W. Allan Nix

Outbreak of Neurologic Enterovirus Type 71 Disease: A Diagnostic Challenge

BACKGROUNDnSimilar to poliovirus, enterovirus type 71 (EV71) causes severe disease, including aseptic meningitis, encephalitis, acute flaccid paralysis, and acute cardiopulmonary dysfunction. Large epidemics of EV71 infection have been reported worldwide.nnnMETHODSnAfter recognition of a cluster of cases of EV71 disease, we reviewed records of patients with EV71 disease who required hospitalization at The Childrens Hospital in Denver, Colorado, from 2003 through 2005. The presence of enterovirus was detected by reverse-transcriptase polymerase chain reaction (PCR) and/or viral culture of specimens from multiple sources, and the virus was typed as EV71 using genetic sequencing.nnnRESULTSnEight cases of EV71 disease were identified in both 2003 and 2005. Fifty-six percent of patients with EV71 disease were < or = 6 months of age (range, 4 weeks to 9 years). All 16 patients had EV71 central nervous system infection. Enterovirus PCR (EV-PCR) of cerebrospinal fluid specimens yielded positive results for only 5 (31.2%) of the 16 patients; all of these patients were < 4 months of age and had less severe disease. However, EV-PCR of upper respiratory tract specimens yielded positive results for 8 (100%) of 8 patients, and EV-PCR of lower gastrointestinal tract specimens yielded positive results for 7 (87.5%) of 8 patients.nnnCONCLUSIONSnAn outbreak of neurologic EV71 disease occurred in Denver, Colorado, during 2003 and 2005. Likely, EV71 disease remains unrecognized in other parts of the United States, because EV-PCR of cerebrospinal fluid frequently yields negative results. EV-PCR of specimens from the respiratory and gastrointestinal tracts had higher diagnostic yields than did EV-PCR of cerebrospinal fluid. EV71 infection should be considered in young children presenting with aseptic meningitis, encephalitis, acute flaccid paralysis, or acute cardiopulmonary collapse. EV71 infection may be an underrecognized emerging disease in the United States.

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

BACKGROUNDnClusters 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.nnnMETHODSnWe defined a case of neurological disease as any child admitted to Childrens 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).nnnFINDINGSn12 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.nnnINTERPRETATIONnWe 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.nnnFUNDINGnNational Center for Advancing Translational Sciences, National Institutes of Health.

Severe respiratory illness associated with a nationwide outbreak of enterovirus D68 in the USA (2014): a descriptive epidemiological investigation

n Summaryn n Backgroundn Enterovirus D68 (EV-D68) has been infrequently reported historically, and is typically associated with isolated cases or small clusters of respiratory illness. Beginning in August, 2014, increases in severe respiratory illness associated with EV-D68 were reported across the USA. We aimed to describe the clinical, epidemiological, and laboratory features of this outbreak, and to better understand the role of EV-D68 in severe respiratory illness.n n n Methodsn We collected regional syndromic surveillance data for epidemiological weeks 23 to 44, 2014, (June 1 to Nov 1, 2014) and hospital admissions data for epidemiological weeks 27 to 44, 2014, (June 29 to Nov 1, 2014) from three states: Missouri, Illinois and Colorado. Data were also collected for the same time period of 2013 and 2012. Respiratory specimens from severely ill patients nationwide, who were rhinovirus-positive or enterovirus-positive in hospital testing, were submitted between Aug 1, and Oct 31, 2014, and typed by molecular sequencing. We collected basic clinical and epidemiological characteristics of EV-D68 cases with a standard data collection form submitted with each specimen. We compared patients requiring intensive care with those who did not, and patients requiring ventilator support with those who did not. Mantel-Haenszel χ2 tests were used to test for statistical significance.n n n Findingsn Regional and hospital-level data from Missouri, Illinois, and Colorado showed increases in respiratory illness between August and September, 2014, compared with in 2013 and 2012. Nationwide, 699 (46%) of 1529 patients tested were confirmed as EV-D68. Among the 614 EV-D68-positive patients admitted to hospital, age ranged from 3 days to 92 years (median 5 years). Common symptoms included dyspnoea (n=513 [84%]), cough (n=500 [81%]), and wheezing (n=427 [70%]); 294 (48%) patients had fever. 338 [59%] of 574 were admitted to intensive care units, and 145 (28%) of 511 received ventilator support; 322 (52%) of 614 had a history of asthma or reactive airway disease; 200 (66%) of 304 patients with a history of asthma or reactive airway disease required intensive care compared with 138 (51%) of 270 with no history of asthma or reactive airway disease (p=0·0004). Similarly, 89 (32%) of 276 patients with a history of asthma or reactive airway disease required ventilator support compared with 56 (24%) of 235 patients with no history of asthma or reactive airway disease (p=0·039).n n n Interpretationn In 2014, EV-D68 caused widespread severe respiratory illness across the USA, disproportionately affecting those with asthma. This unexpected event underscores the need for robust surveillance of enterovirus types, enabling improved understanding of virus circulation and disease burden.n n n Fundingn None.n n

Parechovirus typing in clinical specimens by nested or semi-nested PCR coupled with sequencing

BACKGROUNDnThe Parechovirus genus (Picornaviridae) contains two known species, Human parechovirus (HPeV) and Ljungan virus (LV). HPeVs cause a wide spectrum of disease, including meningitis, gastroenteritis, encephalitis, respiratory illness, and neonatal sepsis-like disease. LVs are associated with diabetes and myocarditis in bank voles and have been proposed to cause disease in humans. The ability to rapidly and accurately type parechoviruses is critical to understanding their role in human disease.nnnOBJECTIVESnFor parechovirus molecular typing, we sought to develop reverse transcription, nested polymerase chain reaction (RT-PCR) assays to amplify the sequence encoding the VP1 capsid protein from all known members of the Parechovirus genus.nnnSTUDY DESIGNnThe assays consist of a two-step RT-PCR with primers flanking VP1 (PCR1), followed by semi-nested PCR2A and PCR2B reactions that produce overlapping amplicons, encompassing the complete VP1 gene, as well as a nested PCR2C that amplifies a shorter internal VP1 amplicon.nnnRESULTSnAll primer sets are 100% sensitive and 100% specific for the 77 parechovirus culture isolates tested. The semi-nested and nested PCR primer sets are 94% sensitive and 100% specific for detection of parechovirus in original specimens. Viral genotype can be deduced from analysis of amplicon sequences. Parechoviruses of the same type share>or=77% complete VP1 nucleotide sequence identity or >or=87% amino acid identity, while those of different types share<or=73% nucleotide identity and <or=81% amino acid identity.nnnCONCLUSIONSnThe PCR primers described here amplify VP1 sequences from all known parechoviruses, providing a sensitive, reliable system for molecular typing directly from original clinical specimens.

Acute Flaccid Myelitis in the United States, August–December 2014: Results of Nationwide Surveillance

BACKGROUNDnDuring late summer/fall 2014, pediatric cases of acute flaccid myelitis (AFM) occurred in the United States, coincident with a national outbreak of enterovirus D68 (EV-D68)-associated severe respiratory illness.nnnMETHODSnClinicians and health departments reported standardized clinical, epidemiologic, and radiologic information on AFM cases to the Centers for Disease Control and Prevention (CDC), and submitted biological samples for testing. Cases were ≤21 years old, with acute onset of limb weakness 1 August-31 December 2014 and spinal magnetic resonance imaging (MRI) showing lesions predominantly restricted to gray matter.nnnRESULTSnFrom August through December 2014, 120 AFM cases were reported from 34 states. Median age was 7.1 years (interquartile range, 4.8-12.1 years); 59% were male. Most experienced respiratory (81%) or febrile (64%) illness before limb weakness onset. MRI abnormalities were predominantly in the cervical spinal cord (103/118). All but 1 case was hospitalized; none died. Cerebrospinal fluid (CSF) pleocytosis (>5 white blood cells/µL) was common (81%). At CDC, 1 CSF specimen was positive for EV-D68 and Epstein-Barr virus by real-time polymerase chain reaction, although the specimen had >3000 red blood cells/µL. The most common virus detected in upper respiratory tract specimens was EV-D68 (from 20%, and 47% with specimen collected ≤7 days from respiratory illness/fever onset). Continued surveillance in 2015 identified 16 AFM cases reported from 13 states.nnnCONCLUSIONSnEpidemiologic data suggest this AFM cluster was likely associated with the large outbreak of EV-D68-associated respiratory illness, although direct laboratory evidence linking AFM with EV-D68 remains inconclusive. Continued surveillance will help define the incidence, epidemiology, and etiology of AFM.

An Outbreak of Concurrent Echovirus 30 and Coxsackievirus A1 Infections Associated with Sea Swimming among a Group of Travelers to Mexico

BACKGROUNDnEnteroviruses are shed in human stool and can cause a wide spectrum of illness. They are the leading cause of aseptic meningitis.nnnMETHODSnIn 2004, the Connecticut Department of Public Health investigated a meningitis cluster among persons returning from a school-organized trip to Mexico.nnnRESULTSnAmong 29 travelers (25 teenagers and 4 adult chaperones), 21 became acutely ill. Viral culture and nucleic acid amplification testing of stool (n=27) and cerebrospinal fluid (n=4) specimens identified enteroviral infection in 20 of 28 travelers from whom any specimen was obtained; 4 had echovirus 30 only, 11 had coxsackievirus (CV) A1 only, 4 had both echovirus 30 and CVA1, and 1 had CVA5 only. Illness onset dates were tightly clustered 4 days after a prolonged swim in the Gulf of Mexico. Time spent swimming was significantly associated with the odds of enteroviral infection (univariate odds ratio for each additional hour swimming, 14.3; 95% confidence interval, 1.3-154.3). Headache, fever, vomiting, and nausea occurred more frequently among the echovirus 30-infected travelers than among the uninfected control subjects (P< .05). The most frequent symptoms among travelers infected with only CVA1 identified were nausea and diarrhea (36% each), but neither was significantly associated with CVA1 infection; 5 patients with CVA1 infection were asymptomatic.nnnCONCLUSIONSnWe identified multiple enteroviruses among the travelers. Clustered illness onsets suggest point-source exposure, which likely was a sea swim in sewage-contaminated seawater. Novel molecular amplification and sequencing methodologies were required to recognize the rarely identified CVA1, but it is ambiguous whether CVA1 infection caused illness. Travelers should be aware of risks associated with swimming in natural waters when visiting areas where there is limited sewage treatment.

Severe enterovirus 68 respiratory illness in children requiring intensive care management

BACKGROUNDnEnterovirus 68 (EV-D68) causes acute respiratory tract illness in epidemic cycles, most recently in Fall 2014, but clinical characteristics of severe disease are not well reported.nnnOBJECTIVESnChildren with EV-D68 severe respiratory disease requiring pediatric intensive care unit (PICU) management were compared with children with severe respiratory disease from other enteroviruses/rhinoviruses.nnnSTUDY DESIGNnA retrospective review was performed of all children admitted to Childrens Mercy Hospital PICU from August 1-September 15, 2014 with positive PCR testing for enterovirus/rhinovirus. Specimens were subsequently tested for the presence of EV-D68. We evaluated baseline characteristics, symptomatology, lab values, therapeutics, and outcomes of children with EV-D68 viral infection compared with enterovirus/rhinovirus-positive, EV-D68-negative children.nnnRESULTSnA total of 86 children with positive enterovirus/rhinovirus testing associated with respiratory symptoms were admitted to the PICU. Children with EV-D68 were older than their EV-D68-negative counterparts (7.1 vs. 3.5 years, P=0.01). They were more likely to have a history of asthma or recurrent wheeze (68% vs. 42%, P=0.03) and to present with cough (90% vs. 63%, P=0.009). EV-D68 children were significantly more likely to receive albuterol (95% vs. 79%, P=0.04), magnesium (75% vs. 42%, P=0.004), and aminophylline (25% vs. 4%, P=0.03). Other adjunctive medications used in EV-D68 children included corticosteroids, epinephrine, and heliox; 44% of EV-D68-positive children required non-invasive ventilatory support.nnnCONCLUSIONSnEV-D68 causes severe disease in the pediatric population, particularly in children with asthma and recurrent wheeze; children may require multiple adjunctive respiratory therapies.

The complete genome sequences for three simian enteroviruses isolated from captive primates

In a recent study, we used RT-PCR and partial genome sequencing to detect simian enteroviruses SV6, SV19 and SV46, as well as two new enterovirus types (EV92 and EV103) in fecal specimens from rhesus macaques (Macaca mulatta), pigtail macaques (M. nemestrina), and sooty mangabeys (Cercocebus atys) with diarrheal disease at a US primate center. The complete genome sequences of representative SV46, EV92, and EV103 strains, presented here, show that SV46 and EV92 are typical of the simian enteroviruses classified within the species Human enterovirus A, while EV103 appears to belong to an unclassified species that also contains SV6 and N125/N203.

Clinical and epidemiologic characteristics of dengue and other etiologic agents among patients with acute febrile illness, Puerto Rico, 2012-2015.

Identifying etiologies of acute febrile illnesses (AFI) is challenging due to non-specific presentation and limited availability of diagnostics. Prospective AFI studies provide a methodology to describe the syndrome by age and etiology, findings that can be used to develop case definitions and multiplexed diagnostics to optimize management. We conducted a 3-year prospective AFI study in Puerto Rico. Patients with fever ≤7 days were offered enrollment, and clinical data and specimens were collected at enrollment and upon discharge or follow-up. Blood and oro-nasopharyngeal specimens were tested by RT-PCR and immunodiagnostic methods for infection with dengue viruses (DENV) 1–4, chikungunya virus (CHIKV), influenza A and B viruses (FLU A/B), 12 other respiratory viruses (ORV), enterovirus, Leptospira spp., and Burkholderia pseudomallei. Clinical presentation and laboratory findings of participants infected with DENV were compared to those infected with CHIKV, FLU A/B, and ORV. Clinical predictors of laboratory-positive dengue compared to all other AFI etiologies were determined by age and day post-illness onset (DPO) at presentation. Of 8,996 participants enrolled from May 7, 2012 through May 6, 2015, more than half (54.8%, 4,930) had a pathogen detected. Pathogens most frequently detected were CHIKV (1,635, 18.2%), FLU A/B (1,074, 11.9%), DENV 1–4 (970, 10.8%), and ORV (904, 10.3%). Participants with DENV infection presented later and a higher proportion were hospitalized than those with other diagnoses (46.7% versus 27.3% with ORV, 18.8% with FLU A/B, and 11.2% with CHIKV). Predictors of dengue in participants presenting <3 DPO included leukopenia, thrombocytopenia, headache, eye pain, nausea, and dizziness, while negative predictors were irritability and rhinorrhea. Predictors of dengue in participants presenting 3–5 DPO were leukopenia, thrombocytopenia, facial/neck erythema, nausea, eye pain, signs of poor circulation, and diarrhea; presence of rhinorrhea, cough, and red conjunctiva predicted non-dengue AFI. By enrolling febrile patients at clinical presentation, we identified unbiased predictors of laboratory-positive dengue as compared to other common causes of AFI. These findings can be used to assist in early identification of dengue patients, as well as direct anticipatory guidance and timely initiation of correct clinical management.

Enterovirus D68 Infection Among Children With Medically Attended Acute Respiratory Illness, Cincinnati, Ohio, July–October 2014

BackgroundnEnterovirus D68 (EV-D68) caused a widespread outbreak of respiratory illness in the United States in 2014, predominantly affecting children. We describe EV-D68 rates, spectrum of illness, and risk factors from prospective, population-based acute respiratory illness (ARI) surveillance at a large US pediatric hospital.nnnMethodsnChildren <13 years of age with ARI and residence in Hamilton County, Ohio were enrolled from the inpatient and emergency department (ED) settings at a childrens hospital in Cincinnati, Ohio, from 1 July to 31 October 2014. For each participant, we interviewed parents, reviewed medical records, and tested nasal and throat swabs for EV-D68 using real-time reverse- transcription polymerase chain reaction assay.nnnResultsnEV-D68 infection was detected in 51 of 207 (25%) inpatients and 58 of 505 (11%) ED patients. Rates of EV-D68 hospitalization and ED visit were 1.3 (95% confidence interval [CI], 1.0-1.6) and 8.4 per 1000 children <13 years of age, respectively. Preexisting asthma was associated with EV-D68 infection (adjusted odds ratio, 3.2; 95% CI, 2.0-5.1). Compared with other ARI, children with EV-D68 were more likely to be admitted from the ED (P ≤ .001), receive supplemental oxygen (P = .001), and require intensive care unit admission (P = .04); however, mechanical ventilation was uncommon (2/51 inpatients; P = .64), and no deaths occurred.nnnConclusionsnDuring the 2014 EV-D68 epidemic, high rates of pediatric hospitalizations and ED visits were observed. Children with asthma were at increased risk for medically attended EV-D68 illness. Preparedness planning for a high-activity EV-D68 season in the United States should take into account increased healthcare utilization, particularly among children with asthma, during the late summer and early fall.