Ferdaus Hassan

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

Comparison of the BD Veritor System for Flu A+B with the Alere BinaxNOW Influenza A&B Card for Detection of Influenza A and B Viruses in Respiratory Specimens from Pediatric Patients

ABSTRACT The performance characteristics of two commercially available rapid tests for influenza, the BD Veritor System for Flu A+B (BD) and the Alere BinaxNOW influenza A&B card (BN), were evaluated using 200 frozen clinical specimens collected from January 2011 to June 2012 from pediatric patients. Real-time reverse transcriptase PCR (RT-PCR) was used as the gold standard to evaluate the results obtained by the two different assays. Of the 200 specimens tested, real-time RT-PCR assay detected influenza A or B virus in 116 samples, while BD detected 104 samples and BN detected 84 samples as positive. The overall sensitivity and specificity for detection of both influenza A and B virus in comparison to those of real-time RT-PCR were 89.6% (95% confidence interval [CI], 82.2 to 94.3) and 98.8% (95% CI, 92.6 to 99.9) for BD Veritor and 72.4% (95% CI, 63.2 to 80.0) and 100% (95% CI, 94.5 to 100.0) for BinaxNOW. Workflow analysis indicated that overall processing times for a batch size of 10 specimens were virtually identical between both systems. Overall, these results indicate that the BD Veritor assay was more sensitive than the BinaxNOW assay in detection of influenza A and B viruses in respiratory specimens from pediatric patients.

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.

Molecular Evolution and Intraclade Recombination of Enterovirus D68 during the 2014 Outbreak in the United States

ABSTRACT In August 2014, an outbreak of enterovirus D68 (EV-D68) occurred in North America, causing severe respiratory disease in children. Due to a lack of complete genome sequence data, there is only a limited understanding of the molecular evolution and epidemiology of EV-D68 during this outbreak, and it is uncertain whether the differing clinical manifestations of EV-D68 infection are associated with specific viral lineages. We developed a high-throughput complete genome sequencing pipeline for EV-D68 that produced a total of 59 complete genomes from respiratory samples with a 95% success rate, including 57 genomes from Kansas City, MO, collected during the 2014 outbreak. With these data in hand, we performed phylogenetic analyses of complete genome and VP1 capsid protein sequences. Notably, we observed considerable genetic diversity among EV-D68 isolates in Kansas City, manifest as phylogenetically distinct lineages, indicative of multiple introductions of this virus into the city. In addition, we identified an intersubclade recombination event within EV-D68, the first recombinant in this virus reported to date. Finally, we found no significant association between EV-D68 genetic variation, either lineages or individual mutations, and a variety of demographic and clinical variables, suggesting that host factors likely play a major role in determining disease severity. Overall, our study revealed the complex pattern of viral evolution within a single geographic locality during a single outbreak, which has implications for the design of effective intervention and prevention strategies. IMPORTANCE Until recently, EV-D68 was considered to be an uncommon human pathogen, associated with mild respiratory illness. However, in 2014 EV-D68 was responsible for more than 1,000 disease cases in North America, including severe respiratory illness in children and acute flaccid myelitis, raising concerns about its potential impact on public health. Despite the emergence of EV-D68, a lack of full-length genome sequences means that little is known about the molecular evolution of this virus within a single geographic locality during a single outbreak. Here, we doubled the number of publicly available complete genome sequences of EV-D68 by performing high-throughput next-generation sequencing, characterized the evolutionary history of this outbreak in detail, identified a recombination event, and investigated whether there was any correlation between the demographic and clinical characteristics of the patients and the viral variant that infected them. Overall, these results will help inform the design of intervention strategies for EV-D68.

Comparison of three multiplex gastrointestinal platforms for the detection of gastroenteritis viruses

BACKGROUNDnViruses are major etiological agents of childhood gastroenteritis. In recent years, several molecular platforms for the detection of viral enteric pathogens have become available.nnnOBJECTIVE/STUDY DESIGNnWe evaluated the performance of three multiplex platforms including Biofires Gastrointestinal Panel (FilmArray), Luminex xTAG® Gastrointestinal Pathogen Panel (GPP), and the TaqMan Array Card (TAC) for the detection of five gastroenteritis viruses using a coded panel of 300 archived stool samples.nnnRESULTSnThe FilmArray detected a virus in 199 (96.1%) and the TAC in 172 (83.1%) of the 207 samples (187 samples positive for a single virus and 20 samples positive for more than one virus) whereas the GPP detected a virus in 100 (78.7%) of the 127 (97 positive for one virus and three positive for more than one virus) samples. Overall the clinical accuracy was highest for the FilmArray (98%) followed by TAC (97.2%) and GPP (96.9%). The sensitivity of the FilmArray, GPP and TAC platforms was highest for rotavirus (100%, 95.8%, and 89.6%, respectively) and lowest for adenovirus type 40/41 (97.4%, 57.9% and 68.4%). The specificity of the three platforms ranged from 95.6% (rotavirus) to 99.6% (norovirus/sapovirus) for the FilmArray, 99.6% (norovirus) to 100% (rotavirus/adenovirus) for GPP, and 98.9% (astrovirus) to 100% (rotavirus/sapovirus) for TAC.nnnCONCLUSIONnThe FilmArray demonstrated the best analytical performance followed by TAC. In recent years, the availability of multi-enteric molecular testing platforms has increased significantly and our data highlight the strengths and weaknesses of these platforms.

Evaluation of RIDA®GENE norovirus GI/GII real time RT-PCR using stool specimens collected from children and adults with acute gastroenteritis

BACKGROUNDnNorovirus is the leading cause of epidemic and sporadic acute gastroenteritis (AGE) in the United States. Widespread prevalence necessitates implementation of accurate norovirus detection assays in clinical diagnostic laboratories.nnnOBJECTIVEnTo evaluate RIDA®GENE norovirus GI/GII real-time RT-PCR assay (RGN RT-PCR) using stool samples from patients with sporadic AGE.nnnSTUDY DESIGNnPatients between 14u202fdays to 101 years of age with symptoms of AGE were enrolled prospectively at four sites across the United States during 2014-2015. Stool specimens were screened for the presence of norovirus RNA by the RGN RT-PCR assay. Results were compared with a reference method that included conventional RT-PCR and sequencing of a partial region of the 5end of the norovirus ORF2 gene.nnnRESULTSnA total of 259 (36.0%) of 719 specimens tested positive for norovirus by the reference method. The RGN RT-PCR assay detected norovirus in 244 (94%) of these 259 norovirus positive specimens. The sensitivity and specificity (95% confidence interval) of the RGN RT-PCR assay for detecting norovirus genogroup (G) I was 82.8% (63.5-93.5) and 99.1% (98.0-99.6) and for GII was 94.8% (90.8-97.2) and 98.6% (96.9-99.4), respectively. Seven specimens tested positive by the RGN-RT PCR that were negative by the reference method. The fifteen false negative samples were typed as GII.4 Sydney, GII.13, GI.3, GI.5, GI.2, GII.1, and GII.3 in the reference method.nnnCONCLUSIONSnThe RGN RT-PCR assay had a high sensitivity and specificity for the detection of norovirus in stool specimens from patients with sporadic AGE.

Multicenter Clinical Evaluation of the Alere™ i RSV Isothermal Nucleic Acid Amplification Assay

ABSTRACT The Alere i respiratory syncytial virus (RSV) assay is an isothermal nucleic acid amplification test capable of detecting RSV directly from respiratory specimens, with results being available in ≤13 min after test initiation. The objective of this study was to evaluate the performance characteristics of the Alere i RSV assay in a point-of-care setting by using direct nasopharyngeal (NP) swab specimens (direct NP) and nasopharyngeal swab specimens eluted and transported in viral transport medium (VTM NP). The study was a prospective, multicenter, clinical trial conducted at 9 sites across the United States to evaluate the clinical performance of the Alere i RSV assay with respiratory specimens obtained from both children (age, <18 years) and older adults (age, >60 years). The performance of the Alere i RSV assay was compared with that of the reference method, the Prodesse ProFlu+ real-time reverse transcriptase PCR (RT-PCR) assay. All specimens with discrepant test results were tested further by a second FDA-cleared PCR assay (the Verigene respiratory virus plus nucleic acid test; Luminex Inc., TX). A total of 554 subjects with signs and symptoms of respiratory infections were enrolled, and respiratory samples were collected in this study. In comparison with the ProFlu+ real-time RT-PCR, the overall sensitivity and specificity of Alere i RSV assay for the detection of RSV were 98.6% (95% confidence interval [CI], 94.4 to 99.7%) and 98.0% (95% CI, 95.8 to 99.1%), respectively, for direct NP and 98.6% (95% CI, 94.4 to 99.7%) and 97.8% (95% CI, 95.5 to 98.9%), respectively, for VTM NP. The Alere i RSV is a highly sensitive and specific molecular assay ideal for rapid RSV detection in patients in the point-of-care setting due to its minimal hands-on time and rapid result availability.

Multicenter evaluation of the Alere™ i influenza A&B assay using respiratory specimens collected in viral transport media

Rapid and accurate detection of influenza virus is critical for proper patient management. The Alere™ i Influenza A&B assay is an isothermal nucleic acid amplification test capable of detecting influenza A and B viruses directly from respiratory specimens. In this multicenter clinical trial conducted in the US, we evaluated the clinical performance of the Alere™ i Influenza A&B assay against that of the Prodesse ProFlu+ assay. A total of 1243 fresh, leftover nasal or nasopharyngeal swabs eluted in viral transport medium were tested by both assays. Sensitivity and specificity of the Alere™ i Influenza A&B assay were 97.8% (95% CI 94.6-99.2) and 96.6% (95% CI 95.2-97.5) for influenza A and 92.9% (95% CI 85.5-96.9) and 98.3% (95% CI 97.4-98.0) for influenza B. The Alere™ i Influenza A&B assay is an ideal molecular assay for influenza virus detection due to its high sensitivity and specificity with minimal hands-on and turn-around-time.

Clinical Impact of Two Different Multiplex Respiratory Panel Assays on Management of Hospitalized Children aged ≤ 24 months

Abstract Background Highly multiplexed molecular assays are popular in clinical laboratories due their high sensitivity, specificity and relatively rapid turn-around time (TAT) for results. Luminex™ respiratory viral panel (RVP) detects 12 respiratory viruses, while BioFire™ respiratory panel (RP) detects 20 respiratory pathogens (17 viruses, 3 bacteria). The aim of the current study was to compare the impact of RVP and RP assay on management of hospitalized children aged ≤24 months. Methods Retrospective data were collected to compare the clinical impact from two multiplex molecular assays (RVP, December 2008–May 2012; RP August 2012–June 2015) on management and outcomes of hospitalized patients. Patients aged ≤24 months and positive for at least one respiratory virus were included. Patients who were (1) receiving immune suppressive therapy, (2) neonates requiring intensive care, or (3) hospitalized for >7 days were excluded. Results A total of 810 patients in RVP and 2,095 patients in RP group were included. The median TAT for RVP and RP assay were 29 hours (IQR 26–58 hours) and 4 hours (IQR 2–8 hours), respectively (P < 0.001). Significantly higher number of children in RVP group (44%, 357/810) received empiric antibiotic therapy compared with RP group (28%, 595/2095) (P < 0.001). Following PCR test reporting, the rate of antibiotic discontinuation was higher in the RP group (23%, 135/595) vs. RVP group (16%, 56/357) (P < 0.001). Antibiotics were discontinued more often in older children aged 6–24 months (23%, 113/492) compared with children aged < 60 days (11%, 34/297) (P < 0.001). Following positive influenza test results, more children received timely oseltamivir in the RP group (85%, 48/56) compared with the RVP group (17%, 7/41) (P < 0.001). The median length of hospitalization (LOH) was shorter in the RP group (48 hours, IQR 32–76 hours) than in the RVP group (54 hours, IQR 39–89 hours) (P < 0.001). Conclusion Rapid availability of test results from RP assay was associated with reduced antibiotic use, timely antiviral therapy and decreased LOH. The implementation of a more comprehensive respiratory multiplex molecular assay with rapid reporting of test results has the potential to improve management of hospitalized children, decrease unnecessary antibiotic therapy and reduce overall costs. Disclosures R. Selvarangan, BioFire Diagnostics: Board Member and Investigator, Consulting fee and Research grant; Luminex Diagnostics: Investigator, Research grant