contacts, 5 (33%) were M. pneumoniae positive; all MLVA types were identical to those of the corresponding SJS case-patient, although the specimen from 1 contact was macrolide resistant. Overall pneumonia cases as well as those caused by M. pneumoniae specifically peaked in October 2013, coinciding with the SJS outbreak. CONCLUSIONS: The outbreak of M. pneumoniae-associated SJS may have been associated with a community outbreak of M. pneumoniae; clinicians should be aware of the M. pneumoniae-SJS relationship. Household transmission of M. pneumoniae was common within the households investigated.

Cytomegalovirus Retinitis in Pediatric Stem Cell Transplants: Report of a Recent Cluster and the Development of a Screening Protocol.

PURPOSE: The incidence of cytomegalovirus (CMV) retinitis in the pediatric allogeneic hematopoietic stem cell transplant (HSCT) population is unknown. We report a cluster of 5 pediatric patients with CMV retinitis diagnosed in a 12-month period and compare this to the rate of CMV viremia and retinitis in the 4 years prior. Presented is the ophthalmic screening protocol developed in response to this experience. DESIGN: Retrospective cross-sectional study. METHODS: A retrospective chart review was performed on patients at Children's Hospital of Colorado (CHCO) who received allogeneic HSCT between January 2010 and December 2014. Fisher exact test was used to compare the proportion of CMV viremia and CMV retinitis in patients transplanted between January 2010 and December 2013 with those transplanted in 2014. RESULTS: A total of 101 patients underwent allogeneic HSCT from January 2010 to December 2013; 32 (32%) tested positive for CMV viremia. No cases of CMV retinitis were identified. From January 2014 to December 2014, 28 patients underwent allogeneic HSCT; 13 patients (46%) had CMV viremia, not a statistically significant increase (P = .18). There were 5 cases of CMV retinitis diagnosed in those transplanted in 2014, a statistically significant increase compared with those transplanted in 2010-2013 (P = .0004). A multidisciplinary team was formed to review the literature and an ophthalmic screening protocol was developed. CONCLUSION: Our recent cluster of CMV retinitis in pediatric allogeneic HSCT patients may suggest a rise in incidence of CMV retinitis. We propose an ophthalmic screening protocol to diagnose retinitis in pediatric HSCT patients in the early, often asymptomatic stage.

Clinical Impact and Provider Acceptability of Real-Time Antimicrobial Stewardship Decision Support for Rapid Diagnostics in Children With Positive Blood Culture Results.

BACKGROUND: Rapid diagnostic technologies for infectious diseases have the potential to improve clinical outcomes, but guideline-recommended antimicrobial stewardship (AS) strategies are not currently optimized for rapid intervention. We evaluated the clinical impact and provider acceptability of implementing real-time AS decision support for children with positive blood culture results according to the FilmArray blood culture identification panel (BCID [BioFire Diagnostics]) at Children's Hospital Colorado. METHODS: A pre-post quasi-experimental design was used to compare the outcomes of 100 postintervention children with positive blood culture results matched with 200 preintervention control children. Causative organisms in the preintervention group were identified using conventional microbiologic techniques and communicated to providers by a microbiology technologist. Postintervention organisms were identified by the BCID and communicated by an AS provider in real time with interpretation and antimicrobial recommendations. The primary outcome was time to optimal antimicrobial therapy (time from blood culture collection to start of predetermined pathogen-specific regimen or antimicrobial discontinuation for contaminants) compared by a log-rank test and Kaplan-Meier analysis. Provider acceptability of the intervention was assessed via E-mailed surveys. RESULTS: The median time to optimal therapy decreased from 60.2 hours before intervention to 26.7 hours after intervention (P = .001). Among children with blood cultures that contained true pathogens, the time to effective antimicrobial therapy decreased from 6.9 to 3.4 hours (P = .03). Unnecessary antibiotic initiation for children with a culture that contained organisms considered to be contaminants decreased from 76% to 26% (P < .001). Providers reported a change in management as a result of BCID results in 73% of the cases and a mean overall satisfaction rating of 4.8 on a 5-point Likert scale. CONCLUSIONS: Real-time AS decision support for rapid diagnostics is associated with improved antimicrobial use and high satisfaction ratings by providers.

Enterovirus D68 in Critically Ill Children: A Comparison With Pandemic H1N1 Influenza.

OBJECTIVE: In 2014, the Unites States experienced an outbreak of enterovirus D68 associated with severe respiratory illness. The clinical characteristics associated with severe illness from enterovirus D68 during this outbreak compared with those associated with the 2009 H1N1 influenza virus outbreak are unknown. DESIGN AND SETTING: In this retrospective cohort study, we characterized the clinical features of children with enterovirus D68 admitted to the PICU between August 1, 2014, and November 1, 2014, and compared them with critically ill children infected with H1N1 influenza during the pandemic admitted between May 1, 2009, and January 31, 2010. PATIENTS: PICU patients. INTERVENTIONS: None. MEASUREMENTS AND MAIN RESULTS: Ninety-seven severely ill children with enterovirus D68 infections were compared with 68 children infected with H1N1 influenza during the 2009 pandemic. Children with enterovirus D68 were more likely to have asthma (62% vs 23%; p < 0.001) and present with reactive airway disease exacerbations, with greater receipt of albuterol (94% vs 49%) and steroids (89% vs 40%; p < 0.0001 for both). Although more children with enterovirus D68 were admitted to the ICU compared with those with H1N1 influenza, they had a shorter hospital length of stay (4 vs 7 d; p < 0.0001), with lower intubation rates (7% vs 44%), vasopressor use (3% vs 32%), acute respiratory distress syndrome (3% vs 24%), shock (0% vs 16%), and death (0% vs 12%; p < 0.05 for all). Compared with children with other enteroviruses and rhinoviruses, children with enterovirus D68 were more likely to have a history of asthma (64% vs 45%) or multiple prior wheezing episodes (54% vs 34%; p < 0.01 for both). CONCLUSIONS: Critically ill children with enterovirus D68 were more likely to present with reactive airway disease exacerbations, whereas children with H1N1 influenza were more likely to present with pneumonia. Compared with the pandemic H1N1 influenza outbreak, the enterovirus D68 outbreak resulted in more children requiring admission to the ICU, but was associated with less severe outcomes.

Enterovirus D68 Infection in Children with Acute Flaccid Myelitis, Colorado, USA, 2014.

During August 8, 2014-October 14, 2014, a total of 11 children with acute flaccid myelitis and distinctive neuroimaging changes were identified near Denver, Colorado, USA. A respiratory prodrome was experienced by 10, and nasopharyngeal specimens were positive for enterovirus D68 (EV-D68) for 4. To determine whether an association exists between EV-D68 infection and acute flaccid myelitis, we conducted a retrospective case-control study comparing these patients with 2 groups of outpatient control children (1 group tested for acute respiratory illness and 1 for Bordetella pertussis infection). Adjusted analyses indicated that, for children with acute flaccid myelitis, the odds of having EV-D68 infection were 10.3 times greater than for those tested for acute respiratory infection and 4.5 times greater than for those tested for B. pertussis infection. No statistical association was seen between acute flaccid myelitis and non-EV-D68 enterovirus or rhinovirus infection. These findings support an association between EV-D68 infection and acute flaccid myelitis.

Potential clinical impact of the film array meningitis encephalitis panel in children with suspected central nervous system infections.

The FilmArray Meningitis Encephalitis Panel, a multiplex PCR for testing of cerebrospinal fluid, was compared to conventional diagnostic methods in children with suspected central nervous system infections. The panel had comparable diagnostic yield (96% agreement) and improved time-to-diagnosis by 10.3 hours with potential for more judicious antimicrobial use, particularly acyclovir.

Outbreak of Mycoplasma pneumoniae-Associated Stevens-Johnson Syndrome.

BACKGROUND: Stevens-Johnson syndrome (SJS) is an uncommon, sporadic disease and outbreaks are rare. In November 2013, an outbreak of SJS was identified at Children's Hospital Colorado. METHODS: Outbreak cases were children aged 5-21 with a discharge diagnosis of SJS admitted from September 1 to November 30, 2013. Medical charts were reviewed using standardized data collection forms. Respiratory specimens were tested for viruses and Mycoplasma pneumoniae (Mp) by polymerase chain reaction (PCR). We conducted a separate 4-year retrospective case-control study comparing hospitalized SJS cases with and without evidence of Mp infection. RESULTS: During the outbreak, 8 children met SJS criteria. Median age was 11.5 years (range 8-16 years); 5 (63%) were boys and 5 (63%) were Mp-PCR-positive. Of the 5 PCR-positive children, none had preceding medication exposure, and all had radiographic pneumonia. All outbreak Mp isolates were macrolide susceptible. The retrospective case-control analysis showed that Mp-associated SJS episodes (n = 17) were more likely to have pneumonia (odds ratio [OR] 7.5, confidence interval [CI] 1.6­35.1), preceding respiratory symptoms (OR 30.0, CI 3.3­269.4) [corrected] an erythrocyte sedimentation rate ≥35 mg/dL (OR 22.8, CI 2.1-244.9), and ≤3 affected skin sites (OR 4.5, CI 1.2-17.4) than non-Mp-associated SJS episodes (n = 23). CONCLUSIONS: We report the largest outbreak of SJS in children, which was also predominately associated with Mp infection. Mp-associated SJS was associated with a distinct clinical presentation that included less extensive skin disease, an elevated erythrocyte sedimentation rate, and evidence of a preceding respiratory infection.

Viral gastroenteritis in children in Colorado 2006-2009.

Acute gastroenteritis accounts for a significant burden of medically attended illness in children under the age of five. For this study, four multiplex reverse transcription PCR assays were used to determine the incidence of adenovirus, astrovirus, coronavirus, norovirus GI and GII, rotavirus, and sapovirus in stool samples submitted for viral electron microscopy (EM) to the Children's Hospital Colorado. Of 1105 stool samples available, viral RNA/DNA was detected in 247 (26.2%) of 941 pediatric samples (median age = 2.97 years, 54% male) with 28 (3.0%) positive for more than one virus. Adenovirus, astrovirus, norovirus GI, norovirus GII, rotavirus, and sapovirus were detected in 95 (10.0%), 33 (3.5%), 8 (0.9%), 90 (9.6%), 49 (5.2%), and 2 (0.2%) of the pediatric samples, respectively. No coronaviruses were identified. Sequencing of norovirus positive samples indicated an outbreak of norovirus strain GII.4 in 2006 with evidence of numerous circulating strains. Multiple samples from the same immunocompromised patients demonstrated symptomatic shedding of norovirus for up to 32 weeks and astrovirus for 12 weeks. RT-PCR detected 99 of 111 (89%) adenovirus-positive samples versus 12 (11%) by EM, and 186 of 192 (97%) sapovirus/astrovirus/norovirus-positive samples versus 21 (11%) by EM. Noroviruses and adenoviruses are common causes of gastroenteritis in children. Immunocompromised patients can be infected with multiple viruses and shed viruses in their stools for prolonged periods. This data support the superiority of RT-PCR compared to EM for diagnosis of viral gastroenteritis.

A cluster of acute flaccid paralysis and cranial nerve dysfunction temporally associated with an outbreak of enterovirus D68 in children in Colorado, USA.

BACKGROUND: Clusters of acute flaccid paralysis or cranial nerve dysfunction in children are uncommon. We aimed to assess a cluster of children with acute flaccid paralysis and cranial nerve dysfunction geographically and temporally associated with an outbreak of enterovirus-D68 respiratory disease. METHODS: We defined a case of neurological disease as any child admitted to Children's Hospital Colorado (Aurora, CO, USA) with acute flaccid paralysis with spinal-cord lesions involving mainly grey matter on imaging, or acute cranial nerve dysfunction with brainstem lesions on imaging, who had onset of neurological symptoms between Aug 1, 2014, and Oct 31, 2014. We used Poisson regression to assess whether the numbers of cases during the outbreak period were significantly greater than baseline case numbers from a historical control period (July 31, 2010, to July 31, 2014). FINDINGS: 12 children met the case definition (median age 11·5 years [IQR 6·75-15]). All had a prodromal febrile illness preceding neurological symptoms by a median of 7 days (IQR 5·75-8). Neurological deficits included flaccid limb weakness (n=10; asymmetric n=7), bulbar weakness (n=6), and cranial nerve VI (n=3) and VII (n=2) dysfunction. Ten (83%) children had confluent, longitudinally extensive spinal-cord lesions of the central grey matter, with predominant anterior horn-cell involvement, and nine (75%) children had brainstem lesions. Ten (91%) of 11 children had cerebrospinal fluid pleocytosis. Nasopharyngeal specimens from eight (73%) of 11 children were positive for rhinovirus or enterovirus. Viruses from five (45%) of 11 children were typed as enterovirus D68. Enterovirus PCR of cerebrospinal fluid, blood, and rectal swabs, and tests for other causes, were negative. Improvement of cranial nerve dysfunction has been noted in three (30%) of ten children. All ten children with limb weakness have residual deficits. INTERPRETATION: We report the first geographically and temporally defined cluster of acute flaccid paralysis and cranial nerve dysfunction in children associated with an outbreak of enterovirus-D68 respiratory illness. Our findings suggest the possibility of an association between enterovirus D68 and neurological disease in children. If enterovirus-D68 infections continue to happen in an endemic or epidemic pattern, development of effective antiviral or immunomodulatory therapies and vaccines should become scientific priorities. FUNDING: National Center for Advancing Translational Sciences, National Institutes of Health.

Epidemiology and clinical characteristics of infants with human parechovirus or human herpes virus-6 detected in cerebrospinal fluid tested for enterovirus or herpes simplex virus.

Human parechovirus (HPeV) and human herpes virus-6 (HHV-6) are acquired commonly in infancy and associated with central nervous system infection. The prevalence of HPeV and HHV-6 in the cerebrospinal fluid (CSF) of infants tested for enterovirus (EV) and herpes-simplex virus (HSV) is unknown. All stored CSF samples from EV or HSV testing in infants less than 6 months of age at Children's Hospital Colorado between January 1, 2010 and December 31, 2011 were tested for HPeV, HHV-6, EV, and HSV by PCR. Clinical characteristics and epidemiological data were collected using retrospective electronic chart review. Of 239 infants tested, 29 cases of EV (12.1%), 7 cases of HPeV (2.9%), 5 cases of HHV-6 (2.1%), and 5 cases of HSV (2.1%) were identified with no bacterial co-infections. HPeV cases occurred between July and October in infants with median age of 24 days. Infants with HPeV had a median maximum temperature of 39 °C, median fever duration of 3 days and median peripheral white blood cell count of 5.2 × 10(3)/µL. HHV-6 cases occurred in infants with median age of 61 days without seasonality. Five percent of infants less than 6 months of age undergoing testing for EV or HSV have HPeV or HHV-6 in the CSF. Targeting testing of HPeV towards febrile infants less than 2 months of age with leukopenia in the late summer to early fall, and HHV-6 towards older infants may increase diagnostic yield. The clinical and fiscal impact of testing infants for HPeV and HHV-6 needs to be determined.

Isolation, propagation, genome analysis and epidemiology of HKU1 betacoronaviruses.

From 1 January 2009 to 31 May 2013, 15 287 respiratory specimens submitted to the Clinical Virology Laboratory at the Children's Hospital Colorado were tested for human coronavirus RNA by reverse transcription-PCR. Human coronaviruses HKU1, OC43, 229E and NL63 co-circulated during each of the respiratory seasons but with significant year-to-year variability, and cumulatively accounted for 7.4-15.6 % of all samples tested during the months of peak activity. A total of 79 (0.5 % prevalence) specimens were positive for human betacoronavirus HKU1 RNA. Genotypes HKU1 A and B were both isolated from clinical specimens and propagated on primary human tracheal-bronchial epithelial cells cultured at the air-liquid interface and were neutralized in vitro by human intravenous immunoglobulin and by polyclonal rabbit antibodies to the spike glycoprotein of HKU1. Phylogenetic analysis of the deduced amino acid sequences of seven full-length genomes of Colorado HKU1 viruses and the spike glycoproteins from four additional HKU1 viruses from Colorado and three from Brazil demonstrated remarkable conservation of these sequences with genotypes circulating in Hong Kong and France. Within genotype A, all but one of the Colorado HKU1 sequences formed a unique subclade defined by three amino acid substitutions (W197F, F613Y and S752F) in the spike glycoprotein and exhibited a unique signature in the acidic tandem repeat in the N-terminal region of the nsp3 subdomain. Elucidating the function of and mechanisms responsible for the formation of these varying tandem repeats will increase our understanding of the replication process and pathogenicity of HKU1 and potentially of other coronaviruses.

Epidemiology and clinical presentation of parainfluenza type 4 in children: a 3-year comparative study to parainfluenza types 1-3.

BACKGROUND: Human parainfluenza viruses (HPIVs) are among the most common causes of respiratory tract infections in children. Little is known about the epidemiology and clinical presentation of HPIV type 4. METHODS: A retrospective chart review and comparison of patients positive for HPIV types 1-4 by multiplex polymerase chain reaction between 2009 and 2012 at Children's Hospital Colorado was performed. Patients who had only direct fluorescent antibody testing performed or concurrent viral infections were excluded. RESULTS: Of 11,533 samples, 752 (6.5%) were positive for HPIV. After exclusion criteria, 316 samples were included in the study. HPIV-4 had year-round prevalence with biennial peaks in odd-numbered years. HPIV-4 and HPIV-3 had similar clinical presentations. 50.8% and 51.5% of patients with HPIV-3-4 had hypoxia compared to 20.3% and 33.3% of patients with HPIV-1-2 (P < .01). HPIV-1 (23.6%) and HPIV-2 (24.2%) were more associated with stridor than HPIV-3 (6.6%) and HPIV-4 (0%) (P < .01). No patients with HPIV-4 had croup. Patients with HPIV-4 had similar lengths of stay and mortality as those with HPIV-1-3. CONCLUSIONS: This is the first large-scale analysis of HPIV-4 clinical and epidemiologic features. HPIV-4 was most similar to HPIV-3 in clinical presentation. HPIV-4 had year-round prevalence with peaks in the autumn of odd-numbered years. HPIV-4 is a common respiratory pathogen capable of causing significant morbidity in children.